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Journal of Clinical Microbiology, June 1998, p. 1501-1511, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Production and Characterization of Ehrlichia
risticii, the Agent of Potomac Horse Fever, from Snails
(Pleuroceridae: Juga spp.) in Aquarium Culture and Genetic
Comparison to Equine Strains
Gerhard H.
Reubel,
Jeffrey E.
Barlough, and
John E.
Madigan*
Department of Medicine and Epidemiology,
School of Veterinary Medicine, University of California, Davis,
California 95616
Received 17 December 1997/Accepted 24 February 1998
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ABSTRACT |
We report on the production and characterization of Ehrlichia
risticii, the agent of Potomac horse fever (PHF), from snails (Pleuroceridae: Juga spp.) maintained in aquarium culture
and compare it genetically to equine strains. Snails were collected from stream waters on a pasture in Siskiyou County, Calif., where PHF
is enzootic and were maintained for several weeks in freshwater aquaria
in the laboratory. Upon exposure to temperatures above 22°C the
snails released trematode cercariae tentatively identified as virgulate
cercariae. Fragments of three different genes (genes for 16S rRNA, the
groESL heat shock operon, and the 51-kDa major antigen)
were amplified from cercaria lysates by PCR and sequenced. Genetic
information was also obtained from E. risticii strains from
horses with PHF. The PCR positivity of snail secretions was associated
with the presence of trematode cercariae. Sequence analysis of the
three genes indicated that the source organism closely resembled
E. risticii, and the sequences of all three genes were
virtually identical to those of the genes of an equine E. risticii strain from a property near the snail collection site. Phylogenetic analyses of the three genes indicated the presence of
geographical E. risticii strain clusters.
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INTRODUCTION |
Potomac horse fever (PHF), also
called equine monocytic ehrlichiosis, is an important disease of horses
caused by a monocytotropic rickettsia, Ehrlichia risticii
(17-21, 24, 29). It was first observed in 1979 on pastures
along the Potomac River in Maryland (18) and has since been
identified in a number of other states and in Europe (24).
Clinical signs include anorexia, lethargy, variable fever, and
diarrhea; laminitis is a complication in a significant percentage of
animals. Fatalities may result if severely affected horses are not
treated promptly with fluid and antibiotic therapy.
The means by which horses become infected with E. risticii
has remained a mystery (17). There is no evidence for the
spread of the disease by arthropod vectors such as ticks (5, 15, 24-26, 33). It appears, rather, that cases of E. risticii infection are associated with riverine and other aquatic
habitats and hence with potential aquatic vectors. E. risticii-caused diarrhea in horses is enzootic in northern
California and southern Oregon and is known locally as the "Shasta
River crud" (SRC) or "ditch fever," owing to its association with
pastures bordering rivers and irrigation ditches (5, 19).
The hypothesis associating E. risticii with an aquatic
environment is supported by recent phylogenetic studies that
demonstrated a close phylogenetic relationship between E. risticii and three other rickettsiae associated with aquatic
habitats: Neorickettsia helminthoeca, the agent of "salmon poisoning," a frequently fatal enteric disease of canids; the SF
agent, isolated in Japan from trematode metacercariae parasitic on gray
mullet fish; and Ehrlichia sennetsu, the agent of human sennetsu ehrlichiosis in Japan and Malaysia (13, 14, 27, 28,
39). Together these four agents form a distinct cluster or
genogroup separate from the other rickettsiae (9, 28, 30,
39).
This phylogenetic relationship and the association between PHF and
aquatic habitats focused our attention on potential aquatic vectors
that might be involved in the epizootiology of E. risticii in northern California and southern Oregon. We have since amplified fragments of three different E. risticii genes from
operculate snails of the genus Juga (4). The
fragments were virtually identical to the homologous genes of the SRC
agent, an E. risticii strain isolated from a horse residing
only a few miles from the snail collection site (19).
Here we describe the PCR-based detection of E. risticii
genes in trematode cercariae released by operculate snails of the genus
Juga. The snails were collected from stream waters on a pasture in Siskiyou County, Calif., when PHF is enzootic and were maintained for several weeks in freshwater aquaria in our laboratory. We hypothesize that trematodes that use operculate snails as
intermediate hosts may be involved in the life cycle of E. risticii in northern California. We also compare genetic
information obtained from snail-derived E. risticii with
that from E. risticii from horses with PHF. We conclude that
certain E. risticii strains obtained from horses are
identical to those obtained from snail secretions and that geographical
clusters of genetically polymorphic E. risticii strains are
present. This information may have practical implications for current
vaccination strategies.
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MATERIALS AND METHODS |
Snail collection.
Freshwater snails were collected in August
1997 from a pasture in Weed, Siskiyou County, Calif.; some horses
residing on that pasture had a history of PHF. Snails were collected by
hand or with a net from the clear, shallow margins of a stream that
flows through the pasture and that is accessible to horses for
drinking. The stream waters are fed by the nearby Shasta River. A total of about 400 pleurocerid snails of the genus Juga
(6) were collected and transported in chilled stream water
(10 to 12°C) to our laboratory at the University of California,
Davis. On the basis of the current nomenclature derived from classical
shell characters, the majority of the individuals resembled the species Juga hemphilli hemphilli, having ribs (costae) for the most
part limited to the apical whorl of the shell. Recognizing, however, that the systematics of the family Pleuroceridae are in need
of thorough revision (6, 8), identification of these snails to the species level remains presumptive.
Aquarium culture.
