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Serological and demographic evidence for domestic dogs as a source of canine distemper virus infection for Serengeti wildlife
Veterinary Microbiology 72 (2000) 217±227
Serological and demographic evidence for domestic dogs as a source of canine distemper virus infection for Serengeti wildlife S. Cleavelanda,b,*, M.G.J. Appelc, W.S.K. Chalmersd, C. Chillingworthd, M. Kaaree, C. Dyea a
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK b Institute of Zoology, Zoological Society of London, Regent's Park, London NW1 4RY, UK c James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA d Intervet (UK) Ltd., The Elms, Thicket Road, Houghton, Huntingdon PE17 2BQ, UK e Ministry of Agriculture and Cooperatives, P.O. Box 203, Musoma, Tanzania
Received 4 August 1999; received in revised form 30 November 1999; accepted 30 November 1999
Abstract Following an epidemic of canine distemper virus (CDV) in Serengeti lions in 1994, the role of domestic dogs in the epidemiology of the disease was investigated by serological and demographic analyses. From 1992 to 1994, data were collected from two domestic dog populations bordering the Serengeti National Park. Several lines of evidence indicated that patterns of CDV infection differed signi®cantly between higher-density dog populations of Serengeti District to the west of the park and lower-density populations of Ngorongoro District to the south-east: (a) CDV ageseroprevalence patterns differed signi®cantly between years in Ngorongoro District populations but not in Serengeti District populations; (b) CDV seropositive pups (<12 months of age) were detected in Ngorongoro District only in 1994, whereas a proportion of pups in Serengeti District were seropositive in each year of the study; (c) in Ngorongoro District, the proportion of deaths attributed to disease was signi®cantly higher in 1994 than in 1993, whereas in Serengeti District, there was no signi®cant difference in disease-related mortality between years; (d) in Ngorongoro District, signi®cantly more CDV seronegative dogs than seropositive dogs died in 1994, whereas *
Corresponding author. Present address: Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Roslin, Midlothian EH25 9RG, UK. Tel.: 44-131650-6404; fax: 44-131-445-5099. E-mail address: [email protected] (S. Cleaveland). 0378-1135/00/$ ± see front matter # 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 2 0 7 - 2
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there was no difference in survival of CDV seropositives and seronegatives between years in Serengeti District. We concluded that, between 1992 and 1994, CDV persisted in higher-density dog populations of Serengeti District, but occurred only sporadically in lower-density Ngorongoro District populations. Data from Ngorongoro District are consistent with exposure of dogs to CDV in 1991 and 1994, but not in 1992 and 1993. These ®ndings suggest that higher-density domestic dog populations to the west of the Serengeti National Park were a more likely source of CDV infection for wildlife during 1994 than lower-density pastoralist dogs to the south and east of the park. # 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Dog canine distempervirus; Epidemiology; Serology; Wildlife; Serengeti; Kenya; Tanzania
1. Introduction From late 1993 to 1994, an epidemic of canine distemper (CD) was recorded in the lion (Panthera leo) population of the Serengeti ecosystem, with cases con®rmed in the Serengeti National Park and Maswa Game Reserve in Tanzania (Roelke-Parker et al., 1996) and in the Masai Mara National Reserve, Kenya (Kock et al., 1998). Approximately 30% of all known lions within a closely-monitored study population of the Serengeti National Park either died or disappeared. During this epidemic, the disease was also con®rmed in spotted hyaenas (Crocuta crocuta) (Haas et al., 1996) and bat-eared foxes (Otocoyon megalotis) (Roelke-Parker et al., 1996), as well as in domestic dog populations surrounding the Ngorongoro Crater (Cleaveland, 1996). Virus isolates obtained from lions, domestic dog, hyaena and bat-eared fox during the epidemic showed identical monclonal antibody reaction patterns, which were indistinguishable from the pattern obtained for virulent canine distemper virus (CDV) strains isolated from domestic dogs (Cleaveland, 1996; Roelke-Parker et al., 1996). Molecular genetic analyses con®rmed the close similarity between Serengeti lion viruses and virulent, wild-type CDV (Roelke-Parker et al., 1996). These analyses also demonstrated close phylogenetic homology among the Serengeti viruses, with a tendency for isolates to cluster according to geographic rather than host species origin (Haas et al., 1996; Harder et al., 1996; Roelke-Parker et al., 1996; Carpenter et al., 1998). These ®ndings suggest that a single virus strain caused mortality in a range species in the Serengeti and that this strain was transmissible between domestic dogs and wild carnivores. Although molecular studies point to a single virus strain affecting multiple hosts, the exact role of different hosts species in the maintenance and transmission of CDV in the Serengeti remains unresolved. Here, the role of domestic dogs was investigated through comparative serological and demographic analyses of high- and low-density domestic dog populations living adjacent to the park. Data were also presented from cross-sectional and longitudinal studies of two populations comparing (i) CDV ageseroprevalence patterns (ii) disease-related mortality and (iii) survival of CDV seropositives and CDV seronegatives. These ®ndings, in combination with published data from Serengeti wildlife populations, were used to explore the dynamics of CDV infection in the Serengeti.
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Fig. 1. Map showing location of study villages (*) around the Serengeti National Park.
