Efficacy of plague prophylaxis with streptomycin, tetracycline, and rifampicin in simultaneous immunization of white mice by resistant EV NRIEG strain]

Tetracycline, doxycycline, streptomycin and rifampicin were used for prophylaxis of experimental plague in albino mice (Yersinia pestis 231, approximately 1000 LD50). The antibiotics were administered 5 hours after the infection for 5 days. Tetracycline and doxycycline provided survival of 60 to 75% of the animals, while the respective figure for streptomycin and rifampicin was 100%, but streptomycin and rifampicin inhibited development of plague immunity evident from a lower protection index (PI) by 3-4 orders. The PI for the tetracyclines lowered by 2 orders. Simultaneous prophylaxis with the tetracyclines and immunization by Y. pestis EV Rifr R(SmTc) (10(6) microbial cells) provided not only higher percentage of the animal survival (80-90%) but also development of sufficient plague immunity: PI of 1.0 x 10(5)--5.0 x 10(5). When the animals were infected with Y. pestis 231 R(SmTc) the use of the tetracyclines failed, whereas the use of doxycycline and simultaneous vaccination by EV Rifr R(SmTc) provided survival of 70-85% of the animals. Successive use of inefficient streptomycin (for 2 days) and efficient rifampicin (for 3 days) provided survival only of 30% of the mice. A similar regimen of the successive use of the inefficient and efficient antibiotics (the total term of 5 days) started simultaneously with immunization by EV Rifr R(SmTc) provided survival of 80% of the animals. The use of combined specific and urgent prophylaxis of plague infection due not only to antibiotic susceptible but also to antibiotic resistant strains of the plague pathogen was shown promising.     

Doxycycline in the prevention of experimental plague induced by plague microbe variants]

A comparative study was performed on the efficacy of doxycycline in experimental plague infection induced in albino mice by strain 231 of the plague microbe and its variant 231 Fra- deprived of the ability to produce the fraction I antigen. It was shown that the LD50 for strain 231 during animal treatment with doxycycline was significantly higher than that for variant 231 Fra-. Prophylaxis of the plague infection caused by the Fra- forms of the plague microbe required significantly higher doses of doxycycline (ED50) than that of the infection caused by the Fra+ forms. The use of the daily maximum permissible doses of doxycycline (50 to 100 mg/kg a day) for 10 days in treatment of albino mice infected with the strain Fra- did not provide animal survival at the level higher than 60 to 70 per cent while the survival rate in the animals infected with the strain Fra+ of the plague microbe and treated according to the same scheme amounted to 90-100 per cent. The lower therapeutic efficacy of doxycycline in the treatment of the infection caused by the fractionless variant of the plague microbe should be considered in development of rational schemes for prophylaxis and treatment of plague.     

Immunochemical characterization of Fraction I of Yersinia pestis strains by CAF1M gene

The role of caf1M gene in biogenesis of Yersinia pestis capsule was studied in natural strains of the agent with Fra+/- phenotypes and recombinant variants with ycaA (caf1+;caf1M;caf1A+;caf1R+) locus defect. These bacteria did not form a clearly discernible capsule stained by classical methods but synthesized Cafl, whose content in the cells was many times higher than in lysates, in external cell wall, and in the medium with reference Y. pestis EV NIIEG culture (caf1+;caf1M;caf1A+;caf1R+). However Caf1 was not detected on the surface or culture fluid of natural and mutant Y. pestis cells. Exclusive role of Caf1M in Caf1 delivery to Y. pestis cell surface, but not in F1 monomer folding, was proven. Retention of lipopolysaccharide (LPS), a typical SR-LPS configuration and epitope specificity of its components was demonstrated, ensuring similar reactivity in solid-phase enzyme immunoassay with a panel of monoclonal antibodies to Y. pestis LPS. Study of immunochemical properties of antigenic substances derived from caf1M-defective Y. pestis cells by isolation of F1 showed that these substances represent an envelope protein involved in the caf1+ strains (together with Caf1) in assembly of "mature" F1 molecule as a result of posttranslation modification of various genes products. Variants of identification of Y. pestis with Fra+ phenotype by means of monoclonal antibodies to F1, fibrinolysis/coagulase, or LPS in solid-phase enzyme immunoassay are discussed.     

