Reaction of macrophage disappearance as a method for the evaluation of cellular immunity in experimental plague.

The reaction of macrophage disappearance (RMD) was found to develop one day after the immunization of experimental animals with the antigen FIA of Y. pestis and to persist for 21 days, correlating with a pronounced protective effect of the above antigen over this period. There were differences between white mice and guinea pigs in the intensity of the RMD, particularly when immunization was carried out using an FIA preparation without prolonged action. These differences are thought to reflect varying degrees of immunity elicited by FIA immunization. It is recommended that the RMD be used to quantify cellular immunity to Y. pestis.     

A rapid diagnostic test for plague detects Yersinia pestis F1 antigen in ancient human remains

A rapid diagnostic dipstick test (RDT) that detects Yersinia pestis F1 antigen has been recently applied on 18 putative plague victims exhumed from four archaeological burial sites in southeastern France dating back to the 16(th), 17(th) and 18(th) centuries. The Y. pestis antigen F1 was detected in 12 ancient samples out of 18 (67%). Negative controls confirmed their negativity (100%). Our results emphasize that the detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a first retrospective diagnosis of Y. pestis infection in ancient remains, and confirm the high specificity and sensitivity of the assay. Double-blind analyses performed by using two different techniques (RDT and 'suicide PCR') led us to the identification of the Y. pestis F1 antigen and the Y. pestis pla and gplD genes. These data provide clear evidence of the presence of Y. pestis in the examined specimens.     

Technical note: a rapid diagnostic test detects plague in ancient human remains: an example of the interaction between archeological and biological approaches (southeastern France, 16th-18th centuries)

A rapid diagnostic test (RDT) that detects Yersinia pestis F1 antigen was applied to 28 putative plague victims exhumed from seven burial sites in southeastern France dating to the 16th-18th centuries. Yersinia pestis F1 antigen was detected in 19 of the 28 (67.9%) samples. The 27 samples used as negative controls yielded negative results. Soil samples taken from archeological sites related to both positive and negative samples tested negative for F1 antigen. The detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a preliminary retrospective diagnosis of Y. pestis infection in human remains. The high specificity and sensitivity of the assay were confirmed. For two sites positive to F1 antigen (Lambesc and Marseille), Y. pestis-specific DNA (pla gene) had been identified previously by PCR-sequence based analyses. Specifically, the positive results for two samples, from the Lambesc cemetery and the Marseille pit burial, matched those previously reported using PCR. Independent analyses in Italy and France of different samples taken from the same burial sites (Draguignan and Martigues) led to the identification of both Y. pestis F1 antigen and Y. pestis pla and gplD genes. These data are clear evidence of the presence of Y. pestis in the ancient human remains examined in this study.     

免疫酶技术鉴定 El Tor 型霍乱弧菌稳定 L 型

细菌稳定 L 型的形态、培养特性以及生化反应常与原菌不同,其菌落在盐水中不能乳化,故不能通过玻片凝集测定其抗原。对于这种一时不能回复为原菌的 L 型很难进行鉴定。本文采用免疫酶技术对由鳝鱼和鲫鱼胆汁诱导的 El Tor 型霍乱弧菌稳定 L 型进行了鉴定。实验证明 L 型的细胞壁可有不同程度的缺失,稳定 L 型仍可能有少量“O”抗原存在。PAP 法比较敏感,即使少量抗原亦可以检出。     

No evidence of persistent Yersina pestis infection at prairie dog colonies in north-central Montana

Sylvatic plague is a flea-borne zoonotic disease caused by the bacterium Yersinia pestis, which can cause extensive mortality among prairie dogs (Cynomys) in western North America. It is unclear whether the plague organism persists locally among resistant host species or elsewhere following epizootics. From June to August 2002 and 2003 we collected blood and flea samples from small mammals at prairie dog colonies with a history of plague, at prairie dog colonies with no history of plague, and from off-colony sites where plague history was unknown. Blood was screened for antibody to Y. pestis by means of enzyme-linked immunosorbent assay or passive hemagglutination assay and fleas were screened for Y. pestis DNA by polymerase chain reaction. All material was negative for Y. pestis including 156 blood samples and 553 fleas from colonies with a known history of plague. This and other studies provide evidence that Y. pestis may not persist at prairie dog colonies following an epizootic.     

