The surface antigens of Rickettsia prowazekii studied with monoclonal antibodies]

R. prowazekii antigens have been tested with the use of monoclonal antibodies (McAb) to different epitopes of the microorganism. As revealed in these tests, McAb B4/4 and A-3/D, active against species-specific thermolabile antigen, interact with protein having a molecular weight of 90-120 KD. McAb C5/2, active against thermostable group antigen common with that of Rickettsia typhi, interact with LPS-like antigen having a molecular weight of 30 KD. Ultrastructural immunochemical studies have revealed that both R. prowazekii antigens are located on surface structures of rickettsiae, such as the microcapsule and cell wall.     

Seroimmunologic monitoring of microorganisms of Rickettsia and Bartonella species microorganisms in the Moscow region

Serological study of 788 blood sera, taken from residents of the Moscow region was conducted using antigens of microorganisms of the genera Rickettsia and Bartonella. The first group under examination consisted of 355 patients with diagnosed diseases of nonreckettsial nature. The second group includes 433 healthy adults working at a meat processing and packing factory. The main method used for sera survey was the indirect immunofluorescence test. In the sera taken from the first group of subjects specific antibodies to R. prowazekii, R. typhi, B. quintana, B. henselae antigens were detected in 2.3%, 5.1%, 4.0% and 2.9% of serum samples respectively. In the serum samples taken from the second group the proportion of antibodies to R. prowazekii, R. typhi, B. quintana, B. henselae antigens was different: 0.5%, 3.3%, 1.7% and 4.0% respectively. In total, specific antibodies to R. typhi and B. henselae prevailed over specific antibodies to R. prowazekii and B. quintana twofold.     

Immunobiological properties of monoclonal antibodies to Mycoplasma hominis antigens

Approaches to obtaining stable mouse hybridomas synthesizing monoclonal antibodies (McAb) to M. hominis key antigens were developed. 4 clones capable of the stable synthesis of McAb of different IgG classes were obtained. Clones A3/2 and A5/D produced antibodies to the thermostable determinant to with a mol. wt. of 80-120 kD, sensitive to sodium periodate and resistant to potassium proteinase. Clone H9/B2 synthesized McAb which interacted with potassium proteinase-sensitive M. hominis thermolabile determinant with a mol. wt. of 80 kD. McAb of clone A3/2, labeled with fluorescein isothiocyanate and horse-radish peroxidase, specifically reacted with M. hominis antigens in the immunofluorescence test and the immunoenzyme assay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data may serve as prerequisites for the development of diagnostic test systems aimed at the detection of M. hominis antigens in different clinical substances.     

Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae

Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.     

The detection of Rickettsia prowazekii in the vectors of louse-born typhus Pediculus humanus by using monoclonal antibody-based preparations]

The results of this investigation indicate that preparations obtained on the basis of monoclonal antibodies have proved to be suitable for the detection of R. prowazekii antigens in the natural carrier of typhus when used in all types of the enzyme immunoassay; of these, the assay made by the capture method has been found to possess the highest sensitivity. The testing of the sensitivity limits has shown that this method known as ELISA-mu-capture, i.e. ELISA carried out with the use of antibodies to the mu chain of human immunoglobulin, is capable of detecting the antigen in a dose of 0.5-1 ng. Preparations based on monoclonal antibodies to species-specific R. prowazekii antigen permit the identification of the causative agent of typhus in its natural carrier within 24 hours.     

Determination of genome size and restriction pattern polymorphism of Rickettsia prowazekii and Rickettsia typhi by pulsed field gel electrophoresis

Abstract Pulsed field gel electrophoresis (PFGE) of Sma I, Mlu I and Sal I digested DNA was used to estimate genome size and perform restriction fragment length polymorphism analysis for Rickettsia prowazekii and Rickettsia typhi . We concluded that the genome of R. prowazekii and R. typhi consisted of a single chromosomal DNA. The total length of DNA of R. prowazekii was 1,106±54 kb and of R. typhi was 1,133±44kb. It was possibleto differentiate two strains of R. prowazekii , Breinl and EVir, by PFGE analysis after Sal I digestion. Restriction fragment length polymorphism analysis did not reveal intraspecies differences between three human isolates and one Xenopsilla cheopis isolate of R. typhi .     

