Incongruent effects of two isolates of Rickettsia conorii on the survival of Rhipicephalus sanguineus ticks.

Rickettsia conorii, the etiologic agent of Mediterranean spotted fever is widely distributed in Southern Europe, the Middle East, Africa, India and the Caspian region. In the Mediterranean region, the brown dog tick, Rhipicephalus sanguineus, is the recognized vector of R. conorii. To study tick-pathogen relationships and pathogenesis of infection caused in model animals by the bite of an infected tick, we attempted to establish a laboratory colony of Rh. sanguineus persistently infected with R. conorii. Rhipicephalus sanguineus ticks of North American and Mediterranean origin were exposed to R. conorii isolates of African (R. conorii conorii strain Malish) and Mediterranean (R. conorii israelensis strain ISTT) origin. Feeding of ticks upon infected mice and dogs, intra-hemocoel inoculation, and submersion in suspensions of purified rickettsiae were used to introduce the pathogen into uninfected ticks. Feeding success, molting success and the longevity of molted ticks were measured to assess the effects of R. conorii on the survival of Rh. sanguineus. In concordance with previously published results, Rh. sanguineus larvae and nymphs from both North American and Mediterranean colonies exposed to R. conorii conorii Malish experienced high mortality during feeding and molting or immediately after. The prevalence of infection in surviving ticks did not exceed 5%. On the other hand, exposure to ISTT strain had lesser effect on tick survival and resulted in 35-66% prevalence of infection. Rh. sanguineus of Mediterranean origin were more susceptible to infection with either strain of R. conorii than those from North America. Previous experimental studies had demonstrated transovarial and transstadial transmission of R. conorii in Rh. sanguineus; however, our data suggest that different strains of R. conorii may employ different means of maintenance in nature. The vertebrate host may be a more important reservoir than previously thought, or co-feeding transmission between different generations of ticks may obviate or lessen the requirement for transovarial maintenance of R. conorii.

Rickettsial infections and their clinical presentations in the Western Province of Sri Lanka: a hospital-based study.

BACKGROUND: Rickettsial infections are re-emerging. A study of the geographical distribution of rickettsial infections, their clinical manifestations, and their complications would facilitate early diagnosis. METHODS: Thirty-one selected patients from the Western Province of Sri Lanka were studied for rickettsial species, clinical manifestations, and complications. RESULTS: Of 31 patients with possible rickettsioses, 29 (94%) fell into the categories of confirmed, presumptive, or exposed cases of acute rickettsial infections (scrub typhus was diagnosed in 19 (66%), spotted fever group in eight (28%)). Early acute infection or past exposure was suggested in two (7%) cases; cross-reactivity of antigens or past exposure to one or more species was suggested in nine (31%). Seventeen out of 19 (89%) patients with scrub typhus had eschars. Nine out of 29 (32%) patients had a discrete erythematous papular rash: seven caused by spotted fever group, two by scrub typhus. Severe complications were pneumonitis in eight (28%), myocarditis in five (17%), deafness in four (14%), and tinnitus in two (7%). The mean duration of illness before onset of complications was 12.0 (SD 1.4) days. All patients except one made a good clinical recovery with doxycycline or a combination of doxycycline and chloramphenicol. CONCLUSIONS: In a region representing the low country wet zone of Sri Lanka, the main rickettsial agent seems to be Orientia tsutsugamushi. Delay in diagnosis may result in complications. All species responded well to current treatment.

Acute hearing loss due to scrub typhus: a forgotten complication of a reemerging disease.

We describe 6 patients with scrub typhus who presented with acute hearing loss, a forgotten complication of this reemerging disease. They were admitted with fever of 10-14 days' duration and had clinical evidence of deafness and pneumonitis. Five patients had eschars, which prompted the diagnosis of typhus fever and led to early institution of treatment. Deafness has been described as a clue to the diagnosis of scrub typhus; awareness of this symptom facilitated early diagnosis in 4 of 5 patients who recovered. Acute hearing loss or hearing impairment in a febrile patient should arouse strong suspicion of scrub typhus.

