Appendix 22. Vosloo, W. 1*, Dwarka, R.M. 1, Bastos, A.D.S. 2, Esterhuysen, J.J. 1, Sahle, M. 3, Sangare, O. 4

Transcription

1 Appendix 22 Molecular epidemiological studies of Foot-and-Mouth disease virus in sub-saharan Africa indicate the presence of large numbers of topotypes: implications for local and international control Vosloo, W. 1*, Dwarka, R.M. 1, Bastos, A.D.S. 2, Esterhuysen, J.J. 1, Sahle, M. 3, Sangare, O. 4 1 Exotic Diseases Division, Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110, South Africa 2 Mammal Research Institute, Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, South Africa 3 National Animal Health Research Centre, Ethiopia 4 Laboratoire Central Veterinaire, BP 2295, Bamako, Mali Abstract: Six of the seven serotypes of foot and mouth disease (FMD) virus occur on the African continent and numerous topotypes occur for each serotype. Due to underreporting of FMD, the current strains circulating throughout sub-saharan Africa are in most cases not known. For both SAT-1 and SAT-2 the genetic diversity is reflected in antigenic variation and indications are that vaccine strains may be needed for each topotype. This has serious implications for control using vaccines and for choice of strains to include in international vaccine banks. The epidemiology of FMD is further complicated by the presence of large numbers of persistently infected African buffalo (Syncerus caffer) and other wildlife species which together with largely uncontrolled movement of domestic animals may spread the disease over vast distances. This dearth of knowledge on FMD in Africa poses a serious threat to regions free of FMD in the face of increased international travel and the possible smuggling of illegal bushmeat and other livestock products. Introduction: Foot and mouth disease (FMD) virus probably originated from Africa since greater genetic variation occurs in the SAT types (Vosloo et al., 1995; Bastos et al., 2000; Bastos, 2001; Bastos et al., 2001; Bastos et al., 2003a and b) and a sub-clinical cycle occurs in African buffalo (Syncerus caffer) where the virus can persist in a single animal for up to five years (Condy et al., 1985). This is the only species for which long-term maintenance of FMD virus has been described (Hedger, 1972; Hedger et al., 1972; Hedger, 1976; Condy et al., 1985; Thomson, 1994; Thomson et al., 2001; Thomson et al., 2003). During persistent infection in buffalo, the SAT type viruses undergo high rates of mutation, giving rise to genetic and antigenic variants (Vosloo et al., 1996). The disease is endemic to most countries in sub-saharan Africa (Vosloo et al., 2002) and will not be eradicated from southern and East Africa while infected buffalo are present. Disease-free areas are recognised mainly in southern Africa, where a number of countries have been able to control FMD by separating infected buffalo from livestock and by limited use of vaccination (Brückner et al., 2002; Thomson et al., 2003). Lack of movement control within countries and across international borders for both wildlife and domestic animals aggravates the problem, and gives credence to the fact that FMD will remain a problem on the sub-continent for the foreseeable future. With the increase in international travel, the threat from illegally smuggled bushmeat and other livestock products cannot be ignored, and it is imperative to understand the current epidemiology of FMD to predict what strains are currently most likely to pose a threat to disease-free regions. Six of the seven serotypes of FMD virus occur on the African continent (Vosloo et al., 2002), with the exception of Asia-1, which complicates control of the disease by vaccination. In sub-saharan Africa, two cycles of FMD occur, one where virus circulates between wildlife hosts and domestic animals and the other where the virus spreads among domestic animals, without the involvement of wildlife (Vosloo and Thomson, 2004). In southern Africa and to a large extent, eastern Africa, the cycle between wildlife and domestic animals occurs, while in West Africa, due to the low numbers of wildlife, the disease is maintained predominately in domestic animals. However, once disease crosses from wildlife into domestic animals, a domestic cycle could be maintained without the involvement of wildlife. As it is costly to sample wildlife, very little is known about the FMD virus populations circulating in these animals and most information outside southern Africa is based on isolates obtained from domestic animals. Molecular epidemiological studies have contributed in planning control strategies by elucidating historical and current disease transmission patterns within and between countries. Furthermore, it is 149

