Why worry about infectious agents ?

Mouse Pathobiology Environmental + Infectious Diseases ((Phenotypes) yp ) Cory Brayton, D.V.M., D.A.C.L.A.M., D.A.C.V.P. Associate Professor, Molecular and Comparative Pathobiology Director, Phenotyping Core Johns Hopkins University, School of Medicine Baltimore, MD 21205

 They look fine…  The mouse house is house cleaner than my house…  Their diet is healthier than mine…

[email protected] http://www.hopkinsmedicine.org/mcp/PHENOCORE/index.html

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Yikes!  How do I find out more about – My mouse diets, environment – My M microbial i bi l SSurveillance ill – Infectious & other ‘environmental’ phenotypes

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129?

 What agents could impact your research ? Wh t iis iin th i h lth  What their health reports ?  Why is it in their health reports ?

1. 2. 3. 4. 5. 6. 

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Phenotypes are determined by: Nature – Genetics • Genetic Manipulation • Genetic Background

Nurture – Environmental Factors • Non-infectious

• Infectious

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Examples: infectious disease phenotypes

Agent DZ

Susceptible

MHV demyelination

B6, BALB/c

Intermediate

Thymic Lymphoma…

BALB/c

Plasmacytoma etc tumors, heart dz, acallosity, kill each other

C3H

TUMORS - Mammary, Liver

Parvo MPV1 seroconversion

C57BL/6

Microphthalmia, Mi hth l i H Hydrocephalus d h l , MUD MUD, O Osteoporosis, t i P Presbyacusis, b i Amyloidosis, AMP, …

TMEV SJL/J, SWR, DBA/2 CBA, C3H responses to these agents demyelination

DBA

Deaf, seizures, glaucoma, autoimmune

FVB/N?

Blind, seizures, mammary/pituitary dz

Sendai Emphasizes some DBA, 129 Pneumonia

NOD

Diabetes, immunoweird

SJL/J

Lymphoma, muscular dystrophy, kill each other

DEAF

C57BL/6, BALB, DBA, etc

BLIND rd1

C3H, CBA, SJL, SWR, FVB

SJL/J

DISCLAIMER C3H/HeN BALB/c, ICR, DBA

recent data

B6 A, B6, B10, DBA/1

responses A, BALB,with SWR relatively B6, SJL

DBA, BALB/c, C3H, immune def

B6, AKR

Mycoplasma

BALB/c, C3H, A/J DBA/2, AKR

B6, B10

H hepaticus

A/J 3H/HeJ & N Nu scid IL10- Rag2-

B6, FVB/N

Ectromelia

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Examples: experimental infection models

Resistant

 This is an over simplification of strain

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1. Recurrent theme:

Nature & Nurture – Again Environmental variables – Top few Viral agents –Top few Emphasizing Bacteria – Top 3 COMPETENT mice Eukaryotes – Top few ‘Normal’ flora – the microbiome Immunodeficient mice – another lecture

Teratomas (Ter), lung tumors, acallosity, AMP, … Lung tumors, anomalies, amyloid, muscular dystrophy

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 Is there an infection?  Is it a problem?

Environmental/Infectious Phenotypes Discussion Plan

AKR

+ Different susceptibilities to infection and disease !

– Or is it related significantly to environmental factors ?

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Nature: (mostly) genetics  Phenotypes in ‘Normal’? (+/+) Mice

 Is your cool phenotype a PRIMARY effect of the genotype or genetic manipulation ?

Aims of this section:

A/J

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Questions

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The Real Aim:

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Environment, Infectious Phenotypes

Agent DZ

Susceptible

Intermediate

Resistant

H5N1

DBA/2

BxD2 RI

B6

Poxviruses

DBA/2

BxD2 RI

B6

Anthrax h

DBA/2

BxD2 RI

B6

Strep pneumoniae

DBA/2

BxD2 RI

B6

A/J

AxB RI

H hepaticus Salmonella (leishmania, some mycobacteria)

C3H/HeJ, A/J, B6

B6 C3H/HeN 129/S6

RI = Recombinant inbred 9

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NATURE: Genotypes  Phenotypes Strain 129

Color

Color Genotype

Phenotype

Haplotypes

Agouti etc.

Variable: Aw +/- p, d

b, bc..

A

Albino

Tyrc/ Tyrc + a/a Tyrp1b/Tyrp1b

a

AKR

Albino

Tyrc/ Tyrc + a/a Hc0/ Hc0

a

BALB/c

Albino

Tyrc/ Tyrc + A/A Tyrp1b/Tyrp1b

d

C3H

A Agouti i

A/A

k

C57BL/6

Black

a/a

b

DBA/2

Dilute brown

Myo5ad/Myo5ad Tyrp1b/Tyrp1b a/a

d

FVB/N

Albino

Tyrc/ Tyrc + A/A

NOD

Albino

SJL/J

Albino

B6;129

Variable

B6C3F1

Agouti (dark)

Swiss

Albino

q g7

p/p

s

Variable

Var

A/a

b/k

Tyrc/ Tyrc

X

H2

Etc.

Var

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NATURE: more genotypes in ‘competent’ inbred strains

Gene Gene /Locus symbol name

Chrom

Allele Symbol

Hemolytic complement (c5)

2

0 (Hc0)

Mx1

Myxovirus resistance 1

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Mx1-

Tlr4

Toll like receptor 4

4

Lps-d (Tlr4Lps-)d

Hc

solute carrier Slc11a1 family 11 (1) Nramp

Slc11a1r 1 Slc11a1s

Allele name

Deficient

Susceptibility Defective lipopolysaccharide response resistance (Bcg/Ity/Lsh) susceptibility (Bcg/Ity/Lsh)

Mutation

Strains

A/HeJ, AKR/J, DBA/2J, FVB/N; NZB/B1NJ, SWR/J, B10.D2/oSnJ Deletion or C57BL/6, BALB/c, nonsense mutation CBA etc 2 base "TA" deletion

Environment, Infectious Phenotypes X

Nurture: 2. Environment Matters …

If you buy your +/+ control mice from J or Crl or Hsd or Ncr or Tac, how relevant are they to mice from your facility?