An outbreak of Rocky Mountain Spotted Fever associated with a novel tick vector, Rhipicephalus sanguineus, in Arizona, 2004: preliminary report.

This study describes preliminary results of an investigation of RMSF in Arizona associated with the brown dog tick, Rhipicephalus sanguineus. High numbers of dogs and heavy infestations of ticks created a situation leading to human disease.

Sequencing of a new target genome: the Pediculus humanus humanus (Phthiraptera: Pediculidae) genome project.

The human body louse, Pediculus humanus humanus (L.), and the human head louse, Pediculus humanus capitis, belong to the hemimetabolous order Phthiraptera. The body louse is the primary vector that transmits the bacterial agents of louse-borne relapsing fever, trench fever, and epidemic typhus. The genomes of the bacterial causative agents of several of these aforementioned diseases have been sequenced. Thus, determining the body louse genome will enhance studies of host-vector-pathogen interactions. Although not important as a major disease vector, head lice are of major social concern. Resistance to traditional pesticides used to control head and body lice have developed. It is imperative that new molecular targets be discovered for the development of novel compounds to control these insects. No complete genome sequence exists for a hemimetabolous insect species primarily because hemimetabolous insects often have large (2000 Mb) to very large (up to 16,300 Mb) genomes. Fortuitously, we determined that the human body louse has one of the smallest genome sizes known in insects, suggesting it may be a suitable choice as a minimal hemimetabolous genome in which many genes have been eliminated during its adaptation to human parasitism. Because many louse species infest birds and mammals, the body louse genome-sequencing project will facilitate studies of their comparative genomics. A 6-8X coverage of the body louse genome, plus sequenced expressed sequence tags, should provide the entomological, evolutionary biology, medical, and public health communities with useful genetic information.

Mapping of monoclonal antibody binding sites on CNBr fragments of the S-layer protein antigens of Rickettsia typhi and Rickettsia prowazekii.

The 120 kDa surface protein antigens (SPAs) of typhus rickettsiae lie external to the outer membrane in regular arrays and chemically resemble the S-layer proteins of other bacteria. These proteins elicit protective immune responses against the rickettsiae. In order to study the immunochemistry of these proteins, purified SPAs from Rickettsia typhi and Rickettsia prowazekii were fragmented with CNBr. The fragments were separated by SDS-PAGE and were recovered on PVDF membrane following electroblotting. The origin of eight major fragments from R. prowazekii and seven major fragments from R. typhi was determined by automated N-terminal amino acid sequencing and by comparison with the DNA sequence encoding R. prowazekii SPA. The cleavage patterns and protein sequences of the two proteins differed significantly. CNBr fragments corresponding to the C-terminus (amino acid 1372-1612 of the deduced sequence from encoding gene spaP) were not present in both SPAs. This suggests that the corresponding C-terminal region was not synthesized or was removed during SPA translocation to the cell surface. Modified amino acids were detected in each protein. Eighteen monoclonal antibodies selected for varied reactivity with both native and denatured SPA proteins could be classified into eight different types based on western blot analysis of the CNBr fragments. Six of the monoclonal antibody types reacted predominantly with a single region of the SPAs. Two types of antibodies bound to several CNBr fragments which contained both limited sequence similarity and modified amino acids either of which might account for the multisite binding of these antibodies.

The line blot: an immunoassay for monoclonal and other antibodies. Its application to the serotyping of gram-negative bacteria.

