An Outbreak of Pandemic H1N1 Influenza in Turkeys in Ontario DAVOR OJKIC ANIMAL HEALTH LABORATORY UNIVERSITY OF GUELPH

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1 An Outbreak of Pandemic H1N1 Influenza in Turkeys in Ontario 1 DAVOR OJKIC ANIMAL HEALTH LABORATORY UNIVERSITY OF GUELPH

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4 Host and lineage origins for the gene segments of the ph1n1 R J Garten et al. Science 2009;325: Published by AAAS

5 Pandemic H1N1 (ph1n1) Influenza in Animals Pigs Canada (AB, QC, MB), UK, USA (NC, IN, MN, SD), Finland, Indonesia, Taiwan, Iceland, Japan, Norway, Ireland, Singapore, Australia, Argentina, China, Italy, Mexico, Germany, Russia, Denmark, Korea Ferrets USA (OR, NE, CA) Cats USA (UT, OR, IA, CO, CA) Dogs USA (NY), China Skunk Canada (BC) Zoo animals Badger, Asian Bearcat (US) Turkeys Chile, Canada, USA (VA, CA), France 5

6 ph1n1 in Ontario Turkeys

7 ph1n1 in Ontario Turkeys Contract producer of grandparent breeding stock Farm A: two laying barns 45 weeks 52 weeks Toms Farm B: two-barns with young birds 15 weeks 20 weeks 7

8 Friday, October 9, week old flock - 5% decrease in egg production The flock appeared healthy No apparent clinical signs 8

9 Sunday, October 11, 2009 Egg production decreased by additional 35% No apparent clinical signs Toms housed on the farm also appeared healthy Slight increase in mortality 9

10 Monday, October 12, 2009 (Thanksgiving) Egg production decreased by over 50% No apparent clinical signs Slight increase in mortality Self-imposed quarantine 10

11 Tuesday, October 13, 2009 Egg production decreased by 80% No apparent clinical signs Increase in mortality 11 Samples from both Farm A (laying farm) and Farm B (younger birds) sent to the lab

12 Mortality % 0.50% 0.40% 0.30% Weekly % Mortality Weekly Standard % Mortality 0.20% 0.10% 0.00%

13 Production Drop 13

14 Wednesday, October 14, Samples from the affected barn were influenzapositive by matrix rrt-pcr Samples from the affected barn were negative for H5, H7 & H3 Samples from other barns were influenza- negative by matrix rrt-pcr

15 Friday, October 16, 2009 Partial HA gene sequencing ph1n1 influenza virus Partial N gene sequencing ph1n1 influenza virus Samples forwarded for confirmatory testing to National Centre for Foreign Animal Disease in Winnipeg, MB. 15

16 AHL PCR Results Matrix rrt-pcr: 3/14 positive Matrix conventional: 5/14 positive Nucleoprotein conventional : 3/14 positive N1 conventional : 5/14 positive H1 conventional (classic pig primers): 0/14 positive H1 conventional (NML primers): 4/14 positive H1 conventional (AHL modified primers-1): 4/14 positive NCFAD AIV -SIV modified matrix rrt-pcr: 11/14 positives 16

17 HA gene Sequencing Results A/Thailand/CU-B5/2009(H1N1), Identity>99% PB2 gene A/Toronto/T5362/2009(H1N1), Identity>99% PB1 gene A/Netherlands/602/2009(H1N1), Identity>99% NP gene A/Nonthaburi/102/2009(H1N1), Identity>99% NA gene A/Toronto/T0106/2009(H1N1), Identity>99% M gene A/swine/Alberta/OTH-33-21/2009(H1N1), Identity =100% NS gene A/Netherlands/602/2009(H1N1), Identity >99% PA gene A/Toronto/T5362/2009(H1N1), Identity >99% 17

18 Hemagglutinin Amino Acid Substitutions 18 VIRUS HA amino acid position A/Mexico/InDRE4487/2009 (H1N1) D G S S R T Q A/Ty/ON/FAV 110 3/2009 (H1N1) V Q N H A/Ty/ON/FAV 110 4/2009 (H1N1) E R Q H A/Ty/ON/FAV 114/2009 (H1N1) P Q H D aspartic acid; G glycine; S serine; R arginine ; T threonine; Q glutamine V valine; N aspargine; E ; H histidine ; P proline 155 receptor binding site 183 egg adaptation site

