Antiserum against culture filtrate is cross-protective for various serovars of Erysipelothrix rhusiopathiae

The protective effect of porcine antiserum prepared against culture filtrate (CF) of an attenuated strain of Erysipelothrix rhusiopathiae (serovar 2) in mice to challenge with 20 virulent strains of 18 serovars and one type N was investigated. Passively immunized mice survived after challenge with serovars 1a, 1b, 2, 5, 6, 8 (strain Goda), 11, 12, 15, 16, 21 or type N, but 10-30% mortality occurred in immunized mice challenged with each strain of serovars 4, 7, 8 (strain 911), 9, 18 or 19 and 70% mortality to serovar 10 (strain 2179). All immunized mice died after challenge with serovar 20 (strain 2553). Non-treated control mice died after challenge with all serovars and the type tested.     

Isolation and purification of a protective protein antigen of Erysipelothrix rhusiopathiae

The rapid growth and high survival rate of Erysipelothrix rhusiopathiae was determined using a culture of the bacterium in tryptic soy broth supplemented with 0.3% Tris-hydroxymethyl aminomethane and 0.1% Tween 80 (TT-TS broth). High concentrations of 64, 66 and 43 kDa proteins, which are associated with protection against E. rhusiopathiae infection in mice, were obtained by alkaline treatment of whole cells using 0.05-1 N NaOH. The supernatant of alkaline treated cells (alkaline extract; AE) was stable at alkaline or neutral pH. However, aggregates appeared at neutral pH in the absence of sodium dodecyl sulphate (SDS). A high yield of 64, 66 and 43 kDa proteins was obtained from strain Agata (serovar 5). The proteins were eluted from gel bands following SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the AE from strain Agata and designated P64 and P43. The amounts of P64 and P43 isolated were 0.7 and 0.3 mg/16 g of wet bacteria, respectively. In a mouse protection test, 50% protective doses (PD50) of P64 and P43 were 0.58 and 0.63 microgram, respectively. Upon Western blotting of the AE, both anti-P64 and anti-P43 antibodies reacted with the 64 and 43 kDa proteins. From these results, it is suggested that P64 is the most effective protective antigen and that P43 (43 kDa protein) is a degradation product of P64. Therefore, the 64 kDa structural proteins are associated with the induction of a protective activity against E. rhusiopathiae infection in mice.     

Protective activity of the purified protein antigen of Erysipelothrix rhusiopathiae in pigs

We purified the protein antigen (P64), which contains 66 and 64 kDa proteins, from the alkaline extract (AE) of whole cells of Erysipelothrix rhusiopathiae strain Agata (serovar 5) to determine the protective activity of the antigen against E. rhusiopathiae infection in pigs. The serum titre of antibody against P64 rapidly increased in pigs immunized with 500 and 100 micrograms of P64 and reached maximum values at 3 weeks after the first immunization (1 week after the second immunization). However, the serum antibody titres were not increased in pigs immunized with 20 micrograms of P64 and in nonimmunized pigs. In the pigs immunized with live cell vaccine (acriflavin-fast attenuated strain Koganei 65-0.15), the serum titres of antibody against P64 also increased at 1-2 weeks after immunization. In a pig challenge test performed on immunized and nonimmunized pigs, all nonimmunized pigs showed typical clinical signs of swine erysipelas (fever, erysipeloid, arthritis), while all pigs immunized with 500 and 100 micrograms of P64 and live cell vaccine showed no clinical signs of this disease. In Western blot analysis, sera from pigs immunized with P64 and live cell vaccine strongly reacted with the 64 kDa protein. In contrast, the serum from nonimmunized pigs did not react with any proteins. From these results, it was suggested that a specific antibody against the 64 kDa protein could be increased in pigs immunized with P64 or live cell vaccine and that this anti-P64 antibody has a strong protective effect against E. rhusiopathiae infection in pigs.     