A procedure is described for assaying antibodies based on the application of antigen to nitrocellulose as a line with an ink pen point. The method requires no expensive apparatus, is easy to perform, and requires less than 0.25 micrograms of antigen per assay. More than 45 antigens can be assayed simultaneously with a single antibody. Antigens can be applied as purified proteins, extracts, or sodium dodecyl sulfate solubilized extracts. The application of the line blot assay for the detection of monoclonal antibodies which recognize heat-sensitive and insensitive epitopes on the typhus rickettsia surface protein antigen is described. A new serotyping assay for Gram-negative bacteria is also described in which sodium dodecyl sulfate solubilized antigens are applied as lines with and without prior proteinase K digestion. The value of the line blot serotyping assay is demonstrated with Proteus. Rickettsia, Rochalimaea, and Legionella antigens. The line blot immunoassay is a simple, but powerful and flexible, alternative to dot and cross-dot immunoassays.

Genetic analysis of isolates of the spotted fever group of rickettsiae belonging to the R. conorii complex.

The cytoplasmic 120 kDa antigen genes of 9 isolates of Rickettsia conorii (RC), 12 isolates of R. africae (RA), and 3 isolates of Israeli tick typhus rickettsiae (ISTT) were compared for restriction fragment length polymorphisms (RFLP) present in portions of the open reading frame amplified by polymerase chain reaction (PCR). Initially, DNAs from 13 species or serotypes of spotted fever group rickettsiae were used to select restriction enzymes (RE) that detected RFLP in gene fragments amplified with primer pairs 483WF/1514R and 764F/3409R. Among the R. conorii complex isolates, Dpn II gave RFLP differentiating all three serotypes. Unique RE patterns were obtained for RC with Bsr I and Hinf I, for RA with Mwo I, Pst I and Ssp I, and for ISTT with Hpa II. While RFLP typing of the 120 kDa gene permitted rapid separation of R. conorii complex isolates into three groups corresponding to the RC, RA, and ISTT rOmp serotypes, additional intragroup genetic variation was also detected in all three serotypes.

Diagnosis of acute typhus infection using the polymerase chain reaction.

We used the polymerase chain reaction (PCR) for the diagnosis of an acute rickettsial infection. A primer pair derived from the 17-kDa antigen sequence of Rickettsia rickettsii amplified specifically a 434-bp DNA fragment from the genome of Rocky Mountain spotted fever and endemic and epidemic typhus. The assay was able to detect as few as 30 rickettsiae. Detection of PCR-amplified DNA with a digoxigenin-labeled DNA probe confirmed an acute human infection with Rickettsia prowazekii.

Detection of murine typhus infected fleas with an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) for the detection of Rickettsia typhi antigen in homogenates of pooled or individual laboratory infected fleas is described. The assay uses a double sandwich technique, employing a pool of monoclonal antibodies to capture the antigen and a hyperimmune rabbit serum for antigen detection. Using pools of R. typhi infected Xenopsylla cheopis, Ctenocephalides felis, and Leptopsylla segnis, the sensitivity of the ELISA was compared with direct fluorescent antibody examination of individual fleas for rickettsiae and with rickettsial titers determined by plaque enumeration on primary chicken embryo fibroblasts (PFU). Pooled samples with less than 4 PFU of viable rickettsiae gave ELISA results which were not significantly above background. Both ELISA OD and ELISA titer (last dilution giving an OD that was 2 SD above the control) of a 1:10 dilution of homogenate (4 fleas/ml) were linearly related to rickettsial titer up to 10(6.8) PFU/sample. Multiple freeze-thaws of pools of infected fleas led to a rapid loss of ELISA sensitivity. ELISA assays on single fleas demonstrated large individual variability in rickettsial content. This was independent of the number of days postinfectious feeding or the mean number of PFU/flea (10(1.7-6.9) found for pooled fleas in the same cohort. The sensitivity and ease of performance of ELISA should make it usable under field conditions.

First cases of spotted fever group rickettsiosis in Thailand.

The first three cases of spotted fever group rickettsiosis from Thailand are reported. The patients presented with fever, headache, lymphadenopathy, and petechial maculopapular rash. One patient also had an eschar and overt evidence of confusion. An indirect fluorescent antibody test, an indirect immunoperoxidase test, and an enzyme-linked immunosorbent assay demonstrated a broad, strong reactions of the sera of the patients with spotted fever group rickettsia antigens of many species, but not with antigens of typhus or scrub typhus rickettsiae. All three patients responded to treatment with a single dose of doxycycline.

