Rickettsiae and Ehrlichiae Within a City Park: Is the Urban Dweller at Risk?

Introduction

In the United States, tick-borne diseases such as spotted fever group rickettsioses (SFGR) and ehrlichioses have their peak incidence during the late spring and summer when ticks are most active. People afflicted with these infections often fail to recall a recent tick bite, so clinicians often rely on epidemiologic features such as previous travel or residence in endemic rural areas to help distinguish these from other nonspecific febrile illnesses. Untreated infections with these pathogens may be severe and life threatening, so it is imperative to recognize their risk factors, signs, and symptoms (Walker et al. 2008). The recreational escape to rural areas during summer months places individuals at risk for these diseases, but does the lack of an obvious rural excursion preclude exposure to these pathogens? In this study, we chose two popular city parks serving the recreational needs of the citizens of Little Rock, AR, to investigate the presence of these pathogens within ticks collected from these parks.

Materials and Methods

Ticks were collected from Allsopp (park A) and Two Rivers Parks (park B) in Little Rock, AR. Collections were performed by carbon dioxide trapping and flannel flag dragging on July 16, 23, and 24 of 2011. Ticks were stored in 70% isopropyl alcohol at 4°C and later separated into pools by species, sex, and collection site. Adults were grouped into pools of one to five ticks, and nymphs were grouped into pools of six to 10 ticks. Pools were placed in 2-mL microcentrifuge tubes with 100 μL of phosphate-buffered saline and two 4-mm stainless steel grinding balls. Ticks were homogenized with a Retsch MM300 mixer mill (Bio-Rad, Hercules, CA) for 2 min at 30 Hz. DNA was extracted from the homogenate using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA).

Real-time PCR analysis for the rickettsial citrate synthase gene was performed using 18 pmol of forward primer alt-gltA-F (5′-GCTATGGGTATACCGTCGCA-3′), 18 pmol reverse primer alt-gltA-R (5′-CAGGATCTTCGTGCATTTCTTTCC-3′), 5 pmol TaqMan FAM-labeled probe (5′-GCCATCCAACCTACGGTTCTTGC-3′), 10 μL of TaqMan gene expression master mix (Applied Biosystems, Carlsbad, CA), and 2 μL of template DNA per reaction. Thermal cycling consisted of a melting cycle of 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min on a C1000 thermal cycler (Bio-Rad). Positive tick pools were then tested as previously described using rickettsial outer membrane protein A (ompA) primers Rr190.70p and Rr190.602n (Regnery et al. 1991) with subsequent Pst I restriction enzyme fragment length polymorphism (RFLP) analysis (Stromdahl et al. 2008). To confirm the RFLP results, PCR products of the rickettsial outer membrane protein B (ompB) using primers 120-2788 and 120-3599 (Roux and Raoult 2000) and the ompA PCR products of two tick pools were cloned in chemically competent Escherichia coli cells using the TOPO TA Cloning Kit (Invitrogen, Grand Island, NY). Plasmid DNA was purified with the PureLink Plasmid Miniprep Kit (Invitrogen) and sequenced three times using a 3100 Capillary Automated DNA Sequencer (Applied Biosystems).

Ehrlichial screening was performed using primers to amplify the variable-length PCR target (Sumner et al. 1999) and dsb gene (Doyle et al. 2005). PCR products were then cloned and sequenced three times as described above.

Results

A total of 273 ticks were collected (table) of which 172 (63%) were collected from park A and 101 (37%) from park B. Amblyomma americanum was the predominant tick species with 255 (93%) of those collected. The remaining 18 (7%) collected were Dermacentor variabilis. Forty-two of the 43 (98%) A. americanum pools were demonstrated to contain rickettsiae by amplification of the citrate synthase gene. The nine D. variabilis pools contained no rickettsiae. Amplification of a portion of the ompA gene was successful in all but two A. americanum pools that contained Rickettsia. RFLP analysis of these ompA PCR products demonstrated Rickettsia amblyommii to be the species present. This was confirmed by sequencing ompA (KF279539) and ompB (KF279540) PCR amplicons from two different A. americanum pools. One A. americanum pool from park A demonstrated the presence of Ehrlichia. DNA sequences of cloned PCR amplicons of the VLPT (KF279542) and dsb (KF279541) genes confirmed the agent to be Ehrlichia chaffeensis.

Discussion

The parks in which ticks were collected are popular and busy recreational areas with heavily used walking and bicycling trails. Park A also had playgrounds, picnic areas, a softball field, and an outdoor pavilion. We observed abundant tick populations in both locations. R. amblyommii was found within a high percentage of A. americanum tick pools tested. These ticks are prevalent in the southern United States and feed on humans at all life stages (Walker et al. 2008). In a prospective study, the history of tick attachment in areas heavily infested with A. americanum containing R. amblyommii was associated with seroconversion to SFGR (Yevich et al. 1995). The growing number of individuals seropositive for SFGR in the southeastern United States, yet without symptoms compatible with Rocky Mountain spotted fever, likely reflects the prevalence of A. americanum, its feeding habits, and its high rate of infection with R. amblyommii (Walker et al. 2008, Stromdahl et al. 2008, Apperson et al. 2008).

Few D. variabilis ticks, the vector that transmits Rickettsia rickettsii in the southeastern United States, were captured. Rickettsia rickettsii is pathogenic for D. variabilis, which maintains the rickettsiae transovarially (Walker et al. 2008). Consequently, a small number of D. variabilis ticks are found to be infected with this virulent pathogen (Stromdahl et al. 2011).

Molecular evidence of E. chaffeensis, the causative agent of human monocytotropic ehrlichiosis (HME), was found within one A. americanum tick pool caught within park A. E. chaffeensis is maintained transstadially but not transovarially in A. americanum (Walker et al. 2008). Therefore, the presence of this organism indicates that bacteremic hosts such as white tailed deer or dogs have been able to maintain E. chaffeensis infection within the park's tick population. Most importantly, the presence of this pathogen places park visitors at risk for exposure to a potentially severe and fatal disease. The case-fatality rate of 1.9% and hospitalization rate of 49% of those with HME (Dahlgren et al. 2011) make recognition of this illness important in endemic areas. As is evident from these findings, the risk for acquiring HME is not limited to those with rural or suburban tick exposures. Even those in urban areas are at risk for acquiring a tick-borne illness.