Bovine serum and nasal secretion immunoglobulins against Pasteurella haemolytica serotype 1 antigens

Experimental intranasal inoculation of cattle with Pasteurella haemolytica serotype 1 resulted in a group that shed the bacteria in their nasal secretions (colonized) and a group that did not shed (uncolonized). After inoculation, antibody titers in serum and nasal secretions against the total P haemolytica increased significantly, and the proportions of total antibody against specific P haemolytica antigens changed so that the proportion directed against the 94- and 62-kD antigens increased. Prior to inoculation, the proportion of total antibody in the serum against 94- and 62-kD antigens of P haemolytica was higher in calves that remained uncolonized than in those that became colonized with P haemolytica after exposure. Antibody specificity of serum and nasal secretions differed in the relative amounts directed against each P haemolytica antigen. The specificity against P haemolytica antigens differed between IgG and IgA isotypes of serum and nasal secretions, with IgA being directed against fewer antigens than was IgG.     

Production and partial characterization of monoclonal antibodies to Pasteurella haemolytica A1 capsular polysaccharide and lipopolysaccharide

Hybridoma-derived monoclonal antibodies (MAB) against the cell surface antigens of Pasteurella haemolytica serotype 1 were obtained by the fusion of murine myeloma cells (P3 X 63 - Ag 8.653) with splenocytes of BALB/c mice immunized with crude logarithmic growth-phase culture supernatant. Initial screening was performed, using an ELISA, with the same bacterial growth culture supernatant as coating antigens. Further selection was done, using a panel of purified antigens--either capsular polysaccharide or lipopolysaccharide--as the coating antigen in an ELISA, and then performing a leukotoxin-neutralization assay. Two MAB, designated IIB-6 and H-2, reacted specifically with the capsular polysaccharide and the other 3, designated IVG-3, IH-3, and IIC-2, reacted with the lipopolysaccharide. One MAB, designated IH-6, did not react with leukotoxin, capsular polysaccharide, or lipopolysaccharide. The MAB to the capsular polysaccharide (IIB-6 and H-2) were characterized further; both antibodies belonged to the IgM class and were agglutinating. In addition, they promoted neutrophil-mediated opsonophagocytosis and complement-mediated immune bacteriolysis of P haemolytica serotype 1. Results from 3 studies indicated that the MAB IIB-6 and H-2 were specific only to the capsular polysaccharide of serotype 1 of P haemolytica. The MAB to the lipopolysaccharide (IVG-3, IH-3, and IIC-2) were of the IgG1, IgG3, and IgM classes, respectively and were not characterized further. The availability of a MAB identifying a serotype-specific, surface-exposed determinant on the capsule of P haemolytica serotype 1 should facilitate and expand studies concerning the role of the capsular material and lipopolysaccharide in the pathogenicity of P haemolytica infection in cattle.     

Production and characterization of monoclonal antibodies against chicken lymphocyte surface antigens

A panel of monoclonal antibodies (mAbs) with specificity for chicken lymphocyte surface antigens was established and characterized based on their reactivities against chicken lymphoid cells and tumor cell lines on flow cytometry. Three mAbs (7-3G-2, 7-2E-8, and JB-2) reacted preferentially with thymocytes, however, none of them reacted with Marek's disease derived T lymphoblastoid cell lines. Four mAbs (6-27A-1, 4-5C-5, Lc-4, and Lc-6) reacted with spleen cells and peripheral blood leukocytes as well as thymocytes. All seven mAbs reacted with chicken embryonic thymocytes from day 12 of embryonic life onward. All mAbs showed no reactivity against bursal lymphocytes.     

Comparison of antigens of Pasteurella haemolytica serotype 1 grown in vitro and in vivo.

To determine whether antigenic differences exist in Pasteurella haemolytica serotype 1 grown in different culture conditions, the bacteria was grown on solid enriched medium, in broth culture, and in tissue chambers subcutaneously implanted in the flanks of calves. The organisms obtained by each culture method were comparable with respect to encapsulation and lipopolysaccharide content. In the bacteria grown in vivo, several unique high molecular-mass (greater than 150 kDa) protein antigens were found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblotting. Bacteria grown in vitro had higher concentrations of a 49- and a 26-kDa protein than the organisms grown in vivo. The concentration of several major proteins (30, 42, 55, 71, and 100 kDa) were similar among the organisms grown by the three cultural conditions. Although the high molecular-mass antigens were unique for the chamber-grown bacteria, they were recognized by serum from a calf that had been vaccinated with formalin-killed, solid medium-grown P haemolytica and were resistant to challenge exposure with the live organism. This recognition of antigens by serum from the P haemolytica-resistant calf that had been vaccinated with solid-medium-grown bacterium, indicates that the high molecular-mass antigens from chamber-grown P haemolytica may be precursors of or share antigenic determinants with other P haemolytica proteins and may not be important for consideration in vaccine formulation.     