Luminescent-serologic analysis of the antigenic interrelationship between R. canada and classic representatives of rickettsiae of the typhus group

The results of studying the antigenic relationships of R. canada, a new Rickettsia species, and classical Rickettsia species of the typhus group are presented. The study was carried out by luminescent serological analysis with the use of corpuscular antigens and the live infectious agent cultures. R. canada and Rickettsiae of the typhus group were similar in their antigenic structures; this, however, could be revealed only in the study of the live cultures of the infectious agents. The study of corpuscular antigens revealed unilateral relationship: R. prowazeki antigen could be detected with homologous and heterologous sera, R. canada antigen with homologous serum only. In the CFT and the agglutination test corpuscular R. canada antigen reacted with homologous and heterologous sera. The study of the live cultures of the infectious agents revealed that different R. prowazeki and R. typhi strains vary in the degree of their similarity to R. canada.     

Antigenic relations of Rickettsia prowazekii and Rickettsia canada, established in the study of sera of patients with Brill's disease

Sera of patients with Brill's disease and of healthy persons with spotted fever in their past history were examined in the complement fixation reaction (CFR) to determine antigenic relations between R. prowazekii and R. canada. R. canada was found to have common antigenic determinants with R. prowazekii and R. mooseri. However, the antigenic determinants of R. canada differed from those of the mentioned rickettsiae. The titres of complement-fixing antibodies in the sera of patients with Brill's disease with the antigen of R. mooseri were lower than the titres with the homologous antigen within the range of 1-2 twofold dilutions of the serum. However, the oscillations of the titres with the antigen of R. canada in the study of the same sera were expressed in 1-5 twofold dilutions. In serological identification of canada rickettsiosis, antigens of rickettsiae of the spotted fever group should invariably be included in the investigation of the sera.     

Monoclonal autoantibodies to the epithelial basement membrane cells of human skin and thymus obtained through immunization with Rickettsia prowazekii antigens

As the result of immunization of BALB/c mice with the commercial preparation of typhus vaccine and R. prowazekii corpuscular antigen, in 29.2% and 40.3% of cases (respectively) the appearance of hybridomas synthesizing monoclonal antibodies (McAb) to different autologous structures (skin and thymic epithelium, cell nuclei, conjunctive tissue structures and vascular endothelium) has been revealed. The McAb under test have proved to be IgM-autoantibodies. McAb M-6, active against the basal membrane of human skin and thymic epithelium, produce quite a definite picture of disturbances in the differentiation of epithelium and can be used for the diagnosis of dyskeratosis.     

Immunogenicity of a chemical typhus vaccine and of a corpuscular radioantigen obtained from Rickettsia prowazekii

The protective activity of chemical typhus vaccine and R. prowazekii corpuscular radioantigen (CRA) was studied. Guinea pigs were immunized with doses of 32 and 48 antigenic units. Antibody production was assayed in the complement fixation test. On days 7, 15, 21, 30 and 60 after immunization the animals were challenged with R. prowazekii introduced in an amount of 10(5) minimum embryonal infective doses (MEID). On day 30 some of the animals were challenged with 10(3) MEID of R. typhi. The results demonstrated that both preparations were highly immunogenic and capable of protecting most of the animals from 10(5) MEID of R. prowazekii. Immunity developed earlier after immunization with CRA. The guinea pigs immunized with CRA, purified in percoll density gradient, and challenged with 10(3) MEID of R. typhi on day 30 showed a high level of cross immunity. In all control animals high fever and periorchitis were observed.     

Isolation of monoclonal antibodies to antigen Lyt-3.2

A hybridoma producing monoclonal antibodies (McAb) NATF9.9 (F9) was obtained from fusion of murine myeloma X63 and splenocytes of AKR mice immunized with a single intravenous injection of 5 X 10(7) thymocytes of CBA mice. F9 McAb were cytotoxic for 80% thymocytes, 10% splenocytes, 20% lymph node cells, 85% cortical and 32% medullary thymocytes of CBA, C57BL/6, BALB/c, DBA/2 and SJL but not for the cells of C58 and AKR mice. F9 McAb reacted only with T cells and did not react with B cells and EL4 thymoma cells (Thy-1.2+, Lyt-1+2-3-). The proportion of F9+ cells accounts for about 40% among T lymphocytes of the lymph nodes and spleen as tested by flow-type cytometry. Lymph node cells treated with F9 McAb plus complement completely lost their reactivity with rat anti-Lyt-2 McAb and only partly (by 30%) with anti-Lyt-1 McAb. The reactivity pattern of F9 McAb attests to their specificity for Lyt-3.2 antigen.     

