Bacterial Diversity in Amblyomma Americanum (Acari: Ixodidae) with Afocusonmembersofthegenusrickettsia

Home , Ehrlichia ewingii, Rickettsia asiatica, Rickettsia massiliae, Rickettsia raoultii, Rickettsia rickettsii

VECTOR-BORNE DISEASES,SURVEILLANCE,PREVENTION Bacterial Diversity in Amblyomma americanum (Acari: Ixodidae) With aFocusonMembersoftheGenusRickettsia

1 2 1 STEPHANIE R. HEISE, M. S. ELSHAHED, AND S. E. LITTLE

Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078

J. Med. Entomol. 47(2): 258Ð268 (2010); DOI: 10.1603/ME09197 ABSTRACT The lone star tick, Amblyomma americanum (Acari: Ixodidae), is commonly reported from people and animals throughout the eastern U.S. and is associated with transmission of a number of emerging diseases. To better deÞne the microbial communities within lone star ticks, 16S rRNA gene based analysis using bacteria-wide primers, followed by sequencing of individual clones (n ϭ 449) was used to identify the most common bacterial operational taxonomic units (OTUs) present within colony-reared and wild A. americanum.Thecolony-rearedtickscontainedprimarilysequenceafÞl- iated with members of the genus Coxiella (89%; 81/91), common endosymbionts of ticks, and Brevibacterium (11%; 10/91). Similarly, analysis of clones from unfed wild lone star ticks revealed that 96.7% (89/92) of all the OTUs identiÞed were afÞliated with Coxiella-like endosymbionts, as compared with only 5.1Ð11.7% (5/98Ð9/77) of those identiÞed from wild lone star ticks after feeding. In contrast, the proportion of OTUs identiÞed as Rickettsia sp. in wild-caught ticks increased from 2.2% (2/92) before feeding to as high as 46.8% (36/77) after feeding, and all Rickettsia spp. sequences recovered were most similar to those described from the spotted fever group Rickettsia,speciÞcallyR. amblyommii and R. massiliae.AdditionalcharacterizationoftheRickettsialestickcommunitybypolymerase chain reaction, cloning, and sequencing of 17 kDa and gltA genes conÞrmed these initial Þndings and suggested that novel Rickettsia spp. are likely present in these ticks. These data provide insight into the overall, as well as the rickettsial community of wild lone star ticks and may ultimately aid in identiÞcation of novel pathogens transmitted by A. americanum.

KEY WORDS Amblyomma americanum,bacterialdiversity,16Sgene,Rickettsia

Amblyomma americanum (Acari: Ixodidae), the lone et al. 2002, Childs and Paddock 2003, Paddock and star tick, is an important arthropod pest and disease Yabsley 2007). At the same time, a number of novel A. vector that has dramatically increased in both number americanum-associated pathogens and diseases have and geographic distribution in recent years largely been described in the last two decades (Ewing et al. because of expansion of white-tailed deer populations 1971, Goddard and Norment 1986, Anderson et al. (Childs and Paddock 2003, Paddock and Yabsley 1991, Kirkland et al. 1997, Reeves et al. 2008). This 2007). Although deer serve as the keystone host for expanding geographic range together with our in- both immature and adult stages of this aggressive tick, creasing awareness of the pathogens and diseases as- lone star ticks also readily bite humans and a variety sociated with A. americanum lends a sense of urgency of other animals; indeed, A. americanum is the tick to efforts to understand the bacterial diversity present most commonly reported from people in the southern in this arthropod in natural systems. United States, accounting for up to 83% of human tick A. americanum has been associated with several bites in some surveys (Felz et al. 1996). Historically human diseases, including ehrlichioses, spotted fever restricted to relatively isolated pockets primarily in caused by rickettsial agents, and Southern Tick Asso- the southeastern and south-central United States, lone ciated Rash Illness (STARI). Lone star ticks are well star ticks have expanded regionally and nationally in established as the primary vector of at least three recent years, with populations now established as far distinct ehrlichial pathogens: Ehrlichia chaffeensis, the north as Maine (Keirans and Lacombe 1998, Ginsberg causative agent of human monocytotropic ehrlichiosis (HME) (Anderson et al. 1991), E. ewingii, the cause of 1 Department of Veterinary Pathobiology, Center for Veterinary human granulocytic ehrlichiosis (Buller et al. 1999), Health Sciences, and Center for Veterinary Health Sciences, and an E. ruminantium-like organism referred to as the Oklahoma State University, Stillwater, OK 74078 (e-mail: susan. Panola Mountain Ehrlichia (PME) (Reeves et al. [email protected]). 2 Department of Microbiology and Molecular Genetics, Oklahoma 2008). Human ehrlichiosis, particularly when caused State University, Stillwater, OK 74078. by E. chaffeensis, is potentially life-threatening and is

