Molecular Characterization of Foot-and-Mouth Disease Viruses Collected in Tanzania Between 1967 and 2009.

Transboundary and Emerging Diseases

ORIGINAL ARTICLE

Molecular Characterization of Foot-and-Mouth Disease Viruses Collected in Tanzania Between 1967 and 2009 C. J. Kasanga1, J. Wadsworth2, C. A. R. Mpelumbe-Ngeleja3, R. Sallu3, F. Kivaria4, P. N. Wambura1, M. G. S. Yongolo3, M. M. Rweyemamu1, N. J. Knowles2 and D. P. King2 1 2 3 4

Southern African Centre for Infectious Diseases Surveillance, Sokoine University of Agriculture, FVM, Morogoro, Tanzania The Pirbright Institute, Pirbright Woking Surrey, UK Tanzania Veterinary Laboratory Agency, Dar es Salaam, Tanzania National Epidemiology Unit, Ministry of Livestock Development and Fisheries, Dar es Salaam, Tanzania

Keywords: foot-and-mouth disease virus; serotypes; genotypes/topotypes; molecular epidemiology; Tanzania Correspondence: C. J. Kasanga. Southern African Centre for Infectious Diseases Surveillance, P.O. Box 3019, Chuo Kikuu, Morogoro, Tanzania. Tel.: +255 78 6181444; Fax: +255 23 2604647; E-mails: [email protected]; [email protected] Received for publication August 26, 2013 doi:10.1111/tbed.12200

Summary This paper describes the molecular characterization of foot-and-mouth disease viruses (FMDV) recovered from outbreaks in Tanzania that occurred between 1967 and 2009. A total of 44 FMDV isolates, containing representatives of serotypes O, A, SAT 1 and SAT 2 from 13 regions of Tanzania, were selected from the FAO World Reference Laboratory for FMD (WRLFMD) virus collection. VP1 nucleotide sequences were determined for RT-PCR amplicons, and phylogenetic reconstructions were determined by maximum likelihood and neighbour-joining methods. These analyses showed that Tanzanian type O viruses fell into the EAST AFRICA 2 (EA-2) topotype, type A viruses fell into the AFRICA topotype (genotype I), type SAT 1 viruses into topotype I and type SAT 2 viruses into topotype IV. Taken together, these findings reveal that serotypes O, A, SAT 1 and SAT 2 that caused FMD outbreaks in Tanzania were genetically related to lineages and topotypes occurring in the East African region. The close genetic relationship of viruses in Tanzania to those from other countries suggests that animal movements can contribute to virus dispersal in sub-Saharan Africa. This is the first molecular description of viruses circulating in Tanzania and highlights the need for further sampling of representative viruses from the region so as to elucidate the complex epidemiology of FMD in Tanzania and sub-Saharan Africa.

Introduction Foot-and-mouth disease virus (FMDV; family Picornaviridae, genus: Aphthovirus) causes a highly contagious disease of ruminants and swine and exists as seven immunologically distinct serotypes, viz. O, A, C, Asia 1, Southern African Territories (SAT) 1, SAT 2 and SAT 3. FMD is endemic in most of the African countries where serotypes O, A, SAT 1 and SAT 2 predominate (Rweyemamu et al., 2000; Vosloo et al., 2002). Phylogenetic analyses of nucleotide sequence data of the VP1-coding region have been used to define genotypes, which occur in defined geographic areas (topotypes) for each of the FMDV serotypes (Samuel and Knowles, 2001; Knowles and Samuel, 2003; Vosloo et al., 2005).

Since the first documented outbreaks in Tanzania in 1927 and first isolation of the virus in 1954, many FMD outbreaks have occurred on an annual basis. Clinical manifestation of FMD in the country varies from mild in indigenous zebu cattle to more severe and overt clinical signs in exotic breeds. Unrestricted animal movements are an important mechanism by which FMD is spread (Rweyemamu et al., 2008; Di Nardo et al., 2011), and control measures implemented in Tanzania during outbreaks typically consist of quarantine and restriction of animal movements (Eisa and Rweyemamu, 1977; Kivaria, 2003). Previous studies have provided evidence for the presence of four FMDV serotypes (O, A, SAT 1 and SAT 2) in Tanzania (Ferris and Donaldson, 1992; Swai et al., 2009; Kasanga et al., 2012). A serological survey of wildlife in

© 2014 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH. 62 (2015) e19–e29 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Molecular Characteristics of FMDV in Tanzania

