ImmunoDeficient Mice
Options & Challenges
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 [email protected]
http://www.hopkinsmedicine.org/mcp/PHENOCORE/index.html
1
Relevance to you?
Do you use immunodeficient mice?
– Nude ? Scid ?
– NOD/scid ?
– NSG or NOG ?
– Other immune deficient? Do you breed them ?
Do they live with or near competent mice?
Do ID & competent mice share equipment ? Imaging?
Do you inoculate biological materials?
– Do you test them by MAP or PCR? How do you do microbial surveillance?
– Sentinels?
– Direct? 2
AIMS
1.
Select the best mice for your project.
2.
Handle them to get best results with
fewer animals.
3.
Identify and eliminate threats before
they damage your research & budget.
3
4
Immunodeficient mice
Discussion Plan
Options
1. Mutations, Genetic backgrounds, Sources 2. Advantages & disadvantages
– RX: Irradiation Cytoxan etc - another lecture
‘Challenges’
1. Infections (manifesting as Entericor Respiratorydisease, death, sepsis abscesses, sepsis/death, or subclinical but patent infections of uncertain significance to your study)
• Biological Materials
• Viruses
• Bacteria & Fungi
• Eukaryotes:
Protozoa, Metazoan parasites
Immunodeficient mouse
publications Pre 2001
Croy & al 2001
Mutations CellsT CellsB InnateNK Leaky Tumors
Foxn1nu (nude) D N N yes
Prkdcscid(scid) D D N- yes lymphomaThymic
PrkdcscidLystbg
(scid-bg) D D N Reduced by bg lymphomaThymic NOD-Prkdcscid
(NOD-scid) D D D Minimal lymphomaThymic
NOD, Prkdcscid, Il2rgtm…
(NSG, NOG) D D D No
Primary T cell deficient options
LEAKY mice
B cells, T cells, immunoglobulin with age & microbial challenge (AKA conventional housing)
Mice that survive infection & reject Xenografts ….
RX: CHECK CBC + IG levels BEFORE XENOGRAFT
• N = Normal D= Deficient
6
7
Functional (phenotypic effects) of some
mutations in immunodeficient mouse models
B2m NK cell development/activity, CD8+ T cell activity
Foxn1
Nude
nu/nu lack thymus T cell deficient unable to reject allogeneic & xenogeneic grafts
nu/nu females are poor breeders, begin to ovulate late ~ 2.5 mo, & stop early, ~ 4mo
Il2rg NO thymicNO NK cells, lymphomas etc defects on in susceptibleinnate immunity. background, NOD.CB17‐Prkdcscid/SzJ mice. Prf1
perforin 1 NK cell activity, CD8+ T cell activity
Prkdc
scid
scid/scid no mature T & B cells, No cell mediated & humoral adaptive responses,
BUT leaky some ‘functional’ B and T cells as they age. Especially in non‐SPF conditions,
leakiness C57BL/6J, BALB/cByJ backgrounds > C3H/HeSnJSmn > NOD/LtSzJ background
++ radiosensitive cannot be as thoroughly irradiated as other IDmodels before being engrafted
Rag1 Rag1tm1MomOn NOD/Rag1 backgroundtm1Mom NONO diabetes mature, functional T and B cells.
NOD background
susceptible to immune mediated insulitis + diabetes
NK, macrophage, APC, complement activity
Immunodeficient Options (J) &
Background Effects
Back ground Innate
NK etc Leaky H2 NAME Characteristics (Phenotypes) BALB/cBy Normal Yes T d CByJ.Cg-Foxn1nu/J Normal B cells etcExtra thymic T cells with age
POOR breeders, female fertility nu/+ F x nu/nu M
Normal High d CBySmn.CB17-Prkdcscid/J
NO functional B and T cells HIGH NK, complement activity (normal APC function) Thymic lymphomas but < NOD.CB17-Prkdcscid/SzJ RadioSensitive:
C57BL/6J Normal NO b B6.129S7-Rag1tm1Mom/J NO functional B and T cells HIGH NK activity (normal APC, complement)
Normal High b B6.CB17-Prkdcscid/SzJ NO functional B and T cells HIGH NK, complement activity (normal APC function)
NOD/LtSzJ Low NO g7 NOD.129S7(B6)-Rag1tm1Mom/J NO functional B and T cellsPre-B cell > Thymic lymphomas (10.5mo) Somewhat RadioResistant
Low NO g7 NOD.Cg-Rag1tm1MomPrf1tm1Sdz/SzNO functional B &T cells; NO NK cell activityThymic lymphomas (short lifespan ~8.5mo) RadioResistant: survives up to 8 Gy
Low Low g7 NOD.CB17-Prkdcscid/SzJ
NO functional B and T cells; NOD background low NK activity, NO hemolytic complement activity, defects in myeloid development, poor APC functions -- RadioSensitive: tolerates up to 4 Gy
Thymic lymphomas (short lifespan ~8.5mo)
Low Low g7 NOD.Cg-PrkdcscidB2mtm1Unc/J
NO functional B and T cells No MHC 1 expression ~NO NK activity Thymic lymphomas (short lifespan ~6.3mo) Hemachromatosis dt?