2 important to have data on the viral topotypes in both wildlife and domestic animals, information that should be heeded when planning FMD vaccination strategies (Vosloo et al., 1992; Vosloo et al., 1995; Bastos, 1998; Bastos et al., 2001; Bastos et al., 2003a and b; Sangare et al., 2003; Sangare et al., 2004). High levels of genetic diversity will most likely be reflected in antigenic differences and it has been shown that for vaccination to be effective, the viruses incorporated into vaccines need to be antigenically related to viruses circulating in the field (Hunter et al., 1996; Hunter, 1998). This paper summarises the current knowledge of genetic variation of FMD virus in sub-saharan Africa and discusses the antigenic relationships of SAT-1 and SAT-2 isolates from various topotypes measured against vaccine strains obtained in southern Africa. Materials and Methods: Phylogenetic analysis: Most viruses included in this study were propagated on IB-RS-2 cells before RNA extraction, cdna synthesis and amplification of the 1D gene using primers and methodology as described in the references presented in Table 1. The 1D gene was partially sequenced and phylogenetic analysis performed as described in the various references. The summary of topotypes in Table 1 was performed by relating similar strains in various papers and not by comparative analysis of sequencing data and may be different when actual data are utilised. Determination of r-values: The antigenic relationships were determined using cross-neutralisation assays in IB-RS-2 cells and microtitre plates against cattle sera prepared by two consecutive vaccinations (vaccinated at day 0, boosted at day 28 and bled at day 38) with alhydrogel containing vaccines with either one strain per serotype or a combination of both vaccine strains per serotype (SAT-1: SAR9/81 and KNP196/91/1; SAT-2: ZIM7/83 and KNP19/89/2) as described by Rweyemamu et al (1978). The r-values were calculated as: r = serum titre against heterologous virus/ serum titre against homologous virus Interpretation of r-values: r-values were interpreted as proposed by Samuel et al. (1990). Briefly, values between indicated highly significant antigenic variation from the vaccine strains and another vaccine strain should be chosen, values of showed a significant difference, but a potent vaccine may provide protection, while r-values of demonstrated that the vaccine and field strains are similar and the vaccine would provide good protection. Results: Topotype diversity in sub-saharan Africa: Within each serotype viruses grouped together in distinct clusters which were supported by high bootstrap values and these clades corresponded to geographically defined regions in agreement with the FMD topotype concept as described for the European and SAT serotypes (Bastos et al., 2001; Samuel and Knowles, 2001). The number of topotypes identified in sub-saharan Africa is summarised in Table 1. For SAT-1 eight topotypes were identified throughout the region of which most had localised geographic distribution. However, isolates from Zambia, Malawi, Tanzania, Kenya and northern Zimbabwe clustered in topotype III, demonstrating that related viruses were found in southern and East Africa and describing a link between the two regions. Three of the topotypes may be extinct (see below) since no recent viruses belonging to these were isolated. However, FMD is endemic to most of these regions and samples are submitted infrequently for further investigation, suggesting that these topotypes may still be present. Further research on this aspect is urgently needed. Topotype VI consisted of isolates obtained during 1974 only from Uganda, while topotype VII had isolates from Nigeria and Sudan between and finally topotype VIII consisted of viruses from Nigeria and Niger isolated between Uganda had three topotypes exclusive to that country (IV, V and VI) similar to Zimbabwe, but the latter shares topotypes with neighbouring countries (I [South Africa, Mozambique and southern Zimbabwe], II [Botswana, Namibia, Zambia and western Zimbabwe] and III [Zambia, Malawi, Tanzania, Kenya and northern Zimbabwe]; Table 1). SAT-2 demonstrated the most genetic diversity with a total of 14 topotypes with five of these possibly extinct (Table 1). Topotypes VI (Gambia and Senegal ), XI (Angola 1974), XII (Uganda ), XIII (Sudan 1977) and XIV (Ethiopia 1991) did not consist of any recent isolates. Two 150