Preparation and characterization of monoclonal antibodies against bovine B lymphocyte surface antigens

Two monoclonal antibodies (MoAbs; BLMo-4 and BLMo-10) were prepared by immunizing with a cell line established from peripheral blood mononuclear cells (PBMC) of enzootic bovine leukosis (EBL) cattle. The specificities of these MoAbs were assayed using bovine PBMC. BLMo-4 reacted with all surface immunoglobulin-positive cells (SIg+ cells; B lymphocytes) and also recognized monocytes, but did not react with T lymphocytes. BLMo-10 recognized a majority, although not all, B lymphocytes, but did not react with either T lymphocytes or monocytes. The antigens recognized by BLMo-4 and BLMo-10 were not Ig, Fc or C3 receptors on the surface of B lymphocytes. The reactivity of the MoAbs with mononuclear cells from the lymphoid organs of adult cattle was studied. BLMo-4 and BLMo-10 did not react with any bone marrow cells. BLMo-10 reacted with 7.4% of thymocytes, and stained the medulla of the thymus in the immunoperoxidase assay. In the case of PBMC, spleen and lymph node cells, the percentage of cells positive for BLMo-4 was slightly higher than that of SIg+ cells, but BLMo-10 showed a slightly lower value.     

Characterisation and biological activity of monoclonal antibodies specific for Pasteurella haemolytica A1 capsule and lipopolysaccharide.

Monoclonal antibodies (mAb) against both Pasteurella haemolytica A1 capsule and lipopolysaccharide (LPS) were produced. Anti-capsule mAb reacted with the homologous A1 serotype only, whereas mAb against LPS reacted with P. haemolytica serotypes A2, A5, A8, A12, A14 and A16 but not with 33 bacterial species or rough LPS mutant strains tested. Both capsule and LPS antigens were visualised on the surface of bacteria by immunogold electron microscopy. Neither of the mAbs demonstrated antibody-dependent complement-mediated killing in vitro but both facilitated phagocytosis in vitro.     

Sequential development of antigens and toxins of Pasteurella haemolytica serotype 1 grown in cell culture medium.

Pasteurella haemolytica was grown in nonsupplemented cell culture medium, or in medium supplemented with bovine serum albumin (BSA) for 24 hours. The production of leukotoxin (LKT) and endotoxin was sequentially evaluated, as were bacterial antigens associated with bacterial cell lysates and culture supernates. Supplementation of medium with BSA had no effect on bacterial growth curves; however, LKT activity was detected earlier and was greater in culture supernates from BSA-supplemented media than from nonsupplemented medium. Leukotoxin antigen (105 kDa) was detected in culture supernates, using a monoclonal antibody, immunoblot analysis, and densitometry. The relative concentrations of LKT antigen were proportional to LKT activity. Endotoxin activity was initially lowest in the culture supernates from nonsupplemented medium, but increased during the incubation period, whereas endotoxin activity in BSA-supplemented culture supernates decreased with time in culture. In culture supernates from nonsupplemented medium, the number of antigenic bands identified by immunoblot analysis with hyperimmune anti-P haemolytica and densitometry was greater than in culture supernates from supplemented media. In bacterial lysates, a 95-kDa antigen was the major antigen detected, using the anti-LKT monoclonal antibody. The concentration of that antigen varied among lysates from nonsupplemented medium and BSA-supplemented media. Using hyperimmune anti-P haemolytica serum, minor differences were seen in the relative quantities of lysate-associated antigens dependent on time in culture and medium used. Among the major antigens seen, differences were most apparent for 150-, 100-, and 87-kDa antigens, whereas differences were not obvious for 42- 40-, and 30-kDa antigens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vaccination of neonatal colostrum-deprived calves against Pasteurella haemolytica A1.

Colostrum-deprived Holstein calves were vaccinated at 2 and 4 wk of age with a Pasteurella haemolytica A1 culture supernatant vaccine to determine whether active immune responses and protection could be induced in this age group in the absence of maternal antibodies. All calves responded to vaccination with high titers of IgM antibodies to capsular polysaccharide within 1 wk of primary vaccination. Mean titers of IgG1 and IgG2 antibodies to this antigen increased significantly by 2 wk after secondary vaccination, but peak antibody titers were low. All of the vaccinated calves seroconverted with production of leukotoxin-neutralizing antibodies, but peak antibody titers were low. Vaccinated calves experienced considerable lung damage after experimental challenge, but survival rate, clinical scores, and percent lung involvement were significantly better than those of control (placebo-injected) calves.     

