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Three aquareovirus strains isolated from grass carp (Ctenopharyngodon idellus), geoduck clams (Panope abrupta) and herring (Clupea harengus) in North America and Asia were examined by RNA-RNA blot hybridization to determine their genogroup. The isolates from clams and herring were identified as members of genogroup A, but the isolate from grass carp did not hybridize to any of the known genogroups, suggesting that this virus probably represents a new, seventh genogroup.

The relative mobilities of the 11 dsRNA genomic segments of 22 aquareovirus isolates from fish and shellfish obtained from different geographical areas of the world were compared by PAGE. Using reciprocal RNA-RNA dot blot hybridization, a new sixth genetic group of aquareovirus (genogroup F) was identified. Genogroup A was represented by eight and genogroup B by 12 isolates. The remaining two isolates represented the new sixth genogroup (genogroup F). The genetic relationship of these aquareoviruses with mammalian rotavirus group A (SA 11) was also examined by reciprocal RNA-RNA blot hybridization but none was found under any of the stringency conditions used.

In this study, the phenotypic, antigenic, and virulence characteristics of 32 Citrobacter freundii strains of fish, human, and veterinary origin were comparatively analyzed. In addition, the spread of drug resistance factors by conjugation was investigated. Regardless of the source of isolation, the strains exhibited variable reactions mainly for arginine dihydrolase, ornithine decarboxylase, and fermentation of sucrose, melibiose, amygdalin, and salicin. Total fatty acid methyl ester analysis by gas chromatography proved to be useful for an intratypic differentiation within the C. freundii strains studied. In fact, although all of the isolates exhibited similar fatty acid methyl ester profiles, significant differences in the major fatty acids 16:1 and 16:0 and in the 17:0 delta region were observed between the isolates from salmonids and the remaining strains. Serological studies using agglutination tests, analysis of lipopolysaccharides (LPS), and the corresponding immunoblots with 13 antisera indicated a great antigenic diversity among the strains. Common LPS patterns were shared only by some isolates showing high cross-agglutination titers. In contrast, although all strains exhibited very similar surface protein patterns, only two common outer membrane proteins of 54 and 58 kDa were immunologically related. Infectivity trials performed in mice and rainbow trout indicated that all of the C. freundii strains were not pathogenic for mice (50% lethal dose of > 5 x 10(7)). Although the isolates displayed a low degree of virulence for trout, inoculated strains were always recovered from the survivors in pure culture.(ABSTRACT TRUNCATED AT 250 WORDS)

The relative mobility of the 11 dsRNA genomic segments of 19 Aquareovirus isolates from fish and shellfish were compared by polyacrylamide gel electrophoresis. This study revealed distinct variations of electrophoretic profiles (electropherotypes) of many aquareovirus isolates. No correlation was observed between the electropherotype and the species from which the isolates were obtained, but there was correlation with the geographic site of isolation. Using reciprocal RNA-RNA dot blot hybridization under high-, medium-, and low-stringency conditions five different genetic groups (genogroups) could be established (designated A through E). RNA-RNA hybridization showed that segment 10, the genome segment that codes for the major outer capsid protein, was the most variable gene. No genetic relationship was observed between these aquareoviruses and rotaviruses in groups A and C.

A nucleic acid hybridization assay was developed to rapidly detect small quantities of aquareovirus RNAs in infected cells and organs. Cloned cDNA copies were synthesized from the genomic RNA of the SBR strain of aquareovirus. By using cloned cDNA probes, aquareovirus RNAs were detected in spleen and kidney tissues of experimentally infected fish.

A number of new virus isolates from finfish have been reported in the scientific literature during the past five years. These include nine aquareoviruses, eight picornaviruses, six iridoviruses, five herpesviruses, three rhabdoviruses, three retroviruses, a paramyxovirus, and a coronavirus. Not all of these agents have been isolated in cell culture or established as etiologic agents of disease by controlled transmission studies. The burgeoning number of fish viruses is a reflection of the increased interest in fish diseases, particularly those occurring in aquaculture facilities, and the number will surely grow as fish farming intensifies on a global scale. This review chronicles the new virus isolates and lists them with other members of their virus family where appropriate., (Copyright © 1993 Published by Elsevier Ltd.)