C to A substitution C3H/HeJ in 3rd exon 129/Sv C3H/HeJ BALB/c C57BL/6J

carcinoembryonic Mouse hepatitis Hc2-r Deletion  23 aa antigen-related 7 SJL/J virus (MHV-4) Ceacam1 cell adhesion (Ceacam1Hv2-r) substitution resistance molecule 1 Search gene, allele, strain updates at MGI http://www.informatics.jax.org/ See complement video http://highered.mcgrawhill.com/sites/0072507470/student_view0/chapter22/animation__activat ion_of_complement.html 11

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NURTURE

Monday morning June 11, 2001 Most water pumped out.

Hi water Level

Some environmental challenges are difficult to control.

Baylor’s Main Entrance at 6:47am Saturday, June 9, 2001 Water had been up to the top step.

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Environment 14

Nurture (Environment): Housing

Nurture (Environment): ENRICHMENT

Vibration (production) Air quality (%H, Temp) Materials (bisphenyls etc) Barrier/Containment $$$ (man hours vs equip costs)

Dust (wind tunnel) Breeding/production Barbering Aggression $$$ Effects vary …..

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Nurture (Environment): Housing / Population density Purpose – Single sex for study, maintenance ? – Breeding / production ?

Conspecifics = – Friends = Social Enrichment •  happier? Healthier? Mice or

– Enemies = social stressors

Dust (nu, hairless) 16

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•  stressed & dead mice 17

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Nurture (Environment): Noise, Temperature  Noise – – – – –

IVC Cage change/wash Human Radio Other spp

 Hearing loss?  Behavior tests

 Temperature  Transport – transient

 Nude/hairless mice – Tumor growth?

Nurture (Environment): BEDDING Dust/allergens Palatability – Restricted or special diets

Absorbance – Bioburden … – Humidity

– BactrimTMS, – Ivermectin – etc

Contaminants Endocrine disrupters $$$ 20

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 Restricted vs ad lib  Fat/Prot/Carb/Fiber  Contaminants  Endocrine disrupters  Rx (Fbz etc)  Special diets

– & their ONLY protein source – – – –

• Prefer bedding ?

 $$$

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X

BOTTOM LINE: What diet & Why ?

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X

MICROBES!

Cardiovascular disease? Cancer ? Uterus - endometrial hyperplasia Bone – marrow ? NOTE – these are from estrogenized mice

Environmental/Infectious Phenotypes Discussion Plan

NURTURE

– How much is in diets for your current cancer studies ? – For F your previous i cancer studies t di ?

 $$$

 Effects on

– Palatability

How much animal products do you want in your rodent diets ?

– Chemical – Microbial

 But are they good for your research ?  Alfalfa & soy = typical plant protein sources

• Loss of nutrients

Nitrosamines ?

– Can they reach it ?

 Drowning  Contaminants

Phytoestrogens are good for you … ?

– Special handling

Diet

ISSUES  Dehydration

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Nurture (Environment): DIET

NURTURE

Nurture (Environment): WATER

OPTIONS  Auto Water  Bottles  RO  Acid/Cl  Rx

– Asthma/resp studies

 Thermoregulatory challenge

Environment, Infectious Phenotypes

1. 2. 3. 4. 5. 6. 

Nature & Nurture – Again Environmental variables – Top few Viral agents –Top few Emphasizing Bacteria – Top 3 COMPETENT mice Eukaryotes – Top few ‘Normal’ flora – the microbiome Immunodeficient mice – another lecture

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Environment, Infectious Phenotypes

Most infections don’t kill them, What’s the problem? Consider: If an infectious agent does NOT kill them, it elicited an effective immune response, ‘immunomodulated’ or ‘immunomodulated’….  Immune phenotypes, e.g. cytokine gene expression, cytokine effects, leukocyte migration & proliferation, antibodies, etc…  Other cool phenotypes too …. 28

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Infectious/Infesting Agents

Commensals Emerging Opportunists

FBZ, TMZ etc

Serum/plasma chemistry – for LDV PCR (feces or specific tissues) – Requires patent infection, shedding

Mouse serology: results – 2009 Agent (assay abbreviation)

PC/CRL Prevalence % N

NA

Europe

Total

Ectromelia (ECTRO)

246,857

0.02

0.00

0.02

Hantavirus (HANT)

144,946

0.00

0.00

0.00

K virus (K)

225,353

0.00

0.00

0.00

Lymphocytic choriomeningitis virus (LCMV)

241,453

0.01

0.02

0.01

Mouse adenovirus 1 and 2 (MAV)

230,351

0.02

0.22

Liang

W EU

Taiwan

Prevalent &/or pathogenic Zoonotic

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MHV

Parvoviruses

Parvoviruses

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MRV(EDIM) ~30%

MNV

PVM

SEN

MRV (EDIM)

MRV (EDIM)

5

Sen ~20%

SEN

TMEV

TMEV

TMEV

6

PVM ~20%

TMEV

0.02

146,511

0.04

0.00

0.04

558,673

1.57

3.25

1.59

5.5

Mouse Norovirus (MNV)

44,876

32.64

24.03

32.37

31.8

Parvovirus generic assay (NS-1)

578,464

1.65

1.92

1.65

- Mouse parvovirus 1 and 2 (MPV)

594,539

1.83

3.64

1.86

- Mouse minute virus (MMV, MVM)

595,903

0.33

0.46

0.33

Pneumonia virus of mice (PVM)

447,656

0.01

0.01

0.01

Polyoma virus (POLY)

225,868

0.02

0.20

0.02

Reovirus 3 (REO, REO-3)

428,821

0.01

0.05

0.01

Rotavirus (EDIM)

466,572

0.56

0.35

0.56

462,209

0.00

0.00

0.00

435,772

0.26

0.27

0.26

1 Less testing of positive areas 1.

1.0

>20%

~3%

>20%

20%

– Single Stranded RNA virus – Human Noroviruses = primary cause of non bacterial human gastroenteritis

 Seroprevalence: p 30%-(60%?!) ( ) in research colonies – 0- low in vendor production colonies

 Transmission: Fecal / oral – Like other Norwalk viruses

 Control: Why ? Does it do anything important?

