Animal Experiments in

Medical Microbiology
 Introduction
 Laboratory animals long been used for various
experiments in medical microbiology

 In-vitro techniques to replace animal

experiments(more reproducible results)

 Some animal experiments will always be

required(tests depending on physiological
interaction of the organs of intact animal)
“Biomedical research involving human subjects must
conform to generally accepted scientific principles and
should be based on adequately performed laboratory
and, where appropriate, animal experimentation and on
a thorough knowledge of the scientific literature.”

(Declaration of Helsinki)
 The 3 Rs –The guiding principles in
animal research today:
1. REDUCE the number of animals used to a
minimum
2. REFINE the way experiments are carried
out
3. REPLACE animal experiments with non-
animal techniques where possible
 Animals Used in Medical
Microbiology
 Rodents and Lagomorphs
 Mouse
 Rat
 Voles
 Gerbils
 Guinea pigs
 Hamsters
 Rabbits
 Ungulates-Sheep,goat,horses,pig
 Carnivores-Cat,dog
 Primates
-Monkeys,Chimpanzees
 Birds
-Fowl
 Amphibia-Frogs,toads
 Other mammals-E.g. Armadillo
 Mice in Medical Microbiology
 Mouse(pl-Mice) – Mus musculus
 “Mouse” from Sanskrit “Mush”
 Most commonly used lab animal
 Mammal with very similar genetic
makeup to humans
 Manifold genetic variations
 Convenient size
 High fertility rate
 Rapid generation time(9 wks)
 Cheap and easily available
 History of mice in research

 1800s-Mice fanciers
 1900-Retired school teacher Abbie Lathrop housed over
11,000 mice for their unusual appearance
 1902-Lucien Cuenot,in France first to demonstrate
Mendelian ratios for inheritance of coat colour
characteristics in mice
 1921-Clarence Cook Little-credited with conceiving of
and creating the first inbred strains of lab mice(DBA-
Dilute Brown Agouti)..credited with establishing Jackson
laboratory
 1977-First mouse gene isolated
 2002-First draft of mouse genome published
 Of Mice and Men
 Mouse genome project completed in 2002(strain
used C57BL)

 Mouse one of the 5 central model organisms to

have its genome sequenced

 99% of the genes in mice have an equivalent

gene (or homologue) in humans
 Uses of Mice in Medical
Microbiology
 Bacteriology
 Mouse pathogenicity testing for S.pneumoniae- classic
experiment by Griffith.Demonstration of Transformation
 Bacillus anthracis –Confirmatory test
Differentiate from B. cereus and other related species
 Clostridium botulinum- Animal pathogenicity testing
Mouse bioassay for demonstration of neurotoxin
 Bordetella pertussis-
 Animal pathogenicity test-intranasal inhalation of B.pertussis
causes severe interstitial pneumonia
 Monitor effectiveness of B.pertussis vaccines
 Borrelia diagnosis-intraperitoneal inoculation
 Clostridium tetani-pathogenicity
 Mycobacterium leprae-foot pads of mice
 Intracranial inoculation of adult mice-
 Herpes simplex virus
 Influenza virus
 Rabies virus
 Cryptococcus neoformans
 Intracerebral inoculation of suckling mice
 Arboviruses
 Coxsackie viruses-distinguish Coxsackie A and B
 HSV
 Intraperitoneal inoculation
 Bacterial pathogenicity testing
 Toxoplasma gondii –To maintain live tachyzoites
for Sabin-Feldman test
 IV inoculation-
 Virulence of Nocardia asteroides
 Blastomyces dematitidis
 Toxigenicity of C.tetani
 Preparation of animals

 Handling
 Identification
 Marking with stains(Dye marking scheme by Lumsden
1973)
 Holes in ear with ear punch
 Anaesthesia
 Short acting –Ether
 Long acting-Phenobarbitone i.p
 Ketamine i.m
 Atropine to reduce secretions s.c or i.m
 Administration of materials

 Body fluids
 Blood
 Urine
 CSF
 Serous fluid
 Cultures
 Fluid cultures
 Growth on solid media

