Animal Experiments in Animal Experiments in Medical MicrobiologyMedical Microbiology

IntroductionIntroduction

Laboratory animals long been used for various Laboratory animals long been used for various experiments in medical microbiologyexperiments in medical microbiology

In-vitro techniques to replace animal In-vitro techniques to replace animal experiments(more reproducible results)experiments(more reproducible results)

Some animal experiments will always be Some animal experiments will always be required(tests depending on physiological required(tests depending on physiological interaction of the organs of intact animal)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 The 3 Rs3 Rs –The guiding principles in –The guiding principles in animal research today:animal research today:

1.1. REDUCE the number of animals used to a REDUCE the number of animals used to a minimumminimum

2.2. REFINE the way experiments are carried REFINE the way experiments are carried outout

3.3. REPLACE animal experiments with non-REPLACE animal experiments with non-animal techniques where possibleanimal techniques where possible

Animals Used in Medical Animals Used in Medical MicrobiologyMicrobiology

Rodents and LagomorphsRodents and Lagomorphs MouseMouse Rat Rat VolesVoles GerbilsGerbils Guinea pigsGuinea pigs HamstersHamsters RabbitsRabbits

Ungulates-Sheep,goat,horses,pigUngulates-Sheep,goat,horses,pig Carnivores-Cat,dogCarnivores-Cat,dog

PrimatesPrimates

-Monkeys,Chimpanzees-Monkeys,Chimpanzees BirdsBirds

-Fowl-Fowl Amphibia-Frogs,toadsAmphibia-Frogs,toads Other mammals-E.g. ArmadilloOther mammals-E.g. Armadillo

Mice in Medical MicrobiologyMice in Medical Microbiology

Mouse(pl-Mice) – Mouse(pl-Mice) – Mus musculusMus musculus ““Mouse” from Sanskrit “Mush”Mouse” from Sanskrit “Mush” Most commonly used lab animalMost commonly used lab animal

Mammal with very similar genetic Mammal with very similar genetic makeup to humansmakeup to humans Manifold genetic variationsManifold genetic variations Convenient sizeConvenient size High fertility rateHigh fertility rate Rapid generation time(9 wks)Rapid generation time(9 wks) Cheap and easily availableCheap and easily available

History of mice in researchHistory of mice in research

1800s-Mice fanciers1800s-Mice fanciers 1900-Retired school teacher Abbie Lathrop housed over 1900-Retired school teacher Abbie Lathrop housed over

11,000 mice for their unusual appearance11,000 mice for their unusual appearance 1902-Lucien Cuenot,in France first to demonstrate 1902-Lucien Cuenot,in France first to demonstrate

Mendelian ratiosMendelian ratios for inheritance of coat colour for inheritance of coat colour characteristics in micecharacteristics in mice

1921-Clarence Cook Little-credited with conceiving of 1921-Clarence Cook Little-credited with conceiving of and creating the first inbred strains of lab mice(DBA-and creating the first inbred strains of lab mice(DBA-Dilute Brown Agouti)..credited with establishing Jackson Dilute Brown Agouti)..credited with establishing Jackson laboratorylaboratory

1977-First mouse gene isolated1977-First mouse gene isolated 2002-First draft of mouse genome published2002-First draft of mouse genome published

Of Mice and MenOf Mice and Men Mouse genome project completed in 2002(strain Mouse genome project completed in 2002(strain

used C57BL)used C57BL)

Mouse one of the 5 central model organisms to Mouse one of the 5 central model organisms to have its genome sequencedhave its genome sequenced

99% of the genes in mice have an equivalent 99% of the genes in mice have an equivalent gene (or homologue) in humansgene (or homologue) in humans

Uses of Mice in Medical Uses of Mice in Medical MicrobiologyMicrobiology

BacteriologyBacteriology Mouse pathogenicity testing for Mouse pathogenicity testing for S.pneumoniae-S.pneumoniae- classic classic

experiment by Griffith.Demonstration of experiment by Griffith.Demonstration of TransformationTransformation

Bacillus anthracisBacillus anthracis –Confirmatory test –Confirmatory test

Differentiate from Differentiate from B. cereus B. cereus and other related speciesand other related species Clostridium botulinumClostridium botulinum- Animal pathogenicity testing- Animal pathogenicity testing

Mouse bioassay for demonstration of neurotoxinMouse bioassay for demonstration of neurotoxin Bordetella pertussis-Bordetella pertussis-

Animal pathogenicity test-intranasal inhalation of Animal pathogenicity test-intranasal inhalation of B.pertussisB.pertussis

causes severe interstitial pneumoniacauses severe interstitial pneumonia Monitor effectiveness of Monitor effectiveness of B.pertussisB.pertussis vaccines vaccines

