Detection of Rickettsia Species,and Coxiella -Like and Francisella -Like Endosymbionts in Amblyomma americanum and Amblyomma maculatum from a Shared Field Site in Georgia,United States of America

Abstract

Two abundant species of aggressive ticks commonly feed on humans in Georgia: the Gulf Coast tick (Amblyomma maculatum) and the Lone Star tick (A. americanum). A. maculatum is the primary host of Rickettsia parkeri, “Candidatus Rickettsia andeanae,” and a Francisella-like endosymbiont (AmacFLE), whereas A. americanum is the primary host for R. amblyommatis, Ehrlichia chaffeensis, E. ewingii, and a Coxiella-like endosymbiont (AamCLE). Horizontal transmission of R. parkeri from A. maculatum to A. americanum by co-feeding has been described, and R. amblyommatis has been found infrequently in A. maculatum ticks. We assessed the prevalence of these agents and whether exchange of tick-associated bacteria is common between A. maculatum and A. americanum collected from the same field site. Unengorged ticks were collected May–August 2014 in west-central Georgia from a 4.14 acre site by flagging and from humans and canines traversing that site. All DNA samples were screened with quantitative PCR assays for the bacteria found in both ticks, and the species of any Rickettsia detected was identified by species-specific TaqMan assays or sequencing of the rickettsial ompA gene. Only R. amblyommatis (15) and AamCLE (39) were detected in 40 A. americanum, while the 74 A. maculatum only contained R. parkeri (30), “Candidatus Rickettsia andeanae” (3), and AmacFLE (74). Neither tick species had either Ehrlichia species. Consequently, we obtained no evidence for the frequent exchange of these tick-borne agents in a natural setting despite high levels of carriage of each agent and the common observance of infestation of both ticks on both dogs and humans at this site. Based on these data, exchange of these Rickettsia, Coxiella, and Francisella agents between A. maculatum and A. americanum appears to be an infrequent event.

Introduction

Ticks are responsible for transmitting a large diversity of pathogens to both humans and wildlife and are best known for their role as vectors of bacteria, parasites, and viruses (Jongejan and Uilenberg 2005, Boulanger et al. 2019). This study examines two species of ticks known to harbor and transmit rickettsial agents and which commonly feed on humans in Georgia and in an expanding range in the United States: Amblyomma maculatum (Gulf Coast tick) and A. americanum (lone star tick) (Merten and Durden 2000, Sonenshine 2018, Allerdice et al. 2019).

Many species of Rickettsia in the spotted fever group (SFGR) cause infections in humans, but the prevalence and severity of disease depend largely on the causative infectious agent. Rickettsia parkeri was first confirmed as a cause of human infection in 2004 (Paddock et al. 2004); its primary U.S. vector is A. maculatum, but this agent and closely related types are also present in different tick species from the United States to Argentina (Nieri-Bastos et al. 2018). Estimates of R. parkeri prevalence in A. maculatum ticks from different U.S. locations vary greatly, from 11.5% to 55.7% (Sumner et al. 2007, Paddock et al. 2010, Fornadel et al. 2011, Nadolny et al. 2014). A. maculatum may also carry Candidatus Rickettsia andeanae; while this agent is not yet known to be infectious in the United States, a related agent was originally detected in association with a rickettsiosis in Peru (Blair et al. 2004, Jiang et al. 2005). Although the prevalence of Candidatus Rickettsia andeanae (hereafter R. andeanae) in A. maculatum is typically as low as 1–7% (Paddock et al. 2010, Fornadel et al. 2011, Varela-Stokes et al. 2011, Ferrari et al. 2012, Noden et al. 2020), rates as high as 47% and 73% have been detected in A. maculatum ticks from Kansas and Oklahoma, respectively, where R. parkeri is found infrequently (Paddock et al. 2015). One of the most common Rickettsia sp. in A. americanum is R. amblyommatis (Karpathy et al. 2016), and there has been speculation that R. amblyommatis might be associated with human illness (Billeter et al. 2007, Apperson et al. 2008, Hardstone Yoshimizu and Billeter 2018). The prevalence of R. amblyommatis in A. americanum also varies greatly, with carriage rates ranging from 26% to 89% in different populations (Mixson et al. 2006, Jiang et al. 2010, Moncayo et al. 2010, Smith et al. 2010, Fritzen et al. 2011, Killmaster et al. 2014, Nadolny et al. 2014, Santanello et al. 2018, Lydy et al. 2020).

