Flinders Island spotted fever rickettsioses caused by "marmionii" strain of rickettsia honei, eastern Australia.

Australia has 4 rickettsial diseases: murine typhus, Queensland tick typhus, Flinders Island spotted fever, and scrub typhus. We describe 7 cases of a rickettsiosis with an acute onset and symptoms of fever (100%), headache (71%), arthralgia (43%), myalgia (43%), cough (43%), maculopapular/petechial rash (43%), nausea (29%), pharyngitis (29%), lymphadenopathy (29%), and eschar (29%). Cases were most prevalent in autumn and from eastern Australia, including Queensland, Tasmania, and South Australia. One patient had a history of tick bite (Haemaphysalis novaeguineae). An isolate shared 99.2%, 99.8%, 99.8%, 99.9%, and 100% homology with the 17 kDa, ompA, gltA, 16S rRNA, and Sca4 genes, respectively, of Rickettsia honei. This Australian rickettsiosis has similar symptoms to Flinders Island spotted fever, and the strain is genetically related to R. honei. It has been designated the "marmionii" strain of R. honei, in honor of Australian physician and scientist Barrie Marmion.

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Australia has several endemic rickettsial diseases. In addition, epidemic typhus arrived with the first fleet in 1788 (1), but the disease did not become established in Australia. The current endemic rickettsial diseases are murine typhus (Rickettsia typhi), scrub typhus (Orientia tsutsugamushi), and the spotted fever group (SFG) diseases--Queensland tick typhus (QTT; R. australis) and Flinders Island spotted fever (FISF; R. honei) (2).

QTT, first described in 1946, was characterized as a relatively mild disease with symptoms of fever, headache, malaise, enlarged lymph nodes, and a maculopapular (sometimes vesicular) rash. Most patients have an eschar and some have a slight cough, myalgia, and chills (3,4). Cases of QTT have been detected only on the eastern seaboard of mainland Australia, with most originating in late winter (5). FISF was described in Australia, in 1991. It is found in southeastern Australia and is characterized by fever, headache, myalgia, transient arthralgia, maculopapular rash, and cough in some cases (6,7). Most cases occur in summer. Both QTT and FISF are transmitted to humans by tick bites. Ticks of the genus Ixodes, especially I. holocyclus, are the main arthropod hosts of QTT and Bothriocroton hydrosauri (formerly Aponomma hydrosauri) are the main hosts of FISF (8-10).

We describe 7 cases of a rickettsial disease similar to FISF, which occurred in the eastern half of Australia. The etiologic agent of this disease is an SFG rickettsia, genetically related to R. honei and less closely related to R. australis. The etiologic agent of the rickettsiosis has been designated the "marmionii" strain of R. honei.

Case Reports

Patient 1

A 37-year-old woman from Port Willunga, South Australia, sought treatment in February 2003, with a 2

week history of headache, fever, and sweats. No rash or eschar was seen, and she had no recollection of arthropod exposure. She had traveled to Kangaroo Island 2-3 weeks before the onset of illness. Laboratory tests showed elevated levels of liver function test enzymes, mild leukopenia, and thrombocytopenia. Her health improved after receiving oral doxycycline for 5 days. Rickettsial serology later showed an increase in antibody titer. Both PCR and culture results were positive for an SFG rickettsia (Table 1).

Patient 2

A 9-year-old girl sought treatment at the Darnley Island Health Clinic, Torres Strait, Queensland, in February 2003. She was febrile (38.5[degrees]C) and reported headache, nausea, and abdominal pain. She had no eschar or rash. She was initially thought to have a viral illness; however, after 3 days she was still febrile (39.0[degrees]C), and the provisional diagnosis was changed to scrub typhus; a regimen of oral doxycycline, 100 mg per day, was begun. She was not seen by medical or nursing staff between day 3 and 8 of the illness, but was afebrile and well by day 8. Her SFG title increased, despite a 6-month delay in obtaining the convalescent-phase serum. Results of culture and PCR of the blood sample taken on day 8 were positive for an SFG rickettsia (Table 1).