0022-2585/10/0258Ð0268$04.00/0 ᭧ 2010 Entomological Society of America March 2010 HEISE ET AL.: BACTERIAL DIVERSITY IN A. americanum 259 considered the most common tick-borne disease of Materials and Methods people in the southern U.S. (McQuiston et al. 2003, Samples/Source of Ticks. Colony-reared adult A. Olano et al. 2003, Manangan et al. 2007, Miura and americanum were obtained from the Oklahoma State Rikihisa 2007, Wimberly et al. 2008). The historical University Tick Laboratory (Stillwater, OK) that has literature also links Rocky Mountain spotted fever maintained the same line of lone star ticks since 1976 caused by R. rickettsii with A. americanum (McDade with the last introduction of new colony material 2 yr and Newhouse 1986), but more recent data suggest another rickettsial agent, such as R. amblyommii, may before this study. (L. Coburn, personal communica- be associated with a spotted fever like illness in people tion). Wild adult lone star ticks were collected from (Apperson et al. 2008). Lake Carl Blackwell Recreational Area, OK (site 1) Perhaps the most perplexing of the lone star tick- and Panola Mountain State Park, GA (site 2) in May associated diseases is STARI, a Lyme borreliosis-like of 2008 using dry ice traps and drags as previously condition in which people develop an erythema mi- described (Lockhart et al. 1997). A subset of 100 adult grans rash after the bite of a lone star tick which cannot lone star ticks from site 1 were fed in two groups of 50 be attributed to infection with Borrelia burgdorferi, (25 males and 25 females) on each of two pathogen- the only known cause of Lyme borreliosis in North free class A beagle dogs that had been shown to be free America (Wormser et al. 2005a,b, Philipp et al. 2006). of infection with all pathogens of interest before tick In endemic areas, erythema migrans after a tick bite is feeding as determined by speciÞc polymerase chain considered pathognomonic for Lyme borreliosis and a reaction (PCR) for Ehrlichia spp., Anaplasma spp., physician-diagnosed erythema migrans is considered Rickettsia spp., and Borrelia spp. on whole blood as sufÞcient evidence to warrant report as a conÞrmed described below. In addition, dogs were evaluated for case for surveillance purposes (Centers for Disease evidence of antibodies to Ehrlichia, Anaplasma, and B. Control [CDC] 1997) However, in areas where Lyme burgdorferi via a commercial ELISA (SNAP 4Dx, borreliosis is not known to be endemic, such as the IDEXX Laboratories Inc., Westbrook, ME) according southern U.S., the etiology of A. americanum-associ- to manufacturerÕs instructions, and by indirect ßuo- ated erythema migrans remains elusive (Blanton et al. rescent antibody assay for R. rickettsii using a com- 2008, Masters et al. 2008). Attempts to attribute STARI mercial laboratory service (Oklahoma Animal Disease to infection with B. lonestari, arelapsingfever-like Diagnostic Laboratory, Stillwater, OK). A subset of spirochete present in wild lone star ticks and associ- 100 colony-reared lone star ticks were fed in two ated with a single case of STARI, or with R. amblyo- groups of 50 (25 males, 25 females) of each of two mmii, have not been conÞrmed with clinical micro- speciÞc-pathogen free New Zealand white rabbits. biological studies to date (James et al. 2001, Wormser Ticks were allowed to feed until females were replete et al. 2005a,b, Billeter et al. 2007, Nicholson et al. 2009). and then collected for PCR. Afullcharacterizationofthespectrumofthebac- Nucleic Acid Extraction. From each of the pools of terial community in A. americanum is an important unfed adults, 100 adults (50 males and 50 females) Þrst step to establish a basis for further investigations were dissected and all internal tissues transferred into of this tick as a vector of known and yet to be discov- asterile1.5mltubecontaining0.8ml1ϫ phosphate- ered pathogens. Recently, multiple studies have con- buffered saline (PBS) and total DNA was extracted vincingly demonstrated the ubiquitous presence of a using a commercially available kit (FastDNA SPIN Kit Coxiella-endosymbiont in wild and colony-reared lone for Soil, MP Biomedicals, Solon, OH) according to the star ticks (Jasinskas et al. 2007, Klyachko et al. 2007, manufacturerÕs instructions. From the two pools of fed Zhong et al. 2007, Clay et al. 2008). However, attempts ticks (site 1, colony-reared), ticks were dissected and to characterize complete microbial communities in all internal tissues and contents from feeding trans- wild lone star ticks have not included colony-reared A. ferred into a sterile vial containing 1ϫ PBS. Because americanum as controls or evaluated changes in di- of large differences in body mass post feeding, fed versity of microbes induced by tick feeding (Clay et males and fed females were pooled and evaluated al. 2008). For the study reported here, our goal was to separately. The volume of PBS used was adjusted document differences in the degree of microbial di- according to body weight of the ticks dissected to versity present in colony-reared ticks and wild caught reach the same ratio (body weight:PBS). Total DNA ticks before and after feeding. The population of vec- was extracted from fed ticks via a modiÞed lysis bead- tor-borne pathogens increases within arthropod vec- beating protocol as previously described (Dojka et al. tors in response to blood feeding (de Silva and Fikrig 1998). Total nucleic acid was extracted from 100 ␮lof 1995, Azad and Beard 1998), and so we used blood whole blood using the GFX genomic blood DNA pu- feeding as a means to increase the proportion of bac- riÞcation kit (Amersham Biosciences, Buckingham- teria that were perhaps more likely to be transmitted shire, United Kingdom) according to manufacturerÕs to hosts. Our results show that microbial diversity is directions. severely limited in colony-reared ticks, and somewhat PCR Ampliﬁcation, Cloning, and Sequencing. Mul- restricted in unfed, wild caught ticks, but that feeding tiple DNA targets were used to identify organisms in greatly increases the microbial diversity detected, par- these samples, including bacteria-wide 16S rRNA ticularly for the members of the Rickettsiales, in wild gene, Rickettsia-speciÞc 17 kDa fragment, and rickett- caught ticks that presumably fed on infected verte- sial citrate synthase gene (gltA). All pools were also brates in their immature stages. tested for individual organisms using PCR protocols 260 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 47, no. 2

