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Letter
Ehrlichia
Prevalence in Amblyomma americanum, Central Texas
Scott Wesley Long,*
J. Mathews Pound,† and Xue-jie Yu*
*University of Texas Medical Branch, Galveston, Texas, USA; and †United
States Department of Agriculture – Agricultural Research Service, Kerrville,
Texas, USA
Suggested citation
for this article:
Long SW, Pound JM, Yu X. Ehrlichia prevalence in Amblyomma
americanum, central Texas [letter]. Emerg Infect Dis [serial on
the Internet]. 2004 Jul [date cited].Available from: http://www.cdc.gov/ncidod/EID/vol10no7/03-0792.htm
Ehrlichia chaffeensis and E. ewingii, agents of human monocytic
ehrlichiosis and ehrlichiosis ewingii, respectively, are transmitted by
the lone star tick, Amblyomma americanum, which is found from west-central
Texas northward to Iowa, and southeastward to the Atlantic Coast (1).
In A. americanum, E. chaffeensis has been found in several
states, while E. ewingii has only been found in North Carolina,
Florida, and Missouri (1,2). E. ewingii infection
in white-tailed deer (Odocoileus virginianus), a potential reservoir,
has been found in the states mentioned previously as well as in Kentucky,
Georgia, and South Carolina (3,4).
Human ehrlichioses are underdiagnosed in the United States and may be
as prevalent as Rocky Mountain spotted fever in some areas (1).
Ehrlichioses are prevalent in Texas, and fatal cases have been reported
(1,5). This study was conducted to examine ticks from
central Texas for Ehrlichia and provide information to increase
public health awareness of this problem. Adult A. americanum ticks
were collected from a 38.8-hectare game fenced-pasture (Plot #8) in the
Kerr Wildlife Management Area, Kerr County, Texas. Ticks were trapped
by using blocks (approximately 85 g) of dry ice centered on smooth, white,
nylon cloths measuring approximately 1 m2. These traps were
placed on the ground in the brush or in areas under tree canopies for
approximately 1 h.
Trapped adult A. americanum were frozen in liquid nitrogen and
then bisected with a sterile scalpel. Halves of the bisected ticks were
stored at –80ºC. The other halves were pooled in groups of six. DNA was
extracted from these pools by using the QIAamp DNA Mini Kit (Qiagen Inc.,
Valencia, CA), and evaluated by using a nested species-specific 16S rRNA
gene polymerase chain reaction (PCR) for E. chaffeensis and E.
ewingii, with E. canis as a negative control. The first-round
primers were genus-specific for Ehrlichia (ECC and ECB). The forward
primers of the nested PCR were HE1, EE72, and Ecan, which were specific
to E. chaffeensis, E. ewingii, and E. canis, respectively.
The reverse primer is a common primer (HE3) for all species (6–8).
An aliquot of the negative control reaction containing no DNA template
was carried through both rounds of the nested PCR with every reaction
set. A dilution series of stock E. chaffeensis DNA mixed with tick
DNA showed no substantial inhibition of the PCR, even with DNA concentrations
as low as 0.2 ng/mL.
Tick pools positive for E. chaffeensis or E. ewingii by
PCR were examined by using DNA from the individual tick halves. DNA was
extracted by using the Nucleobond DNA/RNA Isolation Kit (BD Biosciences
Clontech, Palo Alto, CA). To confirm positive PCR results for individual
ticks, first-round amplicons (primers ECB and ECC) were separated by electrophoresis.
The 478-bp band was recovered using the QIAquick Gel Extraction Kit, then
cloned into the pCR2.1-TOPO vector with the TOPO TA cloning kit (Invitrogen,
Carlsbad, CA). DNA sequences were obtained from both directions of the
insert in the recombinant plasmids by using PE Applied Biosystems (Foster
City, CA) 373XL automated DNA sequencers in the UTMB Sequencing Core.
