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.     

Comparison of antibody responses in cattle to outer membrane proteins from Pasteurella haemolytica serotype 1 and from eight untypeable strains.

Membrane associated proteins from 8 untypeable Pasteurella haemolytica strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with those of P haemolytica serotypes 1 and 2. Cattle antisera obtained from P haemolytica serotype 1 vaccine trials were used in immunoblotting assays to compare the membrane proteins from the 8 untypeable strains with those from P haemolytica serotypes 1 and 2. Densitometry was used to identify bands, and using linear regression analyses, the peak area optical densities (measuring antibody response) were correlated to lesion scores from the vaccinated calves. Significant antibody responses to proteins of 99, 69, 60, 55, 47, 45, 39, 33, 30, 16, and 14.5 kDa were detected for 4 or more of the 8 P haemolytica untypeable strains. Serotypes 1 and 2 of P haemolytica contained a comigrating 30-kDa protein. Antibody responses to proteins of 39, 33, and 32.5 kDa were significant for 3 of the untypeable strains and had significant correlation to lesion scores. Antibody responses to various other proteins were significant for 2 untypeable strains each.     

Aerosol vaccination of calves with pasteurella haemolytica against experimental respiratory disease.

Three experiments were conducted on calves in which the efficacy of vaccination with live Pasteurella haemolytica in aerosol was tested by challenge with sequential aerosol exposure to bovine herpesvirus 1 and P. haemolytica. Neither single nor multiple aerosol vaccinations protected against the experimental disease. Macroscopically recognizable rhinitis, tonsillitis, tracheitis and pneumonia occurred in both controls and vaccinates. In one experiment as many as three aerosol vaccinations with live P. haemolytica for up to 20 minutes failed to elicit clinical signs in exposed calves. Pasteurella haemolytica was isolated less frequently from tissues of vaccinated calves than from those of nonvaccinated calves. Pasteurella haemolytica was isolated from deep nasal swabs of 4/14 vaccinated calves five and six days after viral exposure. It was concluded that although bovine herpesvirus 1 vaccination has been shown previously to prevent the experimental disease produced by bovine herpesvirus 1-P. haemolytica, live P. haemolytica vaccination by aerosol will not provide the same protection.     

Sialoglycoprotease of Pasteurella haemolytica A1: detection of antisialoglycoprotease antibodies in sera of calves.

Log phase culture supernate from Pasteurella haemolytica biotype A, serotype 1 contains a proteolytic enzyme specific for O-sialoglycoproteins. Using two methods, Western immunoblotting and enzyme neutralization assay, it was demonstrated that certain bovine sera from two previous P. haemolytica A1 vaccination and challenge trials contained antibodies (Ab) (isotypes IgG1 and IgG2 on Western immunoblot) to the sialoglycoprotease (Gcp). In these trials, selected calves were vaccinated twice with either the commercial culture supernate vaccine Presponse or given phosphate-buffered saline (PBS). One trial was conducted during spring, P. haem XIX, and the other during the winter, P. haem XXI. Although there was no clear evidence for induction of anti-Gcp in response to vaccination, several calves seroconverted following intrapulmonary challenge with live P. haemolytica A1. This is the first report of anti-Gcp Ab in bovine sera. The results indicated that the Gcp is immunogenic and that the bacterium produces the enzyme in vivo. Further, animals with an anti-Gcp response had less pneumonia at necropsy, suggesting the Gcp may induce protective immunity.     

Serum antibodies to Pasteurella haemolytica lipopolysaccharide: relationship to experimental bovine pneumonic pasteurellosis

An enzyme-linked immunosorbent assay was used to determine the serum antibody response to Pasteurella haemolytica lipopolysaccharide (LPS) for calves vaccinated with saline solution, a formalin-killed P haemolytica bacterin, or live P haemolytica. Bacterin-vaccinated calves had a lower antibody response to LPS than did calves vaccinated with live P haemolytica. Calves vaccinated with either saline solution or the bacterin were more susceptible to intrapulmonic challenge exposure with P haemolytica than were calves vaccinated with liver organisms. Serum antibody responses to P haemolytica LPS did not seem important for resistance to challenge exposure, because there was no significant correlation (P greater than 0.05) between the lung lesion score and antibody response to P haemolytica LPS. There was a highly significant correlation (P less than 0.001) between antibody detected against P haemolytica LPS and that against formalin-killed P haemolytica. Competitive binding studies indicated that P haemolytica LPS is a major antigenic determinant on the surface of P haemolytica. There did not seem to be substantial cross-reaction between LPS from P haemolytica and that from Escherichia coli (serotype O26:B6).     

