David L. Suarez Research Leader EEAV

Size: px

Start display at page:

Download "David L. Suarez Research Leader EEAV"

Allen Park
5 years ago
Views:

1 David L. Suarez Research Leader EEAV Southeast Poultry Research Laboratory United States National Poultry Research Center U.S. National Poultry Research Center

2 Past, ongoing and future research in the areas of poultry health, food safety, product quality and feeds and grains November 2, pm Russell Research Center RSVP required-please see me for contact information

4 35% architectural plans completed (Merrick) Mortenson was selected as the Design Build Contractor Groundbreaking Ceremony on Nov 3 rd Actual construction in January Driveway consolidation between SEPRL and RRC has been funded separately and firm has been selected The new driveway will keep construction traffic and normal traffic separate

6 Laszlo Zsak, Research Leader for Endemic group has retired Taejoong Kim has joined the Endemic group working on Marek s disease Patti Miller has left from the Exotic group Two scientist vacancies at SEPRL The Research Leader Position for the Endemic group is advertised now!

7 Thermal inactivation of AIV in litter Goal: To reduce/eliminate virus in a poultry house during the initial stages of disinfection after an outbreak Reduces costs and potential for virus spread during disinfection process Days to inactivate LPAIV and HPAIV by temperature (F) and moisture level. Inactivation was considered complete when all 3 replicates were negative. Virus Moisture 120 F 110 F 100 F 90 F 80 F 70 F 60 F 50 F LPAIV Wet Dry HPAIV Wet N/A 1 N/A 1 N/A 2 N/A 3 Recommended treatment time (days) Dry N/A 1 N/A 1 N/A 2 N/A 5 Funded by USPEA grant #BRU Spackman research 2017

8 Effect of IBDV on AIV vaccination Chickens exposed to VarE IDBV at hatch Vaccinated for AIV at 2 weeks of age with and inactivated vaccine Challenged at 5 weeks of age IBDV Exposure Vaccination Status 50% bird infectious dose 50% bird lethal dose Not exposed Not vaccinated Exposed Not vaccinated Not exposed Vaccinated 2.4 >6.0 Exposed Vaccinated IBDV only affected the 50% bird lethal dose Infectious dose and shed were not affected Eliminated the protective effect of vaccine to clinical disease The vaccine which elicits serum antibody is only preventing systemic spread, not infection or local replication Spackman research 2017

A/Chicken/TN/17-007431-3/2017 (H7N9) HPAIV 4-week-old SPF White Leghorn

9 Infectious dose and transmission of Tennessee 2017 H7N9 LP and HP viruses in chickens Isolates: A/Chicken/TN/ /2017 (H7N9) LPAIV A/Chicken/TN/ /2017 (H7N9) HPAIV 4-week-old SPF White Leghorn chickens Infectious dose/transmission Pathogenesis M. Pantin-Jackwood, preliminary data

10 Chickens - Infectious dose and transmission H7N9 viruses H7N9 LPAIV H7N9 HPAIV Log 10 Dose Infected/ total Dead/total (MDT) BID 50 Contacts infected/ total 2 0/5 0/5 3.6 log 10 0/3 4 2/5 0/5 0/3 6 5/5 0/5 0/3 2 3/5 3/5 (2.3) <3 log 10 0/3 4 5/5 5/5 (2.4) 0/3 6 5/5 5/5 (2.2) 1/3 (dead) Birds considered infected if they seroconverted and/or shed virus. MDT = mean death time (days) BID 50 = mean bird infectious dose Pathogenesis groups: LPAIV: 9/10 birds infected HPAIV: 10/10 birds infected and died (MDT= 2.1days) M. Pantin-Jackwood, preliminary data

11 Pathogenicity of Indiana 2016 H7N8 LPAI and HPAI viruses in chickens, turkeys and mallards Turkeys the most susceptible to both viruses In all three species it took less virus to cause infection with the HPAIV The HPAI virus did not cause disease or death in the mallards like in the chickens and turkeys, however all mallards became infected and transmitted the virus to contacts, indicating that wild waterfowl can also spread non-h5nx HPAI viruses.

