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Biotechnological interventions to animal
skin – implications to the leather industry
G. Dhinakar Raj
Professor
Department of Animal Biotechnology
Madras Veterinary College
Tamil Nadu Veterinary and Animal Sciences University
Chennai – 600 007
Skin
• Skin is the largest organ of the body
• Functions in protection, sensation,
thermoregulation and metabolism• Primary barrier to pathogens
• Vehicle for delivery of vaccines
• Biological testing
Structure of skin
• Epidermis with its appendices, the hairs.
• It consists of a variety of cell layers produced in the germinative basalcell region located at the base of the dermis. The descendents of thesecells move to the surface of the epidermis while undergoingkeratinization.
• At the final stage they form a horny layer on the surface of theepidermis. This is a continuous process with new cells, produced by thegerminative layer, replacing the horny layer as it breaks into flakes andsloughs off.
• Dermis, a three-dimensional weave of collagen fibers composed ofconnective tissue providing support to the structures associated with theskin, such as hair, sweat glands, nerves and blood vessels.
• It is rich in antigen presenting cells (APCs), called dendritic cells (DCs)and Langerhans cells (LCs) that process and present the antigensdelivered through the skin to initiate immune responses.
• The leather is animal skin treated such that the natural properties of theskin are retained.
Scanning Electron Micrograph of
bovine epidermis; epidermis
loosened after acidic
incubation of skin;
magnification: '2000, length of
bar: 10 mm. Arrow marks
dividing cell.
Scanning Electron Micrograph
of bovine skin, cut after acidic
incubation. Epidermis has
separated from the grain
revealing its surface structure,
magnification: '700, length of
bar: 25 mm.
Epi = epidermis; G = grain.
Skin section of normal sheep.
Normal epidermis without
hyperkeration, parakeration or
asnthosis. Normal hair follicles
(HF) without dermis.
H & E X 160.
Leather grain surface of normal
goat. The surface shows normal
goat grain pattern-undisturbed by
any distortions or contours.
Goat - scabby surface and
unmistakable lichenification
resulting in extensive folds
and wrinkles.
Dermatophilosis/epidermoid
Cyst.
Wool break due to mite
infestation
Biotechnological interventions that could
improve the quality of animal skin
• Diagnosis of diseases
• Vaccines
• Needle free delivery
• Nanotechnology
• Enhancing immunology of the skin
• Alternatives to skin testing methods
PCRPCR• Specifically targets and amplifies a single
sequence from within a complex mixture of
DNA
• In vitro DNA amplification technique invented
by Kary Mullis in 1985
• In the last decade PCR has revolutionized the
detection of infectious pathogens because of
its high sensitivity and specificity
• Automated for routine use in laboratories
worldwide
• PCR is DNA replication in a test tube
Why should we use PCR in diagnosis?
• Very sensitive (1 copy – 10 copies of DNA)
• Can detect organisms that cannot be isolated
• Rapid
Disadvantages of PCR
• Technically demanding
• Can be expensive
• Risk of contamination
• Need rigid QC
BTV PCR
Group-specific primers
BTV S7 (VP7)
BTV S10 (NS3)
BTV S8 (NS2)
BTV S9 (VP 6)
BTV S 6 (NS 1)
Serotype-specific primers
BTV S2 (VP2)
274 bp
101 bp
1156 bpNS1 PCR
VP 7 PCR
274 bp
1156 bp
101 bp
274 bp
1156 bp
Pathogen isolation and identification
Vs
Pathogen identification and isolation
Speed, sensitivity and discriminating
power
Conventional approach – takes a long
time, hence only retrospective diagnosis
Is there a paradigm shift?
