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Foot-and Mouth Disease Ecological Studies In Endemic Settings: Ongoing Studies in Vietnam and Pakistan Luis Rodriguez (Jonathan Arzt) Research leader, USDA-ARS Plum Island

PAKISTAN

CHARACTERIZATION OF LOCAL ISOLATES OF FMDV AND DEVELOPMENT OF VECTOR BASED VACCINES

58-1940-7-161F; 057 002S

9/1/2007 - 8/31/2012

REAL TIME DATA ANALYSIS AND RESEARCH CAPACITY BUILDING

TOWARDS FMD CONTROL IN PAKISTAN

1940-32000-052-14S 09/27/20 12 - 09/27/2014

Dr. Khalid Naeem – NARC Dr. Umer Farooq – NARC Dr. Muhammadimam Afzal, FAO Dr. Manzoor Hussain, NVL Dr. Zaheer Ahmed Dr. Anna Ludi

Study Design

– Acute Clinical Samples • Clinical case reports, geographic location, demographics, etc • Clinical samples, viruses, sequence

– Longitudinal Field Study • Population: buffalo premises – all located near Islamabad • Serological survey of 40 farms for NSP positive animals, probang, history • Selection of 29 farms (300 buffalo) for sampling (serum and probang) 4X

year for 1 year • NSP-ELISA, RT-PCR and virus isolation – Establish panel of reference sera for in country vaccine evaluation • Vaccination of 10 cattle and 10 buffalo with commercial vaccine to be used

in field • Serum collection at 0, 21 dpv, boost vaccination • Serum collection at 42 dpv • Carry out vaccine matching studies

Significant amino acid and nucleotide variation, wide spatial distribution of genetic lineages, no with differences in host species, all consistent with frequent multispecies infection of this serotype O FMDV in a highly mobile population

22/51 Animals VI positive 2 or more times, yielding 47

virus isolates

8 /22 Animals

2 viruses of same serotype isolated at different sampling time points from same animal

[8x2=16 viruses]

ASIA 1 = 14

O = 2

TOTAL = 16

3/22 Animals

3 viruses of same serotype isolated from each Id at different sampling time points

[3x3=9 viruses isolated]

ASIA 1 = 9

TOTAL = 9

9/22 Animals

2 viruses of 2 different serotypes isolated from each animal at different sampling time points

[9x2=18 viruses isolated]

ASIA 1/A 8X2=16

ASIA 1/O 1X2=2

TOTAL =18

1/22 Animal

(3 viruses of 2 different serotypes isolated from each Id at different sampling time points)

[1x3=3 viruses isolated]

ASIA 1/A =3

Obtained multiple viral strains from persistent buffalo – opportunity to look mechanism of strain emergence

Green: Carrier viruses Red: Clinical local Blue: Clinical non-local

Phylogeny of Carrier vs Clinical Serotype A Viruses

•Found close relationship between persistent and acute lineages suggesting transmission •Directionality of transmission not yet determined

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Pak-O 2010, 2012, 2012

SGD/PAK/19/2011 JX170755

KHI/PAK/42/2011 JX170757

ISR/7/2007 AJ294910

OKAB/AFG/L2826/2009 HQ439234

MAY/2/2004 HQ116194

MAY/2/2006 HQ116205

PAK/39/2008 GU384685

PAK/63/2007 FJ798183

BAG/AFG/L1494/2009 HQ439234

MAY/11/2009 HQ116217

PAK/1/2008 FJ798190

PAK/29/2008 GU384684

CAM/1/2008 HQ116174

VIT/124/2010

VIT/169/2010

VIT/17/2005 HQ116283

VIT/7/2002 HQ116273

UKG/3802/2001 DQ164982***

SKR/1/2002 DQ164972

VIT/3/2005 HQ116277

TUR/2/2001 DQ164982

Ankara/TUR/377/10/02 DQ296523

TUR/4/2005 FJ561321

TUR/2/2000 DQ164982

PAK/18/2002 DQ164982

IRN/20/2004 DQ164982

Mersin/TUR/13/01/04 DQ164982

IND/53/73_AF292107

O1 Manisa iso87 AY593823 – Vaccine Virus representative

India/R2/75 AF204276

CAM/3/98 AJ294910

99

97

99

76

99

90

83

82

80

80

97

82

99

0.02

Phylogeny Reconstruction Statistical Method -------------- Maximum Likelihood Test of Phylogeny --------------- Bootstrap method (500 replicates) Substitutions Type -------------- Nucleotide Model/Method ------------------ General Time Reversible model Rates among Sites --------------- Gamma distributed with Invariant sites (G+I) No of Discrete Gamma Categories - 5 ML Heuristic Method ------------- Nearest-Neighbor-Interchange (NNI) Initial Tree for ML ------------------Make initial tree automatically No. of Seqs : 33 No. of Sites : 623 *** - reference strain

SEA CAM-94

ME-

SA

Pan

Asi

a

Pan

Asi

a 2

Vaccine Evaluation Serotype O FMDV

Serotype O Vaccine Matching against O1 Manisa

O/I

SB/2

55

/12

O/F

SD/2

66

/12

O/J

GH

/6/1

2

O/K

HI/

41

/11

BT neut. r1-value BT neut. r1-value BT neut. r1-value BT neut. r1-value

2.33 1.8 0.71 2.18 1.65 0.5 2.48 1.95 1 2.25 1.95 1

1.8 1.5 0.5 2.4 1.95 0.5 1.5 1.5 0.5 2.03 1.35 0.35

1.5 1.8 1

Conclusion: • r1-values are above 0.3 suggesting the vaccine is protective against the serotype O viruses tested.

