lab animal health monitoring introduction-國衛院 …lac.nhri.org.tw/files/slides/960910.pdf ·...

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Lab Animal Health MonitoringLab Animal Health Monitoring

Cho-Hua Wan, D.V.M., Ph.D.Dept. of Veterinary MedicineNational Taiwan University

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Routine Health MonitoringRoutine Health Monitoring

Why to do it? What to do? When to do it?Where/who to do it?How to interpret the test results?What should we do after infectious agents are detected? (inputs from lab animal veterinarian)

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Why To Perform Health MonitoringWhy To Perform Health Monitoring

Public health problem– Zoonotic and human pathogens carried by animals

Animal health problem– Pathogens fatal for animals– Pathogenic, but not fatal for animals– Opportunistic pathogens

Affected research dataCurrent animal problem to be solvedIndicators of microbiologic status of an animal/colony

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What To DoWhat To Do

Routine health monitoring– Gross/behavior (Clinical signs)

observations – Parasitology monitoring – Pathology monitoring– Serology testing– Molecular biology testing– Microbiology testing

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When to Perform Health MonitoringWhen to Perform Health Monitoring

Routine Health Monitoring – How frequently?– What assay?– How many animals?

Random samplessentinels

Problem solving

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Who/Where to do itWho/Where to do it

Gross/behavior monitoring (Clinical signs)– By animal care taker/user/diagnostic lab staff (lab

animal center/diagnostic lab)Parasitology/serology/microbiology/pathology monitoring– By experienced diagnostic lab staff– By lab animal pathologist

Result interpretation

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How to do it How to do it

Routine Health Monitoring – How frequently?– What assay?– How many animals?

Random samplessentinels

Problem solving Important decision! Discuss w/ lab animal veterinarian

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Lab Animal Health Monitoring Lab Animal Health Monitoring

Behavior/activity observationRoutine health monitoring– Parasitology monitoring – Pathology monitoring– Serology testing– Molecular biology testing– Microbiology testing

Genetic monitoring/phenotyping

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Behavior/Activity monitoringBehavior/Activity monitoring

Behaviors in natural habitatsBehaviors in good lab environmentsBehaviors in stress lab environmentsBehaviors of mutant/modified strain

PostureMovement

– Nude mutants (ICR nude) jump more

Barbering– C57BL, C3H, CDF

Aggression– male vs female – C57BL/10 vs CBA/Ca

*Relied on animal care staff/users/veterinarian

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Direct signs– Death, diarrhea, conjunctivitis, hunched posture,

rough hair coat, weight loss, decreased activities, respiratory noise, swollen lymph nodes….

Indirect signs– Reduced diet or water consumption…

Changes in established reaction patterns– Anesthetic period, changed immunological

patterns/data*Relied on animal care staff/users/veterinarian

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Most infections are subclinicalInfectious agents are easily transmitted by subclinical infected animals

Diagnosis of the subclinical infectious diseases mostly relies on laboratory diagnostic methods!

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Genetic MonitoringGenetic Monitoring

Genetic contaminationIncomplete inbreeding/residual heterozygosityGenetic drift/substrain variationGenetically engineered strainsStrains with mixed genetic backgroundUsu. performed in research institutes or in animal breeder companies

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PhenotypingPhenotyping

Phenotyping of Genetically Engineered Modified (GEM) Mice and Rats – An investigator may have created a knockout

mouse intending to produce a model for diabetes, yet the mouse demonstrates a cardiovascular lesion

– IL-k/o mice with typhlitis/colitis lesionInfectious disease?Abnormality caused by ‘Genetic modification’ ?