Patient 3

A 27-year-old man sought treatment at the Darnley Island Health Clinic in March 2003. His temperature was 37.4[degrees]C, and he reported headache, arthralgia, and cough. He exhibited no eschar or rash. The provisional diagnosis was of viral upper respiratory tract infection. He was seen again on days 3 and 4 with persisting symptoms and a sore throat. On the latter visit his condition was diagnosed as tonsillitis, and treatment with penicillin V was begun. Blood tests for malaria and scrub typhus were initiated. He returned on day 29 with fever (37.6[degrees]C), cough, pharyngitis, and arthralgia. Results of serologic investigations for Plasmodium falciparum and rickettsia (taken on day 3) were negative. Antibiotics were not given because the illness was thought to be viral. His symptoms resolved within the following 2 weeks. Antirickettsial antimicrobial agents were not given at any stage during the illness. Day 3 serum and follow-up serum specimens obtained 6 months later were both negative for rickettsial antibodies; however, results of PCR and culture on the day 3 blood specimen were positive for SFG rickettsiae (Table 1).

Patient 4

A 10-year-old boy was brought to the Yam Island Health Clinic, Torres Strait, Queensland, in May 2003, five days into an illness with manifestations of fever (38.1[degrees]C), headache, and cough. Diagnostic tests for scrub typhus, malaria and leptospirosis were initiated but he was given no specific antimicrobial therapy. Two days later, he seemed improved, and a provisional diagnosis of viral upper respiratory tract infection was made. However, when he was seen on day 14, some symptoms remained (cough and headache), and treatment with amoxicillin was begun. He was well when examined on day 22. At no stage was he given antirickettsial therapy. His day 5 blood sample was negative for SFG/typhus group (TG) rickettsial antibodies, but results of PCR and culture were positive for a SFG rickettsia. Follow-up serum taken 14 months later was negative for rickettsial antibody (Table 1).

Patient 5

A 50-year-old man was admitted to Innisfail Hospital, Innisfail, Queensland, in June 2003. He reported a 7-day history of fever and rigors and a 4-day history of maculopapular rash. He also reported myalgia, arthralgia, conjunctivitis, swollen hands, dry cough, and constipation. An eschar was found on the right side of his neck. His temperature was 38.5[degrees]C and blood pressure 95/60 mm Hg. Serum chemistry showed elevated levels of total bilirubin (23; normal range 2-20 [micro]mol/L), alkaline phosphatase (276; normal range 30-115 units/L), gamma-glutamyl transpeptidase (199; normal range 0-70 units/L), aspartate transaminase (AST) (301; normal range 5-40 units/L), alanine transaminase (ALT) (129; normal range 5-40 units/L), and lactate dehydrogenase (LDH) (701, normal range 100-225 units/L). Further investigation showed proteinuria, moderate thrombocytopenia (59; normal range 150-400x[10.sup.9]/L), mild neutrophilia with left shift (7.9; normal range 2.0-7.5 x [10.sup.9]/L), and lymphopenia (0.7; normal range 1.0-4.0 x [10.sup.9]/L). Examination of convalescent-phase serum showed seroconversion to SFG rickettsia. Results of rickettsial PCR and culture were positive for a member of the SFG (Table 1). He recovered after treatment with oral doxycycline (100 mg twice per day) for 5 days.

Patient 6

A 33-year-old man from Lilydale, a small town in northeastern Tasmania, sought treatment from his general practitioner in May 2003 (day 1) after a recent fishing trip. His symptoms included fever (38.3[degrees]C) and headache. On day 6 the patient was improving but had developed cervical lymphadenopathy. His illness was thought to be viral in origin so he was not treated with any antibiotics. The patient's condition improved, and he had a symptom-free period of [approximately equal to]10 days. Fever developed again 33 days after onset of the earlier illness with the same symptoms including aches and pains. Three days later, he was admitted to Launceston General Hospital. He appeared markedly ill with a blanching maculopapular rash that had not been evident before, over his trunk, inguinal lymphadenopathy, neutropenia (0.9; normal range 2.0 7.5 x [10.sup.9]/L) and slightly elevated levels of C-reactive protein (10; normal range 0-8 mg/L). At this time the possibility of rickettsial disease was raised, and appropriate tests were performed.

