Detection of Rickettsia amblyommatis and Ehrlichia chaffeensis in Amblyomma americanum Inhabiting Two Urban Parks in Oklahoma

Abstract

For the past 30 years, the number of people infected with causative agents of ehrlichiosis, Rocky Mountain spotted fever, and spotted fever group rickettiosis (SFGR) has increased in Oklahoma. However, there is a lack of data on pathogen prevalence within urban environments. To assess the prevalence of tick-borne pathogens in different environments, 434 Amblyomma americanum (lone star) ticks were collected from the environment in two parks in Edmond, Oklahoma. The presence of Ehrlichia spp. and spotted fever group (SFG) Rickettsia spp. was determined using quantitative real-time polymerase chain reaction (qPCR). 33.6% (146/434) of the A. americanum ticks were positive for Rickettsia amblyommatis and 15.2% (66/434) were positive for Ehrlichia chaffeensis. No ticks were positive for other SFG Rickettsiae (R. rickettsii, R. parkeri) or other Ehrlichiae (E. ewingii, and Panola Mountain Ehrlichia). These studies provide increased understanding of the potential risk for encountering tick-borne pathogens in urban environments.

Rickettsial and ehrlichial disease occurrence has been increasing among humans in the United States for the past few decades (Biggs et al. 2016). However, few studies have looked at these pathogens in urban environments or city parks (Noden et al. 2016). In Oklahoma, high rates of spotted fever group (SFG) rickettsiosis, ehrlichiosis, and tularemia occur on an annual basis in the population (Noden et al. 2016). In 2017, a total of 431 cases of Rocky Mountain spotted fever and ehrlichiosis were reported to the Oklahoma State Department of Health (OSDH 2018). Reported cases of the two diseases occurred in all months of the year (OSDH 2018).

Amblyomma americanum (lone star tick) is an established tick species in 68 out of 77 counties in Oklahoma (Barret et al. 2015). A. americanum is a pest across most of the southeastern United States, and transmits Rickettsia spp., Francisella tularensis, Ehrlichia chaffeensis, Ehrlichia ewingii, and Heartland virus (Barrett et al. 2015). Its most common activity times are from mid-March to mid-August, and it feeds on a variety of hosts (Bouzek et al. 2013). In many studies, pathogen prevalence is determined by grouping ticks together and testing them as pools. Although testing of pooled tick samples provides evidence for the presence or absence of a pathogen in a given area, it does not accurately describe pathogen prevalence in tick populations. In this study, individual A. americanum ticks that were previously collected from the environment for a full year in two Edmond, Oklahoma parks (Small et al. 2019) were tested for Rickettsia spp. and E. chaffeensis by multiplex quantitative real-time polymerase chain reactions (qPCR). The two parks were in areas with different city zoning, residential (latitude 35° 41′ 8.79″ N, longitude −97° 30′ 36.37″ W) and agricultural (latitude 35° 38′ 40.20″ N, longitude −97° 21′ 45.13″ W).

Ticks were collected from June of 2016 through June of 2017 and stored in 70% ethanol at −20°C until qPCR testing. Adult and nymphal A. americanum ticks were dissected individually and tissues, including the midgut, were removed with a #11 scalpel blade (Varela-Stokes 2007). The tissues were placed into individual centrifuge tubes with lysis buffer and incubated at room temperature for 12–18 h (Halos et al. 2004). A GE Illustra extraction kit (GE Healthcare Life Sciences, Pittsburgh, PA) was used to extract DNA from the tissues of individual ticks. An alternative lysis buffer (NaCl 0.1 M, Tris-HCL 0.21 M, pH8 ethylenediaminetetraacetic acid [EDTA] 0.05 M, sodium dodecyl sulfate [SDS] 0.5%) was used in place of the buffer provided by GE and the remaining extraction procedure followed the protocol provided by the manufacturer (Halos et al. 2004). Extracted DNA was stored at −20°C.

Multiplex qPCR testing was performed on a Bio-Rad CFX96 Touch Real-time PCR Detection System (Bio-Rad, Hercules, CA) and was used to detect Ehrlichia spp. and Rickettsia spp. using previously described methods and primers (Gaines et al. 2014). A 50 μL total volume reaction was used and included Bio-Rad iQ Multiplex Powermix with 10 μL of DNA. Five randomly selected positive Rickettsia amblyommatis samples and five randomly selected positive E. chaffensis samples were sequenced to confirm their identities. Primers used for sequencing were from previously published data (Kocan et al. 2000, Blair et al. 2004). Sequencing of PCR products was done by Eton Bioscience, Inc., San Diego, CA. Chi-squared tests were performed in R and used to determine statistically significant differences in bacteria prevalence within and between the two parks (R Core Team 2018). No other Ehrlichia or Rickettsia spp. were detected.

