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.     

The detection of Salmonella infection in calves by the fluorescent antibody test

The fluorescent antibody test (FAT) was used for the direct detection of salmonellas in 182 lymph nodes from adult cattle and from calves submitted for autopsy or used for experimental purposes. Salmonellas were detected in 22 samples by the FAT and in 18 by culture examination. The predictive value of the FAT was 68% and it was concluded that the test could be used for the rapid presumptive diagnosis of bovine salmonellosis.     

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.     

应用HEV ORF2蛋白McAb间接免疫荧光法检测HEV的研究

应用抗猪戊型肝炎病毒（HEV）ORF2蛋白单克隆抗体和猪戊型肝炎病毒多克隆抗血清对感染猪戊型肝炎病毒的A549细胞培养物进行了间接免疫荧光检测病原的比较研究。单克隆抗体（McAb）进行的间接免疫荧光染色后,HEV感染细胞培养物中多数细胞在其胞浆和细胞膜上出现黄绿色荧光,未接种病毒的细胞培养物被伊文思蓝染成红色,未见有黄绿色荧光染色的细胞存在;多克隆抗血清对接种病毒的细胞培养物染色后,荧光强度高,呈现亮绿色,但未接种HEV的细胞培养物亦有一定的非特异性荧光反应。结果表明,利用所研制的抗HEVORF2蛋白McAb进行间接免疫荧光法对病原检测具有很高的特异性。     

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.     

In situ hybridization for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods.

Archived formalin-fixed, paraffin-embedded tissues from 25 pigs naturally infected with transmissible gastroenteritis virus (TGEV) were examined by in situ hybridization for TGEV nucleic acid using a nonradioactive digoxigenin-labeled cDNA probe that targeted the nucleocapsid sequence of TGEV strains. The results of in situ hybridization for the detection of TGEV were compared with virus isolation (VI), a fluorescent antibody test (FAT), and transmission electron microscopy (TEM). VI, FAT, and TEM were tested over a course of time before the in situ hybridization was performed. Positive hybridization signals were detected in duodenal, jejunal, and ileal enterocytes from 21 pigs. Hybridization signals were confined to the cytoplasm. Intestinal specimens from 25 piglets were evaluated by 4 tests. Twenty-one of 25 were positive by in situ hybridization. Of these 21 samples, 5 (24%) were positive for TGEV by all 4 tests, 15 (71%) were positive by FAT, 14 (67%) were positive by VI, and 6 (29%) were positive by TEM. In situ hybridization for the detection of TGEV in formalin-fixed, paraffin-embedded tissues provides a rapid means of confirmation of a histopathological diagnosis of TGEV without virus isolation, or when only formalin-fixed intestinal specimens were available.     

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.     

Comparison of fluorescent antibody tests for tuberculosis and paratuberculosis with antigens coupled to insoluble spheres or taken up by macrophages

Sera of 106 cattle from farms with histories of Mycobacterium johnei infection and sera from 15 human tuberculous patients as well as a number of control sera were examined by means of two different fluorescent antibody tests (FAT) for the occurrence of antibodies against M. johnei and M. tuberculosis respectively. The antigens used were PPD johnin and PPD tuberculin. In the macrophage uptake FAT (MU/FAT) mouse macrophages after phagocytosis of the tuberculins served as the matrix; in the tests performed using the defined antigen substrate spheres (DASS) system, Sepharose beads activated by CNBr were used for the matrix. A good correlation was found between the results of the DASS/FAT and those of the MU/FAT, which is known to be a sensitive and specific test in the diagnosis of Johne's disease in cattle. It is suggested that the FAT, with utilization of the DASS system, might have good prospects for routine examination for antibodies against species of Mycobacterium.     

Diagnosis and prevalence of leptospira infection in aborted and stillborn horses

A study was conducted to evaluate a recently available fluorescent antibody test (FAT) conjugate for the detection of leptospires in tissues of aborted and stillborn horses, to determine the leptospira antibody titers and compare serologic test results with FAT results, and to determine the prevalence of leptospira-induced abortions and stillbirths in the equine population of central Kentucky. From July 1, 1988 through June 30, 1989, 15 (2.5%) of 594 submissions (fetuses, stillborn foals, and/or placentas) were diagnosed as leptospirosis by the FAT (14 of 15 tested) and/or microscopic agglutination test (12 of 14 tested). Of the 12 serologically positive fetal fluids, 10 had high tigers against Leptospira interrogans serovar pomona and 2 against serovar grippotyphosa.     

