Incorporation of antigens from Mannheimia haemolytica culture supernatant, and recombinant bovine C3d into ISCOM matrix using neutravidin-biotin interaction.

The aim of this study was to incorporate antigens from Mannheimia haemolytica culture supernatant, and an immune modulatory molecule, recombinant bovine C3d (rBoC3d), into immune stimulating complexes (ISCOMs) using neutravidin-biotin interaction. Biotinylated ISCOM matrix was generated using a commercial kit. The biotinylated ISCOM matrix was incubated with neutravidin and then centrifuged in a sucrose density gradient. The rBoC3d was expressed as an in vivo biotinylated protein and with a c-Myc tag (EQKLISEEDL) engineered to facilitate detection. The neutravidin-coated ISCOM matrix was incubated with biotinylated antigens from M. haemolytica culture supernatants and rBoC3d. To test the association among the neutravidin-coated ISCOM matrix, biotinylated antigens and rBoC3d, an analytical sucrose density gradient (10-40%, w/w) was performed. The experimental formulations were run in SDS-PAGE gels under reducing conditions. For Western immunoblot analysis, polyclonal bovine antiavidin, monoclonal anti-c-Myc, monoclonal antileukotoxin, and anti-GS60 antibodies were used to detect the presence of neutravidin, rBoC3d, leukotoxin, and GS60 antigens, respectively. By taking advantage of the biotin-neutravidin interaction, not only leukotoxin but also the recombinant immunomodulatory molecule, rBoC3d, was incorporated into ISCOM particles.

Challenges in mucosal vaccination of cattle.

Recognition of the mucosal portal of entry for many infectious diseases and of the relevance of mucosal immune response to protection has encouraged the development of vaccines administered by mucosal routes, principally oral and intranasal, for stimulation of intestinal and nasopharyngeal lymphoid tissues respectively. The oral route is problematic in cattle and other ruminants where antigen degradation in the rumen is likely, prior to transit to the intestine. On the other hand, rumination can be exploited for exposure of nasopharyngeal tissues during cudding if vaccine antigen is expressed by a fibrous feed like alfalfa. An increase in anti-leukotoxin (Lkt) IgA was demonstrated in nasal secretions of calves following feeding of alfalfa expressing a truncated Lkt50 from Mannheimia haemolytica, and there is evidence suggesting that such vaccination may protect against experimentally induced pneumonia. Intranasal vaccination is an alternative approach for use in pre-ruminating calves. Intranasal administration of ISCOMs carrying soluble antigens of M. haemolytica, including native Lkt, induced Lkt specific IgA in nasal secretions after vaccination at 4 and 6 weeks of age. Subcutaneous (s.c.) administration of the same vaccine induced Lkt specific IgG in both serum and nasal secretions, whereas s.c. administration of a commercial M. haemolytica vaccine did not. Regardless of the vaccination strategy employed it is difficult to assess the immunogenicity of mucosally administered vaccines because production of secreted antibodies tends to be transient, and they do not persist on the mucosal surface in the absence of ongoing antigenic stimulation. An additional challenge is demonstration of vaccine efficacy in response to experimental infection. Protection of the mucosally vaccinated animal will most probably result from recall response, which may not amplify sufficiently to counter the effects of experimental pulmonary delivery of a large bolus of virulent bacteria, even though the response would suffice over the more prolonged and gradual infection that occurs in natural induction of pneumonia.

Performance of a whole blood interferon-gamma assay for detection and eradication of caseous lymphadenitis in sheep.

Caseous lymphadenitis (CLA), a chronic bacterial disease of sheep and goats caused by Corynebacterium pseudotuberculosis, could be controlled by eradication of infected carriers. This study aimed at validation of a whole blood interferon-gamma (IFN-gamma) enzyme immunoassay (EIA) (Bovigam, Pfizer) in naturally infected sheep for use in eradication of infection from a flock. This assay used formalin-inactivated whole bacterial cells as antigen. The sensitivity of the whole cell assay was improved by increasing both the volume of blood and the number of bacterial cells. The assay was validated in experimentally infected sheep and in a flock of known-negative sheep, as well as in a naturally infected flock, a proportion of which was vaccinated with a commercial CLA vaccine. An optical density (540nm) (OD) cut-off of 0.09 was effective in classifying animals as test positive or negative in the naturally infected flock, although there was variation in OD between visits, notably with weakly reacting animals. The test had a sensitivity of 91% and a specificity of 98%. Postmortem data supported the results in test-negative animals. Visit-to-visit variation in IFN-gamma EIA OD in the naturally infected flock as well as CLA disease status was used to develop an algorithm for the eradication of CLA from a known infected flock. The whole blood IFN-gamma assay shows promise for eradication of caseous lymphadenitis from sheep flocks.