Isepamycin in prophylaxis and treatment of experimental plague due to FI+ and FI- variants of plague microbe]

The efficacy of isepamycin vs. other aminoglycosides was studied in vitro and on albino mice with experimental plague due to natural antigen valuable strains of the plague microbe and the pathogen variants deprived of the ability to produce the capsular antigen fraction I (FI- phenotype). The MICs of isepamycin for the strains of the plague microbe (20 FI+ and 20FI-) were 1.0-4.0 mg\l, that did not differ from those of streptomycin, kanamycin, amikacin and tobramycin. The ED50 of isepamycin in the prophylaxis and treatment of the experimental plague of the mice had no statistically significant differences from the ED50 of the other aminoglycosides. The efficacy index of isepamycin was > 10(4), that did not differ from that of streptomycin, amikacin and gentamicin, irrespective of the strain phenotype (Y. pestis 231 FI+ or Y. pestis 231 FI-). The same as the other aminoglycosides, isepamycin in doses equivalent to the human average daily doses, protected 80-100% of the albino mice from death when used in the prophylaxis and therapy of plague irrespective of the strain phenotype. The results of the study made it possible to consider isepamycin as an agent promising for the prophylaxis and treatment of plague.     

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.     

Evaluation of outcomes of combined specific prophylaxis with the antibiotic resistant immunogenic plague pathogen strain and emergency prophylaxis with aminoglycosides in the experimental plague model in white mice]

It was shown that aminoglycosides (streptomycin, kanamycin, gentamicin, sisomicin, tobramycin, amikacin) prevented manifestation of postvaccine immunity in albino mice immunized by vaccine strain Yersinia pestis EV. Avirulent strain Y. pestis 363 Monr with chromosome resistance to aminoglycosides of the 1st, 2nd and 3rd generations provided manifestation of antiplague immunity when streptomycin, kanamycin, gentamicin and amikacin were administered for prophylaxis. ED50 achieved 1.0-1.2 x 10(3) CFU and in control group (without treatment) 9.3 x 10(2) CFU. Gentamicin and amikacin were highly effective for experimental plague prophylaxis (90-100% animal survival), but inhibited development of postinfective immunity. Protective index (PI) value was 1.1 x 10(2). It was demonstrated that combination of specific prophylaxis (Y. pestis 363 Monr) and emergency prophylaxis with aminoglycosides in albino mice infected with approximately 1000 LD50 of virulent strain Y. pestis 358 (5 hours after infection) was highly effective and provided protective effect against subsequent infection with plague pathogen. Value of PI was 1.1 x 10(5) and practically did not differ from PI (1.7 x 10(5)) in control group (intact mice, immunized with strains EV [symbol: see text] 363 Monr).     

Antigenic profiling of yersinia pestis infection in the Wyoming coyote (Canis latrans)

Although Yersinia pestis is classified as a "high-virulence" pathogen, some host species are variably susceptible to disease. Coyotes (Canis latrans) exhibit mild, if any, symptoms during infection, but antibody production occurs postinfection. This immune response has been reported to be against the F1 capsule, although little subsequent characterization has been conducted. To further define the nature of coyote humoral immunity to plague, qualitative serology was conducted to assess the antiplague antibody repertoire. Humoral responses to six plasmid-encoded Y. pestis virulence factors were first examined. Of 20 individual immune coyotes, 90% were reactive to at least one other antigen in the panel other than F1. The frequency of reactivity to low calcium response plasmid (pLcr)-encoded Yersinia protein kinase A (YpkA) and Yersinia outer protein D (YopD) was significantly greater than that previously observed in a murine model for plague. Additionally, both V antigen and plasminogen activator were reactive with over half of the serum samples tested. Reactivity to F1 was markedly less frequent in coyotes (35%). Twenty previously tested antibody-negative samples were also examined. While the majority were negative across the panel, 15% were positive for 1-3 non-F1 antigens. In vivo-induced antigen technology employed to identify novel chromosomal genes of Y. pestis that are up-regulated during infection resulted in the identification of five proteins, including a flagellar component (FliP) that was uniquely reactive with the coyote serum compared with immune serum from two other host species. Collectively, these data suggest that humoral immunity to pLcr-encoded antigens and the pesticin plasmid (pPst)-encoded Pla antigen may be relevant to plague resistance in coyotes. The serologic profile of Y. pestis chromosomal antigens up-regulated in vivo specific to C. latrans may provide insight into the differences in the pathogen-host responses during Y. pestis infection.     

