Sabeta_pp95-100.qxd

INTRODUCTION

Rabies virus (RV) is the prototype member of the
Lyssavirus genus, Rhabdoviridae family, of the
order Mononegavirales (Wunner, Larson, Dietz-
schold & Smith 1988; Tordo & Kouknetzoff 1993).
Rabies virus possesses a non-segmented, nega-
tive-stranded RNA genome of ~12 kb in length. The
viral genome contains information for five proteins:
nucleoprotein (N), matrix protein (M), phosphopro-
tein (P), glycoprotein (G) and polymerase (L).

Epidemiologically, RV is found virtually worldwide
and is perpetuated in several domestic and wild
carnivore species. In the southern African countries
of Zimbabwe and South Africa, rabies virus exists
as two epidemiologically separate lineages (referred
to as canid and mongoose rabies biotypes), which
has been confirmed by antigenic and genetic stud-
ies (Foggin 1988; King, Meredith & Thomson 1993,
1994; Von Teichman, Thomson, Meredith & Nel
1995; Nel, Jacobs, Jaftha & Meredith 1997). Prior
to the era of antigenic and genetic tools for study-
ing the epidemiology of rabies, the existence of the
two rabies biotypes was postulated from historical
records and case surveillance data.

In this paper, we demonstrate the use of reverse
transcription polymerase chain reaction (RT-PCR)
for confirming a clinical diagnosis of rabies infection
in a 2-year-old Thoroughbred colt imported in July
2003, from Golden Acres, Harare in Zimbabwe to
the Ashburton Training Centre, South Africa (see
Fig. 1 for approximate locations of the two sites).

Onderstepoort Journal of Veterinary Research, 72:95–100 (2005)

RESEARCH COMMUNICATION

Importation of canid rabies in a horse relocated
from Zimbabwe to South Africa

C.T. SABETA1 and J.L. RANDLES2

ABSTRACT

SABETA, C.T. & RANDLES, J.L. 2005. Importation of canid rabies in a horse relocated from Zim-
babwe to South Africa. Onderstepoort Journal of Veterinary Research, 72:95–100

In July 2003 a 2-year-old Thoroughbred colt was imported from Harare, Zimbabwe to the Ashburton
Training Centre, Pietermaritzburg, South Africa. Five months after importation, the colt presented
with clinical signs suggestive of rabies: it was uncoordinated, showed muscle tremors and was bit-
ing at itself. Brain tissue was submitted for analysis and the clinical diagnosis was confirmed by the
fluorescent antibody test and reverse-transcription polymerase chain reaction (RT-PCR). Phylo-
genetic analysis of the nucleotide sequence of the cytoplasmic domain of the glycoprotein and the
G-L intergenic region of the rabies virus confirmed it to be an infection with a canid rabies virus, orig-
inating from an area in Zimbabwe endemic for the domestic dog (Canis familiaris) and side-striped
jackal (Canis adustus) rabies.

Keywords: Canid rabies biotype, epidemiology, lyssavirus, rabies virus, South Africa, Zimbabwe

1 Onderstepoort Veterinary Institute, Rabies Unit, Private Bag
X05, Onderstepoort, 0110 South Africa
E-mail: SabetaC@arc.agric.za

2 Rabies Diagnostics Unit, Allerton Provincial Laboratory,
Private Bag X2, Cascades, Pietermaritzburg, 3202 South
Africa

Accepted for publication 20 July 2004—Editor

Canid rabies in horse relocated from Zimbabwe to South Africa

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FIG. 1 Map of Zimbabwe and South Africa showing the approx-
imate geographical locations of Harare (where the 2-year-
old colt originated) and Pietermaritzburg (where it sub-
sequently developed clinical signs compatible with rabies)