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 fewer +

2. More testing to confirm negative status 3. Outbreak testing 

MNV - Murine Norovirus(es)

– Rederive -- Foster ? Depopulate ?

Mouse Viruses

 Similar findings & recommendations in different countries.  Most ‘Prevalences’ seem pretty low (< 5%) - or are they?  Why test for an agent that’s not excluded?

Mouse hepatitis virus (MHV)

Monthly short screen for common Twice /year long screen for less excluded agents likely agent ALSO includes DO YOU know WHAT your surveillance program screens for?

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MHV

MRV (EDIM)

NH J 07

 Calicivirus (MNV1-4…..) Henderson & al 2008

14. as MNVgood as its Any Barrier is only Exclusion policy (& practices) …

Parvoviruses

Parvoviruses

X

Mahler

Mouse cytomegalovirus (MCMV)

JHU Health Monitoring Dirty bedding sentinels

12.IS A MCMV WHAT WHEN THERE POSITIVE FINDING ? + Parasites (fur,HAPPENS tape, float) + helicobacters (Neg areas) 13. Mycoplasma

MRV (EDIM)

TMEV ~ 35%

2009 Mahler

* % of positive non–spf facilities by survey  Norovirus (MNV) is new to the list  Sendai & PVM are less common

Pritchett-Corning, Cosentino, Clifford (2009) (Charles River Laboratories) Mahler & Kohl (2009) [Western Europe]; Liang - Taiwan; Hayashimoto – Japan

Sendai virus (SEND)

6. Mad2 (+1) WHEN? 7. Ect MHV MRV (EDIM) 8. LCMV WHY? MMV(MVM) ( ) [[Parvo]] 9. Sen MPV 1&2 [Parvo] 10. PVM WHAT AGENTS ARE EXCLUDED? TMEV 11. Reo

2009 Pritchett Corning MNV

– Isolation by ventilated caging ?

TMEV, GD-VII 32

1. 2. 3. 4. 5.

Parvos ~ 40%

3

2001 FELASA

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Test Lab/location 

• adequate exposure + • Sufficient immune response

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PATHOGENS

Mouse Virus Testing – ELISA, IFA, MFIA – multiplex – Requires

MHV

2003 Livingston & al MHV

Zoonoses

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Serum/plasma antibodies

1

1998 Jacoby & Lindsay * MHV > 70%

Potential Confounders TREATMENTS!

Top Few Viral Agents of Concern

more – more – (or more + ?)

 Usually detected by serology now, – NOT by obvious clinical disease…  Surveillance (Sentinel & Quarantine testing) represent a lot of time & $$£€ to test for agents that rarely kill, or even cause disease 33

X

MNV - Murine Norovirus(es)

In Immune Sufficient mice • Subclinical seroconversion in Immune Deficient mice • Subclinical in most or  • Pneumonia,, hepatitis, p , vasculitis,, ((encephalitis) p ) in severelyy innate immune deficient mice e.g. Rag2–/–/Stat1–/– Rag1−/−/Stat1−/− Rag1−/−/IFNγR−/− Ward & al.; Wobus & al Is it a problem ? X • Innate immunity ? Macrophage function? Paik & al • IBD, enterohepatic phenotypes ? Paneth cells Cadwell & al • Cell culture contaminant 36

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Environment, Infectious Phenotypes MNV - Murine Norovirus(es) H&E

IHC

 H&E Typical mouse liver  IHC  MNV  Tac:SW sentinel mice = Competent Swiss mice  Perdue, & al. 2007. 37

MHV Mouse Hepatitis Virus(es)

MNV?? Sick Nude mouse Small intestine

 Coronaviruses – mutable  many ‘strains’ • Enterotropic, pneumotropic (polytropic) – useful classifications?

– SSRNA, enveloped

– Necrosis + crypt abscesses elsewhere – Paneth cell degeneration

 ‘seroprevalence’ ~ 5% – Was > 50 % / US, Can, EU in 1980’s – 1990’s – Transmission: fecal oral - Highly infectious (~ TGE, FIP) • direct contact, aerosol, fomites • dirty bedding sentinels should detect • Iatrogenic (inoculated): can contaminate hybridoma, ES cells etc

– Similar to Cadwell 2010 ?

 Control: Rederive

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– Foster ? Depopulate ? Breeding Cessation ??

Ceacam1 & MHV

MHV

carcinoembryonic antigen-related cell adhesion molecule 1

Resistance – Susceptibility Nature: Genetic resistance

 Mouse hepatitis virus (MHV) receptor  Normal ‘wild type’ Ceacam 1 facilitates entry of MHV (some strains) into cells:

– WT Ceacam1 glycoproteins bind MHV spike glycoprotein (S)  membrane fusion (& Syncytia) – SJL/J with defective Ceacam1 resist infection

– Binds MHV spike protein (S protein) – Activates S protein  virus-cell membrane fusion

Nurture: Dietary cholesterol /lipid – Hi cholesterol diet increases susceptibility – In vitro too (cell susceptibility) – Lipid dependent, ceacam independent fusion

 SJL/J have mutant Ceacam 1 (Ceacam1Hv2-r) – Tough for MHV to enter cells without its receptor – > 10,000 fold higher lethal dose than B6, BALB/c etc Bergmann et al. Nature Reviews Microbiology 4, 121–132 (February 2006) | doi:10.1038/nrmicro1343

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MHV Famous for – LIVIM = Lethal intestinal virus of infant mice with sometimes epizootic pup mortality • Enterotropic strains in young mice

– chronic h i wasting, ti h hepatitis, titi d death th iin immunodeficient mice • Usually poly tropic (aka respiratory) strains

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MHV in Immune deficient

MHV  Some competent strains  Subclinical ?   LET’s

 D Percy

‘wasting phenotype’ of Prkdcscid or Foxn1nu etc immunodeficient … Liver Li necrosis i