Influence of in-feed virginiamycin on the systemic and mucosal antibody response of chickens.

Subtherapeutic and prophylactic doses of virginiamycin are capable of altering the intestinal microbiota as well as increasing several growth parameters in chickens. In spite of the fact that the microbiota plays a role in shaping the host's immune system, little information is available on the effects of in-feed antibiotics on the chicken immune system. The objective of this study was to examine the effects of an antibiotic, virginiamycin, on the development of antibody responses. Chickens were fed diets containing no antibiotics, along with either subtherapeutic (11 ppm) or prophylactic (22 ppm) doses of virginiamycin. Chickens were then immunized with keyhole limpet hemocyanin (KLH) and sheep red blood cells systemically, and with BSA and KLH orally. Although antibodies were detected against BSA in the intestinal contents of birds that were orally immunized, there was no difference among different treatment groups. Systemic IgG, and to a lesser extent IgM, antibody responses to KLH were greater (P < 0.05) in birds fed a diet containing 11 or 22 ppm of virginiamycin compared with control birds fed no antibiotic. No treatment effect was found in the sheep red blood cell-immunized birds. Results of the present study implicate virginiamycin in enhancing antibody responses to some antigens in chickens. Further studies are required to determine to what extent these effects on antibody response are mediated through changes in the composition of the microbiota.

Mannheimia haemolytica and bovine respiratory disease.

Mannheimia haemolytica is the principal bacterium isolated from respiratory disease in feedlot cattle and is a significant component of enzootic pneumonia in all neonatal calves. A commensal of the nasopharynx, M. haemolytica is an opportunist, gaining access to the lungs when host defenses are compromised by stress or infection with respiratory viruses or mycoplasma. Although several serotypes act as commensals, A1 and A6 are the most frequent isolates from pneumonic lungs. Potential virulence factors include adhesin, capsular polysaccharide, fimbriae, iron-regulated outer membrane proteins, leukotoxin (Lkt), lipopolysaccharide (LPS), lipoproteins, neuraminidase, sialoglycoprotease and transferrin-binding proteins. Of these, Lkt is pivotal in induction of pneumonia. Lkt-mediated infiltration and destruction of neutrophils and other leukocytes impairs bacterial clearance and contributes to development of fibrinous pneumonia. LPS may act synergistically with Lkt, enhancing its effects and contributing endotoxic activity. Antibiotics are employed extensively in the feedlot industry, both prophylactically and therapeutically, but their efficacy varies because of inconsistencies in diagnosis and treatment regimes and development of antibiotic resistance. Vaccines have been used for many decades, even though traditional bacterins failed to demonstrate protection and their use often enhanced disease in vaccinated animals. Modern vaccines use culture supernatants containing Lkt and other soluble antigens, or bacterial extracts, alone or combined with bacterins. These vaccines have 50-70% efficacy in prevention of M. haemolytica pneumonia. Effective control of M. haemolytica pneumonia is likely to require a combination of more definitive diagnosis, efficacious vaccines, therapeutic intervention and improved management practices.

Chlamydia psittaci infection in sheep: a paradigm for human reproductive tract infection.

Chlamydiae are important reproductive tract pathogens in a wide variety of animals. In humans, chronic or repeated infection of the female genital tract with Chlamydia trachomatis has been identified as a significant factor in the development of occlusive infertility or increased risk of ectopic pregnancy. The spectrum of reproductive disease recognized in sheep to be caused by Chlamydia psittaci has been primarily restricted to pregnant animals because the organism was clearly identified as a major cause of infectious abortion. However, following pregnancy failure, a chronic chlamydial infection can become established in the reproductive tracts of experimentally infected ewes. Persistent infection of the ewe's reproductive tract may eventually result in pathology, similar to that observed in women infected with C. trachomatis, thus decreasing the breeding life of affected ewes. Furthermore, ewes that experienced C. psittaci induced abortion provide a unique opportunity to study the host: parasite dynamic as it relates to persistent infection. This natural model of persistent infection may, in some ways, be superior to more contrived models in which the chlamydial isolate is not a normal reproductive pathogen of the study animal. Thus, the study of persistent chlamydial infection in sheep may be used for the benefit of both human and veterinary medicine.

The detection of the sialoglycoprotease gene and assay for sialoglycoprotease activity among isolates of Pasteurella haemolytica A1 strains, serotypes A13, A14, T15 and A16.