TABLE 1 Virus isolates included in the phylogenetic comparison

Lab. ref Species Locality and country of origin Year Accession no.
no. isolated

22642a Jackal (C. adustus)b Glendale, Zimbabwe 1994 AF177088
21467 Caninec Goromonzi, Zimbabwe 1993 AF177066
21111 Jackal (C. adustus) Bromley, Zimbabwe 1992 AF177083
19649 Jackal (C. adustus) Marondera, Zimbabwe 1991 AY605006
28498 Jackal (C. adustus) Marondera, Zimbabwe 2002 AY605041
23357 Jackal (C. adustus) Wedza, Zimbabwe 1995 AF177090
24465 Canine Wedza CLd, Zimbabwe 1996 AF177074
23367 Jackal (C. adustus) Marondera, Zimbabwe 1995 AF177093
24299 Canine Musikavanhu CL, Zimbabwe 1996 AF177073
23652 Canine Mutare, Zimbabwe 1995 AF177071
A03/646a Equine Pietermaritzburg, South Africa 2003 AY605005
29263 Lion (Panthera leo) Marondera, Zimbabwe 2003 AY605014
24505 Canine Gutu, Zimbabwe 1996 AF177075
16387 Canine Zhombe CL, Zimbabwe 1986 AF177057
16254 Canine Odzi, Zimbabwe 1986 AF177055
22547 Canine Kumutsenzere, Zimbabwe 1994 AF177070
16347 Canine Troutbeck, Nyanga, Zimbabwe 1986 AF177056
21869 Canine Nyakasoro, Pfungwe CL, Zimbabwe 1993 AF177069
21057 Canine Muzarabani CL, Zimbabwe 1992 AF177064
20519 Canine Lower Gweru, Zimbabwe 1992 AF177060
A00/413 Canine Pietermaritzburg, South Africa 2000 AY605042
A90/57 Canine Durban, South Africa 1990 AF177101
A90/352 Canine Durban, South Africa 1990 AF177100
A95/755 Canine Amanzimtoti, South Africa 1995 AF303081
28522 Jackal (C. mesomelas) Gwanda, Zimbabwe 2002 AY605038
23374 Jackal (C. mesomelas) Bulawayo, Zimbabwe 1995 AF177091
21579 Jackal (C. mesomelas) Tsholotsho CL, Zimbabwe 1993 AF177086
17722 Jackal (C. mesomelas) Gwanda, Zimbabwe 1988 AF177079
20034 Canine Bvumba, Mutare, Zimbabwe 1991 AF177059
16838 Canine Shangani, Zimbabwe 1987 AF177058
27792 Jackal (C. mesomelas) Fort Rixon, Zimbabwe 2001 AY605032
27890 Jackal (C. mesomelas) Insiza, Zimbabwe 2001 AY605039
19671 Civettictis civetta Rusape, Zimbabwe 1991 AF304188

a Laboratory reference numbers: Isolates from Zimbabwe use the Harare Central Veterinary Laboratory rabies reference; isolates
prefixed by “A” indicate the Allerton Provincial Veterinary Laboratory reference numbers, Pietermaritzburg

b Where the jackal species was not definitively identified at collection, the species zone that it originated in, and hence the princi-
pal host, is given in parenthesis (see Bingham et al. 1999b)

c We use the term “canine” to refer to the domestic dog, Canis familiaris
d CL stands for communal lands

C.T. SABETA & J.L. RANDLES

The colt presented with clinical signs suggestive of
rabies on 6 December 2003. It was uncoordinated,
manifested muscle tremors and was biting at itself.
It was subsequently admitted to the Summerfield
Equine Hospital on the same day where it was
examined by a veterinary surgeon. It was euthan-
ased the following day and brain tissue was sub-
mitted for rabies testing to the Allerton Provincial
Laboratory in Pietermaritzburg, KwaZulu-Natal.
Although there was no recorded history of a dog
bite and no skin wounds were detected, the brain
sample tested positive by the fluorescent antibody
test (FAT). The rabies virus (laboratory reference
no. A03/646) was then subjected to molecular char-
acterization in order to establish its origin.

