Bivalent vaccination against pneumonic pasteurellosis in domestic sheep and goats with modified-live in-frame lktA deletion mutants of Mannheimia haemolytica.

A temperature-sensitive shuttle vector, pBB80C, was utilized to generate in-frame deletion mutants of the leukotoxin structural gene (lktA) of Mannheimia haemolytica serotypes 1, 2, 5, 6, 7, 8, 9, and 12. Culture supernatants from the mutants contained a truncated protein with an approximate molecular weight of 66 kDa which was reactive to anti-leukotoxin monoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at the molecular weight 100-105 kDa of native LktA. Sheep and goats vaccinated intramuscularly with a mixture of serotypes 5 and 6 mutants were resistant to virulent challenge with a mixture of the wild-type parent strains. These vaccinates responded serologically to both vaccine serotypes and exhibited markedly-reduced lung lesion volume and pulmonary infectious load compared to control animals. Control animals yielded a mixture of serotypes from lung lobes, but the proportion even within an individual animal varied widely from 95% serotype 5-95% serotype 6. Cultures recovered from liver were homogeneous, but two animals yielded serotype 5 and the other two yielded serotype 6 in pure culture.

Mucosal and parenteral vaccination against pneumonic pasteurellosis in cattle with a modified-live in-frame lktA deletion mutant of Mannheimia haemolytica.

A new temperature-conditional shuttle vector, pBB80C, was constructed and utilized to generate an in-frame deletion in the leukotoxin structural gene of Mannheimia haemolytica serotype 1. Culture supernatants from the mutant contained no detectable cytotoxicity to BL-3 lymphocyte targets, and contained a new protein with an approximate molecular weight of 66 kDa which was reactive to anti-leukotoxin monoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at the molecular weight 100-105 kDa of native LktA. Calves vaccinated mucosally by top-dressing the live mutant onto feed, or parenterally by subcutaneous injection, were resistant to virulent challenge with the parent strain. Serologic antibody response, reduction in lung lesion, and reduction in pulmonary infectious load were greater among calves mucosally vaccinated than those which were vaccinated by injection.

Epidemic pasteurellosis in a bighorn sheep population coinciding with the appearance of a domestic sheep.

A pneumonia epidemic reduced bighorn sheep (Ovis canadensis) survival and recruitment during 1997-2000 in a population comprised of three interconnected wintering herds (Kenosha Mountains, Sugarloaf Mountain, Twin Eagles) that inhabited the Kenosha and Tarryall Mountain ranges in central Colorado, USA. The onset of this epidemic coincided temporally and spatially with the appearance of a single domestic sheep (Ovis aires) on the Sugarloaf Mountain herd's winter range in December 1997. Although only bighorns in the Sugarloaf Mountain herd were affected in 1997-98, cases also occurred during 1998-99 in the other two wintering herds, likely after the epidemic spread via established seasonal movements of male bighorns. In all, we located 86 bighorn carcasses during 1997-2000. Three species of Pasteurella were isolated in various combinations from affected lung tissues from 20 bighorn carcasses where tissues were available and suitable for diagnostic evaluation; with one exception, beta-hemolytic mannheimia (Pasteurella) haemolytica (primarily reported as biogroup 1(G) or 1(alphaG)) was isolated from lung tissues of cases evaluated during winter 1997-98. The epidemic dramatically lowered adult bighorn monthly survival in all three herds; a model that included an acute epidemic effect, differing between sexes and with vaccination status, that diminished linearly over the next 12 mo best represented field data. In addition to the direct mortality associated with epidemics in these three herds, lamb recruitment in years following the pneumonia epidemic also was depressed as compared to years prior to the epidemic. Based on observations presented here, pasteurellosis epidemics in free-ranging bighorn sheep can arise through incursion of domestic sheep onto native ranges, and thus minimizing contact between domestic and bighorn sheep appears to be a logical principle for bighorn sheep conservation.

Effect of vaccinating lambs against pneumonic pasteurellosis under New Zealand field conditions on their weight gain and pneumonic lung lesions at slaughter.