A protective antigen for Turkeys purified from a type 1 strain of Pasteurella multocida

A protective antigen was purified from a saline extract of a Type 1 strain of Pasteurella multocida by chromatographic methods, and its chemical and immunological ccharacteristics were studied. Three protein peaks were obtained from crude extract by gel filtration with Sephadex G-200. A bacteria-specific antigen was detected only in the first peak fraction, which, after passing through an immunoadsorbent column to remove any components originating from the growth medium, was adsorbed onto DEAE-cellulose followed by elution with a gradient of NaCl. From the first peak fraction of the gel filtration, 4 protein peaks were obtained, the second and third peaks being the major ones. Carbohydrate/protein ratios of the peak fractions varied from 0.06 to 1.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 2 proteins of molecular weights 44 000 and 25 000 were present in all the fractions. The 4 DEAE-cellulose fractions (DP-1 to DP-4) contained a single antigenically identical material, and induced protective immunity in turkeys against challenge exposure. The second peak fraction from DEAE-cellulose (DP-2) protected turkeys when subcutaneously injected as 2 doses of 10 μg protein with a 14-day interval between doses. The DP-2 fraction induced antibodies in rabbits which formed a single precipitin line against the crude extract. The purified antigen (DP-2) from a Type 1 strain was antigenically distinct from a similar antigen purified from a Type 3 strain; there was no significant cross protection in turkeys between the 2 antigens. These results indicate that protective antigens purified from soluble extracts of a Type 1 or Type 3 strain possess similar physicochemical properties, but that they are immunologically distinct from each other.     

Quantitative diversity of 67 kda protective antigen among serovar 2 strains of Erysipelothrix rhusiopathiae and its implication in protective immune response

Mouse monoclonal antibodies (MAbs), raised against the NaOH-extracted antigen of Erysipelothrix rhusiopathiae strain Kyoto (serovar 2), recognized two different epitopes on a single protein of molecular weight 67 kDa. The MAbs were classified as protective or non-protective against strain Fujisawa (serovar 1). In immunoblotting analysis using the MAbs, fifteen wild strains were shown to contain different amounts of 67 kDa protective antigen. Each formalin-killed whole cell vaccine (bacterin) prepared from the fifteen wild strains conferred different levels of protection against strain Fujisawa in mice. Bacterins prepared from wild strains with larger amounts of 67 kDa protective antigen tended to give high levels of antigen-specific antibody and better protection to mice. These results indicate that the amount of 67 kDa protective antigen which influences the induction of protective immune responses may vary substantially among the strains of E. rhusiopathiae (serovar 2).     

Mechanism of protection induced in mice against Erysipelothrix rhusiopathiae infection by treatment with porcine antiserum to the culture filtrate of an attenuated strain

The mechanism of protection induced in mice against challenge with a virulent strain of Erysipelothrix rhusiopathiae by porcine antiserum to the culture filtrate (CF) of an attenuated strain was investigated. Death and bacterial growth in the spleens of mice challenged with the virulent strain were completely prevented by treatment with the antiserum. The protective effect of the serum was markedly decreased in mice in which polymorphonuclear leucocytes (PMN) were depleted by cyclophosphamide (CY) treatment but not in mice in which macrophages were blocked selectively by carrageenan (CG). The phagocytic rate of PMN and the number of bacteria ingested by PMN were significantly higher in mice treated with the antiserum than in mice treated with normal serum. These results indicate that anti-CF serum exerts its protective effect by opsonic activity and that opsonized E. rhusiopathiae are eliminated mainly by PMN.     

Cross protection of mice and swine inoculated with culture filtrate of attenuated Erysipelothrix rhusiopathiae and challenge exposed to strains of various serovars

Mice and swine inoculated subcutaneously with culture filtrate vaccine prepared from acriflavine-fast attenuated Erysipelothrix rhusiopathiae strain Koganei 65-0.15 (serovar 2), were challenge exposed to 20 pathogenic strains of E rhusiopathiae of 18 serovars and type N. Vaccinated mice survived after challenge exposure to serovars 1b, 2, 8 (strain Goda), and type N, but mortality occurred in vaccinated mice challenge exposed to other strains: 20% to 30% mortality in mice challenge exposed to serovars 1a, 11, 12, 15, 16, or 21; 40% to 50% mortality in mice challenge exposed to serovars 4, 5, 6, 7, or 8 (strain 911); and 60% to 80% mortality in mice challenge exposed to serovars 9, 10, 18, or 19. All vaccinated mice died after challenge exposure with strain 2553 (serovar 20). Non-vaccinated control mice died after challenge exposure to all strains. Of 2 vaccinated swine challenge exposed to strain 2553, 1 developed a local urticarial lesion at the site of intradermal exposure. Vaccinated swine challenge exposed to serovars 1a, 1b, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 18, 19, 21, or type N did not have clinical signs of acute erysipelas. Nonvaccinated control swine developed acute generalized erysipelas or localized urticarial lesions at the site of intradermal exposure.     

