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Dispatch
Cervids as Babesiae Hosts,
Slovenia
Darja Duh,*
Miroslav Petrovec,* Andrej Bidovec,† and Tatjana Avsic-Zupanc*
*Institute of Microbiology and Immunology, Ljubljana, Slovenia; and †Institute
of Wildlife Pathology, Ljubljana, Slovenia
Suggested
citation for this article
We describe cervids
as potential reservoir hosts of Babesia EU1 and B. divergens.
Both babesial parasites were found in roe deer. Sequence analysis of
18S rRNA showed 99.7% identity of roe deer Babesia EU1 with the
human EU1 strain. B. divergens detected in cervids was 99.6%
identical to bovine B. divergens.
Human babesiosis is an emerging tick-transmitted disease caused by intraerythrocytic
parasites of the genus Babesia. A bovine parasite, Babesia divergens
has been implicated as the most common agent of this dangerous zoonosis
in Europe (1). The life cycle of B. divergens
is determined by cattle, the vertebrate host, and by European sheep ticks,
Ixodes ricinus. Ticks are not only the vectors of B. divergens
but also its most important nonbovine reservoir (2).
Many questions regarding parasite epidemiology and biology and the host
response to infection remain to be answered. Furthermore, molecular data
for B. divergens are scarce; only 1 DNA sequence of this parasite
from humans from mainland Europe has been recently deposited (3).
Recently, 2 cases of human babesiosis have been reported in Italy and
Austria. The etiologic agent was identified as Babesia EU1, a pathogen
closely related to, but clearly distinct from, B. divergens (4).
The distinction was based on analysis of the complete babesial 18S rRNA
gene, which also showed that EU1 is most closely related to B. odocoilei,
a parasite of white-tailed deer (Odocoileus virginianus) in the
United States (5). I. ricinus, the most prevalent
and widely distributed tick species in Europe, has already been implicated
as the vector of EU1 (4,6). Moreover, I. ricinus
has a wide range of vertebrate hosts and readily bites humans. Rapidly
and accurately identifying the reservoir of Babesia EU1 will enable
appraisal of the full range of disease control options.
The Study
We investigated 2 species of cervids shot by professional hunters from
1996 to 2000 in the vicinity of Ljubljana, Slovenia. DNA was extracted
from spleen samples of 51 roe deer (Capreolus capreolus) and 30
red deer (Cervus elaphus), as previously described (6).
Babesiae were detected in cervids by using specific nested polymerase
chain reaction (PCR) that allowed discrimination between B. divergens
and EU1. Primers were designed on the basis of alignment of complete 18S
rRNA gene sequences of EU1, B. divergens, and B. odocoilei.
With primers PIRO-A (7) and BABSr, a 600-bp babesial
18S rRNA gene was amplified with 5 μL of DNA and PCR Master Mix (Promega,
Madison, WI, USA). One microliter of PCR product was used for nested PCR
with either primer set PIRO-B/BOD and PIRO-B/BDV to detect 240 bp of 18S
rRNA of EU1 and B. divergens, respectively. Both babesial parasites
were detected in roe deer (76.5%); however, more animals were infected
with B. divergens (54.9%) than Babesia EU1 (21.6%). Only
16.7% red deer were infected with B. divergens alone. Infection
with babesial parasites did not differ significantly between sexes in
either roe or red deer.
To assess DNA sequence homologies with EU1 from human and ticks, distinctive
amplicons of the complete babesial 18S rRNA gene derived from cervids
were cloned and sequenced. Parasite DNA from 2 roe deer that were found
positive with a different set of nested primers was used in PCR with CRYPTO
F and CRYPTO R (6). Amplicons were ligated into a plasmid
vector (TOPO TA Cloning Kit for Sequencing, Invitrogen, Groningen, the
Netherlands), and Escherichia coli–competent cells were transformed
as instructed by the manufacturer. Plasmid DNA was purified from overnight
cultures of selected colonies (Wizard Plus Minipreps DNA Purification
System, Promega) and analyzed for inserts by restriction analysis with
EcoR1 (Promega). Sequencing on both strands was carried out in an automated
sequencer using BigDye Terminator Cycle Sequencing Ready Reaction Kit
(PE Applied Biosystems, Foster City, CA, USA). Two clones were included
in reactions with T3, T7, and internal primers to obtain complete gene
sequence. All primers designed and used for this study are listed in the
Table. Sequences were analyzed with computer programs
of the Lasergene 1999 software package (DNASTAR, Madison, WI, USA) and
submitted to GenBank to determine accession numbers. Homology search and
alignment of the complete sequence of the babesial 18S rRNA gene from
1 roe deer showed 99.7% (5 nucleotide [nt] differences) identity with
EU1 from a human patient and 99.8% (4 nt differences) identity with EU1
present in I. ricinus ticks from Slovenia. The complete sequence
of the babesial 18S rRNA gene from another roe deer was, however, nearly
identical (99.6%, 7 nt differences) to babesial parasite MO1 and B.
divergens. Phylogenetic relationships of babesiae from roe deer and
from other sources are shown the Figure. By using TREECON software (8),
a phylogenetic tree was constructed with the neighbor-joining method,
and topology of the tree was obtained with the K80 model. Support for
the tree nodes was calculated with 1,000 bootstrap replicates.
Conclusions
Babesia EU1, a zoonotic pathogen, was the cause of human babesiosis
recently reported by Herwaldt et al. (4). While I.
ricinus was already implicated as a vector of EU1, no other information
about biology, ecology, or geographic distribution of EU1 exists (4,6).
