New Orientia tsutsugamushi strain from scrub typhus in Australia.

In a recent case of scrub typhus in Australia, Orientia tsutsugamushi isolated from the patient's blood was tested by sequence analysis of the 16S rDNA gene. The sequence showed a strain of O. tsutsugamushi that was quite different from the classic Karp, Kato, and Gilliam strains. The new strain has been designated Litchfield.


Dispatches
Scrub typhus has been recognized as an important cause of fever among inhabitants of tropical Queensland (northeast Australia) for the past 60 years (1)(2)(3)(4)(5)(6). All three groups of rickettsial diseases occur in Australia (Figure). 1) Spotted fever group (SFG): i) Rickettsia australis (Queensland tick typhus) occurs along the eastern side of the continent to the east of the Great Dividing Range (7,8); ii) R. honei (Flinders Island spotted fever) is only now recognized on Flinders Islanda small island off the southeast corner of Australia (9,10); iii) an unidentified SFG rickettsial infection in Tasmania, a large island state to the southeast of the Australian mainland, has been identified through serologic evidence, but no isolate has been obtained. 2) Typhus group (TG): R. typhi (murine typhus) occurs throughout Australia, with foci recognized in southwest Australia, southcentral Australia (first described by Hone [11] in Adelaide, South Australia), and central Queensland (northeast Australia). 3) Scrub typhus: Orientia tsutsugamushi infection, well recognized in tropical coastal Queensland, has probably long been endemic in this area. As Europeans gradually settled the area, the etiologies of numerous febrile illnesses were determined. Serologic evidence (the Weil-Felix serologic test) of scrub typhus became available (1-3) and later isolates of O. tsutsugamushi were made by inoculating mice with patients blood (4). A serologic analysis of the Queensland strains of O. tsutsugamushi suggested that most strains were Karp-like (12). (The Karp strain was isolated from Papua New Guinea [13].) The geographic distribution of scrub typhus in the rest of Australia is less clear. The disease was not considered to occur west of coastal Queensland. However, in the tropical region of the Northern Territory, six possible cases from 1937 to 1939 and one in 1957 were reported (all presumptively diagnosed). Since 1990 , however, nine cases of scrub typhus have been reported in Litchfield Park, a discrete area of rain forest in the In a recent case of scrub typhus in Australia, Orientia tsutsugamushi isolated from the patient's blood was tested by sequence analysis of the 16S rDNA gene. The sequence showed a strain of O. tsutsugamushi that was quite different from the classic Karp, Kato, and Gilliam strains. The new strain has been designated Litchfield.

Dispatches
Northern Territory opened as a park in 1986 (16). All patients had eschars, and all cases were confirmed by specific O. tsutsugamushi serologic tests. A case of scrub typhus was also reported from a similar rain forest pocket in the tropical Kimberley region of northwestern Australia (17).

Case Report
In mid-August 1996, a 38-year old man working on the construction of a tourist path in the rain forest fringe of Litchfield Park (15) became ill with fever, sweats, headache, sore throat, cough, lethargy, and confusion during his second week on the job. His condition worsened over at least a week. He eventually received amoxycillin and clavulanate from a local medical practitioner but contracted diarrhea and was admitted to Royal Darwin Hospital with fever, rigors, but no evident focus of infection. A diagnosis of septicemia was made, and he was started on ceftriaxone and gentamicin.
Over the next day, he became increasingly confused, and his fever persisted. He became hypotensive, hypoxemic, and oliguric and was transferred to the intensive care unit. On day 3 after admission, he required fluid loading, inotrope therapy, intubation, and ventilation. At that time, his work history was obtained from relatives, and a 6-mm sore with a necrotic dark center was noted on his upper right buttock. Scrub typhus was suspected, and he was given intravenous doxycycline. However, his condition continued to deteriorate with hypotension, renal failure, and adult respiratory distress syndrome. Mucosal and gastrointestinal bleeding developed, and the patient died 6 days after admission.

Mouse Inoculation
Three female outbred white mice were inoculated with blood taken from the patient 4 days before he died. The mice were inoculated intraperitoneally with 0.1 ml, 0.2 ml, and 0.5 ml of whole blood in EDTA. The mice were housed in a flexible film isolator in a high security microbiology laboratory at the Australian Animal Health Laboratory, Geelong, Australia. None of the mice died.
Subsequently three additional mice were inoculated with very concentrated preparations of the O. tsutsugamushi strain isolated from the patient; none died. This strain of O. tsutsugamushi appears not to be virulent for mice despite being highly virulent for the patient. No attempt was made to isolate the strain from mouse organs.
Seroconversion of the inoculated mice to O. tsutsugamushi was confirmed by microimmunofluorescence.

