Molecular and Biochemical Parasitology, 54 (1992) 165 174 165

© 1992 Elsevier Science Publishers B,V. All rights reserved. / 0166-6851/92/$05.00

MOLBIO 01784

Genetic variants within the genus Echinococcus identified by

mitochondrial DNA sequencing
J o s e p h i n e Bowles a, D a v i d Blair b a n d D o n a l d P. M c M a n u s a
aTropical Health Program, Queensland Institute of Medical Research, Brisbane, Australia; and
bDepartment of Zoology, James Cook University, Townsville, Australia
(Received 2 January 1992; accepted 16 April 1992)

The pattern of species and strain variation within the genus Echinococcus is complex and controversial. In an attempt to
characterise objectively the various species and strains, the sequence of a region of the mitochondrial cytochrome c oxidase
subunit I (COl) gene was determined for 56 Echinococcus isolates. Eleven different genotypes were detected, including 7
within Echinococcus granulosus, and these were used to categorise the isolates. The 4 generally accepted Echinococcus species
were clearly distinguishable using this approach. In addition, the consensus view of the strain pattern within E. granulosus,
based on a variety of criteria of differentiation, was broadly upheld. Very little variation was detected within Echinococcus
multilocularis. Remarkable intra-strain homogeneity was found at the D N A sequence level. This region of the rapidly evolving
mitochondrial genome is useful as a marker of species and strain identity and as a preliminary indication of evolutionary
divergence within the genus Echinococcus.

Key words: Echinococcus; Intra-specific variation; Mitochondrial DNA; Cytochrome c oxidase I; Direct sequencing;
Polymerase chain reaction

Introduction Considerable intra-specific or 'strain' [2] varia-

Of the 16 species which have been described
within the genus Echinococcus (causative agent
of hydatid disease) only 4, Echinococcus
granulosus, Echinococcus multilocularis, Echi-
nococcus vogeli and Echinococcus oligarthrus,
are now recognised as taxonomically valid [1].
Correspondence address: D.P. McManus, Tropical Health
Program, Queensland Institute of Medical Research, 300
Herston Rd, Brisbane, Queensland 4029, Australia. Tel.:
+ 61-7-3620401; Fax: + 61-7-3620104.
Note: Nucleotide sequence data reported in this paper have
bgen submitted to the GenBank T M data base with the accession
numbers M84661 to M84671.
Abbreviations." PCR, polymerase chain reaction; COl, cyto-
chrome c oxidase subunit 1; G, E. granulosus; M, E.
multilocularis; V, E. vogeli; 0, E. oligarthrus; RFLP, restriction
fragment length polymorphism; rDNA, ribosomal DNA;
mtDNA, mitochondrial DNA.
tion has been observed, particularly within E.
granulosus and to a lesser extent in E.
multilocularis [3]. The term 'strain' refers, in
the case of Echinococcus, to an intra-specific
group of genetically distinguishable isolates
which also share biological features of actual
or potential significance in the control of
hydatid disease [1]. E. granulosus strains vary
in features such as morphology, biochemistry,
physiology, pathogenicity, developmental pat-
terns and infectivity to humans and domestic
animals [1] with important implications for the
epidemiology of hydatid disease. Such differ-
ences necessitate some reliable means of
identifying to the level of strain, isolates
collected in the field or at surgery.
Identification of isolates of E. granulosus has
depended heavily upon a limited number of
morphological features and there has been
much disagreement as to the value of this
166
approach [4]. Furthermore, it has been demon-
strated that morphological features, including
those commonly studied, can vary as a result
of environmental influences [4,5]. Other meth-
ods of identification, such as those based on
immunological and biochemical analysis, are
also limited in their usefulness because of the
potential for host- and environmentally in-
duced variation.
The problem of grouping isolates into
biologically relevant sub-specific categories is
basically one of determining genetic identity.
Approaches which investigate the genetic
composition of an organism directly, by
examining its DNA, overcome problems of
life-cycle stage variability and external influ-
ences on phenotype. It is not known to what
extent epidemiologically significant differences
(such as host range, infectivity, virulence and
drug susceptibility) reflect genetic heterogene-
ity within Echinococcus populations. This
reservation is particularly valid in the light of
recent findings [4] which suggest that host
factors can significantly influence the parasite
phenotype. However, if genetically homoge-
neous sub-groups can be shown to be linked to
features of biological and epidemiological
importance, then the usefulness of these
methods for differentiation and characterisa-
tion cannot be disputed.
In this study, we have attempted to
distinguish and group isolates of Echinococcus
genetically, and have included representative
isolates from many of the proposed intra-
specific groups. Isolates were analysed for
sequence variation within a region of the
mitochondrial cytochrome c oxidase subunit I
(CO1) gene. We chose to study mitochondrial
DNA (mtDNA) since it generally evolves more
rapidly than nuclear DNA and, because it is
haploid, the allele haplotypes can be deter-
mined unambiguously. It is also advantageous
that m t D N A does not recombine, a feature
which simplifies analysis. The functional
significance of variation in the CO1 gene was
not considered in this study. Rather, it was
anticipated that variation in important biolo-
gical characteristics could be linked to parti-
cular mitochondrial genotypes.
Materials and Methods
Total genomic DNA was prepared from
vertebrate tissues and from fresh, frozen or
alcohol preserved isolates of Echinococcus
species using standard extraction techniques
[6]. For the purposes of this study, an E.
granulosus 'isolate' refers to the protoscoleces
obtained from a single hydatid cyst or an
individual adult worm. Isolates of E.
multilocularis from China, Alaska and North
America originated from alveolar cyst material
surgically resected from infected human livers.
The European isolate of E. multilocularis was
obtained from a naturally infected rodent in
Germany. All isolates were passaged by
intraperitoneal injection in jirds (Meriones
unguiculatus) or cotton rats (Sigmodon
hispidus). The E. oligarthrus and E. vogeli
isolates were similarly laboratory maintained,
following original field isolation in Central
America (Panama) and South America, re-
spectively. In all cases, DNA was isolated from
protoscoleces obtained several months follow-
ing passage. It was not necessary to isolate
mitochondrial DNA from total DNA, and the
quality and quantity of DNA extracted from
all samples was suitable for analysis by
polymerase chain reaction (PCR).
Primers suitable for PCR and sequencing
were designed, based on evolutionarily con-
served regions of the CO1 sequence published
for Fasciola hepatica [7]. The sequences of the
primers used in the study were as follows:
Forward 5 ' T T T T T T G G G C A T C C T G A G G T -
TTAT3'(2575) and
Reverse 5'TAAAGAAAGAACATAATGAA-
AATGY(3021).
The numbers in brackets refer to the position
of the 5' end of the primer in the published F.
hepatica sequence [7].
Double-stranded fragments were produced
by standard PCR methods [8] except that one
primer was previously phosphorylated at the 5'
end [6]. Single-stranded sequencing templates
were prepared by lambda-exonuclease diges-
tion [9] and sequenced using Sequenase (USB).
Sequences were verified as being CO1 by
inference from similarity with mouse [10] and
 167