2. Materials and methods 2.1. Study areas The study area was the Serengeti ecological region of Northwestern Tanzania (358 to 368E, 18 300 to 38 70 S). Villages were selected from two districts adjacent to the Serengeti National Park with the aim of sampling dogs from as broad a geographic range as possible. Study villages were located within Ngorongoro District and Serengeti District, which are de®ned by administrative boundaries (Fig. 1). The Ngorongoro District, comprising the Loliondo Game Controlled Area and the Ngorongoro Conservation Area, is a multiple-use controlled wildlife area, inhabited predominantly by Maasai people practising traditional pastoralism and limited cultivation. Human population density was estimated as 2.89 people/km2 in 1992, based on 1988 human census data with a projected population growth rate of 3.4% per annum (Bureau of Statistics, 1991). Estimates of domestic dog densities, determined from the number of dogs per Maasai boma (household) and the aerial distribution of bomas
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(Tanzanian Wildlife Conservation Monitoring, 1991), ranged from 0.38 to 0.46 dogs/km2 from 1992 to 1994 (Cleaveland, 1996). In Serengeti District, agropastoralism is the dominant pattern of land use, with production systems based on livestock and cultivation of crops, such as cassava and maize. Human and domestic dog population densities were much higher than in Ngorongoro District. The human density in 1992 was estimated as 30.10 km2 , based on 1988 census data and projected growth rates of 2.9% per annum (Bureau of Statistics, 1991). Domestic dog densities, determined from the human:dog ratio, ranged from 5.72 to 7.17 dogs/km2 (Cleaveland, 1996). 2.2. Data collection Data were collected during three ®eld periods: from September 1992 to February 1993 (referred to subsequently as 1992); September to December 1993; August to December 1994. Demographic data on the domestic dog population were collected through questionnaire surveys of householders. In rural villages of Serengeti District, households were sampled within each of four quadrants, using the village centre (site of administrative of®ces) as the central reference point. One in ®ve households situated along a transect from the village centre to the periphery was visited within each quadrant. In low-density areas of Ngorongoro District, practical considerations restricted sampling to those households that were accessible by vehicle and/or 30 min walking. Questionnaires were drawn up along WHO guidelines (WHO, 1987), to obtain information on the age, sex and clinical history of each dog sampled. Dogs were identi®ed using the owner's name, dog's name, and a written description which included colour, markings, coat length and texture, size, tail conformation and ear notches. In 1993 and 1994, survival was determined for as many dogs as possible that had been identi®ed in 1992. If a dog had died, information was obtained from owners on the month of death and the suspected cause of mortality. 2.3. Serum samples Blood samples were collected from dogs during questionnaire surveys in each year and from dogs brought to central village locations during trial rabies vaccination progammes (1993 and 1994). Dogs were manually restrained and muzzled using a simple tape muzzle while blood was collected from the cephalic vein. Blood samples were centrifuged within 24 h of collection, serum was stored below ÿ188C in kerosene freezers and transported to laboratories on dry ice. 2.4. Serology A sub-set of serum samples (n407) representative of different age classes, years and areas was analysed at Cornell University for serum neutralising antibodies to CDV using a microneutralisation test (Appel and Robson, 1973). Samples from a further 344 dogs were subsequently analysed at Intervet using a microneutralisation test (Chalmers and Baxendale, 1994). Samples from 84 dogs were analysed at both laboratories.
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In each test serum dilutions were prepared in tissue culture medium before incubating with an equal volume of virus suspension containing 100±300 TCID50/ml neutralising antigen. Neutralisation was allowed to occur for 1 h at 378C. In the Cornell test, CDVOnderstepoort strain was used as neutralising antigen and in the Intervet test, CDV-Bussel strain was used. A suspension of freshly-seeded Vero cells were inoculated with each aliquot of diluted serum/antigen suspension in 96-well mictrotitre plates, the plates incubated at 378C for 3±5 days and then examined for viral infectivity of the Vero cell monolayer. Titration end points were determined microscopically by observation of cytopathic effect (i.e. the presence of multinucleated giant cells) in both instances. 2.5. Statistical analysis To investigate the effect of age, region, and year on mortality, CDV seropositivity and cause of death, logistic regression analyses was carried out. Seropositivity, mortality and cause of death were classi®ed as binary variables. It was often not possible to determine the exact cause of deaths from owner questionnaires, therefore, deaths were assigned to two broad categories which could be distinguished with greater accuracy: (a) all deaths attributed to disease, and (b) all other causes, the most common of which were predation by wild animals and dogs killed by people (poisoning, beating, spearing or road accidents). For each analysis a logistic curve was ®tted to the binary data using a logit link function with binomial errors. The parameters of the logistic models were estimated by maximum likelihood using GENSTAT 5.3 (Payne et al., 1993). The process of model-®tting was based on stepwise deletion, as described in detail by Crawley (1993). Any non-signi®cant variables and interaction terms were removed from the model to produce a minimum adequate model. The effect of variables in the minimum adequate model was measured by the change in deviance following deletion of the term from the model, hence controlling for the effects of all other signi®cant variables. Age was included as a continuous variable (age) or a categorical variable (age class), the classi®cation explaining a greater proportion of the total deviance being used in the ®nal model. 3. Results The frequency distribution of antibody titres measured by both microneutralisation tests demonstrated a demarcation between seropositives and seronegatives at a dilution threshold of >1.2 log10 (Fig. 2). Using this cut-off point, there was agreement between 83 out of 84 sera (98.8%) analysed by both techniques. This threshold was, therefore, adopted throughout the study and seroprevalence data combined for subsequent analyses. Fig. 3(a) and (b) demonstrate cross-sectional age-seroprevalences of Serengeti and Ngorongoro District dog populations for each year of the study, with standard errors calculated using the angular transformation. Seropositivity was detected in pups in
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Fig. 2. Distribution of antibody titres to canine distemper virus in Serengeti domestic dogs.