In a field trial, 9174 lambs from seven commercial sheep flocks with a history of subclinical pneumonia were either vaccinated with Ovipast Plus (Intervet) or given a placebo by systematic random allocation; they were vaccinated twice at an interval of four to six weeks and grazed on pasture in the same paddocks. They were weighed at the first vaccination, 11 and 23 weeks later, and one to three days before they were slaughtered. The extent of the pneumonic lesions in their lungs was scored visually postmortem. A subset of pneumonic lung samples was examined bacteriologically and histopathologically. There were no statistically significant differences between the pneumonic lesions at slaughter or the mean average daily weight gains of the vaccinated and placebo-treated lambs between 11 and 23 weeks or between first vaccination and slaughter. The vaccinated lambs had a lower mean daily gain between first vaccination and 11 weeks. The extent of pneumonic lesions at slaughter was negatively correlated with the mean daily gain between first vaccination and slaughter. There were no significant differences between the frequency of isolation of Mannheimia (Pasteurella) haemolytica and Pasteurella trehalosi or the histopathological classification of disease between pneumonic lung samples from the placebo-treated and vaccinated lambs.

Comparison of immunogenic properties of native and inactivated Mannheimia haemolytica Lkt in calves.

The aim of this study was to compare the immunostimulatory properties of Lkt of M. haemolytica inactivated by formaldehyde and glutaraldehyde and to evaluate the neutralizing properties of anti-Lkt antibodies. The experiment was conducted on 20 Black-and-White Lowland calves of 100 kg body weight, assigned to 4 experimental groups. The animals were given subcutaneous vaccine injections with native Lkt, Lkt inactivated by formaldehyde or Lkt inactivated by glutaraldehyde. The anti-Lkt antibody titres were measured using an enzyme-linked immunosorbent assay (ELISA), based on absorbance of the sera obtained from the animals immunized with the different forms of Lkt. The protective effects of the antibodies present in the sera isolated from the vaccinated animals were estimated using an MTT assay. Analysis of the ELISA absorbance values in the sera from calves in the vaccinated groups did not show any significant differences between the groups. The highest increase in absorbance of sera was observed in calves from the group that received formaldehyde-inactivated Lkt. In the case of calves immunized with native Lkt, the absorbance values were lower than in the group immunized with Lkt inactivated by formaldehyde. The lowest absorbance values were observed in sera obtained from calves vaccinated with Lkt inactivated by glutaraldehyde. Analysis of the MTT assay results revealed the greatest Lkt-neutralizing properties of antibodies in the sera of calves immunized with two doses of a vaccine containing native Lkt and Lkt inactivated with formaldehyde.

Mannheimia haemolytica in bovine respiratory disease: immunogens, potential immunogens, and vaccines.

Mannheimia haemolytica is the major cause of severe pneumonia in bovine respiratory disease (BRD). Early M. haemolytica bacterins were either ineffective or even enhanced disease in vaccinated cattle, which led to studies of the bacterium's virulence factors and potential immunogens to determine ways to improve vaccines. Studies have focused on the capsule, lipopolysaccharide, various adhesins, extracellular enzymes, outer membrane proteins, and leukotoxin (LKT) resulting in a strong database for understanding immune responses to the bacterium and production of more efficacious vaccines. The importance of immunity to LKT and to surface antigens in stimulating immunity led to studies of individual native or recombinant antigens, bacterial extracts, live-attenuated or mutant organisms, culture supernatants, combined bacterin-toxoids, outer membrane vesicles, and bacterial ghosts. Efficacy of several of these potential vaccines can be shown following experimental M. haemolytica challenge; however, efficacy in field trials is harder to determine due to the complexity of factors and etiologic agents involved in naturally occurring BRD. Studies of potential vaccines have led current commercial vaccines, which are composed primarily of culture supernatant, bacterin-toxoid, or live mutant bacteria. Several of those can be augmented experimentally by addition of recombinant LKT or outer membrane proteins.

Pasteurella multocida and bovine respiratory disease.

