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.     

Preparation and partial characterization of monoclonal antibodies against the protective protein antigen of Erysipelothrix rhusiopathiae

Thirteen mouse monoclonal antibodies (Mabs) against the protective protein antigen (P64) of Erysipelothrix rhusiopathiae were prepared and partially characterized. The titres of the Mabs varied from 200 to 1,638,400 as determined by enzyme-linked immunosorbent assay (ELISA). Of the 13 Mabs 10, two and one belonged to the IgG2a, IgG1 and IgM subclasses, respectively. All Mabs reacted strongly with the 64 kDa protein and weakly with the 43 kDa protein upon Western blotting of the alkaline extract (AE) of E. rhusiopathiae. The protective activity (PD50/ml) of the 13 Mabs against E. rhusiopathiae infection in mice varied from < 50 to > 50,000. These Mabs were classified into three groups, highly protective Mabs, moderately protective Mabs and Mabs which did not possess protective activity, based on the protective index (ratio of the PD50/ml to the antibody titre). These results suggest that the 64 kDa protein is an effective protective antigen, which is easily cleaved into many small proteins, including the 43 kDa protein, and possesses at least two epitopes related to its protective activity and at least one epitope which is not related to protection of mice against E. rhusiopathiae infection.     

Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae.

Culture filtrate and alkaline-extracted antigens from whole cells of an attenuated strain of Erysipelothrix rhusiopathiae (strain Koganei: serovar 1a) were fractionated with ammonium sulfate; both induced protective immunity in mice. Sephadex G-200 gel filtration revealed three protein fractions in the alkaline-extracted antigen and four protein fractions in the culture filtrate antigen. A fraction in the alkaline extract (NaOH P-2) and in the culture filtrate (CF P-2) induced protection in mice against challenge with a different serovar strain (strain Agata: serovar 5). Anti-NaOH P-2 and anti-CF P-2 mouse sera were protective against different serovars. Glycoprotein fraction derived from CF P-2 antigen by affinity chromatography with Con A-Sepharose 4B did not show protective activity. Western blotting between the antisera (anti-NaOH P-2, Anti-CF P-2 and anti-Koganei strain) and the antigens (NaOH P-2, and sonicated antigens of Agata, Fujisawa and Koganei strains) showed strong recognition of the same bands at 62, 42 and 41 kDa.     

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).     

Neuraminidase production by Erysipelothrix rhusiopathiae

In order to characterise neuraminidase activity by Erysipelothrix, 85 isolates of Erysipelothrix spp. from a variety of sources including human clinical, marine and terrestrial animals, and the environment were investigated for neuraminidase production. Neuraminidase activity was detected by a peanut lectin haemagglutination method. The effects of media, incubation conditions and pH on the production and activity of neuraminidase were also investigated. Enzyme activity was detected only in the supernatants of the isolates of Erysipelothrix rhusiopathiae which had been incubated in cooked meat broth and Todd Hewitt broth supplemented with horse serum after 16 and 36 h incubation at 37 degrees C. The maximum titres were reached at 40 h in cooked meat broth and 56 h in Todd Hewitt broth supplemented with horse serum. All 58 isolates and the type strain (ATCC 19414) of E. rhusiopathiae produced detectable neuraminidase activity with titres between 10 and 320. The optimal pH for the enzyme activity varied among the isolates with a pH between 6.0 and 7.0 covering the highest enzyme activity of the most. There was no statistically significant difference in the level of neuraminidase activity between isolates from different sources (p > 0.05). Neuraminidase activity was not detected in the non-pathogenic Erysipelothrix spp. such as E. tonsillarum. Neuraminidase was detected only in E. rhusiopathiae suggesting its possible role as a virulence factor. Enzyme production and activity were medium and pH dependent. The peanut lectin haemagglutination assay is a simple, rapid and sensitive method and is particularly useful for the analysis of multiple samples.     

Comparison of pig, rabbit and mouse IgG response to Streptococcus suis serotype 2 proteins and active immunization of mice against the infection.