Transmission experiments with adult soft-shell clams (Mya arenaria) demonstrated that clam sarcomas are transmissible with hemolymph from neoplastic animals but not with cell-free ultrafiltrates. Non-neoplastic clams were injected with either hemolymph from neoplastic clams or a cell-free ultrafiltrate prepared from a subsample of the same hemolymph. Injected clams were held in separate flow-through aquaria and examined for sarcomas by histocytology and histology. Data at 17 weeks showed a 44% prevalence of sarcomas in clams injected with neoplastic inoculum. No sarcomas were observed either in clams injected with a cell-free ultrafiltrate or in the control animals. The lack of sarcomas in clams injected with the ultrafiltrate argues against a viral etiology for the disease.

A comparative analysis of the phenotypic and serological properties of Carnobacterium strains associated with mortalities of cultured striped bass and channel catfish and the properties of isolates from wild brown bullhead catfish in the Chesapeake Bay area in Maryland was conducted. All of the strains were gram-positive, facultatively anaerobic, nonmotile, non-spore-forming rods occurring singly or in short chains. They did not produce cytochrome oxidase or catalase, did not reduce nitrate, failed to produce H2S, were unable to grow on acetate medium, and did not produce gas from glucose or gluconate. The temperature and salinity ranges for most of the strains were 10 to 37 degrees C and 0 to 6% NaCl, respectively. The strains all fermented mannitol and inulin and were arginine dihydrolase positive; these are typical characteristics of Carnobacterium piscicola. The carbohydrate fermentation pattern exhibited by all of the isolates with the API-50 CHL system was also very similar to that shown by C. piscicola. Acid was produced from ribose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, amygdaline, arbutin, esculin, salicin, cellobiose, maltose, sucrose, trehalose, and gentiobiose. The Carnobacterium strains did not show proteolytic, lipolytic, amylolytic, or hemolytic activity. Eighteen drugs were tested; all strains proved to be resistant to chloramphenicol, gentamicin, kanamycin, streptomycin, trimethoprim, quinolones, and nitrofurans. The analysis of membrane proteins supported the phenotypic similarities, two main patterns were established, one shared by the striped bass isolates and the reference strain of C. piscicola and another shared by most of the catfish strains. However, the agglutination assays demonstrated that only one Carnobacterium strain from striped bass was serologically related to C. piscicola ATCC 35586.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical characteristics of five rotavirus-like viruses isolated from striped bass (Morone saxatilis), turbot (Scophthalmus maximus), smelt (Osmerus mordax) and Atlantic salmon (Salmo salar) in North America and Europe were compared. The genome of each isolate was composed of 11 segments of dsRNA and each isolate had a unique electropherotype in polyacrylamide gels. Agarose gel electrophoresis showed similar RNA profiles for all four isolates from North America, whereas the RNA profile of the isolate from Europe was different. Analysis of virion proteins revealed that each virus had five structural proteins ranging in Mr from 130,000 to 34,000. Each isolate had a unique polypeptide profile but their overall polypeptide patterns were similar. Reciprocal RNA-RNA blot hybridization demonstrated that all these rotavirus-like viruses cross-hybridized with each other except for the isolate from Europe which did not hybridize with the RNA from any of the other isolates. No genetic relationship was found between these rotavirus-like viruses of fish and a true group A rotavirus (SA11).

The characteristics of a rotaviruslike (SBR) virus isolated from striped bass (Morone saxatilis) were examined following purification of viruses from infected cell cultures. Virions had a double-layered capsid of icosahedral symmetry and a diameter of 75 nm. Purified viruses contained five polypeptides ranging in molecular mass from 130 to 35 kDa. None of the structural proteins were glycosylated. Treatment with EDTA did not remove the outer capsid. By using enzymes and a chaotropic agent, it was shown that VP5 was the most external polypeptide. The genome of SBR virus was composed of 11 segments of double-stranded RNA (dsRNA). The electrophoretic pattern of the dsRNA of SBR virus was different from that of reovirus type 1 (Lang) and rotavirus (SA11) dsRNA. The SBR virus was compared with reovirus type 1 and SA11 virus by RNA-RNA blot hybridization. There was no cross-hybridization between any of the genome segments of the SBR, reovirus type 1, or SA11 viruses. Antigenic comparison of SBR virus and SA11 virus by cross-immunoprecipitation and cross-immunofluorescence tests did not show any relationship. These results suggest that SBR virus could represent a new genus within the family Reoviridae.