Table 1. Gene targets, primer sequences, and references for PCR protocols used in this study

Targeted gene Primer Sequence Reference Bacteria-wide 16S rDNA 8F 5Ј-AGAGTTTGATCCTGGCTCAG-3Ј Elshahed et al. 2003 805R 5Ј-GACTACCAGGGTATCTAATCC-3Ј Borrelia ßagellin (ßaB) FLALL 1Њ 5Ј-ACATATTCAGATGCAGACAGAGGT-3Ј Barbour et al. 1996 FLARL 5Ј-GCAATCATAGCCATTGCAGATTGT-3Ј FLALS 2Њ 5Ј-AACAGCTGAAGAGCTTGGAATG-3Ј FLARS 5Ј-CTTTGATCACTTATCATTCTAATAGC-3Ј Ehrlichia and Anaplasma ECB 1Њ 5Ј-CGTATTACCGCGGCTGCTGGCA-3Ј Dawson et al. 1996, Anderson 16S rDNA ECC 5Ј-AGAACGAACGCTGGCGGCAAGCC-3Ј et al. 1992a, Anderson et al. E. chaffeensis 16S rDNA HE3 2Њ 5Ј-TATAGGTACCGTCATTATCTTCCCTAT-3Ј 1992b HE1 5Ј-CAATTGCTTATAACCTTTTGGTTATAAAT-3Ј E. ewingii 16S rDNA HE3 2Њ 5Ј-TATAGGTACCGTCATTATCTTCCCTAT-3Ј EE72 5´-CAATTCCTAAATAGTCTCTGACTATT-3´ A. phagocytophilum 16S GE9f 2Њ 5Ј-AACGGATTATTCTTTATAGCTTGCT-3Ј rDNA GA1UR 5Ј-GAGTTTGCCGGGACTTCTTCT-3Ј A. platys 16S rDNA DGA 2Њ 5Ј-TTATCTCTGTAGCTTGCTACG-3Ј GA1UR 5Ј-GAGTTTGCCGGGACTTCTTCT-3Ј E. ruminantium citrate Ehr3CS-185F 1Њ 5Ј-GCCACCGCAGATAGTTAGGGA-3Ј Yabsley et al. 2008 synthase Ehr3CS-777R 5Ј-TTCGTGCTCGTGGATCATAGTTTT-3Ј Ehr3CS-214F 2Њ 5Ј-TGTCATTTCCACAGCATTCTCATC-3Ј Ehr3CS-619R 5Ј-TGAGCTGGTCCCCACAAAGTT-3Ј Rickettsia 17 kDa 17k-5 1Њ 5Ј-GCTTTACAAAATTCTAAAAACCATATA-3Ј Stothard 1995 17K-3 5Ј-TGTCTATCAATTCACAACTTGCC-3Ј 17KD1 2Њ 5Ј-GCTCTTGCAACTTCTATGTT-3Ј 17KD2 5Ј-CATTGTTCGTCAGGTTGGCG-3Ј Rickettsia citrate RpCS.877 5Ј-GGGGGCCTGCTCACGGCGG-3Ј Regnery et al. 1991 synthase (gltA) RpCS.1258 5Ј-ATTGCAAAAAGTACAGTGAACA-3Ј established for Borrelia spp., E. chaffeensis, E. ewingii, same PCR components with the exception of the prim- E. ruminantium, A. platys, and A. phagocytophilum. All ers 17 kD1 and 17 kD2 (Table 1) and cycling condi- primers, ampliÞcation protocols, and references are tions in the secondary reaction were 95ЊCfor3min listed in Table 1. followed by 40 cycles of 95ЊCfor15s,50ЊCfor30s, A16SrRNAgenefragment(Ϸ800 bp) was ampli- 70ЊCfor30s,followedby72ЊCfor7min. Þed as previously described (Elshahed et al. 2003) To detect the citrate synthase (gltA) fragment (380 from the bulk community DNA of each tick pool using bp) of Rickettsia sp., PCR primers RpCS.877 and primers8Fand805R(Table1)in50␮lreactions RpCS.1258 (Table 1) were used in a 25 ␮lreactionthat containing the following (Þnal concentrations): 26.7 contained 17.5 ␮lmolecularbiology-gradewater,2.5 ␮lpurewater,10␮lof5ϫ buffer (Promega, Madison, ␮l25␮MMgCl2(Roche),2.5␮l10XPCRGoldBuffer