Low NO g7 NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJNO functional B and T cellsNO NK cell activity Lymphoma-resistant and long-lived > 16m NIH stock Normal Yes T q NU/J (Foxn1nu) NIH outbred nude stock inbred at TJL
8 adapted from Jax notes #501, 2006
More Foxn1
nu
options
Source Background Name INbred AKA
Tac Bom B6N/Tac B6.Cg/NTac-Foxn1nu + B6 nude BALB/cAnN C.Cg/AnNTac-Foxn1nu + BALB/c nude BALB/cBom C.Cg/AnBomTac-Foxn1nu + BALB/cA nude BALB x NIH(S) CrTac:NCr-Foxn1nu NIH Nude NMRI (S) BomTac:NMRI-Foxn1nu Swiss nude NIH(S) NTac:NIHS-Foxn1nu Swiss Nude Crl BALB/cAnN CAnN.Cg-Foxn1nu/Crl + BALB/c Nude
CD1 (S) Crl:CD1-Foxn1nu CD1 nude NIH Nu Crl:NU-Foxn1nu Nu/nu NIH nude Hsd Ola NIH Nu Hsd:Athymic Nude-Foxn1nu NIH nude
NMRI (S) HsdCpb:NMRI-Foxn1nu Swiss Nude CD1 (S) HsdHli:CD1-Foxn1nu CD1 nude ICR (S) HsdOla:ICRF-Foxn1nu Swiss Nude MF1 HsdOla:MF1-Foxn1nu MF1 nude BALB/cOla BALB/OlaHsd-Foxn1nu + BALB nude
9
[email protected] 2013
More Prkdc
scidoptions
Source Background Name INbred AKA
Tac Bom C.B17 C.B-Igh-1b/IcrTac-Prkdcscid + CB17 scid
IcrTac IcrTac:ICR-Prkdcscid Scid - Swiss
C.B-17 C.B-Igh-1b/GbmsTac-Prkdcscid-Lystbg + scid-beige
NOD + NOD/MrkBomTac-Prkdcscid + NODscid
NOD NOD.Cg-PrkdcscidIl2rgtm1Sug/JicTac + CIEA NOG
Crl C.B17 CB17/Icr-Prkdcscid/IcrCrl + CB17 scid
Crl:SHO-PrkdcscidHrhr Scid hairless outbred
Hsd Ola BALB/cJ BALB/cJHan™Hsd-Prkdcscid + BALB/c Scid
C.B-17 C.B-17/IcrHsd-Prkdcscid + CB17 scid
C3H C3H.C-Prkdcscid/IcrSmnHsd + C3H scid
ICR (S) HsdIcr:Ha(ICR)-Prkdcscid Scid
NOD CB17 NOD.CB17/JHliHsd-Prkdcscid + NODscid
IF the name begins with the SOURCE, it is OUTbred (or not inbred) May not be as homogeneous as inbred BUT May be more robust & easier to breed / maintain
10
More other options
Source Background Name INbred AKA
Tac C.B17 C.B-Igh-1b/GbmsTac-Prkdcscid-Lystbg + Scid beige NIH(S) Tac:NIHS-LystbgFoxn1nuBtkxid NIHIII nude NOD NOD.Cg-PrkdcscidIl2rgtm1Sug/JicTac + CIEA NOG
Crl C.B-17 CB17.Cg-PrkdcscidLyst bg/Crl + Scid beige
NIH(S) Crl:NIH-LystbgFoxn1nu Btkxid NIHIII nude
HSD CBA CBA/HNOlaHsd-Btkxid + XID
NIH(S) Hsd:NIHS-LystbgFoxn1nuBtkxid NIHIII nude
B6 C57BL/6OlaHsd-Lystbg + Beige
C.B-17 C.B-17/IcrHsd-PrkdcscidLystbg + Scid beige
NSG or NOG?
NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ
– NSG- Il2rg null – Schultz at J - (Shultz & al. 2005).
NOD.Cg-PrkdcscidIl2rgtm1Sug/JicTac
– NOG - Il2rg deficient – CIEA (Jic) – Japan (Ohbo & al. 1996
11
Immunodeficient options
Conclusion: LOTS OF OPTIONS.