On the basis of cultural and biochemical properties as well as DNA homology assays, 81 Vibrio strains isolated from diseased striped bass and from Chesapeake Bay water were assigned to eight distinct groups. All organisms belonging to two of the groups were pathogenic for striped bass and were identified as Vibrio anguillarum, whereas organisms classified in the other six groups were nonpathogenic and were designated as Vibrio spp. Unlike the pathogenic V. anguillarum strain 775 isolated in the Pacific Northwest, strains pathogenic for striped bass did not contain any plasmids; however, they were similar to the Northwest isolates in that virulence was correlated with their ability to grow in the presence of nonimmune striped bass serum or under conditions of iron limitation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of outer membranes showed that additional proteins were induced in those organisms capable of growth under conditions of iron limitation. It was of interest that 22 of the nonpathogenic isolates harbored one or more plasmids which, by restriction endonuclease analyses, were shown to be clearly different from the virulence plasmid pJM1.

An immune electron microscopy agglutination technique in which emphasis is placed upon the importance of antigen-antibody equivalence has been developed as a possible method for the serotyping of avian infectious bronchitis viruses. The Connecticut and Massachusetts 41 serotypes were used as a model system. Stock virus concentrations were standardized by physical particle counts of virions sedimented directly onto electron microscope specimen grids. Suspensions containing approximately 150 virions per grid square were allowed to react with dilutions of homologous and heterologous antisera. Virions in these constant virus-variable serum mixtures were sedimented directly onto electron microscope specimen grids, and the relative degree of aggregation per grid was determined from the mean percent aggregation of five randomly selected grid squares. In homologous assays, regions of relative antibody excess, of equivalence, and of relative antigen excess were clearly evident. At equivalence, the mean percent aggregation was significantly higher than in the regions of relative antibody or antigen excess. In the heterologous systems, the degree of aggregation differed little from that of the virus controls containing no antiserum.

Japanese encephalitis virions contain a 58 X 10-3-molecular-weight envelope glycoprotein antigen that can be solubilized with sodium lauryl sulfate and separated from other virion structural polypeptides and viral ribonucleic acid by gel filtration chromatography. The 58 X 10-3-molecular-weight envelope protein is the major antigen responsible for cross-reactivity of the virion in complement fixation tests with other closely related arboviruses. A naturally occurring soluble complement-fixing antigen is found in Japanese encephalitis mouse brain preparations after removal of particulate antigens. After partial purification by gel filtration and isoelectric focusing, the 53 X 10-3-molecular weight soluble complement-fixing antigen is more type specific than the Japanese encephalitis envelope antigen in complement fixation tests. Further, the Japanese encephalitis soluble complement-fixing antigen is stable to treatment with sodium lauryl sulfate and 2-mercaptoethanol, whereas virion complement-fixing antigens are unstable after this treatment.

C3H/He mice immunized by subcutaneous infection with Rickettsia tsutsugamushi Gilliam were examined for the production of immune interferon after intravenous administration of irradiated strain Gilliam antigen, in supernatants of immune lymphocytes stimulated with specific antigen, and after a secondary challenge with viable rickettsiae. Mice administered various doses of irradiated whole-organism antigen 28 days after immunization showed circulating levels of interferon which peaked 4 h after inoculation and were antigen dose dependent. The interferon produced was pH 2 sensitive and stable at 56 degrees C for 1 h and was neutralized by antiserum directed against immune, but not against alpha/beta, interferon. The production of another lymphokine, macrophage migration inhibition factor, paralleled that of interferon. The interferon produced by cultures of spleen cells obtained from immune animals was antigen specific and dose dependent. Peak levels were obtained 48 to 72 h after the addition of antigen. The interferon produced by spleen cell cultures after stimulation with Gilliam antigen was characterized as immune interferon by the same physical and antigenic criteria used for serum interferon. Interferon was produced in vitro by the Thy-1.2+ lymphocyte and required the presence of a spleen-adherent cell population. Immune mice produced high circulating levels of immune interferon after intraperitoneal challenge with viable rickettsiae, which suggested a possible role for interferon in the resistance of immune mice to rechallenge with R. tsutsugamushi.