NOW usually subclinical – detected by seroconversion –  Why worry about it ? 43

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MHV Liver Phenotypes Necrosis  LE’s (e.g. AST, ALT, LDH…..) Syncytia = histologic ‘hallmark’

Ascending colon target Distal SI too Syncytia, Necrosis

MHV CNS Phenotypes

Inflammation  demyelination Model for MS etc demyelinating Dz

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X

MHV Gut Phenotypes

Environment, Infectious Phenotypes

MHV Research Interference

 Death (in epizootics in susceptible mice)  Immunomodulation in survivors  Liver phenotypes - Necrosis/syncytia  Gut phenotypes – diarrhea, diarrhea necrotizing enterocolitis, syncytia  CNS phenotypes - meningoencephalitis, demyelination –  Experimental - model for multiple sclerosis (MS)

 Other phenotypes in spleen, lymph nodes, GALT, marrow, vascular endothelium, brain 49

 non enveloped, SSDNA viruses – Tough & tiny viruses

X

MHV Phenotypes

Disease Models ► Necrotizing MeningoEncephalitis + Demyelination ► Model for multiple sclerosis & immune mediate demyelinating disease

In Immune Deficient mice ► Adult wasting + necrosis/syncytia liver, spleen, lymph nodes, GALT, marrow, brain (nude, scid) ► Don’t clear virus i.e. persistent infection ► Other e.g. FIP-like granulomatous peritonitis/ pleuritis in Ifn-

► ► ► ►

X

• NS1, NS1 NS2 - Non structural (non specific) antigens • VP1, VP2 …Structural/capsid antigens (specific)

 ‘seroprevalence’ ~ 5% - sneaky – tough to detect – Transmission: fecal oral

Parvoviruses in developing (proliferating) tissues

Mouse Parvoviruses – Subclinical infections – Slow & variable seroconversion

 Lymphoid targets  immunomodulation  Oncolytic / oncosuppressive in vitro / in vivo

Problem for you ? Liver enzymes, liver, gut, CNS phenotypes Immune phenotypes Wasting in immunodeficient in chronic studies, or breeding Cell culture contaminant including ES cells

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Challenges to detection & control

• Require S phase for infection / cytolysis

In utero infections affect developing tissues E.g. cerebellar hypoplasia – – – – –

 C57BL/6 &DBA/2 may be slow or NOT seroconvert

• direct contact, fomites - VERY persistent in environment • dirty bedding sentinels CAN detect but challenging .. • Iatrogenic (inoculated): Common biological contaminants

– Transient fecal shedding • Negative fecal PCR, but infected,

 Control: Rederive

 Seronegative or seropositive

• Some infections are cleared, but seropositive

– Foster ? Depopulate ?

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Rat  Hamster Cat Cow Mouse (experimental) D Percy Image

 With PCR negative fecal & mesenteric nodes 52

MHV Phenotypes

In Immune Sufficient mice Disease spectrum depends on virus strain, mouse strain & age ► No infection (no receptor) ► Subclinical seroconversion +/- syncytia ► Suckling diarrhea/death  100% with Necrotizing enterocolitis l

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Mouse Parvoviruses

Coley et al. 2005. J Virol. 79(5):3097-106

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Parvoviruses & Cancer proliferating cells in S phase …. Oncotropic – oncolytic? – Riolobos & al. (2010). "Viral oncolysis … – Wollmann et al. 2005 … viruses with potential oncolytic potential. – Raykov et al. 2005 – Moehler et al. 2003, 2001 – Clement et al. 2002. oncotropic vectors, from MVM(p)

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Mouse Parvoviruses Phenotypes MMV (MVM) (c, i, m, p strains)

MPV (MPV1-4)

In immune sufficient mice Subclinical, Immunomodulation Subclinical, Immunomodulation (T May not persist (Sero+/PCR-) tropic) MVMm MVMm  most prevalent, persistent P i t t/l t t iin mesenteric t i Persistent/latent MVMi  disrupt hematopoiesis in nodes C3H In Immune deficient mice (scid, nude) MVMi exp  Lethal leucopenia in scid

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Environment, Infectious Phenotypes X

Mouse Parvoviruses Phenotypes

Experimental / model phenotypes Neonate  multisystem infection  cerebellar hypoplasia, renal infarct anemia Oncotropic, oncolytic agents Gene therapy vectors Is it a problem for you ? Targets cells in S phase (developing embryo) Oncolytic in cancer models  Poor tumor growth ? Competent sentinels may not seroconvert fast or reliably Cell culture contaminant 57

MRV (EDIM)

MRV (?)

Murine Rotavirus (Reoviridae family) • Double stranded RNA virus, enveloped • Type A rotaviruses  diarrhea in neonates (many species) • EDIM = Epizootic p Diarrhea of Infant Mice

‘seroprevalence’ > CBA, C3H >> A, B6, B10, DBA/1 MHC – plays a role

Sendai Virus

Is it a problem for you ?  Immune modulation  CNS phenotypes  Cell culture contaminant

    

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Proliferative tumor like ‘Adenomatous change’ in change chronic disease & immunodeficient mice

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Sendai Virus Phenotypes

Sendai Virus In nude, scid etc – No T cell-mediated necrosis – Not much Inflammation • Some neutrophils …

– Proliferation – Syncytia – Intranuclear Inclusions 73

In Immune Sufficient mice  Clinical: Chattering, Dyspnea, Hunched, runting; Neonate/suckling death,  Gross: Lung Discoloration/consolidation; splenomegaly, Lymphadenopathy  Histo: Lungs Syncytia, cytoplasmic inclusions  necrotizing bronchiolitis  hyperplasia (sq ( metaplasia))  min dz - bronchiolitis obliterans  Strain susceptibility: DBA, 129 > A, BALB, SWR > B6, SJL resistant In Immune Deficient mice  Progressive wasting, dyspnea, death  Proliferative (tumor-like) lesions instead of necrotizing lung lesions; syncytia, inclusions  Not much inflammation

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2 viral agents not in the ‘top few’

