1. Serotype Specificity of Antibodies against FMDV in Cattle in Selected Districts in Uganda FRANK NORBERT MWIINE Department of Biomolecular Resources & Biolab Sciences (BBS) School of Biosecurity, Biotechnical & Laboratory Sciences (SBLS) College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB) Ph.D dissemination of research findings

2. Introduction Foot-and-mouth disease (FMD) is one of the most important livestock diseases globally due to its economic impact Affects cattle, pigs, sheep, goats, buffalos, and all cloven hoofed wildlife species. FMD virus (FMDV): classified within the Aphthovirus genus as a member of the Picornaviridae family Extremely contagious, difficult and expensive to control and eradicate

3. Introduction FMDV: 7 immunologically distinct serotypes:- O, A, C, Asia 1, SAT 1, SAT 2 & SAT 3 In Africa, 6 serotypes exist apart from Asia 1 that has never been reported In Uganda FMD is endemic, first confirmed in 1953 Control measures based on vaccination and quarantine of livestock

4. Clinical signs of FMD Characterized by fever and vesicles in the mouth, muzzle, teats, and feet. Excess salivation (drooling) is obvious

5. Clinical signs of FMD Severe erosions on the dental pad and gums. (mouth lessions) Raw tongue

6. FMD, Ruptured tongue

7. FMD lesion on the tongue

8. Classical FMD, raw tongue, flothing

9. Clear FMD wounds on coronary band

10. FMD lesion on the gum

11. FMD, Raw gum

13. FMD classical sign

14. FMD, lesion on nostril

15. FMD Lesion on the tongue

16. Healing tongue, FMD

17. Burning FMD infected cattle

18. Study objective To determine the serotype-specificity of the circulating FMDV antibodies in cattle in selected districts in Uganda during the major FMD outbreak in 2006.

19. Map of Uganda showing FMD post-outbreak study districts, 2006.  study carried out in 7 districts; Bushenyi, Isingoro, Mbarara, Kasese, Mpigi, Kiboga and Kiruhura  A total of 349 cattle sera were collected from 28 herds  30 OP collected from 9 herds (Mb,Ks)

21. Methods Serum antibody assays i.Screening for antibodies to FMDV Non Structural Proteins (NSP) & Structural Proteins (SP) was performed using Ceditest ® FMDV-NS & FMDV type O test kits (Cedi Diagnostics BV, Lelystad, The Netherlands). ii.Solid Phase Blocking ELISA (SPBE) an in-house system set up at DTU, National Veterinary Institute, Lindholm Denmark/MAAIF-Uganda FMD Virus isolation /Ag ELISA

22. ELISA TEST

23. Colour development, FMD serotype O After addition of stop solution FMD serotype O

24. Results  From 22 cattle herds with visible typical clinical signs to FMD, 285 sera were obtained; NSP= 78%, SP- O=82%  From 6 cattle herds that had no visible c/s to FMD, 64 sera were obtained; NSP= 5%, SP-O=6%  SPBE; serotypes O (38%, 22/58), SAT 1 (11%, 5/45), SAT 2 (2%, 1/49) and SAT 3 (9%, 7/79) at cut-off of ≥160  Only FMD serotype O virus was isolated (GBACN:EF611987)

25. DistrictVillageHerd no.No. of samp. testedNSP Positive (%)SP-O Positive (%) BushenyiKitweBs699 (100) KitweBs5109 (90)10 (100) RwenjeruBs31110 (91)11 (100) KashoziBs1100 (0) BusheregyenyiBs2100 (0) MashongaBs490 (0) IsingiroKagogoIs155 (100) KaseseKahenderoKs2*65 (83)6 (100) KahenderoKs3*87 (88) KahenderoKs42722 (81)25 (93) KahenderoKs1321 (50) KahenderoKs1531 (33)3 (100) Kasomoro IIKs5*2115 (71)14 (67) Kasomoro IIKs6167 (44)15 (94) NyabubaleKs7*44 (100) NyabubaleKs81413 (93)11 (79) NyabubaleKs91313 (100) NyabubaleKs10*99 (100) NyabubaleKs11*π1916 (84)18 (95) National parkKs123019 (63)2 (7) KibogaButembeKb164 (67) MbararaIshanyuMb188 (100) KafunjoMb2*2111 (52)18 (86) MukoMb3*1210 (83)12 (100) MukoMb4*ß107 (70)10 (100) MpigiMaduMp12318 (78)21 (91) KiruhuraRurambiraKr3150 (0)1 (7) KazoKr4 Ω 182 (11) Total349225 (65)239 (69) Total for herds with clinical signs of FMD285222 (78)235 (82) Total for herds without clinical signs of FMD643 (5)4 (6)

