Evaluation of the specificity of Pasteurella multocida somatic antigen-typing antisera prepared in chickens, using ribosome-lipopolysaccharide complexes as inocula

Purified lipopolysaccharides (LPS) from 16 serotypes of Pasteurella multocida were complexed with Aspergillus fumigatus ribosomes. The complexes were used as inocula to prepare antisera, in chickens, for somatic antigen typing by the gel diffusion precipitin test (GDPT). Antisera made against 15 of 16 LPS reacted with their respective specific heat-stable antigens in the GDPT and homologous LPS in the passive hemagglutination test. Antisera could not be made against serotype 15 LPS. Correlation was not observed between intensity of the precipitin reaction in the GDPT and titer to homologous LPS in the passive hemagglutination test. Most antisera cross-reacted with other heat-stable antigens of other serotypes in the GDPT. Many of these cross-reactions were eliminated by dilution. Cross-reactions that occurred in the GDPT with antisera made against LPS of serotypes 2, 5, 7, and 8 could not be eliminated by dilution.     

Comparisons of serologic responses of white Leghorn and New Hampshire red chickens to purified lipopolysaccharides of Pasteurella multocida

White leghorn and New Hampshire red chickens were inoculated with purified lipopolysaccharides of 14 serotypes of Pasteurella multocida to determine their ability to produce serotype-specific antisera for somatic antigen typing. Specific antisera were made by both breeds of chicken to lipopolysaccharides of serotypes 1, 3, 4, 6, 8, and 16. No specific antisera were made against lipopolysaccharides of serotypes 2, 5, 7, 12, and 14. Lipopolysaccharides of serotypes 10 and 11 failed to stimulate antibody production. White leghorns were more responsive than New Hampshire red chickens. White leghorn antisera had higher titers to lipopolysaccharides in passive hemagglutination tests and produced more intense precipitin reactions with heat-stable antigens in the gel-diffusion-precipitin test.     

Polypeptides associated with Pasteurella multocida infection in rabbits.

Polypeptides from whole cell preparations of Pasteurella multocida serotypes A:12 and A:3 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Antigens were detected by immunoblot analysis, using sera from 3 groups of rabbits. Sera were obtained from rabbits inoculated intranasally with P multocida serotype A:12 or A:3, from rabbits maintained in a rabbitry with enzootic P multocida A:12 infection, and from rabbits maintained in a rabbitry with enzootic P multocida A:3 infection. Immunoblot analyses of pre- and postinoculation sera from experimentally infected rabbits, using serotype A:12 antigen, revealed 3 polypeptides with approximate molecular mass of 28, 30, and 37 kDa that consistently detected antibodies after P multocida-induced infection. Sera from rabbits naturally infected with either serotype, tested against serotype A:12 and A:3 antigens, detected the same polypeptides in both serotypes. Thus, immunologic reactivity to these polypeptides may be useful for serologic detection of P multocida infection.     

Serologic examination of the Westphal-type lipopolysaccharides of Pasteurella multocida.

The serologic specificities of the Westphal-type lipopolysaccharides from 16 serotypes of Pasteurella multocida were compared with those reported for the heat-stable typing antigens in the gel diffusion precipitin test. Like the heat-stable typing antigen, the Westphal-type lipopolysaccharide from each of the serotypes reacted only with its homologous antiserum in 14 of the 16 serotypes. The lipopolysaccharide from type strains representing serotypes 2 and 5 reacted with either of the corresponding typing sera, as did field isolates of these serotypes to a lesser extent. Although differences among the properties of these antigens were minor, the lipopolysaccharide appeared to be a major component of the heat-stable antigen responsible for the type specificity.     

