Comparisons of the serologic response of chickens to Pasteurella multocida and its lipopolysaccharide

Chickens were inoculated with serotype 3 Pasteurella multocida cells or purified lipopolysaccharide (LPS), and their serologic responses to LPS and heat-stable antigens of 16 serotypes were compared. Chickens inoculated with cells or LPS had antibodies against LPS as determined by indirect hemagglutination tests; titers were highest 2-4 weeks after the initial inoculation. Sera from chickens inoculated with cells reacted with unheated and heated cell antigen in a tube-agglutination test. Sera from chickens inoculated with LPS reacted only with heated cell antigen in the tube-agglutination test. Nonspecific reactions with heat-stable antigens of other serotypes occurred in the gel-diffusion-precipitin test with sera from chickens inoculated with cells but not with sera from chickens inoculated with LPS. Antisera prepared against LPS could be used for serotyping field isolates of P. multocida.     

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.     

Investigations on different Ornithobacterium rhinotracheale "ORT" isolates

The aim of the present investigation was to determine the antigenic relationship between different Ornithobacterium rhinotracheale (ORT) isolates and to serotype field isolates obtained from turkey and chickens. Different antigen extractions (heat-stable, proteinase K-stable [lipopolysaccharide], and sodium dodecyl sulfate [SDS] extractions) were prepared from each serotype (A, B, C, D, E, and G) as well as from 21 ORT field isolates and examined in agar gel precipitation (AGP) and enzyme-linked immunosorbent assay (ELISA) tests. The field isolates were cultured from turkey (16 isolates) and chicken (5 isolates) flocks showing respiratory manifestations. Monospecific reactions were obtained with heat-stable as well as proteinase K-stable antigens prepared from serotypes A, C, D, E, and G in AGP tests. On the other hand, with the same antigen preparations from a strain of serotype B in AGP tests, cross-reactions with antisera prepared against serotypes A and E could be detected. The cross-reactions were observed mostly between 48 and 72 hr. In applications of SDS-antigen preparations in AGP tests, cross-reactions between all serotypes except serotype C were detected between 24 and 72 hr. Testing all antigen preparation in ELISA, different cross-reactions were observed and the evaluation of the results is very difficult. Serotyping of the field isolates in AGP tests by using heat-extracted antigens showed after 24 hr that 10 out of 16 isolates from turkey belonged to serotype B, five to serotype A, and one to serotype E. Results obtained after 48-72 hr revealed cross-reactions between serotype B and E in 11 cases and between A and B in two cases. All five isolates obtained from chicken reacted after 24 hr only with serum against serotype A. After 48-72 hr, two isolates showed cross-reaction with antiserum against serotype B. Similar results were obtained with proteinase K-stable antigen.     

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.     

Antigenic structure and relationship between serotypes 1 and 2 of Haemophilus paragallinarum.

Antigenic structure and relationship between serotypes 1 and 2 of Haemophilus paragallinarum were analyzed by the rapid plate agglutination and cross-absorption tests. Encapsulated strain forming iridescent colony type of both serotypes 1 and 2 had at least three antigens: heat-labile and trypsin-sensitive (L), heat-labile and trypsin-resistant (HL), and heat-stable and trypsin-resistant (HS). The L was a major antigen located in a surface and divided serologically into three parts: L1, L2, and L3. The L1 was specifici to serotype 1, the L2 was specific to serotype 2, and the L3 was a common antigen shared by serotypes 1 and 2. The HL and HS were common antigens between serotypes. By trypsinization or heating at 121 C, L antigen lost its agglutinability and agglutinin-producing ability. Nonencapsulated organisms forming noniridescent colony type lacked the L antigen. These results suggested that antigenic structure of H paragallinarum serotypes 1 was L1, L3, HL and HS, while serotype 2 was L2, L3, HL, and HS.     

Serologic studies of Actinobacillus (Haemophilus) pleuropneumoniae strains of serotype-3 and their antigenic relationships with other A pleuropneumoniae serotypes in swine

Serotype-3 strains of Actinobacillus (Haemophilus) pleuropneumoniae possess epitopes shared with almost all other Actinobacillus serotypes. Common epitopes detected in particulate antigens were heat-labile and heat-stable and were of minor nature. Additional cross-reactive epitopes were detected in soluble and particulate antigens prepared from strains of serotypes 3, 6, and 8. Cross-reactions occurring between serotype-3 antigens and those of other serotypes were of 2 types: one associated mainly with 2-mercaptoethanol (2-ME)-sensitive IgM antibodies and the other associated mainly with 2-ME-resistant IgG antibodies. The cross-reactivity between serotypes 3, 6, and 8 was associated mainly with IgG antibodies, as shown by the results of 2-ME tube agglutination, 2-ME-indirect hemagglutination, and coagglutination tests. None of the tests was entirely satisfactory for serotyping serotype-3 isolates, mainly as a result of overlapping of type-specific antigenic determinants of serotypes 3, 6, and 8 in different combinations in the same strain. A combination of tests, using particulate and soluble antigens, may be necessary for typing of serotype-3 isolates.     

