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.     

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 the antigens associated with saline solution, potassium thiocyanate, and sodium salicylate extracts of Pasteurella haemolytica serotype 1

Pasteurella haemolytica antigenic extracts were made, using saline solution, potassium thiocyanate (KSCN), and sodium salicylate (SS) extraction procedures. Of the 3 techniques, saline solution extraction resulted in the lowest protein concentration and lowest ribonucleic acid-to-protein ratio. The extracts varied in protein:carbohydrate ratios, with the KSCN extract being highest and the saline solution extract the lowest. Each extract contained lipopolysaccharide, as determined by detectable quantities of 2-keto, 3-deoxyoctonate. The saline solution extract contained the fewest protein bands by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, but contained the highest molecular weight proteins. All 3 extracts were reasonably similar antigenically, as detected by immunoblotting. Many of the protein bands present in the KSCN or SS extracts did not seem to be antigenic. Each extract was subjected to chromatofocusing, and the greatest antigenic peak, for each extract, failed to bind to the exchanger. These highly antigenic peaks, designated as saline solution, KSCN, or SS antigens, were similarly high in carbohydrate content, had similar antigenic-profiles, and contained high molecular weight (greater than 200,000) antigenic material, most likely carbohydrate in nature, as detected by immunoblotting. Inoculation of mice with 1 of the 3 extracts or the saline solution antigen resulted in marked antibody responses; however, protection against intraperitoneal challenge exposure to P haemolytica was minimal.     

鸡鲍氏3型志贺菌脂多糖及O抗原多糖的提纯与鉴定

为获得高纯度的鸡鲍氏3型志贺菌脂多糖（lipopolysaccharide,LPS）及其亚单位O抗原多糖(O-polysaccharide,O-PS),采用改良热酚水法提取鸡鲍氏3型志贺菌的LPS,并联合使用葡聚糖凝胶层析得到高纯度的LPS;采用酸水解法联合葡聚糖凝胶层析获得高纯度的O-PS。生化方法检测结果显示所提纯LPS和O-PS纯度高,兔回肠襻试验结果显示所提纯的LPS活性好。本试验结果表明提取的LPS和O-PS可进行特异性研究及制备有效的候选疫苗。     

Identification of multiple equine infectious anemia antigens by immunodiffusion reactions.

Equine infectious anemia (EIA) cell antigens prepared from infected equine spleen, equine leukocyte cultures or a persistently infected equine dermis cell line contained at least two serologically reacting components. For convenience one component was designated as soluble antigen (SA) and the other as cell-associated antigen (CAA). The SA appeared as a single component when it was prepared from EIA virus precipitated from infectious tissue culture fluid with polyethylene glycol and ether treated but it was mixed with CAA when the source was infected cells. Cytolytic or mechanical disruption of infected cells appeared to accelerate the release of CAA. Reaction to each component could be identified in double and radial immunodiffusion tests by increasing the concentrations of SA in a two-component antigenic mixture. The CAA component does not appear to affect the value of the immunodiffusion test as a diagnostic aid.     

丙型副伤寒沙门氏菌脂多糖提取纯化及活性分析

为了提取、纯化天鹅源丙型副伤寒沙门氏菌脂多糖（LPS）并检测其活性,试验通过热酚水法提取丙型副伤寒沙门氏菌LPS,采用DNaseⅠ、RNase A和蛋白酶K及醇沉法纯化LPS,测定LPS提取物中多糖、蛋白及核酸的含量,鲎试剂检测凝集活性,显色基质法测定其活性。结果显示,纯化的丙型副伤寒沙门氏菌LPS平均产率为1.48%,多糖含量为3.84%,蛋白含量为1.49%,核酸含量为 5.45%,且核酸片段低于100 bp,SDS-PAGE电泳和银染结果显示条带主要集中在10～15 ku范围内,与2 EU/mL鲎试剂的最小凝集浓度是10.99 ng/mL,显色基质法测定其活性为9.82×10⁵ EU/mg。该试验提取、纯化的丙型副伤寒沙门氏菌LPS纯度较高,生物活性良好。     