Borrelia Borrelia diagnosis-intraperitoneal inoculationdiagnosis-intraperitoneal inoculation ClostridiumClostridium tetanitetani-pathogenicity-pathogenicity Mycobacterium leprae-Mycobacterium leprae-foot pads of micefoot pads of mice

Intracranial inoculation of adult mice-Intracranial inoculation of adult mice- Herpes simplex virusHerpes simplex virus Influenza virusInfluenza virus Rabies virus Rabies virus Cryptococcus neoformansCryptococcus neoformans

Intracerebral inoculation of suckling miceIntracerebral inoculation of suckling mice ArbovirusesArboviruses Coxsackie viruses-distinguish Coxsackie A and BCoxsackie viruses-distinguish Coxsackie A and B HSVHSV

Intraperitoneal inoculationIntraperitoneal inoculation Bacterial pathogenicity testingBacterial pathogenicity testing Toxoplasma gondii –Toxoplasma gondii –To maintain live tachyzoitesTo maintain live tachyzoites

for Sabin-Feldman testfor Sabin-Feldman test

IV inoculation-IV inoculation- Virulence of Virulence of Nocardia asteroidesNocardia asteroides Blastomyces dematitidisBlastomyces dematitidis Toxigenicity of Toxigenicity of C.tetaniC.tetani

Preparation of animalsPreparation of animals

HandlingHandling IdentificationIdentification

Marking with stains(Dye marking scheme by Lumsden Marking with stains(Dye marking scheme by Lumsden 1973)1973)

Holes in ear with ear punchHoles in ear with ear punch AnaesthesiaAnaesthesia

Short acting –EtherShort acting –Ether Long acting-Phenobarbitone i.pLong acting-Phenobarbitone i.p Ketamine i.mKetamine i.m Atropine to reduce secretions s.c or i.mAtropine to reduce secretions s.c or i.m

Administration of materialsAdministration of materials

Body fluidsBody fluids BloodBlood UrineUrine CSFCSF Serous fluidSerous fluid

CulturesCultures Fluid culturesFluid cultures Growth on solid mediaGrowth on solid media

TissuesTissues Brain,liver,spleen,liver-Glass tissue grinderBrain,liver,spleen,liver-Glass tissue grinder Muscle,lung,skin,lymph nodes-electrically powered blenderMuscle,lung,skin,lymph nodes-electrically powered blender

Routes of inoculationRoutes of inoculation Subcutaneous-0.2 mlSubcutaneous-0.2 ml IV-0.7ml(max)IV-0.7ml(max) Intraperitoneal-2mlIntraperitoneal-2ml

Prior withdrawal of food overnight with access to water to Prior withdrawal of food overnight with access to water to decrease chances of injection into visceradecrease chances of injection into viscera

Intracerebral-0.03mlIntracerebral-0.03ml Intranasal-0.1mlIntranasal-0.1ml Injection of infant miceInjection of infant mice

10X 0.5 mm needle used10X 0.5 mm needle used Subcutaneous-0.03mlSubcutaneous-0.03ml Intraperitoneal-0.05mlIntraperitoneal-0.05ml Intracerebral-0.03 mlIntracerebral-0.03 ml

Collection of bloodCollection of bloodFrom tail-0.3 mlFrom tail-0.3 mlRetroorbital plexus-0.7 mlRetroorbital plexus-0.7 mlHeart-upto 1.5 mlHeart-upto 1.5 ml

EuthanasiaEuthanasiaCervical dislocationCervical dislocationCO2/Coal gas cabinetCO2/Coal gas cabinetChamber containing cotton wool saturated Chamber containing cotton wool saturated

with Chloroformwith Chloroform

CHOICE OF MICECHOICE OF MICE

GENETIC STATUSGENETIC STATUS Inbred miceInbred miceOutbred miceOutbred miceMutant miceMutant mice

Nude miceNude miceSCID miceSCID mice

Hybrid miceHybrid miceKnockout miceKnockout miceTransgenic miceTransgenic mice

MICROBIOLOGICAL STATUSMICROBIOLOGICAL STATUSConventionalConventionalSpecific Pathogen Free(SPF)Specific Pathogen Free(SPF)Gnotobiotic(Germ Free)Gnotobiotic(Germ Free)

Inbred MiceInbred Mice

Produced by Brother X Sister matings for Produced by Brother X Sister matings for atleast atleast 20 generations 20 generations in such a way that in such a way that

all individuals trace back to a single common all individuals trace back to a single common ancestorancestor

Inbreeding –F(Coefficient of Inbreeding)Inbreeding –F(Coefficient of Inbreeding) Inbred strains –Isogenic(genetically identical)Inbred strains –Isogenic(genetically identical) AdvantagesAdvantages