Cross protection of mice and swine given live-organism vaccine against challenge exposure with strains of Erysipelothrix rhusiopathiae representing ten serovars

Mice and swine vaccinated (subcutaneous inoculation) with live acriflavine-fast attenuated Erysipelothrix rhusiopathiae, strain Koganei 65-0.15 (serovar 2), were challenge exposed with 10 strains of E rhusiopathiae pathogenic for swine; the latter strains comprised serovars 9 and 10 and other previously undetermined. Vaccinated mice did not die after they were challenge exposed (subcutaneous inoculation) with serovars 4, 6, 7, 8, 9, 10, 15, 16, or N, but vaccinated mice challenge exposed with strain 2553 (serovar 20) had 30% mortality. Nonvaccinated control mice died after they were challenge exposed with all serovars tested. One of 2 vaccinated swine challenge exposed (intradermal inoculation) with each of strains 911 (serovar 8), 2179 (serovar 10), or 2553 developed localized urticarial lesion at the site of intradermal inoculation. Vaccinated swine challenge exposed with serovars 4, 6, 7, 9, 15, 16, or N did not have clinical signs of acute swine erysipelas. Nonvaccinated control swine developed localized lesions at the site of intradermal challenge inoculation.     

Vaccination of turkeys with cell-free culture filtrate of Pasteurella multocida: effects of ultrafiltration and endotoxin removal.

Cell-free culture filtrate (CCF) of Pasteurella multocida strain R44/6 (serotype 3/4/9/12) was fractionated by ultrafiltration into fractions of less than 10,000, greater than 10,000, greater than 30,000, and 10,000 to 30,000 molecular weight (MW). The less-than-10,000-MW fraction contained little endotoxin comparable to bacteriologic medium; the 10,000-to-30,000-MW fraction had a moderate amount of endotoxin, whereas the greater-than-10,000- and greater-than-30,000-MW fractions contained high levels of endotoxin. Following ultrafiltration, each fraction, except the less-than-10,000-MW fraction, was divided into two equal parts, and endotoxin was removed from one part. Turkeys were vaccinated with the various MW fractions of CCF, with and without endotoxin, via the air sacs at 6 and 9 weeks of age and compared with negative controls given bacteriologic medium and positive controls vaccinated with a commercial bacterin. Before oral challenge with strain P-1059 (serotype 3) at 12 weeks of age, antibody titers were detected only in positive control turkeys. Protection against challenge, as measured by post-challenge mortality and body-weight gain, was provided by the greater-than-10,000-, greater-than-30,000-, and 10,000-to-30,000-MW fractions containing endotoxin and the commercial bacterin. Turkeys that had been vaccinated with bacteriologic medium and the four different fractions without endotoxin were not protected. Results indicated that endotoxin in CCF of P. multocida is critical in protecting turkeys from pasteurellosis.     

Evaluation of relationship among three purified antigens from Pasteurella multocida strain P-1059 and of their protective capacities in turkeys

Three antigens were prepared from Pasteurella multocida strain P-1059, and their immunogenicity and antigenic relationships were investigated. The 3 antigens were a soluble antigen purified from a 2.5% NaCl extract (2.5S), a similar antigen purified from an extract in 0.3% formalin solution containing 0.85% NaCl (FS), and lipopolysaccharide (LPS). The antigens were treated with various chemicals and enzymes to study their antigenic and immunogenic determinants. Antigenic analyses with ELISA inhibition tests indicated that 2.5S and FS were similar LPS-protein complex antigens. The 2.5S and FS antigens induced protective immunity in turkeys with high antibody titers against LPS antigen. Although LPS was a component of 2.5S and FS, LPS itself was poorly immunogenic in turkeys. The antigenicity of protein compounds in 2.5S was deteriorated by protease treatment, which, however, did not significantly diminish the protective immunogenicity. Treatment of 2.5S with sodium periodate, altering its carbohydrate moieties, decreased its immunogenicity. The immunogenicity of 2.5S also was abolished by phenol-water treatment, owing to dissociation of the LPS-protein complex. These findings suggest that a certain form of LPS-protein complex is essential for the induction of immunity against the P multocida infection in turkeys.     