Immunogenicity of Yersinia pestis grown on nutrient media at 28 and 37 degrees C

The immunogenicity of Y. pestis strain EV, grown in yeast-casein medium, yeast medium with Hottinger digest and yeast medium with sunflower-seed protein at 28 degrees C and 37 degrees C, for guinea pigs and white mice has been studied. As revealed in this study, these media ensure the formation of highly immunogenic populations of Y. pestis strain EV and, therefore, can be used for growing Y. pestis vaccine strains. Considerable fluctuations in the content of such highly protective antigen as fraction 1 do not affect the immunogenicity of live cultures of Y. pestis strain EV. This is due to the leveling of differences in the content of this antigen in the process of the multiplication of these bacteria in laboratory animals.     

A horizontally acquired filamentous phage contributes to the pathogenicity of the plague bacillus

Yersinia pestis, the plague bacillus, has an exceptional pathogenicity but the factors responsible for its extreme virulence are still unknown. A genome comparison with its less virulent ancestor Yersinia pseudotuberculosis identified a few Y. pestis-specific regions acquired after their divergence. One of them potentially encodes a prophage (YpfPhi), similar to filamentous phages associated with virulence in other pathogens. We show here that YpfPhi forms filamentous phage particles infectious for other Y. pestis isolates. Although it was previously suggested that YpfPhi is restricted to the Orientalis branch, our results indicate that it was acquired by the Y. pestis ancestor. In Antiqua and Medievalis strains, YpfPhi genome forms an unstable episome whereas in Orientalis isolates it is stably integrated as tandem repeats. Deletion of the YpfPhi genome does not affect Y. pestis ability to colonize and block the flea proventriculus, but results in an alteration of Y. pestis pathogenicity in mice. Our results show that transformation of Y. pestis from a classical enteropathogen to the highly virulent plague bacillus was accompanied by the acquisition of an unstable filamentous phage. Continued maintenance of YpfPhi despite its high in vitro instability suggests that it confers selective advantages to Y. pestis under natural conditions.     

Interleukin-1-inducing activity of the polysaccharide-containing antigens of the cell wall in Yersinia pestis

The induction of the synthesis of interleukin-1 (IL-1) in human monocytes under the influence of the endotoxic preparations (LPS) and Y. pestis basis somatic antigen has been experimentally studied. The results obtained in this study make it possible to come to the conclusion that the capacity of the endotoxin of Y. pestis cell wall, consisting of LPS of type R, for inducing the synthesis of IL-1 in human monocytes is not different from the corresponding capacity of Salmonella and Shigella LPS, type S. Y. pestis O-specific polysaccharide in a discrete state has considerably greater IL-1-inducing activity in comparison with other preparations used in this experimental study. Such typical changes, characteristic of the initial stage of Y. pestis infection, as a sharp rise in temperature, transitory neutropenia, significant primary suppression of the oxygen-dependent metabolism of polymorphonuclear neutrophils are probably due to the induction of the synthesis of IL-1 by the polysaccharide-containing antigen of Y. pestis cell wall (LPS, basic somatic antigen) in cells of the mononuclear phagocytizing system.     

Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes

The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage lambda-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.     

A new purification strategy for fraction 1 capsular antigen and its efficacy against Yersinia pestis virulent strain challenge

F1 antigen is an attractive candidate for the development of a subunit vaccine against plague. In previous study, the extraction of this antigen from Yersinia pestis is characterized by using organic solvents. In this work, a new purification strategy that produced high-purity F1 antigen from Y. pestis EV76 was developed by the substitution of physical disruption for organic solvent one, followed by a combination of ammonium sulfate fractionation and Sephacryl S-200HR column filtration chromatography. As revealed in this study, this purification procedure is simple and effective, and avoids potential adverse effect on the antigen by organic solvents. Highly purified F1 that adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to F1 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10(4) CFU of Y. pestis virulent strain 141.     

