A study on foot and mouth disease (FMD) virus serotypes circulating in Ethiopia. Esayas Gelaye, Gelagay Ayelet, Berhe G/Egziaber and Aschalew Zeleke

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1 Appendix 17 Abstract: A study on foot and mouth disease (FMD) virus serotypes circulating in Ethiopia Esayas Gelaye, Gelagay Ayelet, Berhe G/Egziaber and Aschalew Zeleke National Veterinary Institute, Debre Zeit, Ethiopia POBox: 19 gelagayayelet@yahoo.com The study was conducted on the samples which were collected from 1982 to 2005 from Bovine and Swine found in the disease outbreak areas of the country and submitted to the National Veterinary Institute, Foot and Mouth Disease Laboratory, for virus isolation and serotype identification. From the 215 submitted samples Cytopathic effect (CPE) was observed in 191 samples in primary calf kidney, BHK21 or IBRS 2 cell culture and identification of serotype was made on these samples using Complement Fixation Test. The four serotypes identified were O, A, C and SAT 2 (South African Territories) with 73.93%, 19.68%, 1.59% and 4.79% respectively. From Swine samples only type O was identified and SAT 2 was identified for the first time in 1989 from a Bovine sample collected from Leben Ranch, Borena area, southern Ethiopia. Serotype SAT 1 and SAT 3 has not been identified during the recorded period. 68 samples were sent to Per bright, UK for further molecular characterization and phylogenetic analysis. And serotype O from 22 samples, serotype A from 9 and type C from 4 samples were isolated and confirmed. New topotype of serotype O is identified from the samples collected in Mizan Tefi area. This study highlighted distinct lineages of serotype O found in Ethiopia and needs further investigation. The complex epidemiological situation of the serotypes of the Foot and Mouth Disease Virus circulating in Ethiopia indicated that development of polyvalent vaccine and design and implement an appropriate vaccine-based control measure of the disease are essential. Introduction: Foot and mouth disease (FMD) is caused by a virus of the genus Aphthovirus, family Picornaviridae. There are seven serotypes of FMD virus, namely, O, A, C, SAT 1 (South African Territories), SAT 2, SAT 3 and Asia 1, it is the most contagious disease of mammals and has a great potential for causing severe economic loss in susceptible cloven hoofed animals (Boxton, 1977; OIE, 1990; OIE, 1990; OIE, 2000; Richard, 1998). Infection with one serotype does not confer immunity against another. With in serotypes, many subtypes can be identified by biochemical and immunological tests (OIE, 2000; Richard, 1998). Infection of susceptible animals with FMD virus leads to the appearance of vesicles on the feet, in and around the oral cavity, and on the mammary glands of females. Vesicles can occur in other sites, such as inside the nostrils and at pressure points on the limbs especially in pigs (OIE, 1990; OIE, 2000). Diagnosis of the FMD is by the demonstration of FMD viral antigen in samples of tissue or fluid. When a cytopathic effect (CPE) appears in the cultures, the fluids can be used in CFT or ELISA for identification of serotypes (Buxton, 1977; OIE, 1990; OIE, 2000; Richard 1998; Yoseph et al., 1991). The Epidemiology of FMD in sub-saharan Africa is probably more complicated than in any other region of the world. Not only are six of the seven serotypes prevalent in Africa (only Asia 1 has never been recorded), but