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1.
Inactivated infectious haematopoietic necrosis virus (IHNV) vaccines   总被引：1，自引：0，他引：1  
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.  相似文献   

2.
Three strains of infectious pancreatic necrosis (IPN) virus of salmonid fishes were found to be closely related antigenically when compared by cross plaque neutralization. They differed significantly, however, in stability during freezing and storage at ?60°C. The American reference strain VR-299, which was the most stable, required consistently less antibody for a given degree of neutralization than the other two, regardless of whether the antiserum was specific for that strain or for either of the others. The CTT strain was the most unstable of the three, and required the greatest amount of antibody for a comparable degree of neutralization. The COHO strain was intermediate both in stability and in antibody requirement.The results seem to indicate that the CTT virus required the most antibody because it contained the largest amount of non-infectious virus (to chinook salmon embryo cells), which would still bind antibody and render it unavailable for neutralizing activity. The VR-299 virus contained the least amount of non-infectious virus, and consequently required the smallest amount of antibody.It is suggested that the relative stability of viral strains and possible variation in the content of non-infectious virus should be taken into account or controlled when quantitative neutralization data are used to differentiate or identify viral serotypes.  相似文献   

3.
Abstract. A double antibody enzyme-linked immunosorbent assay (ELISA) for the detection of infectious pancreatic necrosis virus (IPNV) is described. The sensitivity of the assay reached 102 TCID50 per 0·1 ml of culture fluid. The specificity of anti-IPNV sera and of the assay was confirmed by agar-gel immunodiffusion, by the direct immunoperoxidase technique for the deletion of IPNV in tissue cultures and by the ELISA inhibition test. High values of specific inhibition (over 90% at serum dilutions 1:40–1:2560) and low values of non-specific inhibition (8·4% at serum dilution 1:160) demonstrated the quality of the rabbit anti-IPNV serum. The results of ELISA agreed well with those of virological examinations. The potential of ELISA for investigations of a large series of field samples is discussed.  相似文献   

4.
Abstract. The pathogenicity of the agent causing viral nervous necrosis (VNN) of striped jack, Pseudocaranx dentex (Bloch & Schneider), was examined in striped jack and other selected marine fish species. Fish were exposed to purified striped jack nervous necrosis virus (SJNNV) (0·1–100 ng ml−1) or homogenates of diseased striped jack larvae. Striped jack larvae (3·5 and 4·4 mm total length) were susceptible to the virus, but juveniles (78 mm) were not. The viral antigens were detected by indirect ELISA and the characteristic pathological changes, i.e. vacuolation in the retina and brain, were reproduced in the affected larvae. The infection was also established in healthy larvae by cohabitation with the diseased larvae. Larvae of red sea bream, Pagrus major Temminck & Schlegel, yellowtail, Seriola quinqueradiata Temminck & Schlegel, and goldstriped amberjack, Seriola lalandi Valenciennes, were not susceptible to SJNNV.  相似文献   

5.
Abstract. The isolation and characterization of infectious pancreatic necrosis (IPN) virus from a goldfish, discus fish and bream is described. The fish from which the isolates were recovered showed no pathological signs of IPN. All three virus isolates were neutralized by antiserum to IPN, strain Ab, but not by antiserum to the Sp or VR-299 strains. They were morphologically identical to IPN virus in negative stain electron microscopy, grew in the cytoplasm of BF-2 cells, as shown by immuno-fluorescence and, like IPNV, were stable to heating, lipid solvents and acid pH.  相似文献   

6.
Abstract. The immuno dot blot technique is a rapid, sensitive and quantitative method for the diagnosis of infectious pancreatic necrosis virus ( ipnv ) in both infected tissue culture lysates and tissue homogenates of fish. Uninfected tissue culture lysates and fish homogenates do not react in the peroxidase dot blot system after appropriate treatment. The immuno dot blot method takes 4h to complete, and the dot blot membrane can be stored in the dark for a long period. The viral proteins in the infected tissue culture cells could be detected at 3h post-infection if an initial high MOI was used. The minimum dose of ipnv-ab strain which could be detected using homologous serum was 2·5 ng purified ipnv protein/dot. The smallest infectious dose of VR-299, SP, AB, EVE and CY-1 ipnv which could be detected by the immuno dot blot system was 104 to 105 TCID50. When one μg of purified ipnv was used, a 1·6 × 104 dilution of rabbit anti-AB antisera could be detected.  相似文献   

