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TCS Tree Based on L-Fragment TCS tree based on L-fragment Kosti village 11 nt changes 12LPN1 (IP 1) 4 nt changes TUR/18/2010 BUL/1/2010 35 nt changes (Gümüşhane) (wild boar) 09/07/2010 30/12/2010 12LPN3 (IP 4) 14 nt changes Rezovo village putative common ancestor of the Bulgarian viruses All nucleotide substitutions are unique Neighbor-joining tree based on Leader+P1 TUR36/2010 TUR18/2010 BUL 12LPN3 (Rezovo) 979 BUL 12LPN4 (Rezovo) 1000 BUL 12LPN1 (Kosti) 767 BUL 12LPN2 (Kosti) BUL/12010 (wild boar) 0.001 Conclusions • The FMD outbreaks in Kosti and Rezovo villages are unlikely to be directly linked. • Both have a common ancestor close to the virus from wild boar. • The long branches between the putative common ancestor and each of the two outbreaks examined (IP1 and IP4) suggest possible intermediate hosts (either wildlife or domesticated animals). FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 Every think started from …. FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 FAO-EU FMD /EC/OIE Tripartitt Group Meeting,Plovdiv, Bulgaria, 25 March,2011 One shot wild boar FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 One shot wild boar FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 Not FMD in wild boar FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 What tests we used? Аg FMDV LFD FMFV Ag ELISA WRL FMDV Svanova One- step rRT-PCR (Reid, S. et all, 2002,) (Callahan et all., 2002) FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 Chronology • 29th December 2010 –wild boar with lesions in the feet, shot 2 km from the Turkish border; (42° 00‘409"N; 27°46'49.33"E) • 3th Jan – the sample sent to NRL • 4th Jan 2011 – Wild boar positive for - FMDV type O, • 9th Jan – 12 goats, 16 sheep,1 bovine and 8 pigs from the village of Kosti confirmed positive for NSP (42° 3'32.01"N; 27°46'49.33"E) FMDV type O • 15th Jan – clinical signs of FMD in bovines in the village of Rezovo; • 17th Jan – FMDV type O - Rezovo village, diagnosis confirmed by NRL – start of killing and burial on the spot FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 Chronology 31 January 2011 –FMDV type O in Gramatikovo 15 th February 2011, 10 h am. Samples sent to WRL Pirbright, UK . All confirmed O-type FMDV 18-th March 2011 samples from suspected new outbreak in village G.Bukovo arrived at the NRL – 22h am. 19-th March FMDV type O confirmed G.Bukovo 22-th March 2011 samples sent to WRL Pirbrihgt, UK 22-th March 2011 started culling of animals in village G.Dabovo 22-th March 2011 samples from suspected new outbreak in village Granichar arrived at the NRL 23-th March 2011 FMDV type O confirmed Granichar Village FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 SEQUENSING ANALYZIS AT WRL UK • The analysis made from the samples by the OIE Reference Laboratory Institute for Animal Health, Pirbright, UK, indicate this FMDV is a serotype O, Topotype ME-SA, Strain PanAsia-2ANT-10 which has more than 99.5% affinity with samples isolated in 2010 in Iran and Turkey. FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 The sub-lineage of the O-PanAsia strain was identified in Pakistan and Iran and named O- Pan-Asia-2 and become widespread in the Middl East A number of distinct sub-lineages have recently evolved and have been named as follows: • PanAsia-2YAZ-09 • PanAsia-2FAR-09 • PanAsia-2ANT-10 • PanAsia-2BAL-09 • PanAsia-2SAN-09 • PanAsia-2PUN-10 • PanAsia-2Kosti-10 FAO-EU FMD /EC/OIE Tripartite Group Meeting, P lovdiv, Bulgaria, 25 March,2011 FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 © Daniel Dalet / d-maps.com 500 km 300 mi Closed Sequences of Isolates of FMDV О-Pan Asia 2010 in Turkey and BG O/BUL/1/2010 (Burgas 30/12/2010) O/TUR/926/2010* (Bursa 26/07/2010) Bulgaria O/TUR/1086/2010* (Antalya 16/08/2010) O/TUR/840/2010* (Agri 15/07/2010) O/TUR/868/2010* (Eskisehir 20/07/2010) Kastamonu O/TUR/1003/2010* (Sivas 10/08/2010) Gümüşhane O/TUR/18/2010 (Gumushane 09/07/2010) Bursa Eskişehir O/TUR/883/2010* (Kastamonu 23/07/2010) Sivas Ağrı O/TUR/1094/2010* (Giresun 11/08/2010) Turkey Iran Antalya O/TUR/35/2010 (Erzincan 13/08/2010) O/TUR/153/2010* (Gaziantep) O/TUR/154/2010* (Gaziantep) O/IRN/94/2010 (West Azerbaijan 15/04/2010) O/IRN/92/2010 (West Azerbaijan 14/04/2010) 100 O/AFG/59/2010 (Samangan 14/05/2010) 97 O/PAK/36/2010 (Gilgit-Baltistan 29/07/2010) O/TUR/36/2010 (Kocaeli 13/08/2010) O/UKG/12/2001 (AJ311724) O1/Manisa/TUR/69 (AJ251477) FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 0.01 • The closest sequences of these strains isolated during 2010 from 7 different provinces in Turkey was differed by only a single nucleotide from each other and