In vitro and in vivo detection of infectious pancreatic necrosis virus in fish by enzyme-linked immunosorbent assay

A double antibody enzyme-linked immunosorbent assay (ELISA) was used to detect infectious pancreatic necrosis virus (IPNV). The ELISA detected VR299 strain of IPNV at a dose of 10 to 20 ng of purified IPNV protein or 10(4) TCID50 in tissue culture fluid. Specificity of ELISA was demonstrated by an ELISA inhibition test. The ELISA did not detect infectious hematopoietic necrosis virus. Normal cell culture fluid and virus-non-inoculated rainbow trout (Salmo gairdneri Richardson) homogenate did not react in the test system. The IPNV was detected in rainbow trout fry inoculated with IPNV. Although infective virus titer in fish decreased rapidly 1 week after inoculation, IPNV antigen was detected by ELISA for 15 days. The IPNV antigen was detected in the fish tissue after inactivation of infective virus. The ELISA is a rapid and reliable method for the diagnosis of IPNV infection.     

The Lethal Dose of Largemouth Bass Virus in Juvenile Largemouth Bass and the Comparative Susceptibility of Striped Bass

Abstract

Largemouth bass virus (LMBV) is an iridovirus that was isolated from wild adult largemouth bass Micropterus salmoides in the southeastern United States in 1994. Although originally isolated from moribund wild fish, its virulence to juvenile largemouth bass is uncertain. To help clarify this point, two LMBV titrations were made in juvenile largemouth bass. Titers of LMBV in fathead minnow cells were 10^4.8 and 10^5.8 tissue culture infectious doses—50% cytopathic endpoint (TCID50) per milliliter, respectively. Tenfold serial dilutions of LMBV employed in each cell culture titration, injected intraperitoneally (0.1 mL/fish) into largemouth bass produced calculated lethal dose—50% mortality endpoints (LD50s) of 282 (10^2.45) and 288 (10^2.46) infectious doses in two consecutive infectivity trials. Virus yield of assayed infected fish averaged 10^8.5 TCID50/g and 10^7.7 TCID50/g in viscera of moribund and dead fish in the two trials and 10^6.5 TCID50/g in surviving exposed fish 14 d after infection. In a second experiment, largemouth bass had 100% mortality 5 d after injection while virus immersed fish had a significantly (P ≤ 0.005) lower mortality of 17% at 14 d. Similarly treated juvenile striped bass Morone saxatilis suffered 63% mortality after injection and significantly (P ≤ 0.005) lower mortality of 10% after immersion. In a third study of 25 d, 100% of injected largemouth bass died by 5 d after injection, and all of them were virus-positive. Injected striped bass had a significantly (P ≤ 0.005) lower mortality of 24%; all three fish were virus-positive initially, two fish were virus-positive at 18 d, and none were positive at 25 d. Juvenile largemouth bass were highly susceptible to LMBV injection and striped bass were moderately susceptible, but both species were only mildly susceptible when exposed by immersion.     

Immunosuppressive effect of infectious pancreatic necrosis virus on rainbow trout (Oncorhynchus mykiss)

The infectivity of infectious pancreatic necrosis virus (IPNV) for rainbow trout (Oncorhynchus mykiss) mononuclear leukocyte subpopulations was investigated to determine the mechanisms of immunosuppression caused by the virus. IPNV was recovered from nylon wool-adherent, surface immunoglobulin (Ig)-positive leukocytes of head kidney, spleen and peripheral blood collected from virus-inoculated fish with higher titers than non-adherent, Ig-negative cells. Non-adherent cell population showed mitogenic response to phytohemagglutinin and concanavalin A but not to lipopolysaccharide. Conversely, the responses of adherent cells to these mitogens were weak. Mitogenic response and non-specific cytotoxicity of head kidney leukocytes significantly decreased by the inoculation of fish with the virus. These results suggest that the suppression of immune responses is involved in the establishment of carrier state in fish after infection with IPNV.     

