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Haemoparasite prevalence and Theileria parva strain diversity in Cape buffalo (Syncerus caffer) in Uganda
Veterinary Parasitology 175 (2011) 212–219
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Haemoparasite prevalence and Theileria parva strain diversity in Cape buffalo (Syncerus caffer) in Uganda C.A.L. Oura a,b,c,∗ , A. Tait b , B. Asiimwe a,d , G.W. Lubega a , W. Weir b a b c d
Department of Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Makerere, P.O. Box 7062, Kampala, Uganda Division of Veterinary Infection and Immunity, University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, UK Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU240NF, UK Department of Medical Microbiology, College of Health Sciences, Makerere University, P.O. Box 7072, Kampala, Uganda
a r t i c l e
i n f o
Article history: Received 25 July 2010 Received in revised form 9 October 2010 Accepted 12 October 2010 Keywords: Theileria parva East Coast fever Cattle Buffalo Reverse line blot
a b s t r a c t Cape buffalo (Syncerus caffer) are considered to be an important reservoir for various tick-borne haemoparasites of veterinary importance. In this study we have compared the haemoparasite carrier prevalence in buffalo from four geographically isolated national parks in Uganda [Lake Mburo National Park (LMNP), Queen Elizabeth National Park (QENP), Murchison Falls National Park (MFNP) and Kidepo Valley National Park (KVNP)]. Differences were seen in haemoparasite prevalence in buffalo from the four national parks. All the buffalo sampled in LMNP were carriers of Theileria parva however, buffalo from MFNP and KVNP, which are both located in the north of Uganda, were negative for T. parva. Interestingly, 95% of buffalo in the northern part of QENP were T. parva positive, however all buffalo sampled in the south of the park were negative. A high multiplicity of infection was recorded in all the buffalo found to be carrying T. parva, with evidence of at least nine parasite genotypes in some animals. Most of the buffalo sampled in all four national parks were carriers of T. mutans and T. velifera, however none were carriers of T. taurotragi, Babesia bovis, Babesia bigemina, Ehrlichia bovis or Ehrlichia ruminantium. All the buffalo sampled from LMNP were positive for T. buffeli and T. sp. (buffalo) however, buffalo from the parks in the north of the country (KVNP and MFNP) were negative for these haemoparasites. Anaplasma centrale and Anaplasma marginale were circulating in buffalo from all four national parks. T. parva gene pools from two geographically separated populations of buffalo in two of the national parks in Uganda (LMNP and QENP) were compared. The T. parva populations in the two national parks were distinct, indicating that there was limited gene flow between the populations. The results presented highlight the complexity of tick-borne pathogen infections in buffalo and the significant role that buffalo may play as reservoir hosts for veterinary haemoparasites that have the potential to cause severe disease in domestic cattle. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction Tick-borne diseases constitute a major constraint on cattle production and the expansion of the dairy industry in
∗ Corresponding author at: Vector-borne Diseases Programme, Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU240NF, UK. Tel.: +44 1483 232441; fax: +44 1483 232448. E-mail address: [email protected] (C.A.L. Oura).
many countries across Africa (Uilenberg, 1995). The most serious tick-borne disease in eastern and central Africa is East Coast fever (ECF) caused by the intracellular protozoan parasite Theileria parva. The disease is associated with high levels of mortality, primarily in exotic and crossbred cattle, but also in indigenous calves and adult cattle in endemically unstable areas (Perry and Young, 1995). Cattle in East Africa are also exposed to a range of other tickborne pathogens in addition to Theileria species, including Ehrlichia, Anaplasma and Babesia species. Recently a reverse
0304-4017/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.10.032
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line blot (RLB) assay has been described (Gubbels et al., 1999; Bekker et al., 2002) that is able to simultaneously detect each of the Theileria, Babesia, Anaplasma and Ehrlichia species in ruminants in one assay. This assay has been applied in a field study in Uganda (Oura et al., 2004b) and it has also been used to identify haemoprotozoa in Minorcan cattle (Almeria et al., 2002) and for the integrated molecular diagnosis of Theileria and Babesia species present in cattle in Italy (Sparagano et al., 2000). Very little is known about the role of African Cape buffalo (Syncerus caffer) as a reservoir for haemoparasites, however buffalo are thought to be the major natural host of T. parva in which the parasite appears not to cause clinical disease (Grootenhuis et al., 1987). Co-evolution of African Cape buffalo with T. parva populations must have occurred prior to infection of cattle (Epstein, 1971) and it is thought that ticks infected with T. parva from buffalo first came into contact with Bos indicus cattle in Sub-Saharan Africa approximately 4500 years ago (Epstein, 1971). ECF is caused by T. parva parasites that can be transmitted between cattle by ticks, but a different clinical syndrome associated with high mortality levels in cattle, Corridor disease, is thought to be caused by the transmission of T. parva directly from buffalo to cattle via ticks. Cattle–tick–cattle transmission is thought to be limited as erythrocytic piroplasms are either absent or at an insufficient level to infect new ticks. The use of molecular characterisation tools has revealed a high level of diversity among buffalo-derived T. parva stocks compared to cattle-derived T. parva stocks (Bishop et al., 1994; Collins and Allsopp, 1999; Geysen et al., 2004; Oura et al., 2004a). Thus, there is growing evidence that only a limited subset of the total T. parva gene pool present within buffalo has become established in cattle. It is unknown whether the transfer of buffaloderived strains to cattle resulting in ECF was a single event or whether there is a constant trickle of new strains transferring from buffalo to cattle. This has very important implications since it has been demonstrated that there is incomplete cross-protection between animals immunised with cattle-derived T. parva on challenge with parasites from buffalo (Young et al., 1973). This suggests that any live vaccination programme may not protect against Corridor disease in areas where cattle and buffalo co-graze. In this study we report the application of an RLB assay to compare the carrier prevalence of tick-borne haemoparasites in Cape buffalo in four geographically isolated national parks in Uganda: Lake Mburo National Park (LMNP), Queen Elizabeth National Park (QENP), Murchison Falls National Park (MFNP) and Kidepo Valley National Park (KVNP). The results presented herein provide evidence for a complex pattern of infection of tick-borne pathogens in buffalo in Uganda and demonstrate distinct gene pools of T. parva parasites in populations derived from buffalo in two of the Ugandan national parks. 2. Materials and methods 2.1. Buffalo sampling Cape buffalo were sampled as part of the Pan African programme for the control of epizootics (PACE). In total 19
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adult buffalo were sampled from LMNP, 12 adult buffalo were sampled from MFNP and 25 adult buffalo were sampled from KVNP. In QENP 18 adult buffalo were sampled from the northern part of the park and nine adult buffalo were sampled from the southern part of the park. The northern and southern parts of QENP are separated by the Kazinga channel which connects lakes George and Edward and by the 32 km2 Maramagambo Forest, which appear to form a barrier for migration of buffaloes between the northern and southern sectors of the park (Woodford, 1982). The location of the four national parks from which buffalo were sampled is shown on a map of Uganda (Fig. 1). 2.2. Haemoparasite detection Blood samples were processed and analysed for the presence of haemoparasites by an RLB assay (Gubbels et al., 1999) with modifications previously described (Oura et al., 2004b). All samples were also assayed for the presence of T. parva by a nested PCR assay using mini-satellite marker MS 16 (Oura et al., 2004a). The RLB assay had a detection threshold of between 4.4 × 10−5 and 1.4 × 10−5 % parasitaemia that is equivalent to around 1–2 parasites/l of blood. The MS 16 mini-satellite assay was more sensitive with a detection threshold of between 1.4 × 10−5 % parasitaemia and 4.6 × 10−6 % parasitaemia that is equivalent to between approximately 1 and 0.3 parasites/l of blood (Oura et al., 2004b). 2.3. Micro- and mini-satellite PCR assay 2.3.1. Sample preparation Buffalo blood was collected in EDTA vacutainers (Becton Dickinson), aliquoted into 1.5 ml tubes and stored at −20 ◦ C. DNA was purified from bovine blood samples spotted on to FTA filter paper (Whatman BioScience) according to the protocol previously described in Oura et al. (2005). 2.3.2. PCR amplification A nested PCR reaction was carried out on DNA from buffalo blood samples immobilised on FTA filter paper. The inner and outer nested primers were designed from sequences in the flanking region of three micro- and 10 mini-satellite repeats (ms 2, ms 5, ms 7, MS 7, MS 8, MS 15, MS 16, MS 19, MS 25, MS 27, MS 30, MS 33 and MS 40). The copy number and consensus repeat sequences as well as the sequences of the inner nested primers are as described (Oura et al., 2003) and the sequences of the outer nested primers are as previously described (Oura et al., 2004a, 2005). The conditions used in the nested PCR amplifications have also been described previously (Oura et al., 2004a). 2.3.3. High-resolution amplicon separation using “Spreadex” gels The use of Spreadex gels to define different mini- and micro-satellite alleles of T. parva at high resolution has been described previously (Oura et al., 2003, 2004a). Under optimal conditions these gels provide a resolution of three base pairs (bp). Allele sizes were estimated by direct comparison with the M3 marker (Elchrom Scientific), which contains more than 50 DNA fragments ranging between 75
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Fig. 1. Map of Uganda showing the location of the four national parks, Lake Mburo National Park (LMNP), Queen Elizabeth National Park (QENP), Murchison Falls National Park (MFNP) and Kidepo Valley National Park (KVNP) from which buffalo were sampled during this study.