2. Mouse retroviruses

In Immune Sufficient mice  Susceptible: Disseminated Dz, facial edema, conjunctivitis, multisystem necrosis: liver, spleen, lymphoid tissue, gut, skin  death  Semisusceptible: rash, ectromelia, long term shedding; splenic fibrosis  Cytoplasmic inclusions – skin/mucosa (Cowdry A); Cowdry B (Basophilic) in liver etc  Resistant: Subclinical rapid resolution, minimal shedding Sneaky – silent in resistant strains  Susceptibility: varies with strain, age, etc: DBA, BALB, C3H, immune deficient  acute lethal dz >> B6, AKR (resistant) In Immune Deficient mice  DEATH - Many immunodeficient Hily susceptible to acute lethal dz  Few Gross lesions (die too quickly)  +/- liver spleen necrosis with inclusions 79

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Is it a problem for you ? Not so likely today – contained by microisolator caging Morbidity mortality in susceptible strains Respiratory phenotypes, and Immunomodulation Wasting dyspnea in Immune deficient in chronic studies, breeding  Cell culture contaminant    

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 ‘seroprevalence’ ~ 0 • Vaccinia vectors ectors (e.g. (e g in gene therapy therap studies) st dies) can ca cause se seroconversion

 Transmission: inoculation / trauma • Important Biological contaminant • NOT reliably detected by sentinels  Unless they fight/cannibalize (contact sentinels)  Not highly contagious

 Control: TEST Biologicals (serum, cell lines) 77

Ectromelia Virus Phenotypes

Experimental/model phenotypes  Similar to clinical disease  Model for human parainfluenza 1

• BIG double stranded DNA • Mousepox is the name of the disease • Ectromelia refers to shortened limbs that may result

 The Original genetic engineers

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Sendai Virus Phenotypes

Ectromelia Virus

• Important part of mouse genome (endogenous) • Also potentially infectious (as exogenous agents) • We don’t test for them

X

 Orthopoxvirus (~ vaccinia)

1. Ectromelia virus (ECTV) • Historical – devastating outbreaks before 1980 • (Few) recent outbreaks associated with contaminated biologicals

Environment, Infectious Phenotypes

X

– Depopulate; Rederive; test & cull

Ectromelia Virus Phenotypes

Experimental model phenotypes  Model of orthopoxvirus infection  Gene therapy vector  seroconversion Is it a problem for you ?  Biological materials  Sentinels do not reliably seroconvert  Seroconversion from gene therapy vectors can be confusing

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Ectromelia

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D Percy

Mouse Retroviruses MMTV’s, EMV’s etc  Important today as endogenous viruses – ‘provirus’, ‘retroelements’ , loci, genes in mouse genome

 Endogenous – proviruses, retrotransposons, IAP etc  – named genes (Mtv1….; Mlv1…; Emv1… (Akv1..) – d, d rd1 rd1, hr etc – 100% prevalence, strain variations

 Many are not oncogenic  Exogenous viruses  salivary, milk, semen – eliminated from most commercially avail strains – C3H strain with exogenous MMTV avail from NCI & few others

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Mouse retrovirus – related phenotypes  Some Lymphomas e.g.

Genetic engineering by retroviruses:

Retroviruses & Lymphoma

Hrhr - hairless

– AKR (C58 etc) thymic lymphoma – Moloney sarcoma, Friend Leukemia, Abelson virus

 Color - Myo5ad  Dilute in DBA, etc h  Hairless H i l phenotype h - Hr H hr  Vision - Pde6brd1  rd blindness in C3H, SJL, FVB etc. – Viral insertion (Xmv-28) in intron 1 – + nonsense mutation (C  A) that truncates the protein

 i.e. Important part of mouse genomes

82

Environment, Infectious Phenotypes

functional genes/alleles, markers Retroelements (transposons, IAP etc) ~ 30% of mouse genomes

Retroviruses & Lymphoma

Hrrh - rhino  allele defined by NON complementation with hr  similar to Hr/Hr except no hair Normal coat up to ~ 10 do, then lose regeneration & skin becomes all hair, then waves of sparse fuzzy progressively thickened and growth hair growth… i kl d M t i wrinkled More extensive Cysts from hair canals, sheaths or hyperkeratosis in follicles  sebaceous glands  sebaceous large hair canal cysts with transformation, later keratinization plugs/balls of keratin Abnormal mammae, nails …  Autoimmune ? Leukemia

 Retroviral integration –

 

one or more MLV proviruses closely linked to hr allele.

   UV Irradiation Resistance?

83

84

mouse retroviruses as infectious agents

MMTV Bittner agent Early onset mammary tumors in C3H mmtv+

Hartley & al. 2008

– Vertical transmission via milk – Eliminated by fostering/rederivation

– RAW264.7 cells…. – Common mouse macrophage cell line (ATCC TIB71) – Newborn mice developed lymphoma following inoculation

Later onset mammary tumors in C3H mmtv mmtv– dt endogenous Mtv’s (Mtv1-56)

Mtv’s ‘recombine’, are B lymphocytotropic

Thymic T cell lymphoblastic lymphoma 85

86

Don’t forget/ignore these viruses completely:  DNA viruses – Adenoviruses MAV1, MAV2 (~8% J&L 1998) – Herpesviruses (~5% J&L 1998) • MCMV, MTV Thymic virus, gammaherpesviruses

– Papovaviridae – Poxviridae P i id

 INCLUSIONS

PyV (MPV), K (polyomaviruses) ECTV (O (Orthopoxvirus) th i )

Probably in a freezer, or in feral mice near you….  RNA viruses

88

– – – – – –

Arteriviridae Arenaviridae Hantaviruses Paramyxoviridae Picornaviridae Reoviridae

LDV - chemistry test – not ‘serology’ LCMV (bunyavirus?) (~5% J&L 1998) Wild mouse reservoirs Sendai, PVM, K – Respiratory … TMEV, EMCV , Ljungan? Reo3 (~5% J&L 1998)