3 topotypes occurred in West Africa (IV [Burundi, Malawi, Kenya, Tanzania and Ethiopia] and V [Nigeria, Senegal, Liberia, Ghana, Mali and Cote d Ivoire]), with topotype IV including countries from East and southern Africa. Four were found in southern Africa (I [South Africa, Mozambique and southern Zimbabwe], II [Namibia, Botswana, northern and western Zimbabwe], III [Botswana, Zambia and Zimbabwe] and XI [Angola]; Table 1). All the other topotypes were found in East Africa, with a single isolate from the Democratic Republic of Congo grouping with a virus from Uganda (topotype X; Table 1). Most of the topotypes in East Africa consisted of single isolates from one country (VII Eritrea; VIII Rwanda; IX Kenya, Uganda; XII Uganda; XIII Sudan; XIV Ethiopia) and could have wider geographic distribution if more isolates were available from these regions. Zimbabwe shared three different topotypes with neighbouring countries (I, II and III), Botswana shared two (II and III), while three topotypes were found in Uganda (IX, X, and XII), two consisting solely of isolates from this country (Table 1). Although SAT-3 has the most restricted distribution and is the type least frequently recovered from African buffalo, 6 topotypes were found with 25 genotypes of which four occurred in southern Africa and two were unique to East Africa (Bastos et al., 2003a). The latter two (V and VI) consisted of one virus each obtained from a single game park in Uganda isolated 37 years apart (Bastos et al., 2003a). Zimbabwe had the most diversity with three topotypes (I [South Africa, southern Zimbabwe], II [Namibia, Botswana and western Zimbabwe] and III [Malawi and northern Zimbabwe]; Table 1). The topotype distribution of SAT-3 demonstrated similarities with SAT-1 and SAT-2 distribution and as more information becomes available, better correlation will become evident. The SAT-3 topotype IV consisted of isolates obtained solely from Zambia since the genetic diversity is of such an extent that they were assigned to a separate topotype, while with both SAT-1 and SAT-2, the Zambian viruses grouped with neighbouring countries (Table 1). The number of topotypes and the geographical distribution was concluded by extracti9ng information form various publications (Knowles and Samuel, 2003; Sahle, 2003; Samuel and Knowles, 2001; Sangare et al., 2001) and the sequencing data needs to be evaluated to ensure the conclusions are correct. However, in the absence of these analyses, it was found that eight topotypes for serotype O exist in Africa of which a number could be ascribed to exotic introductions from elsewhere in the world. During 2000 South Africa experienced an outbreak caused by the Pan-Asian serotype O virus (topotype VII; Table 1) which was also closely related to the strain causing the later outbreak in the United Kingdom (Sangare et al., 2001; Knowles and Samuel, 2003). Similarly, the outbreak that occurred in Angola during (topotype VIII; Table 1) grouped with a virus isolate in Venezuela during 1951 (Sangare et al., 2001). A number of the isolates from East Africa grouped with North Africa in topotype I (Ethiopia, Eritrea, Kenya, Somalia, Sudan, Tunisia, Egypt, Algeria, Morocco, Libya and Tanzania) while viruses from West Africa grouped together in a single topotype similarly with north African countries (topotype II [Algeria, Cote d Ivoire, Guinea, Morocco, Niger, Ghana, Burkina Faso, Tunisia and Sudan]). All other topotypes consisted of viruses from East African countries (III [Uganda, Kenya, Sudan], IV [Uganda], V [Uganda], VI [Tanzania, Uganda]). Uganda demonstrated the most diversity with four topotypes, while Sudan formed the link between East and West Africa in topotype II (Table 1). Sudan also acted as link between East and West Africa when investigating serotype A distribution by grouping in the same topotype which consisted of all the West African viruses investigated so far (topotype I [Mauritania, Mali, Cote d Ivoire, Ghana, Niger, Nigeria, Cameroon, Chad, Senegal, Gambia, Sudan] Table 1; Knowles et al., 1998). Topotype II consisted of isolates from southern Africa (Angola and Malawi) and North Africa (Algeria, Morocco, Libya