Genetic stabilityGenetic stability UniformityUniformity

Characteristics of inbred miceCharacteristics of inbred mice Isogenicity-all mice of a particular inbred Isogenicity-all mice of a particular inbred

strain-identical or isogenic at 99% genetic locistrain-identical or isogenic at 99% genetic lociHomozygosityHomozygosityStability(genetic)Stability(genetic)Uniformity-only few inbred animals required Uniformity-only few inbred animals required

for statistical calculations compared to for statistical calculations compared to outbredoutbred

Sensitivity-to experimental t/t and change in Sensitivity-to experimental t/t and change in environmental conditionsenvironmental conditions

UsesUsesUsed in cancer research for over 60 yearsUsed in cancer research for over 60 yearsHybridoma technologyHybridoma technologyTissue transplantation studies(transfer of Tissue transplantation studies(transfer of

tumours b/w syngeneic and xenogeneic tumours b/w syngeneic and xenogeneic animals)animals)

Specific strains developed forSpecific strains developed forHigh incidence of tumoursHigh incidence of tumoursUnique pattern of behaviourUnique pattern of behaviourSpecific immune responsesSpecific immune responsesSusceptibility to particular diseasesSusceptibility to particular diseases

List of Inbred strains List of Inbred strains Albino White BALB/cAlbino White BALB/cAgouti black CBAAgouti black CBAAgouti C3HAgouti C3HDilute Brown DBA/1Dilute Brown DBA/1Albino white NZWAlbino white NZWWistar Furth,Wistar KyotoWistar Furth,Wistar Kyoto

Nomenclature:Inbred strains identified by 1 to 4 Nomenclature:Inbred strains identified by 1 to 4 letters(code) followed by procurement or letters(code) followed by procurement or breeding nucleusbreeding nucleus

Outbred MiceOutbred Mice

““Genetically undefined”-Genetic makeup totally Genetically undefined”-Genetic makeup totally unknown at a given locusunknown at a given locus

Widely used-experiments where Widely used-experiments where considerations of specific genotype of lesser considerations of specific genotype of lesser importanceimportance

< 1% inbreeding per generation< 1% inbreeding per generation Advantages-Advantages-

Healthy,vigorousHealthy,vigorous CheapCheap Breed well (because of heterozygosity of genetic Breed well (because of heterozygosity of genetic

characters)characters)

Strains: Swiss, NMRI, WistarStrains: Swiss, NMRI, WistarName of stock written in capital lettersName of stock written in capital lettersCode of person/institute maintaining the Code of person/institute maintaining the

animals precedes stock nameanimals precedes stock nameE.g. Nii:SWISSE.g. Nii:SWISS

Hybrid MiceHybrid Mice F1 hybrids-1F1 hybrids-1stst generation of a cross b/w 2 inbred generation of a cross b/w 2 inbred

strainsstrains Many of the useful features of inbred strainsMany of the useful features of inbred strains Preferred to inbred strains for some experimentsPreferred to inbred strains for some experiments

Many of the useful features of inbredsMany of the useful features of inbreds Hybrid vigorHybrid vigor Better survival rate under stress as compared to Better survival rate under stress as compared to

inbredinbred Longer lifespan and larger litter sizeLonger lifespan and larger litter size

F1 hybrids- accept grafts from either parentF1 hybrids- accept grafts from either parent

(converse not true)(converse not true)

Mutant MiceMutant Mice

Provide opportunity to study biological Provide opportunity to study biological mechanismsmechanisms

Mutants-Changes in the genome of an individualMutants-Changes in the genome of an individual Environmental factorsEnvironmental factors Subjecting animal to mutagenic agentsSubjecting animal to mutagenic agents

Some mutations affect immune system of the Some mutations affect immune system of the mice(ideal models to study immunological mice(ideal models to study immunological parameters) parameters)

Mice mutants with immunological defectsMice mutants with immunological defects

Nude(nu)-chromosome 11Nude(nu)-chromosome 11

Beige(bg)-13 Beige(bg)-13 (homozygotes closely resemble Chediak (homozygotes closely resemble Chediak –Higashi disease in man;defective cytotoxic T cell and –Higashi disease in man;defective cytotoxic T cell and cytotoxic ab response)cytotoxic ab response)

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

X linked immunedeficiency(Xid)-Chr 10X linked immunedeficiency(Xid)-Chr 10

SCID (scid)-Chr 16SCID (scid)-Chr 16

Mutant mice can be inbred or outbredMutant mice can be inbred or outbredTypes of Mutant inbred miceTypes of Mutant inbred mice

Isogenic-2 inbred strains genetically identicalIsogenic-2 inbred strains genetically identicalCoisogenic-single locus change due to point Coisogenic-single locus change due to point

mutationmutationCongenic-strains differing from normal Congenic-strains differing from normal

counterpart by small fragment of single counterpart by small fragment of single chromosomechromosome

