Homologous Serotype Research Articles

Cross-Protection Factor(s) of Pasteurella multocida: Passive Immunization of Turkeys against Fowl Cholera Caused by Different Serotypes

An antiserum cross-protective against different serotypes of Pasteurella multocida was made in turkeys by inoculating them with killed serotype 3 organisms grown in vivo and then exposing them to live serotype 3 organisms. In passive-immunization studies, the antiserum protected young turkeys against the homologous and heterologous serotypes 1, 4, 5, 9, and 12. In addition, the antiserum protected against P. multocida of a heterologous capsule serogroup, serogroup F. A globulin and two IgG fractions purified from the antiserum protected against heterologous challenge with serotype 1. Turkey-grown P. multocida were chemically lysed and separated into soluble and insoluble components to make immunoadsorbents. Antibodies from the cross-protective antiserum isolated by the immunoadsorbents passively protected young turkeys against heterologous serotype I challenge.

Immunization of salmonids against Yersinia ruckeri: significance of humoral immunity and cross protection between serotypes.

Brook trout (Salvelinus fontinalis) were immunized with bacterins containing either Serotype 1 or 2 isolates of Yersinia ruckeri to determine the relative degree of cross-protection afforded when the fish were challenged with the homologous or heterologous serotype. While fish immunized with pH-lysed bacterins produced highly specific agglutinins that did not cross-react with antigens derived from a heterologous serotype of Y. ruckeri all fish were protected against experimental challenge, regardless of which serotype was used for bacterin production and experimental challenge. Other experiments indicated that brook trout injected intraperitoneally with highly specific antibodies could not be passively immunized against experimental challenge.

Antibodies against the two serotypes of vesicular stomatitis virus measured by enzyme-linked immunosorbent assay: immunodominance of serotype-specific determinants and induction of asymmetrically cross-reactive antibodies

The serological relationship between the two vesicular stomatitis virus (VSV) strains Indiana (VSV-Ind) and New Jersey (VSV-NJ) were analyzed by using an enzyme-linked immunosorbent assay (ELISA). Immunoglobulin G responses, defined by their resistance to treatment with 2-mercaptoethanol, were assessed by ELISA by using sucrose gradient-purified VSV or purified VSV glycoproteins (G) as antigens. When low doses (10(6) PFU) of live VSV or 10(8) PFU of UV-inactivated virus were given intraperitoneally (i.p.), only non-cross-reactive antibody responses were observed in a primary immune response. However, when 10(6) PFU of live VSV were injected intravenously (i.v.), cross-reactive antibodies were generated; anti-VSV-NJ antibodies cross-reacted more against VSV-Ind than did anti-VSV-Ind antibodies against VSV-NJ. When 10(8) PFU of live VSV or UV-inactivated VSV mixed with complete Freund adjuvant was given i.p., high levels of cross-reactive antibodies detectable by ELISA were induced in primary and secondary responses. When purified G protein was used instead of purified whole virus in the ELISA, the cross-reactivity was found to be asymmetrical after immunization with live VSV given i.v. but not after i.p. inoculation; anti-VSV-NJ sera bound almost equally well to VSV-Ind G protein, whereas anti-VSV-Ind sera bound virtually exclusively to the G protein of the homologous serotype. The data suggest that immunization with VSV given i.p. results in a more specific, i.e., less cross-reactive, response than that either after i.v. infection or after the virus antigen is made available in great amounts or if it persists for prolonged periods when given i.p. together with complete Freund adjuvant. The unique determinants were immunodominant because they induced antibodies preferentially, whereas partially shared determinants induced antibody responses asymmetrically, more slowly, and with lower titers. Interestingly, the asymmetric cross-reactivity of anti-VSV antibodies, as measured by ELISA, against purified VSV G was opposite that observed for cytotoxic T cells.