Polymerase chain reaction (PCR) using specific primers to the sialoglycoprotease gene (gcp) of Pasteurella haemolytica biotype A, serotype 1 amplified a 1-kb fragment from each of P. haemolytica serotypes A7, A13, A14 and A16, but not T15; which was confirmed by Southern blot hybridization analysis. Using a sialoglycoprotease (Gcp) activity assay, Gcp activity was found in serotypes A13, A14 and A16. Inclusion of these three serotypes confirms that all recognized A biotypes are positive for both gcp gene and activity, with the exception of serotype A11 (which has a different genetic organization and exhibits no Gcp activity). Furthermore, all recognized T biotypes are negative for both the gene and Gcp activity.

Automated colorimetric assay for the detection of Pasteurella haemolytica leucotoxin.

A rapid and easily performed colorimetric method has been adapted for the detection of the Pasteurella haemolytica leucotoxin using the lysis of bovine leucocyte BL-3 cells (a bovine lymphoid cell line obtained from Dr. G. Theilen, University of California, Davis). The method involves incubating BL-3 cells in the presence of P. haemolytica culture supernatant and assessing cell survival at the end of the assay by staining remaining BL-3 cells with the dye neutral red. Solubilization of the cells with concomitant dye release allows the assay to be read by measuring optical density (540 nm) with an automated spectrophotometer. This assay can also be used for the determination of neutralizing antiserum titres.

The effect of Pasteurella haemolytica A1 leukotoxic culture supernate on the in vitro proliferative response of bovine lymphocytes.

The effect of sublethal concentrations of the Pasteurella haemolytica leukotoxic culture supernate on bovine lymphocyte blastogenesis was investigated. Blastogenesis in cultures stimulated with either concanavalin A (Con A) or pokeweed mitogen (PWM) was inhibited in the presence of the supernate, as was the response to purified protein derivative in lymphocytes from BCG-vaccinated cattle. Partially purified leukotoxin had a similar effect. Pre-incubation of the leukotoxic supernate with a polyclonal rabbit antiserum raised to the immunogenic molecule of recombinant leukotoxin (r LktA) abrogated this effect, implicating leukotoxin as the factor responsible for the inhibition. B cell enriched cultures tended to be more sensitive to leukotoxic effects than did T cell enriched cultures. Although only ruminant cells are susceptible to the lethal effects of P. haemolytica leukotoxin, the toxin did inhibit both Con A- and PWM-induced proliferation of human and dog lymphocytes. As well, at high leukotoxin doses, Con A-stimulated pig lymphocyte proliferation was reduced. Rabbit lymphocytes were not affected by leukotoxin in either Con A- or PWM-stimulated cultures.

Preliminary investigation of the mechanism of inhibition of bovine lymphocyte proliferation by Pasteurella haemolytica A1 leukotoxin.

Pasteurella haemolytica A1 leukotoxic culture supernate has been shown to inhibit bovine lymphocyte blastogenesis induced by concanavalin A (Con A), pokeweed mitogen (PWM) and purified protein derivative (PPD). The various mechanisms by which this inhibition could be overcome were investigated in an effort to determine at which stage of cell activation the leukotoxin exerted its inhibitory effect. For both Con A and PWM stimulated cultures, the addition of partially purified bovine interleukin 1 reduced the leukotoxin-induced inhibition. Recombinant interleukin 2 had a similar effect. Addition of the glycolipid, monosialoganglioside was also able partially to overcome the inhibition.

Preparturient vaccination to enhance passive immunity to the capsular polysaccharide of Pasteurella haemolytica A1.

The potential to increase passive serum antibody titres to a polysaccharide antigen in neonates, by preparturient vaccination of the dams was investigated. Dairy cows in five private herds were vaccinated with a commercial Pasteurella haemolytica culture supernatant vaccine (Presponse, Langford Inc.), at 6 and 3 weeks before their calculated due dates. Dams' sera, colostral whey, and post-colostral calf sera were assayed for antibodies of the IgG1 isotype binding purified capsular polysaccharide of P. haemolytica A1, using an enzyme immunoassay. Antibody titres were analyzed using the General Linear Model procedure (Statistical Analysis Systems Institute Inc.). Vaccinated dams had a significant increase in serum antibody titre after vaccination compared with non-vaccinates (P <0.01), and their antibody titres in colostral whey were significantly higher (P <0.05). Calves of vaccinated dams had significantly higher passive antibody titres than those of non-vaccinates (P <0.01) in all herds.

Safety, immunogenicity, and efficacy of two Escherichia coli cya crp mutants as vaccines for broilers.