Evaluation of an enzyme-linked immunosorbent assay in Thai scrub typhus patients.

We report the development of an improved enzyme-linked immunosorbent assay (ELISA) to detect Orientia (formerly Rickettsia) tsutsugamushi antibody in human sera. Results were compared with a standard test, the indirect immunoperoxidase assay (IIP). Control serum samples were collected from 96 American soldiers and 198 Royal Thai Army soldiers with no recent history of clinical illness. Sera were examined from 79 febrile, Thai scrub typhus patients presenting at Chiang Rai (76) and Bangkraui Nontaburi (3) Provincial hospitals (cases confirmed by elevated IIP IgG levels > or = 1:1,600, IgM levels > or = 1:400, or presence of an eschar). The mean + 2 SD, used for the upper limit of normal reactions in the IgG ELISA, was 0.10 for U.S. soldiers and 0.42 for Thai soldiers. Using the 0.10 cutoff value, 29% of the asymptomatic Thai soldiers would be designated as antibody positive. Variability of IgG ELISA values was greater in the Thai soldier group than in American soldiers, possibly reflecting previous exposure to O. tsutsugamushi. In the Thai patients, there was a significant correlation between IIP titers and single serum dilution (1:100) ELISA values (IgG, r = 0.75, n = 104; P < 0.0005; IgM, r = 0.70, n = 75; P < 0.0005) and between IIP titers and ELISA titers (IgG, r = 0.87, n = 103; P < 0.0005; IgM, r = 0.76, n = 75; P < 0.0005). The single serum dilution ELISA was as effective as the titration in determining presence of specific antibodies. The O. tsutsugamushi ELISA is a rapid and objective test amenable to accurately testing the large numbers of sera often obtained in seroepidemiologic investigations.

Effectiveness of a dot-blot immunoassay of anti-Rickettsia tsutsugamushi antibodies for serologic analysis of scrub typhus.

We compared a commercially available dot-blot immunoassay system with the indirect immunofluorescence assay (IFA) in tests of known negative and known positive sera from scrub typhus cases. Using a panel of 100 sera from patients with various rickettsial and nonrickettsial infections, we observed that the IFA was 99% specific and the dipstick assay was 98% specific. In tests of 91 sera (30 negative and 61 positive for scrub typhus antibodies) from a study of febrile patients in Malaysia, using the standard of an IFA titer < 1:64 as negative, an IFA titer > 1:128 as positive, and an IFA titer = 1:64 as either positive or negative (supported by clinical records), dipsticks were 83% specific and 90% sensitive. The quantitative correlation of the dipsticks to IFA titers was confirmed by significant differences in geometric means of inverse IFA titers corresponding to the number of positive dipstick spots (no dots = 8.5, one dot = 43.3, two dots = 206.7, and three dots = 676.9). The assay would enable physicians and public health workers who deal with patients to quickly diagnose and appropriately treat most cases of the disease, especially in areas of high prevalence where the proportion of false-positive results to true-positive results would be low.

Pathology of Rickettsia tsutsugamushi infection in Bandicota savilei, a natural host in Thailand.

