Immunoblot analysis of cyanogen bromide-cleaved Moraxella bovis pilin reveals presence of shared antigenic determinants on pili from heterologous strains

Moraxella bovis pilus proteins, collected and purified from four strains of M. bovis, were cleaved with cyanogen bromide. Two major fragments were produced. Antisera were produced in rabbits to the pilin protein fragments and to whole uncleaved pili from these strains. Immunoblots of whole and cyanogen bromide-cleaved pilin were reacted with the homologous and heterologous antisera to whole pili and cleaved pilin. Antisera to whole pili reacted strongly with homologous pilin. Weaker and inconsistent reactions were detected with heterologous pilin. Antisera produced to cyanogen bromide-cleaved pilin proteins reacted strongly with homologous and heterologous pilin fragments and uncleaved pilin proteins. These findings demonstrate the presence of conserved antigenic determinants on pili from heterologous strains that are non-immunogenic in the intact pilus but are immunogenic after treatment with cyanogen bromide. Cyanogen bromide-treated pilus preparation might have potential as a vaccine because antibodies are induced against heterologous strains of M. bovis, whether these cross-reactive antibodies are protective remains to be determined.     

Detection of shared magnetic antigenic determinants on whole Moraxella bovis pili by use of antisera to cyanogen bromide-cleaved M. bovis pilus protein

OBJECTIVE: To determine the ability of antisera against cyanogen bromide-cleaved pili from 4 strains of Moraxella bovis to react with whole or nondenatured pili. SAMPLE POPULATION: Antisera to 4 strains of M. bovis produced by New Zealand White rabbits. PROCEDURE: Pili from 4 strains of M. bovis were collected and purified. Pilus proteins (pilin) were cleaved, using cyanogen bromide. Whole pilus and cyanogen bromide-cleaved pilin were injected into rabbits. Antisera were serially diluted, reacted with 4 strains of M. bovis, and examined by immunoelectron microscopy and indirect immunofluorescence. RESULTS: Antisera to whole pili aggregated and distorted pili from homologous strains, but pili from heterologous strains were unaffected. Antisera to cleaved pilin fragments resulted in partial aggregation and thickening of homologous and heterologous pili, suggestive of heterospecific antibodies. Attachment of antibodies to pili was detected by indirect immunofluorescence, indicating a strong reaction of antisera to whole pili with homologous pili. Weak cross-reactions were evident with certain heterologous strains. In contrast, antisera to cleaved pilin fragments reacted strongly with pili from homologous and heterologous strains. CONCLUSIONS AND CLINICAL RELEVANCE: We detected shared antigenic determinants on pili from various strains of M. bovis that were not immunogenic in intact pili. These sites were immunogenic after cleavage of pilus protein with cyanogen bromide, and antisera produced to protein fragments reacted with whole pili from heterologous strains of the organism. Vaccines produced from cyanogen bromide-treated pili may induce broader immunity against infectious bovine keratoconjuctivitis than that provided by currently available vaccines.     

Serological reactivity to Mycobacterium bovis protein antigens in cattle.

The serological response to 12 purified Mycobacterium bovis antigens were examined in an ELISA assay. These antigens included the majority of M. bovis protein antigens described to date and in most cases they were very similar to the M. tuberculosis antigens of the same molecular mass.

The purified antigens were tested against sera from M. bovis infected cattle, M. bovis culture-negative cattle from infected herds and animals infected with related microorganisms, mainly other mycobacterial species. All the antigens gave strong reactions with at least some sera from the M. bovis infected group and showed cross-reactivity with some of the sera from the other two groups. The antigen with the highest specificity reacted strongly with only 60% of the M. bovis infected sera. Antigens that reacted with most or all of the M. bovis infected sera also gave the highest cross-reactivity with sera from the other two groups. These results indicate that a serological test based on any one or a combination of these antigens, without removal of the cross-reacting epitopes, would be unsatisfactory.     