and 622 bp. The M3 marker has been specifically designed for the accurate sizing of micro- and mini-satellite alleles that may differ in size by as little as three base pairs and contains a range of markers that are three to five base pairs apart.
object of the analysis was to identify underlying trends within the dataset of genotypic profiles. The Microsoft Excel plug-in “Genalex6 (Peakall and Smouse, 2006) was used to implement PCA. 3. Results
2.4. Data analysis A ‘genotypic profile’ was generated for every buffalo sample and this was achieved by recording the presence or absence of each allele for every marker. Jaccard’s coefficient (Jaccard, 1908) was used to calculate pair-wise similarities between each buffalo’s genotypic profile and the data was utilised for principal component analysis (PCA). PCA reduces the number of dimensions in a dataset while retaining those characteristics that contribute most to its variance by generating a number of uncorrelated variables called principal components. The first principal component accounts for as much of the variability in the data as possible, with each successive component accounting for as much of the remaining variability as possible. The
3.1. Haemoparasite carrier prevalence in buffalo from four national parks in Uganda Blood samples were taken from 19 buffalo in Lake Mburo National Park (LMNP), 27 buffalo in Queen Elizabeth National Park (QENP), 12 buffalo from Murchison Falls National Park (MFNP) and 25 buffalo from Kidepo Valley National Park (KVNP). The samples were analysed by a RLB assay (Fig. 2) and results are summarised in Table 1. The results for each haemoparasite are detailed below. Theileria parva: The RLB assay revealed that 17 out of 19 (90%) of the buffalo sampled inside LMNP were positive for T. parva, however, the more sensitive nested mini-satellite PCR assay (MS 16) revealed that all of the buffalo sampled in
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Fig. 2. Reverse line blot of (a) buffalo from Lake Mburo National park (LMNP), (b) buffalo from the northern and southern sectors of Queen Elizabeth National park (QENP), (c) buffalo from Murchison Falls National park (MFNP) and (d) buffalo from Kidepo Valley National park (KVNP). Species-specific oligonucleotide probes were applied to the horizontal rows of the RLB and are shown to the left of the blot (T/B catch-all, Theileria/Babesia catch-all; E catch-all, Ehrlichia catch-all; E, Ehrlichia; A, Anaplasma; B, Babesia; T, Theileria).
LMNP were positive for T. parva (Fig. 2 and Table 1). Buffalo in the northern section of MFNP and in KVNP were negative for T. parva by both RLB as well as the mini-satellite PCR (Fig. 2). Interestingly, 17 out of 18 (95%) of the buffalo from the northern part of QENP were positive for T. parva whereas all nine buffalo sampled in the south of QENP were negative for T. parva. Theileria mutans: All 58 buffalo sampled in LMNP, QENP (north and south sectors) and MFNP were RLB positive for T. mutans and 92% (23 out of 25) of buffalo in KVNP were positive for T. mutans. Theileria velifera: The pattern of T. velifera prevalence in buffalo was similar to that for T. mutans. All 58 buffalo sampled in LMNP, QENP and MFNP were RLB positive for T. velifera and 80% (20 out of 25) of buffalo in KVNP were positive for T. velifera. Theileria buffeli: There were marked differences in prevalence of T. buffeli in the different national parks in Uganda. All 19 buffalo sampled in LMNP were carriers of T. buffeli, however T. buffeli was not present in any of the 37 buffalo sampled from MFNP or KVNP. In QENP only 11% (2
out of 18) of buffalo were positive in the north of the park and no buffalo were positive in the south of the park. Theileria sp. (buffalo) (Allsopp et al., 1993): All 19 (100%) of buffalo sampled from LMNP were carriers of T. sp. (buffalo) but this parasite was absent in buffalo from MFNP and KVNP. All 18 buffalo in the north of QENP were positive for T. sp. (buffalo) although only 33% (3 out of 9) of sampled buffalo in the south of the park were positive. Anaplasma centrale: 12 out of 19 (63%) of the buffalo in LMNP were carriers of A. centrale, 10 out of 12 (84%) of buffalo were carriers in MFNP and 14 out of 25 (56%) of buffalo were positive in KVNP. In the northern part of QENP 14 out of 18 (78%) of buffalo were carriers of A. centrale as opposed to 3 out of 9 (33%) in the southern part of the park. Anaplasma marginale: 14 out of 19 (74%) of the buffalo in LMNP were positive for A. marginale and in QENP, MFNP and KVNP 40%, 50% and 68% of buffalo were positive for A. marginale. None of the sampled buffalo in the four national parks were carriers of T. taurotragi, E. bovis, E. ruminantium, B. bovis or B. bigemina.
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n, number of buffalo sampled; RLB, reverse line blot; MS 16, mini-satellite (MS 16) PCR; T, Theileria; B, Babesia; E, Ehrlichia.
0 0 0 0 0 19 (100%) 18 (100%) 9 (100%) 12 (100%) 23 (92%) 19 (100%) 17 (95%) 0 0 0 17 (90%) 17 (95%) 0 0 0 19 18 9 12 25 Lake Mburo National Park Queen Elizabeth National Park (North) Queen Elizabeth National Park (South) Murchison Falls National Park (North) Kidepo Valley National Park
RLB
MS 16
T. taurotragi T. mutans T. parva n Sampling site
Table 1 Prevalence of haemoparasites in buffalo in four national parks in Uganda.