12 Pathogenesis of IN 2016 H7N8 LP and HPAIV Species Species LPAIV BID 50 (log 10 ) HPAIV BID 50 (log 10 ) Chickens Turkeys 3.0 <2.0 Mallards LPAIV % Mortality % Transmission to contacts HPAIV % Mortality % Transmission to contacts Chickens Turkeys Mallards BID 50 = 50% bird infectious dose in log 10 50% egg infectious doses

13 Conclusions Both the LP and HP H7N8 viruses had the lowest infectious doses in turkeys, and disease from the LPAIV infection was only observed in this species Clinical signs caused by the HPAIV differed between chickens and turkeys; neurological signs were only observed in turkeys Mallards were infected with both viruses and transmitted them to contacts, but neither virus caused clinical disease This study corroborates the high susceptibility of turkeys to AIV, and demonstrates that mallards can be asymptomatically infected and potentially transmit AIV to poultry Pantin-Jackwood et al. PLOS One 2017.

14 Experimental Design: Study 2, Adult WL Chickens Study 2 61w 62w 63w 64w 65w 66w 67w rgh5n1 or RP-H5 Challenge clade H5N2 HPAIV EID 50 /0.1ml, A/Tk/MN/12582/2015 OP swabs for qrt-pcr Serum for HI Adult WL chickens Vax: 61w Challenge: 64w Terminate: 66w Parameters: survival, OP & CL shedding, HI serology

Experimental Design: Study 3, Adult WL Chickens Study 3 61w 62w 63w 64w.. 84w 85w 86w rgh5n1 rgh5n1 Challenge clade 2.3.4.4 H5N2 HPAIV 10 6.9 EID 50 /0.

15 Experimental Design: Study 3, Adult WL Chickens Study 3 61w 62w 63w 64w.. 84w 85w 86w rgh5n1 rgh5n1 Challenge clade H5N2 HPAIV EID 50 /0.1ml, A/Tk/MN/12582/2015 OP swabs for qrt-pcr Serum for HI Adult WL chickens Vax: 61w & 64w Challenge: 84w Terminate: 86w Parameters: survival, OP & CL shedding, HI serology (biweekly)

16 lo g 2 G M T Length of Immunity: Studies 2 and 3 rgh5n1 group: 20/20 sero+ at challenge (5.9log2) and 20/20 at termination (6.4log2) RP-H5 group: 15/20 sero+ at challenge (5.6log2) and 19/20 at termination (6.4log2) b o o s t H A U c h a lle n g e te rm in a tio n 2x rgh5n1 group: 20/20 sero+ at boost (7log2), 20w later (9.4log2) and at termination (10.6 log2) 5 p rim e H A U w 6 4 w 6 6 w 6 8 w 7 0 w 7 2 w 7 4 w 7 6 w 7 8 w 8 0 w 8 2 w 8 4 w 8 6 w a g e

17 Results: Studies 2 & 3 Survival, Adult WL Chickens P e r c e n t s u r v iv a l rg H 5 N 1 rg H 5 N 1 x R P d p c s h a m s h a m x2 0% survival in sham controls by 3 & 4 dpi 100% survival in rgh5n1 vaccinated (1x & 2x) 95% survival in RP-H5 vaccinated

