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Molecular epidemiology of rabies: Focus on domestic dogs (Canis familiaris) and black-backed jackals (Canis mesomelas) from northern South Africa
Virus Research 140 (2009) 71–78
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Molecular epidemiology of rabies: Focus on domestic dogs (Canis familiaris) and black-backed jackals (Canis mesomelas) from northern South Africa G.C. Zulu a,b,∗ , C.T. Sabeta a , L.H. Nel b a b
Rabies Unit, ARC-Onderstepoort Veterinary Institute, Private Bag X 05, Onderstepoort, Pretoria 0110, South Africa Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa
a r t i c l e
i n f o
Article history: Received 20 August 2008 Received in revised form 6 November 2008 Accepted 7 November 2008 Available online 20 December 2008 Keywords: Rabies virus Black-backed jackal Domestic dog G-L intergenic region South Africa
a b s t r a c t Phylogenetic relationships of rabies viruses recovered from black-backed jackals (Canis mesomelas) and domestic dogs (Canis familiaris) in northern South Africa were investigated to determine whether the black-backed jackal is an emerging maintenance host species for rabies in this region. A panel of 123 rabies viruses obtained from the two host species between 1980 and 2006 were characterised by nucleotide sequencing of the cytoplasmic domain of the glycoprotein gene and the non-coding G-L intergenic region. Through phylogenetic analysis a viral cluster specific to black-backed jackals and spanning a 5-year period was delineated in western Limpopo. Virus strains associated with domestic dogs prevail in densely populated communal areas in north-eastern Limpopo and in south and eastern Mpumalanga. The data presented in this study indicated the likelihood that black-backed jackals are capable of sustaining rabies cycles independent of domestic dogs. It is proposed that wildlife rabies control strategies, in synergy with domestic animal vaccination should be considered for effective control of rabies in South Africa. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Rabies is a fatal viral disease that affects all warm blooded vertebrates. Despite the availability of vaccines to prevent this disease, it is still a significant public and veterinary health problem in many countries particularly in Asia and Africa (Meslin et al., 1994; Cleaveland, 1998; WHO, 1999; Knobel et al., 2005). The virus causing the disease belongs to genotype 1 of the Lyssavirus genus in Rhabdoviridae family. The rabies virus (RABV) genome consists of a single-stranded, non-segmented negative sense RNA of approximately 12 kb (Tordo et al., 1986). At first, the invasive form of canid rabies was noted in the Limpopo province in South Africa in 1950 following the outbreaks which spread from Angola in 1947 (Swanepoel et al., 1993). During this time, rabies spread primarily in domestic dogs in the densely populated rural areas in Limpopo (LP), Mpumalanga (MP) and KwaZulu Natal (KZN). The virus has since spread widely in domestic dogs throughout the country. To date dog rabies is endemic in many communal areas in Eastern Cape (EC), MP and the entire KZN (Bishop et al., 2002; Sabeta et al., 2007; Coetzee and Nel, 2007). As a consequence, human rabies is also common in these areas.
∗ Corresponding author at: Rabies Unit, ARC-Onderstepoort Veterinary Institute, Private Bag X 05, Onderstepoort, Pretoria 0110, South Africa. Tel.: +27 12 529 9122; fax: +27 12 529 9390. E-mail address: [email protected] (G.C. Zulu). 0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2008.11.004
In addition to domestic dogs, canid rabies is currently sustained by black-backed jackals (Canis mesomelas) in the LP province and bat-eared foxes (Otocyon megalotis) in the Cape region (Sabeta et al., 2007). The black-backed jackal has co-existed with the domestic dog as primary hosts of rabies in LP since 1950 (Swanepoel, 2004). Large black-backed jackal populations are common in bushveld ranches in western LP and in commercial farmland in the north and central areas of this province. Here black-backed jackals have become established as primary carnivores and are thought to reach densities that may be high enough to sustain continuous rabies cycles (Bingham et al., 1999; Cumming, 1982). Jackal rabies has been a serious concern to farmers in LP due to a close correlation between rabies cases in this canid species (C. mesomelas) with those in cattle (Brückner and Hurter, 1978; Barnard, 1979). Moreover, the frequent transmission of RABV between black-backed jackals and domestic dogs (Nel et al., 1993; Sabeta et al., 2003) imposes a serious public health threat. Although the number of human rabies cases caused by jackals is relatively low (Bingham et al., 1999), the consideration of jackals as a rabies problem is linked to its association with domestic dogs. Domestic dogs in their close association with man are the strongest link between rabies in jackals and rabies in humans (McKenzie, 1993). The attempts to control rabies in black-backed jackals that involved the use of meat baits laced with strychnine as poison only provided temporary and localised control of the disease (Mansvelt, 1956). In the case of domestic dogs, parenteral vaccination remains the most effective approach in reducing the spread of rabies in rural African regions (Cleaveland et al., 2003, 2006), hence the risk
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Table 1 Rabies viruses from Limpopo, North-West and Mpumalanga provinces of South Africa included in this study. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76
Reference 1403/80* 1627/80* 1265/80 788/80 47/81 3065/81 101/90* 598/90* 378/90* 45/94 820/94* 446/95* 1034/95 479/96* 504/96* 536/96 373/97* 528/97 224/98* 306/98* 194/99 208/99 223/99 418/99 549/99 557/99 590/99 596/99* 609/99 669/99* 673/99* 675/99* 717/99* 733/99* 1004/99 1024/99 1041/99 1064/99 80/00 171/00 207/00 273/00 354/00 438/00 572/00 596/00 641/00 685/00 714/00 35/00 344/00 390/00 426/00 907/00 27/01 582/01 644/01 42/01 127/01 191/01 433/01 131/02 307/02 422/02 629/02 110/02 136/02 202/02 213/02 193/03 211/03 224/03 343/03 543/03 778/03 64/04
Year of isolation 1980 1980 1980 1980 1981 1981 1990 1990 1990 1994 1994 1995 1995 1996 1996 1996 1997 1997 1998 1998 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2001 2001 2001 2001 2001 2001 2002 2002 2002 2002 2002 2002 2002 2002 2003 2003 2003 2003 2003 2003 2004
Species C. mes C. mes C. fam C. mes C. mes C. mes C. mes C. mes C. mes C. mes C. fam C. mes C. mes C. fam C. mes C. fam C. fam C. mes C. fam C. mes C. mes C. fam C. mes C. mes C. mes C. mes C. mes C. fam C. mes C. mes C. mes C. fam C. mes C. fam C. fam C. fam C. fam C. fam C. mes C. mes C. fam C. fam C. mes C. fam C. fam C. mes C. mes C. mes C. mes C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. mes C. mes C. mes C. fam C. mes C. mes C. mes C. fam C. fam C. fam C. fam C. mes C. mes C. fam C. mes C. mes C. mes C. mes
Province LP LP NW LP NW LP LP LP LP LP NW NW NW LP LP MP MP LP MP LP LP LP LP LP LP LP LP MP LP LP LP NW LP NW LP MP MP MP LP LP LP LP LP LP LP LP LP LP LP MP MP MP MP MP MP MP MP LP LP LP LP MP LP LP LP MP MP MP MP LP LP LP LP LP LP LP
Locality Soutpansberg Soutpansberg Brits Musina Brits Musina Soutpansberg Soutpansberg Potgietersrus Warmbad Vryburg Vryburg Kudumane Thabazimbi Warmbad Carolina Barberton Thabazimbi Ermelo Warmbad Messina Waterberg Polokwane Polokwane Polokwane Polokwane Polokwane Piet Retief Soutpansberg Polokwane Potgietersrus Mankwe Polokwane Brits Polokwane Piet Retief Wakkerstroom Ermelo Potgietersrus Thohoyandou Potgietersrus Polokwane Potgietersrus Soutpansberg Potgietersrus Ellisras Polokwane Potgietersrus Potgietersrus Barberton Barberton Barberton Barberton Piet Retief Barberton Piet Retief Barberton Musina Potgietersrus Soutpansberg Ellisras Piet Retief Ellisras Ellisras Ellisras Barberton Piet Retief Piet Retief Piet Retief Polokwane Louis Trichardt Ellisras Ellisras Ellisras Ellisras Musina
Coordinates (long.–lat.) ◦
◦