F. hepatica [7] sequences. The partial mito- Results

chondrial CO1 sequences (366 bp) obtained
were aligned by eye (Fig. I) and isolates with For each Echinococcus isolate examined, a
identical COl genotypes grouped together single double-stranded PCR product of ap-
(Table I). proximately the expected size (446 bp) was
Amino acid sequences were predicted using visualised on an ethidium-bromide stained
known mitochondrial modifications to the agarose gel. Unambiguous sequences of at
universal genetic code [7] (Fig. 2). least 366 bp were obtained for each sample.
The extent of variation amongst the detected Vertebrate tissue D N A was not amplified
mitochondrial genotypes was estimated by under the PCR conditions used for the
pairwise comparison of nucleotide and amino Echinococcus isolates.
acid sequences (Table II). Eleven distinct partial CO1 sequences were
detected amongst the 56 Echinococcus isolates
examined (Fig. 1). Out of the 366 surveyed

G1 CCT GGA TTT GGT ATA ATT AGT CAT Aq'T TGT TTG AGT ATT AGT C,C T AAT TT? GAT GCG T'I'F GGG TTC TAT GGG T'PG TTG "FFF
G2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . T . . . . . . . . . . . . . . .
G3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . .
G4 ..... G . . . . . . . . . . . . . . . . . . . . . . . . . . A . .G . . . . . . . . . . . . . . G .... T . . . . . . . . . T . . . . . . . . . . . . . . .
G5 ..... G ...... G.T . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . G .... ~ ........ T . . . . . . . . . . . A ...
G6 . . . . . . . . . . . . G.T . . . . . . . . . . . . . . . . . . . . G ...... T . . . . . . . G .... TT ........ T . . . . . . . . . . . . . . .
G7 ..C ......... G.T . . . . . . . . . . . . . . . . . . . . G ...... T . . . . . . . G .... "IT ........ T . . . . . . . . . . . . . . .
M1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A ..... A .... G . . . . . . . . . . . . . . . . . . . . . T ..... T .........
M2 . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . A ..... A .... G . . . . . . . . . . . T . . . . . . . . . T ..... T .........
V ..... G . . . . . . . . . . . . . . . . . . . . A ........ G G . . . . . . . . . . . . . G .... T . . . . . . . . . T ........ A ......
O ..... T ........ T . . . . . . . . . . . A ... C.T . . . . . . . . . . . . . . . . CG .... TT ..... A ..T . . . . . . . . . . . . . . .

