Overview of results of serosurvey in Transcaucasian region - buffer zones

Emiliana Brocchi Santina Grazioli

FMD NRL Istituto Zooprofilattico Sperimentale Brescia - Italy

IZSLER BRESCIA Serosurvey in Transcaucasian region

Objectives Vaccinal immunity • level and distribution of antibody to SP Investigate on possible virus circulation • antibodies to NSP

N° Villages / Sampling N° Country conditions N° Counties dates sera 16-17 30 / 12 June 900 Good 31 / 9 11-13 July 929 Very good

AZERBAIJAN 30 / 8 6-7 June 948 Bad (leaking)

Arrival Brescia →→→ end July IZSLER BRESCIA

138

TESTS

Tests Sampling dates Serum dilut NOTES

O Manisa LPB-ELISA 1/10 →1/2430 In-house Mabs-based A 96 SPC-ELISA 1/10 →1/2430 Recognise correctly Asia 1 SPC-ELISA 1/10 →1/2430 RS POS retested by NSP-3ABC 3ABC-ELISA 1/100 IZS-Brescia & Ceditest

Sera treated 56 °C, 45 minutes  Testing conducted during August • holidays time, less human resources, less routine serology  15 full (wet) working days • 60 plates/day SP ELISAs or 20 plates/day NSP-ELISA IZSLER  Data entry & elaboration BRESCIA O Manisa Asia 1 A Iran 96 SP-ELISA

IZSLER →→→ Serum dilution: range 1/10 1/2430 BRESCIA

139

age NSP-Ab N° Region County Tow n, village range cattle Pos % Pos years 30 2 2 6,7 Overall 131 sera/896 30 1-2 2 6,7 Ararat Shagap 30 1-2 1 3,3 POS NSP-Ab (15 %) Avshar 30 1-2 0 0 Ararat 30 3-7 11 36,7 30 1-9 0 0 N° 9/30 villages Berkhanush 30 1-3 0 0 Artashat Mxchyan 30 1-2 0 0 (all regions) Akhavnatun 30 1-3 28 93,3 Echmiadzin Lernamerc 30 1-3 3 10 High seroprevalence Echmiadzin t. 30 1-3 2 6,7 → V. 30 1-3 2 6,7 17% 93% (clusters) Armavir Eghegnut 31 1-3 2 6,5 Armavir Lenughi 30 1-3 0 0 29 1-3 0 0 N° 14/30 villages Bagramyan 30 1-4 6 20 ARMENIA N.Sasnashen 30 1 10 33,3 1 → 3 seropositive/vill. Aragacotn Talin Davtashen 30 1-2 3 10 → Mastara 30 1-2 2 6,7 3,3% 10% 28 1-10 7 25,0 Syuniq Meghri lower virus circulation? Lehbaz 30 3-7 3 10 Vogchi 30 1-7 19 63,3 Amasia animal movement? Alvar 28 1-5 0 0 Qarabert 30 1-6 10 33,3 Jrapi 30 1-4 2 6,7 Shirak Musaelyan 30 1-4 5 16,7 Maisyan 30 2-7 3 10 Akhuryan Sariar 30 1-9 2 6,7 Pograshen 30 1-7 1 3,3 IZSLER Gyumry Gyumry 30 1-5 5 16,7 BRESCIA

O A Iran age Asia 1 Town, N° NSP-Ab Manisa 96 Region County range village cattle years Pos % Pos % Pos % Pos % Pos

Vedi 30 2 2 6,7 73 73 93 Surenavan 30 1-2 2 6,7 83 90 100 Ararat Shagap 30 1-2 1 3,3 93 93 93 Avshar 30 1-2 0 0 70 100 100 Ararat Hovtashat 30 3-7 11 36,7 100 100 100 Masis Darbnik 30 1-9 0 0 97 100 100 Berkhanush 30 1-3 0 0 90 90 93 Artashat Mxchyan 30 1-2 0 0 100 100 100 Akhavnatun 30 1-3 28 93,3 100 100 100 Echmiadzin Lernamerc 30 1-3 3 10 70 70 80 Echmiadzin t. 30 1-3 2 6,7 93 93 93 V. Armavir 30 1-3 2 6,7 90 97 87 Armavir Eghegnut 31 1-3 2 6,5 81 90 81 Armavir Lenughi 30 1-3 0 0 53 67 90 Arevik 29 1-3 0 0 76 97 100 Bagramyan Myasnikyan 30 1-4 6 20 50 87 73 N.Sasnashen 30 1 10 33,3 87 90 90 Aragacotn Talin Davtashen 30 1-2 3 10 77 100 100 ARMENIA Mastara 30 1-2 2 6,7 60 63 53 Meghri 28 1-10 7 25,0 89 96 89 Syuniq Meghri Lehbaz 30 3-7 3 10 87 97 100 Vogchi 30 1-7 19 63,3 87 93 93 Amasia Alvar 28 1-5 0 0 57 86 82 Qarabert 30 1-6 10 33,3 83 100 97 Ani Jrapi 30 1-4 2 6,7 87 100 100 Shirak Musaelyan 30 1-4 5 16,7 90 97 100 Maisyan 30 2-7 3 10 93 100 100 Akhuryan Sariar 30 1-9 2 6,7 90 90 93 Pograshen 30 1-7 1 3,3 83 93 97 Gyumry Gyumry 30 1-5 5 16,7 97 93 100 IZSLER TOTAL / AVERAGE % 896 131 15 % 83 % 92 % 93 % BRESCIA

