Antibody levels following vaccination of beef calves with a tetanus toxoid.

Tetanus is a preventable, yet often fatal, disease affecting many species, including beef cattle. Vaccination for tetanus is recommended for calves at high risk of disease, but typical beef cattle management practices often make adherence to vaccine manufacturers' guidance for a second (booster) dose of vaccine difficult. This study examined the antibody response following a single dose of tetanus toxoid, as well as following booster vaccination at various intervals. Anti-tetanus IgG antibodies were detectable 25 days (D25) after a single dose, and rose following booster at either D25 D109 after initial vaccination. Antibody levels then declined numerically from D109 to D179 for calves boostered at D25 but rose on D179 for those receiving a second dose on D109. The relatively rapid response in IgG production, even in the absence of a booster vaccine, may suggest value in vaccinating calves for tetanus at time of greatest risk, even if a booster cannot be administered. The study also provides support for priming of the immune response lasting at least until D109 after primary immunization.

Clinical disease and lung lesions in calves experimentally inoculated with Histophilus somni five days after metaphylactic administration of tildipirosin or tulathromycin.

OBJECTIVE: To compare clinical disease and lung lesions in calves experimentally inoculated with Histophilus somni 5 days after metaphylactic administration of tildipirosin or tulathromycin. ANIMALS Twenty-four 3-month-old Holstein and Holstein-crossbreed steers. PROCEDURES: Calves were randomly allocated to 3 groups of 8 calves. On day 0, calves in group 1 received tildipirosin (4 mg/kg, SC), calves in group 2 received tulathromycin (2.5 mg/kg, SC), and calves in group 3 received isotonic saline (0.9% NaCl) solution (1 mL/45 kg, SC; control). On day 5, calves were inoculated with 10 mL of a solution containing H somni strain 7735 (1.6 × 10(9) CFUs/mL, intrabronchially; challenge). Calves were clinically evaluated on days 5 through 8 and euthanized on day 8. The lungs were grossly evaluated for evidence of pneumonia, and bronchial secretion samples underwent bacteriologic culture. RESULTS: The mean clinical score for each group was significantly increased 12 hours after challenge, compared with that immediately before challenge, and was significantly lower for tildipirosin-treated calves on days 6, 7, and 8, compared with those for tulathromycin-treated and control calves. The mean percentage of lung consolidation for tildipirosin-treated calves was significantly lower than those for tulathromycin-treated and control calves. Histophilus somni was isolated from the bronchial secretions of some tulathromycin-treated and control calves but was not isolated from tildipirosin-treated calves. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that metaphylactic administration of tildipirosin to calves 5 days prior to H somni challenge prevented subsequent culture of the pathogen from bronchial secretions and was more effective in minimizing clinical disease and lung lesions than was metaphylactic administration of tulathromycin.

Proteomic analysis and immunogenicity of Mannheimia haemolytica vesicles.

Mannheimia haemolytica, a major causative agent in bovine respiratory disease, inflicts extensive losses each year on cattle producers. Commercially available vaccines are only partially efficacious. Immunity to M. haemolytica requires antibodies to secreted toxins and outer membrane proteins (OMPs) of the bacterium. Gram-negative bacteria produce membrane blebs or vesicles, the membrane components of which are primarily derived from OMPs. Accordingly, vesicles have been used as immunogens with various degrees of success. This study characterized components of M. haemolytica vesicles and determined their immunogenicity in mice and cattle. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of vesicles from this bacterium identified 226 proteins, of which 58 (25.6%) were OMPs and periplasmic and one (0.44%) was extracellular. Vesicles were used to vaccinate dairy calves and BALB/c mice. Analyses of sera from calves and mice by enzyme-linked immunosorbent assay (ELISA) showed that circulating antibodies against M. haemolytica whole cells and leukotoxin were significantly higher on days 21 and 28 (P < 0.05) than on day 0. For control calves and mice, there were no significant differences in serum anti-whole-cell and leukotoxin antibody levels from days 0 and 21 or 28, respectively. Lesion scores of lungs from vaccinated calves (15.95%) were significantly (P < 0.05) lower than those from nonvaccinated calves (42.65%). Sera from mice on day 28 and calves on day 21 showed 100% serum bactericidal activity. Sera from vesicle-vaccinated mice neutralized leukotoxin.