While A. maculatum is the primary vector of R. parkeri, R. parkeri has been detected in field-collected A. americanum ticks at frequencies of <1% (Cohen et al. 2009, Gaines et al. 2014), higher in engorged ticks (Lydy et al. 2020), but not at all in other studies (Hudman et al. 2018, Egizi et al. 2020). Recently, laboratory experiments have shown that horizontal transmission of R. parkeri from A. maculatum ticks to A. americanum by co-feeding is possible (Goddard 2003, Wright et al. 2015). Lee et al. (2018) were able to demonstrate co-feeding transmission of R. parkeri, but not R. andeanae between capillary infected adult A. maculatum and uninfected nymphs fed on cattle. In addition, R. amblyommatis has been documented infrequently in field-collected A. maculatum ticks, at the low rate of 1.42% (Fornadel et al. 2011) and in 1 of 59 adult A. maculatum (8 with R. andeanae and 2 with R. parkeri) from Kentucky (Lockwood et al. 2018), but not detected at all in other studies (Nadolny et al. 2014, Pagac et al. 2014). Harris et al. (2017) demonstrated a loss of fitness by A. maculatum exposed to R. amblyommatis and R. parkeri, and while transovarial transmission occurred, diminished transstadial transmission from F1 larvae to F1 adults occurred with both rickettsiae. On the other hand, while the presence of R. amblyommatis in A. americanum ticks reduced the ability of R. rickettsii co-infected larvae to transmit the latter transstadially, no effects of R. rickettsii acquisition by engorgement on nymphs or adults were observed and R. rickettsii transovarial transmission was comparable in the two cohorts (Levin et al. 2018).

In addition to SFGR, Ehrlichia spp. are also tick-borne intracellular bacteria (Anaplasmataceae) that can cause ehrlichiosis in humans. In the United States, Ehrlichia chaffeensis and E. ewingii are the major agents of human ehrlichiosis. A. americanum is the primary vector for both E. ewingii and E. chaffeensis (Anderson et al. 1993, Varela-Stokes 2007). Prevalence of E. ewingii and E. chaffeensis in A. americanum is estimated at 3.5–3.7% and 4.7–5.6%, respectively, but varies widely among different tick populations (Mixson et al. 2006, Fritzen et al. 2011, Sayler et al. 2016). The Panola Mountain Ehrlichia is also found in low abundance in A. americanum throughout its range (Loftis et al. 2008, 2016).

A. americanum and A. maculatum also each have their own distinct endosymbionts. Detection of a Coxiella-like endosymbiont (hereafter referred to as AamCLE) in A. americanum is almost ubiquitous, typically occurring at rates from 89% to 100% (Jasinskas et al. 2007, Heise et al. 2010). This endosymbiont is thought to provide essential vitamins and amino acids that are insufficient with an obligate hematophagous diet (Zhong 2012). A. maculatum also contains a Francisella-like endosymbiont (hereafter referred to as AmacFLE) (Scoles 2004) in high abundance (Budachetri et al. 2014, Varela-Stokes et al. 2018), and it also appears to contribute essential nutrients to its host (Gerhart et al. 2016).