Pasteurella multocida is a pathogenic Gram-negative bacterium that has been classified into three subspecies, five capsular serogroups and 16 serotypes. P. multocida serogroup A isolates are bovine nasopharyngeal commensals, bovine pathogens and common isolates from bovine respiratory disease (BRD), both enzootic calf pneumonia of young dairy calves and shipping fever of weaned, stressed beef cattle. P. multocida A:3 is the most common serotype isolated from BRD, and these isolates have limited heterogeneity based on outer membrane protein (OMP) profiles and ribotyping. Development of P. multocida-induced pneumonia is associated with environmental and stress factors such as shipping, co-mingling, and overcrowding as well as concurrent or predisposing viral or bacterial infections. Lung lesions consist of an acute to subacute bronchopneumonia that may or may not have an associated pleuritis. Numerous virulence or potential virulence factors have been described for bovine respiratory isolates including adherence and colonization factors, iron-regulated and acquisition proteins, extracellular enzymes such as neuraminidase, lipopolysaccharide, polysaccharide capsule and a variety of OMPs. Immunity of cattle against respiratory pasteurellosis is poorly understood; however, high serum antibodies to OMPs appear to be important for enhancing resistance to the bacterium. Currently available P. multocida vaccines for use in cattle are predominately traditional bacterins and a live streptomycin-dependent mutant. The field efficacy of these vaccines is not well documented in the literature.

Immunization of bighorn sheep against Mannheimia haemolytica with a bovine herpesvirus 1-vectored vaccine.

Mannheimia haemolytica is an important pathogen of pneumonia in bighorn sheep (BHS), consistently causing 100% mortality under experimental conditions. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, a vaccine containing leukotoxin and surface antigens of M. haemolytica induced 100% protection in BHS, but required multiple booster doses. Vaccination of wildlife is difficult. BHS, however, can be vaccinated at the time of transplantation, but administration of booster doses is impossible. A vaccine that does not require booster doses, therefore, is ideal for vaccination of BHS. Herpesviruses are ideal vectors for development of such a vaccine because of their ability to undergo latency with subsequent reactivation which obviates the need for booster administration. The objective of this study was to evaluate the potential of bovine herpesvirus 1 (BHV-1) as a vector encoding M. haemolytica immunogens. As the first step towards this goal, the permissiveness of BHS for BHV-1 infection was determined. BHS inoculated with wild-type BHV-1 shed the virus following infection. The lytic phase of infection was superseded by latency, and treatment of latently-infected BHS with dexamethasone reactivated the virus. A recombinant BHV-1-vectored vaccine encoding a leukotoxin-neutralizing epitope and an immuno-dominant epitope of the outer membrane protein PlpE was developed by replacing the viral glycoprotein C gene with a leukotoxin-plpE chimeric gene. Four of six BHS vaccinated with the recombinant virus developed significant leukotoxin-neutralizing antibodies at day 21 post-vaccination, while two of six BHS developed significant surface antigen antibodies at day 17 post-vaccination. These antibodies, however, were inadequate for protection of BHS against M. haemolytica challenge. These data indicate that BHV-1 is a suitable vector for immunization of BHS, but additional experimentation with the chimeric insert is necessary for development of a more efficacious vaccine.

Virus detection by PCR following vaccination of naive calves with intranasal or injectable multivalent modified-live viral vaccines.

We evaluated duration of PCR-positive results following administration of modified-live viral (MLV) vaccines to beef calves. Twenty beef calves were randomly assigned to either group 1 and vaccinated intranasally with a MLV vaccine containing bovine alphaherpesvirus 1 (BoHV-1), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza virus 3 (BPIV-3), or to group 2 and vaccinated subcutaneously with a MLV vaccine containing bovine viral diarrhea virus 1 and 2 (BVDV-1, -2), BoHV-1, BRSV, and BPIV-3. Deep nasopharyngeal swabs (NPS) and transtracheal washes (TTW) were collected from all calves, and whole blood was collected from group 2 calves and tested by PCR. In group 1, the proportions of calves that tested PCR-positive to BVDV, BoHV-1, BRSV, and BPIV-3 on any sample at any time were 0%, 100%, 100%, and 10%, respectively. In group 1 calves, 100% of calves became PCR-positive for BoHV-1 by day 3 post-vaccination and 100% of calves became PCR-positive for BRSV by day 7 post-vaccination. In group 2, the proportions of calves that tested positive to BVDV, BoHV-1, BRSV, and BPIV-3 on any sample at any time were 50%, 40%, 10%, and 0%, respectively. All threshold cycle (Ct) values were >30 in group 2 calves, irrespective of virus; however, Ct values <25 were observed in group 1 calves from PCR-positive results for BoHV-1 and BRSV. All calves were PCR-negative for all viruses after day 28. Following intranasal MLV viral vaccination, PCR results and Ct values for BRSV and BoHV-1 suggest that attempts to differentiate vaccine virus from natural infection is unreliable.

