A sensitive chemiluminescence assay for pertussis toxin and for evaluation of cell-free pertussis toxoids

Abstract Pertussis toxin (PT) inhibited luminol-enhanced chemiluminescence induced in rabbit peritoneal neutrophils by N'-formyl- l -methionyl- l -leucyl- l -phenylalanine (fMLP) at doses as low as 0.8 ng·ml⁻¹, even in the presence of a 10-fold higher concentration of filamentous haemagglutinin (FHA). A cell-free extract of Bordetella pertusis , containing predominantly PT and FHA, suppressed the neutrophil response to fMLP. After toxoiding with carbodiimide, the inhibitory activity of the extract was abolished and an enhancement of neutrophil chemiluminescence was observed due to FHA activity. Abrogation of the chemiluminescent response of neutrophils to fMLP is proposed as a sensitive, in vitro assay for PT, and may be useful for monitoring the residual toxin activity in pertussis toxoids and for determining the anti-toxic effects of anti-PT antibodies.     

百日咳杆菌去内毒素及保护性抗原的纯化

内毒素(Endotoxin,简称ET)是百日咳全菌苗(Bordetellapertussis vaccine)产生副作用的主要毒素之一,且不易除去。现有的分离方法,如蔗糖密度梯度离心法,较繁琐,成本高。本文采用Sepha-cryl S-300凝胶层折法可以简便有效的去除大部分内毒素。初步毒素试验结果表明:已达到日本生物制品规格的要求。两种保护性抗原FHA和LPF-HA也得到进一步分离纯化,为今后研制高效的百日咳组分菌苗提供了实验条件。     

Examination of the genus Bordetella for specific human IgA protease activity

Abstract Three laboratory strains and 3 clinical isolates of Bordetella pertussis were found to have no IgA protease activity when incubated with radio-labelled IgA. In addition, no IgA protease activity was detected in the Second British Reference Preparation for Pertussis Vaccine, an acellular vaccine produced in Japan, or one strain each of Bordetella parapertussis and Bordetella bronchiseptica .     

Conserved sequence motifs in the unorthodox BvgS two-component sensor protein ofBordetella pertussis

The unorthodox two-component sensor protein BvgS ofBordetella pertussis contains several interesting sequence motifs of unknown functional relevance, such as a histidine motif in its output domain, which is conserved among several unorthodox sensor proteins, a putative nucleotide binding site [Walker box type A] in its linker region, and a region in its periplasmic domain with significant homology to the TonB protein ofEscherichia coli. We investigated potential functions of these sequences by constructingB. pertussis strains that express mutant BvgS derivatives. The His₁₁₇₂ residue in the output domain was exchanged for Gln, and the Walker motif was mutated either by the replacement of Lys₆₂₅ by Arg, or of Gly₆₂₄ by Val and Lys₆₂₅ by Leu. To analyse the TonB motif, the periplasmic domain of BvgS was replaced with the corresponding domain of EvgS, anE. coli sensor that is highly homologous to BvgS but lacks the similarity with TonB. All mutations except the conservative Lys/Arg exchange in the Walker box caused the inactivation of BvgS, indicating the functional importance of the conserved motifs. The activity of the mutant proteins could be restored by complementation in trans with various separately expressed, truncated parts of BvgS. Mutations in the BvgS receiver domain could be complemented not only by a construct expressing the wild-type receiver and output domains, but also by the derivative containing the His-Gln exchange. Therefore, the histidine motif, although important for BvgS function, is not essential for complementation of BvgS mutants. The mutations in the Walker box and in the periplasmic domain could be complemented by a truncated BvgS derivative lacking the receiver and output domains. The characterization of a spontaneous revertant of the strain expressing the originally inactive EvgS/BvgS hybrid protein revealed the presence of a mutation in the BvgS linker region, conferring constitutive activity on the protein. As TonB energizes transport processes across the outer membrane ofE. coli, the strain expressing the constitutive EvgS/BvgS hybrid protein lacking the TonB motif was used in preliminary investigations of a possible direct involvement of BvgS in transport processes.     