Following rodent surveys in a rice-growing area of central Thailand where we found Bandicota savilei, B. indica, and Rattus rattus infected with Rickettsia tsutsugamushi, we performed a study of pathogenesis of R. tsutsugamushi in laboratory-reared B. savilei. Eight animals were injected with saline and 19 animals were injected with 4.0 x 10(6) mouse 50% lethal dose units of a strain of R. tsutsugamushi isolated from a human in central Thailand. Animals were evaluated at intervals for IgG and IgM antibodies to R. tsutsugamushi by an indirect immunoperoxidase assay, the presence of the pathogen in liver and spleen by murine inoculation, and the pathology of representative tissues by gross and microscopic examination. The infected animals began to show internal evidence of mild illness 7-14 days after inoculation, and exhibited no changes in behavior. Total white blood cell counts decreased on day seven (including lymphocytes and polymorphonuclear leukocytes), followed by an almost equal increase on day 14. Gross pathology noted at necropsy was limited to slight liver and spleen enlargement accompanied by low numbers of abscesses and fibrinous tags present in the abdominal cavity. In addition to the gross morphologic changes, histopathologic lesions noted were all mild, consisting of vasculitis of the lung, activation of the mononuclear phagocyte system, abdominal mesothelial cell hyperplasia, and peritonitis. Rickettsiae were isolated from liver and spleen on days 0, 7, and 14, but not thereafter. Specific antibody response was first observed on day 14, peaked on day 21, and it decreased to levels observed in uninfected animals between days 120 and 180.(ABSTRACT TRUNCATED AT 250 WORDS)

Short report: surveillance of rickettsial infections in Indonesian military personnel during peace keeping operations in Cambodia.

Indonesian peacekeepers in Cambodia provided a unique study population to estimate the threat of rickettsial exposure to Rickettsia typhi (murine typhus), Orientia tsutsugamushi, (scrub typhus), and R. conorii (spotted fever) for the region. Prescreening prevalence measure showed a large proportion (36%) of soldiers with antibodies to R. typhi. Predeployment prevalence for antibodies to O. tsutsugamushi was 8%, with no evidence of background R. conorii infections. Actual seroconversions of R. typhi (3) and O. tsutsugamushi (1), attributed to exposure(s) in Cambodia, translated into annualized incidence rates of 24 and 8 per 1,000 per year, respectively. Surveillance of rickettsial infections and/or disease is particularly warranted in Cambodia with recent recognition of drug-resistant scrub typhus in neighboring Thailand.

Seroepidemiologic evidence for murine and scrub typhus in Malang, Indonesia.

Indonesian military personnel stationed in Malang, East Java were among troops deployed to central Cambodia as part of the United Nations' Transition Authority Cambodia peace-keeping operation in 1992. Predeployment blood samples obtained from a cohort of Indonesian soldiers indicated a high prevalence of antibodies to antigens of Rickettsia typhi or Orientia (formerly Rickettsia) tsutsugamushi, the etiologic agents for murine and scrub typhus, respectively. To evaluate the potential risk of these rickettsial diseases in the Malang area, a subsequent seroepidemiologic survey was conducted. This study involved civilian personnel residing within one of three Malang kelurahans (neighborhoods) representing urban, suburban, and rural communities. The heads-of-households from 197 homes completed a detailed epidemiologic survey. In addition, blood samples were collected from 464 individuals residing within the households surveyed. Examination of civilian blood samples disclosed that 34.7% and 1.3% of the study participants were seroreactive to R. typhi and O. tsutsugamushi, respectively. These results were similar to those obtained earlier from the military samples. In addition, assessment of 78 blood samples obtained from peridomestic rodents trapped from within or near the households surveyed showed that 28 were reactive to R. typhi antigens and four were reactive to O. tsutsugamushi antigens. These data indicate that military and civilian personnel living in the Malang area of East Java are at risk of infection with rickettsiae that are antigenically indistinguishable from those that cause murine and scrub typhus.

Western blotting analysis of heat shock proteins of Rickettsiales and other eubacteria.