Detection of antigenic fractions from Brucella abortus S45/20 which bind to non-agglutinating antibodies using electroblotting and enzyme-linked antibody probes

Outer membrane antigens which bind to non-agglutinating antibodies (NAAb) elicited by smooth (S19) and rough (S45/20) Brucella abortus strains, were extracted from S45/20 by stirring in cold 2.5% NaCl and then analyzed by SDS-PAGE, electroblotting and enzyme-linked antibody test. Eight bands were observed in the gel stained with Coomassie blue. Seven antigenic fractions were transferred to nitrocellulose by blotting. A 27-kd band was recognized by bovine anti-S45/20 non-agglutinating serum and not by purified NAAb against surface antigens. Bands 10 kd and 14.3 kd bound to bovine anti-S45/20 NAAb from calves immunized with either S19 or S45/20. A 12.0-kd band was recognized by the serum and NAAb from calves immunized with S45/20 but not by those injected with S19. There are thus antigenic fractions shared by S19 and S45/20 which bind in vitro to NAAb.     

Soluble fractions of Pasteurella multocida: their protective qualities against fowl cholera in turkeys

Soluble fractions of Pasteurella multocida strain P1059 were extracted from a single source by four methods, and their immunogenicity was evaluated by challenge exposure in turkeys. The fractions were extracted by 1) heating in 2.5% NaCl, 2) 0.5M potassium thiocyanate, 3) 1.0M sodium salicylate, and 4) prolonged stirring in formalin solution followed by pelleting (LPS-protein antigen). Eighty percent to 90% of infected turkeys were protected in two trials by vaccination with the saline extract or LPS-protein antigen, whereas less consistent protection was associated with the other two preparations. Endotoxin content was the highest in LPS-protein antigen, followed by KSCN, Na salicylate, and saline extract in that order. The four fractions contained at least one common antigen, which had previously been shown to be a surface-protective antigen.     

Studies on the antigenic structure of Bacteroides nodosus.

The antigenic mosaics of three Bacteroides nodosus isolates (198, 199 and 127) were studied to elucidate the nature of the protective immunogen. In vaccinated sheep the three isolates induced high homologous serum agglutinin titres but it was also apparent that 198 and 199 shared a major surface antigen not present on 217. This major cross-reacting antigen was not detected with rabbit antisera. The fimbriae, consisting predominantly of protein, induced high homologous titres in rabbits and represented the type-specific antigen. Lipopolysaccharide (LPS) from each of the isolates induced low agglutinin titres and high 2-mercaptoethanol-sensitive indirect haemagglutinating antibody titres. The heat-stable LPS contained at least two common carbohydrate O antigen determinants but no type-specific O antigens were detected.     

Isolation and biological characterization of two lipopolysaccharides and a capsular-enriched polysaccharide preparation from Haemophilus pleuropneumoniae