The stability of poliovirus 1 in estuarine water and sediment was examined. The present data indicated that a 2 log reduction in virus titer at 15 degrees c occurred within 6-7 days in water samples taken from estuarine waters on both sides of the Atlantic Ocean. The antiviral effect decreased significantly when the seawater was subjected to autoclaving but not when it was filtered. That the antiviral activity activity of the seawater was related to the growth activities of microorganisms was corroborated by the isolation of antibiotic-producing marine bacteria that had marked activity against poliovirus (net inactivation greater than or equal to 2 logs within 6-8 days). These organisms retained this activity following repeated subcultivation on laboratory media. Since comparable inactivation rates were observed in cell-free filtrates from these marine strains, extracellular products appear to be involved in the virus-inactivation process. Other enteric viruses, Coxsackie B-5 and ECHO-6, were also inactivated by these marine bacteria. The addition of sediment to natural seawater increased the length of poliovirus survival more than three times over that in seawater alone. However, this was not found under sterile conditions, suggesting that the sediment can protect the viruses from inactivation by the marine microflora.

A human T-cell line (Molt-4) was shown by viral hemagglutination and infectivity assays to support the replication of rat virus (RV) and H-1 virus. In addition, H-1 virus, but not RV, multiplied in two human B-cell lines, AV-1 and NC-37. The ability to bind radioactively labeled RV was demonstrated for each of the cell lines, but viral adsorption occurred to a greater degree with Molt-4 cells than with either AV-1 or NC-37 cells. After challenge with RV, virus-specific antigens were detected in cells of the B-cell lines by the indirect immunofluorescence technique. Infection of AV-1 or NC-37 cells by RV apparently results in an abortive cycle of virus replication. Differences among the three cell lines that might influence with H-1 virus or RV are discussed.

It was demonstrated that gamma-irradiated L-929 cells support plaque formation by three strains of Rickettsia tsutsugamushi and representative species of the spotted fever and typhus group rickettsiae. Sensitivity of the plaque assay for detection of viable scrub typhus rickettsiae was similar to that achieved with intraperitoneal inoculation of random-bred mice. The concentration of irradiated cells and the temperature and length of incubation were all found to affect plaque size. A technique combining terminal dilution and plaque purification was used to obtain clones of three strains of scrub typhus rickettsiae.

The ability of antigen-responsive, thymus-derived lymphocytes to produce immune (gamma) interferon was investigated during the development and expression of cellular immunity to Rickettsia tsutsugamushi. C3H/HeDub mice infected subcutaneously with the Gilliam strain developed the ability to produce serum interferon in response to intravenously inoculated antigen which correlated with the development of resistance to intraperitoneal rechallenge. Antigen-responsive lymphocytes, measured by interferon production and proliferation, were first apparent in draining lymph node cells, but spleen cell responses were detectable relatively soon after the appearance of reactive lymph node cells. The peak spleen cell response was of a greater magnitude and was found to be relatively long-lived. Reactivity to heterologous strains of R. tsutsugamushi also developed after immunization and paralleled the homologous responses, although reactivity was greatest to homologous antigens. Responses to heterologous strains differed in magnitude and time of appearances; however, immune mice resisted challenge with all strains of R. tsutsugamushi tested.

The macrophage migration inhibition (MMI) test was found to be a satisfactory procedure for distinguishing between adenovirus types 1, 4, 5, and 7. Highly purified virus preparations were used for the sensitization of Hartley strain guinea pigs, whereas the MMI test antigen consisted of crude virus preparations grown in KB cells. With all four virus types, a significantly greater MMI response was noted when peritoneal exudate cells were exposed to the homologous sensitizing antigen as compared to that obtained with the three heterologous antigens. Studies with adenovirus type 1 indicate that sensitizing doses between 70 and 150 mug of viral protein per guinea pig gave the optimal MMI response. Doses below 70 mug did not stimulate the delayed response, whereas doses above 120 mug produced MMI reactions which were nonspecific, as differences between homologous and heterologous antigens were not demonstrable.

