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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
8
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
10
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