G1 GCT ATG TTT TCT ATA GTG TGT T'PG GGT AGC AGG Gq'~ TGG GGT CAT CAT ATG q'Fl" ACT GTT GGG TTG GAT GTG AAG ACG GCT
G2
G3
G4
G5 . . . . . . . . . . . . . . . . . . . . . . . A . . . . T , .T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G6 . . . . . . . . . . . . . . . . . . . . . . . A . . . . T . .T ........ A . . . . . . . . . . . . . . . . . . . . A . .A . . . . . . . . . . . T . . .
G7 . . . . . . . . . . . . . . . . . . . . . . . A . . . . T • .T ........ A . . . . . . . . . . . . . . . . . . . . A . .A . . . . . . . . . . . T . . .
M1 . . . . . . . . . . . . . . . . . . . . . . . A . .G .T , .T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G
M2 . . . . . . . . . . . . . . . . . . . . . . . A . .G .T , .T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G
V . . . . . . . . . . . . . . . . . A ..... A . .A .T ........ A . .G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
O . . . . . . . . . . . . . . . . . . . . . . . A . . . . A . .A ..... A . .G . . . . . . . . . . . . . . . . . . . . . . . A . .C . .T .........

G1 GTT TTT ~ AGC TCT GTT ACT ATG ATT ATA GGG GTT CCT ACT GGT ATA AAG GTG TTT ACT TGG TTA TAT AT(] TTG TTG AAT
G2
G3
G4
G5 . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . A . . . . . .
G6 . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . A . . . . . .
G7 . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G ........ A ......
M1 . . . . . . . . . . . T ........ G . . . . . . . . . . . T ..... G . . . . . . . . . . . . . . . . . . . . . . . . . . G ......... C.T . . .
M2 . . . . . . . . . . . T ........ G . . . . . . . . . . . T ..... G . . . . . . . . . . . . . . . . . . . . . . . . . . G ......... C.T . . .
V . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . .
O . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . T ........ A . . . . . . . . . . . . . . . . . . . . A . ,G ........ A ......

G1 TCG AGT GTT AAT GTT AGT ~AT CCG GTT q~PG TGA TGG Gq'T G ~ TCT TTT ATA GTG TTG TTT ACG TTT GGG GGA GTT ACG GGT
G2 . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G3 . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . . .
G4 .... A . . . . . . AAG ........ T . . . . . . . . . . . . . . G . . . . . . . . . . . . . . T ........ T ..... A ........ T . . .
G5 ..T .A . . . . . . CG . . . . . . . . . T ........ G ...... A . . . . . . . . . . . . . T ..A ..C ........ T ..T ..... T ...
G6 ..T .A . . . . . . . C . . . . . . . . . T ........ G ...... A . . . . . . . . . . . . . T ..A . . . . . . . . . . . . . . C . .C . .T ...
G7 • .T .A . . . . . . . C . . . . . . . . . T ........ G ...... A . . . . . . . . . . . . . T . .A . . . . . . . . . . . . . . C , .C . .T . . .
M1 • .T ..... A .. AAG ........ T A ....... G ...... A . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . .T ..... T ...
M2 • .T ..... A .. AAG ........ T A ....... G ...... A . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . .T ..... T ...
V ........ G .. AAG G . . . . . . . . . . . . . A . .G . .A . .G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . .T ..... T ...
O .... A. . .A .. AAG ........ T ........ G ..... G A .... G ........ A ........ A ..... T . .T ..... T ...