Among the 434 A. americanum ticks tested, R. amblyommatis, formerly Rickettsia amblyommii (Karpathy et al. 2016) was more prevalent in adults and nymphs at both sites, with a prevalence of ∼33.6% (Table 1). E. chaffeensis was more prevalent in the adult A. americanum ticks at the nonresidential site. Statistical analyses showed no significant difference in bacteria prevalence between the two parks (data not shown). However, there was a significant difference in prevalence of these bacteria within each park (p < 0.05). The prevalence of R. amblyommatis was significantly higher than E. chaffeensis at each park. A greater prevalence of R. amblyommatis in A. americanum ticks than E. chaffeensis has been reported by others as well (Sanchez-Vicente et al. 2019). Whether the differences in A. americanum infection rates with R. amblyommatis and E. chaffeensis is biologically relevant is unclear.

Table 1.

Rickettsia amblyommatis and Ehrlichia chaffeensis Prevalence Among Amblyomma americanum Ticks at a Residential and Nonresidential Park in Edmond, Oklahoma, USA as Determined by qPCR

Park	Stage (No. of ticks tested)	R. amblyommatis (No. of positive ticks)	E. chaffeensis (No. of positive ticks)
Residential	Adults (144)	33.3% (48)	14.6% (21)
	Nymphs (145)	36.6% (53)	13.1% (19)
Nonresidential	Adults (93)	29.0% (27)	21.5% (20)
	Nymphs (52)	34.6% (18)	11.5% (6)
Total	434	33.6% (146)	15.2% (66)

Higher case rates of Rocky Mountain spotted fever (RMSF) and SFG rickettiosis (SFGR) than ehrlichiosis in Oklahoma coupled with lower prevalence of Dermacentor variabilis ticks, the primary vector of Rickettsia rickettsii, may be an indication that R. amblyommatis is responsible for some of the RMSF or RMSF-like illnesses reported in Oklahoma. In 2008, Apperson et al. proposed that the high R. amblyommatis positivity rates in A. americanum ticks in North Carolina may be contributing to the rise in reported RMSF cases in North Carolina (Apperson et al. 2008). Therefore, additional studies are warranted to address the possibility that R. amblyommatis is more than a symbiont. Of the A. americanum ticks tested, two were positive for both R. amblyommatis and E. chaffeensis.

The residential park is surrounded by neighborhoods and a school. It provides paved walking trails, a disc golf course, and several playgrounds. In contrast, the nonresidential park provides access to Arcadia Lake, in addition to camping spots, hiking trails, and mountain biking trails. At both sites, the two bacteria were found in both adult and nymphal A. americanum ticks. Rickettsia spp. can be transmitted transovarially and transstadially (Biggs et al. 2016), therefore, it is likely that A. americanum may be acting as a reservoir host and maintaining the prevalence of this potential pathogen. Rickettsia species were found to have a higher prevalence rate in nymphal A. americanum ticks tested than adults. E. chaffeensis is conserved by ticks transstadially, but not transovarially (Long et al. 2003). The prevalence of this pathogen in an area must be maintained by a reservoir host, specifically, the white-tailed deer (Odocoileus virginianus) or potentially dogs (Blanton et al. 2014).

The prevalence rate of E. chaffeensis was highest among A. americanum adults at the nonresidential site. Grund et al. (2002) found that in a park woodland area (similar to the nonresidential site in this study) within a season, the home range of white-tailed deer was larger than the home range of white-tailed deer in a residential woodland (similar to the residential park in this study) (Grund et al. 2002). Therefore, the greater prevalence of E. chaffeensis in A. americanum ticks from the nonresidential park in this study may be due to an increased presence of white-tailed deer in the nonresidential park and surrounding areas, which provides A. americanum ticks more opportunities to feed on infected deer.

RMSF, SFGR, and ehrlichiosis are potential life-threatening diseases among the human population (Biggs et al. 2016). As E. chaffeenis is an established human pathogen and R. amblyommatis remains a possible but unconfirmed human pathogen of SFGR or a RMSF-like disease, it is important to inform the general public of areas where these bacteria are present in the community and how to avoid their vectors. Additional studies of white-tailed deer and small mammal populations in both park types are warranted to better understand pathogen prevalence. Pathogen prevalence among other tick species such as D. variabilis (American dog tick), Amblyomma maculatum (Gulf Coast tick), and Ixodes scapularis (deer tick) that are present in both parks would provide more information on the potential for encountering additional tick-borne pathogens.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This study was supported by the Oklahoma Idea Network of Biomedical Research Excellence, 655 Research Parkway, Suite 2000 Oklahoma City, OK 73104, and The University of Central Oklahoma Office of Research and Grants, 100 North University Drive, Edmond, OK 73034.