Both domestic and wild carnivore species are commonly diagnosed with rabies virus (RABV) infection in South Africa. Although the majority of confirmed rabies cases in wild carnivore species are reported from the yellow mongoose (
The wild dog (
Increasingly, spillover of canine pathogens such as rabies virus (RABV) (Kat et al.
Rabies contributed significantly to wild dog mortalities in the 1990s in the Serengeti, in Tanzania (Cleaveland & Dye
According to Haydon et al. (
Serological evidence lends support to exposure of some wildlife species to a plethora of pathogens. For example, it was shown that spotted hyaenas in East Africa, black-backed jackals and captive cheetahs in Namibia, as well as the African wild dog, were exposed to bacterial pathogens such as
Serological evidence of exposure to RABV and CDV in domestic animals and wildlife demonstrated that domestic dogs were maintenance hosts for RABV and that exposure was more likely in this species than in the wild carnivores (Prager et al.
The potential threat of zoonotic disease transfer is increasing with the expanding global human population, along with increasing global tourism, the decrease in natural habitat areas for wildlife and the increasing human–wildlife interface in and around game parks. There is therefore not only the need to adopt appropriate countermeasures for the protection of endangered wildlife species such as wild dogs but also the associated need to diminish opportunities for zoonotic disease transfer to humans.
Conservation of wild carnivores has been successful by means of the application of mass domestic dog parenteral vaccination. Data from the field demonstrated that annual vaccination of at least 67% of the domestic dog population resulted in the control of rabies in Ngorongoro in Tanzania (Fitzpatrick et al.
A study was conducted by Van Heerden et al. (
An outbreak of rabies occurred in 2000 in a pack of wild dogs in the Madikwe Game Reserve, South Africa (Hofmeyr et al.
Recently, wild carnivore species are increasingly being devastated by emerging and re-emerging zoonotic diseases. This report describes recent laboratory-confirmed RABV infections in two wild dogs and a hyaena in the Madikwe Game Reserve (North West province of South Africa). Through antigenic and genetic characterisation as well as background Madikwe predator and environmental data, the report attempts to identify the possible sources of infection in the park.
Madikwe Game Reserve is situated in the North West province of South Africa and is bordered on the west by Botswana and was announced in 1991. It is spread over 680 km2, which has been expanded to 750 km2 by newly incorporated private reserves. It was previously degraded farm land and has recovered as a result of sustainable use by a diversity of wildlife including 66 large animal species. The perimeter is 150 km of electrified fencing. The geography is large open grassland plains, woodlands with rocky outcrops and single mountains (
On 26 December 2014, a wild dog was found biting at light bulbs in a tourist lodge in Madikwe Game Reserve (
Map showing the North West province of South Africa.
Half of the brain from the carcasses of the first male wild dog (Agricultural Research Council – Onderstepoort Veterinary Institute [ARC-OVI] reference number# 889/14, i.e. sample number 889 of 2014), the second wild dog (ARC-OVI reference number# 66/15) and the spotted hyaena (ARC-OVI reference number# 113/15) were submitted on ice to the OIE Rabies Reference Laboratory at the ARC-OVI to exclude lyssavirus infection. Immunohistochemistry (IHC) was carried out at the Section of Pathology, Department of Paraclinical Sciences at the Faculty of Veterinary Science, University of Pretoria, Onderstepoort on formalinised, wax-embedded brain tissues from the first wild dog and the hyaena. On receipt of the specimens at ARC-OVI, composite brain smears were prepared and subjected to the direct fluorescent antibody test (dFAT) as described previously (Dean, Abelseth & Ataanasiu
The lyssaviruses confirmed in the 1997 wild dog rabies outbreak together with the most recent viruses from wild dogs and a hyaena from the Madikwe Game Reserve and adjacent localities were genetically characterised by amplifying and sequencing a partial region of the highly conserved nucleoprotein (N) gene. In brief, Trizol-extracted viral ribonucleic acid (RNA) from original RABV-infected brain tissues (approximately 200 ng of tissue) and freeze-dried infected mouse brain material (for wild dog viruses recovered prior to 2000, see
Rabies virus isolates used in the study including wild and domestic animal isolates isolated over a 19-year period.