G1 ATA GTT TTG TCT GCT TGT GTG TTA GAT AAT Aq'l~ TTG CAT GAT
G2
G3
G4
G5 . . . . . . . . . . . G . . . . . . . . . . . G .... G . . . . . . . . . . .
G6 . . . . . . . . . . . . . . . . . . . . . . . G .... G .... A ......
G7 . . . . . . . . . . . . . . . . . . . . . . . G .... G .... A ......
M1 ........ A . . . . . . . . . . . . . . G .... G .... A . .C . . ,
M2 ........ A . . . . . . . . . . . . . . G .... G .... A . .C . . .
V . . . . . . . . . . . . . . . . . . . . A . .G .... G . . . . . . . . . . .
O ........ A . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . .

Fig. 1. Nucleotide sequences of a 366-bp fragment of the mitochondrial CO1 gene, for a number of species and strains within
the Echinococcus genus• GI to G7 represent the 7 variants identified within E. granulosus, M1 and M2 are E. rnultilocularis
variants, and V and O represent E. vogeli and E. oligarthrus respectively. Dots denote homology with the G1 sequence. The
first and last nucleotides correspond to nucleotides 2611 and 2979, respectively, of the F. hepatica COl sequence [7].
168

TABLE I
Host and geographical origins of Echinococcus isolates examined
Host (number of Origin
isolates examined)
G1 Sheep (19) UK, Spain, China, New South Wales, Kenya, Uruguay, Turkey, Jordan, Lebanon, Italy
Tasmania
Human (4) Queensland, Tasmania, China
Kangaroo (2) Queensland
Dingo (2)a Queensland
Cattle (5) UK, China, Spain
Camel (1) China
Pig (1) China
Goat (2) China, Masailand (Kenya)

G2 Sheep (2) Tasmania

G3 Buffalo (2) India

G4 Horse (2) UK, Spain

Donkey (1) Ireland

G5 Cattle (1) Holland

G6 Camel (2) Somalia, Sudan

Goat (1) Turkana (Kenya)

G7 Pig (2) Poland

M1 (3) China, Alaska, North America

M2 (1) Europe

V (2) South America

O (1) Panama
G1 G7, E. granulosus; M1, M2, E. multilocularis; V, E. vogeli; O, E. oligarthrus.
~Adult worms.
Isolates are arranged in the genotypic groups suggested by mitochondrial cytochrome c oxidase 1 gene sequence data.

nucleotide positions, 76 variant nucleotide
positions were found. G1 to G7 represent
variants of E. granulosus, M1 and M2 are
variants of E. multilocularis, and E. vogeli and
E. oligarthrus are denoted by V and O,
respectively. The 4 Echinococcus species could
be clearly distinguished. A significant amount
of CO1 sequence variation was found within E.
granulosus. The 49 E. granulosus isolates
examined could be divided into 7 discrete
groups (G1-G7) (Fig. 1). The CO1 sequences
of some of these groups are very similar. The
geographical and host origins of the isolates
examined are shown in Table I, along with
their genotypic grouping as designated by
virtue of their CO1 sequence.
All the sheep isolates, with the exception of a
sample (pool of 2 isolates taken from different
sheep on the same farm) from Tasmania, were
found to have the G1 genotype, which was
used as a reference sequence. All human, cattle
(apart for an isolate from Holland), Australian
sylvatic and Chinese isolates tested also fell
into this category as did a single goat isolate
from Kenya. The sequence obtained with the
Tasmanian sheep sample (G2) differed from
the standard sheep sequence at 3 of the 366
nucleotide sites examined, and 2 of these
variants should correspond to an amino acid
change (Fig. 2). A common sequence (G4) was
found in E. granulosus isolates of horse and
donkey origin. A unique CO1 sequence (G5)
was found with the single cattle isolate
examined from Holland. Camel isolates from
 169

G1 PGFGI/MISH I CLS I SANFDAFGFYGLLFAMFSI/MVCLGSSVWGHHMFTVGLDVKTAVFFSTVT

G2 . . . . . . . . . . . . . . . . . . V ..........................................
G3 .... . ... . ... • ............--.... • -...---.-...--..-.--.........
G4 . . . . . . . . . . . . . . . . L.V ..........................................
G5 .... V . . . . . . . . . . . L.V ..........................................
G6 .... V . . . . . . . . . S.L.V ..........................................
G7 .... V . . . . . . . . . S.L.V ..........................................

M1 . . . . . . . . . . . . I/M.G ..............................................
M2 ..... V . . . . . . I/M.G...V ..........................................

V . . . . . . . . I/M... V ...L.V ..........................................