Of the 66 adult A. americanum ticks examined, 5 were positive
for E. ewingii (7.6%). The 16S rRNA gene sequences from these five
positive samples were most similar to the E. ewingii 16S rRNA gene
sequence (GenBank accession no.U96436). Sequence variations are summarized
in the Table. These mutations may result from polymerase
errors prior to cloning. E. ewingii has never been cultured or
handled by our laboratory, and all negative controls for the nested PCR
were negative, minimizing the possibility of false-positive results.
This is the first report of ticks infected with E. ewingii in
states other than North Carolina, Florida, or Missouri. Ticks are found
in damp wooded areas (1,9). Seasonal population changes
have been associated with climatic factors, including precipitation, temperature,
and day length (9–11). These ticks were collected during
August, one of the hottest months of the year in Texas, with temperatures
averaging 33°C. Adult ticks are more abundant earlier in the summer,
and the actual prevalence of E. ewingii infection may be higher.
August is a dry month in Texas, averaging 2.32 inches of an annual rainfall
of 26 to 30 inches (12). Although cases of E. ewingii
infection have not been reported from Texas, this study shows the presence
of ticks infected with E. ewingii in Texas.
No ticks infected with E. chaffeensis were found in this sample.
The prevalence of E. chaffeensis may be so low that it was not
detected in the small sample size. Also, E. chaffeensis may not
survive well at this extreme of the host range. Infection exclusion may
occur in the tick or reservoir hosts (or both), such that an established
population of one ehrlichial species prevents another ehrlichial species
from establishing itself. This phenomenon has been noted in the related
rickettsial organisms Rickettsia peacockii and R. rickettsii
in the Rocky Mountain wood tick, Dermacentor andersoni (13).
Another finding involves using nested 16S rRNA PCR to identify ehrlichial
infection. These primers are not as specific as thought previously. Arthropods
should be carefully cleaned to prevent contamination by Shigella
and other soil contaminants. A single positive-nested PCR reaction should
not be considered sufficient for positive identification of the organism.
Sequencing of the outer PCR product, or another confirming method, should
be used to positively identify the organism. Primers directed to more
divergent sequences, such as the dsb gene, should be utilized in
place of, or in addition to, 16S rRNA gene PCR (14).
This study was supported
by a fellowship for Scott W. Long from the Sealy Center for Vaccine
Development, University of Texas Medical Branch, and a grant from the
National Institute of Allergy and Infectious Diseases (AI45871).
References
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Missouri. Vector Borne Zoonotic Dis. 2002;2:53–60.
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et al. Ehrlichia
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Emerg Infect Dis. 2002;8:668–71.
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JA, et al. Detection
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by PCR assay and serologic analysis. J Clin Microbiol. 2003;41:1263–5.
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of notifiable diseases, United States 2000. MMWR Morb Mortal Wkly
Rep. 2002;49:1–100.
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et al. Detection
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WR, et al. Susceptibility
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- Oregon State University, Spatial Climate Analysis Service, 2000. Texas.
Average annual precipitation, 1961-1990. Available from: http://www.ocs.orst.edu/pub/maps/Precipitation/Total/States/TX/tx.gif
- Niebylski ML, Schrumpf ME, Burgdorfer W, Fischer ER, Gage KL, Schwan
TG. Rickettsia
peacockii sp. nov., a new species infecting wood ticks, Dermacentor
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| Table.
Sequence variation of the 16S rRNA gene in Ehrlichia ewingii
detected in Amblyomma americanum ticks from central Texas,
compared to the partial 16S rRNA gene sequence of Ehrlichia ewingii
in Genbank (accession U96436)a |
|
| |
G16
|
A93G
|
A157G
|
T190C
|
A429G
|
C474
|
|
|
Tick B5
|
–
|
–
|
–
|
–
|
–
|
–
|
|
Tick B7
|
+
|
–
|
–
|
+
|
–
|
–
|
|
Tick D1
|
–
|
+
|
+
|
–
|
+
|
+
|
|
Tick D2
|
–
|
+
|
+
|
–
|
–
|
–
|
|
Tick D4
|
–
|
+
|
+
|
–
|
–
|
–
|
|
| a+, mutation present, –, mutation
not present. |
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