Changing Pathways: Lessons from Recent Pathogen Migrations for FMD Risk Assessment

Transcription

1 Changing Pathways: Lessons from Recent Pathogen Migrations for FMD Risk Assessment Executive Director, Prof. Soren Alexandersen National Centres for Animal Disease (NCAD) Winnipeg and Lethbridge Laboratories Canadian Food Inspection Agency 30 October Her Majesty the Queen in Right of Canada (Canadian Food Inspection Agency), all rights reserved. Use without permission is prohibited.

2 L W NCAD = L+W National Centres for Animal Disease NCAD: Lethbridge Laboratory + National Centre for Foreign Animal Disease (NCFAD)-Winnipeg 2

3 NCFAD established in 1998 in Winnipeg in the Canadian Science Centre for Human and Animal Health as a partner with the Public Health Agency of Canada (PHAC) One Facility: Two Labs together: One Health UNIQUE FACILITY with both human and animal CL 4 labs Only CL 4 labs in Canada A unique approach that encourages collaboration and provides better science and policy 3

4 National Centres for Animal Disease (NCAD) NCFAD-Winnipeg and Lethbridge Laboratory of the Canadian Food Inspection Agency (CFIA) Containment Level 2-25% Containment Level 4-5% Can handle all FAD, zoonotic and emerging infections CL 3+/ag - 70 % Staff of ~ 65 + facility Expertise: virology, mol biol, pathology, immunology/serology and reagents LETHBRIDGE LABORATORY ~35 staff + facility. Expertise: BSE, TSE, Anthrax, endemic viruses, CL3 large animals etc 4

5 INDIRECT CONTACT Traditionally: looked mainly at potential international/transboundary transmission as happening by specific (biological) products and a few things like vectors or boots or clothing 2010 Now: Movement from A to B to. Z of ANYTHING by ANY route Amount and complexity are increasing and contact to susceptible species by all possible routes very complex Transport and travel are increasing Farming changing/intensifying Improbable are changing to possible! 5

6 25 February 2001 FMD UK 2007 escape from site faulty effluent lines? 6 6

7 Canadian Examples FMD Saskatchewan 1952 Foreign worker/on purpose?? Rabbit Hemorrhagic Disease A single pet rabbit in apartment Winnipeg 2011 How??? ph1n into swine in Alberta Likely from a returning foreign worker H5N2 AIV into poultry in Manitoba 2010 close match to circulating wild bird sequences Also anaplasmosis in Manitoba in 2010 and in Ontario and Manitoba in 2013 BTV 11 BC 2013 Likely culicoides (wind) EHDV 2 Alberta Deer crossing 7

8 Danish Example International Examples Schmallenberg virus Culicoides? MERS-CoV Dromedary camels??? BSE MBM 8

9 PORCINE EPIDEMIC DIARRHEA VIRUS (PEDV) IN THE USA AND THEN IN CANADA An Alphacoronavirus, first seen in the UK in 1971, then other parts of Europe and later Asia. Affects only swine; not zoonotic - maybe originated from bats! From late April 2013, Iowa State University (ISU) got samples from farms with diarrhea/vomiting in all ages and 90 95% mortality in suckling piglets. Severe atrophy of villi in the small intestines, rota- and TGE virus negative; Coronavirus-like particles by EM, pan-coronavirus RT-PCR positive. Sequencing: % identity among RT-PCR products from first 4 farms, highest identity (>99%) to PEDV strains from China in NVSL: confirmed using 3 nested PCRs; close to 2012 China strains. Whole genome sequences of virus from 2 farms in 2 states had 99.5% identity with PEDV China strains. The nearly simultaneous outbreaks and high degree of homology ( %) between strains from unrelated farms, suggested a common source of virus. Source into USA still not identified but source into Canada is! 9

