Antigenic stability of fimbriae of Bacteroides nodosus

Successful vaccination of sheep against footrot and attempts to eradicate the disease depend on there being a limit to the antigenic diversity of the causative bacterium, Bacteroides nodosus. Fimbrial antigenic variation was therefore investigated in vivo, both under conditions of chronic infection and under the pressure of a vaccine-induced immune response, to ascertain whether this represented an obstacle to such goals. Material was available from 5 experiments and although B. nodosus appeared to have undergone changes in its fimbrial antigens in one of these, the possibility that superinfection was responsible for the variation detected could not be ruled out because all sheep in this case were maintained at pasture. Overall, the results provided no evidence of fimbrial antigenic shift in B. nodosus in vivo and in conclusion, the survival of the organism in the sheep's foot, both in long-term natural infection and following vaccination, must therefore be related to factors other than the ability to undergo antigenic variation in order to evade the host's immune response.     

Classification of Bacteroides nodosus by agglutination tests

One thousand two hundred and sixty seven isolates of Bacteroides nodosus from 292 sheep in 58 flocks were examined. Of these, 1260 could be classified by slide agglutination into 8 serogroups designated A to H. Up to 6 serogroups were detected in individual flocks, with up to 4 serogroups being detected in a single foot. Of the 292 sheep examined, 38 (13%) carried mixed serogroup infections. Determination of the range of serological types infecting a flock frequently required the examination of a number of isolates from each of a number of sheep. Cross-tube agglutination tests carried out on 44 isolates and their antiserums indicated that members of some serogroups could be divisible into subgroups or serotypes. These results suggested that 16 or more serotypes of B. nodosus might exist. The nature of the antigens responsible for both slide and tube agglutination reactions needs to be determined.     

Effect of pilus dose and type of Freund's adjuvant on the antibody and protective responses of vaccinated sheep to Bacteroides nodosus

Groups of sheep were immunised twice with one or other of six vaccines consisting of purified pili from Bacteroides nodosus at three dose levels (10, 38 and 154 micrograms) and emulsified with either complete (CFA) or incomplete Freund's adjuvant (IFA). Beginning one month after vaccination the sheep were homologously challenged on irrigated pasture, with naturally transmitted foot rot for a period of 26 weeks. Statistical analyses of the number of feet per sheep with severe foot rot demonstrated that there was a significant effect of vaccinal dose but neither an adjuvant effect nor an interaction between dose and adjuvant. Similar conclusions were reached when the titres of antipilus agglutinins in the serum were analysed. By both criteria the responses to doses of 154 and 38 micrograms of pili were significantly better than to 10 micrograms, but not significantly different from each other. The IFA vaccines caused less reaction at the sites of injection than the CFA vaccines and within the former the vaccines containing 10 and 38 micrograms pilus produced less reaction than those containing 154 micrograms. Hence a vaccine containing 38 micrograms of purified pili in IFA is nearly optimal for homologous protection against severe foot rot and is acceptable in terms of the reaction at the injection site.     

Motility in relation to virulence of Bacteroides nodosus

Fourteen Bacteroides nodosus isolates from footrot lesions of sheep were examined microscopically and all were found to have twitching motility. The mean percentage of cells showing motility was 40% and 9% for virulent and benign strains, respectively. This corresponded with mean agar colony diameters of 17 mm and 7 mm, respectively, for these strains. Two strains of intermediate virulence had values of motility and colony diameter similar to the benign strains. However, the intermediate and the virulent strains produced relatively stable protease compared to the benign strains. All virulent, benign and intermediate strains produced abundant pili. Included for comparison in this study was an avirulent variant strain which was highly motile, formed large colonies and produced stable protease, but showed no pili on electron microscopy. It was concluded that the properties of motility and protease stability may be used to distinguish, in the laboratory, wild-type virulent, benign and intermediate strains of B. nodosus.     