Protectivity of an immunoaffinity-purified 39 kDa capsular protein of avian Pasteurella multocida in mice

A 39 kDa protein of avian Pasteurella multocida strain P-1059 (serovar A:3) was purified from a crude capsular extract by immunoaffinity chromatography by using a ligand of purified mouse monoclonal antibody to the 39 kDa capsular protein of the strain. Protective activity of the purified 39 kDa protein antigen was determined by inoculation of ddY mice twice with 25 or 125 mug of the protein and challenge-exposure with 10 or 50 LD(50) of strains P-1059 or X-73 (serovar A:1). The results showed that the antigen gave high protection (60 to 100%). These results indicated that the 39 kDa protein of avian P. multocida is a cross-protective antigen over serovars A:1 and A:3.     

Binding patterns of five monoclonal antibodies to Actinobacillus (Haemophilus) pleuropneumoniae

Five monoclonal antibodies were obtained after immunising mice with superficial antigens of three strains representing serovars 1 to 3 of Actinobacillus (Haemophilus) pleuro-pneumoniae. When tested in ELISA against the standard strains representing serovars 1 to 10, the monoclonal antibody raised against the standard strain of serovar 1 reacted only with that strain. Of the three monoclonal antibodies raised against the standard strain of serovar 3, one reacted with serovars 3 and 8 only, another with serovar 7 only and the third with the strains representing serovars 7, 9 and 10. The monoclonal antibodies produced with the serovar 2 strain also reacted with a wide spectrum of strains, representing serovars 7 to 10.     

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.     

Characterization of a 39kDa capsular protein of avian Pasteurella multocida using monoclonal antibodies

The role of a 39kDa protein of avian Pasteurella multocida in pathogenesis of fowl cholera was investigated using monoclonal antibodies (Mabs). Mabs were prepared by immunization of BALB/c mice with a crude capsular extract (CCE) of P. multocida strain P-1059 (serovar A:3). Totally eight hybridomas producing Mab were obtained. Immunoblot analysis of the hybridomas revealed that all the Mabs recognized a 39kDa protein of CCE. Treatment of CCE antigen with proteinase K or periodic acid indicated that the epitope recognized was proteinaceous. The Mabs reacted with a major 39kDa protein of CCE from encapsulated strains but not with any protein of non-capsulated strains indicating that a direct correlation between encapsulation and the 39kDa protein. Immunoelectron microscopy on strain P-1059 and the non-capsulated derivative P-1059B (serovar -:3) reacting with the Mabs and gold-labeled anti-mouse IgG indicated that the protein is associated with the capsule. The Mabs significantly inhibited the adherence of encapsulated P. multocida strains to chicken embryo fibroblast cells, but only slightly that of non-capsulated strains. Mice passively immunized with the Mabs were protected from lethal challenge with virulent strains P-1059 and X-73 (serovar A:1). Thus the capsular 39kDa protein was determined to be an adherence factor and a cross-protective antigen of avian P. multocida type A strains.     

Extraction, separation, and partial characterization of Brucella abortus antigens

Brucella abortus isolates strain 19 (a vaccinal strain) and 458 (a virulent field isolate) were analyzed for antigenic differences. Whole cell preparations were extracted with detergents, salt solutions, and phenol-acetic acid-water (PAW). Antigens were separated by starch block electrophoresis and sucrose density gradient centrifugation, serotested, and chemically analyzed. Six distinct precipitin lines were observed for the PAW extract against hyperimmune bovine antisera. With sucrose density centrifugation or starch block electrophoresis, antigens were separated into (i) complement-fixing (CF) antigens and (ii) antigens that did not fix complement but formed precipitin bands. Reciprocal CF tests with purified CF antigen could not differentiate between the antigens from either isolate of Brucella.     