MATERIALS AND METHODS

For molecular characterisation of the rabies virus
(A03/646), it was decided to target the variable
glycoprotein gene. RT-PCR and DNA sequence
analyses were carried out at the Rabies Unit, Onder-
stepoort Veterinary Institute, Pretoria as described
previously (Von Teichman et al. 1995; Sabeta, Bing-
ham & Nel 2003). An amplicon of the expected size
(~850 bp) was obtained with the G/L primer set and
purified with a Wizard PCR CleanUp System (Pro-
mega, USA). An aliquot (~50 ng) of the purified
amplicon was sequenced using the BigDye Term-
inator system (Perkin Elmer) with the same primer
pair (G/L) as in the previous RT-PCR steps. A con-
sensus sequence of the equine rabies virus was
assembled after comparison of the forward and re-
verse sequences with Sequence Navigator Software
(PE Applied Biosytems).

Phylogenetic analysis of the nucleotide sequence of
the rabies virus together with nucleotide sequences
of other virus isolates from our database (Table 1)
was done using the Phylip software package (Phyl-
ogeny Inference Package).

In brief, a multiple alignment of a 592-nucleotide
region of the nucleotide sequences included in the
analysis was generated with ClustalW (Thompson,
Higgins & Thompson 1994). Distance calculations
were done using the Kimura 2-parameter model for
evolutionary rate (Kimura 1980). For construction
of the phylogenetic trees, the Neighbour Joining
(NJ) method combined with a 1 000 bootstrap iter-
ations was used (Saitou & Nei 1987; Hills & Bull
1993). The program TREEVIEW was used to dis-
play the graphical output of the tree (Fig. 2) (Page
1996).

RESULTS AND DISCUSSION

Both the direct immunofluorescent antibody test
(FAT) and RT-PCR confirmed the rabies infection.
The rabies virus was successfully amplified by the
G/L primer pair to yield an expected product of
approximately 850 bp (not shown). Nucleotide se-
quencing of the purified PCR amplicon and phylo-
genetic comparison with other nucleotide sequences
from our database of Zimbabwean and South Afri-
can rabies G-L nucleotide sequences demonstrated
it to be a canid rabies virus (genotype 1) originating
from Zimbabwe (see Fig. 2).

Despite the close genetic relatedness of the canid
rabies virus isolates compared here [mean se-
quence homology of 96.3 % calculated in MEGA
(Kumar, Tamura, Jakobsen & Nei 2001)], the rabies
virus (A03/646) was found to cluster with Zimbab-
wean canid rabies virus isolates (Cluster 1) endemic
within the domestic dog (Canis familiaris) and side-
striped jackal (Canis adustus) (Bingham, Foggin,
Wandeler & Hill 1999a; Bingham, Foggin, Wandeler
& Hill 1999b; Sabeta et al. 2003). Cluster 1 was sta-
tistically supported with a bootstrap value of 98 %.
Cluster 2 is composed of virus isolates exclusively
from domestic dogs in north-eastern Zimbabwe.
Clusters 1 and 2 consist of Zimbabwean viral iso-
lates and can be distinguished from isolates from
domestic dogs in KwaZulu-Natal (cluster 3), the
region in which the colt developed clinical rabies 5
months after it had been imported from Harare.
Cluster 4 is made up of viruses exclusively from
black-backed jackal (Canis mesomelas) and clus-
ter 5 from both domestic dogs and C. mesomelas.
Both clusters 4 and 5 represent viral isolates that
are associated with rabies cycles in the C. meso-
melas-zone of southern Zimbabwe and northern
South Africa (Sabeta et al. 2003).