Reactivity of sera from sheep immunised with individual outer membrane proteins of Bacteroides nodosus against heterologous bacterial strains

In order to identify those bacterial antigens which might be involved in immunity against ovine footrot, antisera were raised in sheep to 6 proteins in the outer membrane complex (OMC) of one strain of Bacteroides nodosus. Examination of the specificity of these antisera by Western blotting, crossed immunoelectrophoresis (XIEP) and IEP, revealed that they recognized the homologous OMC protein, but did not precipitate either undenatured pili or OMC, nor could they agglutinate the homologous bacteria. In contrast, anti-OMC and anti-pili sera could precipitate OMC or pili respectively, and agglutinate whole bacteria. Subsequent analysis of these sera against 5 strains of B. nodosus from different serogroups revealed that Proteins 1, 3 and 4 had a similar antigenic structure in all strains examined. The reactivity of anti-pili sera was restricted to homologous bacteria whereas anti-pilin sera (raised against denatured pili) also reacted with pilin from 2 of 3 heterologous strains. However, none of the patterns of staining or absorption of any of these sera matched the spectrum of cross-protection afforded by vaccination of sheep with B. nodosus strain 198 cells. The results question the role of individual OMC proteins in cross-protective immunity and may imply that interactions between several bacterial components are involved in the phenomenon.     

Cross-protection from Bacteroides nodosus vaccines and the interaction of pili and adjuvants

The effects of vaccination of Merino sheep with the purified pili or the whole cells of Bacteroides nodosus strain 198, either in oil or alum-oil adjuvant, on the severity of foot-rot induced with the homologous strain (198) and a heterologous strain (217) were determined in a field experiment, on flood irrigated pasture. The efficacy of the whole cell vaccines was comparable to that of purified pili vaccines, against homologous challenge, when both had a similar content of pilus antigen although the purified pili vaccines induced significantly greater homologous pilus agglutinating antibody titres than the whole cell vaccines. However, against heterologous challenge, the whole cell vaccines in oil (CO) or alum-oil (CAO) provided significantly greater protection than a purified pili-in-oil (PPO) vaccine, the number of severely affected feet in sheep vaccinated with PPO being similar to that of the unvaccinated group. The group vaccinated with purified pili in alum-oil (PPAO) was intermediate between these two extremes. The superior performance of the PPAO in comparison to the PPO vaccine, against heterologous challenge, was associated with significantly higher mean ELISA titres to the outer membrane complex. Western blot analyses implicated a role in cross-protection for outer membrane proteins, in particular a protein Mr 78,000. The PPO vaccine produced fewer, smaller and less persistent vaccination reactions at the inoculation sites than did the other vaccines. Bodyweight gains in the period prior to challenge were much lower for the groups vaccinated with CO and CAO than for the controls and those vaccinated with purified pili, due presumably to the larger vaccination reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analyses of lipopolysaccharides, outer membrane proteins and DNA fingerprints reveal intraspecies diversity in Moraxella bovis isolated in Argentina

Intra-specific diversity within Moraxella bovis was investigated analysing DNA fingerprints, outer membrane proteins (OMP) and lipopolysaccharides (LPS) profiles. Three collection strains and 57 isolates of M. bovis, collected during 3 years from cattle with infectious bovine keratoconjunctivitis (IBK) symptoms, from diverse geographical locations of Argentina, were examined. The LPS and OMP profiles were studied through SDS–PAGE analysis and genotype was determined by PCR-DNA fingerprinting. Genotyping identified five DNA types while analysis of LPS and OMP profiles identified three rough LPS types and three OMP types among the 60 isolates of M. bovis including the three collection strains. None of the three methods employed to assess diversity was discriminating when used alone because the degree of heterogeneity in each group of surface structures was limited, but when data of each typing method were combined, 15 distinct subgroups were determined. This subgrouping was clearly able to differentiate isolates of the same genotype. These typing methods appear to be useful to assess different aspects of the disease such as the diversity within a population of M. bovis associated to epidemic conditions, track the causal agent in an outbreak of the disease, monitoring vaccination programs and studies on virulence.     