Efficacy of β-lactamase-resistant penicillin and influence of penicillin tolerance in eradicating streptococci from the pharynx after failure of penicillin therapy for group A streptococcal pharyngitis

We studied the ability of dicloxacillin, a beta-lactamase-resistant penicillin, to eradicate persistent group A streptococci from the upper respiratory tract of children previously given penicillin V, and concomitantly assessed the role of antibiotic (penicillin and dicloxacillin) tolerance as a contributing factor. During a group A streptococcal pharyngitis outbreak, 66% of 324 children were found to be culture positive for group A streptococci. Ninety percent of these isolates were serotype M-1, T-agglutination pattern T-1. The treatment failure rate after initial orally administered penicillin treatment was 21% (42 of 204). These 42 children then received either a second course of penicillin V orally or a course of dicloxacillin. The resulting rates of failure to eradicate the homologous streptococcal serotype were 83% (20 of 24) and 50% (9 of 18), respectively (P less than 0.02). Of 189 streptococcal isolates tested by the gradient replicate plate method for penicillin tolerance, 18 (10%) were tolerant. In this study, penicillin-tolerant strains of group A streptococci were no more frequently isolated from children in whom initial penicillin treatment failed than from those who were successfully treated. Dicloxacillin tolerance was not a factor in failure of dicloxacillin therapy. These data, when evaluated with data from previous studies, suggest that reasons for failure to eradicate group A streptococci from the upper respiratory tract are complex, but dicloxacillin may be beneficial in some patients who fail to respond to orally administered penicillin therapy.

Cell-associated hemagglutinin-deficient mutant of Vibrio cholerae.

Cell-associated hemagglutinin-negative mutants were derived from cholera enterotoxin-negative Vibrio cholerae JBK70 by Tn5 mutagenesis. One of the mutants identified, SB001, was characterized in greater detail. Its ability to colonize ilea of adult rabbits was determined by feeding approximately 10(8) V. cholerae to each animal. At 17 h after feeding, the numbers of viable vibrios in the ilea were determined. There was a significant, 4 log, decrease in the ability of the hemagglutinin-negative mutant to colonize ileal tissue compared with the parent strain JBK70. In addition, the higher levels of colonization attained by JBK70 and the wild-type parent of JBK70, N16961, were associated with intestinal fluid accumulation and death. Rabbits immunized orally with approximately 10(8) SB001, when challenged 3 weeks later with either homologous biotype and serotype El Tor Inaba N16961 or heterologous Classical Ogawa 395, were protected to the same extent as those animals immunized with either the challenge strain or JBK70. This was evidenced by decreases in both the number of animals showing detectable colonization and the level of colonization achieved. A hemagglutinin-negative mutant of V. cholerae may therefore be of potential use as a live oral vaccine against cholera.

Antibody response of swine to outer membrane components of Haemophilus pleuropneumoniae during infection.

Sera from pigs infected with Haemophilus (Actinobacillus) pleuropneumoniae were tested for antibodies to outer membrane proteins (OMPs) of the organism by immunoblotting. Convalescent sera were produced in naturally born, colostrum-fed pigs and in cesarean-derived, colostrum-deprived pigs given H. pleuropneumoniae serotype 5 intranasally twice at 5-week intervals. Sera, collected at weekly intervals, were reacted with Sarkosyl-insoluble, OMP-enriched preparations of H. pleuropneumoniae which had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. Antibodies were detected to OMPs with an apparent molecular weight of 16,500 (16.5K OMP); to 29K, 38.5K, 43.5K, 45K, 49.5K, and 66.5K OMPs; and to several high-molecular-weight (greater than or equal to 94,000) OMPs, but not to the major 42K OMP. Antibodies to the heat-modifiable OMP (29K/43.5K) and the 38.5K OMP were detected in sera from noninfected pigs. Antibodies were also detected to two broad 54,000- and 95,000-molecular-weight bands which did not stain with Coomassie blue, stained with silver nitrate, resisted proteinase K digestion, and were eliminated by oxidation with sodium metaperiodate. This indicates that the 54,000- and 95,000-molecular-weight bands represent polysaccharide, possibly capsular or lipopolysaccharide immunogens. Adsorption of sera with cells from the homologous serotype 5 strain removed antibodies to the 45K, 49.5K, 66.5K, and greater than or equal to 94K OMPs and to the two polysaccharide bands, indicating that these antibodies were directed primarily to surface-exposed epitopes. When tested with OMP preparations from other serotype 5 strains, heterogeneity was apparent, both in the reactions with OMPs and with the polysaccharide bands. Silver staining of proteinase K-treated, whole-cell lysates from serotype 5 strains also indicated variable expression of the polysaccharide bands. Sera also reacted with OMPs from H. pleuropneumoniae serotypes 1 and 7; however, several OMPs and the lipopolysaccharide or polysaccharide determinants of these serotypes appeared to be type specific.