 Tissues
 Brain,liver,spleen,liver-Glass tissue grinder
 Muscle,lung,skin,lymph nodes-electrically powered blender
 Routes of inoculation
 Subcutaneous-0.2 ml
 IV-0.7ml(max)
 Intraperitoneal-2ml
 Prior withdrawal of food overnight with access to water to
decrease chances of injection into viscera
 Intracerebral-0.03ml
 Intranasal-0.1ml
 Injection of infant mice
 10X 0.5 mm needle used
 Subcutaneous-0.03ml
 Intraperitoneal-0.05ml
 Intracerebral-0.03 ml
 Collection of blood
 From tail-0.3 ml
 Retroorbital plexus-0.7 ml
 Heart-upto 1.5 ml

 Euthanasia
 Cervical dislocation
 CO2/Coal gas cabinet
 Chamber containing cotton wool saturated
with Chloroform
 CHOICE OF MICE
 GENETIC STATUS
 Inbred mice
 Outbred mice
 Mutant mice
 Nude mice
 SCID mice
 Hybrid mice
 Knockout mice
 Transgenic mice
 MICROBIOLOGICAL STATUS
 Conventional
 Specific Pathogen Free(SPF)
 Gnotobiotic(Germ Free)
 Inbred Mice
 Produced by Brother X Sister matings for atleast
20 generations in such a way that
all individuals trace back to a single common
ancestor
 Inbreeding –F(Coefficient of Inbreeding)
 Inbred strains –Isogenic(genetically identical)
 Advantages
 Genetic stability
 Uniformity
 Characteristics of inbred mice
 Isogenicity-all mice of a particular inbred
strain-identical or isogenic at 99% genetic loci
 Homozygosity
 Stability(genetic)
 Uniformity-only few inbred animals required
for statistical calculations compared to
outbred
 Sensitivity-to experimental t/t and change in
environmental conditions
 Uses
 Used in cancer research for over 60 years
 Hybridoma technology
 Tissue transplantation studies(transfer of
tumours b/w syngeneic and xenogeneic
animals)
 Specific strains developed for
 High incidence of tumours
 Unique pattern of behaviour
 Specific immune responses
 Susceptibility to particular diseases
 List of Inbred strains
 Albino White BALB/c
 Agouti black CBA
 Agouti C3H
 Dilute Brown DBA/1
 Albino white NZW
 Wistar Furth,Wistar Kyoto

 Nomenclature:Inbred strains identified by 1 to 4

letters(code) followed by procurement or
breeding nucleus
 Outbred Mice
 “Genetically undefined”-Genetic makeup totally
unknown at a given locus
 Widely used-experiments where
considerations of specific genotype of lesser
importance
 < 1% inbreeding per generation
 Advantages-
 Healthy,vigorous
 Cheap
 Breed well (because of heterozygosity of genetic
characters)
 Strains: Swiss, NMRI, Wistar
 Name of stock written in capital letters
 Code of person/institute maintaining the
animals precedes stock name
 E.g. Nii:SWISS
 Hybrid Mice
 F1 hybrids-1st generation of a cross b/w 2 inbred
strains
 Many of the useful features of inbred strains
 Preferred to inbred strains for some experiments
 Many of the useful features of inbreds
 Hybrid vigor
 Better survival rate under stress as compared to
inbred
 Longer lifespan and larger litter size
 F1 hybrids- accept grafts from either parent
(converse not true)
 Mutant Mice
 Provide opportunity to study biological
mechanisms
 Mutants-Changes in the genome of an individual
 Environmental factors
 Subjecting animal to mutagenic agents
 Some mutations affect immune system of the
mice(ideal models to study immunological
parameters)
 Mice mutants with immunological defects
 Nude(nu)-chromosome 11

 Beige(bg)-13 (homozygotes closely resemble Chediak

–Higashi disease in man;defective cytotoxic T cell and
cytotoxic ab response)

 Hairless(hr)-14(High incidence of leukaemia)

 X linked immunedeficiency(Xid)-Chr 10

 SCID (scid)-Chr 16
 Mutant mice can be inbred or outbred
 Types of Mutant inbred mice
 Isogenic-2 inbred strains genetically identical
 Coisogenic-single locus change due to point
mutation
 Congenic-strains differing from normal
counterpart by small fragment of single
chromosome
 Nude Mice(Athymic Mice)
 Well studied model of primary immunodeficiency
 Genetic mutation that causes deteriorated or absent
thymus →inhibited immune system
-greatly ↓ no. of T cells
 Genetic trait designated nu controlled by a recessive
gene(FOXN1 gene) on chromosome 11
 Homozygous mice(nu/nu)→
hairless ;vestigeal thymus
 Animals maintained under
conditions protecting them from
infections
 Nude mice-lack CMI responses
Unable to make abs to most antigens
 Tolerate both allografts and xenografts