Brucella abortus RB51: enhancing vaccine efficacy and developing multivalent vaccines

Brucella abortus vaccine strain RB51 is an attenuated, stable rough mutant that is being used in many countries to control bovine brucellosis. Our earlier study demonstrated that the protective efficacy of strain RB51 can be significantly enhanced by overexpressing Cu–Zn superoxide dismutase (SOD), a homologous protective antigen. We have also previously demonstrated that strain RB51 can be engineered to express heterologous proteins and mice vaccinated with such recombinant RB51 strains develop a strong Th1 type of immune response to the foreign proteins. The present study is aimed at combining these two characteristics to generate new recombinant RB51 vaccines with enhanced abilities to protect against brucellosis and simultaneously able to protect against infections by Mycobacterium spp. We constructed two recombinant RB51 strains, RB51SOD/85A which overexpresses SOD with simultaneous expression of the 85A, a protective protein of Mycobacterium spp., and RB51ESAT which expresses ESAT-6, another protective protein of M. bovis, as a fusion protein with the signal sequence and few additional amino terminal amino acids of SOD. Mice vaccinated with these recombinant strains developed specific immune responses to the mycobacterial proteins and significantly enhanced protection against Brucella challenge compared to the mice vaccinated with strain RB51 alone.     

Genomic, protein and antigenic variability of mycoplasma bovis

Restriction endonuclease analysis (REA) with three enzymes SmaI, PstI, BamHI- was used to identify 13 different genomic groups among 37 Mycoplasma bovis strains. One genomic group was comprised of 14 strains. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) patterns for one strain chosen from each genomic group and an international reference strain PG45 were all similar. Antigenic variability in M. bovis species was investigated by immunoblotting, using serum from a calf that had been naturally infected with M. bovis and three M. bovis-specific monoclonal antibodies — mAbs N₂, I₂ and 5D7. Twenty M. Bovis field strains were tested, comprising one from each genomic group, six from the same genomic group and the reference strain. Antigenic profiles obtained with calf serum differed markedly one from the other, the heterogeneity being equally great among the strains belonging to the same genomic group as those coming from different groups. A stable antigen common to 164 out of 168 strains was detected by mAb N₂, whilst with mAbs I₂ and 5D7, two different membrane antigenic systems were demonstrated that were strikingly variable. These variations in expression occurred not only from one strain to another, but also within the same lineage of clones from a single cell.     

新疆石河子地区牛支原体的分离鉴定及药物敏感性分析

【目的】确定引起新疆石河子地区集约化牛场常发性肺炎的主要病原同时进行病原的体外药物敏感性分析。【方法】采集有典型咳嗽、流涕症状的牛鼻拭子10份和病死牛肺脏组织1份,用牛支原体特异性引物进行PCR检测,将检测为阳性的样本进行病原培养纯化,对纯化后的分离株菌落进行形态学观察、Dienes染色、生化试验及16S rRNA测序和进化分析,通过测定颜色变化单位(CCU)测定分离株生长曲线,并对分离株进行药物敏感性试验。【结果】PCR结果显示,10份鼻拭子中检测出7份牛支原体阳性样本,1份病死牛肺脏组织也检测为阳性;在涂有肺脏组织研磨液培养液的PPLO固体培养基上长出针尖状的菌落,纯化后分离株菌落形态为典型的煎蛋状;Dienes染色可见明显的深蓝色中心脐;生化试验结果显示,分离株不水解明胶、精氨酸、七叶苷,不发酵乳糖、葡萄糖和甘露醇,不分解尿素,可还原氯化三苯基四氮唑;16S rRNA测序结果显示,分离株与牛支原体国际标准株PG45相似性为99.7%,与国内牛支原体地方流行株XBY01、Ningxia-1、NM2012、Tibet-10的相似性最高,均为99.9%;生长曲线测定结果显示,分离株在培...     

