Monoclonal antibodies to bovine coronavirus glycoproteins E2 and E3: demonstration of in vivo virus-neutralizing activity.

Six monoclonal antibodies (MAbs) to bovine coronavirus (BCV, Quebec isolate) E2 and E3 glycoproteins which were found previously to be neutralizing in vitro were examined for virus-neutralizing activity in vivo. Surgically ligated intestinal loops of newborn colostrum-deprived calves were virus-inoculated, mock-infected or inoculated with a mixture of virus and antibody. Of the six BCV-specific MAbs, four were found to be protective against a virulent field isolate of BCV, as indicated by a reduction in villous atrophy. These MAbs were specific to antigenic domain A and antigenic domains A1 and A2 on the E2 and E3 glycoproteins respectively. MAbs to antigenic domains B and C on the E2 and E3 glycoproteins, respectively, were not protective.

Preliminary characterization of an epitope involved in neutralization and cell attachment that is located on the major bovine rotavirus glycoprotein.

The 38,200-molecular weight (unreduced)/41,900-molecular-weight (reduced) glycoprotein of bovine rotavirus, isolate C486, was identified as the major neutralizing antigen. This glycoprotein as well as the corresponding glycoprotein of another bovine rotavirus serotype also specifically attached to cell monolayers under normal conditions for virus adsorption in vitro. Further support for this glycoprotein being directly responsible for virus attachment to cells was that (i) infectious virus of both serotypes could compete with the C486 glycoprotein for cell surface receptors, and (ii) neutralizing monospecific antiserum and neutralizing monoclonal antibodies directed toward the glycoprotein could block this virus-cell interaction. Preliminary epitope mapping of the glycoprotein with monoclonal antibodies further localized the neutralization-adsorption domain to a peptide with an approximate molecular weight of 14,000. The effect of two protein modifications, glycosylation and disulfide bridging, on the reactivity of this peptide with antibodies and cell surface receptors was investigated. It was demonstrated that, whereas glycosylation did not appear to affect these reactivities, disulfide bridging seemed to be essential.

Effect of recombinant DNA-produced bovine interferon alpha (BoIFN-alpha 1) on the interaction between bovine alveolar macrophages and bovine herpesvirus type 1.

Treatment of bovine alveolar macrophages (BAM) with bovine leukocyte interferon (BoIFN-alpha 1) resulted in reduced bovine herpesvirus type 1 replication and spread. This was demonstrated by reduced virus yields and number of cells infected. BoIFN-alpha 1 treatment of BAM also induced enhanced Fc receptor expression and/or avidity by the cells, increased their activity in antibody-dependent cellular cytotoxicity, and augmented their extrinsic antiviral activity as measured by a reduction in the development of plaques in a susceptible cell line. These results are discussed in the context of the possible role of interferon in activation of AM during the early phases of a virus infection.

Comparison of helium leak test and vacuum leak test using canned foods: collaborative study.

Two can leak tests were compared by 7 collaborators. In the helium leak test, pressurized helium is applied to the outside of the container, and a headspace gas sample from the can is then analyzed for the presence of helium. The vacuum test is described in the Bacteriological Analytical Manual. Ninety No. 303 cans of creamed-style corn, green beans, carrots, fruit cocktail, and whole-kernel corn were shipped in 3 groups. Two groups of 30 cans had 10 dented flat cans, 5 flat controls (nondented), 10 dented swollen cans, and 5 swollen control cans (nondented). The third group had 10 dented swollen cans and 5 swollen control cans. Of 600 cans analyzed, 37 (6.2%) were deleted from the analysis because results were not available for both tests. One laboratory was constrained by scheduling to analyze 15 of 45 swollen cans. The helium leak test found 12 (13%) positives of 92 nondented swollen cans. One pressurization test yielded 7 of those 12 positives. Of the 400 dented cans sent as possible leakers, the helium test found 267 positives, and the vacuum test found 181. Five of the 7 analysts had significantly (alpha = 0.05) higher percent positive helium results. One analyst found more leakers by the vacuum leak test. Both tests found fewer positives in the swollen dented cans than in the flat dented cans. After exposure to pressurized helium, all cans with greater than 8 psi headspace pressure were positive helium leakers. The method was adopted official first action.

Interactions of monoclonal antibodies and bovine herpesvirus type 1 (BHV-1) glycoproteins: characterization of their biochemical and immunological properties.