and Tunisia) but it is not clear whether there is a link between these outbreaks and should be investigated further. Type A is not endemic to southern Africa and outbreaks have only been documented in Namibia, Malawi and Zambia of which a number were most probably introductions from outside the continent (Knowles et al., 1998). The largest number of topotypes were identified in East Africa (III [Tanzania, Burundi, Malawi]; IV [Kenya, Somalia, Ethiopia]; V [Sudan, Eritrea]; VI [Uganda, Kenya, Ethiopia]). Serotype C isolates from Africa grouped into three distinct topotypes (I [Kenya]; II [Ethiopia, Kenya] and III [Angola]; Table 1), but it is possible that this serotype is extinct and recent outbreaks are vaccine related. Antigenic relationships between FMD virus from various topotypes: Vaccine strains from southern Africa (SAT-1 and SAT-2) were antigenically compared to viruses from various topotypes in sub-saharan Africa to determine whether these vaccines would be applicable to other regions of the sub-continent. The two SAT-1 viruses (SAR9/81 and KNP196/91/1) both belong to topotype I (Table 1) and demonstrated r-values generally higher than 0.4 when compared with other isolates belonging to the same topotype (Figure 1). However, differences were observed between the two vaccine strains when compared to isolates such as KNP41/95/1 where to 151

4 SAR9/81 had an r-value of 0.24 while the KNP196/91/1 had a value of Antiserum from animals vaccinated with a combination of the two vaccine strains had higher values on most comparisons (Figure 1). The r-values were markedly lower when viruses from other topotypes were investigated and were generally below 0.2. The two isolates from topotype II (BOT14/98/1 and NAM307/98/1) both had r-values of approximately 0.1, demonstrating highly significant antigenic differences between the vaccine strains and field isolates. Similarly low values were observed with one of the topotype III viruses, ZIM14/90/1, but the SAR9/81 had better, albeit low r-values than the to KNP196/91/1 when compared to other topotype III viruses (ZIM25/90/1, ZIM6/94/1, ZAM2/93/1, ZAM18/96/1 and TAN1/99/1; Fig. 1). An exception was ZIM 6/94/1, a buffalo isolate from Hwange National Park in the west of Zimbabwe where values of 0.35 (against SAR9/81 ), 0.36 (KNP196/91/1) and 0.48 (combination of the two vaccine strains) were observed. The isolate from Uganda (UGA1/97/1 topotype V) demonstrated values of 0.23 (SAR9/81) and 0.16 (KNP196/91/1), while one from Nigeria (NIG5/81/1 topotype VI) had lower values (0.05 against SAR9/81 and 0.07 against KNP196/91/1) indicating that a high potency vaccine may still protect animals in the field in Uganda, but not in Nigeria. The two SAT-2 vaccine strains belong to different topotypes, KNP19/89/2 to topotype I and ZIM7/83 to II (Table 1). The antigenic variation for SAT-2 was more pronounced with r-values below 0.4 even when vaccine and field strains from the same topotype were compared (Figure 2). As with the SAT-1 data, the prepared with both vaccine strains presented higher r-values, but prepared against ZIM7/83 alone reacted better against all other viruses compared to prepared against KNP19/89/2 alone (Figure 2). Discussion: Due to the endemic nature of the disease, outbreaks are often not reported in sub-saharan Africa and samples not submitted for further study. Therefore the results do not represent the current situation and some of the topotypes may be extinct, but this can only be confirmed with improved sampling strategies. The results indicate that the genetic variation is significant with each of the six serotypes displaying numerous topotypes. Consequently, vaccination strategies should not only consider the fact that numerous serotypes could be occurring simultaneously, but that the genetic variation within serotypes can have serious implications on the choice of strain to include in antigen banks and vaccines. It also demonstrates that frequent surveillance investigating circulating strains is necessary. Although the topotypes demonstrated discrete geographic distribution, overlaps occurred with several countries having more than one topotype circulating within their borders. Uncontrolled movement within countries and across borders could lead to greater dissemination of different topotypes, further complicating control by vaccination. Using molecular data it was shown that Zimbabwe suffered four different