each of the three study years in Serengeti District, but only in 1994 in Ngorongoro District. For the analysis of age-seroprevalence data, the greatest proportion of total deviance was explained when age was classi®ed into three groups: 0±1 years, 1±2 years and >2 years. This classi®cation was, therefore, used in the ®nal model. In both study areas, CDV seropositivity differed among age classes, with a greater proportion of seropositives in older age classes (Serengeti, w22 80:4, p<0.001; Ngorongoro w22 46:7, p<0.001). Controlling for age, seropositvity differed signi®cantly between years in Ngorongoro District (w22 33:4, p<0.001), but not in Serengeti District (w22 1:2, p>0.05). The magnitude of the year effect was greatest in young dogs with signi®cantly more seropositive pups recorded in 1994 (for the year/age class interaction term w24 20:3, p<0.001). Over the study period, the annual mortality from all causes was similar in Serengeti District (34.5%, n342) and Ngorongoro District (37.5%, n368). However, in explaining the variation in deaths attributed to disease, both geographical area and year were signi®cant (Fig. 4). Thus, the difference in disease-associated mortality between years was signi®cant in Ngorongoro District, but not in Serengeti District (for the year/ area interaction term, w21 12:6, p<0.001). A survival analysis was conducted for dogs with known CDV seroprevalence status in Serengeti (n159) and Ngorongoro Districts (n160). In Serengeti District, there was no signi®cant difference in the survival of CDV seropositive and CDV seronegative dogs (w22 0:65, p>0.05). In contrast, there was signi®cantly higher mortality in Ngorongoro District dogs that were seronegative (w21 24:03, p<0.001) and the magnitude of the difference was signi®cantly greater in 1993±1994 than in 1992±1993 (for the year/ seropositive interaction term, w21 3:94, p<0.05) (Fig. 5(a) and (b)).
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Fig. 3. Canine distemper virus age-seroprevalence curves for (a) Serengeti District and (b) Ngorongoro District domestic dog populations.
4. Discussion Several lines of evidence presented in this study indicate that CDV infection patterns differed signi®cantly between higher-density domestic dog populations to the west of the Serengeti National Park (in Serengeti District) and lower-density populations to the south and east (in Ngorongoro District). In low-density populations, age-seroprevalence patterns are consistent with the interpretation that domestic dogs were exposed to CDV in 1991 and 1994, but not in 1992 and 1993. Thus, in 1992 and 1993, seropositive dogs were
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Fig. 4. Proportion of deaths attributed to disease in domestic dogs of Serengeti and Ngorongoro District from 1992 to 1994.
found only among those animals that were alive in 1991. This interpretation is in line with clinical observations of canine distemper in this population in late 1991 and in late 1994 (Cleaveland, 1996). The absence of seropositive pups in 1992 and 1993 suggests that the virus was unable to persist in this low-density population between outbreaks. In contrast, the ®nding of seropositive pups among Serengeti District dogs in each of the 3 years of the study provides evidence for more prolonged viral persistence in higher-density populations. In Serengeti District, the lack of signi®cant variation in age-seroprevalence patterns between years also suggests that CDV occurred as a more stable infection than in the Ngorongoro District population. Comparison of mortality in Serengeti and Ngorongoro District dogs provides further evidence for differing CDV infection patterns in the two populations. Although canine distemper could not be determined reliably as a cause of death from owner questionnaires, a comparison of mortality rates in seropositive (immune) and seronegative (susceptible) dogs provides an indication of the relative impact of CDV. Thus, in Ngorongoro District, the increase in mortality of CDV seronegative dogs in 1994 is consistent with clinical observations of a CDV epidemic in that year. In contrast, the relatively constant survival rate of CDV seronegatives across years in Serengeti District is consistent with a more stable pattern of infection. In line with these interpretations, disease-associated mortality increased signi®cantly in Ngorongoro dogs in 1994 whereas disease-associated mortality remained relatively stable in Serengeti District. Serological and survival data thus indicate that between 1992 and 1994, CDV persisted as a relatively stable infection in higher-density dog populations to the west of the park but occurred only sporadically in lower-density dog populations to the southeast.
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Fig. 5. Survival of CDV seropositive and CDV seronegative dogs in (a) Serengeti District and (b) Ngorongoro District from 1992 to 1994.
With respect to the 1994 lion epidemic in the Serengeti, serological and demographic data indicate that Maasai dogs of Ngorongoro were not likely to be the source of CDV infection for wildlife. This interpretation is supported by the temporal sequence of con®rmed cases during the epidemic, with infection in wildlife spreading south through the park before occurring as an epidemic in Ngorongoro dogs in late 1994. There is also anecdotal evidence that domestic dogs in Shinyanga Region, to the southwest of the park, were infected from wildlife, as a canine distemper epidemic was reported in the Region early in 1995, several months after the last observed cases in lions (M. Kaare, unpublished data). In contrast, CDV was circulating in the Serengeti District domestic
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dog population prior to the lion epidemic and no increase in disease-associated mortality was recorded after the epidemic. We, therefore, consider the Serengeti District dog population the more likely source of infection for wildlife. While molecular analyses of Serengeti CDV isolates demonstrate close similarity with virulent domestic dog strains of virus, we cannot rule out the possibility of virus maintenance in wildlife reservoirs. Serological data from wild animal populations in the Serengeti are currently too limited to draw conclusions about the role of wild carnivores in the epidemiology of canine distemper. However, published data from lion and hyaena populations in the ecosystem provide no evidence for independent maintenance of CDV infection in these populations. Thus, age-seroprevalence data from the Serengeti lion population are consistent with exposure of the population to CDV in the early 1980s with no new infection recorded until late 1993 (Packer et al., in press). Similarly, in the Masai Mara in Kenya (the northern extension of the Serengeti ecosystem), the presence of seropositive hyaenas in some years (1980±1982), but not others (1979) (Alexander et al., 1995) is suggestive of sporadic exposure rather than persistent infection. Although the exact routes of transmission betwen dogs and wildlife are unknown, there are many opportunities for CDV to be transmitted from Serengeti District dogs to wildlife. Direct contact between dogs and lions may occur and, during the course of this study, lions were observed in at least two of the study villages in Serengeti District. It is more likely, however, that lions were infected indirectly through chains of transmission in other species. Potential vectors of CDV, such as jackals, spotted hyaenas and mongooses, are frequently recorded within Serengeti District villages in close proximity to domestic dogs (Cleaveland, 1996). Frequent interactions among jackals, hyaenas and lions at kills provide a potential mechanism for subsequent transmission of infection to lions. Supportive evidence for a link between domestic dogs and hyaenas comes from the Masai Mara, Kenya, where low-ranking hyaenas that scavenge more frequently in villages had higher CDV seropositivity than high-ranking animals (Alexander et al., 1995). If dog populations to the west of the park were indeed the source of infection for lions, the question remains as to why an epidemic occurred only in 1993, when CDV was present in the dog population from at least late 1991. One possible explanation is that a severe drought in late 1993 increased the probability of contact between dogs and wildlife. At this time, large numbers of wildebeest were forced to ®nd grazing in higherrainfall areas of Serengeti District, which led to high mortality from local hunting (Tanzanian Wildlife Conservation Monitoring, 1994). In one 30 km2 area outside the park, a single survey recorded 1188 fresh wildebeest carcasses (Dr. Simon Mduma, pers. commun.). The abundance of fresh carcasses in proximity to villages clearly has the potential to bring scavenging dogs and wildlife into close contact, increasing the probability of inter-speci®c transmission. The conclusions of this study have important implications for CDV control in the Serengeti. If higher-density dog populations to the west of the park are indeed a major source of CDV infection for wildlife, elimination of infection in these populations should reduce the risk of future outbreaks in wildlife. To test this hypothesis, a vaccination programme against canine distemper has recently been initiated in Serengeti District dogs and it is hoped that results of this trial will provide further insights into the epidemiology of CDV in the Serengeti.