10 From: Status Update on Swine Coronaviruses Recently Identified in US Swine (Iowa State University) Huang, Dickerman, Piñeyro et al. mbio 4(5):doi: /mBio Origin, Evolution, and Genotyping of Porcine Epidemic Diarrhea Virus Strains in the United States Initial European strain from 70ties from late 2013/ early 2014 Plus swine deltacoronavirus from early 2014 Reported to the OIE as emerging infection by USA, Japan and Canada on 21, 24 and 26 of April 2014, respectively 10

11 NCFAD preparedness: Heads-up from NVSL-Ames on 17 May 2013 Work closely with NVSL and our CAHSN labs Set up conventional RT-PCRs (NVSL but modified primers) and partial sequencing AHL-Guelph develop real time RT-PCR which we all test for suitability Get positive RNA from ISU and an European virus isolate (BR1/87) and positive serum from helpful Danish colleagues grow virus in Vero cells and ship positive RNA to several labs Set up serology by IFA and an ELISA using mabs from The Netherlands. All done very quickly but without dedicated resources or reportable mandate. Canada (Ontario) case 1 Late on 22 Jan 2014 heads up from AHL-Guelph receive samples at NCFAD on 23 Jan late afternoon Report preliminary positive rrt-pcr before midnight (within 8 hours) Report positive conventional RT-PCR results (3 targets) on 24 Jan at noon (within 20 hours) Report confirmatory sequences on 25 Jan early morning - consistent with initial USA strain (within 40 hours) Subsequent full S gene sequenced (4.2 kb) and virus isolated! 11

12 Further testing/confirmation done at NCFAD Ontario cases 2-11 (partial S sequence 14 Feb) Manitoba case 1 (13, 14 and 15 Feb) PEI case 1 (14, 14 and 18 Feb) Quebec case 1 (26, 26 and 27 Feb) Follow up epidemiological investigation on the first 11 Ontario cases and the PEI case pointed to feed and one lot of spray dried porcine plasma from the USA as a common risk factor Ontario feed & dried plasma RT-PCR POSITIVE BIOASSAY Other feed and feed ingredients mainly negative, a very few considered non-negative/dubious 12

13 Direct testing on Ontario plasma and feed (PEDV real time and small S gene RT-PCR) 5 plasma samples all weak positives (Ct ) Also positive in S gene RT-PCR and sequence as initial USA strain 3 plasma samples for bioassay. Only 1/5 feed samples had a dubious reaction in the rrt-pcr (Ct 42.88). This sample had a dubious reaction (wrong size) in conventional S RT-PCR while a third sample was clearly positive. The clearly positive sample and the real time RT-PCR reactive sample selected for bioassay The one feed sample S amplicon sequence is PEDV but not sufficient for good/quality sequence 13

14 BIOASSAY 1 and BIOASSAY 2 BIOASSAY 1 (started 14 Feb 2014) 40 piglets, 10 positive control, 10 negative control, 3 x 4 piglets for plasma and 2 x 4 piglets for feed 3-4 piglets as contacts from day 7 Given a 10% suspension 25 ml by gastric tube and 25 ml orally RESULTS TO BE DISCUSSED BIOASSAY 2 (started 21 Feb) 60 piglets, 12 negative controls 3 x 8 piglets given feed as above and 1 x 8 piglets also fed this feed for two more days 4 contact piglets introduced from day 2 BIOASSAY 2 ALL NEGATIVE FOR 7 DAYS 14

15 BIOASSAY 1 Clinical observations No clinical signs observed in negative controls. Positive control group: depression, off feed and diarrhea/soft faeces from 1 day after inoculation; temperatures within normal range. Plasma group: a few piglets with diarrhea/soft faeces from day 2; temperatures within normal range. Feed group: mildly depressed and one or two piglets with diarrhea/soft faeces from day 1 and decreased feed intake day 3-7; temperatures within normal range. Such mild signs may be caused by many things can only be related to a specific cause by testing and should not be used for any conclusions! 15