WI), 5 ␮lMgCl2,2␮ldeoxynucleosidetriphosphate (Roche), 0.25 ␮l10␮MdNTPs(Promega),0.5␮lof mixture (10 ␮M), 2 ␮lofthe8Fand805Rprimers each primer (10 ␮l), 0.25 ␮lAmpliTaqGold(Roche), (Invitrogen Corp., Carlsbad, CA), and 1 ␮l(1.5U)of and 1 ␮lofsampleDNA.Cyclingconditionswere95ЊC GoTaq Flexi DNA polymerase (Promega), and 2 ␮l for 5 min followed by 35 cycles of 94ЊCfor30s,60ЊC DNA. PCR ampliÞcation was carried out on a Gene for 30 s, 72ЊCfor45s,followedby72ЊCfor5min. Amp PCR system 9700 thermocycler. The 16S rRNA The amplicons produced in all reactions were di- gene ampliÞcation used a protocol involving initial rectly cloned using a TOPO-TA cloning kit according denaturation for 5 min at 94ЊCand39cyclesof92ЊC to the manufacturerÕs instructions (Invitrogen). Plas- for 0.5 min, 50ЊCfor1min,and72ЊCfor2min,followed mids from a total of 96 clones from each ampliÞcation by a Þnal extension at 72ЊCfor20min.PCRproducts protocol and each pool were submitted for sequencing obtained were visualized on a 2% agarose electro- (1,152 total sequence requests); sequencing and as- phoresis gel. sembly procedures were performed at the Advanced AmpliÞcation of the 17 kDa fragment (434 bp) of Center for Genome Technology at the University of Rickettsia sp. was achieved using a nested PCR pro- Oklahoma as previously described in detail (Elshahed tocol using the external primers 17K-5 and 17k-3 (Ta- et al. 2003). ble 1) in a 25 ␮lreactionthatcontained13.5␮lmo- Phylogenetic Analysis. For each of the three gene lecular biology-grade water, 2.5 ␮l25␮MMgCl2 targets (16S, gltA, 17 kDa), sequences were initially (Roche Applied Biosystems, Indianapolis, IN), 2.5 ␮l compared with entries in GenBank database using 10X PCR Gold Buffer (Roche), 0.25 ␮l10␮MdNTPs BLASTnr (Altschul et al. 1997) for rough determina- (Promega), 0.5 ␮lofeachprimer(10␮M), 0.25 ␮l tion of phylogenetic afÞliation. Sequences were also AmpliTaq Gold (Roche), and 5 ␮lofsampleDNA. aligned in Greengenes NAST aligner to a 7,862-char- Cycling conditions in the primary reaction were 95ЊC acter global alignment (DeSantis et al. 2006b), and ran for 3 min followed by 40 cycles of 95ЊCfor15s,48ЊC through Greengenes classiÞer (DeSantis et al. 2006a). for 30 s, 70ЊCfor30s,followedby72ЊCfor7min.For In addition to Greengenes classiÞer output, the NAST- the secondary reaction, 5 ␮lofprimaryproductwas aligned sequences were imported to Greengenes May used as template in a 25 ␮lreactioncontainingthe 2007 ARB database and added to the ARB universal March 2010 HEISE ET AL.: BACTERIAL DIVERSITY IN A. americanum 261

A 100% 90% 80% 70% 60% Brevibacterium 50% Rickesia 40% Acinetobacter 30% Coxiella 20% 10% 0% Colony (n=91) Georgia (n=90) Oklahoma (n=92) B 100%