Importance of pilus-associated antigen in Bacteroides nodosus vaccines

The performances of two cellular vaccines, one sparsely piliated and the other well piliated, were compared on irrigated pasture with those of vaccines containing their respective, purified pili. There were statistically significant differences among the four vaccines in the mean numbers of feet per sheep which developed severe foot rot during 27 weeks of exposure. The incidence of severe foot rot was significantly lower in the two pili-vaccinated groups than in the group vaccinated with well-piliated cells, which were in turn substantially more effective than the sparsely piliated. However, the two pili vaccines did not prevent infection of the interdigital skin so that the differences in vaccine performance were much less marked when interdigital skin lesions were included. The number of feet affected by blowfly strike was closely associated with the presence of the most severe lesions and consequently there were far fewer struck feet in the two pili vaccine groups than in the two cellular vaccine groups. Analysis of serum agglutinin titres led to the same assessment of vaccinal efficacy as that derived from the analysis of severe foot rot lesions. Individually the agglutinin response to vaccination was not universally associated with resistance or susceptibility to severe foot rot but mean titres were significantly higher in the two pili vaccine groups than in the highly piliated-cell vaccine group, which were all in turn significantly higher than in the poorly piliated-cell vaccine group.     

Susceptibility of Bacteroides nodosus to various antimicrobial agents

The susceptibility of 18 strains of Bacteroides nodosus to 21 antimicrobial agents was tested in vitro. Penicillin was the most effective antibiotic tested. Other antibiotics tested, in order of relative efficacy, were cefamandole, clindamycin, tetracycline, chloramphenicol, erythromycin, sodium cefoxitin, tylosin tartrate, nitrofurazone, tinidazole, and dihydrostreptomycin sulfate. Tests of solutions of 4 antibiotics in 70% ethanol indicated that ethanol served primarily as a diluent and did not contribute substantially to the curative effect of such topical medications on foot rot lesions in sheep. Of the chemicals commonly used in foot baths for treatment of ovine foot rot, copper sulfate was most effective, followed by zinc sulfate, then formalin. Several commercial disinfectants and iodine were quite effective against B nodosus, whereas 5.25% sodium hypochlorite and 70% ethanol alone were relatively ineffective.     

Characterisation of virulent and benign strains of Bacteroides nodosus

The extracellular proteases of 395 isolates of B. nodosus from ovine, bovine and caprine foot lesions were classified as either thermostable or thermolabile. Stable protease was associated with one and unstable protease with four distinctive isoenzyme patterns, each pattern differentiated by the relative mobility of paired isoenzymes. Pathogenicity tests on 64 isolates showed a correlation between the production of stable protease and the production of virulent ovine footrot lesions. The mean values for total protease activity, twitching motility and colony diameter were significantly higher for virulent compared to benign isolates, but the range of values overlapped. SDS-PAGE whole-cell electrophoretic profiles of virulent isolates were similar to the profiles of some benign isolates.     

Colostral transfer of Bacteroides nodosus antibodies in sheep.

Ovine contagious foot rot may cause lameness in sheep, resulting in decreased wool growth and low weight gain. Affected neonatal lambs are difficult to treat, and treatment is labor intensive; thus, a method of prevention is warranted. Vaccination of ewes with a multivalent vaccine in an oil adjuvant induced development of antibody to the somatic O antigen of Bacteroides nodosus, and this antibody was detected in serum of newborn lambs after consumption of colostrum from the vaccinated ewes. Antibody titers were determined in 48 unvaccinated ewe/lamb pairs, and in 50 once-vaccinated and 78 twice-vaccinated pairs. Serum and colostrum O-agglutinin titers to B nodosus were determined by a microtitration agglutination test. Lambs from vaccinated ewes had significantly (P less than 0.05) higher O-agglutinin titers than those from unvaccinated ewes, and double vaccination of ewes resulted in the highest potentially protective titers (greater than 1:2,400) in ewes and lambs.     

Diversity of pilin of serologically distinct Bacteroides nodosus.