also marked regional differences in the distribution and prevalence of serotypes and intratypic variants occur (OIE, 2002). The evolutionary changes of viruses are determined by comparing genomic material from more than one virus with each other. The basic process in the evolution of DNA/RNA sequence is the substitution of one nucleotide for another over evolutionary time. Changes in nucleotide sequences are used in molecular evolutionary studies both for estimating the rate of evolution and for re-constructing the evolutionary history of organisms (Graur and Wen-Hsiung, 2000). At present, 116

2 DNA sequencing and phylogenetic trees are widely used to illustrate the genetic relationships between viruses. In order to construct evolutionary trees assumptions are made about the substitution process and these assumptions are stated in the form of a model. Phylogenetic analysis of the VP1 region of FMD viruses has been used extensively to investigate the molecular epidemiology of the disease worldwide. These techniques have assisted in studies of the genetic relationships between different FMD virus isolates, geographical distribution of lineages and genotypes, and the establishment of genetically and geographically linked topotypes and tracing the source of virus during outbreaks (Mesfine, 2004). The occurrence of FMD in Ethiopia is increasing and in 1999, almost 10% of cattle were under risk of infection (Asfaw, et al., 2000), and in 2000 and 2001 a total of 27 and 88 disease outbreaks were reported (OIE, 2002). Therefore, continuous research is needed to establish the geographic relatedness between isolates, the genetic variation, and molecular evolution of viruses in carriers. During outbreaks, it is also important to identify the origin of infection and its relationship to vaccines available for protection which will assist in planning a control program in the country. The objectives of the present paper, therefore, are to give retrospective disease outbreak result on those samples submitted to the National Veterinary Institute, to illustrate the importance of FMD infection and to demonstrate the epidemiology of FMD virus infection in Ethiopia. And to show the molecular epidemiology of the FMD virus circulating in Ethiopia in comparison with the rest of the world. Materials and Methods: Sample collection : Samples were collected from veterinary clinics, regional veterinary laboratories, animal health and production research centers and farms and submitted to the National Veterinary Institute, Foot and Mouth Disease Laboratory, between 1982 and The outbreak samples were collected from different areas of the country for virus isolation and identification of the circulating serotypes responsible for that particular outbreak. Bovine samples were collected from different areas of the country whereas Swine samples were collected only from Alagae swine farm, Zeway-eastern Shoa, in 1986 and Epithelial tissues and vesicular fluids (which contain high titer of virus) from clinically sick animals were collected from unruptured and/or freshly ruptured vesicles of the tongue, buccal mucosa and feet (Buxton, 1977; OIE, 1990; Richard, 1998; Yoseph, et al., 1991). The samples were transported from the collection site to the diagnostic laboratory using transport medium (glycerol) or under a refrigerator at +4 0 c (Buxton, 1977; OIE, 1990; Richard, 1998; Yoseph, et al., 1991). Laboratory diagnosis Virus isolation: Established cell