Lyssavirus antigen was confirmed in the brain tissues of the two adult wild dogs and the hyaena using dFAT and for the first wild dog and hyaena using IHC. Typical apple-green fluorescence virus–infected particles were observed in 100% of the field of the composite brain smear and in addition, antigen was observed immunohistochemically in glandular epithelium of the mandibular salivary gland of the first wild dog (889/14) (see
(a) Wild dog hippocampus; rabies antibody immunohistochemical labelling with haematoxylin counterstain (1000× magnification). Viral inclusion bodies, Negri bodies, present within hippocampal neurons and small viral inclusions (rabies dust) present within the neuropil (arrows). (b) Wild dog (case 889/14) mandibular salivary gland, immunohistochemical rabies antibody labelling with haematoxylin counterstain (400× magnification). Numerous small red-brown viral inclusion bodies (arrows) within salivary gland epithelium. This corroborates the known route of transmission via saliva and indicates the advanced stage of infection with rabies virus.
Nucleotide sequencing and phylogenetic analysis of the wild dog RABV partial nucleoprotein gene sequences together with other previously characterised domestic dog and wildlife RABV sequences from South Africa confirmed that canid rabies variant was circulating in these wild dogs (
Rabies trends in the North West province of South Africa over a 10-year period (2005–2015) showed that significant numbers of cases, for example, 44.4% (2007), 58.8% (2012) and 18.3% (2014) of the total annual confirmed animal cases, were positive wildlife cases as recorded at the OIE Rabies Reference Laboratory at the Onderstepoort Veterinary Institute. Whereas the RABVs from wildlife species such as jackals and hyaena in 2007 were of the mongoose rabies biotype, those in 2012 and 2014 were of the canid rabies biotype (Onderstepoort Veterinary Institute records), highlighting the biological variations within the classical RABV variants found in southern Africa and the occurrence of spillover events between wildlife and domestic host species (Nel et al.
A phylogenetic tree showing the genetic placement and relationships of the recent wild dog rabies virus (clade 1) with rabies virus from other wild carnivores and domestic dogs associated with rabies outbreaks in the North West province.
Numbers of positive rabies cases in various species from 2012 to 2015 from the North West province.
Utilising primary diagnostic tests such as the dFAT and IHC, we were able to confirm lyssavirus infection in two wild dogs and a hyaena from Madikwe. The lyssaviruses identified in the current 2014–2015 outbreak inside the park were canid rabies biotype, which delineated into clade 1b of two sub-clusters, 1a and 1b, both consisting of a mixture of domestic (dog) and wildlife (jackal) viruses (
In Madikwe Game Reserve in 1997 (Hofmeyr et al.
According to Haydon et al. (
In South Africa, ‘predator proof’ fencing around small reserves has been very effective at keeping wild dogs confined inside the reserves, provided the fence is properly maintained, but such fencing has also been reported not to be 100% effective (Davies-Mostert, Mills & Macdonald
Only 2 out of 39 radio-collared jackals managed to pass through the fence to the outside of Madikwe, using holes made by aardvarks (
In the past 15 years, there has been no record or memory of free-roaming foreign domestic dog sightings within the park. Occasionally, however, domestic cats have been seen and were immediately euthanised by park officials, but not submitted for rabies testing. Cats appear unlikely as a source of RABV infection but cannot be ruled out. Between 2010 and 2016, six cats from the North West province but outside of Madikwe Game Reserve were found to be lyssavirus positive by the OIE Rabies Reference Laboratory, Onderstepoort Veterinary Institute, but only one of these was typed further and found to be mongoose biotype, as were the two feline cases in
Pet dogs belonging to park personnel in Madikwe are supposed to remain enclosed within fencing at staff housing sections; however, occasionally these dogs do escape temporarily into the park. However, all these dogs were vaccinated annually by a veterinarian, including for rabies, with certificates kept up to date. The outlying domestic dog and cat population, for example, those in Molatedi village which lies close to Madikwe, and a subsistence farmer known to have dogs living adjacent to the southern boundary fence should have had their dogs vaccinated as part of a campaign driven by the state; however, on enquiry, vaccination records were not available.
Based on the preceding information, transmission of RABV from jackals to other wildlife carnivores within Madikwe is more likely than from rabid animals occurring outside of Madikwe. Although territorial jackals are generally more spatially confined than dispersing jackals, they have been regularly recorded in Madikwe to travel straight line distances in excess of 8 km from core areas to seek food and water resources. In contrast, dispersal and floater jackals have been recorded to wander over the whole of Madikwe in their quest to seek those areas of least competition from dominant jackals for space and food resources and to facilitate breeding opportunities (field records of RH-W). In this respect, where wild carnivores like lions, hyaenas or wild dogs have made substantial-sized kills, jackals are the most frequently observed mammalian scavengers (field data of RH-W, Madikwe). In Etosha National Park, jackals are the most frequently observed scavengers at anthrax-confirmed carcasses (Bellan et al.