Production and characterization of monoclonal antibodies to serotype specific antigens of Haemophilus pleuropneumoniae serotype 2

Mouse monoclonal antibodies were produced to Haemophilus pleuropneumoniae bacteria of the most common serotype 2. 11 hybridoma cultures were recovered that produced antibodies with moderate to strong reactivity to the antigen. 10 of these antibodies were specific to isolated capsular antigens from H. pleuropneumoniae of serotype 2, while one antibody reacted with capsular antigens from bacteria of all 8 serotypes. One hybridoma producing antibodies with a titre of 1:1000 was cloned and the antibody specificity studied further. The binding of this antibody (1F3) to whole bacteria, and capsular extracts isolated at different temperatures indicate that the antibody is specific for a thermostable polysaccharide antigen present in the cellular capsule of H. pleuropneumoniae of serotype 2.     

Monoclonal antibodies against surface antigens of Pasteurella multocida strain P-1059

Four monoclonal antibodies (MAbs) were developed against serotype 3:A, P-1059 strain of Pasteurella multocida. Enzyme-linked immunosorbent assays were used to screen those hybridomas producing antibodies to either a surface protective (2.5 S) or lipopolysaccharide (LPS) antigen. MAbs 6EE11, D7H10, E11E3, and C11H2 were positive against 2.5 S antigen, and two of them, E11E3 and C11H2, were positive for the LPS antigen. MAbs 6EE11 and D7H10 reacted with a major protein band of molecular weight of 35,500, whereas E11E3 and C11H2 recognized a band with a molecular weight of 12,500 of the 2.5 S antigen. Treatment of the 2.5 S antigen with periodic acid abolished epitopes reacting with E11E3 but not with 6EE11. MAb 6EE11 did not recognize any band in Western blot after proteinase K treatment of the 2.5 S antigen, whereas antibody activity of E11E3 did not change. MAb 6EE11 reacted with serotypes 3, 4, 9, 10, 11, 12, and with M-9 strains in the immunofluorescence test. MAb E11E3 was positive only with serotype 3 or 10 strains, excluding M-9 strain. Electron microscopic studies with P-1059 strain indicated that antigens binding to 6EE11 and/or E11E3 were present in the capsule.     

A crude cytotoxin vaccine protects sheep against experimental Pasteurella haemolytica serotype A2 infection

Three vaccines containing Pasteurella haemolytica serotype A2 antigens were tested for their ability to protect sheep against a homologous challenge. A crude cytotoxin preparation in combination with a sodium salicylate extract (SSE) or crude cytotoxin alone were highly protective (98 and 86%, respectively), whereas SSE alone was poorly (47%) protective. These findings indicated that the crude cytotoxin was an essential component of a protective vaccine. Protection correlated with serum cytotoxin-neutralising (CN) titres and bactericidal activity, which were stimulated by antigens in the crude cytotoxin preparation.     

Pasteurella haemolytica bacteriophage: identification, partial characterization, and relationship of temperate bacteriophages from isolates of Pasteurella haemolytica (biotype A, serotype 1)

Pasteurella haemolytica (biotype A, serotype 1) isolates (n = 15) from the upper respiratory tract of clinically normal cattle, as well as from lung lesions from cases of fatal bovine pasteurellosis, were examined for the presence of bacteriophage after irradiation with UV light. Treatment of all P haemolytica isolates with UV irradiation resulted in lysis of bacteria due to the induction of vegetative development of bacteriophages. The extent of growth inhibition and bacterial lysis in irradiated cultures was UV dose-dependent. Bacterial cultures exposed to UV light for 20 s reached peak culture density between 60 and 70 minutes after irradiation; thereafter, culture density declined rapidly, so that by 120 minutes, it was approximately 60% of the original value. When examined ultrastructurally, lytic cultures from each isolate revealed bacteriophages with an overall length of approximately 200 nm and that appeared to have a head with icosahedral symmetry and a contractile tail. Cell-free filtrate from each noninduced bacterial isolate was inoculated onto the other bacterial isolates in a cross-culture sensitivity assay for the presence of phages lytic for the host bacterial isolates. Zones of lysis (plaques) did not develop when bacterial lawns grown from the different isolates were inoculated with filtrates from the heterologous isolates.     