Study Design 5-10, 9-wk-old WL chickens and 8-wk-old Pekin

(tranquilized) Hard-scald Defeathering Evisceration Clean-up

Exposure to chickens, ducks and/or ferrets in same airspace to

A/Vietnam/1203/2004 Human 1 A/chicken/Vietnam/NCVD-878/2011

2 A/chicken/Egypt/102d/2010 Poultry 2.2.1 A/Egypt/N6658/2011 Human 2.

18 Study Design 5-10, 9-wk-old WL chickens and 8-wk-old Pekin ducks IN, 8 H5N1 Gs/GD HPAI viruses Processed 24 hpi (ck)/2.5 dpi (dk). 5 steps in halal slaughter process (6-7 min): Kill (tranquilized) Hard-scald Defeathering Evisceration Clean-up Air sampling: NIOSH cyclone air sampler or -ionizing sampler Exposure to chickens, ducks and/or ferrets in same airspace to assess transmission [ Isolate Host/Source Genetic clade A/Vietnam/1203/2004 Human 1 A/chicken/Vietnam/NCVD-878/2011 Poultry 1.1 A/chicken/West Java-Subang/29/2007 Poultry A/whooper swan/mongolia/244/2005 Waterfowl 2.2 A/chicken/Egypt/102d/2010 Poultry A/Egypt/N6658/2011 Human A/chicken/Vietnam/NCVD-675/2011 Poultry A/chicken/Vietnam/093/2009 Poultry 7.2

19 3 Table Processing site 2. Scald site 3. Kill can 4. BC 251 Cyclone air sampler (>4µm, 1-4 µm and <1 µm) or - ionizing sampler: HEPA ventilated flexible film isolator in BSL-3E Certified Building

20 Results: Virus Detection in Air Samples All chickens and ducks were infected based on virus detection or isolation from OP swabs Chickens: Air samples were positive with 4 viruses (VI); 3 had higher titers in large droplet than aerosol fraction Ducks: air samples were positive with 3 viruses (VI): less consistent droplet size pattern a lo g 1 0 /m 3 lo g 1 0 /m V N /0 4 -H 5 V N /8 7 8 /1 1 -H 5 W J /0 7 -H 5 C h ic k e n s E g /1 0 -H 5 E g /1 1 -H 5 V N /6 7 5 /1 1 -H 5 V N /0 9 -H 5 Clades: b D u c k s > 4 µ m 1-4 µ m < 1 µ m > 4 µ m 1-4 µ m < 1 µ m V N /0 4 -H 5 V N /8 7 8 /1 1 -H 5 W J /0 7 -H 5 E g /1 1 -H 5 Clades:

21 Results: Transmission All IN inoculated chickens were infected with clade 1 or 2.2 HPAI viruses All naïve chickens exposed to same airspace as processing of clade 1 and 2.2 infected chickens became infected and died 3 of 4 naïve ferrets exposed to same airspace as processing of clade 1 infected chickens became infected and died Virus Mong/05- H5 (2.2) VN/04-H5 (1) VN/04-H5 (1) Intranasal infected birds processed Chickens (n=10) Chickens (n=10) Duration of the slaughter process 1 h 1 h Naïve exposed hosts* Chickens (n=5) Chickens (n=5) Mortality of exposed hosts (MDT) 5/5 (4.4 dpe) 5/5 (4.0 dpe) Chickens (n=10) 1 h Ferrets (n=4) 3/4 (8.3 dpe) Virus detection in exposed hosts (log 10 EID 50 /ml) 5/5 at time of death 5/5 at time of death Seroconversio n in surviving exposed hosts na na 1/4 on 3 dpe (3.0) 0/1

22 lo g 1 0 /m l Results: Transmission IN inoculated ducks were infected based on +rrt-pcr OP swabs All airborne particle exposed ducks had minimal quantity of virus detected in swabs, but only 1 of 5 seroconverted None of airborne particle exposed ferrets (n = 3) become infected /5 o ra l s w a b s c lo a c a l s w a b s 1 /5 3 /5 4 /5 3 /5 4 /5 5 /5 3 / d p e Virus Intranasal infected birds processed Duration of the slaughter process Naïve exposed hosts* Mortality of exposed hosts (MDT) Virus detection in exposed hosts (log 10 EID 50 /ml) Seroconversion in surviving exposed hosts VN/04-H5 Ducks (n=5) 30 min Ducks (n=5) 0/5 5/5 (1.6) (Fig. 3) 1/5 VN/04-H5 Ducks (n=5) 30 min Ferrets (n=3) 0/3 0/3 0/3

23 Basic NGS workflow From unknown sample to its identification I. TEMPLATE PREPARATION AND ENRICHMENT II. LIBRARY PREPARATION SISPA AMPLIFICATION dsdna RNA IV. DATA ANALYSIS Unknown sample III. SEQUENCING