Lipopolysaccharides of the Heddleston serotypes of Pasteurella multocida

Lipopolysaccharides (LPS) were extracted from 13 of the 16 Heddleston serotypes of Pasteurella multocida by phenol-chloroform-petroleum ether (PCP). Serotypes 3, 9, and 13 were extracted only by phenol-water (PW). After extraction of LPS of serotype 9 by PW, an additional LPS was isolated by PCP. All LPS contained glucose, 2-keto-3-deoxyoctonate, and heptose. Two isomers of heptose, D-glycero-D-mannoheptose and L-glycero-D-mannoheptose, were found in serotypes 2 and 5. Antisera made against purified LPS of serotypes 2 and 5 reacted with both heat-stable antigens and LPS from serotypes 2 and 5 in the gel-diffusion precipitin test. Antisera against serotype 2 LPS protected turkeys against challenge with capsulated serotype 5, indicating that a structural relationship exists between LPS of strains that cause hemorrhagic septicemia and fowl cholera. Rhamnose was a component of serotype 9 LPS, and galactose was found in all LPS, except for serotype 11. The LPS of serotype 13 contained an isomer of heptose that has not been identified. The LPS had buoyant densities in CsCl of 1.40 +/- 0.0148 g/ml, and all hemagglutinated chicken and turkey, but not sheep or horse, RBC.     

Proteins and antigens of Pasteurella multocida serotype 1 from fowl cholera.

The protein profiles of Pasteurella multocida serotype 1 isolates and the response of chickens to serotype 1 antigens were investigated using SDS-PAGE. Patterns obtained with Coomassie blue staining of soluble protein extracts were similar. The major difference between isolates was the position of one of the major proteins in the 34-38 kDa region. When chickens were experimentally infected with a clinical isolate of P. multocida serotype 1 various proteins were recognised by immunoblotting, including one with a relative molecular weight of 34 kDa; however, no reactions were observed in the region where LPS is known to migrate. When these infection sera were used in an EIA with purified LPS obtained from Heddleston serotype 1 type strain (X-73) they reacted strongly. Serum used for serotyping isolates in the gel diffusion precipitin test recognised many antigens in common with sera from infected birds, but some antigens were specific to typing sera.     

Effects of the lipopolysaccharide-protein complex and crude capsular antigens of Pasteurella multocida serotype A on antibody responses and delayed type hypersensitivity responses in the chicken.

The effects of the lipopolysaccharide-protein complex (LPS) and crude capsular antigen (CCA) prepared from Pasteurella multocida serotype A isolated from a duck in the Philippines, on antibody responses to sheep red blood cells (SRBC) and Brucella abortus (BA) and delayed type hypersensitivity (DTH) responses to bovine serum albumin (BSA) in the chickens were studied. Chickens injected subcutaneously with LPS and CCA at 1 and 2 weeks of age and immunized intravenously with the mixed antigens of SRBC and BA, at 3 and 4 weeks of age showed significantly increased antibody responses against both SRBC and BA, when evaluated at 7 days after each immunization. In addition, these chickens sensitized intramuscularly with the emulsion of BSA in complete Freund's adjuvant at 5 weeks of age, and then injected into the wattle with BSA at 7 weeks of age also showed significantly increased DTH responses against BSA, when evaluated at 24 and 48 hr after challenge. These results indicate that LPS and CCA of P. multocida serotype A have a property enhancing humoral and cell-mediated immune responses.     

Protection of chickens by ribosomal vaccines from Pasteurella multocida: dependence on homologous lipopolysaccharide

Chickens were protected against fowl cholera by ribosomal vaccines prepared from noncapsulated Pasteurella multocida. Passive hemagglutination (PHA) titers to lipopolysaccharide (LPS) and the degree of protection conferred by ribosomal vaccines were diminished or abolished when ribosomes were chromatographed on an immunoadsorbent column. Addition of subimmunogenic amounts of serotype 1 (homologous) LPS to highly purified ribosomes resulted in vaccines that protected against challenge exposure and produced PHA titers to homologous LPS. Addition of serotype 5 LPS to highly purified ribosomes did not protect chickens against challenge exposure with serotype 1 P multocida, but produced PHA titers to serotype 5 LPS. Combinations of serotype 1 ribosomal RNA and serotype 1 (homologous) LPS did not protect chickens or produce PHA titers to LPS. Purified ribosomes from Brucella abortus, Aspergillus fumigatus, and chicken liver were combined with LPS from P multocida and were evaluated as vaccines. Brucella abortus and A fumigatus ribosomes combined with LPS protected chickens as well as did bacterin made from whole cells of P multocida. Chicken liver ribosomes combined with LPS did not provide protection. To determine whether a protein carrier would substitute for ribosomes, methylated bovine albumin (MBA) was combined with LPS and evaluated as a vaccine. A serologic response to LPS was induced by MBA-LPS vaccine, but the vaccine offered no better protection than when LPS was used alone as vaccine. Ribosome-LPS vaccines produced serologic responses to LPS that were at least 5-fold greater than those produced by MBA-LPS vaccine.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody (IgG) to Pasteurella haemolytica