Immunogenicity of Mannheimia haemolytica recombinant outer membrane proteins serotype 1-specific antigen, OmpA, OmpP2, and OmpD15.

We previously identified Mannheimia haemolytica outer membrane proteins (OMPs) that may be important immunogens by using immunoproteomic analyses. Genes for serotype 1-specific antigen (SSA-1), OmpA, OmpP2, and OmpD15 were cloned and expressed, and recombinant proteins were purified. Objective 1 of this study was to demonstrate immunogenicity of the four recombinant OMPs in mice and cattle. Objective 2 was to determine if the addition of individual recombinant OMPs or combinations of them would modify immune responsiveness of mice to the recombinant chimeric protein SAC89, containing the main epitope from M. haemolytica outer membrane lipoprotein PlpE and the neutralizing epitope of M. haemolytica leukotoxin. Mice vaccinated with recombinant OmpA (rOmpA), rSSA-1, rOmpD15, and rOmpP2 developed significant antibody responses to M. haemolytica outer membranes and to the homologous recombinant OMP. Cattle vaccinated with rOmpA and rSSA-1 developed significant antibodies to M. haemolytica outer membranes by day 28, whereas cattle vaccinated with rOmpD15 and rOmpP2 developed only minimal responses. Sera from cattle vaccinated with each of the recombinant proteins stimulated complement-mediated killing of the bacterium. Concurrent vaccination with SAC89 plus any of the four rOMPs singly resulted in increased endpoint anti-SAC89 titers, and for the SAC89/rSSA-1 vaccinees, the response was increased significantly. In contrast, the SAC89/P2/SSA-1 and SAC89/OmpA/P2/D15/SSA-1 combination vaccines resulted in significant decreases in anti-SAC89 antibodies compared to SAC89 vaccination alone. In conclusion, under the conditions of these experiments, vaccination of mice and cattle with rOmpA and rSSA-1 stimulated high antibody responses and may have protective vaccine potential.

Identification and immunogenicity of Mannheimia haemolytica S1 outer membrane lipoprotein PlpF.

Immunity against Mannheimia haemolytica requires antibodies against leukotoxin (LKT) and bacterial cell surface antigens, most likely immunogenic outer membrane proteins (OMPs). Five immunogenic outer membrane lipoproteins identified and characterized in M. haemolytica were designated Pasteurella lipoproteins (Plp) A, -B, -C, -D and -E. Using immunoproteomics, we identified a heretofore-uncharacterized M. haemolytica immunogenic outer membrane lipoprotein that we designated PlpF, which was previously designated in the published sequence as a conserved hypothetical protein. We cloned and expressed rPlpF from two M. haemolytica serotype 1 strains (SAC159 and SAC160) and demonstrated a variable number of perfect (KKTEED) or imperfect (KKaEEa) repeats between residues 41 and 76 on the N-terminus. Antigenicity plots predicted the N-terminus repeat region to be highly antigenic. The plpF gene in multiple M. haemolytica S1, S2, and S6 isolates varied in the number of repeats from three to seven. C-terminal region was highly conserved. Immunization of mice with SAC159 or SAC160 demonstrated immunogenicity in a dose-response manner. Immunization of calves demonstrated an increase in antibodies to PlpF, and rPlpF antibodies stimulated complement-mediated killing of M. haemolytica. Because calves had pre-existing anti-M. haemolytica antibodies due to prior natural exposure, functionality of the anti-PlpF antibody responses were demonstrated by marked reduction of complement-mediated killing by blocking of anti-PlpF antibodies with rPlpF In conclusion, PlpF might have vaccination potential against M. haemolytica infection in cattle.

Serum antibody responses in horses and mice following immunization with Actinobacillus equuli outer membrane proteins and recombinant Aqx toxin.