Nude Mice(Athymic Mice)Nude Mice(Athymic Mice) Well studied model of primary immunodeficiencyWell studied model of primary immunodeficiency Genetic mutation that causes deteriorated or absent Genetic mutation that causes deteriorated or absent

thymus thymus →inhibited immune system→inhibited immune system -greatly ↓ no. of T cells-greatly ↓ no. of T cells Genetic trait designated nu controlled by a recessive Genetic trait designated nu controlled by a recessive

gene(FOXN1 gene) on chromosome 11gene(FOXN1 gene) on chromosome 11 Homozygous mice(nu/nu)→Homozygous mice(nu/nu)→ hairless ;vestigeal thymushairless ;vestigeal thymus Animals maintained under Animals maintained under conditions protecting them fromconditions protecting them from infectionsinfections

Nude mice-lack CMI responsesNude mice-lack CMI responses Unable to make abs to most antigensUnable to make abs to most antigens Tolerate both allografts and xenograftsTolerate both allografts and xenografts

USESUSES Hybridomas or solid tumour from any origin grown as Hybridomas or solid tumour from any origin grown as

ascites or implanted tumor in nude mouseascites or implanted tumor in nude mouse Animal model in study of autoimmune diseasesAnimal model in study of autoimmune diseases Demonstrate helper T cell functionDemonstrate helper T cell function Demonstrate func of T lymphocytes in transplant Demonstrate func of T lymphocytes in transplant

rejectionrejection Cultivation of lepra bacilli and to detect its drug Cultivation of lepra bacilli and to detect its drug

sensitivitysensitivity

SCID MouseSCID Mouse

Model for primary immunodeficiencyModel for primary immunodeficiency SCID mouse-Early B and T lineage cells presentSCID mouse-Early B and T lineage cells present Virtual absence of lymphoid cells in Virtual absence of lymphoid cells in

thymus,spleen,lymph nodes and gut tissuethymus,spleen,lymph nodes and gut tissue Neither make abs nor carry out DTH or graft Neither make abs nor carry out DTH or graft

rejection rejection Useful in studies of cellular immunologyUseful in studies of cellular immunology

Window into possible causes of combined T and B Window into possible causes of combined T and B cell immunodeficiencycell immunodeficiency

1980s-SCID mouse 1980s-SCID mouse →→

In vivo model of the humanIn vivo model of the human

immune systemimmune system Human fetal thymus and Human fetal thymus and

lymph nodes transplanted →lymph nodes transplanted →

then embryonic human immunethen embryonic human immune

cells injectedcells injected SCID mouse circulation containsSCID mouse circulation contains

Ig of human originIg of human origin SCID mouse reconstituted with human lymphoid tissueSCID mouse reconstituted with human lymphoid tissue

(SCID Hu-mouse)(SCID Hu-mouse)→animal model to test therapeutic →animal model to test therapeutic strategies against strategies against HIV infectionHIV infection of transplanted human of transplanted human lymphoid tissuelymphoid tissue

HumouseHumouse ““Humanized” mouse modelsHumanized” mouse models Original impetus-Animal model for HIV pathogenesisOriginal impetus-Animal model for HIV pathogenesis Dengue virus pathogenesisDengue virus pathogenesis

in vivo and development ofin vivo and development of

vaccinesvaccines Human specific enteric pathogensHuman specific enteric pathogens

→→implanting human fetal intestinalimplanting human fetal intestinal

Xenografts into subscapular region of Xenografts into subscapular region of

SCID miceSCID mice(SCID-HU-INT(SCID-HU-INT model)model) Study of enteric pathogens likeStudy of enteric pathogens like

Entamoeba histolytica,Shigella flexneri,Entamoeba histolytica,Shigella flexneri,

Cryptosporidium parvumCryptosporidium parvum ““Oncohumouse”-Oncohumouse”-harbour both a harbour both a

human immune system and a human cancerhuman immune system and a human cancer

Transgenic MiceTransgenic Mice

Contain additional artificially introduced Contain additional artificially introduced genetic material in every cellgenetic material in every cellMost often leads to a gain of functionMost often leads to a gain of function

Transgenic mouse-very useful system for Transgenic mouse-very useful system for studying mammalian gene function and studying mammalian gene function and

regulationregulationAlso to model human diseases that involve Also to model human diseases that involve

overexpression or misexpression of a overexpression or misexpression of a particular proteinparticular protein

Necessary to introduce the DNA into cells of the Necessary to introduce the DNA into cells of the very early mouse embryo that will contribute to very early mouse embryo that will contribute to germline germline