29 24 –22 32 29◦ 13 –22◦ 38 27◦ 34 –25◦ 22 29◦ 47 –22◦ 17 27◦ 28 –25◦ 17 29◦ 35 –22◦ 36 28◦ 55 –22◦ 48 29◦ 53 –22◦ 50 28◦ 31 –23◦ 10 27◦ 44 –24◦ 52 24◦ 45 –26◦ 08 24◦ 36 –26◦ 20 23◦ 23 –26◦ 48 27◦ 24 –24◦ 43 27◦ 44 –24◦ 56 30◦ 33 –25◦ 57 31◦ 48 –25◦ 42 26◦ 49 –24◦ 43 29◦ 59 –26◦ 31 28◦ 07 –24◦ 51 29◦ 33 –22◦ 33 28◦ 21 –24◦ 35 29◦ 17 –23◦ 56 29◦ 50 –23◦ 45 29◦ 24 –24◦ 04 29◦ 24 –24◦ 05 29◦ 25 –24◦ 13 30◦ 45 –27◦ 15 30◦ 00 –23◦ 22 29◦ 27 –23◦ 47 28◦ 36 –22◦ 43 27◦ 23 –25◦ 06 29◦ 29 –23◦ 42 27◦ 33 –25◦ 14 29◦ 14 –23◦ 43 30◦ 43 –27◦ 07 30◦ 32 –27◦ 04 30◦ 33 –26◦ 43 28◦ 33 –23◦ 00 29◦ 46 –23◦ 58 29◦ 19 –22◦ 48 29◦ 24 –24◦ 03 28◦ 19 –23◦ 29 29◦ 50 –23◦ 25 28◦ 10 –22◦ 58 27◦ 19 –23◦ 39 29◦ 27 –23◦ 36 28◦ 18 –23◦ 17 28◦ 16 –23◦ 31 31◦ 47 –25◦ 45 31◦ 36 –25◦ 48 31◦ 37 –25◦ 46 31◦ 50 –25◦ 53 30◦ 36 –27◦ 09 31◦ 48 –25◦ 45 30◦ 48 –27◦ 42 31◦ 35 –25◦ 37 29◦ 47 –22◦ 33 29◦ 12 –24◦ 21 30◦ 10 –22◦ 33 27◦ 23 –23◦ 39 30◦ 43 –27◦ 04 27◦ 37 –23◦ 29 27◦ 24 –23◦ 41 27◦ 33 –23◦ 23 31◦ 57 –25◦ 26 30◦ 48 –27◦ 00 31◦ 19 –27◦ 17 30◦ 48 –27◦ 00 29◦ 24 –23◦ 56 29◦ 29 –22◦ 50 28◦ 12 –22◦ 42 27◦ 54 –24◦ 03 27◦ 11 –23◦ 48 28◦ 12 –23◦ 38 29◦ 33 –22◦ 29
Genbank Acc. No. AF177116 AF177104 EF686047 EF686048 EF686049 EF686050 AF079902 AF177117 AF177106 EF686064 AF177118 AF177107 EF686051 AF303070 AF177108 EF686057 AF303069 EF686062 AF177098 AF177105 EF686046 EF686061 EF686059 EF686070 EF686069 EF686054 EF686053 AF303063 EF686055 AF303062 AF303061 AF303071 AF303064 AF303067 EF686052 EF686068 EF686058 EF686080 EF686056 EF686076 EF686063 EF686065 EF686112 EF686060 EF686066 EF686110 EF686075 EF686109 EF686100 EF686077 EF686103 EF686079 EF686071 EF686106 EF686096 EF686072 EF686078 EF686067 EF686074 EF686073 EF686084 EF686140 EF686081 EF686107 EF686083 EF686086 EF686101 EF686113 EF686097 EF686092 EF686108 EF686111 EF686144 EF686088 EF686090 EF686105
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Table 1 (Continued ) No 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
Reference 133/04 187/04 315/04 567/04 606/04 622/04 370/05 391/05 409/05 416/05 449/05 499/05 507/05 535/05 537/05 561/05 565/05 572/05 575/05 686/05 698/05 721/05 728/05 729/05 819/05 757/05 84/06 91/06 98/06 136/06 236/06 288/06 266/06 264/06 262/06 294/06 108/06 296/06 367/06 364/06 272/06 315/06 221/06 304/06 370/06 341/06 197/06
Year of isolation 2004 2004 2004 2004 2004 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006
Species C. mes C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. mes C. fam C. fam C. mes C. fam C. mes C. fam C. mes C. fam C. fam C. mes C. mes C. fam C. mes C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. mes C .fam C. fam C. fam C. mes C. fam C. mes C. fam C. fam C. fam C. fam C. fam
Province LP MP MP MP MP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP MP LP LP LP LP MP LP LP LP LP LP LP LP LP LP LP LP LP LP MP LP LP LP LP
Locality Louis Trichardt Nkomazi Piet Retief Piet Retief Piet Retief Polokwane Thohoyandou Thohoyandou Thohoyandou Thohoyandou Thohoyandou Sibasa Sibasa Sibasa Louis Trichardt Thohoyandou Polokwane Giyani Polokwane Louis Trichardt Sibasa Louis Trichardt Louis Trichardt Skukuza Soutpansberg Ellisras Sibasa Thohoyandou Ermelo Thohoyandou Louis Trichardt Giyani Phalaborwa Louis Trichardt Sibasa Louis Trichardt Thohoyandou Louis Trichardt Louis Trichardt Phalaborwa Louis Trichardt Polokwane Nkomazi Louis Trichardt Louis Trichardt Louis Trichardt Sibasa
Coordinates (long.–lat.) ◦
◦
29 54 –23 2 31◦ 40 –25◦ 21 27◦ 17 –31◦ 22 31◦ 12 –27◦ 17 31◦ 08 –27◦ 11 29◦ 28 –23◦ 54 30◦ 43 –22◦ 48 30◦ 43 –22◦ 48 30◦ 53 –22◦ 45 30◦ 30 –22◦ 55 30◦ 36 –22◦ 49 30◦ 36 –22◦ 49 30◦ 28 –22◦ 58 30◦ 43 –22◦ 48 29◦ 57 –22◦ 25 30◦ 31 –22◦ 51 29◦ 28 –23◦ 54 (not known) 29◦ 28 –23◦ 54 29◦ 17 –23◦ 2 30◦ 31 –22◦ 51 29◦ 45 –23◦ 15 29◦ 45 –23◦ 15 31◦ 36 –24◦ 59 29◦ 10 –22◦ 43 28◦ 17 –22◦ 47 30◦ 31 –22◦ 51 30◦ 30 –22◦ 55 30◦ 07 –26◦ 05 30◦ 28 –22◦ 58 29◦ 54 –23◦ 2 31◦ 8 –23◦ 56 31◦ 8 –23◦ 56 29◦ 54 –23◦ 2 30◦ 28 –22◦ 54 29◦ 54 –23◦ 2 30◦ 28 –22◦ 58 29◦ 54 –23◦ 2 30◦ 16 –23◦ 6 29◦ 32 –23◦ 55 30◦ 2 –22◦ 54 29◦ 32 –23◦ 55 31◦ 53 –25◦ 33 30◦ 16 –23◦ 6 30◦ 16 –23◦ 6 29◦ 54 –23◦ 2 30◦ 28 –22◦ 58
Genbank Acc. No. EF686099 EF686102 EF686151 EF686104 EF686131 EF686115 EF686082 EF686089 EF686091 EF686098 EF686095 EF686114 EF686085 EF686093 EF686133 EF686094 EF686130 EF686116 EF686117 EF686132 EF686119 EF686129 EF686118 EF686120 EF686121 EF686122 EF686123 EF686124 EF686125 EF686126 EF686127 EF686128 EF686134 EF686135 EF686136 EF686137 EF686138 EF686139 EF686141 EF686142 EF686143 EF686145 EF686146 EF686147 EF686149 EF686150 EF686152
Key: LP, Limpopo; MP, Mpumalanga; NW, North-West; C. mes, Canis mesomelas; C. fam, Canis familiaris. RABV isolates denoted with asterisks were sequenced previously (Sabeta et al., 2003) and were retrieved from GenBank.
of human infection. However, this does not address the control of rabies in wildlife. Thus, this raises concerns about the eradication of the disease given the possibility that wildlife vectors can theoretically act as sources of infection for domestic animals (Bingham et al., 1999; Nel et al., 1997; Rupprecht et al., 2008). Although the black-backed jackal has become a potentially important vector throughout southern Africa, the epidemiology of the disease in this species has not been elucidated. Reports from southern Africa have been contradictory with regard to the issue as to whether black-backed jackals are capable of sustaining rabies infection cycles independently of domestic dogs. Some authors advocated that jackal populations do not reach high enough densities to sustain the infection cycles in the absence of domestic dogs (Rhodes et al., 1998; Haydon et al., 2002). Other authors are of opposing opinion – speculating that domestic dogs introduced rabies infection into jackals, where after the latter maintained their own infection cycles (Cumming, 1982; Bingham et al., 1999). In light of this controversy, the current study was aimed to determine the molecular epidemiological relationships of rabies viruses from domestic dogs and C. mesomelas in order to clarify the com-
plexity of dog and C. mesomelas rabies cycles. Precise knowledge of the distribution of canid rabies in the region and a better understanding of rabies transmission dynamics between domestic dogs and C. mesomelas is needed to underpin effective rabies control strategies. 2. Materials and methods 2.1. Viruses A panel of 123 rabies viruses [domestic dogs n = 69 and blackbacked jackals n = 54] from LP (n = 89), MP (n = 27) and NW (n = 7) provinces collected between 1980 and 2006 were included in this study. Samples of brain material from rabies-suspect animals were submitted to the Rabies Laboratory at the ARC-Onderstepoort Veterinary Institute in Pretoria, South Africa. The samples were tested for the presence of lyssavirus antigen using the fluorescent antibody test (FAT) (Dean et al., 1996) and original infected brain material was stored at −20 ◦ C prior to molecular characterisation. The rabies viruses collected between 1980 and 1998 were passaged once in
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suckling mice and stored as 20% lyophilised mouse brain fractions. Viruses collected after 1998 were not passaged, but stored as original brain material. Full details of the species of origin, year of isolation and area of origin of these virus isolates are presented in Table 1. 2.2. Viral RNA extractions, RT-PCR and sequencing Total RNA was extracted using Trizol reagent (Sigma–Aldrich, USA) as per manufacturer’s recommended protocol. RT-PCR and direct sequencing were performed as described earlier (Sacramento et al., 1991). A consensus sequence was obtained after alignment of the forward and reverse sequences using MEGA 3.1 software. The sequences were trimmed to 592 bp nucleotides which included the cytoplasmic domain of the glycoprotein gene and the G-L intergenic region. All sequences were submitted to GenBank (accession numbers are given in Table 1). 2.3. Phylogenetic analysis Phylogenetic analyses were based on the 592 bp nucleotide sequence region. Nucleotide sequences were aligned using Clustal X (Higgins and Sharp, 1989; Higgins, 2003) and genetic distances calculated using Kimura’s two-parameter method in MEGA 3.1 software (Kumar et al., 2004). A phylogenetic tree was constructed using the Neighbour-joining method (NJ) (Saitou and Nei, 1987). Bootstrap support values were determined using 1000 replicates to evaluate the significance of the branching pattern and values of ≥70% were regarded as providing statistical evidence for phylogenetic groupings (Hills and Bull, 1993). The results were validated by the maximum parsimony method and the Tree Explorer module of MEGA v 3.1 was used to obtain the graphic output.