The immune responsiveness of mice (without prior natural exposure) and mares (with naturally acquired antibodies) was determined following vaccination with Actinobacillus equuli outer membrane proteins (OMPs) and/or recombinant A. equuli toxin (rAqx). Mice were vaccinated subcutaneously on days 0 and 21 with one of three doses (5, 25 or 50µg) of A. equuli OMPs, rAqx or both, together with Freund's incomplete adjuvant (FIA). Antibodies against formalin-killed whole bacterial cells (WBCs), OMPs and Aqx were determined on days 0, 21 and 42. Mares were vaccinated subcutaneously on days 0 and 21 with 100µg OMPs, 100µg rAqx or a combination of 50µg of each antigen, together with FIA. Antibodies against WBCs, OMPs and Aqx were determined at 7day intervals for the first 42days, as well as on days 56, 70, 154 and 238. Vaccination of mice stimulated an apparent dose response to OMPs and Aqx. Antibodies against OMPs and Aqx were enhanced following vaccination of mares that had naturally acquired pre-existing antibodies. There was no evidence of interference with antibody responses to the individual antigens when OMPs and rAqx were combined prior to vaccination.

Mannheimia haemolytica chimeric protein vaccine composed of the major surface-exposed epitope of outer membrane lipoprotein PlpE and the neutralizing epitope of leukotoxin.

Mannheimia haemolytica is commonly identified in cattle with shipping fever pneumonia. Vaccines currently available do not provide complete protection against the disease. In an effort to develop a vaccine that delivers the immunogenic regions of leukotoxin (LKT) A and the outer membrane protein (OMP) PlpE, a total of four chimeric proteins were constructed. Mice were subcutaneously immunized with 25, 50 and 75 microg quantities of each chimeric protein. The specificity of the immune response was confirmed by Western blot analysis and enzyme-linked immunosorbent assays (ELISA). Moreover, the hyperimmune sera were bactericidal to M. haemolytica in the presence of complement and neutralized LKT. While all of the chimeric proteins induced some level of immune response two, SAC87 and SAC89, were most promising. These results demonstrate that a functional immune response against M. haemolytica can be induced by vaccination with recombinant chimeric proteins created from specific immunogenic regions of the LKT and PlpE proteins.

Vaccination with Pasteurella multocida recombinant OmpA induces strong but non-protective and deleterious Th2-type immune response in mice.

Pasteurella multocida OmpA (PmOmpA) belongs to the major and multifunctional Escherichia coli OmpA family of proteins. We have previously reported that the protein is conserved, immunogenic and an adhesin that binds host cells and host cell extracellular matrix molecules [Dabo SM, Confer AW, Quijano-Blas RA. Molecular and immunological characterization of Pasteurella multocida serotype A:3 OmpA: evidence of its role in P. multocida interaction with extracellular matrix molecules. Microb Pathog 2003;35(4):147-157]. In this study, we found that immunization of mice with the recombinant PmOmpA elicited strong Th2-type immune response, characterized by high immunoglobulin G(1) (IgG(1)) antibodies production. Subsequent intraperitoneal homologous challenge of the immunized mice resulted in lack of protection associated with the high IgG(1) titers in anti-rPmOmpA sera. Furthermore, the protection afforded by vaccination with P. multocida OMPs alone was adversely affected by the addition of the rPmOmpA to the vaccine preparations. The results demonstrate that PmOmpA has a detrimental effect on the efficacy of vaccination with OMPs in mice. Targeted inactivation of pmOmpA gene in P. multocida 232 represents a potential mean towards the development of an effective vaccine candidate.

Recombinant Mannheimia haemolytica serotype 1 outer membrane protein PlpE enhances commercial M. haemolytica vaccine-induced resistance against serotype 6 challenge.