In the laboratory, the snails were rinsed
briefly with tap water and were distributed into three freshwater
aquaria (10 gal each). The floor of each tank was covered with washed
sand pebbles and the tanks were filled with tap water (pH 8.1) treated
with 5 ml of water conditioner (Amquel; Kordon, Hayward, Calif.) per 10 gal to eliminate possible ammonia, chloramine, and chlorine contamination. The water tested negative for copper, which is known to
be lethal for snails. The lids of the tanks were fitted with daylight
lamps that were used for about 8 to 10 h each day. Two tanks with
75 snails each were kept at room temperature (RT; about 22 to 24°C),
and one tank with about 250 snails was kept at 8°C. The larger snails
(about 2.0 to 2.5 cm) were kept at RT. The sizes of snails maintained
at 8°C ranged from about 0.5 to 1.5 cm. Snails were fed alga pellets
and fish flakes ad libitum. The tank water was continually filtered
through activated carbon filters (Whisper Power Filter; Tetra Sales,
Blacksburg, Va.) and was replaced weekly with fresh tap water. The
filters were replaced weekly as well.
Source and preparation of E. risticii strains used
for phylogenetic comparison.
Peripheral blood leukocytes (PBLs)
were obtained from 10 horses clinically diagnosed with PHF and were
used as the source of template DNA for PCR. Five horses (Buck, Bunn,
Danny, Tate, and Thorenberg) were from Klamath Falls, Oreg. Three
horses (Doc, Dr Pepper, and Ms Annie) were from three locations (Horse
Creek, Weed, and Montague, respectively) in northern California. One horse (Eclipse) was from Elizabethville, Pa., and one horse (Mostly Memories) was from Richland, Mich. One additional horse (Shotgun), from
Mount Shasta City, Calif., had previously been reported to be a source
of the SRC agent (19). PBL lysates were prepared as
described elsewhere (3, 5). Briefly, 10 ml of venous blood
collected into tubes containing acid citrate dextrose were centrifuged
at 700 × g for 5 min, and the buffy coat cells were removed and frozen overnight at 
20°C. Erythrocytes were lysed with
0.2% NaCl, and the buffy coat cells were washed three times with
phosphate-buffered saline. The buffy coat cells were pelleted and lysed
for 3 h at 56°C in 100 µl of lysis buffer (10 mM Tris hydrochloride [pH 8.3], 0.45% Nonidet P-40, 0.45% Tween 20, 100 µg of proteinase K per ml). The proteinase K was subsequently heat
inactivated for 15 min at 95°C. Three microliters of the cell lysate
was used as template for the PCR. Three E. risticii strains
obtained from snails were also used for phylogenetic comparison. Two
strains (the Shasta snail-1 [SHSN-1] and Shasta snail-2 [SHSN-2] strains) have been characterized previously (4). One strain (the Klamath Falls snail [KLSN] strain) originated from a pool of
lymnaeid snails, genus Stagnicola, collected in August 1996 near Klamath Falls, Oreg. DNA was extracted from the snails as described below.
Processing of snails, snail secretions, and tank water for
PCR.
For further investigation, three separate experiments were
performed. In experiment 1, snail secretions and tank water samples were obtained at different time points and were examined for the presence of E. risticii DNA by PCR. Snails were sampled on
days 2, 6, 7, 13, 14, 17, and 21 after snail collection. Secretions were collected with sterile pipet tips from the anterior shell aperture
of individual snails in the holding tanks, placed onto glass microscope
slides, and examined under a light microscope at ×200 to ×400
magnification for the presence of trematode cercariae. Thereafter, the
secretions were transferred from the slides into 2-ml microcentrifuge
tubes and spun at top speed in a microcentrifuge for 1 min, and the
supernatant was removed. Five hundred microliters of DNA lysis buffer
was added, and the pellets were lysed as described above. Three
microliters of the lysate was used for PCR without further DNA
extraction. Tank water was prepared at different time points (days 2, 8, and 13 after the beginning of the experiment) for PCR examination by
centrifuging various volumes (15, 70, 400 ml) at 1,500 × g for 20 min and subsequent lysis of the resulting pellet as
described above.
In experiment 2, selected snails were removed from the holding tanks,
rinsed for 2 min in running tap water, assigned to three groups of five
snails each, and kept in petri dishes (8-cm-diameter petri dishes
filled with 20 ml of tap water) for 3 days at different temperatures
(8, 22, and 37°C). At the end of the experiment, the snails and water
were frozen inside the petri dishes at 20°C for several days and
were then thawed at RT. The snails and water were separated and
processed as follows. The snails were dissected from their shells with
sterile scissors and placed into 2-ml microcentrifuge tubes, and the
tissues were mechanically disrupted for 1 min on a BeadBeater (Biospec
Products, Bartlesville, Okla.). The tubes were spun at top speed in a
microcentrifuge for 1 min. Excess crude extract was removed until
approximately 1 ml remained in each microcentrifuge tube. The tubes
were filled with 1.0 ml of DNA extraction buffer (10 mM Tris [pH
8.0], 2 mM EDTA, 0.1% sodium dodecyl sulfate [SDS], 500 µg of
proteinase K per ml), vortexed well, placed in a heating block (56°C)
for 3 h, vortexed again, and then heated to 97°C for 15 min to
inactivate the proteinase K. The water from the petri dish was
centrifuged at 1,500 × g for 30 min, and the pellet
was resuspended in 1.5 ml of DNA extraction buffer and treated as
described above for the snail tissues. DNA was then extracted from
these samples by standard procedures (31). The DNA content
was checked with a UV spectrophotometer, and each sample was adjusted
so that it contained approximately 300 ng of DNA per µl. One
microliter was used per PCR mixture.
