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Journal of Clinical Microbiology, November 2001, p. 4204-4207, Vol. 39, No. 11
Onderstepoort Veterinary Institute,
Onderstepoort 0110, South Africa
Received 2 April 2001/Returned for modification 23 July
2001/Accepted 2 September 2001
DNA samples from dogs presenting with symptoms suggestive of canine
ehrlichiosis, but with no morulae detected on blood smears, frequently
failed to give a positive reaction with a North American Ehrlichia canis-specific PCR assay targeting the 16S
rRNA gene. We suspected the presence of a pathogen genetically
different from North American E. canis,
and we performed experiments to test this hypothesis. DNA from one
canine blood sample was subjected to PCR with primers designed to
amplify Ehrlichia (Cowdria)
ruminantium ruminantium 16S and
map1 genes. Amplicon sequencing yielded 16S and
map1 sequences which were more closely related to other
E. ruminantium sequences than to those of
any other Ehrlichia species. Fifty canine DNA samples
were subjected to a PCR assay, previously found to be
Cowdria-specific, which targets the pCS20 gene.
Thirty-seven (74%) gave a positive signal, and 16 (32%) also gave
visible amplicons after gel electrophoresis, suggesting that this
E. ruminantium organism is common in the
Pretoria-Johannesburg area. The organism has not been isolated in
culture, so we cannot definitively state that it was responsible for
the canine ehrlichiosis symptoms, although the occurrence of several
similar cases suggests this to be so. Most importantly, we also do not
yet know whether the organism is infective for, or causes heartwater
in, ruminants.
Canine ehrlichiosis is a disease
syndrome common and widespread in dogs in tropical and subtropical
regions. Animals infected only with Ehrlichia canis may not
exhibit acute symptoms (12), but hemorrhagic forms of the
disease may be fatal (14). In South Africa, dogs
frequently become coinfected with both Babesia canis and
E. canis. Infection with the former species is
usually readily recognized by the presence of piroplasms in blood
smears, but ehrlichial morulae are usually difficult to detect because
of low parasite numbers (10). If there are no acute
symptoms, then anorexia and generalized debility, coupled with
characteristic but nonspecific hematological changes, are taken to be
suggestive of ehrlichiosis. Various serological tests are available for
the diagnosis of canine ehrlichiosis (13), but
cross-reactions with other Ehrlichia species preclude
definitive diagnosis. Cell culture isolation of the parasite is both
sensitive and specific (15), but it requires 1 to 4 weeks
before results are obtained, so it cannot be used for routine
diagnosis. A highly specific and sensitive PCR assay for E. canis, based on the 16S sequence of the Louisiana isolate of
E. canis, has been developed in the United States
(18). This test is used at Onderstepoort to confirm the
presence of E. canis in dogs with clinical
symptoms typical of ehrlichiosis but without morulae on blood smears.
Very few of the samples tested yielded a positive result.
The genus Ehrlichia (Fig. 1)
comprises a group of organisms of which the classification has recently
been revised (11). The genus includes several recently
identified organisms: the human pathogen Ehrlichia
chaffeensis (24); the dog pathogen Ehrlichia
ewingii (5), later found to also infect humans
(7); and the mouse pathogen Ehrlichia muris
(26). The genus includes Ehrlichia
(Cowdria) ruminantium, which was originally
thought to be a single species which causes heartwater in ruminants
(8). It is now known that E. ruminantium comprises a clade (Fig. 1) of several closely
related organisms (3), some of which may not cause
heartwater and which may therefore need to be reclassified when more
information is at hand. The growing number of new species being
recognized in the genus Ehrlichia led us to propose that the
dogs which presented with symptoms suggestive of canine ehrlichiosis, but which were negative for E. canis Louisiana,
were carrying a previously unrecognized pathogen. To test this
hypothesis, we amplified and probed for 16S genes from blood samples of
50 such dogs.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.4204-4207.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Novel Ehrlichia Genotype Detected in
Dogs in South Africa
ABSTRACT
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FIG. 1.
Maximum likelihood phylogenetic tree of some ehrlichial
species based on comparison of 16S ribosomal RNA genes. The
phylogenetic position of the canine E.
ruminantium is not differentiated from that of
E. ruminantium Mara 87/7. Abbreviations:
E., Ehrlichia; A.,
Anaplasma; N.,
Neorickettsia; and R.,
Rickettsia.
The samples used in this study came from several sources. Many were from apparently healthy animals held in quarantine kennels, which were the subject of routine testing after importation into South Africa or which required clearance certificates for export. Other samples came from animals presenting with clinical symptoms of ehrlichiosis but with no evidence of Ehrlichia morulae, either from the University of Pretoria Veterinary Faculty's companion animal clinic or from private veterinary clinics in Pretoria. The samples from the kennels and from the companion animal clinic had been tested and found negative using the E. canis Louisiana-specific PCR test (18).
DNA was purified from canine blood using the QIAamp DNA mini kit
(Qiagen, Hilden, Germany) according to the manufacturer's protocol and
was eluted from the columns with 100 µl of the proprietary elution
buffer supplied with the kit. Using primers (Table
1) which amplify the V1 hypervariable
region of Ehrlichia 16S genes (3) and a
protocol described elsewhere (2), a PCR (50-µl reaction
volume) was carried out on 5-µl aliquots of each sample. Positive
controls consisted of cloned, near full-length 16S genes of organisms
of South African origin related to E. canis.