The aim of this study was to compare the IgG response of different animal species to Streptococcus suis serotype 2 proteins and to evaluate the immunogenic potential of these proteins in the mouse experimental model of infection. The protein profiles of ten different S. suis capsular type 2 isolates were compared by Western blotting using antisera produced in mice, rabbits and pigs against the reference strain. Strains were grown overnight in Todd-Hewitt broth, harvested by centrifugation, processed in a French press cell and digested with lysozyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was then performed and proteins transferred to nitrocellulose. The rabbit antiserum recognized seventeen common immunoreactive proteins, of which, proteins of 33, 44, 96, 122 kDa were present in all strains. Two, 128 and 136 kDa proteins were recognized by swine serum in many strains. An additional protein of 30 kDa was recognized by the mouse antiserum. These seven proteins, originating from the reference strain, were excised directly from polyacrylamide gels, mixed with incomplete Freund's adjuvant and given to groups of five mice on days 0 and 10. Immunoglobulin G response to each protein was monitored on day 20 using Western blots. Mice were then experimentally infected on day 21. Results indicated that vaccination with proteins of 33, 44, 128 and 136 kDa resulted in an IgG response and protection against the challenge with the reference strain, but gave only a partial protection against another virulent S. suis serotype 2 strain.     

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.     

Identification of RSVP14 and RSVP20 components by two-dimensional electrophoresis and Western-blotting.

We have already shown that RSVP14 and RSVP20, two ram seminal plasma (SP) proteins postulated to be involved in sperm capacitation and gamete interaction can protect spermatozoa against cold-shock. In this study, we use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the analysis of SP proteins of Rasa Aragonesa rams, using enhanced protein solubilization in the presence of tributyl phosphine (TBP) and a polyacrylamide linear gradient gel with a narrow pH range (4-7). The image analysis of the 2D map detected 195 protein spots, with isoelectric points (pIs) ranging from 4.5 to 6.6, and molecular weight (M(r)) from 11.7 to 90.4. Staining of 2D gels with Pro-Q Emerald 300 Glycoprotein Stain revealed that most significant proteins in ram SP are glycosylated. The removing of protein N-linked oligosaccharides improved the gel resolution. 2D-PAGE analysis of the whole fraction 6 (F6) separated from ram SP by exclusion chromatography showed six main protein spots, four (a, b, c, d) in the 14 kDa and two (e, f) in the 20 kDa region. Western-blot analyses indicated that the anti-P14 antibody recognized four spots on the SP map, 4, 5, 6 and 7, that matched with spots a, b, c, d of F6 map. The anti-P20 antibody recognized spots 13 and 14 of SP map that corresponded to spots e, f of F6 map. The deduced sequences by de novo sequencing evidenced that protein spots 7 and 13 have significant similarities to BSP family, while protein spots 4 and 14 did not appear to be homologous with any reported protein in the current mammalian Proteinbank databases.     

Vaccination of ducks with recombinant outer membrane protein (OmpA) and a 41 kDa partial protein (P45N') of Riemerella anatipestifer.

The generation of protective immunity against Riemerella anatipestifer infection in ducks were investigated by immunizations with recombinant glutathione sulfatransferase (GST) fusion's proteins of OmpA, a 42kDa major outer membrane protein, and P45N', a 41kDa N-terminal fragment of a newly identified 45kDa potential surface protein from R. anatipestifer. The DNA encoding OmpA and P45N' were isolated from R. anatipestifer serotype 15 (field strain 110/89) and serotype 19 (reference strain 30/90), respectively. Immunoblotting and ELISA results showed that the purified recombinant proteins induced the production of antibodies in immunized ducks. However, neither was protective against subsequent challenge with the virulent serotype 15 strain, 34/90. All the five ducks immunized with formalinized R. anatipestifer strain 34/90 survived the challenge with the homologous strain whereas six out of seven ducks in the non-immunized control group died within a week following the challenge.     