Monoclonal antibodies to Mycoplasma pneumoniae antigens]

Approaches to obtaining stable mouse hybridomas, capable of producing monoclonal antibodies (McAb) to M. pneumoniae key antigens, were developed. As the result of hybridization experiments, 7 clones were obtained; of these, 4 clones stably synthesized IgG McAb. Clones H1/H9 and H9/B2 synthesized antibodies to thermolabile, proteinase-sensitive K protein, produced by cytoplasmic membranes of M. pneumoniae cells. The molecular weight of this protein was found to be 90 kD. McAb of clone H1/H9, labeled with horse-radish peroxidase and fluorescein isothiocyanate, specifically reacted with M. pneumoniae antigens in the immunofluorescence test and the enzyme immunoassay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data are prerequisites for the development of diagnostic test systems for the detection of M. pneumoniae antigens in different biological substances obtained from patients with respiratory pathology.     

Serological evidence of typhus group rickettsia in a homeless population in Houston, Texas

We tested sera from 176 homeless people in Houston for antibodies against typhus group rickettsiae (TGR). Sera from 19 homeless people were reactive to TGR antigens by ELISA and IFA. Two people had antibodies against Rickettsia prowazekii (epidemic typhus) and the remaining 17 had antibodies against Rickettsia typhi (murine typhus).     

Cytotoxicity of monoclonal antibodies to a subset of Giardia isolates

Previous studies showed that some Giardia lamblia isolates differ and can be categorized on the basis of their DNA banding patterns after digestion with endonuclease restriction enzymes, surface antigens, and excretory-secretory (E-S) products. In the present study, monoclonal antibodies (McAb) were produced that reacted with one specific group of Giardia isolates. These McAb recognized a 170,000 dalton antigen, which was present on the surface of these Giardia and released into the medium as an E-S antigen. This antigen was previously characterized and found to distinguish this subgroup of Giardia. In addition, these McAb were cytotoxic only for this subgroup of Giardia. Immobilization occurred immediately, and killing was documented by 7 min. The mechanism(s) of killing remains unknown but was shown to be complement independent and did not occur with Fab'. These McAb identifies certain isolates and can be used to type Giardia.     

Immunological characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test and reactivity with patient sera of tsutsugamushi diseases.

Immunological analyses of lipopolysaccharides (LPS) isolated from Proteus strains OX2, OX19, and OXK used as antigens of Weil-Felix (WF) test, were performed by quantitative agglutination, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Antisera against LPS and whole cells (WC) of the three Proteus strains reacted with homologous LPS but not with heterologous LPS, and the reaction was inhibited by the O-polysaccharide fraction isolated from the homologous LPS except OX19-LPS, which lacked O-polysaccharide moiety. The immunological data support the findings that the O-polysaccharide moieties of LPS from OX2 and OXK strains possess different chemical composition (Mizushiri, Amano, Fujii, Fukushi, and Watanabe, Microbiol. Immunol. 34: 121-133, 1990). Antisera against Proteus strains reacted weakly with WC of Rickettsia prowazekii, Rickettsia typhi, and Rickettsia tsutsugamushi. Antisera from patients with tsutsugamushi disease reacted with OXK-WC by WF test when the sera were obtained 13 days after onset of fever. The immunoperoxidase (IP) test titers of these antisera began to rise 6 days after the onset of fever. By ELISA tests these antisera reacted with OXK-WC and OXK-LPS independently of the titers of WF or IP tests.     