The role of infectious agents in sudden infant death syndrome

Abstract Epidemiological factors associated with susceptibility to respiratory infections are similar to those associated with Sudden Infant Death Syndrome. Here we review the evidence that respiratory pathogens might be involved in some cases of Sudden Infact Death Syndrome in the context of factors identified in epidemiological studies of cot deaths: the age range affected; mother's smoking; respiratory viral infections; immunisation status. Both laboratory and epidimiological evidence suggests that vulnerability of infants to infectious agents depends on interactions between genetic, developmental and environmental factors that contribute to colonisation by microorganisms, the inflammatory and specific immune responses and the infants' physiological responses to inflammatory mediators. A model is proposed to explain how microorganisms might trigger a series of events resulting in some of these unexpected deaths and discusses how the present recommendations regarding child care practices might help reduce the numbers of Sudden Infant Death Syndrome cases associated with infectious agents.     

Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

Inhibition of Bordetella pertussis filamentous hemagglutinin-mediated cell adherence with monoclonal antibodies

Abstract Filamentous hemagglutinin (FHA), a 220-kDa protein located on the surface of Bordetella pertussis , is one of the major cell adhesins of this bacterium. We have produced three hybridoma cell lines that express monoclonal antibodies (mAbs) against FHA: X3C, X3E and X4B. The anti-FHA mAbs X3C and X3E reacted with 220-kDa FHA protein bands on Western blots. The mAb X4B, which reacted with FHA in ELISA, did not bind to FHA in a Western blot assay. All three mAbs seemed to be directed to the same epitope or to epitopes in close proximity as suggested by competition ELISAs. All three mAbs were able to inhibit the adherence of Chinese hamster ovary cells to purified FHA, and they could also inhibit the FHA-mediated agglutination of goose red blood cells. The attachment of B. pertussis to epithelial cell monolayers was inhibited by the mAb X3C. These antibodies are very useful probes to identify the presence of FHA in bordetellae species and in clinical reagents such as pertussis vaccines, and to characterize the functional domains of this important bacterial adhesin.     

Epitope mapping the Fim2 and Fim3 proteins of Bordetella pertussis with sera from patients infected with or vaccinated against whooping cough

Abstract Antibody-binding epitopes on the Fim2 and Fim3 proteins of Bordetella pertussis , which have been associated with the induction of protective antibody, were located using sera from 12 patients with whooping cough and 4 vaccinated children. Fifteen epitopes were identified on both Fim2 and Fim3. In each case 9 were recognised by serum antibody from 11 or more infected patients. Epitopes associated with the highest IgG activity were not the same as those associated with the highest IgA activity. None of the vaccinated patients had detectable IgA. Most epitopes showed little or no evidence of serotype-specific responses, suggesting this is largely directed towards conformational epitopes. The reactivity of all but two epitopes was confirmed in an ELISA with patients' sera in which epitopes were re-synthesised as free soluble peptides. The short linear epitopes described may therefore be useful in the development of serodiagnostic assays but are unlikely vaccine candidates.     

林麝源支气管败血波氏杆菌FMDBb1株的分离鉴定及全基因组序列分析

[背景] 呼吸系统疾病是圈养麝常见疾病之一,其中部分由支气管败血波氏杆菌引起,但目前关于林麝源支气管败血波氏杆菌的研究甚少。[目的] 对分离自患病林麝鼻黏液的一株疑似支气管败血波氏杆菌进行分离鉴定和全基因组序列分析,为林麝相关疾病的防治奠定基础。[方法] 将病原菌纯化培养后,通过菌落形态和生化试验初步鉴定病原菌类型,然后进行药敏试验和小鼠致病性试验以观察其耐药和毒力表型,最后对其进行全基因组测序,通过平均核苷酸一致性（Average Nucleotide Identity,ANI）比对在全基因组水平进一步评估物种间的亲缘关系,并进行基因功能注释和遗传进化分析。[结果] 该病原菌经ANI分类属于经典波氏杆菌亚种,结合菌落形态和生化结果综合鉴定为支气管败血波氏杆菌,菌株命名为FMDBb1,药敏表型显示其对林可霉素、利福平及大多数β-内酰胺类抗生素耐药,对小鼠的半数致死量为8.55×10⁶ CFU。全基因组测序显示该菌基因组大小为5 133 936 bp,基因功能注释显示其拥有强代谢能力,基因组内检测到65个经典波氏杆菌毒力因子,以及1个粉霉素和1个利福平的靶向耐药基因,同时发现19个插入序列和1个噬菌体区域的存在。序列类型为ST33,克隆复合体类型为CC6,管家基因联合建树分析显示与分离于韩国短毛猫的KVNON-570株相似性最高。[结论] 研究分离了林麝源支气管败血波氏杆菌并对其进行了全基因组测序及分析,为该病原菌感染的防治提供了宝贵的信息。