[email protected] Rev Feb 2012

X Phenotype

87

Viral phenotypes oversimplified

Also consider

Enteric / Enterohepatic

MNV MHV MRV (mCMV)

Helicobacters C piliforme Salmonella Giardia? Spironucleus? Pinworms  rectal prolapse

Respiratory primarily

y murina Pneumocystis Sendai, PVM Pasteurella pneumotropica MPV (pneumotropic virus) Klebsiella oxytoca etc Bordetella avium, hinzii

Death & necrosis

Ectromelia CMV

MHV C piliforme;, Salmonella

Subclinical immunomodulation etc

Parvoviruses

Many possibilities

Subclinical + inclusion bodies

Adenoviruses, Herpesviruses, Polyomaviruses Most common agents in red

‘infected’ B cells traffic thru Mammary G  transform  polyclonal mammary tumors Thymic L in GR mice B Cell lymphoproliferative Dz in SJL/J

Environmental/Infectious Phenotypes Discussion Plan

X

Viruses

– – – –

1. 2. 3. 4. 5. 6. 

Nature & Nurture – Again Environmental variables – Top few Viral agents –Top few Emphasizing Bacteria – Top 3 COMPETENT mice Eukaryotes – Top few ‘Normal’ flora – the microbiome Immunodeficient mice – another lecture

90

10

Environment, Infectious Phenotypes

Mouse Bacteriology: Results – 2009

Health Monitoring recommendations

Pritchett-Corning, Cosentini & Clifford 2009 PREVALENCE % Bacterium

Method

Bordetella bronchiseptica Cilia-associated respiratory bacillus

N

NA

Europe

Culture

109,802

0.00

0.00

0.00

Serology

158,741

0.01

0.00

0.01

Citrobacter rodentium

Culture

82,337

0.00

0.00

0.00

Corynebacterium kutscheri

Culture

109,804

0.00

0.00

0.00

Helicobacter genus (any sp.)*

PCR

91,119

15.88

21.28

16.08

NH 07

> 1% Gram+

EU FELASA 2001 http://www.felasa.eu/recommendations/r ecommendation/

Pritchett Corning & al 2009 % POSITIVE tests

- Helicobacter hepaticus

PCR

91,463

12.45

10.23

12.37

1. Citrobacter rodentium

1. Helicobacter spp – 16% 2. P pneumotropica – 13%

- Helicobacter bilis

PCR

91,386

2.20

1.49

2.17

2. Cl. piliforme

3.

S aureus – 6%

Klebsiella oxytoca

Culture

185,937

0.38

1.32

0.38

Klebsiella pneumoniae

Culture

186,667

0.10

0.85

0.10

3 C. C kutscheri k t h i 3.

Culture

61,592

0.00

nt

0.00

Serology

455,102

0.01

0.16

0.01

PCR

43,777

0.00

nt

0.00

Pasteurella multocida

Culture

109,376

0.00

0.00

0.00

Pasteurella pneumotropica

Culture

109,403

13.20

4.00

12.90

• • • • •

Klebsiella oxytoca Klebsiella pneumoniae Mycoplasma CARbacillus Streptococcus

Other Pasteurella species

Culture

106,232

0.31

0.00

0.31

Any Salmonella species

Culture

109,655

0.00

0.00

0.00

Staphylococcus aureus

Culture

6.03

11.56

Mycoplasma pulmonis

Culture

Streptobacillus moniliformis

107,002 2842

0.00

0.00

6.07

Culture

109,804

0.00

0.00

0.00

Streptococcus sp. – β-haemolytic, Group B

Culture

106,971

0.24

0.00

0.24

Streptococcus sp. – β-haemolytic, Group G

Culture

109,733

0.00

0.11

0.00

X

6%

4. Mycoplasma spp. 5. Pasteurellaceae 6. Salmonella spp. 7. Streptococci

7%

Morbidity Mortality in mice X Likely bacterial causes (today)

Enteric /enterohepatic

Respiratory primarily

Helicobacters

Pasteurella pneumotropica

Staphylococcus aureus

Citrobacter rodentium

K pneumoniae (oxytoca)

Klebsiella oxytoca

Clostridium piliforme

Mycoplasma pulmonis

Streptococcus

Salmonella

CARBacillus

Strepto. moniliformis

Bordetella spp

Pseudomonas

K pneumoniae

C bovis

Enterococcus?

Other

K oxytoca

8. Helicobacter spp. 9. Streptobacillus moniliformis

23%

0.00

Streptococcus pneumoniae

X

Bacteria

J facilities Total

Likely bacterial phenotypes (especially in immunodeficient)

92

Bacteria

Also consider

Enteric /enterohepatic

Helicobacters Citrobacter rodentium Clostridium piliforme Salmonella Enterococcus?

MNV? MHV, MRV, mCMV Giardia? Spironucleus? Pinworms  rectal prolapse

Respiratory primarily

Pasteurella pneumotropica Klebsiella oxytoca etc Bordetella avium, hinzii

Pneumocystis murina M pulmonis, CARbacillus Sendai, MPV

Death & Sepsis

Pseudomonas Strep/enterococcus spp

Klebsiella oxytoca Proteus mirabilis Endo/enterotoxemia

Abscesses primarily

Staphylococcus spp

Streptococcus Gram negatives

Skin disease

Corynebacterium bovis MUD + opportunists

Check for mites! Ringworm ?

93

Helicobacter, Pasteurella, Staph aureus

X

Phenotype

 Prevalent &/or Pathogenic  Usually commensal/ opportunist  Not so likely – Historical …  Zoonosis

What can they do. Mouse strain susceptibilities. Research impact.

 Prevalent &/or Pathogenic  Usually commensal / opportunist  Not so likely – Historical …  Zoonosis

Rectal prolapse MOST likely cause today?

Helicobacters  Pinworms?  Citrobacter rodentium ?  Tumors?  Other phenotype ?