Can group- and serovar-specific proteins be detected in Ureaplasma urealyticum?

Ureaplasma urealyticum has been subspeciated by a number of serologic methods. Eight serotypes have been identified by modified metabolic inhibition, growth inhibition and indirect hemagglutination. Fourteen serovars have been identified by immunofluorescence and 16 by the mycoplasmacidal assay. The present studies were performed to determine if group-specific antigens could be detected by immunofluorescence and if group- or serovar-specific antigens could be detected by enzyme-linked immunosorbent assay and immunoblotting. Reaction of rabbit antisera to U. urealyticum with homologous and heterologous serotypes based upon end point immunofluorescent titration did not differentiate the serovars into the two biotypes. In attempts to quantitate the number of a given serovar present in clinical specimens, the total number of colonies that were stained often exceeded 100%, suggesting either that the serovars represented in stock cultures are not truly representative of those present in humans or that certain strains express multiple serovar specificities. End point titration in an enzyme-linked immunoadsorbent assay using either whole cells or cell lysates and rabbit antisera failed to distinguish between group and serovars (i.e. in many cases end point titers were only 2-fold lower with heterologous antigens). Furthermore convalescent sera from patients infected with a single serovar failed to induce a serovar-specific response detectable in an enzyme-linked immunosorbent assay. Likewise immunoblotting of one dimensional electrophoretograms with sera from humans known to be infected with a single serovar showed that antibodies against that serovar recognized peptides present in all serovars and reacted with those bands with various degrees of intensity.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphoprotein NS of vesicular stomatitis virus: Phosphorylated states and transcriptional activities of intracellular and virion forms

The phosphorylation and transcriptional competence of the free cytoplasmic form and the virion form of NS protein of vesicular stomatitis virus (VSV-Indiana/Mudd-Summers) were compared. NS protein is known to exist in two distinct phosphoylated states. NS1 and NS2, that are resolvable by gel electrophoresis. In vitro phosphorylation of virion NS protein by the viral L protein-associated protein kinase resulted in the phosphorylation of both NS1 and NS2. However, in the presence of the N-RNA complex, the NS2 form was preferentially phosphorylated. A cellular protein kinase activity, found in cytoplasmic extracts from VSV-infected or uninfected cells, preferentially phosphorylated NS1, which did not undergo dephosphorylation by cellular phosphotase and also did not convert to NS2. In contrast, the virion or cellular NS2 which had been phosphorylated in vivo or in vitro could be rapidly dephosphorylated by a cellular phosphatase. Cytoplasmic NS protein was found to be fully capable of binding to the virion N-RNA template, and in conjunction with L protein, it participated in synthesis of the leader RNA and five mRNA species of VSV. Moreover, under these conditions, neither cellular phosphate nor cellular ribonuclease was able to bind to reconstituted nucleocapsids. Binding of cytoplasmic NS to the virion N-RNA template in the presence of L protein resulted in a large and preferential enhancement of NS2 phosphorylation. A protein kinase activity, which phosphorylated NS protein in vitro, was found to be associated with the N-RNA template. This activity appeared to by very tightly bound to N-RNA and exhibited absolute specificity for NS protein of the homologous serotype.

Antibody mediated suppression of secondary IgM response in nude mice against vesicular stomatitis virus.

Nude mice immunized with either of the two serotypes of vesicular stomatitis virus (VSV), VSV Indiana (VSV-Ind) or VSV-New Jersey (VSV-NJ), showed an early T cell independent immunoglobulin (Ig) M antibody response comparable with normal euthymic mice. Unlike euthymic mice, however, nude mice reinjected with the homologous serotype were unable to mount a second measurable serum neutralizing (SN) antibody response; a second injection with the heterologous serotype induced a normal primary type of SN antibody response. The serotype-specific refractoriness to a second challenge recovered at about 10 wk after primary infection. When antibody responses were assayed by enzyme-linked immunoabsorbent assay (ELISA), suppressive effects by previous immunization could be observed even after challenge with the heterologous serotypes; this finding probably reflects the known existence of common nonneutralizing determinants shared between the two serotypes. A weak 2-mercaptoethanol (2-ME)-resistant anti-VSV IgG SN antibody response was noticed during the primary response in nude mice and was also found in ELISA; after second infections, this 2-ME-resistant response did not develop. Serum transfer studies in nude and +/+ mice confirmed that the serotype-specific transitory refractoriness of a second response in nude mice was mediated through the anti-VSV-specific IgM antibodies.