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.     

Comparison of Pasteurella multocida serotyping systems.

Thirty-two type specific cultures used in four typing systems for serologically classifying Pasteurella multocida were compared as originally described for: (1) Little and Lyon's plate agglutination test; (2) Carter's indirect hemagglutination test, hyaluronidase decapsulation test, and acriflavine reaction; (3) Namioka's plate and tube agglutination tests; and (4) Heddleston's gel diffusion precipitin test. In addition, seven cultures from Robert's five passive protection groups were included. When reference cultures were examined by the typing system from which they were described, the results generally correlated with those results published. However, serotypes determined by one typing system generally did not correlate with serotypes determined by another system. Cultures of a single serotype in one system often represented more than one serotype in another system. Results indicated that cultures with one or two serotyping antigens in common may differ in other antigens. Because of the antigenic complexity of P multocida and the nature of the antigens involved in each test, a reliable correlation or equality between serotypes determined by different typing systems could not be made.     

Characterisation and biological activity of monoclonal antibodies specific for Pasteurella haemolytica A1 capsule and lipopolysaccharide.

Monoclonal antibodies (mAb) against both Pasteurella haemolytica A1 capsule and lipopolysaccharide (LPS) were produced. Anti-capsule mAb reacted with the homologous A1 serotype only, whereas mAb against LPS reacted with P. haemolytica serotypes A2, A5, A8, A12, A14 and A16 but not with 33 bacterial species or rough LPS mutant strains tested. Both capsule and LPS antigens were visualised on the surface of bacteria by immunogold electron microscopy. Neither of the mAbs demonstrated antibody-dependent complement-mediated killing in vitro but both facilitated phagocytosis in vitro.     

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.     

Antigenic properties of four serotypes of Pasteurella multocida determined by enzyme-linked immunosorbent assay

Broiler minibreeder hens were used to produce monovalent antisera to bacterins prepared from serotypes 1, 3, 4, and 3 X 4 cross (CU strain) of P. multocida and to a polyvalent fowl cholera bacterin containing serotypes 1, 3, and 4. Antiserum to the CU strain (live vaccine) was also produced. Monovalent enzyme-linked immunosorbent assay (ELISA) plate antigens were prepared by separately sonicating each of the strains. Polyvalent plate antigen (Poly 3) was prepared by combining, in equal amounts after sonication, antigens from serotypes 1, 3, and 4. Each antiserum was assayed against its homologous ELISA plate antigen and against all other heterologous plate antigens, including Poly 3. The strongest reactions, as indicated by the highest absorbance values, were observed in homologous ELISAs. The CU strain may be the best monovalent ELISA plate antigen for detecting antibodies formed in response to a commercial polyvalent bacterin and to vaccinations with the live CU strain. Overall, monovalent serotype 1 (strain X-73) antiserum did not react well with any other heterologous ELISA plate antigen, whereas monovalent antisera of serotypes 4 (strain P-1662) and 3 X 4 (CU strain) reacted equally strongly with monovalent serotype 4 ELISA plate antigen. Background binding of negative serum was significantly lower (P less than 0.05) when using CU plate antigen than when using any of the other plate antigens.     

Production and characterization of monoclonal antibodies to serotype specific antigens of Haemophilus pleuropneumoniae serotype 2

Mouse monoclonal antibodies were produced to Haemophilus pleuropneumoniae bacteria of the most common serotype 2. 11 hybridoma cultures were recovered that produced antibodies with moderate to strong reactivity to the antigen. 10 of these antibodies were specific to isolated capsular antigens from H. pleuropneumoniae of serotype 2, while one antibody reacted with capsular antigens from bacteria of all 8 serotypes. One hybridoma producing antibodies with a titre of 1:1000 was cloned and the antibody specificity studied further. The binding of this antibody (1F3) to whole bacteria, and capsular extracts isolated at different temperatures indicate that the antibody is specific for a thermostable polysaccharide antigen present in the cellular capsule of H. pleuropneumoniae of serotype 2.     

Intestinal carriage of Campylobacter jejuni and Salmonella by chicken flocks at slaughter.