The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.     

Opsonic monoclonal antibodies against lipopolysaccharide (LPS) antigens of Pasteurella multocida and the role of LPS in immunity.

A panel of six monoclonal antibodies (MAbs) produced from mice immunized with Pasteurella multocida (M1404) (Heddleston serotype 2) reacted with homologous lipopolysaccharide, as indicated by enzyme immunoassay and immunoblotting. All six MAbs reacted with serotypes 2 and 5 of the 16 Heddleston serotypes. The reactive epitopes were localized on the bacterial cell surface by immunogold labelling. The antibodies could agglutinate P. multocida only if cells were first treated with 1 N HCl. All six MAbs opsonized P. multocida for phagocytosis by mouse macrophages but were not bactericidal in the presence of complement. They afforded only partial protection against infection in mice. The results, together with those of active immunization experiments with LPS, suggest a subordinate role for LPS in protection from experimental infection in mice.     

Evaluation of different antigens in the complement-fixation test for diagnosis of Haemophilus pleuropneumoniae (parahaemolyticus) infections in swine

The identification of new serotypes of Haemophilus pleuropneumoniae (parahaemolyticus) and the frequency of pleural adhesions due to contagious pleuropneumonia in many fattening swine herds have prompted the study of the complement-fixation (CF) test as a diagnostic tool for use in swine. Whole cell antigens, mixed antigens, autoclaved antigens, and phenol-water-extracted antigens derived from different serotypes were prepared and tested with immunized-swine sera by the CF test. Mixed antigen consisting of whole cells from all known serotypes was the best screening antigen for routine use. This antigen gave positive titers with all sera in which a positive reaction against the separate serotype antigen was registered. The most highly serotype-specific reactions were obtained with antigens prepared by phenol-water extractions of whole cells. When whole-cell antigens were used in the CF test, antibodies to superficial serotype-specific and common species-specific antigens could be detected.     

Natural killer cell activity in chickens exposed to Marek's disease virus: inhibition of activity in susceptible chickens and enhancement of activity in resistant and vaccinated chickens

Chickens of 2 genetic lines (lines P and N) were inoculated with a pathogenic strain of Marek's disease (MD) virus (MDV) and chronologically examined for disease response and natural killer (NK) cell expression. The NK cell reactivity was assayed in an in vitro cytotoxicity assay in which effector cells from the spleen of test chickens were reacted with 51Cr-labeled LSCC-RP9 target cells. Chickens of line P developed progressive debilitating disease and a high incidence of gross tumors and death. The NK cell reactivity of line-P chickens infected with MDV was significantly lower than that of uninfected control hatchmates. In contrast, NK cell levels were significantly elevated in MDV-inoculated line-N chickens that were resistant to MD and in chickens of lines P or N that had been inoculated with herpesvirus of turkeys (HVT). NK cell levels were also elevated in line P if chickens were vaccinated with HVT before infection with MDV. Inhibition of NK reactivity in susceptible chickens and elevation of reactivity in naturally resistant or vaccinated chickens may indicate a role for the NK cell system in regulating resistance to MD.     