24 WORKFLOW FOR NEXT GENERATION SEQUENCING OF SMALL RNA VIRUSES Applied de novo assembly

25 Summary: NGS-based Random sequencing Allantoic fluids NGS-based random sequencing of total RNA from AF quickly, effectively, and simultaneously characterized the genomic sequences of multiple viruses and bacteria. Sample processing, library preparation, sequencing, and analysis took 72 hours and the cost of all steps was estimated to be 106 USD per sample Twenty-eight Newcastle disease viruses (NDV, APMV-1), one APMV-13, four avian influenza and two infectious bronchitis virus complete or nearly complete genome sequences were obtained from the single run (a total of 30 samples). The median sequencing depth (> 2500x) across samples was sufficient for deep variant analysis. The complete de novo approach did not require any previous knowledge of the agent, use of primers, or specific method of agent identification

26 SEQUENCING OF NDV GENOME FROM PARAFFIN EMBEDDED TISSUES

27 Isolate Filtered reads NDV reads Mean fragm ent length # of cont igs % cover. IHC - 2 pathologists Raw reads ECDO/USA/MT/ /Kidney/ ECDO/USA/MT/ /Liver/ ECDO/USA/MT/ /Spleen/ ECDO/USA/NM/ /Kidney/ ECDO/USA/TX/ /Kidney/ ECDO/USA/TX/ /Kidney/ ECDO/USA/TX/ /Liver/ ECDO/USA/UT/ /Kidney/ ECDO/USA/UT/ /Spleen/ ECDO/USA/TX/ /Kidney/ ECDO/USA/TX/ /Spleen/ ECDO/USA/TX/118

COMPLETE FUSION GENE PHYLOGENETI C TREE CLEAVAGE SITE 113RKKR F117 95.

ECDO/USA(TX)/1182-spleen/26877-2-C/2016 ECDO/USA(UT)/1181-spleen/26594-1-A/2015 ECDO/USA(UT)/1181-kidney/26594-1-D/2015 100 100 ECDO/USA(UT)/1181-liver/26594-1-D/2015

28 COMPLETE FUSION GENE PHYLOGENETI C TREE CLEAVAGE SITE 113RKKR F % ECDO/USA(TX)/1185-kidney/ A/ ECDO/USA(TX)/1184-kidney/ C/2015 ECDO/USA(TX)/1182-kidney/ C/ ECDO/USA(TX)/1182-spleen/ C/2016 ECDO/USA(UT)/1181-spleen/ A/2015 ECDO/USA(UT)/1181-kidney/ D/ ECDO/USA(UT)/1181-liver/ D/2015 ECDO/USA(KS)/1191-spleen/W /2016 ECDO/USA(KS)/1191-kidney/W /2016 ECDO/USA(KS)/1195-kidney/W / ECDO/USA(KS)/1194-kidney/W /2016 ECDO/USA(KS)/1193-kidney/W /2016 ECDO/USA(TX)/1180-kidney/ C/2014 ECDO/USA(TX)/1180-liver/ C/2014 ECDO/USA(KS)/1192-kidney/W / ECDO/USA(TX)/1180-spleen/ C/2014 ECDO/USA(TX)/1179-kidney/ C/2014 VI a/turkey/usa(la)/ /2004 VI a/ecdo/usa(tx)/4156/2005 VI a/pigeon/usa(ct)/0708/2007 VI a/pigeon/usa(me)/0714/2007 VI a/pigeon/usa(ny)/0603/ ECDO/USA(MT)/1177-spleen/ B/2010 ECDO/USA(MT)/1177-liver/ B/2010 ECDO/USA(MT)/1177-kidney/ B/ VI a/pigeon/usa(oh)/0709/2007 VI a/pigeon/usa(mn)/723 /2009 VI a/pigeon/usa(pa)/0723/2007 VI a/chicken/usa(ma)/ /2004 VI a/pigeon/usa(nj)/0812/2008 VI a/pigeon/usa(mn)/511296/2007 VI a/pigeon/usa(md)/0719/2007 VI a/chicken/usa/pa/0503/2005 VI a/rodo/usa(md)/nd / RODO/USA(MA)/1188-spleen/ B/ RODO/USA(MA)/1188-kidney/ G/ RODO/USA(PA)/1189-kidney/ C/ VI a/rodo/usa(mi)/nd / VI a/rodo/usa(pa)/nd /2013 VI a/dove/italy/vir24/2008 VI a/pigeon/china/sms12/2012 VI a/pigeon/belgium/ / VI a/pigeon/china/guangdong/gz290/2013 VI a/chicken/usa(ca)/fontana/1083/ % 98.2 % TX, UT, KS TX, LA MT, PA, MA CT, ME, NY, OH, MN, NJ, MD, PA, MI AVMA Convention 2017, AAAP session, July 21-25, Indianapolis, IN, USA

