Measurement of Pasteurella haemolytica-specific lung and serum antibodies by ELISA.

The modified enzyme-linked immunosorbent assay (ELISA) was used to determine the relative quantities of class-specific antibodies to Pasteurella haemolytica. IgG1, IgG2 and IgA were present in significantly higher quantities in bronchoalveolar washings (BAW), but in decreasing quantities, respectively; IgM was present in very low amounts. IgM, IgG1 and IgG2 were present in serum, again in decreasing quantities, respectively. IgA antibody quantities were lowest in serum. The indirect antibody ELISA was found to be superior to the indirect bacterial agglutination (IBA) technique for determining antibody titres against P. haemolytica.

Colorimetric assay using XTT for assessing virulence of avian Pasteurella multocida strains.

A colorimetric assay using sodium 3,3'-[1[(phenylamino)carbonyl]3,4- tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) was adapted to quantitate bactericidal activity of chicken macrophage HD 11 cell line against five Pasteurella multocida strains and an avirulent transposon insertion mutant. The strains used were virulent P1059, and D92, and four avirulent strains including a streptomycin resistant mutant of P1059 (P1059 SmR), two live vaccine strains namely, the Clemson University (CU) and M9, and a transposon insertion mutant PmTn-294. Percentage of bacteria killed by chicken macrophage (HD 11) cells was determined by extrapolation from a standard formazan curve derived by incubating XTT with known bacterial cell numbers of each strain. The amount of formazan as measured by absorption at 450 nm was directly related to the number of viable bacterial cells. The percentages of P1059 SmR, CU, M9 and PmTn-294 killed by HD 11 cells were approximately 50%, 61%, 25% and 34%, respectively. By contrast, the virulent P1059 and D92 strains were resistant to killing, and were able to replicate inside the HD 11 cells. Association of virulence with resistance to phagocytic killing by HD 11 cells as assessed by the colorimetric bactericidal assay, was validated with resistance to complement (C')-mediated killing and a turkey mortality test. Strains P1059 and D92 were resistant to C'-mediated killing, whereas strains P1059 SmR, CU, M9 and PmTn-294 strains were susceptible. All turkeys challenged with P1059 or D92 were dead within 18 hrs. Mortality did not occur in turkeys challenged with strains of P1059 SmR, M9 and PmTn-294. The mortality among CU challenged turkeys ranged from 0 to 40%. The results suggest that the colorimetric bactericidal assay using XTT can be used to quantitate chicken macrophage phagocytic killing of P. multocida strains, and may be a valuable assay to differentiate virulent from avirulent strains of avian P. multocida.

Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida.

The SDS-PAGE patterns of the outer membrane protein (OMP) extracts of Pasteurella multocida strain P1059, grown under iron-restricted, iron-replete and in vivo conditions, were examined. The results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 76 kDa, 84 kDa, and 94 kDa were expressed by bacteria grown in iron-restricted media. They were also expressed by in vivo grown P. multocida. Convalescent-phase sera, obtained from turkeys which had survived pasteurellosis, contained antibodies that reacted intensly with th three IROMPs. This indicated that these proteins were expressed in vivo. Bacteria expressing the IROMPs showed greater binding to Congo Red when compared to cells not expressing IROMPs. Cells expressing the IROMPs or its OMP extracts grown in iron-restricted media also showed greater binding to 59Fe-pasteurella siderophore (multocidin) when compared to bacteria or its extracts not expressing IROMPs. Convalescent-phase sera, which contained antibodies against the IROMPs, blocked this specific 59Fe-multocidin binding to IROMPs. Autoradiography was used to determine which of these IROMPs functioned as a receptor for the iron-multocidin complex. The results suggested that these three IROMPs have specific epitopes for binding to the iron multocidin complex.

Susceptibility of Pasteurella haemolytica to the bactericidal effects of serum, nasal secretions and bronchoalveolar washings from cattle.

Several isolates of logarithmic-phase organisms of Pasteurella haemolytica were shown to be sensitive to an antibody and complement-mediated killing mechanism in adult bovine serum. Data suggested that the classical complement pathway was important in the induction of bactericidal activity of serum. Sera from calves after colostrum feedings (post-colostral sera) killed only 30% of the bacteria in spite of the presence of high levels of antibodies against P. haemolytica. Addition of post-colostral serum to heat-inactivated adult bovine serum decreased the bactericidal capacity of the latter. It was speculated that this inhibition may have been caused by the presence of blocking antibodies (IgA) found in the post-colostral serum. Undiluted nasal secretions collected from adult cattle were not bactericidal to P. haemolytica. The results also suggest that the bronchoalveolar washings (BAW) from vaccinated calves, in spite of having a high antibody titer, were less bactericidal to P. haemolytica than BAW from sham-vaccinated calves (71.12% vs. 83.12%). The bactericidal factor(s) present in BAW from sham-vaccinated calves was heat stable, not complement dependent, and was not related to lysozyme concentration.

