Serum antibody response in the immunized goats indirect ELISA

1
Biotechnological Approaches in Animal Health
Indian Perspective
Dr R. K. Singh Head, Division of Virology Indian
Veterinary Research Institute Mukteswar-263 138
2
Preamble

• ? Largest Population
• Productivity ? Economy
• ? Infectious Animal diseases-
• ? Influence the production trade
• ? May jeopardize human health
• ? Improvement in productivity
• ? Synergistically dependent on AH
• ? Needs to be supported by better healthcare
• Management
• ? Improvement can be achieved by
• ? Improved diagnostics
• ? Immunoprophylactics (Vaccine)
• ? Therapeutics
• ? Nutraceuticals

3
OUR STRENGTHS

• Adequate expertise in AH Biotech.
• Exposure to technological advances
• Cross-discipline linkages Zoonoses VPH
• of AH related research products are Commercial
  attractive
• Strong Information Technology Base
• Enormity of biological data/resource
• Potential for herbal medicine

4
Challenges Before Us (i)

• Improving productivity of livestock
• Ensuring food safety nutritional security for
  human population
• Management of
• Emerging infectious production diseases
• Exotic diseases
• Zoonoses public health concerns
• Drug resistance of animal origin
• Organic Animal Farming
• Economically viable disease control strategies
• Development of simpler vaccine delivery system

5
Challenges Before Us (ii)

• Inadequate resources for RD
• Non-availability of diagnostic kits at affordable
  price
• Ethical Issues of animal welfare
• Biosecurity concerns
• Improving animal nutrition qualitatively
  quantitatively

6
Opportunities (i)

• Veterinary Public Health
• Disease Modeling Forecasting Systems
• Predictive Epidemiological assessment using
  molecular tools
• Development of mathematical models for disease
  spread
• Establish strategic integrated disease control
  programmes
• Develop Novel vaccines/Diagnostics/Adjuvants
• Better vaccine delivery system

7
Opportunities (ii)

• Genetic selection for disease resistance
• Improved diagnostics
• Molecular surveillance for infections in living
  animals
• Conservation of native breeds improved
  genotypes
• Zoo/Wildlife disease management
• Use of Bioinformatics in A. H. management

8
Biotechnology Issues in Perspective Current
Focus 1. Molecular Diagnosis - PCR -
Recombinant antigens - NA Probes 2.
Molecular Epidemiology - Molecular tools 3.
Molecular Pathogenesis - Molecular tools 4.
Hybridoma Technology - Monoclonal antibodies 5.
Peptide Technology - Diagnostic reagents
vaccines 6. Vaccine Development -
Conventional/Recombinant 7. Antimicrobial
therapeutics - Non-antibiotic/RNAi/Ind. Med.
plants 8. Transgenic Research - Therapeutic
proteins/ edible vaccines - Exptl Animal
models
9
Areas of Innovation
1. Diagnostics (i) Morphology based EM (ii)
Ag/Ab-based assays (iii) NA-based assays 2.
Vaccines (i) Conventional (ii) Recombinant
(Rec. protein/gene vaccine/Edible
vaccine/vector vaccines) (iii) Vaccine
delivery 3. Therapeutics Ind. Med.
Plants/RNAi 4. Molecular Epidemiology
Pathogenesis 5. Animal Cloning Transgenics
10
Diagnostics (Antigen/Antibody-based)
11
A. Serial Design
Samples
Rapid Test-1
Non-reactive
Reactive
Rapid Test-2
Report Negative
Non-reactive
Reactive
Non-reactive
Rapid Test-3
Report Positive
Reactive
Singer et al. (2005). J. Clin. Microbiol. 43
(10) 5312-5315
12
B. Parallel Design
Sample
Rapid Test-1 Rapid Test-2
Concordant Reactive
Concordant Non-Reactive
Discordant Results
Report Positive
Report Negative
Rapid Test-3
Non-reactive
Reactive
Singer et al. (2005). J. Clin. Microbiol. 43
(10) 5312-5315
13
Diagnostics
Detection of antigen
Detection of antibody
Detection of nucleic acid

