Distribution of immunogenic epitopes on the two major immunodominant proteins (rOmpA and rOmpB) of Rickettsia conorii among the other rickettsiae of the spotted fever group.

Forty-four monoclonal antibodies were raised against strain Seven, the type strain of Rickettsia conorii. Of these 44 monoclonal antibodies, 13, 27, and 4 were demonstrated to be directed against the 116-kDa protein (rOmpA), the 124-kDa protein (rOmpB), and lipopolysaccharide-like antigen, respectively. The antiprotein monoclonal antibodies were found to be directed against 29 distinct epitopes, which were located on the two major immunodominant proteins discussed above. Further analysis showed that strain-specific epitopes were located on the rOmpA protein and species- and subgroup-specific epitopes were located on the rOmpB protein. R. conorii Manuel, Indian tick typhus rickettsia, and Kenya tick typhus rickettsia also possessed all 29 epitopes, whereas the other rickettsiae of the spotted fever group (SFG) expressed between 3 and 25 epitopes, with the exception of Rickettsia helvetica, R. akari, and R. australis which did not possess any epitopes. Additional analyses by Western immunoblotting confirmed that the epitopes shared among the SFG rickettsiae were located on the same two high-molecular-mass proteins as on R. conorii. However, although epitopes on the R. conorii rOmpB protein were expressed on the rOmpB proteins of most other SFG rickettsiae, some were found on the rOmpA proteins of R. aeschlimannii, R. rickettsii, and R. rhipicephali. Both proteins possessing the common epitopes were found to have different sizes in the SFG rickettsial species. The different distributions of common epitopes in the SFG rickettsiae were also used to build a taxonomic dendrogram, which demonstrated that all the R. conorii strains formed a relatively independent cluster within the SFG rickettsiae and was generally consistent with previously proposed taxonomies.     

Evaluation of a PCR assay for quantitation of Rickettsia rickettsii and closely related spotted fever group rickettsiae

A spotted fever rickettsia quantitative PCR assay (SQ-PCR) was developed for the detection and enumeration of Rickettsia rickettsii and other closely related spotted fever group rickettsiae. The assay is based on fluorescence detection of SYBR Green dye intercalation in a 154-bp fragment of the rOmpA gene during amplification by PCR. As few as 5 copies of the rOmpA gene of R. rickettsii can be detected. SQ-PCR is suitable for quantitation of R. rickettsii and 10 other genotypes of spotted fever group rickettsiae but not for R. akari, R. australis, R. bellii, or typhus group rickettsiae. The sensitivity of SQ-PCR was comparable to that of a plaque assay using centrifugation for inoculation. The SQ-PCR assay was applied successfully to the characterization of rickettsial stock cultures, the replication of rickettsiae in cell culture, the recovery of rickettsial DNA following different methods of extraction, and the quantitation of rickettsial loads in infected animal tissues, clinical samples, and ticks.     

Rapid fingerprinting of Helicobacter pylori by polymerase chain reaction and restriction fragment length polymorphism analysis.

A simple and reliable technique was developed for differentiating Helicobacter pylori strains by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified DNAs. Oligonucleotide primer pairs developed to the urease, 48-kDa stress protein (htrA), and 26-kDa antigen-encoding genes were used to amplify fragments of the appropriate size from crude boiled cell preparations. The PCR-amplified products were digested with Sau3A, HaeIII, MspI, AluI, MluI, HinfI, and XbaI restriction endonucleases. Restriction fragment length polymorphisms were particularly evident within the urease and htrA genes and were easily detected by Sau3A, HaeIII, MspI, and AluI restriction endonuclease analysis. Double digestion of these separately amplified products or restriction analysis of multiple PCR-amplified fragments was found to discriminate 17 of 17 (100%) H. pylori strains which had unique genomic DNA fingerprints. Results of an investigation of multiple isolate sets obtained from patients before and after therapy was consistent with the hypothesis that treatment failures were due to the persistence of the same strain but did not discount the possibility that the patients were reinfected with a strain shared by family members or close contacts. The results indicate that the PCR-restriction endonuclease analysis method can be applied directly to biopsy samples, has the potential to fingerprint H. pylori isolates rapidly, and may permit detailed epidemiological investigations on the transmission of this important pathogen.     

Detection of Rickettsia japonica in Haemaphysalis longicornis ticks by restriction fragment length polymorphism of PCR product.