Tanzania suggested infection with O, A, SAT 1 and SAT 2 may be widespread in free-living African buffalo in many parts of the country, and FMDV infection of other wildlife species was also detected (Hamblin et al., 1990). Samples (n = 233) from cases of suspected FMD in cattle occurring in Tanzania between 1967 and 2008 were submitted to the WRLFMD for virus isolation and serotyping (WRLFMD Records). A total of 35 (15%), 15 (6.4%), 47 (20.2%) and 27 (11.6%) were identified as FMDV types O, A, SAT 1 and SAT 2, respectively. FMDV genome was detected in a further 25 (10.7%) samples using real-time RT-PCR, and 84 (36.1%) samples were negative for FMDV in all tests. Thus, the occurrence of multiple FMDV serotypes in Tanzania complicates the control of the disease in the country through vaccination. The use of appropriate vaccines for control of FMD is dependent upon knowledge about the antigenic and genetic diversity of the circulating serotypes in the region. However, no detailed molecular epidemiological studies have been conducted on FMDV isolates from the country. The aim of this study was to characterize FMD viruses recovered from Tanzanian FMD outbreaks from 1967 to 2009 using VP1 sequence data, and the genetic relationships of these viruses were subsequently compared with other viruses collected from neighbouring countries. Materials and Methods Viruses A total of 44 FMD viruses isolated from samples collected from FMD outbreaks in Tanzania (1967–2009) were selected for use in this study. Sampling locations and other sample details are shown in Table 1 and Fig. 1. RNA extraction, RT-PCR and DNA sequencing Total RNA was extracted from cell culture passage viruses using RNeasyâ kit (Qiagen Ltd., Crawley, West Sussex, UK). The VP1 region was amplified using a one-step RTPCR kit (Qiagen) as previously described (Knowles et al., 2009). Oligonucleotide primers used for PCR amplification were selected based on serotype (Table 2). For type O, forward primers were either O-1C244F or O-1C272F and reverse primer EUR-2B52R. Primers used for serotype A were either A-1C562F or A-1C612F as a forward primer, with EUR-2B52R as a reverse primer. For serotype SAT 1, forward primers were either SAT1-P1-1228F, SAT11C559F or SAT1U-OS, each with SAT-2B208R as a reverse primer. SAT 2 RT-PCR used either SAT2-1C445F or SAT2P1-1223F as a forward primer and SAT-2B208R as a reverse primer. For serotype A, amplification conditions were as previously described (Knowles et al., 2009), while the annealing temperatures of 50°C and 60°C were used for e20

C. J. Kasanga et al.

SAT 2 and O viruses, respectively. RT-PCR products were sequenced using BigDyeâ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) using the following oligonucleotides (Table 2): NK72 for all serotypes; O-1C499F, O-1C583F, O-1C605eF, O-1D296F, O-1D296bF and O-1D628R for serotype O; A-1C612F for serotype A, SAT1-1C559F, SAT1U-OS, SAT1-1D394R and SAT1-1D200F for serotype SAT 1 and SAT2-1C445F, SAT2VP3-AB, SAT2-1C523F, SAT-1D209F and SAT2-D for SAT 2. DNA sequencing was performed on an ABI 3730 DNA Analyzer (Applied Biosystems). Nucleotide sequences covering the complete VP1-coding region were assembled using SeqMan Pro™ part of the Lasergene 8.0 software package (DNAStar Inc., Madison, WI, USA). Phylogenetic analysis Complete VP1 nucleotide sequences were aligned using BioEdit 7.0.5.3 (Hall, 1999) and Clustal W 1.83 (Thompson et al., 1994). These alignments were used to construct distance matrices using the Kimura-2-parameter nucleotide substitution model (Kimura, 1980) as implemented in the program MEGA 5.2 (Tamura et al., 2011). Midpointrooted neighbour-joining (NJ) trees were then constructed using MEGA 5.2. To construct maximum likelihood (ML) phylogenies (Nei and Kumar, 2000), each data set was tested for 24 common nucleotide substitution models using MEGA 5.2. The model with the lowest Bayesian information criterion (BIC) score was chosen to construct each tree. All positions with less than 95% site coverage were eliminated. The robustness of each tree topology (NJ and ML) was assessed with 1000 bootstrap replicates. Individual FMD viruses were classified into geographically restricted clusters, also known as topotypes, as previously described (Knowles and Samuel, 2003; Vosloo et al., 2004). Results The geographic distribution of FMDV serotypes detected from 1967 to 2009 is shown in Fig. 1. Additional FMDV, which were available on the public sequence databases, were also included in the analyses. This included four VP1 sequences of Tanzanian viruses (two type O and two type SAT 2). The NJ trees showed essentially the same phylogenetic relationships between the viruses as the ML trees (data not shown). Phylogenetic analysis of serotype A The evolutionary history was inferred using the maximum likelihood method based on the Hasegawa–Kishino–Yano (HKY) model (Nei and Kumar, 2000). The tree with the highest log likelihood ( 7343.3387) is shown (Fig. 2). A

© 2014 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH. 62 (2015) e19–e29



Table 1. Details of the Tanzanian foot-and-mouth disease virus isolates examined

Serotype

Topotype/ Lineage

WRLFMD Ref. No.

