Amblyomma imitator Ticks as Vectors of Rickettsia rickettsii, Mexico

Real-time PCR of Amblyomma imitator tick egg masses obtained in Nuevo Leon State, Mexico, identified a Rickettsia species. Sequence analyses of 17-kD common antigen and outer membrane protein A and B gene fragments showed to it to be R. rickettsii, which suggested a potential new vector for this bacterium.

held in 16-mL glass vials (Wheaton Glass, Millville, NJ, USA) with a mesh top over a supersaturated solution of potassium nitrate. Larvae and nymphs obtained blood meals from mice, and adults were fed on pathogen-free rabbits. Only the A. imitator colony was successfully established.
Real-time PCR analysis of eggs laid by 1 full generation of laboratory-reared females identifi ed rickettsial DNA in 2 egg masses. DNA from 5 egg masses laid by different females was extracted by using a DNeasy Kit (QIAGEN, Valencia, CA, USA). Real-time PCR was performed by using Rickettsia spp.-specifi c primers CS5A and CS6 (5) for amplifi cation of a 150-bp fragment of the citrate synthase gene in an iCycler thermocycler (Bio-Rad, Hercules, CA, USA) as described (5). R. australis DNA and water served as the positive and negative controls, respectively, and serial dilutions of a plasmid that contained the R. prowazekii citrate synthase gene were used as standards.
We selected egg masses for isolation of the rickettsial agent in Vero cells by using shell vials as described (6). The shell vials were incubated at 34°C and monitored daily by Diff-Quik staining (Dade International Inc., Miami, FL, USA) for rickettsiae. Slides that contained >4 rickettsiae were considered positive. The monolayer from the corresponding shell vial was removed manually, placed in a T-25 fl ask containing Vero cells (in Dulbecco minimal essential medium containing 3% heat-inactivated bovine calf serum). Samples were then placed in 150-cm 2 fl asks containing Vero cells for propagation of the agent.
For characterization of isolates, partial sequences of Rickettsia-specifi c genes were amplifi ed and analyzed. Nested PCR was performed by using primers 17K3 and 17K5 (5) for the fi rst reaction and 17KD1 and 17kD2 (7) for the second reaction of amplifi cation of 17-kD common antigen (htrA) gene fragments. For amplifi cation of an outer membrane protein B (ompB) gene fragment, primers 120-M59 and 120-807 were used (8). Seminested PCR with primers 120-M59 and 120-807 for the fi rst reaction and 120-607 and 120-807 for the second reaction was performed for 1 of the samples (8). An ompA fragment was amplifi ed by using primers Rr190.70F and Rr190.602R (9). For 1 of the samples, a seminested PCR was necessary; primers Rr190.70F and Rr190.701R (10) were used for the fi rst reaction and Rr190.70F and Rr190.602R for the second reaction.
Fragments were cloned into pCR4-TOPO (Invitrogen, Carlsbad, CA, USA), and plasmids from selected clones were sequenced >3× by using universal primers M13F and M13R. Nucleotide sequences were edited with SeqMan (www.dnastar.com/t-sub-products-lasergene-seqmanpro. aspx) and used for BLAST analysis (11). Amplifi cation of the ompB fragment was achieved for only 1 of the samples. This fi nding was probably the result of a mutation at the site of primer annealing because amplifi cation of another region of the gene was possible.
Analysis of sequences obtained from the htrA gene fragments (434 bp) amplifi ed from both samples showed 99% identity with spotted fever group Rickettsia sequences, including the R. rickettsii sequence from a fatal case of RMSF in southwestern Mexico, Yucatan State (GenBank accession no. DQ176856.1) (12), with only a 1-nt difference. Analysis of the partial sequence of ompB (856 bp) and ompA (533 bp) genes showed 100% identity with R. rickettsii Sheila Smith strain (CP000848.1). Sequences obtained in this study were submitted to GenBank under accession nos. GU723476 and GU723477 for htrA fragments, GU723478 and GU723479 for ompA fragments, and GU723475 for the ompB fragment.
For detection of rickettsiae in A. imitator ticks, salivary glands, midguts, and ovaries were dissected from unfed adult ticks and fi xed in modifi ed Ito fi xative (13), postfi xed in 1% osmium tetroxide for 1 h, stained en bloc with 2% aqueous uranyl acetate for 20 min at 60°C, dehydrated in ethanol, and embedded in epoxy resin (Poly/Bed 812). Ultrathin sections were cut with an Ultracut S Ultramicrotome (Reichert, Vienna, Austria), placed on copper grids, and stained with lead citrate. Ticks were tested for Rickettsia spp. by nested PCR with primers 17K3/17K5 and 17KD1/17KD2, and sections of organs from PCR-positive samples were examined in a Philips CM-100 electron microscope (FEI, Hillsboro, OR, USA) at 60 kV.
Single rickettsial cells were found in highly vacuolated cytoplasm of midgut epithelial cells of 1 male tick (no. 5) (Figure, panel A). Cells had typical ultrastructure for gram-negative bacteria and were surrounded by 2 trilaminar membranes (Figure, panel B): an inner cytoplasmic membrane and an outer cell wall membrane. Their size was 0.6 × 0.2 μm. Rickettsial organisms were not visualized in either salivary gland or ovaries probably because of a low number of rickettsiae, as suggested by the fact that detection of rickettsial DNA by PCR required a nested PCR.

Conclusions
This study demonstrated that R. rickettsii was present in egg masses of A. imitator ticks. Because eggs were laid by fi eld-collected, unfed, adult ticks that were fed in the laboratory on pathogen-free rabbits for 1 full generation, R. rickettsii in eggs documents their transovarial transmission by naturally infected ticks and suggests a role for this tick species in the maintenance of R. rickettsii in nature.
Because hosts of A. imitator ticks are various species of birds and mammals (3), the notable fi nding of this study is the potential participation of A. imitator ticks in a zoonotic cycle of R. rickettsii. According to Kohls (2), RMSF transmission studies performed with supposed A. cajennense ticks by Parker et al. (14) were actually performed with A. imitator ticks, which suggests that this species of tick could be a vector of R. rickettsii. On the basis of the results of our study and the aggressive nature of the tick for humans, we suggest that A. imitator ticks are a potential vector or at least involved in maintenance of R. rickettsii in nature in Mexico.