outbreaks of SAT-2 since 2001 that belonged to the three topotypes occurring in that country (Records of the Exotic Diseases Division, Onderstepoort Veterinary Institute) and vaccines containing strains from various topotypes were used to ensure sufficient protection (S. Hargreaves, personal communication). However, the use of different vaccines in various districts confounded logistics significantly. It has also been shown that sequence data of FMD viruses can be used under certain conditions to trace the origin of animals (Vosloo et al., 2001), but not when movement is unrestrained therefore affecting the possible tracing of outbreaks negatively and confounding control strategies. The topotype distribution also indicated that movement between southern and East Africa as well as between East and West Africa has occurred in the past. Since wildlife does not occur in large numbers between East and West Africa, dissemination of disease most likely occurred through uncontrolled movement of livestock. Large numbers of African buffalo and other antelope move between the northern borders of southern and East Africa, and the most likely explanation would be due to persistently infected buffalo or other infected species moving between the two regions. However, movement of livestock cannot be ruled out completely. Uganda demonstrated the most diverse genetic range of viruses with three SAT-1, three SAT-2, two SAT-3, four O and one A topotypes with several of these occurring in Uganda only. The reason for this diversity is not clear and it could be that a more intensive study of the region will find similar results in neighbouring countries. Currently there is a dearth of knowledge in countries with large cattle populations such as Sudan, Somalia and further afield in Nigeria and Chad and it is possible that the topotypes found in Uganda is a reflection of what is circulating in other countries or alternatively that many more topotypes are present of which there is no information. It is therefore imperative that the situation in sub-saharan Africa be studied more intensively with emphasis on countries in the Horn of Africa and North Africa, the regions posing the biggest threat to southern Europe and the Middle East. It is generally accepted that r-values higher than 0.4 demonstrate a good antigenic relationship and vaccine strains would protect against those field isolates (Samuel et al., 1990). The antigenic variation 152

5 within SAT-1 was less marked than that of the SAT-2 viruses. The SAT-1 topotype I vaccine strains demonstrated good antigenic relationships with viruses from the same topotype, but significantly poorer against viruses from other topotypes. The SAT-2 vaccine strains had poor antigenic relationships with most other isolates, even within the same topotype. It was shown during persistent infection in buffalo that a plaque-purified SAT-2 virus demonstrated higher rates of change over time than a SAT-1 isolate and the genetic changes in SAT-2 were reflected in greater antigenic changes than for SAT-1 (Vosloo et al., 1996). With both SAT-1 and SAT-2 one strain reacted consistently better with different viruses, albeit not at a high enough value to guarantee protection. This could indicate that certain strains may have better cross-reactivity across topotypes and these should be investigated or developed to include into vaccines. A combination of more than one strain per serotype presented better r-values and the possibility of combinations of strains should be further investigated. The amount of antigenic variation has serious implications on the choice of vaccine strain, as it seems that each topotype may need to have its own vaccine strain. However, more data is needed where viruses from different topotypes are compared before reaching a final conclusion. The topotypes that have occurred in the Horn of Africa and North Africa up to are summarised in Table 2. Uganda is included in this list as it may represent disease occurrence in neighbouring countries in the Horn of Africa. For SAT-1 three topotypes have occurred in Tanzania and Kenya (III) and 2 in Uganda (IV and V) up to No antigenic profile has been performed for topotype IV viruses by the authors of this paper and data is limited for the other topotypes present in these regions. More focus should be placed on these topotypes by countries at risk from introduction from this area. Similarly, 5 topotypes of SAT-2 may