A A A A A A A

AFRICA/G-I AFRICA/G-I AFRICA/G-I AFRICA/G-I AFRICA/G-I AFRICA/G-I AFRICA/G-I

TAN/2/68 TAN/3/68 TAN/4/80 TAN/11/2008 TAN/12/2008 TAN/4/2009 TAN/9/2009

A

AFRICA/G-I

TAN/11/2009

A

AFRICA/G-I

TAN/42/2009

A

AFRICA/G-I

TAN/45/2009

A

AFRICA/G-I

TAN/47/2009

A A A

AFRICA/G-VI AFRICA/G-I AFRICA/G-I

GHA/16/73 KEN/42/66 (K18/66) KEN/7/2008 (K10/08)

A

AFRICA/G-I

KEN/8/2008 (K13/08)

A A

AFRICA/G-I AFRICA/G-I

KEN/28/2008 (K73/08) KEN/22/2009 (K63/09)

A A O O

AFRICA/G-V AFRICA/G-VII EA-2 EA-2

NGR/2/73 UGA/13/66 TAN/1/85 TAN/3/96

O

EA-2

TAN/7/98

O

EA-2

TAN/9/98

O

EA-2

TAN/1/2004

O

EA-2

TAN/2/2004

O O

EA-2 EA-2

TAN/3/2004 TAN/12/2004

O

EA-2

TAN/14/2004

O

EA-2

TAN/17/2004

O O O

EA-2 EA-2 EA-2

TAN/16/2008 TAN/5/2009 TAN/44/2009

SAT1 SAT1 SAT1

I I I

T155/71 TAN/2/71 TAN/2/77

Geographic origin

Species

Collection date

Accession

Reference

Tanzania Tanzania Arusha Region, Tanzania Iringa Region, Tanzania Iringa Region, Tanzania Morogoro Region, Tanzania Njombe District, Iringa Region, Tanzania Kibaha District, Pwani Region,

Cattle Cattle Not known Cattle Cattle Cattle Cattle

1968 1967 1980 01/08/2008 01/08/2008 01/05/2009 13/06/2009

KF561687 KF561688 KF561689 KF561690 KF561691 KF561692 KF561693

This study This study This study This study This study This study This study

Cattle

21/06/2009

KF561694

This study

Cattle

07/09/2009

KF561695

This study

Cattle

06/11/2009

KF561696

This study

Cattle

01/11/2009

KF561697

This study

Not known Not known Cattle

1973 1966 09/02/2008

KF561698 KF561699 KF561700

This study This study This study

Cattle

19/02/2008

KF561701

This study

Cattle Cattle

01/08/2008 01/03/2009

KF561702 KF561703

This study This study

Cattle Not known Cattle Cattle

1973 1966 1985 25/07/1996

KF561704 KF561705 KF561676 EU919241

Cattle

22/10/1998

AJ296320

Cattle

22/10/1998

KF561677

This study This study This study M. Chitray, F. F. Maree and W. Vosloo, unpub. Samuel and Knowles (2001) This study

Cattle

2004

KF561678

This study

Cattle

2004

KF561679

This study

Cattle Cattle

2004 2004

KF561680 KF561681


Cattle

2004

KF561682

This study

Cattle

2004

KF561683

This study

Cattle Cattle Cattle

25/09/2008 01/05/2009 27/10/2009

KF561684 KF561685 KF561686



1971 1971 1977

KF561706 KF561707 KF561708


Tanzania Mpwapwa, Dodoma Region, Tanzania Iringa Rural District, Iringa Region, Tanzania Bagamoyo, Pwani Region, Tanzania Ghana Kenya Wei, Nakuru North, Rift Valley, Kenya Gathanji, Kiambu West, Central, Kenya Loitokitok, Rift Valley, Kenya Olulunga, Narok South, Rift Valley, Kenya Niamey, Niger Uganda Iringa Region, Tanzania Kibaha District, Pwani Region, Tanzania

Kyela, Mbeya Region, Tanzania Kyela, Mbeya Region, Tanzania Bagamoyo District, Pwani Region, Tanzania Kibaha District, Pwani Region, Tanzania Iringa Region, Tanzania Chunya District, Mbeya Region, Tanzania Songea, Ruvuma Region, Tanzania Nkasi District, Rukwa Region, Tanzania Morogoro Region, Tanzania Morogoro Region, Tanzania Makete District, Iringa Region, Tanzania Tanzania Tanzania Tanzania


e21



Table 1. (continued)

Serotype

Topotype/ Lineage

WRLFMD Ref. No.

SAT1

I

TAN/3/80

SAT1 SAT1 SAT1

I I I

TAN/1/96 TAN/5/96 TAN/6/99

SAT1

I

TAN/18/99

SAT1 SAT1

I I

TAN/19/99 TAN/21/99

SAT1

I

TAN/25/99

SAT1

I

TAN/26/99

SAT1 SAT1 SAT1

I I I

TAN/37/99 TAN/43/99 TAN/51/99

SAT1

I

TAN/60/99

SAT2

IV

TAN/1/75

SAT2

IV

TAN/1/86

SAT2 SAT2 SAT2

IV IV IV

TAN/4/2004 TAN/6/2004 TAN/9/2004

SAT2

IV

TAN/18/2004

SAT2

IV

TAN/21/2004

SAT2

IV

TAN/43/2009

Geographic origin

Species

Collection date

Accession

Reference

Sumbawanga District, Rukwa Region, Tanzania Arusha, Arusha Region, Tanzania Koibate, Arusha Region, Tanzania Mtwara Urban, Mtwara Region, Tanzania Sumbawanga District, Rukwa Region, Tanzania Musoma, Mara Region, Tanzania Simanjiro, Manyara Region, Tanzania Musoma District, Mara Region, Tanzania Hai District, Kilimanjaro Region, Tanzania Iringa, Iringa Region, Tanzania Iringa, Iringa Region, Tanzania Kinondoni District, Dar es Salaam Region, Tanzania Serengeti National Park, Mara Region, Tanzania Sanya Juu, Moshi, Kilamanjaro Region, Tanzania Mtawanya, Mtwara Region, Tanzania Arusha Region, Tanzania Arusha Region, Tanzania Nkoanrua, Arumeru District, Arusha Region, Tanzania Nkasi District, Rukwa Region, Tanzania Kihonda, Morogoro Urban District, Morogoro Region, Tanzania Makete District, Njombe District, Iringa Region, Tanzania