Strengths Vs Weaknesses of PCR in
diagnosis
• Speed
• Sensitivity
• Specificity
• Origin of virus in a
outbreak (topotype)
• Sometimes differentiation
between vaccine and field
virus
• Phylogenetic relationships
• Does not detect live virus
per se
• Need live virus for cell
culture, animals and
vaccine studies
• Problems of high
sensitive tests in relation
to laboratory
contamination and GLP
Real time PCR
• Monitor the accumulation of the PCR
product while amplification is
occurring
• Analyze cycle to cycle changes in
fluorescent signal generated during the
three phases of PCR
• The fewer cycles to see detectable
fluorescence signal the greater the
amount of template DNA
Real-Time PCR
Real-time PCR monitors the
fluorescence emitted during the reaction
as an indicator of amplicon production
at each PCR cycle (in real time) as
opposed to the endpoint detection
GAPDH standards GAPDH standard
curve; Y=0.24x + 8.19;
r2=0.994
GAPDH sample curves
IFN g - StandardsIFN g standard curve
Y=0.29x +11.20
r2=0.987
IFN g sample curves
Melting curve –
standard, sample,
control
Vaccination
• Oral or injected vaccine
– induces the host to generate antibodies
against the disease-causing organism
– subsequent infections do not establish the
disease
– infectious agent is inactivated by the
antibodies and immune response
Conventional Vaccines
• Inactivated agent
– killed organism used
• Attenuated agent
– live, nonvirulent form of organism
New generation vaccines
• Subunit Vaccines
• Peptide Vaccines
• Genetic Immunization (DNA Vaccines)
• Vector Vaccines
• Anti-idiotype vaccines
• Edible vaccines
Subunit Vaccines
• Generally whole pathogenic agent is
used to construct attenuated or
inactivated vaccine
• Immune response generally elicited by
interaction with proteins on outer
surface of pathogen
Schematic of an animal virus
Envelope
Proteins
Envelope
Nucleic Acid
Capsid
Subunit Vaccine
• So, is the entire pathogen required?
Subunit Vaccine
• No, only outer surface proteins are
needed to elicit an immune response
• Vaccines that use components of a
pathogen rather than the whole
organism are “subunit” vaccines
Subunit Vaccine
• Advantages
– Using a purified protein ensures that thevaccine is safe and stable
• Disadvantages
– Purification may be costly
– Isolated protein may not have the sameconformation as in the pathogen, so maynot have the same antigenicity
Development of Subunit
Vaccine against HSV
HSV
Not Protected Protected
clone gD
gene transfect
inject
purify &
concentrateinfect
infect
CHO cell secreted protein
Multiprotein Subunit Vaccine
Vaccines
• Subunit Vaccines
• Peptide Vaccines
• Genetic Immunization (DNA Vaccines)
• Vector Vaccines
• Anti-idiotype vaccines
• Edible vaccines
Peptide Vaccines
• Subunit vaccine uses entire protein
– Contains several antigenic determinants
• Peptide Vaccine
– vaccine from a specific domain of an
antigenic protein
– single epitope or antigenic determinant
Generalized membrane-bound
protein with external epitopes
5 possible
antigenic
epitopes
Structure of a peptide
vaccine
Linker
Short
PeptidesCarrier
Protein
KLH or HBcAg
Limitations of Peptide
Vaccines– Epitope must consist of a contiguous stretch of
amino acids
– Not all peptides are effective in eliciting an
immune response (may need 2 or more)
– Peptide must have the same conformation as in
pathogen
– Amount of peptide required to elicit an immune
response may be 1000X more than for inactivated
pathogen
Vaccines
• Subunit Vaccines
• Peptide Vaccines
• Genetic Immunization (DNA Vaccines)
• Vector Vaccines
• Anti-idiotype vaccines
• Edible vaccines
DNA Vaccines
plasmidMuscle cell
Gene
for
antigen
Muscle cell
expresses protein -
antibody made
CTL response
DNA Vaccines
• Plasmids are easily manufactured in large amounts
• DNA is very stable
• Storage and transport are straight forward
• DNA sequence can be changed easily in the laboratory.
This means that we can respond to changes in the
infectious agent
• By using the plasmid in the vaccinee to code for antigen
synthesis, the antigenic protein(s) that are produced are
processed (post-translationally modified) in the same
way as the proteins of the virus against which protection
is to be produced. This makes a far better antigen than
purifying that protein and using it as an immunogen.