The large wild dog pack inhabited mainly the eastern region of the Madikwe Game Reserve and always hunted along the eastern boundary fence (unpublished field data of RH-W). A small number of bat-eared foxes were also recorded in the north-eastern region of the park. Aggressive interactions between wild dogs inside the park and canid species outside the park may occur through the fence but had not been recorded up to 2016.
Wild dogs usually engorge themselves on fresh carcass kills, mostly kudu in Madikwe; they then lie bloated a little distance from the carcass remains. Although jackals are generally wary of wild dogs with wild dogs often chasing jackals from kills (field observations of RH-W), the disorientation and aggressive behaviour of jackals in the terminal phases of rabies may encourage close physical contact interactions between such rabid jackals and wild dogs.
A factor of likely significance in the period prior to the 2014 wild dog rabies outbreak in Madikwe was the effect on the wild dog behaviour of the chemical contraception applied by the park in 2014 as a result of their rapidly expanding population. Wild dogs numbered 34–36 in February 2014 and 26 in September 2014, at which time contraception was introduced. Their numbers thereafter dropped to 13 when counted on 16 February 2015 because of the subsequent rabies outbreak (field data of RH-W). The reproductive intervention had resulted in fragmentation of the northern and southern packs and creation of smaller groups, which appeared to increase antagonistic encounters between these splinter groups. These aggressive contact encounters would likely have exacerbated intra-specific, wild dog rabies transmission. Mills (
Between 2008 and 2010, the Madikwe jackal population was drastically reduced as a consequence of the high lion numbers and densities. The latter resulted in significant opportunistic lion predations on jackals and also an inability of jackals to access lion kills, which in turn were devoured within a short space of time. Lion numbers were reduced steadily between 2010 and 2015 as a result of management initiatives. Jackal numbers are positively favoured when lion numbers are low and when the percentage of lions under 2 years in the population is small, as young lions have been regularly recorded to kill jackals (field observation data of RH-W, Madikwe).
On 15 of January 2015, a video was recorded of a wild dog male eating a dead wild dog (data of RH-W), highly likely at that time to have died of rabies. The last wild dog of the larger pack, which was the second positively diagnosed wild dog case, was shot and submitted to Onderstepoort on 26 January 2015.
The devastating effect of rabies on the wild dog population in Madikwe during the latter half of 2014 was not paralleled in the jackal population (data of RH-W), although data suggest that jackal breeding pairs declined by 15.7% between 2013 and 2014 (pre-rabies outbreak) and by a further 6.2% between 2014 and 2015. In this respect, severe mange, small jackal litter sizes, poor jackal pup survivals and a continued lack of good jackal representations at large carcasses and lion kills between 2013 and 2015 implicate the progressively worsening drought conditions and associated food deprivations as pivotal factors in the poor performance of jackals during this period. Although it was not possible in 2014 to confirm whether all adult jackal pairs associated with pup mortalities were alive after the loss of some or all their pups, some jackal pairs were recorded in their territorial areas after these after the death of these pups. These data challenge rabies as an implicating factor in jackal population mortalities during the latter half of 2014, when rabies probably peaked in the Madikwe wild dog population. This hypothesis is further supported by the observed increase in the numbers of jackals reported at large carcasses during 2016, which suggests that jackal pup survival rates from 2015 jackal litters had improved. The increased availability of food from the extraordinarily large amount of ungulate drought-related mortalities that were present in the field during the latter half of 2015 would have favoured jackal pup survival. The quandary with respect to the origins of the rabies outbreak and as to whether there is a possibility that rabies is present and maintained in certain wild species populations as reservoir hosts within Madikwe needs further investigation (field data and interpretation of RH-W).
Spread of rabies amongst members of a spotted hyaena clan as well as inter-clan spread depends on the number of clans, clan size, their density in an area and on the ranks of the affected animals, which would affect behaviour and potential contacts with other hyaenas. In general, widely distributed clans or clans with few members would show slow intra-clan and inter-clan spread. Spotted hyaenas rarely meet with all clan members and individuals tend to be solitary or in small groups. They are more territorial than wild dogs: they scent-mark and defend their territories from other clans (Mills
In February 2015, the remaining five adult wild dogs and eight pups in Madikwe were vaccinated against rabies but six pups still died, although none were tested for rabies. Wild dog pups in Kruger National Park have been recorded to have a high mortality rate, many because of unknown cause but several by lion predation (Mills
As human populations increase around reserve borders, the risk to wild dogs which venture outside or escape from parks is also likely to increase. Humans in game parks are at considerable risk of contact with rabid carnivores because several, as in this situation, are shot within tourist lodges or camps or in close proximity to them (Bellan et al.