Cattle

1980

KF561709

This study


17/05/1996 1996 01/06/1999

KF561710 KF561711 KF561712


Cattle

1999

KF561713

This study

Cattle Cattle

1999 1999

KF561714 KF561715


Cattle

1999

KF561716

This study

Cattle

1999

KF561717

This study


1999 1999 04/08/1999

KF561718 KF561719 KF561720


Wildebeest

1999

KF561721

This study

Cattle

1975

AY343970

Cattle

07/05/1986

AY343971


2004 2004 2004

KF561722 KF561723 KF561724

Sahle et al. (2007) Sahle et al. (2007) This study This study This study

Cattle

2004

KF561725

This study

Cattle

2004

KF561726

This study

Cattle

27/10/2009

KF561727

This study

discrete gamma distribution was used to model evolutionary rate differences among sites [five categories (+G, parameter = 0.6032)]. The rate variation model allowed for some sites to be evolutionarily invariable [(+I), 42.5518% sites]. Eleven serotype A virus isolates detected in Tanzania between 1967 and 2009 all fell into genotype I (G–I) within the AFRICA topotype. In this genotype, the Tanzanian viruses were grouped in two major subclusters, one with older viruses detected between 1967 and 1980, while the other comprised of viruses detected between 2008 and 2009. Tanzanian viruses from 2009 fell into two groups differing by 84.8–85.3% nt identity. One group (A/TAN/9/ 2009) was closely related to two Tanzanian viruses from the previous year (94.5% nt id); all three viruses were from the Iringa Region. The second group, consisting of A/TAN/4/ e22

2009, A/TAN/11/2009, A/TAN/42/2009, A/TAN/45/2009 and A/TAN/47/2009, from four contiguous regions of Tanzania (which included Iringa), had an intragroup variability of 97.5–99.2% nt id and were closely related to two Kenyan viruses (A/KEN/28/2008 and A/KEN/22/2009) with nt id values of 97.5–99.2%. Intermediate between the two groups were two Kenyan viruses (A/KEN/7/2008 and A/KEN/8/ 2008) with 84.5–85.0% nt id and 91.7–93.3% nt id to the first and second groups, respectively. Phylogenetic analysis of serotype O The evolutionary history was inferred using the maximum likelihood method based on the Tamura–Nei (TN93) model (Nei and Kumar, 2000). The tree with the highest




Legend

A O SAT 1 SAT 2

Mara 3

Kagera

Arusha 1 2 3

Mwanza

Kilimanjaro 1 1

Shinyanga

1 Manyara

Kigoma

Singida Rukwa 1 2 1 Mbeya 3

N

Pemba

Tanga

Tabora

Zanzibar

Dodoma 1 Iringa 3

4

1 Dar es Salaam Morogoro 2 1

Pwani 3 2

1

2 1

Africa 2 (EA-2) topotype, (i) O/TAN/1/85; (ii) O/TAN/3/ 96; (iii) O/TAN/7/98; O/TAN/9/98 (closely related to a virus from Malawi (O/MAL/1/98; 99.2–99.5% nt id); (iv) O/TAN/1/2004; (v) O/TAN/2/2004, O/TAN/3/2004 and O/ TAN/12/2004 [closely related to a virus from Kenya (O/ K48/05; 98.1-98.4% nt id)]; (vi) O/TAN/14/2004 and O/ TAN/17/2004; (vii) O/TAN/16/2008; and (viii) O/TAN/5/ 2009 and O/TAN/44/2009 [most closely related to viruses from Uganda in 2005–2006; 95.8–96.4% nt id (Fig. 3)].

Lindi

Ruvuma 1

1

1 Mtwara

Fig. 1. Map of Tanzania showing geographic location of the 48 virus isolates (1967–2009) analysed in this study.

log likelihood ( 11962.9132) is shown (Fig. 3). A discrete gamma distribution was used to model evolutionary rate differences among sites [five categories (+G, parameter = 0.6762)]. Thirteen serotype O viruses detected in various geographic locations of Tanzania between 1985 and 2009 were clustered in eight lineages within the East