Heat shock proteins (Hsp) of four Rickettsia species, three Bartonella species, two Ehrlichia species, Orientia tsutsugamushi and seventeen other eubacterial species were characterized by the enhanced chemiluminescence Western blotting (WB) technique with antibodies raised against recombinant Hsp from Escherichia coli and purified GroES from R. typhi. Although E. coli DnaK and GroEL have epitopes that are highly conserved among the homologous proteins found in Rickettsia, Ehrlichia, O. tsutsugamushi, Bartonella and other Proteobacteria, anti-E. coli DnaK and GroEL monoclonal antibodies (Dasch et al. (1990) Ann. N.Y. Acad. Sci. 590, 352-369) recognize less conserved epitopes. In contrast, epitopes on E. coli DnaJ, GrpE and GroES are much less conserved since anti-E. coli DnaJ, GrpE and GroES polyclonal antibodies did not recognize DnaJ, GrpE or GroES homologues in Rickettsia, Bartonella, Orientia, Ehrlichia and Legionella. Polyclonal antiserum prepared against GroES from R. typhi reacted strongly with purified 10 kDa GroES peptide from Rickettsia and Bartonella, and strongly bound to proteins of varying electrophoretic mobility from Wolbachia, Legionella, Proteus and Shigella flexneri and more weakly to other GroES homologues including that found in E. coli. Consequently, commercially available anti-DnaJ, anti-GrpE and anti-GroES polyclonal antibodies and anti-DnaK monoclonal antibody raised against their respective recombinant E. coli Hsp are not suitable for detection and identification of homologues of these proteins in a wide range of eubacteria.

Immunoblot cross-reactions among Rickettsia, Proteus spp. and Legionella spp. in patients with Mediterranean spotted fever.

Sera from patients suffering from Mediterranean spotted fever (i.e. an infection due to Rickettsia conorii) were studied by immunoblot to investigate cross-reactivity. A prevalence of IgM antibodies to Proteus OX 19, Proteus OX 2, to the Rickettsia typhus group, to Legionella pneumophila serovars 4 and 5, to L. bozemanii Wiga and to L. micdadei Tatlock was found. Western blot confirmed that the antibodies were directed against the lipopolysaccharide as demonstrated by proteinase K digestion of the antigens. Cross-adsorptions showed that there is a common cross-reacting epitope among L. bozemanii Wiga, R. typhi and Proteus OX 19 but cross-reacting antibodies to L. micdadei and OX 2 were distinct and independent. This IgM cross-reaction could lead to a misdiagnosis.

Annotated list of ticks (Acari: Ixodidae, Argasidae) reported in Peru: distribution, hosts, and bibliography.

A literature review and compilation of the tick specimens found in Peru and now held in the National Tick Collection was carried out to develop a working list of the tick species likely to be found in Peru. Evidence of 44 species (29 Ixodidae, 15 Argasidae), was found; representatives of 40 species are held as reference specimens. This report adds 14 species to the previously published list.

Detection and characterization of Rickettsia tsutsugamushi (Rickettsiales: Rickettsiaceae) in infected Leptotrombidium (Leptotrombidium) fletcheri chiggers (Acari: Trombiculidae) with the polymerase chain reaction.

We developed a method for detecting and characterizing the DNA of Rickettsia tsutsugamushi in chiggers (larval trombiculid mites) by polymerase chain reaction (PCR). Three procedures for extracting DNA from frozen chiggers were compared by evaluating the yield of PCR amplicand obtained with nine oligonucleotide primer pairs derived from the rickettsial 22 kD, 47 kD, groESL, 56 kD, and 110 kD antigen genes. Although extracts and primer pairs differed in amplification efficiency, R. tsutsugamushi DNA was successfully detected in extracts of colonized infected Leptotrombidium (Leptotrombidium) fletcheri (Wormersley & Heaslip) chiggers and in uninfected chigger extracts seeded with known amounts of Karp-strain rickettsiae. The 22 kD gene restriction fragment length polymorphisms (RFLP) observed in PCR amplicands from five rickettsial isolates obtained from the infected chigger colony over a 26-yr period were identical to those of PCR amplicands derived directly from infected chiggers taken from the same colony. This suggests that stable transmission of R. tsutsugamushi occurs in mites (62 generations), and isolates encompass the full genetic heterogeneity found in the chigger. PCR/RFLP analysis is an important new tool for investigating the complex epidemiology of scrub typhus rickettsiae in their mite vectors.