Molecular typing of papillomatous digital dermatitis-associated Treponema isolates based on analysis of 16S-23S ribosomal DNA intergenic spacer regions

Papillomatous digital dermatitis (PDD), an emerging infectious disease of cattle, is characterized by painful, ulcerative foot lesions. The detection of high numbers of invasive spirochetes in PDD lesions suggests an important role for these organisms in the pathogenesis of PDD. PDD-associated spirochetes have phenotypic characteristics consistent with members of the genus TREPONEMA: Partial 16S ribosomal DNA (rDNA) sequence analysis of clonal isolates from California cattle showed that they comprise three phylotypes which cluster closely with human-associated Treponema spp. of the oral cavity (T. denticola and T. medium/T. vincentii) or genital area (T. phagedenis). The goal of our study was to apply 16S-23S rDNA intergenic spacer region (ISR) sequence analysis to the molecular typing of U.S. PDD-associated Treponema isolates. This methodology has potentially greater discriminatory power for differentiation of closely related bacteria than 16S rDNA analysis. We PCR amplified, cloned, and sequenced the ISRs from six California PDD-associated Treponema isolates and, for comparative purposes, one strain each of T. denticola, T. medium, T. vincentii, and T. phagedenis. Two ISRs that varied in length and composition were present in all the PDD-associated Treponema isolates and in T. denticola, T. medium, and T. phagedenis. ISR1 contained a tRNA(Ala) gene, while ISR2 contained a tRNA(Ile) gene. Only a single ISR (ISR1) was identified in T. vincentii. Comparative analyses of the ISR1 and ISR2 sequences indicated that the California PDD-associated Treponema isolates comprised three phylotypes, in agreement with the results of 16S rDNA analysis. PCR amplification of the 16S-tRNA(Ile) region of ISR2 permitted rapid phylotyping of California and Iowa PDD-associated Treponema isolates based on product length polymorphisms.     

Use of highly variable intergenic spacer sequences for multispacer typing of Rickettsia conorii strains

By use of the nearly perfectly colinear genomes of Rickettsia conorii and Rickettsia prowazekii, we compared the usefulness of three types of sequences for typing of R. conorii isolates: (i) 5 variable coding genes comprising the 16S ribosomal DNA, gltA, ompB, and sca4 (gene D) genes, which are present in both genomes, and the ompA gene, which is degraded in R. prowazekii; (ii) 28 genes degraded in R. conorii but intact in R. prowazekii, including 23 split and 5 remnant genes; and (iii) 27 conserved and 25 variable intergenic spacers. The 4 conserved and 23 split genes as well as the 27 conserved intergenic spacers each had identical sequences in 34 human and 5 tick isolates of R. conorii. Analysis of the ompA sequences identified three genotypes of R. conorii. The variable intergenic spacers were significantly more variable than conserved genes, split genes, remnant genes, and conserved spacers (P < 10(-2) in all cases). Four of the variable intergenic spacers (dksA-xerC, mppA-purC, rpmE-tRNA(fMet), and tRNA(Gly)-tRNA(Tyr)) had highly variable sequences; when they were combined for typing, multispacer typing (MST) identified 27 different genotypes in the 39 R. conorii isolates. Two batches from the same R. conorii strain, Malish (Seven), with different culture passage histories were found to exhibit the same MST type. MST was more discriminatory for strain genotyping than multiple gene sequencing (P < 10(-2)). Phylogenetic analysis based on MST sequences was concordant with the geographic origins of R. conorii isolates. Our study supports the usefulness of MST for strain genotyping. This tool may be useful for tracing a strain and identifying its source during outbreaks, including those resulting from bioterrorism.     

Genetic differentiation of Chinese isolates of Rickettsia sibirica by partial ompA gene sequencing and multispacer typing

Current data on rickettsiae and rickettsial diseases in China remain limited. Using partial ompA gene sequencing and multispacer typing, we identified 15 rickettsial isolates from China. All isolates were found to belong to Rickettsia sibirica subsp. sibirica. Four isolates from Dermacentor sinicus collected in Beijing, China, were fully identical to strain BJ-90, previously demonstrated to belong to R. sibirica subsp. sibirica despite antigenic and genotypic specificities. All 11 remaining isolates were similar to the R. sibirica subsp. sibirica type strain, 246. These were widely distributed in China in humans and different tick species. We emphasize the importance of surveying the distribution of R. sibirica in China.     

