NEWER APPROACHES FOR

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NEWER APPROACHES FOR DIAGNOSIS AND CONTROL OF
CSF
G Sai Kumar, I/C National CSF Referral
Laboratory, Indian Veterinary Research Institute,
Izatnagar.
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Etiology
BVD, CSFV, Border disease
Pestiviruses
GENOME ssRNA, VE POLARITY, 12 KB, 1 ORF, 2
UTR. PROTEINS 11 5 STRUCTURAL, 6 NON-STRUCTURAL
Flaviviridae
Flaviviruses
Hepatitis C virus
Japanese Encephalitis Dengue West Nile virus St.
Louis encephalitis
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Geographic Distribution
Higher prevalence East and Southeast Asia, India,
China, South and Central America Eradicated in
U.S.(1978) Australia (1961) New
Zealand Canada Parts of Europe
CSF in South Africa after 87 years absence.
Sandvik et al 2005, Vet Rec,157267.
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Prevalence in India

• Map depicting States where CSF outbreaks(red
  dots) have been recorded.
• The density of pig population is highest in the
  NE region.
• Besides Northeast, the disease is endemic in
  Uttar Pradesh where pig husbandry is popular
  among the backward classes and scheduled tribes.

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Courses of disease - Postnatal and Prenatal
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Pathological Lesions and CSF Diagnosis

• Since 1960's moderate and low virulent strains
  that cause
• milder illness are more common in Mexico, Central
  America and Europe.

• LESIONS IN ACUTE CSF ARE NOT PATHOGNOMONIC.
• IN CHRONIC FORM, ONE ANATOMICAL SYSTEM IS
  INVOLVED.

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Comparative Analysis of Diagnostic Assays
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Laboratory Diagnosis Methods
DIRECT
INDIRECT
ANTIBODY DETECTION - NEUTRALISATION TEST
- ELISA ANTIBODIES
1. VIRUS ISOLATION 2. ANTIGEN DETECTION -
FAT - ELISA(Ag) 3. NUCLEIC ACID DETECTION
- RT-PCR
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Laboratory Diagnosis Approach Requirements
SECONDARY OUTBREAK
PRIMARY OUTBREAK
CLINICAL SIGNS OF CSF
CLINICAL SIGNS, EPIDEMIOLOGY
AgELISA, FAT, PCR ONE TEST POSITIVE
AgELISA OR FAT OR PCR ONE test ve
FAT PCR OR FAT AgELISA OR AgELISA
PCR POSITIVE
Serology AbELISA, VNT
VIRUS ISOLATION
CSF
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Characterization of CSFV isolates

• Pathogenicity testing of isolates.
• Development of a panel of Mabs against CSFV
  specific antigenic domains
• Characterization of antigenic domains of Erns, E2
  and NS3 using panel of Mabs

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Monoclonal Antibody based Diagnostics

• Use of MAbs for CSFV antigen detection
• Application of MAbs in First tier tests Lateral
  flow and Flow through tests
• Application of MAbs in Second tier tests ELISA

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ELISA Kits In the market
IDEXX Laboratories Herdchek CSFV/Ab
(E2) Blocking ELISA for the detection of
CSFV antibodies in swine serum or
plasma.HerdChek CSFV Antigen Test
Kit Antigen-capture enzyme immunoassay designed
to detect CSFV antigen in porcine peripheral
blood leukocytes, whole blood, cell culture and
tissue samples Chekit-CSF-Sero This kit can
reliably distinguish pigs infected by BVDV-BDV
virus (mild and self-limiting) from those
infected by CSFV Based on the detection of
antibody against the glycoprotein E2 CHEKIT CSF
Marker This is officially approved in the EU
Member States to differentiate the animals
vaccinated with an E2 subunit vaccine (Erns
negative) from CSF-infected animals
(Erns-positive).
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ELISA Kits In the market
Prionics AG, Switzerland (Marketing products
developed by Cedi Diagnostics, Lelystad) The
Ceditest CSFV detects antibodies against the E2
envelope protein of the CSFV. The Ceditest
CSFV 2.0 also detects antibodies against the E2
envelope protein of the CSFV. An important
advantage of the test is its high specificity for
CSFV because the test does not cross-react with
related viruses.The Ceditest CSFV Erns can
also discriminate between vaccinated and infected
pigs when used in combination with the E2 marker
vaccine.Ceditest CSFV-Ag detects the CSF virus
itself.
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RT-PCR Phylogenetic Analysis
RT-PCR based methods - Quick and
Precise Sequencing viral genes
Phylogenetic analysis
China -- 1.1 Germany - 2.1, 2.2, 2.3 France
- 2.3 Czech Rep -- 2.2, 2.3 Austria --
2.3 Brazil - 1.1 Taiwan -- 2.1, 3.4 Thailand --
1.1, 3.3 INDIA -- 1.1, 2.1
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RT-PCR ... Novel Pestiviruses Identified