T. velifera
19 (100%) 18 (100%) 9 (100%) 12 (100%) 20 (80%)
T. buffeli
19 (100%) 2 (11%) 0 0 0
19 (100%) 18 (100%) 3 (33%) 0 0
T. spa (buffalo)
12 (63%) 14 (78%) 3 (33%) 10 (84%) 14 (56%)
A. centrale
14 (74%) 9 (50%) 2 (22%) 6 (50%) 17 (68%)
A. marginale
0 0 0 0 0
E. bovis
3.2. Allelic variation among buffalo genotypes of T. parva in two geographically separated national parks Buffalo grazing in LMNP and QENP are separated by over 300 km and historically are not thought to have mixed. In order to investigate to what extent the gene pools of T. parva genotypes in these two populations of buffalo are separate, blood samples from 19 buffalo in LMNP and 17 buffalo from the northern part of QENP were PCR amplified with a panel of 13 mini- and micro-satellite primers and the amplified DNA was separated on Spreadex gels. Spreadex gels showing a high level of multiple alleles amplified with five of the 13 mini- and micro-satellites are shown in Fig. 3 (left panel – LMNP buffalo, right panel – QENP buffalo). The high frequency of multiple alleles in the buffalo samples from each park made construction of multi-locus genotypes (MLGs) impossible, so similarity analyses were instead carried out comparing full ‘genotypic profiles’ between each of the buffalo in both parks. Jaccard’s coefficient of similarity was calculated and the results are shown as a PCA plot (Fig. 4). The plot indicates that the combinations of alleles present in the buffalo in LMNP are distinct from those in QENP and this indicates that there is geographical sub-structuring between T. parva genotypes in the buffalo present in these two national parks. A high multiplicity of infection (MOI) was revealed in both buffalo populations with seven samples from the QENP population found to contain as many as nine alleles using the marker MS 7, whereas a maximum of seven alleles were found at any one locus in individual samples from the LMNP population. The maximum number of alleles found at a single locus (MS 7) was used as a proxy for MOI (Oura et al., 2005) and it was confirmed that the mean MOI for each population was different, with the value of 7.38 for QENP being significantly greater than that of LMNP which was 5.89 (Fig. 5, Students t-test, p < 0.01). 4. Discussion The carrier prevalence of all the major tick-borne haemoparasites in a selection of 83 buffalo from four national parks in Uganda was measured using a reverse line blot (RLB) assay. The carrier prevalence of T. parva was also compared using an RLB and a mini-satellite (MS 16) PCR assay and the latter was found to be more sensitive at detecting T. parva in agreement with a previous study (Oura et al., 2004b). Very different carrier prevalences of haemoparasites were observed in buffalo from the four national parks in Uganda. Buffalo from the two national parks in the northern part of Uganda (MFNP and KVNP) were negative for T. parva indicating that the risk of T. parva parasites spreading Corridor disease from buffalo to cattle grazing in and around these parks in the north of Uganda is very low. In contrast, all the buffalo sampled from LMNP were carriers of T. parva and thus pose a risk to the domestic cattle population grazing in and around the park. Interestingly, all the buffalo from the northern part of QENP were positive for T. parva whereas the nine buffalo sampled in the south of QENP were negative for T. parva. There is a diffuse buffalo/cattle interface in the northern sector of QENP where cattle are
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Fig. 3. Spreadex gels showing PCR products generated using mini-satellite primers from (a) MS 27, (b) MS 40, (c) MS 16 and (d) MS 33 and (e) microsatellite ms 5 to amplify DNA from a selection of samples from buffalo grazing in Lake Mburo National park (LMNP) (left panel) and buffalo grazing in Queen Elizabeth National Park (QENP) (right panel). Alleles were sized by direct comparison with the M3 marker shown in the left and right of each gel and alleles of identical sizes were allocated a letter.
grazed illegally and utilise watering holes within the park. The buffalo in the southern sector of the park, however, are not thought to have contact with cattle (Woodford, 1982) as cattle do not graze in this area. As it is very unlikely that cattle are the source of buffalo infection, it is likely that the buffalo in the northern sector of the park have a pre-existing buffalo-derived population of T. parva which
is unrelated to the buffalo/cattle interface. The buffalo from the north and south of QENP are separated by a large channel of water (the Kazinga channel) separating Lake Edward and Lake George together with the 32 km2 Maramagambo Forest and therefore historic mixing between the two populations of buffalo is considered to be extremely unlikely.
Fig. 4. PCA showing Theileria parva genotypes present in buffalo in Lake Mburo National park (LMNP) and Queen Elizabeth National park (QENP). This diagram is based on a difference matrix derived from Jaccard’s similarity among genotypic profiles from buffalo grazing in Lake Mburo National park (LMNP) and buffalo grazing in Queen Elizabeth National park (QENP).
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Fig. 5. Box plot representing the multiplicity of infection in buffalo sampled from Lake Mburo National Park (LMNP) and Queen Elizabeth National Park (QENP). (IQR – inter-quartile range).
The carrier prevalence of co-infecting tick-borne haemoparasites was also measured using the RLB assay. A high carrier prevalence of T. mutans and T. velifera was seen in the buffalo from all four parks. The patterns of T. velifera and T. mutans prevalence were similar to each other in buffalo from each of the national parks which is unsurprising as both haemoparasites are transmitted by the same tick species (Amblyomma spp.). This high prevalence may be due either to a high and continuous challenge with T. mutans and T. velifera or the fact that these species are carried for longer periods of time at high levels after infection. It is generally considered that T. mutans and T. velifera are non-pathogenic in cattle, however an immunisation study carried out in Uganda (Robson et al., 1977) showed that, despite high levels of protection against ECF, zebu cattle still died during field trials if other tick-borne diseases were not also controlled. Field trials have also shown that T. mutans may cause severe anaemia and death in susceptible cattle (Snodgrass et al., 1972; Irvin et al., 1972). T. taurotragi, which is considered to be primarily a parasite of eland (Grootenhuis et al., 1980), was not present in any of the sampled buffalo, suggesting that buffalo are resistant to this parasite. Eland are present in all the sampled parks except QENP and, in a recent study of animals from LMNP (Oura et al., 2010), 100% of the eland were infected with T. taurotragi. Additionally, a previous study showed that T. taurotragi was present in a significant percentage of cattle in Uganda (Oura et al., 2004b) leading to the conclusion that the parasite was likely to be circulating directly amongst cattle populations in that country. None of the buffalo were positive for B. bovis, B. bigemina, E. bovis or E. ruminantium suggesting that these parasites do not circulate in buffalo or that a state of endemic instability exists for these parasites in buffalo. The infection of most buffalo with T. mutans and T. velifera demonstrates the presence of the Amblyomma sp. vector tick and the lack of infection with E. ruminantium transmitted by the same vector species is therefore significant. Experimentally buffalo have been shown to be susceptible to E. ruminantium