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PhenotypingPhenotyping MethodsMethods

Primary assessments– Clinical characteristics (activity, gross

anatomy, reproduction, life span)– Pathology (clinical & histopathologic)

Secondary assessments– Embryonic evaluation– Physiology– Biochemistry– Behavior

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Routine Health Monitoring Routine Health Monitoring

Parasitology monitoringPathology monitoringSerology testingMolecular biology testingMicrobiology testing

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ParasitologyParasitology

Diagnosis– Based on morphology (frequently)– Molecular diagnosis (less frequently)

Ectoparasite– fur between ears, shoulders, or hip

(dorsal area)—fur mitesEndoparasite– Mucosal/content smear– protozoa– Perianal Tape Test– pin worms– Fecal test—pin worms & tapeworms

Photos obtained from RADIL website

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ParasitologyParasitology

Endoparasite– Tissue gross/histo exam– Cysticercoid cyst

Cysticercus fasciolaris(in liver, cyst of cat tape worm)

– Trichosomoidescrassicauda


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Parasitology monitoringPathology monitoring– Infectious diseases– Non-infectious disorders

Serology testingMolecular biology testingMicrobiology testing

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Pathology TestingPathology Testing

Histopathology– Interpretation of tissue morphology (gross and histo

lesions)Disease diagnosisHealth monitoring

Clinical Pathology– Clinical chemistry (liver, kidney, and multi-organ functions)– Hematology– Cytology

Phenotyping of genetically-modified animals

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Histopathology TestingHistopathology Testing

Disease Diagnosis– Rely on typical histo lesions & staining

characteristicsRoutine Health monitoring

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Rodent MicrobesRodent Microbes

ParvovirusCoronavirusCytomegalovirusSendai virusPVMLCMMousepoxReovirusHantavirusHerpesvirusRotavirus

Clostridium piliformeMycoplasma pulmonisPseudomonas aeruginosaCorynebacteriumkutscheri & bovisStreptobacillusmoniliformisPasteurella pneumotropicaCitrobacter rodentiumStaphylococcus aureus

Helicobacterspp.CAR bacillusPneumocystisProteus mirabilisStreptococcusSalmonellaChlamydiaKlebsiellaDermatophytes

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Gross LesionGross Lesion

SDAV/RCV infection (coronavirus)– Cervical swelling– Nasal/ocular discharges (porphyrin stained)

Photos obtained from RADIL & Merck websites

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HistopathologyHistopathology

SDAV/RCV infection– Inflammation of salivary

glands and Harderian glands

Photos obtained from AFIP website

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Mouse Hepatitis Virus (MHV) (coronavirus)


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Tyzzer’s Disease (C. piliforme)


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Helicobacter spp.


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Chronic Respiratory Disease– Mycoplasma pulmonis– Cilia-Associated Respiratory (CAR) Bacillus


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Encephalitozoon cuniculi


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Ectromelia virus (mousepox)


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Emerging DiseaseEmerging Disease

Rat Respiratory Virus (RRV)– A hantavirus – Idiopathic interstitial pneumonia of rat


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Clinical PathologyClinical Pathology

Urinalysis– Kidney, liver,

Hematology & Clinical ChemistrySpecies/strain variation

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Problems in Problems in HistopathHistopath MonitoringMonitoring

Typical gross/histopathology lesions are uncommon for most infectious agentsAnimal sacrifice is required

Serology/molecular biology/microbiology diagnostics are the choice

Histopath is the great method to identify new pathogens or disorders

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Serologic TestSerologic Test

Traditional Assays– Enzyme-Linked ImmunoSorbent Assay (ELISA)– Indirect Fluorescent Antibody Test (IFA)– Hemagglutination Inhibition Assay (HAI)

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Serology TestingSerology Testing

Traditional Assays– ELISA

Ag-coated microplates(polystyrene or polyvinyl)Primary AbyEnzyme-conjugated secondary AbyChromogenic enzyme substrate

* Sensitive, Specific?

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Serology Testing (continual)Serology Testing (continual)

Traditional Assays– IFA

Infected and uninfected cells fixed to wells in a glass slidePrimary AbyFluorescent labeled secondary Aby

* Sensitive, Specific

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Traditional Assays– HAI

The ability of specific Abyto inhibit virus-mediated hemagglutinationV-bottom microplateRodent parvoviruses, Reovirus type 3, Pneumonia virus of mice, Sendai virus

* Low sensitivity, High Specificity

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Traditional Assays– Advantage

Cheap equipmentAutomated (ELISA)Sensitive (ELISA, IFA)Specific (IFA, HAI)

– DisadvantageIndividual Assay (inefficient)Manual (IFA & HAI) (labor-intensive)Insensitive (HAI)

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Improvement in DetectionImprovement in Detection

Multiplex & Automation– Multiplex Fluorescent

Immunoassay (MFI)Microspheresinternally dyed with red and intraredfluorophores

100 different microspheres -++Result

Aby α CAby α BAby α A

C#101 #120 #125A

B

B

B B

B

CC

CC

A AA

A

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Improvement in Detection (Continual)Improvement in Detection (Continual)

Automation & Multiplex– ElectroChemiLuminescence

Coated nitrocellulose membranethe light emitting chemiluminescentreaction is preceded by an electrochemical reaction.