16 BIOASSAY 1 rrt-pcr on rectal swabs Piglet number, Ct (Y axis) and day (X axis). Contact piglets from day 7 as black. Negative as Ct 45. All piglets at beginning of trial, all controls and all contact piglets at day 7 tested negative. All feed inoculated piglets NEGATIVE (BUT!) - also negative in Bioassay 2. 16

17 S gene RT-PCR and amplicon sequence on BIOASSAY 1 rectal swabs at day 3 All 12 plasma inoculated piglets strongly positive - sequence similar to the initial USA strain One feed inoculated piglet (35) had very weak band of right size - also PEDV sequence but poor quality. Cloned and sequenced: 1/ 8 clones PEDV sequence like plasma piglets. A few others had weak bands of a different size - unspecific amplification unrelated to PEDV 35 17

18 Colorado Iowa 1 PEI Plasma direct Feed 9 piglet Ontario case Plasma direct Quebec case Plasma piglets Ontario case Manitoba case Ontario Case 1 Plasma piglets Ontario cases Plasma piglet Minnesota Indiana SK environmental Second USA strain 18

19 Positive control (5 days post contact) Plasma feed group (7 days post inoculation) Negative control Carissa Embury-Hyatt 19

20 Additional evidence Electron microscopy Plasma piglet d7 Pos cont contact d5 Lynn Burton Serology clear seroconversion in plasma inoculated and positive control piglets! Positive Control Group Spray Dried Plasma Group Done using a monoclonal antibodies-based ELISA to detect antibodies to the PEDV spike (S) protein and supported by immunofluorescence assay (IFA) 20

21 Conclusions! This imported lot of spray-dried porcine plasma contains infectious PEDV: Virus excretion for many days by rrt-pcr and S gene sequence typical for the USA/Canada PEDV strain Spread to contact piglets on day 7 & seroconversion Tested feed is non-conclusive/not possible to determine as infectious by laboratory trial (Bioassay): Weak positive by RT-PCR, but variable results and PEDV sequence obtained not of good quality A single piglet weak positive band day 3 sequence weak but typical PEDV of the same sequence Repeat (Bioassay 2) using more piglets and contacts: all negative but old feed sample However, strong indication that PEDV was introduced into Canada by contaminated porcine plasma in feed! 21

22 +MB 19 & 24 Sept week 35/36 *30 *9 *13 *21 & 30 April May June July 2 nd strain *Jan 2014 CSHIN 12, 24 June, 21, 30 July

23 Swine/Porcine deltacoronavirus (SDCV/PDCV) detected in the USA SDCV/PDCV initially described by Hong Kong scientists in 2012 based on sequence findings (not disease) related to Asian Leopard Cat and Sparrow deltacoronavirus Detected in the USA by Ohio State University in early 2014 and by AHL-Guelph in March 2014 in samples from 6 premises in Ontario. We have confirmed SDCV in 2 of their first submissions using rrt-pcr and a pan-cov RT-PCR followed by sequencing. Speculation that this virus may also have been in feed: We tested feed and plasma samples received including those positive for PEDV: All clearly negative for SDCV in our test. All 40 piglets from Bioassay 1 also negative for SDCV. 23

24 SUMMARY Constant risk of introduction of new or emerging infections Importance and impacts may be both health and economic Economic impacts can be from production losses, treatment and control costs, loss of international market access or loss of domestic consumer confidence Effects are highly inter-connected and inter-dependant and global events can quickly come close to home Consider: Movement from A to..z of ANYTHING by ANY route (spray dried milk powder next???) It is important for everyone to have optimal: Awareness, bio-security and a good veterinarianclient relationship and excellent laboratory services. 24

25 Acknowledgements I thank all staff at NCAD for their contributions and dedication and other staff at CFIA and at NML for productive collaboration. Also Thank: NVSL&ISU-Ames, Lindholm- Denmark, Lelystad-Netherlands, CAHSN labs (in particular AHL-Guelph), Biovet and many others Thanks for listening! 25

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