Multisite Monoclonal Immunoassay for Dengue Viruses: Detection of Viraemic Human Sera and Interference by Heterologous Antibody

A monoclonal radioimmunoassay (RIA) was developed for detection of dengue virus in infected cell culture fluids and blood samples from dengue patients. Antibodies used to construct the RIA were selected on the basis of high binding avidity, the demonstration of synergism in competitive binding assays and empirical trials with different antibody combinations. Optimal binding of all four dengue virus serotypes was achieved by use of a flavivirus group-reactive and a dengue virus complex-reactive antibody as radiolabelled probe. A 'simultaneous sandwich' format and prolonged (18 h) incubation at 37 degrees C yielded optimal results. The limit of sensitivity of the RIA for detection of dengue type 2 virus was 2.7 log10 mosquito 50% infectious doses (MID50). The assay was tenfold more sensitive for dengue type 2 than for dengue types 1 and 3 viruses and 100-fold more sensitive than for dengue type 4 virus. Specificity, assessed using over 500 disease control human sera, was increased by addition of monoclonal anti-tetanus blocking antibodies, resulting in a false positive rate of only 0.2%. Heterologous dengue virus antibodies were shown to inhibit the RIA in assays performed with artificial immune complexes. Acute phase human sera containing 10(4.2) to 10(7.6) MID50 but no detectable antigen by RIA, were also shown to inhibit binding of the homologous dengue virus serotype; this effect was attributed to heterologous antibody from a prior infection. Among 116 viraemic sera from dengue patients, the RIA was positive in 43 to 47% of patients with dengue type 1, 2 or 3 infections but in only 10% of the dengue type 4 cases. Virus was more frequently detected in cases of primary infection (54%) than in cases of superinfection (16%). Despite the limitations imposed by immunological interference, the antigen capture RIA appears useful as a rapid diagnostic technique for dengue surveillance.

Rapid detection ofVibrio cholerae O∶1 by motility inhibition and immunofluorescence with monoclonal antibodies

Monoclonal antibodies against the group and type specific antigens of Vibrio cholerae O:1 lipopolysaccharide were used for the rapid detection of Vibrio cholerae strains by motility inhibition and immunofluorescence. Motility inhibition of live Vibrio cholerae O:1 was obtained with group specific monoclonal antibodies. Monoclonal antibodies against the type specific antigens B (Ogawa) and C (Inaba) inhibited motility of strains of homologous serotypes only. Indirect immunofluorescence of heatfixed bacteria with monoclonal antibodies and fluorescein-isothiocyanate conjugated rabbit anti-mouse immunoglobulin was also shown to be suitable for the rapid detection of Vibrio cholerae O:1. Both tests were highly specific and no cross-reactions were observed with strains of non-O:1 vibrios, Escherichia coli or Salmonella spp. tested. However, a weak fluorescence of some Ogawa strains was observed when high concentrations of Inaba specific monoclonal antibodies were used.

Antigenic variation is associated with DNA rearrangements in a relapsing fever borrelia

Borrelia hermsii, an agent of relapsing fever, undergoes antigenic variation in its host. Surface-exposed proteins with differing primary structures determine the serotype of each organism. Using amino acid sequence data from two of these variable proteins, we synthesized two mixed-sequence oligonucleotides and then used the oligonucleotides to probe mRNA and DNA of three isogenic serotypes of B. hermsii. In Northern blots the probes were specific for the mRNA of the homologous serotype. Southern blots revealed two classes of hybridizing fragments: those common to the three serotypes and those specific for a particular serotype. A serotype-specific DNA fragment, which had hybridized to both oligonucleotide probes, was cloned. Subsequent use of the cloned fragment as a probe provided further evidence that antigenic variation in B. hermsii is associated with DNA rearrangements and with occurrence of expression-linked copies of all, or part, of an antigen-specifying gene.

Enzyme-linked immunosorbent assay for immunoglobulin G antibody to Pasteurella multocida in rabbits.