A single dilution enzyme-linked immunosorbent assay for the quantitative detection of antibodies to bovine herpesvirus type 1

Three serological assays were compared for detection of antibodies to bovine herpes-virus type 1. These were virus neutralization (VN), enhanced complement fixation (CF) and enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using an infected cell lysate antigen and purified virus and was optimized in relation to antigen and antisera dilutions. The CF assay was enhanced by the addition of bovine complement. These 3 assays were compared for detection of: specific virus antibody titers; sero-conversions; early antibody response in experimentally-infected cattle. Both ELISA end-point titers and single dilution values were found to be more sensitive than the CF or VN assays for specific antibody level quantitation. With a single dilution ELISA test procedure a correlation was obtained between ELISA values and VN titers. Using the single dilution ELISA test the assay also detected antibodies in experimentally-infected cattle before either the VN or CF assays, and agreed with the VN test in 35/38 seroconversions found by 4-fold or more VN changes between acute and convalescent paired sera from naturally-infected animals. The single dilution ELISA was a rapid and sensitive test for routine antibody detection in bovine sera.     

Comparison of three methods of differentiating bovine mycoplasma.

Growth inhibition (GI) by specific antisera, direct fluorescent antibody technique (FAT), and fatty-acid profile analysis by gas-liquid chromatography (GLC) were compared as methods for identifying 13 strains of bovine mycoplasma. By the FAT, there were 8 bilateral and 13 unilateral cross reactions. In the GI study, there were 4 bilateral and 7 unilateral cross reactions. In both FAT and GI studies, there were fluorescence and inhibition, respectively, with 13 homologous antisera. Analysis by GLC revealed the 13 mycoplasmas could be placed into 4 distinct chromatographic groupings. The GLC profiles of 2 organisms were sufficiently unique that they could be used for specific identification.     

Laboratory decision-making during the classical swine fever epidemic of 1997-1998 in The Netherlands

The National Reference Laboratory for classical swine fever (CSF) virus in the Netherlands examined more than two million samples for CSF virus or serum antibody during the CSF epizootic of 1997–1998. The immense amount of samples and the prevalence of border disease (BD) virus and bovine viral diarrhoea (BVD) virus infections in Dutch pig herds necessitated the diagnostic efforts of the laboratory to be focused on generating CSF specific test results throughout the eradication campaign.

Detection of 82% of the 429 outbreaks was achieved through the combined use of a direct immunofluorescence and peroxidase assay (FAT/IPA) with samples (tonsils) collected from clinically-suspected pigs. This suggests that in the majority of the outbreaks, the pigs had clinical signs that were recognised by the farmer and/or veterinarians, indicating the presence of CSF virus in a pig herd. A positive diagnosis of 74% of all the tissue samples (tonsils) collected at infected pig holdings was established by FAT. More than 140,000 heparinised blood samples were examined by virus isolation, resulting in the detection of 4.5% of the infected herds. CSF virus was isolated in approximately 29% of all the blood samples collected from pigs at infected or suspected farms.

Several serological surveys — each done within a different framework — led to the detection of 13.5% of the total number of outbreaks. The detection of CSF virus antibody in serum was carried out by semi-automated blocking ELISA. Approximately 28.5% of the sera which reacted in the ELISA were classified as CSF virus-neutralising antibody positive and 26.5% as positive for other pestiviruses following the virus neutralisation test (VNT).

We concluded that two of the CSF laboratory diagnostic methods described were determinative in the eradication campaign: first, the FAT for the screening of diseased pigs; and second, the ELISA and VNT when millions of predominantly healthy pigs needed to be screened for the presence of CSF serum antibody. Decision-making on the basis of results generated by either method can, however, be seriously hindered when samples are examined from pig herds with a high prevalence of non-CSF pestiviruses.     

A rapid and sensitive PCR-based diagnostic assay to detect bovine herpesvirus 1 in routine diagnostic submissions

We describe a rapid, sensitive and specific polymerase chain reaction (PCR) assay for the detection of BHV1 DNA in a range of routine diagnostic submissions without the need for prior virus isolation. The assay, which is based on the selected amplification of a portion of the viral tk gene, detected both BHV1.1 and BHV1.2 subtypes in a panel of 15 characterised field isolates, and its sensitivity was estimated to be <0.125 TCID(50). BHV2, alcephaline herpesvirus, BHV4, equine herpesvirus 1 (EHV1), EHV4 and pseudorabies virus were not detected confirming the specificity of the assay. One hundred and five diagnostic submissions, including tissues, nasal secretions and nasal swabs were taken from cattle with respiratory disease and tested using the routine methods of virus isolation (VI) and the fluorescent antibody test (FAT), and the results were compared with those obtained by PCR. The PCR assay detected BHV1 DNA in all samples that were positive by VI. BHV1 DNA was also detectable by PCR in raw and extended semen samples at a sensitivity of 1 TCID(50) per 50microl. The assay also detected BHV5, permitting differentiation between it and BHV1 by virtue of the size of the amplified PCR product. The PCR assay is more sensitive and independent of sample quality than either virus isolation or FAT, and it is faster than virus isolation. The sample preparation method is simple with few steps involved. There are no extra post-amplification blotting/hybridisation steps and the assay is not based on a nested PCR strategy that might otherwise exacerbate the problem of oversensitivity/contamination in the routine use of such a test in a diagnostic laboratory. This assay would permit discrimination between those animals naturally infected with wild type BHV1 and those vaccinated with tk-BHV1 strains.     