Attenuated derivatives (delta cya delta crp mutants) of an O2 and an O78 avian septicemic Escherichia coli strain were used to immunize broiler chickens by spray to determine the safety, immunogenicity, and efficacy of the derivatives in single- and double-dose regimens. In the safety and immunogenicity studies, groups of 10 chickens were vaccinated by spray (droplet size approximately 20 microm) with the parent E. coli, the mutant organisms, or phosphate-buffered saline (PBS) at 14 days of age and euthanatised 21 days later. There was no deaths or gross pathologic finding in any of the chickens immunized with the vaccine strains. Compared with the levels in chickens exposed to PBS, there were significantly higher levels of immunoglobulin (Ig) G antibody in serum and air sac washings and of IgA antibody in air sac washings in response to the virulent parent strains than to the vaccine strains. In efficacy studies, chickens were immunized with the O2 or the O78 vaccine strain or PBS at day 14 and with the O2 vaccine strain or PBS at days 10 and 14 and challenged with the parent strain 10 days after the last vaccination. There was no significant difference in local IgA and IgG and serum IgG responses between vaccinated and control groups. Chickens vaccinated with the O2 strain, but not the O78 strain, had significantly lower air sac lesion scores compared with those of the unvaccinated groups in both single- and double-dose regimens. We conclude that the mutant O2 strain provided moderate protection against airsacculitis.

Experimentally induced pneumonia in scid/beige mice, using a bovine isolate of Pasteurella haemolytica.

BACKGROUND AND PURPOSE: Intranasal challenge of immunocompetent mice with Pasteurella haemolytica results in little or no pulmonary inflammation. The study reported here was designed to investigate the inflammatory response in the lungs of immunodeficient scid/beige mice after similar challenge. METHODS: Fifty-five scid/beige mice were challenged intranasally with saline or one of three doses (2.8 x 10(6), 3.4 x 10(9), or 3.3 x 10(11) colony-forming units [CFU]/ml) of P. haemolytica. The lungs were examined for changes in weight, bacterial count, and presence of gross and microscopic lesions at 24, 48, or 96 hours after challenge. RESULTS: Intranasal challenge with concentrations > or = 3.4 x 10(9) CFU/ml of P. haemolytica induced significantly heavier lung weight, with severe pulmonary lesions, and development of suppurative and fibrinous bronchopneumonia in dose- and time-dependent manner 48 hours after challenge. Pasteurella haemolytica was consistently isolated from the lungs at 24 hours after challenge. CONCLUSIONS: Bronchopneumonia was induced by P. haemolytica in mice without manipulation of the mouse or the bacteria. The lesions were similar to those that develop in the lungs of cattle infected with P. haemolytica and indicate potential use of the model for the study of this host/bacterial interaction.

Inhibition of the blastogenic response of peripheral blood mononuclear cells to mitogen and antigens by bovine pulmonary macrophages and their culture supernatants.

Bovine pulmonary macrophages were shown to inhibit blastogenic response of peripheral blood mononuclear cells to high and low level stimulation with phytohaemagglutinin P. The blastogenic response of cells from calves sensitised to Pasteurella haemolytica or BCG, when stimulated with the corresponding antigen, was also suppressed by the addition of autologous pulmonary macrophages. Twenty-four-hour-old macrophage culture medium was likewise inhibitory to the blastogenic response. Determination of the arginine content of culture medium before and after 24 and 48 hours' incubation with macrophages showed a progressive decrease in arginine content. Dilution of the arginine deficient medium 1:16 with fresh culture medium did not reverse the inhibition, thus making arginine deficiency unlikely to account for the inhibition.

Passive immunity to Pasteurella haemolytica A1 in dairy calves: effects of preparturient vaccination of the dams.

Dairy cows from five herds were assigned to receive a commercial Pasteurella haemolytica vaccine or no vaccine at all, administered at six and three weeks before parturition. Vaccination was associated with increased leukotoxin neutralizing serum antibody titers in the dams (p < 0.001), and with increased titers in colostrum (p < 0.001). Vaccination of dams also had a significant association with increased passive leukotoxin neutralizing antibody titers in their calves (p < 0.001). Vaccination was also associated with increased indirect agglutinating antibody titers in serum of the dams (p < 0.001). In the analysis of agglutinating antibody titers in colostral whey the interaction "vaccination*herd" was found to be significant (p < 0.001), indicating that the effects of vaccination on colostral titers were not consistent from herd to herd. The analysis was repeated, stratifying by herd. Vaccination was associated with increased agglutinating antibody titers in colostrum (p < 0.05) in three herds of the five in the study. In two of these three herds there were significant increases in passive neonatal titers associated with vaccination. In the remaining herd the mean IgG1 level in the calves was consistent with failure of passive transfer of immunoglobulins (IgG1 < 8.0 g/L). These results suggest that preparturient vaccination of dairy cows can induce modest increases in passive antibody titers to antigens of Pasteurella haemolytica in their calves, but the antigen of interest and the population being studied can affect the outcome.