Phylogenetic analysis based on comparing the complete 18S rRNA gene sequence
of EU1 derived from humans and ticks with other babesial parasites showed
that EU1 is more closely related to B. odocoilei than B. divergens
(4). B. odocoilei, which is transmitted by I.
scapularis, primarily infects white-tailed deer in the United States
(5). Cases of fatal babesiosis were described in 2 other
species of cervids, namely a zoo-housed caribou (Rangifer tarandus
caribou) and an elk (C. elaphus elaphus) (9).
Therefore, we tested 2 species of cervids from Slovenia as potential reservoir
hosts of EU1. By using specific nested PCR, the presence of EU1 was established
in roe deer (21.6%) but not in red deer.
In Slovenia, roe deer are widely distributed, and the population density
is high. Their pasture comprises woodland, bushes, and even open meadows
and fields (10). However, red deer were nearly extinct
in Slovenia in the beginning of the 19th century. Although they were later
imported from Austria, Poland, and Hungary, they are still less numerous
and therefore harbor fewer ticks (10). The identity of
babesial parasites from roe deer from Slovenia with EU1 was confirmed
by cloning and sequencing the complete babesial 18S rRNA gene. The sequences
obtained were 99.8% and 99.7% identical to the 18S rRNA genes of EU1 from
ticks and humans, respectively. Since the habitat of roe deer is expanding
in other European countries (10), additional studies
are needed to determine whether roe deer are reservoir hosts of EU1 elsewhere
in Europe.
Whereas the presence of EU1 in cervids was anticipated, detection of
B. divergens in roe and red deer was surprising. With the exception
of a single report of naturally acquired babesiosis caused by B. divergens
in reindeer (R. tarandus tarandus), no data about cervids as reservoirs
of B. divergens were available at the time of our research (11,12).
Although B. divergens can infect cervids experimentally, animals
experience only mild infections with low parasitemia (2,11).
However, 54.9% of roe deer and 16.7% of red deer were infected with B.
divergens in this study. Further cloning and sequencing of the complete
18S rRNA gene of the parasite indicated 99.6% (7 nt differences) identity
with babesial parasite MO1 and B. divergens. MO1 was described
as an etiologic agent of human babesiosis acquired in Missouri and was
genetically almost identical to B. divergens (99.9% identity, 2
nt differences), but the authors claimed that the parasites probably differ
(13). However, piroplasms in abnormal hosts or hosts
that are not generally considered primary hosts may have morphologic differences
(12). In addition, high molecular identity of piroplasms
does not necessarily mean that they have the same infectivity for different
hosts.
A Babesia sp., tentatively called B. capreoli, was observed
and described in red deer in Scotland (14) and sika
deer (C. nippon) in Ireland (15). The parasite
resembled B. divergens morphologically and antigenically. B.
capreoli was suggested to be transmitted by I. ricinus ticks.
The main difference between bovine B. divergens and these deer
parasites is in their host specificity. Whereas B. divergens can
infect a wide range of animals after splenectomy, including some deer
species, various nonhuman primates, gerbils, and humans, B. capreoli
can apparently infect only cervids and perhaps sheep (15).
Nevertheless, with no deposited sequence of 18S rRNA of B. capreoli,
the identity of B. divergens from roe and red deer from Slovenia
is uncertain.
The finding that roe and red deer may be reservoirs for B. divergens
has serious implications. Future research must determine if parasites
from cervids share biologic characteristics with B. divergens,
such as infectivity to cattle and humans and transmission by I. ricinus.
Acknowledgments
We thank Jeremy
S. Gray for critical review and helpful comments on the manuscript.
Ms. Duh is a PhD
student at the Medical Faculty, Ljubljana, Slovenia. She is currently
working at the Institute of Microbiology and Immunology. Her research
is primarily based on babesiae and other tickborne pathogens.
References
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Table. Nucleotide sequences
and optimum annealing temperatures of the primers designed and used
for nested polymerase chain reaction (PCR) and those for amplifying
and sequencing the complete babesial 18S rRNA gene
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Primer
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Nucleotide sequence (5´→3´)
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Annealing temperature (°C)
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BABSr*
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CTC CAA TCC CTA GTC GGC A
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60
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BOD*
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GTT ATT GAC TCT TGT CTT TAA
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53
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BDV*
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AAT ATT GAC TGA TGT CGA GAT
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53
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CRYPTO F
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AAC CTG GTT GAT CCT GCC AGT AGT CAT
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60 (6)
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PIRO A*
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AAT ACC CAA TCC TGA CAC AGG G
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(7)
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PIRO B*
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TTA AAT ACG AAT GCC CCC AAC
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(7)
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PIRO C
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GTT GGG GGC ATT CGT ATT TAA
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60
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1055 F
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GGT GGT GCA TGG CCG
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60
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1055 R
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AAC GGC CAT GCA CCA C
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60
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1200F
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CAG GTC TGT GAT GCC
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60
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1200 R
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GGG CAT CAC AGA CCT G
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60
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CRYPTO R
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GAA TGA TCC TTC CGC AGG TTC ACC TAC
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60 (6)
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*Primers used for nested PCR.
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Suggested citation
for this article:
Duh D, Petrovec M,
Bidovec A, Avsic-Zupanc T. Cervids as babesiae hosts, Slovenia. Emerg
Infect Dis [serial on the Internet]. 2005 Jul [date cited]. Available
from http://www.cdc.gov/ncidod/EID/vol11no07/04-0724.htm
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