In experiment 3, a group of 60 snails was removed from the holding tank
(8°C) 10 weeks after collection from the field. The
snails were
rinsed for 2 min in running tap water, placed in a
beaker with 50 ml of
Amquel-treated tap water, and kept for 24
h at 29°C in a water
bath. Snail secretions were sampled at 0,
1, 2, 3, 4, 5, 6, 7, 8, and
24 h after the start of the experiment
and were microscopically
examined for the presence of cercariae.
One milliliter of the
secretions was collected at each time point
for PCR examination and was
processed as described above.
Scanning electron microscopy.
Snail secretions were obtained
as described above and centrifuged at 1,500 × g, and
the supernatant was removed. The remaining pellet was fixed overnight
at 4°C in modified Karnovsky's fixative (2.0% paraformaldehyde and
2.5% glutaraldehyde in 0.06 M Sorensen's phosphate buffer [pH 7.2])
and was attached to a 12-mm coverslip with 0.1% polylysine. The sample
was rinsed briefly in 0.1 M Sorensen's phosphate buffer and dehydrated
in a graded acetone series (50 to 100%), 10 min per step; the 100%
acetone step was repeated three times. The critical dry point was
achieved with bone-dry-grade liquid carbon dioxide. The specimen was
mounted on support stubs with silver suspension paste, coated with 5-nm
gold particles in a Polaron E5000 sputter coater, and viewed and
photographed in a Philips PSEM501 scanning electron microscope at 10 to
15 kV.
Nested PCR assays.
A nested PCR that amplifies a 5' segment
(527 bp) of the 16S rRNA gene of E. risticii was used as an
initial screen for the presence of E. risticii DNA in snail
secretions and in PBLs of horses. The components and conditions of this
PCR have been described in detail elsewhere (5). Current
cycling parameters were preheating at 94°C for 5 min and then 35 cycles of 94°C for 1 min, 60°C for 2 min, and 72°C for 1.5 min,
followed by a final extension at 72°C for 7 min. The PCR products
were visualized in ethidium bromide-stained 1.5% agarose minigels.
Segments of two additional ehrlichial genes were amplified by PCR. Sets
of nested primers were designed to detect portions
of the
E. risticii homolog of the
Escherichia coli groESL heat
shock operon gene (
35) and the
E. risticii 51-kDa
major antigen
gene (
10,
11,
37). Primer sequences for
amplifying the heat
shock operon were 5'-ACCAGGCTACCTCACAGGC-3'
and 5'-TTGACCCTCGCATCAATG-3'
(outer primers) and
5'-CACAAGTTGGTTCAATTTCTGC-3' and
5'-CCGAGATCTTCAACAGTAAGGC-3'
(inner primers). The amplified
sequence was entirely contained
within the
groEL portion of
the operon. Primer sequences for amplifying
the 51-kDa major antigen
gene were 5'-GGATCGATAACTGCGATGCT-3'
and
5'-ACCGGCCTGACCACTAAAG-3' for the outer primers and
5'-TCCTATAATGGCACCACTAGCG-3'
and
5'-CCATCCGCAGTAGAGTTTGAG-3' for the inner primers.
Predicted molecular sizes for the
groESL fragment were 823 bp (first-round product) and 526 bp (nested product). The nested
product comprised nucleotides 500 through 1025 of the operon (numbering
relative to that for GenBank accession no.
U96732). The predicted
sizes
for the 51-kDa major antigen gene fragment were 818 bp (first-round
product) and 569 bp (nested product). The nested product comprised
nucleotides 1303 through 1871 of the gene (numbering relative
to that
for GenBank accession no.
U85784). Components and conditions
of the PCR
assays for
groESL and the 51-kDa major antigen genes
were
similar to those for the standard 16S rRNA gene amplification,
except
that the annealing temperatures varied from 45 to 55°C.
Cloning and sequencing of amplified PCR products.
For the
majority of the equine E. risticii strains and for the
strain obtained from the Klamath Falls snails, a 5' segment of the 16S
rRNA gene was amplified by nested PCR with primers ER-3
(5'-ATTTGAGAGTTTGATCCTGG-3') (5, 7) and PC-5
(5'-TACCTTGTTACGACTT-3') (40) for the first round
and primers ER-3 and ER-2 (5'-GTTTTAAATGCAGTTCTTGG-3') (5) for the second round. Nearly complete sequences of
the 16S rRNA gene were obtained from Juga snail secretions
and from two of the equine E. risticii strains (the strains
from horses Bunn and Eclipse) by amplifying and cloning the gene in
three overlapping fragments. The majority of the gene (ca. 1,440 bp) was amplified in the first round with primers ER-3 and PC-5. In the
nested round the 5' segment of the gene was amplified with primers ER-3
and ER-2; the middle segment was amplified with primers ER-2a(R)
(5'-CCCGTAAGTTAGGTGTG-3') and ER-X
(5'-CATCTCACGACACGAGC-3'), and the 3' segment was amplified
with primers ER-Y (5'-CCAACACAGGTGTTGC-3') and ER-Z2
(5'-ACCCCAGTCACCCACCCC-3'). Cycling conditions were as
described above for the standard nested PCR, except that annealing was
performed at 52°C and the 72°C extension was lengthened to 2 min.
The PCR products of the 16S rRNA,
groESL, and 51-kDa major
antigen genes were purified by spin chromatography (PCR SELECT-II
spin
columns; 5'
3' Inc., Boulder, Colo.) and cloned with the
pNoTA/T7
shuttle vector and competent
E. coli (Prime PCR Cloner
Cloning System; 5'
3' Inc.). Double-stranded DNA was isolated
with a
PERFECTprep Plasmid DNA Preparation Kit (5'
3' Inc.). One
microgram
of plasmid DNA was used for restriction endonuclease
digestion with
BamHI (New England Biolabs, Beverly, Mass.) to
verify insert
sizes. Inserts were sequenced with the universal
M13 forward and
reverse sequencing primers present in the vector.
