biotechnological interventions to animal skin – implications to the … · 2010-01-29 · dna...

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