Functional and metabolic activity of bovine pulmonary lavage cells phagocytically stimulated with pathogenic isolates of Pasteurella haemolytica

Live Pasteurella haemolytica biotype A, serotype 1 isolates (n = 3) and Escherichia coli K-12, strain W3110, were reacted with bovine pulmonary lavage cell (PLC) suspensions. The comparative effects of the different bacteria on the functional and metabolic activity of alveolar macrophages (AMO) in the PLC suspensions were assessed simultaneously by use of 51Cr release, luminol-dependent chemiluminescence (LDCL), and AMO bactericidal assays. The bovine PLC responded differently to E coli, than to the 3 P haemolytica isolates in each of the 3 experimental test systems; however, responses to each of the P haemolytica isolates were not found to be significantly different. Unopsonized live P haemolytica cells adversely affected the functional and metabolic response of PLC, whereas there was no evidence of a cytotoxic (cytocidal) influence of E coli. A difference in 51Cr release for reaction mixtures containing E coli and P haemolytica was not detected at zero time; however, at each subsequent time, reaction mixtures phagocytically stimulated with P haemolytica had significantly increased amount of 51Cr release (P less than 0.05), compared with those mixtures containing E coli. Bovine AMO in the PLC suspensions were able to effectively kill E coli in vitro, but were unable to prevent survival and subsequent growth of P haemolytica. The luminol-dependent chemiluminescence profiles for reaction mixtures phagocytically stimulated with E coli provided evidence of sustained production of oxygen radicals with antimicrobial capabilities by bovine AMO in the PLC. Production of these highly reactive antimicrobial oxidants appeared initially in cultures containing P haemolytica but, subsequently, their production declined precipitously and ceased altogether.     

Efficacy of an outer membrane protein of Pasteurella haemolytica A2, A7 or A9-enriched vaccine against intratracheal challenge exposure in sheep

The outer membrane proteins (OMP) were extracted from the P. haemolytica A2, A7 and A9 to determine their potential as immunogens and their capability for cross-protection. Sixty lambs of approximately 9 months old were divided into four main groups. Animals in Group 1 were vaccinated with 2ml vaccine containing 100microg/ml of the outer membrane proteins of P. haemolytica A2. Animals in Group 2 were similarly vaccinated with the OMPs of P. haemolytica A7 while Group 3 with OMPs of P. haemolytica A9. Animals in Group 4 were unvaccinated control. During the course of the study, serum was collected to evaluate the antibody levels toward each OMP. There appeared to be good immune responses. However, high antibody levels did not necessarily result in good protection of the animals, particularly against cross-infection with P. haemolytica A9 in animals vaccinated with the OMPs of P. haemolytica A2. It seemed that the antibody responses were more specific toward the homologous challenge but generally did not cross-protect against heterologous serotype challenge. However, the OMPs of P. haemolytica A7 produced good in vivo cross-protection and excellent correlations when good antibody responses against all serotypes led to successful reductions of the extent of lung lesions following homologous and heterologous challenge exposures. Thus, the OMPs of P. haemolytica A7 was effective in protecting animals against homologous and heterologous infection by live P. haemolytica A2, A7 and A9.     

