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…
phenocore@jhmi.edu
http://www.hopkinsmedicine.org/mcp/PHENOCORE/index.html
1
Yikes!
 How do I find out more about
– My mouse diets, environment
– My
M microbial
i bi l SSurveillance
ill
– Infectious & other ‘environmental’
phenotypes
4
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.

X
Phenotypes are determined by:
Nature
– Genetics
• Genetic Manipulation
• Genetic Background
Nurture
– Environmental Factors
• Non-infectious
• Infectious
6
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
X
Examples:
experimental infection models
Resistant
 This is an over simplification of strain
8
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
cbrayton@jhmi.edu Rev Feb 2012
 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 ?
3
5
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
7
Questions
2
The Real Aim:
X
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
1
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
10
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
15
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
12
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.
13
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 …..
15
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
cbrayton@jhmi.edu Rev Feb 2012
•  stressed & dead mice
17
18
2
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
$$$
20
19
 Restricted vs ad lib
 Fat/Prot/Carb/Fiber
 Contaminants
 Endocrine disrupters
 Rx (Fbz etc)
 Special diets
– & their ONLY protein source
–
–
–
–
• Prefer bedding ?
 $$$
23
X
BOTTOM LINE:
What diet & Why ?
24
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
21
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
27
cbrayton@jhmi.edu Rev Feb 2012
3
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
X
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
cbrayton@jhmi.edu Rev Feb 2012
MHV
Parvoviruses
Parvoviruses
4
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%
<1%
~8%
1.7
>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?
35
 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?
34
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
30
Test Lab/location 
• adequate exposure +
• Sufficient immune response
2
PATHOGENS
Mouse Virus Testing
– ELISA, IFA, MFIA – multiplex
– Requires
MHV
2003
Livingston
& al
MHV
Zoonoses
29
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
4
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
38
39
– 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
40
41
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
42
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
cbrayton@jhmi.edu Rev Feb 2012
44
45
5
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
47
46
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
51
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 ?
cbrayton@jhmi.edu Rev Feb 2012
53
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
50
Mouse Parvoviruses
Coley et al. 2005. J Virol. 79(5):3097-106
48
54
6
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)
55
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
56
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’ <1% (from ~30% )
Transmission: fecal oral
• direct contact, fomites – pretty fragile
• dirty bedding sentinels should detect well
Control: Rederive
58
X
– Foster ? Depopulate ?
MRV (EDIM)
Phenotypes
In immuno sufficient mice
Subclinical, or Suckling diarrhea - yellow, watery with milk in
stomach
Pups usually survive (EDIM live, LIVIM die)
Transient hydropic swelling of villus TIP epithelium
(resembles normal suckling)
Acidophilic cytoplasmic inclusions ??
Can  Malabsorption + E coli overgrowth; dry skin, fecal impaction,
runting
In immuno deficient mice
Transient neonatal diarrhea – like competent mice
Nudes clear it ; SCID, Rag etc  persistent infection + shedding
Experimental /model phenotypes
Neonatal diarrhea – model for rotavirus infection in human etc spp
Is it a problem for you ?
Breeding colonies, runting
61Immunomodulation
cbrayton@jhmi.edu Rev Feb 2012
Mouse diarrhea
photo scoring system.
system
a. negative / normal stool
b. Category 2 stool
c. liquid yellow stool
from mouse inoculated
with RRV
d. similar stool expressed
from mouse inoculated
with PI-RRV (d).
 Transient Apical
epithelial
vacuolation
 Looks like normal
‘nursing’ pup gut
with absorption
of hi fat milk….
• AFIP WSC 14
1/14/98
SHAW R & al. Rotavirus Diarrhea Is Caused by Nonreplicating Viral
Particles. J Virol, 1995, p. 5946–5950 Vol. 69, No. 10
59
60
TMEV (GD7, GDVII)
Theiler’s Mouse Encephalitis Virus
TMEV demyelination
 Picornaviridae; Cardiovirus group
– SS RNA virus, non enveloped
– Was called Mouse Polio due to RARE paralytic clinical Dz
– Other PicoRNAvirus of interest: EMCV, Ljungan virus
 ‘seroprevalence’
seroprevalence <1%
1% (from much higher)
• May persist at low prevalence in breeding colonies without strict
surveillance / exclusion …
 Transmission: fecal oral
• direct contact, fomites? – not highly infectious
• dirty bedding sentinels should detect well
• Biological contaminant
 Control: Rederive (or test & cull)
62
– Foster ? Depopulate ?
