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Emerging tick-borne Rickettsia and Ehrlichia at joint base Langley-Eustis, fort Eustis, Virginia.

Rickettsiae and related ehrlichial organisms are obligate intracellular bacteria carried by mites, fleas, ticks, and lice and are the agents of numerous tick-borne diseases found in Virginia, such as Rocky Mountain spotted fever (Rickettsia rickettsii), Tidewater spotted fever (Rickettsia parkeri), Human monotropic ehrlichiosis (Ehrlichia chaffeensis), and Ewingii ehrlichiosis (Ehrlichia ewingii). These and other rickettsial diseases have affected military activities and public health throughout the world for more than 2,000 years. (1) Rickettsial diseases, generally incapacitating and sometimes fatal, are frequently unrecognized or misdiagnosed. If recognized early, they can be treated effectively with antibiotics such as doxycycline, the treatment of choice. Delayed treatment is often associated with a more serious disease outcome, often with complications. (1)

In the United States, there are many tick-borne rickettsiae, most of which belong to the spotted fever group of rickettsiae (SFGR). Among them are R rickettsii, R parkeri, Rickettisa montanensis, and Rickettsia amblyommii. The first two are known to be pathogenic to humans and the latter two have limited evidence suggesting possible pathogenicity. (2) Over the past few decades, rickettsiology has undergone significant changes and many new and some previously characterized rickettsiae have been found to be pathogenic. (2,3) As of 2012, 26 Rickettsia species with validated and published names have been reported, the vast majority of which are considered tick-borne rickettsiae. (2)

For most of the 20th century, R rickettsii, the causative agent of Rocky Mountain spotted fever, was considered the only tick-borne rickettsial agent pathogenic to humans in the Americas. Rocky Mountain spotted fever (RMSF) has been consistently described as a potentially fatal disease. In the early 20th century, 63% of RMSF diagnosed patients from Montana died from the disease. (4) In the late 1940s, antimicrobial therapy was developed for RMSF4 and doxycycline is now considered the drug of choice for all tick-borne rickettsial diseases in children and adults. (5) While the fatality rate of RMSF has diminished to 1.4% in the United States in the 21st century, it is higher in South American countries (greater than 20%) despite therapy. (4,6) One of the reasons for the fatalities due to RMSF is the difficulty correctly diagnosing the rickettsiosis. Diagnosis of RMSF is problematic due to nonspecific signs and symptoms associated with the disease, which include fevers, headaches, rashes, and the lack of commercially available species-specific assays. (2) Recently there has been an increase in reported RMSF cases in the United States. Only 495 cases were reported to the Centers for Disease Control and Prevention (CDC) in 2000, (7) but 2,288 cases were reported in 2006 and 2,016 in 2007, marking the highest recorded levels in over 80 years. (8) However, most of these cases have been described as suspect RMSF cases (ie, clinical presentation and a single serum positive test which is group--but not species-specific). The prevalence of the SFGR antibody is known to exist in about 10% of the US population. (9) Thus, the presence of a single positive SFGR-specific serological assay will not specifically diagnose RMSF. Since the assay is nonspecific, the positive serologic reaction could represent an infection with another SFGR pathogen (eg, R parkeri, R akari) or a rickettsia of unknown pathogenicity (eg, R amblyommii, R montanensis). Thus, the lower fatality rates associated with RMSF may actually be due to the misdiagnosis of other rickettsioses with lower fatality rates than RMSF.

Tidewater spotted fever, also known as Rickettsia parkeri rickettsiosis or American boutoneuse fever, has been a recently described human disease even though the causative agent, R parkeri, has been known since its isolation in 1937. Ralph Robinson Parker isolated R parkeri from Amblyomma maculatum, commonly known as the Gulf Coast tick. Rparkeri was considered a nonpathogenic rickettsia and received little attention until 2004, when the first case of Rparkeri human infection, which was similar to yet distinct from RMSF, was reported. (3,10) A second case of Rparkeri human infection was documented 3 years later. (11) Both cases were from the Tidewater region of Virginia, in the same region as Fort Eustis. New research has also revealed that multiple tick species within the Amblyomma genus can harbor R parkeri (3,12) The many novel findings concerning R parkeri human infection indicate that much is still unknown about many human rickettsioses. Infection with R parkeri poses a significant threat to public health (2) because at least one-third of reported RMSF cases are believed to be caused by R parkeri. (3,4)