Pili from 11 distinct serotypes of Bacteroides nodosus were examined for diversity of pilin polypeptide subunits among serotypes and for purity of the pilin preparations. The pilin of all 11 samples was shown to be homogeneous. Mean +/- SD molecular weight of the pilin of 7 serotypes (A198, IV, V, VI, IX, XVII, and XVIII) was 18,500 +/- 100. The pilin of serotypes I, III, and VIII had molecular weight of 17,600, 19,400, and 19,000, respectively. Serotype XV differed greatly from the other 10 serotypes in that 2 distinct polypeptide bands with molecular weight of approximately 7,800 and 6,200 were detected. We suggest that these 2 low molecular weight bands resulted from proteolytic cleavage of the pilin protein.     

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.     

Studies on the ultrastructural morphology of Bacteroides nodosus.

The morphology of Bacteroides nodosus was examined with the electron microscope. B nodosus stained with solium phosphotungstate and uranyl acetate possessed fimbriae and in addition organisms negatively stained with sodium phosphotungstate often possessed rings on their surface. Phage-like particles were also observed in negatively stained preparations. In thin sections, B nodosus had a multilayered cell envelope and the type of cell division characteristic of Gram-negative bacteria. The cytoplasmic region contained a diffuse nucleoid area, ribosomes and, sometimes, concentrically arranged membranous lamellae. Fimbriae and capsular material were also seen in sections of B nodosus fixed with glutaraldehyde-osmium. Their visualisation appeared to be enhanced when ruthenium red was incorporated n the glutaraldehyde-osmium fixative but only when sections were stained with heavy metal salts, indicating that the fimbriae and capsule were not predominantly polysaccharide in nature.     

The protection given by pilus and whole cell vaccines of Bacteroides nodosus strain 198 against ovine foot-rot induced by strains of different serogroups

A highly purified pilus vaccine prepared from cells of Bacteroides nodosus strain 198 provided a high level of protection against homologous challenge and small, not statistically significant, levels of protection against challenge with 4 other strains each from different serogroups. In a second experiment, a partially purified pilus vaccine from strain 198 induced significant immunity to 1 of 4 heterologous strains which were different from those used in the first experiment. In a third experiment a strain 198 whole cell vaccine produced significant immunity against 3 of 6 heterologous strains used in the first 2 experiments. There was no obvious relationship between the colony type, degree of piliation and level of cross-protection obtained against a particular strain. The results provide further evidence that immunogens associated with, but distinct from, the pilus are involved in cross-protection and that cross-protective antigens are common to some, but not all, strains.     

Differences in morplioiogy of Bacteroides nodosus attributable to culture media

A single strain of Bacteroides nodosus was cultured under controlled conditions on hoof agar or in either biphasic medium or hoof broth. The gross and ultrastructural appearances of organisms were compared one with the other and with B. nodosus as seen in necrotic detritus obtained from a case of non-progressive foot rot. The possible imphcations of those morphological differences with regard to the antigenic structure of cells and their suitability for vaccine production, are briefly discussed.

The ‘rough’ colony form was predominant on hoof agar and considered to be normal but both ‘smooth’ and intermediate colony forms were also observed either when plates were dried insufficiently or after repeated subculture of organisms in hoof broth.

B. nodosus organisms seen in smears of necrotic detritus, were surrounded by a clear halo, bore filamentous appendages thought to be pili and possessed a layered cell envelope typical of Gram-negative bacteria. Electron-dense polychromatic granules were either small and scattered through the nucleoplasm or were much larger and occurred singly, often near the poles.

B. nodosus cells grown on hoof agar were sometimes longer but in other morphological respects were similar to those seen in necrotic detritus. No capsular material was demonstrable to account for the clear zone surrounding the cell envelope.

After 24-hr incubation in either biphasic medium or hoof broth, B. nodosus showed evidence of suboptimal cultural conditions as indicated by absence of pili, wrinkling of the cell envelope, appearance of amorphous extracellular structures, cytoplasmic vacuolation and cell lysis.     

Monoclonal antibodies against pili of serologically distinct Bacteroides nodosus.