140

ARMENIA – REGION ARARAT – 30 samples/village 2 Y 1,5-2 Y 1-2 Y 1-2 Y

10000 100

Arat/ Vedi Arat /Avshar Arat /Surenavan Arat / Shagap 90

80

1000 70

60

50 100 40

30 A Asia O NSP 20 10 cut-off NSP 10

0

1 -10

3-7 Y 2-9 Y 1-3 Y 1-2 Y 10000 100

Masis/Hovtashat Masis/Darbnik Artashat/Berkhanus Artashat/Mxchyan 90

80

1000 70

60

50 100 40

30

10 A Asia O NSP 20 cut-off NSP 10

0 1 -10 ARMENIA – REGION ARMAVIR – 30 samples/village 1-3 Y 1-3 Y 1-3 Y 1-4 Y

10000 100 Bagramyanr/Myasnikyan Echmiadzin/Achavnatun A Asia O NSP 90

Echmiadzin/Lernamer Echmiadzin/Echmiadzi 80 1000 70

60

50 100 40

30

20 10 cut-off NSP 10

0

1 -10

1-3 Y (>1) 1-3 Y 1-3 Y 1-3 Y 10000 100 Armavir/Eghegnut Armavir/Arevik Armavir/Lenughi Armavir/V.Armavir 90 A Asia O NSP 80 1000 70

60

50 100 40

30

20 10 cut-off NSP 10

0 1 -10

141

ARMENIA – REGION SHIRAK– 30 samples/village 1-4 Y 1-6 Y 1-5 Y 1-7 Y 10000 Ani/Jrapi Ani/Qarabert Amasia/Alvar Amasia/Vogchi 100 90

80 1000 70

60

50 100 40

30

20 10 A Asia O NSP cut-off NSP 10

0

1 -10 1-7 Y 2-7 Y 1-4 Y 1-9 Y 1-5 Y 10000 100 Akhuryan/Pograshen Akhuryan/Maisyan Akhuryan/Musaelyan Akhuryan/Sariar Gymry 90 80 1000 70 60 50 100 40 30 20 10 A Asia O NSP cut-off NSP 10 0 1 -10 ARMENIA – REGIONS ARAGACOTN, SYUNIQ - 30 samples/village

1 Y 1 Y (2) 1 Y (2) 3-7 Y 1-10 Y

10000 100

Talin/N.Sasnashen Talin/Mastara Talin /Devtashen Meg/Leh Meg/Meg 90

A Asia O NSP 80 1000 70

60

50 100 40

30

20 10 cut-off NSP 10

0

1 -10

IZSLER BRESCIA

142

age NSP-Ab N° County Town, village range cattle years Pos % Pos Overall 23 sera/929 Gogadzeebi 30 1 3,3 30 0 0 → Shuakevi POS NSP-Ab 2.5 % Nenia 30 0 0 Brili 30 0 0 Dilipi 30 0 0 Udjmana 30 0 0 N° 1/31 villages Spasovka 30 0 0 Ganakhleba 30 3 10 High seroprevalence Vernebi 30 0 0 Gurta 30 0 0 33.3 % (cluster) Bodzauri 30 0 0 Atskvita 30 0 0 Saxudabeli 30 2 6,7 N° 8/31 villages Tmogvi 30 0 0 Sulda 30 10 33,3 3seropositive/vill. Alastini 30 0 0