Sequencing was
performed with a fluorescence-based automated sequencing
system
(Applied Biosystems, Foster City, Calif.). The sequences
of both DNA
strands were determined for all inserts.
Analysis of sequence data.
The sequences were subjected to
BLAST analysis (1) of GenBank nucleic acid sequences for
similarity rank, percent identity, and deduced amino acid sequences
(the last one for the groESL and 51-kDa genes only).
Multiple sequence alignments were made with ClustalW (36)
and in some cases with GeneWorks (IntelliGenetics Inc., Mountain View,
Calif.). Phylogenetic analyses were performed by the DNA maximum
likelihood method (DNAML) of PHYLIP (12), which allows
unequal expected frequencies of the four nucleotides, with the
frequencies determined empirically from those present in the sequences
analyzed, and unequal rates of transitions and tranversions. A single
rate of change was assumed for all sites. Phylogenetic trees based on
these analyses were generated by Treeview (PHYLIP) (12).
Nucleotide sequence accession numbers.
Most 16S rRNA gene
sequences were obtained from the GenBank database and have the
following accession numbers: E. risticii Illinois (type
strain), M21290; E. sennetsu, M73225; E. canis,
M73221; E. chaffeensis, U23503; E. equi, M73223; human granulocytic ehrichiosis agent, U02521; SHSN-1, AF037210; and
SHSN-2, AF037211. The sequences for the Kentucky, Ohio 081, and SRC
strains of E. risticii were derived from published sources
(19, 38). The groESL (35) and 51-kDa
major antigen (11) gene sequences of E. risticii
(the strain from a horse in Maryland) (16) had GenBank
accession nos. U96732 and U85784, respectively. The GenBank accession
numbers of other relevant groESL sequences were as follows:
E. risticii, U24396; E. sennetsu, U88092;
E. chaffeensis, L10917; SHSN-1, AF037212; SHSN-2, AF037213;
and the SRC agent, AF037214. The GenBank accession numbers for the
51-kDa major antigen gene sequences of SHSN-1, SHSN-2, and the SRC
agent were AF037215, AF037216, and AF037217, respectively.
The sequences of strains from horses and snails newly reported in this
paper have been assigned the following GenBank accession
numbers: for
the 16S rRNA gene fragments, Buck,
AF036648; Bunn,
AF036649; Danny,
AF036650; Doc,
AF036651; Dr Pepper,
AF036652; Eclipse,
AF036653; Juga,
AF036654; KLSN,
AF036655;
Mostly Memories,
AF036656; Ms Annie,
AF036657; Tate,
AF036658;
and Thorenberg,
AF036659; for
groESL heat shock operon fragments,
Bunn,
AF036660; Danny,
AF036661; Doc,
AF036662; Dr Pepper,
AF036663; Eclipse,
AF036664; Juga,
AF036665; KLSN,
AF036666;
Mostly Memories,
AF036667; Ms Annie,
AF036668; Tate,
AF036669;
and Thorenberg,
AF036670; and for 51-kDa
major antigen gene
fragments, Doc,
AF036671; Dr Pepper,
AF036672;
Eclipse,
AF036673; Juga,
AF036674; Ms Annie,
AF036675; and Thorenberg,
AF036676.
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RESULTS |
Aquarium culture.
Snails collected from stream waters
readily adapted to the artificial situation in the aquarium. Within a
few hours many of the snails attached with their foot to the glass
walls of the aquarium as well as to rocks and pebbles. The two
tentacles arising from the base of the head (proboscis) were slowly
undulating, and both proboscis and foot protruded from the shell. The
majority of the snails kept in water at a temperature of 8°C remained
alive for more than 10 weeks after collection. In RT water most snails survived for as long as 3 weeks, with only a few surviving up to 5 weeks. Metabolism of snails, as measured by the amount of feces and
other secretions produced, was visibly higher at RT than at 8°C. The
carbon-activated filters in the tanks kept at RT clogged more easily
and had to be replaced weekly to prevent clouding of the water, whereas
the filter in the tank kept at 8°C had to be replaced at 3-week
intervals.
Light microscopic examination.
After several hours in RT
water, snails frequently released tubiform feces and other cloudy,
white secretions from their orifices. Microscopic examination of both
types of secretions showed various numbers of rapidly motile,
sperm-like organisms of about 0.1 to 0.15 mm in length (Fig.
1A). The organisms had a characteristic tail with a dorsoventral finfold that was shorter than the body and
that was used at high revolutions for locomotion. The body had a
bilobed virgula organ located in the region of the oral sucker. A
smaller ventral sucker was also visible. On the basis of morphology and
behavior, the cercariae were tentatively identified as virgulate
cercariae (32). Cercariae were readily seen for about 7 days
in secretions from snails maintained in water at RT and in concentrated
and nonconcentrated RT water samples (experiment 1). Some cercariae
were also observed in water secretions of experiment 2 when the snails
were kept at RT. No cercariae were observed in experiment 2 in the
secretions of snails kept at 8 and 37°C or in experiment 3 in any of
the samples. Cercariae were also not observed in secretions of snails
kept in the holding tank at 8°C. After about 7 days, cercariae were
no longer observed in either secretions or water samples from tanks
kept at RT.
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FIG. 1.
Light-microscopic (A) and scanning electron microscopic
(B) pictures of a virgulate cercaria released into aquarium water by
pleurocerid snails of the genus Juga. Bar, 0.01 mm.