 USES
 Hybridomas or solid tumour from any origin grown as
ascites or implanted tumor in nude mouse
 Animal model in study of autoimmune diseases
 Demonstrate helper T cell function
 Demonstrate func of T lymphocytes in transplant
rejection
 Cultivation of lepra bacilli and to detect its drug
sensitivity
 SCID Mouse
 Model for primary immunodeficiency
 SCID mouse-Early B and T lineage cells present
 Virtual absence of lymphoid cells in
thymus,spleen,lymph nodes and gut tissue
 Neither make abs nor carry out DTH or graft
rejection
 Useful in studies of cellular immunology
 Window into possible causes of combined T and B
cell immunodeficiency
 1980s-SCID mouse →
In vivo model of the human
immune system
 Human fetal thymus and

lymph nodes transplanted →

then embryonic human immune
cells injected
 SCID mouse circulation contains

Ig of human origin
 SCID mouse reconstituted with human lymphoid tissue

(SCID Hu-mouse)→animal model to test therapeutic

strategies against HIV infection of transplanted human
lymphoid tissue
 Humouse
 “Humanized” mouse models
 Original impetus-Animal model for HIV pathogenesis
 Dengue virus pathogenesis

in vivo and development of

vaccines
 Human specific enteric pathogens

→implanting human fetal intestinal

Xenografts into subscapular region of
SCID mice(SCID-HU-INT model)
 Study of enteric pathogens like

Entamoeba histolytica,Shigella flexneri,

Cryptosporidium parvum
 “Oncohumouse”-harbour both a

human immune system and a human cancer

 Transgenic Mice
 Contain additional artificially introduced
genetic material in every cell
 Most often leads to a gain of function
 Transgenic mouse-very useful system for
studying mammalian gene function and
regulation
 Also to model human diseases that involve
overexpression or misexpression of a
particular protein
 Necessary to introduce the DNA into cells of the
very early mouse embryo that will contribute to
germline

 2 major methods
 Pronuclear microinjection-foreign DNA
introduced directly into mouse egg just after
fertilisation
 Introduction of DNA into embryonic stem cells
 Knockout Mice
 Technique in which a desired gene targeted to specific
sites within the germ line of a mouse
 First knockout mouse-Mario Capecchi,Martin Evans and
Olivier Smithies in 1989
 Primary use-replace a normal gene with a mutant allele
or disrupted form of the gene,knocking out the gene’s
function
 Infering the probable function of a gene
 Mouse models named after the gene which is targeted
e.g.-p53 knockout mouse-named after p53 gene
simulates Li-Fraumeni syndrome seen in humans
 Gene targeted knockout mice
Isolation,culture of ES cells from inner cellmass of mouse blastocyst
↓
Introduction of mutant or disrupted gene into ES cells
↓
Selection of homologous recombinant cells in which gene of interest
knocked out
↓
Inj of homologous recombinant ES cells into recipient mouse
blastocyst
↓
Surgical implantation of blastocyst into pseudopregnant mouse
↓
Mating of chimeric offsprings heterozygous for the disrupted gene
produce homozygous knockout mice
 Hybridoma Technology
 Large scale production of monoclonal antibodies against
any desired antigen

 Kohler and Milstein -1975(hybridoma technology)

 Hybridomas-somatic cell hybrids→fusion of ab producing

spleen cells with myeloma cells
↓
Hybrid with ab producing capacity of spleen cell
And ability of myeloma cells to multiply indefinitely
 Mouse monoclonals humanised-chimeric abs(murine
variable regions and human constant regions
 E.g.Infliximab
 Gnotobiotic Animals
 Conventional animals:Undefined flora
 Specific pathogen Free (SPF) animals:
 Free from a list of possibly pathogenic organisms
 Caesarean section;reared in extremely clean but not
sterile conditions
 Gnotobiotic Animals:
 “Gnotobiotic”-Gnotos-Well known(Greek)
Biota-all life
 Germ-free or associated with any number of
organisms of established identity
 Gnotobiotic experiment:tool in study of
host microbial relationships :
 Portrays the host either when free from germs
and left to its own resources or when modified
by known microbiota
 Permits study of interflora relationship within
host organism
 Study of any endogenous
factors(nutrition,immune reactions to various
stimuli)
 Embryos developing inside an egg or the mammalian
uterus –microbiologically sterile(provided from healthy
parent stock)
 Mammals-aseptically derived by hysterectomy or
hysterotomy as late as possible before term
 Gnotobiotic animals
 Mice/Rats
 Guinea pigs
 Rabbits
 Beagle dogs
 Piglets
 Chicken
 Gnotobiotic Animals