7.
目前检测卵黄抗体的方法很多,但IHA方法具有简便快速的优点,本研究的目的是为了建立抗犬瘟热高免卵黄抗体检测方法。采用犬瘟热病毒和犬细小病毒二联疫苗处理作为疫苗抗原,用犬瘟热病死犬的肝脏部分处理后作为组织抗原,制备敏化绵羊红细胞,用IHA方法确定了犬二联疫苗抗原和犬瘟热病毒组织抗原的最适含量。二联疫苗提取抗原的含量为0.7 mg/mL致敏红细胞时,与犬瘟热单克隆抗体反应显著,抗体效价为1∶256;而患犬瘟热松狮犬的肝脏组织抗原蛋白浓度为0.5mg/mL,与犬瘟热单克隆抗体反应显著,抗体效价为1∶128。经过IHA验证,制备的松狮犬瘟热肝组织抗原,蛋白含量0.5 mg/mL 1%致敏红细胞,对应的单抗、鸡血清均出现了凝集,阳性对照也出现凝集,阴性对照未出现凝集。建立的IHA抗体检测方法,为准确检测制备抗犬瘟热高免卵黄抗体的效价奠定了基础。  相似文献   

8.
A monoclonal antibody (designated MAb-007) was produced against the pathogenic haemoflagellate Cryptobia salmositica Katz. This IgG3 antibody recognized the 47-kDa antigenic polypeptide of C. salmositica (SDS-PAGE and Western immuno-blotting). The antibody did not agglutinate live parasites, and there was no change in the staining intensity of the 47-kDa band on Western immunoblots after immunoabsorption of MAb-007 with live intact parasites. The 47-kDa antigen recognized by MAb-007 was localized in the cytoplasm of the parasite (immunogold labelling and electron microscopy). The monoclonal antibody cross- reacted with the 47-kDa polypeptides of C. bullocki Srrout and C. catostomi Bower & Woo. It was used in an antigen-capture ELISA for the detection of parasite antigen in the plasma of rainbow trout inoculated with the parasite, or with an attenuated vaccine strain of C. salmositica. All pre-infection plasma were negative while all infected fish with detectable parasitaemias were positive for antigen at 1–9 weeks after infection. Parasite antigen was even detected in vaccinated fish that were negative for parasites using the wet mount microscopic technique. The antigen-capture ELISA detected C, salmositica antigen in whole cell lysate preparations at concentrations as low as 0.5 μg ml-1. Fifty microlitres of fish plasma was required in the antigen-capture ELISA, and the use of a plate reader and 96-well plates facilitated rapid analysis of a large number of plasma samples. The sensitivity of the assay makes it a potentially useful tool for detection of Cryptobia infections.  相似文献   

9.
Abstract. One thousand each of pond and hatchery reared 0+ brown trout, Salmo trutta L., were fin-clipped and released in a 1300-m2 large earthen pond. The pond was drawn down 5 days after the introduction, and descending individuals were caught in a trap at the outlet. A total of 904 pond-reared and 890 hatchery-reared fish were recaptured, i.e. a loss of 9·6 and 110% respectively, A total of 25 pond-reared and 16 hatchery reared fish were recorded stranded in the pond during the draw-down, accounting for 2·7 and 1·8% of total recoveries.
Pond-reared fish descended significantly earlier than did hatchery fish. Most individuals descended during the first 4·5h (75-83%). However, the final recoveries were made 10 days (233h) after the first descent.  相似文献   