Sequence diversity of the immunogenic outer membrane lipoprotein PlpE from Mannheimia haemolytica serotypes 1, 2, and 6.

Mannheimia haemolytica serotype 1 (S1), S6 and S2 are the most common bacterial isolates found in shipping fever pneumonia in beef cattle. Currently used vaccines against M. haemolytica do not provide complete protection against the disease. Research with M. haemolytica outer membrane proteins (OMPs) has shown that antibodies to one particular OMP from S1, PlpE, may be important in immunity. Recombinant PlpE (rPlpE) is highly immunogenic in cattle, and the acquired immunity markedly enhanced resistance to experimental challenge. We previously demonstrated that the immunodominant epitope (R2) is located between residues 26 and 76 on the N-terminus of PlpE from a reference S1 strain (). This region consists of eight hexapeptide repeats. The potential of this epitope as a vaccine or supplement to commercial vaccines is dependant on its state of conservation amongst isolates of the three serotypes. To determine this, we sequenced plpE genes from 32 isolates. The sequences from S1 and S6 were identical with one exception. Substantial variation was observed among sequences from S2 strains, particularly in the R2 region of the protein. These variations in S2 isolates range from 3 to 28 hexapeptide repeats. Calculated molecular weight of PlpE from S1 and S6 isolates was 37 kDa, where as PlpE from S2 strains ranged from 30 to 50 kDa. These similarities and differences were demonstrated by western blot. Competitive binding assay was used to determine that antibody against rPlpE from S1 binds native PlpE on surfaces of both S1 and S2 cells.

Pasteurella multocida and its role in porcine pneumonia.

Pasteurella multocida has been recognized as a contributor to debilitating and fatal porcine pneumonia for at least 120 years and there continues to be sustained, unabated high prevalence of the organism in cases submitted for diagnostic work up. Understanding of its role in disease has been limited, in part because of difficulty in reproducing the disease experimentally with capsular type A strains of P. multocida, the predominant type associated with porcine pneumonia. This limitation has stymied the development of improved methods for disease control. In this review, the reports of efforts to reproduce the disease are compared. Reports have indicated induction of pneumonia in combined infections with agents such as hog cholera virus, pseudorabies virus and Mycoplasma hyopneumoniae. Pneumonia has been induced with intratracheal or endobronchial inoculation of anesthetized swine using capsular type A strains. Substantial recent progress in understanding the putative virulence attributes and molecular genetics of P. multocida will likely lead to better understanding of the host-parasite and parasite-parasite interactions in porcine pneumonia associated with this organism. In particular, it seems important to consider the role of biofilm formation in the pathogenesis of this disease. Ultimately, this understanding should provide a foundation for better methods for induction of the experimental disease, development of improved diagnostics, development of better therapeutic/prophylactic pharmaceutical approaches and development of immunoprophylactic products.

A chimeric protein comprising the immunogenic domains of Mannheimia haemolytica leukotoxin and outer membrane protein PlpE induces antibodies against leukotoxin and PlpE.

Mannheimia haemolytica is a very important pathogen of pneumonia in ruminants. Bighorn sheep (BHS, Ovis canadensis) are highly susceptible to M. haemolytica-caused pneumonia which has significantly contributed to the drastic decline of bighorn sheep population in North America. Pneumonia outbreaks in wild BHS can cause mortality as high as 90%. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, an experimental vaccine containing leukotoxin and surface antigens of M. haemolytica developed by us induced 100% protection of BHS, but required multiple booster injections. Vaccination of wild BHS is difficult. But they can be vaccinated at the time of transplantation into a new habitat. Administration of booster doses, however, is impossible. Therefore, a vaccine that does not require booster doses is necessary to immunize BHS against M. haemolytica pneumonia. Herpesviruses are ideal vectors for development of such a vaccine because of their ability to undergo latency with subsequent reactivation. As the first step towards developing a herpesvirus-vectored vaccine, we constructed a chimeric protein comprising the leukotoxin-neutralizing epitopes and the immuno-dominant epitopes of the outer membrane protein PlpE. The chimeric protein was efficiently expressed in primary BHS lung cells. The immunogenicity of the chimeric protein was evaluated in mice before inoculating BHS. Mice immunized with the chimeric protein developed antibodies against M. haemolytica leukotoxin and PlpE. More importantly, the anti-leukotoxin antibodies effectively neutralized leukotoxin-induced cytotoxicity. Taken together, these results represent the successful completion of the first step towards developing a herpesvirus-vectored vaccine for controlling M. haemolytica pneumonia in BHS, and possibly other ruminants.