3. Results 3.1. Rabies prevalence in Limpopo, Mpumalanga and North-West provinces A high proportion of the virus isolates [n = 89 (72%)] used in this study was obtained from the LP province – 51 were recovered from C. mesomelas and 38 from domestic dogs. The majority of RABV isolates associated with domestic dogs were obtained from north-eastern LP and south to eastern MP. Those from jackals were obtained mainly from the north, west and central areas in LP. Rabies trend analysis for the past decade shows that the disease was generally more predominant in the LP in comparison to NW and MP provinces (Fig. 1) (Records of the Onderstepoort Veterinary Institute, 1995–2006). Since this trend analysis was based on passive surveillance it is possible that it might not truly reflect the actual rabies prevalence in the region. During the period under review, dog rabies in LP appeared to have been under control prior to 2005. Since 1995 less than 10 dog rabies cases were reported from LP province annually but a substantial increase was noted from 2005 (Fig. 2). On the other hand, C. mesomelas had a comparatively higher prevalence with an intense outbreak in 1998 (Fig. 2). Other than jackals and dogs, livestock species are the only other species included and numbers of rabies cases in these animals rose in concert with those in canids. 3.2. Genetic characterisation of virus isolates For all the viruses studied here, the G/L primer pair yielded the desired PCR amplicon of approximately 850 bp. Nucleotide sequencing on average yielded approximately 700–750 bases which were then trimmed to 592 bp to include the cytoplasmic domain of the glycoprotein gene and the G-L intergenic region.
Fig. 1. Laboratory confirmed rabies cases in the northern South Africa in the provinces of Limpopo (LP), Mpumalanga (MP), and North-West (NW) from 1995 to 2006 (Records of the Onderstepoort Veterinary Institute, 1995–2006).
Fig. 2. Laboratory confirmed rabies cases in black-backed jackals, domestic dogs and other species (mainly livestock) in the Limpopo province from 1995 to 2006 (Records of the Onderstepoort Veterinary Institute, 1995–2006).
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Fig. 3. Neighbour-joining tree constructed based on a multiple alignment of 592 bp nucleotide sequences encompassing the cytoplasmic domain of the glycoprotein and the G-L intergenic region of 92 canid rabies viruses from Limpopo (LP), North-West (NW) and Mpumalanga (MP) provinces. Bootstrap values based on 1000 replicates are shown on the major branches. The horizontal branch lengths are indicative of evolutionary distances. The scale indicates nucleotide substitutions per site. The Pasteur virus (PV) strain was used to root the tree. The nucleotide sequences are preceded by the prefix d or j to denote species of origin, i.e. d = dog and j = jackal. The species prefix is followed by a second prefix; LP, MP, NW which was used to denote the region where viruses were obtained.
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Fig. 4. Map showing the geographical distribution of the RABV clusters identified in Limpopo, Mpumalanga and North-West provinces. Only the representative virus isolates for the clusters were plotted. Filled symbols denote domestic dogs and empty ones denote black-backed jackals.
Through a pairwise alignment of the sequence data it was demonstrated that for this region of the genome rabies viruses from northern South Africa were closely related – supported by an intrinsic nucleotide sequence identity of 96.7% determined by Kimura 2-parameter model (Kumar et al., 2004). RABV isolates from this region differed on average by 18.7% from the PV strain which was used as a reference. Similar to previously characterised rabies viruses from South Africa and Zimbabwe (Coetzee and Nel, 2007; Sabeta et al., 2003) the isolates characterised in this investigation lacked the first transcription, termination and polyadenylation signal which was found to be present for the G gene of the vaccine (PV and ERA) strains (Sacramento et al., 1992). Virus isolates from the same geographical areas and with 100% nucleotide sequence similarity were removed from further analysis, to improve the clarity of phylogenetic trees. A Neighbour-joining tree (Fig. 3) was thus constructed for 92 rabies viruses to determine the evolutionary relationships between viruses from C. mesomelas and domestic dogs. The topology of the NJ tree was similar to that obtained using a Maximum Parsimony method (results not shown). The branching of the NJ tree demonstrated that rabies viruses from this region could be divided into six clusters (Fig. 3). Cluster LP/NW-I was composed of viruses from LP and NW spanning the period from 1994 to 2006. These viruses were found in the north, central and south western LP as well as in the western part of the NW province (Fig. 4). Cluster LP/NW-I consisted of the largest number of virus isolates from both canid species – this viral cluster may represent the core of the LP epidemic since the early 1990’s. More than 70% of virus isolates in this lineage originated from C. mesomelas. Such observation demonstrates that this viral cluster has been well established in the C. mesomelas population and highlights the important role played by jackals in the transmission of the virus. The most significant finding of this investigation was the identification of cluster LP-I that was composed of viruses exclusively obtained from C. mesomelas and spanning a 5-year period (2000–2005). Our data shows that this virus variant has segregated into a distinct C. mesomelas
rabies cluster. Viruses in this cluster originated from the bushveld ranches and commercial farmland in western LP and were very closely related as shown by 99.8% average nucleotide sequence similarity. RABV isolates recovered between 1980 and 1999 grouped into a distinct cluster LP/NW-II (Fig. 3), there were however no recent viruses obtained from the region that belonged to this cluster. Viruses in cluster LP/NW-II were distributed from the north to southern LP and into the adjacent Brits town in the NW province. The two clusters LP/NW-I and LP/NW-II were composed of viruses from both domestic dogs and C. mesomelas. Cluster LP/MP/NWI defines a tight group or viruses (average nucleotide sequence identity of 99.6%) and was clearly distinct from other virus clusters identified in the region (Fig. 3). Viruses in this cluster were obtained exclusively from domestic dogs from north-eastern LP (Fig. 4). This virus variant was later associated with at least 30 human deaths in northern LP (Cohen et al., 2007). The distribution of this viral strain showed that this outbreak also spread to MP province (Fig. 4). Isolate dNW733/99 from Brits in NW province was an outlier in this group of viruses. Viruses from MP represented a separate canid rabies lineage that was divided into two clusters. Clusters MP/LP-I and MP-I were composed of viruses obtained from domestic dogs except one virus isolate (378/90) in cluster MP/LP-I that was obtained from a C. mesomelas from Potgietersrus in central LP. These virus variants were generally distributed in the eastern areas along the borders with Mozambique and Swaziland and in the south eastern areas along the periphery with KwaZulu Natal province.