80% Bacillaceaea Micrococcaceae 60% Methylobacterium Rickesia 40% Enterobacteriaceae Stenotrophomonas 20% Pseudomonas Coxiella 0% Fed Males (n = 98) Fed Females (n=77) Fig. 1. (A) Bacterial community structure in colony reared and wild A. americanum from two different locations. (B) Effect of feeding on bacterial community structure within male and female wild A. americanum collected from Lake Carl Blackwell, OK (site 1). dendogram using ARB parsimony function to deter- sentatives of closely related reference sequences. Dis- mine their position in the global phylogenetic tree tance neighbor joining trees with no corrections, F-84 (Ludwig et al. 2004). corrections, and Jukes-Cantor corrections were con- For OTU assignment and phylogenetic tree constructed using PAUP, and all gave similar tree topol- struction, sequences were aligned using ClustalX ogies. (Thompson et al. 1997), and the alignments were Nucleotide Sequence Accession Number. Sequences exported to PAUP (Version 4.01b10; Sinauer associ- generate in this study were deposited in GenBank ates, Sunderland, MA). A pair-wise distance matrix database under the accession numbers GQ302888- generated in PAUP was exported to DOTUR (Schloss GQ302959. and Handelsman 2005), and used for assignment of operational taxonomic units (OTUs) at the standard Results 97% sequence similarity cutoff for analysis of the 16S sequences, 98% for Rickettsia-speciÞc 17 kDa gene, Bacterial Diversity in Unfed Colony-Reared and and 99% for the gltA Rickettsia gene. The cutoff for 16S Wild-Caught A. americanum. In the clone library from rRNA gene sequences was based on widely accepted colony-reared lone stars, the majority of 16S rRNA species cutoff values for such gene (DeSantis et al. gene sequences obtained (80/91 clones, 20 OTUs, 2006b). The cutoffs for 17 kDa and gltA rickettsial 87.9%) were members of the genus Coxiella, class ␥- genes was based on typical average sequence diver- Proteobacteria (Fig. 1). These clones were most gence between gltA and 17 kDa genes identiÞed in closely related (94Ð98% sequence similarity) to Cox- various ricketssial geneomes and PCR based analysis iella endosymbionts of A. americanum (Fig. 2) re- of pure cultures of rickettsial species. Basic diversity ported in previous studies examining bacterial di- measurements, for example, Shannon index, average versity in lone star ticks (Clay et al. 2008, Jasinskas nucleotide diversity, and GoodÕs coverage were cal- et al. 2007, Klyachko et al. 2007, Zhong et al. 2007). culated as previously described (Good 1953, Martin In addition to Coxiella-afÞliated clones, a few 16S 2002, Magurran 2004). Phylogenetic trees were con- rRNA gene sequences (10/91 clones, 3 OTUs) be- structed using OTUs from this study as well as repre- longed to the family Brevibacteriaceae, within the 262 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 47, no. 2

Fig. 2. Distance neighbor-joining tree based on the 16S rRNA gene sequences of bacterial OTUs in colony reared and wild-caught lone star ticks (site 1) before and after feeding encountered in clone libraries constructed in this study (excluding members of the order Rickettsiales). Bold circles indicate a bootstrap value of Ͼ50%. Bootstrap values were based on 1,000 replicates. The frequency of occurrence of each OTU is reported in parentheses. (Online Þgure in color.) phylum Actinomycetes, with their closest relatives Both populations of wild lone star ticks evaluated being members of the aerobic, heterotrophic genus from two distant geographical regions within the U.S. Brevibacterium (97% sequence similarity to Bre- (Oklahoma, GA) had similar bacterial communities, vibacterium avium strain 3055, isolated from dis- with 16S rRNA gene sequences of Coxiella endosym- eased poultry (Y17962) (Pascual and Collins 1999) bionts again being the most abundant (94.4% Georgia (Figs. 1 and 2). and 96.7% Oklahoma) (Fig. 1). While the most abun- March 2010 HEISE ET AL.: BACTERIAL DIVERSITY IN A. americanum 263

