CONICET | Buscador de Institutos y Recursos Humanos

Introduction: Amblyomma triste (Acari: Ixodidae) is a tick distributed in the Neotropical and Nearctic regions, but most records are from South America. In Argentina, A. triste became a tick of public health relevance because of the finding of specimens infected with Rickettsia parkeri and the recently reported clinical cases of rickettsiosis in humans with a history of tick bites in localities where A. triste prevails. Furthermore, Romer et al. (2011) identified R. parkeri DNA in cutaneous lesions in two of the clinical cases documented in the Paraná River Delta region. Rickettsia parkeri was previously isolated from A. triste in Uruguay and Brazil. In Uruguay, human disease allegedly caused by R. parkeri has been recurrently reported since 1990 but it was not until recently that a case of R. parkeri infection in a human was confirmed. In Brazil, surprisingly, not a single human case of rickettsiosis associated with A. triste bite has been reported so far. However and notwithstanding all the information available about the distribution of this bacterium, there are no previous reports inquiring about the infection levels of R. parkeri in naturally infected A. triste ticks. Material and Methods: Questing adults ticks were collected from the vegetation by drag flagging in fields in the vicinity of Campana city (34°9.5′S, 58°51.8′W), Buenos Aires Province, Argentina. Collected ticks were brought alive to the laboratory, identified by using standard taxonomic keys and disinfected in iodine alcohol. One half of each tick was processed for DNA extraction and the other half was immediately frozen at 80°C for rickettsia isolation. All tick samples were screened for rickettsial infection through testing them individually by a real-time PCR assay using primers RR190.547 and RR190.701. Ticks positive for ompA real time PCR were subjected to further amplification of a larger fragment of ompA gene by routine PCR using primers RR190.70 and RR190.701. For those samples that presented low levels of rickettsial DNA, a nested PCR using primers RR190.107 and RR190.602 was attempted. Isolation of rickettsiae was attempted on four of the ompA real-time PCR positive A. triste ticks by the shell vial technique. Results: A total of 201 adults (74 males, 127 females) of A. triste were collected. Forty one ticks (20.4%) were found to contain DNA of the rickettsial gene ompA by real-time PCR, 26 of which presented high rickettsial concentration (from 4.3×105 to 9.6×106 ompA copies per tick), while the remaining 15 presented much lower rickettsial levels (from 4.4×101 to 1.3×104 ompA copies per tick). A larger fragment of ompA was amplified using DNA obtained from ticks with high (four samples) and low (two samples, nested PCR) levels of rickettsial DNA, and the resulting nucleotide sequences were 100% identical to the ompA sequence of R. parkeri (U43802) in all cases (592/592, nested: 435/435). No amplification was observed in the negative controls (Ct>50). Isolation assays by the shell vial technique were attempted from four of the ompA real time PCR positive ticks. Rickettsiae were successfully isolated and established in Vero cell culture from two of the R. parkeri-positive ticks. These isolates were designated At97ARG and At114ARG. DNA extracted from infected cells of the third passage was subjected to PCR targeting the gltA, ompA, ompB and htrA genes. The corresponding gene sequences of the two isolates were 100% identical to each other, and the gltA, ompA, ompB and htrA sequences were 100% (1106/1106), 100% (590/590), 100% (817/817) and 99.6% (495/497) similar to the corresponding sequences of R. parkeri strain Maculatum from North America (U59732, U43802, AF123717, U17008, respectively), moreover the htrA sequence was 100% (497/497) similar to the corresponding sequence of R. parkeri isolate At24 from Brazil (EF102237). Discussion: In the present study, we report the first in vitro isolation of R. parkeri from Argentina. Rickettsia parkeri isolates At97ARG and At114ARG obtained herein showed no genetic differences with those R. parkeri isolates reported in Uruguay and Brazil, both obtained from A. triste, and only a slight difference was observed when compared to R. parkeri isolated from Amblyomma maculatum from the United States. Furthermore, these new isolates were achieved from A. triste ticks collected in a region where cases of human R. parkeri-rickettsiosis were confirmed. Previously, Nava et al. (2008) reported R. parkeri infecting 8.4% of the A. triste ticks collected from the vegetation in the Paraná River Delta. In the present study, a higher R. parkeri infection rate was found in questing ticks (20.4%) in the same location. The differences observed in infection rates could be in part due inter-annual variation or to the high sensitivity reported for the real-time PCR assay used in our study, compared with the PCR approach used by Nava et al. (2008). However, we cannot rule out the possibility that infection incidence of R. parkeri is indeed increasing, posing a risk for public health. In this respect, further research should be aimed at elucidating whether climatic conditions, soil management, cattle presence or host abundance is affecting R. parkeri infection rate of A. triste ticks. Interestingly, the distribution of R. parkeri infection intensity observed in A. triste ticks was distinctly bimodal, with approximately 60% of the infected ticks presenting high rickettsial loads and the remainder with low rickettsial levels. Although very pertinent for our understanding of the eco-epidemiology of rickettsial pathogens, the distribution of the levels of infection in the ticks has not been previously assessed. The bimodal distribution found herein could have important epidemiological implications, as the infection intensity in the tick must be directly related with the exposure dose after a tick bite. Elucidating the determinants of this distinct distribution is also crucial to better understand the ecology of this disease (e.g. circumstances that cause a predominance of high infection intensities in ticks would consequently increase the risk of infection in humans). In addition, this bimodality in R. parkeri infection intensity in ticks could determine not only differences in the severity of the disease, but also differential mechanisms of transmission. Finally, the isolates of R. parkeri obtained herein will also provide us with a valuable tool to develop diagnostic tests which will allow to assess the serological status of livestock and wildlife populations and to confirm human cases suspicious of rickettsiosis in our region.