On day 7 after the second onset of fever, the patient was able to work but still felt ill and had a slight fever (37.6[degrees]C). On day 27 after the second onset of fever, more rickettsial tests were performed before he received treatment with a 14-day course of doxycycline. He made a complete recovery without further relapse. He showed a raised rickettsial SFG titer and a positive SFG rickettsial PCR results for both blood samples tested (days 34 and 60) (Table 1).

Patient 7

A 55-year-old man, an entomologist, at Iron Range, Cape York Peninsula in far north Queensland, removed a tick from the left ventrolateral side of his abdomen in late May 2002. Five days after removing the tick (day 5), an influenzalike illness with myalgia and arthralgia developed. On day 6, a high fever developed, and on the following day, he experienced persisting severe lethargy and severe muscle cramps in major muscle groups of his upper and lower legs. On day 8, an eschar appeared at the site of the tick bite. It was oval in shape and [approximately equal to]150 mm by 75 mm. A widespread maculopapular/petechial rash also appeared over his body. High fever, severe lethargy, and myalgias continued. On day 9, he visited his doctor in Brisbane where the examination confirmed a widespread maculopapular/petechial rash with generalized lymphadenopathy and myalgias affecting large muscle groups. A large eschar was found on his left lower abdomen. An SFG illness was suspected, and treatment with doxycycline, 100 mg twice per day, was begun His doctor reexamined him on day 20, and his condition had improved. His myalgia had decreased, and the rash faded over 5 weeks.

Laboratory testing on day 10 showed lymphopenia (0.8; normal range 1.0-4.0 x [10.sup.9]/L) and mild thrombocytopenia (146; normal range 150-400 x [10.sup.9]/L). Liver function tests showed slightly elevated AST (48; normal range 5-40 units/L) and ALT (44, normal range 5-40 units/L) and mildly elevated LDH (325; normal range 100-225 units/L). Rickettsial serology was negative on day 10 but convalescent-phase serology on day 23 showed an SFG seroconversion. A real-time PCR on the day 10 serum specimen showed a positive result for the SFG/TG gltA gene, but the 17-kDa PCR result was negative (Table 1).

The removed tick was subsequently identified as Haemaphysalis novaeguineae. DNA was extracted from the tick and PCRs performed targeting the rickettsial rrs, ompA and ompB genes. PCR products were sequenced, aligned, searched with BLAST (available from http://130.14.29.110/BLAST/), and submitted to GenBank (accession nos. AJ585043, AJ585044, and AJ585045 for the rrs, ompA, and ompB genes, respectively). Phylogenetic analysis of all 3 genes showed that the closest relatives were R. honei strain TT-118 (Thai tick typhus) and R. honei strain RB (FISF) (11).

Methods

Rickettsial Serology

Serologic testing was performed on human serum specimens by using a goat anti-human IgM, IgG, and IgA fluorescein isothiocyanate-labeled secondary antibody (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA), by an indirect immunofluorescence assay (IFA) as described (7). Antigens used included R. honei, R. australis, R. akari, R. conorii, R. sibirica, and R. rickettsii from the SFG; and R. typhi and R. prowazekii from the TG. All titers >128 were considered positive.

Rickettsia Isolation from Blood

Rickettsial isolation was performed with Vero cell cultures as previously described (12). Cultures were observed microscopically weekly for a cytopathic effect and monthly by immunofluorescence. IFA-positive cultures had their DNA extracted and their rickettsial status confirmed by PCR. Positive cultures were passaged onto confluent XTC-2 cell monolayers and grown at 28[degrees]C in Leibovitz L-15 media (Invitrogen, Melbourne, Victoria, Australia) supplemented with 5% heat-inactivated fetal bovine serum, 0.4% tryptose phosphate (Oxoid, Basingstoke, UK), and 200 mmol/L-glutamine (Invitrogen).

Rickettsial PCR on Blood

Rickettsial real-time PCR was performed on buffy coat (except for serum for case 7). DNA was extracted by using a DNA extraction kit (Gentra, Minneapolis, MN, USA) and the primers CS-F and CS-R and the probe CS-P (Table 2; Biosearch Technologies Inc., Novato, CA, USA) as previously described (13).