Absorption of Rickettsia rickettsii antibodies by Rickettsia rickettsii antigens in four diagnostic tests.

Immunoabsorption profiles were determined for Rickettsia rickettsii antigens used in four confirmatory tests for detection of antibodies to Rocky Mountain spotted fever. A human serum reactive in the four tests was absorbed with each test antigen and then reexamined by all four tests. The results indicated that the whole organism and complement-fixation antigen absorbed the whole array of antibodies to R. rickettsii, whereas erythrocyte-sensitizing substance coated on latex or erythrocytes did not. With erythrocyte-sensitizing substance coated on latex there was a decrease in titer when tested by the latex, indirect hemagglutination, and complement fixation tests but none when tested by the microimmunofluorescence test. With erythrocyte-sensitizing substance coated on sheep erythrocytes there was a decrease in titer when tested by indirect hemagglutination or complement fixation but none when tested by latex or microimmunofluorescence. These findings confirm that erythrocyte-sensitizing substance contains more than a single antigenic substance and plays a major role in the complement fixation test and a minor role in the microimmunofluorescence test.     

Genomic comparison of virulent Rickettsia rickettsii Sheila Smith and avirulent Rickettsia rickettsii Iowa

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. To identify genes involved in the virulence of R. rickettsii, the genome of an avirulent strain, R. rickettsii Iowa, was sequenced and compared to the genome of the virulent strain R. rickettsii Sheila Smith. R. rickettsii Iowa is avirulent in a guinea pig model of infection and displays altered plaque morphology with decreased lysis of infected host cells. Comparison of the two genomes revealed that R. rickettsii Iowa and R. rickettsii Sheila Smith share a high degree of sequence identity. A whole-genome alignment comparing R. rickettsii Iowa to R. rickettsii Sheila Smith revealed a total of 143 deletions for the two strains. A subsequent single-nucleotide polymorphism (SNP) analysis comparing Iowa to Sheila Smith revealed 492 SNPs for the two genomes. One of the deletions in R. rickettsii Iowa truncates rompA, encoding a major surface antigen (rickettsial outer membrane protein A [rOmpA]) and member of the autotransporter family, 660 bp from the start of translation. Immunoblotting and immunofluorescence confirmed the absence of rOmpA from R. rickettsii Iowa. In addition, R. rickettsii Iowa is defective in the processing of rOmpB, an autotransporter and also a major surface antigen of spotted fever group rickettsiae. Disruption of rompA and the defect in rOmpB processing are most likely factors that contribute to the avirulence of R. rickettsii Iowa. Genomic differences between the two strains do not significantly alter gene expression as analysis of microarrays revealed only four differences in gene expression between R. rickettsii Iowa and R. rickettsii strain R. Although R. rickettsii Iowa does not cause apparent disease, infection of guinea pigs with this strain confers protection against subsequent challenge with the virulent strain R. rickettsii Sheila Smith.     

Immunological Properties of Rickettsia rickettsii Purified by Zonal Centrifugation

The properties of Rickettsia rickettsii purified from infected chicken yolk sacs or mouse L cell cultures by sucrose density gradient centrifugation in a zonal rotor were examined in various ways. Rickettsiae derived from both L cells and yolk sacs had similar compositions: about 12% nitrogen, 1.5% phosphorus, 5% carbohydrate, and 30% fatty acids. On a dry-weight basis, purified rickettsiae were at least 2,000 times as effective as a commercial spotted fever vaccine in protecting guinea pigs against infection with spotted fever rickettsiae and mice against death from toxin of R. rickettsii. Gradient-purified rickettsiae (0.6 μg) induced a serological response in guinea pigs of the same magnitude as that stimulated by 1,600 μg of the commercial vaccine. Gradient-purified rickettsiae had little group reactivity in complement fixation tests but became anti-complementary upon storage. Microagglutination and hemagglutination tests with the purified antigen gave promise of usefulness in diagnosis of spotted fever. These results suggest that zonal centrifugation may be a valuable procedure for the preparation of R. rickettsii vaccine and diagnostic reagent.     

Detection of Rickettsia rickettsii DNA in clinical specimens by using polymerase chain reaction technology.