(reviewed in Peter et al., 2002) so this implies that this ricketsia is not present. Similarly, the widespread infection with Anaplasma marginale and A. centrale indicates the presence of Boophilus (Rhipicephalus) microplus or a related species of tick and, as these ticks are also vectors of Babesia bigemina and B. bovis, the absence of these infections suggests this parasite is not present in the region. Experimentally buffalo have been infected with B. bigemina, but infections with B. bovis have not been reported (reviewed by Penzhorn, 2006) and a survey of 190 buffalo in Botswana found no evidence for infection with Babesia spp. Thus the published data suggest that infections with these parasite species are rare but whether this is due to inherent resistance is not clear. Previous data (Oura et al., 2010) from cattle grazing in or near LMNP which are also not infected with E. ruminantium, B. bovis or B. bigemina, despite being susceptible to these pathogens, supports the view that these pathogens are not present rather than their absence from buffalo reflecting inherent resistance. Each of the buffalo samples which were positive for T. parva infection was found to contain a number of genotypes. Although up to nine alleles were found at the MS 7 locus indicating the presence of at least nine haploid genotypes, the true number of genotypes may be much higher when recombinants are considered. Although a higher mean multiplicity of infection was measured in QNEP buffalo, the significance of this finding is unclear. Studies in P. falciparum have found that MOI correlates with transmission intensity in an area (Arnot, 1998; Babiker et al., 1999) and it may be hypothesised that disease challenge is subtly different between the two parks. It is clear, however, that MOI in buffalo populations is far higher than that recorded in Ugandan cattle (Oura et al., 2005). In a previous study, it was demonstrated that 46% of T. parva positive adult cattle from the Mbarara area showed evidence of only one or two genotypes using the MS 7 marker and less than 10% of calves under a year had three or more genotypes. In contrast, the MS 7 data from this study demonstrates that over 90% of buffalo sampled had three or more T. parva genotypes and a high multiplicity of infection is a common feature of both buffalo populations (Fig. 5). As the parasitaemia in such highly susceptible crossbred cattle is likely to be comparable to that in buffalo, this difference in multiplicity of infection is likely to reflect a larger number of distinct genotypes in buffalo. Furthermore, given the high sensitivity of the PCR amplification of the microsatellite loci (Oura et al., 2004a), it is unlikely that additional genotypes are present in the cattle samples, unless they are at very low abundance. McKeever (2009) has speculated that the low pathogenicity of T. parva in buffalo may be associated with evasion of CTL mediated immunity, giving rise to a protracted period of infection. This would, in turn, be expected to promote co-infection with a comparatively higher number of parasite genotypes in buffalo. A high level of genetic diversity has previously been observed in buffalo samples (Bishop et al., 1993, 1994; Collins and Allsopp, 1999) and this has lead to speculation that the buffalo-associated T. parva population may represent a more diverse gene pool (Collins and Allsopp, 1999). Thus, a high background level of diversity in the buffalo-
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associated population may make it easier to discriminate between multiple co-infecting genotypes. The inability to generate predominant MLGs precludes formal population genetic analysis, however the similarity analysis of genotypic profiles (Fig. 4) clearly demonstrates the presence of two discrete parasite populations and these correspond perfectly with the two buffalo sampling sites. This finding suggests that the parasite populations are maintained in relative isolation, with any gene flow between areas being insufficient to homogenise the overall population. The nature of the tick life-cycle would suggest that self-propelled tick dispersal is limited to the local area and so for T. parva genotypes to disperse over wide areas host movement would be required. The results of this study suggest that there is no buffalo or other host migration between the southern and northern sectors of QENP. These sectors are separated by the Kazinga channel, which connects lakes George and Edward, and by the Maramagambo Forest which are highly likely to act as a barrier for migration of buffaloes and other wildlife between the northern and southern sectors of the park (Woodford, 1982). In conclusion the results presented in this paper highlight the complexity of tick-borne pathogen infections in buffalo in Uganda and emphasise the significant role that buffalo may play as reservoir hosts for tick-borne haemoparasites that have the potential to cause severe disease in domestic cattle. Acknowledgements C.A.L. Oura was funded by a Tropical Research Fellowship from the Wellcome Trust. We are grateful to the Uganda Wildlife Authority (UWA) for collecting the blood samples from the buffalo in the four national parks in Uganda We are especially thankful to Joseph Okori from UWA for his help and we would like to thank the PACE programme for allowing us access to the buffalo blood samples for this study. References Allsopp, B.A., Baylis, H.A., Allsopp, M.T., Cavalier-Smith, T., Bishop, R.P., Carrington, D.M., Sohanpal, B., Spooner, P., 1993. Discrimination between six species of Theileria using oligonucleotide probes which detect small subunit ribosomal RNA sequences. Parasitology 107 (Pt 2), 157–165. Almeria, S., Castella, J., Ferrer, D., Gutierrez, J.F., Estrada-Pena, A., Sparagano, O., 2002. Reverse line blot hybridization used to identify hemoprotozoa in Minorcan cattle. Ann. N. Y. Acad. Sci. 969, 78–82. Arnot, D., 1998. Unstable malaria in Sudan: the influence of the dry season. Clone multiplicity of Plasmodium falciparum infections in individuals exposed to variable levels of disease transmission. Trans. R. Soc. Trop. Med. Hyg. 92, 580–585. Babiker, H.A., Ranford-Cartwright, L.C., Walliker, D., 1999. Genetic structure and dynamics of Plasmodium falciparum infections in the Kilombero region of Tanzania. Trans. R. Soc. Trop. Med. Hyg. 93 (Suppl. 1), 11–14. Bekker, C.P., de Vos, S., Taoufik, A., Sparagano, O.A., Jongejan, F., 2002. Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization. Vet. Microbiol. 89, 223–238. Bishop, R.P., Sohanpal, B.K., Allsopp, B.A., Spooner, P.R., Dolan, T.T., Morzaria, S.P., 1993. Detection of polymorphisms among Theileria parva stocks using repetitive, telomeric and ribosomal DNA probes and anti-schizont monoclonal antibodies. Parasitology 107 (Pt 1), 19–31.