Confirmatory PCR was performed on the 17-kDa gene (orf17) by using the primers MTO-1 and MTO-2 (Table 2; Invitrogen) (14), with an annealing temperature of 51[degrees]C and a total of 45 cycles. PCR products were visualized by electrophoreses on a 1% Tris-acetate EDTA agarose gel (Amresco, Solon, CA, USA) stained with ethidium bromide. PCR-positive samples had their DNA cleansed using the QIAquick DNA clean up kit (QIAGEN, Dusseldorf, Germany) and were sequenced at Newcastle DNA (Newcastle University, Newcastle, New South Wales, Australia). Phylogenetic analysis of DNA sequences was performed with DNADIST and NEIGHBOR computer programs of the PHYLIP version 3.63 software package (available from http://evolution.genetics. washington.edu/phylip.html). Sequences were compared to those of the rickettsial strains considered to be valid species (19). Phylogenetic trees and bootstrap analyses were performed with 100 alignments by using the SEQBOOT and CONSENSE programs of PHYLIP.

Rickettsial Molecular Characterization

Rickettsial isolates had portions of their gltA, I6S rRNA, ompA, and Sca4 antigen genes amplified and sequenced to supplement the 17-kDa gene analysis done on buffy coat and cultures. The primer pairs CS-162-F with CS-731-SR and CS-398-SF with RPCS1258 (Table 2) were used to amplify the 5' and 3' ends of gltA, respectively (15).

The 16S rRNA gene (rrs) was amplified by using the primer pairs rRNA1 with rRNA3 and rRNA2 with rRNA4 (Table 2) (17). The PCR contained 1 [micro]mol of each respective primer, 200 [micro]mol/L of each dNTP, 10x reaction buffer, 2 mmol/L Mg[Cl.sub.2], 2 U Taq polymerase, and 4 gL of rickettsial DNA extract. The amplification was performed in a thermocycler (Rotor-Gene 3000, Corbett Research, Sydney, New South Wales, Australia) with an initial denaturation of 95[degrees]C for 3min, followed by 40 cycles of denaturation at 95[degrees]C for 30 s, annealing at 51[degrees]C for 30 s, and extension at 72[degrees]C for 1 min; with a final extension of 10 min. PCR products were visualized and sequenced as described above.

The ompA gene (ompA) was amplified by using the primers Rr190.70p and Rr190.602n (Table 2) by using the above protocol but with an annealing temperature of 48[degrees]C (16). The Sca4 antigen gene (sca4) was amplified by using the primer pairs D1f and D928r, D767f and D1390r, D1219f and D1876r, and D1738f and D2482r, following the specified protocol (18). The final segment of the gene was amplified with the primers D2338f and D3069r following the same protocol and an annealing temperature of 48[degrees]C (18).

Results

Seroconversion, defined as a 4-fold increase in antibody titer, occurred in only 2 of the 7 patients (patients 5 and 7), although positive titers were seen in 5 of 7 patients (Table 1). In 5 of 6 patients a rickettsia was isolated from blood (in EDTA-vacutainers; Table 1) in Vero cell culture, however, 4 of these 5 isolates did not persist in cell culture after their third passage. The remaining isolate, from patient 5, has been maintained in continuous culture in only the XTC-2 cell line.

Patients 1-6 had rickettsial DNA detected in their buffy coat DNA extracts by real-time PCR. Patient 7 had rickettsial DNA detected in his serum by using real-time PCR. Of the 7 cases, all but 1 (patient 7), were PCR positive for the 17-kDa gene and all 5 positive rickettsial cultures were also PCR positive for the same gene (Table 1). The 17-kDa PCR sequences for the buffy coats of cases 1-6 and cultures of patients 1-5 were found to be 100% homologous to one another and to the Japanese Haemaphysalis tick sequences Hf151 and H1550 (20) (GenBank accession nos. AB114816 and AB114807, respectively). A 399-bp sequence also exhibited 99.2% homology with R. honei strains RB and TT-118 (GenBank accession nos. AF060704 and AF060706, respectively) as shown in the phylogenetic tree (Figure).