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Scanning electron microscopy.
A scanning electron micrograph
of a cercaria is shown in Fig. 1B. The tail with the dorsoventral
finfold and the oral sucker containing a so-called stylet are clearly
visible.
PCR.
The results of the nested PCRs for the E. risticii 16S rRNA, groESL, and 51-kDa major antigen
genes are summarized in Table 1. The 5'
end of the 16S rRNA gene was readily amplified from all horse and snail
samples (15 of 15). With the exception of the sample from one horse
(Buck from Oregon), groESL gene fragments also were obtained
from all samples. The 51-kDa major antigen gene was amplified from 9 of
15 samples. No amplification of this gene fragment was obtained from
the Klamath Falls snail sample, samples from four horses from Oregon
(Bunn, Buck, Danny and Tate), and a sample from a horse from Michigan
(Mostly Memories).
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TABLE 1.
Results of nested PCRs for the 16S rRNA,
groESL, and 51-kDa major antigen genes of E. risticii with equine PBL- and snail-derived DNA as templates
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In snail experiment 1, the secretions from and the tank water of snails
maintained at RT were positive for all three genes
at days 2 (Fig.
2), 6, and 7 after the start of the
experiment
and were negative on days 8, 13, 14, 17, and 21 (data not
shown).
The snail tissues derived from experiment 2 were PCR negative
for the
E. risticii 16S rRNA gene. Secretions obtained from
snails
kept at RT and at 37°C were faintly PCR positive for the 16S
rRNA
gene; secretions obtained from snails kept at 8°C were negative
(experiment 2; data not shown). Secretions serially taken from
snails
kept at RT in experiment 3 were all PCR negative.
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FIG. 2.
Nested PCR of E. risticii for the 16S rRNA
(527 bp), groESL heat shock operon (526 bp), and 51-kDa
major antigen (569 bp) genes with DNA from Juga secretions
(lanes 1), concentrated tank water (lanes 2), and positive (lanes +)
and negative (lanes ) E. risticii DNA controls.  ,
X174 replicative-form DNA HaeIII digest (molecular size
marker).
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16S rRNA sequences.
BLAST searches with the 16S rRNA gene
sequences amplified from horse and snail samples consistently resulted
in 98 to 100% homology with corresponding fragments of the 16S rRNA
genes of E. risticii Illinois (data not shown). E. sennetsu was the next closest organism (about 1% less identity
than E. risticii Illinois), followed by N. helminthoeca with about 94% identity (data not shown). For the
Juga snail secretions and for samples from two horses
(Eclipse from Pennsylvania and Bunn from Oregon), the majority (1,251 bp) of the 16S rRNA gene was available for sequence comparison. The 16S
rRNA gene fragment amplified from Juga snail secretions was
identical to the homologous fragment of the SRC agent. It had one
nucleotide change each compared to the two sequences obtained for
operculate snails from Siskiyou County (4). Compared to the
type strain of E. risticii, strain Illinois, it had the same four nucleotide changes as the SRC agent. The 16S rRNA gene fragment amplified from a sample from Eclipse (Pennsylvania) was closely related
to the Illinois type strain of E. risticii (two nucleotide changes), whereas that from a sample from Bunn (Oregon) was
genotypically highly polymorphic (14 nucleotide changes). With the
exception of the fragment from a sample from Eclipse all other
sequences had the same four nucleotides changes (at positions 956, 1221, 1230, and 1246) in comparison to the sequence of the
Illinois type strain of E. risticii. The data are summarized
in Table 2.
For isolates from nine horses (Buck, Bunn, Danny, Tate, and Thorenberg
from Oregon; Doc, Dr Pepper and Ms Annie from California;
and Mostly
Memories from Michigan) and one snail pool (KLSN from
Oregon), partial
sequences (527 bp) from the 5' end of the 16S
rRNA gene were available
for comparison. The sequence of this
genomic region for isolates from
two horses in California (Ms
Annie and Dr Pepper) and one horse in
Oregon (Thorenberg) were
identical to that for the Illinois type strain
of
E. risticii;
isolates from two other horses in Oregon
(Danny and Tate) had
one nucleotide change, the isolate from one horse
in California
(Doc) had four nucleotide changes; the isolate from a
horse in
Michigan (Mostly Memories) had three nucleotide changes. The
E. risticii strains obtained from the Oregon snail sample
(KLSN)
and from a horse (Buck) residing in this area had four and five
nucleotide changes, respectively, relative to the sequence of
the type
strain, and these changes were identical to those seen
in
E. risticii Ohio 081. The data are summarized in Table
3.
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TABLE 3.
Nucleotide differences at the 5' end of the 16S rRNA
genes of 11 equine and one snail E. risticii strains
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groESL heat shock operon sequences.
Partial
nucleotide sequences of the groESL heat shock operon gene
were obtained from nine horse and two snail samples and were used for
BLAST searches. The groESL gene nucleotide sequences had
between 91 and 100% identity to the corresponding groESL
sequences of a Maryland strain of E. risticii
(35). The next closest related groESL sequence
was that from E. sennetsu (ranging between 89 and 96%
identity), followed by E. chaffeensis (about 65% identity) (Table 4). The deduced amino acid
sequences of groESL were compared to those for a reference
equine E. risticii strain (Maryland), the SRC agent
(19), and two previously characterized snail E. risticii strains (4) (Fig.
3). Most of the nucleotide mutations were
silent changes which resulted in very similar amino acid sequences for
all strains. The isolates from horses Danny, Doc, and Thorenberg had
one amino acid change each relative to the sequence of the Maryland
strain, while the amino acid sequence obtained from the Klamath Falls
snail had two changes.