In this study, we examined questing A. maculatum and A. americanum collected at the same field site from vegetation and from dogs and humans traversing this site for several intracellular bacteria. We tested all the tick samples for Rickettsia, E. chaffeensis, E. ewingii, AamCLE, and AmacFLE to determine their prevalence in this population of A. americanum and A. maculatum and to determine if any exchange of their agents between the two tick species could be detected. Such exchange of transovarially maintained Rickettsia and endosymbionts could have occurred in ancestral lineages of these ticks or in previous larva and nymph life stages for Ehrlichia, which are transstadially maintained. Finally, we discuss some factors that may influence how frequently these bacterial agents may be exchanged between A. americanum and A. maculatum and the potential importance of such exchanges in human risk of infection.

Materials and Methods

Tick collection and identification

Ticks were collected as a convenience sample during a bird-banding study from May to August 2014 at least once per month from a 4.14 acre site in west-central Georgia (Lamar County), United States of America. Questing ticks were collected by running a 1 m² flannel cloth over vegetation and augmented with questing ticks donated by infested individuals and dog owners that were collected with forceps before extended attachment for a total of 114 samples. Samples were stored in 70% ethanol at 4°C. Ticks were identified to life stage, sex, and species using morphological characteristics (Strickland et al. 1976, Keirans and Litwak 1989, Keirans and Durden 1998).

DNA extraction

Before DNA extraction, ticks were washed sequentially with 10% bleach, 70% ethanol, and three distilled water rinses to reduce surface contamination. Adults were bisected, frozen by immersion of the tube in liquid nitrogen, and pulverized with a Kontes pestle. DNA was extracted from one half of each adult individual; the nymphs were pulverized and extracted without bisection (Bermudez et al. 2009). DNA extraction was performed with the Promega Wizard SV 96 Genomic DNA Purification System (Promega, Madison, WI). Purified tick DNAs were stored at 4°C for the duration of this study.

Molecular analyses

The concentration of all tick DNAs was quantified using the Qubit dsDNA HS Assay Kit on the Qubit 2.0 Fluorometer (ThermoFisher, Waltham, MA) following DNA extraction. All DNA samples were then tested with real-time PCR assays for various infectious agents: Rickettsia spp. ompA (Eremeeva et al. 2003); Rickettsia spp., E. chaffeensis, and E. ewingii (Killmaster et al. 2014); R. parkeri ompB (Jiang et al. 2012, Denison et al. 2014); FLE 16S (Dergousoff and Chilton 2012); and Coxiella-like endosymbiont (AamCLE) fusA (Jasinskas et al. 2007). Primer and probe sequences for these assays are listed in Table 1. Duplicates of each sample, including positive and negative controls, were screened with each quantitative PCR (qPCR) assay. Nuclease-free water was used as a no-template control in all experiments.

Table 1.