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Bishop, R.P., Spooner, P.R., Kanhai, G.K., Kiarie, J., Latif, A.A., Hove, T., Masaka, S., Dolan, T.T., 1994. Molecular characterization of Theileria parasites: application to the epidemiology of theileriosis in Zimbabwe. Parasitology 109 (Pt 5), 573–581. Collins, N.E., Allsopp, B.A., 1999. Theileria parva ribosomal internal transcribed spacer sequences exhibit extensive polymorphism and mosaic evolution: application to the characterization of parasites from cattle and buffalo. Parasitology 118 (Pt 6), 541–551. Epstein, H., 1971. On the Classification of Cattle. The Origin of Domestic Animals of Africa. 1. African Publishing Corporation, New York, p. 187. Geysen, D., Bazarusanga, T., Brandt, J., Dolan, T.T., 2004. An unusual mosaic structure of the PIM gene of Theileria parva and its relationship to allelic diversity. Mol. Biochem. Parasitol. 133, 163–173. Grootenhuis, J.G., Leitch, B.L., Stagg, D.A., Dolan, T.T., Young, A.S., 1987. Experimental induction of Theileria parva lawrencei carrier state in an African buffalo (Syncerus caffer). Parasitology 94 (Pt 3), 425–431. Grootenhuis, J.G., Morrison, W.I., Karstad, L., Sayer, P.D., Young, A.S., Murray, M., Haller, R.D., 1980. Fatal theileriosis in eland (Taurotragus oryx): pathology of natural and experimental cases. Res. Vet. Sci. 29, 219–229. Gubbels, J.M., de Vos, A.P., van der, W.M., Viseras, J., Schouls, L.M., de Vries, E., Jongejan, F., 1999. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. J. Clin. Microbiol. 37, 1782–1789. Irvin, A.D., Brown, C.G., Burridge, M.J., Cunningham, M.P., Musoke, A.J., Pierce, M.A., Purnell, R.E., Radley, D.E., 1972. A pathogenic theilerial syndrome of cattle in the Narok District of Kenya. I. Transmission studies. Trop. Anim. Health Prod. 4, 220–229. Jaccard, P., 1908. Bull. Soc. Vaudoise Sci. Nat. 44, 223–270. McKeever, D.J., 2009. Bovine immunity – a driver for diversity in Theileria parasites? Trends Parasitol. 25, 269–276. Oura, C.A., Tait, A., Asiimwe, B., Lubega, G.W., Weir, W., 2010. Theileria parva genetic diversity and haemoparasite prevalence in cattle and wildlife in and around Lake Mburo National Park in Uganda. Parasitol. Res., doi:10.1007/s00436-010-2030-8. Oura, C.A., Asiimwe, B.B., Weir, W., Lubega, G.W., Tait, A., 2005. Population genetic analysis and sub-structuring of Theileria parva in Uganda. Mol. Biochem. Parasitol. 140, 229–239. Oura, C.A., Bishop, R., Wampande, E.M., Lubega, G.W., Tait, A., 2004a. The persistence of component Theileria parva stocks in cattle immunized with the ‘Muguga cocktail’ live vaccine against East Coast fever in Uganda. Parasitology 129, 27–42. Oura, C.A., Bishop, R.P., Wampande, E.M., Lubega, G.W., Tait, A., 2004b. Application of a reverse line blot assay to the study of haemoparasites in cattle in Uganda. Int. J. Parasitol. 34, 603–613. Oura, C.A., Odongo, D.O., Lubega, G.W., Spooner, P.R., Tait, A., Bishop, R.P., 2003. A panel of microsatellite and minisatellite markers for the characterisation of field isolates of Theileria parva. Int. J. Parasitol. 33, 1641–1653. Peakall, R., Smouse, P.E., 2006. GENALEX6: genetic analysis in Excel. Population genetic software for teaching and research. Mol. Ecol. Notes 6, 288–295. Penzhorn, B.L., 2006. Babesiosis of wild carnivores and ungulates. Vet. Parasitol. 138 (1–2), 11–21. Perry, B.D., Young, A.S., 1995. The past and future roles of epidemiology and economics in the control of tick-borne diseases of livestock in Africa: the case of theileriosis. Prevent. Vet. Med. 25, 107–120. Peter, T.F., Burridge, M.J., Mahan, S.M., 2002. Ehrlichia ruminantium infection (heartwater) in wild animals. Trends Parasitol. 18 (5), 214–218. Robson, J., Pedersen, V., Odeke, G.M., Kamya, E.P., Brown, C.G., 1977. East Coast Fever immunisation trials in Uganda: field exposure of zebu cattle immunized with three isolates of Theileria parva. Trop. Anim. Health Prod. 9, 219–231. Snodgrass, D.R., Trees, A.J., Bowyer, W.A., Bergman, J.R., Daft, J., Wall, A.E., 1972. East Coast Fever: field challenge of cattle immunised against Theileria parva (Muguga). Trop. Anim. Health Prod. 4, 142–151. Sparagano, O., Loria, G.R., Gubbels, M.J., de Vos, A.P., Caracappa, S., Jongejan, F., 2000. Integrated molecular diagnosis of Theileria and Babesia species of cattle in Italy. Ann. N. Y. Acad. Sci. 916, 533–539. Uilenberg, G., 1995. International collaborative research: significance of tick-borne hemoparasitic diseases to world animal health. Vet. Parasitol. 57, 19–41. Woodford, M.H., 1982. Tuberculosis in wildlife in the Ruwenzori National Park Uganda (part I). Trop. Anim. Health Prod. 14, 81–88. Young, A.S., Brown, C.G., Burridge, M.J., Cunningham, M.P., Kirimi, I.M., Irvin, A.D., 1973. Observations on the cross-immunity between Theileria lawrencei (Serengeti) and Theileria parva (Muguga) in cattle. Int. J. Parasitol. 3, 723–728.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Haemoparasite prevalence and Theileria parva strain diversity in Cape buffalo (Syncerus caffer) in Uganda C.A.L. Oura a,b,c,∗ , A. Tait b , B. Asiimwe a,d , G.W. Lubega a , W. Weir b a b c d
Department of Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Makerere, P.O. Box 7062, Kampala, Uganda Division of Veterinary Infection and Immunity, University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, UK Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU240NF, UK Department of Medical Microbiology, College of Health Sciences, Makerere University, P.O. Box 7072, Kampala, Uganda
a r t i c l e
i n f o
Article history: Received 25 July 2010 Received in revised form 9 October 2010 Accepted 12 October 2010 Keywords: Theileria parva East Coast fever Cattle Buffalo Reverse line blot
a b s t r a c t Cape buffalo (Syncerus caffer) are considered to be an important reservoir for various tick-borne haemoparasites of veterinary importance. In this study we have compared the haemoparasite carrier prevalence in buffalo from four geographically isolated national parks in Uganda [Lake Mburo National Park (LMNP), Queen Elizabeth National Park (QENP), Murchison Falls National Park (MFNP) and Kidepo Valley National Park (KVNP)]. Differences were seen in haemoparasite prevalence in buffalo from the four national parks. All the buffalo sampled in LMNP were carriers of Theileria parva however, buffalo from MFNP and KVNP, which are both located in the north of Uganda, were negative for T. parva. Interestingly, 95% of buffalo in the northern part of QENP were T. parva positive, however all buffalo sampled in the south of the park were negative. A high multiplicity of infection was recorded in all the buffalo found to be carrying T. parva, with evidence of at least nine parasite genotypes in some animals. Most of the buffalo sampled in all four national parks were carriers of T. mutans and T. velifera, however none were carriers of T. taurotragi, Babesia bovis, Babesia bigemina, Ehrlichia bovis or Ehrlichia ruminantium. All the buffalo sampled from LMNP were positive for T. buffeli and T. sp. (buffalo) however, buffalo from the parks in the north of the country (KVNP and MFNP) were negative for these haemoparasites. Anaplasma centrale and Anaplasma marginale were circulating in buffalo from all four national parks. T. parva gene pools from two geographically separated populations of buffalo in two of the national parks in Uganda (LMNP and QENP) were compared. The T. parva populations in the two national parks were distinct, indicating that there was limited gene flow between the populations. The results presented highlight the complexity of tick-borne pathogen infections in buffalo and the significant role that buffalo may play as reservoir hosts for veterinary haemoparasites that have the potential to cause severe disease in domestic cattle. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction Tick-borne diseases constitute a major constraint on cattle production and the expansion of the dairy industry in
∗ Corresponding author at: Vector-borne Diseases Programme, Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU240NF, UK. Tel.: +44 1483 232441; fax: +44 1483 232448. E-mail address: [email protected] (C.A.L. Oura).