• SNT
• MAb based C-ELISA
• Indirect ELISA
• Lateral flow assay

• RT-PCR
• PCR-ELISA
• N.A. Hybridization
• LAMP

• AGPT
• CIE
• Immunocapture ELISA
• MAb based S-ELISA
• Dot ELISA
• Lateral flow assay

EM, DNA Micro-array, FACS
14
Competitive ELISA Kits

• 1. c-ELISA kit for detection of RPV Abs
• PPR MAB- anti H protein (D272b)
• Validated by OIE Reference Lab at CIRAD, France
  Accepted by FAO Permitted by GoI for use in NPRE
  
• Validated - gt 2,00,000 samples
• 2. c-ELISA kit for detection of PPRV Abs
• PPR MAB- anti H protein (4B11)
• Compared with VNT
• Sensitivity - 92.2
• Specificity - 98.84
• Validated - gt 1,50,000 samples

15
Sandwich ELISA Kit

• 3. s-ELISA kit developed for detection of PPR Ag
  in clinical samples.
• PPR MAb anti N protein (4G6)
• Compared with IC ELISA (BDSL)
• Sensitivity- 89
• Specificity- 93
• Validated- 20,000 samples

16
Diagnostic Kits in Field use

• Kits extensively used in field
• 1. RP c-ELISA kits 125 (1500 samples in
  duplicate)
• 2. PPR c-ELISA kits 174 (500 samples in
  duplicate)
• 3. PPR s-ELISA kits 166 (100 samples in
  duplicate)

17
4. PAb - based i-ELISA for detection of PPRV
Antibodies

• Validated gt2000 serum samples from small
  ruminants
• DSp/DSn
• 1. Compared with VNT
• Relative specificity of Indirect ELISA 100
  (388 of 388)
• Relative sensitivity of Indirect ELISA 79
  (195 of 247)
• 2. Compared with C-ELISA
• Relative specificity 98.4 (952 of 967)
• Relative sensitivity 92.2 (676 of 733)

18
Recombinant antigen based kits

• Three new kits developed
• Rec. PPRV H protein Antigen based kit
• Rec. RPV H protein Antigen based kit
• Rec. BTV VP7 protein Antigen based kit

19
Pen-side Diagnostic tests

• Developed for detection of PPRV Abs/antigen in
  clinical samples
• Developed at TANUVAS/IVRI
• Validation
• (a) TANUVAS tests validated at IVRI
• (b) IVRI tests validated at TANUVAS
• Rapid Results - within 30 min
• Works efficiently
• User-friendly
• Need to be commercialized

20
Visual Immuno-dot comb test for Sero-diagnosis of
PPR
21
PR Serum
PPR - Serum
PPR - Serum
PPR Control
PPR - Serum
PPR Serum
PPR Serum
PPR Serum
Standardization of comb test
PPR Positive samples by C-ELISA
Screening of field serum samples in parallel
with C-ELISA kit
22
Salient Features of Comb Test

• Clearly differentiate PPR from RP
• Not limited to species barriers (can detect Abs
  in serum sample of any species)
• No equipment requirement
• No pipetting devices required
• Results obtained in 30 min
• Can be performed at RT
• Easy to use farmer friendly
• Scaling-up possible ? synthetic peptides used as
  Ag
• Used combs ? archived indefinitely for record

23
Representation of IC-Test for PPRV Antigen
detection
24
Salient Features of IC-test

• The IC-test is Rapid (results obtained within 2
  minutes)
• The test is free from Hook Effect Matrix effect
• This test clearly differentiates between PPR RP
  viral antigen (No cross reactivity)
• The test is easy to perform and is well suited
  for field use
• Test consumes very less amount of clinical sample
  to declare results

25
Diagnostics (Nucleic acid-based)
26
NA based diagnostics

• PCR RT PCR, Multiplex PCR and PCR-RFLP
• Most commonly used molecular tool
• Many variants
• Real time PCR
• More specific sensitive than PCR
• Overcomes problem of cross contamination
• PCR-ELISA
• Highly specific Sensitive
• NA Hybridization Automation
• Many Formats Dot/slot/ISH/NASBA-Microarray