Isolator for Gnotobiotic animals Gnotobiotic mice

 USES
 Early incentive for gnotobiotic experiments-Study of
body defenses
 Oral disease(caries,periodontal disease)
 Oldest,best documented and most fruitful
application of this biological tool
 Rats used
 Enteric infection models-V.cholerae,Shigella,
Salmonella typhimurium
 Study of EPEC(O140:K)-in gnotobiotic piglets
 Model for H .pylori infection-Gnotobiotic piglet
 Other commonly used animals in
Microbiology
 Guinea pig(Cavia porcellus)
 C.diphtheriae toxigenicity
 M.tuberculosis and M.bovis pathogenicity
 Brucella-most susceptible animal
 Clostridium tetani toxigenicity
 C.perfringens and C.novyi lab diagnosis
 Pneumonic plague lab diagnosis
 Standardisation of toxins and antitoxins(Diphtheria)
 Source of complement
 Animal model for anaphylaxis
 Paul Bunnel test for IM
 Sereny’s test(EIEC,Shigella)
 Neil Mooser or tunica reaction for rickettsiae
 Leptospira-blood and urine inoculation intraperitoneally
 Strauss reaction for Pseudomonas mallei
 Rat(Ratus norvegicus)
 Diagnosis of Plague
 Animal model in study of bacterial
endocarditis
 Rats treated with steroids for study of
spontaneous fungal infections
 Rabbit(Oryctolagus caniculus)
 Source of blood for blood agar
 To raise high titre antisera and amboceptor
 Maintain Nichol’s strain of T. pallidum intratesticularly
 Toxigenicity testing(Ileal loop assay)
 Sereny’s test
 Propagation of vaccinia virus
 Paul’s test(Scarification of rabbit cornea with Variola
virus-keratitis and guarneri bodies)
 Differentiate M tuberculosis and M bovis
 Fowl
 RBCs for hemagglutination(V.cholerae)
 Golden Hamster
 Leishmania brasiliensis
 Leishmania tropica(Syrian Hamster)
 L donovani-Chinese hamster(i.p)
 Ferrets-Influenza
 Monkey(Aotus triroroatus)-cultivation of
P.falciparum
 Chimpanzee- cultivation of Hepatitis virus
Animal model for gonococcal urethritis
 Armadillo(nine banded)-cultivation of M .leprae
 Generation of Antisera
 Antibody formation-2 phases
 Induction phase
 Production phase
 Choice of animals
 Rabbits,goats,sheep,horses
 Form and dose of ags
 Soluble and particulate proteins,viruses,subcellular
particulates,entire cells
 Adjuvants
 Freund’s adjuvant
 LPS
 Bordetella pertussis
 Route of immunization-
i.d,subcutaneous,i.m,i.p,intravenous,footpad
 Immunization and Bleeding schedule
 Chicken Antibodies
 Egg yolk as a source of immunoglobulins for
diagnostic,prophylactic and therapeutic
purposes
 While egg still in the ovary, transfer of serum Igs
into the yolk
 IgM and IgA transferred with other proteins in oviduct
into egg white
 IgG(IgY in chickens) in egg follicle passed by
receptors selectively into egg yolk
 IgY conc in yolk comparable to conc in serum
 Evaluation of IgY against Rotavirus
 Animals in Vaccine Research
 Polio vaccine-
 Mice used to study stages of Polio and in subsequent development of
vaccines
 TB vaccine-New DNA vaccine developed in mice
 Protection against mice getting infected
 Also works as a t/t by stimulating immune system
 SARS-A DNA vaccine induces SARS coronavirus neutralization and
protective immunity in mice