95

96

Rectal prolapse

Silver stains lots of bacteria

Perineal skin Perianal glands

97

[email protected] Rev Feb 2012

Colitis – Proctitis Inflammatory Bowel Disease phenotypes

98

99

11

Environment, Infectious Phenotypes Helicobacters in mice Liver  Chronic (lymphocytic) and active (neutrophilic) inflammation  Cholangiolar proliferation  Anisocytosis, y , anisokaryosis, y , aneuploidy, hepatocytomegaly,  intranuclear ‘inclusions’ of invaginated cytoplasmic material can be common in older mice.  relation ship with helicobacter or other 100 infections is not clear

Helicobacters in mice Liver  BALB/c sentinels  Necrosis + inflammation  With bacteria (WS)  This slide is very faded.  Don’t see necrosis in some strains  Can see necrosis (infarct?) without any agents 101

H hepaticus hepatitis Strain & Sex influence AxB RI mice 14 mpi B = Resistant

– Inflammation – Biliary hyperplasia

X

Helicobacters often subclinical

H hepaticus & tumors

H hepaticus  strain/sex dependent dz – SUBCLINICAL with no significant pathology OR – Hepatitis + Liver tumors

– discovered in A/J dt liver tumors

– WNL

103

Liver  Modified Steiner’s Silver stain  Sensitivity is low compared to PCR  Careful examination is time consuming  Silver stains are expensive  Rodent helicobacters are small  Liver Histology is terminal – for the mouse

102

Hepatocellular adenoma, carcinoma

A = Susceptible

Males more susceptible to liver dz

Helicobacters in mice

• MALES more susceptible

– Typhlocolitis in immunodeficient + competent mice,

Lymphoma Hemangiosarcoma

• FEMALES more susceptible

– OTHER TUMORS ? Mammary & Gut • Rao et al. 2006: - Rag2-deficient C57BL/6 Apc(Min/+)

Ihrig & al. 1999

LOTS of rodent helicobacters

H bilis, H hepaticus, H typhlonius  chronic typhlocolitis in immunodeficient immunoweird H bilis ‘associated associated with with’ human biliary Dz ?? H muridarum – chronic gastritis Many others: H rodentium, H typhlonius, (Flexispira) rappini, ganmani, etc sp…

X

[email protected] Rev Feb 2012

105

Helicobacters in Research

Liability or asset ? – Inflammation / Immune responses

Mouse Respiratory Disease Likely cause TODAY Pasteurella pneumotropica

• Cytokine etc immune responses

– Typhlocolitis in Susceptible mice – Hepatitis in Susceptible mice

– 4-14% ‘prevalence’ – isolated from submissions

Consider:

• elevated liver enzymes

– Bordetella (B hinzii etc – recent reports) – Klebsiella (K oxytoca – recent reports) – Mycoplasma & CARBacillus

– Liver tumors in Susceptible mice – Other tumors ? Mammary & Gut tumors

– H ganmani – most common in rats (& at JHU) 106

Ihrig & al. 1999

104

• Historical concern ? Unlikely now? 107

108

12

Environment, Infectious Phenotypes

URI: Rhinitis Mice are obligate nose breathers This could kill them Who dunnit ?

Who dunnit ? – P pneumotropica – B hinzii, avium ? – Klebsiella (oxytoca) ?

– P pneumotropica hi ii avium i ? – B hinzii, – Klebsiella (oxytoca) ?

Otitis

URI: Tracheitis

Pretty common in our mouse submissions Effects on – Hearing? – Behavior? – Immune?

• Immunodeficient ?

– Mycoplasma ?

• Immunodeficient ?

• Not so common

– Mycoplasma ?

– Streptococci ?

• Not so common

– Streptococci ? 109

110

111

Conjunctivitis Blepharoconjunctivitis

Bronchopneumonia (+ AMP)

Otitis Opportunists ? Or Pathogens ? – – – –

P pneumotropica? K oxytoca ? B hinzii/avium ? M Mycoplasma l ?

Likely causes?  Strain-related? e.g. Microphthalmia, entropion, KCS?  P pneumotropica ?  Bordetella spp ?

• Historical ?

– May isolate a lot of things

– Pseudomonas ?

NOT so likely:  Ectromelia virus ?

• Historical ? • Only? in immunodeficient ? • Neutrophil deficient

– Common finding in recent outbreaks

112

113

Phenotype: Infertility Pyometra

X

– P pneumotropica? – M pulmonis? – Imperforate vagina

X

 Also isolated from & implicated in – Conjunctivitis, otitis, pneumonia, cystitis, metritis ( fertility), preputial adenitis – Bronchopneumonia (with PcP ?) – Gram neg – pure cultures, can’t see it with tissue gram or silver stains

 Opportunists in immuno-deficient immunoweird D Percy

[email protected] Rev Feb 2012

116

Staphylococcus spp.

 S aureus (1 of few coagulase POSITIVE species) – Lymph node abscesses, botryomycosis, ulcerative dermatitis in competent mice – Furunculosis, abscesses in immunodeficient

– ‘Normal’? gut microflora, – isolated from healthyy nasopharynx p y

• Vaginal septa

P pneumotropica pyometra

Pasteurella pneumotropica

 Gram negative bacillus  Usually considered Commensal – opportunist

 Likely Causes?

115

114

 S xylosus, sciureus, hominis, hyicus etc ‘commensal’ (coagulase Negative) isolated from abscesses + Botryomycosis lesions in immunodeficient & GEM

117

13

Fungi

Abscesses

of concern / interest

Often one end or the other

 Usually Commensals, Opportunists in Compromised animals – Or experimental infections

 Pneumocystis murina (P carinii) – DISCUSSED WITH IMMUNODEFICIENT MICE  Trichophyton mentagrophytes – skin - ringworm

Environment, Infectious Phenotypes Pneumocystis murina

X

If you see death and pneumonia with these lung lesions, your mice probably are significantly immune deficient/ suppressed

 Aspergillus, Paecilomyces – opportunists

 Blastomyces, Histoplasma etc – opportunist yeast forms

 Torulopsis / Kazachstania ? 118

119

– ON gastric mucosa (PAS positive Easter eggs)

However … GMS

120

Environmental/Infectious Phenotypes Discussion Plan 1. 2. 3. 4. 5. 6. 