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.

Efficacy of tulathromycin in the treatment and prevention of natural outbreaks of bovine respiratory disease in European cattle.

The efficacy of tulathromycin in the treatment (phase 1) and prevention (phase 2) of bovine respiratory disease (BRD) was evaluated on commercial farms in France, Germany, Italy, and Spain. In phase 1, commingled cattle with clinical BRD were treated with tulathromycin (n = 128) or florfenicol (n = 125) on day 0. Similar percentages of animals showed sustained clinical improvement at day 14 (tulathromycin 83.3% versus florfenicol 81.0%) and had not relapsed by day 60 (tulathromycin 63.3% versus florfenicol 58.4%). In phase 2, healthy in-contact cattle were treated with tulathromycin (n = 492), tilmicosin (n = 494), or saline (n = 265) on day 0. Significantly more (P = .0001) tulathromycin-treated cattle remained healthy to day 14 (92.4%) than tilmicosin-treated (83.7%) or saline-treated (63.7%) cattle, and this was maintained through day 60 (tulathromycin 85.4% versus tilmicosin 75.1% and saline 56.2%). Tulathromycin was highly effective in the treatment and prevention of BRD.

Cross-protection study of a Mannheimia haemolytica serotype 1 vaccine against acute pasteurellosis in lambs induced by a serotype 2 strain.

Ovine pneumonia is an economic important disease worldwide for the sheep industry. Multiple serotypes (S) of Mannheimia haemolytica are involved in the disease and S2 and S1 are the most frequent isolates associated with lung lesions in sheep. Vaccines based on some M. haemolytica S2 strains have been shown to have poor immunogenicity. The objective of this study was to determine the cross-protection effect of an S1 strain based vaccine, Bovilis MH, in sheep against an experimental challenge with an S2 strain. Lambs (n=12) in the vaccine group were injected subcutaneously with 1 ml of the Bovilis MH vaccine, and revaccinated 4 weeks later, while the control group (n=12) received 1 ml of saline at each occasion. Two weeks after revaccination, all lambs were challenged intratracheally with parainfluenza virus 3, and with an M. haemolytica S2 strain at day 7 post-viral challenge. The proportion of animals having pyrexia in the first 2 days post-bacterial challenge was significantly less in the vaccine group compared to the control group (P<0.05). The animals in the vaccine group had significantly lower dyspnoea scores and lung/bodyweight ratio than those in the control group (P<0.05). The vaccine provided 49.1% overall protection. Prior to the challenge, the vaccinated animals had significantly higher titres of antibodies to S1 and S2 whole cell antigens and to leukotoxins produced by S1 and S2 strains compared to the control animals (P<0.05). The S1 strain vaccine provided considerable cross-protection against the S2 strain challenge.

Application of beta-1,3-glucan to prevent shipping fever in imported heifers.

Particulated beta-1,3-glucan from Saccharomyces cerevisiae was evaluated to prevent shipping fever in imported heifers during the 15 days following their arrival to Cuba. Seventy seven animals received a single subcutaneous dose (5 mg/kg body weight) during the first 12 h following their arrival, whereas 44 served as untreated controls. Clinical symptoms were observed in 3 treated and 19 untreated animals (p < 0.001). One untreated heifer died. These observations confirm the usefulness of beta-1,3-glucan to prevent shipping fever.

Evaluation of efficacy of three commercial vaccines against experimental bovine pneumonic pasteurellosis.