Mapping of IS6110 insertion sites in Mycobacterium bovis isolates in relation to adaptation from the animal to human host

The physiological role and impact of IS6110 insertions on the biology of Mycobacterium tuberculosis complex is not well understood. Insertion of IS6110 in coding regions can cause loss of gene activity, while homologous recombination between two copies of IS6110 can result in the deletion of genes or in rearrangement of genomic regions involved. In addition to these genomic changes, IS6110 can also activate flanking genes through acting as a mobile promoter.

In order to determine the possible role of IS6110 transposition in the adaptation to humans, we selected Mycobacterium bovis isolates from endogenous reactivation cases in elderly people in The Netherlands. The human isolates contained higher number of IS6110 copies in comparison to the bovine M. bovis strains. These additional integration sites of IS6110 were sequenced and analyzed. From 12 of such IS6110 insertion sites, 6 loci were located in the intergenic regions, and 6 other occurred within coding regions. IS6110 was inserted in a position where it might serve as a promoter in two cases. We conclude that IS6110 transpositions in M. bovis may be a driving force in the adaptation from the animal to the human host.     

分枝杆菌分型三重PCR检测方法的建立及应用

本研究旨在建立一种快速鉴定分枝杆菌的三重PCR方法,并比较分析其在临床检测中的可靠性。根据已发表的结核分枝杆菌、牛分枝杆菌和非洲分枝杆菌rv 3036c基因,结核分枝杆菌rv 1970f基因(RD7)和牛分枝杆菌pncA基因的序列,改造并设计合成了3对特异性扩增引物,建立了一种能对分枝杆菌样品进行初步鉴定的三重PCR方法。结果显示该方法可针对rv 3036c、rv 1970f和pncA基因分别扩增出大小为500、125和249 bp的目的片段,能特异性检测出结核分枝杆菌(500和125 bp两条带)和牛分枝杆菌(500和249 bp两条带),并可将结核分枝杆菌、牛分枝杆菌与其他分枝杆菌加以区分。本方法的检测灵敏度为50 pg/μL模板基因组DNA。对86株抗酸染色阳性菌进行三重PCR鉴定,鉴定结果与细菌16S rDNA和ITS序列测定结果一致,检测准确度为100%,优于生长特征和生化试验鉴定。     

Establishment and Application of Mycobacterium Typing Triple PCR Detection Method

This study was aimed to establish a triple PCR method to rapidly identify Mycobacterium species, and evaluate its testing reliability.Three pairs of primer that were respectively specific to rv 3036c, rv 1970f and pncA genes of Mycobacterium were designed to establish a triple PCR for preliminary identification of Mycobacterium tuberculosis(M.tuberculosis), Mycobacterium bovis(M.bovis) and other Mycobacterium spp.PCR products were the expected sizes of 500(rv3036c), 125(rv1970f) and 249 bp(pncA), and contained two DNA bands(500 and 125 bp) with M.tuberculosis DNA template, two DNA bands(500 and 249 bp) with M.bovis DNA template.No band or non-specific band appeared with Mycobacterium spp.except M.tuberculosis and M.bovis DNA templates.The sensitivity of the triple PCR was calculated to 50 pg/μL template of genomic DNA.86 acid-fast bacteria were detected by the triple PCR, 16S rDNA and ITS gene sequencing, growth test and biochemical test, and the results were consistent between triple PCR and 16S rDNA and ITS gene sequencing.The detecting accuracy of triple PCR was 100%, and higher than growth test and biochemical test.     