many countries across Africa (Uilenberg, 1995). The most serious tick-borne disease in eastern and central Africa is East Coast fever (ECF) caused by the intracellular protozoan parasite Theileria parva. The disease is associated with high levels of mortality, primarily in exotic and crossbred cattle, but also in indigenous calves and adult cattle in endemically unstable areas (Perry and Young, 1995). Cattle in East Africa are also exposed to a range of other tickborne pathogens in addition to Theileria species, including Ehrlichia, Anaplasma and Babesia species. Recently a reverse
0304-4017/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.10.032
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line blot (RLB) assay has been described (Gubbels et al., 1999; Bekker et al., 2002) that is able to simultaneously detect each of the Theileria, Babesia, Anaplasma and Ehrlichia species in ruminants in one assay. This assay has been applied in a field study in Uganda (Oura et al., 2004b) and it has also been used to identify haemoprotozoa in Minorcan cattle (Almeria et al., 2002) and for the integrated molecular diagnosis of Theileria and Babesia species present in cattle in Italy (Sparagano et al., 2000). Very little is known about the role of African Cape buffalo (Syncerus caffer) as a reservoir for haemoparasites, however buffalo are thought to be the major natural host of T. parva in which the parasite appears not to cause clinical disease (Grootenhuis et al., 1987). Co-evolution of African Cape buffalo with T. parva populations must have occurred prior to infection of cattle (Epstein, 1971) and it is thought that ticks infected with T. parva from buffalo first came into contact with Bos indicus cattle in Sub-Saharan Africa approximately 4500 years ago (Epstein, 1971). ECF is caused by T. parva parasites that can be transmitted between cattle by ticks, but a different clinical syndrome associated with high mortality levels in cattle, Corridor disease, is thought to be caused by the transmission of T. parva directly from buffalo to cattle via ticks. Cattle–tick–cattle transmission is thought to be limited as erythrocytic piroplasms are either absent or at an insufficient level to infect new ticks. The use of molecular characterisation tools has revealed a high level of diversity among buffalo-derived T. parva stocks compared to cattle-derived T. parva stocks (Bishop et al., 1994; Collins and Allsopp, 1999; Geysen et al., 2004; Oura et al., 2004a). Thus, there is growing evidence that only a limited subset of the total T. parva gene pool present within buffalo has become established in cattle. It is unknown whether the transfer of buffaloderived strains to cattle resulting in ECF was a single event or whether there is a constant trickle of new strains transferring from buffalo to cattle. This has very important implications since it has been demonstrated that there is incomplete cross-protection between animals immunised with cattle-derived T. parva on challenge with parasites from buffalo (Young et al., 1973). This suggests that any live vaccination programme may not protect against Corridor disease in areas where cattle and buffalo co-graze. In this study we report the application of an RLB assay to compare the carrier prevalence of tick-borne haemoparasites in Cape buffalo in four geographically isolated national parks in Uganda: Lake Mburo National Park (LMNP), Queen Elizabeth National Park (QENP), Murchison Falls National Park (MFNP) and Kidepo Valley National Park (KVNP). The results presented herein provide evidence for a complex pattern of infection of tick-borne pathogens in buffalo in Uganda and demonstrate distinct gene pools of T. parva parasites in populations derived from buffalo in two of the Ugandan national parks. 2. Materials and methods 2.1. Buffalo sampling Cape buffalo were sampled as part of the Pan African programme for the control of epizootics (PACE). In total 19
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adult buffalo were sampled from LMNP, 12 adult buffalo were sampled from MFNP and 25 adult buffalo were sampled from KVNP. In QENP 18 adult buffalo were sampled from the northern part of the park and nine adult buffalo were sampled from the southern part of the park. The northern and southern parts of QENP are separated by the Kazinga channel which connects lakes George and Edward and by the 32 km2 Maramagambo Forest, which appear to form a barrier for migration of buffaloes between the northern and southern sectors of the park (Woodford, 1982). The location of the four national parks from which buffalo were sampled is shown on a map of Uganda (Fig. 1). 2.2. Haemoparasite detection Blood samples were processed and analysed for the presence of haemoparasites by an RLB assay (Gubbels et al., 1999) with modifications previously described (Oura et al., 2004b). All samples were also assayed for the presence of T. parva by a nested PCR assay using mini-satellite marker MS 16 (Oura et al., 2004a). The RLB assay had a detection threshold of between 4.4 × 10−5 and 1.4 × 10−5 % parasitaemia that is equivalent to around 1–2 parasites/l of blood. The MS 16 mini-satellite assay was more sensitive with a detection threshold of between 1.4 × 10−5 % parasitaemia and 4.6 × 10−6 % parasitaemia that is equivalent to between approximately 1 and 0.3 parasites/l of blood (Oura et al., 2004b). 2.3. Micro- and mini-satellite PCR assay 2.3.1. Sample preparation Buffalo blood was collected in EDTA vacutainers (Becton Dickinson), aliquoted into 1.5 ml tubes and stored at −20 ◦ C. DNA was purified from bovine blood samples spotted on to FTA filter paper (Whatman BioScience) according to the protocol previously described in Oura et al. (2005). 2.3.2. PCR amplification A nested PCR reaction was carried out on DNA from buffalo blood samples immobilised on FTA filter paper. The inner and outer nested primers were designed from sequences in the flanking region of three micro- and 10 mini-satellite repeats (ms 2, ms 5, ms 7, MS 7, MS 8, MS 15, MS 16, MS 19, MS 25, MS 27, MS 30, MS 33 and MS 40). The copy number and consensus repeat sequences as well as the sequences of the inner nested primers are as described (Oura et al., 2003) and the sequences of the outer nested primers are as previously described (Oura et al., 2004a, 2005). The conditions used in the nested PCR amplifications have also been described previously (Oura et al., 2004a). 2.3.3. High-resolution amplicon separation using “Spreadex” gels The use of Spreadex gels to define different mini- and micro-satellite alleles of T. parva at high resolution has been described previously (Oura et al., 2003, 2004a). Under optimal conditions these gels provide a resolution of three base pairs (bp). Allele sizes were estimated by direct comparison with the M3 marker (Elchrom Scientific), which contains more than 50 DNA fragments ranging between 75
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Fig. 1. Map of Uganda showing the location of the four national parks, Lake Mburo National Park (LMNP), Queen Elizabeth National Park (QENP), Murchison Falls National Park (MFNP) and Kidepo Valley National Park (KVNP) from which buffalo were sampled during this study.
and 622 bp. The M3 marker has been specifically designed for the accurate sizing of micro- and mini-satellite alleles that may differ in size by as little as three base pairs and contains a range of markers that are three to five base pairs apart.
object of the analysis was to identify underlying trends within the dataset of genotypic profiles. The Microsoft Excel plug-in “Genalex6 (Peakall and Smouse, 2006) was used to implement PCA. 3. Results
2.4. Data analysis A ‘genotypic profile’ was generated for every buffalo sample and this was achieved by recording the presence or absence of each allele for every marker. Jaccard’s coefficient (Jaccard, 1908) was used to calculate pair-wise similarities between each buffalo’s genotypic profile and the data was utilised for principal component analysis (PCA). PCA reduces the number of dimensions in a dataset while retaining those characteristics that contribute most to its variance by generating a number of uncorrelated variables called principal components. The first principal component accounts for as much of the variability in the data as possible, with each successive component accounting for as much of the remaining variability as possible. The
3.1. Haemoparasite carrier prevalence in buffalo from four national parks in Uganda Blood samples were taken from 19 buffalo in Lake Mburo National Park (LMNP), 27 buffalo in Queen Elizabeth National Park (QENP), 12 buffalo from Murchison Falls National Park (MFNP) and 25 buffalo from Kidepo Valley National Park (KVNP). The samples were analysed by a RLB assay (Fig. 2) and results are summarised in Table 1. The results for each haemoparasite are detailed below. Theileria parva: The RLB assay revealed that 17 out of 19 (90%) of the buffalo sampled inside LMNP were positive for T. parva, however, the more sensitive nested mini-satellite PCR assay (MS 16) revealed that all of the buffalo sampled in
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Fig. 2. Reverse line blot of (a) buffalo from Lake Mburo National park (LMNP), (b) buffalo from the northern and southern sectors of Queen Elizabeth National park (QENP), (c) buffalo from Murchison Falls National park (MFNP) and (d) buffalo from Kidepo Valley National park (KVNP). Species-specific oligonucleotide probes were applied to the horizontal rows of the RLB and are shown to the left of the blot (T/B catch-all, Theileria/Babesia catch-all; E catch-all, Ehrlichia catch-all; E, Ehrlichia; A, Anaplasma; B, Babesia; T, Theileria).
