The melanophore aggregating response of isolated fish scales: a very rapid and sensitive diagnosis of whooping cough

Pertussis toxin (PT) has been found to block noradrenaline-induced pigment aggregation in fish melanophores, and, based on this, a rapid and highly sensitive assay for PT was developed. Some preliminary results have also indicated that it may be possible to detect PT-like activity in saliva samples from patients with clinically suspected pertussis. In the present study the diagnostic value of the fish melanophore method was evaluated in 70 patients suspected of having pertussis; culture, serology and physician diagnosis were used as reference methods. In 60 of the patients, pertussis was verified by at least one of the reference methods. The melanophore test showed PT-like activity in saliva samples from 58 of the patients. Three patients with reference-verified pertussis showed no PT-like activity in the test; among these, one patient had been immunized and had also been treated with erythromycin during 3 days immediately prior to visiting the hospital. The melanophore test has three major advantages: it allows detection of pertussis in the early and curable stage of the disease; it takes only 2 h to perform; and it requires no sophisticated equipment.     

百日咳杆菌69KDa外膜蛋白的分离纯化及生物学特性研究

本文发展了一种从百日咳杆菌Ⅰ相菌株中纯化６９ＫＤａ外膜蛋白的简易方法,将细菌体经加热浸提、乙醇沉淀蛋白、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ５０柱层析精制而成。用ＳＤＳ－ＰＡＧＥ、免疫印迹、光密度仪扫描分析,证明纯化制剂为均一的、特异性的６９ＫＤａ外膜蛋白,其收率为５４．２％,纯度达９９．２％,每微克６９ＫＤａ蛋白制剂中的内毒素含量低于０．８５ＥＵ;ＰＴ残留量小于０．１０５ｎｇ。抗６９ＫＤａ蛋白抗血清能     

Simple and specific detection of Bordetella holmesii by using a loop-mediated isothermal amplification assay

A loop-mediated isothermal amplification (LAMP) assay for simple detection of Bordetella holmesii was developed. This assay discriminates between B. holmesii and other Bordetella species and successfully detect B. holmesii DNA in nasopharyngeal swab samples from subjects with suspected pertussis. The LAMP assay results were in complete agreement with the results of previously published real-time PCR assay, indicating that the former is a powerful tool for the accurate diagnosis and surveillance of B. holmesii.     

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.     

Immune responses to the pertussis toxin in Balb/c and C57B1/6 mice

Abstract Immunization of Balb/c and C57B1/6 mice with the pertussis toxin (Ptx), purified from the culture supernatant of Bordetella pertussis (the whooping cough bacillus) resulted in different immune reactions in these genetically different strains of mice. Antibody responses to Ptx were detected only in Balb/c, whereas both Balb/c and C57B1/6 produced anti-Ptx antibodies when immunized with detoxified Ptx. Also, delayed-type hypersensitivity reactions differ strongly according to the use of Ptx or detoxified Ptx as eliciting antigen.     

Recombinant acellular pertussis vaccine—from the laboratory to the clinic: improving the quality of the immune response

Abstract Vaccination is the most effective way to prevent infectious diseases. Recombinant DNA technologies have provided powerful new tools to develop vaccines that were previously impossible or difficult to make, and to improve the vaccines that were already available but had been developed using old technology. In the case of whooping cough, an effective vaccine (composed of killed bacterial cells) is available, but its use is controversial because of the many side effects that have been associated with it. An improved vaccine against this disease should contain pertussis toxin, a molecule that needs to be detoxified in order to be included in the vaccine. Classical methods of detoxification, such as formaldehyde treatment have been used to inactivate this toxin. We have used recombinant DNA technologies to clone the pertussis toxin gene, express it in bacteria, map the B and T cell epitopes of the molecule, and to identify the amino acids that are important for enzymatic activity and toxicity. Finally, we have used this information to mutate the gene in the chromosome of Bordetella pertussis in order to obtain a strain that produces a molecule that is already non-toxic. This genetically inactivated pertussis toxin was tested extensively in animal models and clinical trials and was found to induce an immune response that is superior in quality and quantity to that induced by the vaccines produced by conventional technologies.     

