Animal Models in Microbiology

1. Animal Models
Dr. Kanwal Deep Singh
Lyall
M.D. Microbiology

2. TOPICS
• Definition
• Animal selection
• History
• Preparation, administration of material
• Rabbit, Guinea Pig, Mouse, Rat-
- living conditions
- handling
- diseases
- collection of specimens
- dosing
- uses
• Sheep
• Horse

3. Animal Models
• non-human animal that has a disease
or injury that is similar to a human
condition
• test conditions are termed as animal
models of disease
• similar in etiology and function to
the human equivalent
• spontaneous or induced by physical,
chemical or biological means

4. Animal Experimentation
• use of non-human animals in
scientific experimentation.
• 50 to 100 million vertebrate animals
are used annually.
• Research such as genetics,
developmental biology, behavioral
studies, biomedical research, drug
testing , toxicology tests etc.

5. History
• Aristotle and Eristratus – first to
perform experiments on living
animals
• Louis Pasteur – Germ theory of
Medicine by inducing Anthrax in
sheep in 1880
• Rudolph Jaenisch – first transgenic
animal in 1974
• Mason – used Guinea pig in 1940

6. Animal selection
• Physiology
• Reaction to infection
• Nature of infection
• Closely resemble humans

7. Choice Of Animal
• Species
- known susceptibility to disease
- size in relation to the yield required
- ability to afford a statically
significant no of
animals
• Genetic status
- Out bred
- Inbred

8. • Microbiological status – 3 types
1.Conventional animals
2.Specific Pathogen Free (SPF)
3.Gnotobiotic animals

9. Preparation Of Animals
• Handling
• Anaesthesia:
Ether- jar method convenient
- irritant
methoxyflurane
Fentanyl –fluanisone-
neuroleptanalgesia
+ diazepam - Intraperitoneal
or I/M

10. Administration of material
• Body fluids- syringe and needle
• Cultures - fluid with syringe
-growth scrapped off
and
suspended in broth or
saline
• Tissues - homogenized

11. RABBIT
• Oryctolagus cuniculus
• Temperate conditions
• Life span 15 yrs
• Signs of ageing by 6-7yrs
• Room to accommodate 50-60 animals
• Temp-10-18˚C
• Relative humidity -50%
• Marking-rings, tattooing in ear, ear
notching, gentian blue
• Diet- green herbs and other plant material
• Energy requirement – 120 Kcal for 1 kg

12. Diseases
• Intestinal- enteritis, mucoid
enteritis, diarrhoea
• Coccidiosis
• Respiratory- snuffles, pneumonia

13. Handling
• Young – grasping firmly over the
loins
• Larger – grasping ears together with
the fold of skin over the shoulder
• Non slippery surface
• Restraint boxes

14. Lab procedures
• Anaesthesia- Pentobarbitone sodium
with ether or halothane
• Euthanasia- dislocation of neck
- carbon dioxide
-for large rabbits- 1.5ml
Pentobarbitone sodium

15. Collection of specimens
• Blood – marginal ear vein
• Urine – slight pressure over the
bladder
• Faeces – hard pellets from cage and
soft from rectum

16. Dosing and injection
procedures
• Oral – stomach tube
• subcutaneous – max vol 1ml
- Flanks or nape of neck
• Intravenous – max vol 5ml
- marginal ear vein
• Intramuscular – max vol 0.5ml
- posterior aspect of thigh
• Intraperitoneal – max vol 20ml
- 10mm from the midline on
the left
side of abdomen at the
level of
umbilicus

17. USES
• General
• Cardiac surgery
• hypertension
• Infectious diseases
• virology
• embryology
• breeding of tsetse flies
• oral contraceptives
• teaching
• research on reproduction
• Pregnancy testing not done

18. • Microbiological :
• Blood, plasma cells, complement,
liver, brain powder- for lab purposes
• rabies vaccine
• Plasma – for coagulase test
• Schwartzman reaction
• Ileal loop and skin- LT, ST detection
• Antiserum

19. Antiserum production
• Responds well to immunization
• Early antibodies more specific
• Particulate antigens- IgG Ab
I/V
• Soluble antigens- IgG Ab
- S/C or I/M

20. Particulate antigens
• Killed whole bacteria
• I/V
• 4-6 doses, 3 day interval
• 0.5 ml
• Antiserum collected after 5 days of
last dose

21. Soluble antigens
• Stable
• Serum proteins, toxoids
• Water-in-oil emulsion, Freund’s
adjuvant
• S/C, I/M
• 1 mg of serum protein
• Ab response peaks at 8-10 weeks,
plateau level for 1-2 yrs
• Antiserum – 5-6 days after booster

22. • Labile
• Tissue homogenates, cell suspensions
• Water-in-oil emulsions
• Repeat dose at weekly intervals
• Booster after 3 weeks, without
adjuvant
• Antiserum – 5 days after booster

23. GUINEA PIG
• Cavia porcellus
• 18-22˚C
• 50-70%
• 12-16hrs light/day
• Diet- green
foods,seeds,roots,pellets,hay
• Marking – coat patterns, stains,
metal ear tags

24. Handling
• Easy
• one hand around the shoulders and
other supporting the hindquarters

25. Diseases
• Parasitic
• Viral- LCM, HSV, Leukemia
• Bacterial – Salmonellosis
Pseudotuberculosis

26. LAB PROCEDURES
• Anaesthesia-Ether, Pentobarbitone
sodium ,
Alphaxalone and
Alphadolone
• Euthanasia - carbon dioxide,
sharp blow on back of
head

27. Collection of specimens
• Blood
-marginal ear vein(0.5ml)
-cardiac puncture – upto 5ml at
weekly intervals
• Urine-
-pressure over posterior abdominal
area
-metabolism cage

