Hydrocortisone suppresses the chemiluminescent response of striped bass phagocytes.

Phagocytic cells obtained from the pronephros of striped bass (Morone saxatilis) were exposed in vitro to various levels of hydrocortisone acetate. This treatment reduced the normal ability of the cells to generate a chemiluminescent response when exposed to bacteria or phorbol myristate acetate. Although the levels of hydrocortisone were higher than is physiological for fish, suppression was dose dependent and was not attributable to a reduction in cell viability. Whereas phagocytic chemiluminescence has been linked to the respiratory burst and bactericidal activity, the possibility exists that stress-induced elevations in serum corticosteroids lead to increased susceptibility to infection.

Chemiluminescence of phagocytic cells isolated from the pronephros of striped bass.

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.

Alteration of lymphocyte function due to anesthesia: in vivo and in vitro suppression of mitogen-induced blastogenesis by sodium pentobarbital.

The mechanism of decreased lymphocyte responsiveness after major surgery is unclear. Because sodium pentobarbital, and intermediately long-acting barbiturate, will reproducibly induce anesthesia in experimental animals, we utilized a canine model to investigate its effect on lymphocyte proliferation induced by the mitogenic lectins erythroagglutinating phytohemagglutinin (E-PHA) and leukoagglutinating phytohemagglutinin (L-PHA). Although no effect was observed at 10 minutes or 1 hour after an anesthetic dose of sodium pentobarbital, after 1 and 3 hours of anesthesia, canine lymphocytes were significantly suppressed, as demonstrated by decreased responsiveness to E-PHA and L-PHA mitogen stimulation. After 3-hours the majority of animals had mitogenesis values of less than 50% of the preanesthetic control values. Recovery, as measured by a return to at least 70% of the preanesthetic mitogenesis value, was noted in the majority of animals at 24, 48, and 72 hours. In order to investigate the machanisms of the in vivo capability of sodium pentobarbital to induce immunosuppression of lymphocyte transformation, in vitro studies were carried out. Sodium pentobarbital was found to significantly inhibit mitogen-induced canine mononuclear cell blastogenesis at anesthetic (1.5 to 3.0 mg%) drug concentrations in vitro. Lymphocytes pretreated with barbiturate and washed prior to plating did not show this inhibiting effect. Our findings suggest that depression of the immune response reported in patients after operation could result from short-acting barbiturates administered during the induction phase of clinical anesthesia. Furthermore, the suppression may involve in vivo metabolism of pentobarbital, hormones or other in vivo factors, since washed lymphocytes from the in vivo but not the in vitro experiments demonstrated suppression. These results indicate that anesthesia may be an important factor in the immunosuppression reported after major surgery.

Effects of metals on the chemiluminescent response of rainbow trout (Salmo gairdneri) phagocytes.

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.

Monoclonal antibodies to the Sp strain of infectious pancreatic necrosis virus.

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.

Association of toxic capsule and cell wall mucopeptide with virulence in Gaffkya tetragena.

Nine strains of organisms morphologically and physiologically identified as Gaffkya tetragena were obtained from various sources to study their pathogenicity. Initial virulence analysis of all strains by mouse intraperitoneal injection of viable cells revealed that only three strains, recently isolated from and associated with respiratory infections in hospitalized patients, caused death of mice within 48 hr. The ld(50) for these virulent, encapsulated strains was 1 x 10(7) to 6 x 10(7) viable organisms. To associate virulence with a toxic component, the following fractions were purified from all strains: capsular material, cell walls, mucopeptide preparations from cell walls and whole cells, grouplike material, cytoplasmic material, and culture filtrate with and without added reducing agent. Rabbit and mouse dermal toxicity testing of these fractions revealed that the capsular material, cell walls, and mucopeptide preparations of the virulent strains were toxic. None of the nonvirulent strains contained toxic components, with the exception of one strain which yielded capsular material equal in toxicity to that of the virulent strains. The capsular material induced a soft pustular lesion persisting for approximately 22 days. Cell walls and mucopeptide preparations produced a hard nodular lesion, identical to that produced by autoclaved whole cells, that persisted for 25 to 30 days. One strain may represent a virulence intermediate between the virulent and nonvirulent strains, since it contains toxic capsular material but nontoxic cell wall mucopeptide. The results indicate that the virulence of this organism is associated with toxic capsular material and cell wall mucopeptide.