• Characterisation of a classical swine fever
  virus-like ovine pestivirus isolate
• Sandvik T, Hurtado A, Moore S and Ibata G.
• UK and Spain
• An ovine pestivirus resembled CSF when typing was
  done based on partial
• 5' UTR sequence. Sequencing of Npro and E2
  revealed that its different from CSFV,
• BVDV and BDV. Immunoreactivity with MABs also
  showed lot of variation.
• Full-length genomic sequencing and phylogenetic
  analysis placing the pig
• bovine viral diarrhoea virus strain ZM-95 into a
  novel genetic subgroup
• Xu X, Zhang Q, Yu X, Liang L, Hu R, Xiao C, Tu C.
• China
• Strain ZM-95 is the first pig BVDV isolated in
  China in 1995 from a sick pig with
• signs resembling CSF. This report showed that pig
  BVDV could form an isolated
• cluster in BVDV-1 group and likely have a new
  origin than cattle source.

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Real-time RT-PCR

• Detection and identification of CSFV in general
  and specific strain/genotype of CSFV.
• Sequence analysis of CSFV to identify consensus
  and unique sequences.
• Designing multiple sets of primers and dual
  labeled probes (TaqMan, Mol. Beacons) tailored to
  SPECIFIC isolates/genotypes of CSFV.

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Recent Advancements Real-Time RT-PCR
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Latest Trend...... Real-Time RT-PCR
Method of detection of classical swine
fever USPTO Patent Application
20060177818Patent Title Method of detection of
classical swine fever Patent Abstract The
invention relates to a multiplex real-time RT-PCR
assay with a heterologous internal control system
(i.e., EGFP-RNA) for the simple and fast
diagnosis of classical swine fever virus (CSFV).
Primers and FAM-labeled TaqMan probes, specific
for CSFV were selected by analyzing the consensus
sequence of the 5'-non translated region of
various CSFV strains.
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Real-Time RT-PCR Diagnostic Kits
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CSF DATABASE
)
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Research Efforts in Japan
But this test does not discriminate BVDV and CSFV
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Latest Trend...... Bio-Chip for CSF Diagnosis
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Latest Trend...... Surface Plasmon Resonance
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Latest Trend.....Non-Conventional Diagnostic
Specimens
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Real-Time RT-PCR ... Work done in India
Viral Load Estimation
Handling many samples made easy
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Real-Time RT-PCR ... Work done in India
CSF in pigs sampled at slaughterhouse
Out of 112 cases tested by Real-Time RT-PCR,
positive reaction was observed in 86 cases
(76.78).
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CSF Control .... In European Union

• THE PROBLEM OF RECURRENT OUTBREAKS IN EUROPE
• IS DUE TO INFECTION SPILLING OVER FROM FERAL
  SWINE.
• In the European Union, CSF outbreaks are
  controlled by stamping-out policy.
• Vaccination against CSF is banned. This decision
  is based entirely on economical reasons.

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CONTROL THROUGH USE OF VACCINES...