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TABLE 4.
Percent nucleotide identities of E. risticii
groESL heat shock operon nucleotide sequences (526 bp) from nine
horses and two snails
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FIG. 3.
Alignment of the deduced amino acid sequences of
groESL heat shock operon gene fragments amplified from
E. risticii strains from 11 horses and four snails (the
snail designations are underlined). The sequences of an equine E. risticii strain (from a horse in Maryland; GenBank accession no.
U24396), the SRC agent (GenBank accession no. AF037214), and two
snail-derived E. risticii strains (SHSN-1 and SHSN-2;
GenBank accession nos. AF037212 and AF037213, respectively) are
included for comparison. Identical amino acids are represented by
periods; differences in sequence relative to the sequence of the strain
from a horse in Maryland are indicated by capital letters.
|
|
Sequences of the 51-kDa major antigen genes.
The nucleotide
sequences of the 51-kDa major antigen genes were more diverse than
those of the groESL heat shock operon and ranged from 92 to
99% nucleotide sequence identity and 90 to 97% amino acid sequence
identity (Table 5). The sequence of the
strain obtained from the Juga secretions was virtually
identical to those of two previously characterized operculate snail
strains collected in Siskiyou County (4), a strain from a
horse in California (Dr Pepper), and the equine SRC agent
(19) (Fig. 4). The sequence of
another strain from a horse in California (Ms Annie) was closely related to that of the strain from Juga secretions (one
amino acid change at position 149). The 51-kDa major antigen gene
sequences of the Maryland reference strain, the strain from a horse in
Pennsylvania (Eclipse), and two strains from horses in Oregon (Doc and
Thorenberg) differed from those of the rest of the strains. Two hot
spots of amino acid changes were apparent. The first was located at the
5' end of the examined sequence and revealed up to eight amino acid
changes compared to the sequences of strains from California. The
second hot spot was located around amino acid position 135 and revealed
up to seven amino acid changes. With five exceptions, the sequences of
the strains from horses in Maryland and Pennsylvania were identical.
The sequences of the strains from the horses Doc (California) and
Thorenberg (Oregon) appeared to be more closely related to each other
than to those of the other strains from horses in California and
Oregon. Common to both strains were several amino acid changes that
were not seen in the other strains. They also shared at position 139 one unique amino acid insertion which was not present in any of the
other strains. The strain from Doc was the most diverse strain, with 18 amino acid changes relative to the Maryland strain of E. risticii.
View this table:
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TABLE 5.
Nucleotide and amino acid sequence
identitiesa of E. risticii
51-kDa major antigen gene sequences (569 bp) from five horses and
one snail in comparison to the sequences of a Maryland equine
strainb of E. risticii
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FIG. 4.
Alignment of the deduced amino acid sequences of 51-kDa
major antigen gene fragments amplified from E. risticii
strains from seven horses and three snails (the snail designations are
underlined). The sequences of an equine E. risticii strain
(from a horse in Maryland; GenBank accession no. U24396), the SRC agent
(GenBank accession no. AF037217), and two snail-derived E. risticii strains (SHSN-1 and SHSN-2; GenBank accession nos.
AF037215 and AF037216, respectively) are included for comparison.
Identical amino acids are represented by periods, differences in
sequence relative to the sequence of the strain from the horse in
Maryland are indicated by capital letters; insertions are shown by
underlined italics.
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|
Phylogenetic analysis of the 16S rRNA gene.
The nucleotide
changes observed in the multiple alignments were reflected in the
outcome of the phylogenetic analysis. The phylogenetic relationship
among equine and snail E. risticii strains inferred from the
5' end sequences of the 16S rRNA genes was characterized by the
presence of two main clusters and one clear outlier. The sequence
divergence was generally very low, but a cluster consisting of an
Oregon snail strain (strain KLSN), an Oregon equine strain (from the
horse Buck), and the E. risticii Ohio 081 equine reference strain was observed. This cluster of strains was clearly remote from
the majority of the other strains (Fig.
5). The strain from the horse Bunn
(Oregon) showed the highest sequence divergence.
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FIG. 5.
DNA maximum-likelihood phylogram inferred from the
nucleotide sequences of 18 16S rRNA gene fragments (527 bp at the 5'
end) originating from E. risticii strains from 14 horses and
four snails (the snail designations are underlined). Four of the
sequences are for equine E. risticii reference strains:
E. risticii Illinois (type strain; GenBank accession no.
M21290), E. risticii Kentucky (38), E. risticii Ohio 081 (38), and the E. risticii
SRC agent (19). Two snail-derived E. risticii
strains (SHSN-1 and SHSN-2; GenBank accession nos. AF037210 and
AF037211, respectively) are also included.
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|
A similar picture was seen for those equine and snail
E. risticii strains for which the majority of the 16S rRNA genes were
available for analysis (Fig.
6). The
sequence divergence was again
very low, but the strain from horse Bunn
(Oregon) and the
E. risticii Ohio 081 strain were clearly
separated from the remainder of the
strains.
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FIG. 6.
DNA maximum-likelihood phylogram inferred from the
nucleotide sequences of nine 16S rRNA gene fragments (1,251 bp)
originating from E. risticii strains from six horses and
three snails (the snail designations are underlined). The divergence of
16S rRNA gene fragments from the strains from aquarium Juga
snails is compared to the divergence of two newly obtained equine
E. risticii strains from Oregon (from Bunn) and Pennsylvania
(from Eclipse) and to four equine E. risticii reference
strains (E. risticii Illinois [GenBank accession no.