Primers and Quantitative PCR Assays Employed in the Study

Amplicon target	Assay type	Oligo name	Primer sequences (5′-3′)	References
Rickettsia spp. ompA	EvaGreen	RR190.547-F	CCT GCC GAT AAT TAT ACA GGT TTA	Eremeeva et al. (2003)
Rickettsia spp. ompA	EvaGreen	RR190.701-R	GTT CCG TTA ATG GCA GCA TCT	Eremeeva et al. (2003)
Francisella and AmacFLE 16S rDNA	EvaGreen	NC-Fran16S-F	CAA CAT TCT GGA CCG AT	Dergousoff and Chilton (2012)
Francisella and AmacFLE 16S rDNA	EvaGreen	NC-Fran16S-R	TGC GGG ACT TAA CCC AAC AT	Dergousoff and Chilton (2012)
Rickettsia 17-kDa	TaqMan	R17K135-F	ATG AAT AAA CAA GGK CAN GGH ACA C	Killmaster et al. (2014)
Rickettsia 17-kDa	TaqMan	R17K249-R	AAG TAA TGC RCC TAC ACC TAC TC	Killmaster et al. (2014)
Ehrlichia16S rDNA	TaqMan	ECH 16s-17	GCG GCA AGC CTA ACA CAT G	Killmaster et al. (2014)
Ehrlichia16S rDNA	TaqMan	ECE 16s-99	CAC CCG TCT GCC ACT AAC AAY TAT	Killmaster et al. (2014)
Rickettsia spp. 17 kDa antigen	TaqMan	R17KBC-Pr	Cal Red-TT GGT TCT CAA TTC GGT AAG GGT AAA GG-BHQ2	Killmaster et al. (2014)
Ehrlichia ewingii 16S rDNA	TaqMan	EEW 16s-40-74	HEX-TC GAA CGA ACA ATT CCT AAA TAG TCT CTG ACT ATT-BHQ2	Killmaster et al. (2014)
E. chaffeensis 16S rDNA	TaqMan	ECH 16s-38-70	FAM-AG TCG AAC GGA CAA TTG CTT ATA ACC TTT TGG T-BHQ2	Killmaster et al. (2014)
R. parkeri ompB	TaqMan	Rpa129F	CAA ATG TTG CAG TTC CTC TAA ATG	Jiang et al. (2012)
R. parkeri ompB	TaqMan	Rpa224R	AAA ACA AAC CGT TAA AAC TAC CG	Jiang et al. (2012)
R. parkeri ompB	TaqMan	Rpa188probe	FAM-CGC GAA ATT AAT ACC CTT ATG AGC AGC AGT CGC G-BHQ1	Jiang et al. (2012)
AamCLE fusA	TaqMan	AAMFUSA-F	AGC CTT ATT AGA TGC TGT GGT TGA	Jasinskas et al. (2007)
AamCLE fusA	TaqMan	AAMFUSA-R	CGT CTG CTT CTT CAC CTC GAA	Jasinskas et al. (2007)
AamCLE fusA	TaqMan	AAMFUSA-Pr	FAM-ATT TAC CTG CAC CTA CTG ATA TAC CTG AT-BHQ1	Jasinskas et al. (2007)
Rickettsia spp. ompA	Sequence	RR190.70-F	ATG GCG AAT ATT TCT CCA AAA A	Eremeeva et al. (2003)
Rickettsia spp. ompA	Sequence	RR190.701-R	GTT CCG TTA ATG GCA GCA TCT	Eremeeva et al. (2003)
R. amblyommatis polyC region	Amplicon/Sequence	Ram1490F_1160175	CAT CAC TGC CTC GCT TAT TT	This study
R. amblyommatis polyC region	Amplicon/Sequence	Ram1491R_1160586	AAT TTC TTT CTT CCT CTT CAT CAA	This study
R. parkeri #77 variable site	Amplicon/Sequence	RAF-3650_846F	ACC TGA CAA TGA GAA GTT ACA	This study
R. parkeri #77 variable site	Amplicon/Sequence	RAF-3650_1128R	CGT AGT AGA TTC TAC AGG CT	This study
R. parkeri #89 variable site	Amplicon/Sequence	RAF-475_3861F	AAC TGA AAT AGG GCA AGA AGT A	This study
R. parkeri #89 variable site	Amplicon/Sequence	RAF-475_4517R	GGT TGT GTA TCA TCC TTT GTT	This study

Conditions for each assay are provided in the “Materials and Methods” section.

Tick DNAs were tested for rickettsial ompA gene using the primers RR190.547-F and RR190.701-R with each reaction containing 10 μL of SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA), 5.75 μL of nuclease-free water, 0.125 μM of each primer, and 4 μL of template DNA for a total of 20 μL. The reaction was performed in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using the following thermocycler conditions: one step of 95°C (3 min), 50 cycles of 95°C (20 s), 57°C (30 s), 72°C (15 s), one step of 55°C (1 min), and a melt curve analysis of 55–95°C (increments of 0.5°C for 10 s). Samples were considered positive for SFGR Rickettsia if they had a C _q value ≤35 and the expected melting temperature for the amplicon (EvaGreen assays only).