• Inactivated vaccines, such as phenol/glycerol,
  formalin or crystal violet, were used in the
  early period.
• Lapinized C-strain vaccine attenuated by repeated
  passages (480) in rabbits. The C-strain was
  developed from virulent Shimen strain in China in
  the mid-1950s.
• Many countries have successfully controlled or
  eradicated the disease by mass vaccination with
  C-strain (van Oirschot, 2003).
• The Japanese guinea-pig exaltation-negative
  strain (GPE-) derived from virulent ALD was first
  used in Japan in 1969 and the disease was
  controlled by 1993 (Suzuki, 2001). Protection
  after 3 days.

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CONTROL THROUGH USE OF VACCINES...

• The French Thiverval strain, derived from
  virulent Alfort strain, is a cell culture adapted
  strain.
• The Chinese C strain adapted to SK6 cells is
  marketed as "Cedipest, and is used in many
  European countries. It confers protection for a
  period of 6 months.
• The C-strain is also commercially available as
  Pestiffa (Merial, France), which is a IR04 cell
  line adapted C-strain.
• Other commercial strains derived from the
  C-strain are SUVAC (Hungary), Lapest (Poland),
  Suiferin C (former East Germany), JIK (former
  USSR), VADIMUN (USA) and Reims (Germany).

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CONTROL THROUGH USE OF VACCINES...

• The C-strain vaccine has been known to be 100
  efficacious, protection lasts for 6-18 months, or
  even lifelong.
• With regards to emergency vaccination, this
  vaccine is known to protect the animals after 1
  week.
• The C-strain vaccine is considered to be very
  safe as it prevents congenital infection,
  complete cessation of challenge virus
  replication, and does not induce disease in young
  piglets or pregnant sows.
• Presently a lapinized vaccine strain developed at
  Weybridge, UK is being used in India.

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New Generation Marker Vaccines ...

• The E2 glycoprotein based subunit vaccine
  marketed by Intervet as Porcilis Pesti is
  being used in Eastern Europe and Central America.
  This is a baculo virus expressed E2 glycoprotein.
  The vaccine has also been licenced in The
  Netherlands for use as emergency vaccination.
• A similar product from Bayer is marketed as
  Bayovac CSF Marker.
• The antibody response after vaccination with
  Bayovac CSF Marker vaccine was more pronounced
  than after vaccination with Porcilis Pesti
  (Depner et al. 2001).

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New Generation Marker Vaccines ...

• The E2 subunit vaccines confer complete
  protection after booster vaccination following 4
  weeks of the first vaccination. Efficacy is
  variable as viraemia has been detected in the
  vaccinated animals following challenge.
• A study conducted in China demonstrated that an
  amino acid sequence, TAVSPTTLR, residing in
  glycoprotein E2, had the capacity to induce
  neutralizing antibodies. The results showed that
  the 6-repeated chimeric recombinant protein
  managed to elicit epitope-specific neutralizing
  antibodies.

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New Generation Marker Vaccines ...

• Live marker vaccines have been developed using
  viral vectors such as poxvirus, pseudorabies,
  porcine adenovirus and bovine viral diarrhea
  virus vectors containing CSFV RNA.
• DNA vaccine or a recombinant deletion vaccine
  developed out of an infectious DNA copy of viral
  RNA is also being tried. Results were encouraging
  (Wehrle et al. 2007).
• Oral bait vaccines for controlling CSF in feral
  pigs.
• Delivery of E2 subunit vaccine encapsulated in
  PLGA (polylactide-co-glycolide) microspheres
  administered through mucosal route showed that
  systemic immune response after nasal
  administration was more intense and less variable
  than oral route.

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Gaps Constraints.. In India

• Easy access to diagnostic laboratories
• Specific monoclonal antibodies - Not available in
  India.
• More simpler tests that can be used on the farm
  Not Available
• Real-time RT-PCR assays - Not available in India.
• Lack of adequate epidemiological information on
  CSF
• Easy access to cheap and effective vaccines Not
  Available
• There is no commercially available vaccine that
  can be bought by needy farmers or farm owners and
  the efforts of some international firms to market
  vaccine(s) in the country are yet to bear fruit.

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Thanks for your attention