Three antigen preparations of Pasteurella multocida, lipopolysaccharide antigen, boiled-cell extract antigen, and boiled whole-bacterium antigen, were used in an enzyme-linked immunosorbent assay (ELISA) to detect rabbit immunoglobulin G antibody to P. multocida. The sensitivity of each antigen preparation was compared by using sera from P. multocida-infected and uninfected rabbits and sera from two rabbits immunized with different serotypes of P. multocida. In the ELISA, all three antigen preparations detected high titers of antibodies in infected rabbits and markedly lower levels in uninfected rabbits. When whole-bacterium or boiled-cell extract antigens were used, the ELISA detected antibodies in sera from both immunized rabbits, but with lipopolysaccharide antigen, only antibody to the homologous serotype was detected. Sera absorbed with P. multocida and Bordetella bronchiseptica, another respiratory pathogen of rabbits, revealed that antibodies detected in the ELISA did not cross-react. Since the lipopolysaccharide antigen was more difficult to prepare and may be type specific, and since the whole-bacterium antigen was the least sensitive, the boiled-cell extract was chosen as the best antigen preparation to use in the ELISA.

Use of a purified outer membrane protein F (porin) preparation of Pseudomonas aeruginosa as a protective vaccine in mice.

The outer membrane protein F (porin) from the PAO1 strain of Pseudomonas aeruginosa was purified by two different methods. One procedure involved separation by column chromatography of proteins extracted from isolated outer membranes, whereas the other involved extraction from gels after slab polyacrylamide gel electrophoresis of proteins extracted from cell envelopes. Both procedures yielded protein F preparations which successfully immunized mice from subsequent challenge with the PAO1 strain. The protein F preparations contained small quantities of lipopolysaccharide (LPS). This level of LPS contamination protected immunized mice from challenge with the homologous LPS serotype strain. However, immunization of mice with protein F preparations from the PAO1 strain also afforded protection against challenge with two different LPS serotype strains. This protective ability was lost when the protein F preparation was treated with papain before use as a vaccine. These observations support the conclusion that protein F has protective ability, which is not due to LPS contamination, when given as a vaccine. After immunization with the protein F preparation, mice showed an increase in antibody titer to the purified protein F preparation by enzyme-linked immunosorbent assay. Mice were protected passively by administration of rabbit antisera raised to the protein F preparation. These results indicate that the protein F preparation elicits a specific humoral antibody response in immunized animals. Our results suggest that purified protein F has potential as an effective vaccine for P. aeruginosa.

Effects of Subtype Variations in the Holland Strain of Infectious Bronchitis Virus When Applied as a Vaccine

Massachusetts-type infectious bronchitis viruses (IBV) designated H120, from three sources, have been evaluated as vaccines. Significant subtype variations were observed with the different vaccines based upon challenge virus infection and typical IB lesions induced where homologous and heterologous serotypes were used. In numerous cases, there was little agreement between challenge virus recovery from the respiratory tract and the presence or absence and severity of IB lesions. The study indicates that histopathological observations provide a sensitive, reliable indicator of the immune response to IB vaccination and augment the use of challenge virus isolation attempts. The data suggest that the use of the latter criterion alone was insufficient in evaluating IB immunity.The study also demonstrated that IB vaccine strains, although labeled similarly, varied in their immunogenic properties. Manipulation of infectious bronchitis virus (IBV) and other factors should be considered when obtaining and employing vaccine strains from different sources. Complete characterization of IBV vaccine strains used commercially should be stressed together with an appropriate labeling of each.

Assay of type-specific M antigens on whole group A streptococci.

A novel radioimmunoassay of type-specific M antigens on whole group A streptococcal cells is described. Absorbed rabbit anti-M antisera directed against M types 12 and 49 were used for determining M antigens on intact bacterial organisms. Staphylococcal protein A labelled with 125I was used as an anti-antibody reagent. The absorbed antisera were tested against ten homologous and 48 heterologous serotypes. All homologous serotypes gave an unequivocal reaction distinct from the weaker reaction with the heterologous serotypes. The type-specificity of the reaction was confirmed by the removal of type-specific antibodies after absorption to purified M protein coupled to Sepharose 4B. The results indicate that the described method is a simple and reliable technique for the recognition of M types of group A streptococci and offers a valuable tool for studies of M antigen in situ.

Conjugation of meningococcal lipopolysaccharide R-type oligosaccharides to tetanus toxoid as route to a potential vaccine against group B Neisseria meningitidis.