Tick DNAs were tested for AmacFLE using the primers NC_Fran16S-F and NC-Fran16S-R, which are specific for AmacFLE and F. tularensis bacterial 16S ribosomal RNA gene. Each reaction contained 10 μL of SsoFast EvaGreen Supermix, 5.75 μL of nuclease-free water, 0.125 μM of each primer, and 4 μL of template DNA for a total of 20 μL. The reaction was performed in a CFX96 Touch Real-Time PCR Detection System using the following thermocycler conditions: one step of 95°C (5 min), 30 cycles of 95°C (30 s), 52°C (30 s), 72°C (30 s), one step of 72°C (5 min), and a melt curve analysis of 70–90°C (increments of 0.2°C for 10 s).

Using a Triplex (multiplex) TaqMan assay, tick DNAs were tested for rickettsial 17-kDa antigen gene, E. ewingii 16S, and E. chaffeensis 16S using the primers R17K135-F, R17K249-R, ECH 16s-17, and ECE 16s-99 and using the probes R17KBC-Pr, EEW 16s-40-74, and ECH 16s-38-70. Each reaction contained 10 μL of iTaq Universal Probes Supermix (Bio-Rad), 4.6 μL of nuclease-free water, 0.8 μM of each primer, 0.1 μM of each probe, and 4 μL of template DNA for a total of 20 μL. The reaction was performed in a CFX96 Touch Real-Time PCR Detection System using the following thermocycler conditions: one step of 95°C (3 min), 40 cycles of 95°C (15 s), and 57°C (1 min).

Tick DNAs positive for Rickettsia spp. based on the ompA EvaGreen and theTriplex TaqMan assay were tested for R. parkeri ompB gene using the primers Rpa129F and Rpa224R and using the probe Rpa188probe. Each reaction contained 10 μL of iTaq Universal Probes Supermix, 7.4 μL of nuclease-free water, 0.2 μM of each primer, 0.2 μM of probe, and 2 μL of template DNA for a total of 20 μL. The reaction was performed in a CFX96 Touch Real-Time PCR Detection System using the following thermocycler conditions: one step of 95°C (3 min), 45 cycles of 94°C (15 s), and one step of 60°C (1.5 min).

Tick DNAs were assayed for AamCLE endosymbiont fusA gene using the primers AAMFUSA-F and AAMFUSA-R and using the probe AAMFUSA-Pr. Each reaction contained 10 μL of iTaq Universal Probes Supermix, 5.4 μL of nuclease-free water, 0.8 μM of each primer, 0.1 μM of probe, and 4 μL of template DNA for a total of 20 μL. The reaction was performed in a CFX96 Touch Real-Time PCR Detection System using the following thermocycler conditions: one step of 95°C (4 min), 40 cycles of 95°C (30 s), 50°C (30 s), and 72°C (1 min), and one step of 72°C (7 min).

Genotyping of Rickettsia-positive samples

A 412 bp amplicon was obtained from A. americanum tick DNAs that contained a Rickettsia using 1490F–1491R primers (RR1124–RR1125) derived from the genome of R. amblyommatis GAT-30V between coordinates 1160175 and 1160586 (Table 1). This region of the R. amblyommatis genome contains a polymorphic poly-C domain (10 in the prototype Georgia strain GAT-30V). A. maculatum DNAs positive for R. parkeri in the screening qPCR assays were tested with primers #77 and #89 (Table 1) to assess tandem repeat differences in R. parkeri DNAs. Primer sequences were derived from the R. africae ESF-5 genome and previously found to detect sequence differences in R. parkeri reference strain DNAs (not shown). The amplifications were performed on an Eppendorf MasterGradient PCR machine using the following thermocycler conditions: 95°C for 5 min, 35 cycles of 95°C for 30 s, 57 (assays #89, 1490–1491) or 50°C (assay #77) for 30 s, and 70°C for 60 s, followed by a 10-min extension at 72°C. The amplicons were purified from a 1% agarose gel and sequenced as described below using the amplification primers.