M21290], E. risticii Kentucky [38],
E. risticii Ohio 081 [38], and the E. risticii SRC agent [19]). Two snail-derived
E. risticii strains (SHSN-1 and SHSN-2; GenBank accession
nos. AF037210 and AF037211, respectively) are also included.
|
|
Phylogenetic analysis of the groESL heat shock
operon.
The close similarity in the deduced amino acid sequences
among all E. risticii strains was reflected in a generally
tight phylogenetic clustering (Fig. 7).
However, three strains from horses in the eastern states (Eclipse from
Pennsylvania, the strain from a horse in Maryland, and Mostly Memories
from Michigan) formed a separate cluster. The strain from the Oregon
snail sample showed the highest sequence divergence and was, with
E. sennetsu, farther apart from the rest of the strains.
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FIG. 7.
DNA maximum-likelihood phylogram inferred from the
nucleotide sequences of 16 groESL heat shock operon gene
fragments originating from E. risticii strains from 11 horses and four snails (the snail designations are underlined) and from
an E. sennetsu reference strain (GenBank accession no.
U88092). groESL heat shock operon genes of a Maryland strain
of E. risticii (GenBank accession no. U24396), the SRC agent
(GenBank accession no. AF037214), and two snail-derived E. risticii strains (SHSN-1 and SHSN-2; GenBank accession nos.
AF037212 and AF037213, respectively) are included for comparison.
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|
Phylogenetic analysis of the 51-kDa major antigen gene.
The
fairly high nucleotide and amino acid sequence diversity among the
51-kDa major antigen genes was mirrored in their phylogenetic relationships (Fig. 8). Two strains from
horses in the eastern states (Eclipse from Pennsylvania and the strain
from a horse in Maryland) formed one group and the majority of the
California equine and snail strains formed another. Two strains from
horses (Doc from California and Thorenberg from Oregon) were separate from the rest of the strains and were on individual branches.
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FIG. 8.
DNA maximum-likelihood phylogram inferred from the
nucleotide sequences of 10 51-kDa major antigen gene fragments
originating from E. risticii strains from seven horses and
three snails (the snail designations are underlined). An equine
E. risticii strain (from a horse in Maryland; GenBank
accession no. U24396), the SRC agent (GenBank accession no. AF037217),
and two snail-derived E. risticii strains (SHSN-1 and
SHSN-2; GenBank accession nos. AF037215 and AF037216, respectively) are
included for comparison.
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|
| |
DISCUSSION |
Here we report the detection of E. risticii DNA in
secretions from snails that were collected in the field and that were
kept for several weeks in freshwater aquaria in the laboratory. For the
first time it was possible to examine snails as potential vectors for
the transmission of E. risticii and possibly other infectious organisms in a defined laboratory setting. The presence of
trematode virgulate cercariae in snail secretions was surprising but
not completely unexpected since many snail species are known to harbor
trematodes (32). Virgulate cercariae, for example, can
develop in operculate snails and are produced by trematodes of the
family Lecithodendriidae (32). However, the
association between the presence of cercariae and PCR positivity for
three E. risticii gene sequences in snail secretions is the
first description of a possible link between snails, trematodes, and
the epizootiology of PHF in horses.
For more than 15 years it has remained a mystery as to how E. risticii infection is spread among horses (17).
Transmission of the organism through arthropod vectors has been widely
considered but never experimentally proved (5, 15, 23-25,
32). The disease has, however, been associated from the very
beginning with riverine habitats (18), and it has frequently
been noted by veterinary clinicians that horses kept on dry lot in
areas where the disease is enzootic usually do not develop the disease. The hypothesis that freshwater snails are involved in the life cycle
and transmission of E. risticii would provide an explanation for these observations. The field-collected snails in our study readily
released cercariae upon exposure to water temperatures above 22°C.
This would help to explain the seasonality of the disease, with cases
often occurring on irrigated pastures after heat spells. Many
trematodes have life cycles with several intermediate hosts
(32). Among these is the trematode Nanophyetus
salmincola, the vector for N. helminthoeca, which
causes "salmon poisoning" disease in dogs (22).
Transmission of E. risticii might similarly be achieved with
other intermediate hosts, perhaps arthropod vectors such as ants or
beetles that feed on snail secretions. In this regard the observed
presence of darkling beetles and their larvae on farms where PHF is
enzootic might be noteworthy (24).
The ease by which E. risticii DNA was detected in secretions
from snails in aquaria is in contrast to our previous attempts to
amplify E. risticii DNA from snail tissues, which were
tedious and time-consuming and which resulted in the detection of only a few positive snails (4). By focusing on cercariae rather than whole snail tissues, the sensitivity of the PCR is likely to be
increased. This could explain why we were able to detect E. risticii DNA in secretions at a much higher rate than in snail pools or individual whole snails. Because the DNA from entire snails is
extracted, it is likely that dilution of rickettsial DNA occurs. The
E. risticii load in the cercariae was obviously high enough
for its DNA to be detectable by PCR with lysed secretions without the
need for DNA extraction.
The fact that cercariae were not observed in secretions of snails
maintained for several weeks at 8°C can be attributed to two facts.
First, most of the snails kept at this temperature were smaller than
those releasing cercariae. It is known for other trematodes that only
large snails (those with a size of 2 cm or larger) will harbor
cercariae (22). Second, the larger snails in the holding
tank might have released cercariae after the water was replaced with
fresh tap water (temperature, about 22°C). The fresh tap water may
have temporarily increased the water temperature to a level inducing an
unnoticed shedding of cercariae. We hypothesize that once cercariae are
released from the snails, new cercariae are no longer produced and a
new trematode life cycle (i.e., infection of the snail by miracidia)
must start before the production of cercariae can begin again.