Immunogenicity of iron-regulated outer membrane proteins of Pasteurella multocida A:3 in cattle: molecular characterization of the immunodominant heme acquisition system receptor (HasR) protein

The iron-regulated outer membrane proteins (IROMPs) of Pasteurella multocida A:3 strain 232 (Pm232), a bovine isolate, were investigated as potential immunogens in cattle. We addressed the ability of P. multocida IROMP-enriched fractions to induce antibody responses in cattle by different vaccination strategies and the protective efficacy of these antibodies using a P. multocida-induced pneumonia challenge model. Vaccination of cattle with outer membrane-enriched fractions derived from Pm232 grown on either iron-depleted (IROMPs) or iron-sufficient (OMPs) conditions induced significant antibody responses; however, the correlation with lung lesion scores was not significant (P = 0.01 and P < 0.07, respectively). SDS-PAGE, Western blots and densitometric analyses of Pm232 grown under iron-deficient conditions revealed five major IROMPs including an immunodominant 96 kDa protein band. Mass spectrometry analysis of the 96kDa protein band suggested homology with the heme acquisition system receptor (HasR) of avian P. multocida (strain Pm70) and was confirmed by DNA sequence analysis of the cloned Pm232 hasR gene. Further analyses indicated that Pm232 HasR is a surface-exposed OMP and conserved among most P. multocida isolates investigated. In addition, cattle vaccinated with live Pm232 or IROMPs had significantly higher antibody responses to the 96 kDa protein band and the correlation with lung lesion scores approached significance (P = 0.056). These results indicate that antibody responses in cattle are induced by P. multocida IROMPs, and that the 96 kDa HasR protein is an immunodominant IROMP.     

Investigation of outer membrane proteins of Pasteurella. 2: Iron-regulated outer membrane proteins of Pasteurella multocida and Pasteurella haemolytica

Pasteurella multocida and Pasteurella haemolytica produce specific proteins in the outer membrane under iron-depleted conditions. Pasteurella multocida serovar A expresses these proteins of molecular masses of 76 and 96 kDa as determined by electrophoresis. The analogous serovar D produces a further iron-regulated protein of 85 kDa. The Pasteurella haemolytica strains of serovar A1, A6 and T contain iron-regulated outer membrane proteins of molecular masses of 71, 77 and 100 kDa. These proteins possess binding positions for iron ions. Both Pasteurella multocida and Pasteurella haemolytica strains utilize iron from porcine and bovine transferrin, but not from haemin and haemoglobin.     

Cloning of a gene fragment encoding bovine complement component C3d with expression and characterization of derived fusion proteins

The gene fragment coding for bovine C3d gene (boC3d) was cloned and expressed as a component of fusion proteins destined for use in vaccine studies in cattle, and for in vitro experiments. This fragment of complement protein C3 (C3d) has been shown to enhance B cell responses when complexed with antigen. Three potential vaccine constructs were engineered to contain one, two or three boC3d units linked to a fragment of the leukotoxin of Mannheimia haemolytica A1, an economically important pathogen of cattle that causes a fibrinous pneumonia in calves. A recombinant biotinylated boC3d protein (for use in in vitro studies) was generated by endogenous biotinylation in Escherichia coli by means of the BirA holoenzyme synthetase. All recombinant proteins incorporated polyhistidine tags and were purified by nickel-agarose chromatography, then analyzed by SDS-PAGE and Western immunoblot. The identity of boC3d was confirmed by mass spectrometry, since monoclonal antibodies to boC3d were not available. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice and rabbits, using antigens unrelated to diseases occurring naturally in these species. The boC3d fusion proteins expressed in this study will provide the basis for immunization trials in cattle and studies of receptor binding and cell activation of bovine lymphocytes.     

In vitro lymphocyte proliferative responses and gamma-interferon production as measures of cell-mediated immunity of cattle exposed to Pasteurella haemolytica.