Hybridoma cell lines producing monoclonal antibodies to bovine herpes virus type 1 (BHV-1) were established. The monoclonal antibodies were characterized with respect to their antigen specificities and biological activities. One group of eight monoclonal antibodies precipitated the glycoproteins GVP 3 (180K) and GVP 9 (91K), a second group of thirteen monoclonal antibodies precipitated GVP 6 (130K), GVP 11 (74K) and GVP 16 (55K), and one monoclone secreted antibodies specific for GVP 7 (105K). Analysis of the immune precipitates by electrophoresis under nonreducing conditions suggested that GVP 3 is a dimer of GVP 9. It also indicated that GVP 11 and GVP 16 are components of a disulfide-linked complex, GVP 6. The results, obtained by immunoprecipitation were confirmed by Western blot analysis and an enzyme-linked immunosorbent assay (ELISA), using electrophoretically separated viral glycoproteins. In addition, these techniques demonstrated differential reactivities of the monoclonal antibodies with GVP 11 and GVP 16. The monoclonal antibodies were used to analyze the biological roles of these three sets of glycoproteins. Monoclonal antibodies directed against GVP 3/GVP 9 did not neutralize viral infectivity, but most of them mediated complement-dependent lysis of the infected cell. Individual monoclonal antibodies directed against GVP 6/GVP 11/GVP 16 could neutralize virus as well as participate in complement-mediated lysis. The only available monoclone against GVP 7 did not show any biological activity in the above two assays. Thus, GVP 6/GVP 11/GVP 16 may contain the attachment site of the virion.

Herpesvirus-induced "early" glycoprotein: characterization and possible role in immune cytolysis.

Glycoprotein GVP-11 (molecular weight, 71,500), induced by bovine herpesvirus type 1, was detected on the external surface of infected cells. It could be categorized as an "early" or "beta" class protein since it was synthesized early in the infectious process and its expression was not dependent upon prior viral DNA replication in the infected cells. Monoclonal antibodies directed against GVP-11 immunoprecipitated that glycoprotein and some low-molecular-weight polypeptides from infected cells labeled with either [35S]methionine or [3H]glucosamine. Immunoprecipitation of extracts from cells surface labeled with 125I yielded an additional 138,000-molecular-weight polypeptide. Tunicamycin- or bromovinyl deoxyuridine-treated infected cells yielded polypeptides that were smaller in size than corresponding glycoproteins in untreated cells. Tunicamycin-sensitive glycosylation appeared to be necessary for the expression of the glycoproteins on the surface of the infected cells. The monoclonal antibodies directed against GVP-11 and serum from an immune cow could participate in antibody- and complement-mediated immunocytolysis of infected cells, and this immunocytolysis could be enhanced by arresting cells in the early phase of viral gene expression by treatment with inhibitors of viral DNA synthesis.

Comparison of yeast and mold counts by spiral, pour, and streak plate methods.

Foods with naturally occurring yeast and mold were used in a comparative study of 4 plating techniques. Oats, green beans, and cheese were incubated for 3 and 5 days at 25 degrees C. Although 3 days of incubation would be sufficient for yeast, 5 days were necessary for the mold count. The percent recovery of yeast and molds by the spiral and streak methods ranged from 200 to 357% compared with the pour plating method described in the Bacteriological Analytical manual (BAM). The spiral method had the highest overall recovery and the lowest replicate plating error.

Piliation and colonial morphology among laboratory strains of meningococci.

Colonial morphology and piliation were studied on twelve strains from various serogroups of Neisseria meningitidis. Six different colony types (M1 to M6) were identified. Most strains elaborated only an M1 colonial type, which is similar to gonococcus T4. Several combinations of piliation and colonial morphology were observed: (i) colonial variation in which neither parent nor variant were piliated; (ii) colonial variation involving piliated and nonpiliated cells; (iii) dissociation of piliated from nonpiliated cells with no colonial change; and (iv) colonial variation in which both variants were piliated but with distinctly different pili. Results of this study demonstrate that correlations between piliation and colony morphology within N. meningitidis are exceptions rather than the rule.

Collaborative study comparing the spiral plate and aerobic plate count methods.

The spiral plate count method is a semiautomated plating technique that greatly reduces manpower and material costs normally associated with the pour plating technique. In this collaborative study, 8 laboratories compared the spiral and pour plating techniques, using 4 samples each of 3 products: frozen pumpkin pie, frozen chicken pot pie, and shampoo. The results show that 10 of the 12 comparisons of means of the pour and spiral methods were not significantly different; 2 values were significant at alpha = 0.01. Overall, the components of variance were less than that of the current milk standard, and the replicate per cent coefficient of variation was satisfactory. This study indicates that the spiral plate method is an acceptable alternative to the pour plate method; the spiral plate method has been adopted as official first action.

Pathology in rabbits treated with leukocyte-degraded meningococci in combination with meningococcal endotoxin.