2 major methods2 major methodsPronuclear microinjection-foreign DNA Pronuclear microinjection-foreign DNA

introduced directly into mouse egg just after introduced directly into mouse egg just after fertilisationfertilisation

Introduction of DNA into embryonic stem cellsIntroduction of DNA into embryonic stem cells

Knockout MiceKnockout Mice Technique in which a desired gene targeted to specific Technique in which a desired gene targeted to specific

sites within the germ line of a mousesites within the germ line of a mouse First knockout mouse-Mario Capecchi,Martin Evans andFirst knockout mouse-Mario Capecchi,Martin Evans and Olivier Smithies in 1989Olivier Smithies in 1989 Primary use-replace a normal gene with a mutant allele Primary use-replace a normal gene with a mutant allele

or disrupted form of the gene,knocking out the gene’s or disrupted form of the gene,knocking out the gene’s functionfunction

Infering the probable function of a geneInfering the probable function of a gene Mouse modelsMouse models named named after the gene which is targetedafter the gene which is targeted

e.g.-p53 knockout mouse-named after p53 genee.g.-p53 knockout mouse-named after p53 gene simulates Li-Fraumeni syndrome seen in humanssimulates Li-Fraumeni syndrome seen in humans

Gene targeted knockout miceGene targeted knockout mice

Isolation,culture of ES cellsIsolation,culture of ES cells from inner cellmass of mouse blastocyst from inner cellmass of mouse blastocyst

↓↓

Introduction of mutant or disrupted geneIntroduction of mutant or disrupted gene into ES cells into ES cells

↓ ↓

SelectionSelection of homologous recombinant cells in which gene of interest of homologous recombinant cells in which gene of interest knocked outknocked out

↓ ↓

Inj of homologous recombinant ES cells into Inj of homologous recombinant ES cells into recipient mouserecipient mouse blastocystblastocyst

↓ ↓

Surgical implantation of blastocystSurgical implantation of blastocyst into pseudopregnant mouse into pseudopregnant mouse

↓ ↓

Mating of chimeric offspringsMating of chimeric offsprings heterozygous for the disrupted gene heterozygous for the disrupted gene

produce homozygous knockout miceproduce homozygous knockout mice

Hybridoma TechnologyHybridoma Technology Large scale production of monoclonal antibodies against Large scale production of monoclonal antibodies against

any desired antigenany desired antigen

Kohler and Milstein -1975(hybridoma technology)Kohler and Milstein -1975(hybridoma technology)

Hybridomas-somatic cell hybridsHybridomas-somatic cell hybrids→fusion of ab producing →fusion of ab producing spleen cells with myeloma cellsspleen cells with myeloma cells

↓ ↓

Hybrid with ab producing capacity of spleen cellHybrid with ab producing capacity of spleen cell

And ability of myeloma cells to multiply indefinitelyAnd ability of myeloma cells to multiply indefinitely Mouse monoclonals humanised-Mouse monoclonals humanised-chimericchimeric absabs(murine (murine

variable regions and human constant regionsvariable regions and human constant regions E.g.InfliximabE.g.Infliximab

Gnotobiotic AnimalsGnotobiotic Animals

Conventional animals:Undefined floraConventional animals:Undefined flora Specific pathogen Free (SPF) animals:Specific pathogen Free (SPF) animals:

Free from a list of possibly pathogenic organismsFree from a list of possibly pathogenic organisms Caesarean section;reared in Caesarean section;reared in extremely clean but notextremely clean but not

sterilesterile conditions conditions

Gnotobiotic AnimalsGnotobiotic Animals:: ““Gnotobiotic”-Gnotos-Well known(Greek)Gnotobiotic”-Gnotos-Well known(Greek)

Biota-all lifeBiota-all life Germ-free or associated with any number of Germ-free or associated with any number of

organisms of established identityorganisms of established identity

Gnotobiotic experiment:tool in study of Gnotobiotic experiment:tool in study of host microbial relationships :host microbial relationships :Portrays the host either when free from germs Portrays the host either when free from germs

and left to its own resources or when modified and left to its own resources or when modified by known microbiotaby known microbiota

Permits study of interflora relationship within Permits study of interflora relationship within host organismhost organism

Study of any endogenous Study of any endogenous factors(nutrition,immune reactions to various factors(nutrition,immune reactions to various stimuli)stimuli)

Embryos developing inside an egg or the mammalian Embryos developing inside an egg or the mammalian uterus –microbiologically sterile(provided from healthy uterus –microbiologically sterile(provided from healthy parent stock)parent stock)

Mammals-aseptically derived by hysterectomy or Mammals-aseptically derived by hysterectomy or hysterotomy as late as possible before termhysterotomy as late as possible before term