Several monoclonal antibodies (McAbs) against pili of Bacteroides nodosus were examined to determine their reactivity with 11 different serotypes. One McAb was identified by enzyme-linked immunosorbent assay (ELISA) analysis that bound to nine of the 11 serotypes and another that bound to the remaining two serotypes tested. In addition, some McAbs demonstrated specificity for a single serotype, while others displayed specificities for up to five other serotypes. Comparison of immunoblot analysis with the ELISA revealed that the former method was not as sensitive in that all McAbs positive by the ELISA, were not positive by immunoblot. Possible explanations of these findings are discussed. There appear to be several antigenic determinants on B. nodosus pili and considerable sharing of these determinants between pili types. The 11 serotypes analyzed by the McAbs in this report are representative of all 20 US serotypes as well as the A-set and D-set categories of Australia. Therefore, the two epitopes recognized by two of the McAbs reported herein encompass all of the currently characterized B. nodosus serotypes and may provide a basis for bivalent vaccines efficacious for all types of B. nodosus induced footrot in sheep.     

Biochemical and biological studies on the lipopolysaccharide of Bacteroides nodosus

Lipopolysaccharides (LPS) were extracted from three Bacteroides nodosus isolates by the aqueous phenol method and purified by ultracentrifugation. The structure of B nodosus LPS appears to be similar to enterobacterial LPS, with the polysaccharide component joined to the lipid A moiety through an acid-labile 2-keto-3-deoxyoctonic acid (KDO) linkage. B nodosus LPS contained glucose, galactose, hexosamine, KDO and heptose and no sugars apart from ribose, which was possibly a nucleic acid contaminant, were unique to any of the isolates. Electron microscopic examination indicated a similar morphology to LPS derived from other Gram-negative bacteria. B nodosus LPS was found to exhibit biological properties characteristic of endotoxins, such as pyrogenicity, leucopenic and leucocytotic activity, production of the primary inflammatory response in rabbit skin and Shwartzman reactivity. However, the toxicity of B nodosus LPS was low.     

奶牛腐蹄病节瘤拟杆菌PCR检测法研究

腐蹄病是指侵害指（趾）间皮肤及皮肤更深层软组织的急性或亚急性炎症。患病皮肤常坏死、裂开，炎症从指（趾）间皮肤蔓延到蹄冠、系部和球节，病肢明显跛行，并伴有全身性症候。腐蹄病是奶牛蹄病中危害性极为严重的一类疾病，病畜食欲减退、泌乳量下降、繁殖能力降低，病情严重者将被迫淘汰，严重影响奶牛业的发展，造成严重的经济损失。鉴于该病对奶牛业的严重危害，奶牛出现蹄病时的及时、准确临床诊断，以及与其他蹄病的鉴别诊断，对于奶牛蹄病的临床治疗和预防具有重要的实际指导意义。本试验根据节瘤拟杆菌纤毛蛋白基因序列的特异性，设计1对引物BPP并建立了PCR检测方法。试验结果表明，引物BPP具有相对较高的特异性和敏感性，其敏感性达到了152.5 pg，且引物FLP具有很好的特异性，能够准确诊断出节瘤拟杆菌的存在。     

Cross-protective immunity and the serological classification system for Bacteroides nodosus

The relationship between the serological classification system for serogroup B and for serogroup H of Bacteroides nodosus and cross-protection between subgroups within these serogroups was examined. Protection against ovine footrot following vaccination was achieved against other subgroup strains provided sufficient cross-reactive antibody was induced by shared pilus antigens. Within serogroup B, better cross-protection against one subgroup was obtained with a pili vaccine than a whole cell vaccine which correlated with higher pilus antibody titres induced by the former. For serogroup H, a lack of cross-protection and serological reactivity between subgroups was demonstrated, which indicates that the prototype strain of subgroup H2 should be designated a new serogroup.     

Electron microscopic study of Bacteroides nodosus pili and associated structures.

Surface structures of Bacteroides nodosus were examined by electron microscopy. Collodion film and chrome shadowing were used for maximizing the visualization of B nodosus pili and ring structures. The existence of B nodosus pili in foot rot lesions was confirmed. Contrary to previous reports, it was found that B nodosus pili production can be retained through serial broth transfer under certain conditions. Capsule production by B nodosus was irregular in that it could be either lacking or variable in thickness. A bacteriophage capable of infecting B nodosus also was detected.