Isolation and identification of Rickettsia massiliae from Rhipicephalus sanguineus ticks collected in Arizona

Abstract

Twenty Rhipicephalus sanguineus ticks collected in eastern Arizona were tested by PCR assay to establish their infection rate with spotted fever group rickettsiae. With a nested PCR assay which detects a fragment of the Rickettsia genus-specific 17-kDa antigen gene (htrA), five ticks (25%) were found to contain rickettsial DNA. One rickettsial isolate was obtained from these ticks by inoculating a suspension of a triturated tick into monolayers of Vero E6 monkey kidney cells and XTC-2 clawed toad cells, and its cell culture and genotypic characteristics were determined. Fragments of the 16S rRNA, GltA, rOmpA, rOmpB, and Sca4 genes had 100%, 100%, 99%, 99%, and 99%, respectively, nucleotide similarity to Rickettsia massiliae strain Bar29, previously isolated from R. sanguineus in Catalonia, Spain (L. Beati et al., J. Clin. Microbiol. 34:2688-2694, 1996). The new isolate, AZT80, does not elicit cytotoxic effects in Vero cells and causes a persistent infection in XTC-2 cells. The AZT80 strain is susceptible to doxycycline but resistant to rifampin and erythromycin. Whether R. massiliae AZT80 is pathogenic or infectious for dogs and humans or can cause seroconversion to spotted fever group antigens in the United States is unknown.

FIG. 1.

Nucleotide sequence comparison of 17-kDa antigen gene fragment from AZ ticks and its nearest relatives. Reference sequences of R. rhipicephali (AF483196), ARANHA (AY360215), and ATT (AF483196) are from NCBI GenBank; sequences from AZ ticks are from this study.

FIG. 2.

RFLP typing of spotted fever group rickettsiae in R. sanguineus ticks. The 70- to 602-nucleotide fragment of the rOmpA gene was amplified using seminested PCR, followed by restriction enzyme digestion with RsaI (A) and PstI (B). Restriction patterns of the homologous fragments from other spotted fever group rickettsiae found in R. sanguineus were published previously (25). Lanes 1, 1-kb Plus DNA molecular size ladder (Invitrogen); lanes 2, AZT68; lanes 3, AZT80. Other abbreviations: Bar29, R. massiliae strain Bar29; Mas, R. massiliae strain Mtu1; RH, R. rhipicephali strain 3-7-6♀; RC, R. conorii Malish; RR, R. rickettsii Sheila Smith. Arrows indicate positions of the 100-bp fragment of the ladder.

FIG. 3.

Acridine orange staining of AZT80 isolate released from VERO E6 cell monolayers (A) and XTC-2 cells (B). Photographs were taken using Zeiss fluorescent microscope with a 10× ocular and a 100× objective.

FIG. 4.

Kinetics of AZT80 growth in XTC-2 cells (A) and VERO E6 cells (B) determined by the SYBR green PCR assay. Data are the averages (± standard error) of two independent measurements for each of two 33-mm dishes infected under the same conditions and are expressed as numbers of copies of rickettsial DNA normalized to total DNA concentration in each sample (solid lines and black symbols). Broken lines and open symbols correspond to medians of the same experimental variables.

FIG. 5.

Effects of varied antibiotics on growth of AZT80 isolate. The data are shown as the average ± standard error of two independent measurements for each of two 33-mm dishes infected under the same conditions and are expressed as numbers of copies of rickettsial DNA normalized to total DNA concentration in each sample. The inset illustrates the effects of the antibiotics on R. rickettsii.

FIG. 6.

Sequence variability of the rOmpA gene fragment among isolates of R. massiliae. Differences in nucleotide sequence (A) and derived amino acid sequence (B) are shown. The following reference sequences were from the NCBI GenBank: isolate Mtu1 (U43799), isolate Bar29 (U43792), and isolate GS (U43793).