4. Discussion Canid rabies viruses from northern South Africa displayed limited sequence divergence in the cytoplasmic domain of the glycoprotein and the non-coding G-L intergenic region, confirming a recent and common evolutionary history of these viruses. Genetic
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characterisation, in accord with surveillance data has showed that C. mesomelas and domestic dogs are the principal hosts involved in the transmission of rabies in the northern South Africa. Phylogenetic analysis showed that the viruses from this region could be divided into six variants. The geographical distribution of these virus variants is determined mainly by ecological conditions and land use. For example, rabies variants associated with domestic dogs; LP/MP/NW-I, MP/LP-I and MP-I were identified in communal areas in LP and MP. The virus variants maintained by both canid species were located mainly in commercial farmlands in LP. A distinct virus variant that was only found associated with C. mesomelas, was identified in western LP. The identification of this variant corroborate arguments that C. mesomelas species are capable of maintaining continuous rabies infection cycles independent of domestic dogs, under the specific ecological conditions described here. Based on surveillance data similar findings were made in Zimbabwe for the side-striped jackals (Canis adustus) and black-backed jackals (Bingham et al., 1999). Through trend analysis it has been demonstrated that, despite the effectiveness of dog vaccination approaches, rabies has remained uncontrolled in LP as it is being continuously disseminated by C. mesomelas. With the exception of the period of dog rabies outbreak C. mesomelas has often accounted for most of rabies cases in LP. This observation is suggestive of a maintenance role for the jackal which our phylogenetic analysis reinforces. Since C. mesomelas rabies has always been a concern to many farmers in LP, a rabies suspect jackal would elicit an immediate attention and hence, the largest number of jackal cases is reported from these areas. On the other hand, western LP is sparsely inhabited and is predominantly composed of the bushveld ranches. It is highly likely that the reported cases from this part of LP underestimate the actual prevalence of jackal rabies in the area. Considering its social behaviour, that of a potentially aggressive, territorial carnivore that can occur in relatively high densities in a wide range of habitats (McKenzie, 1993), the black-backed jackal appear to be an ideal rabies vector. Aggressive interactions during territorial defence create an ideal opportunity for intra-species transmission of the virus. Furthermore, the wide ranging movements of these animals together with the sharing of resources such as water and large carcasses facilitate close contact between remote individuals in the population (McKenzie, 1993; Meredith, 1982). It has previously been argued that jackals are unable to maintain continuous rabies cycles because of low population densities compared to domestic dogs (Rhodes et al., 1998; Haydon et al., 2002). However, this concept of long-term persistence is discredited by the wave occurrence nature of rabies epidemics in any vector population (Bingham, 2005) which is determined by various factors, i.e. replenishment of susceptible populations, the emergence of new strains, etc. The high intrinsic population growth rates of C. mesomelas also allow rapid recovery of populations decimated by persecution or disease (Mansvelt, 1956; McKenzie, 1993). It was noted that in commercial farming areas, jackal rabies may not necessarily be independent of rabies in domestic dogs. The existence of rabies cycles involving both domestic dogs and C. mesomelas species clearly demonstrates that the virus is freely communicable between the two species. The overlap of urban and sylvatic rabies cycles has also been demonstrated in other rabies endemic countries in Africa and Latin America for terrestrial cycles of RABV (Nel et al., 1993; Nel and Rupprecht, 2007; Bingham et al., 1999; Clark et al., 1994; Rupprecht et al., 1995; De Mattos et al., 1999; Krebs et al., 1999; Cleaveland and Dye, 1995; Gascoyne et al., 1993). Such cross-species transmission produces the regional diversity of RABV and increases the chances of virus transmission between different canid groups (Carnieli et al., 2006). The recent reemergence of dog rabies in north-eastern LP further illustrated the opportunism shown by RABV through a population of susceptible
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hosts. In MP dog rabies is endemic in the south and eastern areas adjoining Mozambique and Swaziland, the rest of the province has a much lower rabies incidence. Our study provides evidence to suggest that black-backed jackals, given appropriate ecological conditions, are capable of sustaining rabies infection cycles independently of domestic dogs. This conclusion indicates that despite the immunization of domestic dogs, rabies epizootics could be sustained in jackal populations, with the inevitable likelihood of the re-infection of domestic dogs. Hence, wildlife rabies control strategies to complement the existing domestic animal vaccination programs are urgently required to ensure effective control of rabies in the relevant territories of southern Africa. Acknowledgements This work was funded by Department of Science and Technology, South Africa, Department of Agriculture, South Africa (grant # OVI4/16/c171) and Poliomyelitis Research Foundation (grant #05/45). We would also like to thank the previous heads of the OIE Rabies Reference Laboratory; Courtney Meredith, John Bingham and Antoinette Liebenberg for maintaining an archive of rabies viruses. Wilna Vosloo and Melvyn Quan are thanked also for critically reviewing the manuscript and recommendations they have made. References Barnard, B.J.H., 1979. The role played by wildlife in the epizootiology of rabies in South Africa and South West Africa. Onderstepoort J. Vet. Res. 46, 155–163. Bingham, J., Foggin, C.M., Wandeler, A.I., Hill, F.W.G., 1999. The epidemiology of rabies in Zimbabwe. 2. Rabies in jackals (Canis adustus and Canis mesomelas). Ondersterpoort J. Vet. Res. 66, 11–23. Bingham, J., 2005. Canine rabies ecology in Southern Africa. Emerg. Infect. Dis. 11 (9), 1337–1342. Bishop, G.C., Durrhein, D.N., Kloeck, P.E., Godlonton, J.D., Bingham, J., Speare, R., Rabies Advisory Group, 2002. Rabies-Guide for the Medical, Veterinary and Allied Professions. Department of Agriculture, South Africa. Brückner, G.K., Hurter, L.R.B.J.N., 1978. Field observations on the occurrence of rabies in cattle in the magisterial districts of Soutpansberg and Messina. J. S. Afr. Vet. Assoc. 49, 33–36. Carnieli Jr., P., Brandão, P.E., Carrieri, M.L., Castilho, J.G., Macedo, C.I., Machado, M.L., Rangel, N., Cavalcanti de Carvalho, R., Carvalho, V.A., Montebello, L., Wada, M., Kotait, I., 2006. Molecular epidemiology of rabies virus strains isolated from wild canids in North-eastern Brazil. Virus Res. 120, 113–120. Clark, K.A., Neill, S.U., Smith, J.S., Wilson, P.J., Whadford, V.W., McKirahan, G.W., 1994. Epizootic canine rabies transmitted by coyotes in south Texas. J. Am. Vet. Med. Assoc. 204, 536–540. Cleaveland, S., Dye, C., 1995. Maintenance of a microparasite infecting several host species: rabies in the Serengeti. Parasitology 111 (Suppl.), S533–S547. Cleaveland, S., 1998. Epidemiology and control of rabies. The growing problem of rabies in Africa. Trans. R. Soc. Trop. Med. Hyg. 92, 131–134. Cleaveland, S., Kaare, M., Tiringa, P., Mlengeya, T., Barrat, J., 2003. A dog rabies vaccination campaign in rural Africa: impact on the incidence of dog rabies and human dog-bite injuries. Vaccine 21, 1965–1973. Cleaveland, S., Kaare, M., Knobel, D., Laurenson, M.K., 2006. Canine vaccinationproviding broader benefits for the disease control. Vet. Microbiol. 117, 43–50. Cohen, C., Sartorius, B., Sabeta, C., Zulu, G., Paweska, J., Mogoswane, M., Sutton, C., Nel, L.H., Swanepoel, R., Leman, P.A., Grobbelaar, A.A., Dyason, E., Blumberg, L., 2007. The re-emergence of rabies in Limpopo province, South Africa: epidemiology and viral molecular characterization. Emerg. Infect. Dis. 13, 1879–1886. Coetzee, P., Nel, L.H., 2007. Emerging epidemic dog rabies in coastal South Africa: a molecular epidemiological analysis. Virus Res. 126, 186–195. Cumming, D.H.M., 1982. A case history of the spread of rabies in an African country. S. Afr. J. Sci. 78, 443–447. Dean, D.J., Abelseth, M.K., Atanasiu, P., 1996. The fluorescent antibody test. In: Meslin, F.-X., Kaplan, M.M., Koprowski, H. (Eds.), Laboratory Techniques in rabies. World Health Organization, Geneva, pp. 88–95. De Mattos, C., Loza-Rubio, E., Aguilar-Setien, A., Orciari, L.A., Smith, J.S., 1999. Molecular characterisation of rabies virus isolates from Mexico: implications for transmission dynamics and human risk. Am. J. Trop. Med. Hyg. 61, 587–597. Gascoyne, S.C., Laurenson, M.K., Lelo, S., Borner, M., 1993. Rabies in African wild dogs (Lycaon pictus) in the Serengeti region, Tanzania. J. Wildlife Dis. 29, 396–402. Haydon, D.T., Cleaveland, S., Taylor, L.H., Laurenson, M.K., 2002. Identifying reservoirs of infection: a conceptual and practical challenge. Emerg. Infect. Dis. 8, 1468–1473.