LMNP were positive for T. parva (Fig. 2 and Table 1). Buffalo in the northern section of MFNP and in KVNP were negative for T. parva by both RLB as well as the mini-satellite PCR (Fig. 2). Interestingly, 17 out of 18 (95%) of the buffalo from the northern part of QENP were positive for T. parva whereas all nine buffalo sampled in the south of QENP were negative for T. parva. Theileria mutans: All 58 buffalo sampled in LMNP, QENP (north and south sectors) and MFNP were RLB positive for T. mutans and 92% (23 out of 25) of buffalo in KVNP were positive for T. mutans. Theileria velifera: The pattern of T. velifera prevalence in buffalo was similar to that for T. mutans. All 58 buffalo sampled in LMNP, QENP and MFNP were RLB positive for T. velifera and 80% (20 out of 25) of buffalo in KVNP were positive for T. velifera. Theileria buffeli: There were marked differences in prevalence of T. buffeli in the different national parks in Uganda. All 19 buffalo sampled in LMNP were carriers of T. buffeli, however T. buffeli was not present in any of the 37 buffalo sampled from MFNP or KVNP. In QENP only 11% (2
out of 18) of buffalo were positive in the north of the park and no buffalo were positive in the south of the park. Theileria sp. (buffalo) (Allsopp et al., 1993): All 19 (100%) of buffalo sampled from LMNP were carriers of T. sp. (buffalo) but this parasite was absent in buffalo from MFNP and KVNP. All 18 buffalo in the north of QENP were positive for T. sp. (buffalo) although only 33% (3 out of 9) of sampled buffalo in the south of the park were positive. Anaplasma centrale: 12 out of 19 (63%) of the buffalo in LMNP were carriers of A. centrale, 10 out of 12 (84%) of buffalo were carriers in MFNP and 14 out of 25 (56%) of buffalo were positive in KVNP. In the northern part of QENP 14 out of 18 (78%) of buffalo were carriers of A. centrale as opposed to 3 out of 9 (33%) in the southern part of the park. Anaplasma marginale: 14 out of 19 (74%) of the buffalo in LMNP were positive for A. marginale and in QENP, MFNP and KVNP 40%, 50% and 68% of buffalo were positive for A. marginale. None of the sampled buffalo in the four national parks were carriers of T. taurotragi, E. bovis, E. ruminantium, B. bovis or B. bigemina.
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n, number of buffalo sampled; RLB, reverse line blot; MS 16, mini-satellite (MS 16) PCR; T, Theileria; B, Babesia; E, Ehrlichia.
0 0 0 0 0 19 (100%) 18 (100%) 9 (100%) 12 (100%) 23 (92%) 19 (100%) 17 (95%) 0 0 0 17 (90%) 17 (95%) 0 0 0 19 18 9 12 25 Lake Mburo National Park Queen Elizabeth National Park (North) Queen Elizabeth National Park (South) Murchison Falls National Park (North) Kidepo Valley National Park
RLB
MS 16
T. taurotragi T. mutans T. parva n Sampling site
Table 1 Prevalence of haemoparasites in buffalo in four national parks in Uganda.
T. velifera
19 (100%) 18 (100%) 9 (100%) 12 (100%) 20 (80%)
T. buffeli
19 (100%) 2 (11%) 0 0 0
19 (100%) 18 (100%) 3 (33%) 0 0
T. spa (buffalo)
12 (63%) 14 (78%) 3 (33%) 10 (84%) 14 (56%)
A. centrale
14 (74%) 9 (50%) 2 (22%) 6 (50%) 17 (68%)
A. marginale
0 0 0 0 0
E. bovis
3.2. Allelic variation among buffalo genotypes of T. parva in two geographically separated national parks Buffalo grazing in LMNP and QENP are separated by over 300 km and historically are not thought to have mixed. In order to investigate to what extent the gene pools of T. parva genotypes in these two populations of buffalo are separate, blood samples from 19 buffalo in LMNP and 17 buffalo from the northern part of QENP were PCR amplified with a panel of 13 mini- and micro-satellite primers and the amplified DNA was separated on Spreadex gels. Spreadex gels showing a high level of multiple alleles amplified with five of the 13 mini- and micro-satellites are shown in Fig. 3 (left panel – LMNP buffalo, right panel – QENP buffalo). The high frequency of multiple alleles in the buffalo samples from each park made construction of multi-locus genotypes (MLGs) impossible, so similarity analyses were instead carried out comparing full ‘genotypic profiles’ between each of the buffalo in both parks. Jaccard’s coefficient of similarity was calculated and the results are shown as a PCA plot (Fig. 4). The plot indicates that the combinations of alleles present in the buffalo in LMNP are distinct from those in QENP and this indicates that there is geographical sub-structuring between T. parva genotypes in the buffalo present in these two national parks. A high multiplicity of infection (MOI) was revealed in both buffalo populations with seven samples from the QENP population found to contain as many as nine alleles using the marker MS 7, whereas a maximum of seven alleles were found at any one locus in individual samples from the LMNP population. The maximum number of alleles found at a single locus (MS 7) was used as a proxy for MOI (Oura et al., 2005) and it was confirmed that the mean MOI for each population was different, with the value of 7.38 for QENP being significantly greater than that of LMNP which was 5.89 (Fig. 5, Students t-test, p < 0.01). 4. Discussion The carrier prevalence of all the major tick-borne haemoparasites in a selection of 83 buffalo from four national parks in Uganda was measured using a reverse line blot (RLB) assay. The carrier prevalence of T. parva was also compared using an RLB and a mini-satellite (MS 16) PCR assay and the latter was found to be more sensitive at detecting T. parva in agreement with a previous study (Oura et al., 2004b). Very different carrier prevalences of haemoparasites were observed in buffalo from the four national parks in Uganda. Buffalo from the two national parks in the northern part of Uganda (MFNP and KVNP) were negative for T. parva indicating that the risk of T. parva parasites spreading Corridor disease from buffalo to cattle grazing in and around these parks in the north of Uganda is very low. In contrast, all the buffalo sampled from LMNP were carriers of T. parva and thus pose a risk to the domestic cattle population grazing in and around the park. Interestingly, all the buffalo from the northern part of QENP were positive for T. parva whereas the nine buffalo sampled in the south of QENP were negative for T. parva. There is a diffuse buffalo/cattle interface in the northern sector of QENP where cattle are
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Fig. 3. Spreadex gels showing PCR products generated using mini-satellite primers from (a) MS 27, (b) MS 40, (c) MS 16 and (d) MS 33 and (e) microsatellite ms 5 to amplify DNA from a selection of samples from buffalo grazing in Lake Mburo National park (LMNP) (left panel) and buffalo grazing in Queen Elizabeth National Park (QENP) (right panel). Alleles were sized by direct comparison with the M3 marker shown in the left and right of each gel and alleles of identical sizes were allocated a letter.
grazed illegally and utilise watering holes within the park. The buffalo in the southern sector of the park, however, are not thought to have contact with cattle (Woodford, 1982) as cattle do not graze in this area. As it is very unlikely that cattle are the source of buffalo infection, it is likely that the buffalo in the northern sector of the park have a pre-existing buffalo-derived population of T. parva which
is unrelated to the buffalo/cattle interface. The buffalo from the north and south of QENP are separated by a large channel of water (the Kazinga channel) separating Lake Edward and Lake George together with the 32 km2 Maramagambo Forest and therefore historic mixing between the two populations of buffalo is considered to be extremely unlikely.
Fig. 4. PCA showing Theileria parva genotypes present in buffalo in Lake Mburo National park (LMNP) and Queen Elizabeth National park (QENP). This diagram is based on a difference matrix derived from Jaccard’s similarity among genotypic profiles from buffalo grazing in Lake Mburo National park (LMNP) and buffalo grazing in Queen Elizabeth National park (QENP).
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Fig. 5. Box plot representing the multiplicity of infection in buffalo sampled from Lake Mburo National Park (LMNP) and Queen Elizabeth National Park (QENP). (IQR – inter-quartile range).