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Acknowledgements S.C. was supported by a grant from the BBSRC and by a Wellcome Research Fellowship in Tropical Medicine. We thank Tanzania National Parks, Ngorongoro Conservation Area Authority and Serengeti Wildlife Research Institute for permission to work in the Serengeti and Dr. F. Gulland, Dr. S. Mkumbo, Dr. P. Loomu, Mr. P. Tiringa, Mr. Mosha, Mr. Magoto, Mr. Miran and Mr. Sarmo for ®eld assistance. The Frankfurt Zoological Society and British Development Division, East Africa provided logistic support for which we are most grateful. Prof. C. Packer and Dr. A. MacColl provided useful comments on the manuscript. References Alexander, K.A., Kat, P.W., Frank, L., Holekamp, K.E., Smale, L., House, C., Appel, M.J.G., 1995. Evidence of canine distemper virus infection among free-ranging spotted hyaenas (Crocuta crocuta) in the Masai Mara, Kenya. J. Zoo Wild. Med. 26, 201±206. Appel, M.J.G., Robson, D.S., 1973. A microneutralization test for canine distemper virus. Am. J. Vet. Res. 34, 1459±1463. Bureau of Statistics, 1991. Tanzania Sensa 1988. President's Of®ce, Planning Commission, Dar es Salaam, Tanzania. Carpenter, M.A., Appel, M.J.G., Roelke-Parker, M.E., Munson, L., Hofer, H., East, M., O'Brien, S.J., 1998. Genetic characterization of canine distemper virus in Serengeti carnivores. Vet. Immun. Immunopathol. 65, 259±266. Chalmers, W.S.K., Baxendale, W., 1994. A comparison of canine distemper vaccine and measles vaccine for the prevention of canine distemper in young puppies. Vet. Rec. 135, 349±353. Cleaveland, S., 1996. The epidemiology of rabies and canine distemper in the Serengeti, Tanzania. Ph.D. Thesis, University of London. Crawley, M.J., 1993. GLIM for Ecologists. Blackwell Scienti®c Publications, Oxford, pp. 188±210. Haas, L., Hofer, H., East, M., Wohlsein, P., Liess, B., Barrett, T., 1996. Canine distemper virus infection in Serengeti spotted hyaenas. Vet. Microbiol. 49, 147±152. È rvell, C., Barrett, T., Appel, Harder, T.C., Kenter, M., Vos, H., Sieblink, K., Huisman, W., van Amerongen, G., O M.J.G., Osterhaus, A.D.M.E., 1996. Canine distemper virus from diseased large felids: biological properties and phylogenetic relationships. J. Gen. Virol. 77, 397±405. Kock, R., Chalmers, W.S.K., Mwanzia, J., Chillingworth, C., Wambura, J., Coleman, P.G., Baxendale, W., 1998. Canine distemper antibodies in lions of the Masai Mara. Vet. Rec. 142, 662±665. Packer, C., Altizer, S., Appel, M.J.G., Brown, E., Martenson, J., O'Brien, S.J., Roelke-Parker, M., HofmannLehmann, R., Lutz, H. Viruses of the Serengeti: patterns of infection and mortality in African lions. J. Anim. Ecol., in press. Payne, R.W., Lane, P.W., Digby, P.G.N., Harding, S.A., Leech, P.K., Morgan, G.W., Todd, A.D., Thompson, R., Tunnicliffe Wilson, G., Welham, S.J., White, R.P., 1993. Genstat 5 Release 3 Reference Manual, Clarendon Press, Oxford. Roelke-Parker, M.E., Munson, L., Packer, C., Kock, R., Cleaveland, S., Carpenter, M., O'Brien, S.J., Pospischil, A., Hofmann-Lehmann, R., Lutz, H., Mwamengele, G.L.M., Mgasa, M.N., Machange, G.A., Summer, B.A., Appel, M.J.G., 1996. A canine distemper virus epidemic in Serengeti lions (Panthera leo). Nature 379, 441± 445. Tanzanian Wildlife Conservation Monitoring, 1991. Ngorongoro Conservation Area/Loliondo Boma Census. Preliminary report, distribution maps and data summary. Tanzania Wildlife Conservation Monitoring. Tanzanian Wildlife Conservation Monitoring, 1994. Status and trends of wildebeest in the Serengeti Ecosystem. Tanzania Wildlife Conservation Monitoring. WHO, 1987. Guidelines for dog rabies control. World Health Organization, WHO/VPH/83.43, Geneva, Switzerland.
