ISOLATION OF Rickettsia bellii FROM Amblyomma ovale AND Amblyomma incisum TICKS FROM SOUTHERN BRAZIL

Objective. To isolate and characterize rickettsiae from the ticks Amblyomma ovale and Amblyomma incisum collected in the state of São Paulo. Materials and methods. Adult, free-living A . ovale and A . incisum were collected in an Atlantic rainforest area in the state of São Paulo, Brazil. Each tick was tested using the hemolymph assay; samples from positive ticks were placed in shell vials in order to isolate rickettsiae and subsequently grown in Vero cells. Amplification of three rickettsial genes ( gltA, htrA and ompA ) was attempted using polymerase chain reaction (PCR) for each isolate obtained. Amplicons were subsequently sequenced. Results . A total of 388 A . incisum and 50 A. ovale were collected. Only one A . incisum and one A. ovale were hemolymph-test positive. Rickettsiae were successfully isolated from these ticks; however establishment in Vero cell culture was successful only for the isolate from A. ovale . Bacterial contamination in the first cell passage of the A. incisum isolate precluded successful isolation of the organism. PCR products were obtained with the gltA and htrA primers for the two isolates, but no product was obtained with the ompA primers. By BLAST analysis, partial gltA and htrA sequences of isolates from A. ovale and A. incisum were similar to the corresponding sequences of R. bellii . Conclusions . This is the first report of R. bellii infecting A. incisum and the first successful isolation from A. ovale.

with Rickettsia canadensis (3).R. bellii, the most common rickettsia found in ticks in America, was isolated for the first time in embryonated chicken eggs from a triturated pool of unfed adult Dermacentor variabilis in the United State, one of the most important vectors of R. rickettsii in that country, but there is no evidence that R. bellii can infect humans (4).It has been found in various other hard ticks, including species of Amblyomma, Ixodes, Haemaphysalis, and in soft ticks, including species of Argas and Ornithodoros (4)(5)(6)(7)(8)(9)(10)(11)(12).
In Brazil, despite R. bellii was also the most common rickettsial species identified in ticks from the state of Rondônia, Western Amazon, it was only successfully isolated and established in continuous cell passages from Amblyomma scalpturatum (7) and in Amblyomma aureolatum, Amblyomma dubitatum, and Haemaphysalis juxtakochi in the state of São Paulo (10)(11)(12).In the present study, we tested samples of

INTRODUCTION
Rickettsiae are pathogenic bacteria that require eukaryotic cells in order to grow.They are gram-negative, small, rod-shaped, a-proteobacteria causing mild to severe diseases in humans and animals.The genus subdivides into the conventionally well defined typhus group (TG) and spotted fever group (SFG) based on phylogenetic analyses.The TG includes the louse-borne Rickettsia prowazekii (the agent of epidemic typhus) and the flea-borne Ricketsia typhi (the agent of endemic typhus).The SFG includes tick-borne Rickettsia rickettsii (the agent of Rocky Mountain spotted fever), Rickettsia conorii (the agent of Mediterranean spotted fever), Rickettsia africae (the agent of African tick bite fever), Rickettsia sibirica (the agent of North Asian tick-borne fever), mite-borne Rickettsia akari (the agent of rickettsialpox), and flea-borne Rickettsia felis (the agent of flea-borne spotted fever) (1,2).Rickettsia bellii is the most divergent species, being representative of a distinct group within the genus Rickettsia, together Amblyomma ovale and Amblyomma incisum ticks collected in the state of São Paulo (southeastern Brazil), attempting to isolate rickettsiae in cell culture and establishment in continuous cell passages.(13).Ticks were brought alive to the laboratory and before testing live ticks by the hemolymph test, ticks were held in an incubator at 32 o C and 95% RH for 3 to 5 days, in order to stimulate rickettsial growth (14).