Evidence of bovine immunoglobulin G1 (IgG1) protease activity in partially purified culture supernate of Pasteurella haemolytica A1.

In the bovine respiratory tract, IgG1 is a major secretory immunoglobulin (Ig), and both IgG1 and IgG2 are believed to be important in defense against pneumonic pasteurellosis (shipping fever) in calves. Here we provide evidence for hydrolysis of IgG1 in the presence of partially purified culture supernate (ppCS) from the respiratory pathogen Pasteurella haemolytica A1. Bovine IgG1 was hydrolysed sequentially into three distinct bands (approximately 39, 12, and 7 kDa respectively). Furthermore, partial hydrolysis of bovine IgG2 was observed, but neither bovine IgA nor IgM were affected by incubation with ppCS. These findings suggest that the production of an IgG1-specific protease by P. haemolytica A1 may be a virulence mechanism contributing to the pathogenesis of bovine pneumonic pasteurellosis.

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.

Vaccination of calves with leukotoxic culture supernatant from Pasteurella haemolytica.

In three experiments subcutaneous vaccination of calves with adjuvanted bacteria-free leukotoxic culture supernatant from log phase cultures of Pasteurella haemolytica A1 (toxin 1) was shown to induce some protection against intrabronchial challenge with live P. haemolytica A1. This toxin 1 vaccine was as effective as a whole cell bacterin in stimulating agglutinating antibody to P. haemolytica. Induction of leukotoxin neutralizing activity was variable; in some cases vaccination only primed the animal to produce an anamnestic response after challenge, whereas in other instances antitoxic activity increased in response to immunization. Two doses of vaccine were shown to be more effective than a single immunization. Vaccination with leukotoxic culture supernatant from the nonpathogenic P. haemolytica serotype 11 was as effective as vaccination with toxin 1 in stimulating antitoxic activity but was not protective. This implies that both serospecific agglutinating activity and an antitoxic response are needed for immunity.

Chlamydia psittaci infection and associated infertility in sheep.

Nineteen ewes were injected subcutaneously with the agent of enzootic ovine abortion, Chlamydia psittaci serovar 1, at 50 days gestation. Placental and fetal tissues were examined at 15 days postinfection and thereafter at ten day intervals. Placental infection was detected at 15 days postinfection. Only postinoculation sera collected from postinfected ewes contained antibodies reactive to C. psittaci. Five (26%) chlamydial infected ewes experienced inapparent fetal loss before day 105 of gestation. This finding is significant since C. psittaci infection in sheep is commonly associated with abortion and not infertility.

Comparison of protection of experimentally challenged cattle vaccinated once or twice with a Pasteurella haemolytica bacterial extract vaccine.

Three groups of calves (15-18 per group) were injected twice at a 3-week interval with 2 doses of phosphate buffered saline (PBS, CONTROL group), 2 doses of PRESPONSE, a Pasteurella haemolytica A1 bacterial extract vaccine (PRESPONSE-2 group) or 1 dose of PBS followed by a 2nd vaccination with 1 dose of PRESPONSE (PRESPONSE-1 group). Three weeks after the 2nd vaccination, the calves were challenged intratracheally with P. haemolytica A1. Calves were evaluated clinically for 3 days prior to challenge and for 5 days after challenge. Six days postchallenge, calves were either euthanized or sent to slaughter and the lungs were evaluated for percent pneumonic tissue. There was a significant effect of single or double application of vaccine on clinical scores (P = 0.0409). Percent pneumonic tissue at necropsy was significantly affected by vaccine group (P = 0.014). Calves in the CONTROL group had significantly higher percent pneumonic tissue after arcsine transformation (45.30%) than calves in any group receiving PRESPONSE, regardless of vaccination frequency (25.18% and 25.78%, for calves receiving 2 doses or 1 dose of PRESPONSE, respectively). Both serum toxin neutralizing and direct agglutinating titers were negatively correlated with percent pneumonic tissue. Most importantly, 1 dose of PRESPONSE was as efficient as 2 doses at eliciting a protective immune response. It is concluded that the presence of P. haemolytica as a natural commensal in the upper respiratory tract of the calf can effectively prime the animal, and allow the animal to respond in an anamnestic nature to only 1 dose of this vaccine.