Twelve hybrids secreting antibody to the Sp serotype of infectious pancreatic necrosis virus (IPNV) were isolated from the fusion of murine myeloma cells and spleen cells from mice immunized with pelleted virus. All of the monoclonal antibodies possessed the kappa (K) light chain isotype. Nine contained the mu (M), two had the gamma 2a (G2a), and one had the gamma 1 (G1) heavy chain isotype. Using an enzyme-linked immunosorbent assay (ELISA), 10 antibodies were found to be broadly reactive against partially purified representatives of the three serotypes of IPNV, the Sp, Ab, and VR-299 strains. The other two antibodies reacted with the Sp serotype alone. Characterization by immunostaining of viral polypeptides electrophoretically transferred to nitrocellulose sheets was possible only with IgG type antibodies. One of the specific monoclonal antibodies was shown to be directed against the major capsid protein while the other specific monoclonal antibody and the broadly reacting one reacted with the low molecular weight viral polypeptides.

Plasmid profiles of representative fish pathogens, Aeromonas salmonicida, Aeromonas hydrophila, Vibrio anguillarum, Pasteurella piscicida, Yersinia ruckeri, Edwardsiella tarda, and Renibacterium salmoninarum, were determined by agarose gel electrophoresis with four different plasmid detection methods. A combination of two methods was required to detect the plasmids present in these strains and to calculate precisely the molecular weights of the plasmids. Of 38 strains, 28 harbored one or more plasmids, with the majority of strains demonstrating multiplasmid banding. Similarity in plasmid banding between strains was noted and related to geographic source. Five strains of A. salmonicida possessed six plasmid bands having molecular weights of 8.6 X 10(6), 8.4 X 10(6), 8.1 X 10(6), 3.6 X 10(6), 3.5 X 10(6), and 3.4 X 10(6). Four P. piscicida isolates shared three plasmid bands having molecular weights of 37 X 10(6), 15 X 10(6), and 5 X 10(6), and five A. hydrophila strains harbored a common plasmid having a molecular weight of ca. 20 X 10(6) to 30 X 10(6). The highest-molecular-weight plasmids (145 X 10(6) and 130 X 10(6) were detected in V. anguillarum. From curing experiments, it was found that in A. hydrophila strain 79-62, a loss of resistance to tetracycline was associated with loss of plasmid content in all susceptible derivatives, suggesting plasmid-mediated tetracycline resistance. Cell surface characteristics and metabolic properties were also modified in cured derivatives of A. hydrophila strain 79-62.

Corticosteroids have been reported to induce immunosuppression in fish exposed to many types of bacterial antigens. We document a similar phenomenon in fish exposed to infectious pancreatic necrosis virus (IPNV). Fingerling striped bass that were injected with the steroid triamcinolone acetonide (100 mg/kg body weight) 24 hours before receiving intraperitoneal inoculation with IPNV became viremic 3 days post inoculation (dpi) and virus was still detected in the buffy coat cells 14 dpi. In contrast, viremia could not be detected after 7 dpi in fish that received virus but not steroids. Circulating virus neutralizing antibodies were first detected in steroid treated fish at 10 dpi compared to 7 dpi for the virus injected fish and titers were consistently lower in the steroid group. Steroid treatment of chronic IPNV-carriers did not induce detectable viremia nor alter circulating antibody levels in chronic IPNV-carriers. None of the striped bass demonstrated clinical signs of viral disease.

Quantification of an induced chemiluminescent (CL) response in phagocytes is currently being evaluated as an indicator system for determining those environmental pollutants that may predispose fish to disease. A CL assay was developed using phagocytes from the pronephros of rainbow trout (Salmo gairdneri). The CL response of phagocytes to phorbol myristate acetate, a chemical inducer of CL, was shown to be dose-dependent. The response to five species of bacteria was also evaluated. Staphylococcus aureus and Aeromonas hydrophila produced the most intense CL responses and the longest duration of response (100 min.) Yersinia ruckeri induced an immediate strong CL response of short duration (20 min.) whereas Vibrio anguillarum and Aerococcus viridans failed to stimulate CL under the test conditions employed. The effect of sub-toxic levels of Cu, Al, and Cd on the CL response of phagocytes to S. aureus was examined using phagocytes exposed to the metals immediately before assay or after 1 hr or 24 hr exposure times. Copper caused a significant decrease in CL to the baseline level under all treatment conditions upon stimulation with S. aureus. Similar results were obtained with Al except that the decrease in CL, although significant, was not to the baseline level. In contrast, Cd caused a significant increase in CL when added 1 hr prior to or immediately before the assay; but, following a 24 hr exposure, the results were variable, in that either no change or a decrease was observed. The addition of Cu to phagocytes already exhibiting a strong CL response to S. aureus caused an immediate decrease in CL to that seen with the negative controls.