 Rabies vaccine-

 JE vaccine-Formalin inactivated mouse brain vaccine used for

human immunization in Japan(using Nakayama strain)
 Malaria vaccines-Rhesus monkey
 Infected mice shown to generate immune
responses against malaria parasite
 Studying the proteins of parasite causing this
immune response may lead to development
of a vaccine
 HIV vaccine research: Mice used
 Pre-clinical safety studies on animals
 Actual vaccine production process –only blood or serum
of animals may be required for culturing media
 Extensive use of animals(especially mice) for quality
control
 Safety Testing
 Specific toxicity
 Test for residual live virus-eg-Rabies virus causes death on
intracerebral inoculation
 Identity test-Serum antibody assay
 Antimicrobial Research
 Testing of potential antimicrobial agents in animal
models of infectious disease-
Essential prerequisite of anti-infective therapy
 Bridging of gap b/w invitro characterization and clinical
evaluation of antimicrobial agents
 In-vivo testing
 Invitro activities of antimicrobial agents altered by host factors
(metabolic processes,anti-infective defense mechanisms)
 Animal testing-prerequisite to clinical trials
 Toxicity testing in animals- clear indications of possible
short term and long term toxic effects and maximum
tolerable doses
 Efficacy evaluation:
 Various types of models used
 Basic antimicrobial screening
 Model most commonly used-systemic inf in mice
 Results evaluated on basis of “all-or-none parameters”
 E.g.-Meningitis,Pneumonia in mice
 Ex vivo models
 Foreign body implanted subcutaneously,animal subsequently infected
 Accumulations in foreign body sampled
 Valuable for determining capacity of antibiotic to penetrate specific site of
infection
 Discriminative models
 Complicated;mimic the initiation and progress of infection in humans
 Multiple parameters of efficacy measured
 Have delineated effective therapeutic strategies now adopted clinically
 Rifampicin + Va for t/t of Osteomyelitis
 Beta lactams + aminoglycosides in Endocarditis
 Adjunctive Dexamethasone therapy for bacterial meningitis
 CPCSEA guidelines for laboratory
animal facilities
 Committee for the Purpose of Control and
Supervision of Experiments on Animals

 Ministry of Social Justice and Empowerment,

Govt. of India

 Good Laboratory Practices(GLP) for animal

facilities
 Goal
 To promote the humane care of animals used
in biomedical and behavioural research

 Testing with the basic objective of providing

specifications that will enhance animal well
being,quality in the pursuit of advancement of
biological knowledge that is relevant to
humans and animals
 Veterinary care
 Animal procurement
 Quarantine,Stabilization and Separation
 Surveillance,Diagnosis,t/t and control of diseases
 Animal care and technical personnel
 Personal hygiene
 Animal experiments involving hazardous agents
 Multiple surgical procedures on single animal
 Duration of experiments
 Environment
 Food and water
 Sanitation and cleanliness
 Waste disposal
 Pest control
 Record keeping
 Personnel and training
Conflicting Views
 Alternatives
to
animal experiments
Replacement
Reduction
Refinement
 Replacement
· in vitro techniques · human material
· audio-visual aids
· slaughterhouse
material
· invertebrates
 Reduction
• pre-screening
• choice animal
• experimental set-up
model
(standardisation/
statistics)
• ethical verification
 Refinement
• animal care
• study performed by skilled
persons (bio-technicians)
• analgesia, anaesthesia,
euthanasia
• animal behaviour
 Hierarchy of substrates
· human · subcellular fractions
· animal species / strains · molecular fractions,
· surviving - embryo DNA
- organ · epidemiological data
- tissue · computer model
- cells · physical / chemical
· cell culture model
· bacteria, yeasts,
protozoa
 Conclusion
 Use of animals in medical microbiology and
biomedical research
 New variety of animal models in immunology
and cancer research

 Need for an optimum balance between use of

animal models and alternatives

 Animal research necessary in vaccine and drug

development
 References
 Practical Medical Microbiology-Mackie and McCartney- 14th edition

 Handbook of practical and clinical Immunology-Talwar

 Kuby Immunology-6th edition

 CPCSEA guidelines

 Gnotobiotic animal as a tool in the study of host microbial

relationships-Gordon and Pesti,Bacteriological Reviews,Dec 1971
 www.jax.org-Jackson laboratory
 www.harlan.com