* All values given in log10

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PAK/CHK/11/2012*

PAK/KCH/15/2012*

PUN/PAK/L1354/2009_HQ439251

PAK/76/2009_GU384686

SIN/PAK/L694/2009_HQ439247

SAR/AFG/L1435/2009_HQ439274

IRN/5/2008_FJ755068

TUR/1/2008_FJ755133

IRN/1/2005_EF208769*** vaccine virus representative

PAK/1/2006_FJ755082

PAK/ICT/2/2008*

PAK/ICT/1/2008

PAK/ICT/3/2008

PAK/KCH/5/2009*

PAK/KCH/6/2009

PAK/KCH/7/2009

AFG/131/2004_EF457981

IRN/41/2003_FJ655020

PUN/PAK/L1364/2009_HQ439253

IRN/22/99_EF208772***

IRN/2/87_EF208770***

A22 Iraq64 iso86_AJ251474*** vaccine virus representative

IRN/1/96_EF208771***

IRN/5/2003_FJ775018 100

100

87

100

99

0.05

Vaccine Evaluation Serotype A FMDV

Phylogeny Reconstruction Statistical Method -------------- Maximum Likelihood Test of Phylogeny --------------- Bootstrap method (500 rep) Substitutions Type -------------- Nucleotide Model/Method ------------------ General Time Reversible model Rates among Sites --------------- Gamma distributed with Invariant sites (G+I) No of Discrete Gamma Categories - 5 ML Heuristic Method ---------Nearest-Neighbor-Interchange (NNI) Initial Tree for ML ------------- Make initial tree automatically No. of Seqs : 24 No. of Sites : 623 *** Ref Strains * used for vaccine matching

Serotype A Vaccine Matching against A-IRN05 and A22 IRQ

A/I

CT/

2/2

00

8

A/K

CH

/5/2

00

9

A/C

HK

/11

/20

12

A/K

CH

/15

/12

BT neut. r1

A22 R1 IRN BT neut.

r1 A22

r1 IRN BT neut BT neut

1.65 1.65 1.41F 1F 1.73 1.95 1.41 1 1.8 <1.2 1.95 <1.2

2.03 1.65 0.7 0.25 2.25 1.65 1.41 0.5 2.03 <1.2 2.48 <1.2

1.58 <1.2

Conclusion: • VN titers above 1.6 observed for 2008-2009 isolates •VN titers below 1.2 for 2012 isolates, suggesting that the vaccine is not a good match to these strains.

* All values given in log10

VIETNAM MOLECULAR EPIDEMIOLOGY, SURVEILLANCE AND PREDICTIVE TOOLS

FOR FMD CONTROL IN VIETNAM

58-1940-0-070F, 057 14S

6/1/2010-9/30/2012

Collaborators: Dr. Jonathan Arzt Dr. Helena Ferreira Dr. Thanh Long Ngo, DAH, HCMC Dr. Ho Huu Dung, DAH, Hanoi Dr. Carla Huston, Mississippi State University

Objectives

• 1- Molecular epidemiology of FMDV in local livestock including cattle, buffaloes and pigs

• 2- To better understand the transmission mechanism of persistently infected to susceptible livestock in natural setting

• 3- Enhance strategies for identification of persistently infected animals using new technology

Design

Objective 1 – Acute Clinical Samples – Samples collected during outbreak – Pigs, buffalo and cattle – Northern and Southern Vietnam

Objective 2 – Longitudinal Field Study • HCMC region – tranmsission cells (9 farms) • Hanoi-SonLa region – carrier cattle and buffalo (field necropsy)

Objective 3 – Persistent buffalo studies • Field necropsies • Molecular characterization (tissue level, cytokine mRNA, protein expression)

Objective 1. Phylogenetics of novel strains of FMDV

•Multiple lineages in multiple species no species specific lineages •Novel strains (bold) found in this study

Objective 2. Ecology of FMDV carriers

• Two Provinces identified with history of FMD outbreaks in cattle, buffalo – SonLa (north)

• Jan 2011

– LongAn (south)

• Feb. 2011

• Established study sites: – Targeted surveillance study

– Buffalo necropsy study / cattle transmission study

Targeted surveillance study

• Risk of being FMDV infected and/or FMDV carrier: • Buffalo - highest risk of being FMDV-infected, intermediate carrier risk

• Dairy cattle –lower risk of being FMDV-infected (OR=0.2), lowest carrier risk (OR=0.2)

• Beef cattle - lowest risk of being FMDV-infected (OR=0.4), highest carrier risk (OR=4.9)

Carrier infected : 3ABC+ and Probang +; Ever inf.: 3ABC+; Never inf. : 3ABC neg

• Study site in southern Vietnam – no clinical activity >1yr

• Individual small premises - pastures, surrounding premises vaccinated bi-annually

• 9 farms – 2 donor carrier buffalo or cattle (NSP +, Probang +) housed in direct contact with 2 naïve cattle (NSP -, Probang -)

• Direct contact - one year study – serum and probang every 2-3 months

• Probang samples from naïve cattle all tested NEGATIVE for FMDV by rRT-PCR, VI

• Serum from naïve animals all tested for negative NSP -

Objective 3. Transmission Study From Carrier Cattle to Sentinel Cattle

Preliminary Results

• All donors shed virus intermittently throughout the exposure time

• None of the naïve cattle developed antibodies (NSP)

• Naïve cattle remained probang negative until the end of the study

• Multiple viral sequences were obtained from persistent animals

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SUPPORT:

Thank you!