Cell-mediated immune mechanisms may play a role in the pathogenesis and prevention of pneumonia in cattle caused by Pasteurella haemolytica serotype A1. To determine the circumstances required to stimulate and identify cell-mediated immune responses, calves were vaccinated with a commercial P. haemolytica bacterin or a live commercial P. haemolytica vaccine, or were infected intratracheally with virulent P. haemolytica. All calves were challenge-exposed intratracheally with P. haemolytica 31 d after vaccination or prior infection. Peripheral blood mononuclear cells and mediastinal and superficial cervical lymph node cells were stimulated with antigens prepared from P. haemolytica to evaluate in vitro proliferative responses and gamma-interferon production as measures of cell-mediated immunity. Strong proliferative responses and gamma-interferon production were detected in lymph node cells from calves vaccinated with the live vaccine and from infected calves, especially in response to stimulation with an outer membrane protein preparation from P. haemolytica. Greater proliferative responses and gamma-interferon production were associated with the lymph node nearer the site of bacterin administration (superficial cervical lymph node) or the site of infection (mediastinal lymph node), whereas greater proliferative responses and gamma-interferon production were associated with the more distant lymph node (mediastinal lymph node) in calves vaccinated with the live vaccine. Neither proliferative responses nor gamma-interferon production were detected in peripheral blood mononuclear cells from calves that were vaccinated for or infected with P. haemolytica. Antileukotoxin antibody titers were determined by a serum neutralization assay, and protection against pneumonic lesions was more closely correlated with antileukotoxin antibody responses than with lymphocyte proliferation or gamma-interferon responses.     

A putative iron-regulated TonB-dependent receptor of Mannheimia (Pasteurella) haemolytica A1: possible mechanism for phase variation.

A recombinant plasmid that codes for a novel iron receptor protein (Irp) of Mannheimia (Pasteurella) haemolytica A1 was isolated by the partial complementation of an Escherichia coli fur mutant. The deduced amino acid sequence of Irp exhibited characteristics typical of TonB-dependent receptors. These include: a TonB-box at the N-terminal; a 50 amino acid region homologous to the "plug" domain of the E. coli FhuA and FepA receptors; and a C-terminal TonB-dependent signature which likely functions as an outer membrane anchoring domain. Previously uncharacterized Irp homologues were detected by BLAST analysis of available databases and incomplete microbial genomes. When the irp homologues from Neisseria gonorrhoeae and N. meningitidis were cloned by PCR and expressed in E. coli, novel proteins of the predicted size (84kDa) were detected in cell lysates, demonstrating that these are functional genes. The M. haemolytica A1 irp gene undergoes phase variation at a nucleotide region which contain the sequence AAAAAAATTAAAA (7A-2T-4A) flanked by a short inverted repeat. Site-specific mutagenesis of the 7A-2T-4A sequence as well as replacement of the inverted repeats resulted in a stable construct that expressed the Irp protein without phase variation. The expression of irp in M. haemolytica A1 was regulated by iron concentrations and most likely a Fur homologue, consistent with the proposed function of Irp in iron metabolism. The irp genes may represent contingency loci that play a role in iron acquisition during infection.     

Bovine pneumonic pasteurellosis: chemiluminescent response of bovine peripheral blood leukocytes to living and killed Pasteurella haemolytica, Pasteurella multocida, and Escherichia coli

A luminol-dependent chemiluminescence (LDCL) assay was used to evaluate the response of bovine polymorphonuclear leukocytes; (neutrophils [PMN]) to living and heat-killed Escherichia coli, Pasteurella multocida (type A, serotype 3), and P haemolytica (biotype A, serotype 1), and to heat-killed P haemolytica and sterile culture supernatant from living P haemolytica. Control cultures containing PMN that had not been phagocytically stimulated with bacteria had a modest increase in LDCL during the initial 10 minutes of incubation, followed by a gradual decline throughout the 120-minute incubation period. Bovine PMN emitted LDCL more efficiently when the cells were exposed to living E coli or P multocida than when they were exposed to the same bacteria killed by heat. The mean LDCL values for reaction mixtures containing living E coli or P multocida peaked at 30 minutes of incubation and remained above values for mixtures containing the same heat-killed bacteria. Kinetics of the LDCL response of bovine PMN to heat-killed P haemolytica were similar (although reduced in amplitude) to that observed with killed E coli or P multocida. The LDCL response of bovine PMN to living P haemolytica was not like that for E coli or P multocida, and was characterized by the development of a peak response at 10 minutes followed by a precipitous decrease in responsiveness and a subsequent complete cessation of LDCL. Addition of sterile culture supernatant from living P haemolytica to test samples containing heat-killed P haemolytica induced a response similar to that obtained with the living microorganism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of vaccination with live or killed Pasteurella haemolytica on resistance to experimental bovine pneumonic pasteurellosis