O . . . . . . . . I/M . . . . . . . S.V ..........................................

GI MII/MGVPTGI/MKVFTWLYMLLNSSVNVSDPVLWWVVSFI/MVLFTFGGVTGI/MVLSACVLDLILHD
G ~ .o o o o o . o o . o o o . ~ o o . o o o . o o a o . o o o . . o o . o o . o o o . ° . o o o . o o o o , . o o o o . . o

G 3 .o . o . . , . . Q . . . . . . . , ° . . . o . A , . . . . , . . . . o ° , . . . o . o . ° o ° o . . o . . , o . . . ° o

G4 . . . . . . . . . . . . . . . . . . . . . N..K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V...
G5 . . . . . . . . . . . . . . . . . . . . . N..R ........ I . . . . . . . . . . . . . . . . . . . . . . . V...
G6 . . . . . . . . . . . . . . . . . . . . . N..A ........ I . . . . . . . . . . . . . . . . . . . . . . . V...
G7 . . . . . . . . . . . . . . . . . . . . . N..A ........ I . . . . . . . . . . . . . . . . . . . . . . . V...

M1 . . . . . . . . . . . . . . . . . . . . . . . . K. . .I .... I . . . . . . . . . . . . . . . . . . . . . . . V. . .
M2 . . . . . . . . . . . . . . . . . . . . . . . . K. . .I .... I . . . . . . . . . . . . . . . . . . . . . . . V. . .

V . . . . . . . . . . . . . . . . . . . . . . . . KG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V...

O . . . . . . . . . . . . . . . . . . . . . N..K ........ I . . . . . . . . . . . . . . . . . . . . . . . V...

Fig. 2. Predicted partial amino acid sequences of mitochondrial cytochrome c oxidase I for each of the genetically distinct
groups of Echinococcus. A dot indicates an amino acid that is conserved relative to the G1 sequence. Modifications of the
universal genetic code were used, based on knowledge of the mitochondrial genetic code in other organisms [7]. G 1 - G 7
represent the 7 variants identified within E. granulosus, MI and M2 are E. rnultilocularis variants, and V and O represent E.
vogeli and E. oligarthrus respectively.

Somalia and the Sudan and a goat isolate shown in Fig. 2. By considering the third base
originating from the Turkana region of north- changes that have occurred in the sequences of
west Kenya shared the same sequence (G6), some of these isolates, it is probable that A G A
which differed from the sequence found in pig and A G G c o d e for serine (rather than
isolates from Poland (G7) at only 1 nucleotide arginine) as they do in F. hepatica [7] and
position. nematodes [11]. T G A is not a stop codon, and
Minor variation was detected in the CO1 so probably codes for tryptophan, as has been
sequences of the E. multilocularis isolates, documented in other metazoan mitochondrial
which fell into two groups (M1 and M2). D N A (mtDNA) [7]. ATA specifies isoleucine
Sequence for the European isolate examined in the universal code but in mammals and
(M2) varied from the others at 2 nucleotide Drosophila it codes for methionine. By max-
sites, both of which should cause amino acid imising the conservation of amino acid
changes. The 2 E. vogeli isolate sequences (V) sequence it is more likely that ATA codes for
were identical, and distinct from all other isoleucine than methionine in the Echinococcus
isolates. The CO1 sequence determined for E. organisms but both alternatives have been
oligarthrus (O) was unique. included in the predicted amino acid sequen-
The derived amino acid sequences for this ces shown.
region of the mitochondrial CO1 protein are Pair-wise nucleotide and amino acid se-
170

TABLE II
Levels of pairwise sequence differences among the 11 mitochondrial CO1 genotypes found within the genus Echinococcus
Genotype G1 G2 G3 G4 G5 G6 G7 M1 M2 V O

G1 3 2 30 32 33 34 34 36 31 42
G2 2 1 28 30 30 31 34 34 29 40
G3 1 1 29 31 31 32 33 35 30 41
G4 5 4 5 30 30 31 32 32 21 33
G5 7 6 7 3 17 18 31 31 33 32
G6 8 6 7 4 2 1 35 35 37 35
G7 8 6 7 4 2 0 - 36 36 38 36
M1 5/6 6/7 5/6 6/7 7/8 7/8 7/8 2 35 39
M2 7/8 6/7 7/8 6/7 7/8 7/8 7/8 2 - 35 39
V 6/7 5/6 6/7 3/4 6/7 7/8 7/8 7/8 7/8 34
O 6/7 5/6 6/7 2/3 3/4 4/5 4/5 4/6 5/7 5