Table 2. Diversity estimates obtained from bacterial 16S Characterization of Rickettsial Populations in Wild rRNA clone libraries generated in this study A. americanum Before and After Feeding Using 17 kDa and gltA as Group Speciﬁc Targets. Because Clones Goods Shannon ACE avg (n) coverage members of the Rickettsia sp., a lineage with multiple pathogenic members, was stimulated by feeding, we Colony-reared 91 2.01 48.2 87.9 Georgia 90 1.73 19.7 92.2 decided to further characterize the rickettsial popu- Oklahoma 92 1.44 24.8 93.5 lation in ticks using two Rickettsiales-speciÞc phylo- Fed ticks (OK) 175 2.59 68.9 64.3 genetic markers. To this end, we cloned and se- quenced PCR products of genes encoded outer membrane protein (17 kDa) and citrate synthase (gltA) in DNA pools from wild ticks (site 1) before and dant bacterial group within both colony-reared and after feeding. wild ticks from two distinct locations were similar, the In general, both phylogenetic trees from both gene less abundant members were completely different. No sequences gave similar topologies, with rickettsial Brevibacteriaceae-afÞliated sequences were identiÞed populations identiÞed that belong to two main lin- in the wild caught ticks. Instead, sequences afÞliated eages. The Þrst lineage is comprised of close relatives with the genus Acinetobacter (Order Pseudomonales, of the R. amblyommii and R. japonica group, and the ␥-Proteobacteria), as well as spotted fever group Rick- other is a relatively minor component that may rep- ettsia sp. (closely afÞliated to R. amblyommii, and R. resent a novel rickettsial species. Analysis of results massiliae)wereidentiÞedastheminorcomponentsof using both targets revealed a higher proportion of the bacterial communities (Figs. 1 and 2). No signif- clones within the R. amblyommii group. In unfed ticks, icant differences in diversity estimates were observed 56.3% (17 kDa) to 80% (gltA) were R. amblyommii between wild and colony reared ticks (Table 2). afÞliated; upon feeding, this proportion increased to Change in Microbial Population Distribution Upon 95.2% (17 kDa) to 95.6% (gltA). Feeding Adult Ticks. Analysis of sequence of 175 Other Pathogenic Microbes Described From Lone clones (98 clones from pooled male ticks and 77 clones Star Ticks. All tick pools (before and after feeding) from pooled female ticks) from fed, wild-caught ticks were tested for speciÞc pathogens using established (site 1) showed that blood feeding led to signiÞcantly PCR protocols and were found to be PCR negative for higher diversity, when compared with unfed controls. E. chaffeensis, E. ewingii, E. ruminantium, Anaplasma In addition to observing previously undetected phy- sp., B. burgdorferi, and B. lonestari. In addition, both logenetic groups upon feeding (Fig. 1b), diversity dogs failed to seroconvert to Ehrlichia, Anaplasma, B. estimates clearly indicate that feeding results in an burgdorferi, or Rickettsia. increase in the community diversity within lone star ticks, as evident by increase in diversity indices (Sh- annon, ACE), as well as the decrease in the library Discussion coverage (Table 2). In this study, we characterized the spectrum of In both male and female wild ticks, feeding resulted bacteria present in A. americanum from an established in a signiÞcantly lower proportion of Coxiella (Fig. 1), laboratory colony and compared it to that found in and hence a more even bacterial distribution resulting wild lone star ticks before and after feeding to identify in a higher ACE estimate. In addition, feeding resulted both common and less frequent components of the in the identiÞcation of various OTUs not detected in natural tick microfauna. As expected from previous clone libraries from unfed ticks, including members of reports (Jasinskas et al. 2007, Klyachko et al. 2007), the genus Pseudomonas (Order Pseudomonales, ␥-Pro- Coxiella was very commonly identiÞed in both colony- teobacteria), members of the genus Methylobacterium reared and wild caught ticks although the proportion (class ␣-Proteobacteria), as well as sequences afÞliated decreased upon tick feeding because of increase in with the genus Stentrophomonas, family Enterobacte- other organisms, most notably Rickettsia sp. (Fig. 1). riaceaea, and families Micrococcaceaea, and Bacillace- The near-ubiquitous presence of Coxiella sp. in mul- aea within the Phylum Firmicutes. Most importantly, tiple-organ systems suggests that these might be ob- the proportion of clones afÞliated with the genus Rick- ligate endosymbionts for lone star ticks. A reduced ettsia, order Rickettsiales, within the ␣-Proteobacteria genome compared with the pathogen C. burnetii, as signiÞcantly increased, particularly in the fed females well as reduced reproductive success of lone star ticks (2.2 to 46.8%). Rickettsial clones belonged to the spot- without Coxiella, support this hypothesis (Jasinskas et ted fever group, with R. amblyommii, R. massiliae, and al. 2007, Klyachko et al. 2007, Zhong et al. 2007). R. rickettsii str. Iowa the closest relatives to our se- Colony ticks, but not wild ticks, also harbored Bre- quences (Figs. 1 and 3). Although Coxiella (20%) and vibacterium, agram-positivesoilorganismnotprevi- Bacillaceaea (23.6%), and a diverse array of other bac- ously described from lone star ticks (Clay et al. 2008). teria were present in the colony raised ticks after Because it was absent from wild ticks, we suspect this feeding, no sequences associated with members of organism may be present or maintained because of the the genus Rickettsia, Pseudomonas, Methylobacterium, colony rearing process. Stenotrophomonas, Enterobacteriaceaea, or Micrococ- Wild lone star ticks, which presumably feed on a caceaea were found in the colony-raised fed ticks (data variety of wild vertebrates as larvae and nymphs and not shown). thus acquire a complex microfauna, harbored bacteria 264 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 47, no. 2