Monoclonal antibodies against surface antigens of Pasteurella multocida strain P-1059

Four monoclonal antibodies (MAbs) were developed against serotype 3:A, P-1059 strain of Pasteurella multocida. Enzyme-linked immunosorbent assays were used to screen those hybridomas producing antibodies to either a surface protective (2.5 S) or lipopolysaccharide (LPS) antigen. MAbs 6EE11, D7H10, E11E3, and C11H2 were positive against 2.5 S antigen, and two of them, E11E3 and C11H2, were positive for the LPS antigen. MAbs 6EE11 and D7H10 reacted with a major protein band of molecular weight of 35,500, whereas E11E3 and C11H2 recognized a band with a molecular weight of 12,500 of the 2.5 S antigen. Treatment of the 2.5 S antigen with periodic acid abolished epitopes reacting with E11E3 but not with 6EE11. MAb 6EE11 did not recognize any band in Western blot after proteinase K treatment of the 2.5 S antigen, whereas antibody activity of E11E3 did not change. MAb 6EE11 reacted with serotypes 3, 4, 9, 10, 11, 12, and with M-9 strains in the immunofluorescence test. MAb E11E3 was positive only with serotype 3 or 10 strains, excluding M-9 strain. Electron microscopic studies with P-1059 strain indicated that antigens binding to 6EE11 and/or E11E3 were present in the capsule.     

Detection of serum antibodies in Eimeria tenella-infected chickens by enzyme linked immunosorbent assay (ELISA) with merozoite and oocyst antigens.

Soluble antigens prepared from sporulated oocytes and second generation merozoites of E. tenella were used for enzyme linked immunosorbent assay (ELISA) to investigate antibody in sera of two breeds of chickens, i.e. commercial broilers and SPF single comb white leghorn layers, which were experimentally infected with E. tenella. In broilers inoculated with oocysts at 15 days of age, ELISA values increased rapidly after day 19 post inoculation (PI) and reached the maximum lebel on days 29 and 32 PI against both merozoite and oocyst antigen. The values against merozoite antigen were significantly higher than those against oocyst antigen. In SPF layers infected at 15 days of age, the values increased gradually after 7 days PI. There were no significant differences between values against two antigens. Generally, the values in broilers tended to be higher than those in SPF layers, especially against merozoite antigen. In broilers inoculated with oocysts at 1 and 15 days of age, ELISA values increased rapidly and reached the maximum level on days 11 and 20 post second inoculation (PSI) against merozoite and oocyst antigens respectively and then the values against merozoite antigen decreased. The values against merozoite antigen were markedly higher than those against oocyst antigen. In SPF layers inoculated twice, the values reached the highest on day 11 PSI as in the case of broiler; however, after that day, the values against both antigens decreased. The sera reacted similarly against both antigens. The values against merozoite antigen were significantly higher in broilers than in SPF layers.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Elimination of mycoplasmal plate agglutination cross-reactions in sera from chickens inoculated with infectious bursal disease viruses

Sera from chickens inoculated with various challenge infectious bursal disease viruses or infectious bursal disease vaccines were found to cross-react in the Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) serum plate agglutination (SPA) tests. Two-fold dilutions of these cross-reacting sera with phosphate-buffered saline before retesting eliminated all non-specific agglutination in the MG and MS SPA tests. Cross-reactions were observed in the SPA test using sera from chickens inoculated with either MG or MS. Dilutions of these sera 1:2 had little effect on the number of these cross-reactions. At 1:4 serum dilutions, however, the number of cross-reactions between MG and MS was reduced. At 1:8 dilution of test sera, cross-reactions between MG and MS were further reduced. Some reduction in specific MG and MS SPA reactions, however, also occurred at the 1:8 dilution of sera with some of the plate antigen.     

Overexpression and immunogenicity of the Oma87 outer membrane protein of Pasteurella multocida

The outer membrane protein of Oma87 from Pasteurella multocida A:1 has significant similarity to the D15 protective antigen of Haemophilus influenzae (Ruffolo and Adler, 1996). Four fragments of Oma87 from a P. multocida serotype D strain were cloned into a pGEX expression vector and transformed into E. coli JM105. Western blot analysis revealed that convalescent chicken sera reacted with only GST-F1 fusion protein which contained amino acids 18 through to 130 of Oma87 fused to the GST protein. Vaccination with the GST-F1 protein failed to protect chickens against challenge with a virulent P. multocida serotype A.     