B6
SJL
nice example of (experimental) strain
dependent susceptibility
63
– Lipton & Dal Canto (1979).
7
Environment, Infectious
Phenotypes
TMEV
TMEV
Phenotypes
TMEV susceptibility & MHC
TMEV demyelination – MS model since 1970’s
– Spectrum of strain susceptibility – Resistance
– Why are B6, B10 so resistant ?
– 1985 Rodrigues & David – breeding study
• on C57BL/10 background
 Susceptible haplotypes: s, f, p, r, v, or q
 Resistant haplotypes: b, k, or d
CBA
C3H
 Lipton & Dal Canto (1979). "Susceptibility of inbred
mice to chronic central nervous system infection by
Theiler's murine encephalomyelitis virus." Infect Immun
26(1): 369-74.
64
– Confirmed with transgenics in after 2000
65
 Susceptible q haplotype (FVB/N)
 Intermediate k, d, s haplotypes
 Resistant b haplotype (B6, B10)
TMEV
Phenotypes
X
Experimental/model phenotypes
 Acute encephalitis with neuronolysis (virulent strains e.g. GD7, FA)  “Early Dz’ (less virulent strains e.g. TO DA) Encephalomyelitis,
neuronolysis, meningitis, perivasculitis, poliomyelitis with
neuronolysis neuronophagia  paralysis
neuronolysis,
 ‘Late Dz’ (less virulent strains e.g. TO DA) Demyelination (immune
mediated) – spastic
67
Paramyxovirus - respirovirus genus
• ~ human parainfluenza 1
• SS RNA enveloped
• Infection / Disease are usually evident
Transmission: aerosol fecal oral
• Rederive ? Foster ? – difficult because hily infectious
Competent mice
Acute disease
RADIL images
69
Sendai Virus
Sendai Virus
Competent mice
Chronic / repair phase
– Proliferative
– Syncytia
– Inclusions
– D Percy
cbrayton@jhmi.edu Rev Feb 2012
Sendai
Paramyxovirus
Control: Depopulate
– Necrotizing
– Syncytia
– Inclusions
70
66
• Hily Infectious (mouse to mouse) -- But
• Not so reliably detected by dirty bedding sentinels
• Biological contaminant
Sendai Virus
Acute
In Immune deficient mice
 Nu exp dz Early Encephalomyelitis (neuronolytic)  demyelination
(oligo-lytic)
‘seroprevalence’
p
<<1% ((from much higher)
g )
68
Competent mice
In immune sufficient mice
Usually subclinical, persistent infection, intermittent shedding
RARE clinical dz, posterior paresis with poliomyelitis
Virus Virulence: GD7,
GD7 FA >>> TO,
TO DA strains
Strain Susceptibility: SJL, SWR, DBA/2 >> 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





71
Proliferative
tumor like
‘Adenomatous
change’ in
change
chronic disease
& immunodeficient mice
72
8
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
74
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
cbrayton@jhmi.edu Rev Feb 2012
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




75
 ‘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
76
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
80
Ectromelia
78
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
81
9
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)
cbrayton@jhmi.edu 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
cbrayton@jhmi.edu 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
cbrayton@jhmi.edu 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
cbrayton@jhmi.edu 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
cbrayton@jhmi.edu Rev Feb 2012
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 << 0.1%
0 1% Pathogenic?
0.08
123
>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
cbrayton@jhmi.edu 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
– <20 egg/d ; 24d cycle
• Sneakier  low level infestation is hard to detect
– Cecum, colon, larvae in crypts
136
138
137
NOT how you want to diagnose
pinworms in a clean barrier …..
Do you expect
this burden in
competent
mice?
Pinworms
Protozoa
Flagellates
139
140
X
 FUR
– Myobia musculi
– Radfordia affinis
– Myocoptes musculinus
Gravid Pinworm
Gut
Eggs
Protozoa
Flagellates
Entamoeba
141
NOT how you want to diagnose
mites in a clean barrier …..
Arthropod Parasites
Mites
Pinworms + protozoa
Immunodeficient? or dirty ?