Immunization of black-tailed prairie dog against plague through consumption of vaccine-laden baits

Prairie dogs (Cynomys spp.) are highly susceptible to Yersinia pestis and, along with other wild rodents, are significant reservoirs of plague for other wildlife and humans in the western United States. A recombinant raccoon poxvirus, expressing the F1 antigen of Y. pestis, was incorporated into a palatable bait and offered to three groups (n = 18, 19, and 20) of black-tailed prairie dogs (Cynomys ludovicianus) for voluntary consumption, either one, two, or three times, at roughly 3-wk intervals. A control group (n = 19) received baits containing raccoon poxvirus without the inserted antigen. Mean antibody titers to Y. pestis F1 antigen increased significantly in all groups ingesting the vaccine-laden baits, whereas the control group remained negative. Upon challenge with virulent Y. pestis, immunized groups had higher survival rates (38%) than the unimmunized control group (11%). The mean survival time of groups ingesting vaccine-laden baits either two or three times was significantly higher than that of animals ingesting vaccine-laden baits just one time and of animals in the control group. These results show that oral immunization of prairie dogs against plague provides some protection against challenge at dosages that simulate simultaneous delivery of the plague bacterium by numerous (3-10) flea bites.     

Detection of viable Yersinia pestis by fluorescence in situ hybridization using peptide nucleic acid probes

A successful method has been developed for the detection of live Yersinia pestis, the plague bacillus, which incorporates nascent RNA synthesis. A fluorescent in situ hybridization (FISH) assay using peptide nucleic acid (PNA) probes was developed specifically to differentiate Y. pestis strains from closely related bacteria. PNA probes were chosen to target high copy mRNA of the Y. pestis caf1 gene, encoding the Fraction 1 (F1) antigen, and 16S ribosomal RNA. Among Yersinia strains tested, PNA probes Yp-16S-426 and Yp-F1-55 exhibited binding specificities of 100% and 98%, respectively. Y. pestis grown in the presence of competing bacteria, as might be encountered when recovering Y. pestis from environmental surfaces in a post-release bioterrorism event, was recognized by PNA probes and neither hybridization nor fluorescence was inhibited by competing bacterial strains which exhibited faster growth rates. Using fluorescence microscopy, individual Y. pestis bacteria were clearly differentiated from competing bacteria with an average detection sensitivity of 7.9x10(3) cells by fluorescence microscopy. In the current system, this would require an average of 2.56x10(5) viable Y. pestis organisms be recovered from a post-release environmental sample in order to achieve the minimum threshold for detection. The PNA-FISH assays described in this study allow for the sensitive and specific detection of viable Y. pestis bacteria in a timely manner.     

Yersinia pestis lacZ expresses a beta-galactosidase with low enzymatic activity

Although very little, if any, beta-galactosidase activity is detected in Yersinia pestis by a standard Miller assay, we found that Y. pestis KIM6+ cells formed blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). Searches of the Y. pestis genome databases revealed the presence of noncontiguous sequences highly homologous to Escherichia coli lacZ, lacY, and lacI. Yersinia pestis lacZ is predicted to encode a 1060 amino-acid protein with 62% identity and 72% similarity to beta-galactosidase from E. coli. A deletion in the Y. pestis lacZ gene caused the formation of white colonies on X-gal-containing plates and beta-galactosidase activity was at background levels in the KIM6+lacZ mutant, while the complemented strain expressed about 190 Miller units. The Y. pestis lacZ promoter was not regulated by isopropylthiogalactoside or glucose. Finally, uptake of lactose by Y. pestis may be impaired.     

Antigenuria in plague-infected great gerbils

In the urine of plague-infected great gerbils Yersinia pestis capsular antigen was detected by means of diagnostic preparations, both commercial and experimental (based on monoclonal antibodies). The antigen was detected in many urine samples taken from the animals over a prolonged period. The incidence and duration of antigenuria were found to be related to the survival time of great gerbils after infection and the level of antibodies in their blood. The number of animals with antigenuria markedly exceeded the number of animals from which Y. pestis was isolated, especially at a later period after infection. Examinations of urine samples from live great gerbils trapped in natural foci appears to be a method more effective in epizootiological survey than the bacteriological analysis of the animals.     

The Yersinia pestis YscY protein directly binds YscX, a secreted component of the type III secretion machinery

Human pathogenic yersiniae organisms export and translocate the Yop virulence proteins and V antigen upon contact with a eukaryotic cell. Yersinia pestis mutants defective for production of YscX or YscY were unable to export the Yops and V antigen. YscX and YscY were both present in the Y. pestis cell pellet fraction; however, YscX was also found in the culture supernatant. YscY showed structural and amino acid sequence similarities to the Syc family of proteins. YscY specifically recognized and bound to a region of YscX that included a predicted coiled-coil region. These data suggest that YscY may function as a chaperone for YscX in Y. pestis.     