GEORGIA Khorenia 30 0 0 3,3% → 6.7% Zanakidzeebi 30 1 3,3 1 - 2 years 2 - 1 Shua 29 0 0 low virus circulation? Ortabatumi 30 0 0 Kveda chkhutuneti 30 0 0 animal movement? Kulalisi 30 2 6,7 false-positive? (1.4 %) Sviri 30 2 6,7 Zikilia 30 0 0 Kikibo 30 1 3,3 Patara Zanavi 30 1 3,3 Chorchali 30 0 0 Zeda agara 30 0 0 Tsoniaresi 30 0 0 Keda IZSLER Koromkheti 30 0 0 Dzesopeli 30 0 0 BRESCIA

O age NSP-Ab A Iran 96 Asia 1 N° Manisa County Town, village range cattle years Pos % Pos % Pos % Pos % Pos

Gogadzeebi 30 1 3,3 50 37 40 Vani 30 0 0 17 0 17 Shuakevi Nenia 30 0 0 77 40 57 Brili 30 0 0 17 10 10 Dilipi 30 0 0 37 33 50 Ninotsminda Udjmana 30 0 0 40 30 37 Spasovka 30 0 0 57 33 57 Ganakhleba 30 3 10 70 80 33 Vernebi 30 0 0 50 20 0 Khulo Gurta 30 0 0 67 63 20 Bodzauri 30 0 0 30 3 10 Atskvita 30 0 0 17 3 3 Aspindza Saxudabeli 30 2 6,7 63 40 13 Tmogvi 30 0 0 20 17 0 Sulda 30 10 33,3 87 63 67 Akhalkalaki Alastini 30 0 0 67 67 63 Khorenia 30 0 0 73 73 67 GEORGIA Zanakidzeebi 30 1 3,3 63 60 27

Shua makhinjauri 29 1 - 2 years Khelvachauri 0 0 66 100 83 Ortabatumi 30 0 0 93 100 97 Kveda chkhutuneti 30 0 0 100 93 13 Kulalisi 30 2 6,7 30 20 10 Akhaltsikhe Sviri 30 2 6,7 17 7 10 Zikilia 30 0 0 30 17 7 Kikibo 30 1 3,3 23 33 23 Adigeni Patara Zanavi 30 1 3,3 10 23 17 Chorchali 30 0 0 13 47 17 Zeda agara 30 0 0 17 40 17 Tsoniaresi 30 0 0 7 0 100 Keda Koromkheti 30 0 0 60 63 50 Dzesopeli 30 0 0 80 67 57 IZSLER TOTAL / AVERAGE 929 23 2.5 % 47 % 41 % 34 % BRESCIA

143

GEORGIA - ANIMALS AGE 1 – 2 YEARS – 30 SAMPLES/VILLAGE

10000 100 Akhalk/Alastini Akhalk/Sulda Khulo/Vernebi Khulo/Gurta Khulo/Ganakhleba 90 A Asia O NSP Khulo/Bodzauri Akhalki/Khorenia 80 1000 70

60

50 100 40

30

20 10 cut-off NSP 10

0

1 -10 ↑↑↑ REGIONS KHULO, ALKHALSIKHE ↓↓↓ REGION KHELVACHAURI 10000 100 Khelv./Zanakidzeebi Khelv./ShuaMachinjauri Khelv./Ortabatumi Khelv./Kveda chkhutuneti 90 80 1000 70 60 50 100 40 30

10 A Asia O NSP 20 cut-off NSP 10 0 1 -10 REGION KEDA GEORGIA - CATTLE AGE 1 – 2 YEARS

10000 100

Keda/Zeda agara Keda/Tsoniaresi Keda/Koromkheti Keda/Dzesopeli 90 A Asia O NSP 80 1000 70

60

50 100 40

30

10 20 cut-off NSP 10

0

1 -10 REGION SHUAKEVI 30 samples/village 10000 100

Shuakhevi/Vani /Nenia Shuakhevi/Gogadzeebi Shuakhevi/Brili 90 A Asia O NSP 80 1000 70

60

50 100 40

30

20 10 cut-off NSP 10

0 1 -10

144

REGION ADIGENI, NINOTSMINDA GEORGIA 10000 100

Adigeni/Chorchali Adigeni/Kikibo Adigeni/Patara Ninotsm/Dilipi Ninotsm/Spasovka 90 A Asia O NSP Ninotsm/Udjmana 80 1000 70

60

50 100 40

30

10 20 cut-off NSP 10

0

1 -10 REGION ASPINDZA, AKHALTSIKHE 30 samples/village, age 1-2 years

10000 100 Aspindza/Atskvit Aspindza/Tmogvi Akhaltsikhe/Kulalisi Akhaltsikhe/Zikila 90 Aspindza/Saxudabeli A Asia O NSP Akhaltsikhe/Sviri 80 1000 70