马立克氏病毒单克隆抗体的研究

获得了4株分泌马立克氏病毒(MDV)特异性单克隆抗体(McAb)的杂交瘤细胞:4BS10对MDV所有毒株呈阳性反应;4CN8 对MDV血清1,3型毒株发生反应;2BN90和4CN24只对MDV血清1型毒株有阳性反应。3个McAb属IgG1,1个为IgG2b,均不中和MDV,免疫扩散试验也无沉淀线。对禽白血病毒(ALV)无交叉反应。 以2BN90和辣根过氧化物酶、异硫氰酸荧光素的结合物进行直接酶联免疫吸附试验和直接荧光抗体试验,均获得成功。抗体滴度前者为1/51,200,后者为1/640。对ALV无交叉反应。     

Monoclonal antibodies to Chlamydia psittaci: their isolation, characteristics and use]

A panel of 22 hybridomas producing monoclonal antibodies (McAb) to C. psittaci was obtained. 15 hybridomas produced IgG1 antibodies, 4 hybridomas produced IgM antibodies and 3 hybridomas produced IgG2b, IgG3 or IgA antibodies. IgG1 antibodies and 2 IgM antibodies did not bind complement in the complement fixation test. All McAb were reactive in the enzyme immunoassay and the indirect immunofluorescence test and did not precipitate specific antigens. Peroxidase conjugates on the basis of McAb effectively detected Chlamydia antigen, prepared from the crude suspension of chick embryo yolk sack infected with different strains of C. psittaci and C. trachomatis, in different modifications of EIA.     

Isolation of monoclonal antibodies reacting with peribacteriod membranes and other components of pea root nodules containing Rhizobium leguminosarum

Plant and bacterial antigens contributing to nodule development and symbiosis in pea (Pisum sativum L.) roots were identified after isolation of a set of monoclonal antibody (McAb)-producing hybridoma lines. Rats were immunised with the peribacteriod material released by mild osmotic shock treatment from membrane-enclosed bacteroids of Rhizobium leguminosarum bv. viceae. In order to diversify the range of McAb specificities, this material was either used as immunogen directly (method 1), or after immunodepletion of a set of glycoprotein and lipopolysaccharide antigens (method 2), or after deglycosylation (method 3). After fusion and screening of cloned hybridoma lines, these three immunisation methods gave respectively 4, 2 and 1 classes of McAb with unique antigen specificities. Ultrastructural immunogold localisation studies showed four different antigens to be present on peribacteriod and plasma membranes (identified by MAC 64, 202, 206 or 209); in addition, a glycoprotein of plant origin but present in the infection-thread matrix was identified by MAC 204. Although none of the epitopes recognised by these McAb was nodule-specific, several were found to be more abundant in extracts of nodule tissue than in uninfected roots (MAC 64, 202, 204, 206). Two McAb reacted with new bacterial antigens: MAC 203 identified a bacterial antigen expressed upon infection but not in free-living cultures of Rhizobium, and MAC 115 identified a bacterial polypeptide (55 kdaltons) that was present in both free-living and bacteroid forms. There were also some McAb of broader specificity that react with antigens present in both plant and bacterial cytoplasms.Abbreviations ELISA enzyme-linked immunosorbent assay - Ig inmunoglobulin - kDa kilodalton - LPS lipopolysaccharide - McAb monoclonal antibody - PBM peribacteroid membrane - SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis - TFMS trifluoromethane sulfonic acid     

Lysis of cells infected with typhus group rickettsiae by a human cytotoxic T cell clone

Cytolytic human T cell clones generated in response to the intracellular bacterium Rickettsia typhi were characterized. Growing clones were tested for their ability to proliferate specifically in response to antigens derived from typhus group rickettsiae or to lyse targets infected with R. typhi or Rickettsia prowazekii. Two clones were able to lyse targets infected with typhus group rickettsiae. One of these clones was more fully characterized because of its rapid growth characteristics. This cytolytic clone was capable of lysing an autologous infected target as well as a target matched for class I and II histocompatibility leukocyte antigens (HLA). It was not capable, however, of lysing either a target mismatched for both class I and II HLA or a target partially matched for class I HLA. In addition, the clone exhibited specificity in that it was able to lyse an autologous target infected with typhus group rickettsiae, but did not lyse an autologous target infected with an antigenically distinct rickettsial species, Rickettsia tsutsugamushi. These results demonstrate, for the first time, that cells infected with intracellular bacteria can be lysed by human cytotoxic T lymphocytes.