a/h1n1 pdm in a turkey breeder farm in northeastern italy · a/h1n1 pdm in turkey breeder •first...

A/H1N1 pdm in a Turkey Breeder Farm in Northeastern Italy

Federica Gobbo

IZSVe SCS6 Specialized Virology Laboratory EURL

25 th Annual meeting of the NRLs for Avian Influenza and Newcastle Disease of European Union Member States

19-21 June Padova, Italy

A/H1N1 pdm in turkey breeder

• First detection in humans in Mexico 2009

• Described in the turkey breeders: Chile 2009, Canada 2009, USA 2010, UK 2010 , Norway 2016

Marked Egg Drop w/o mortality

• High genetic similarity with circulating strains of H1N1 pdm(2009) suggested humans as source of infection and likely during Artificial Insemination procedures

Federica Gobbo 20th June 2019 2

• Respiratory exposure failed to reproduce infection

• Intrauterine exposure in turkey hens (53 wks): eggdrop 5 days p.i.

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Experimental infections with pA/H1N1 in turkey breeder

Clinical case

• On monday 15th April 2019 a Turkey Breeder Company located in Northeastern Italy

called the Official Veterinary Authority complaining for a sudden and drastic drop in egg

production in barns 1-2-3 (hens of 48 weeks), the male breeders were located in barn 4

nome cognome - data della presentazione 4

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Barn 1Barn 2Barn 3

Sampling for AI suspect:

60 tracheal swabs/barn 1-2-3-4 -rRT-PCR M-gene-rRT-PCR and r-PCR H5 -rRT-PCR and r-PCR H7-rRT-PCR H9

30 blood sera/barn 1-2-3-4 Serology ELISA (IDVet®) HI for H5, H7, pH1N1 (A/Verona/IT/2810/09)

Confirmed case: 15th April 2019


• ELISA and HI (pH1N1) Serology (30 blood

sera/barn): Barn 1: positive (1:1024-1:2048)Barn 2: positive (1:1024-1:2048)Barn 3: 50% sera positive (1:32-1:2048)Barn 4: 2 positive sera (1:16-1:64)

Laboratory resultsdemonstrated a previous infectionin barn 1-2 and tracheal sheddingin barn 3 and barn 4

• 6 pool of 10 tracheal swabs collected in barn 1-2-3-4

Barn 1 (hens): negative

Barn 2 (hens): negative

Barn 3 (hens): positive (3 pools out of 6)

Barn 4 (males): positive (1 pool out of 6)

99-100% similarity A/H1N1 pdm09

rRT-PCR M-gene (Spackman et al., 2002) rRT-PCR M-gene* (Spackman et al., 2002-modifiedrRT-PCR H5 (VLA SOP VI.492)rRT-PCR H7 (VLA SOP VI.536)rRT-PCR H9 (Monne et al., 2008)rRT-PCR H1-N1( Hooffman et al., 2016)

Poon et al., 2009

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RESEARCH ACTIVITIES

• Different stage of infection between hens barns (1-2-3)

• Acute infection in stags (barn 4)

• Autolimiting infection

• Monitoring viral sheeding & Serology in barn 3-4

• Study of tissue tropism via necropsy of dead birds

• Rule out vertical transmission to the progeny and egg-borne dissemination

• Monitoring 1 day poults for viral detection and serology

Field and laboratorry activities

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16th April 2019Barn 1: 2 fresh carcassesBarn 3 (hens): 20 trachealand 20 cloacal swabsBarn 4 (males): 10 trachealand 10 cloacal swabs,1 spermatic pool (5 birds)

23rd April 2019Barn 4: 20 tracheal and 20 cloacal swabs20 blood sera4 spermatic pools (5 birds)Barn 3: 2 fresh carcasses20 blood sera • 30 pipped eggs with dead in shell

13rd May 2019• 47 one-day poults for serologyand virus detection • 34 poults dead on arrival (DAO)• 22 pipped eggs with dead inshell

13rd June 2019Barn 3: 10 tracheal and 10 cloacal swabs10 blood seraBarn 4:10 tracheal and 10 cloacal swabs10 blood sera

15th April 2019

pH1N1 detection

Results: viral shedding in infected hens (barn 3) and stags (barn 4)

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16 th April 2019Barn 1: 1 carcass out of 2 resulted positive in trachea, ovary, oviduct, kidney, pancreas and cecal tonsilBarn 3 (hens): 19 hens out of 20 resulted positive in rRT-PCR12 birds were simultaniously positive in tracheal and cloacalspecimensBarn 4 (males): 5 males resulted positive in rRT-PCR