Phytohemagglutinin stimulation of enhanced immunoglobulin G production in mice inoculated with type III pneumococcal polysaccharide.

In BALB/c mice previously inoculated intraperitoneally with an immunogenic dose of the T-independent antigen type III pneumococcal polysaccharide, the intravenous administration of the T-cell activating agent phytohemagglutinin P causes a pronounced increase in the number and relative proportion of immunoglobulin G-producing cells. These results, detected by a modified hemolytic plaque assay, were supported by finding increased levels of serum immunoglobulin G anti-type III pneumococcal polysaccharide in the treated mice. A comparable stimulation of immunoglobulin G antibody-producing cells was not induced in phytohemagglutinin P-treated nude mice, indicating that the change in class of the predominant antibody is attributable to the activation by the phytohemagglutinin P of a T-cell population. Under the conditions of these experiments, phytohemagglutinin P also promotes a progressive suppression of the antibody-forming cells during the response to type III pneumococcal polysaccharide.

Serological relationships among budding, prosthecate bacteria.

The somatic antigens of 25 strains of budding bacteria were typed and 14 serologically distinct groups were identified, suggesting considerable antigenic diversity among hyphomicrobia. Ten of the groups were represented by a single isolate, 2 contained two isolates, 1 three isolates, and 1 eight isolates. The strains in the largest group of eight isolates each shared at least one common antigen. However, there was also considerable antigenic heterogeneity within this cluster. Serological activity resided in the lipopolysaccharide (LPS) portion of cell walls and also in a heat labile component of Hyphomicrobium neptunium. The amino acid utilizing isolates, H. neptunium and Hyphomonas polymorpha, were serologically unrelated and it is suggested that the two organisms could be grouped as members of the same genus but not the same species.

Cadmium chloride susceptibility, a characteristic of Campylobacter spp.

We report a simple diagnostic characteristic useful in the presumptive identification of Campylobacter jejuni and Campylobacter coli. Filter paper disks impregnated with cadmium chloride were placed on streaked agar medium. Zones of growth inhibition for Campylobacter spp. occurred at 1.25 micrograms per disk. Other enteropathogens (Salmonella spp., Shigella spp., Vibrio cholerae, Vibrio parahaemolyticus, Escherichia coli, and Yersinia enterocolitica) were resistant to at least 40 micrograms per disk, with the exception of a strain of Shigella flexneri, which showed first susceptibility at 10 micrograms per disk. Most of the 52 Campylobacter strains, which were isolated from human clinical and animal sources, showed zones of inhibition greater than 10 mm with 2.5 micrograms of cadmium chloride per disk. At 20 micrograms per disk, Campylobacter isolates from clinical sources were significantly (P less than 0.01) more susceptible to cadmium chloride inhibition than were those from meat samples.

Positive fluorescent treponemal antibody reactions in diabetes.

In a survey of 129 diabetic patients and 142 normal individuals, a significantly higher percentage of positive reactions in the fluorescent treponemal antibody-200 (FTA-200) test was found among diabetic patients than in the normal population. Absorption of all FTA-200-reactive sera with an extract of Reiter's treponeme eliminated most of the positive reactions in sera from diabetic patients, and three of the five positive reactions detected in sera from apparently normal subjects. On immunoelectrophoresis, precipitin bands developed most frequently between the Reiter sorbent and sera from diabetic patients positive in the FTA-200 test. Serum components responsible for FTA reactivity and precipitin reactions against the sorbent were resistant to treatment with mercaptoethanol, suggesting antibody of the IgG class. Cross-reacting antibodies produced in response to normal treponemal flora, and perhaps acquiring enhanced reactivity by means of nonspecific interacting substances in sera peculiar to the altered physiological state of diabetes, are suggested as possible causes of positive reactions of unabsorbed sera. No correlation could be made between age of the diabetic patient, treatment or duration of the disease, and FTA or precipitin reactivity of the patient's serum.

