Applications of Biotechnology in Animal Health

B.Rajashekar M.V.Sc I Yr

Vety.Biotechnology dept.

Biotechnology

• “Biotechnology" means any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.

New tools and products developed by biotechnologists are useful in research, agriculture, industry and the clinic.

Applications to the animal health

• 1. Biotechnology-derived veterinary vaccines • 2. Veterinary diagnostic systems• 3. Biotechnology-derived therapeutics• 4. Prebiotics• 5. Enzymes• Other techniques

Biotechnology-derived veterinary vaccines

Biotechnology-derived veterinary vaccines

• RECOMBINANT VACCINES

• The vaccines are produced using recombinant DNA

technology or genetic engineering.

• Recombinant vaccines are those in which genes for

desired antigens of a microbe are inserted into a vector.

DNA vaccines

Subunit vaccines

Gene deleted vaccines

VLPS

Synthetic peptides

Types of rDNA vaccines

• GE Vaccines are non infectious

• Production can easily be scaled to produce cheap vaccines.

• Most vectors are safe, easy to grow and store.

• Antigens which do not elicit protective immunity or which elicit

damaging responses can be eliminated from the vaccine, e.g.

Cholera toxin A from cholera vaccine.

• Genes for protective antigens of even non-cultivable viruses/

bacteria/ parasites can used to produce a vaccine.

Advantages of GE Vaccines

• Genetically engineered bacteria might transfer their new gene efficiently to

indigenous bacteria in gut.

• Naked DNA may persist in the environment, upon release or escape to the

wrong place at the wrong time, horizontal gene transfer with unpredictable

long- and short-term biological and ecological effects are unpredictable.

• Consequences are not known of probable random insertions of vaccine

constructs into cellular genomes in target or non-target species in

environment

RISK associated with GE vaccines

DNA vaccines

DNA vaccine is DNA sequence used as a vaccine.

This DNA Sequence code for antigenic protein of pathogen.

As this DNA inserted into cells it is translated to form antigenic protein.

As this protein is foreign to cells, so immune response raised against this protein.

In this way, DNA vaccine provide immunity against that pathogen.

DNA vaccines Vs Traditional vaccines

֍ Uses only the DNA from infectious organisms.

֍ Avoid the risk of using actual infectious organism

֍ Provide both Humoral & Cell mediated immunity

֍ They are temperature stable, Refrigeration is may

not be required and safe to transport

֍ They have the potential to be less expensive than

other commercial vaccines

DNA vaccines Traditional vaccines

֍ Uses weakened or killed form of infectious

organism.

֍ Create possible risk of the vaccine being

fatal.

֍ Provide primarily Humoral immunity

֍ Usually requires Refrigeration.

DNA vaccines In INDIA

• DNA vaccines

• FMD• Brucellosis• Rabies • IBR• PPR• Calf diarrhoea vaccines

DISADVANTAGES

Limited to protein immunogen only

Extended immune stimulation leads to chronic inflammation

Chance of developing auto immune disorders

Risk of transfer of antibiotic resistance gene

Due to integration of foreign DNA into host, lead to

insertional mutagenesis

GENE-DELETED VACCINES

Gene-deleted organisms can be used as vaccines as they retain the immunogenic features of the wild-type organism but cannot cause disease.

This approach has been successfully used to create several live attenuated vaccine strains of bacterial pathogens

That are genetically stable, safe to use and induce better protection than killed vaccines.

Virulence Gene

Gene deletion by homologous recombination

KanRSuicide plasmid

Homologous recombination with

suicidal plasmid and bacterial chromosome

(virulence gene)

Gene replacement

Bacterial chromosome

Bacterial chromosome

KanR

Virulence Gene

KanR

They are now available against the herpesvirus that causes pseudorabies in swine.

The enzyme thymidine kinase (TK) is required by herpes viruses to replicate in non dividing cells such as neurons.

Viruses from which the TK gene has been removed can infect nerve cells but cannot replicate and cannot cause disease.

As a result, these vaccines not only confer effective protection but also block cell invasion by virulent pseudo rabies viruses and so prevent the development of a persistent carrier state

SUBUNIT VACCINES

Subunit vaccines contain purified antigens instead of whole organisms.

Such a preparation consists of only those antigens that elicit protective

immunity.