29 Sequence Independent Single Primer Amplification - SISPA 5`-GACCATCTAGCGACCTCCACNNNNNNNN-3` I. RT-PCR II. dsdna synthesis 5`-GACCATCTAGCGACCTCCAC-3` III. PCR amplification

30 Results The whole genome sequencing of H5N1 and H5N2 AIVs SISPA NGS: 99.8% of AIVs genome coverage

31 Results The whole genome sequencing of NDV and IBV NDV, vaccine strain Genome coverage: 100% IBV Genome coverage: 100%

32 Results III. Limit of detection Determine the range of detection 10-fold dilutions of know virus standard applied to SISPA- NGS technique What is the viral load necessary in a sample to identify (isolate ID) versus to detect (+/-).

33 Results Limit of detection - D: - D: +/- Viral genome: detectable Diagnosis (D): + v

34 NDV LaSota vaccine strain-based infectious laryngotracheitis virus (ILTV) recombinant viruses expressing the glycoproteins B (gb) or D (gd) was developed that worked in SPF chickens. Commercial broiler chickens were vaccinated at 1 or 10 days of age (DOA) or boosted at 10 DOA and challenged at 21 DOA with virulent ILTV. Vaccination reduced ILTV shedding approximately 3 log 10 TCID 50 compared to the unvaccinated group Vaccination at 1 or 10 DOA did not make a significant difference in protection and boost vaccination did not improve protection The rls/iltv-gd appears to provide better protection than rls/iltv-gb in this experiment

35 Table 1. NDV maternal antibody and immunoresponses following vaccination NDV HI titer Treatment 1 day of 10 days of 21 days of Groups age age age PBS 6.42 ± ± ± 0.87 rls/iltv-gd D ± 0.99 rls/iltv-gd D ± 1.56 rls/iltv-gd D ± 1.50 rls/iltv-gb D1 3.62± 0.74 rls/iltv-gb D ± 1.34 rls/iltv-gb D ± 0.83 FP-LT 2.60± 1.07 CEO 3.11± 0.66 Table 2. ILTV maternal antibody and immunoresponses following vaccination 1 day of age 21 days of age Treatment # of POS Mean VN titer ± SD # of POS Mean VN titer ± SD groups birds Log 10 birds Log 10 No treatment 8/ ± 0.10 rls/iltv-gd D1 12/ ± 0.15 CEO D10 13/ ± 0.15

36 The poultry gut virome and the enteric picornaviruses: The enteric picornaviruses are common in poultry in the U.S. and abroad Avian picornaviruses group geographically and are distinct from archived samples Comparative metagenomics suggests role for picornavirus in enteric disease/performance problems Infections with two or more picornaviruses even in the same flock or bird are common Further pathogenesis work with turkey and chicken picornaviruses (concomitant infections with common enteric viruses) Full characterization of the novel turkey phacoviruses

37 Novel phacoviruses VP1

38 Exotic and Emerging Avian David Suarez David Swayne Erica Spackman Mary Pantin- Jackwood Darrell Kapczynski Claudio Afonso Endemic Avian Disease Group Stephen Spatz Qing Yu Taejoon Kim Michael Day

SEPRL NC1180 Station Report

SEPRL NC1180 Station Report Project: Control of emerging and re-emerging poultry respiratory diseases in the united states Avian influenza group: David E. Swayne, David L. Suarez, Darrell R. Kapczynski,