Cecum
Fur mites  itchy mice
hypersensitivity  dermatitis
Lice
 Polyplax serrata – fur – nits
Fleas
 Xenopsylla, Leptopsylla
 Follicles
– Demodex musculi
– Psorergates simplex
 Biting – sucking
– Ornithonyssus bacoti
– Laelaps echidnina
Psocids, Psocoptera
 book lice, dust mites, in
mouse rooms, storage
areas
 NOT parasites on mice
 Cellulose & humidity
 Allergenic?
142
cbrayton@jhmi.edu Rev Feb 2012
Don’t like nude or hairless mice
Myocoptes musculinus (A.),
143
Myobia musculi (B.),
Radfordia affinis (C.)
144
16
Environment, Infectious
Phenotypes
Mesostigmatid mites
Mesostigmatid mites
Diagnosis
 NOT on mice
Disease
transmission
X
 Enteric
–
–
–
–
– Eat & leave
 1mm moving
ov g spot
on a filter top
Ornithonyssus bacoti
• Spiny rat mite
• Tropical rat mite
145
–
–
–
–
 Or rash on
investigator
146
Lice
• Book (Bark) lice
− 6 legs + antennae
− Wings in some adults
– Pediculus humanus
capitis
Direct microscopic
Fur plucks
Pelt digestion
Histo
 PCR
– Also identify viruses,
bacteria – in patent
infections
– Immunodeficient
– Improve? Accelerate?
Quarantine
Whatsthatbug.com
NOT Parasites
Psocids (on tape)
Psocids
Human Head lice
Direct microscopic
Fecal flotation
Tape test
Histo
 Fur mites
0.5 mm
Laelaps echidnina
How to Diagnose Parasites
NOT Parasites
Psocids
Book lice ‘Dust mites’
Moving critters in room
Not on mice
Like Cellulose &
Humidity
Allergenic ?
 hypersensitivity ?
http://entoplp.okstate.edu/labs/go/psocid/p_key.
h l
148
149
150
Environmental/Infectious
Phenotypes Discussion Plan
NOT Parasites
Dust mites (8 legs)
 Dermatophagoides
farinae (American
house dust mite)
~ 10% of your 2 year old pillow !?
151
1.
2.
3.
4.
5.
6.

 Dermatophagoides
pteronyssinus (European
house dust mite) &
+ Bed predator mite
(Cheyletus sp.)
cbrayton@jhmi.edu Rev Feb 2012
152
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
153
17
Environment, Infectious
Phenotypes
What is/are normal flora ?
What is in your GEM ?
What is in your control mice?
Does it matter ?
X
Schaedler’s flora
various recipes
~ 8 bacterial spp e.g.
• 2 lactobacilli
• 2 clostridia
• 1 Bacteroides
• 1 Eubacterium
• 2 ASF#’s
Commensal or Opportunist ?
– No problem in competent or control…
– Problem ? in immuno weird or other mutant
– Some may enhance immunity & survival (SFB)
154
Common findings
??? significance
What is/are Defined flora ?
No fungi
Stomach - yeast
Small Intestine - Segmented Filamentous Bacteria
Large intestine – Flagellates + Entameobae
No protozoa
No SFB
THUS you should NOT see
these in gnotobiotic, or in
clean ID from isolator…
You should NOT see these in gnotobiotic,
or in clean immunodeficient mice from isolators…
You should not see protozoa in
clean rederived
You should not see protozoa in clean rederived mice.
Most Vendors test for & exclude the
155
156
Kazachstania spp.
(Torulopsis, Candida)
NOT NEW:
Savage & Dubos. 1967
Stomach: Yeasts
protozoa.
Surface of gastric mucosa
Non pathogen
Find it in hamsters, rats etc. rodents….