Subunit vaccines are composed of toxoids, subcellular fragments, or

surface antigens

Sub unit vaccine

Advantages:

They can safely be given to immune suppressed animals They are less likely to induce side effects. Disadvantages:

Antigens may not retain their native conformation, so that antibodies produced against the subunit may not recognize the same protein on the pathogen surface.

Isolated protein does not stimulate the immune system as well as a whole organism vaccine

oRecombinant vaccines are available for respiratory pathogens such as Mannheimia haemolytica and Actinobacillus pleuropneumoniae based upon the leukotoxins produced by these organisms

VIRUS-LIKE PARTICLES

Virus-like particles (VLPs) are supra-molecular structures composed of one or more recombinant proteins

Several vaccines for veterinary application in development. These include vaccine for bluetongue virus, rota and parvovirus.

Similar to viruses and bacteria multiple copies of the vaccine antigens are displayed in a highly repetitive and ordered, which can cross-link the B cell receptor resulting in activation of the B cell and subsequent induction of T-independent IgM responses

• VLPs offer the advantage of• formulating the vaccine antigen in a particulate structure, thereby

increasing the immunogenicity of the vaccine.

• VLPs are similarity to viral and bacterial structures, the ability for large-scale production and the possibility of combining the VLPs with other adjuvants.

• High safety profile

• VLPs can either be used as vaccine itself or be used as carrier for recombinant antigens, either incorporated, directly, genetically fused or covalently linked.

ex; bovine rotavirus virus protein 6 (VP6) forms VLPs that are highly immunogenic and confer protection against challenge infection

Examples of VLPs used as carriers1.well characterised hepatitis B core antigen VLPs as carrier for the influenza a M2 protein

Synthetic Peptides

• If the structure of a protective epitope is known it may be chemically synthesized and used alone in a vaccine.

• The procedures involved include a complete sequencing of the antigen of interest, followed by identification of its important epitopes.

• The epitopes may be predicted by the use of computer models of the protein or by the use of monoclonal antibodies to identify the critical protective components.

• Experimental synthetic vaccines have been developed against hepatitis B, diphtheria toxin, Foot-and-mouth-disease virus, canine parvovirus, andinfluenza A, and they provoke some protective immunity

Chimera means unrelated combination

Vector is a self replicating carrier moleculeViruses like pox ,adeno and bacteria like salmonella

are used as vectors

Vector is introduced with gene of interest

Such molecule can replicate along with our gene producing enough

amount of antigens required for adequate immune response

Chimeric live vaccines

• A chimeric vaccine based on yellow fever virus that includes the pre membrane (preM) and envelope (E) proteins of West Nile virus was used for protective immunization of horses.

Veterinary diagnostic systems

Veterinary diagnostic systems

• The development of molecular biology has opened up huge possibilities in diagnostic techniques which are fast becoming widespread in veterinary diagnostic laboratories

• Single DNA sequences provide a high degree of specificity in the diagnosis and control of pathogenic microorganism species and subspecies

Diagnosis

All traditional diagnostic tests take long time, may yield ambiguous results, and some of them cannot be applied in certain cases.

Ex; antibody titre estimation in the case of latent viral infections

False diagnosis Consequences No proper treatment Animal will suffer Economic losses, farmer will expense so much money for

treatment

DNA / RNA Probes

Nucleic acid-based probes are Small oligonucleotide sequence used to detect the presence complementary sequence in nucleic acid sample

Probe Both DNA and RNA used as probesThe probe can be composed of an oligonucleotide, a DNA fragment,

single-stranded DNA, or plasmid DNA

Probe either radio actively or non radioactively labelled consisting of a enzyme, or chemiluminescent substrate is attached to probeProbes are being used to detect micro organisms in the samples for disease diagnosis Probes used as follows;i) Hybridizationii) Ligation reaction

Hybridization

• Southern blot• Dot blot• For dot blot analyses test samples like blood samples are generally

lysed directly on nitrocellulose membrane. a probe can hybridize with a test DNA sample only when the latter contains the complementary sequence.• The probes used in diagnostic assays are highly specific to

concerned pathologic micro organism. Therefore, a positive hybridization signal of a test DNA sample with given probe reveals the presence of concerned micro organism

Some selected micro organisms against which probes are available

Protozoa Helminths Bacteria Virus

Leishmania(kala-azar)Trypanosoma(sleeping sickness)PlasmodiumE. histolyticaToxoplasma gondii