19 Neuraminidase Amino Acid Substitutions major ph1n1 NA groups: V106/N248 I106/D248 I106/N VIRUS NA amino acid position A/Mexico/InDRE4487/2009 H1N1 V N A/Ty/ON/FAV 110 3/2009 H1N1 S D A/Ty/ON/FAV 110 4/2009 H1N1 S D A/Ty/ON/FAV 114/2009 H1N1 S D V valine; N aspargine; S serine; D aspartic acid

20 Typing by Hemagglutination Inhibition 20 VIRUS TITER A/Ck/BC/3/98 (H1N1) hyperimmune chicken serum 320 A/DK/ON/629/05 (H1N1) hyperimmune chicken serum 128 A/Mexico/InDRE4467/09 (H1N1) hyperimmune turkey serum 1024 A/Turkey/ON/FAV 110/09 (H1N1) turkey field serum 512 A/SW/Iowa/31 (H1N1) hyperimmune chicken serum 160

21 Turkey Infectious Dose 50 * DPI TCID 50 /ml 2x10 6 2x10 5 4x10 4 2x10 4 Cloacal Sheding 2 4/8 1/7 1/6 0/5 4 5/7 1/6 1/5 0/4 6 4/6 1/5 0/4 0/3 8 2/5 1/4 0/3 0/2 14 0/5 0/4 0/3 0/2 21 DPI celisa/hi celisa HI celisa HI celisa HI celisa HI 0 0/8 0/8 0/7 0/7 0/6 0/6 0/6 0/6 7 1/5 0/4 0/3 0/3 14 3/5 1/4 0/3 0/3 21 3/5 3/5 1/4 1/4 0/3 0/3 0/3 0/3 28 3/5 3/5 1/4 1/4 0/3 0/3 0/3 0/3 *A/Mexico/InDRE/4467/2009 (H1N1)

22 Design ph1n1 Pathogenesis & Transmission 3 groups of 8 to 10-week-old influenza A free turkey pullets cloacal group oronasal group control group Monitoring for clinical signs (twice daily) for cloacal and oropharyngeal shedding of the virus by rrt- PCR for seroconversion by ELISA & HIs necropsies (3, 5 & 7 DPI) 22 *A/Mexico/InDRE/4467/2009 (H1N1)

23 ph1n1 Pathogenesis & Transmission 23 DPI Cloacal Group Oronasal Group Contact Group Oral shedding Cloacal shedding Oral shedding Cloacal shedding Oral shedding Cloacal shedding 2 2/10 8/10 6/10 2/10 0/10 0/10 3 2/10 8/10 6/10 0/10 0/10 0/10 5 1/10 8/10 6/9 0/9 0/9 0/9 7 1/9 6/9 4/8 1/8 0/8 0/8 9 0/8 3/8 1/7 0/7 0/7 0/7

24 ph1n1 Pathogenesis & Transmission 24 DPI Cloacal Group Oral Group Contact Group celisa HI celisa HI celisa HI 0 0/10 0/10 0/10 0/10 0/10 0/10 7 1/9 0/9 0/8 0/8 0/8 0/8 14 4/8 0/8 3/7 0/7 0/7 0/7 21 5/8 6/8 3/7 3/7 1/7 1/7 28 5/8 6/8 4/7 3/7 1/7 1/7

25 ph1n1 Pathogenesis & Transmission Inoculated birds: Clinical Signs None observed Shedding & Seroconversion >50% of turkeys inoculated with 2x10 6 Histopathology & Immunohistochemistry Three turkeys in cloacal inoculation group Naive birds 25 Clinical signs not observed Shedding not detected One naive bird seroconverted

26 Conclusions First case of ph1n1 in turkeys in North America Drop in egg production Slight increase in mortality Highly related to human ph1n1 One worker on the farm was positive for ph1n1 Turkey Infectious Dose 50 between 1x10 5 and 2x 10 6 ph1n1 Pathogenesis & Transmission No clinical signs Shedding & Seroconversion >50% of turkeys inoculated with 2x10 6 Limited virus presence in lymphoid tissues Bird-to-bird transmission not demonstrated Low bird density? Low level of virus shed? Cleaning? 26

27 Acknowledgments OMAFRA CFIA Hybrid Turkeys Dr Susy Carman

Swine Influenza Virus Surveillance Canada John Pasick Guelph, Ontario, Canada

OFFLU swine influenza virus meeting 27 28 March 2017 FAO Headquarters, Rome, Italy Swine Influenza Virus Surveillance Canada John Pasick Guelph, Ontario, Canada Yohannes Berhane National Centre for Foreign