QUIZ:
–
Do you order from the same vendor ?
–
Do you order exactly
the same mice ?
–
Do you check the health reports?
–
Do you request Klebsiella negative?
–
Do you order from the same room/isolator?
–
Every time?
12 13Immunodeficient mice
Discussion Plan
1.
Options
1. Mutations, Genetic backgrounds, Sources
2. Advantages & disadvantages
– RX: Irradiation Cytoxan etc - another lecture
2.
‘
Challenges’
1. Infections(manifesting as Enteric or Respiratory disease, death, sepsis abscesses, sepsis/death, or subclinical but patent infections of uncertain significance to your study) • Biological Materials
• Viruses • Bacteria & Fungi • Eukaryotes:
Protozoa, Metazoan parasites
Competent vs Immunodeficient
Infectious Phenotypes
Really Oversimplified
Competent Immunodeficient •Immune response •Innate + acquired•Antibody response seroconversion •immune mediated lesions, necrosis •Control/eliminate infection.
•Less inflammation •Cytopathic effect /necrosis •Failure to seroconvert •Failure to clear heavy burden
GREAT RESERVOIR & risk to others IMPLICATIONS
Immunodeficient don’t seroconvert reliably
hard to detect by serology seroconversion ID don’t clear many infections - if they survive:
Heavier burdens
Easier to detect agents
Harder to eliminate agents
Source of agents that may be hazard to others 14
Competent vs Immunodeficient
Infectious Phenotypes
Really Oversimplified
Competent Immunodeficient •Immune response •Innate + acquired•Antibody response seroconversion •immune mediated lesions, necrosis •Control/eliminate infection..
•Less inflammation •Cytopathic effect / necrosis •Failure to seroconvert •Failure to clear heavy burden
GREAT RESERVOIR & risk to others Implications for Microbial surveillance
Competent sentinels
– Should seroconvert but may not (e.g. Parvoviruses)
– May clear opportunist infections to undetectable levels
– May pose a risk to immunodeficient neighbors
Direct testing (e.g. fecal PCR) of immunodeficient residents, may be a useful adjunct
15
Likely Infectious Phenotypes & Agents
in immunodeficient mice
Enteric
enterohepatic Respiratory Other
MNV Sendai Parvoviruses
MHV PVM (paramyxo-) Ectromelia viruses
MRV mCMV
Helicobacters Pasteurella pneumotropica Staph spp
Clostridium piliforme Bordetella spp Klebsiella oxytoca Salmonella Klebsiella o or p? Strep/enterococcus
Pseudomonas Giardia ? Pneumocystis murina Proteus mirabilis
Spironucleus? C bovis
Pinworms Etc….
More Common and or Problematic agents in red 16
NOT primarily enteric or respiratory
(Oversimplified)
Phenotype Agents
Death & Necrosis Ectromelia virus - orthopoxvirusCytomegalovirus mCMV Death & Sepsis
Gram Positive: Strep/enterococcus spp etc.
Gram negative: Pseudomonas Klebsiella Proteus etc Endotoxemia, enterotoxemia
Abscesses Staphylococccus spp, etc. Skin disease Corynebacterium bovis MUD + opportunists Subclinical
immunomodulation etc Parvoviruses Subclinical +
inclusion bodies Adenoviruses, Herpesviruses, Polyomaviruses
17
Likely viral
phenotypes in
immunodeficient mice
Phenotype Viruses Also consider
Enteric / Enterohepatic MNV MHV MRV (mCMV) Helicobacters C piliforme Salmonella Giardia? Spironucleus? Pinwormsrectal prolapse Respiratory primarily Sendai, PVMMPV (pneumotropic virus)
Pneumocystis murina
Pasteurella pneumotropica
Klebsiella oxytoca etc Bordetella avium, hinzii Death & necrosis EctromeliaCMV MHVC piliforme;, Salmonella Subclinical
immunomodulation etc Parvoviruses Many possibilities Subclinical + inclusion bodies Adenoviruses, Herpesviruses, Polyomaviruses 18
[email protected] 2013
Likely bacterial
phenotypes in
immunodeficient mice
Phenotype Bacteria Also consider
Enteric /enterohepatic Helicobacters Citrobacter rodentium Clostridium piliforme Salmonella Enterococcus? MNV? MHV, MRV, mCMV Giardia? Spironucleus? Pinworms rectal prolapse
Respiratory primarily Pasteurella pneumotropicaKlebsiella oxytocaetc Bordetella avium, hinzii
Pneumocystis murina Sendai, MPV Death & Sepsis Pseudomonas
Strep/enterococcus spp
Klebsiella oxytoca Proteus mirabilis Endo/enterotoxemia Abscesses primarily Staphylococccus spp Streptococcus
Gram negatives Skin disease Corynebacterium bovis
MUD + opportunists Ringworm ?