layers of primary calf kidney, BHK21 or IBRS 2 were inoculated with the suspension of suspected material. Cytopathic effect (CPE) was observed after 48 hours (or even less) in positive cases. If no CPE was detected, the cells were frozen and thawed, used to inoculate fresh cultures and examined for CPE for another 48 hours before the samples were declared to be negative (Buxton, 1977; OIE, 1990; Yoseph, et al., 1991). Serotype identification: Typing of FMD virus were made by applying Complement Fixation Test (CFT) from the fluids of fresh samples or cell cultures showing CPE.100% haemolysis technique was applied for typing of the virus (Buxton, 1977;OIE 1990; Yoseph, et al., 1991). In those samples which were not enough to inoculate directly into cell culture or to do a Complement Fixation Test, we made a passage of the samples on Guinea pigs and collect fluid and epithelial tissues from the lesions and this extract was used for typing of FMD virus (Yoseph, et al., 1991). Phylogenetic analysis: A total of 68 tissue culture FMD virus samples were submitted to Perbright UK for further molecular characterization and phylogenetic analysis. The 1D gene characterization was used to study phylogenetic relationships between 22 serotype O foot-and-mouth disease (FMD) viruses in Ethiopia as well as with other O-type isolates from East, South and West Africa, the Middle East, Asia and Europe. 117

3 Results: The records of ministry of agriculture and rural development (MOARD) from 2000 to 2006 indicated that FMD outbreak occurred every year with the highest in 2001 with 88 outbreaks (figure 1). Of the total 215 outbreak samples examined, cytopathic effect (CPE) was observed in 191 samples in primary calf kidney or IBRS 2 cell culture for the FMD virus (Table 1). The CPE was characterized by a fast destruction of the cell monolayer and infected cells were round and formed singly. Complete destruction of the cell sheet was mostly seen with in 48 hours of inoculation. On those samples that showed CPE further examination were done to identify the type of the virus with complement fixation test; thus serotype O, A, C and SAT 2 were recorded. Type O was the dominant serotypes identified with 73.93% rate, while type A (19.68%), C (1.59%) and SAT 2 (4.79%) rate were detected. Only type O was identified from a swine samples that were collected from Alagae Swine farm, Zeway-eastern Shoa, from 1986 and 1998 (Table 1 and 2). SAT 2 was recorded for the first time in Ethiopia in 1989 from a Bovine sample collected from Borena area (Third Livestock Development Project), southern Ethiopia (Table 2). Figure 1: Number of FMD outbreaks per year (MOARD report) No of outbreak Series1 2000G.C 2001G.C 2002G.C 2003G.C 2004G.C 2005G.C 2006G.CYears Table 1: Species of animals affected and FMD serotypes identified Animal Species Sample Tested Serotype Positive Samples (CPE) O C A SAT 2 Bovine Swine

4 Table 2: Serotypes of Foot and Mouth Disease Virus isolated in Ethiopia between 1982 and Year Serotype O*, 8A O, 3C A A O, 2A O O O, 2SAT O, 2SAT O, 4SAT O, 1A O O, 12A O O ** O O O, 1SAT O O O O 119