Rickettsial DNA Detection by Polymerase Chain Reaction (PCR)
One ml of the patients blood, taken 4 days before death, was lysed; DNA was extracted (18); and part of the 56kDa-type specific antigen (an outer membrane protein) gene was amplified by PCR. Primers a (5'-TACATTAGCTGCGGGTATGACA-3') and b (5'-CCAGCATAATTCTTCAACCAAG-3') were used as previously described (19), except that a nested procedure was not used. The PCR product was electrophoresed on agarose gel, stained with ethidium bromide, and detected by UV fluorescence. Positive and negative controls were run with the unknown samples. The PCR product from the patients blood had the same molecular weight as a PCR product from a known O. tsutsugamushi strain.

Tissue Culture Infection
Three 25-cm 2 flasks containing a confluent monolayer of Vero cells were infected with whole blood (in EDTA) from the patient (0.1 ml, 0.5 ml, and 1.0 ml, respectively). After sitting at 35°C for 4 hours without centrifugation, the blood was removed, and the monolayers were washed twice with Hanks balanced salt solution. The medium used for culture was RPMI 164O with 10% fetal calf serum (heat-inactivated) at 35°C (without CO 2 ). Cultures were examined weekly for abnormalities, and the pH was adjusted as necessary. On day 60 postinfection, a 3+ cytopathogenic effect was observed in one flask, with most Vero cells floating in the supernatant. These cells were tested for bacterial contamination (Gram stainnegative; growth on horse Dispatches blood agarnegative) and for rickettsiae (positive for Giminez stain; positive for scrub typhus, as determined by immunofluorescence staining with convalescent-phase human serum; and positive as determined by PCR for 56 kDa antigen gene). The isolated strain was subcultured and grown for DNA extraction.

16S rDNA Sequencing
The 16S rDNA was amplified by PCR with primers A and H* (20), and the products were cloned into pGEM-T (Promega). The complete nucleotide sequences of both strands of several clones were determined by using internal primers (20,21); sequencing reactions were analyzed on an ABI 310 Genetic Analyser by using the ABI Big-Dye chemistry. The sequence has been submitted to GenBank under the accession number AF062074.
Comparison with the databases confirmed that the sequence from the novel isolate was O. tsutsugamushi but indicated that it differed greatly from the four type strains (Karp, Kuroki, Gilliam, Kato). The novel isolate has five unique base substitutions in the 16S rDNA and thus differs more from the reference strains than they do from each other. On this basis, it was designated a new strain and was called Litchfield.
The Litchfield strain of O. tsutsugamushi is the first isolated in culture from the Northern Territory of Australia, although previous isolates have been made from Queensland (4,12). O. tsutsugamushi has probably been present in the mites and native mammals of northern Australia for millennia. One of the most common mite vectors, Leptotrombidium deliense, has been detected on several species of native rat in Litchfield Park (22), although O. tsutsugamushi has not yet been isolated from native mammals from the Northern Territory. In Queensland, however, O. tsutsugamushi has been isolated from native mammals and mites (23)(24)(25).
The emergence of scrub typhus in Litchfield Park in the Northern Territory of Australia is probably due to increasing contact between humans and this scrub and rain forest environment and its animals. Tourism is a major industry in Australia, and increasing numbers of visitors are traveling to remote parts to experience the tropical out-back. Thus, public health officials and local health providers need to be aware of foci of endemic diseases in remote parts of Australia. One problem with rickettsial diseases is that they do not respond to ß-lactam antibiotics (e.g., penicillins and cephalosporins) because of their unusual bacterial cell wall or to aminoglycoside antibiotics (e.g., gentamicin) because of their intracellular location. Treatment of patients with undiagnosed sepsis in Australian hospitals is very likely to involve the use of a cephalosporin plus gentamicin, a combination that is not effective against scrub typhus or any other rickettsial disease. The travel history of a febrile patient is essential if tropical infections are to be included in the differential diagnosis of febrile illness.
The 16S rDNA evolves relatively slowly. Hence, despite geographic proximity, the key difference between the sequences from the Karp strain (isolated from a patient in New Guinea) and the Litchfield isolate implies a long period of isolation between these strains. Few general conclusions can be drawn from one sequence for a single Australian isolate. However, the prediction is that at least some Australian strains of O. tsutsugamushi will differ substantially from type strains.
Dr. Odorico is a molecular biologist with a broad range of interests, including molecular phylogenetics. His present research involves the functional analysis of eukaryotic promoters by use of site-directed mutagenesis and reporter gene techniques.