19
Infectious Agents in
Biological Materials
Tumor xenografts, cell lines, sera, etc.
In a freezer, in specimens, or in mice
near you …
Risk to projects & personnel.
ATCC does NOT test for most of these.
MAP Test or PCR test to detect them
20
Biological Materials
Recent (also historical) contaminants
Agents found recently… Some Phenotypes in immunodeficient Mycoplasma sp. Respiratory dz +shedding
LDV Immunomodulate LE’s +shedding
Human herpesviruses BIOHAZARD + shedding
HIV BIOHAZARD + shedding
Hepatitis B BIOHAZARD + shedding
Parvoviruses Immunomodulate, Tumor growth ? +shedding
Polyoma virus Immunomodulate, Tumors +shedding
LCMV Immunomodulate, BIOHAZARD + shedding
Mouse adenovirus Immunomodulate – INIB? Kid, Gut +shedding
Mouse hepatitis virus Wasting + enterohepatic DZ +shedding
mCMV) Death – hepatic etc necrosis +shedding
Ectromelia virus Death &shedding
Pneumonia virus of mice (PVM) Respiratory DZ +shedding
Murine norovirus Enteritis encephalitis n some +shedding
Sendai virus Wasting + respiratory Dz +shedding
E. cuniculi Kid + CNS ? +shedding
from CBW RADIL & CC CRL – AALAS 2010
21
Rodent Agents in Biomaterials
from CBW RADIL & CC CRL – AALAS 2010
22
Agent % Positive
Mycoplasmasp. ~8.0%
Lactic dehydrogenase elevating virus (LDV) > 1%
Parvoviruses < 1%
Polyoma virus < 1%
Lymphocytic choriomeningitis virus (LCMV) < 1%
Mouse adenovirus < 1%
Mouse hepatitis virus < 1% Mouse cytomegalovirus (mCMV) < 1%
Ectromelia virus < 1%
Pneumonia virus of mice (PVM) < 1%
Murine norovirus < 1%
Sendai virus < 1%
E. cuniculi < 1%
Human & other viruses found in
‘human’ biomaterial submissions
(2009-10)
23
Infectious agent Percent positive
HIV <1%
Hepatitis B <1%
Human herpesvirus 6 >1%
Human herpesvirus 8 <1%
Epstein Barr virus >1%
Reo, LDV, LCMV, parvo <1%
from CBW LR RADIL & CC CRL – AALAS 2010
Compmed May 16, 2011
…Inoculated mice became ill, had ascites
and splenomegaly …
The (commercial mouse) serum was
positive with high titer to
mouse
hepatitis virus, Theiler's
meningoencephalitis virus, mouse
rotavirus, mouse norovirus and Sendai
virus….
–
Comparative Medicine List
[[email protected]]
–
Monday May 16, 2011
24
Enteric +/- hepatic disease in
Immunodeficient mice
Viruses
MNV - Hepatitis etc in stat deficient MRV – neonatal diarrhea like competent mice MHV – Hepatitis enterocolitis wasting
CMV – mouse cytomegalovirus death with liver, necrosis etc
Bacteria
Helicobacters
Clostridium piliforme Salmonella typhimurium
Protozoa Giardia, Spironucleus ? proliferative enteritis ? Bigger Pinworms– heavy burdens rectal prolapse
NOT associated with disease
– Segmented Filamentous Bacteria – Large intestine protozoa 25
MNV - Murine Norovirus(es)
in immunodeficient mice
Phenotypes (pathology) in Immune deficient mice•Subclinical in most or
•Pneumonia, hepatitis, vasculitis, (encephalitis) in severely
innate immune deficient mice e.g. Rag2–/–/Stat1–/–
Rag1−/−/Stat1−/−Rag1−/−/IFNγR−/−Ward & al.; Wobus & al Is it a problem for you ?
•Innate immunity ? Macrophage function? Paik & al •IBD, enterohepatic phenotypes ? Paneth cells Cadwell & al
•Cell culture contaminant
26
MNV?
Sick Nude mouse
Small intestine
–
Necrosis + crypt
abscesses
elsewhere
–
Paneth cell
degeneration
–
Similar to Cadwell
2010 ?
27[email protected] 2013
Mouse Rotavirus (MRV - EDIM)
in immunodeficient mice
Phenotypes (pathology) in Immune deficient mice•Neonatal diarrhea – as in competent mice Is it a problem for you ?