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Higgins, D.G., Sharp, P.M., 1989. Fast and sensitive multiple sequence alignments on a microcomputer. Cabios 5, 151–153. Higgins, D., 2003. Multiple alignments. In: Salemi, M., Vandamme, A. (Eds.), The Phylogenetic Handbook: A Practical Approach to DNA and Protein Phylogeny. Cambridge University Press, London, pp. 45–69. Hills, D.M., Bull, J.J., 1993. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst. Biol. 42, 182–192. Knobel, D.L., Cleaveland, S., Coleman, P.G., Fevre, E.M., Meltzer, M.I., Miranda, E.G., Shaw, A., Zinsstag, J., Meslin, F., 2005. Re-evaluating the burden of rabies in Africa and Asia. Bull. World Health Organ. 83 (5), 1–13. Krebs, J.W., Smith, J.S., Rupprecht, C.E., Childs, J.E., 1999. Rabies surveillance in the United States during 1998. J. Am. Vet. Med. Assoc. 215, 1786–1798. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5, 150–163. Mansvelt, P.R., 1956. Rabies in the northern Transvaal (1950) outbreak. J. S. Afr. Vet. Med. Assoc. 27, 167–178. McKenzie, A.A., 1993. Biology of the black-backed jackal Canis mesomelas with reference to rabies. Onderstepoort J. Vet. Res. 60, 367–371. Meredith, C.D., 1982. Wildlife rabies: past and present in South Africa. S. Afr. J. Sci. 78, 411–415. Meslin, F.X., Fishbein, D.B., Matter, H.C., 1994. Rationale and prospects for rabies elimination in developing countries. Curr. Top. Microbiol. Immunol. 187, 1–26. Nel, L.H., Thomson, G.R., Von Teichman, B.F., 1993. Molecular epidemiology of rabies virus in South Africa. Onderstepoort J. Vet. Res. 60, 301–306. Nel, L., Jacobs, J., Jaftha, J., Meredith, C., 1997. Natural spillover of a distinctly Canidaeassociated biotype of rabies virus into an expanded wildlife host range in southern Africa. Virus Genes 15, 79–87. Nel, L.H., Rupprecht, C.E., 2007. Emergence of lyssaviruses in the old world: the case of Africa. In: Childs, J. (Ed.), Current Topics in Microbiology and Immunology: Wildlife and Emerging Zoonotic Diseases: The Biology, Circumstances and Consequences of Cross-Species Transmission. Springer-Verlag, pp. 161–193. Onderstepoort Veterinary Institute Records, 1980–2006. Onderstepoort, Pretoria, South Africa.
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Molecular epidemiology of rabies: Focus on domestic dogs (Canis familiaris) and black-backed jackals (Canis mesomelas) from northern South Africa G.C. Zulu a,b,∗ , C.T. Sabeta a , L.H. Nel b a b
Rabies Unit, ARC-Onderstepoort Veterinary Institute, Private Bag X 05, Onderstepoort, Pretoria 0110, South Africa Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa
a r t i c l e
i n f o
Article history: Received 20 August 2008 Received in revised form 6 November 2008 Accepted 7 November 2008 Available online 20 December 2008 Keywords: Rabies virus Black-backed jackal Domestic dog G-L intergenic region South Africa
a b s t r a c t Phylogenetic relationships of rabies viruses recovered from black-backed jackals (Canis mesomelas) and domestic dogs (Canis familiaris) in northern South Africa were investigated to determine whether the black-backed jackal is an emerging maintenance host species for rabies in this region. A panel of 123 rabies viruses obtained from the two host species between 1980 and 2006 were characterised by nucleotide sequencing of the cytoplasmic domain of the glycoprotein gene and the non-coding G-L intergenic region. Through phylogenetic analysis a viral cluster specific to black-backed jackals and spanning a 5-year period was delineated in western Limpopo. Virus strains associated with domestic dogs prevail in densely populated communal areas in north-eastern Limpopo and in south and eastern Mpumalanga. The data presented in this study indicated the likelihood that black-backed jackals are capable of sustaining rabies cycles independent of domestic dogs. It is proposed that wildlife rabies control strategies, in synergy with domestic animal vaccination should be considered for effective control of rabies in South Africa. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Rabies is a fatal viral disease that affects all warm blooded vertebrates. Despite the availability of vaccines to prevent this disease, it is still a significant public and veterinary health problem in many countries particularly in Asia and Africa (Meslin et al., 1994; Cleaveland, 1998; WHO, 1999; Knobel et al., 2005). The virus causing the disease belongs to genotype 1 of the Lyssavirus genus in Rhabdoviridae family. The rabies virus (RABV) genome consists of a single-stranded, non-segmented negative sense RNA of approximately 12 kb (Tordo et al., 1986). At first, the invasive form of canid rabies was noted in the Limpopo province in South Africa in 1950 following the outbreaks which spread from Angola in 1947 (Swanepoel et al., 1993). During this time, rabies spread primarily in domestic dogs in the densely populated rural areas in Limpopo (LP), Mpumalanga (MP) and KwaZulu Natal (KZN). The virus has since spread widely in domestic dogs throughout the country. To date dog rabies is endemic in many communal areas in Eastern Cape (EC), MP and the entire KZN (Bishop et al., 2002; Sabeta et al., 2007; Coetzee and Nel, 2007). As a consequence, human rabies is also common in these areas.
∗ Corresponding author at: Rabies Unit, ARC-Onderstepoort Veterinary Institute, Private Bag X 05, Onderstepoort, Pretoria 0110, South Africa. Tel.: +27 12 529 9122; fax: +27 12 529 9390. E-mail address: [email protected] (G.C. Zulu). 0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2008.11.004
In addition to domestic dogs, canid rabies is currently sustained by black-backed jackals (Canis mesomelas) in the LP province and bat-eared foxes (Otocyon megalotis) in the Cape region (Sabeta et al., 2007). The black-backed jackal has co-existed with the domestic dog as primary hosts of rabies in LP since 1950 (Swanepoel, 2004). Large black-backed jackal populations are common in bushveld ranches in western LP and in commercial farmland in the north and central areas of this province. Here black-backed jackals have become established as primary carnivores and are thought to reach densities that may be high enough to sustain continuous rabies cycles (Bingham et al., 1999; Cumming, 1982). Jackal rabies has been a serious concern to farmers in LP due to a close correlation between rabies cases in this canid species (C. mesomelas) with those in cattle (Brückner and Hurter, 1978; Barnard, 1979). Moreover, the frequent transmission of RABV between black-backed jackals and domestic dogs (Nel et al., 1993; Sabeta et al., 2003) imposes a serious public health threat. Although the number of human rabies cases caused by jackals is relatively low (Bingham et al., 1999), the consideration of jackals as a rabies problem is linked to its association with domestic dogs. Domestic dogs in their close association with man are the strongest link between rabies in jackals and rabies in humans (McKenzie, 1993). The attempts to control rabies in black-backed jackals that involved the use of meat baits laced with strychnine as poison only provided temporary and localised control of the disease (Mansvelt, 1956). In the case of domestic dogs, parenteral vaccination remains the most effective approach in reducing the spread of rabies in rural African regions (Cleaveland et al., 2003, 2006), hence the risk