Gnotobiotic animalsGnotobiotic animals Mice/RatsMice/Rats Guinea pigsGuinea pigs RabbitsRabbits Beagle dogsBeagle dogs PigletsPiglets ChickenChicken

Gnotobiotic AnimalsGnotobiotic Animals

Isolator for Gnotobiotic animals Gnotobiotic miceIsolator for Gnotobiotic animals Gnotobiotic mice

USESUSES Early incentive for gnotobiotic experiments-Study of Early incentive for gnotobiotic experiments-Study of

body defensesbody defenses Oral disease(caries,periodontal disease)Oral disease(caries,periodontal disease)

Oldest,best documented and most fruitful Oldest,best documented and most fruitful application of this biological toolapplication of this biological tool

Rats usedRats used Enteric infection models-Enteric infection models-V.choleraeV.cholerae,,ShigellaShigella,,Salmonella typhimuriumSalmonella typhimurium Study of EPEC(O140:K)-in gnotobiotic pigletsStudy of EPEC(O140:K)-in gnotobiotic piglets Model for Model for H .pyloriH .pylori infection-Gnotobiotic piglet infection-Gnotobiotic piglet

Other commonly used animals in Other commonly used animals in MicrobiologyMicrobiology

Guinea pig(Guinea pig(Cavia porcellus)Cavia porcellus) C.diphtheriae C.diphtheriae toxigenicitytoxigenicity M.tuberculosis and M.bovis M.tuberculosis and M.bovis pathogenicitypathogenicity Brucella-most susceptible animalBrucella-most susceptible animal Clostridium tetaniClostridium tetani toxigenicity toxigenicity C.perfringens C.perfringens and and C.novyi C.novyi lab diagnosislab diagnosis Pneumonic plague lab diagnosisPneumonic plague lab diagnosis Standardisation of toxins and antitoxins(Diphtheria)Standardisation of toxins and antitoxins(Diphtheria) Source of complementSource of complement Animal model for anaphylaxisAnimal model for anaphylaxis Paul Bunnel test for IMPaul Bunnel test for IM Sereny’s test(EIEC,Shigella)Sereny’s test(EIEC,Shigella) Neil Mooser or tunica reaction for rickettsiaeNeil Mooser or tunica reaction for rickettsiae Leptospira-blood and urine inoculation intraperitoneallyLeptospira-blood and urine inoculation intraperitoneally Strauss reaction for Strauss reaction for Pseudomonas malleiPseudomonas mallei

Rat(Rat(RatusRatus norvegicus)norvegicus)Diagnosis of PlagueDiagnosis of PlagueAnimal model in study of bacterial Animal model in study of bacterial

endocarditisendocarditisRats treated with steroids for study of Rats treated with steroids for study of

spontaneous fungal infectionsspontaneous fungal infections

Rabbit(Rabbit(OryctolagusOryctolagus caniculus)caniculus) Source of blood for blood agarSource of blood for blood agar To raise high titre antisera and amboceptorTo raise high titre antisera and amboceptor Maintain Nichol’s strain of Maintain Nichol’s strain of T. pallidumT. pallidum intratesticularly intratesticularly Toxigenicity testing(Ileal loop assay)Toxigenicity testing(Ileal loop assay) Sereny’s testSereny’s test Propagation of vaccinia virusPropagation of vaccinia virus Paul’s test(Scarification of rabbit cornea with Variola Paul’s test(Scarification of rabbit cornea with Variola

virus-keratitis and guarneri bodies)virus-keratitis and guarneri bodies) Differentiate Differentiate M tuberculosisM tuberculosis and and M bovisM bovis

FowlFowlRBCs for hemagglutination(RBCs for hemagglutination(V.choleraeV.cholerae))

Golden HamsterGolden HamsterLeishmania brasiliensisLeishmania brasiliensisLeishmania tropicaLeishmania tropica(Syrian Hamster)(Syrian Hamster)LL donovanidonovani-Chinese hamster(i.p)-Chinese hamster(i.p)

FerretsFerrets-Influenza-Influenza Monkey(Monkey(AotusAotus triroroatustriroroatus)-)-cultivation of cultivation of

P.falciparumP.falciparum ChimpanzeeChimpanzee- - cultivation of Hepatitis viruscultivation of Hepatitis virus Animal model for gonococcal urethritisAnimal model for gonococcal urethritis ArmadilloArmadillo(nine banded)-cultivation of (nine banded)-cultivation of M .lepraeM .leprae

Generation of AntiseraGeneration of Antisera Antibody formation-2 phasesAntibody formation-2 phases

Induction phaseInduction phase Production phaseProduction phase

Choice of animalsChoice of animals Rabbits,goats,sheep,horsesRabbits,goats,sheep,horses