Campylobacter jejuni were isolated in large numbers from the majority of birds sampled in colonic swabs from 28 of 60 flocks at slaughter. By contrast only small numbers of birds from 11 of the same 60 flocks yielded Salmonella enteritidis serotypes. Three C. jejuni isolates from each flock were serotyped on the basis of their heat-stable antigens, using antisera prepared against 16 serotypes common in Campylobacter diarrhea in man. The majority (72 of 83) of the chicken isolates could be serotyped.     

Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene and its relationship with fimbrial and enterotoxin markers in E. coli isolates from pigs with diarrhoea

The presence of the astA gene responsible for production of enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) was examined in E. coli strains isolated from pigs with postweaning diarrhoea. Two hundred and seven isolates were tested using PCR for the astA marker and for heat-labile I (LTI), heat-stable I (STI), and heat-stable II (STII) enterotoxin genes. Moreover, the isolates were also analysed for their serotypes (O and K antigens) as well as for fimbrial adhesins using agglutination methods. It was shown that 96 (46.4%) of the isolates possessed the astA genetic determinant. The most common EAST1-positive E. coli serotype was O149:K91 and these strains were mostly LTI/STII-positive. A close correlation between the presence of F4 fimbriae and the EAST1 gene was also observed: 88 of 96 (91.7%) astA(+) isolates tested possessed the F4 antigen. Thus, EAST1 enterotoxin may represent an additional virulence determinant playing a role in the pathogenesis of porcine colibacillosis.     

Capsular polysaccharide antigens for detection of serotype-specific antibodies to Actinobacillus pleuropneumoniae.

Capsular polysaccharides (CPS) of serotypes 1, 2, 5 and 7 of Actinobacillus (Haemophilus) pleuropneumoniae were obtained from 18 h culture supernatants by precipitation with hexadecyltrimethyl-ammonium bromide (Cetavlon) followed by extraction with sodium chloride and reprecipitation in ethanol. These crude extracts, and portions purified further by phenol extraction to remove contaminating proteins, were evaluated as antigens for the detection of serotype-specific antibodies to A. pleuropneumoniae in sera from immunized rabbits and swine by an enzyme-linked immunosorbent assay. The crude extracts reacted strongly with homologous antisera, but except for serotype 1 showed considerable cross-reactivity with antisera to other serotypes. Phenol extraction greatly improved the serospecificity of the antigens from serotypes 1, 7 and, to a lesser extent, 5. The serotype 2 CPS antigen showed poor reactivity following phenol extraction, and did not appear as useful for detection of serotype-specific antibodies.     

A characterization of monoclonal antibodies prepared against Pasteurella haemolytica serotype 1 surface antigens.

The production and characterization of monoclonal antibodies against Pasteurella haemolytica serotype 1 is described. Ten monoclonal antibodies were produced and divided, on the basis of their properties, into six different groups. One produced bacteria agglutination only of P. haemolytica serotype 1. Three antibodies bound with P. haemolytica serotypes 1, 5-8 and 12 and the antigen was identified in immunoblots as lipopolysaccharide. Two antibodies bound P. haemolytica serotypes 1, 2, 5-8 and 12 and P. multocida serotypes 1-7, 9, 12, 15 and 16, recognizing an epitope present on a 29 kDa outer membrane protein. One antibody bound all P. haemolytica and P. multocida serotypes. The antigen was a hexosamine less than 30 kDa which contained a formalin sensitive epitope. One antibody bound only to P. haemolytica serotype 1 and the antigen was identified as a 66 kDa outer membrane protein. Two antibodies bound P. haemolytica serotypes 1, 2, 5-9 and 12 and the antigen, while not identified, was localized on the outer membrane. This study identified antigens which contribute to the cross-reactions among P. haemolytica and P. multocida serotypes and the antibodies may be useful in investigating the pathogenesis of pneumonic pasteurellosis.     

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.     

Broad antigenicity exhibited by some isolates of avian adenovirus.

Ten of 16 isolates of avian adenovirus could be readily classified as members of serotypes 2, 5, or 7. However, 6 exhibited broad neutralization reactions which created typing problems. Detailed studies of these interactions revealed that our prototype for serotype 5 (i.e., T-8; subsequently found to be an "intermediate" strain) contributed to this broad cross-reactivity because it shared antigens with members of serotypes 5 and 7. As a result of this study, TR-59 or 58-1 is recommended as the suitable prototype for the serotype 5 viruses. The 6 broadly reactive isolates were classified as serologic "variants" of serotypes 5 and 7.     

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.     

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.