Phylogenetic analysis of serotype SAT 1 The evolutionary history was inferred using the maximum likelihood method based on the TN93 model (Nei and Kumar, 2000). The tree with the highest log likelihood ( 8127.7373) is shown (Fig. 4). A discrete gamma distribution was used to model evolutionary rate differences among sites [five categories (+G, parameter = 0.7745)]. The rate variation model allowed for some sites to be evolutionarily invariable [(+I), 32.2379% sites]. The 16 serotype Tanzanian SAT 1 virus isolates from samples collected between 1971 and 1999 all fell into topotype I (aka NWZ) (Fig. 4). One sublineage (with 99.1–100% nt id), represented by SAT1/TAN/19/99 (Mara), SAT1/TAN/25/99 (Mara), SAT1/TAN/21/99 (Manyara), SAT1/TAN/26/99 (Kilimanjaro) and SAT1/TAN/51/99 (Dar es Salaam), was identified in the north of Tanzania and clustered closely

Table 2. Oligonucleotide primers used for RT-PCR and sequencing Serotype

Primer name

Primer sequence (5′–3′)

Use

O and A O O A A SAT 1 and SAT 2 SAT 1 SAT 1 SAT 1 SAT 2 SAT 2 All O O O O O O SAT 1 SAT 1 SAT 2 SAT 2 SAT 2 SAT 2

EUR-2B52R O-1C244F O-1C272F A-1C562F A-1C612F SAT-2B208R SAT1-P1-1228F SAT1-1C559F SAT1U-OS SAT2-1C445F SAT2-P1-1223F NK72 O-1C499F O-1C583F O-1C605eF O-1D296F O-1D296bF O-1D628R SAT1-1D394R SAT1-1D200F SAT2VP3-AB SAT2-1C523F SAT-1D209F SAT2-D

GAC ATG TCC TCC TGC ATC TGG TTG AT GCA GCA AAA CAC ATG TCA AAC ACC TT TBG CRG GNC TYG CCC AGT ACT AC TAC CAA ATT ACA CAC GGG AA TAG CGC CGG CAA AGA CTT TGA ACA GCG GCC ATG CAC GAC AG AAC CTG CAC TTC ATG TAC AC GTG TAT CAG ATC ACA GAC ACA CA GTG TAC CAG ATC ACT GAC AC TGG GAC ACM GGI YTG AAC TC TGA ACT ACC ACT TCA TGT ACA CAG GAA GGG CCC AGG GTT GGA CTC TAC GCG TAC ACC GCG TC GAC GGY GAY GCI CTG GTC GT TAG CTA GCG CCG GCA AGG ACT TCG AG ACA ACA CCA CCA ACC CAA C ACA ACA CCA CCA ATC CAA C GTT GGG TTG GTG GTG TTG T GGY TTG TAC TTR CAR TCA CCG TTG TA TGC GYG CIG CCA CGT ACT AYT TCT C CAC TGC TAC CAC TCR GAG TG GAC ACN CCM GCM ATG GC CCA CAT ACT ACT TTT GTG ACC TGG A GGT GCG CCG TTG GGT TGC CA

RT & PCR PCR PCR PCR PCR & SEQ RT & PCR PCR PCR & SEQ PCR & SEQ PCR & SEQ PCR SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ


e23


A/TAN/42/2009 (KF561695) A/TAN/47/2009 (KF561697) A/KEN/28/2008 (KF561702) 100 A/KEN/22/2009 (KF561703) A/TAN/11/2009 (KF561694) 100 A/TAN/4/2009 (KF561692) 82 A/TAN/45/2009 (KF561696) A/KEN/7/2008 (KF561700) 100 100 A/KEN/8/2008 (KF561701) A/TAN/9/2009 (KF561693) A/TAN/11/2008 (KF561690) 100 100 A/TAN/12/2008 (KF561691) A/KEN/42/66 (K18/66) (KF561699) A/TAN/4/80 (KF561689) A/TAN/2/68 (T626/68) (KF561687) 100 A/TAN/3/68 (T809/67) (KF561688) A/SUD/3/77 (GU566064) A/NGR/2/73 (KF561704) A/EGY/1/72 (EF208756) A/GHA/16/73 (KF561698) A21/Lumbwa/KEN/64 (AY593761) A/UGA/13/66 (KF561705) A/K37/84* (EU414532) A/KEN/15/98 (EF208774) 100 A/KEN/16/98A (EF208775) A23/Kitale/KEN/64 (AY593766)


76

94

99

G-I

AFRICA

G-IV G-V G-II G-VI G-III G-VII

EURO-SA

98 92

A11/GER/29 (AGB)(EU553852) 81

ASIA

0.05

with Kenyan viruses (SAT1/K96/99 and SAT1/K114/99; 98.5–99.4% nt id) isolated in the same year. A single virus (SAT1/TAN/60/99) isolated from a wildebeest in the Serengeti National Park also belonged to this sublineage (98.9–99.4% nt id). However, a genetically distinct second sublineage (87.2–88.1% nt id to the first sublineage and with intravariability of 99.1–99.7% nt id), represented by SAT1/TAN/6/99 (Mtwara), SAT1/TAN/18/99 (Ruwka), SAT1/TAN/37/99 (Iringa) and SAT1/TAN/43/99 (Iringa), was found in southern Tanzania. The most closely related viruses to this group were SAT1/TAN/2/77 and SAT1/ TAN/3/80, which shared 86.9–87.3% and 89.6–90.0% nt id, respectively (Fig. 4). Phylogenetic analysis of serotype SAT 2 The evolutionary history was inferred using the maximum likelihood method based on the HGY model (Nei and Kumar, 2000). The tree with the highest log likelihood ( 11571.3763) is shown (Fig. 5). A discrete gamma distribution was used to model evolutionary rate differences among sites [five categories (+G, parameter = 0.8124)]. The rate variation model allowed for some sites to be evolutionarily invariable [(+I), 33.2835% sites]. The serotype SAT 2 viruses found in Tanzania between 1975 and 2009 all belonged to topotype IV. Within this topotype, five viruses e24