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 biosensor for the detection of Yersinia pestis

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.     

Prophylactic use of ceftriaxone in combination with F I antigen immunization in studies on uninbred albino mice infected by Yersinia pestis. Antiplague immunity development]

Administration of highly immunogenic (ED50 12.6 mcg/mouse) F I antigen (100 mcg/mouse) to albino mice 5 hours after their contamination approximately with 1000 LD50 of Yersinia pestis 231 provided 99-percent survival of same animals (17-50%) and 2-5-day prolongation of the life-span, that was indicative of the phenomenon analogous to the survival phenomenon observed in infected animals immunized by immunogenic strains of the plague microbe. The experiment on the mice confirmed high efficacy of ceftriaxone (100-percent survival) when used prophylactically for 5 days 5 hours after the contamination by Y. pestis 231 (approximately 1000 LD50) in the dose equivalent to the daily dose for humans. However, no antiplague immunity developed in the survivors: the immunity index (II) of 1.5x10. The use of ceftriaxone according to the same scheme simultaneously with single immunization by F I antigen in a dose of 100 mcg/mouse resulted not only in 100-percent survival of the animals but also in development of expressing antiplague immunity (II 2.2x10(5)). The protection level corresponded to the control with the same live-stock of the animals after a single immunization in the analogous dose of F I antigen (II 3.2x10(4)) and the ceftriaxone use (II 1.0x10(5)), as well as after immunization of the mice by 10(6) microbial cells of Y. pestis EV NIIEG (II 1.2x10(5)). The results of the study are indicative of the prospective use of subsingle vaccines of the new generation based on F I antigen for combined specific and urgent prophylaxis.     

Yersinia pestis L-forms in rodents and ectoparasites

Y. pestis L-forms and bacterial forms persist in the body of great gerbils for 40 days. L-forms are poorly phagocytized and can persist in phagocytes for a long time. In guinea pigs immunized with vaccine EV, Y. pestis antigen could be detected till day 160. An unstable L-form was isolated from Ornithodoros mites 3 years after their experimental infection with Y. pestis. Bacterial forms persist in mites for 1-3 years. For 5 years Y. pestis antigen is regularly detected in a high percentage of mites.     

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.     

Biogenesis of the fraction 1 capsule and analysis of the ultrastructure of Yersinia pestis

Analysis of a Yersinia pestis Delta caf1A mutant demonstrated that the Caf1A usher is required for the assembly and secretion of the fraction 1 capsule. The capsule assembled into thin fibrils and denser aggregates on the bacterial surface. Pilus-like fibers were also detected on the surface of Y. pestis. The capsule occasionally coated these fibers, suggesting how the capsule may cloak surface features to prevent host recognition.     

Field trial of an immunoenzyme method using beta-lactamase as the monoclonal antibody marker for the capsular antigen of the plague microbe

Beta-lactamase (penicillinase) has been used as a marker of monoclonal antibodies in the enzyme immunoassay made with a view to the detection of Y. pestis capsular antigen and antibodies to it. The trial of the enzyme immunoassay with the use of the beta-lactamase conjugate in laboratories and under field conditions has revealed the advantage of this assay over hemagglutination tests commonly used for this purpose.     