犊牛支原体肺炎继发牛A型多杀性巴氏杆菌感染的诊治

某奶牛场犊牛相继发生肺炎和关节炎,为确诊该牛场犊牛群发病的原因并提出防控方案,本试验剖检新生犊牛并采集病料,分别开展牛支原体及其他病原菌的分离培养、PCR鉴定及药敏分析;进行牛病毒性腹泻病毒、牛口蹄疫病毒和牛传染性鼻气管炎病毒PCR检测;制作犊牛肺脏组织病理切片并进行观察和评估。从犊牛肺脏组织分离到牛支原体和牛A型多杀性巴氏杆菌;牛病毒性腹泻病毒、牛口蹄疫病毒和牛传染性鼻气管炎病毒检测均为阴性;肺脏组织病理切片可见肺泡结构破坏、出血及大量炎性细胞浸润;药敏试验结果显示,牛支原体和牛A型多杀性巴氏杆菌分别对泰乐菌素和头孢唑啉敏感,但对青霉素、庆大霉素、林可霉素和氨苄西林均呈现耐药。该犊牛群确诊为牛支原体肺炎继发牛A型多杀性巴氏杆菌感染,采用泰乐菌素联合头孢唑啉肌肉注射,配合对症治疗和规范管理,有效控制了该场犊牛疾病。     

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.     

Interaction of antigen presenting cells with mycobacteria

The interaction of mycobacteria with antigen presenting cells is a key feature in the pathogenesis of tuberculosis and the outcome of this interaction is pivotal in determining whether immunity or disease ensues. Human and mouse macrophages and dendritic cells (DC) have been shown to become infected with mycobacteria and to produce a response to infection that reflects their suggested role in immunity. Thus, macrophages elicit anti-microbial mechanisms for elimination of mycobacteria and DC up-regulate expression of molecules that aid their stimulation of T lymphocytes. We have examined the effects of infection with the avirulent strain Mycobacterium bovis BCG and with virulent M. bovis on bovine antigen presenting cells. Differences in the intracellular survival of bacteria within DC and macrophages were observed with higher numbers of bacteria maintained within DC following infection compared to macrophages. BCG was killed more effectively than M. bovis. Alterations in the expression of cell surface molecules involved in antigen presentation and the stimulation of T cells, including MHC II and CD40, were observed following infection of bovine antigen presenting cells. In addition infected DC secreted IL-12, TNF and IL-10 whereas macrophages produced TNF, IL-10 and little IL-12. Generally responses were more marked when virulent M. bovis was used compared to BCG. These studies indicate that infection of bovine antigen presenting cells by mycobacterial species results in the induction of both innate and adaptive immune responses that are critical for the outcome of infection.     

The effect of antisera on porcine enteropathogenic Escherichia coli in ligated segments of pig intestine.

Nineteen antisera produced in pigs against 14 enteropathogenic and five nonenterotoxigenic porcine strains of Escherichia coli were tested for their ability to inhibit gut loop fluid accumulation induced by homologous and heterologous organisms. In addition, four antisera produced in pigs by an intensive series of intravenous inoculations and three by a less intensive series of intramuscular injections of a polyvalent E. coli vaccine were evaluated. Antisera were also produced in rabbits against eight strains of porcine enteropathogens and tested in pig gut loops. Fluid inhibiting activity was detected in prevaccinal sera of pigs but not of rabbits. This activity was significantly increased following immunization. When single strains of E. coli were used for immunization the activity of the antisera against heterologous organisms varied considerably from one test strain to another and was usually much less than that against the homologous organism. The activity against heterologous organisms could not be associated with relatedness of the O, K and H antigens of the vaccine and the test strains. Antisera produced against a vaccine made by combining three strains were shown to exert inhibitory effects on heterologous organisms similar to those against homologous organisms. Considerably less activity against homologous and heterologous organisms was present in antisera produced by the series of intramuscular compared with the series of intravenous injections.     