A polymerase chain reaction (PCR) procedure for detecting rickettsial DNA was developed and shown to be specific for Rickettsia rickettsii and R. conorii, the etiologic agents of Rocky Mountain spotted fever (RMSF) and Boutonneuse fever, respectively. Blood clots were obtained from nine confirmed RMSF patients and six controls and analyzed for the presence of rickettsial DNA by the PCR method. A defined region of the rickettsial genome was successfully amplified from seven of the nine clinical specimens tested; all six control specimens gave negative results. These findings indicate that R. rickettsii can be detected early after the onset of RMSF, possibly facilitating the decision regarding appropriate antibiotic therapy for some patients. Further refinement of PCR technology could make this procedure a mainstay in the clinical laboratory.     

Quantitative analyses of variations in the injury of endothelial cells elicited by 11 isolates of Rickettsia rickettsii.

Eleven isolates of spotted fever group rickettsiae from the blood of patients or ixodid ticks from North and South America were characterized. All isolates were identified as Rickettsia rickettsii using restriction fragment length polymorphism analysis of a 532-bp rOmpA gene fragment obtained by PCR. The ability of the R. rickettsii isolates to elicit cytopathic effects and parameters of oxidative injury were examined in cultured human EA.hy 926 endothelial cells. Cytopathic effects were determined by direct observation of infected cultures, by measuring the release of cytoplasmic lactate dehydrogenase (LDH), and by determination of intracellular pools of peroxide and reduced glutathione. Four biotypes of R. rickettsii were defined. Group I included two highly cytopathic isolates from Montana, Bitterroot and Sheila Smith, and three isolates from Maryland, North Carolina, and Brazil. These isolates rapidly damaged cells, released large amounts of cytoplasmic LDH, caused accumulation of intracellular peroxide, and depleted intracellular pools of reduced glutathione. Group II contained three isolates, two from Montana, Hlp#2 and Lost Horse Canyon, and an isolate from Colombia, which were similar to group I but caused either lower responses in LDH release or smaller changes in intracellular peroxide levels. The group III isolates, Sawtooth from Montana and 84JG from North Carolina, caused lower cellular injury by all measures. Group IV isolate Price T from Montana was the least cytopathic and caused minimal alterations of all parameters measured. Understanding the molecular basis for the varied cellular injury caused by different isolates of R. rickettsii may contribute to improved treatment of Rocky Mountain spotted fever and to the rapid identification of those isolates which are more likely to cause fulminant disease.     

Dynamics of Actin-Based Movement by Rickettsia rickettsii in Vero Cells

Actin-based motility (ABM) is a virulence mechanism exploited by invasive bacterial pathogens in the genera Listeria, Shigella, and Rickettsia. Due to experimental constraints imposed by the lack of genetic tools and their obligate intracellular nature, little is known about rickettsial ABM relative to Listeria and Shigella ABM systems. In this study, we directly compared the dynamics and behavior of ABM of Rickettsia rickettsii and Listeria monocytogenes. A time-lapse video of moving intracellular bacteria was obtained by laser-scanning confocal microscopy of infected Vero cells synthesizing beta-actin coupled to green fluorescent protein (GFP). Analysis of time-lapse images demonstrated that R. rickettsii organisms move through the cell cytoplasm at an average rate of 4.8 +/- 0.6 micrometer/min (mean +/- standard deviation). This speed was 2.5 times slower than that of L. monocytogenes, which moved at an average rate of 12.0 +/- 3.1 micrometers/min. Although rickettsiae moved more slowly, the actin filaments comprising the actin comet tail were significantly more stable, with an average half-life approximately three times that of L. monocytogenes (100.6 +/- 19.2 s versus 33.0 +/- 7.6 s, respectively). The actin tail associated with intracytoplasmic rickettsiae remained stationary in the cytoplasm as the organism moved forward. In contrast, actin tails of rickettsiae trapped within the nucleus displayed dramatic movements. The observed phenotypic differences between the ABM of Listeria and Rickettsia may indicate fundamental differences in the mechanisms of actin recruitment and polymerization.     

Serological typing of spotted fever group Rickettsia isolates from Zimbabwe.