•Breeding colonies, runting
•Immunomodulation
28
MHV Mouse Hepatitis Virus
in immunodeficient mice
Phenotypes (pathology) in Immune deficient mice• Adult wasting with necrosis/syncytia liver, spleen, lymph nodes, GALT, marrow, brain
• Don’t clear virus i.e. persistent infection
• (granulomatous peritonitis/ pleuritis in Ifn Ko – similar
to Feline Infectious Peritonitis? ) Is it a problem for you?
• Wasting in old mice in chronic studies, or breeding
• Liver enzymes, liver, gut phenotypes • Immune phenotypes
• Cell culture contaminant including ES cells 29
MHV
in immunodeficient mice
‘wasting phenotype’ of
Prkdc
scidor
Foxn1
nuetc immunodeficient …
Liver necrosis –
progressive
severe
30MHV
Liver Phenotypes
Necrosis – extensive in
immunodeficient
–
+/-
LE’s
•AST, ALT, LDH, GGT…–
Not much inflammation
in immunodeficient
–
Syncytia = histologic
‘hallmark’
31MHV
Gut Phenotypes
Ascending colon target
Distal SI too
Syncytia, Necrosis
Syncytia in other tissues
too
32Morphologic Diagnosis: _____________
Cause (most likely Today): __________
Helicobacters
Likely sex?
Also consider:
Citrobacter?
Pinworms?
Tumors?
IBD Phenotype ?
33 34Colitis – Proctitis
Inflammatory + Proliferative
Bowel Disease phenotypes
Lymphocyte deficient mice
more
neutrophils + macrophages
Helicobacters
often subclinical
35
Phenotypes (pathology) in Immune deficient mice
• Usually typhlocolitis, often proliferative (IBD)
• H hepaticus, mastomyrinus, typhlonius, etc
• Females more susceptible ? to bowel disease
Is it a problem for you ?
• Immunomodulate
• Gut, Liver phenotypes, Liver enzymes • Liver, Gut, Mammary tumors
Morphologic diagnosis: ____________
Etiology:
____________
36
[email protected] 2013
Liver
– Necrosis +
– INTRACELLULAR filamentous bacteria Giemsa Warthin Starry UCD CCM images 37
Clostridium piliforme
Tyzzer’s disease
(
Bacillus piliformis
)
Kills hamsters + susceptible mice, rats, horses, etc
Gram negative filamentous bacilli
– Warthin Starry silver stains it best
Obligate intracellular parasite
– Cultured in eggs or cell culture
Historically significant agent in research rodents –
low prevalence now.
38
Segmented Filamentous
bacteria
Florid growth
suggests
immunoweird ?
Competent mice
typically clear to
very low levels by ~
1mo
Good for your
mice? Th17 etc
development…
39Giardia muris
Nude sentinel
–
Abundant in
nu/nu
but NOT
nu/+
cagemates
–
nu/+
cleared to
undetectable levels
–
0-minimal
inflammation
–
Expect these in
hamsters …
40Spironucleus muris
(Hexamita muris)
Nude sentinel
–
Abundant in
nu/nu
but NOT
nu/+
cagemates
–
nu/+
cleared to
undetectable levels
–
0-minimal inflammation
–
Expect these in
hamsters …
41 In CryptsLarge Intestine Flagellates
42
Entamoeba muris
43 Amoebae ~>10 u –probably has helicobacters too
Pinworms
in
Immunodeficient mice
Expect this mouse to
be immunodeficient
because of heavy load
easy to detect
shedding
lots of infectious eggs
in environment, on
equipment …
44 45
Nematodes
Pinworms (oxyurids)
Adults, larvae in cecum, colon
– Detect by (terminal) direct examination…
Syphacia obvelata (muris)
– LOTS OF asymmetric |) eggs on perineum
– Detect by tape test
Aspicul
uris
tetraptera
– Sneakier…
– FEWER symmetric () eggs in feces
– Detect by fecal flotation
RADIL images
Respiratory disease in
Immunodeficient mice
Viruses
Sendai virus – Paramyxovirus
Pneumonia virus of mice – Paramyxovirus Mouse pneumotropic virus – Polyomavirus
Bacteria
Pasteurella pneumotropica etc opportunists Mycoplasma CARbacillus
Bordetella avium, hinzii etc Klebsiella oxytoca or pneumoniae? Fungi Pneumocystis murina
Clinical Phenotypes
– Dyspnea – maybe chattering
– Sick- dead
– Wasting in chronic disease
46
Sendai Virus
in Immunodeficient mice
47
Phenotypes (pathology) in Immune deficient mice
• Progressive wasting, dyspnea, death
• Proliferative (tumor-like) lesions instead of necrotizing
lung lesions; syncytia, inclusions
• Not much inflammation
Is it a problem for you ?