Two urease-positiveVibrio spp. were isolated from a brown shark (Carcharhinus plumbeus) that died in captivity at a national aquarium. Morphological, biochemical, and molecular genetic studies revealed one of the isolates to beV. damsela; the other isolate was unique and has been classified asV. carchariae sp. nov. BothV. damsela andV. carchariae were found to be virulent for spiny dogfish (Squalus acanthias), causing death in less than 18 hours after intraperitoneal injection of ca. 4×10(6) cells.V. damsela was strongly cytotoxic for Y1 adrenal cell monolayers;V. carchariae exhibited weak cytotoxicity for Y1 cells.V. damsela contained cryptic plasmids and both isolates were urease positive.V. carchariae was able to utilize urea as sole source of carbon and nitrogen.

Inoculation of a human thymus-derived lymphocyte cell line (Molt-4) with the Kilham rat virus resulted in persistently infected cultures, that released infectious virus for periods up to seven months. At any time following infection, a small percentage of the cells were positive for rat virus antigens as determined by immunofluorescene assay.

Hemagglutinating DNA viruses of 20 nm diameter were isolated from bovine adenovirus types 1 and 2. The isolates were heat stable, chloroform resistant, and defective. Their densities were 1.38 to 1.39 g/cm3, and they were found to be serologically identical to the bovine adeno-associated virus strain X7. A partial antigenic relationship was found between these and the canine adeno-associated virus.

The mechanism of enterovirus inactivation by marine bacteria was investigated using poliovirus type 1 as a model virus and with strains of Pseudomonas and Vibrio isolated from the marine environment. Treatment of virus with cell-free filtrates from late log phase bacterial cultures produced alterations in the viral capsid as shown by a reduction in efficiency of adsorption to host cells, increased sensitivity to ribonuclease, and by the release of ribonucleic acid from the treated virions. Filtration of 14C-labelled, treated virus through 25-nm filters revealed that the majority of the isotope (85-96%) passed the filters, indicating extensive capsid disruption. However, the most rapid and pronounced change observed during virus inactivation was the loss of infectivity, suggesting that enzymatic degradation is not the first event in the poliovirus inactivation process by marine bacteria.

Infection of striped mullet (Mugil cephalus) with Aeromonas hydrophila results in an acute septicemic disease. The disease can be experimentally induced by intramuscular injection, skin or gill scarification or by the oral route using pellets purposely seeded with bacteria. The organism was isolated from the blood 1-2 days after infection and from all organs 24 hr or longer after infection. The disease is characterized by early inflammatory and proliferative changes and later necrotic changes. Enteritis and hepatic necrosis are constant findings in aeromonad disease of M. cephalus but surface lesions are not pathognomic for these infections in mullet. Death of infected fish may be attributed to bacterial toxins which cause necrosis of parenchymal organs and soft tissue structure.

Exposure of rainbow trout to sublethal levels of copper in water increased their susceptibility to infectious hematopoietic necrosis virus. In most instances, the percent mortality was twice as great in the stressed groups compared with those groups which were not stressed but received the same virus dose. Although the level of copper in the water influenced the mortality rates, the length of exposure did not prove to be critical, as similar results were obtained after 1, 3, 5, 7, or 9 days of exposure. When different virus challenges were employed, the percent mortalities were again greater in the stressed fish at all virus doses tested, and at one dose level mortalities were noted in the stressed group but not in the untreated group.

Several antischistosomal drugs and their metabolites were found to transform rat embryo cell cultures persistently infected with the Rauscher leukemia virus. The transformed cells produced fibrosarcomas when implanted into newborn rats. Cell cultures treated with either virus or chemical alone were not transformed nor were they invasive in rats. The advantages of early screening of developmental drugs in cell culture systems are discussed.