Sequencing

Amplicons from Rickettsia spp.-positive tick DNAs from A. maculatum were sequenced to confirm their origin. These samples were sequenced with PCR primers for ompA, RR190.701-R and RR190.70-F (Table 1), which amplify a 632 bp fragment (Eremeeva et al. 2003).

The amplicons were visualized on a 2% agarose gel, and bands were excised and purified using the Wizard SV Gel and PCR Clean-Up System (Promega). Purified amplicons were sequenced bidirectionally using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on an ABI 3130xl Genetic Analyzer. Sequences were aligned in Geneious (Kearse et al. 2012) and compared to sequences in GenBank by BLAST.

Results

Sixteen female A. americanum (14.0%, n = 114), 20 male A. americanum (17.5%, n = 114), 4 nymph A. americanum (3.5%, n = 114), 45 female A. maculatum (39.5%, n = 114), and 29 male A. maculatum (25.4%, n = 114) were collected from May to August 2014 (Table 2). About 35% of the A. americanum ticks (similar numbers of females and males) were collected from hosts, while all but two of the A. maculatum ticks were from hosts (Table 2). Ticks were not allowed to feed to full engorgement as daily tick checks were performed. DNAs were extracted from all ticks and assayed for Rickettsia spp., E. ewingii, E. chaffeensis, AmacFLE, and AamCLE (Table 3). A total of 74 A. maculatum ticks were positive for AmacFLE (100%, n = 74); AmacFLE was not detected in any A. americanum (0%, n = 40). A total of 39 ticks were positive for AamCLE (34.2%, n = 114), of which all were A. americanum (97.5%, n = 40) and none was A. maculatum (0%, n = 74). No ticks were positive for either E. ewingii (0%, n = 114) or E. chaffeensis (0%, n = 114).

Table 2.

Summary of Georgia Ticks Collected For Quantitative PCR Assays

Tick species (no. from host)	Female	Male	Nymph	Total
Amblyomma americanum	16 (7)	20 (7)	4 (0)	40 (14)
Amblyomma maculatum	45 (43)	29 (29)	0	74 (72)
Total	61 (50)	49 (36)	4 (0)	114 (86)

Table 3.

Prevalence of Bacterial Agents Detected in Amblyomma americanum and A. maculatu m

Tick species	Number positive/total samples (%, mean C _q)
Tick species	Rickettsia spp. ompA (EvaGreen)	AmacFLE (EvaGreen)	E. ewingii (Triplex TaqMan)	E. chaffeensis (Triplex TaqMan)	Rickettsia spp. (Triplex TaqMan 17 kDa antigen)	R. parkeri (TaqMan)	AamCLE (TaqMan)
A. americanum	15/40 (37.5%, 25.0)	0/40 (0, NA)	0/40 (0, NA)	0/40 (0, NA)	15/40 (37.5%, 25.1)	0/15 (0, NA)	39/40 (97.5%, 23.7)
A. maculatum	33/74 (44.6%, 24.1)	74/74 (100%, 22.3)	0/74 (0, NA)	0/74 (0, NA)	33/74 (44.6%, 23.6)	30/33 (90.9%, 24.4)	0/74 (0, NA)
Total	48/114 (42.1%)	74/114 (64.9%)	0/114 (0, NA)	0/114 (NA)	48/114 (42.1%)	30/48 (62.5%)	39/114 (34.2%)

Conditions for each assay are provided in the “Materials and Methods” section.

NA, not applicable.