26. Res. T2: Titration of serotype-specific antibodies against seven Foot -and-mouth disease (FMDV) serotypes Titre*OACAsia 1SAT 1SAT 2SAT 3 < 1.9019/5815/158/82/237/4544/4967/79 ≥1.9, < 2.2017/580/150/80/23/454/495/79 ≥ 2.2022/580/150/80/25/451/497/79 10 * Expressed on log 10 and cut-off ≥160. serotypes O (38%, 22/58), SAT 1 (11%, 5/45), SAT 2 (2%, 1/49) and SAT 3 (9%, 7/79)

27. Conclusions Study shows that majority of the FMD outbreaks in 2006 were caused by FMDV serotype O Evidence of antibodies to both SAT 1 and SAT 3 in one FMD outbreak in a non-vaccinated herd (ks12) inside Queen Elizabeth national park area

28. © 2010 Blackwell Verlag GmbH Transboundary and Emerging Diseases. 57 (2010) 365–374

29. Serotype-specificity of antibodies towards FMD in cattle herds surrounding Lake Mburo National Park in 2008

30. Study area  Lake Mburo National Park (LMNP) in the South-Western part of Uganda-Kiruhura District  211 sera samples from 23 herds of long horned Ankole breeds aged more than four years were collected with a history of previous FMD outbreaks

31. Methods Antibody assays i.Screening for antibodies to FMDV Non Structural Proteins (NSP) & Structural Proteins (SP) was performed using Ceditest ® FMDV-NS & FMDV type O test kits (Cedi Diagnostics BV, Lelystad, The Netherlands). ii.Solid Phase Blocking ELISA (SPBE) an in-house system set up at DTU, National Veterinary Institute, Lindholm Denmark/MAAIF-Uganda

32. Results Out of 211 cattle sera samples:- – 42.7% (90/211) were positive for antibodies against NSP of FMDV – 75.4% (159/211) were positive for SP of FMDV serotype O  FMDV Titres ≥ 1:160 in SPBEs were:- – Serotype O, 61% (19/31), – Serotype A, 33% (5/15), – SAT 1, 67% (20/30), – SAT 2, 37% (10/27) – SAT 3, 2% (4/33)

33. conclusions FMD outbreaks in cattle herds around LMNP were probably caused by FMDV serotype O, A and/ or SAT-serotype(s). Usage of non-purified, multivalent vaccines obscures the serological diagnosis of FMDV outbreaks

34. General Recommendations Regional studies should be carried out to establish FMDV serotypes and/or strains involved in disease outbreaks and emphasis should be on virus recovery. For future serological work, circulating field viruses in Uganda should be obtained and homologous reagent be made for SPBE. Post outbreak sampling for serological diagnosis should be focused on young unvaccinated stock of 6- 12-months of age. Carrier status should be evaluated in districts where FMD outbreaks are frequent.

35. FMD Control Vaccination of animals –cattle and sheep goats and pigs Strict quarantine of animals Establish FMD free zones in Uganda/regions Observe all-in-all out rule of animals Restriction of persons and trucks and proper sanitation(animal owners, vets, trucks)

36. Acknowledgements This study was funded by DANIDA under the Livestock Wildlife Diseases in East Africa Project (LWDEA), grant number: P104.Dan.8.1.316. 1.National Animal Disease Diagnostics and Epidemiology Centre, Ministry of Agriculture Animal Industry and Fisheries, Entebbe, Uganda 2.Makerere University Institute of Environment and Natural Resources, Kampala, Uganda 3.National Veterinary Institute, Danish Technical University, Kalvehave, Denmark 4.Department of Biology, University of Copenhagen, Denmark My former supervisors, William Olaho-Mukani, Kirsten Tjørnehøj Project coordinators and scientists/advisors Anna Rose Ademun Okurut, Prof. Søren Alexandersen, Vincent Muwanika,, Laurids Siig Christensen, Hans Redlev Siegismund, Charles Masembe, Karl Johan Sørensen, Graham Belsham, Jorn Klien, Bøtner Annete Former PhD scientists on LWDEA project : Chrisostom Ayebazibwe, Sheila Nina Balinda, Abraham Kiprotich Sangula Technical team: Esau Martin, Eugene Arinaitwe, Zac Duluga (deceased), Patrick Atim Nedi, Preben Norman, Jani Christensen, Jane Borch and Tina Fredricksen

37. Thank you