Fig. 3. (A) Distance neighbor-joining tree based on the 16S rRNA gene sequences of OTUs afÞliated with members of the order Rickettsiales encountered in clone libraries constructed in this study. Bold circles indicate a bootstrap value of Ͼ50%. Bootstrap values were based on 1,000 replicates. The frequency of occurrence of each OTU is reported in parentheses. (Online Þgure in color.) not found in the colony ticks, namely spotted fever closely related genera Pseduomonas and Stentroph- group Rickettsia spp. and Acinetobacter spp. Another omonas are widespread gram-negative bacteria that recent study investigated the bacterial community of can induce clinically relevant opportunistic infections unfed adult lone star tick and identiÞed four bacterial (Toleman et al. 2007, Crossman et al. 2008, Looney et components: the Coxiella endosymbiont, R. amblyo- al. 2009); both genera have been previously reported mii, Pantoea agglomerans, and an Arsenophonus endo- from Ixodid ticks (Benson et al. 2004, Moreno et al. symbiont (Clay et al. 2008). However, numbers of 2006). To our knowledge, this is the second report of clones evaluated, relative proportion of the popula- Pseudomonas spp. (Clay et al. 2008) and the Þrst of tion comprised by each bacteria, and overall diversity Stentrophomonas from lone star ticks. Methylobacte- estimates were not reported (Clay et al. 2008), pre- rium spp. are commonly found in soil and on the cluding direct comparison to our results. Acineto- surface of plants; infection of immunocompromised bacter, agram-negativesoilorganismoccasionallyas- individuals has been reported but this genus has not sociated with nosocomial infections, was previously been previously identiÞed in ticks although another reported from larval but not adult lone star ticks (Clay Rhizobiales (Ochrobactrum spp.) was reported from et al. 2008). larval lone star ticks (Clay et al. 2008). Fed adult A. Upon blood feeding, the microbial diversity in the americanum in the current study also harbored bac- wild A. americanum increased, allowing detection of teria in the families Bacillaceaea, Enterobacteraceaea, organisms not identiÞed in the ticks before feeding, and Micrococcaceaea. Two other members of the En- including those in the genera Pseudomonas, Stentro- terobacteraceaea, Enterobacter agglomerans, and an Ar- phomonas, and Methylobacterium. Members of the senophonus endosymbiont were reported from unfed March 2010 HEISE ET AL.: BACTERIAL DIVERSITY IN A. americanum 265