5 Figure 2: The epidemiology of FMD virus serotypes isolated in Ethiopia FMD virus serotypes isolated C serotype 1983 O serotype A serotype SAT2 serotype 1989/91 24 Phylogenetic analysis A total of 68 tissue culture FMD virus samples were submitted to Perbright UK for further molecular characterization and phylogenetic analysis. And serotype O from 22 samples, serotype A from 9 and type C from 4 samples were isolated and confirmed. The 1D gene characterization was used to study phylogenetic relationships between 22 serotype O foot-and-mouth disease (FMD) viruses in Ethiopia as well as with other O-type isolates from East, South and West Africa, the Middle East, Asia and Europe. Most of the O serotype isolates of Ethiopia lies on East Africa Lineage III, and also new topotype of O serotype was identified from Samples of Mizan Tefri, which is located South West of Addis Ababa bordering Kenya and Sudan and also two National Parks (Omo and Mago) are adjust to this location. But the serotype A and C are the same as African Types (see figure 3, 4 and 5). 120

6 Figure 3: Neighbour joining tree comparing the complete VPI coding sequence of type O FMD virus Euro-SA EA-1 WA? ME-SA EA-3 Cathay EA-2 Unrooted Neighbor-joining tree based on a comparison of the complete VP1 gene. The tree was outgroup-rooted using O1/Kaufbeuren/FRG/66. *, not a WRLFMD ref. no. N.J. Knowles, J. Wadsworth & K.G. Swabey, 4 September % O1/Kaufbeuren/FRG/66 [X00871] O/HKN/6/83 O/MAU/19/2000 O/BKF/1/200 O/NGR/11/2001 O/ALG/2/99 2 O/CIV/8/99 O/CAR/6/89 O/GHA/5/93 O/CAR/16/2000 O/CAR/17/2000 O/ETH/58/2005 <<< O/ETH/59/2005 <<< O/ETH/60/2005 <<< O/IND/R2/75* [AF204276] O1/Manisa/TUR/69 O/IND/53/79 [AF292107] O/SAR/1/2000 O/BUN/3/2003 O/BUN/7/2003 O/BUN/6/2003 O/TAN/12/200 O/TAN/2/ O/TAN/3/2004 O/KEN/5/2002 O/KEN/7/2002 O/KEN/3/2002 O/UGA/5/2002 O/UGA/4/2004 O/UGA/18/2004 O/UGA/1/2004 O/UGA/3/2004 O/UGA/6/2004 O/UGA/8/2004 O/UGA/5/2004 O/UGA/7/2004 O/UGA/3/2002 O/UGA/6/2002 O/TAN/1/2004 O/MAL/1/98 O/TAN/7/98 O/ZAM/1/2000 O/ZAM/2/2000 O/TAN/3/96 O/RWA/2/2004 O/RWA/3/2004 O/TAN/14/2004 O/TAN/17/2004 O/K40/84* O/KEN/102/60 O/UGA/5/96 O/K83/79* O/K70/77* O/KEN/5/95 [K36/95] O/K77/78 O/KEN/2/95 [K10/95] O/UGA/3/72 O/SUD/2/86 O/ERI/3/2004 O/ERI/22/2004 O/ERI/21/2004 O/ERI/1/2004 O/ERI/2/2004 O/SUD/1/99 O/SUD/12/2004 O/SUD/3/2004 O/SUD/16/2004 O/SUD/1/2004 O/SUD/26/2004 O/SUD/15/2004 O/SUD/4/2004 O/SUD/14/2004 O/SUD/30/2004 O/SUD/9/2004 O/SUD/25/2004 O/SUD/3/2005 O/SUD/1/2005 O/SUD/2/2005 O/ETH/11/2005 [1996] <<< O/ETH/1/79 [OVI] O/K201/83* O/K22/82* Debre Birhan O/K101/80* O/K8/84* O/YEM/48/2004 O/YEM/1/2004 O/YEM/22/2003 O/YEM/28/2003 O/ETH/22/01* O/ETH/1/2005 <<< [OVI] Awassa O/ETH/67/2005 <<< O/ETH/66/2005 <<< O/ETH/54/2005 <<< O/ETH/57/2005 <<< O/ETH/65/2005 <<< O/ETH/63/2005 <<< O/ETH/64/2005 <<< O/ETH/55/2005 <<< O/ETH/56/2005 <<< O/YEM/70/2004 O/ETH/38/2005 [2003/4] <<< O/YEM/57/2004 O/YEM/66/2004 O/YEM/21/2004 O/YEM/28/2004 O/YEM/35/2004 O/ETH/48/2005 <<< O/ETH/61/2005 O/ETH/49/2005 <<< <<< O/ETH/51/2005 <<< O/ETH/52/2005 <<< O/ETH/53/2005 <<< O/ETH/62/2005 <<< Mizan Teferi-Maji Arsi-Robe Addis Ababa North-Wollo, Gubalafto 121

7 Figure 4: Neignbor joining tree comparing the complete VPI coding sequence of type A FMD virus Euro-SA Africa Asia 1% Unrooted Neighbor-joining tree based on a comparison of the complete VP1 gene. The tree was outgroup-rooted using members of the Euro-SA topotype. A/ARG/1/2001 A/BRA/1/200 1 A24/Cruzeiro/BRA/55 A/URU/1/2001 [AJ251476] A5/Allier/FRA/60 [AY593780] A/EGY/1/2006 A/EGY/2/2006 A/KEN/15/98 [K62/98] A/KEN/16/98A [K67/98] A/ETH/7/92 A/ETH/23/94 A/ETH/1/94 A/YEM/2/98 A/UGA/13/6 6 A/SUD/1/82 A/K37/84* A/UGA/20/76 A/K35/80* A/KEN/6/96 [K15/96] A/ERI/3/97 A/ERI/3/98 A/K49/84* A/UGA/1/2002 A/GAM/44/98 A/SEN/34/97 A/MAI/2/97 A/MAU/3/97 A/EGY/1/72 A/K5/80* A/SAU/23/86 