A smooth-type lipopolysaccharide (HpS-LPS), a rough-type lipopolysaccharide (HpR-LPS), and a capsular-enriched polysaccharide preparation (HpC-PS) were extracted and purified from Haemophilus pleuropneumoniae serotype 5, strain J45, by the use of phenol-water (HpS-LPS) and phenol-chloroform-petroleum-ether (HpR-LPS) techniques. Chemical analysis of the HpS-LPS and HpR-LPS indicated that they contained 0.7% and 4.4% (w/w) 2-keto-3-deoxyoctonate, 11.8% and 10.4% phosphate, 0.8% and 0.8% nucleic acid, and 0.8% and 1.1% protein, respectively. The HpC-PS contained 0.3% 2-keto-3-deoxyoctonate, 1.4% phosphate, 0.2% nucleic acid, and 0.8% protein. With sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the HpS-LPS banded as a smooth-type LPS and the HpR-LPS banded as a rough-type LPS. Electrophoresis of HpC-PS indicated the presence of a broad high molecular weight band. Gelation of Limulus amoebocyte lysate developed at a minimum concentration of 8 ng/ml of HpS-LPS, 0.3 ng/ml of HpR-LPS, and 35 ng/ml of HpC-PS. The lipopolysaccharide preparations provoked a positive dermal Shwartzman reaction in rabbits and swine, a biphasic febrile response in rabbits, and a monophasic response in swine. Responses were typical of endotoxic activity with swine having greater sensitivity than rabbits. The chick embryo 50% lethal dose was calculated to be 7.3 ng for HpS-LPS, 1.6 ng for HpR-LPS, 5.1 ng for the lipopolysaccharide of Escherichia coli 0111:B4; and the HpC-PS was not toxic. In all assays, HpR-LPS was significantly more toxic than was HpS-LPS. The HpC-PS preparation was not toxic, even at high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Comparison of the Antibody Response in Adult Cattle Against Different Epitopes of Brucella abortus Lipopolysaccharide

The comparison of serological responses in a sample of adult, vaccinated and field‐infected bovines with Brucella abortus is reported. Indirect enzyme immunoassay (EIA) titration curves and Western blotting tests for smooth‐type lipopolysaccharide (S‐LPS), rough‐type LPS (R‐LPS) and lipid A were performed. In the initial screening of sera, an overall prevalence of 20.5 % was found, which corresponds to a country with a high incidence of brucellosis. End‐point EIA titres against LPS antigens from vaccinated and field‐infected cows were not significantly different. However, the absorbance values in the titration curves were significantly higher for S‐LPS as compared with the other antigens. A high correlation coefficient (r=0.933) was obtained when the titres to R‐LPS versus lipid A were compared. Western blotting reactions of vaccinated and field‐infected animals were indistinguishable. S‐LPS, R‐LPS and lipid A epitopes were recognized in a heterogeneous manner. In general, the number of bovines that reacted against LPS was higher in the field‐infected group, with a stronger binding to S‐LPS. Based on our observations, the vaccinated and field‐infected bovines are capable of producing similar antibody responses to the Brucella main outer surface antigen, LPS. It should be emphasized that the humoral response of cattle to Brucella LPS contains significant amounts of antibodies to other antigenic moieties of this important surface molecule, which may contribute to the immunity to brucellosis.     

猪附红细胞体可溶性抗原的分析

为了解附红细胞体可溶性抗原的特性,本试验对解离下的猪附红细胞体,经超声波裂解制备粗抗原,再经SephadexG-200分离提纯后,应用对流免疫电泳(CIEP)定位特异性抗原蛋白峰,再进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及免疫印迹(Westernblot),对猪附红细胞体粗提及纯化的可溶性抗原进行了分析。结果表明,猪附红细胞体可溶性抗原的电泳图谱上有四条蛋白带,其分子量分别为77Ku、62Ku、58Ku、29Ku,其中特异性抗原分子量为58Ku和29Ku。从而为附红细胞体抗原成分的进一步分析及该病的免疫学诊断等研究奠定了基础。     

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.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anaplasma centrale and Anaplasma marginale

An enzyme-linked immunoassay (ELISA) was applied to detect antibodies to A. centrale and A. marginale using homologous and heterologous antigens. The assay was compared with the indirect fluorescent antibody (IFA) test, and although a similar degree of sensitivity was obtained, the ELISA test had several advantages. Partially purified Anaplasma initial bodies used for antigen preparations contained negligible amounts of residual erythrocytic material, and did not interfere with the specificity of the ELISA. The antigenic similarity between A. marginale and A. centrale was further substantiated by cross-reactivity obtained with heterologous antigens in both ELISA and IFA tests, and antibodies produced during natural infection with A. marginale were indistinguishable in both tests from those produced following vaccination with A. centrale.     