Expression of the envelope antigen F1 of Yersinia pestis is mediated by the product of caf1M gene having homology with the chaperone protein PapD of Escherichia coli.

The effective synthesis of the envelope antigen F1 of Y. pestis in E. coli HB101 is mediated by the expression of the caf1M gene. This gene was sequenced, and the protein encoded was found to have a significant homology with the chaperone protein PapD of uropathogenic E. coli. The data presented allow one to suppose Caf1M and PapD proteins perform similar functions in the biogenesis of the Y. pestis capsule and E. coli P-pili, respectively.     

Radioimmunological analysis in studying the persistence of the plague antigen in the body of rodents and ectoparasites in natural foci in Siberia

To study the persistence of Y. pestis capsular antigen, or fraction 1 (F1), in the body of less important plague carriers in the Mountain Altai and Transbaikal natural foci, as well as in experimentally infected ticks, the liquid-phase competitive radioimmunoassay (RIA) was used for the first time. In this study RIA showed, due to its sensitivity, doubtless advantages over traditional methods, such as the passive hemagglutination (PHA) test and the antibody neutralization (AN) test, and made it possible to detect F1 in picogram amounts. RIA revealed that F1 persisted in Siberian long-tailed gophers for up 14 months after the infection of the animals in diffusion chambers and for 7 months after their infection by subcutaneous injection. Experiments on Daurian pikas confirmed that, in comparison with the PHA and AN tests, RIA ensured fourfold effectiveness in the detection of antigen F1. The study of infected mites revealed that antigen F1 could be retained in them for more than a year and detected by RIA techniques in 10% of cases. The data obtained in this investigation indicate that the persistence of microorganisms should be studied mainly with the use of new-generation tests, and RIA, being one of the most sensitive techniques, deserves wide approval and introduction into the practical work of institutions intended for plague control.     

Cloning and detailed mapping of the fra-ymt region of the Yersinia pestis pFra plasmid]

The genetical libraries of the pFra plasmid of Yersinia pestis genes were obtained by insertion into the PstI, SalGI, EcoRI, XhoI restriction sites of the cosmid vector pHC79. The immunochemical analysis of the recombinant clones has revealed the clones synthesizing the antigen Fl (fraction I) and mouse toxin (Ymt--Yersinia pestis murine toxin). The restriction analysis of the plasmids from antigen synthesizing clones has permitted to construct the detailed physical map of the fra-ymt region of the pFra plasmid the size of 22 kb. The recombinant F1 positive clones of Escherichia coli are able to form at 37 degrees C the capsule-like structure peculiar for Yersinia pestis. The antigen F1 and the mouse toxin were isolated, purified and characterized. The antigen F1 is an 1-2 Md polymer containing a 16 kDa protein subunit. The mouse toxin a 240 kDa protein consisting of 61 kDa subunits. The nucleotide sequence of ymt gene has been defined.     

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.     

Identification of Yersinia pestis with varied plasmid composition using monoclonal and polyclonal fluorescent immunoglobulins

Abstract The efficiency of serological identification of Yersinia pestis strains which contain different plasmids was assessed with polyclonal and monoclonal immunoglobulin preparations in the direct fluorescent antibody method. Plague polyclonal luminescent immunoglobulins recognize only those Y. pestis strains which contain pPst, pFra plasmids or both. Anticapsular plague monoclonal antibodies interact only with capsule-forming plague agent strains (pFra+) grown at 37°C. With plague monoclonal lipopolysaccharide antibodies one can identify all Y. pestis strains irrespective of their plasmid content and cultivation temperature. However, these antibodies cross-react with Yersinia pseudotuberculosis bacteria in 60% of cases. The problem of laboratory diagnosis of the plague organism, whatever its plasmid profile, can be solved through the development of a test kit involving two preparations such as plague lipopolysaccharide monoclonal luminescent antibodies and pseudotuberculosisspecific luminescent adsorbed immunoglobulins.