Fig. 3. Continued. (B) Distance neighbor-joining tree based on the 17 kDa gene sequences obtained in this study. Bold circles indicate a bootstrap value of Ͼ50%. Bootstrap values were based on 1,000 replicates. The frequency of occurrence of each OTU is reported in parentheses. (C) Distance neighbor-joining tree based on the gltA gene sequences obtained in this study. Bold circles indicate a bootstrap value of Ͼ50%. Bootstrap values were based on 1,000 replicates. The frequency of occurrence of each OTU is reported in parentheses. (Online Þgure in color.) lone star ticks previously (Clay et al. 2008) but the dermal rash, which can be combined with an eschar at sequences from our A. americanum were distinct. the site of the tick bite (Parola et al. 2009). Sequences Male and female ticks differed, in some instances from our ticks were most closely related to R. amblyo- dramatically, in both type and the relative proportion mmii, R. massiliae, and R. rickettsii. Additionally, our of bacteria present. Overall, fed male lone star ticks analysis revealed a higher proportion of clones within had a comparatively smaller proportion of Rickettsia the R. amblyommii group in the fed ticks, suggesting spp. and more bacteria that are thought to be envi- that this group is selectively stimulated by feeding or ronmental or commensals. For example, Stentroph- that it has a higher relative growth rate than other omonas was much more common in male (81.6% of Rickettsia species. Further, it appear that feeding se- sequences) than female (2.6% of sequences) lone star lects for a single or very few OTUs within this group, ticks, suggesting surface contamination may have as evident by the prevalence of a single OTU in fed been responsible for its presence. In contrast, Rick- libraries of both genes, as opposed to a relatively more ettsia spp. and the Coxiella endosymbiont comprised a diverse, even distribution within unfed libraries of larger share of the sequences found in fed female ticks, both genes. Thus feeding appears to stimulate only an observation consistent with the fact that these or- speciÞc strains with the rickettsial community of A. ganisms are transovarially transmitted and increase in americanum. number with tick feeding (de Silva and Fikrig 1995, R. amblyommii is widely distributed in A. america- Azad and Beard 1998, Macaluso et al. 2001, Stromdahl num populations, and is suspected to account for a et al. 2008, Zanettii et al. 2008). high rate of seropositivity to the spotted fever group Analysis of Rickettsia spp. by three different genes because of cross-reactivity (Apperson et al. 2008, Stro- (16S, 17 kDa, gltA) showed at least three distinct hmdahl et al. 2008). This agent has been associated spotted fever group Rickettsia were present in these with a rash in a human patient after a tick bite (Billeter ticks. Known pathogens in this group share a common et al. 2007) although its role as a human pathogen has clinical presentation, including fever, headache, and not yet been fully established (Parola et al. 2009). To 266 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 47, no. 2 date, R. massiliae has only been associated with Rhi- Acknowledgments picephalus sanguineus, R. turanicus, and Ixodes ricinus This research was supported by the Krull-Ewing Endow- (Eremeeva et al. 2006, Fernandez-Soto et al. 2006, ment at Oklahoma State University. Outstanding technical Ogawa et al. 2006, Blanc et al. 2007). To our knowl- support and assistance was provided by M. West, A. C. Ed- edge, this is the Þrst report of R. massiliae in lone star wards, J. Davis, N. Youssef, and Lisa Coburn. Sequences were ticks. generated by F. Z. Najar in the Advanced Center for genome In North America, R. rickettsii, the causative agent Technology University of Oklahoma, Norman, OK. of Rocky Mountain spotted fever (RMSF), is vectored by Dermacentor spp. and, occasionally, R. sanguineus (Demma et al. 2006). However, Amblyomma spp. ticks References Cited infected with R. rickettsii have been reported from South America (Guedes et al. 2005). Historic reports Altschul, S., T. Madden, A. Schaffer, J. Zhang, Z. Zhang, W. of lone star ticks as a vector of RMSF in North America Miller, and D. Lipman. 1997. Gapped BLAST and PSI- are thought largely because of the presence of related BLAST: a new generation of protein database search spotted fever group Rickettsia spp. in A. americanum, programs. Nucl. Acids Res. 25: 3389Ð3402. such as R. amblyommii (Burgdorfer 1969). Our Þnding Anderson, B. E., J. E. Dawson, D. C. Jones, and K. H. Wilson. 1991. Ehrlichia chaffeensis, anewspeciesassociatedwith in this study of sequences that most closely resemble, human ehrlichiosis. J. Clin. Microb. 29: 2838Ð2842. yet are distinct from, R. rickettsii (96%) suggests novel Anderson, B. E., C. E. Greene, D. C. Jones, and J. E. Dawson. spotted fever group Rickettsia species may be present 1992a. NOTES: Ehrlichia ewingii sp. nov., the etiologic in lone star ticks although additional speciÞc data are agent of canine granulocytic ehrlichiosis. Int. J. Syst. Bac- required to conÞrm that assertion. Novel spotted fever teriol. 42: 299Ð302. group Rickettsia continue to be reported (Fournier et Anderson, B. E., J. W. Sumner, J. E. Dawson, T. Tzianabos, al. 2006, Fujita et al. 2006, Mediannikov et al. 2008), C. R. Greene, J. G. Olson, D. B. Fishbein, M. Olsen- underscoring the importance of continuing to pursue Rasmussen, B. P. Holloway, and E. H. George. 1992b. the role of these organisms as human and veterinary Detection of the etiologic agent of human ehrlichiosis by pathogens. polymerase chain reaction. J. Clin. Microb. 30: 775Ð780. Apperson, C. S., B. Engber, W. L. Nicholson, D. G. Mead, J. SpeciÞc PCRs optimized for detection of Ehrlichia Engel, M. J. Yabsley, K. Dail, J. Johnson, and D. W. spp., Anaplasma spp., and Borrelia spp. did not identify Watson. 2008. Tick-borne diseases in North Carolina: is these individual pathogens in these lone star ticks. “Rickettsia amblyommii” apossiblecauseofrickettsiosis These results were somewhat surprising as E. chaffeen- reported as Rocky Mountain Spotted Fever? Vector sis, E. ewingii, and B. lonestari are known to be present Borne Zoonotic Dis. 8: 1Ð9. in white-tailed deer and lone star ticks from this area Azad, A. F., and C. B. Beard. 1998. Rickettsial pathogens and (Mixson et al. 2006). Pooling of the ticks before DNA their arthropod vectors. Emerg. Infect. Dis. 4: 179Ð186. extraction may have suppressed detection of infre- Bacon, R. M., M. A. Pilgard, B.J.B. Johnson, J. Piesman, B. J. quent organisms. As a basic rule, the greater the di- Biggerstaff, and M. Quintana. 2005. Rapid detection versity within sequences isolated from a distinct mi- methods and prevalence estimation for Borrelia lonestari glpQ in Amblyomma americanum (Acari: Ixodidae) pools crobial population, the more likely minor populations of unequal size. Vector Borne Zoonotic Dis. 5: 146Ð156. are masked (Schloss and Handelsman 2006). This con- Barbour, A. G., G. O. Maupin, G. J. Teltow, C. J. Carter, and cept is supported in the current study by the statistics J. Piesman. 1996. IdentiÞcation of an uncultivable Bor- shown in Table 2. Pooling ticks before analysis has relia species in the hard tick Amblyomma americanum: been previously demonstrated to decrease prevalence possible agent of a Lyme disease-like Illness. J. Infect. 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Audic, J.-M. Claverie, and tick. Environmental inßuences appear to have a less D. Raoult. 2007. Lateral gene transfer between obligate signiÞcant inßuence on the proportion inhibited by intracellular bacteria: evidence from the Rickettsia mas- the different groups of bacteria compared with the siliae genome. Genome Res. 17: 1657Ð1664. feeding process. While some bacteria are acquired Blanton, L., B. Keith, and W. Brzezinski. 2008. Southern from the skin of the host, which is incorporated in the tick-associated rash illness: erythema migrans is not al- ways Lyme disease. South. Med. J. 101: 759Ð760. feeding lesion, feeding causes pathogens, especially Buller, R. S., M. Arens, S. P. Hmiel, and C. D. Paddock. 1999. Rickettsia spp. to multiply. The Þndings of this study Ehrlichia ewingii, anewlyrecognizedagentofhuman establish a basis for further investigations on the back- ehrlichiosis. N. Engl. J. Med. 341: 148Ð155. ground of various diseases in human and veterinary Burgdorfer, W. 1969. 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