These were Anaplasma sp. strain Omatjenne (previously
Ehrlichia sp. strain Omatjenne), Ehrlichia sp.
strain Germishuys, and the Welgevonden isolate of
E. ruminantium (9). Amplification
products were slot blotted onto nylon membranes (Hybond N+; Amersham
International) and probed with radioactively labeled oligonucleotides
(Table 2) which detect (i) any
Anaplasma species, (ii) any Ehrlichia sp. other
than E. ruminantium, and (iii) E. ruminantium only (3). Fifty samples were examined,
and none gave a signal with the probes for any Anaplasma sp.
or any Ehrlichia sp. other than E. ruminantium. Thirty six (72%) gave a positive signal with
the E. ruminantium-specific 16S probe
(3).
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A PCR assay which targets the pCS20 gene, and which has previously been found to be Cowdria-specific (17), was performed. Visible amplicons of the expected size were obtained from 16 (32%) of the samples, indicating a relatively high level of target DNA, whereas a total of 37 (74%) samples gave positive hybridization signals with the pCS20 probe.
One DNA sample, which gave strong signals with both pCS20 and E. ruminantium 16S probes, was amplified using primers (Table 1) to obtain a near full-length 16S gene. Amplicons of the appropriate size (~1,400 bp) were gel purified and cloned into pGEM-T according to the manufacturer's protocol (pGEM-T Vector Systems; Promega Corporation, Madison, Wis.), and transformants were screened for E. ruminantium 16S recombinants using the E. ruminantium-specific probe (Table 2). Positive recombinants were sequenced, and the sequence of the canine Ehrlichia 16S gene was deposited in GenBank.
The DNA sample noted in the previous paragraph was also amplified with degenerate primers (Table 1) which amplify the polymorphic map1 gene of any E. ruminantium strain. A band of ~870 bp, the expected size of the coding region of the map1 gene, was excised from the gel and cloned into pGEM-T, and transformants were screened for map1 recombinants using an E. ruminantium Welgevonden map1 probe (Table 2). Positive recombinants were sequenced from both ends, and the sequence of the canine Ehrlichia map1 gene was deposited in GenBank.
Alignments of the new 16S and map1 sequences were carried out with a range of published homologues using CLUSTALW (22) and manually adjusted using the SEQLAB multiple alignment program (version 10-1; GCG). A maximum likelihood phylogeny of the MAP1 derived amino acid sequence was inferred using the PROTML program from the MOLPHY (version 2.3) package (1), and the 16S phylogeny was inferred using the FastDNAML algorithm (20). Phylogenetic trees were drawn using "njplot" (21).
The canine Ehrlichia 16S gene sequence was identical over
the hypervariable V1 loop with that of the Mara 87/7 isolate of E. ruminantium (4), but elsewhere in
the sequence were single nucleotide polymorphisms which differentiated
this canine isolate from E. ruminantium Mara 87/7
in the 16S phylogenetic tree (Fig. 1). These 16S sequence data, and the
E. ruminantium-specific pCS20 probe hybridization
information (4), show that the organism detected in 37 of
the 50 dogs is phylogenetically E. ruminantium and differs from E. canis Louisiana. The
map1 gene sequence differed by 1 bp from, and the deduced
MAP1 sequence was identical to that of, a Ghanaian E. ruminantium isolate (Pokoase). The new canine isolate, as
well as E. ruminantium Pokoase, clustered with
E. ruminantium Senegal in the inferred MAP1
phylogeny (Fig. 2).
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Of 17 samples from dogs presently held in quarantine, 2 were from Ethiopia, 2 were from Mali, 3 were from Uganda, and 2 were from Kenya. Eight of these dogs were pCS20 probe positive, suggesting that they had acquired an E. ruminantium infection before their arrival in the country. Other workers have shown that dogs infected experimentally with E. ruminantium Crystal Springs did not become sick but showed visible pCS20 amplicons for up to 3 weeks after infection (16). This indicates that, although some dogs may show symptoms suggestive of ehrlichiosis, apparently healthy dogs may be asymptomatic carriers of E. ruminantium.
This E. ruminantium organism has not been isolated in culture or tested for its infectivity to dogs. Until this has been done, we cannot state definitively that E. ruminantium was the cause of the illness in the animal from which the 16S and map1 sequences were derived. It is known that several closely related organisms comprise the clade known as E. ruminantium (Fig. 1) and that some of them may not cause heartwater, even in ruminants (3). The finding of a genotype of E. ruminantium which infects dogs further emphasizes the multiplicity of organisms which molecular techniques have been able to reveal in the diverse genus Ehrlichia.
This E. ruminantium organism infecting dogs must be established in culture before its pathogenicity and host specificity can be determined, and the vector must also be identified. If it is found to be confined to canids, it cannot be described as E. ruminantium and should receive a different specific name. If, on the other hand, it is found to cause heartwater symptoms in ruminants, and/or is transmittable by Amblyomma species ticks, then dogs carrying the organism could represent a potential reservoir of heartwater.
Nucleotide sequence accession numbers. The sequence of the canine Ehrlichia 16S gene was deposited in GenBank under accession no. AF325175, and the sequence of the canine Ehrlichia map1 gene was deposited in GenBank under accession no. AF325176.
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ACKNOWLEDGMENTS |
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We thank S. W. Vogel for dog blood samples from private veterinary clinics and A.-M. Bosman for canine DNA samples.
This research was funded by the Agricultural Research Council of South Africa.
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FOOTNOTES |
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* Corresponding author. Mailing address: Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, South Africa. Phone: 27 12 5299205. Fax: 27 12 5299431. E-mail: maria{at}ovisun.ovi.ac.za.
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Journal of Clinical Microbiology, November 2001, p. 4204-4207, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.4204-4207.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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