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Table 1 Rabies viruses from Limpopo, North-West and Mpumalanga provinces of South Africa included in this study. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76
Reference 1403/80* 1627/80* 1265/80 788/80 47/81 3065/81 101/90* 598/90* 378/90* 45/94 820/94* 446/95* 1034/95 479/96* 504/96* 536/96 373/97* 528/97 224/98* 306/98* 194/99 208/99 223/99 418/99 549/99 557/99 590/99 596/99* 609/99 669/99* 673/99* 675/99* 717/99* 733/99* 1004/99 1024/99 1041/99 1064/99 80/00 171/00 207/00 273/00 354/00 438/00 572/00 596/00 641/00 685/00 714/00 35/00 344/00 390/00 426/00 907/00 27/01 582/01 644/01 42/01 127/01 191/01 433/01 131/02 307/02 422/02 629/02 110/02 136/02 202/02 213/02 193/03 211/03 224/03 343/03 543/03 778/03 64/04
Year of isolation 1980 1980 1980 1980 1981 1981 1990 1990 1990 1994 1994 1995 1995 1996 1996 1996 1997 1997 1998 1998 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2001 2001 2001 2001 2001 2001 2002 2002 2002 2002 2002 2002 2002 2002 2003 2003 2003 2003 2003 2003 2004
Species C. mes C. mes C. fam C. mes C. mes C. mes C. mes C. mes C. mes C. mes C. fam C. mes C. mes C. fam C. mes C. fam C. fam C. mes C. fam C. mes C. mes C. fam C. mes C. mes C. mes C. mes C. mes C. fam C. mes C. mes C. mes C. fam C. mes C. fam C. fam C. fam C. fam C. fam C. mes C. mes C. fam C. fam C. mes C. fam C. fam C. mes C. mes C. mes C. mes C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. mes C. mes C. mes C. fam C. mes C. mes C. mes C. fam C. fam C. fam C. fam C. mes C. mes C. fam C. mes C. mes C. mes C. mes
Province LP LP NW LP NW LP LP LP LP LP NW NW NW LP LP MP MP LP MP LP LP LP LP LP LP LP LP MP LP LP LP NW LP NW LP MP MP MP LP LP LP LP LP LP LP LP LP LP LP MP MP MP MP MP MP MP MP LP LP LP LP MP LP LP LP MP MP MP MP LP LP LP LP LP LP LP
Locality Soutpansberg Soutpansberg Brits Musina Brits Musina Soutpansberg Soutpansberg Potgietersrus Warmbad Vryburg Vryburg Kudumane Thabazimbi Warmbad Carolina Barberton Thabazimbi Ermelo Warmbad Messina Waterberg Polokwane Polokwane Polokwane Polokwane Polokwane Piet Retief Soutpansberg Polokwane Potgietersrus Mankwe Polokwane Brits Polokwane Piet Retief Wakkerstroom Ermelo Potgietersrus Thohoyandou Potgietersrus Polokwane Potgietersrus Soutpansberg Potgietersrus Ellisras Polokwane Potgietersrus Potgietersrus Barberton Barberton Barberton Barberton Piet Retief Barberton Piet Retief Barberton Musina Potgietersrus Soutpansberg Ellisras Piet Retief Ellisras Ellisras Ellisras Barberton Piet Retief Piet Retief Piet Retief Polokwane Louis Trichardt Ellisras Ellisras Ellisras Ellisras Musina
Coordinates (long.–lat.) ◦
◦
29 24 –22 32 29◦ 13 –22◦ 38 27◦ 34 –25◦ 22 29◦ 47 –22◦ 17 27◦ 28 –25◦ 17 29◦ 35 –22◦ 36 28◦ 55 –22◦ 48 29◦ 53 –22◦ 50 28◦ 31 –23◦ 10 27◦ 44 –24◦ 52 24◦ 45 –26◦ 08 24◦ 36 –26◦ 20 23◦ 23 –26◦ 48 27◦ 24 –24◦ 43 27◦ 44 –24◦ 56 30◦ 33 –25◦ 57 31◦ 48 –25◦ 42 26◦ 49 –24◦ 43 29◦ 59 –26◦ 31 28◦ 07 –24◦ 51 29◦ 33 –22◦ 33 28◦ 21 –24◦ 35 29◦ 17 –23◦ 56 29◦ 50 –23◦ 45 29◦ 24 –24◦ 04 29◦ 24 –24◦ 05 29◦ 25 –24◦ 13 30◦ 45 –27◦ 15 30◦ 00 –23◦ 22 29◦ 27 –23◦ 47 28◦ 36 –22◦ 43 27◦ 23 –25◦ 06 29◦ 29 –23◦ 42 27◦ 33 –25◦ 14 29◦ 14 –23◦ 43 30◦ 43 –27◦ 07 30◦ 32 –27◦ 04 30◦ 33 –26◦ 43 28◦ 33 –23◦ 00 29◦ 46 –23◦ 58 29◦ 19 –22◦ 48 29◦ 24 –24◦ 03 28◦ 19 –23◦ 29 29◦ 50 –23◦ 25 28◦ 10 –22◦ 58 27◦ 19 –23◦ 39 29◦ 27 –23◦ 36 28◦ 18 –23◦ 17 28◦ 16 –23◦ 31 31◦ 47 –25◦ 45 31◦ 36 –25◦ 48 31◦ 37 –25◦ 46 31◦ 50 –25◦ 53 30◦ 36 –27◦ 09 31◦ 48 –25◦ 45 30◦ 48 –27◦ 42 31◦ 35 –25◦ 37 29◦ 47 –22◦ 33 29◦ 12 –24◦ 21 30◦ 10 –22◦ 33 27◦ 23 –23◦ 39 30◦ 43 –27◦ 04 27◦ 37 –23◦ 29 27◦ 24 –23◦ 41 27◦ 33 –23◦ 23 31◦ 57 –25◦ 26 30◦ 48 –27◦ 00 31◦ 19 –27◦ 17 30◦ 48 –27◦ 00 29◦ 24 –23◦ 56 29◦ 29 –22◦ 50 28◦ 12 –22◦ 42 27◦ 54 –24◦ 03 27◦ 11 –23◦ 48 28◦ 12 –23◦ 38 29◦ 33 –22◦ 29
Genbank Acc. No. AF177116 AF177104 EF686047 EF686048 EF686049 EF686050 AF079902 AF177117 AF177106 EF686064 AF177118 AF177107 EF686051 AF303070 AF177108 EF686057 AF303069 EF686062 AF177098 AF177105 EF686046 EF686061 EF686059 EF686070 EF686069 EF686054 EF686053 AF303063 EF686055 AF303062 AF303061 AF303071 AF303064 AF303067 EF686052 EF686068 EF686058 EF686080 EF686056 EF686076 EF686063 EF686065 EF686112 EF686060 EF686066 EF686110 EF686075 EF686109 EF686100 EF686077 EF686103 EF686079 EF686071 EF686106 EF686096 EF686072 EF686078 EF686067 EF686074 EF686073 EF686084 EF686140 EF686081 EF686107 EF686083 EF686086 EF686101 EF686113 EF686097 EF686092 EF686108 EF686111 EF686144 EF686088 EF686090 EF686105
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Table 1 (Continued ) No 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
Reference 133/04 187/04 315/04 567/04 606/04 622/04 370/05 391/05 409/05 416/05 449/05 499/05 507/05 535/05 537/05 561/05 565/05 572/05 575/05 686/05 698/05 721/05 728/05 729/05 819/05 757/05 84/06 91/06 98/06 136/06 236/06 288/06 266/06 264/06 262/06 294/06 108/06 296/06 367/06 364/06 272/06 315/06 221/06 304/06 370/06 341/06 197/06
Year of isolation 2004 2004 2004 2004 2004 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006
Species C. mes C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. mes C. fam C. fam C. mes C. fam C. mes C. fam C. mes C. fam C. fam C. mes C. mes C. fam C. mes C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. fam C. mes C .fam C. fam C. fam C. mes C. fam C. mes C. fam C. fam C. fam C. fam C. fam
Province LP MP MP MP MP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP MP LP LP LP LP MP LP LP LP LP LP LP LP LP LP LP LP LP LP MP LP LP LP LP
Locality Louis Trichardt Nkomazi Piet Retief Piet Retief Piet Retief Polokwane Thohoyandou Thohoyandou Thohoyandou Thohoyandou Thohoyandou Sibasa Sibasa Sibasa Louis Trichardt Thohoyandou Polokwane Giyani Polokwane Louis Trichardt Sibasa Louis Trichardt Louis Trichardt Skukuza Soutpansberg Ellisras Sibasa Thohoyandou Ermelo Thohoyandou Louis Trichardt Giyani Phalaborwa Louis Trichardt Sibasa Louis Trichardt Thohoyandou Louis Trichardt Louis Trichardt Phalaborwa Louis Trichardt Polokwane Nkomazi Louis Trichardt Louis Trichardt Louis Trichardt Sibasa
Coordinates (long.–lat.) ◦
◦
29 54 –23 2 31◦ 40 –25◦ 21 27◦ 17 –31◦ 22 31◦ 12 –27◦ 17 31◦ 08 –27◦ 11 29◦ 28 –23◦ 54 30◦ 43 –22◦ 48 30◦ 43 –22◦ 48 30◦ 53 –22◦ 45 30◦ 30 –22◦ 55 30◦ 36 –22◦ 49 30◦ 36 –22◦ 49 30◦ 28 –22◦ 58 30◦ 43 –22◦ 48 29◦ 57 –22◦ 25 30◦ 31 –22◦ 51 29◦ 28 –23◦ 54 (not known) 29◦ 28 –23◦ 54 29◦ 17 –23◦ 2 30◦ 31 –22◦ 51 29◦ 45 –23◦ 15 29◦ 45 –23◦ 15 31◦ 36 –24◦ 59 29◦ 10 –22◦ 43 28◦ 17 –22◦ 47 30◦ 31 –22◦ 51 30◦ 30 –22◦ 55 30◦ 07 –26◦ 05 30◦ 28 –22◦ 58 29◦ 54 –23◦ 2 31◦ 8 –23◦ 56 31◦ 8 –23◦ 56 29◦ 54 –23◦ 2 30◦ 28 –22◦ 54 29◦ 54 –23◦ 2 30◦ 28 –22◦ 58 29◦ 54 –23◦ 2 30◦ 16 –23◦ 6 29◦ 32 –23◦ 55 30◦ 2 –22◦ 54 29◦ 32 –23◦ 55 31◦ 53 –25◦ 33 30◦ 16 –23◦ 6 30◦ 16 –23◦ 6 29◦ 54 –23◦ 2 30◦ 28 –22◦ 58
Genbank Acc. No. EF686099 EF686102 EF686151 EF686104 EF686131 EF686115 EF686082 EF686089 EF686091 EF686098 EF686095 EF686114 EF686085 EF686093 EF686133 EF686094 EF686130 EF686116 EF686117 EF686132 EF686119 EF686129 EF686118 EF686120 EF686121 EF686122 EF686123 EF686124 EF686125 EF686126 EF686127 EF686128 EF686134 EF686135 EF686136 EF686137 EF686138 EF686139 EF686141 EF686142 EF686143 EF686145 EF686146 EF686147 EF686149 EF686150 EF686152
Key: LP, Limpopo; MP, Mpumalanga; NW, North-West; C. mes, Canis mesomelas; C. fam, Canis familiaris. RABV isolates denoted with asterisks were sequenced previously (Sabeta et al., 2003) and were retrieved from GenBank.