A substantial portion of this study was concerned with analyses of
genome sequences obtained for strains from snail cercariae and strains
from horses with PHF. The E. risticii sequences from the
Juga snail secretions were nearly identical to those
obtained from operculate snails previously collected at the same site
as the aquarium snails (4) and to sequences derived from
strain from a horse with PHF residing in close vicinity to the snail collection site (19). We feel that the presence of such
highly similar genotypes in both species is sufficient evidence to
conclude that horses and snails harbor the same or very similar strains of E. risticii. This conclusion is supported by the fact
that strains from horses from different geographical areas such as the
eastern United States appeared to have different genotypes. It is well
documented that the antigenic profiles or restriction enzyme patterns
of E. risticii strains originating from different geographical areas are quite diverse (7, 37, 38).
Previous studies of the genetic diversity of E. risticii
strains used solely the 16S rRNA gene sequences for analysis (7, 37, 38). The sequences of this gene are known to vary in an orderly manner throughout the phylogenetic tree and hence represent desirable targets for PCR and phylogenetic analyses (40).
Differences in the 16S rRNA gene sequences of different strains of the
same species of rickettsial organisms are very small (0 to 0.1%)
(2, 34), which can sometimes make conclusive phylogenetic
analysis difficult. We conclude from the analysis of two additional
E. risticii gene sequences (the groESL heat shock
operon gene and the 51-kDa major antigen gene) that genetic diversity
is more extensive in genes coding for antigenic determinants that serve as targets for antibody- or cell-mediated immune selection. Further studies of the molecular epidemiology of E. risticii should
therefore focus on these or similarly diverse genes. Similar
suggestions have recently been made for other members of the
rickettsiae, such as Ehrlichia equi and Ehrlichia
phagocytophila (35).
The presence of geographical clusters of E. risticii strains
was evident from phylogenetic analyses of all three examined gene
fragments. However, the most profound differences were observed in the
51-kDa major antigen genes. The 51-kDa major antigen sequences of
strains from the Shasta and Juga snails, the SRC agent, and strains from the horses Dr Pepper and Ms Annie were virtually identical. These strains all originated in the same geographical area
with the same river drainage (Shasta) and the same ecosystem. The
strains from horses in Pennsylvania and Maryland are geographically associated as well and share significant genotypic homology to each
other. A close association between the strains from the horses Thorenberg and Doc was noticed; both strains came from the same geographical area (the drainage here is the Klamath River). We were not
able to amplify 51-kDa gene sequences from a few E. risticii strains that were more divergent (geographically clustered), even though PCR conditions (for example, the primer annealing stringency) were modified. We consider this to be additional evidence for the
widespread genetic diversity of E. risticii, which may be a
major factor in recognized vaccine failures (7, 23, 37).
The known geographical distribution of Juga spp. encompasses
northern California, northern Nevada, Oregon, and Washington (6), an area similar to that of the pleurocerid host of
N. salmincola. If snails have a role in the life cycle of
E. risticii in other areas of the United States, different
snails must certainly be involved. Preliminary evidence in this regard
is the successful amplification of two E. risticii gene
fragments from a pool of lymnaeid snails, genus Stagnicola.
It is possible that strains of ehrlichiae causing PHF may eventually be
found in a variety of snail genera, a host range that may be reflected
in the genetic and antigenic variation observed among E. risticii isolates. Thus, the "agent" of PHF may in actuality
represent an array of closely related ehrlichial strains differing in
their particular snail hosts and vectors and perhaps in their virulence
for horses as well.
The information derived from this study should aid future
investigations of snails as vectors of disease-causing organisms and
greatly simplify the detection of E. risticii in these
mollusks. The cost-effectiveness of aquarium culture versus the
collection of large numbers of snails with subsequent laborious DNA
extraction is an important factor for consideration. Similar procedures
might also be applied to other suspected cercaria-transmitted
infections.
We conclude by emphasizing that cercarial transmission of E. risticii infection remains a hypothesis worthy of further testing. Because of a ubiquitous bacterial flora, the isolation of E. risticii directly from snail secretions appears to be difficult.
An alternative method would be to attempt experimental transmission of
E. risticii to horses through snail-derived cercariae, with
subsequent isolation of the organism from peripheral blood cells. It
will be interesting to see whether results similar to ours are obtained
with snail species from other areas of the country where PHF is known
to occur.
| |
ACKNOWLEDGMENTS |
We thank Stacia Hoover and Eric Bowman for nucleotide sequencing;
Mike Ronne, Jon Goodell, Paul Miller, Tom Sampson, Amy Finken, and Hal
Schott for providing equine blood specimens; and Larisa Vredevoe,
Elfriede DeRock, and Inderpal Kaur W. Singh for generous help in
collecting snails. We thank Bob Munn for electron microscopy, Carlos
Munos for valuable information regarding snail husbandry, Stewart
Schell for advice on cercarial identification, Steve Holloway for help
with phylogenetic analyses, and Yasuko Rikihisa for valuable discussions and inspiration. The support of Nancy East is greatly appreciated.
This work was supported by grants from the Center for Equine Health,
University of California, Davis, with funds provided by the Oak Tree
Racing Association, the State of California satellite wagering fund,
and contributions from private donors, and by discretionary funds from
the Department of Medicine and Epidemiology, School of Veterinary
Medicine, University of California, Davis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: School of
Veterinary Medicine, University of California, Davis, CA 95616. Phone:
(530) 752-6513. Fax: (530) 752-0414. E-mail: jemadigan{at}ucdavis.edu.
| |
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Journal of Clinical Microbiology, June 1998, p. 1501-1511, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