60

50 100 40

30

10 20 cut-off NSP 10

0 1 -10

age NSP-ELISA N° County Town, village range cattle years Pos % Pos Overall 79 sera/939 Anbabiu 47 1-2 3 6,4 POS NSP-Ab → 8.4 % Astara Motalayag 14 1-8 0 0 Tahtakaran 35 1-3 0 0 Almew 32 0.3-1.2 0 0 Lerik Kokoniu 26 1-2 0 0 N° 5/30 villages Arta 15 1-2 0 0 Orengala 28 0.5-1.3 0 0 (4 counties) Tiurkler 33 0.5-1.5 0 0 Beyliagan High seroprevalence Khachynabad 32 0.6-1.6 0 0 Gadekler 31 0.8-1.2 0 0 19% → 41% (clusters) Fioletovka 32 0.9-4 8 25 Belasuar Tazakend 34 2-4 10 29,4 Arkhangelovka 28 1-2 1 4 Jeferly 26 0.5-1.3 5 19 N° 14/30 villages YukharyGaragiuvendly 33 0.6-1.8 3 9,1 → Imishli Khelfaly 34 1-1.9 2 6 1 4 seropositive/vill. Sarykhanly 34 0.3-2 2 6 2.6% → 12% Badgyravan 27 0.8-1.9 0 0 Khalilly 30 1-2 9 30 lower virus circulation? Khajejavadly 33 1-2 4 12,1 Laken 29 1-3 3 10,3 animal movement? Khoviuzbiulag 30 1-2 2 6,7 Jalilabad Adnaliu 48 0.7-2 2 4,2 Biuravar 21 1-2.5 1 4,8 Shykhlar 28 1-2 1 3,6 N° 2 counties Lezran 30 1-2 0 0 NO virus circulation Alhanbeyly 39 1-5 16 41 Fyziuli Kerimbeyly 39 1-7 4 10 Unash 34 0.5-2 2 6 IZSLER Yardimly Avin 39 0.7-2 1 2,6 BRESCIA

145

O A Iran age NSP-ELISA Asia 1 N° Manisa 96 County Town, village range cattle years Pos % Pos % Pos % Pos % Pos

Anbabiu 47 1-2 3 6,4 89 98 100 Astara Motalayag 14 1-8 0 0 64 93 100 Tahtakaran 35 1-3 0 0 91 91 51 Almew 32 0.3-1.2 0 0 44 84 84 Lerik Kokoniu 26 1-2 0 0 65 88 92 Arta 15 1-2 0 0 7 100 93 Orengala 28 0.5-1.3 0 0 89 100 100 Tiurkler 33 0.5-1.5 0 0 82 94 79 Beyliagan Khachynabad 32 0.6-1.6 0 0 34 97 84 Gadekler 30 0.8-1.2 0 0 90 100 100 Fioletovka 32 0.9-4 8 25 78 97 100 Belasuar Tazakend 34 2-4 10 29,4 91 94 94 Arkhangelovka 28 1-2 1 4 71 75 93 Jeferly 26 0.5-1.3 5 19 100 100 96 YukharyGaragiuvendly 33 0.6-1.8 3 9 88 97 100 Imishli Khelfaly 34 1-1.9 2 6 47 85 79 Sarykhanly 34 0.3-2 2 6 68 82 76

AZERBAIJAN Badgyravan 27 0.8-1.9 0 0 85 96 96 Khalilly 30 1-2 9 30 100 100 100 Khajejavadly 33 1-2 4 12,1 27 48 52 Laken 29 1-3 3 10,3 41 100 100 Khoviuzbiulag 30 1-2 2 6,7 73 97 83 Jalilabad Adnaliu 48 0.7-2 2 4,2 33 85 90 Biuravar 21 1-2.5 1 4,8 90 100 95 Shykhlar 28 1-2 1 3,6 64 100 89 Lezran 30 1-2 0 0 47 93 100 Alhanbeyly 39 1-5 16 41 90 100 100 Fyziuli Kerimbeyly 39 1-7 4 10 67 97 92 Unash 34 0.5-2 2 6 65 100 100 Yardimly Avin 38 0.7-2 1 2,6 68 97 97 TOTAL / AVERAGE 939 79 8.4 % 69 % 93 % 90 % IZSLER BRESCIA