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MFI & ECL Assays– Advantage

Automated MultiplexSensitive & SpecificLess amount of test article required

– DisadvantageExpensive equipment

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Molecular Biology Diagnostics Molecular Biology Diagnostics

Application of molecular biology methodologies in nucleic acid detectionMonitoring – The nucleic acids from microbes (virus,

bacterium, parasite)– The genetic materials from biotics– The expression status of genes (mRNA)

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Molecular Biology MethodologiesMolecular Biology Methodologies

Nucleic acid amplification– PCR, RT-PCR, nested PCR (in test tubes) – In-situ PCR, in-situ RT-PCR

Nucleic acid hybridization– Southern blotting (detecting DNA)– Northern blotting (detecting RNA)– In-situ hybridization

SequencingRestriction enzyme digestion– Restriction fragment length polymorphism (RFLP)

Nucleic acid amplificationNucleic acid amplification

PCRRT-PCRIn situ PCRIn situ RT-PCR

Forward Primer

Reverse Primer

1st cycle

nth cycle

2n-1 ampliconn=30

109 amplicon

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Nucleic Acid HybridizationNucleic Acid Hybridization

Southern blotNorthern blotIn situ hybridization– DNA– RNA

Add probe

Target gene

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Molecular Biology MethodologiesMolecular Biology Methodologies

Sequencing– Time consuming– Cost– Effort

Restriction digestion (RFLP)

A BE E E

E E

Strain A

Strain BE: EcoRI

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Molecular Biology DiagnosticsMolecular Biology Diagnostics

Goals – Sensitive– Accurate– Fast

AmplificationPCR/RT-PCR

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PCR/RTPCR/RT--PCRPCR

Primer design– Generic v.s. Specific– Conserved region v.s. Diverse regionEX. Rodent parvoviruses:

Generic rodent parvovirus PCRNS region: ~90% similarity

MMV, MPV, KRV, H-1 specific PCRVP region: ~70-75% similarity

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PCR/RTPCR/RT--PCRPCR

Specimen quality– Clinical samples

Feces, urine, blood, bio-swab, tissues– Inhibition factors

– Cultural & environmental specimensCells, media, cage swab

– Amount of specimen to screen – Inhibition factors

False Negative!

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Rodent Rodent ParvovirusesParvoviruses

Mouse– Mice Minute Virus (MMV)– Mouse Parvovirus 1 (MPV-1)

Rat– Kilham Rat Virus (KRV)– Toolan’s H-1 Virus (H-1)– Rat Parvovirus 1 (RPV-1)– Rat Minute Virus 1 (RMV-1)

Hamster– Hamster Parvovirus (HaPV)

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Diagnosis of Parvovirus InfectionDiagnosis of Parvovirus Infection

PCR assays (Mesenteric LN, Spleen)– Generic PCR assay– Serotype specific PCR assays

KRV specificH-1 specific RMV-1 specificMMV specificMPV/HaPV specific

RPV-

1aRM

V-1a

RMV-

1c

RMV-

1bKR

V

HaPV

MPV

-1

H-1

MM

V–

tissu

e

RMV-1 Specific

–co

ntro

l

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Rodent Rodent CoronavirusesCoronaviruses

Mouse– Mouse Hepatitis Virus

Multiple strains– Polytrophic (respiratory) : JHM, 1, 2, 3, A59, S

Usually more virulent– Enteric: D, Y, DVIM

Highly mutable (esp. the S gene)Pathogenic vs. nonpathogenic

Rat– Rat Coronavirus/Sialodacryoadenitis virus

(RCV/SDAV)Multiple strains

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Rodent Rodent CoronavirusCoronavirus