Serological and demographic evidence for domestic dogs as a source of canine distemper virus infection for Serengeti wildlife S. Cleavelanda,b,*, M.G.J. Appelc, W.S.K. Chalmersd, C. Chillingworthd, M. Kaaree, C. Dyea a
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK b Institute of Zoology, Zoological Society of London, Regent's Park, London NW1 4RY, UK c James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA d Intervet (UK) Ltd., The Elms, Thicket Road, Houghton, Huntingdon PE17 2BQ, UK e Ministry of Agriculture and Cooperatives, P.O. Box 203, Musoma, Tanzania
Received 4 August 1999; received in revised form 30 November 1999; accepted 30 November 1999
Abstract Following an epidemic of canine distemper virus (CDV) in Serengeti lions in 1994, the role of domestic dogs in the epidemiology of the disease was investigated by serological and demographic analyses. From 1992 to 1994, data were collected from two domestic dog populations bordering the Serengeti National Park. Several lines of evidence indicated that patterns of CDV infection differed signi®cantly between higher-density dog populations of Serengeti District to the west of the park and lower-density populations of Ngorongoro District to the south-east: (a) CDV ageseroprevalence patterns differed signi®cantly between years in Ngorongoro District populations but not in Serengeti District populations; (b) CDV seropositive pups (<12 months of age) were detected in Ngorongoro District only in 1994, whereas a proportion of pups in Serengeti District were seropositive in each year of the study; (c) in Ngorongoro District, the proportion of deaths attributed to disease was signi®cantly higher in 1994 than in 1993, whereas in Serengeti District, there was no signi®cant difference in disease-related mortality between years; (d) in Ngorongoro District, signi®cantly more CDV seronegative dogs than seropositive dogs died in 1994, whereas *
Corresponding author. Present address: Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Roslin, Midlothian EH25 9RG, UK. Tel.: 44-131650-6404; fax: 44-131-445-5099. E-mail address: [email protected] (S. Cleaveland). 0378-1135/00/$ ± see front matter # 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 2 0 7 - 2
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there was no difference in survival of CDV seropositives and seronegatives between years in Serengeti District. We concluded that, between 1992 and 1994, CDV persisted in higher-density dog populations of Serengeti District, but occurred only sporadically in lower-density Ngorongoro District populations. Data from Ngorongoro District are consistent with exposure of dogs to CDV in 1991 and 1994, but not in 1992 and 1993. These ®ndings suggest that higher-density domestic dog populations to the west of the Serengeti National Park were a more likely source of CDV infection for wildlife during 1994 than lower-density pastoralist dogs to the south and east of the park. # 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Dog canine distempervirus; Epidemiology; Serology; Wildlife; Serengeti; Kenya; Tanzania
1. Introduction From late 1993 to 1994, an epidemic of canine distemper (CD) was recorded in the lion (Panthera leo) population of the Serengeti ecosystem, with cases con®rmed in the Serengeti National Park and Maswa Game Reserve in Tanzania (Roelke-Parker et al., 1996) and in the Masai Mara National Reserve, Kenya (Kock et al., 1998). Approximately 30% of all known lions within a closely-monitored study population of the Serengeti National Park either died or disappeared. During this epidemic, the disease was also con®rmed in spotted hyaenas (Crocuta crocuta) (Haas et al., 1996) and bat-eared foxes (Otocoyon megalotis) (Roelke-Parker et al., 1996), as well as in domestic dog populations surrounding the Ngorongoro Crater (Cleaveland, 1996). Virus isolates obtained from lions, domestic dog, hyaena and bat-eared fox during the epidemic showed identical monclonal antibody reaction patterns, which were indistinguishable from the pattern obtained for virulent canine distemper virus (CDV) strains isolated from domestic dogs (Cleaveland, 1996; Roelke-Parker et al., 1996). Molecular genetic analyses con®rmed the close similarity between Serengeti lion viruses and virulent, wild-type CDV (Roelke-Parker et al., 1996). These analyses also demonstrated close phylogenetic homology among the Serengeti viruses, with a tendency for isolates to cluster according to geographic rather than host species origin (Haas et al., 1996; Harder et al., 1996; Roelke-Parker et al., 1996; Carpenter et al., 1998). These ®ndings suggest that a single virus strain caused mortality in a range species in the Serengeti and that this strain was transmissible between domestic dogs and wild carnivores. Although molecular studies point to a single virus strain affecting multiple hosts, the exact role of different hosts species in the maintenance and transmission of CDV in the Serengeti remains unresolved. Here, the role of domestic dogs was investigated through comparative serological and demographic analyses of high- and low-density domestic dog populations living adjacent to the park. Data were also presented from cross-sectional and longitudinal studies of two populations comparing (i) CDV ageseroprevalence patterns (ii) disease-related mortality and (iii) survival of CDV seropositives and CDV seronegatives. These ®ndings, in combination with published data from Serengeti wildlife populations, were used to explore the dynamics of CDV infection in the Serengeti.
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Fig. 1. Map showing location of study villages (*) around the Serengeti National Park.