Schistosomes Fasciola hepaticaonchocercaTaeniaTrichinella spiralis

MycobacteriumMycoplasma

Herpes virus

Advantages• Fast Sensitive and specific (PCR) • Rapid and much simpler• It can detect even a single molecule in

the test sample• Probes can detect latent viral infection• Automatable

Dis advantages• Expensive• Multistep• Detects dead organisms• Possible false-negatives from PCR

inhibitors• Possible false-positives from

carryover contamination (PCR)

PCR in Diagnosis of Infection

• The PCR is the most sensitive of the existing rapid methods to detect microbial pathogens in clinical specimens• In particular, when specific pathogens that are difficult to culture in

vitro or require a long cultivation period are expected to be present in specimens, the diagnostic value of PCR is known to be significant.  • Since a variety of clinical specimens, such as blood, urine, sputum,

CSF and others• DNA extraction from specimens,• PCR amplification, Detection of amplicons.

PCR

Commercially available PCR kitsspecies Bacterial diseases Viral diseases Parasitic diseases

Bovine pcr kits

Ovine pcr kits

brucella abortus bovine herpesvirus 2 theileria lestoquardi

mastitis(multiplex kit pcr)

anaplasma marginale

sheep pox virus pcr theileria parva

Avian pcr kitssalmonella sp marek s disease

canine pcr kitscanine parvovirus type 2

demodex canis

Detection of PCR products

 Traditional electrophoresis  method on an ethidium bromide-containing agarose gel.

Southern hybridization with a specific probe, labelled with a radioisotope

Gel electrophoresis method

Southern hybridization

RT PCR

• RT  PCR is preceded with conversion of sample RNA into cDNA with enzyme Reverse Transcriptase

Sources of enzyme

• Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) is an RNA-dependent DNA polymerase that can be used in cDNA synthesis• Avian Myeloblastosis Virus Reverse Transcriptase

Commercially available kits SPECIES

VIRUS

Bovine Bovine viral diarrhoea virus RT-PCR Detection KitBovine ephemeral fever RT-PCR Detection Kit

Ovine RT-PCR KITS

BT virus RT-PCR Detection Kit

Avian RT-PCR KITS: 

Influenza A virus RT-PCR Detection KitInfectious bursal disease virus RT-PCR Detection Kit 

Real-time polymerase chain reactionReal-Time PCR a specialized technique that allows a PCR reaction to

be visualized “in real time” as the reaction progresses.Identical to standard PCR except that the progress of reaction is

monitored by a detector during each PCR cycle.Use of fluorescent marker that binds to DNA.As gene copy number increases, fluorescence also increases.Quantification is achieved by measuring the fluorescence during

exponential phase of PCR.

Real-Time PCR allows us to measure minute amounts of DNA sequences in a sample

Real-time PCR fluorescence detection systems

Specific detection: Taqman probe

• The 5’nuclease domain has the ability to degrade DNA bound to the template, downstream of DNA synthesis. • A second key element in the 5’nuclease assay is a

phenomenon called FRET: fluorescent resonance energy transfer. In FRET, the emissions of a fluorescent dye can be strongly reduced by the presence of another dye, often called the quencher,

Non-specific detection: SYBR green IDNA binding dye.Binds to dsDNA.Emits light when bound.More dsDNA=more fluorescence. But it is unspecific as it binds to any dsDNA.

Quantification:

• The light emitted from the dye in the excited state is received by a computer and shown on a graph display, such as this, showing PCR cycles on the X-axis and a logarithmic indication of intensity on the Y-axis

• The threshold cycle (Ct ) is the cycle number at which the fluorescent signal of the reaction crosses the threshold

The advantages of real-time PCR include• Ability to monitor the progress of the PCR reaction as it occurs in real time • Ability to precisely measure the amount of amplicon at each cycle, which allows highly accurate quantification of the amount of starting material in samples • Amplification and detection occurs in a single tube, eliminating post-PCR manipulations

Commercially available kits

Bovine ANAPLASMA PHAGOCYTOPHILUM PCR KITBOVINE VIRALE DIARRHEA PCR KIT

ovine BTV – BLUETONGUE VIRUS PCR KITCOXIELLA BURNETII PCR KIT

Avian KIT FOR THE DETECTION OF THE MAREK VIRUSAVIAN MYCOPLASMA PCR KITS

Swine INFLUENZA VIRUS TYPE A PCR KIT FOR SWINE HOSTCLASSICAL SWINE FEVER PCR KIT

Microarrays

• DNA microarrays are typically composed of DNA “probes” that are bound to a solid substrate such as glass. • Each spot (50 to 150 µm) in the array lattice is composed of many

identical probes that are complementary to the gene of interest. • During hybridisation DNA “targets” diffuse passively across the

glass surface, when sequences complementary to a probe will anneal and form a DNA duplex. • Hybridised targets can then be detected using one of many reporter

molecule systems.