Preparation of monoclonal antibodies against cell surface antigens in cattle

The BALB/c mice were immunized three times with bovine lymphocytes and thrombocytes in vivo. Besides, dissociated spleens of the mice were immunized with bovine lymphocytes in vitro. Hybridomas producing monoclonal antibodies were formed as a result of the fusion of the spleen cells with the murine myeloma cells. On the whole, four fusions were performed after immunization in vivo and three fusions after immunization in vitro, and 70 stable hybridoma clones were obtained. Five monoclonal antibodies exhibited an identical specific reaction only with bovine thrombocytes, two antibodies reacted only with a certain limited population of bovine spleen cells. The remaining monoclonal antibodies exhibited no tissue specificity and were bound to the lymphocytes of peripheral blood, lymph nodes, thymus, and spleen, to thrombocytes, liver cells, and spermatozoa, but never to erythrocytes. As for the amount of the obtained hybridomas and specific antibodies, no significant difference was observed in the effectiveness of in vivo and in vitro immunization.     

Mannheimia haemolytica serotype 1 and Pasteurella trehalosi serotype 10 culture supernatants contain fibrinogen-binding proteins

Fibrinogen-binding proteins were found in the culture supernatants of Mannheimia haemolytica serotype 1 (ATCC 43270) and Pasteurella trehalosi serotype 10 (ECO-100). Sheep fibrinogen was biotinylated and shown to bind to proteins in the culture supernatants by modified western blot. Fibrinogen-binding proteins in the culture supernatant may be important virulence factors leading to the characteristic fibrinous pneumonia caused by these organisms and may be critical antigenic targets for immune prophylaxis.     

Genomic distribution of a serotype 1-specific antigen-coding DNA fragment of Pasteurella haemolytica.

A genomic fragment of Pasteurella haemolytica biotype A coding for a serotype 1-specific agglutinating antigen was used as a probe in a series of hybridization experiments to determine distribution of the fragment in various P. haemolytica serotypes as well as other bacteria. Results showed presence of the fragment in seven out of the 12 serotypes tested, all of which belonged to biotype A. Two other serotypes belonging to biotype A, all three serotypes belonging to biotype T, two Pasteurella multocida isolates and Escherichia coli did not have the fragment in their genome. Thus the expression of the P. haemolytica biotype A serotype 1-specific agglutinating antigen (PHA1SAA) seems to be due to serotype-specific regulation of protein expression rather than to genetic deletion. Differences in methylation of the PHA1SAA-coding fragment was also noted in DpnI and Sau3AI genomic DNA digests from the various serotypes analyzed by Southern blot. However, no apparent correlation was observed between methylation and PHA1SAA expression. E. coli with a recombinant plasmid containing a homologous genomic fragment derived from P. haemolytica serotype 2 also expressed PHA1SAA.     

Characterization of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5.

Two monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5, designated as 5MAb-1 and 5MAb-6, were characterized. Enzyme-linked immunosorbent assay-inhibition tests with whole-cell antigens obtained from strains of serotype 1 through 12 of A pleuropneumoniae revealed that 5 MAb-1 bound to only serotype-5 strains. The epitope recognized by 5MAb-1 was a carbohydrate that was sensitive to periodate oxidation and resided on the structure of beta-1,6-linked D-galactose in an O-antigen polysaccharide of serotype-5 lipopolysaccharide. Analysis of these results revealed that the O-antigen polysaccharide of lipopolysaccharide was 1 of the antigenic determinants responsible for the serotype specificity of A pleuropneumoniae. On the other hand, 5MAb-6 reacted with strains of serotype 1 through 10 in varying degrees and its epitope was located on polypeptides sensitive to proteinase K. In an immunoblotting analysis, 5MAb-6 reacted with 2 polypeptide bands, with molecular weights of approximately 41,500 and 28,000, in the outer membrane protein-rich fraction obtained from strains of serotype 1 through 10. These results indicated that outer membrane proteins from several serotype strains of A pleuropneumoniae possessed common antigenic determinants.     

Pasteurella haemolytica serotype 1 colonization of the nasal passages of virus-infected calves

Nasal passages of calves with a virus-induced respiratory tract disease became colonized by Pasteurella haemolytica serotype 1 after they were inoculated intranasally with P haemolytica. Inoculation with infectious bovine rhinotracheitis virus caused a more severe clinical illness and resulted in a greater degree of colonization with P haemolytica than developed after inoculation with parainfluenza-3 virus. Nasal passages of parainfluenza-3 virus-inoculated calves were colonized to a greater degree with P haemolytica than were those of healthy, nonstressed calves. Calves were susceptible to P haemolytica colonization during or shortly after virus-induced illness, even though they had been previously exposed to P haemolytica and had serum antibody and nasal secretion antibody to P haemolytica.     

Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus

Two foot-and-mouth disease virus (FMDV) monoclonal antibodies (mAbs) were produced from mice immunized with either FMDV serotype A, subunit (12S) or FMDV serotype O, whole virus (140S). Both mAbs (F1412SA and F21140SO) recognized all seven serotypes of FMDV in a double antibody sandwich (DAS) ELISA, suggesting that the binding epitopes of the two mAbs are conserved between serotypes. These mAbs are IgG1 isotype and contain kappa light chains. In order to define the mAb binding epitopes, the reactivity of these mAbs against trypsin-treated and denatured FMDV were examined using an indirect ELISA. The binding site of the mAb, F1412SA is trypsin sensitive and the epitope is linear. Both ELISA and Western blot results suggested that the polypeptide VP2 contributed to the immunodominant site. This mAb showed reactivity to VP2 peptide (DKKTEETTILEDRIL). The mAb, F21140SO, recognized an epitope which is trypsin resistant and discontinuous. This mAb binding to FMDV is dependent on conformational structures of intact viral (140S) or subunit (12S) particle, since it failed to recognize any viral protein in Western blot. This conformational and highly conserved epitope is the first identified epitope among all seven FMDV serotypes. Because the use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal antisera, these two mAbs with different specificities are suitable for type-independent diagnosis of FMDV, such as DAS ELISA, or could be adapted to immuno-chromatographic or flow-through rapid test.     

Serum antibody response to carbohydrate antigens of Pasteurella haemolytica serotype 1: relation to experimentally induced bovine pneumonic pasteurellosis

The antibody responses to the capsular carbohydrate (CC) purified from Pasteurella haemolytica serotype 1 were determined by an ELISA, using 135 sera from 6 calves vaccinated with phosphate-buffered saline solution, formalin-killed P haemolytica bacterins, live P haemolytica, or an extract of P haemolytica referred to as carbohydrate-protein subunit (CPS). Calves vaccinated with live P haemolytica, bacterins, or CPS developed serum antibodies to CC. Bacterins containing Freund incomplete adjuvant or Freund complete adjuvant induced higher antibody responses than did bacterins containing aluminum hydroxide. In 4 of 6 experiments, high antibody responses to CC were significantly (P less than 0.05) correlated with resistance to transthoracic challenge exposure with P haemolytica. When calves were challenge exposed with a dose of P haemolytica that was 4.5 times greater than the standard challenge exposure dose or when calves that had been vaccinated with CPS were challenge exposed, antibody responses did not significantly (P greater than 0.05) correlate with resistance to challenge exposure. The amount of serum antibodies to CPS increased significantly (P less than 0.05) when calves were vaccinated with live or killed P haemolytica or with CPS, compared with that in calves given saline solution. In 5 of 6 experiments, correlation between high antibody responses and resistance to challenge exposure was significant (P less than 0.05). The correlation between those variables was not significant (P less than 0.07) for CPS-vaccinated calves. In the ELISA, treatment of CPS with sodium m-periodate, to oxidize periodate-sensitive carbohydrate epitopes, failed to markedly alter the antibody response to CPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of acute bronchopneumonia in mice by intrabronchial inoculation of Pasteurella haemolytica serotype 1.

Dose dependent pulmonary lesions of acute bronchopneumonia were induced in male, outbred Swiss Webster mice by intrabronchial inoculation of Pasteurella haemolytica. Five exponential dilutions ranging from 5 x 10(4) to 5 x 10(8) colony forming units per mL (CFU/mL) of Pasteurella haemolytica serotype 1 were inoculated into five groups of mice. Mice were killed by cervical dislocation 24 hours postinoculation. Pulmonary lesions occurred in mice of all five groups, however, 5 x 10(7) CFU/mL was the minimal dose which consistently produced lesions. Focal parenchymal necrosis, suppurative bronchiolitis, and flooding of interalveolar septa and alveoli by edema fluid, fibrin, neutrophils and macrophages, were observed microscopically. We conclude that outbred Swiss Webster mice can be used as a model for the study of selected disease mechanisms of acute lung inflammation and that this model may be used to determine some of the pathogenic mechanisms involved in the development of pulmonary lesions in bovine pneumonic pasteurellosis.