PCR was applied to the detection of Rickettsia japonica, the causative agent of Oriental spotted fever (OSF), in ticks collected at two sites of the Muroto area on Shikoku Island, a major area in Japan where OSF is endemic. Primer pair Rr190.70p and Rr190.602n of the R. rickettsii 190-kDa antigen gene sequence of Regnery and others (R.L. Regnery, C.L. Spruill, and B.D. Plikaytis, J. Bacteriol. 173:1576-1589, 1991) primed the DNA extracted from Haemaphysalis longicornis ticks but not those extracted from Haemaphysalis formosensis, Haemaphysalis flava, Haemaphysalis hystricis, or Amblyomma testudinarium ticks. Digestion of the amplification product with the restriction endonucleases PstI and AluI produced the restriction fragment length polymorphism pattern specific to R. japonica. The HindIII and MspI digests gave restriction fragment length polymorphism patterns identical to those of the PCR product from R. japonica DNA. Hemolymph preparations of H. longicornis ticks were demonstrated to contain rod-shaped organisms that were detected by immunofluorescence with the monoclonal antibody specific to R. japonica species. The primer pair did not amplify the DNA of a laboratory colony of H. longicornis ticks originally collected at an area where OSF is not endemic. Our results provided evidence that H. longicornis ticks might be an arthropod reservoir for R. japonica and a vector of OSF.     

Polymerase chain reaction-based restriction fragment length polymorphism analysis of a fragment of the ribosomal operon from Rochalimaea species for subtyping.

Restriction endonuclease analysis of a polymerase chain reaction-amplified DNA fragment which included the spacer region between the genes coding for 16S and 23S rRNAs and a portion of the gene coding for 23S rRNA (spacer + 23S) was done on 10 previously characterized clinical isolates of Rochalimaea henselae, one clinical isolate of Rochalimaea quintana, and the type strains of R. henselae, R. quintana, Rochalimaea vinsonii, and Bartonella bacilliformis. Brucella abortus DNA was not amplified by the primer set used. The clinical isolates of Rochalimaea were obtained from blood or tissue from patients with and without preexisting disease. The amplicon from each strain was digested with five endonucleases (AluI, HaeIII, TaqI, HinfI, and MseI). AluI and HaeIII were useful in species differentiation and subtyping of R. henselae. R. henselae isolates showed six different restriction patterns with AluI and four patterns with HaeIII. TaqI, HinfI, and MseI were useful only in species differentiation. These observations indicate that PCR amplification of the spacer + 23S region of the ribosomal DNA of Rochalimaea spp., along with restriction endonuclease analysis, allows differentiation of Rochalimaea spp. from closely related genera, differentiation among the species within Rochalimaea, and differentiation of strains within R. henselae. The subtyping potential of this method may be useful for further clinical and epidemiologic studies of the spectrum of diseases caused by R. henselae.     

Strain analysis of hepatitis B virus on the basis of restriction endonuclease analysis of polymerase chain reaction products.

To assess the value of classifying hepatitis B virus (HBV) strains at the genomic level with products from the polymerase chain reaction (PCR), an HBV PCR DNA typing procedure was developed. The design of this method was based on the selective sensitivity of the PCR product to digestion with different restriction endonucleases and on the size of the fragment resulting from a specific nuclease digestion. On the basis of published nucleotide sequences of different HBV subtypes, a set of primers was selected within the preS-S region. One of the primers was 1679F containing 25 nucleotides (5'-GGGTGGAGCCCTCAGGCTCAGGGCA-3'), and the other was 2254R containing 24 nucleotides (5'-GAAGATGAGGCATAGCAGCAGGAT-3'). All of the reactive sera produced the same sized 575-bp identification band. The product was subjected to digestion by a selected panel of restriction endonucleases. By using prototype HBV of known subtype as a model, these restriction nuclease maps of the PCR product were subtype specific. Most adr subtype clinical samples produced predicted PCR DNA restriction nuclease fragmentation in accordance with the nucleotide sequence of the prototype virus. Occasionally, samples of either the adw or the ayw subtype gave discrepant results in which they appeared to be a different subtype or were resistant to the restriction nuclease digestion. This genetic alteration was consistent in the paired specimens from sexual transmission cases. These results show that the described procedures are applicable to HBV strain assessment.     

Genomic and proteinic characterization of strain S, a rickettsia isolated from Rhipicephalus sanguineus ticks in Armenia.

Strain S, a spotted fever group (SFG) rickettsia isolated from Rhipicephalus sanguineus ticks collected in Armenia, was identified. Microimmunofluorescence, sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis and Western immunoblotting, PCR and then restriction fragment length polymorphism analysis, pulsed-field gel electrophoresis, and 16S rRNA gene sequencing were used to compare strain S with reference isolates. Strain S was found to possess proteinic, antigenic, and genomic patterns which were unique among SFG rickettsiae. Strain S is characterized by its high degree of pathogenicity for experimental animals, but its role as a potential human pathogen should be determined. The role of R. sanguineus ticks in the epidemiology of SFG rickettsiae is discussed.     