Evidence for an intracellular niche for Bordetella pertussis in broncho-alveolar lavage cells of mice

Bordetella pertussis can attach, invade and survive intracellularly in human macrophages in vitro. To study the significance of this bacterial feature in vivo, we analyzed the presence of viable bacteria in broncho-alveolar lavage (BAL) cells of mice infected with B. pertussis. We found B. pertussis to be present in a viable state in BAL fluid cells until at least 19 days after infection, suggesting B. pertussis to be able to survive in those cells. This intracellular niche may play an important role in the pathogenesis of pertussis. Pertussis toxin and the RGD sequence of the virulence factor filamentous hemagglutinin (FHA) both play a role in the attachment of B. pertussis to human and mouse macrophages in vitro and we hypothesized these virulence factors to be required for invasion and subsequent intracellular survival of B. pertussis in macrophages in vivo. A B. pertussis double mutant, in which the FHA RGD motif was changed to RAD and the ptx genes were deleted, was also found in a viable state in BAL fluid cells, albeit at lower levels than the wild-type strain. In our model, uptake of B. pertussis by alveolar phagocytes in vivo is thus, at least in part, determined by the bacterial virulence factors FHA and pertussis toxin.     

Growth and siderophore production by Bordetella pertussis under iron-restricted conditions

It has been demonstrated that under iron-restricted conditions Bordetella pertussis can obtain iron from iron-saturated human transferrin. Direct contact between B. pertussis and transferrin was not required as B. pertussis was able to acquire iron from transferrin when they were separated by a dialysis membrane. Siderophore activity was detected in supernatants from iron-restricted cultures of B. pertussis, B. bronchiseptica and B. parapertussis. Siderophores were identified as hydroxamates and were produced by both virulent and avirulent strains of B. pertussis.     

The antibacterial action of cupric ions in Pseudomonas syringae

It has been demonstrated that under iron-restricted conditions Bordetella pertussis can take up iron from human transferrin within 30 min of exposure. B. pertussis utilizes two mechanisms for acquiring iron from human transferrin, a direct contact method and a siderophore mediated system. Both systems are shown to result in bacterial internalization of iron from transferrin. However, direct contact between B. pertussis and transferrin provides far more effective iron uptake than siderophore activity alone.     

Antimicrobial effect of human milk on Bordetella pertussis

It has been demonstrated that human milk, unlike bovine milk, can reduce the viability of Bordetella pertussis. This antibacterial activity was not due to the presence of antibiotics or antibodies in the human milk. Reducing the level of available iron or increasing the concentration of lysozyme in bovine milk did not induce anti-B. pertussis activity. Analysis of total fatty acids revealed that human milk contained significantly more linoleic acid than bovine milk. However, the addition of linoleic acid to bovine milk did not inhibit the growth of B. pertussis.     

百日咳杆菌粘着素的分子克隆、表达、纯化及生物学特性研究

大肠杆菌中克隆了编码百日咳杆菌粘着素（Ｐｅｒｔａｃｔｉｎ,Ｐｒｎ）１．９ｋｂ的结构基因,获得了４株表达Ｐｒｎ的重组菌株,以融合蛋白的形式,对Ｐｒｎ进行了表达。Ｐｒｎ融合蛋白的表达量占细胞总蛋白量的４６．４％。融合蛋白纯度达８９．２％。免疫印迹证明,抗天然Ｐｒｎ的单克隆抗体ＢＰＥ３能识别重组菌中及纯化的Ｐｒｎ融合蛋白。Ｐｒｎ融合蛋白能与抗百日咳杆菌Ⅰ相血清发生特异凝集反应。Ｐｒｎ融合蛋白及重组菌免疫小鼠后,对百日咳杆菌毒力株１８３２３的脑内攻击,保护率达６０～６２．５％。     

Cholesterol-dependent attachment of human respiratory cells by Bordetella pertussis

Bordetella pertussis is a re-emerging human respiratory pathogen whose infectious process is not fully understood, hampering the design of effective vaccines. The nature of bacterial attachment to host cells is a key event in the outcome of the infection. However, host cell receptors involved in B. pertussis colonization of the respiratory tract are still under investigation. Here, we report that cholesterol-rich domains are involved in B. pertussis adhesion to epithelial cells. Treatment of A549 cells with cholesterol-sequestering drugs such as methyl-β-cyclodextrin, nystatin, or filipin resulted in a significant decrease of B. pertussis attachment. Confocal laser microscopy studies showed B. pertussis associated with cholesterol-rich domains. Accordingly, B. pertussis was found in detergent-resistant membrane domain fractions isolated from bacterial-infected A549 cells. Our results indicate a main role of filamentous hemagglutinin, an environmentally regulated virulence factor, in this interaction, and a specific affinity for cholesterol, one of the major components of traqueal secretions, which might additionally contribute to the effective colonization of the respiratory tract.