The objective of this study was to evaluate the efficacy of three commercial vaccines against experimental pneumonic pasteurellosis in cattle. The three vaccines were: (a) One Shot (SmithKline Beecham, West Chester, PA.), (b) Presponse (Langford Laboratories, Guelph, Ontario) and (c) Once PMH (BioCor, Omaha, NE.). Protective immunity was evaluated in terms of lower clinical and pneumonic lesion scores after endobronchial challenge with virulent P. haemolytica. The results indicate that One Shot elicited antibodies against leukotoxin (Lkt), capsular poly-saccharide (CP) and surface antigens (SA), while Presponse and Once PMH elicited antibodies against CP and SA. There was significant correlation between lung and serum antibody levels against Lkt (P < 0.0001), CP (P < or = 0.0001) and IROMPs (P < or = 0.035). Animals that received the One Shot had significantly (P < or = 0.05) lower mean pneumonic lesion score (36.6 +/- 10.97) as compared to the control group (48.6 +/- 25.92). A significant negative correlation (-0.41; P < or = 0.008) existed between serum antibody levels against Lkt and pneumonic lesion score. High serum antibodies against SA did not correlate with reduction in pneumonic lesion score. In addition, high antibody levels against CP did not correlate consistently with reduced pneumonic lesion scores. The results from this study demonstrates that commercial vaccines evaluated in this trial did not confer optimal protection in vaccinated calves, against experimental pneumonic pasteurellosis. However, One Shot vaccinates showed a better protective immunity compared to the other two vaccine groups.

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.

Construction and vaccine potential of an aroA mutant of Pasteurella haemolytica.

The aroA gene, encoding 5-enolpyruvylshikimate 3-phosphate synthase, from Pasteurella haemolytica biotype A, serotype 1 was cloned by complementation of the aroA mutation in Escherichia coli strain AB2829 after electroporation with a DNA library constructed in pUC18. The cloned P. haemolytica aroA gene was inactivated by insertion of a kanamycin resistance gene and reintroduced by allelic exchange into the chromosome of the parental P. haemolytica using PbluescriptII SK+. The P. haemolytica aroA mutant was highly attenuated in a mouse septicaemic model. Mice immunized intraperitoneally with two doses of live P. haemolytica aroA mutant were protected against a lethal parental strain challenge.

Comparative evaluation of antibodies induced by commercial Pasteurella haemolytica vaccines using solid phase immunoassays.

The objective of this study was to evaluate the ability of four commercial vaccines to elicit antibodies against the leukotoxin (Lkt), capsular polysaccharide (CP), iron regulated outer membrane proteins (IROMPs), and whole cell (WC) antigens of Pasteurella haemolytica A1. Modified double antibody sandwich enzyme linked immunosorbent assays (ELISAs) were developed to measure antibody levels against Lkt, CP and IROMPs. An indirect ELISA was developed to measure the levels of antibody against WC antigens. The ideal cut off points for ELISAs were determined on receiver operating characteristic curves, using sera from 30 calves injected subcutaneously with a live P. haemolytica 12296 strain as positive control and sera from 30 colostrum-deprived calves as negative control. The vaccines evaluated were: 'One Shot' (SmithKline Beecham, West Chester, PA) a bacterin-toxoid, 'Presponse' (Langford Laboratories, Guelph, Ontario) a Lkt-rich culture supermatant, 'Once PMH' (BioCor Inc., Omaha, NE) a modified live vaccine, and 'Septimune' (Fort Dodge laboratories, Fort Dodge, IA) an outer membrane extract. Thirty, 4-6 week old Holstein calves were randomized into 5 groups to receive one of the four vaccines or a placebo (sterile phosphate buffered saline). The calves were vaccinated intramuscularly on day 0 and on day 14, and bled on days, 0, 14, and 28 to measure antibody levels against Lkt, CP, IROMPs, and WC antigens of P. haemolytica Al. 'One Shot', and 'Once PMH' vaccinates showed a significant (P < 0.05) increase in antibody levels against Lkt at 28 days. 'Once PMH' vaccinates also showed significant (P < 0.05) increase in antibody levels against IROMPs at 28 days compared to the other four groups but this increase was not significant over time within the 'Once PMH' group. 'Presponse', 'Once PMH' and 'One Shot' vaccinates showed a significant (P < 0.05) increase in antibody levels against CP over time. These groups also had significantly higher antibody levels against CP, compared to controls and 'Septimune' vaccinates at 14 and 28 days (P < 0.05).