Phylogenetic Placement of Rickettsiae from the Ticks Amblyomma americanum and Ixodes scapularis

A rickettsial isolate (isolate MOAa) belonging to the spotted fever group (SFG) was obtained from the lone star tick Amblyomma americanum. We used PCR to characterize the genes for the rickettsial outer membrane proteins rOmpA and rOmpB. We sequenced the PCR products (domains I of both the rompA gene and the rompB gene) of MOAa and WB-8-2, another rickettsial isolate from A. americanum. To place MOAa and WB-8-2 and two other nonpathogenic isolates (Rickettsia rickettsii Hlp2 and Rickettsia montana M5/6) with respect to their putative sister species, we included them in a phylogenetic analysis of 9 Rickettsia species and 10 Rickettsia strains. Our phylogenetic results implied three evolutionary lineages of SFG rickettsiae and that WB-8-2 and MOAa were most closely related to R. montana. New World isolates were not the most closely related to each other (they did not form a clade). Rather, our results supported four independent origins (introductions) of rickettsiae into North America from different Old World regions. The results of our phylogenetic analysis did not support the hypothesis of a stable coevolution of rickettsiae and their tick hosts. Finally, we examined the rompA gene of a nonpathogenic rickettsial symbiont isolated from the tick Ixodes scapularis. In a phylogenetic analysis, the symbiont was placed as the sister to R. montana and its isolates. The relationship of this symbiont to R. montana raised questions as to the potential origin of pathogenic SFG rickettsiae from nonpathogenic tick symbionts, or vice versa.     

Determination of genome size and restriction fragment length polymorphism of four Chinese rickettsial isolates by pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was used to determine the genome size and the restriction fragment length polymorphism (RFLP) of four new Chinese isolates of spotted fever group (SFG) rickettsiae. The genome size of the isolates Ha-91, BJ-90, 054 and 036 was 1,253 kb, 1,236 kb, 1,272 kb, and 1,272 kb, respectively. The isolates 054 and 036 had identical RFLP profiles. All the isolates differed in the properties under study from the so far known SFG rickettsiae. The unique RFLP profiles of the isolates supported our opinion that they are new strains of SFG rickettsiae or even new species of SFG rickettsiae.     

Evaluation of PCR-based assay for diagnosis of spotted fever group rickettsiosis in human serum samples

A nested PCR assay was developed for the detection of spotted fever group (SFG) rickettsiae in serum samples. The assay was based on specific primers derived from the rickettsial outer membrane protein B gene (rompB) of Rickettsia conorii. An SFG rickettsia-specific signal is obtained from R. akari, R. japonica, R. sibirica, and R. conorii. Other bacterial species tested did not generate any signal, attesting to the specificity of the assay. As few as seven copies of the rompB gene of R. conorii could be detected in 200 microl of serum sample. The assay was evaluated with a panel of sera obtained from patients with acute-phase febrile disease tested by immunofluorescent antibody assay (IFA). The SFG rickettsia-specific DNA fragment was detected in 71 out of 100 sera, which were proven to have immunoglobulin M antibodies against SFG rickettsial antigen by IFA. The results were further confirmed by restriction fragment length polymorphism and sequencing analysis of the DNA fragments. The results indicated that this PCR assay is suitable for the diagnosis of spotted fever group rickettsiosis in Korea.     