AZERBAIJAN 15 →→→ 40 SAMPLES/VILLAGE 1-2 Y 1-8 Y 1-3 Y 0.3-1.2 Y 1-2 Y 1-2 Y 10000 100

Astara/Anabiu Astara/Motala Astara/Tahtakaran Lerik/Almev Lerik/Kokoniu 90 Lerik/Arta A Asia O NSP 80

1000 70

60

50 100 40

30

20 10 cut-off NSP 10

0

1 -10 0.5 1.3 Y 0.5-1.5 Y 0.6-1.6 Y 0.8-1.2 Y 0.5-2 Y 0.7-2 Y 10000 100 Beyliag/Orengala Beyliag/Tiurkler Beyliag/Khachin Beyliag/Gadekler Yardimly/Unash Yardimly/Avin 90 A Asia O NSP 80 1000 70 60 50 100 40 30 10 20 cut-off NSP 10 0 1 -10

146

AZERBAIJAN 30 →→→ 40 SAMPLES/VILLAGE 1-4 Y 2-4 Y 1-2 Y 1-4,5,6,7 Y 1→→→5 Y 10000 100 Bel//Fioletokva Bel/Tazakend Bel/Arkhangelovka Fyziuli/Keribeyly Fyziuli/Alhanbeyly 90 A Asia O NSP 80 1000 70 60 50 100 40 30 10 20 10 cut-off NSP 0 1 -10

0.8-1.9 Y 1.5-3 Y 0.6-1.8 Y 0.3-2 Y 1-1.9 Y 10000 100 Imishli/Badgyrava Imishli/Jeferly A Asia O NSP Imishli/Sarykhanly Imishli/Khelfaly 90 Imishli/Yukh-Gar 80 1000 70 60 50 100 40 30 10 20 cut-off NSP 10 0 1 -10 AZERBAIJAN (21 →→→ 48 samples/village) COUNTY JALILABAB

1-2.5 Y 1-2 Y 1-2 Y 1-2 Y 10000 100 Jelilabab/Biuravar Jelilabab/Khajejavadli Jelilabab/Khalill Jelilabab/Lezra 90 A Asia O NSP 80 1000 70 60 50 100 40 30 10 20 cut-off NSP 10 0 1 -10

0.7-2 Y 1-2 Y 1-2 Y 2-3 Y 10000 100 Jelilabab/Adnaliu Jelilabab/Shykhlari Jelilabab/Khoviuzbiula Jelilabab/Laken 90 A Asia O NSP 80 1000 70 60 50 100 40 30 10 20 cut-off NSP 10 0 1 -10

147

General prevalence of anti-NSP antibodies and vaccinal coverage

N° NSP-ELISA O Manisa A Iran 96 Asia 1 Summary cattle Pos % Pos % Pos % Pos % Pos 15 % 83 % 92 % 93 % ARMENIA 900 131 (0-93) (50-100) (63-100) (53-100) 2.5 % 47 % 42 % 34 % GEORGIA 929 23 (0-33.3) (7-100) (0-100) (0-100) 8.4 % 69 % 93 % 90 % AZERBAIJAN 939 79 (0-41) (7-100) (48-100) (51-100)

 Virus Neutralisation test for type O Manisa performed on ≈ 100 sera Sera were selected from the three countries and represented various immune conditions (different titres, positive or negative to NSP, ….)

 Evidence of neutralising antibodies in sera positive by ELISA;  Correlation between ELISA and VNT titres;  Vaccinal immunity is likely to be protective IZSLER BRESCIA

Conclusions

IZSLER BRESCIA

148

1000000 100 A Asia O NSP 3 >4 vacc. 2 >4 vacc. 90 ISRAEL 2004 100000 80 >4 vacc. 70 10000 60 →→→ 50 2 4 VACC. 1000 40 NO clin. Signs 30 100 20 Titres O cut-off NSP 10 10 3 →→→ 4 0 10 10 1 -10

1000000 100 A Asia O NSP 1 vacc. 90 100000 80 1 VACC. 70 10000 NO clin. Signs 60 50 Titres O 1000 40 2 →→→ 3 30 40% 10 10 100 20 3 → 4 cut-off NSP 45% 10 →→ 10 10 10 4 5 0 15% 10 →→→ 10 1 -10

1000000 100 A Asia O NSP 1 vacc 90 100000 80 70 1 VACC. 10000 60 50 Clinical signs 1000 40 Titres O 30 100 20 3 →→→ 4 cut-off NSP 10 10 10 10 0 1 -10

149