Fig. 2. Midpoint-rooted maximum likelihood tree showing the relationships between the serotype A viruses collected from Tanzania. The three serotype A topotypes are labelled AFRICA, ASIA and EURO-SA. Seven proposed African genotypes, G-I to G-VII, are also shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. *reference number not assigned by the WRLFMD.

collected in 2004 (intra-isolate 98.5–100% nt) were closely related to a Kenya isolate from the same year (SAT2/K120/ 04; 97.8–98.3% nt id), whereas an isolate from 2009 was not closely related to any of the other viruses (maximum nt id 89.2% with SAT2/KEN/9/99). Older Tanzanian viruses from 1975 (SAT2/TAN/1/75) and 1986 (SAT2/TAN/1/86) were not closely related to any other viruses, but grouped with Kenyan viruses from 1974 (SAT2/K183/74; 91.5% nt id) and 2006 (SAT2/K6/06; 90.0% nt id), respectively (Fig. 5). Discussion This study describes the first molecular analysis of FMD virus isolates collected from Tanzania. Since 1954, only four of the seven FMDV serotypes (O, A, SAT 1 and SAT 2) have been detected. This observation is consistent with previous recent serotyping studies that reported the presence of serotypes O, A, SAT 1 and SAT 2 in various locations in Tanzania (Swai et al., 2009; Kasanga et al., 2012) and indicate that the epidemiology of FMD in the country is complicated by the presence of multiple serotypes. This study used phylogenetic analysis to define the genetic relationships between Tanzanian FMD viruses and those that have been collected from neighbouring countries (Figs 2–5). From the phylogenetic trees constructed, it is possible to infer the genetic relationship of isolates, and




O/K150/00* (HM756613) O/K147/00* (HM756612) O/Kumi/UGA/EL-1/02* (FJ461344) O/K141/00* (HM756610) O/K117/00* (HM756607) 86 O/K145/00* (HM756611) O/Kapchorwa/UGA/KPR-1/02* (FJ461345) O/UGA/3/2002 (DQ165077) O/UGA/7/03* (EU919243) O/U13B/04* (HM756618) 100 94 O/U17B/04* (HM756620) 98 O/U14B/04* (HM756619) O/U20B/04* (HM756621) O/U12/05* (HM756625) O/TAN/5/2009 (KF561685) 100 99 O/TAN/44/2009 (KF561686) O/U312/06* (HM191257) O/U18/06* (HM756627) 96 O/KSE/UGA/2006 (EF611987) O/U25/06* (HM756628) O/K28/07* (HM756631) 83 O/K30/07* (HM756632) O/K82/07* (HM756634) 94 O/K2/07* (HM756629) 100 O/K6/07* (HM756630) O/K31/05* (HM756623) O/K31/08* (HM756639) O/K63/00* (HM756605) 88 O/K61/01* (HM756615) 78 O/K79/02* (HM756616) 70 O/K130/00* (HM756608) 73 O/KEN/5/2002 (DQ165073) O/K5/05* (HM756622) 90 O/K50/06* (HM756626) O/K14/08* (HM756638) O/K45/01* (HM756614) O/K109/00* (HM756606) O/K82/98* (HM756602) O/K131/00* (HM756609) O/K117/99* (HM625676HM756603) O/TAN/16/2008 (KF561684) O/K1/08* (HM756635) 76 100 O/K4/08* (HM756636) O/K32/08* (HM756640) O/K48/05* (HM756624) 100 O/TAN/12/2004 (KF561681) O/TAN/2/2004 (KF561679) 90 O/TAN/3/2004 (KF561680) O/K11/08* (HM756637) O/K55/03* (HM756617) O/TAN/1/2004 (KF561678) O/TAN/7/98 (AJ296320) O/MAL/1/98 (DQ165074) 100 O/TAN/9/98 (KF561677) O/TAN/14/2004 (KF561682) 100 O/TAN/17/2004 (KF561683) O/TAN/3/96 (EU919241) O/K131/85* (HM756593) O/TAN/1/85 (KF561676)

98

96

91

Fig. 3. Midpoint-rooted maximum likelihood tree showing the relationships between the serotype O viruses collected from Tanzania. The 11 serotype O topotypes are labelled EURO-SA, CATHAY, ME-SA, SEA, ISA-1, ISA-2, WA and EA-1, EA-2, EA-3 and EA-4. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. *reference number not assigned by the WRLFMD.