G, E. granulosus; M, E. multilocularis; V, E. vogeli; O, E. oligarthrus. For each pairwise comparison, the number of nucleotide
sequence differences is shown above the diagonal and the number of amino acid sequence differences below the diagonal
Where 2 alternative values are given for the number of amino acid sequence differences, the second value refers to the number
of amino acid sequence differences if ' A T A ' codes for methionine rather than isoleucine (see Fig. 2).

quence comparisons (Table II) provide some
quantitative information about the relation-
ships amongst the genotypic groups. The
similarity of the G1/G2/G3, G6/G7 and M1/
M2 groups is evident. In addition, G5 is quite
similar to G6 and G7. The levels of difference
between the recognised species of Echinococcus
are not appreciably greater than those between
genetically-defined subspecific groups of E.
granulosus. This is the case for both nucleotide
and amino acid sequence comparisons.
Discussion
Previous studies have shown that E.
granulosus is made up of a number of
genetically distinct subspecific forms [1,12]. In
this study we have obtained part of the
mitochondrial CO1 sequence for a number of
isolates of E. granulosus and have been able to
use this information to group them into 7
distinct intra-specific categories (Table I). Prior
conclusions of strain patterns, based on a wide
variety of intrinsic and extrinsic criteria [1] are
broadly upheld by our findings.
The G 1 group represents the widespread and
well recognised 'sheep' strain of E. granulosus.
The remarkable uniformity of this strain is
confirmed at the DNA sequence level, with
isolates from sheep and a number of other
hosts from geographically diverse regions
sharing an identical mitochondrial CO1 geno-
type.
In biological and epidemiological features,
the sheep strain is considered to be homo-
geneous except in Tasmania where morpholog-
ical distinctiveness and a significantly
shortened prepatency period have been report-
ed [1,13]. Two (group G2) out of a total of 9
Tasmanian sheep isolates examined in this
study differed slightly in CO1 sequence from
all the other sheep isolates examined. These
same 2 Tasmanian isolates could not be
distinguished from UK and Australian main-
land sheep isolates by restriction fragment
length polymorphism (RFLP) analysis of the
ribosomal DNA (rDNA) repeat region [14]
although the well-established strain groups can
be clearly differentiated using this approach
[12]. Even if the characteristic features of the
Tasmanian form of E. granulosus are the result
of some fundamental genetic difference,
knowledge of the general genetic similarity
indicates that it has only very recently diverged
from the common sheep strain.
Gill and Rao [15] and Rao [16] have
proposed that a morphologically and devel-
opmentally distinct E. granulosus strain exists
in India, producing large fertile cysts in the
lungs of buffaloes. It has been suggested [1]
that this form of E. granulosus may be the same
as the bovine-adapted strain reported in
Switzerland, South Africa and various other