• Wasting dyspnea in chronic studies, breeding
• Open cages – contained by microisolator caging • Immunomodulate
• Cell culture contaminant
Sendai Virus
In nude, scid etc
–
No T cell-mediated
necrosis
–
Not much
Inflammation
•Some neutrophils …–
Proliferation
–
Syncytia
–
Intranuclear
Inclusions
48Sendai Virus
Proliferative
tumor like
‘Adenomatous
change’ in
immuno-deficient mice
49Upper Respiratory Disease (URI)
Mice are obligate nose breathers
Rhinitis can kill.
–
P pneumotropica?
–
B hinzii, avium?
–
Klebsiella oxytoca ?
K pneumoniae
?
–
Mycoplasma ?
•Not so common–
Streptococci ?
•Not as likely 50Upper Respiratory Disease (URI)
Otitis
Common
Gram 0
– ???? – MycoplasmaGram+
– Strep/Enterococcus Gram-– Pasteurella – Bordetella – Klebsiella 51Upper Respiratory Disease (URI)
Otitis
Common
Gram+
– Strep/Enterococcus Gram-– Pasteurella Bordetella KlebsiellaGram 0
– ? – Mycoplasma 52Upper Respiratory Disease (URI)
Otitis
Common
Myd88
Gram+
– Strep/Enterococcus 53Bronchopneumonia (+ AMP)
Lobar consolidation with bronchitis,
broncho-pneumonia
Also acidophilic macrophage pneumonia
–
P pneumotropica?
–
B hinzii, avium?
–
Klebsiella oxytoca ?
K pneumoniae
?
54[email protected] 2013
Pasteurella pneumotropica
Gram negative bacillus
Usually considered Commensal – opportunist
– ‘Normal’? gut microflora,
– isolated from healthy nasopharynx
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
55
Bordetella spp
Gram negative bacillus
B avium, B hinzii
B bronchiseptica
– historic? in mice
– Rabbit & Guinea pig problem
Isolated from implicated in respiratory disease
with upper respiratory lesions (rhinitis
tracheitis) in mice
Short gram negative rods
Look among cilia in respiratory epithelium
Warthin starry silver stain
– cf B bronchiseptica, Kennel cough
56
Bordetella
spp
Gram negative Bacilli adhere to cilia
Hard to identify on histo
SEM of
B bronchiseptica
on cultured rabbit
tracheal epithelium
Edwards & al 2005
57Respiratory disease in
immunodeficient mice
Sudden Death
Diffuse consolidation
interstitial
pneumonia
58Respiratory disease in
immunodeficient mice
Consolidations
Intra-alveolar
eosinophilic foamy
material
0-Minimal inflammation
Special stain ?
Other diagnostic tests ?
59
Respiratory disease in
immunodeficient mice
Pneumocystis murina (P carinii)
GMS silver stains
Cysts
Fungus
60
NOT primarily enteric or respiratory
Phenotypes - Oversimplified
Death with Necrosis - Viral
Death with Necrosis – bacterial (sepsis)
Abscesses – bacterial
Skin disease
No obvious disease
61
Death, Necrosis, Sepsis in
Immunodeficient mice
Viruses
Ectromelia virus - orthopoxvirus Cytomegalovirus mCMV
(also MHV – with necrosis in liver) Bacteria
Gram Positive: Strep/enterococcus spp etc. Gram negative: Pseudomonas Klebsiella Proteus etc (Also c piliforme, Salmonella – with necrosis in liver)
Fungi Opportunists? –.g. Paecilomyces Trichosporon, zygomycetes
62
Ectromelia Virus
63
Phenotypes (pathology) in Immune deficient mice
• DEATH
• Many immunodeficient Hily susceptible to acute lethal dz • Few Gross lesions (die too quickly)
• +/- liver spleen necrosis with inclusions
Is it a problem for you ?