Certain strains of yeast were cultured frequently from the feces of adult CFW mice and LongEvans and Sprague-Dawley rats, but not from infants of those murine strains, or from adults
or infants of NCS or NCS-D mice. When the yeasts could be cultured from the
feces, they could also be grown from all areas of the digestive tracts of the
animals, but especially from the stomachs, where they formed layers on the
epithelium of the glandular mucosa. Three of the yeast isolates, one each
from the three murine colonies,, were p
provisionallyy classified in the g
genus
Torulopsis of the asporogenous yeasts. …. When established in NCS-D mice, the
yeast strains did not affect qualitatively or quantitatively the growth of the animals or the
composition of the bacterial flora in the gastrointestinal tracts. Moreover, they did not elicit
an unusual inflammatory response in the digestive tracts; nor were they pathogenic for NCS
mice when injected by the intraperitoneal or intravenous routes. The yeasts thus appear to be
harmless saprophytes that are able to flourish in the environment of the surface of the
secreting epithelium of the murine stomach. The findings conform with the view that some
types of microorganisms of the gastrointestinal tract are not just mixed randomly but rather
occupy microenvironments in almost pure culture. This concept is important to the
understanding of the ecology of the gut microflora.
157
158
159
Distal small intestine:
Segmented Filamentous bacteria
e.g. Degu stomach
Segmented Filamentous Bacterium (SFB)
of the Small Bowel of mammals and birds






Described by Savage, Dubos etc 1970’s & earlier
Gram Positive
Spore former
Attach to brush border
Role in Mucosal immunity?
Distal SI – esp in immunodeficient out of isolators
– RP Orcutt
 Cannot be grown in pure culture
 not in Schaedler’s flora
PAS positive Easter eggs
160
cbrayton@jhmi.edu Rev Feb 2012
161
162
18
Large intestine
Flagellates + Entamoebae
Common
Don’t seem to
cause a problem
Some agents might
‘invade’ damaged
mucosa ?
NOT in ASF.
Normal flora
Conclusions (?)
X
Defined flora (ASF) may not be ‘normal’,
but are defined, and limited.
– Some intruders are pretty obvious
Agents in your ‘control’ mice, are ‘normal’
for your studies.
Environment, Infectious
Phenotypes
Is CLEANEST BEST?
for the mice? for the project?
‘Barrier’ defined by the agents excluded, and
the exclusion practices
‘SPF’
‘Clean’ moving target, as new issues/pathogens
emerge or are rediscovered
– Clearly different flora suggest different origin
– Also strain variations in enteric populations
Pyriform, undulating membrane
Compatible with Trichomonad
163
Collegiality and Collaboration
are Great …
165
X
Conclusions
Environmental variables and infectious
agents contribute to phenotypes & results
Some can seriously impact mice,
mice
projects, budgets …
Some may help your projects !
– Cool molecular mechanisms
But be careful what you share…
166
Environmental/Microbial ‘challenge’
may expose a phenotype
Interest in the microbiome is increasing –
stay tuned.
164
defined by the agents excluded, and
the exclusion practices
Learn more!
Infectious
REFERENCES
http://pixdaus.com/
Baker, D. G. (2007). FLYNN’S PARASITES OF LABORATORY ANIMALS. 2nd ed. ACLAM,
Blackwell Publishing.
Baker, D. G. (2003). Natural Pathogens of Laboratory Animals: Their Effects on
Research. Washington, D.C., ASM Press (American Society of Microbiology)
Brayton, Mahler, Nicklas. (2004). Viral Infections. The Laboratory Mouse. H. J.
Hedrich, Elsevier: 357-391.Hedrich H. Ed. THE HANDBOOK OF EXPERIMENTAL
ANIMALS SERIES: THE LABORATORY MOUSE.
MOUSE Elsevier.
Elsevier
Clifford – in the new book – coming soon
Percy, D. H. and S. W. Barthold (2007). PATHOLOGY OF LABORATORY RODENTS AND
RABBITS. Ames, IA, Iowa State University Press. 3rd ed
Various authors, specific diseases and agents. 2006. In Fox & al. Ed’s. THE MOUSE
IN BIOMEDICAL RESEARCH 2nd Ed. ACLAM series. Elsevier.
Various Auhors 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
167
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
Thanks! Σε ευχαριστώ! Merci! Danke!
Gracias! Grazie! Kiitos! 谢谢你!
 Organizers !!
That’s all folks!
 Mice & GEM
 Nadine
N di F
Forbes
b
 MCP & core faculty
 LAM & Vet path trainees on
– NIH T32 RR0077022 &
– ACVP STP coalition support
 Email cbrayton@jhmi.edu
 Google Hopkins + Phenotyping
169
cbrayton@jhmi.edu Rev Feb 2012
170
19