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Journal of Clinical Microbiology, January 2000, p. 448-449, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Natural Infection of Domestic Goats with
Ehrlichia chaffeensis
Vivien G.
Dugan,1
Susan E.
Little,1,*
David E.
Stallknecht,2 and
Ashley D.
Beall1
Department of Medical Microbiology and
Parasitology1 and Southeastern
Cooperative Wildlife Disease Study,2
College of Veterinary Medicine, The University of Georgia, Athens,
Georgia 30602
Received 21 July 1999/Returned for modification 31 August
1999/Accepted 19 October 1999
 |
ABSTRACT |
Thirty-eight domestic goats from an area of Ehrlichia
chaffeensis endemicity were tested for antibodies reactive to
E. chaffeensis and for E. chaffeensis-specific
16S rRNA gene fragments by an indirect fluorescent antibody test and a
nested PCR assay, respectively. Twenty-eight of 38 (73.7%) goats had
antibodies reactive to E. chaffeensis (
1:128), and 6 of
38 (15.8%) goats were positive by diagnostic PCR; E. chaffeensis was isolated in cell culture from one goat. Our data
indicate that goats in areas of endemicity are naturally exposed to and
infected with E. chaffeensis.
| |
TEXT |
Human monocytic ehrlichiosis is a
newly described tick-borne disease caused by Ehrlichia
chaffeensis. The disease presents as a nonspecific, febrile,
flu-like illness; characteristic clinical pathology findings include
thrombocytopenia and leukopenia (1). Since human monocytic
ehrlichiosis was first detected in 1986, approximately 750 cases have
been reported to the Centers for Disease Control and Prevention
(13), with most occurring in the southeastern and
south-central United States (15). Previous research has
demonstrated that E. chaffeensis is maintained in nature
through a cycle involving white-tailed deer, Odocoileus virginianus, as the cardinal reservoir host and the lone star tick, Amblyomma americanum, as the primary vector (5,
9, 10). Additional studies have shown that dogs are also
naturally infected with E. chaffeensis in areas of
endemicity (4). Because domestic goats are parasitized by
the lone star tick (6), we evaluated domestic goats from
Clarke County, Georgia, an area of E. chaffeensis endemicity
(9), for exposure to this organism.
Blood samples were collected from 38 domestic goats from an established
herd pastured at a farm maintained by the College of Veterinary
Medicine, University of Georgia (Athens). Serum samples were tested for
antibodies reactive to E. chaffeensis by indirect
fluorescent antibody assay (IFA) as previously described (2). Serum or plasma samples initially were tested at 1:64 and 1:128 dilutions, with positive samples further tested in twofold serum dilutions to an endpoint. A comparative IFA performed on serum
and plasma samples from the same goat confirmed that plasma samples are
viable substitutes for serum samples in our system.
For PCR, DNA was extracted from 100 µl of EDTA-anticoagulated whole
blood by using the USB Isolation Kit (Amersham Life Science) or the GFX
Genomic Blood DNA Purification Kit (Amersham Pharmacia Biotech)
following the manufacturers' protocol for fast DNA extraction. The
final pellet was resuspended in 50 µl of molecular-biology-grade water, and 5 µl was tested by nested PCR as previously described (8) for the presence of the E. chaffeensis 16S
rRNA gene (rDNA), by using primers ECC and ECB in a primary reaction
followed by primers HE1 and HE3 in a secondary reaction. DNA
extraction, DNA amplification, and analysis of the results were
performed in different laboratories to prevent contamination. A
negative control was included in each step of the assay; a positive
control was included with each set of reactions.
Culture isolation was performed with blood from 10 goats as previously
described (3). Briefly, 7 to 10 ml of EDTA-anticoagulated blood was transferred into a sterile 50-ml centrifuge tube containing 25 ml of ACE lysing buffer (150 mM NH4Cl, 0.7 mM
KH2PO4, 3 mM EDTA-Na2), and the
tube was gently inverted. After 5 min at room temperature, the
suspension was centrifuged at 160 × g for 5 min, the
supernatant was removed, and the leukocyte fraction was washed in ACE
lysing buffer. After a second centrifugation, the pellet was suspended
in 1 ml of minimal essential medium supplemented with 5% fetal bovine
serum. Confluent cultures of DH82 canine macrophages in a
12.5-cm2 flask with 5 ml of minimal essential medium were
inoculated with the suspended cells. After 72 h the supernatant
was decanted, and 5 ml of fresh minimal essential medium was added.
Fresh medium was added to each culture twice weekly. Cultures were
tested by direct fluorescent antibody assay (FA) as previously
described (2) if cytopathic effect was observed or
approximately 60 days following inoculation if cytopathic effect was
not observed. Nucleic acid was extracted from FA-positive culture
material by using InstaGene purification matrix (Bio-Rad; Hercules,
Calif.) according to the manufacturer's directions and was tested by
nested PCR as described above. Representative amplicons from blood and
from cell culture material were sequenced as previously described
(7).
Twenty-eight of the 38 (73.7%) goats had antibodies reactive to
E. chaffeensis at a titer of 128, with a maximum titer of 2,048. The overall geometric mean titer (14) for positive
reciprocal titers was 453. On PCR assay, 6 of 38 (15.8%) samples
showed evidence of 16S rDNA characteristic of E. chaffeensis. E. chaffeensis was successfully isolated
from one goat on two separate occasions; the interval between
isolations was 40 days. The sequence of the 16S rDNA fragment amplified
from goat blood and from cell culture was identical to previously
published sequences of E. chaffeensis.
These data provide serologic and PCR evidence and cell culture
confirmation that domestic goats in an area of E. chaffeensis endemicity are naturally exposed to and infected with
E. chaffeensis. To date, white-tailed deer are the only
confirmed natural reservoir host of E. chaffeensis proven
capable of infecting ticks (5, 9). Previous serological
surveys of wildlife also have demonstrated E. chaffeensis-reactive antibodies in raccoons (Procyon
lotor) and opossums (Didelphis virginianus)
(10). In contrast, E. chaffeensis-reactive antibodies have not been found in wild rodent populations
(11) and immunocompetent rodents are difficult to infect
experimentally (12). Although both serologic and PCR
evidence of E. chaffeensis in dogs has been reported
(4), to our knowledge this organism has not been isolated in
cell culture from a naturally infected dog. Thus, this work is the
first report of isolation of E. chaffeensis from a naturally
infected vertebrate other than a deer. Further characterization of
E. chaffeensis infection dynamics in domestic goats,
including tick transmission studies, may provide a model reservoir host
for use in long-term studies.
| |
ACKNOWLEDGMENTS |
We thank Anneke Brandsma and Kathy Maraist for excellent laboratory
support and Nat Seney for generous assistance with animal handling.
This work was supported by a Georgia Veterinary Medical Experiment
Station grant.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology and Parasitology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602. Phone: (706) 542-8447. Fax: (706) 542-0059. E-mail:
slittle{at}calc.vet.uga.edu.
| |
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Journal of Clinical Microbiology, January 2000, p. 448-449, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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