of human infection. However, this does not address the control of rabies in wildlife. Thus, this raises concerns about the eradication of the disease given the possibility that wildlife vectors can theoretically act as sources of infection for domestic animals (Bingham et al., 1999; Nel et al., 1997; Rupprecht et al., 2008). Although the black-backed jackal has become a potentially important vector throughout southern Africa, the epidemiology of the disease in this species has not been elucidated. Reports from southern Africa have been contradictory with regard to the issue as to whether black-backed jackals are capable of sustaining rabies infection cycles independently of domestic dogs. Some authors advocated that jackal populations do not reach high enough densities to sustain the infection cycles in the absence of domestic dogs (Rhodes et al., 1998; Haydon et al., 2002). Other authors are of opposing opinion – speculating that domestic dogs introduced rabies infection into jackals, where after the latter maintained their own infection cycles (Cumming, 1982; Bingham et al., 1999). In light of this controversy, the current study was aimed to determine the molecular epidemiological relationships of rabies viruses from domestic dogs and C. mesomelas in order to clarify the com-
plexity of dog and C. mesomelas rabies cycles. Precise knowledge of the distribution of canid rabies in the region and a better understanding of rabies transmission dynamics between domestic dogs and C. mesomelas is needed to underpin effective rabies control strategies. 2. Materials and methods 2.1. Viruses A panel of 123 rabies viruses [domestic dogs n = 69 and blackbacked jackals n = 54] from LP (n = 89), MP (n = 27) and NW (n = 7) provinces collected between 1980 and 2006 were included in this study. Samples of brain material from rabies-suspect animals were submitted to the Rabies Laboratory at the ARC-Onderstepoort Veterinary Institute in Pretoria, South Africa. The samples were tested for the presence of lyssavirus antigen using the fluorescent antibody test (FAT) (Dean et al., 1996) and original infected brain material was stored at −20 ◦ C prior to molecular characterisation. The rabies viruses collected between 1980 and 1998 were passaged once in
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suckling mice and stored as 20% lyophilised mouse brain fractions. Viruses collected after 1998 were not passaged, but stored as original brain material. Full details of the species of origin, year of isolation and area of origin of these virus isolates are presented in Table 1. 2.2. Viral RNA extractions, RT-PCR and sequencing Total RNA was extracted using Trizol reagent (Sigma–Aldrich, USA) as per manufacturer’s recommended protocol. RT-PCR and direct sequencing were performed as described earlier (Sacramento et al., 1991). A consensus sequence was obtained after alignment of the forward and reverse sequences using MEGA 3.1 software. The sequences were trimmed to 592 bp nucleotides which included the cytoplasmic domain of the glycoprotein gene and the G-L intergenic region. All sequences were submitted to GenBank (accession numbers are given in Table 1). 2.3. Phylogenetic analysis Phylogenetic analyses were based on the 592 bp nucleotide sequence region. Nucleotide sequences were aligned using Clustal X (Higgins and Sharp, 1989; Higgins, 2003) and genetic distances calculated using Kimura’s two-parameter method in MEGA 3.1 software (Kumar et al., 2004). A phylogenetic tree was constructed using the Neighbour-joining method (NJ) (Saitou and Nei, 1987). Bootstrap support values were determined using 1000 replicates to evaluate the significance of the branching pattern and values of ≥70% were regarded as providing statistical evidence for phylogenetic groupings (Hills and Bull, 1993). The results were validated by the maximum parsimony method and the Tree Explorer module of MEGA v 3.1 was used to obtain the graphic output.
3. Results 3.1. Rabies prevalence in Limpopo, Mpumalanga and North-West provinces A high proportion of the virus isolates [n = 89 (72%)] used in this study was obtained from the LP province – 51 were recovered from C. mesomelas and 38 from domestic dogs. The majority of RABV isolates associated with domestic dogs were obtained from north-eastern LP and south to eastern MP. Those from jackals were obtained mainly from the north, west and central areas in LP. Rabies trend analysis for the past decade shows that the disease was generally more predominant in the LP in comparison to NW and MP provinces (Fig. 1) (Records of the Onderstepoort Veterinary Institute, 1995–2006). Since this trend analysis was based on passive surveillance it is possible that it might not truly reflect the actual rabies prevalence in the region. During the period under review, dog rabies in LP appeared to have been under control prior to 2005. Since 1995 less than 10 dog rabies cases were reported from LP province annually but a substantial increase was noted from 2005 (Fig. 2). On the other hand, C. mesomelas had a comparatively higher prevalence with an intense outbreak in 1998 (Fig. 2). Other than jackals and dogs, livestock species are the only other species included and numbers of rabies cases in these animals rose in concert with those in canids. 3.2. Genetic characterisation of virus isolates For all the viruses studied here, the G/L primer pair yielded the desired PCR amplicon of approximately 850 bp. Nucleotide sequencing on average yielded approximately 700–750 bases which were then trimmed to 592 bp to include the cytoplasmic domain of the glycoprotein gene and the G-L intergenic region.
Fig. 1. Laboratory confirmed rabies cases in the northern South Africa in the provinces of Limpopo (LP), Mpumalanga (MP), and North-West (NW) from 1995 to 2006 (Records of the Onderstepoort Veterinary Institute, 1995–2006).
Fig. 2. Laboratory confirmed rabies cases in black-backed jackals, domestic dogs and other species (mainly livestock) in the Limpopo province from 1995 to 2006 (Records of the Onderstepoort Veterinary Institute, 1995–2006).
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Fig. 3. Neighbour-joining tree constructed based on a multiple alignment of 592 bp nucleotide sequences encompassing the cytoplasmic domain of the glycoprotein and the G-L intergenic region of 92 canid rabies viruses from Limpopo (LP), North-West (NW) and Mpumalanga (MP) provinces. Bootstrap values based on 1000 replicates are shown on the major branches. The horizontal branch lengths are indicative of evolutionary distances. The scale indicates nucleotide substitutions per site. The Pasteur virus (PV) strain was used to root the tree. The nucleotide sequences are preceded by the prefix d or j to denote species of origin, i.e. d = dog and j = jackal. The species prefix is followed by a second prefix; LP, MP, NW which was used to denote the region where viruses were obtained.
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Fig. 4. Map showing the geographical distribution of the RABV clusters identified in Limpopo, Mpumalanga and North-West provinces. Only the representative virus isolates for the clusters were plotted. Filled symbols denote domestic dogs and empty ones denote black-backed jackals.
Through a pairwise alignment of the sequence data it was demonstrated that for this region of the genome rabies viruses from northern South Africa were closely related – supported by an intrinsic nucleotide sequence identity of 96.7% determined by Kimura 2-parameter model (Kumar et al., 2004). RABV isolates from this region differed on average by 18.7% from the PV strain which was used as a reference. Similar to previously characterised rabies viruses from South Africa and Zimbabwe (Coetzee and Nel, 2007; Sabeta et al., 2003) the isolates characterised in this investigation lacked the first transcription, termination and polyadenylation signal which was found to be present for the G gene of the vaccine (PV and ERA) strains (Sacramento et al., 1992). Virus isolates from the same geographical areas and with 100% nucleotide sequence similarity were removed from further analysis, to improve the clarity of phylogenetic trees. A Neighbour-joining tree (Fig. 3) was thus constructed for 92 rabies viruses to determine the evolutionary relationships between viruses from C. mesomelas and domestic dogs. The topology of the NJ tree was similar to that obtained using a Maximum Parsimony method (results not shown). The branching of the NJ tree demonstrated that rabies viruses from this region could be divided into six clusters (Fig. 3). Cluster LP/NW-I was composed of viruses from LP and NW spanning the period from 1994 to 2006. These viruses were found in the north, central and south western LP as well as in the western part of the NW province (Fig. 4). Cluster LP/NW-I consisted of the largest number of virus isolates from both canid species – this viral cluster may represent the core of the LP epidemic since the early 1990’s. More than 70% of virus isolates in this lineage originated from C. mesomelas. Such observation demonstrates that this viral cluster has been well established in the C. mesomelas population and highlights the important role played by jackals in the transmission of the virus. The most significant finding of this investigation was the identification of cluster LP-I that was composed of viruses exclusively obtained from C. mesomelas and spanning a 5-year period (2000–2005). Our data shows that this virus variant has segregated into a distinct C. mesomelas
rabies cluster. Viruses in this cluster originated from the bushveld ranches and commercial farmland in western LP and were very closely related as shown by 99.8% average nucleotide sequence similarity. RABV isolates recovered between 1980 and 1999 grouped into a distinct cluster LP/NW-II (Fig. 3), there were however no recent viruses obtained from the region that belonged to this cluster. Viruses in cluster LP/NW-II were distributed from the north to southern LP and into the adjacent Brits town in the NW province. The two clusters LP/NW-I and LP/NW-II were composed of viruses from both domestic dogs and C. mesomelas. Cluster LP/MP/NWI defines a tight group or viruses (average nucleotide sequence identity of 99.6%) and was clearly distinct from other virus clusters identified in the region (Fig. 3). Viruses in this cluster were obtained exclusively from domestic dogs from north-eastern LP (Fig. 4). This virus variant was later associated with at least 30 human deaths in northern LP (Cohen et al., 2007). The distribution of this viral strain showed that this outbreak also spread to MP province (Fig. 4). Isolate dNW733/99 from Brits in NW province was an outlier in this group of viruses. Viruses from MP represented a separate canid rabies lineage that was divided into two clusters. Clusters MP/LP-I and MP-I were composed of viruses obtained from domestic dogs except one virus isolate (378/90) in cluster MP/LP-I that was obtained from a C. mesomelas from Potgietersrus in central LP. These virus variants were generally distributed in the eastern areas along the borders with Mozambique and Swaziland and in the south eastern areas along the periphery with KwaZulu Natal province.