Isolation and characterization of monoclonal antibodies against pili of Corynebacterium renale and Corynebacterium pilosum

Five monoclonal antibodies against pili of Corynebacterium renale 115 P+ (piliated clone) and two monoclonal antibodies against pili of C. pilosum 92 P+ (piliated clone) were produced. These antibodies bound to pili of the homologous strain in in enzyme-linked immunosorbent assay (ELISA) and agglutinated P+ but not P- (non-piliated clone) of each homologous strain. The five monoclonal antibodies against C. renale 115 P+ pili were divided into 2 groups, comprising 16/5, 160/1 and 32/6 and 13/4 and B20/3, based on the results of a competitive binding assay. The results may indicate the presence of at least 2 distinct antigenic areas on the pilus of C. renale 115 P+. The monoclonal antibodies of the first group inhibited adhesion of C. renale 115 P+ bacteria to the epithelial cells of bovine vulva, while the second group did not. Two monoclonal antibodies against C. pilosum 92 P+ pili recognized the same area on the pilus of C. pilosum 92 P+, and inhibited the adhesion of C. pilosum 92 P+ bacteria to the epithelial cells of bovine vulva. The adhesion of these bacteria was inhibited by the monoclonal antibodies in the form of IgG as well as by the Fab fragment. The strains of C. renale and C. pilosum which reacted with each of the anti-C. renale 115 P+ pili and anti-C. pilosum 92 P+ pili monoclonal antibodies were small in number and of restricted distribution.     

Immunodiffusion reactions among porcine enteroviruses and other picornaviruses

Antisera raised in rabbits against the porcine enterovirus strains V13 and T80 produced two precipitin lines in immunodiffusion tests with the homologous crude antigens, and a single precipitin line with each of ten heterologous porcine enteroviruses which were tested, and with poliovirus type 1, coxsackievirus B4 and equine rhinovirus type 1, but not with a bovine rhinovirus. C and D antigens prepared from V13 virus by density gradient centrifugation produced single precipitin lines with V13 antiserum. A single precipitin line was also formed when the heated crude antigens of V13 and T80 viruses were reacted with the homologous antisera, indicating destruction of the D antigen, and these lines fused with those produced by the heterologous viruses. It was concluded that porcine enteroviruses contain C and D antigens, and that the C antigen is responsible for the serological cross-reactivity demonstrated among porcine enteroviruses and other picornaviruses by immunodiffusion.     

Infectivity and virulence of Australian strains of Moraxella bovis for the murine and bovine eye in relation to pilus serogroup sub-unit size and degree of piliation

The degree of piliation of 29 haemolytic and 4 non-haemolytic Australian strains of Moraxella bovis representing 7 different pilus antigen groups was determined. The infectivity and virulence for the eye was measured in steroid-treated mice and in cattle. Non-piliated strains failed to infect the murine eye. Most moderately or heavily piliated strains reproducibly produced the highest infectivity and virulence scores in mice when compared with lightly or very lightly piliated strains (p less than 0.05). Non-haemolytic, piliated strains were infective and in one instance virulent for mice. Almost similar levels of infectivity and virulence were observed for 7 representative haemolytic strains tested in both cattle and mice. The relative molecular weight of pilin sub-units was compared using sodium dodecyl-sulphate polyacrylamide gel electrophoresis. Three classes of pili, alpha, beta and gamma of ascending sub-unit size were identified among the 7 pilus antigen serogroups. Pilin sub-unit size bore no relationship to the degree of piliation but most strains that were highly virulent in mice and cattle expressed alpha and gamma sub-units. Some strains appeared capable of switching from alpha to beta or form beta to gamma sub-unit production.     

Isolation of mycoplasmas from milk, lungs and prepuce of cattle

M. bovis was mainly isolated from 6.25% of milk samples collected from cows with clinical and sub-clinical mastitis. The main mycoplasma strain isolated from the respiratory tract of calves with symptoms of bronchopneumonia was M. bovirhinis (12.1% of samples). M. bovigenitalium was most frequently recovered from bull's prepuce (67.1% of samples).