Isolation of monoclonal antibodies for the identification of strains of the causative agent of plague that do not contain a capsular antigen

In most cases the immunological identification of Y. pestis strains is based on the use of capsular antigen as an immunological marker. However, there are Y. pestis strains without capsular antigen. For the immunological identification of such strains, homogeneous antigen with a molecular weight of 43 KD has been isolated and monoclonal antibodies to it have been obtained. The enzyme-linked immunosorbent assay, carried out with the use of these monoclonal antibodies and intended for the detection of antigen with a molecular weight of 43 KD, has been developed. The sensitivity of the assay is about 10 ng/ml.     

Magnetic immunoenzyme analysis of Yersinia pestis antigens

The detection of Y. pestis cells in magnetic enzyme immunoassay is carried out with the use of magnetic polyacrylamide microgranules. In the assay system for the determination of the antigen commercial Y. pestis antigens, peroxidase-labeled antibodies, the substrate mixture consisting of sodium salt of 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid and H2O2 in citrate-phosphate buffer solution, pH 4.5, are used. The sensitivity of the method is 5 X 10(4) microbial bodies per ml.     

鼠疫耶尔森氏菌环二鸟苷酸代谢及生物被膜形成调控研究进展

鼠疫耶尔森氏菌(Yersinia pestis,以下简称"鼠疫菌")是烈性传染病鼠疫的病原菌,以鼠蚤作为传播媒介。鼠疫菌在其传播媒介鼠蚤的前胃中形成生物被膜从而促进其在宿主间传播。鼠疫菌生物被膜的形成受第二信使分子环二鸟苷酸(c-di-GMP)的正向调控。鼠疫菌中c-di-GMP由二鸟苷酸环化酶(DGC)HmsT和HmsD合成,由磷酸二酯酶(PDE)HmsP降解。文中主要介绍影响鼠疫菌环二鸟苷酸代谢及生物被膜形成的调控因子,并对其作用机制进行讨论和总结。     

Constructing a test system for the identification of plague microbe based on genetic probes]

DNA probes for detection of the plague agent Yersinia pestis were made on a basis of its three typical extrachromosomal replicons. The recombinant plasmid pBS2 including pBR327 vector and SalGI-BspRI fragment of the plasmid pFra was constructed. The above fragment is connected with synthesis of Y. pestis capsular antigen and it is a 400 bp species-specific DNA probe called F1 which is suitable for identification of Y. pestis species that bears the 60 mdal plasmid. The DNA probes called P1 was made on a basis of the plasmid pPst; it is the 460 BglII-BamHI fragment of the fibrinolysin-coagulase gene suitable for species-specific detection of Y. pestis species that bears the 60 mdal plasmid. The P1 fragment was cloned into the pAT153 vector and the constructed recombinant plasmid was called pEK7. The recombinant plasmid pCL1, including the pBR325 vector and the 6th BamHI fragment of Y. pestis EV plasmid pCad was constructed. The above fragment includes the replication origin of the pCad and it is hybridized to the pCad-bearing strains of Y. pestis and Y. tuberculosis only. Thus, it may be a basis for a bi-species-specific DNA probe making. These three recombinant plasmids are considered as a test-system for detection of both typical and atypical strains of Y. pestis.     

Immunoelectrophoretic analysis of the antigenic composition of Yersinia

The antigenic composition of 24. Y. pseudotuberculosis newly isolated and reference strains, 7 Y. enterocolitica strains, as well as Y. pestis vaccine strain EV, has been studied by the method of immunoelectrophoresis in agar. The antigenic composition of these bacteria has been found to be complicated and to comprise not less than 8-11 antigens, and among them nonspecific protein antigens common for enterobacteria, the common generic antigen, the antigen common with Y. pestis, as well as O-antigens specific for each serovar are identified. Immunoelectrophoretic study has shown the possibility of Y. pseudotuberculosis O-antigen, serovar I, with Salmonella sera, serogroup A, and Y. enterocolitica 09 with brucellar and cholera sera.