Eight rickettsialike organisms were isolated in tissue culture from ticks of dogs and cattle from various areas of Zimbabwe. These isolates and a reference strain, Rickettsia conorii Simko, were tested by microimmunofluorescence against homologous and heterologous antisera raised in mice. From the titers obtained by this method, specificity differences (SPDs) were calculated between each of the rickettsiae. Only small serological differences were detected among the isolates from ticks obtained from dogs (mean SPD, 0.5) and also among the isolates from ticks obtained from cattle (mean SPD, 0.3). However, when isolates from ticks obtained from dogs and cattle were compared, the serological differences were greater (mean SPD, 1.3). The isolates from ticks obtained from dogs were found to be very similar serologically to the Simko strain of R. conorii (mean SPD, 0.8), while three of four isolates from ticks obtained from cattle were different enough (SPD, greater than or equal to 3) to be identified as separate serotypes. These findings indicate that there is a high degree of antigenic heterogeneity among the tick-transmitted spotted fever group rickettsiae in Zimbabwe.     

Human endothelial cell culture plaques induced by Rickettsia rickettsii.

Primary cultures of human umbilical vein endothelial cells were inoculated with plaque-purified Rickettsia rickettsii. After adsorption of rickettsiae, monolayers were overlaid with medium containing 0.5% agarose. Small plaques appeared on day 4 postinoculation, and distinct 1- to 2-mm plaques were observed on day 5. Plaquing efficiency was less than that of primary chicken embryo cells in the same medium. Human endothelial cell monolayers were susceptible to infection by R. rickettsii and underwent necrosis as demonstrated by supravital staining. The topographic association of endothelial cell necrosis and rickettsial infection in the plaque model confirmed the direct cytopathic effect of R. rickettsii on human endothelium. Uninfected cells appeared normal by supravital staining and transmission electron microscopy. This model offers the possibility of investigating rickettsial pathogenesis and mechanisms of enhanced severity of Rocky Mountain spotted fever in specific genetically determined conditions.     

Heparin protects human endothelial cells infected by Rickettsia rickettsii.

Routine culture of endothelial cells currently includes the use of heparin, which significantly reduces cell doubling time and increases cell population size. Heparin protects cultured arterial endothelial cells from damage by toxic oxygen metabolites produced by the action of xanthine and xanthine oxidase. Because of our hypothesis implicating free radicals in cell injury caused by Rickettsia rickettsii, we have carried out a series of experiments to examine the effects of heparin on injury to endothelial cells infected by this microorganism. These studies showed that heparin does not inhibit replication of R. rickettsii in the cytoplasm of endothelial cells. Furthermore, heparin appears to exhibit a protective effect on the infected host cell as measured by (i) reduced plaque size, (ii) increased longevity of the cell monolayer, (iii) reduction in the amount of lactic dehydrogenase released from infected cells, and (iv) reduction in the levels of intracellular peroxides formed in infected cells. Electron microscopic studies also show a significant reduction in dilatation of the rough-surfaced endoplasmic reticulum of the infected cells in the presence of heparin. These observations appear to lend additional support to involvement of an oxidative mechanism in human endothelial cell injury caused by R. rickettsii.     

Molecular typing of echovirus serotype 4 isolates

We report on clinical samples Stuttgart/97, Berlin/99 and Jasi/99 associated with aseptic meningitis. All three samples contained echovirus 4 (E4) but Stuttgart/97 was simultaneous infected with echovirus 30 (E30). The genetic relationship of the E4 strains was assessed using RT-PCR and direct sequencing of amplicons derived from the genomic region encoding the capsid protein VP1. The sequences have been compared with each other and with sequences of further E4 strains obtained from GenBank. The analysis confirms that sequences of recent isolates have drifted away from elderly strains over a longer period of time. Several amino acid changes in assumed antigenic sites of the VP1 gene may be sufficient to cause changes in antigenic specificity and therefore they may be a reason for failure of serological typing of some new antigenic E4 variants.     

Molecular typing of Neisseria perflava clinical isolates

Multilocus sequence typing and pulsed‐field gel electrophoresis were used to type 22 commensal isolates of Neisseria perflava collected by swabbing from neutropenic patients. High genetic diversity was found among our N. perflava clinical isolates.     

Role of Phospholipase-Associated Penetration Mechanism in Cell Injury by Rickettsia rickettsii

The cholesterol receptor-binding drugs amphotericin B and digitonin reduced rickettsial plaque formation, presumably by blocking rickettsial attachment, even when added to the plaque model on day 4 post-inoculation when infected foci were well established. The phospholipase inhibitor phentermine reduced plaque formation, presumably by inhibiting the phospholipase-associated entry step of penetration.     