Comprehensive typing of DR52 (DRB3)-associated DRB1 and DRB3 alleles by PCR-RFLP

Abstract: The DR52-associated DRB1 and DRB3 alleles were resolved by PCR-RFLP. Second exon was amplified using four primer pairs (groups 1–4) for DRB1 and a pair for DRB3 alleles. Except for three endonucleases, all others had either none or only one site for a specific amplified product. Group 1 primers amplify 10 DRB1 alleles (DRB1*0302, 1101, 1302, 1303, 1305, 1307, 1402, 1403, 1407 and 1409). All but one pair, DRB1*1402 from 1409, could be resolved using seven endonucleases (ApaI, SacII, FokI, AvaII, BsaAI, BsrBI and SfaNI). Group 2 consisted of four alleles (DRB1*1201, 1202, 1404 and 1411) that can be resolved along with co-amplified DRB1*0804 and 0806 using five endonucleases (AvaII, SacII, FokI, HaeII and RsaI). Group 3 primers amplify 15 DRB1 alleles (DRB1*0301, 0303, 1102, 1103, 1104, 1107, 1301, 1304, 1306, 1308, 1401, 1405, 1406, 1408 and 14-New), which can be resolved using nine enzymes (KpnI, AvaII, FokI, SacII, HaeII, BsrBI, SfaNI, DdeI and RsaI). BsrBI, a new endonuclease, can resolve DRB1*1301 from 1306 and the previously unresolved allele DRB1*1103 from 1104. DRB1*1410, co-amplified with DR4 group-specific primers, is resolved with PstI which cleaves all DR4 alleles but not DRB1*1410. All four DRB3 alleles (DRB3*0101, 0201, 0202 and 0301) and their heterozygotes are resolved using two endonucleases, RsaI and HphI. Thirty-four DR52-associated alleles and their heterozygotes can be unambiguously resolved, except for DRB1*1402 from 1409. Thus, PCR-RFLP remains an effective method for high-resolution HLA-DR typing. Furthermore, PCR-RFLP can complement the evolving PCR-SSP method for allele-specific typing using a minimal number of restriction endonucleases.     

Detection and identification of spotted fever group rickettsiae in Dermacentor species from southern California

Dermacentor occidentalis Marx and Dermacentor variabilis (Say) commonly bite humans in California. These Dermacentor species may play a role in transmitting spotted fever group (SFG) rickettsiae to humans in many parts of the state where Dermacentor andersoni Stiles, a known vector for the etiologic agent of Rocky Mountain spotted fever, Rickettsia rickettsii, is absent. However, the specific rickettsial agents present in these ticks and their current prevalence are poorly understood. In total, 365 D. occidentalis and 10 D. variabilis were collected by flagging vegetation at 16 sites in five counties of southern California. The presence of SFG rickettsial DNA in these ticks was detected with rOmpA and GltA gene polymerase chain reaction (PCR) assays. The rickettsial species were identified by sequencing PCR amplicons. Of 365 D. occidentalis, 90 (24.7%) contained R. rhipicephali DNA, 28 (7.7%) contained DNA of unclassified genotype 364D, two (0.55%) contained R. bellii DNA, and one (0.3%) contained R. rickettsii DNA. Of 10 D. variabilis, four (40%) contained only R. rhipicephali. Four new genotypes of R. rhipicephali were discovered. For the first time, we detected R. rickettsii in D. occidentalis. Our study provides the first molecular data on the prevalence and species identification of SFG rickettsiae circulating in populations of these California ticks. Because neither D. variabilis nor R. rickettsii were abundant, 364D should be evaluated further as a potential cause of human SFG rickettsioses in southern California.     

Differentiation of Shigella flexneri strains by rRNA gene restriction patterns.

We studied the restriction endonuclease cleavage patterns of rRNA genes (ribotypes) of 72 clinical isolates of Shigella flexneri representing eight serotypes to determine whether ribotyping could be used to distinguish S. flexneri strains and to compare the discriminating ability of the method with that of serotyping. By using a cloned Escherichia coli rRNA operon as the probe, Southern blot hybridization of restriction endonuclease-digested total DNA was carried out. Ribotyping of the isolates with each of the five restriction endonucleases BamHI, EcoRI, HindIII, PstI, and SalI generated reproducible restriction patterns. However, HindIII produced the optimum digestion pattern of the rRNA genes and was more useful than the other enzymes used in differentiating strains. Analysis of the 72 isolates showed 11 different HindIII cleavage patterns of their rRNA genes. Four of these HindIII-generated ribotypes could be further differentiated into two to four subribotypes by using PstI. The results indicate that ribotyping has an application for differentiation of S. flexneri strains and can complement serotyping. Definition of strains in terms of both serotype and ribotype may be of greater use in epidemiological studies.     

Evaluation of PCR-Based Assay for Diagnosis of Spotted Fever Group Rickettsiosis in Human Serum Samples

A nested PCR assay was developed for the detection of spotted fever group (SFG) rickettsiae in serum samples. The assay was based on specific primers derived from the rickettsial outer membrane protein B gene (rompB) of Rickettsia conorii. An SFG rickettsia-specific signal is obtained from R. akari, R. japonica, R. sibirica, and R. conorii. Other bacterial species tested did not generate any signal, attesting to the specificity of the assay. As few as seven copies of the rompB gene of R. conorii could be detected in 200 μl of serum sample. The assay was evaluated with a panel of sera obtained from patients with acute-phase febrile disease tested by immunofluorescent antibody assay (IFA). The SFG rickettsia-specific DNA fragment was detected in 71 out of 100 sera, which were proven to have immunoglobulin M antibodies against SFG rickettsial antigen by IFA. The results were further confirmed by restriction fragment length polymorphism and sequencing analysis of the DNA fragments. The results indicated that this PCR assay is suitable for the diagnosis of spotted fever group rickettsiosis in Korea.     

