Biotechnology vaccines,Biotechnology diagnosis, Biotechnology treatment

1. Applications of Biotechnology in Animal Health
B.Rajashekar
M.V.Sc I Yr
Vety.Biotechnology dept.

3. 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.

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

5. Biotechnology-derived veterinary vaccines

6. 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.

7. DNA vaccines
Subunit vaccines
Gene deleted vaccines
VLPS
Synthetic peptides
Types of rDNA vaccines

8. • 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

9. • 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

10. 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.

12. 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.

14. DNA vaccines In INDIA
• DNA vaccines
• FMD
• Brucellosis
• Rabies
• IBR
• PPR
• Calf diarrhoea vaccines

15. 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

16. 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.

17. 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

18. 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

19. 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

20. Sub unit vaccine

21. 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

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

23. 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

24. • 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

25. • 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 carriers
1.well characterised hepatitis B core antigen VLPs as carrier for the
influenza a M2 protein

26. 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.

27. • 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

28.  Chimera means unrelated combination
Vector is a self replicating carrier molecule
Viruses 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

29. • 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.

30. Veterinary diagnostic systems

31. 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

32. 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

33. 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 probes
The probe can be composed of an oligonucleotide, a DNA fragment,
single-stranded DNA, or plasmid DNA

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

35. 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

36. Some selected micro organisms against which probes are available
Protozoa Helminths Bacteria Virus
Leishmania(kala-azar)
Trypanosoma
(sleeping sickness)
Plasmodium
E. histolytica
Toxoplasma gondii
Schistosomes
Fasciola hepatica
onchocerca
Taenia
Trichinella spiralis
Mycobacterium
Mycoplasma
Herpes virus

37. 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 carryov
contamination (PCR)

38. 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.

39. PCR

40. Commercially available PCR kits
species 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 kits
salmonella sp marek s disease
canine pcr kits
canine parvovirus
type 2
demodex canis

41. Detection of PCR products
 Traditional electrophoresis method on an ethidium
bromide-containing agarose gel.
Southern hybridization with a specific probe, labelled with
a radioisotope

42. Gel electrophoresis method

43. Southern hybridization

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

45. 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

46. Commercially available kits
SPECIES
VIRUS
Bovine Bovine viral diarrhoea virus RT-PCR Detection Kit
Bovine 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 Kit
Infectious bursal disease virus RT-PCR Detection
Kit

47. Real-time polymerase chain reaction
Real-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

48. 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,

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

51. 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

52. 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

53. Commercially available kits
Bovine ANAPLASMA PHAGOCYTOPHILUM
PCR KIT
BOVINE VIRALE DIARRHEA PCR KIT
ovine BTV – BLUETONGUE VIRUS PCR KIT
COXIELLA BURNETII PCR KIT
Avian KIT FOR THE DETECTION OF THE
MAREK VIRUS
AVIAN MYCOPLASMA PCR KITS
Swine INFLUENZA VIRUS TYPE A PCR KIT
FOR SWINE HOST
CLASSICAL SWINE FEVER PCR KIT

54. 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.

55. • 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.

56. Biotechnology-derived therapeutics

57. 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.

58. 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.

59. 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.

60. • 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

61. 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

62. 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

63. Enzyme Reaction catalysed Application
Papain Protein hydrolysis Deworming
Lysozyme Bacterial cell wall
hydrolysis
As antibiotics
Collagenase Hydrolysis of
collagen
Treating skin ulcers

64. 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.

65. 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.

66. 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/GUI
DE_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

67. THANK YOU