Detection of Rickettsia rickettsii antibodies in human sera by crossed immunoelectrophoresis

To identify Rickettsia rickettsii antigens of immunological importance, we examined sera from patients with serologically confirmed cases of Rocky Mountain spotted fever by crossed immunoelectrophoresis for antibodies to antigens extracted from the R strain of R. rickettsii with the detergent Triton X-100. Sixteen antigens were identified in the detergent extract by crossed immunoelectrophoresis with a hyperimmune rabbit serum raised against whole rickettsiae. When the rabbit antiserum was placed in the reference gel and patient sera were placed in the intermediate gel, antibodies to one or more antigens were detected in 61 of 71 North Carolina sera, all of 7 Oklahoma sera, and 9 of 10 Montana sera obtained from 1 day to 40 years after onset of Rocky Mountain spotted fever. Antibodies to antigens 1 and 16 were found as early as 1 day after onset of illness, and antibody to 16 was found in 20 of 29 sera obtained within the first 7 days of illness. Antibodies to antigens 2 and 3 generally did not appear until the third week of illness but were found in six of seven serum samples collected 4 to 40 years after onset of Rocky Mountain spotted fever. Antibodies to R. rickettsii antigens 1, 7, 8, and 16 were found in sera from patients with illnesses caused by other etiological agents. Four of the Oklahoma and Montana sera from Rocky Mountain spotted fever patients, but none of the North Carolina sera, had antibodies to antigen 12. Sera containing antibodies against antigens 3 and 14 prevented death of mice challenged with two 50% lethal doses of R. rickettsii.     

Detection of fibrils associated with Rickettsia rickettsii.

The ultrastructural appearance of the "halozone" formed at the interface between the spotted fever agent Rickettsia rickettsii and the cytoplasm of persistently infected cultured vole cells (Microtus pennsylvanicus) was studied by transmission electron microscopy. In sections of epoxy-embedded specimens stained with uranyl acetate and lead citrate, the halozone appeared clear and devoid of ultrastructural features. However, when unembedded preparations of whole infected cells were examined at 1,000 kV, fine structural features were observed within the halozone. These features, associated with the rickettsial outer membrane, were more clearly detectable when the infected cells were extracted with the detergent Triton X-100 before fixation. Under such conditions, long extensions of the rickettsial outer membrane, microfilament-like structures attached to that membrane, and extensive attachments between adjacent rickettsiae were seen. The fine structural features within the rickettsial halozone were also seen at 75 kV when unembedded sections were prepared from polyethylene glycol-embedded specimens. Thus, epoxy-embedding medium obscures the fine structural features within the halozone surrounding the rickettsiae in infected cells.     

Molecular cloning, sequence and characterization of cjsT, a putative protease from Rickettsia rickettsii

The cloning and sequencing of a gene from Rickettsia rickettsii which confers haemolytic activity on Escherichia coli strain TB1 is described. The open reading frame of the haemolysis-promoting gene, cjsT, is 1041 bp and encodes a putative protein with a molecular mass of 33 825 Da. CjsT has high sequence similarity to several bacterial proteases, particularly type IV signal peptidases. Cell lysates from an E. coli clone containing cjsT in pUC19 (pJON1) exhibited greater protease activity in functional assays than found in E. coli containing pUC19 alone. Disruption of the cjsT gene by insertional inactivation with a kanamycin cassette reduced both the protease and haemolytic activities conferred by cjsT. The protease inhibitors antipain and diisopropylfluorophosphate (DFP) both reduced the proteolytic activity of pJON1. The mechanism by which the R. rickettsii cjsT promotes haemolysis in E. coli remains unclear.     

Demonstration of Rickettsia rickettsii in the Rhesus Monkey by Immune Fluorescence Microscopy

Indirect immune fluorescence was used to detect rickettsiae in the tissues of a primate inoculated subcutaneously with Rickettsia rickettsii. Rickettsiae were identified by indirect immune fluorescence predominantly in skin, skeletal muscle, scrotum, testicles, nares, heart, kidney, liver, brain, spleen, pancreas, and larynx. Cell culture assay confirmed the presence of infectious organisms in those specimens.