The pathogenic role of Yersinia enterocolitica serotypes O:3, O:8, and O:9 in human infections is well documented. Whereas the virulence of the O:8 strains can be readily demonstrated in mice by 50% lethal dose determinations, the O:3 and O:9 strains have no lethal effect on mice by any route of inoculation. A mouse virulence test for the O:3 and O:9 strains is described. Y. enterocolitica strains were first tested for the presence of virulence-associated plasmid characteristics by auto-agglutination and gel electrophoresis procedures before mouse virulence determinations. The 50% lethal dose of the O:3 strains injected intraperitoneally with 2.5% mucin was about 10(7) colony-forming units. However, histological examinations showed that mucin allowed the growth of Y enterocolitica on the surface of the livers and spleens of the mice without internal lesions. The 50% lethal dose of the same O:3 strains injected intraperitoneally with 1 ml of 10% iron dextran in saline was about 10(5) to 10(6) colony-forming units, and the nonlethal infective dose with typical lesion development was 20 to 200 colony-forming units. The infected mice developed symptoms and extensive liver and spleen lesions which differed from those in mice infected intraperitoneally with the virulent O:8 strains. These results showed that the virulence of the O:3 Y. enterocolitica strains can be measured by intraperitoneal injection with iron dextran. This procedure was used to test the virulence of food isolates, plasmidless strains, and the effect of growth temperatures.

Phagocytosis of bacterial fish pathogens by cells isolated from the pronephros of striped bass (Morone saxatilis) was measured using an assay of chemiluminescence. Results of the assay, which proved to be quite reproducible, indicated that the degree of phagocytosis was related to the number of bacteria employed and to the species of bacteria eliciting the response. Cells from individual fish gave similar phagocytic responses but of different magnitudes.

A method was developed for production of a freeze-dried Western equine encephalomyelitis vaccine from virus propagated in chick embryo cell culture monolayers maintained with a serum-free medium. A sufficient concentration of virus accumulated in the cell culture fluids prior to the occurrence of viral cytopathology to permit the production of a vaccine relatively free from serum and cellular proteins. Inoculation with two mouse ld(50) doses of virus per 100 tissue culture cells was found to yield reproducible high virus titers at a convenient harvest time. These harvests were inactivated at 22 C by 0.05% formalin within 48 hr. Potency test results, as measured by the protection of immunized guinea pigs against an intracerebral virus challenge, indicated that the vaccine produced from the virus propagated in cell culture was equal in potency to a lot of whole chick embryo vaccine used to immunize laboratory and field workers subject to a high risk of infection.

The Kilham rat virus (RV) was found to vary in infectivity and antigenic characteristics when propagated in two different host systems. Both cross hemagglutination-inhibition (HI) and cross virus-neutralization tests revealed that a nonreciprocal or one way cross exists between RV propagated in rat embryo cell cultures (RE-RV) and RV propagated in suckling hamsters (H-RV). Specifically, immune serum to RE-RV inhibited hemagglutination and infectivity by both viruses to the same extent. Immune serum to H-RV, however, exhibited higher HI and neutralization titers to H-RV than to RE-RV. Infectivity studies demonstrated that, although both viruses were able to infect either host, the virus showed a higher infectivity titer for the last host in which it had been propagated. Serological studies indicated that H-RV possesses an antigen(s) not found on the RE-RV. This host-controlled variation did not result after a single passage in the new host, but rather required at least three passages for a complete conversion to occur, and did not appear to result from the incorporation of unaltered host antigens into the virus particle. Solubilization of purified RV propagated in each host with sodium dodecyl sulfate, 2-mercaptoethanol, and urea followed by electrophoresis in polyacrylamide gels indicated that the number of components in the protein of each virus was not the same.

Chikungunya virus vaccines prepared by Tween 80 and ether inactivation of virus grown in green monkey kidney cell cultures were shown to be as immunogenic as comparable Formalin-inactivated vaccines. Both types of vaccine stimulated hemagglutination-inhibiting, complement-fixing, and neutralizing antibody and afforded protection to mice against a live virus challenge. It was shown after Tween-ether treatment of chikungunya virus that the infectivity of the virus was lost and the hemagglutinin titer was increased. By characterization of the resultant hemagglutinin by sucrose and cesium chloride density gradient centrifugation, it was found that the extracted particle was smaller in size and greater in density than the parent virus particle. Removal of lipid may account for the alterations in the physical characteristics of the infectious virus particle. Conditions for treatment of chikungunya virus with Tween and ether were found that preserved high titers of hemagglutinin as well as the immunogenicity of the virus preparations.