A/BHU/27/2003 A/BHU/41/2002 A/BHU/7/2003 A/IND/78/2000* [AF390664] A/IND/60/2000* [AF390656] A/IND/104/2000* [AF390592] A/IND/192/2000* [AF390619] A/IND/53/2000* [AF390654] A/IND/67/2000* [AF390658] A/IND/24/2001* [AF390624] A/IND/126/2000* [AF390599] A/IND/128/2000* [AF390600] A/IND/172/2000* [AF390614] A/IND/38/2000* [AF390643] A/IND/84/2000* [AF390668] A/IRN/33/200 A/IRN/5/ A/IRN/9/2003 A/IRQ/2/2002 A/IRQ/100/2002 A/IRQ/99/2002 A/IRQ/24/2002 A/IRQ/59/2002 A/IRQ/33/2002 A/IRQ/60/2002 A/IRQ/107/2002 A/IRQ/108/2002 A/IRN/32/2004 A/IRN/17/2005 A/IRN/1/9 6 A/TUR/2/2003 A/TUR/14/2002 A/TUR/4/2003 A/SYR/5/2002 A/TUR/17/2001 A/IND/17/77* [AF204108] A22/IRQ/24/64 [AJ251474] A/TAI/118/87* [D] A/MAY/2/2002 A/TAI/7/2002 A/TAI/8/2003 A/MAY/1/2003 A/MAY/3/2003 A/MAY/4/2003 A/TAI/11/2003 A/TAI/12/2003 A/TAI/10/2003 A/TAI/4/2003 A/TAI/7/2003 A/TAI/1/2001 A/TAI/2/97 A/TAI/3/2001 A/TAI/2/2002 A/IND/68/2001* [AF390659] A/IND/7/82 [1980] A/WBN 17/82* [A/IND/490/97*] [AF390652] A/IND/17/82* [IndImm] A/IRN/87 A/SAU/41/91 A/IRN/22/99 A/TUR/5/2003 A/IRN/10/2003 A/PAK/28/2002 A/PAK/9/2003 A/IRN/32/2001 A/IRN/34/200 1 A/IRN/2/2002 A/IRN/7/2003 A/IRN/41/2003 A/IRN/7/2004 A/IRN/7/2005 A/SAU/15/2005 A/SAU/16/2005 A/IRN/4/2005 A/IRN/1/2005 A/IRN/2/2005 A/IRN/5/2005 A/TUR/2/2006 A/TUR/1/2006 A/TUR/3/2006 A/IRN/13/200 5 A/IRN/10/200 5 A/IRN/14/2005 A/IRN/18/2005 A/IRN/16/200 5 Irn96 Irn99 N.J. Knowles, J. Smith & K. Swabey, 23 February

8 Figure 5: Neignbor joining tree comparing the complete VPI coding sequence of type C FMD virus 1 % Neighbor-joining tree based on a comparison of the complete VP1 gene ( nt). The tree was outgrouprooted using C 1 /GER/c.26. * Not a WRLFMD Ref. No. C1/GER/c.26 [CGC] [M90368] C/UKG/149/34 [AY593810] C3/Goias/BRA/88 C3/ARG/85 [AJ007347] C/ANG/3/73 C3/Indaial/BRA/71 [K01202] C2/Pando/URU/44 C4/TDF/ARG/66 [AY593808] C2/997/UK/53 C1/Bombay/IND/64 [IVRI] C1/Santa Pau/SPA/70 [AJ133357] C1/Oberbayern/FRG/60 [X00130] C1/Loupoigne/BEL/53 C1/Noville/SWI/65 [AY593804] C3/PAR/69 C5/ARG/69 [AY593809] C/PHI/1/79 C/PHI/7/76 C3/ARG/84 [M19761] C3/ARG/8 3 C3/Resende/BRA/55 [M90381] C/PHI/3/94 C/PHI/3/88 C/PHI/6/89 C/ETH/1/71 C/ETH/6/2005 [1983] <<< C/ETH/7/2005 [1983] <<< C/KEN/1/2004 [K6/04] C/KEN/5/96 [K14/96] C/KEN/32/70 [K267/67] C/K221/83 [Kenya] C/UGA/35/71A C/UGA/1/71 C/UGA/18/70 C/UGA/19/70 C/UGA/20/7 0 C/SRL/1/84 C/CEY/4/71 C/SRL/4/78 C/KUW/2/82 C/SAU/1/84 C/SAU/12/84 C/NEP/35/96 C/IND/63/96* [1991-IVRI] C/IND/147/93* [IVRI] C/IND/26/93* [IVRI] C/IND/146/93* [IVRI] C/NEP/10/93 C/IND/65/96* [1991-IVRI] C/IND/64/96* [1991-IVRI] C/IND/67/96* [1991-IVRI] C/IND/66/96* [1991-IVRI] C/NEP/124/90 C/BAN/1/92 C/BAN/2/92 C/BHU/7/91 C/BHU/10/91 C/IND/89/92* [1991-IVRI] C/IND/8/93* [1992-IVRI] C/IND/9/93* [1992- C/IND/7/92* IVRI] [1991-IVRI] C/IND/136/92* [IVRI] N.J. Knowles, J. Wadsworth & K.G. Swabey, 5 September

9 Discussion: The result indicated that the occurrence of Foot and Mouth Disease outbreak has been serious challenge every year in Ethiopia and this statement agrees with Asfaw (2000) that 12, 129 and 305 outbreaks were reported in 1997, 1998 and 1999 respectively; similarly 27 and 88 outbreaks were also reported in 2000 and 2001 respectively (OIE, 2002). Out of the seven serotypes of FMD virus the existence of serotype O, A, C and SAT 2 were recorded between 1982 and 2005 from a Bovine and Swine samples collected from outbreak areas of the country. Most of the outbreaks were occurred by serotype O followed by A, SAT 2 and