While R parkeri was newly identified as a pathogenic species, many other rickettsiae have emerged as possible pathogens, the most notable of which is R amblyommii. There have been tick bite rashes and probable RMSF cases associated with R amblyommii, though none have been confirmed. (13,14) R amblyommii has been found in large percentages of Amblyomma americanum ticks, commonly known as lone star ticks, and is believed to be the most common rickettsia infecting A americanum ticks. (13,15-17) R amblyommii and Borrelia lonestari have been suspected at one time to play a role in southern tick-associated rash illness (STARI), however the true causative agent of STARI has yet to be confirmed. (18-20) A americanum ticks are notoriously aggressive, nonspecific feeders whose geographic range covers a large portion of the continental United States. (21-23) A confirmation that R amblyommii is a human pathogen would be of great interest and concern.

Other tick-borne obligate intracellular bacteria pathogenic to humans are E chaffeensis and E ewingii, members of the order Rickettsiales, and agents of human monocytotropic ehrlichiosis (HME) and ewingii ehrlichiosis, respectively. (21) Human monocytotropic ehrlichiosis is a mild-to-fatal febrile illness with a case fatality rate of 2.7%. (24) A majority of HME patients require hospitalization; in one HME study, 85% of patients were hospitalized and many had serious complications. Older patients were more likely to develop complications and have longer hospitalizations. (25) Ehrlichia ewingii is known to cause a mild febrile illness in humans and may account for as much as 7% of all human ehrlichiosis cases in the United States. (26) A americanum is the main vector of E chaffeensis and E ewingii, while whitetail deer (Odocoileus virginianus) are the preferred vertebrate hosts of A americanum (21) Vertebrate hosts infected with ehrlichiae are bacteremic for prolonged periods, (27) which increases the chance for transmission to a tick host. Human monocytotropic ehrlichiosis has been reported in 47 states, with the highest reported average annual incidence rates in Arkansas, North Carolina, Missouri, and Oklahoma. Additionally, two-thirds of HME cases occur between May and July. (24)

Fort Eustis is an approximately 3,197 hectare military installation located in the Tidewater region of coastal Virginia. Over 2,100 hectares of this property are in natural areas including pine-mixed hardwood forests, wetlands, and early successional habitat. These natural areas provide abundant opportunities for military training and recreational activities like golfing, camping, hiking, and hunting. These types of activities often center around the warmer months when many tick species are at their most active. A large component of the overall force health protection plan on Fort Eustis involves familiarity with natural hazards such as vector-borne diseases, which may be transmitted to personnel by the bite of an infected tick. Because numerous tick-borne rickettsiae like R parkeri, the etiologic agent of Tidewater spotted fever, are emerging as pathogenic bacteria, evaluation of ticks in the Fort Eustis area of Virginia was undertaken to determine the risk of tick-borne disease to residents and visitors. A large-scale surveillance effort was begun in October of 2007 to assess the tick fauna of designated portions of the property. A concise but limited portion of this effort is presented here.

MATERIALS AND METHODS

Specimens. Questing ticks were collected from 5 preselected locations by dragging a one meter square cloth for approximately 100 meters at each site once per month from May through August 2009. Site selection was based on suitable tick habitat, and individual sites were divided between areas of high human use such as golf course edges and walking trails, and low-human use such as gated natural areas. Additionally, the sites were not treated with any type of pesticide prior to or during this study.

Nucleic acid purification. Adult and nymph ticks were identified and individually placed in 300 [micro]L of Tissue Lysis Buffer (Qiagen, Valencia, CA). Larvae were similarly handled with the exception that 15 larvae were placed in each 300 [micro]L of lysis buffer. Ticks were bisected with a sterile knife and incubated with the addition of proteinase K prior to nucleic acid purification according to kit directions with the DNeasy Blood & Tissue Kit (Qiagen). Purified nucleic acids were eluted with 100 [micro]L of elution buffer. The bisected ticks, remaining lysate, and purified nucleic acids were stored at -80[degrees]C for future analysis.

Polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) assays. Purified nucleic acid preparations from individual (adults and nymphs) and pooled (larvae; n=15) A americanum ticks were analyzed for R amblyommii by the Rambl qPCR assay as previously described. (16) In addition, the A americanum samples were screened for Ehrlichia species in a 20 [micro]L real-time multiplex reaction designed to amplify and differentiate a segment of the heat shock protein operon groEL of E chaffeensis and E ewingii (28) using the Light-Cycler FastStart DNA Master HybProbe kit (Roche) and 2 [micro]L of sample. To be determined as positive, the sample melting peak was compared with a known standard and only samples that had an equivalent melting temperature were considered positive. Positive E ewingii samples were confirmed to species in a 25 [micro]L conventional PCR reaction targeting the p28 gene (29) using the PuReTaq Ready-To-Go PCR Beads (GE Healthcare Biosciences, Piscataway, NJ) and 2 [micro]L of sample. Samples positive for E chaffeensis were confirmed in a 20 [micro]L qPCR reaction targeting the 16s rRNA sequence (30) using the LightCycler TaqMan Master kit (Roche) and 5 [micro]L of sample. Borrelia lonestari infection was determined by screening with a SYBR Green I assay that amplified and detected a portion of the glpQ gene. (31) Real-time PCR with a melting curve was performed in a 20 [micro]L reaction using LightCycler SYBR Green I master mix (Roche) and 5 [micro]L of sample. Samples that produced a melting peak and equivalent Tm to the known standard were further analyzed with a conventional PCR that amplified a portion of the flagellen gene of B lonestari. (32)

Individual D variabilis, A maculatum, and pooled or individual A americanum nucleic acid preparations were screened for rickettsiae by the genus-specific Rick17b

qPCR assay as previously described. (33) The screen positive A maculatum nucleic acid preparation was assessed by the species-specific Rpark and Rande qPCR assays for R parkeri and Candidatus Rickettsia andeanae, as previously described. (33) The single adult I scapularis (black-legged tick) was tested for Borrelia and Anaplasma with a real-time multiplex (34) using the Light-Cycler TaqMan Master Kit (Roche) in a 20 [micro]L reaction with 5 [micro]L of sample. Conventional PCR was performed on a MJ Research PTC 200 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA), and real-time PCR reactions were performed on the LightCycler 2.0 instrument (Roche).

Standard PCR for sequencing. Standard and nested PCR assays were used to amplify outer membrane protein B (ompB) and A (ompA), and the surface cell antigen 4 (sca4) genes of Rickettsia. (34) The master mix was composed of Platinum PCR SuperMix High Fidelity (Invitrogen), 0.3 [micro]M of primers, and one [micro]L of template. All nested PCR were followed by gel electrophoresis run on a 1.5% agarose gel at 150 volts for 30 minutes.

Purifying PCR products. Nested PCR products were purified using either QIAquick PCR Purification Kit or DNA Gel Extraction Kit (Qiagen) when multiproducts were produced, 20 [micro]L of buffer was used to elute the final DNA product.

Multilocus sequence typing was performed as previously described. (35) Briefly, purified PCR products were sequenced for both strands by using the Big-Dye terminator reagent (Applied Biosystems; Foster City, CA). Cycling temperatures were 25 cycles at 96[degrees]C for 10 seconds, 50[degrees]C for 5 seconds, and 60[degrees]C for 4 minutes. Sequencing reactions were cleaned up by using gel cartridges and run on a 3130 automated sequencing analyzer (Applied Biosystems). To obtain the final sequence data, Chromas software (Technelysium; Queensland, Australia) and Vector NTI software (Invitrogen; Fredrick, MD) were used.

RESULTS

Ticks collected. Eight hundred sixty-one ticks (0.4 per square meter) were collected by dragging a one meter square cloth for 100 meters at each of the sample sites on each date for a total of approximately 2,000 meters. Three hundred forty A americanum, 33 D variabilis, and 2 A maculatum were assessed for evidence of rickettsiae, B lonestari, E ewingii, and E chaffeensis. One I scapularis was assessed for Borrelia and Anaplasma species. Four hundred eighty-two A americanum larvae and 3 nymphs collected from 3 sites were not included in this study. All 4 tick species collected are considered man-biting pests and vectors of disease.

Amblyomma americanum ticks assessed for Rickettsia, Borrelia and Ehrlichia: Purified nucleic acid preparations from the 340 (81 single adults, 34 single nymphs, and 15 pools of larvae) A americanum ticks selected for analysis were subject to qPCR testing for R amblyommii, B lonestari, E ewingii, and E chaffeensis (Table 1). Borrelia lonestari was detected in 2 male and 2 female A americanum adults for an infection rate of 5% of the adults tested. Ehrlichia chaffeensis was detected in one (1%) of the adult ticks tested, and E ewingii was detected in 2 (2%) adult and 2 (6%) of the nymph samples. Neither of these organisms were detected in the pools of larval ticks. Rickettsia amblyommii was detected in a minimum of 65 samples, and no further analysis was performed.