Interactions between radioresistant T cells from plasmacytoma-bearing mice and radiosensitive T cells from normal mice leading to suppression.

Spleens from BALB/c mice bearing the plasmacytoma SPQC 11 contain a population of T cells capable of suppressing the production of antibodies when cultured with normal BALB/c splenocytes, but not with splenocytes from nude BALB/c mice. Inhibition was characteristically the most effective at low suppressor cell numbers using unirradiated T cells. Maximum suppression occurred when a radiosensitive population of T cells from normal mice interacted with a population of radioresistant T cells from plasmacytoma-bearing animals. Accurate characterization of the suppressor T cell activity required testing over a wide range of suppressor/indicator cell ratios. Suppressor activity was eliminated by treatment with anti-Thy 1.2 plus complement, was radioresistant and present over a full range of doses when irradiated cells were used.

Cell-mediated immune response to products of Actinomyces viscosus cultures.

Hartley strain guinea pigs were sensitized with 0.5 ml of concentrated cell-free Actinomyces viscosus culture supernatant fluids mixed with Freund complete adjuvant. Fourteen to 16 days later the animals were challenged by intradermal injection with 0.1 ml of the culture supernatant, and the reactions were observed at 4, 8, 16, 24, and 48 h. Peritoneal exudate cells from sensitized animals were used for determination of migration inhibition factor, and guinea pig peripheral blood served as a source of cells for determining the induction of mitogenesis by antigenic material. Skin responses were consistently positive to challenge with the test material, whereas reactions to noninoculated culture medium were negative. Sensitized cells, challenged with antigen, resulted in 60% or greater inhibition of migration of indicator cells in migration inhibition factor experiments. Tests for mitogenesis showed a greater than fourfold increase in isotope uptake when sensitized cells were challenged with test material. The data are consistent with the suggestion that A. viscosus culture supernatants contain substances that induce cell-mediated immune responses in guinea pigs.

Response of Campylobacter jejuni to combinations of ferrous sulphate and cadmium chloride.

On Mueller Hinton (MH) agar, Campylobacter jejuni showed 20.0 and 30.9 mm zones of inhibition surrounding discs impregnated with 2.5 and 20 micrograms CdCl2 respectively. The minimal inhibitory concentration (MIC) ranged from 0.64 to 3.2 micrograms CdCl2/ml of MH agar for four C. jejuni strains. In the presence of 23 micrograms FeSO4/ml of MH the MIC increased to a range of 1.5-5.4 micrograms CdCl2/ml of MH. Moreover, the numbers of colonies present on MH supplemented with FeSO4 were greater than on MH without iron. The growth response of C. jejuni in the presence of 0.025% (w/v) FeSO4 in MH broth was increased about 10,000 fold in three of four strains when compared with the growth in unsupplemented MH broth. Zones of inhibition surrounding 20 micrograms discs of CdCl, were 50.6 and 24.4 mm on MH and Campy-BAP media respectively, with cells grown on MH. These results suggest that the blood-containing medium 'neutralized' the biocidal influence of the CdCl2. In comparison, C. jejuni inoculum from fluid thioglycollate (FT) medium showed smaller zones of inhibition. These decreased from 34.9 mm on MH agar to 19.6 mm on Campy-BAP agar, suggesting that components in the FT growth medium ameliorated the toxic influence of CdCl2. Atomic absorption spectroscopy analysis indicated mean values (mg/100 g dry weight) of selected metals bound by C. jejuni as: Cu, 10.4; Mg, 146; Na, 2385; Fe, 45.1; Zn, 13.0; and K, 172.

Biochemical and serological characterization of Carnobacterium spp. isolated from farmed and natural populations of striped bass and catfish.

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)

Comparison of the taxonomy, serology, drug resistance transfer, and virulence of Citrobacter freundii strains from mammals and poikilothermic hosts.

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)