DNA Vaccines
• Mixtures of plasmids could be used that encode many
protein fragments from a virus/viruses so that a broad
spectrum vaccine could be produced
• The plasmid does not replicate and encodes only the
proteins of interest
• No protein component so there will be no immune
response against the vector itself
• Because of the way the antigen is presented, there is a
CTL response that may be directed against any antigen in
the pathogen.
DNA Vaccines
Possible Problems
• Potential integration of plasmid into host
genome leading to insertional mutagenesis
• Induction of autoimmune responses (e.g.
pathogenic anti-DNA antibodies)
• Induction of immunologic tolerance (e.g. where
the expression of the antigen in the host may
lead to specific non-responsiveness to that
antigen)
Vaccines
• Subunit Vaccines
• Peptide Vaccines
• Genetic Immunization (DNA Vaccines)
• Vector Vaccines
• Anti-idiotype vaccines
• Edible vaccines
Transfer vector for
Homologous Recombination
Plasmid
Cloned Antigen
Gene
Vaccinia virus
thymidine kinase
DNA
Vaccinia virus
promoter
Vaccinia virus
thymidine kinase
DNA
Homologous Recombination
into the Vaccinia Genome
Plasmid
Vaccinia virus
DNA
Cloned antigen
gene
Vaccinia virus DNA
Vaccinia
virus promoter
Vaccines
• Subunit Vaccines
• Peptide Vaccines
• Genetic Immunization (DNA Vaccines)
• Vector Vaccines
• Anti-idiotype vaccines
• Edible vaccines
antibody
Anti-idiotype vaccine
epitope
Antibody
with
epitope
binding site
Virus
antibody
Anti-idiotype vaccine cont
Make antibody
against antibody
idiotype
Anti-
idiotype
antibody
Anti-idiotype
antibody mimics
the epitope
Anti-anti-idiotype
antibody
Anti-idiotype antibodycont 2Use anti-idiotype antibody as
injectable vaccine
Binds and
neutralizes virus
Anti-idiotype
antibody
Anti-anti-idiotype
antibody
Anti-anti-idiotype
antibody
Use as
vaccine
Vaccines
• Subunit Vaccines
• Peptide Vaccines
• Genetic Immunization (DNA Vaccines)
• Vector Vaccines
• Anti-idiotype vaccines
• Edible vaccines
A subunit based edible vaccine?
• economical
• heat stable
• oral administration
– no more needles
– mucosal immune response
• sub-unit vaccine
– unable to replicate
– overcome maternal antibodies
Needle-free technology – Why?
• Better quality of hides and skin
• Consistent vaccine delivery
• Lower vaccine volume
• Higher antigen dispersion
• Elimination of broken needles
• Elimination of needle disposal
• Less pain and stress to animal
The skin is rich in antigen-presenting cells. Dendritic cells (DCs) can be
found at high density in the dermis whereas Langerhans cells (LCs) are
mostly localized in the epidermis.
• Comparative MRI’s show good subcutaneous
delivery
• Slightly broader dispersion than conventional needle
Needle & SyringeNeedle-Free
Needle free technologies available
• Spring loaded jet injector
• Battery powered jet injector
• Gas powered jet injector
Schematic overview of the main cellular mechanisms
involved in nasal vaccination.
NANOTECHNOLOGY
Nanotechnology is science of seeing, measuringand manipulating matter at atomic, molecularand supramolecular levels.
At this scale, the physical, chemical andbiological properties of materials differfundamentally and often unexpectedly fromthose of the corresponding bulk materials.
Nanobiotechnology, is the biomedicalapplications of nano-sized systems
Nanoparticles are defined as particles whichmeasures 1-100 nm
Nano…• Nanotechnology (Greek word nano means ‘dwarf’) is the
creation and utilization of materials, devices, and systemsthrough the control of matter on the nanometer length scale
• It is the popular term for the construction and utilization offunctional structures with at least one characteristicdimension measured in nanometer scale –a nanometer(nm) is one billionth of a meter (10 –9 m).
• This is roughly four times the diameter of an individual atom.
• Width of DNA is approximately 2.5 nm and proteinmolecules measure 1–20 nm.