Forty-eight ticks were Rickettsia positive using the triplex TaqMan assay for Rickettsia 17 kDa antigen (42.1%, n = 114), of which 15 were A. americanum (37.5%, n = 40) and 33 were A. maculatum (44.6%, n = 74). The same 48 ticks were also Rickettsia positive using the OmpA qPCR assay. The amount of Rickettsial DNA detected for each tick was very comparable as well as the average amount from all positive ticks (Table 3). Of the 48 Rickettsia spp.-positive ticks, 30 (62.5%, n = 48) were positive for R. parkeri using the ompB assay, all of which were A. maculatum (90.9%, n = 33). The R. parkeri qPCR assay was slightly less sensitive than the triplex and ompA assays (Table 2). The A. americanum ticks were confirmed to contain variant types of R. amblyommatis by isolate typing using a polymorphic polyC region (GenBank #MT712884–MT712891). The sequence of this region contains many variant nucleotides compared to the homologous region from R. parkeri (Portsmouth genome and GenBank #MT712883), thus further confirming their origin from R. amblyommatis. The other A. americanum-positive ticks were shown to contain R. amblyommatis by using the R. parkeri typing primers as they produce different size amplicons from R. parkeri (not shown). One of two typing regions of R. parkeri (Primer#77, rickA) detected several variant genotypes (GenBank #MT712901–MT712919); typing of sca2 (Primer #89) detected only sequences identical to that of R. parkeri Portsmouth (GenBank #MT712892–MT712900). The R. parkeri sequence regions are divergent from the homologous regions of R. amblyommatis, further confirming that they indeed originated from R. parkeri DNA in the tick samples.

Ten of the ompB assay R. parkeri-positive A. maculatum ticks were sequenced with ompA fragment primers, and identification of R. parkeri was confirmed (GenBank #MT712869–MT712878). In addition, the three R. parkeri-negative, but Rickettsia spp.-positive A. maculatum were confirmed to be R. andeanae by ompA sequencing (GenBank #MT712879-MT712881). The ompA fragment of one A. americanum Rickettsia-positive sample was sequenced and it had the expected R. amblyommatis sequence (GenBank #712882).

Discussion

We obtained no evidence for exchange of Rickettsia, AmacFLE, and AamCLE between A. americanum and A. maculatum in a natural setting where both A. americanum and A. maculatum were common. This failure of exchange occurred, despite very high levels of carriage of each of these bacterial agents (Table 3) and the common observance of infestations by both ticks on both dogs and humans at this site (Table 2). Such exchange could have occurred in previous generations or life stages at this site. Based on this number of ticks, exchange of these Rickettsia, Coxiella, and Francisella agents between A. maculatum and A. americanum would have to have occurred at a frequency >1% to be detectable. This infrequent exchange is corroborated by existing literature, which detected R. amblyommatis in A. maculatum at rates of 0–2% (Egizi et al. 2020) and R. parkeri is usually found in A. americanum <1% of the time (Cohen et al. 2009, Gaines et al. 2014, Lydy et al. 2020); however, this estimate is without any consideration of the relative prevalence of each tick or their respective rickettsial agents at those sample sites since that is unknown. Moreover, assuming this low prevalence is typical, our sample size (n = 114) could have been too small to detect such a rare exchange of agents because a prevalence of 1.42% or <1% would require at least a sample size of 210 or >300, respectively, for a 95% chance of finding one positive tick.

While this study was focused on ticks collected from humans and dogs during a mist-netting and banding effort in the field where the samples were collected, both A. americanum and A. maculatum are rather promiscuous in their host vertebrate species (discussed in Lydy et al. 2020) at different life stages. Consequently, multiple opportunities for the exchange of the agents examined in this study could have occurred during larval and nymphal feeding, or indeed during feeding of previous generations of ticks since the Rickettsia, CLE, and FLE agents are maintained transovarially in contrast to the Ehrlichia agents surveyed. The important point is that these ticks were collected at the same site and during the same summer months and no exchange of these agents was detected.

Allerdice et al. (2019) surveyed the prevalence of R. parkeri and R. andeanae in A. maculatum collected from six other counties close to Atlanta, but not Lamar County. The sample sizes varied from 4 to 90 ticks and the prevalence of R. parkeri from 12% to 73% (40.5% found for Lamar). R. andeanae varied from 12% to 80%, while we found 4% in Lamar. We found microheterogeneity in both R. amblyommatis and R. parkeri at our site and had previously observed this in R. amblyommatis samples from Sweetwater and Panola Mountain State Parks. One cannot a priori exclude physiological differences in the ability of both R. amblyommatis and R. parkeri strains to be exchanged between different species of ticks or potentially to or from different vertebrate hosts.