ME-SA

81

91 91 99 100

99 100 100 72

96 100

EA-2

SEA EA-3 EA-4 WA EA-1 ISA-1 ISA-2 EURO-SA CATHAY

0.05


e25


81

100

95

80

SAT1/TAN/60/99 (KF561721) SAT1/TAN/26/99 (KF561717) SAT1/TAN/19/99 (KF561714) 84 SAT1/TAN/25/99 (KF561716) 100 SAT1/TAN/21/99 (KF561715) SAT1/TAN/51/99 (KF561720) SAT1/K114/99* (HQ267524) 80 SAT1/K96/99* (HQ267523) SAT1/K67/05* (HQ267526) SAT1/K57/05* (HQ267525) 100 99 SAT1/K10/06* (HQ267527) SAT1/K110/81* (HQ267521) SAT1/KEN/5/98 (DQ009721) 100 SAT1/KEN/4/98 (AY441993) 100 SAT1/KEN/9/91 (AY441995) 100 SAT1/KEN/11/91 (AY441994) SAT1/K66/80* (HQ267520) SAT1/TAN/2/71 (KF561707) 99 SAT1/T155/71 (KF561706) 99 99 SAT1/TAN/5/96 (KF561711) SAT1/TAN/1/96 (KF561710) SAT1/TAN/3/80 (KF561709) SAT1/TAN/2/77 (KF561708) SAT1/TAN/18/99 (KF561713) SAT1/TAN/43/99 (KF561719) 100 SAT1/TAN/6/99 (KF561712) SAT1/TAN/37/99 (KF561718) SAT1/ZIM/23/2003 (KF219690)

II (SEZ) IV (EA-1) III (WZ) VIII (EA-3) IX

100 100 100

88

V 99 90 100

VII (EA-2) VI

0.1

how FMD viruses might be dispersed between countries in sub-Saharan Africa at different times and places. These findings indicate that serotype A viruses (Fig. 2) are constantly evolving with time and geographic location and give rise to variant viruses that are genetically unrelated. Figure 2 shows that the group containing the Tanzanian strains isolated between 2008 and 2009 also contained viruses detected from Kenya in 2008 (A/KEN/8/2008) suggesting viruses from Tanzania and Kenya share common epidemiological links. Furthermore, a group of viruses from Tanzania (A/TAN/3/68, A/TAN/2/68 and A/TAN/4/ 80) also was most closely related to an older virus from Kenya (A/KEN/42/66), implying that the ancestral history of this lineage is shared in those East African countries. The phylogenetic tree for serotype O isolates (Fig. 3) features the close genetic relationship of Tanzanian viruses and FMD viruses collected elsewhere in the region (Kenya and Uganda), suggesting that long-distance animal e26


I (NWZ)

Fig. 4. Midpoint-rooted maximum likelihood tree showing the relationships between the serotype SAT 1 viruses collected from Tanzania. The nine serotype SAT 1 topotypes are labelled from I to IX. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. *reference number not assigned by the WRLFMD.

movements through trade and pastoralism could contribute to the spread of FMD across East Africa. It is also clear that FMD viruses occasionally spread into the northern parts of some Southern African countries (as evidenced by the close relationship between O/MAL/1/98 and Tanzanian viruses from the same year). Studies conducted elsewhere (Kivaria, 2003; Habiela et al., 2010; Di Nardo et al., 2011) described further that the spread of FMDV can also be associated by animal movement, especially in the pastoral livestock systems in Africa and Asia. SAT strains are known to be endemic in most Southern African countries with African buffalo being incriminated as reservoirs for these viruses (Vosloo et al., 2004, 2007). Phylogenetic analysis of the SAT viruses (Figs 4 and 5) shows high degree of nucleotide heterogeneity in SAT 1 viruses that were isolated from different geographic areas and times in Tanzania. Interestingly, in 1999, SAT 1 viruses belonging to topotype I were clustered in two dif-




SAT2/K17/07* (HM623705)

80

SAT2/K20/07* (HM623706) SAT2/K15/07* (HM623704) SAT2/K12/07* (HM623703) SAT2/K42/07* (HM623707)

100

SAT2/K67/07* (HM623709) SAT2/K59/07* (HM623708)

100

SAT2/K120/04 *(HM623700) SAT2/TAN/18/2004 (KF561725)

95

SAT2/TAN/21/2004 (KF561726)

79

SAT2/TAN/6/2004 (KF561723) SAT2/TAN/4/2004 (KF561722)

98

SAT2/TAN/9/2004 (KF561724) SAT2/TAN/43/2009 (KF561727) SAT2/K13/02* (HM623699) SAT2/KEN/8/99 (DQ009729)

87

SAT2/KEN/16/98 (AY343962) SAT2/KEN/7/99 (AF367132)

100

IV

SAT2/KEN/5/99 (AF367131) 84

SAT2/KEN/9/99 (AF367133) SAT2/TAN/1/86 (AY343971) SAT2/K6/06 (HM623702) SAT2/TAN/1/75 (AY343970) SAT2/KEN/2/76 (K183/74)(AY343940)

SAT2/K151/83* (HM623683)

71

SAT2/K52/84* (HM623685)

100

SAT2/K46/82* (HM623679)

100

SAT2/KEN/1/86 (AY343943) SAT2/K34/84* (HM623684) SAT2/KEN/2/87 (AY343945) SAT2/K81/81 (HM623678) 79

SAT2/KEN/1/87 (AY343944) SAT2/KEN/1/84 (K7/84) (AY344505) SAT2/ETH/1/90 (1989)(AY343935) SAT2/KEN/122/2009 (JX570630)

99 100 76 72

Ken-09

SAT2/KEN/13/2009 (JX570629)

III I

95

Fig. 5. Midpoint-rooted maximum likelihood tree showing the relationships between the serotype SAT 2 viruses collected from Tanzania. The 14 serotype SAT 2 topotypes are labelled from I to XIV. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. *reference number not assigned by the WRLFMD.