C (Table 2). This shows that type O has highly prevalent and a dominant serotype causing an outbreak in Ethiopia and this observation agrees with the survey of Buxton (1977) that there is a tendency for type O strain to occur most frequently in the outbreak area. The first isolation of SAT 2 was in 1989 a sample collected from Bovine that were reared in Leben Ranch, Borena Zone southern Ethiopia operated by Third Livestock Development project (TLDP); These animals were purchased from an area called Wachle, which is around 100 km far from the broader of Kenya. This statement suggests that SAT 2 might be introduced from Kenya along with cattle movement since SAT 1 and SAT 2 are endemic in Kenya (OIE, 2002). The findings of this record are also similar to the previous report by Martel (1974) that SAT 2 was not isolated in Ethiopia. This statement explains the probable existence of numerous types of the virus and recent introduction of new serotype to the country has existed like SAT 2. Sample collected from swine revealed only type O and this explains swine can act as a source of infection for other cloven-hoofed animal species. Serotype C was isolated from the samples collected in 1983 since then it has not been isolated and this indicates that type C seems to have disappeared from Ethiopia. This statement agrees with OIE (2002) that serotype C has been disappeared elsewhere in the world and it has been postulated that the continue occurrence of this serotype in east Africa may be vaccine-related rather than due to natural persistence. Like serotype C, SAT 1 and SAT 3 were not identified and this finding agrees with the result of Martel (1974) that he did not identified these two (SAT 1 and SAT 3) FMD virus types in Ethiopia. Molecular Phylogeny This finding demonstrated the presence of new distinct topotype of serotype O in Ethiopia, which is in contrast with previous studies where a limited number of isolates from the country were included and only one topotype from Ethiopia was indicated (Mesfin., 2004). Mezan Teferi is bordering to Keny and Sudan and also the area is known with large number of wild lives, since it is in close proximity to Mago and Omo National Parks. It illustrates the importance of performing comprehensive studies for molecular epidemiology and to include representative samples from all regions in the analysis. However, even this study may not be a true reflection of the number of topotypes present in Ethiopia, as it is a well known fact that all outbreaks are not reported or investigated due to the endemic nature of the disease in country. The molecular epidemiology of serotype O has been investigated and reviewed by Samuel and Knowles, (2001) and Knowles and Samuel, (1997) who demonstrated the existence of 8 topotypes within samples collected around the world based on the comparison of sequence data of the VP1 gene. Among these topotypes two were found in Africa, one in East Africa and one in West Africa. The genetic diversity of type O viruses from West Africa has also been determined by Sangare et al. (2001) who identified four genotypes from the African isolates. Conclusions and Recommendations: During study period serotype O, A, C and SAT 2 were recorded with highest rate for serotype O. SAT 2 was isolated in 1989 for the first time from a sample collected from Bovine that were reared in Leben Ranch, Borena Zone- southern Ethiopia. From 68 samples which were sent to Per bright, UK for