threaten disease safety (Table 2) and limited antigenic data is available for only 3 (Fig. 2). The distribution of SAT-3 is more limited and only one topotype has been found in Uganda during 1997 from buffalo (Bastos et al., 2003a, Table 2). Serotype O is currently the most prevalent in the risk areas and 5 topotypes may be present. Serotype A demonstrates the most genetic and antigenic variation of the classical European serotypes and four topotypes may still be circulating in the North, West and East African regions (Table 2). Antigenic data and cross protection studies of the strains included in the antigen banks need to be performed to ensure that they will confer protection to the various topotypes present in the field. Most outbreaks in southern Africa can be attributed to SAT-2, followed by SAT-1 and then SAT-3 (Thomson, 1994). This information is not available for the rest of the sub-continent, but SAT-2 has recently occurred in Cameroon, a country where this serotype has not occurred before (Bronsvoort et al., 2004). SAT-2 has also caused outbreaks in the Middle East in 1990 and 2000, while incursions of SAT-1 has occurred during and 1970 (Ferris and Donaldson, 1992; Bastos et al., 2003b). Currently there is widespread occurrence of serotype O in East Africa, with reports from Kenya, Uganda, Rwanda and southern Sudan (records of the Exotic Diseases Division, Onderstepoort Veterinary Institute, D. Paton and G.R. Thomson, personal communication). However, despite the genetic variation observed for serotype O viruses world-wide, the antigenic variation is not extensive and the current vaccine strains can protect against most outbreaks (Knowles and Samuel, 2003). Therefore, it seems probable that SAT-2 may pose a greater threat to the rest of the world than other serotypes in terms of ability to cause outbreaks and together with the antigenic data presented here, more attention should be given to this serotype to determine what strains should be included in international antigen banks to protect countries currently free from disease. The endemic nature of FMD on the African continent and the low priority for controlling a disease that does not cause high rates of mortality is a serious threat to regions free of FMD. References: Bastos, A.D., Anderson, E.C., Bengis, R.G., Keet, D.F., Winterbach, H.K. and Thomson, G.R. 2003a. Molecular epidemiology of SAT3-type foot-and-mouth disease. Virus Genes. 27: Bastos, A.D.S., Boshoff, C.I., Keet, D.F., Bengis, R.G. and Thomson, G.R Natural transmission of foot-andmouth disease virus between African buffalo (Syncerus caffer) and impala (Aepyceros melampus) in the Kruger National Park, South Africa. Epid. Infect. 124: Bastos, A.D.S Detection and characterisation of foot-and-mouth disease virus in sub-saharan Africa. Onderstepoort J. Vet. Res. 65: Bastos, A.D.S Molecular epidemiology and diagnosis of SAT-type foot-and-mouth disease in southern Africa. PhD thesis. University of Pretoria, pp Bastos, A.D.S., Haydon, D.T., Forsberg, R., Knowles, N.J., Anderson, E.C., Bengis, R.G., Nel, L.H. and Thomson, G.R Genetic heterogeneity of SAT-1 type foot-and-mouth disease viruses in southern Africa. Arch. Virol. 146:

6 Bastos, A.D.S., Haydon, D.T., Sangare, O., Boshoff, C.I., Edrich, J.L. and Thomson, G.R. 2003b. The implications of viral diversity within the SAT-2 serotype for control of foot-and-mouth disease in sub-saharan Africa. J. Gen. Virol. 84: Bronsvoort, B.M., Radford, A.D., Tanya, V.N., Nfon, C., Kitching, R.P. and Morgan, K.L Molecular epidemiology of foot-and-mouth disease viruses in the Adamawa province of Cameroon. J. Clin. Microbiol. 42: Brückner, G.K., Vosloo, W., Du Plessis, B.J.A., Kloeck, P.E.L.G., Connoway, L., Ekron, M.D., Weaver D.B., Dickason, C.J., Schreuder F.J., Marais, T. and Mogajane, M.E Foot and mouth disease: the experience in South Africa. Rev. Sci. Tech. OIE. 21: Condy, J.B., Hedger, R.S., Hamblin, C. and Barnett, I.T.R The duration of the foot-and-mouth disease carrier state in African buffalo (i) in the individual animal and (ii) in a free-living herd. Comp. Immunol. Microbiol. Infect. Dis. 8: Ferris, N.P. and Donaldson, A.I The World Reference Laboratory for Foot and Mouth Disease: a review of thirty-three years of activity ( ). Rev. Sci. Tech. 11: Hedger, R.S Foot-and-mouth disease and the African buffalo (Syncerus caffer). J. Comp. Path. 82: Hedger, R.S Foot-and-mouth disease in wildlife with particular reference to the African buffalo (Syncerus caffer). In: Wildlife Diseases. L.A. Page, ed. Pp New York, Plenum Publishing. Hedger, R.S., Condy, J.B. and Golding, S.M Infection of some species of African wildlife with foot-andmouth disease virus. J. Comp. Pathol. 