• Microarray technology has been utilized in the identification of various infectious disease pathogens such as Avian influenza (H5N1), FMD, Viral Haemorrhagic fever.

• Advantages microarray systems have been developed that have the potential for simultaneous detection of many pathogens.

Biotechnology-derived therapeutics

Monoclonal antibodies:

Monoclonal antibodies have several therapeutic applications 1 To provide passive immunity against diseases

2 Used in diagnosis, used to detect antigen and antibody Ex; Elisa (Anthrax,brucellosis,BTV, Theileriosis, Fasciolosis) Monoclonal antibodies have been developed that identify common antigenic determinants on the surface of several strains

3. In treatment of diseases like lymphoma in dogs.

Stem cell therapy• Stem cells can be classified as an adult (bone marrow, adipose tissue,

tendon) and embryonic stem cells (inner cell mass of the blastocyst)• In adults, stem cells act as a repair system by replenishing tissues of

the body • In embryonic stem cells differentiate into all the specialized cells and

develop into adult.

Tendon and ligament repair

The quality of the tendon and ligament healing can be improved with altered therapeutic strategies which include stem cell therapy.

Tendinitis in the superficial digital flexor tendon treated with adipose derived nucleated cells (ADNC) injection.

In race horses, the adipose derived MSCs were used to successfully treat experimental tendinitis.

• Wound Healing• Caprine Wharton's jelly mesenchymal stem cells of umbilical cord

were used to treat cutaneous wounds in goat.• Treated group with less inflammation, thinner granulation tissue

formation with minimum scar

Immuno castration• Surgical castration must be performed by specialist personnel, is

irreversible, causes infections and can later lead to inguinal hernias and immunosuppression, in some cases resulting in death. • The administration of steroids causes side effects detrimental to

animal health.• For these reasons, animal immune castration has been tested using

peptides similar to gonadotropin releasing hormone (GnRH), combined with proteins, to trigger antibodies that neutralise the function of GnRH

Enzymes as antimicrobials and therapeutics• Several enzyme based products and multi enzyme formulations are or

enzyme mediated strategies are already available in health care.• The enzymes that target different microbial cellular components and

biofilm development are increasingly investigated for applications to bacterial control in health care.• There is a surge of interest in the use of bacteriophage or their gene

products to control bacterial pathogens as alternatives to traditionally or currently used antibiotics

Enzyme Reaction catalysed Application

Papain Protein hydrolysis Deworming

Lysozyme Bacterial cell wall hydrolysis

As antibiotics

Collagenase Hydrolysis of collagen

Treating skin ulcers

Prebiotics• Prebiotics are food ingredients that improve the host’s health by

selectively stimulating the growth and/or activity of probiotic bacteria in the intestinal tract.• The leading prebiotics are non-digestible oligosaccharides, including:

fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), malto oligosaccharides (MOS) and xylo-oligosaccharides (XOS). • The most typical prebiotics are FOS and inulin and they are sold

widely for use in monogastric animals.

Conclusions• 1.Technologies based on modern biotechnology offer enormous

potential for the production of vaccines, medicinal products and other veterinary products. • 2. The development and use of these technologies is concentrated in a

few countries of the region, while in others they are still not in widespread use. • 3. There is a need to publicise and provide training in these

technologies• 4. It is necessary to faster the establishment of a comprehensive and

effective regulatory framework for the safe use of these technologies from the dual standpoints of biosafety and the regulations established in the veterinary register.

REFERENCES

• 1. https://scholar.google.co.in/• 2. http://www.ncbi.nlm.nih.gov/• 3.Veterinary Immunology, 9th Edition Author :I Tizard • 4.Textbook of Animal Biotechnology by B. Singh (Author), S.K. Gautam

(Author))• 5.http://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/

GUIDE_3.3_VACCINES_NEW_TECH.pdf• 6.http://www.biomerieux-industry.com/veterinary-diagnostics/adiavet-

real-time-PCR-diagnostic-kits-for-bovine-species• 7. http://www.kitpcr.com/products/6-Bovine

THANK YOU