28. Dosing and injection
procedures
• I/D-0.1ml,flank or side of abdomen
• I/P – 10ml,right of midline about
2.5cm in front of pubis
• I/M- 0.2 ml, posterior aspect of thigh
• I/V- marginal ear vein, saphenous
vein
• S/C – 1 ml, skin over back or flank

29. Uses
• General
• biochemical
• toxicological
• physiological
• pharmacological investigations
• study of TB
• otology
• ascorbic acid metabolism

30. • Microbiological
• production of anti bodies
• Source of complement
• Shultz Dale phenomena
• Invitro toxigenicity tests in C.diptheriae
• Straus reaction in B.mallei
• Classical and South Indian type of
M.tb
• Koch’s phenomena in M.tb
• RBCs for Haemadsorption test

31. MOUSE
• Mus musculus
• Cheapest and most readily available lab
animal
• Temp 20-25 ˚C
• Relative humidity 45-65%
• 12-15 air changes per hour
• Life span- 1.5-2.5 yrs
• Diet- cereals, seeds, stored food stuffs
• Marking- splitting or clipping the ears

32. Handling
• from proximal part and never tail tip

33. Diseases
• Viral - Infantile Diarrhoea
• Bacterial – Salmonellosis
• Parasitic – Pin worm

34. Lab Procedures
• Anaesthesia –ether for short term,
fentanyl-fluanisone
• Euthanasia –carbon dioxide

35. Collection of specimens
• Blood
-From the tail :0.3ml
-From the retro orbital plexus – most
efficient for repeated samples.
Vol from 20g mouse-0.7ml
-From the heart- 1.5ml
• Urine – metabolism cage
• Faeces – metabolism cage

36. Collection of blood
• Tail
• cleaned with alcohol
• dilation by immersing the tail in
warm water for 5 to 10 seconds
• small blood sample - capillary action
using a microhematocrit tube inserted
into the hub of a small needle
previously placed into the tail vein
• Larger blood samples - small incision
over the vessels 0.5 to 2 cm from the tail
base

38. • Cardiac puncture
• needle is inserted under the xyphoid
• needle is advanced at a 20 to 30
degree angle from the horizontal axis
to the sternum to enter the heart.

40. Dosing and Injection
procedures
• S/C – max vol 0.2ml, skin over back or
flank
• I/V – 0.7ml, tail vein
• I/P – 2ml
• I/C – 0.03 ml, midway b/w outer
canthus of eye and attachment of ear
• I/N – 0.1ml
• For infant mice- S/C-0.03ml, I/P
0.05ml, I/C 0.03ml

41. Uses
• General
• bioassay
• toxicity tests
• screening of new compounds
• cancer research
• behaviour research
• Nude mouse – tissue immunity and
transplantation research

42. • Microbiological
• Monoclonal antibodies
• Hemolytic plaque assay
• Tolerance induction through thymic
transplantation
• Immunotherapy for treating metastatic
cancers
• Antitumor effect of immune RNA and
transfer factor
• Xenodiagnosis

43. • T cell dependence for graft rejection
• Animal pathogenecity testing for
Pneumococci
• Toxigenicity testing for Tetanus
• Steroid production in Y1 adrenal cells
• Foot pad for leprosy
• Infant suckling mouse- for isolation of
arbovirus and coxsackie virus
• Infant mouse intragastric test for ST
detection, sealed adult mouse model for ST
• For study of transformation by Griffith
• Best animal for fungal diseases

44. Murine models for B and T cell
immunodeficiency disorders
• Nude mouse- autosomal recessive mutant
- Model congenital T-cell
immunodeficiency disorder
- Tissue immunity ,
Transplantation research
• XID MOUSE – partial B cell defect
- X linked
immunodeficiency
disorder

45. • SCID mouse- defect on a site
designated scid
- serves as a model T
and B cell
ID disorder
- helps to explore the
mechanisms
of gene
rearrangement

46. Transgenic Mice

47. RAT
• Rattus rattus
• Rattus norvegicus
• Handling – tail
- hand round the thorax
• Dosing and injections
• S/C - 0.4ml, neck
• I/V- 0.5ml, tail vein
• I/P-12ml

48. Collection of specimens
• Blood-
-Retro orbital plexus- 1 ml
-Heart – 5ml
-Tail vein – 0.2-0.3 ml
• Urine – metabolism cage
• Faeces – metabolism cage

49. Uses
• Genetics
• Effect of drugs
• Psychological studies
• To study diseases
• Xenodiagnosis

50. Knockout Rat
• Single gene disruption
• Mimics human disease
• Gene function , drug discovery and
development

51. SHEEP
• Ovis aries
Blood- media preparation
RBCs – immunological reactions
Antiserum
• HORSE -Equus ferus caballus
Antiserum

52. Collection of sheep blood
• Restraint: hold the head at about a 30
degree angle to the side
• Shearing: shave an area about 3
inches by 5 inches over the jugular
groove on the neck.

53. • Locating the vein: apply pressure
ata place about half way down the
shaved area, the vein will pop up
• Blood collection: Either a syringe
and needle or a vacutainer needle and
vacutainer may be used.

54. • Syringe and Needle Method:
• pull the plunger back
• depress completely to remove the air
• guide the needle into the vein at the
lowest point on the shaved area
• Gently pull back on the plunger
• blood appears in the syringe

55. • Vacutainer Method:
• With the left hand hold off the vein .
• Aim for the center of the vessel.
• apply pressure to break through the
skin and enter the vessel
• apply pressure so that the tube is
pushed up onto the vacutainer needle.
• blood should flow into the tube.