Form and dose of agsForm and dose of ags Soluble and particulate proteins,viruses,subcellular Soluble and particulate proteins,viruses,subcellular

particulates,entire cellsparticulates,entire cells AdjuvantsAdjuvants

Freund’s adjuvantFreund’s adjuvant LPSLPS Bordetella pertussisBordetella pertussis

Route of immunization-Route of immunization-i.d,subcutaneous,i.m,i.p,intravenous,footpadi.d,subcutaneous,i.m,i.p,intravenous,footpad

Immunization and Bleeding scheduleImmunization and Bleeding schedule

Chicken AntibodiesChicken Antibodies

Egg yolk as a source of immunoglobulins for Egg yolk as a source of immunoglobulins for diagnostic,prophylactic and therapeutic diagnostic,prophylactic and therapeutic purposespurposes

While egg still in the ovary, transfer of serum Igs While egg still in the ovary, transfer of serum Igs into the yolkinto the yolk IgM and IgA transferred with other proteins in oviduct IgM and IgA transferred with other proteins in oviduct

into egg whiteinto egg white IgG(IgY in chickens) in egg follicle passed by IgG(IgY in chickens) in egg follicle passed by

receptors selectively into egg yolkreceptors selectively into egg yolk IgY conc in yolk comparable to conc in serumIgY conc in yolk comparable to conc in serum Evaluation of IgY against RotavirusEvaluation of IgY against Rotavirus

Animals in Vaccine ResearchAnimals in Vaccine Research

Polio vaccine-Polio vaccine- Mice used to study stages of Polio and in subsequent development of Mice used to study stages of Polio and in subsequent development of

vaccinesvaccines TB vaccine-New DNA vaccine developed in miceTB vaccine-New DNA vaccine developed in mice

Protection against mice getting infectedProtection against mice getting infected Also works as a t/t by stimulating immune systemAlso works as a t/t by stimulating immune system

SARS-A DNA vaccine induces SARS coronavirus neutralization and SARS-A DNA vaccine induces SARS coronavirus neutralization and protective immunity in miceprotective immunity in mice

Rabies vaccine-Rabies vaccine-

JE vaccine-Formalin inactivated mouse brain vaccine used for JE vaccine-Formalin inactivated mouse brain vaccine used for human immunization in Japan(using Nakayama strain)human immunization in Japan(using Nakayama strain)

Malaria vaccines-Rhesus monkeyMalaria vaccines-Rhesus monkey Infected mice shown to generate immune Infected mice shown to generate immune

responses against malaria parasiteresponses against malaria parasiteStudying the proteins of parasite causing this Studying the proteins of parasite causing this

immune response may lead to development immune response may lead to development of a vaccineof a vaccine

HIV vaccine research: Mice usedHIV vaccine research: Mice usedPre-clinical safety studies on animalsPre-clinical safety studies on animals

Actual vaccine production process –only blood or serum Actual vaccine production process –only blood or serum of animals may be required for culturing mediaof animals may be required for culturing media

Extensive use of animals(especially mice) for quality Extensive use of animals(especially mice) for quality controlcontrol

Safety TestingSafety Testing Specific toxicitySpecific toxicity Test for residual live virus-eg-Rabies virus causes death on Test for residual live virus-eg-Rabies virus causes death on

intracerebral inoculationintracerebral inoculation Identity test-Serum antibody assayIdentity test-Serum antibody assay

Antimicrobial ResearchAntimicrobial Research

Testing of potential antimicrobial agents in animal Testing of potential antimicrobial agents in animal models of infectious disease-models of infectious disease-

Essential prerequisite of anti-infective therapyEssential prerequisite of anti-infective therapy Bridging of gap b/w invitro characterization and clinical Bridging of gap b/w invitro characterization and clinical

evaluation of antimicrobial agentsevaluation of antimicrobial agents In-vivo testingIn-vivo testing

Invitro activities of antimicrobial agents altered by host factorsInvitro activities of antimicrobial agents altered by host factors (metabolic processes,anti-infective defense mechanisms)(metabolic processes,anti-infective defense mechanisms)

Animal testing-prerequisite to clinical trials Animal testing-prerequisite to clinical trials Toxicity testing in animals- clear indications of possible Toxicity testing in animals- clear indications of possible

short term and long term toxic effects and maximum short term and long term toxic effects and maximum tolerable dosestolerable doses

Efficacy evaluation:Efficacy evaluation: Various types of models usedVarious types of models used Basic antimicrobial screeningBasic antimicrobial screening

Model most commonly used-systemic inf in miceModel most commonly used-systemic inf in mice Results evaluated on basis of “all-or-none parameters”Results evaluated on basis of “all-or-none parameters” E.g.-Meningitis,Pneumonia in miceE.g.-Meningitis,Pneumonia in mice