areas. The Indian buffalo isolates examined in
this study (G3) are clearly quite distinct from
the Dutch cattle isolate (G5), and only slightly
different from the common sheep strain (G1).
Our genetic investigations suggest that, like the
Tasmanian sheep isolates, these Indian buffalo
isolates are either variants of the common
sheep strain or represent a genetically distinct
but very closely related group.
All of the hydatid material analysed from
Chinese hosts (human, sheep, cattle, camel and
pig) is representative of the common 'sheep'
strain. These samples were collected from the
Northern part of Xinjiang province in North
West China. Other strains of E. granulosus
may occur in this and other areas but, from the
public health point of view, it is of significance
that a range of animal hosts in this part of
China harbour the sheep strain which is known
to be infective to man.
Evidence of morphological, biochemical and
developmental differences between isolates of
E. granulosus of domestic and sylvatic origin
on mainland Australia led to their proposed
designation as distinct strains [17]. However,
this hypothesis has been questioned following
new morphological studies [4,18] and RFLP
analysis [14,19]. The current study found no
genetic evidence to support the theory that a
distinct Australian sylvatic strain of E.
granulosus exists, at least in Queensland.
Based on their host and geographical
origins, groups G4, G5, G6 and G7 probably
represent the well-characterised and distinct
'UK horse' [20], 'Swiss cattle' [21], 'African
camel' [22,23] and 'European pig' [24] strains,
respectively. Unlike G2 and G3, these groups
are quite different genetically from the sheep
strain (G1). This result complements earlier
reports which have documented the biological
distinctiveness of these strains. The G5
genotype ('Swiss cattle') is reasonably similar
to the G6 ('African camel') and G7 ('European
pig') group genotypes, but is still clearly
identifiable. The G4 genotype ('UK horse')
does not appear particularly close to any other
genotype.
There is some evidence of intra-specific
variation in E. multilocularis. As reviewed by
171
Thompson and Lymbery [1], there have been
reports of variability in morphology, patho-
genicity, developmental characteristics and
host specificity. Furthermore, RFLPs have
been detected among E. multilocularis isolates
originating from different endemic areas [25].
Three geographically distinct subspecies of E.
multilocularis have been described. The nomi-
nate subspecies (E. m. multilocularis) is found
in central Europe whilst E. m. sibiricensis is
reported in Alaska and E. m. kazakhensis in
Kazakhstan [26]. The E. multilocularis isolates
examined here from Alaska, China and North
America were genetically identical in the CO 1
region and were very slightly different from the
European isolate. Further sequence informa-
tion will be necessary to determine whether
stable genetic distinctions, which parallel
biological and morphological dissimilarities,
can be found.
The amount of between group nucleotide
sequence change we have detected is very slight
in some cases. G1, G2 and G3 are obviously
very closely related, and may simply represent
polymorphism within the common 'sheep'
strain. If so, the genotype G1 is probably the
most common within that strain. It may be
significant that variation, albeit slight, is only
found in isolates which, for other reasons, are
thought to be distinct from the common sheep
strain of E. granulosus. It is because of this
correlation with epidemiological differences
that we decided to assign even minor variants
to distinct groups. Similarly, the 'camel' (G6)
and 'pig' (G7) strains, and the 2 E.
multilocularis groups (M1 and M2) have been
kept separate because other evidence [12,26]
exists to support their distinctiveness. Further
investigations may uncover more clear-cut
genetic differences between these groups.
We have chosen to examine and compare
Echinococcus isolates using the most objective
and unambiguous method available, that of
direct DNA sequence analysis. The mitochon-
drial CO1 gene is sufficiently variable in the
Echinococcus organisms to allow discrimina-
tion at the intraspecific level. This is despite the
fact that this has been shown in other
organisms to be one of the least variable of
172
the mitochondrial protein-encoding genes [7].
Additional sequence information obtained
from the more variable genes may help clarify
whether the minor sequence variants detected
in this study represent polymorphisms within
strain groups or genetically definable strains in
their own right. Perhaps by chance, we have
not constructed biologically irrelevant groups
using this approach as may have been antici-
pated [27]. Although, as expected, the E.
granulosus strain pattern has been shown to
be complex, the variation is not continuous.
The mtDNA types found in the G1/G2/G3,
G4, G5 and G6/G7 clusters appear to be not
only distinct but also discrete, suggesting that
the categorisation of isolates into a number of
well-defined intra-specific groups is not an
oversimplification.
This study reinforces the usefulness of direct
genetic approaches in the study of variation.
We have no data to suggest that we will be
unable to identify unambiguously strains and
species of Echinococcus using the DNA
sequencing approach, as long as the DNA
regions targeted are carefully selected. The
gene examined need not have any direct
relevance to the nature of the infection. An
advantage of the approach is that sequence
data provides information about the types and
magnitude of evolutionary changes which have
become fixed, and supplies a large number of
characters suitable for evolutionary and phy-
logenetic analysis. We are currently using the
COl and other sequence data in phylogenetic
studies aimed at determining the actual
interrelationships and taxonomic status of
species and strains of Echinococcus.
Acknowledgements
We would like to thank the National Health
and Medical Research Council of Australia,
the European Economic Community, the
Tropical Health Program and the Australian
Meat and Livestock Development Corpora-
tion (Junior Research Fellowship awarded to
J.B.) for financial support. We would also like
to thank the following colleagues who collect-
ed or assisted in the collection of the various
Echinococcus isolates analysed in the study: F.
van Knapen, Z. Pawlowski, C. Macpherson,
C. Cuesta-Bandera, G. Macchioni, T. Wiker-
hauser, A. Nieto, I. Reisin, R. Matossian, M.
Abo-Shehada, R. Lorenzini, S. Gulen, C.
Hatch, H. Ross, P. Schantz, D. Obendorf, J.
Goldsmid, O. Sousa, B. Gottstein, Z.X. Ding,
W.G. Yang.
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