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.

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).

Increased tumor necrosis factor-alpha and interleukin-1 beta expression in the lungs of calves with experimental pneumonic pasteurellosis.

We used a well characterized pneumonic pasteurellosis model in calves to determine whether increased proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) expression and secretion were associated with pneumonic lesions. Bronchoalveolar lavage fluids, lavage cells consisting of alveolar macrophages and neutrophils with degenerative changes, and lung tissues were analyzed for the presence of TNF-alpha and IL-1 beta approximately 48 h following endobronchial inoculation of logarithmic phase Pasteurella haemolytica 12296 organisms. Levels of TNF-alpha and IL-1 beta mRNA were significantly increased in lavage cells of P. haemolytica-infected animals but not in cells from phosphate buffered saline (PBS) inoculated controls based on in situ hybridization analysis. Significantly increased levels of TNF-alpha, and IL-1 beta mRNA were also expressed within the pneumonic lesions from P. haemolytica-infected calves. In contrast, lung tissues from PBS-inoculated control calves had cytokine mRNAs expressed at extremely low levels. Increased levels of bioactive IL-1 and immunoreactive (not bioactive) TNF-alpha were found in lavage fluids from P. haemolytica-infected calves compared with lavage fluids from PBS-inoculated calves. These findings indicate that the proinflammatory cytokines TNF-alpha and IL-1, may be associated with pathogenesis of lung injury in bovine pneumonic pasteurellosis.

Effects of Pasteurella haemolytica A1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals.

To further define the role of Pasteurella haemolytica A1 leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis, its in vitro effects on bovine neutrophils were investigated. Leukotoxin-containing culture supernatant, from P. haemolytica, stimulated a neutrophil respiratory burst as measured by the generation of oxygen-derived free radicals O2- and H2O2. This effect was immediate because preincubation of neutrophils with the culture supernatant for 5 min or longer substantially suppressed this respiratory burst. This suppression was due to cytolysis of the neutrophils. Prolonged incubation of neutrophils with the same culture supernatant caused further cytolysis and degranulation. Heat-inactivated P. haemolytica culture supernatant that had lost its cytotoxic properties failed to stimulate respiratory burst by neutrophils. Furthermore, the respiratory burst, cytolysis and degranulation were abrogated only by leukotoxin-neutralizing monoclonal and polyclonal antibodies, but not by antibodies against the lipopolysaccharide. These studies show that the leukotoxin component in the culture supernatant was responsible for the generation of oxygen-derived free radicals and proteolytic enzymes from neutrophils which may participate in direct lung injury.

Serotyping of Mannheimia (Pasteurella) haemolytica isolates from the upper Midwest United States.

Mannheimia (Pasteurella) haemolytica biotype A serotype1 (A1) is the primary bacterial agent responsible for the clinical signs and pathophysiologic events in bovine pneumonic pasteurellosis. The goal of this study was to determine the prevalence of other serotypes of M. haemolytica biotype A organisms obtained from the upper Midwest diagnostic laboratories. A total of 147 M. haemolytica isolates were collected from Minnesota, South Dakota, and Michigan. Isolates were tested against M. haemolytica antisera obtained from the National Animal Disease Center, Ames, Iowa. Results indicated that M. haemolytica serotype 1 represented approximately 60%, serotype 6 represented 26%, and serotype 2 represented 7% of the total examined isolates. In addition, 7% of the isolates were serotype 9, 11, or untypable. This finding suggests that M. haemolytica serotypes other than serotype 1 can be isolated from the lung lesions of diseased cattle and seem to be capable of causing the pathologic changes observed in the lung with pneumonic pasteurellosis.

Immune profile of infectious bursal disease: I. Effect of infectious bursal disease virus on peripheral blood T and B lymphocytes of chickens.

A comparison was made of absolute numbers of peripheral blood B and T lymphocytes, as defined by the anti-bursal and anti-thymus sera, in chickens infected with infectious bursal disease virus at one day and 3 weeks of age by age-matched controls. Birds were evaluated sequentially at weekly intervals for a period of 8 weeks postinfection. The severity of depressions of B-lymphocyte numbers was found to be age-dependent: the birds infected at one day of age showed a more severe depletion than those infected at 3 weeks of age. The T-lymphocyte numbers were less markedly affected in infected chickens of either age group.

Immune profile of infectious bursal disease (IBD). II. Effect of IBD virus on pokeweed-mitogen-stimulated peripheral blood lymphocytes of chickens.