10.
Abstract. An indirect fluorescent antibody (IFA) test was developed to detect viral antigen in tissue sections prepared from rainbow trout experimentally inoculated with infectious pancreatic necrosis virus (IPNV). Specific fluorescence was present in the pancreatic acinar tissue and occurred as brilliant fluorescence throughout the mesentery surrounding the pyloric caeca and intestines. In addition, multiple foci of fluorescent cells were seen occasionally in the kidneys and liver of infected fry. Fluorescence was not observed in tissues other than the pancreas, kidneys, and liver. The IFA test was found to be quite specific and offers a rapid means of diagnosing IPN during acute outbreaks.  相似文献   

11.
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12.
Cyprinid herpesvirus 2 (CyHV‐2) is the main pathogen responsible for causing haematopoietic necrosis disease in Carassius auratus gibelio. Although many nucleic acid‐based diagnostic methods have been applied, no stable and sensitive immunological diagnostic approaches have been reported. In this study, to detect CyHV‐2 in clinical samples using immunological methods, recombinant ORF72 protein (pORF72), encoded by the CyHV‐2 ORF72 gene, was used as a capture antigen to identify blood and tissues infected with CyHV‐2. First, ORF72 gene was amplified from the CyHV‐2 genome and cloned into a PGEX‐4t‐3 expression vector to produce pORF72 in Escherichia coli. The purified pORF72 was used as an immunogen to prepare monoclonal antibodies. The Western blotting assays revealed that the monoclonal antibody could specifically identify the pORF72. Furthermore, an immunohistochemical protocol and a blood smear method were established to detect CyHV‐2 in carps. The results indicate that the monoclonal antibody against pORF72 could be utilized as an effective detection tool for haematopoietic necrosis disease in Carassius auratus gibelio.  相似文献   

13.
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14.
Abstract. In 1969, a new kind of epizootic occurred among eels in Japan and virological investigation was initiated. A new virus designated eel virus european (EVE) was isolated and biological, cytological, serological tests and infectivity trials were carried out. In its CPE on RTG-2 cells and additionally in its biological properties and particle size, EVE was found to be similar to infectious pancreatic necrosis virus (IPNV). Serologically, EVE was similar to the ď Honnincthun, France, strain of IPNV. However, infectivity trials showed that EVE and IPNV differed; EVE killed Japanese eels Anguilla japonica but not rainbow trout fry Salmo gairdneri , while IPN virus killed rainbow trout fry but not eels. We consider EVE to be the primary agent causing the new epizootic and propose the name viral kidney disease for the resulting clinical condition.  相似文献   

15.
Abstract. Cyprinid herpesvirus 1 (CHV) or Herpesvirus cyprini was virulent for carp, Cyprinus carpio L., fry following 1 h immersion in water at 20 °C. Cumulative mortality for carp fry was 86–97% in 2-week-old common carp, 20% in 4-week-old fancy carp, and 0% in both 8-week-old common and fancy carp. The virus did not produce mortality in fry of crucian carp, grass carp or other cyprinids. It was also oncogenic in carp, inducing papillomas to the extent of 55% among both common and fancy carp fry. The neoplasms appeared 5–6 months after carp had been exposed to the virus by immersion and recurred at an incidence of 83% in carp 7·5 months post-desquamation of the tumour. The CHV was reisolated from all moribund fish and from all survivors. It also induced papillomas at an incidence of 13% in adult mirror carp and at 10% in adult fancy carp 5 months after intraperitoneal inoculation of 105 TCID50 ml-1 fish. The virus was rcisolated only from the ncoplastic tissue and not from internal organs. The neoplasms were normally located on fin, skin or mandible, at the intraperitoneal inoculation site. Specific fluorescence for CHV antigen was frequently detected in the gills, liver, kidneys and intestine of 2-week-old fry from 3 to 21 days following challenge with CHV. It was found in greater concentrations in experimentally induced papillomata on 2-week-old carp fry survivors examined 24 weeks after challenge than in naturally occurring neoplasms.  相似文献   