APPLICATION OF NANOPARTICLES
Vaccine delivery
Carriers for various proteins
Targeted drug delivery
Gene therapy using vectors
Gene delivery
Transfection of genes to cells
Tumor imaging, in vivo biomolecular profiling
of cancer biomarkers
Therapeutic use
Morphology of CaP nanoparticles in
SEM
• Spherical, smooth and tendency to
agglomerate
Morphology of CaP nanoparticles in TEM
CaP nanoparticles
appeared spherical,
smooth and
uniformly
distributed
Size of CaP nanoparticles using SEM
• The mean (±
SE) size of
CaP
nanoparticles
was 557.44 ±
18.62 nm
(n=40).
Humoral immunity - Mean (± SE) HI titers (log2)
in the serum of chickens vaccinated with CaP
nanoparticles coupled NDV
2.00a ±0.001.67a ±0.684.00 b ±0.584
2.17a ± 0.314.17b ± 0.314.20b ± 0.543
2.33a ± 0.214.00b ± 0.454.20b ±0.712
2.75 a ± 0484.00 a ± 0.413.00 a ± 0.411
IIIIII
Treatment GroupsWeeks
PV
Prolonged immunity in CaP coupled NDV vaccinated chickens (Group 1)
Cell mediated immunity - Mean (± SE) stimulation index of
splenocytes stimulated with antigen (NDV) following
vaccination of chickens with CaP nanoparticles coupled
NDV
0.75a ±0.03001.29 b ±0.01001.41b ±0.07004
0.52a± 0.00200.87 b±0.01701.38 b ±0.0363
0.50 a± 0.00050.99 b±0.00061.11b±0.00042
0.59a ±0.00210.72a ±0.00261.08b ±0.00211
IIIIII
Treatment Groups
Weeks
PV
Early increase in stimulation index in CaP groupcompared to conventional vaccine
Gelatin nanoparticles
Components of a modern vaccine
Caudal Fold Tuberculin
Test• The Purified Protein
Derivative (PPD)
tuberculin is injected
intradermally
(between the layers
of skin) of the caudal
tail fold, under the
animal’s tail
• The injection site is examined 72 hours
later, plus or minus 6 hours
• If there is any response at the injection
site
– swelling
– redness
– hardness
• The animal is considered suspect
(responder) and further testing is required
Comparative
Cervical
Tuberculin (CCT)
Test
• This test is only done on animals that respond to
the caudal fold test (CFT)
• Once the animal is secured a section of the neck
is shaved in two places before administering the
CCT test between (intradermally) the skin layers
on the animal’s neck
• Skin thickness is measured using a special
caliper and then the veterinarian will inject avian
(bird) tuberculin and bovine (cattle) tuberculin
into two shaved sites on the neck
• Swelling is often the reaction to the avian
TB injection.
– Producers should not be alarmed
• Test results are plotted on a scattergram.
Based on where the results fit on the
graph, the animals will be classified as
negative, suspect or reactor
Alternative to skin testing
• Interferon assay
• Component of cell mediated
immune response
• Antigen specific secretion
• Measurable
• Associated with TB exposure
QuantiFERON® kitWhole Blood CultureWhole Blood Culture
Measure [IFN- ] & Interpret] & Interpret
Centrifuge 5 minutes to
separate plasma above gelCollect 1mL of
blood in 3
tubes
Incubate at
37ºC for 16-24
hours.
Nil
Mtb
PHA
TMBTMB
COLORCOLOR
Measure [ IFN-Measure [ IFN- ] in] in
‘‘SandwichSandwich’’ ELISA ELISASoftware calculates
results and prints
report
Collect 50 L of
plasma for
ELISA
Nil
Mb
PHA
TB diagnosis using ELISPOT
• Collect blood
• Recover, wash, & countPBMCs
• Aliquot 250,000 PBMCs to4 wells with anti-IFN-
• Add saline, PHA, ESAT-6 orCFP-10 & incubate
• Wash away cells
• Develop & count spotswhere cells produced IFN-
INF-
Antibody
Sensitized T
cell
INF-
Captured
Detection
Antibody
Chromogen
Spot
Saline ESAT-6 CFP-10 PHA
Sum up….
• Biotechnological methods offer ways and
means for improving the overall health of
animals and in particular the quality of skin
?