Computerized comparison of the protein compositions of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum strains

Protein profiles of six Erysipelothrix rhusiopathiae strains, five Erysipelothrix tonsillarum strains and three Erysipelothrix strains of uncertain taxonomic position were studied by sodium dodecyl sulphate-polyactylamide gel electrophoresis (SDS-PAGE). In a computerized comparison of the protein patterns of the strains, the level of similarity between the strains was determined. The SDS-PAGE protein bands were divided into 14 groups based on molecular weight. The relative distribution of proteins within these groups was used to characterize the strains. These distribution patterns were analysed by computing Pearson's correlation coefficient between strains, and by cluster analysis based on Euclidean distances and the unweighted pair-group method of arithmetic averages (UPGMA). The geometric mean of the similarities calculated by Pearson's correlation coefficient was 0.980 +/- 0.018 between the E. rhusiopathiae strains and 0.979 +/- 0.013 for E. tonsillarum strains. The value was 0.932 +/- 0.036 between the strains belonging to different species. However, a threshold value applicable for identification of a given strain to a species could not be established. Of the three strains of uncertain taxonomic position, the strains designated Rotzunge and Iszap 4 had a protein composition more similar to that of E. tonsillarum than to that of the E. rhusiopathiae type strain. The strain designated Pécs 56, which may be a member of a new species according to literature data, gave inconsistent results by the two methods used. The computerized evaluation method developed here is suitable for the comparison of the protein composition of the strains and for the construction of the protein similarity tree by cluster analysis.     

Ginseng extract in aluminium hydroxide adjuvanted vaccines improves the antibody response of pigs to porcine parvovirus and Erysipelothrix rhusiopathiae

Ginseng, the dry extract prepared from the Panax ginseng C.A. Mayer-root contain immunomodulators named ginsenosides, which in the pig enhance the antibody response to viral and bacterial antigens. The enhancing effect of ginseng was demonstrated vaccinating pigs against porcine parvovirus (PPV) and Erysipelothrix rhusiopathiae infections, using commercially available vaccines. The potency of the licensed, aluminium hydroxide adjuvanted; vaccines were compared with those supplemented with ginseng. The antibody response to PPV was measured by the haemagglutination inhibition (HI) test whereas the mouse potency test and ELISA evaluated the immune response to E. rhusiopathiae. Antibodies to the 64-66 kDa glycoprotein of the E. rhusiopathiae were demonstrated by immunoblotting. The qualitative antibody responses were evaluated by means of ELISA(s) using monoclonal antibodies to swine IgG1 and IgG2. The addition of 2mg ginseng per vaccine dose, potentiate the antibody response of the commercial vaccines without altering their safety. Significantly higher (P<0.001) antibody titres were achieved to both PPV and to E. rhusiopathiae by the supplementation with ginseng. Aluminium hydroxide adjuvanted vaccines favoured the production of IgG1 antibodies. Interestingly, the vaccines supplemented with ginseng favoured IgG2. The vaccines used in the evaluations varied in their immunogenic potency. However, after the addition of ginseng the less immunogenic vaccine proved to be as potent as the better one without ginseng. Thus, the use of ginseng as a co-adjuvant provides a simple, safe and cheap alternative for improving the potency of aluminium hydroxide adjuvanted vaccines.     

Co-migrating and shared antigens of selectedPasteurella haemolytica untypable strains

Bacterial cell envelope preparations from eight untypable strains ofPasteurella haemolytica were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting with rabbit antisera prepared against the eight untypable strains (one untypable strain per rabbit) and with cattle antisera prepared againstP. haemolytica serotypes 1, 2, 5, 6, 9 and against one heterologous untypable strain. Numerous comigrating and shared antigens were recognized by the eight rabbit antisera and theP. haemolytica serotype cattle antisera. Comigrating antigens at 43 and 30 kilodaltons (kDa) were recognized by all eight rabbit antisera. Shared antigens, detected by all eight rabbit antisera when reacting againstP. haemolytica serotype 1, were recognized at 43, 32, 30, 20 and 15 kDa.     