Purification and characterization of a 45-kDa concealed antigen from the midgut membranes of Ornithodoros erraticus that induces lethal anti-tick immune responses in pigs

Ornithodoros erraticus is an argasid tick that can transmit severe diseases such as human relapsing fever and African swine fever. In the search for a vaccine against this parasite, a crude extract of tick midgut membranes (GME) was obtained that in pigs and mice induced a protective response able to kill up to 80% of the nymphs in the first 72 h post-feeding and to reduce the fecundity of females by more than 50%. To identify the protective antigens, the GME was subjected to successive biochemical fractionations and the resulting simpler protein fractions were inoculated in pigs. A 45-kDa antigen, the so-called Oe45, was detected, purified and demonstrated to be responsible for the protection induced by the GME. Oe45 seems to be a membrane protein that is presumably expressed on the luminal membrane of midgut epithelial cells. Oe45 consists of at least two differently charged bands (cationic and neutral), which show antigenic cross-reactivity. The possibility that these bands might be different isoforms of the same protein is discussed. Although Oe45 is constitutively expressed at low levels throughout the trophogonic cycle, its expression is up-regulated by the ingestion of blood, as suggested by the higher levels observed between 6 and 72 h post-feeding.     

Partial purification of extracellular toxic material of fish Vibrio

Hemolytic activity of 3 pathogenic strains, of fish Vibrio commonly associated with vibriosis (V anguillarum NCMB6 and NCMB571 strains, and Vibrio sp N7802 strain) was examined, using chicken and mammalian erythrocytes. Vibrio strains NCMB6 and NCMB571 and their culture filtrates had hemolytic activity against 8 kinds of erythrocytes tested, whereas Vibrio strain N7802 produced only a little amount of hemolysin. Strain NCMB571 culture filtrate and its material partially purified by column chromatography were lethal in mice. From 2 peaks of protein, hemolysin was recovered from the 1st peak, which coincided with toxicity in mice. Heat-inactivation of culture filtrate indicated that hemolytic materials may be thermolabile proteins, but that toxic material may be comparatively thermostable.     

Evaluation of recombinant proteins of Haemophilus parasuis strain SH0165 as vaccine candidates in a mouse model

The recently completed genome sequence of Haemophilus parasuis strain SH0165 allowed us to screen putative OMPs for the development of recombinant vaccines. The objective of this study was to evaluate the immunogenicity and protective efficacy of three OMPs of H. parasuis. Three putative OMPs (SmpA, YgiW and FOG) were cloned, expressed and purified by Ni affinity chromatography using nitriloacetic acid resin. Mice were immunized either individually (individual protein, IP) or synergistically (synergistic protein, SP) with the recombinant proteins. A significant increase in IgG titer was detected in all protein-immunized mice. Isotyping studies revealed that the antibodies produced were predominantly IgG2a-type, indicating a predominant Th1 response. A significant increase was observed in IL-2, IL-4 and IFN-γ levels in the culture supernatants of splenocytes isolated from immunized mice. Furthermore, mice were challenged intraperitoneally with 6×10(9)CFU (5×LD(50)) of highly virulent homologous serovar 5 strain (SH0165) or 7.0×10(9) CFU (5×LD(50)) of the heterologous serovar 4 strain (MD0322) at fourteen days after the last immunization. All of the recombinant proteins enhanced survival and reduced histopathological lesions. Our results indicated that the three OMPs showed protection both individually and synergistically against infection with the highly virulent H. parasuis in mice.     

Inhibition of capsular protein synthesis of Pasteurella multocida strain P-1059

A mutant strain, PBA322, was constructed by electroporation of a phagemid containing the coding region of antisense RNA of the ompH gene, encoding 39 kDa capsular protein or OmpH, into the parental strain P-1059 (serovar A:3) of Pasteurella multocida, and the pathogenicity was determined in mice and chickens. Grayish colonies of the mutant, indicating loss of capsule synthesis, were observed under a stereomicroscope using obliquely transmitted light, while iridescent colonies were observed for the parental strain. Moreover, strain PBA322 showed a low amount of OmpH compared with the parental strain on SDS-PAGE. Additionally, the capsule of strain PBA322 was thinner than that of the parental strain according to electron microscopy, correlating to the attenuation against chickens. In conclusion, strain PBA322, the mutant of P. multocida strain P-1059, was completely attenuated for chickens.     

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.