Contact between some wild carnivores such as jackals and domestic dogs is a common phenomenon in southern Africa and provides an ideal opportunity for pathogen transmission across species boundaries (Bingham
Molecular epidemiology, the scientific investigation that focuses on the contribution of potential genetic and environmental risk factors, has now become useful in tracing routes and origins of infection, as well as in identifying the emergence of new variants, as exemplified by lyssavirus evolution; a phenomenon illustrated in the current study. A database of nucleotide sequences for the N gene for South African domestic and wildlife carnivore species will be useful in future in order to assess origins and mapping of movement of rabies outbreaks. Similarly, nucleotide sequences of the rabies viral N and glycoprotein (G) genes from virus isolates of four wild dogs (including an individual from Tanzania) indicated that a viral variant common amongst domestic dogs was responsible for RABV infection in wildlife carnivores in Kenya and Tanzania (Kat et al.
Molecular techniques have been shown to be useful in confirming RABV infection even in decomposed and exhumed carcasses, as recently demonstrated by two independent groups (Markotter et al.
The value of IHC for rabies diagnosis in formalin-fixed samples was clearly demonstrated in the current study. If fresh tissues cannot be submitted for whatever reason, as commonly happens in remote areas of South Africa and other parts of the continent, or where other preservatives such as glycerol saline may be in short supply, IHC can be considered as an equivalent alternative method for rabies diagnosis in animal tissues where diagnostic facilities offer such tests. Furthermore, with IHC, the virus inhabiting cells and any associated pathological changes in the brain are clearly observable, in comparison with other diagnostic methods such as the dFAT or molecular techniques. Formalin fixation of the brain preserves tissue architecture and allows histological evaluation to formulate a differential diagnosis (Stein et al.
Personnel and laboratory employees should treat all fresh samples from suspect rabies cases as potentially rabies-infected and when appropriate throughout the dFAT procedure, as RABV is not inactivated with acetone fixation (Jarvis, Franke & Davis
In conclusion, in order for effective control of rabies outbreaks in domestic mammals, non-endangered and endangered wildlife and humans, the epidemiology of RABV infection in these species and in varying circumstances requires ongoing intensive study. This implies that study of predator interactions, movements, behaviour, food sources, competition and the dynamics which alter with human interventions and natural environmental changes within the confines of game parks should be ongoing. Close monitoring of species numbers and submission of material for diagnosis when carcasses are found or when animals are euthanised as a result of aberrant behaviour are essential. Because of its multi-host nature, RABV is an important threat to ecologically threatened animal populations such as the wild dog. RABV is a pathogen that can be effectively controlled or eliminated at the primary host animal source by vaccination. Parenteral vaccination is key to the potential elimination of some viral diseases from domestic dogs and cats and further mitigates transmission of rabies and other pathogens to both wildlife and humans. The diligent ongoing implementation of suitable, effective vaccination of wild carnivores against RABV for continued prevention of the disease and the preservation of these species, although controversial, is recommended.
The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.
C.T.S. initiated the study and was in control of the initial diagnosis, molecular testing and interpretation of PCR results at the World Organisation for Animal Health (OIE) rabies reference laboratory; he wrote up most of the introduction, molecular biology methods and results; compiled the table and map; and wrote much of the discussion. D.D.J.v.R. contributed the histopathology of the second wild dog and the hyaena case, wrote the section on immunohistochemistry with its discussion and contributed the histopathology images and legends. B.P. amplified some of the viruses after accepting the fresh tissue samples submitted to the Onderstepoort Veterinary Institute OIE rabies reference laboratory and sequenced and generated phylogenetic trees. B.P. also contributed to the writing of the manuscript. D.M. antigenically typed the viruses involved and generated trends analyses of rabies in the North West. R.F.H-W. contributed all the field data pertaining to the numbers and environmental observations in Madikwe Game Reserve during the time prior to and during and after the rabies outbreak and interpreted those findings – he supplied information on the running of the park and maintenance of the fence; the movement of radio-collared jackals mentioned is data from his individual project. C.E. was Field Ecologist in Madikwe at the time and provided the information on the situation around the behaviour and shooting of the two wild dogs as well as pack numbers and movements just prior to the losses. She also provided field information on the hyaena that was found dead. She also provided precise information on vaccination and control measures taken against rabies and other infectious diseases in the pets belonging to Park staff. J.H.W. took the specimens from and wrote the histopathology of the first wild dog case and did further research for both introduction and discussion, specifically with respect to possible epidemiology and reservoir hosts, especially jackal; she also recruited both C.E. and later R.F.H-W. to contribute to the background field data and its interpretation, and helped in refining and co-ordinating the final preparation of the manuscript to reach its current form.