Pokeweed-mitogen-stimulated peripheral blood lymphocytes from chickens infected with infectious bursal disease virus (IBDV) at 1 day and 3 weeks of age, together with those from uninfected age-matched control chickens, were examined at weekly intervals for their capacity to undergo in vitro terminal differentiation. This study included the determination of cytoplasmic-immunoglobulin (CIg)-containing peripheral blood lymphocytes and those bearing the surface membrane immunoglobulin (SmIg) cells to detect any defect in immunoglobulin synthesis and secretion, respectively. The results of our study indicated that there was a highly significant and pronounced progressive depletion of CIg-containing cells in chickens infected at 1 day of age. These findings suggest that IBDV affected the "immature" or precursor B cells to a far greater extent than mature B lymphocytes.

Immune profile of infectious bursal disease. III. Effect of infectious bursal disease virus on the lymphocyte responses to phytomitogens and on mixed lymphocyte reaction of chickens.

A whole-blood-culture technique was used to sequentially evaluated peripheral blood lymphocyte responses to phytohemagglutinin (PHA) and concanavalin A (Con A) of normal chickens and chickens infected at 1 day or 3 weeks of age with infectious bursal disease virus (IBDV). This method had numerous advantages over the more conventional techniques. A comparative study was made on the percentage of inhibition of responses of peripheral blood lymphocytes to PHA and Con A of 1-day- and 3-week-old IBDV-infected chickens. In both groups, there was a minimum inhibition between 3 and 4 weeks postinfection (PI) and a maximum inhibition at 6 weeks PI. A one-way mixed lymphocyte reaction (MLR) was performed using mitomycin-C-treated cells as stimulator cells obtained from chickens of genetically different strains. Lymphocytes from the experimental birds (control, 1-day-infected, and 3-week-infected groups) were used as the responder cells. The results showed that MLR response of the IBDV-infected chickens was significantly reduced compared with those of the uninfected controls. The degree of lowered response was much more severe in chickens infected at 1 day of age than in those infected at 3 weeks of age.

Studies on Pasteurella multocida. VI. Nature of systemic immunity and analysis of the correlation between levels of immunity induced by various fowl cholera vaccines and protection against challenge.

The nature of the systemic immunity induced by various vaccines against fowl cholera disease in turkeys was investigated. Used as vaccines were three avirulent strains (CU, M2283, and P1580) of Pasteurella multocida administered in the drinking water and a bacterin administered parenterally. A passive hemagglutination test was developed to measure levels of systemic humoral immunity. Systemic cell-mediated immune response was assayed by an in vitro immunostimulation microculture system utilizing peripheral blood lymphocytes. The bacterin induced high levels of systemic humoral and cell-mediated immune responses which respectively persisted for 8 and 6 weeks. The CU strain of live vaccine induced average levels of systemic immunity (humoral and cell-mediated) which respectively persisted for 4 and 6 weeks. The M2283 strain of live vaccine also induced average levels of systemic immunity (less than induced by the CU strain) which respectively persisted for 4 and 6 weeks. The P1580 strain of live vaccine induced low levels of systemic immunity which persisted for only 2 weeks. Results from data generated from an analysis of correlation between levels of immunity and protection indicate that systemic immunity (humoral and cell-mediated) played a significant role.

Studies on Pasteurella multocida. VII. Dynamics and temporal development of local humoral immunity induced by a live avirulent fowl cholera vaccine.

Vaccination of turkeys by administering the CU strain of Pasteurella multocida in drinking water induced local antibodies in the tracheal secretions. Their appearance and persistence were demonstrated by the indirect immunofluorescence technique. Local antibodies were induced by the 10th day and persisted up to the 42nd day postvaccination, whereas none were induced by an oil-adjuvanted bacterin throughout the observation period (56 days). Thus, the CU strain of P. multocida appeared to generate a local humoral immunity in the respiratory system whereas the bacterin did not.

Purification and identification of turkey immunoglobulin-A.

Turkey immunoglobulin-A (IgA) was isolated from bile, intestinal secretions, and serum by affinity chromatography using monospecific anti-turkey IgA coupled to CNBr-activated Sepharose. The isolated immunoglobulin was antigenically distinct from IgM and IgG. The purity of IgA was demonstrated by immunodiffusion, immunoelectrophoresis, and analytical ultracentrifugation. The predominant forms of polymeric IgA in bile and intestinal secretions had respective So20w values of 16.1 and 15.2. Larger polymers (25-26S) were also present. Two molecular forms (8.5S and 17S) were found in serum. The 8.5S peak was higher than the 17S, indicating a greater concentration of 8.5 S.

Comparison of enzyme-linked immunosorbent assay with dot immunobinding assay for detection of antibodies against Pasteurella multocida in turkeys.