Detection of serum antibodies against Mycoplasma gallisepticum and Mycoplasma synoviae by a dot-immunobinding technique

Both Mycoplasma gallisepticum (MG) and M. synoviae (MS) antigens prepared for the routine haemagglutination inhibition (HI) test were diluted and absorbed to the separate pieces of durapore membrane for the measurement of dot-immunobinding (DIB) titers of test sera. Besides, durapore strips bearing both antigens were employed for a DIB test with chicken sera definitely diluted 100-fold. Shortening of reaction time of chicken sera with antigens as well as with the secondary serum markedly eliminated non-specific DIB reactions exhibited at low dilutions although the same condition was not so effective on the elimination of non-specific reactions among rabbit hyperimmune sera. Rapid and specific development of DIB antibody which continued at high titer up to 1:640 for 10 weeks postinoculation was proved in the sera of SPF chickens inoculated with MG or MS, while DIB titers of sera from uninoculated chickens remained 1:20 or lower. Non-specific reactions, which occurred in the routine serum plate agglutination test with a part of sera from the inoculated chickens, were not exhibited in the DIB as well as in the HI test with the same sera. Results of the DIB test with serum samples from 287 conventionally reared chickens definitely diluted 100-fold coincided with the results of HI test at a level of 90% with MG and 89% with MS antigen. This technique seems to be useful for a rapid, simple and specific diagnosis of avian mycoplasmosis.     

Enzyme-linked immunosorbent assay to detect subgroup-specific antibodies to avian leukosis viruses

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies against avian leukosis viruses (ALV), using antigens extracted from Rous-associated virus-inoculated chicken embryo fibroblast (CEF) cells by Nonidet P-40 treatments. The antigens reacted strongly to sera of chickens immunized with antigenically homologous viruses, but weakly to those of chickens immunized with heterologous viruses. Antigens extracted from noninoculated CEF cells by the same procedures did not react to either of the immune sera. Normal control sera did not react to any of the antigens. Reactivities of immune sera were decreased markedly by the sera adsorbing with homologous Rous-associated virus-inoculated CEF cells, but not with heterologous CEF cells. The ELISA-specific optimal doses (the differences between the optimal doses with antigens from ALV-inoculated and noninoculated CEF cells) were correlated strongly with the virus-neutralization titers (r = 0.876, P less than 0.01). Examination of the antibody response from ALV-inoculated chickens revealed that ELISA detected antibodies at the same time or several weeks earlier than did the virus-neutralization test.     

Development and evaluation of a mixed long-chain lipopolysaccharide based ELISA for serological surveillance of infection with Actinobacillus pleuropneumoniae serotypes 2, 6 and 12 in pig herds

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of antibodies against Actinobacillus pleuropneumoniae (Ap) serotypes 2, 6 and 12. The assay was designated MIX-ELISA. Lipopolysaccharide (LPS) from Ap serotypes 2, 6 and 12 was purified using hot phenol-water extraction followed by fractionation by size-exclusion chromatography. A mixture of fractions containing molecules with molecular weight above 50 kDa from all three serotypes was used as antigen. The MIX-ELISA was evaluated with sera from pigs experimentally infected with the serotypes 1, 2, 5b, 6, 7, 8, 10 and 12 of Ap biotype 1. In addition to reaction with sera from pigs inoculated with Ap serotypes 2, 6 and 12, reaction was observed with sera from pigs inoculated with serotype 8. Furthermore, the sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6 or 12 and with sera from herds free of infection with any Ap serotype of biotype 1. The ELISA showed a high herd sensitivity (0.98; 95% confidence interval: 0.89-1.00) and specificity (0.95; 0.88-0.99). The high diagnostic sensitivity and specificity of the assay indicate that screening of herds for Ap infection can be performed using this ELISA. Efficient serological surveillance can be achieved by using such mixed antigen ELISAs coated with size-selected LPS-antigens from the most prevalent serotypes.