23rd April 2019

Barn 3:2 fresh carcasses: positive in trachea, ovary, oviduct, kidney, pancreas and cecal tonsilBarn 4: positive 2 tracheal and 4 cloacalswabs

13rd June 2019

Barn 3: 10 tracheal and 10 cloacal swabs: negativeBarn 4:10 tracheal and 10 cloacal swabs: negative

15th April 2019

pH1N1 detection

Results: viral shedding and serology integrated with egg production

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Results: vertical transmission and egg-borne dissemination

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16 th April 20191 spermatic pool(5 birds/pool):-rRT-PCR (M-gene*): negative

23rd April 20194 spermatic pools (5 birds/pool): -rRT-PCR (M-gene*): negative30 pipped egg with dead in shell:-rRT-PCR (M-gene*) from external surface of

shell: negative-rRT-PCR (M-gene*) from embryo’s organs

pool: negative

13 rd May 201947 one-day poults:-Serology (ELISA and HI ): 11 out of 47 poults presented maternalantibody (1:64-1:128)

-rRT-PCR (M-gene*) via naso-tracheal swab and cloacal swab: negative34 poults dead on arrival (DOA): -rRT-PCR (M-gene*) via naso-tracheal swab and cloacal swab: negative22 pipped eggs with dead in shell:-rRT-PCR (M-gene*) from external surface of shell: negative-rRT-PCR (M-gene*) from embryo’s organs pool: negative

15th April 2019

pH1N1 detection

Results: virus tropism in hens carcasses

• rRT-PCR

• Gross patology

• Histopatology/IHC

• Ovary/Oviduct

• Trachea

• Cecal tonsil

• Pancreas

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Tracheal specimens


Oviductal specimens


Cecal tonsil


• The topology of the 8 phylogenetic trees show that the analyzed viruses cluster together with H1N1 pdm09 human strains detected in 2018-2019

Barn 1Barn 3Barn 4

NGS

HA PB2 PB1 NP M NS2

POSITION H1 numbering 130 162 216 223 450 453 497 298 456 643 101 213 89

POSITION H3 numbering (133) (165) (219) (226) (453)

HUMAN pdm09 K N T Q R T S I H A D V T

A/turkey/Italy/19VIR2658-46/2019_H1N1 Barn 3 K K T Q K T S V H T D V T

A/turkey/Italy/19VIR2658-48/2019_H1N1 Barn 3 K N R Q K A S V H A D V T

A/turkey/Italy/19VIR2658-68/2019_H1N1 Barn 3 K N R Q K A S V H A D V T

A/turkey/Italy/19VIR2795-12/2019_H1N1 Barn 3 K N R Q K A S V H A D V A

A/turkey/Italy/19VIR2658-75/2019_H1N1 Barn 4 R N R R (40%) K A N V N (70%) A G I T

• Q226R

-Mutation related to host specificity shift: switch in receptor binding preference from the α2,6-sialoglycan to the α2,3-sialoglycan receptor

(Chen et al., JVI 2010; He et al., J Gen Virol. 2014; Suptawiwat et al., Acta Virol. 2013)

• N165K

- Mutation HA N165K removes a potential N-glycosylation site NQT KQT

- The majority of avian H1 viruses does not present this N-glycosylation site

NGS


• Egg drop production in turkey breeder was associated to human A/H1N1pdm (Biosecurity issues )

• Tracheal swabs collected during the Egg Drop resulted negative in r-RT PCR (Barn1 and 2)

• Infected hens and stags presented tracheal and cloacal shedding up 8 days post case confimation (15th April) and cloacal sheeding seems to be predominat in late stage of infection

• Although viral dissemination in tissues and specific tropism for reproductivetract, vertical transmission and egg-borne dissemination of pH1N1 seems not tobe a potential threat

• In1-day poults maternal antibody were detected in 23% of birds

Discussion and conclusion

Discussion and conclusion

• The behaviour of viral dissemination in the affected barns, serology data and viral infection in stags (barn 4) suggest a possible horizzontal transmission ofvirus between barns

• NGS of clinical samples (tracheal swabs) put in evidence a potentialmechanism of viral adaptation in hens of barn 3 and a change in receptorbinding preference in stags of barn 4


Thanks to…….


Company and field practitioner

IZSVe SCS3 Histopatology:

• Zanardello Claudia

• Scarin Giulia

IZSVe SCS4 Epidemiology:

• Bonfanti Lebana

• Scolamacchia Francesca

• Azzolini Alessandra

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a/h1n1 pdm in a turkey breeder farm in northeastern italy · a/h1n1 pdm in turkey breeder •first...

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