In contrast, the very high prevalence of AmacFLE and AamCLE in their respective hosts suggests that a greater likelihood of exchange of these agents might be expected by co-feeding. No such exchange was observed in our samples. This question has not been previously addressed. The likelihood that these agents can be shared among ticks is based on their random association with different species of both soft and hard ticks, recent suggestions that the AmacFLE agents are phylogenetically intermediate between free-living Francisella isolates found in many hosts (including ticks) and diverse environments, and the possibility that Coxiella-like tick endosymbionts are readily shed in saliva, tick feces, and coxal fluids. To the best of our knowledge, this shedding of CLEs has not been examined for hard ticks, but was reported for the soft tick Carios capensis (Reeves et al. 2005). Ultimately, the vector potential of A. americanum for AmacFLE and A. maculatum for AamCLE will need to be established experimentally as well as their capacity for acquiring these agents by salivary co-feeding.

Conclusion

Using sensitive contemporary qPCR methods, we have demonstrated that the five intracellular bacteria (three Rickettsia species, a CLE, and a FLE) found in the Amblyomma species commonly co-occurring at the same location in Georgia are not frequently exchanged between these tick species. While this does not preclude this exchange from occurring in nature, the probability of detecting such an exchange is greatly enhanced by laboratory manipulations. Such manipulations may be atypical of such events in nature (possibly an uncommon happenstance event that might have characteristics similar to conditions found in laboratory manipulations). Horizontal transmission of R. parkeri from A. maculatum to A. americanum by co-feeding has been described (Wright et al. 2015), and R. amblyommatis has been found infrequently in A. maculatum ticks (Fornadel et al. 2011, Lockwood et al. 2018). The prevalence of Rickettsia agents we detected in A. americanum and A. maculatum was comparable to the levels found in nearby counties in Georgia by Allerdice et al. (2019). Our methodological approach differed from that study by our emphasis on specific qPCR assays rather than exclusively on DNA sequencing. Our sequence data affirmed the results we obtained from the qPCR assays. We also demonstrated that both R. parkeri and R. amblyommatis populations at the field site exhibited genetic heterogeneity. Consequently, we obtained no evidence for the frequent exchange of these tick-borne agents in a natural setting, despite high levels of carriage of each agent and the common observance of infestation of both ticks on both dogs and humans at this site.

Footnotes

Acknowledgments

We would like to thank Thomas Gillespie, PhD, Department of Environmental Sciences at Emory University in Atlanta, GA, for his mentoring and support of J.R.H. as part of this study. We also thank Minh Tang for her assistance with performing the Rickettsia genotyping assays. The research reported in this study was supported, in part, by appointment of J.R.H. and A.J.W.-N. to the Research Participation Program at the Centers for Disease Control and Prevention (CDC) administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the United States Department of Energy and CDC. A.J.W.-N. was also supported by an Association of Public Health Laboratories Bioinformatics Fellowship under the guidance of G.A.D., PhD. Charlie Muise kindly provided access to the tick field collection sites on his property in Lamar County and ticks he removed from his dogs.

This article was presented as a poster as follows: J.R. Hensley, M.L. Zambrano, A.J. Williams-Newkirk, and G.A. Dasch. 2016. Assessing exchange of Rickettsia, Coxiella, and Francisella agents between Amblyomma americanum and A. maculatum ticks in Georgia. ASM Microbe 2016 Meeting, Boston June 16–20, 2016.

Disclaimer

The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

The research reported here was supported in part by appointments of Jasmine R. Hensley (JRH) and Amanda Jo Williams-Newkirk (AJW-N) to the Research Participation Program at the Centers for Disease Control and Prevention (CDC) administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and CDC. Other funding was provided by intramural programs of the Centers for Disease Control and Prevention.

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