Ex vivo modelsEx vivo models Foreign body implanted subcutaneously,animal subsequently infected Foreign body implanted subcutaneously,animal subsequently infected Accumulations in foreign body sampledAccumulations in foreign body sampled Valuable for determining capacity of antibiotic to penetrate specific site of Valuable for determining capacity of antibiotic to penetrate specific site of

infectioninfection Discriminative modelsDiscriminative models

Complicated;mimic the initiation and progress of infection in humansComplicated;mimic the initiation and progress of infection in humans Multiple parameters of efficacy measuredMultiple parameters of efficacy measured Have delineated effective therapeutic strategies now adopted clinicallyHave delineated effective therapeutic strategies now adopted clinically

Rifampicin + Va for t/t of OsteomyelitisRifampicin + Va for t/t of Osteomyelitis Beta lactams + aminoglycosides in EndocarditisBeta lactams + aminoglycosides in Endocarditis Adjunctive Dexamethasone therapy for bacterial meningitisAdjunctive Dexamethasone therapy for bacterial meningitis

CPCSEA guidelines for laboratory CPCSEA guidelines for laboratory animal facilitiesanimal facilities

Committee for the Purpose of Control and Committee for the Purpose of Control and Supervision of Experiments on AnimalsSupervision of Experiments on Animals

Ministry of Social Justice and Empowerment,Ministry of Social Justice and Empowerment,

Govt. of IndiaGovt. of India

Good Laboratory Practices(GLP) for animal Good Laboratory Practices(GLP) for animal facilitiesfacilities

GoalGoalTo promote the humane care of animals used To promote the humane care of animals used

in biomedical and behavioural researchin biomedical and behavioural research

Testing with the basic objective of providing Testing with the basic objective of providing specifications that will specifications that will enhance animal wellenhance animal well beingbeing,quality in the pursuit of advancement of ,quality in the pursuit of advancement of biological knowledge that is relevant to biological knowledge that is relevant to humans and animalshumans and animals

Veterinary careVeterinary care Animal procurementAnimal procurement Quarantine,Stabilization and SeparationQuarantine,Stabilization and Separation Surveillance,Diagnosis,t/t and control of diseasesSurveillance,Diagnosis,t/t and control of diseases Animal care and technical personnelAnimal care and technical personnel Personal hygienePersonal hygiene Animal experiments involving hazardous agentsAnimal experiments involving hazardous agents Multiple surgical procedures on single animalMultiple surgical procedures on single animal Duration of experimentsDuration of experiments EnvironmentEnvironment Food and waterFood and water Sanitation and cleanlinessSanitation and cleanliness Waste disposalWaste disposal Pest controlPest control Record keepingRecord keeping Personnel and trainingPersonnel and training

Conflicting ViewsConflicting Views

AlternativesAlternatives

toto

animal experimentsanimal experiments

AlternativesAlternatives

toto

animal experimentsanimal experiments

Replacement

Reduction

Refinement

· audio-visual aids· slaughterhouse

material · invertebrates

Replacement

· in vitro techniques · human material

ReductionReduction

• pre-screening

• experimental set-up (standardisation/statistics)

• ethical verification

• choice animal model

• animal care

• study performed by skilled persons (bio-technicians)

• analgesia, anaesthesia, euthanasia

• animal behaviour

RefinementRefinement

· human· animal species / strains· surviving - embryo

- organ - tissue - cells

· cell culture· bacteria, yeasts, protozoa

· subcellular fractions· molecular fractions,

DNA· epidemiological data · computer model· physical / chemical

model

Hierarchy of substratesHierarchy of substrates

ConclusionConclusion

Use of animals in medical microbiology and Use of animals in medical microbiology and biomedical researchbiomedical research

New variety of animal models in immunology New variety of animal models in immunology and cancer researchand cancer research

Need for an optimum balance between use of Need for an optimum balance between use of animal models and alternativesanimal models and alternatives

Animal research necessary in vaccine and drug Animal research necessary in vaccine and drug developmentdevelopment

ReferencesReferences Practical Medical Microbiology-Mackie and McCartney- 14Practical Medical Microbiology-Mackie and McCartney- 14 thth edition edition

Handbook of practical and clinical Immunology-TalwarHandbook of practical and clinical Immunology-Talwar

Kuby Immunology-6Kuby Immunology-6thth edition edition

CPCSEA guidelinesCPCSEA guidelines

Gnotobiotic animal as a tool in the study of host microbial Gnotobiotic animal as a tool in the study of host microbial relationships-Gordon and Pesti,Bacteriological Reviews,Dec 1971relationships-Gordon and Pesti,Bacteriological Reviews,Dec 1971

www.jax.org-Jackson laboratorywww.jax.org-Jackson laboratory www.harlan.comwww.harlan.com