from the Bulgarian FMDV sequence (isolated January 2011 from wild boar in Kosti village Burgas district) FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 TCS tree based on L-fragment 11 nt changes Kosti village 12LPN1 (IP 1) 4 nt changes TUR/18/2010 BUL/1/2010 35 nt changes (Gümüşhane) (wild boar) 09/07/2010 30/12/2010 12LPN3 (IP 4) 14 nt changes Rezovo village putative common ancestor of the Bulgarian viruses All nucleotide substitutions are unique Neighbor-joining tree based on Leader+P1 TUR36/2010 TUR18/2010 BUL 12LPN3 (Rezovo) 979 BUL 12LPN4 (Rezovo) 1000 BUL 12LPN1 (Kosti) 767 BUL 12LPN2 (Kosti) BUL/12010 (wild boar) 0.001 Conclusions made at WRL • The FMD outbreaks in Kosti and Rezovo villages are unlikely to be directly linked. • Both have a common ancestor close to the virus from wild boar. • The long branches between the putative common ancestor and each of the two outbreaks examined (IP1 and IP4) suggest possible intermediate hosts (either wildlife or domesticated animals). Sequencing data DTU 23 March 2011 O/BUL/2010 (wild boar) 73 O/Kosti/2/2011 99 O/Kosti/1/2011 BUL isolates 2010/ 2011 100 O/Rezovo/3/2011 95 O/Rezovo/4/2011 71 O Iran 1 2010 (HQ450645) 98 O/PAK/3/2008 (FJ798192) 39 O/PAK/70/2007 (FJ798188) O/BUL/1/96 (EU553839) 100 99 O/TUR/1/96 99 O/TUR/2/2005 FJ561319 O/TUR/7/2003 (DQ164991) 100 O IRN/1/2000 (DQ164892) 74 O/IRQ/30/2000 (DQ165057) 78 O/Konya/TUR/512/10/99 (DQ296515) 96 O/UKG/11/2001 (DQ404180) O1/Manisa/Turkey/69 (AJ251477) O/BUL/1/91 (EU553837) 100 O/BUL/1/93 (EU553838 0.01 Neighbor-joining tree, showing the relationships between FMDV isolates in Bulgaria 2010 – 2011, 77 O/BUL/2010 100 Rezovo/3 BUL isolates 2010/ 2011 71 Kosti/1 O/ISL/PAK/L1573/2009 (HQ113232) 100 O/PAK/48/2008 (GU384683) O/UKG/11/2001 (DQ404180) O Campos/Bra/58 (AJ320488) 0.01 Decision voted at the SCoFCAH meeting on 12th Jan Abd r Shumen Varna Sliven Burgas Yambol Haskovo Annex I Kardjali Annex II FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Additional regions to Annex II Bulgaria, 25 March,2011 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 bovines 194 Kosti – the village of the bones sheep 117 goats 149 pigs 72 FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 Rezovo – the most southerly point on the Bulgarian Black sea coast bovines 92 sheep 68 goats 9 pigs 28 FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 21 Turkish buffalos FMD positives: FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 LPBL ELISA and Mono-O-type ELISA Prionics Results from Buffaloes Sera • Linear end-point titration mono O-type FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 TURKISH BUFFALOES - SEROLOGY RESULTS FMDV O, A and Asia-1 LIQUID PHASE BLOCKING ELISA (IAH) O-type LPBL A-type LPBL ASIA-1 LPBL Buffaloes ELISA ELISA ELISA 1 (>1:256) (1:16) (1:32) 2 (1:256) (-) (1:16) 3 (>1:256) (1:16) (1:64) 4 (>1:256) (1:64) (1:64) 5 (>1:256) (1:16) (1:32) 6 (1:256) (1:16) (1:32) FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 TURKISH BUFFALOES - SEROLOGY RESULTS FMD MONO-O-TYPE PRIONICS LINEAR TITRATION ELISA RESULTS Buffaloes Mono - O – type Priocheck Linear Titration 1 1:1024 2 1:256 3 1:1024/1:2048 4 1:1024 5 1:1024 6 1:256 FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 FMDV MONO-O-TYPE PRIONICS LINEAR TITRATION ELISA and rRT- PCR RESULTS Mono - O - type Buffaloes Prionics linear One Step r RT-PCR titration 1 1:1024 neg 2 1:128 neg 3 1:1024 neg 4 1 : 64 neg 5 1:256 POS (ct 31.58) 6 1:256 neg 7 1:128 neg 8 1:128 neg FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 CATTLE AND BUFFALOES VIROLOGY RESULTS FROM WRL IAH PIRBRIGHT, UK SPECIES EAR TAG LOCATION PCR R CELL RESULT CULTURE (ct value) ISOLATION BUFFALO-TR 3953-1887 REZOVO 33.14 POS NVD BUFFALO-TR NO EAR TAG REZOVO 31.58 POS NVD CATTLE NO EAR TAG REZOVO 19.34 POS FMDV - O CATTLE NO EAR TAG REZOVO 18.27 POS FMDV - O CATTLE NO EAR TAG KOSTI 16.86 POS FMDV - O CATTLE NO EAR TAG KOSTI 20.47 POS FMDV - O FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 TURKISH BUFFALOES - SEROLOGY RESULTS FROM WRL IAH PIRBRIGHT, UK Bufalloes Type –O / Type - A / Type – Asia 1 FINAL RESULT VNT VNT VNT 1 POS / 512 POS / 32 POS / 128 POS 2 POS / 128 POS / 40 POS / 16 POS 3 POS / 90 NEG /< 8 NEG / 8 POS 4 POS / 356 POS / 64 POS / 64 POS 5 POS / 178 POS / 178 POS / 45 POS 6 POS / 256 POS / 45 POS / 45 POS 7 POS / 355 POS / 355 POS / 64 POS FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 rRT-PCR Results from last outbreak investigated epithelium smaples FAO-EU FMD /EC/OIE Tripartite Group Meeting, Plovdiv, Bulgaria, 25 March,2011 rRT-PCR Results from last suspicion investigated in village St.
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