16.
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.  相似文献   

17.
Abstract. A commercially prepared vaccine against Edwardsiella ictaluri was used to vaccinate 12-day-old channel catfish fry by immersion, or by immersion plus an oral booster 2 months later. One month after the fish were fed the booster vaccine, they were challenged by waterborne exposure to 2·1 × 106 cells ml−1 of E. ictaluri. Immersion only vaccinated fish suffered 6·7% mortality and immersion plus oral-boosted fish had a 3·3% mortality. Mortality among non-vaccinated controls was 96·7% and was significantly ( P < 0·01) above the vaccinated mortality. The relative per cent survival for the immersion-only fish was 93·1, while it was 96·6 for the immersion plus oral-boosted fish. Agglutinating antibody titres of the vaccinated fish were significantly ( P < 0·05) higher than the control fish. When the ponds were drained 6 months after stocking, 42·7% of non-vaccinated, 56·3% of immersion-only and 70·8% of immersion plus oral-boosted fish were harvested. Survival of immersion plus orally-boosted fish was significantly ( P < 0·05) higher than the controls of immersion-only fish. Duplicate populations of immersion plus oral-booster-vaccinated fish grew 34% and 56% faster, respectively, on an average daily gain than the control fish, while immersion-only fish in one pond grew 20% less per day and fish in the second pond grew 48% faster.  相似文献   

18.
Betanodavirus infection of fish has been responsible for mass mortalities in aquaculture hatcheries worldwide. Betanodaviruses possess a bipartite single-stranded RNA genome consisting of the 3.1 kb RNA1 encoding an RNA-dependent RNA polymerase and the B2 protein, while the 1.4 kb RNA2 encodes the viral nucleocapsid protein, alpha. A panel of six monoclonal antibodies against the alpha protein of greasy grouper nervous necrosis virus (GGNNV) was developed for use in diagnostics. All antibodies reacted with native and recombinant alpha in immunoblot and indirect immunofluorescence assays. Each of the monoclonal antibodies reacted against discrete regions of the alpha protein, though none reacted with the extreme C-terminal region of the protein. One of the monoclonal antibodies, specific for the K151-T246 region of alpha, was used for the development of an antigen capture ELISA. In this assay we could detect 10(3)-10(4) TCID(50) units of virus derived from infected tissue culture supernatants. Head tissue extracts prepared from experimentally infected barramundi, Lates calcarifer, juveniles were assayed for GGNNV using the antigen capture assay and a clear increase in alpha antigen was detected from 5 to 15 days post-challenge. The assay thus represents a useful method for field-based detection of betanodavirus in fish hatcheries.  相似文献   

19.
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.  相似文献   

20.
Flavobacterium psychrophilum is the aetiologic agent of bacterial coldwater disease and rainbow trout fry syndrome. In this study, we compared a wild‐type strain (CSF 259‐93) with a rifampicin‐resistant strain and virulence‐attenuated strain of F. psychrophilum (CSF 259‐93B.17). The attenuated strain harboured a mutation in the rpoB gene consistent with resistance to rifampicin. Two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and mass spectrometry demonstrated an altered proteome with eight proteins characteristic for the parent strain and six that were unique to the attenuated strain. Immunoblotting with a diagnostic monoclonal antibody (FL‐43) identified a putative antigen (FP1493) that was subsequently cloned, expressed as a recombinant protein and confirmed as recognized by FL‐43. 2D‐PAGE, immunoblotting with rainbow trout, Oncorhynchus mykiss (Walbaum), convalescent antisera and mass spectrometry of bacterial whole‐cell lysates revealed several uniquely expressed immunoreactive proteins including FP1493. An FP1493 recombinant subunit vaccine was tested, but did not provide protection against challenge with the CSF259‐93 strain. While the exact mechanism responsible for altered protein synthesis and attenuation of CSF 259‐93B.17 is still unknown, the differentially expressed immunoreactive proteins are a valuable resource to develop subunit vaccines and to identify proteins that are potentially involved in disease.  相似文献   

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