Mannheimia haemolytica outer membrane protein PlpE, a major immunogenic outer membrane lipoprotein has identical sequences in serotypes 1 (S1) and S6. Recombinant outer membrane lipoprotein PlpE (rPLpE) from M. haemolytica S1 was added to commercial M. haemolytica S1 vaccines to determine if it would enhance vaccine-induced immunity against heterotypic M. haemolytica S6 challenge. Serum antibody responses to M. haemolytica whole cells, leukotoxin and rPlpE were measured. Experiment 1 consisted of four vaccine groups: controls, 100 microg rPlpE, M. haemolytica Bacterin-Toxoid (One Shot) and M. haemolytica Bacterin-Toxoid + 100 microg rPlpE. Vaccines were given on day 0. On day 21, calves were challenged transthoracically with M. haemolytica S6. Lung lesion scores and percentage lesion reduction were 6.3 +/- 2.0 for controls, 3.6 +/- 2.4 for rPlpE vaccinates (42.9% reduction), 3.4 +/- 1.5 for One Shot-vaccinates (46.0% reduction), and 2.4 +/- 1.4 for One Shot/rPlpE vaccinates (61.9% reduction). Experiment 2 consisted of four vaccine groups: controls, 100 microg rPlpE, M. haemolytica toxoid (Presponse), and M. haemolytica toxoid+100 microg rPlpE. On day 28, calves were challenged transthoracically with M. haemolytica S6. Lung lesion scores and percentage lesion reduction were 8.1 +/- 2.2 for controls, 4.4 +/- 4.7 for the rPlpE vaccinates (45.7% reduction), 4.8 +/- 2.2 for Presponse-vaccinates (40.7% reduction), and 2.0 +/- 1.2 for Presponse/rPlpE vaccinates (75.3% reduction). These results indicate that addition of rPlpE from M. haemolytica S1 can enhance commercial M. haemolytica vaccine-induced resistance against experimental challenge with M. haemolytica S6.

Immunogenicity of recombinant Mannheimia haemolytica serotype 1 outer membrane protein PlpE and augmentation of a commercial vaccine.

Mannheimia haemolytica is the major cause of severe bacterial pneumonia associated with shipping fever in cattle. The gene for M. haemolytica outer membrane protein (OMP) PlpE was cloned into the expression vector pRSETA. The cloned gene was then expressed in BL21(DE3)pLysS and the recombinant PlpE (rPlpE) was purified and used in immunological and vaccination studies. Vaccination of cattle with commercial M. haemolytica vaccines stimulated no significant (P>0.05) antibody responses to rPlpE. Recombinant PlpE in a commercial proprietary adjuvant was highly immunogenic when injected subcutaneously into cattle. Vaccination of cattle with 100 microg of rPlpE markedly enhanced resistance against experimental challenge with virulent M. haemolytica. Addition of 100 microg of rPlpE to a commercial M. haemolytica vaccine, Presponse, significantly enhanced (P<0.05) protection afforded by the vaccine against experimental challenge. Addition of rPlpE to commercial M. haemolytica vaccines could greatly enhance vaccine efficacy.

Relationship of vitamin E supplementation and antimicrobial treatment with acute-phase protein responses in cattle affected by naturally acquired respiratory tract disease.

OBJECTIVE: To correlate serum concentrations of fibrinogen (Fib), haptoglobin (Hap), serum amyloid-A (SAA), and alpha-1 acid glycoprotein (AGP) with clinical respiratory tract disease and response to treatment in transport-stressed feedlot cattle fed vitamin E-supplemented diets. ANIMALS: 387 heifer calves (mean initial weight, 197 kg). PROCEDURE: Calves purchased from an order buyer were delivered to a feedlot to study the effects of dietary supplementation with 2,000 IU of vitamin E for 0, 7, 14, or 28 days after arrival. Serum or plasma Fib, Hap, SAA, and AGP concentrations were measured on days 0, 7, and 28 after arrival as well as at the time of treatment for respiratory tract disease with antimicrobial drugs and after completion of treatment. RESULTS: Vitamin E supplementation was associated with decreased treatment costs. In cattle that were not recognized as sick or responded positively to 1 antimicrobial treatment, serum Hap concentrations were significantly lower on days 0 and 7 than concentrations for cattle that required > 1 treatment. Serum Hap concentrations and ratios of Hap to SAA on day 0 significantly correlated with the number of antimicrobial treatments required. Serum Hap concentrations at the time of initial treatment were significantly lower for cattle that required only 1 treatment, compared with those that required > 1 treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Serum Hap concentrations are of potential value for use in assessing feedlot cattle that may become ill as a result of respiratory tract disease and for use in monitoring treatment efficacy.