Analysis of a 1082 bp gltA sequence from the KB strain exhibited 99.7 and 99.8% homology with R. honei strains RB and TT-118, respectively (GenBank accession nos. AF018074 and U59726, respectively) (Figure). An 1142 bp rrs sequence exhibited 100% homology with the Australian Haemaphysalis novaeguineae tick sequence AL2003 (11) (GenBank accession no. AJ585043) and a 1,388-bp sequence exhibited 99.6% and 99.9% homology with the R. honei strains RB and TT-118, respectively (GenBank accession nos. U17645 and L36220, respectively) (Figure). A 511-bp sequence of ompA exhibited 100% homology with the H. novaeguineae sequence AL2003 (11) (GenBank accession no. AJ585044) and a 513-bp sequence had 99.8% homology with the R. honei strains RB and TT- 118 (GenBank accession nos. AF018075 and U43809, respectively). Only 1 nucleotide substitution was found in a 2,961-bp sequence of the Sca4 gene (100% homology) with R. honei strain RB (GenBank accession no. AF 163004) (Figure).

These R. honei strain "marmionii" sequences have been submitted to GenBank with the accession nos. AY37683 for the 17-kDa gene, AY37684 for the gltA gene, AY37685 for the 16S rRNA gene, DQ309095 for the Sca4 gene, and DQ309096 for the ompA gene.

Discussion

These 7 cases of FISF are an example of many newly emerging rickettsial diseases (21). Its symptoms are consistent with a relatively mild rickettsial SFG disease. The most frequent acute symptoms observed were fever (100%), headache (71%), arthralgia (43%), myalgia (43%), cough (43%), rash (maculopapular/petechial) (43%), nausea (29%), pharyngitis (29%), and lymphadenopathy (29%). In only 2 patients was an eschar evident. The rash did not appear on the palms or soles, unlike previously reported FISF cases (6,12). One patient (patient 7), had a history of a H. novaeguineae tick bite, which may imply an incubation period of 5 days. The cases in this report occurred between February and June (late summer and autumn), in contrast to previously described cases of FISF and QTT, which have their peak onsets in summer and late winter, respectively (5,6).

The biphasic illnesses seen in patients 3 and 6 were unusual for SFG rickettsial diseases. Because no specimens were taken during the initial phase of either patient's illness, that this phase was rickettsial in nature cannot be confirmed. Patient 6's illness may have been rickettsial in nature because of the appropriate incubation time after a fishing trip in an area endemic for ticks. His illness had the longest duration of all the reported cases, with rickettsiae still detectable 27 days after the onset of the second febrile illness. This is possibly the first report of an SFG rickettsia being associated with a chronic infection in a human. Relapsing rickettsial diseases are known to exist, such as Brill disease, a recurrent form of epidemic typhus (22). Rickettsiae persisting in human and animal organs after illness have been reported with scrub typhus and SFG rickettsia (23,24). An Australian case of recurrent rickettsial illness was diagnosed serologically as QTT (25).

The isolation of rickettsiae from patient 2 after antimicrobial drug therapy and while she was clinically well is unusual. The presence of rickettsiae may be due to the bacteriostatic nature of the patient's treatment, which allowed a small number of rickettsiae to survive before being eliminated by her immune system. This phenomenon may also have been the beginning of a chronic infection, as described above in patients 3 and 6.

Apart from patients 5 and 7, antibody levels of paired serum specimens (Table 1) did not show a marked rise in titer. Because the second serum sample from 4 of the case-patients was received in excess of 6 months after illness, the antibody levels may have subsided, explaining the apparent lack of seroconversion in patients 3 and 4. Because most rickettsioses are diagnosed through serologic tests, some cases of rickettsial disease are likely being missed due to a lack of seroconversion, as we have observed with these cases of FISF. This demonstrates the usefulness of PCR for diagnosing acute rickettsial diseases. Cases of rickettsioses without seroconversion or positive serology titers have been previously described with "R. sibirica mongolotimonae" (26). Despite the initial isolation of R. honei strain "marmionii" in Vero and L929 cells at 35[degrees]C, no isolate could be continuously grown in these cell lines. This may be due partially to temperature-dependent growth kinetics, similar to those of R. felis (27).