82: Hunter, P Vaccination as a means of control of foot-and-mouth disease in sub-saharan Africa. Vaccine. 16: Hunter, P., Bastos, A.D.S., Esterhuysen, J.J. and van Vuuren, C. de W.J Appropriate foot-and-mouth disease vaccines for southern Africa. All Africa Conference on Animal Agriculture, Pretoria, South Africa : 1-4. Knowles, N.J. and Samuel, A.R Molecular epidemiology of foot-and-mouth disease virus. Virus Res. 91: Knowles, N.J., Ansell, D.M. and Samuel, A.R Molecular comparison of recent foot-and-mouth disease type A viruses from West Africa with historical and reference virus strains. Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of foot-and-mouth disease, Pirbright, UK, September, Reid, S.M., Ferris, N.P., Hutchings, G.H., De Clercq, K., Newman, B.J., Knowles, N.J. and Samuel, A.R Diagnosis of foot-and-mouth disease by RT-PCR: use of phylogenetic data to evaluate primers for the typing of viral RNA in clinical samples. Arch Virol. 146: Rweyemamu, M.M., Booth, J.C., Head, M. and Pay, T.W.F Microneutralization tests for serological typing and subtyping of Foot-and-Mouth Disease virus strains. Journal of Hygiene, Cambridge. 81, Sahle, M An epidemiological study of the genetic variants of foot and mouth diseases viruses in East Africa. PhD thesis. University of Pretoria. Samuel, A.R. and Knowles, N.J Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes). J. Gen. Virol. 82: Samuel, A.R., Ouldridge, E.J., Arrowsmith, A.E.M, Kitching, R.O. and Knowles, N.J Antigenic analysis of serotype O foot-and-mouth disease virus isolates from the Middle East, Vaccine, 8: Sangare, O Molecular epidemiology of foot-and-mouth disease virus in West Africa. Pp PhD thesis. University of Pretoria. Sangare, O., Bastos A.D.S., Venter, E.H. and Vosloo, W Retrospective genetic analysis of SAT-1 type footand-mouth disease outbreaks in West Africa ( ). Vet. Microbiology. 93: Sangare, O., Bastos A.D.S., Venter, E.H. and Vosloo, W A first molecular epidemiological study of SAT-2 type foot-and-mouth disease viruses in West Africa. Epidemiology and Infection, 132: Sangare, O., Bastos, A.D., Marquardt, O., Venter, E.H., Vosloo, W. and Thomson, G.R., Molecular epidemiology of serotype O foot-and-mouth disease virus with emphasis on West and South Africa. Virus Genes. 22: Thomson, G.R Foot-and-mouth disease, In: Infectious Diseases of livestock with special reference to southern Africa. J.A.W. Coetzer, G.R. Thomson and R.C. Tustin, eds. Pp Cape Town, London, New York: Oxford University Press. 154

7 Thomson, G.R., Bengis, R.G. and Brown, C.C Picornaviruses. In: Infectious diseases of wild mammals, 3 rd Edn. E.S. Williams and I.K. Barker, eds. Pp Iowa University Press, Ames. Thomson, G.R., Vosloo, W. and Bastos, A.D.S Foot and mouth disease in wildlife. Virus Res. 91: Vosloo, W. and Thomson, G.R Natural Habitats in which Foot-and-Mouth Disease Viruses are Maintained. In: Foot-and-mouth disease. Domingo, E. and Sobrino, F. eds. Horizon Scientific Press, pp Vosloo, W., Bastos A.D.S., Michel, A. and Thomson, G.R Tracing movement of African buffalo in southern Africa. Rev. Sci. Tech. OIE. 20: Vosloo, W., Bastos, A.D., Kirkbride, E., Esterhuysen, J.J., Janse van Rensburg, D., Bengis, R.G., Keet, D.F. and Thomson, G.R Persistent infection of African buffalo (Syncerus caffer) with SAT-type foot-and-mouth disease viruses: rate of fixation of mutations, antigenic change and interspecies transmission. J. Gen. Virol. 77: Vosloo, W., Bastos, A.D.S., Sangare, O., Hargreaves, S.K. and Thomson, G.R Review of the status and control of foot and mouth disease in sub-saharan Africa. Rev. Sci. Tech. OIE. 21: Vosloo, W., Kirkbride, E., Bengis, R.G., Keet, D.F. and Thomson, G.R Genome variation in the SAT types of foot-and-mouth disease viruses prevalent in buffalo (Syncerus caffer) in the Kruger National Park and other regions of southern Africa, Epidem. Infect. 114: Vosloo, W., Knowles, N.J. and Thomson, G.R Genetic relationships between southern African SAT-2 isolates of foot-and-mouth disease virus. Epidem. Infect. 109:

8 TABLE 1 Summary of the topotype distribution of FMD serotypes O, A, C and SAT types 1-3 in Africa Serotype Topotype Representative Country / Countries References SAT-1 I South Africa, southern Zimbabwe, Mozambique Vosloo et al II Botswana, Namibia, Zambia, western Zimbabwe III Zambia, Malawi, Tanzania, Kenya, northern Bastos et al Zimbabwe IV Uganda Reid et al V Uganda VI Uganda Sahle 2003 VII Nigeria, Sudan VIII Nigeria, Niger Sangare et al SAT-2 I South Africa, Mozambique, southern Zimbabwe II Namibia, Botswana, northern & western Bastos et al. 2003b Zimbabwe III Botswana, Zambia, Zimbabwe Vosloo et al IV Burundi, Malawi, Kenya, Tanzania, Ethiopia V Nigeria, Senegal, Liberia, Ghana, Mali, Cote d Ivoire VI Gambia, Senegal Sangare 2002 VII Eritrea VIII Rwanda Sahle 2003 IX Kenya, Uganda X Democratic Republic of the Congo, Uganda Sangare et al XI Angola XII Uganda XIII Sudan XIV Ethiopia SAT-3 I South Africa, southern Zimbabwe II Namibia, Botswana, western Zimbabwe Vosloo et al III Malawi and northern Zimbabwe IV Zambia Bastos et al. 2003a V Uganda VI Uganda Reid et al O I Ethiopia, Eritrea, Kenya, Somalia, Sudan, Tunisia, Egypt, Algeria, Morocco, Libya, Tanzania II Algeria, Côte d Ivoire, Guinea, Morocco, Niger, Ghana, Burkina Faso, Tunisia, Sudan Samuel and Knowles 2001 III Uganda, Kenya, Sudan IV Uganda Sangare 2002 V Uganda VI Tanzania, Uganda Sahle 2003 VII South Africa VIII Angola Sangare et al A I Mauritania, Mali, Côte d Ivoire, Ghana, Niger, Nigeria, Cameroon, Chad, Senegal, Gambia, Sudan Knowles and Samuel 2003 II Angola, Algeria, Morocco, Libya, Tunisia, Knowles et al Malawi III Tanzania, Burundi, Malawi IV Kenya, Somalia, Ethiopia V Sudan, Eritrea VI Uganda, Kenya, Ethiopia C I Kenya Reid et al II Ethiopia, Kenya III Angola Knowles and Samuel

9 TABLE 2 Topotypes occurring recently in the Horn of Africa and West Africa that may be a threat to the Mediterranean and Middle East Serotype Topotype Year of Representative Country / Countries isolation SAT-1 III Zambia, Malawi, Tanzania, Kenya, northern Zimbabwe IV 1999 Uganda V 1997 Uganda SAT-2 IV Burundi, Malawi, Kenya, Tanzania, Ethiopia VII Eritrea, Saudi Arabia VIII 2000 Rwanda IX Kenya, Uganda X Democratic Republic of the Congo, Uganda SAT-3 VI 1997 Uganda O I Ethiopia, Eritrea, Kenya, Somalia, Sudan, Tunisia, Egypt, Algeria, Morocco, Libya, Tanzania II Algeria, Côte d Ivoire, Guinea, Morocco, Niger, Ghana, Burkina Faso, Tunisia, Sudan III Uganda, Kenya, Sudan IV Uganda VI Tanzania, Uganda A I Mauritania, Mali, Côte d Ivoire, Ghana, Niger, Nigeria, Cameroon, Chad, Senegal, Gambia, Sudan IV Kenya, Somalia, Ethiopia V Sudan, Eritrea VI Uganda, Kenya, Ethiopia C II Ethiopia, Kenya 157

10 1.2 1 r-value SAR 9/81 KNP 196/91/ KNP 196/91/1 b (I) SAR 9/91 i (I) KNP 148/91/1 b (I) KNP 41/95/1 b (I) HV3/90 b (I) GN13/91 b (I) BOT 14/98/1 b (II) NAM 307/98/1 b (II) ZIM 14/90/1 b (III) ZIM 25/90/1 b (III) Virus and topotype ZIM 6/94/1 b (III) ZAM 2/93/1 b (III) ZAM 18/96/1 b (III) ZAM 29/96/1 b (III) TAN 1/99/1 c (III) UGA 1/97/12 b (V) NIG 5/81/1 c (VI) SAR 9/81 and KNP 196/91/1 Figure 1: : Chart to indicate the r-values of various SAT-1 FMD virus isolates from different topotypes in sub-saharan Africa compared to prepared to the vaccine strains SAR9/81and KNP196/91/1 (both topotype I). The topotype of each virus is indicated in brackets after the virus name. b indicates an isolate obtained from buffalo, i from impala and c from cattle. KNP and SAR indicate isolates made in the Kruger National Park (South Africa), GN Ghonerazhou National Park, HV Hippo Valley (both Zimbabwe), BOT Botswana, NAM Namibia, ZIM Zimbabwe, ZAM Zambia, TAN Tanzania, UGA Uganda and NIG Nigeria ZIM 7/83 r-value KNP 19/89/2 b (I) KNP 2/89/2 i (I) KNP 143/91/2 b (I) KNP 5/92/2 i (I) KNP 51/93/2 i (I) GN 3/91 b (I) GN 10/91 b (I) ZIM 7/83 c (II) ZIM 1/88/2 b (II) ZIM 34/90/2 b (II) ZIM 8/94/2 b (II) SWA 4/89/2 c (II) NAM 1/91/2 c (II) MAL 1/03/2 c (IV) GHA 8/91/2 c (V) RWA 1/01/2 c (VIII) KEN 9/99 c (IX) Virus and topotype KNP 19/89/2 ZIM 7/83 and KNP 19/89/2 Figure 2: Chart to indicate the r-values of various SAT-2 FMD virus isolates from different topotypes compared to prepared to the vaccine strains KNP19/89/2 (topotype I) and ZIM7/83 (topotype II). The topotype of each virus is indicated in brackets after the virus name. b indicates an isolate obtained from buffalo, i from impala and c from cattle. KNP indicates isolates made in the Kruger National Park (South Africa), GN Ghonerazhou National Park (Zimbabwe), ZIM Zimbabwe, SWA and NAM Namibia, MAL Malawi, GHA Ghana, RWA Rwanda and KEN Kenya. 158