27
M-Gene Based Single-Step RT-PCR
28
Multiplex RT-PCR for RP/PPRV
Simultaneous detection/differentiation RPV/PPRV
in clinical samples ?Emergency preparedness in
Post-RP Eradication era Sensitivity 100fg of
viral RNA 10 TCID50 Nasal/Ocular/Oral
discharges from 7th 17th DPI in goats vs 7-12
DPI (S-ELISA).
Lane M, DNA size marker, 100 bp ladder, Lane 1,
purified PPRV RNA, Lane 2, RNA from Vero cell
culture infected with RPV Lane 3 Vero cell RNA
(negative control)
29
RT-PCR-ELISA for Detection of PPR virus PCR
based on M/ N genes of PPRV developed More
sensitive than F gene based PCR Sensitivity
0.01TCID50 Critical samples 6th 17th DPI in
nasal/ocular swabs Vs 7-12 DPI (S-ELISA)
Evaluation 72 clinical samples compared with
S-ELISA Useful for testing critical samples
30
A gene PCR for detection of GPXV/SPPV
M 1 2 3 4 5 6 7 8
9 10 11 12 M
500 bp
192 bp
Lane M 100 bp ladder Lane 1 GPPV ve control
Lane 2 SPPV ve control Lanes 3-6 GPPV
field samples Lane 7-10 SPPV field
samples Lane 11 BPXVLane 12 Camelpox
31
PCR-RFLP of A gene of GTPV/SPPV
M 1 2 3 4 5
6 7
500 bp
192 bp
133 bp
59 bp
M-PCR marker Lane-1 ve sheeppox sample
Lane-2 goatpox sample Lane-3-5 sheeppox field
samples Lane-6-7 goatpox field samples
32
PCR-RFLP of P32 gene of SPPV/GPPV
33
Duplex PCR A single-tube reaction
M 1 2 3 4 5 6 7
8 9 10 11 12 M
472 bp
500 bp
192 bp
(A) PCR amplifies two fragments of 472 bp and 192
bp in GPV samples (B) PCR amplifies only one
fragment of 192 bp in SPV samples Lane 1-
goatpox ve control Lane 2- sheeppox ve
control Lane 3-6 goatpox field samples Lane
7-10 sheeppox field samples Lane 11-
Buffalopox Lane 12 Camelpox M-100 bp
ladder.
34
C18L gene based PCR for diagnosis of Buffalopox
1 2 3 4 5 M
M 1 2 3 4
Agarose gel electrophoresis of PCR products of
C18L gene region of BPXV and CMLV. A-PCR
products from BPXV isolates. Lane 1-5 BPXV-BP4,
VIJ96, AUR03, Pune 03 and Nellore 06 isolates,
respectively. Lane M 100 bp ladder plus
molecular size marker. B- PCR products from CMLV
samples. Lane 1-2 CMLV isolates 1 and 2,
respectively. Lane M 100 bp ladder plus
molecular size marker. Lane 3, Reference
BPXV-BP4, Lane 4, BPXV-Pune 03 isolate from cow.
C- PCR for differentiation of BPXV and CMLV.
Lane 1,3 products in BPXV and CMLV using
BPVC18LF BPVC18LR2 primers. Lane 2 Negative
control. Lane 4. PCR product using CMLV specific
CMPVC18LF CMPVC18LR primes. Lane 5-6 Reference
BPXV-BP4, BPXV-Pune03 isolates from cow,
respectively.
243bp
A
B
C
35
Real-Time PCR for Detection of BPXV




A



B



1



2

M



3

4


C


369bp

Real-time PCR on infected Vero cells with or
without JExt using BXPV specific C18L gene
primers
36
NA Hybridization RPV PPRV
Shaila et al (1989) Vet. Rec., 125 602
37

ISH for Detection of CSFV
Tonsils Cryptal cells showing positive signals.
Note I/C distribution of the blue colored
precipitates. ISH, NBT/BCIP chromogen. Counter
stain- Methyl green 600X
Lymph node RE cells showing I/C positive
signals red). ISH, NBT/BCIP chromogen. Counter
stain- Nuclear fast red 880X
38
Nucleic Acid Probes Foot and Mouth
Disease Pasteurella multocida Infectious Bursal
Disease Clostridium perfringens Bovine Herpes
Virus -1 Mycobacterium avium paratuberculosis Duc
k hepatitis virus Trypanosoma evansi Leptospira
sp. Echinococcus granulosus
39