Rabies in horses is a relatively uncommon disease
and transmission of the disease from horses to
humans is rare (Green, Smith, Vernau & Beacock
1992). However, a potential risk to veterinarians,
horse handlers and horse owners does exist and
should be emphasized. Given the high incidence of
rabies in Zimbabwe and in the sub-region as a
whole, it would be advisable for horse owners and
those in the horse industry alike to vaccinate their
animals against rabies. The absence of wounds in
the 2-year-old colt does not exclude inoculation by
a bite from a rabid animal, because had bite wounds
been present, they might have been very small and
puncture-like and were therefore not detected, or
they could have healed prior to the development of
clinical signs. The rabies virus isolate investigated

Canid rabies in horse relocated from Zimbabwe to South Africa

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FIG. 2 Neighbour Joining tree showing the phylogenetic posi-
tion of the equine rabies virus (A03/646) described in the
paper. The inferred phylogeny is based on sequence
comparison of the cytoplasmic domain of the glycopro-
tein and the G-L intergenic regions of the virus isolates.
Included in the phylogenetic tree are: a typical Zimbab-
wean mongoose rabies virus (ci19671), typical canid
rabies viruses from Zimbabwe (j22642, d21467, j21111,
j19649, j28948, j23357, d24465, j23667, d24299,
d23652, lion29263, d24505, d16387, d16254, d22547,
d16347, d21869, d21057, d20519, j28522, j23374,
j21579, j17722, d20034, d16838, j27792, j27890) and
South Africa (A00/413, A90/57, A90/352, A95/755) (see
Table 1 for epidemiological information of these isolates).
The bootstrap values are shown on the branches. The
horizontal branch lengths are proportional to the similar-
ity of sequences within and between clusters, with the
scale bar indicating nucleotide substitutions per site.
Vertical lines are for clarity of presentation only and the
sequence of PV was included as an outgroup

and described here would most likely have been
transmitted as a result of a dog bite. This is highly
probable considering that the canid rabies variant is
widespread and prevalent, making it the most
threatening variant for humans and domestic carni-
vore species alike (Bingham et al. 1999a). This calls
for stricter import/export regulations for animals in
transit in order to diminish the spread of rabies
across regional borders.

Although horses are known to be moderately sus-
ceptible to rabies infection, long incubation periods
of up to 5 months, as shown in this case, may be
unusual. The clinical signs of rabies are variable in
horses and generally appear after an incubation
period of 2–6 weeks, although in some it may be up
to 3 months (Beran 1981). However, rabies virus
has been isolated from the saliva of a dog that sur-
vived 20 months post-exposure to the virus (Green
et al. 1992). Although it has been shown that rabies
virus remain at the site of a bite for most of the
incubation period, which is generally 10–90 days,
longer incubation periods have been described by
Smith, Fishbean, Rupprecht & Clark (1991).

This investigation illustrates the usefulness of RT-
PCR as a tool for studying animal viral diseases
such as rabies. Furthermore, it increases our under-
standing of epidemiological relationships of lyssa-
viruses not only through establishing their genetic
relationships but also by providing us with informa-
tion on the origin, geographical distribution and paths
of transmission of the various strains of the virus
(Brown 1994).

Several molecular epidemiological studies of rabies
have been conducted in the southern African sub-
region (Nel, Thomson & Von Teichman 1993; Von
Teichman et al. 1995; Nel et al. 1997; Sabeta et al.
2003). Further molecular studies of lyssaviruses in
countries in the sub-region that include Botswana,
Namibia, Mozambique, Swaziland, Lesotho and
Zambia should be encouraged, and are already be-
ing realized. These studies are essential in order to
elucidate the dynamics of rabies in the sub-region.
An expansion of our current nucleotide sequence
database of rabies viruses would thus be useful for
tracing the routes of infection as illustrated by the
equine case described here and also for establish-
ing concrete measures to control the disease.

ACKNOWLEDGEMENTS

This investigation was made possible by funding
from the International Foundation of Sciences (IFS)

(grant #B3179-1). We thank Prof. Berndt Klingeborn
for critically reading this manuscript.

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Canid rabies in horse relocated from Zimbabwe to South Africa