• Biological materials
• Sentinels do not reliably seroconvert
Bacterial sepsis in
Immunodeficient mice
Bacteria Gram Positive Streptococcus Enterococcus Gram Negative Klebsiella oxytoca Proteus mirabilis PseudomonasEnterotoxemias – post antibiotics Any ? bacteria that are not cleared can
bacteremia
Clinical Phenotypes
– Death
– Ill-moribund mice
64
Mortality NSG Post Xenograft
Liver
Gram stain
Usually not so
obvious
Bacteremia
sepsis
65
Mortality in NSG
Kidney
Intravascular
bacteria
66
Mortality in Myd88
Brain
Gram stain
Overwhelming
gram positive
bacteremia and
bacterial
colonization
E faecalis
isolated
from some
67Mortality in LAD
cd11/18 deficient
Meningoencephalitis
Gram positive cocci
Experimental IP streptococcus infection
–
Unexpected mortality
68
Streptococci / Enterococci
Morphology
–
Gram positive cocci
–
Pairs (diplococci)
–
Chains (streptococci)
69
Streptococci / Enterococci
S pneumoniae (beta hemolytic, non A, non B)
– Many mice relatively resistant
– but otitis / meningoencephalitis / mortality in LAD
– Pneumonia/mortality in C3H/HeJ (Tlr4 deficient) Group B (S agalactiae) may rhinitis, meningitis,
encephalomyelitis in nude
Alpha hemolyticssepticemia in scid
Non-hemolytic (= gamma hemolytic) isolates from septic immunodeficient
S zooepidemicus dermatitis (Lancefield type C considered to be non-pathogen)
E faecalis – emerging problem in immunodeficient ? Especially NSG ? Foreman & al 2010
70
Gram Negative
Sepsis bacteremia in
immunodeficient mice
Klebsiella oxytoca – ‘emerging’? NSG etc
Pseudomonas aeruginosa – most famous
Proteus mirabilis
– Immunodeficient mice can have these and do fine – until stressed….
– Do you check vendor health reports?
– Do you request mice that are negative for these agents?
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Klebsiella oxytoca
Gram negative rod
– capsule
Opportunist in NSG etc
Esp in UTI urinary tract infections – Foreman & al 2010
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[email protected] 2013
Klebsiella oxytoca ?
Gram negative rod
– capsule
Opportunist in NSG etc
Esp in UTI urinary tract infections – Foreman & al 2010
Rule out E coli
73 74
K oxytoca
otitis in C3H/HeJ
Macarthur & al 2007
Note ‘capsules
’Pseudomonas aeruginosa
Common isolate from healthy mice
– P aeruginosa etc pseudomonads are common in tapwater control with acidification, hyperchlorination
Opportunist
bacteremia, mortality in
neutropenic
(esp irradiated) mice
– A lot of immunodeficient mice are fine - until irradiated, then succumb (no neutrophils).
– Also urolithiasis, otitis etc
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Proteus mirabilis
Common isolate from healthy mouse GI tract
– Grows rapidly & Overgrows other species in culture – may mask a real cause of disease
– Dx: ‘Proteus overgrowth’
Immunodeficient mice
septicemia,
fibrinosuppurative peritonitis, pyelonephritis,
hepatitis, pneumonitis, mortality
Bone marrow
MDA 468
MCF7 (+ estrogen)
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Happy hematopoietic marrow
Too much bone
Not much marrow
Abscesses in
Immunodeficient mice
Bacteria Also consider
1. Staphylococcus spp
Klebsiella oxytoca E coli
Streptococcus etc
Corynebacterium kutscheri –pseudotuberculosis – historical ?
Etc Gram Neg
Fungal Opportunistsabscesses, granulomas, mycosis • Paecilomyces variotii • Trichosporon beigelii • Zygomycetes 78
Abscesses
Often one end or the other
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S. sciureus
isolated
Head & Neck tumor?
Ulcerated = SCC ?
Facial
Abscesses
Abscesses
S aureus or
Coagulase NEG
Staphylococcus spp
e.g. S xylosus, S hyicus
etc. in immunodeficient
or immunoweird
Botryomycosis
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[email protected] 2013
Staphylococcus spp
.
S aureus
(1 of few coagulase POSITIVE species)
– Lymph node abscesses, botryomycosis, ulcerative dermatitisin competent mice
– Furunculosis, abscesses in immunodeficient
S xylosus, sciureus, hominis, hyicus
etc
‘commensal’ (coagulase Negative) isolated from
abscesses + Botryomycosis lesions in
immunodeficient & GEM
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Skin disease in
Immunodeficient mice
Bacteria
Corynebacterium bovis
Opportunists in ulcerative dermatitis, - likely? to progress to sepsis
Fungi Ringworm ? T mentagrophytes? Historic ? Bigger Demodex muris in follicles
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Skin
Disease
Nude mouse
Etiology?
Histology
findings?
Special Stain?
– c/o RADIL DORA at
– http://www.radil.missouri.edu/info/dora/mousepag/derm.html#III
8585 86
Scurfy skin + pup mortality in
Nude, Rag1, NODscid, NSG etc
Gram
Corynebacterium
Gram POSITIVE irregular short rods
‘Chinese letter’ configuration
Very small
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C. diphtheriae
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Corynebacterium bovis
HAC = Hyperkeratosis Associated Coryneform
bacteria
Orthokeratitic epidermitis, dermatitis
– looks like ‘corn meal’ in Nude mice
Mortality in suckling mice
Also isolated from hyperkeratotic, haired
immunodeficient (and competent ?) mice
Tough to eradicate….