MATERIALS AND METHODS
Thereafter, each tick was subjected to the hemolymph test for detection of Rickettsialike structures, as previously described (14).Briefly, the distal portion of one leg of each tick was cut with scissors and a drop of hemolymph was fixed by air in a glass slide and stained by Gimenez staining (15).Right after the collection of hemolymph, ticks were frozen in a -80 o C freezer.
Attempts to isolate Rickettsia in Vero cell culture were performed with ticks shown to contain Rickettsia-like structures by the hemolymph test.For this purpose, frozen ticks were thawed and subjected to the shell vial technique as previously described ( 16), with some modifications (8).Briefly, individual ticks were thawed in water bath at 37 o C and disinfected for 10 minutes in iodine alcohol followed by several washes in sterile water.Then each tick was triturated in 500 ml of brain heart infusion broth (BHI) and the resultant tick homogenate inoculated into shell vials containing a monolayer of confluent Vero cells.After inoculation, the shell vials were centrifuged for one hour at 700 g at 22 o C. Thereafter, the monolayer was washed once with minimal essential medium containing 5% bovine calf serum, and then incubated at 28 o C with medium containing antibiotics (1% penicillin, streptomycin).After three days, the medium was switched to antibiotic-free medium, and the aspirated medium was checked by Gimenez staining for the presence of Rickettsia like-organisms.If the result was positive, the monolayer of the shell vial was harvested and inoculated into a 25 cm 2 flask containing a monolayer of confluent uninfected Vero cells.Cells of the 25 cm 2 flask were checked by Gimenez staining until more than 90% of the cells were infected, when they were harvested and inoculated into 150 cm 2 flasks of Vero cells.At all instances, inoculated Vero cells were incubated at 28 o C. The level of infection of cells was monitored by Gimenez staining (15), of scrapped cells from inoculated monolayer.A rickettsia isolate was considered established in the laboratory after at least three passages through 150 cm 2 Vero cell flasks, each reaching more than 90% of infected cells (8).
Cell passages of isolates were genotypicaly identified by polymerase chain reaction (PCR) amplification and sequencing the product of the resultant infected cells.For this purpose, DNA was extracted using the Dneasy Tissue Kit (Quiagen ® ), following manufacturer's protocol for isolation of DNA from culture cells samples and DNA from infected cell passages were tested by a battery of PCR using all primer pairs listed in table 1, targeting fragments of tree rickettsial genes: citrate synthase (gltA), 17-kDa outer membrane protein (htrA), and 190-kDa outer membrane protein (ompA).For each set of reactions, a negative control (5 μl of water) and positive control (5 μl of DNA extracted from A. cajennense ticks experimentally infected with R. parkeri) was included (17).
All PCR products of the expected size obtained in the present study were purified using ExoSap (USB) and sequenced in an automatic sequencer (Applied Biosystens/ Perkin Elmer, model ABI Prism 310 Genetic, California, USA) according to the manufacturer's protocol.Partial sequences obtained were submitted to BLAST analysis, (18), to determine similarities to other Rickettsia species.

RESULTS
A total of 388 ticks A. incisum were collected (180 males, 208 females) and 50 ticks A. ovale (17 males, 33 females).By the hemolymph test, one A. incisum male and one A. ovale female that showed to contain rickettsia-like organisms by the hemolymph test were processed by the shell vial technique (8,16).Rickettsia was successfully isolated and established in Vero cell culture from tick A. ovale.Although the establishment of Rickettsia in Vero cell culture from the A. incisum tick was unsuccessful, the initial cultivation of rickettsia was possible due to the presence of numerous Giménez-stained intracellular rickettsia-like organisms from the inoculated shell vials, which could not be cultivated further than the first passage owing to contamination of the cell cultured by extracellular bacteria.
Even though, DNA of contaminated cell culture at first passage was tested by PCR, as described below.The isolate from A. ovale generated in the present study has been deposited in the Rickettsial Collection of the Laboratory of Parasitic Diseases of the Faculty of Veterinary Medicine, in the University of São Paulo, where it is available upon request.

DNA of infected cells at fourth passage of
A. ovale and first passage of A. incisum were subjected to PCR targeting the rickettsial genes gltA, ompA and htrA.PCR products of the expected size were obtained with the gltA and htrA primers for two isolates, but no product was obtained with the ompA primers (these primers have been specific for SFG rickettsiae).We sequenced 1060 and 500 nucleotides of the gltA and htrA genes, respectively, from the A. ovale isolate, and 1096 and 499 nucleotides of the gltA and htrA genes, respectively, from the A. incisum isolate.By BLAST analysis, partial gltA sequence of the A. ovale rickettsia in the present study was 100% (1060/1060) similar to the corresponding sequence of R. bellii strain HJ7 (DQ865204), and partial gltA sequence of the A. incisum rickettsia was 99.9% (1095/1096) similar to the corresponding sequence of Rickettsia bellii RML369-C (CP000087).Partial htrA sequence of the A. ovale rickettsia was 99.6% (497/ 499) similar to the corresponding sequence of R. bellii (AY362702), whereas partial htrA sequence of the A. inicisum rickettsia was 100% (499/499) similar to R. bellii (AY362702).Thus, both rickettsial agents from A. ovale and A. inicisum were genotypically identified as R. bellii.
During 2004-2006, adult free-living A. ovale and A. incisum ticks were collected on the vegetation in an Atlantic rainforest area in the Intervales State Park, Ribeirão Grande Municipality, state of São Paulo (24 o 18'S, 48 o 24'W) by visual inspection of vegetation during another study on ecology of freeliving ticks in the Intervales State Park

Table 1 .
Primer pairs used for amplification of rickettsial genes.