Paradoxical effects of cadmium exposure on antibacterial antibody responses in two fish species: inhibition in cunners (Tautogolabrus adspersus) and enhancement in striped bass (Morone saxatilis)

Previous work in a marine fish, the cunner (Tautogolabrus adspersus), showed that endocytosis of bacteria by cells in the liver and spleen was affected by 96-h exposure of the fish to cadmium at a concentration of 12 micrograms/ml; however, antibody response to sheep erythrocytes was not affected. Since the latter finding was questionable because of short immunization times, and data from more than a single fish species were desirable, both the cunner and an anadromous fish, the striped bass (Morone saxatilis), were examined for antibody responses against the bacterium Bacillus cereus in Freund's complete adjuvant during a 6- to 8-wk time period. Exposure to 12 micrograms/ml cadmium caused significant inhibition of serum antibody titers (P less than 0.007) in cunners. Paradoxically, antibody response in striped bass exposed to 10 micrograms/ml cadmium was enhanced sixfold. This enhancement was weaker but still evident when the antigen was injected 48 d after cadmium exposure. Peritoneal exudate cells from cadmium-exposed striped bass also showed more active migration through microporous filters than cells from non-exposed fish. Since the 96-h cadmium LC50 was 26 micrograms/ml for cunners and 20 micrograms/ml for striped bass, the differences in antibody response could not be explained on the basis of differences in cadmium toxicity. Although geometric mean liver cadmium levels after exposure at 15 degrees C were higher in cunners (163.8 +/- 1.2 micrograms/gm) than in striped bass (64.4 +/- 1.2 micrograms/g), cunners exposed to cadmium at 2 degrees C had lower cadmium levels (46.4 +/- 1.2 micrograms/g) which were still effective in inhibiting antibody production at 8 degrees C. On the other hand, striped bass not exposed to cadmium showed a strong, exponential rise in serum antibody when the temperature at immunization, 14 degrees C, was reduced to 9 degrees C; whereas cunners held in the same tanks exhibited a weaker, biphasic serum antibody response. Regardless of the cause (innate differences in cellular response, or better stress adaptability in an anadromous fish), the data show that chemical-stress effects on the immune system of one fish species cannot be extrapolated to another species.     

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     

Experimental induction of the carrier state in yearling brook trout: a model challenge protocol for IPNV immunization

Brook trout fry (Salvelinus fontinalis) were not protected from infectious pancreatic necrosis virus (IPNV) challenge by immersion vaccination with inactivated, purified virus at concentrations of 10(7) to 10(9) pfu/ml. Mortalities in vaccinated groups were higher than for the unvaccinated control group and appeared to be dose-dependent. A challenge protocol for adult brook trout was developed for future vaccine trials. A single intraperitoneal injection of virulent, purified virus was sufficient to make long-lasting carriers of 16 month-old trout. Fish underwent a transient viremia, identified by virus isolation from plasma and leucocytes. Feces were the most reliable samples for identification of IPNV carriers by non-sacrificial testing. Many fish in the remaining infected group were still carriers 12 and 27 weeks post-infection.     

Characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection

The present study characterized the homologous and heterologous immune response in type-I porcine reproductive and respiratory syndrome virus (PRRSV) infection. Two experiments were conducted: in experiment 1, eight pigs were inoculated with PRRSV strain 3262 and 84 days post-inoculation (dpi) they were challenged with either strain 3262 or strain 3267 and followed for the next 14 days (98 dpi). In experiment 2, eight pigs were inoculated with strain 3267 and challenged at 84 dpi as above. Clinical course, viremia, humoral response (neutralizing and non-neutralizing antibodies, NA) and virus-specific IFN-γ responses (ELISPOT) were evaluated all throughout the study. Serum levels of IL-1, IL-6, IL-8, TNF-α and TGF-β were determined (ELISA) after the second challenge. In experiment 1 primo-inoculation with strain 3262 induced viremia of ≤ 28 days, low titres of homologous NA but strong IFN-γ responses. In contrast, strain 3267 induced longer viremias (up to 56 days), higher NA titres (≤ 6 log₂) and lower IFN-γ responses. Inoculation with 3267 produced higher serum IL-8 levels. After the re-challenge at 84 dpi, pigs in experiment 1 developed mostly a one week viremia regardless of the strain used. In experiment 2, neither the homologous nor the heterologous challenge resulted in detectable viremia although PRRSV was present in tonsils of some animals. Homologous re-inoculation with 3267 produced elevated TGF-β levels in serum for 7–14 days but this did not occur with the heterologous re-inoculation. In conclusion, inoculation with different PRRSV strains result in different virological and immunological outcomes and in different degrees of homologous and heterologous protection.     

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.     

Ultrastructural observations on peritoneal exudate cells from the striped bass

An ultrastructural study was conducted of the principal cells recovered from the peritoneal exudate of striped bass (Morone saxatilis) injected with killed Bacillus cereus. The most frequently observed cells were identified as macrophages, eosinophils, and neutrophils on the basis of comparing their structure with that reported for other fish species and higher vertebrates. All three of the types of cells were phagocytic; however, the numbers of bacteria engulfed were limited. Like other studies of this type in fish, the peritoneum of the striped bass proved to be a good source of cells for morphological investigations of phagocytosis.     

Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.     

The occurrence and transmission characteristics of airborne H9N2 avian influenza virus

To better understand the transmission route of H9N2 avian influenza virus (AIV), two duplicate trials were conducted to observe the process of aerosol infection and direct contact in specific pathogen free chickens. Fifteen chickens (G1) were inoculated with H9N2 AIV and housed together with another 15 chickens (G2) in the same positive-negative-pressure isolator (A). Fifteen chickens (G3) were bred in another isolator (B) which was connected with A so that air could flow unidirectionally from A to B. Air, oropharyngeal and cloacal swabs, and blood samples were collected for the detection of aerosolized virus, virus shedding, and seroconversion. AIV aerosols were initially detected at day 2-3 post inoculation (dpi), reaching peak concentrations at 7 dpi. Virus shedding was detected in all chickens of G2, but only in a part in G3 (T1: 87%, T2: 80%). Antibodies were initially detected at 4-5 dpi, peaking at 14-21 dpi. The results showed that H9N2 AIV could be transmitted by both aerosol exposure and direct contact.     

Porcine circovirus type 2 (PCV2)-infection and re-inoculation with homologous or heterologous strains: virological, serological, pathological and clinical effects in growing pigs

Long-term PCV2 infection and/or concurrent infection with genotypes PCV2a and PCV2b may play a role in the development of clinical porcine circovirus-associated disease (PCVAD). To evaluate this premise, 24 11-week-old specific pathogen-free (SPF) pigs were randomly assigned to 1 of 4 treatments: negative controls, a single inoculation with PCV2a, single inoculation followed by re-inoculation with a homologous PCV2a strain, or repeated inoculations with heterologous strains (PCV2a, PCV2b). Pigs were evaluated for clinical signs daily through 140 days post inoculation (dpi). Serum samples were collected every other day from dpi 0 through 14 and weekly thereafter. PCV2-inoculated pigs were viremic by dpi 2 and 13 of 18 pigs remained viremic at 140 dpi. No statistical differences in the onset, level, or duration of PCV2 viremia were detected among treatment groups. Anti-PCV2 antibodies were detected between 14 and 28 dpi and were present through 140 dpi without statistical differences in antibody response among treatment groups. In the current study, pigs had extended viremia combined with detectable tissue PCV2 antigen levels despite the presence of high levels of anti-PCV2 antibody; however, no clinical disease was observed.     

Transmission of hog hog cholera virus by mosquitoes.

Mosquitoes trapped during an epizootic of hog cholera (HC) in Maryland in 1969 were prepared into 40 pools which were inoculated in pigs. Hog cholera virus was confirmed in pigs inoculated with 8 of 40 pools of mosquitoes. Generally, the pigs contracting HC developed chronic infections with persistent viremia that lasted 30 or more days. Two pigs seemed healthy when euthatized 62 and 80 days after inoculation, yet viremia of high titer was detected in each. Experimental studies were performed with 2 laboratory strains of mosquitoes, Aedes aegypti and Culex tarsalis, to determine if biological and mechanical transmission occur. Biological transmission was not confirmed, but HC virus was retained in A aegypti for 3 days. Mechanical transmission was confirmed with A aegypti in 2 of 9 experiments.     

Detection of infectious pancreatic necrosis virus (IPNV) RNA by hybridization with an oligonucleotide DNA probe

Marine farming of Atlantic salmon (Salmo salar) is growing rapidly in Norway, and fish diseases have now become one of the biggest obstacles for this new industry. Infectious pancreatic necrosis virus (IPNV) is commonly found in fish from Norwegian sea farms. Diagnosis of IPNV infection is, at present, mainly based on virus isolation in cell cultures and identification by neutralization tests (NT). A DNA-RNA hybridization assay was developed using a 24 base DNA oligonucleotide probe. This is homologous to a part of the nucleotide sequence of the IPNV genome coding for a protease. RNA extracted from IPNV and harvest from infected cell cultures were fixed to nylon filters and hybridized with the 32P end-labelled probe. The results showed that the probe specifically identified IPNV from these two materials, both for the three different virus strains (Ab, Sp and VR-299) used, and for several different field isolates. It did not hybridize with reoviruses or non-infected cell cultures used as controls. These results indicate that the probe is not serotype specific, and furthermore that RNA extraction is not required before hybridization. This method may be a useful alternative to NT for routine identification of IPNV, particularly when non-radioactive labelling of the probe is introduced.     