The effects of a preparative dose of the leukocyte egesta containing degraded meningococci and a provocative dose of the meningococcal lipopolysaccharide on development of pathological lesions associated with disseminated intravascular coagulation were studied in tissues of 32 rabbits. These effects were compared with effects of a single dose of meningococcal lipopolysaccharide as well as leukocyte egesta containing degraded Staphylococcus epidermidis. Rabbits injected subcutaneously with egesta containing degraded meningococci followed after 12 h with meningococcal endotoxin (intravenously) exhibited heterophilic leukocytosis and disseminated intravascular coagulation mainly in the pulmonary capillaries and venules; focal necroses occurred in myocardium, lungs, and liver, whereas, cortical renal necrosis developed in lethal cases. Similar lesions, however, but less severe and with less frequency, developed even after a single dose of meningococcal endotoxin or after endotoxin that followed a dose of supernatant fluid from normal leukocytes. Our findings suggest that meningococcal material from polymorphonuclear degradation plays a role in the pathology characteristic of meningococcal septicemia.

A Collaborative Study of the Spiral Plate Method for Examining Milk Samples.

The spiral plating procedure is a rapid method for determining bacteriological counts. Results from a collaborative study indicate that the procedure should be useful in milk analysis. Typical milk samples (homogenized milk, raw milk, chocolate drink, 2% milk, and 20% cream) were sent to six analysts to be examined by standard plate count (SPC) and spiral plate count (SPLPC). Analysis of duplicate samples shows that the SPC and SPLPC values did not differ at the a = 0.01 level. Components of variance for replicate determinations among laboratories and laboratory-sample interaction were computed. The standard deviation was 0.109 compared to the 0.110 estimate reported for SPC in state laboratories. Results from the SPLPC method compared favorably to the results of conventional (SPC) pour procedure.

Spiral plate count method for the examination of raw and pasteurized milk.

The spiral plate count method (SPLPC) was compared with the standard plate count (SPC) method by examining 201 samples of raw and pasteurized milk. Although the means of the two methods differed significantly at alpha = 0.01,the difference was less than 10% and was not considered to be of any practical importance. The pooled replicate variances of both methods were less than 0.003, indicating good agreement between duplicate plates, with the variance of the SPLPC slightly less than that of the SPC. We believe this study indicates that the SPLPC could be substituted for the SPC in the bacteriological examination of milk.

Pili on meningococci from primary cultures of nasopharyngeal carriers and cerebrospinal fluid of patients with acute disease.

The nasopharynx of known meningococcal carriers without signs of acute meningococcal disease as well as cerebrospinal fluid from patients with acute meningococcal disease were cultured on Thayer-Martin agar. Pili were observed in negatively stained preparations of over 80% cells from all primary cultures of both nasopharnyx and cerebrospinal fluids. Although pili were abundant on cells from all primary cultures, all pili were lost on serial subculture in the laboratory. This loss of pili from the cell surface on laboratory subculture was not accompanied by a concomitant loss of cell wall blebs.

Ultrastructure of pili and annular structures on the cell wall surface of Neisseria meningitidis.

In a survey of negatively stained preparations of the prototype strains of Neisseria meningitidis, pili were detected on three strains. However, these pili were detected on fewer than 5% of cells in populations of these three strains. Those individual cells with pili were seldom observed to contain more than two or three pili per cell. In contrast, nearly all cells of the nonprototype group B strain ATCC 13090 had numerous pili on their surfaces. When viewed in frozen-etched replicas, a few pili were observed lying on the cell surface of this latter strain. An annular structure was also found in frozen-etched replicas. This structure usually consisted of a series of concentric rings that were always found on the flat side of these bean-shaped cells. It is concluded that such structures represent a differentiated portion of the cell wall, which is involved in cross-wall formation during synthesis of the cell septum in dividing cells.

Release of endotoxin in the form of cell wall blebs during in vitro growth of Neisseria meningitidis.

Multiple cell wall blebs were observed on the surface of three strains of N. meningitidis taken from log phase cultures. The blebs originated as evaginations of the outer layer of the cell wall. Bleb production was noted on both defined or complex media either as broth or a solid medium. The addition of 10% normal bovine serum to the various media did not affect the production and release of these surface blebs. However, as broth cultures progressed into the stationary phase of growth, the blebs disappeared from the surface of the cells. Blebs were present in substantial quantities in culture supernatant fluids and on cell surfaces and were readily isolated by ultracentrifugation. Analysis for 2-keto-3-deoxyoctonate in cultures revealed that 18% of the total endotoxin of log phase cultures was present in blebs from the cell wall.