Characterization of Mycobacterium paratuberculosis antigenic proteins

The characterization of a purified antigen from Mycobacterium paratuberculosis, recently made commercially available for use in serodiagnosis by enzyme-linked immunosorbent assay (ELISA), of paratuberculosis in cattle was described. This assay had 89% specificity and 83% sensitivity for M paratuberculosis infection. The protein/polypeptide composition of the purified antigen was compared with that of a crude protoplasmic extract of strain 18 M paratuberculosis used in the agar-gel immunodiffusion test and ELISA and with that of sonicated strain 19698 M paratuberculosis organisms grown on Dorset-Henley synthetic liquid medium. The sonicated M paratuberculosis contained 27 major proteins/polypeptides; the crude protoplasmic extract, 18; and the purified antigen contained 14 proteins/polypeptides, using sodium dodecyl-sulfate polyacrylamide-gel electrophoresis analysis. The serologic reactivity of these proteins/polypeptides were defined, using the enzyme-linked immuno-electrotransfer blot technique. The sonicated M paratuberculosis contained 20 serologically reactive proteins/polypeptides (34,000 to 84,000 daltons); the crude protoplasmic extract contained 3 (37,000 to 45,000 daltons); and the purified extract contained a diffuse polypeptide band (34,000 to 38,000 daltons). Identification by enzyme-linked immuno-electrotransfer blot technique of M paratuberculosis antigens reactive in the ELISA will allow us to further study these antigens in the ELISA to improve sensitivity and specificity of the diagnostic test.     

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.     

An indirect enzyme-linked immunosorbent assay for detection of antibodies to Actinobacillus pleuropneumoniae serovar 7 in pig serum.

Lipopolysaccharide (LPS) antigen was purified from Actinobacillus pleuropneumoniae serovar 7 by phenol-water extraction and fractionated on a, S-100 Sephacryl column. High molecular weight fractions of LPS purified from the S-100 column were pooled and used as antigen in an indirect serovar 7 ELISA. The ELISA was evaluated with sera from pigs experimentally infected with 11 different A. pleuropneumoniae serovars of biotype 1. Estimation of sensitivity and specificity of the A. pleuropneumoniae serovar 7 ELISA was performed using pig sera from herds naturally infected with A. pleuropneumoniae serovar 7 as well as sera from herds free of infection with A. pleuropneumoniae serovar 7. When compared to the complement fixation test (CFT) as a reference test, the ELISA showed much higher sensitivity and statistically equivalent specificity.     

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.     

猪传染性胃肠炎华毒弱毒株纤突蛋白B、C抗原位点片段基因的克隆与序列分析

猪传染性胃肠炎华毒（TGEV H）是我国分离的代表毒株,H弱毒株是我国自行培育成功的具有良好免疫原性的疫苗株。应用RT－PCR方法扩增出1．3kb的目的片段,包括S基因的编码的B、C两个主要抗原位点的片段,目的片段与pUC19质粒载体进行连续,转化,鉴定后得到阳性重组质粒,命名为pTS1,将重组质粒插入的目的基因片段进行序列分析和比较,其结果表明TGEV H株和S基因5’端B、C位点片段1190bp的核苷酸序列与国外的TGEV毒株Miller、Purdue-115、FS772／70及台湾省TFI毒株序列均具有很高的同源性,达94．72以上。推导的氨基酸序列同源性为94．70％以上。编码抗原位点C的序列与其它毒株完全一致,但在B位点发生改变,即第97个氨基酸发生改变,由Trp突变为Ser。该核苷酸该片段全长1261bp,包括起始密码子ATG及基因间的共同序列ACTAAAC。本研究首次报道了国内的TGEV分离株－H弱毒株S基因的RT－PCR扩增及其编码的B、C抗原位点片段的分子克隆及序列分析,为进一步揭示TGEV强弱毒差别的分子机制、开展新型疫苗及进行分子诊断的研究奠定了重要基础。