The 7 described cases were distributed widely throughout eastern Australia. Cases have appeared on the eastern seaboard of Australia (including the Torres Strait), Tasmania, and in South Australia. Cases are yet to be reported in Victoria, New South Wales, the Northern Territory, or Western Australia. The discovery of FISF cases in the Torres Strait suggests its possible presence in Papua New Guinea. In comparison, QTT is found only down the eastern seaboard and not south or west of Wilson's Promontory in Victoria. Traditionally, FISF has only been found in the southeastern states, including Tasmania and South Australia (6,12,28).

At present, R. honei has been found on 2 other continents, with potential reservoirs in Ixodes and Rhipicephalus ticks in Asia and in Amblyomma cajennense in North America (29). The only known vector/reservoir of R. honei in Australia is Bothriocroton hydrosauri (10). R. honei strain "marmionii" has not been found in any B. hydrosauri ticks, although H. novaeguineae may be a vector/reservoir, as a H. novaeguineae tick was removed from patient 7 before the onset of illness. Rickettsial rrs and ompA gene sequences within the tick demonstrated 100% homology with R. honei strain "marmionii" (11). H. novaeguineae is known to bite numerous animals including humans and is found in both northern Australia and Papua New Guinea (30). The vectors and reservoirs of R. honei strain "marmionii" in southern Australia are not known.

When compared phylogenetically to other rickettsiae, R. honei strain "marmionii" has the closest homology with Australian R. honei strain RB, which had been isolated from a febrile patient on Flinders Island. When the gltA, rrs, ompA, orf17, and sca4 genes are compared between R. honei strains RB and "marmionii," they are 99.7%, 99.6%, 99.6%, 99.0%, and 100% homologous, respectively. Homologies of 99.8% and 99.9% are seen with the gltA and rrs genes, respectively, when R. honei strains TT-118 and "marmionii" are compared. An 811-bp ompB gene sequence from the H. novaeguineae tick removed from patient 7 also showed 100% homology with R. honei (11). This supports its description as an SFG rickettsia but not a new species by using previously proposed criteria (19). Further analysis is needed to further define the taxonomic position of R. honei strain "marmionii."

The 7 cases of an illness similar to FISF demonstrate that new emerging rickettsioses are present in Australia. These described cases encompass a geographic distribution larger than those of FISF and QTT. The only known tick host of R. honei strain "marmionii" is H. novaeguineae, a tick not previously recognized as a transmitter of human pathogens. Genetically, the etiologic agent of these 7 cases is closely related to R. honei. We propose to name the agent Rickettsia honei strain "marmionii," in honor of the Australian physician and scientist Barrie R Marmion, for his research into Q fever, another important rickettsial disease.

Acknowledgments

Chelsea Nguyen undertook the serologic testing. Christine Bush, Nicholas Richardson, Merilyn Williams, and Nathan Kesteven contributed to the clinical notes. Simone Cough, Brian Milburn, and. John McBride coordinated the sending of specimens from Darnley Island, Thursday Island, and north Queensland, respectively. We sincerely thank these collaborators.

Dr Unsworth is a postdoctoral research associate at Texas A&M University, College Station, Texas, USA. His interests include the epidemiology of Australian rickettsiae and Q fever pathogenesis.

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(30.) Roberts FHS. Australian ticks. Melbourne (Australia): Commonwealth Scientific and Industrial Research Organisation; 1970.

Address for correspondence: John Stenos, Australian Rickettsial Reference Laboratory, Geelong Hospital, PO Box 281 Geelong, Victoria, Australia 3220; email: [e-mail]

Nathan B. Unsworth, * John Stenos, * Stephen R. Graves, * Antony G. Faa, ([dagger]) G. Erika Cox, ([double dagger]) John R. Dyer, ([section]) Craig S. Boutlis, ([paragraph]) Amanda M. Lane, # Matthew D. Shaw, # Jennifer Robson, ** and Michael D. Nissen ([double dagger])

* Australian Rickettsial Reference Laboratory, Geelong, Victoria, Australia; (1) Warwick Hospital (Southern Downs Health Services District), Warwick, Queensland, Australia; ([double dagger]) Launceston General Hospital, Launceston, Tasmania, Australia; ([section]) Fremantle Hospital, Fremantle, Western Australia, Australia; ([paragraph]) Menzies School of Health Research, Darwin, Northern Territory, Australia; # University of Queensland, Brisbane, Queensland, Australia; ** Sullivan Nicolaides Pathology, Brisbane, Queensland, Australia; and ([double dagger]) Royal Brisbane Hospital, Brisbane, Queensland, Australia