4. Discussion Canid rabies viruses from northern South Africa displayed limited sequence divergence in the cytoplasmic domain of the glycoprotein and the non-coding G-L intergenic region, confirming a recent and common evolutionary history of these viruses. Genetic
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characterisation, in accord with surveillance data has showed that C. mesomelas and domestic dogs are the principal hosts involved in the transmission of rabies in the northern South Africa. Phylogenetic analysis showed that the viruses from this region could be divided into six variants. The geographical distribution of these virus variants is determined mainly by ecological conditions and land use. For example, rabies variants associated with domestic dogs; LP/MP/NW-I, MP/LP-I and MP-I were identified in communal areas in LP and MP. The virus variants maintained by both canid species were located mainly in commercial farmlands in LP. A distinct virus variant that was only found associated with C. mesomelas, was identified in western LP. The identification of this variant corroborate arguments that C. mesomelas species are capable of maintaining continuous rabies infection cycles independent of domestic dogs, under the specific ecological conditions described here. Based on surveillance data similar findings were made in Zimbabwe for the side-striped jackals (Canis adustus) and black-backed jackals (Bingham et al., 1999). Through trend analysis it has been demonstrated that, despite the effectiveness of dog vaccination approaches, rabies has remained uncontrolled in LP as it is being continuously disseminated by C. mesomelas. With the exception of the period of dog rabies outbreak C. mesomelas has often accounted for most of rabies cases in LP. This observation is suggestive of a maintenance role for the jackal which our phylogenetic analysis reinforces. Since C. mesomelas rabies has always been a concern to many farmers in LP, a rabies suspect jackal would elicit an immediate attention and hence, the largest number of jackal cases is reported from these areas. On the other hand, western LP is sparsely inhabited and is predominantly composed of the bushveld ranches. It is highly likely that the reported cases from this part of LP underestimate the actual prevalence of jackal rabies in the area. Considering its social behaviour, that of a potentially aggressive, territorial carnivore that can occur in relatively high densities in a wide range of habitats (McKenzie, 1993), the black-backed jackal appear to be an ideal rabies vector. Aggressive interactions during territorial defence create an ideal opportunity for intra-species transmission of the virus. Furthermore, the wide ranging movements of these animals together with the sharing of resources such as water and large carcasses facilitate close contact between remote individuals in the population (McKenzie, 1993; Meredith, 1982). It has previously been argued that jackals are unable to maintain continuous rabies cycles because of low population densities compared to domestic dogs (Rhodes et al., 1998; Haydon et al., 2002). However, this concept of long-term persistence is discredited by the wave occurrence nature of rabies epidemics in any vector population (Bingham, 2005) which is determined by various factors, i.e. replenishment of susceptible populations, the emergence of new strains, etc. The high intrinsic population growth rates of C. mesomelas also allow rapid recovery of populations decimated by persecution or disease (Mansvelt, 1956; McKenzie, 1993). It was noted that in commercial farming areas, jackal rabies may not necessarily be independent of rabies in domestic dogs. The existence of rabies cycles involving both domestic dogs and C. mesomelas species clearly demonstrates that the virus is freely communicable between the two species. The overlap of urban and sylvatic rabies cycles has also been demonstrated in other rabies endemic countries in Africa and Latin America for terrestrial cycles of RABV (Nel et al., 1993; Nel and Rupprecht, 2007; Bingham et al., 1999; Clark et al., 1994; Rupprecht et al., 1995; De Mattos et al., 1999; Krebs et al., 1999; Cleaveland and Dye, 1995; Gascoyne et al., 1993). Such cross-species transmission produces the regional diversity of RABV and increases the chances of virus transmission between different canid groups (Carnieli et al., 2006). The recent reemergence of dog rabies in north-eastern LP further illustrated the opportunism shown by RABV through a population of susceptible
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hosts. In MP dog rabies is endemic in the south and eastern areas adjoining Mozambique and Swaziland, the rest of the province has a much lower rabies incidence. Our study provides evidence to suggest that black-backed jackals, given appropriate ecological conditions, are capable of sustaining rabies infection cycles independently of domestic dogs. This conclusion indicates that despite the immunization of domestic dogs, rabies epizootics could be sustained in jackal populations, with the inevitable likelihood of the re-infection of domestic dogs. Hence, wildlife rabies control strategies to complement the existing domestic animal vaccination programs are urgently required to ensure effective control of rabies in the relevant territories of southern Africa. Acknowledgements This work was funded by Department of Science and Technology, South Africa, Department of Agriculture, South Africa (grant # OVI4/16/c171) and Poliomyelitis Research Foundation (grant #05/45). We would also like to thank the previous heads of the OIE Rabies Reference Laboratory; Courtney Meredith, John Bingham and Antoinette Liebenberg for maintaining an archive of rabies viruses. Wilna Vosloo and Melvyn Quan are thanked also for critically reviewing the manuscript and recommendations they have made. References Barnard, B.J.H., 1979. The role played by wildlife in the epizootiology of rabies in South Africa and South West Africa. Onderstepoort J. Vet. Res. 46, 155–163. Bingham, J., Foggin, C.M., Wandeler, A.I., Hill, F.W.G., 1999. The epidemiology of rabies in Zimbabwe. 2. Rabies in jackals (Canis adustus and Canis mesomelas). Ondersterpoort J. Vet. Res. 66, 11–23. Bingham, J., 2005. Canine rabies ecology in Southern Africa. Emerg. Infect. Dis. 11 (9), 1337–1342. Bishop, G.C., Durrhein, D.N., Kloeck, P.E., Godlonton, J.D., Bingham, J., Speare, R., Rabies Advisory Group, 2002. Rabies-Guide for the Medical, Veterinary and Allied Professions. Department of Agriculture, South Africa. Brückner, G.K., Hurter, L.R.B.J.N., 1978. Field observations on the occurrence of rabies in cattle in the magisterial districts of Soutpansberg and Messina. J. S. Afr. Vet. Assoc. 49, 33–36. Carnieli Jr., P., Brandão, P.E., Carrieri, M.L., Castilho, J.G., Macedo, C.I., Machado, M.L., Rangel, N., Cavalcanti de Carvalho, R., Carvalho, V.A., Montebello, L., Wada, M., Kotait, I., 2006. Molecular epidemiology of rabies virus strains isolated from wild canids in North-eastern Brazil. Virus Res. 120, 113–120. Clark, K.A., Neill, S.U., Smith, J.S., Wilson, P.J., Whadford, V.W., McKirahan, G.W., 1994. Epizootic canine rabies transmitted by coyotes in south Texas. J. Am. Vet. Med. Assoc. 204, 536–540. Cleaveland, S., Dye, C., 1995. Maintenance of a microparasite infecting several host species: rabies in the Serengeti. Parasitology 111 (Suppl.), S533–S547. Cleaveland, S., 1998. Epidemiology and control of rabies. The growing problem of rabies in Africa. Trans. R. Soc. Trop. Med. Hyg. 92, 131–134. Cleaveland, S., Kaare, M., Tiringa, P., Mlengeya, T., Barrat, J., 2003. A dog rabies vaccination campaign in rural Africa: impact on the incidence of dog rabies and human dog-bite injuries. Vaccine 21, 1965–1973. Cleaveland, S., Kaare, M., Knobel, D., Laurenson, M.K., 2006. Canine vaccinationproviding broader benefits for the disease control. Vet. Microbiol. 117, 43–50. Cohen, C., Sartorius, B., Sabeta, C., Zulu, G., Paweska, J., Mogoswane, M., Sutton, C., Nel, L.H., Swanepoel, R., Leman, P.A., Grobbelaar, A.A., Dyason, E., Blumberg, L., 2007. The re-emergence of rabies in Limpopo province, South Africa: epidemiology and viral molecular characterization. Emerg. Infect. Dis. 13, 1879–1886. Coetzee, P., Nel, L.H., 2007. Emerging epidemic dog rabies in coastal South Africa: a molecular epidemiological analysis. Virus Res. 126, 186–195. Cumming, D.H.M., 1982. A case history of the spread of rabies in an African country. S. Afr. J. Sci. 78, 443–447. Dean, D.J., Abelseth, M.K., Atanasiu, P., 1996. The fluorescent antibody test. In: Meslin, F.-X., Kaplan, M.M., Koprowski, H. (Eds.), Laboratory Techniques in rabies. World Health Organization, Geneva, pp. 88–95. De Mattos, C., Loza-Rubio, E., Aguilar-Setien, A., Orciari, L.A., Smith, J.S., 1999. Molecular characterisation of rabies virus isolates from Mexico: implications for transmission dynamics and human risk. Am. J. Trop. Med. Hyg. 61, 587–597. Gascoyne, S.C., Laurenson, M.K., Lelo, S., Borner, M., 1993. Rabies in African wild dogs (Lycaon pictus) in the Serengeti region, Tanzania. J. Wildlife Dis. 29, 396–402. Haydon, D.T., Cleaveland, S., Taylor, L.H., Laurenson, M.K., 2002. Identifying reservoirs of infection: a conceptual and practical challenge. Emerg. Infect. Dis. 8, 1468–1473.
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