Prevalence of Streptococcus iniae in tilapia, hybrid striped bass, and channel catfish on commercial fish farms in the United States

OBJECTIVE: To determine the prevalence of Streptococcus iniae in tilapia (Oreochromis spp), hybrid striped bass (Morone chrysops X M saxatilis), and channel catfish (Ictalurus punctatus) on commercial fish farms in the United States. ANIMALS: 1,543 fish (970 tilapia, 415 hybrid striped bass, and 158 channel catfish). PROCEDURES: The dry-swab technique was used for collection of specimens for streptococcal isolation. Specimens were shipped by overnight delivery and processed by use of standard bacteriologic techniques. RESULTS: Streptococcus iniae was not isolated from market-size channel catfish. Prevalence in tilapia and hybrid striped bass was 37 of 970 (3.81%) and 30 of 415 (7.23%), respectively. Prevalence by farm ranged from 0.0 to 27.4% for tilapia and 0.0 to 21.6% for hybrid striped bass. In tilapia, prevalence was lowest in market-size and nursery fish (4 of 239 [1.67%] and 3 of 339 [0.88%], respectively), with an increase in prevalence for fish in the grow-out stage (30 of 337 [7.96%]). For hybrid striped bass, prevalence was lowest in nursery and market-size fish (3 of 96 [3.12%] and 1 of 47 [2.12%], respectively) and highest in fish in the grow-out stage (26 of 272 [9.56%]). Prevalence in market-size tilapia and hybrid striped bass was 5 of 286 (1.75%). CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study do not support the contention that S iniae is a serious public health threat associated with commercially raised fish; rather, it represents a limited risk for older or immunocompromised people who incur puncture wounds while handling and preparing fish.     

Evaluation of zebrafish Danio rerio as a model for enteric septicemia of catfish (ESC)

Zebrafish (also known as zebra danio) Danio rerio were injected intramuscularly with Edwardsiella ictaluri at doses of 6 x 10(3), 6 x 10(4), or 6 x 10(5) colony-forming units per gram (CFU/g) or sterile phosphate-buffered saline (sham) or were not injected. Mortality occurred from 2 to 5 d postinjection (dpi) at rates of 0, 76.6, and 81.3% for the low, medium, and high doses, respectively, and E. ictaluri was isolated from dead fish. Survivors were sampled at 10 dpi and E. ictaluri was not isolated. Sham-injected and noninjected controls did not suffer mortality. Histopathology trials were performed in which zebrafish were injected with 1 x 10(4) CFU/g or sham-injected and sampled at 12, 24, 48, 72, and 96 h postinjection for histological interpretation. Collectively, these zebrafish demonstrated increasing severity of splenic, hepatic, cardiac, and renal interstitial necrosis over time. To evaluate the progression of chronic infection, zebrafish were injected with 1 x 10(2) CFU/g and held for 1 month postinjection. Beginning at 12 dpi and continuing for an additional 2 weeks, zebrafish demonstrated abnormal spiraling and circling swimming behaviors. Histopathology demonstrated necrotizing encephalitis. In immersion trials, zebrafish were exposed to low, medium, and high doses (averaging 1.16 x 10(5), 1.16 x 10(6), and 1.16 x 10(7) CFU/mL of tank water) of E. ictaluri for 2 h. Mortality occurred from 5 to 9 d postexposure at rates of 0, 3.3, and 13.3% for the low, medium, and high doses, respectively; E. ictaluri was isolated from dead fish. Channel catfish Ictalurus punctatus exposed to the medium doses suffered 100% mortality, and E. ictaluri was isolated from these fish. This study demonstrates the potential use of zebrafish as a model for E. ictaluri pathogenesis.     

Communications: Detection of Anti-Amyloodinium ocellatum Antibody from Cultured Hybrid Striped Bass (Morone saxatilis × M. chrysops) during an Epizootic of Amyloodiniosis

Abstract

After an epizootic of amyloodiniosis (caused by the protozoan Amyloodinium ocellatum) in a commercial aquaculture facility for hybrid striped bass (female striped bass Morone saxatilis × male white bass M. chrysops), sera from these fish, as well as from others that had been experimentally immunized with the parasite, were evaluated by an enzyme-linked immunosorbent assay for antibody specific for the parasite. Titers were similar between the fish infested in culture and the experimentally immunized fish, and were significantly higher in both the cultured and artificially exposed fish than in unexposed fish. These results suggest that an infestation of A. ocellatum can stimulate the production of humoral antibodies to the parasite, providing further evidence that natural infestations of the parasite may confer protective resistance in fish that survive the initial parasitic infestation.     

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