Oligosaccharides were obtained by the mild acid hydrolysis of the lipopolysaccharides from a number of different strains of Neisseria meningitidis, serotypes L2, L3, L4, L5, and L10. The dephosphorylated oligosaccharides were conjugated to tetanus toxoid as their 2-(4-isothiocyanatophenyl)-ethylamine derivatives, which resulted in the incorporation of from 18 to 38 oligosaccharides per molecule of tetanus toxoid. When injected in rabbits, the conjugates produced oligosaccharide-specific antibodies which were predominantly serologically specific but which also exhibited some cross-reactivity. These serological results can be attributed to regions of structural dissimilarity and similarity within the oligosaccharides. The oligosaccharide-specific antibodies were also lipopolysaccharide serotype specific, thus indicating that the oligosaccharides are the determinants associated with this serotype specificity. Consistent with the serological results, the conjugate antisera were bactericidal for the homologous serotype meningococcal organisms and in some cases for heterologous serotype organisms.

Selective intrapartum chemoprophylaxis of neonatal group B streptococcal early-onset disease. II. Predictive value of prenatal cultures.

To determine the value of prenatal cultures in defining maternal colonization status at delivery, 5,586 pregnant women were screened at prenatal visits for vaginal and rectal carriage of group B streptococci (GBS). GBS were isolated from 1,272 (22.8%). At delivery, semiquantitative cultures were obtained from 393 prenatal carriers, of whom 264 (67.2%) retained carriage at delivery. Seventeen (8.5%) of 200 women with negative prenatal cultures acquired carriage. The predictive value of a positive prenatal culture was highest (72.5%) in women with prenatal vaginal and rectal colonization and lowest (59.7%) in women with only rectal colonization. The predictive value varied inversely with the interval between prenatal sampling and delivery. In mothers with prenatal carriage, density of colonization at parturition was not predicted by the sites of prenatal colonization. Density of colonization, however, strongly influenced rates of vertical transmission to neonates and rates of heavy infant colonization. Ten infants born to prenatally cultured mothers developed group B streptococcal early-onset disease; the mothers of eight (80%) of the 10 had prenatal colonization with the homologous GBS serotype.

Successful colonization and immunization of adult rabbits by oral inoculation with Vibrio cholerae O1.

Adult rabbits were inoculated orally (or duodenally) with virulent Vibrio cholerae O1. Jejunal colonization occurred only when hypoperistalsis was induced at the time of inoculation by tincture of opium given intraperitoneally (or by temporary ileal obstruction). For oral inoculation, prior neutralization of gastric acid was also required. Inoculation with 10(9) V. cholerae caused jejunal colonization for 1 to 2 days and ileal colonization for 5 to 6 days. The extent of small bowel colonization 18 h after oral inoculation was related to inoculum size but also reflected limited multiplication of small inoculum sizes and net death, clearance of large inoculum sizes, or both. Serious diarrhea occurred only in rabbits fed large inoculum sizes, i.e., 10(10) V. cholerae, and then rarely. Rabbits colonized once with 10(10) V. cholerae became highly resistant to recolonization with either the same or opposite serotype. After 18 weeks, these rabbits were still partially protected, whereas twice-colonized rabbits were highly protected. Protection against recolonization appeared to be due, at least partly, to interference with the adherence of V. cholerae to the bowel mucosa, thus allowing rapid removal of V. cholerae when peristalsis resumed. Prior colonization also protected against cholera-like diarrhea in rabbits challenged by the removable intestinal tie-adult rabbit diarrhea technique, the 50% effective dose for severe or lethal diarrhea being increased more than 100-fold, and probably more than 10,000-fold, for challenge with either the homologous or heterologous serotype of V. cholerae. The described rabbit model appears well suited for the study of immunity evoked by enteric colonization with V. cholerae O1.

Experimental immunisation of lambs against pneumonic pasteurellosis

Methods of immunising lambs against pneumonic pasteurellosis, caused by several serotypes of Pasteurella haemolytica, were assessed in specific pathogen free lambs. Lambs were vaccinated intramuscularly with sodium salicylate extract (SSE) of P haemolytica, either alone or in combination with heat-killed organisms (HKO). SSE of P haemolytica type A1 protected vaccinated lambs against pneumonia resulting from challenge with the homologous serotype. SSE of type A2 also provided some protection but this was improved by vaccination with a combination of SSE and HKO.