2. Materials and methods 2.1. Study areas The study area was the Serengeti ecological region of Northwestern Tanzania (358 to 368E, 18 300 to 38 70 S). Villages were selected from two districts adjacent to the Serengeti National Park with the aim of sampling dogs from as broad a geographic range as possible. Study villages were located within Ngorongoro District and Serengeti District, which are de®ned by administrative boundaries (Fig. 1). The Ngorongoro District, comprising the Loliondo Game Controlled Area and the Ngorongoro Conservation Area, is a multiple-use controlled wildlife area, inhabited predominantly by Maasai people practising traditional pastoralism and limited cultivation. Human population density was estimated as 2.89 people/km2 in 1992, based on 1988 human census data with a projected population growth rate of 3.4% per annum (Bureau of Statistics, 1991). Estimates of domestic dog densities, determined from the number of dogs per Maasai boma (household) and the aerial distribution of bomas
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(Tanzanian Wildlife Conservation Monitoring, 1991), ranged from 0.38 to 0.46 dogs/km2 from 1992 to 1994 (Cleaveland, 1996). In Serengeti District, agropastoralism is the dominant pattern of land use, with production systems based on livestock and cultivation of crops, such as cassava and maize. Human and domestic dog population densities were much higher than in Ngorongoro District. The human density in 1992 was estimated as 30.10 km2 , based on 1988 census data and projected growth rates of 2.9% per annum (Bureau of Statistics, 1991). Domestic dog densities, determined from the human:dog ratio, ranged from 5.72 to 7.17 dogs/km2 (Cleaveland, 1996). 2.2. Data collection Data were collected during three ®eld periods: from September 1992 to February 1993 (referred to subsequently as 1992); September to December 1993; August to December 1994. Demographic data on the domestic dog population were collected through questionnaire surveys of householders. In rural villages of Serengeti District, households were sampled within each of four quadrants, using the village centre (site of administrative of®ces) as the central reference point. One in ®ve households situated along a transect from the village centre to the periphery was visited within each quadrant. In low-density areas of Ngorongoro District, practical considerations restricted sampling to those households that were accessible by vehicle and/or 30 min walking. Questionnaires were drawn up along WHO guidelines (WHO, 1987), to obtain information on the age, sex and clinical history of each dog sampled. Dogs were identi®ed using the owner's name, dog's name, and a written description which included colour, markings, coat length and texture, size, tail conformation and ear notches. In 1993 and 1994, survival was determined for as many dogs as possible that had been identi®ed in 1992. If a dog had died, information was obtained from owners on the month of death and the suspected cause of mortality. 2.3. Serum samples Blood samples were collected from dogs during questionnaire surveys in each year and from dogs brought to central village locations during trial rabies vaccination progammes (1993 and 1994). Dogs were manually restrained and muzzled using a simple tape muzzle while blood was collected from the cephalic vein. Blood samples were centrifuged within 24 h of collection, serum was stored below ÿ188C in kerosene freezers and transported to laboratories on dry ice. 2.4. Serology A sub-set of serum samples (n407) representative of different age classes, years and areas was analysed at Cornell University for serum neutralising antibodies to CDV using a microneutralisation test (Appel and Robson, 1973). Samples from a further 344 dogs were subsequently analysed at Intervet using a microneutralisation test (Chalmers and Baxendale, 1994). Samples from 84 dogs were analysed at both laboratories.
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In each test serum dilutions were prepared in tissue culture medium before incubating with an equal volume of virus suspension containing 100±300 TCID50/ml neutralising antigen. Neutralisation was allowed to occur for 1 h at 378C. In the Cornell test, CDVOnderstepoort strain was used as neutralising antigen and in the Intervet test, CDV-Bussel strain was used. A suspension of freshly-seeded Vero cells were inoculated with each aliquot of diluted serum/antigen suspension in 96-well mictrotitre plates, the plates incubated at 378C for 3±5 days and then examined for viral infectivity of the Vero cell monolayer. Titration end points were determined microscopically by observation of cytopathic effect (i.e. the presence of multinucleated giant cells) in both instances. 2.5. Statistical analysis To investigate the effect of age, region, and year on mortality, CDV seropositivity and cause of death, logistic regression analyses was carried out. Seropositivity, mortality and cause of death were classi®ed as binary variables. It was often not possible to determine the exact cause of deaths from owner questionnaires, therefore, deaths were assigned to two broad categories which could be distinguished with greater accuracy: (a) all deaths attributed to disease, and (b) all other causes, the most common of which were predation by wild animals and dogs killed by people (poisoning, beating, spearing or road accidents). For each analysis a logistic curve was ®tted to the binary data using a logit link function with binomial errors. The parameters of the logistic models were estimated by maximum likelihood using GENSTAT 5.3 (Payne et al., 1993). The process of model-®tting was based on stepwise deletion, as described in detail by Crawley (1993). Any non-signi®cant variables and interaction terms were removed from the model to produce a minimum adequate model. The effect of variables in the minimum adequate model was measured by the change in deviance following deletion of the term from the model, hence controlling for the effects of all other signi®cant variables. Age was included as a continuous variable (age) or a categorical variable (age class), the classi®cation explaining a greater proportion of the total deviance being used in the ®nal model. 3. Results The frequency distribution of antibody titres measured by both microneutralisation tests demonstrated a demarcation between seropositives and seronegatives at a dilution threshold of >1.2 log10 (Fig. 2). Using this cut-off point, there was agreement between 83 out of 84 sera (98.8%) analysed by both techniques. This threshold was, therefore, adopted throughout the study and seroprevalence data combined for subsequent analyses. Fig. 3(a) and (b) demonstrate cross-sectional age-seroprevalences of Serengeti and Ngorongoro District dog populations for each year of the study, with standard errors calculated using the angular transformation. Seropositivity was detected in pups in
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Fig. 2. Distribution of antibody titres to canine distemper virus in Serengeti domestic dogs.