Using 6- to 8-month-old beef calves, 3 experiments were conducted to compare the effect of vaccination with live or killed Pasteurella haemolytica on resistance to a transthoracic challenge exposure with the organism and to correlate serum antibody response with resistance. In each experiment, calves were vaccinated twice at 1-week intervals and were challenge exposed 21 days after the first inoculation. Lung lesions were evaluated by a system, such that higher scores indicated the more severe lesions. In each experiment, calves immunized with live P haemolytica had lower lesion scores than calves vaccinated with saline solution or bacterin. In 2 of the experiments, the differences were significant (P less than 0.05). In all experiments, calves vaccinated parenterally with a commercial P haemolytica/P multocida bacterin or with a formalin-killed P haemolytica bacterin had lesion scores that were not significantly different (P greater than 0.05) than for control calves vaccinated with saline solution. Live and killed bacterial preparations induced a significant serum antibody response to P haemolytica as measured by a quantitative fluorometric immunoassay. The antibody response to vaccination was not affected by preexisting titers to P haemolytica. Serum antibody titers were not consistently as high for calves vaccinated with bacterins as for calves vaccinated with live organisms. Although high antibody titers correlated with low lesion scores when calves vaccinated with saline solution or live organisms were analyzed collectively, there was not a significant correlation between the 2 variables when calves, vaccinated with saline solution or with bacterin, were analyzed collectively. These data indicate that, although bacterins may induce a detectable serum antibody response, they do not induce protection against transthoracic challenge exposure to P haemolytica.     

Efficacy of a streptomycin-dependent, live Pasteurella haemolytica vaccine against challenge exposure to Pasteurella haemolytica in cattle

A streptomycin-dependent, live Pasteurella haemolytica vaccine was given in 1 or 2 doses to 2 groups of weaned calves; 2 other groups of calves were not vaccinated. All calves in the vaccinated groups and calves in 1 of the nonvaccinated groups were stressed by transport, intratracheally inoculated with bovine herpesvirus type-1 (Cooper strain), and then intratracheally inoculated with P haemolytica type A1. The 4th group of calves (nonvaccinated controls) was not stressed and were not intratracheally inoculated with virus or bacteria. Mean daily weight gains, total clinical sign scores, lung lesion scores, plasma fibrinogen concentrations, and antibody titers against P haemolytica were determined at various intervals. Calves that had been vaccinated twice had greater mean daily weight gains and lower total clinical sign scores and lung lesion scores than did nonvaccinated, challenge-exposed calves, but the difference was not significant (P greater than 0.05). Calves vaccinated once had the greatest mean daily weight gains, the lowest total clinical sign scores, and the lowest lung lesion scores when compared with the other 2 challenge-exposed groups of calves. Mean daily weight gains and total clinical sign scores of calves vaccinated once were significantly different (P less than 0.05) than those of calves vaccinated twice. Nonvaccinated, nonchallenge-exposed control calves did not develop clinical signs of disease, did not develop lung lesions, and had consistently positive daily weight gains, and had scores in these areas that were significantly different (P less than 0.05) from those of all challenge-exposed groups of calves. Increases in plasma fibrinogen concentrations corresponded to infection with P haemolytica.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serological evidence of an Australian bovine lentivirus.

Recombinant 26 kDa capsid (CA) proteins of bovine lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), were expressed in Escherichia coli and utilised as antigens for an enzyme-linked immunosorbent assay (ELISA) and a western immunoblot (WIB) procedure for the detection of antibody in dairy cattle in Western Australia. A total of 690 serum samples, 30 from each of 23 farms, were tested by ELISA with a JDV CA protein antigen, and antibody was detected in 3.8% (p<0.05) of the sera. Nine sera from each farm were also tested by WIB with JDV CA protein antigens and antibody was detected in 15.9% of these samples. All ELISA-positive results were also WIB-positive, and all sera antibody-positive by WIB with JDV CA protein antigens were also antibody-positive by the WIB using recombinant BIV CA antigens. This study showed that recombinant protein antigens can be used for serological tests to detect bovine lentivirus infection in Australia.     