Genetic variation in Staphylococcus aureus coagulase genes: potential and limits for use as epidemiological marker.

To perform coagulase gene typing, the repeated units encoding hypervariable regions of the Staphylococcus aureus coagulase gene were amplified by the PCR technique; this was followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP) patterns. In order to assess the discriminatory power of this typing method, 30 epidemiologically unrelated S. aureus strains which differed by their pulsed-field gel electrophoresis patterns were examined. Although 18 of the 30 strains had unique and unshared AluI RFLP patterns, there were only four observed patterns in the remaining 12 strains. This finding indicated that unrelated strains may share identical AluI RFLP patterns. To elucidate the degree of genetic variation in the C-terminus-encoding loci within the coagulase genes, the PCR products of these 12 strains were subjected to Taq polymerase-mediated sequencing. Sequence analysis confirmed the AluI recognition sites in each of the four RFLP groups and demonstrated that AluI appears to yield the highest RFLP in restriction enzyme analysis. By their DNA sequences the majority of strains sharing common AluI groups could be clearly differentiated from each other and revealed between 93.2 and 98.5% homology. When we determined the nucleotide sequences of two strains after six subcultivations no significant alterations were observed. Because the discriminatory power of the current coagulase gene typing method is not great enough to be used as the sole method to type S. aureus, additional techniques are necessary. Sequence analysis of the repeated unit-encoding region for the typing of S. aureus may be potentially useful as an alternative to other current molecular typing techniques.     

Sensitive detection of Helicobacter pylori by using polymerase chain reaction.

A polymerase chain reaction (PCR) for the specific detection of Helicobacter pylori was developed with a single primer pair derived from the nucleotide sequence of the urease A gene of H. pylori. We achieved specific amplification of a 411-bp DNA fragment in H. pylori. After 35 cycles of amplification, the product could be detected by agarose gel electrophoresis and contained conserved single HinfI and AluI restriction sites. This fragment was amplified in all 50 strains of H. pylori tested, but it was not detected in other bacterial species, showing the PCR assay to be 100% specific. PCR DNA amplification was able to detect as few as 10 H. pylori cells. PCR detected H. pylori in 15 of 23 clinical human gastric biopsy samples, whereas culturing and microscopy detected H. pylori in only 7 of the samples found to be positive by PCR. Additional primer pairs based on the urease genes enabled the detection of H. pylori in paraffin-embedded human gastric biopsy samples. The detection of H. pylori by PCR will enable both retrospective and prospective analyses of clinical samples, elucidating the role of this organism in gastroduodenal disease.     

Differentiation of Helicobacter pylori strains directly from gastric biopsy specimens by PCR-based restriction fragment length polymorphism analysis without culture.

Recent studies have shown the usefulness of PCR-based restriction fragment length polymorphism (RFLP) analysis for differentiating Helicobacter pylori strains isolated by culture. For this study, a PCR-based RFLP assay was developed for directly typing H. pylori strains from gastric biopsy specimens. Nineteen gastric biopsy specimens obtained from patients undergoing endoscopy for gastrointestinal complaints were cultured for isolation of H. pylori. Genomic DNA preparations from these gastric biopsy specimens and the corresponding H. pylori isolates were tested by our PCR-based RFLP assay. The 1,179-bp H. pylori DNA fragments amplified by the PCR assay were digested with the restriction enzymes HhaI, MboI, and AluI and analyzed by agarose gel electrophoresis. HhaI, MboI, and AluI digestion produced 11, 10, and 6 distinguishable digestion patterns, respectively, from the 19 H. pylori isolates tested and generated 13, 11, and 6 different patterns, respectively, from the 19 gastric biopsy specimens. The patterns from 13 of the 19 gastric biopsy specimens matched those of the H. pylori isolates from the corresponding patients. The patterns from the remaining six biopsy specimens appeared to represent infection by two strains of H. pylori; the pattern of one strain was identical to that of the isolate from the corresponding patient. By combining all the restriction enzyme digestion patterns obtained by using HhaI, MboI, and AluI, we observed 19 distinct RFLP patterns from the 19 specimens. The results suggest that the PCR-based RFLP analysis method may be useful as a primary technique to identify and distinguish H. pylori strains directly from gastric biopsy specimens without culture of the organisms.