A dot immunobinding assay (DIA) was developed to detect antibodies against Pasteurella multocida in turkey serum. Five coating antigens, namely, whole-cell (WC) antigen, sonicated cell lysate (SCL), crude capsular extract (CE), formalin extract (FE), and heat-stable antigen (HSA), were compared by enzyme-linked immunosorbent assay (ELISA) and DIA using reference antisera against P. multocida organisms. WC and SCL antigens showed higher sensitivity, whereas FE and HSA antigens were more specific coating antigens in both assays. The specificity of DIA was greater than ELISA by comparing the P/N ratios of HSA against serum prepared from heterologous serotype of P. multocida. The DIA had also several distinct advantages over the ELISA, which included reduction of the manipulation time and more uniform binding of coating antigens onto the nitrocellulose membranes compared with binding of coating antigens to microtiter plates for ELISA.

Effect of infectious bursal disease virus infection on the phagocytosis of Staphylococcus aureus by mononuclear phagocytic cells of susceptible and resistant strains of chickens.

Studies were conducted to determine why chickens infected with infectious bursal disease (IBD) are more susceptible to Staphylococcus aureus. We evaluated the effect of IBD virus on functions of the peripheral mononuclear phagocytes and looked for humoral abnormalities in the serum. An in vitro opsonophagocytic assay using (H3) thymidine-labeled opsonized Staphylococcus aureus was used to measure phagocytosis. Infection of IBD-sensitive (SCWL 420) chickens with IBD virus did not cause any cellular defects, as shown by phagocytic inability, or any humoral abnormalities, as shown by the lack of opsonics. However, IBD virus did cause some humoral defects 2 weeks postinfection in resistant (Hardy Red) chickens. Therefore, the basic mechanisms governing increased susceptibility of IBD-infected chickens to S. aureus is still unknown.

Studies on Pasteurella multocida. III. In vitro assay for cell-mediated immunity.

Peripheral blood lymphocytes (PBL) from turkeys immunized against fowl cholera with a bacterin or a live avirulent vaccine (strain CS-148) were cultured in vitro with various antigenic preparations from Pasteurella multocida (strain P-1059). The degree of lymphocyte stimulation (blastogenesis) was quantitated by measurement of the uptake of (3H) thymidine. Higher stimulation indices were obtained with immune lymphocytes rather than nonimmune lymphocytes. Stimulation was specific since PBL from turkeys immunized against P. multocida failed to react with Escherichia coli or Mycoplasma synoviae antigens. These differences were statistically significant as analyzed with the student's t-test. The lymphocyte transformation assay was emphasized as a convenient and useful in vitro indicator of cell-mediated immunity that should help define the role of cell-mediated immunity in P. multocida infections of turkeys.

Pasteurella haemolytica as a co-pathogen in pullets and laying hens.

Pasteurella haemolytica was isolated from internal organs of laying hens and pullets of four flocks in which five incidents of increased mortality had occurred. There was inflammation of the trachea in all cases, and infectious laryngotracheitis virus was identified in three of the incidents. Rapid response to antimicrobial therapy was seen in two out of three outbreaks in the pullets. P. haemolytica was regarded as a significant secondary invader in these cases.

Morphological and morphometrical analysis of the acute response of the bovine alveolar wall to Pasteurella haemolytica A1-derived endotoxin and leucotoxin.

Endotoxin or leucotoxin derived from Pasteurella haemolytica biotype A serotype 1 or saline was deposited by fibreoptic bronchoscopy into the caudal segment of the right anterior lung lobe of calves, and the lesions were characterized by light and transmission electron microscopy. Morphometric techniques were used to determine if changes in the arithmetic mean thickness of the alveolar wall occurred. Group 1 calves (n = 2) were inoculated with 6 ml saline, groups 2 calves (n = 3) received 6 ml of a partially purified leucotoxin preparation, group 3 calves (n = 3) received 96 micrograms of endotoxin in 6 ml of saline and group 4 calves (n = 3) received 2.5 mg of endotoxin in 6 ml of saline. Calves were killed 4 h after inoculation. Lesions in groups 2, 3 and 4 were similar and we found that (a) endotoxin alone is capable of initiating an inflammatory response in the bovine lung, (b) leucotoxin causes cytotoxic changes in alveolar macrophages but not in parenchymal cells of the lung, (c) neutrophil sequestration and platelet aggregation occur in alveolar capillaries in association with pulmonary intravascular macrophages, (d) neutrophils and fibrin were found in the alveolus in close association with alveolar macrophages, (e) disruption of the alveolar epithelial layer occurred in association with neutrophils and (f) there were no significant increases in the arithmetic mean thickness of the alveolar wall.