Dermacentor variabilis, A americanum and A maculatum ticks analyzed for Rickettsia species. Thirty three adult D variabilis, 2 female A maculatum and 340 adult and immature A americanum ticks were tested for rickettsiae (Tables 1, 2). All D variabilis ticks were negative, however, R amblyommii was detected in a minimum of 63 A americanum ticks. Rickettsia species was detected in one of the 2 female A maculatum with the Rick17b and Rpark qPCR assays (Tables 1 and 2). Subsequently, the Rickettsia species positive DNA sample preparation from the R parkeri positive A maculatum adult tick was analyzed by multilocus sequence typing using ompA, ompB, and sca4 genes. Two fragments of ompA (648 and 856 bp) and ompB (806 and 584 bp), and one fragment of sca4 (812bp) were 100% identical to R parkeri Maculatum 20 (GenBank #AF 123717). The Candidatus Rickettsia andeanae qPCR assay (Rande) was negative for the R parkeri positive sample.

The ubiquitous occurrence of A americanum collected at Fort Eustis along with the aggressive man-biting character of this tick species indicates that the potential risk of rickettsial and ehrlichial human infections could be high in the summer months in this area. Another possible public health threat is STARI, which has been documented following an A americanum tick bite. (35) Even though R amblyommii and B lonestari have been suggested as possible agents of STARI, the true etiologic agent of this disease has yet to be identified. (18,20) An additional potential health concern associated with A americanum is the recent discovery of R parkeri in Lone Star ticks collected in Tennessee and Georgia. (37) In this study, we did not detect R parkeri in any of the A americanum ticks tested.

Dermacentor variabilis ticks are known to carry R rickettsii, the causative agent of RMSF, and R montanensis, a rickettsia of unknown human pathogenicity. (38) No rickettsial agent was identified in the small number of D variabilis ticks assessed. This is not surprising since R rickettsii are rarely found in D variabilis, even in areas highly endemic for RMSF, (39) and R montanensis is usually detected in only 5%-19% of D variabilis evaluated. (9,17,38)

One of the 2 adult A maculatum ticks collected in this study was found to harbor R parkeri. A low number of Gulf Coast ticks identified in Fort Eustis or the Tidewater region is not surprising since its presence in this area and throughout Virginia has only been sporadically encountered. (40,41) With that said, it is interesting that 2 of the first reported R parkeri rickettsiosis cases occurred in the Tidewater area of Virginia. (10-11) R parkeri was initially isolated from A maculatum ticks in 193742 and has recently been found in multiple Amblyomma species such as Amblyomma triste, (12) A americanum, (37,43) Amblyomma nodosum, (44) and experimentally in Amblyomma cajennense. (45) The discovery of R parkeri infected ticks at Fort Eustis (in the Tidewater region of Virginia) combined with 2 Tidewater R parkeri human infections (10,11) from the same region implies that residents of and visitors to the Tidewater area may be at risk of R parkeri infections. In addition, A maculatum, endemic to the southern United States, (40,41,46) may be widening its geographic range, implying that the geographic range of R parkeri is expanding as well. If the endemic region of A maculatum and R parkeri are growing, healthcare providers should be made aware of possible R parkeri rickettsiosis in their areas, and that Tidewater spotted fever has been confused for RMSF. (4) The results of this study reveal the need to learn more about the distribution of this vector in the Tidewater region, the prevalence of R parkeri infection of the Gulf Coast tick, and the incidence of Tidewater spotted fever in this area.

The inherent sampling bias of the cloth drag method is well explained by Schulze et al. (47) However, in this instance it was employed as a surrogate to estimate rates at which a human may encounter ticks at Fort Eustis. Based on this assessment, the potential exists to encounter at least 4 ticks per 10 meters traveled on foot in natural, training, and recreational areas at Fort Eustis. Furthermore, pathogenic R parkeri, E ewingii and E chaffeensis which pose a risk to human health in the region were identified in 5% of the questing adult and nymph ticks collected from Fort Eustis. The predicted rate of encounter of an infected adult or nymph tick may be as high as 5 infected ticks per 100 meters traveled. Human rickettsial and ehrlichial diseases including RMSF and HME (1) are difficult to recognize and may be misdiagnosed due to their often cryptic symptoms. Since R parkeri has been recognized as a human pathogen only for the past few years, there is an even greater chance of misdiagnosis of R parkeri rickettsiosis. Indeed, one-third of supposed RMSF cases are believed to be misdiagnosed R parkeri cases. (4) Clinicians, therefore, should be aware of this condition. Moreover, studies are needed to determine the occurrence, distribution, and seasonality of A maculatum and R parkeri in the mid-Atlantic states of North Carolina, Virginia, and Maryland, which were previously not known to have long-established populations of A maculatum. Likewise, E chaffeensis and E ewingii, the discoveries of which occurred in 1986 and 1999, respectively, are also relatively unknown agents associated with the underreported disease HME and ewingii ehrlichiosis. (24,25) Location of the diseases and their arthropod vectors are essential in informing medical health care providers, preventive medicine personnel, and the general population as to the risk of tick-borne diseases. (48)