Mouse Ulcerative Dermatitis
Isolate anything from
chronic MUD
MUD likely to progress to
sepsis in immunodeficient
Staphylococcus spp are
common
Epidermolytic toxins can
staphylococcal scalded
skin syndrome (SSSS)
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Subclinical Infectious concerns
in Immunodeficient mice
Subclinicalimmunomodulation
etc Parvoviruses
Subclinical +
inclusion bodies Adenoviruses, Herpesviruses, Polyomaviruses Sublinical until stressed, further suppressed …. Gram positives… Gram negatives… Almost anything ? 90
[email protected] 2013
Parvoviruses
in immunodeficient mice
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Phenotypes (pathology) in Immune deficient mice
•Usually No Evidence of Disease •Persistent infection, shedding
•Runting, death, INIB in NOD.Cg-H2H4-Igh-6−/− •MVMi exp Lethal leucopenia in scid
Is it a problem for you ?
•Targets cells in S phase
•Oncolytic in cancer models Poor tumor growth ? •Competent sentinels may not seroconvert fast or reliably •Cell culture contaminant
MVM (MMV) – no lesions?
NOD.Cg-H2H4-Igh-6
−
/
−
mouse
Runting & death - Naugler et al. 2001
Spleen - intranuclear inclusion bodies
IHC positive for
MVM VP2
Frankin 2006
ILAR J
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Immunodeficient Challenges
Conclusion: LOTS OF CHALLENGES.
QUIZ:
–
DO you MAP or PCR test biological materials
that you use in Immunodeficient mice?
–
Do you check the health reports?
–
Do you request Klebsiella negative?
–
Do you assess ill mice for agents that could
be a risk to other mice or experiments?
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Is CLEANEST BEST?
for the mice? for the project?
YES
for immunodeficient mice that are
expected to survive, and accept grafts
from other animals.
Maybe not necessarily?? for GEM
where Environmental/Microbial
‘challenge’ may expose a phenotype &
Rederivation may ‘disappear’ it
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Immunodeficient mice
SUMMARY & Conclusions
Options – Lots
1.
Mutations, Genetic backgrounds, Sources
2.
Advantages & disadvantages
‘
Challenges’ – Lots
1. Beware of Biological Materials!
2. Enteric, Respiratory & OTHER diseases
-What is likeliest in your situation ?
3. Consider direct testing (PCR) as well as
sentinels
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Infectious
REFERENCES
Baker, D. G. (2007). FLYNN’S PARASITES OF LABORATORY ANIMALS. 2nded. ACLAM, Blackwell Publishing.
Hedrich, 2012. THE HANDBOOK OF EXPERIMENTAL ANIMALS SERIES: THE LABORATORY MOUSE. Elsevier.
Percy, D. H. and S. W. Barthold (2007). PATHOLOGY OF LABORATORY RODENTS AND RABBITS. Ames, IA, Iowa State University Press. 3rded
Sellers, R; Clifford; Treuting; Brayton. 2012. Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice. Vet Path. 49(1): p. 32-43.
Treuting, P; Clifford; Sellers; Brayton. 2012. Of mice and microflora: considerations for genetically engineered mice. Vet Path. 44-63.
Various authors, specific diseases and agents. 2006. In Fox & al. Ed’s. THE MOUSE IN BIOMEDICAL RESEARCH 2nd Ed. ACLAM series. Elsevier.
Various Authors ILAR Journal Volume 49(3) Detection and Management of Microbial Contamination in Laboratory Rodents
http://dels-old.nas.edu/ilar_n/ilarjournal/49_3/html/
Various authors, University of Missouri RADIL Diseases of Research Animals (DORA) site http://www.radil.missouri.edu/info/dora/Dora.htm
http://pixdaus.com/
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Sources of mice
and surveillance information …
Charles River (Crl) http://www.criver.com/research_models_and_services /research_models/mice_a_b.html Harlan (Hsd, Ola) http://www.harlan.com/models/usmodels.asp Jax (J) http://jaxmice.jax.org/info/strain_categories.html NCI Frederick (N, Cr) http://web.ncifcrf.gov/research/animal_production_pr ogram/strain_information/ Taconic (Tac) http://www.taconic.com/wmspage.cfm?parm1=579
Research & Reporting
Guidelines
Workman, P., E. O. Aboagye, et al. (2010). "Guidelines for the welfare and use of animals in cancer research." Br J Cancer 102(11): 1555-1577.
Kilkenny, C., W. J. Browne, et al. (2010). "Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research." PLoS Biol 8(6): e1000412.
National Academy of Sciences: Guidance for the Description of Animal Research in Scientific Publications (2011)
– http://dels.nas.edu/Report/Guidance-Description-Animal/13241
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