The carrier prevalence of co-infecting tick-borne haemoparasites was also measured using the RLB assay. A high carrier prevalence of T. mutans and T. velifera was seen in the buffalo from all four parks. The patterns of T. velifera and T. mutans prevalence were similar to each other in buffalo from each of the national parks which is unsurprising as both haemoparasites are transmitted by the same tick species (Amblyomma spp.). This high prevalence may be due either to a high and continuous challenge with T. mutans and T. velifera or the fact that these species are carried for longer periods of time at high levels after infection. It is generally considered that T. mutans and T. velifera are non-pathogenic in cattle, however an immunisation study carried out in Uganda (Robson et al., 1977) showed that, despite high levels of protection against ECF, zebu cattle still died during field trials if other tick-borne diseases were not also controlled. Field trials have also shown that T. mutans may cause severe anaemia and death in susceptible cattle (Snodgrass et al., 1972; Irvin et al., 1972). T. taurotragi, which is considered to be primarily a parasite of eland (Grootenhuis et al., 1980), was not present in any of the sampled buffalo, suggesting that buffalo are resistant to this parasite. Eland are present in all the sampled parks except QENP and, in a recent study of animals from LMNP (Oura et al., 2010), 100% of the eland were infected with T. taurotragi. Additionally, a previous study showed that T. taurotragi was present in a significant percentage of cattle in Uganda (Oura et al., 2004b) leading to the conclusion that the parasite was likely to be circulating directly amongst cattle populations in that country. None of the buffalo were positive for B. bovis, B. bigemina, E. bovis or E. ruminantium suggesting that these parasites do not circulate in buffalo or that a state of endemic instability exists for these parasites in buffalo. The infection of most buffalo with T. mutans and T. velifera demonstrates the presence of the Amblyomma sp. vector tick and the lack of infection with E. ruminantium transmitted by the same vector species is therefore significant. Experimentally buffalo have been shown to be susceptible to E. ruminantium
(reviewed in Peter et al., 2002) so this implies that this ricketsia is not present. Similarly, the widespread infection with Anaplasma marginale and A. centrale indicates the presence of Boophilus (Rhipicephalus) microplus or a related species of tick and, as these ticks are also vectors of Babesia bigemina and B. bovis, the absence of these infections suggests this parasite is not present in the region. Experimentally buffalo have been infected with B. bigemina, but infections with B. bovis have not been reported (reviewed by Penzhorn, 2006) and a survey of 190 buffalo in Botswana found no evidence for infection with Babesia spp. Thus the published data suggest that infections with these parasite species are rare but whether this is due to inherent resistance is not clear. Previous data (Oura et al., 2010) from cattle grazing in or near LMNP which are also not infected with E. ruminantium, B. bovis or B. bigemina, despite being susceptible to these pathogens, supports the view that these pathogens are not present rather than their absence from buffalo reflecting inherent resistance. Each of the buffalo samples which were positive for T. parva infection was found to contain a number of genotypes. Although up to nine alleles were found at the MS 7 locus indicating the presence of at least nine haploid genotypes, the true number of genotypes may be much higher when recombinants are considered. Although a higher mean multiplicity of infection was measured in QNEP buffalo, the significance of this finding is unclear. Studies in P. falciparum have found that MOI correlates with transmission intensity in an area (Arnot, 1998; Babiker et al., 1999) and it may be hypothesised that disease challenge is subtly different between the two parks. It is clear, however, that MOI in buffalo populations is far higher than that recorded in Ugandan cattle (Oura et al., 2005). In a previous study, it was demonstrated that 46% of T. parva positive adult cattle from the Mbarara area showed evidence of only one or two genotypes using the MS 7 marker and less than 10% of calves under a year had three or more genotypes. In contrast, the MS 7 data from this study demonstrates that over 90% of buffalo sampled had three or more T. parva genotypes and a high multiplicity of infection is a common feature of both buffalo populations (Fig. 5). As the parasitaemia in such highly susceptible crossbred cattle is likely to be comparable to that in buffalo, this difference in multiplicity of infection is likely to reflect a larger number of distinct genotypes in buffalo. Furthermore, given the high sensitivity of the PCR amplification of the microsatellite loci (Oura et al., 2004a), it is unlikely that additional genotypes are present in the cattle samples, unless they are at very low abundance. McKeever (2009) has speculated that the low pathogenicity of T. parva in buffalo may be associated with evasion of CTL mediated immunity, giving rise to a protracted period of infection. This would, in turn, be expected to promote co-infection with a comparatively higher number of parasite genotypes in buffalo. A high level of genetic diversity has previously been observed in buffalo samples (Bishop et al., 1993, 1994; Collins and Allsopp, 1999) and this has lead to speculation that the buffalo-associated T. parva population may represent a more diverse gene pool (Collins and Allsopp, 1999). Thus, a high background level of diversity in the buffalo-
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associated population may make it easier to discriminate between multiple co-infecting genotypes. The inability to generate predominant MLGs precludes formal population genetic analysis, however the similarity analysis of genotypic profiles (Fig. 4) clearly demonstrates the presence of two discrete parasite populations and these correspond perfectly with the two buffalo sampling sites. This finding suggests that the parasite populations are maintained in relative isolation, with any gene flow between areas being insufficient to homogenise the overall population. The nature of the tick life-cycle would suggest that self-propelled tick dispersal is limited to the local area and so for T. parva genotypes to disperse over wide areas host movement would be required. The results of this study suggest that there is no buffalo or other host migration between the southern and northern sectors of QENP. These sectors are separated by the Kazinga channel, which connects lakes George and Edward, and by the Maramagambo Forest which are highly likely to act as a barrier for migration of buffaloes and other wildlife between the northern and southern sectors of the park (Woodford, 1982). In conclusion the results presented in this paper highlight the complexity of tick-borne pathogen infections in buffalo in Uganda and emphasise the significant role that buffalo may play as reservoir hosts for tick-borne haemoparasites that have the potential to cause severe disease in domestic cattle. Acknowledgements C.A.L. Oura was funded by a Tropical Research Fellowship from the Wellcome Trust. We are grateful to the Uganda Wildlife Authority (UWA) for collecting the blood samples from the buffalo in the four national parks in Uganda We are especially thankful to Joseph Okori from UWA for his help and we would like to thank the PACE programme for allowing us access to the buffalo blood samples for this study. References Allsopp, B.A., Baylis, H.A., Allsopp, M.T., Cavalier-Smith, T., Bishop, R.P., Carrington, D.M., Sohanpal, B., Spooner, P., 1993. Discrimination between six species of Theileria using oligonucleotide probes which detect small subunit ribosomal RNA sequences. Parasitology 107 (Pt 2), 157–165. Almeria, S., Castella, J., Ferrer, D., Gutierrez, J.F., Estrada-Pena, A., Sparagano, O., 2002. Reverse line blot hybridization used to identify hemoprotozoa in Minorcan cattle. Ann. N. Y. Acad. Sci. 969, 78–82. Arnot, D., 1998. Unstable malaria in Sudan: the influence of the dry season. Clone multiplicity of Plasmodium falciparum infections in individuals exposed to variable levels of disease transmission. Trans. R. Soc. Trop. Med. Hyg. 92, 580–585. Babiker, H.A., Ranford-Cartwright, L.C., Walliker, D., 1999. Genetic structure and dynamics of Plasmodium falciparum infections in the Kilombero region of Tanzania. Trans. R. Soc. Trop. Med. Hyg. 93 (Suppl. 1), 11–14. Bekker, C.P., de Vos, S., Taoufik, A., Sparagano, O.A., Jongejan, F., 2002. Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization. Vet. Microbiol. 89, 223–238. Bishop, R.P., Sohanpal, B.K., Allsopp, B.A., Spooner, P.R., Dolan, T.T., Morzaria, S.P., 1993. Detection of polymorphisms among Theileria parva stocks using repetitive, telomeric and ribosomal DNA probes and anti-schizont monoclonal antibodies. Parasitology 107 (Pt 1), 19–31.
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