A Fluorescent Antibody Test for Detection of the Rickettsia Causing Disease in Chilean Salmonids

Abstract

An indirect fluorescent antibody test (IFAT) was developed for detection of the rickettsia that was causing epizootics among salmonids cultured in seawater net-pens in southern Chile. Antiserum against the rickettsial agent was produced in New Zealand white rabbits with a preparation grown in antibiotic-free chinook salmon embryo (CHSE-214) cell cultures and partially purified by a combination of filtration and centrifugation steps. The IFAT was effectively used on blood films, tissue sections, and smears. Two gram-negative and two gram-positive bacterial pathogens of salmonids did not react in this test. Detection of the rickettsial agent has previously been restricted to examination by light microscopy or isolation in salmonid cells. The IFAT provides a simple, rapid, sensitive method for detection of the agent and diagnosis of the disease. The rickettsia is thought to be a member of the tribe Ehrlichieae and was tested by IFAT with sera from animals infected with other rickettsial agents.     

Detection of rinderpest antibodies

Rinderpest antibodies were detected by employing the fluorescent antibody test (FAT) and the immunoperoxidase test (IPT) and the results were compared with the counterimmuno electrophoresis test (CIE). FAT was found to be the most sensitive in detecting post-vaccinal antibodies followed by IPT and CIE tests.     

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.     

Detection of African malignant catarrhal fever virus antigens in cell cultures by immunofluorescence

A fluorescent antibody (FA) test for antigens of African bovine wildebeest-derived malignant catarrhal fever virus was developed. Serum from one of the few survivors of the experimental disease in steers was used to prepare the conjugate. Both a virulent and an attenuated strain of malignant catarrhal fever virus were used to infect bovine thyroid cell cultures. Cells infected with both strains were readily detected by FA staining as early as 24 h post infection, whereas cytopathic effect could be observed by bright-field microscopy only after days 5 or 6 post infection. Controls consisting of normal bovine thyroid cells or infected cells treated with conjugated normal globulins did not show autofluorescence. The reaction was blocked by treatment of infected cells with homologous positive antisera but not by treatment with normal bovine serum or antisera to foot-and-mouth disease, rinderpest, bovine virus diarrhea, Ibaraki, infectious bovine rhinotracheitis, or bovine herpes mammilitis viruses. Treated with African malgnant catarrhal fever virus conjugate did not react.     

Biochemical and Antigenic Properties of the First Isolates of Infectious Hematopoietic Necrosis Virus from Salmonid Fish in Europe

Abstract

The first isolates of infectious hematopoietic necrosis virus (IHNV) recovered from rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) in France and Italy were compared to six representative strains from North America by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of virion polypeptides and neutralization by monoclonal antibodies (MAbs). All three IHNV isolates from Europe had similar polypeptide profiles when compared by SDS-PAGE. An analysis of the antigenic relatedness of the European isolates to representative strains from North America showed that they were clearly different from viruses obtained from salmonids in California. The RB/B5 MAb, which was developed against virus isolated from adult steelhead (anadromous rainbow trout) reared in central Oregon, neutralized all isolates examined. The 193–110/B4 MAb, developed against IHNV isolated from infected yearling rainbow trout in southern Idaho, neutralized all isolates tested except those from California. The SRCV/A4 MAb, developed against Sacramento River chinook virus (SRCV) isolated from adult spring chinook salmon O. tshawytscha in central California, was the least reactive, and strong neutralization was observed only with the SRCV strain of IHNV from California. However, partial reactivity of the virus isolates from France with the SRCV/A4 MAb distinguished them from the virus recovered from salmonids in Italy.     

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.     

Distribution of rabies virus in infected mice,vaccinated and submitted to P. acnes as immunomodulator

The lethality and distribution of rabies virus were evaluated in swiss mice experimentally infected with street rabies virus, vaccinated and submitted to immunomodulation by P .acnes (formerly Corynebacterium parvum). The animals were sacrificed at different times,when the different tissues were collected and submitted to fluorescent antibody test (FAT) and mouse inoculation test (MIT). The group submitted to vaccination and P. acnes treatment presented a percentage of survival superior to that observed in infected mice only treated with P. acnes. Control infected animals had the lowest survival rates.The distribution of rabies virus in spleen of infected mice, vaccinated and submitted to P. acnes was superior to that verified in infected mice not treated with P.acnes. The increased survival correlated with the distribution of rabies virus in lymphoid tissues, could be interpreted as the consequence of P. acnes activity on macrophages. The results suggest the role of macrophages against rabies virus infection in mice and the importance of vaccination in the post expositive treatment of rabies.