Linear, ss(+) RNAGeneric RT-PCR– Both MHV & RCV/SDAV– MHV: feces or mesenteric LN– RCV/SDAV: Salivary gland or Harderian

gland or lungNo virus species/strain-specific RT-PCR

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Gram-negative, spiralEnterohepatic infection– H. hepaticus– H. bilis– H. typhlonius– H. rodentium

Hepatitis, enterocolitis, hepatocellular neoplasmasModel for H. pylori

1 µm

Helicobacter in RodentsHelicobacter in Rodents

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EnterocolitisEnterocolitis


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Helicobacter Generic PCRHelicobacter Generic PCR

Consensus region of helicobactersß-Actin PCR– Housekeeping gene– extraction control– inhibition factors

monitor* Fecal PCR

Fecal

2hepa bilis typh

102

10 102

10 10 NT10

2Fec

al 1

Helicobacter: 375 bpß-Actin: 272 bp

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Helicobacter SpeciesHelicobacter Species--specific PCRspecific PCR

H. hepaticus specificH. bilis specificH. typhlonius specificH. rodentium specific

* Fecal PCR

106

105

104

103

102

10 1 Control

H. hepaticus: 396 bpSensitivity: 10 copies

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Improvement in AmplificationImprovement in Amplification

Multiplex PCR– ↑ information for

client dollar– ↑ efficiency

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102

10 Fecal

Control

H. typhlonius

H. bilis

Template copies

H. hepaticus

No templat

e

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Signal AmplificationSignal Amplification

PCR-ELISA

Anti-● AntibodyPCRELISA

Forward Primer

Reverse Primernth cycle

●

●

●

●

●

●●

●

●●

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PCR-Oligo hybridization

PCRForward Primer


Hybridization

★

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PCR- RFLPPCR

Forward Primer


RFLP

A B

E E E

E E

Strain A

Strain BE: EcoRI

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Automation– Labeled Probe

TaqMan– Quantitative Real-time v.s. endpoint PCR– The fluorogenic 5' nuclease PCR assay

the 5' --> 3' nuclease activity of Taqthe emission of the reporter dye is quenched by the intact quencher

Q R

QR

…..

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Automation & Multiplex– Multiplex Fluorescent Assay

Microspheres internally dyed with red and intraredfluorophores

100 different microspheres

-++Result

CBAAmplicon

#101 #120 #125

A

B

B

B

B

A

A

A

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Improvement in Detection (Continual)Improvement in Detection (Continual)

Automation & Multiplex– ElectroChemiLuminescence– Oligo-coated membrane – the light emitting

chemiluminescent reaction is preceded by an electrochemical reaction.

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MurineMurine NorovirusNorovirus–– Related to human Norwalk virusRelated to human Norwalk virus–– Primary GI infectionPrimary GI infection–– No clinical signs in naturally infected miceNo clinical signs in naturally infected mice

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Murine Norovirus exp. inoculation– RAG/STAT-/- mice exhibited high Mt with

encephalitis, meningitis, vasculitis, pneumonia, hepatitis

– RAG-/- exhibited low Mt but persistently infected– Immunocompetent mice seroconverted and

transiently infected– Mice with no INFαβ and IFNγ receptors are more

susceptible to MNV infection.

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Microbiology monitoringMicrobiology monitoring

Isolation of microbes in pure culture– Routine monitoring area: Nasopharynx and

cecum (rodents and rabbits)– Area of suspected lesions

Some organisms require specific culture medium and conditions

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Culture MethodsCulture Methods

Sensitive and expressiveResults are dependent on culture media, experience,…Expensive, sometimes time consuming and labor-extensiveFor most bacteria and fungi: the best method

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Techniques for Definite Diagnosis of RodentRodent Infections

Positive results: What can be doneConfirm lab results

Retest the same sampleRetest with other methodRetest in other diagnostic labRetest samples from the same source

Rule out the cross-reactionCross reactivity with related organisms

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How to Interpret the ResultsHow to Interpret the Results

Possible combinations of results of different testing (e.g. serology and PCR for MPV)

－－4＋－3－＋2＋＋1

MPV PCRMPV ELISASample

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Routine Health Monitoring

How frequently to screen?What assay/method to be applied?How to interpret the results?

lab animal health monitoring introduction-國衛院 …lac.nhri.org.tw/files/slides/960910.pdf ·...

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