SAT2/KEN/11/2009 (JX570628)

73

II

89

VI V

100

XI

99

72

XIII XIV 100

99 100

93

VII

VIII IX XII X

0.1

ferent lineages implying a possibility of independent evolutionary events among viruses from a particular geographic location. These observations raise the possibility that epidemiological factors and evolutionary characteristics of viruses in a defined geographic location are related and may contribute significantly to FMD events and emergence of FMDV variants. The general trend for the occurrence of FMDV serotypes in Tanzania shows that serotype A virus has been present for long time in southern and central parts of the country. The type A viruses were incriminated to cause FMD outbreaks up to early 1980s, and there has been no evidence of serotype A detection in Tanzania until very recently (Swai et al., 2009; Kasanga et al., 2012). In part, the apparent disappearance of FMDV type A for almost 30 years (from the 1980s to the 2008) could reflect the low level of livestock sampling in the country. However, it is also possible that this could be ascribed to several aspects, including epidemiological factors related to maintenance and transmission of the virus, vaccination pressure, climatic and ecological

changes in specific geographic areas of disease occurrence. Further studies including in-depth molecular characterization are required to clarify the reoccurrence phenomenon of serotype A in similar environments after long-time disappearance of the virus. In recent years, there appears to be a north–south division of FMD viruses in East Africa with topotypes/genotypes occurring in Ethiopia, Eritrea, Somalia and Sudan (O/EA3, A/G-IV, A/G-VII, SAT1/IX, SAT2/VII, SAT2/XIII) being different to those occurring to the south in the Democratic Republic of the Congo, Kenya, Tanzania and Uganda (O/EA-2, O/EA-4, A/G-I, SAT1/I and SAT2/IV) (Ayelet et al., 2009; Balinda et al., 2010; Habiela et al., 2010; Sangula et al., 2010; Kasambula et al., 2012; Wekesa et al., 2013), although occasionally viruses from one region may appear in the other, for example, O/EA-4 in Ethiopia (Ayelet et al., 2009). Occasionally, viruses normally present in East Africa may spread to other regions such as the recent multiple incursions of SAT 2 into Egypt and the Middle East (Ahmed et al., 2012).


e27


We have sequenced the VP1-coding regions of four FMDV serotypes O, A, SAT 1 and SAT 2, which have been cocirculating in Tanzania between 1967 and 2009. The genetic characteristics of FMDV within each serotype revealed variations in nucleotide sequences; however, the overall genotypes/topotypes to which these viruses belonged were stable and consistent with those present in other countries in East Africa. The presence of multiple serotypes and genotypes/topotypes as well as the complex epidemiology of FMD complicates the control of the disease through vaccination and establishment of FMD-free zones. Therefore, further studies in both domesticated and wild animals are required to determine the phylogeography of viruses, investigate the genetic and antigenic characteristics of the circulating strains so that a rational control method for FMD in Tanzania and neighbouring countries can be recommended. These data provide historical context for a number of ongoing studies that aim to describe the epidemiology of FMD in Tanzania and the factors that influence the maintenance of the disease in the country. Acknowledgements The authors wish to acknowledge the assistance provided by farmers and staff in Veterinary Investigation Centres for sample collection during FMD outbreaks. We appreciate the contribution of staff at the WRLFMD for virus isolation and serotyping of samples. This work was funded by UK Department for the Environment, Food and Rural Affairs (Defra; projects SE2939 and SE2940), Biotechnology and Biological Sciences Research Council, the Department for International Development and the Scottish Government through their support for the Combating Infectious Diseases of Livestock for International Development initiative (project BB/H009302/1), the Wellcome Trust Grant WT087546MA to Southern African Centre for Infectious Diseases & Surveillance and the Ministry of Livestock Development and Fisheries, United Republic of Tanzania. References Ahmed, H. A., S. A. H. Salem, A. R. Habashi, A. A. Arafa, M. G. A. Aggour, G. H. Salem, A. S. Gaber, O. Selem, S. H. Abdelkader, N. J. Knowles, M. Madi, B. Valdazo-Gonzalez, J. Wadsworth, G. H. Hutchings, V. Mioulet, J. M. Hammond, and D. P. King, 2012: Emergence of foot-and-mouth disease virus SAT 2 in Egypt during 2012. Transbound. Emerg. Dis. 59, 476– 481. Ayelet, G., M. Mahapatra, E. Gelaye, B. G. Egziabher, T. Rufeal, M. Sahle, N. P. Ferris, J. Wadsworth, G. H. Hutchings, and N. J. Knowles, 2009: Genetic characterization of foot-and-mouth disease viruses, Ethiopia, 1981–2007. Emerg. Infect. Dis. 15, 1409–1417.

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