ACKNOWLEDGEMENT

This work was supported by the Department of Defense Global Emerging Infections System program work unit # 847705.82000.25GB.A0074.

The authors thank Ben Pagac for his review of this manuscript.

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AUTHORS

Ms Miller is an entomologist and Chief, Laboratory Sciences Division, Public Health Command Region-North, Fort Meade, Maryland.

Ms Jiang is a rickettsiologist in charge of laboratory detection, identification and characterization of rickettsial agents in the Rickettsial Diseases Research Program, Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland.

Ms Truong is an investment banking analyst at Stifel Nicolaus Weisel. She received a BS in Biology (Physiology and Neurobiology) and a BS in Finance from the University of Maryland, College Park, Maryland.

Ms Yarina is a microbiologist and currently a Masters of Public Health candidate focusing on epidemiology at George Washington University, Washington, DC.

Dr Evans is a postdoctoral fellow in the laboratory of Dr Edward Mitre, Department of Microbiology and Immunology, Uniformed Services University, Bethesda, Maryland.

Mr Christensen is a biological scientist overseeing the natural resources and entomology programs for Joint Base Langley-Eustis, Fort Eustis and assigned to the Environmental Element, Civil Engineer Division, 733 Mission Support Group, Fort Eustis, Virginia.

Dr Richards is a senior scientist and rickettsiologist, in charge of the Rickettsial Diseases Research Program, Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, MD. He is also an adjunct professor of the Emerging Infectious Diseases program and Preventive Medicine and Biometrics Department, Uniformed Services University of the Health Sciences, Bethesda, Maryland.

Melissa K. Miller, MS

Ju Jiang

Melissa Truong

Tamasin Yarina

Holly Evans, PhD

Timothy P. Christensen, MS

Allen L. Richards, PhD
Table 1. Amblyomma americanum ticks collected by drag sampling at
Joint Base Langley-Eustis, Fort Eustis, Virginia, May-August 2009.

Life     Number         Number        Borrelia lonestari
Stage   Collected     Tested (N)     Number Positive (%N)

Adult      81             81                4 (5%)
Nymph      37             34                0 (0)
Larva      707      225 (15 pools)          0 (0)

Life    Ehrlichia chaffeensis     E ewingii     Rickettsia amblyommii
Stage   Number Positive (%N)       Number       Number Positive (%N)
                                Positive (%N)

Adult          1 (1%)              2 (2%)             34 (42%)
Nymph           0 (0)              2 (6%)             16 (47%)
Larva           0 (0)              0 (0)              15 (7%) MIR *

* Minimum infection rate, larvae tested in pools of 15 individuals.

Table 2. Ticks collected by drag sampling at Joint Base
Langley-Eustis, Fort Eustis, Virginia, May-August 2009.

Tick          Life     Number     Number   Borrelia/Anaplasma
species       stage   collected   Tested     Number Positive
                                   (N)            (%N)

Amblyomma     Adult       2         2          Not tested
maculatum

Dermacentor   Adult      33         33         Not tested
variabilis

Ixodes        Adult       1         1             0 (0)
scapularis

Tick          Rickettsia species    Rickettsia parkeri
species         Number Positive       Number Positive
                     (%N)                  (%N)

Amblyomma           1 (50%)               1 (50%)
maculatum

Dermacentor          0 (0)                 0 (0)
variabilis

Ixodes            Not tested            Not tested
scapularis
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Author:Miller, Melissa K.; Jiang, Ju; Truong, Melissa; Yarina, Tamasin; Evans, Holly; Christensen, Timothy
Publication:U.S. Army Medical Department Journal
Geographic Code:1U5VA
Date:Oct 1, 2016
Words:5134
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