Prokaryotic Expression and Identification of P66 Gene from Borrelia garinii SZ

In order to obtain the P66 protein from Borrelia garinii (B. garinii) SZ, the first strand cDNA was synthesized based on the total RNA extracted from B. garinii SZ, and then the targeted P66 gene was amplified by PCR. The fragment was linked into the pET-30a(+) vector, and transformed into Escherichia coli BL21(DE3). After identified by PCR, double restriction enzyme digestion, and nucleotide sequencing, the recombinant protein was expressed and purified. Polyclonal antibody was then prepared from New Zealand rabbit immunized with purified recombinant protein. The recombinant protein was about 70 ku in size confirmed by SDS-PAGE, and Western blotting analysis indicated that the recombinant P66 protein could recognize the mouse monoclonal anti-His-tag, positive sera of spirochete from mouse, and anti-P66 polyclonal antibody. Additionally, the anti-P66 polyclonal antibody could recognize native P66 protein. In this study, we successfully expressed the recombinant P66 protein and obtained the anti-P66 polyclonal antibody, which provided the foundation for further functional studies of P66 protein from B. garinii SZ.     

Expression of iron-regulated outer membrane proteins by porcine strains of Pasteurella multocida.

The outer membrane protein (OMP) profiles of two strains of capsular type A Pasteurella multocida isolated from the lungs of pigs with enzootic pneumonia were studied. Sarkosyl extracted OMPs from P. multocida grown under iron-restricted and iron-replete conditions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 74 kDa, 94 kDa, 99 kDa and 109 kDa were expressed by strain A52, while 74 kDa, 82 kDa, 94 kDa and 99 kDa IROMPs were expressed by strain B80. Swine immune sera, obtained from pigs which were first immunized with a polyvalent P. multocida type A and type D bacterin and subsequently challenged with type A strain of P. multocida, contained antibodies against the IROMPs. These antibodies cross-reacted with the IROMPs expressed by avian strain P1059 of P. multocida. Convalescent-phase serum obtained from turkeys which survived fowl cholera, also cross-reacted with the IROMPs from porcine strains of P. multocida. These results suggested that IROMPs from porcine and avian strains of P. multocida may share common epitopes that were recognized by swine immune serum as well as turkey convalescent-phase serum.     

伽氏疏螺旋体尚志株P66基因的原核表达与鉴定

为获得伽氏疏螺旋体尚志株（B. garinii SZ）的P66蛋白,本研究从培养的螺旋体菌液中提取总RNA,反转录合成第一链cDNA,利用PCR扩增P66基因。将目的片段连接表达载体pET-30a（+）,并转化大肠杆菌表达菌BL21（DE3）,经PCR、双酶切及测序验证正确后,进行IPTG诱导表达和纯化,然后将纯化的融合蛋白免疫新西兰大白兔,制备多克隆抗体。SDS-PAGE结果显示,获得约70 ku的表达产物;Western blotting分析表明,表达产物与抗His标签的小鼠单克隆抗体、螺旋体鼠源阳性血清、兔抗P66多克隆抗体均能发生反应,获得的多克隆抗体也可识别天然蛋白。本试验成功表达了B. garinii SZ株P66蛋白,并制备了多克隆抗体,为后续B. garinii SZ株P66蛋白的功能研究奠定了基础。     

猪丹毒QL251弱毒菌株的稳定性试验

猪丹毒ＱＬ２５１弱毒株在马丁肉汤、血液琼脂斜面、小白鼠及猪丹毒敏感猪体内连续传代后，测其安全性和免疫原性。试验证明，ＱＬ２５１无论是在培养基上，还是在动物体内传代后都是稳定的，仍然保持了原有的特性。     

Production of bacteriocin by Leuconostoc mesenteroides 406 isolated from Mongolian fermented mare's milk,airag

The purification and characterization of a bacteriocin produced by Leuconostoc mesenteroides strain 406 that was isolated from traditional Mongolian fermented mare's milk, airag, were carried out. Leuconostoc mesenteroides strain 406 was identified on the basis of its morphological and biochemical characteristics and carbohydrate fermentation profile and by API 50 CH kit and 16S ribosomal DNA analyses. The neutral‐pH cell‐free supernatant of this bacterium inhibited the growth of several lactic acid bacteria and food spoilage and pathogenic organisms, including Listeria monocytogenes and Clostridium botulinum. The bacteriocin was heat‐stable and not sensitive to acid and alkaline conditions, but was sensitive to several proteolytic enzymes such as pepsin, pronase E, proteinase K, trypsin, and α‐chymotrypsin, but not catalase. Optimum bacteriocin production (4000 activity units/mL) was achieved when the strain was cultured at 25°C for 24–36 h in Man Rogosa Sharpe medium. The bacteriocin was partially purified by ammonium sulfate precipitation (80% saturation), dialysis (cut‐off MW: 1000), and gel filtration chromatography. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed that the bacteriocin had a molecular weight of approximately 3.3 kDa. To our knowledge, this is the first report of the isolation of a bacteriocin‐producing Leuconostoc strain from airag. An application to fermented milks would be desired.     