Mouse Eukaryota: Results - 2009 Pritchett-Corning, Cosentino & Clifford 2009 PREVALENCE %

Nature & Nurture – Again Environmental variables – Top few Viral agents –Top few Emphasizing Bacteria – Top 3 COMPETENT mice Eukaryotes – Top few ‘Normal’ flora – the microbiome Immunodeficient mice – another lecture

• Arthropods

2.Endoparasites: • Enteric helminths • Enteric protozoa

FELASA recommendations become an issue i in i US when h we wantt tto export to EU. A lot of places do not evaluate for protozoa considered to be commensal .

 Eimeria, Giardia, Spironucleus  Tritrichomonas (Chilomastix? Hexamastix? Entamoeba?)

124

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NA

Europe

Total

145,053

0.00

0.00

0.00

Direct

126,482

0.00

nr

0.00

0.43

0.12

1.31

0.25

Mites

Direct

130,976

0.11

Aspiculuris tetraptera

Direct

135,860

0.19

Syphacia muris

Direct

128,657

0.01

Syphacia obvelata

Direct

128,657

0.11

Oxyurids*

Protozoa Chilomastix sp.

Wet mount

94,890

3.74

nr

Wet mount

94,890

8.08

nr

8.08

Giardia sp.

Wet mount

102,093

0.00

0.00

0.00

Hexamastix sp.

Wet mount

94,890

4.45

nr

4.45

Monocercomonoides sp.

94,890

0.04

nr

0.04

Wet mount

94,890

0.03

nr

0.03

Spironucleus sp.

Wet mount

102,093

0.08

0.00

Trichomonads

Wet mount

94,890

8.88

nr

Retortamonas sp.

Wet mount

Eukaryota

Pathogenic ?

 Small Intestine (may be pathogenic, but not prevalent) – Giardia muris (not lamblia) – Spironucleus (Hexamita) muris – Cryptosporidium parvum • If you want to see them, look at a hamster »

 Large Intestine (prevalent, probably not pathogenic) – Flagellates (Tritrichomonads, Chilomastix, Hexamastix, etc ) – Entamoeba muris

Metazoa  A tetraptera  S muris  S obvelata

~ 0.2% (colon, float) ~ 0.01-1.3% (cecum, tape) ~ 0.12% (cecum, tape)

 (Fur) Mites

~ 0.1% (direct exam)

125

8.88

Protozoa of concern / interest

X

Pritchett-Corning, Cosentino & Clifford 2009

Protozoa  Entamoeba (muris) ~ 8%  Flagellates > 8%  Giardia or Spironucleus 1%

X

3.74

Entamoeba sp.

Octomitus ?

http://www.lal.org.uk/pdffiles/LAfel2.PDF

1.Ectoparasites:

N

Serology

Lice

122

Eukaryota Recommended Q3 months Testing

Method

Encephalitozoon cuniculi Metazoa

 http://www.radil.missouri.edu/n/1297374302/index.html

Health Monitoring in Accordance with 2001 FELASA recommendations (EU)

Agent

 Very Unlikely (unless wild mouse exposure) – – – –

Cryptosporidium muris - Stomach Eimeria muris – SI Klossiella muris - Kidney Sarcocystis muris - Muscle

126

14

Environment, Infectious Phenotypes Small intestine nu/nu

Cecum, colon B6,129,FVB TmTg

Small intestine

 Giardia muris

 Spironucleus muris

– Flying saucers • ‘On’ mucosa

– (Hexamita muris) – Torpedoes

– Enteritis?

• In crypts

– Enteritis? – Hamsters – Wild mice • Peromyscus

Diagnosis? Significance ?

127

– Hamsters – Wild mice

Diagnosis? Significance ? 129

• Peromyscus

Protozoal Flagellates in large intestine

Typical movement / motility

Commensal – always? Pyriform trophozoites Can fill lumen of cecum, prox colon Characteristic movements

130

X

– – – –

Tritrichomonad Chilomastix Hexamastix Cercomonoides etc

Regarding enteric protozoa

– Giardia  spin/ tumble/rotate – Spironucleus  darts – Trichomonads  slow swimmer – Entameobae  ‘morph’ – amoeboid

131

Amoebae ~>10 u – probably has helicobacters too

Nematodes (Pinworms)  Syphacia spp Easier to Detect – tape test

Where’ss the hamster? Where

Cestodes (tapeworms) ( p ) - Requires cat / carnivore exposure

Large intestine protozoa may be benign, but raise concerns about hygiene or microbial status 133

[email protected] Rev Feb 2012

132

Nematodes: Pinworms (oxyurids)

Metazoan parasites Helminths = worms

X

You should NOT see flying saucers or torpedoes in the small intestine.

– Or wild mouse? Peromyscus spp ….

Enteric protozoa on direct smears

Cecum, Colon

Adults, larvae in cecum, colon – Detect by (terminal) direct examination…

 Aspiculuris spp Sneakier …

Syphacia obvelata (muris) – asymmetric |) eggs on perineum – Detect by tape test

– Cysticercus fasciolaris  Taenia taeniaeformis  Asymptomatic with liver cysts

 Wild mouse exposure – Rodentolepis nana (smallest) -- Small intestine – dwarf tapeworm

 Arthropod intermediate host (found in humans too) 134

– Hymenolepis diminuta -- Small intestine – ‘rat tapeworm’ – R microstoma -- bile pancreatic ducts, duodenum

RADIL images

Aspiculuris tetraptera – symmetric () eggs in feces – Detect by fecal flotation 135

15

Environment, Infectious Phenotypes Mouse Pinworms

Mouse Pinworms

NOT how you want to diagnose pinworms in a clean barrier …..

 Syphacia obvelata (muris) – asymmetric eggs - tape test – > 400 egg/d !!; 12d cycle ! – Cecum, colon, retroinfection/anus? H i iis Enterobius E t bi vermicularis i l i ? Human pinworm – Old report of possible human infection by Syphacia…

 Aspiculuris tetraptera

RADIL images

– symmetric eggs in feces –