further molecular characterization and phylogenetic analysis; serotype O from 22 samples, serotype A from 9 and type C from 4 samples were isolated and confirmed. And also new topotype of serotype O is identified from the samples collected in Mizan Tefi area. In this area a large number of wild life are found since it is adjust to Mago and Omo National Parks, therefore further study is essential particularly in Mizan Tefi area. Since SAT 1 and SAT 2 were highly prevalent in neighboring countries, Kenya and Sudan, a serious attention should be given during importation of cattle breeds and control livestock movement 124

10 across the border areas. Furthermore, the complex epidemiological situation of Foot-and-Mouth Disease in Ethiopia needs more detailed investigation for improved vaccine-based control is to be achieved efficiently so as to make a disease free zone. And also conducting serology using 3ABC and LP B-ELISA is essential to draw the clear picture of the epidemiology of FMD serotypes circlulating in Ethiopia. Acknowledgements: The researchers would like to thank Agricultural Research Fund (ARF) program for funding this project and also we are grateful for National Veterinary Institute (NVI) on providing bank samples collected. Also we would like to extend our gratitude to Per bright laboratory for their help in molecular characterization of the isolates. References: Asfaw, W. and Sintaro, T The status of FMD in Ethiopia, - a growing concern. Ethiopian Veterinary Epidemiology Newsletter. Addis Ababa, Ethiopia Vol. 1, Issue 2, 1-5. Buxton, A. and Faser, G Animal Microbiology. Blackwell scientific publications Ltd., Edinburgh. Vol. 2, Graur D & Wen Hsiung Li, Evolutionary change in Nucleotide Sequences. In Fundamentals of molecular evolution 2 nd Sinauer Associate, INC., Publishers, Sunderland, Massachusetts, P:67. Martel, J. L., Foot and mouth disease in Ethiopia. Distribution of Foot and Mouth Disease Virus Serotypes. Revue Elev. Med. Vet. Pays Trop., 27 (2), Mesfin Sahle (2004): An epidemiological study on the genetic relationships of foot and mouth disease viruses in east Africa. A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa. OIE, Recommended Diagnostic techniques and Requirements for Biological products. Office International des Epizooties (OIE), Paris, France, 1-7. OIE, World Organization for Animal Health. Manual of Standards for Diagnostic Tests and Vaccines. Office International des Epizooties (OIE), Paris, France, OIE, World Animal Health in Reports on the Animal Health Status and Disease Control Methods. Office International des Epizooties (OIE), Paris, France, Richard P. and Kitching, Foot and Mouth Disease. The Merck Veterinary Mnual.8 th edition, Merck and co. Inc., White House Station NJ, USA, SAMUEL A.R. & KNOWLES N.J Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (Topotypes). Journal of General Virology, 82: SAMUEL A.R., KNOWLES N.J., KITCHING R.P. & HAFEZ S.M Molecular analysis of type O foot-and-mouth-disease viruses isolated in Saudi Arabia between 1983 and Epidemiology and Infection, 119: SANGARE O., BASTOS A.D.S., MARQUARDT O., VENTER E.H., VOSLOO W. & THOMSON G.R Molecular epidemiology of serotype O foot-and-mouth disease virus with emphasis on West and South Africa. Virus Genes, 22(3): Yoseph, F., Bayou, K., Geteneh, K., G/Yesus, M., M., Maurice, V. and Tsigie, Z., Veterinary Laboratory Diagnostic Manual. Ministry of Agriculture, Addis Ababa, Ethiopia. Vol.3, Virology,