each of the three study years in Serengeti District, but only in 1994 in Ngorongoro District. For the analysis of age-seroprevalence data, the greatest proportion of total deviance was explained when age was classi®ed into three groups: 0±1 years, 1±2 years and >2 years. This classi®cation was, therefore, used in the ®nal model. In both study areas, CDV seropositivity differed among age classes, with a greater proportion of seropositives in older age classes (Serengeti, w22 80:4, p<0.001; Ngorongoro w22 46:7, p<0.001). Controlling for age, seropositvity differed signi®cantly between years in Ngorongoro District (w22 33:4, p<0.001), but not in Serengeti District (w22 1:2, p>0.05). The magnitude of the year effect was greatest in young dogs with signi®cantly more seropositive pups recorded in 1994 (for the year/age class interaction term w24 20:3, p<0.001). Over the study period, the annual mortality from all causes was similar in Serengeti District (34.5%, n342) and Ngorongoro District (37.5%, n368). However, in explaining the variation in deaths attributed to disease, both geographical area and year were signi®cant (Fig. 4). Thus, the difference in disease-associated mortality between years was signi®cant in Ngorongoro District, but not in Serengeti District (for the year/ area interaction term, w21 12:6, p<0.001). A survival analysis was conducted for dogs with known CDV seroprevalence status in Serengeti (n159) and Ngorongoro Districts (n160). In Serengeti District, there was no signi®cant difference in the survival of CDV seropositive and CDV seronegative dogs (w22 0:65, p>0.05). In contrast, there was signi®cantly higher mortality in Ngorongoro District dogs that were seronegative (w21 24:03, p<0.001) and the magnitude of the difference was signi®cantly greater in 1993±1994 than in 1992±1993 (for the year/ seropositive interaction term, w21 3:94, p<0.05) (Fig. 5(a) and (b)).
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Fig. 3. Canine distemper virus age-seroprevalence curves for (a) Serengeti District and (b) Ngorongoro District domestic dog populations.
4. Discussion Several lines of evidence presented in this study indicate that CDV infection patterns differed signi®cantly between higher-density domestic dog populations to the west of the Serengeti National Park (in Serengeti District) and lower-density populations to the south and east (in Ngorongoro District). In low-density populations, age-seroprevalence patterns are consistent with the interpretation that domestic dogs were exposed to CDV in 1991 and 1994, but not in 1992 and 1993. Thus, in 1992 and 1993, seropositive dogs were
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Fig. 4. Proportion of deaths attributed to disease in domestic dogs of Serengeti and Ngorongoro District from 1992 to 1994.
found only among those animals that were alive in 1991. This interpretation is in line with clinical observations of canine distemper in this population in late 1991 and in late 1994 (Cleaveland, 1996). The absence of seropositive pups in 1992 and 1993 suggests that the virus was unable to persist in this low-density population between outbreaks. In contrast, the ®nding of seropositive pups among Serengeti District dogs in each of the 3 years of the study provides evidence for more prolonged viral persistence in higher-density populations. In Serengeti District, the lack of signi®cant variation in age-seroprevalence patterns between years also suggests that CDV occurred as a more stable infection than in the Ngorongoro District population. Comparison of mortality in Serengeti and Ngorongoro District dogs provides further evidence for differing CDV infection patterns in the two populations. Although canine distemper could not be determined reliably as a cause of death from owner questionnaires, a comparison of mortality rates in seropositive (immune) and seronegative (susceptible) dogs provides an indication of the relative impact of CDV. Thus, in Ngorongoro District, the increase in mortality of CDV seronegative dogs in 1994 is consistent with clinical observations of a CDV epidemic in that year. In contrast, the relatively constant survival rate of CDV seronegatives across years in Serengeti District is consistent with a more stable pattern of infection. In line with these interpretations, disease-associated mortality increased signi®cantly in Ngorongoro dogs in 1994 whereas disease-associated mortality remained relatively stable in Serengeti District. Serological and survival data thus indicate that between 1992 and 1994, CDV persisted as a relatively stable infection in higher-density dog populations to the west of the park but occurred only sporadically in lower-density dog populations to the southeast.
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Fig. 5. Survival of CDV seropositive and CDV seronegative dogs in (a) Serengeti District and (b) Ngorongoro District from 1992 to 1994.
With respect to the 1994 lion epidemic in the Serengeti, serological and demographic data indicate that Maasai dogs of Ngorongoro were not likely to be the source of CDV infection for wildlife. This interpretation is supported by the temporal sequence of con®rmed cases during the epidemic, with infection in wildlife spreading south through the park before occurring as an epidemic in Ngorongoro dogs in late 1994. There is also anecdotal evidence that domestic dogs in Shinyanga Region, to the southwest of the park, were infected from wildlife, as a canine distemper epidemic was reported in the Region early in 1995, several months after the last observed cases in lions (M. Kaare, unpublished data). In contrast, CDV was circulating in the Serengeti District domestic
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dog population prior to the lion epidemic and no increase in disease-associated mortality was recorded after the epidemic. We, therefore, consider the Serengeti District dog population the more likely source of infection for wildlife. While molecular analyses of Serengeti CDV isolates demonstrate close similarity with virulent domestic dog strains of virus, we cannot rule out the possibility of virus maintenance in wildlife reservoirs. Serological data from wild animal populations in the Serengeti are currently too limited to draw conclusions about the role of wild carnivores in the epidemiology of canine distemper. However, published data from lion and hyaena populations in the ecosystem provide no evidence for independent maintenance of CDV infection in these populations. Thus, age-seroprevalence data from the Serengeti lion population are consistent with exposure of the population to CDV in the early 1980s with no new infection recorded until late 1993 (Packer et al., in press). Similarly, in the Masai Mara in Kenya (the northern extension of the Serengeti ecosystem), the presence of seropositive hyaenas in some years (1980±1982), but not others (1979) (Alexander et al., 1995) is suggestive of sporadic exposure rather than persistent infection. Although the exact routes of transmission betwen dogs and wildlife are unknown, there are many opportunities for CDV to be transmitted from Serengeti District dogs to wildlife. Direct contact between dogs and lions may occur and, during the course of this study, lions were observed in at least two of the study villages in Serengeti District. It is more likely, however, that lions were infected indirectly through chains of transmission in other species. Potential vectors of CDV, such as jackals, spotted hyaenas and mongooses, are frequently recorded within Serengeti District villages in close proximity to domestic dogs (Cleaveland, 1996). Frequent interactions among jackals, hyaenas and lions at kills provide a potential mechanism for subsequent transmission of infection to lions. Supportive evidence for a link between domestic dogs and hyaenas comes from the Masai Mara, Kenya, where low-ranking hyaenas that scavenge more frequently in villages had higher CDV seropositivity than high-ranking animals (Alexander et al., 1995). If dog populations to the west of the park were indeed the source of infection for lions, the question remains as to why an epidemic occurred only in 1993, when CDV was present in the dog population from at least late 1991. One possible explanation is that a severe drought in late 1993 increased the probability of contact between dogs and wildlife. At this time, large numbers of wildebeest were forced to ®nd grazing in higherrainfall areas of Serengeti District, which led to high mortality from local hunting (Tanzanian Wildlife Conservation Monitoring, 1994). In one 30 km2 area outside the park, a single survey recorded 1188 fresh wildebeest carcasses (Dr. Simon Mduma, pers. commun.). The abundance of fresh carcasses in proximity to villages clearly has the potential to bring scavenging dogs and wildlife into close contact, increasing the probability of inter-speci®c transmission. The conclusions of this study have important implications for CDV control in the Serengeti. If higher-density dog populations to the west of the park are indeed a major source of CDV infection for wildlife, elimination of infection in these populations should reduce the risk of future outbreaks in wildlife. To test this hypothesis, a vaccination programme against canine distemper has recently been initiated in Serengeti District dogs and it is hoped that results of this trial will provide further insights into the epidemiology of CDV in the Serengeti.