• Targeted PCR Assays (i)
• Viral diseases
• ? FMD ? EDS -76
• ? Bluetongue ? CIA
• ? PPR/RP ? IBD/ ND
• ? BHV 1/EHV ? Duck Plague
• ? CSF ? Reo virus infection
• ? Auzesjkys disease ? Inclusion Body
  Hepatitis
• ? Poxviruses OPV/CaPV ?Exotic
  diseases
• ? Rabies ? Equine Influenza
• ? Hydro-pericardium Syndrome

40
Targeted PCR Assays (ii) (B) Bacterial diseases
Mycobacteria sp, Pasteurella sp.,
Leptospira sp., Salmonella sp., Brucella
sp, Mycoplasma cluster, (C) Mycoplasmal
diseases Mycoplasma cluster, (D)
Parasitic diseases T. evansi, T. annulata,
B. bigemina, T. gondii, H. contortus etc. (E)
Chlamydial infections
41

• Molecular Epidemiology

42
PPRV phylogeny F-Gene based
Dhar et al. (2002). Vet. Microbiol., 88 153-159.
43
PPR Virus Phylogeny
Based on H gene
Based on F gene
Kaul, R. (2003). Ph.D. Thesis, IVRI, Mukteswar
44
Phylogenetic tree of A27L sequences of BPV
45
Phylogenetic analysis of OPV
Unrooted tree constructed d on aa sequence using
neighbor joining method of MEGA 3.1
HA Gene
46
Phylogenetic tree of different poxviruses based
on DNA POL gene sequence
Capripoxvirus
Suipoxvirus
Orthopoxvirus
Parapoxvirus
47
Emerging Infections

• SARS-Corona Virus
• Avian Influenza
• FMD outbreak in UK
• Prion disease
• Hendra/Nipah/WNV outbreaks
• Emergence of Monkeypox outbreaks
• Chikungunya Virus
• Dengue Haemorrhagic fever virus

48
Vaccine Development
49
Conventional vaccines
(i) Live attenuated PPR vaccine (ii) Live
attenuated Goatpox Vaccine (iii) Live Combined
Vaccine (PPR Goatpox) (iv) Live attenuated
Buffalopox Vaccine (v) Avian Influenza (vi) Duck
Plague vaccine (vii) Orf vaccine (viii) Sheeppox
vaccine (ix) Thermo-resistant PPR vaccine
50
Recombinant Vaccines

• 1. Virus-vectored vaccine
• ? SPPV virus harboring FMDV genes
• 2. Vero-cell Expressed Protein vaccine
• ? PPRV H protein
• 3. Baculovirus/Silkworm expressed
• ? RPV/PPRV
• 4. Gene Vaccines ? PPR, GTPV/UTR/IFN?
• 5. Forage (edible) Vaccine ? PPR H gene

51
Bivalent vaccination for GP PPR

• Combined vaccine induced virus-specific immune
  responses
• Safe, immunogenic protective
• Both vaccine viruses are compatible making the
  live bivalent vaccination a feasible approach

52
Local skin reaction at the inoculation site in
the immunized goats
105 TCID 50
102 TCID 50
53
Virus Propagation for Attenuation

• ? BPV/Vij-96 isolate passaged in Vero cells
• ? 50 passages carried over
• ? Virus titre up to 7.63 TCID50/ml (P-28)