Table 1. Rickettsial serology, PCR, and culture results from 7
cases of FISF *

                                    1st serum sample

                             Day after
                              disease      SFG      TG
Patient       Location         onset      titer    titer

1         Port Wilunga, SA      14         128      <128
2         Darnley Is, QLD        8         256      <128
3         Darnley Is, QLD        3        <128      <128
4           Yam Is, QLD          5        <128      <128
5          Innisfail, QLD        7        <128      <128
6         Launceston, TAS       34         256       128
7         Iron Range, QLD        5        <128      <128

                       2nd serum sample

             Day after
              disease
Patient        onset      SFG titer    TG titer

1               190           256        <128
2               186           512        <128
3               179          <128        <128
4               515          <128        <128
5                17         4,096       2,048
6                60           256         128
7                18         1,024        <128

                    Microbial detection of FISF agent by

               Sero-      Serocon-
Patient     positivity    version             PCR           Culture

1                +           -        +                        +
2                +           -        +                        +
3                -           -        +                        +
4                -           -        +                        +
5                +           +        +                        +
6                +           -        + ([dagger])             -
7                +           +        + ([double dagger])      ND

* FISF, Flinders Island spotted fever; SFG, spotted fever group; TG,
typhus group; SA, South Australia, Is, island; QLD, Queensland, TAS,
Tasmania; ND, not done.

([dagger]) PCR positive at both 34 and 60 d after disease onset.

([double dagger]) Performed on serum and real-time PCR positive
sample only.

Table 2. Oligonucleotide primers used for PCR amplification and
sequencing of products

                      Nucleotide sequence
Primer               (5' [right arrow] 3')            Gene    Reference

CS-F             TCG CAA ATG TTC ACG GTA CTT T        gltA       13
CS-R             TCG TGC ATT TCT TTC CAT TGT G        gltA       13
CS-P *       TGC AAT AGC AAG AAC CGT AGG CTG GAT G    gltA       13
MTO-1              GCT CTT GCA ACT CTA TGT T          orf17      14
MTO-2              CAT TGT TCG TCA GGT TGG CG         orf17      14
CS-162-F        GCA AGT ATC GGT GAG GAT GTA ATC       gltA       15
CS-398-SF         5'ATT ATG CTT GCG GCT GTC GG        gltA       15
CS-731-SR          AAG CAA AAG GGT TAG CTC C          gltA       15
RpCS1258p        ATT GCA AAA AGT ACA GTG AAC A        gltA       16
rRNA1              AGA GTT TGA TCC TGG CTC AG          rrs       17
rRNA2              AAG GAG GTG ATC CAG CCG CA          rrs       17
rRNA3              CCC TCA ATT CCT TTG AGT TT          rrs       17
rRNA4               CAG CAG CCG CGG TAA TAC            rrs       17
Rr190.70p         ATG GCG AAT ATT TCT CCA AAA         ompA       16
Rrl90.602n        AGT GCA GCA TTC GCT CCC CCT         ompA       16
D1f                ATG AGT AAA GAC GGT AAC CT         sca4       18
D928r              AAG CTA TTG CGT CAT CTC CG         sca4       18
D767f              CGA TGG TAG CAT TAA AAG CT         sca4       18
D1390r            CTT GCT TTT CAG CAA TAT CAC         sca4       18
D1219f             CCA AAT CTT CTT AAT ACA GC         sca4       18
D1876r             TAG TTT GTT CTG CCA TAA TC         sca4       18
D1738f            GTA TCT GAA TTA AGC AAT GCG         sca4       18
D2482r             CTA TAA CAG GAT TAA CAG CG         sca4       18
D2338f             GAT GCA GCG AGT GAG GCA GC         sca4       18
D3069r             TCA GCG TTG TGG AGG GGA AG         sca4       18

* 5' end labeled with 6-FAM; 3' end labeled with BHQ-1.