Vaccination studies against experimental bovine Pasteurella pneumonia.

Vaccination-challenge experiments were conducted in colostrum-deprived calves to evaluate the efficacy of Pasteurella bacterins and vaccines against experimental pneumonic pasteurellosis. Calves were vaccinated with formalin-killed bacterins and live vaccines, then challenge exposed intratracheally with P. haemolytica or P. multocida. Infectious bovine rhinotracheitis virus was inoculated intranasally three to four days prior to P. haemolytica challenge-exposure. All calves were examined for macroscopic and microscopic lesions after being found dead or following euthanasia four to seven days after challenge exposure with the bacterial pathogen. Clinical, hematological, and pathological responses to challenge exposure in aluminum hydroxide absorbed P. haemolytica and P. multocida bacterin-treated calves were consistent with the pneumonic lesions of pulmonary pasteurellosis in the control calves. An oil-adjuvanted P. haemolytica bacterin limited clinical and pathological responses in the affected calves whereas a P. multocida oil-adjuvanted bacterin did not. Both clinical and pathological responses to challenge exposure in calves vaccinated with live Pasteurella vaccines were less severe than those of the control calves. Vaccine effectiveness appeared to be dose dependent.     

A live Pasteurella haemolytica vaccine efficacy trial

A live Pasteurella haemolytica serotype 1 vaccine was used in an efficacy trial conducted on 100 lightweight feeder calves purchased from a Florida ranch. Forty-one calves were inoculated with the vaccine intradermally in the neck. Fifty-nine calves served as nonvaccinated controls. Fourteen days later, the calves were shipped to an order buyer in eastern Tennessee, where the calves were mixed with 60 local calves in a community sale barn for 72 hours. After 3 additional days, the calves were shipped to a research feedlot in Bushland, Tex. They remained in the feedlot for 56 days, and the test was concluded 76 days after vaccination. The P haemolytica vaccine had no significant effect on performance, morbidity, or mortality. There was no significant difference between the vaccinated and nonvaccinated calves in the number of times Pasteurella was isolated. The calves became seropositive to bovine viral diarrhea virus, respiratory syncytial virus, and infectious bovine rhinotracheitis (IBR) virus during the 76-day experiment. All calves initially were seropositive to parainfluenza-3 virus. A virulent outbreak of IBR occurred 30 days after the calves arrived at the feedlot. Before the onset of IBR, the isolation of P haemolytica serotype 1 from nasal turbinates was rare (2 of 500 nasal swabs). After the IBR outbreak, P haemolytica serotype 1 was isolated from 40 of 92 calves.     

A characterization of monoclonal antibodies prepared against Pasteurella haemolytica serotype 1 surface antigens.

The production and characterization of monoclonal antibodies against Pasteurella haemolytica serotype 1 is described. Ten monoclonal antibodies were produced and divided, on the basis of their properties, into six different groups. One produced bacteria agglutination only of P. haemolytica serotype 1. Three antibodies bound with P. haemolytica serotypes 1, 5-8 and 12 and the antigen was identified in immunoblots as lipopolysaccharide. Two antibodies bound P. haemolytica serotypes 1, 2, 5-8 and 12 and P. multocida serotypes 1-7, 9, 12, 15 and 16, recognizing an epitope present on a 29 kDa outer membrane protein. One antibody bound all P. haemolytica and P. multocida serotypes. The antigen was a hexosamine less than 30 kDa which contained a formalin sensitive epitope. One antibody bound only to P. haemolytica serotype 1 and the antigen was identified as a 66 kDa outer membrane protein. Two antibodies bound P. haemolytica serotypes 1, 2, 5-9 and 12 and the antigen, while not identified, was localized on the outer membrane. This study identified antigens which contribute to the cross-reactions among P. haemolytica and P. multocida serotypes and the antibodies may be useful in investigating the pathogenesis of pneumonic pasteurellosis.