1 2 3 4 5
10-2 10-3 10-4 10-5 10-6 10-7 10-8 CC
Virus Titre 107.63 TCID50 per ml
Uninfected Vero
Infected Vero- 3dpi
54
Immunization of buffalo calves
Local erythema at the site of inoculation of
virus (day 6PI)
55
Results of I/D challenge infection
A-B control animals C BPXV-VIJ96 and D
BPXV-BP4
56
Immune response of the PPRV rec. H protein in
Rabbits
From 21 days Post vaccination onwards PPR
C-ELISA- PI value varies from 80-98 SNT- varies
from 32-128 Immune Response maintained till 8
month (so far tested)
57
Immune response of the PPRV rec. H protein in
Goats
From 7th days Post vaccination onwards, immune
response observed and maintained up to 4 month
(so far tested ) PPR C-ELISA- PI value varies
from 80-99 after 14th DPV Immune Response
maintained till 4 month (so far tested)
58
Approaches to Flu Vaccines
India
59
Impact of Diagnostics
60
Distribution of PPR s-ELISA c-ELISA kits
61
Transfer of Technology C-ELISA
2001-2002 893 2002--2003 985 2003-2004 1015
2004-2005 1507 2005-2006 630 5030
samples NMRL 61500 samples at State
Labs 66,530 (Total)
Testing of samples for PPR ser-diagnosis _at_ Rs
5/test sample
62
Transfer of Technology S-ELISA
2001-2002 325 2002--2003 225 2003-2004 357
2004-2005 3322 2005-2006 1069 5298
samples NMRL 8000 samples at State Labs 13,298
(Total)
Testing of samples for PPR diagnosis _at_ Rs 25/test
sample
63
PPR Sero surveillance
Prevalence of PPR virus antibodies in different
states of the Country as determined by PPR
c-ELISA kit
64
Cost Factor Analysis for PPR-s-ELISA and
PPR-c-ELISA kits
665100
1,09,71650
11636750
The cost factor analysis reveals revenue saving
of ? Rs 21,94,330/- in a year ? Rs
1,09,71,650- in five years

65
Disease Control Programs Strengths Weaknesses
1 mark to each point
66
Table 1 Virus Repository at Division of
Virology, IVRI, Mukteswar

67
Structural Genomics

• SARS Coronavirus
• PPR vaccine virus whole genome sequence
• Many viruses Partial genome sequence
• New Leads
• 1. Sheep pox/goat pox viruses
• ? Phylo-genetically distinct viruses
• 2. Differentiation of GPV/SPV
• ? PCR-RFLP of P32/A genes, Multiplex PCR
• 3. BPXV is VACV-like virus
• 4. Pox in cows in India caused by BPXV
• 5. DNA Pol gene is genus-specific in poxviruses
  - RNAi

68
RNAi Potential in Antiviral Therapy

• Human Viruses
• - HIV, Polio, Hep B, Hep C, RSV
• 2. Animal viruses
• FMD virus
• Duck Plague virus
• PPR virus
• Pox viruses Buffalopox/goatpox viruses
• Pseudorabies virus

69
97.18
95.65
87.60
97.49
40.95
68.38
70
Inhibition of BPV replication with siRNAs in vitro
Inhibition of viral replication to the extent of
46 to 500 times as compared to the control
untreated cells
71
98.852
99.801
97.830
94.98
72
Future Thrust

• Micro-array based diagnostics
• Functional Genomics Proteomics
• Animal/Plant Transgenics
• Disease Resistance
• Non-antibiotic antimicrobials
• One-Medicine concept of Jenner
• ? Viral Vaccines ? Antiviral therapeutics

73
Rinderpest Stamping out 1890
74
Slaughter of cattle herds in a bid to eradicate
Foot-and-mouth disease in South Africa in the
1930s
75
We need to avoid this !
Cattle carcases burning on a farm in
Gloucestershire during the 2001 FMD outbreak.
Such images, widely disseminated by the media,
horrified the public, and had an impact on animal
disease control policies that extended well
beyond the UK.
76
Changing Disease Patterns Demand Cross-Field
Partnerships ( European Med-Vet-Net)
Med-Vet-Net is made up of 16 European partners.
The aim is to provide a network of excellence
for integrated research on the prevention and
control of zoonoses
77
One Medicine Concept of Jenner Still Holds Good
Jenners observation that the zoonotic character
of cowpox could be used to protect against
smallpox, was, Prof. Gibbs suggested, arguably
the most important observation in the history of
infectious disease. In honour of Jenners
scientific approach and the role of the cow in
protecting mankind from smallpox, Louis Pasteur
named the process of inoculation vaccination
(from vacca, latin for cow)-appropriate
recognition for the one medicine for human and
veterinary disease, as practiced by Jenner and
Pasteur.
78
Thank You