Comparative Analysis of the Outer Membrane Protein Profiles of Isolates of the Pasteurella multocida (B:2) Associated with Haemorrhagic Septicaemia

Outer membrane proteins (OMP) of P. multocida (serotype B:2) field isolates (n = 6) and a vaccine strain (P-52) were extracted by a sarkosyl method and characterized using SDS-PAGE and immunoblotting. About 20 polypeptide bands were observed in the profile of the vaccine strain with MW ranging from 16 to 90 kDa and, based on band thickness and intensity of staining, three polypeptides of MW 31, 33 and 37 kDa were considered to be the major OMPs. The profiles of the field isolates showed minor differences when compared with that of the vaccine strain. The OMP of 33 kDa was only expressed by the vaccine strain. Four field isolates expressed an OMP of 39 kDa, which did not appear in the profiles of the remaining two field isolates and the P-52 strain. Similarly, an OMP of 25 kDa was exclusively seen in the profile of a single isolate. By immunoblotting studies, using anti-P. multocida (P-52) whole-cell hyperimmune serum raised in rabbits as well as buffalo immune sera, it became evident that the polypeptide of 37 kDa was the most antigenic OMP in the profiles of all the isolates, including the P-52 strain. Other polypeptides were either weakly antigenic or visible in the profile of only a few of the isolates. The study thus identified the major OMP of P. multocida (B:2) and suggested that this highly antigenic 37 kDa OMP has potential for further protective and immunodiagnostic studies.     

丹毒丝菌spaA基因免疫保护区的克隆与功能研究

本研究以丹毒丝菌XJ1249基因组DNA为模板,根据GenBank已发表的丹毒丝菌表面保护性抗原A (SpaA)的序列设计引物进行PCR扩增,得到大小约1 029 bp的spaA基因N端免疫保护片段(spaA-N)。将spaA-N连接到载体pGEX-4T-1上构建重组原核表达质粒pGEX-spaA-N,对重组质粒进行序列验证后,在IPTG的诱导下,表达和纯化重组蛋白r-SpaA-N,并研究其对小鼠的免疫保护作用。结果显示:分离纯化的重组蛋白具有免疫原性,并对小鼠具有免疫保护性,能够有效防止丹毒丝菌对小鼠的侵染。研究结果为进一步研究丹毒丝菌致病机理,开发新的猪丹毒诊断试剂盒和亚单位疫苗奠定了基础。     

Comparison of inoculation regimes for the experimental production of swine erysipelas arthritis: I Clinical, pathological and bacteriological findings

The arthritic form of swine erysipelas was induced in pigs by multiple intravenous inoculation of 2 strains of Erysipelothrix rhusiopathiae. The strains differed significantly in their arthritogenicity but not in the number of cases of lameness induced. The use of 3 intravenous inoculations instead of 5 did not significantly affect the outcome. In a second trial, the more arthritogenic strain was injected in ten-fold dilutions from 5 x 10(9) to 5 x 10(4) organisms. Pigs receiving the lower doses showed high variability in their arthritic responses that precluded sensitive analysis of the dose effects on the number of arthritic and infected joints. However, slaughter weights showed a significant negative correlation with dose. Mean slaughter weights in treatment groups varied by 14.6 kg per pig, an average weight loss of 3 kg per pig for each ten-fold rise in dose of the highly virulent strain, and significantly correlated with the number of arthritic and infected joints. Culture of homogenised synovial membrane through selective horse meat-serum broth containing kanamycin, neomycin and vancomycin identified 66% and 59% more infected joints than primary blood agar culture of synovial fluid or synovial membrane homogenate, respectively.