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Acknowledgements S.C. was supported by a grant from the BBSRC and by a Wellcome Research Fellowship in Tropical Medicine. We thank Tanzania National Parks, Ngorongoro Conservation Area Authority and Serengeti Wildlife Research Institute for permission to work in the Serengeti and Dr. F. Gulland, Dr. S. Mkumbo, Dr. P. Loomu, Mr. P. Tiringa, Mr. Mosha, Mr. Magoto, Mr. Miran and Mr. Sarmo for ®eld assistance. The Frankfurt Zoological Society and British Development Division, East Africa provided logistic support for which we are most grateful. Prof. C. Packer and Dr. A. MacColl provided useful comments on the manuscript. References Alexander, K.A., Kat, P.W., Frank, L., Holekamp, K.E., Smale, L., House, C., Appel, M.J.G., 1995. Evidence of canine distemper virus infection among free-ranging spotted hyaenas (Crocuta crocuta) in the Masai Mara, Kenya. J. Zoo Wild. Med. 26, 201±206. Appel, M.J.G., Robson, D.S., 1973. A microneutralization test for canine distemper virus. Am. J. Vet. Res. 34, 1459±1463. Bureau of Statistics, 1991. Tanzania Sensa 1988. President's Of®ce, Planning Commission, Dar es Salaam, Tanzania. Carpenter, M.A., Appel, M.J.G., Roelke-Parker, M.E., Munson, L., Hofer, H., East, M., O'Brien, S.J., 1998. Genetic characterization of canine distemper virus in Serengeti carnivores. Vet. Immun. Immunopathol. 65, 259±266. Chalmers, W.S.K., Baxendale, W., 1994. A comparison of canine distemper vaccine and measles vaccine for the prevention of canine distemper in young puppies. Vet. Rec. 135, 349±353. Cleaveland, S., 1996. The epidemiology of rabies and canine distemper in the Serengeti, Tanzania. Ph.D. Thesis, University of London. Crawley, M.J., 1993. GLIM for Ecologists. Blackwell Scienti®c Publications, Oxford, pp. 188±210. Haas, L., Hofer, H., East, M., Wohlsein, P., Liess, B., Barrett, T., 1996. Canine distemper virus infection in Serengeti spotted hyaenas. Vet. Microbiol. 49, 147±152. È rvell, C., Barrett, T., Appel, Harder, T.C., Kenter, M., Vos, H., Sieblink, K., Huisman, W., van Amerongen, G., O M.J.G., Osterhaus, A.D.M.E., 1996. Canine distemper virus from diseased large felids: biological properties and phylogenetic relationships. J. Gen. Virol. 77, 397±405. Kock, R., Chalmers, W.S.K., Mwanzia, J., Chillingworth, C., Wambura, J., Coleman, P.G., Baxendale, W., 1998. Canine distemper antibodies in lions of the Masai Mara. Vet. Rec. 142, 662±665. Packer, C., Altizer, S., Appel, M.J.G., Brown, E., Martenson, J., O'Brien, S.J., Roelke-Parker, M., HofmannLehmann, R., Lutz, H. Viruses of the Serengeti: patterns of infection and mortality in African lions. J. Anim. Ecol., in press. Payne, R.W., Lane, P.W., Digby, P.G.N., Harding, S.A., Leech, P.K., Morgan, G.W., Todd, A.D., Thompson, R., Tunnicliffe Wilson, G., Welham, S.J., White, R.P., 1993. Genstat 5 Release 3 Reference Manual, Clarendon Press, Oxford. Roelke-Parker, M.E., Munson, L., Packer, C., Kock, R., Cleaveland, S., Carpenter, M., O'Brien, S.J., Pospischil, A., Hofmann-Lehmann, R., Lutz, H., Mwamengele, G.L.M., Mgasa, M.N., Machange, G.A., Summer, B.A., Appel, M.J.G., 1996. A canine distemper virus epidemic in Serengeti lions (Panthera leo). Nature 379, 441± 445. Tanzanian Wildlife Conservation Monitoring, 1991. Ngorongoro Conservation Area/Loliondo Boma Census. Preliminary report, distribution maps and data summary. Tanzania Wildlife Conservation Monitoring. Tanzanian Wildlife Conservation Monitoring, 1994. Status and trends of wildebeest in the Serengeti Ecosystem. Tanzania Wildlife Conservation Monitoring. WHO, 1987. Guidelines for dog rabies control. World Health Organization, WHO/VPH/83.43, Geneva, Switzerland.