Download Prevalent Infections of Laboratory Rats and Mice: Implications for

Prevalent Infections of Laboratory
Rats and Mice: Implications for their
Use in Research
Definitions



Animal research model:
An animal in which normative biology or
behaviour can be studied, or in which a spontaneous
or induced pathological process can be investigated,
and in which the phenomenon in one or more
respects resembles the same phenomenon in
humans or other (target) species of animals
[Wessler, 1976]
Timing of discovery
Upfront
• During research
• After research
• Never……..hindsight
•
“Usual” Reasons Against Infected
Rodents
Some diseases are zoonotic
 Some may cause significant morbidity or
mortality
 Accreditation/Regulatory pressure
 Complications in collaboration/sharing
rodents strains and stocks
 Some subclinical infections reported to
alter research results

Prevalence (%)
Agent (assay abbreviation)
Method
N
NA
Europe
Total
Ectromelia (ECTRO)
Serology 246,857 0.02
0.00
0.02
Hantavirus (HANT)
Serology 144,946 0.00
0.00
0.00
K virus (K)
Serology 225,353 0.00
0.00
0.00
Lymphocytic choriomeningitis virus (LCMV)
Serology 241,453 0.01
0.02
0.01
Mouse adenovirus 1 and 2 (MAV)
Serology 230,351 0.02
0.22
0.02
Mouse cytomegalovirus (MCMV)
Serology 146,511 0.04
0.00
0.04
Mouse hepatitis virus (MHV)
Serology 558,673 1.57
3.25
1.59
Mouse norovirus (MNV)
Serology 44,876
24.03
32.37
Parvovirus generic assay (NS-1)
Serology 578,464 1.65
1.92
1.65
Mouse parvovirus
Serology 594,539 1.83
3.64
1.86
Serology 595,903 0.33
0.46
0.33
Pneumonia virus of mice (PVM)
Serology 447,656 0.01
0.01
0.01
Polyoma virus (POLY)
Serology 225,868 0.02
0.20
0.02
Reovirus 3 (REO, REO-3)
Serology 428,821 0.01
0.05
0.01
Rotavirus (EDIM)
Serology 466,572 0.56
0.35
0.56
Sendai virus (SEND)
Serology 462,209 0.00
0.00
0.00
Serology 435,772 0.26
0.27
0.26
1 and 2 (MPV)
Mouse minute virus
(MMV, MVM)
Theiler's murine encephalomyelitis virus
(TMEV,
GD-VII)
32.64
Agent
Citrobacter rodentium
Prevale Disease*
nce (%)
0
Transmissible murine colonic hyperplasia
Clostridium piliforme**
4
Corynebacterium kutscheri
0
Mycoplasma spp.***
0.01–2
Pasteurellaceae
13
Salmonella spp.
0
f3-Haemolytic streptococci
(not
group D)***
0.2
Streptococcus pneumoniae^ 0
Helicobacter spp.
16
Streptobacillus
moniliformis^^
0
Tyzzer’s disease (classical trias enteritis, hepatitis, myocarditis),
multifactorial
Pseudotuberculosis,multifactorial
Most relevant: M. pulmonis (murine respiratory mycoplasmosis),
cofactorial disease
P. pneumotropica causes opportunistic infections, e.g. Suppurative
lesions
Salmonellosis (mainly important as model organism)
Rarely induce clinical disease; group B (Sc. agalactiae), systemic and
suppurative lesions; a report of dermatitis of group G streptococci
Usually subclinical
H. bilis, H. hepaticus:enterohepatic lesions (dependent on strain and
immune-status); H. muridarum: gastritis, H. typhlonius: typhlocolitis
(Il10tm1Cgn mice)
Septicemia,polyarthritis; zoonosis
Bacteria to be monitored in mice according to the FELASA guidelines for health monitoring [1].
* For further reading concerning impact on research and pathology see [2,186].
** Up to 65% in rats.
*** Up to ∼4% in rats.
^ A recent outbreak of subclinical infection was reported at an European vendor in 200.
^^ Determined by culture, which unlikely detects latent infections of this bacterium [187].
A. Bleich, A.K. Hansen / Comparative Immunology, Microbiology and Infectious Diseases 35 (2012) 81– 92
Microbiological assessment of the
health status of laboratory rats and
mice
The science of evaluating representative
sample groups from given units of those
species against a specific listing of etiological
agents of disease to define the health status of
the source colony.
The purpose of this information is to prevent
introduction of disease and to monitor the
microbial status of resident colonies
Principles of pathogen screening
The current standard in health monitoring
A first prerequisite for health monitoring is the definition of a hygienic unit, in which rodents of given
colonies (e.g. at a vendor) are likely to display the same pathogen status, usually because they are
maintained in the same barrier protected area without being exposed directly or indirectly to other rodents
(a). Out of this unit, a predetermined number of animals has to be investigated for pathogens (b). This
sampling size depends on the prevalence of a given agent in a colony of at least 100 animals and the
confidence level at which one would like to detect a pathogen. A further prerequisite is a defined list of
agents that are to be monitored (c) as well as the methods used to detect them. Finally, results need to
be depicted in a formalized manner, usually a health report citing all details including current and
historical results of health monitoring (d).
A. Bleich, A.K. Hansen / Comparative Immunology, Microbiology and Infectious Diseases 35 (2012) 81– 92
Health surveillance programs

detect by examination of 1or more
representative sample groups the presence
(even in a single individual) of any pathogen
from a specific profile of infectious agents.
Development of such data on a repetitive
schedule forms the objective basis on which to



establish and/or confirm the ongoing microbial status of
commercial and institutional rodent production colonies
develop institutional procurement standards for supplier
eligibility based on animal health criteria
continuously monitor the health status of institutional
research animal residents, including recent arrivals
undergoing equilibration or quarantine before use, those
currently involved in research protocols, and those coming
off study.
Testing methods
Clinical Evaluation, Gross and Microscopic
Pathology
 Diagnostic Detection and Identification of
Microorganisms

◦ Microscopy (ecto & endoparasites)
◦ Culture (resp. & enteric)
◦ Serology/PCR (tissue, feces) from animals or
tissues (tumors and cell lines to be introduced)
Factors that can influence the HM results
Sampling method (colony animals, contact
sentinels, bedding sentinels)
 The number of animals (sera or other
samples) in the sample group (Prevalence
and confidence)
 The periodicity of sample group submission
 The age and sex of animals in the
group/room
 The testing method (serology, PCR, type of
culture)

Factors that can influence the HM results
(cont.)
The type of caging (isolators, IVC, static
microiso., open cages)
 The immunological status of the animals
(unknown in GM, stressed from research –
can be used for dexamethasone provoked
testing)
 The agent profile against which to evaluate
the health status

Examples of sample selection errors
Result
Methodology
Falsenegative
*Serology
Falsepositive
Error
Acutely ill, serum antibodies not yet
detectable
Immunodeficient or immunosuppressed,
weak or no antibody response
Bacteriology/parasitolo Older and recovered from infection
gy
Site where organism is not normally
resident
All
Small sample size
Sentinels not adequately exposed via
soiled bedding or contact to infectious
agents carried by principals
Serology
Strain with autoimmune disease
Immunized or inoculated with biological
material (such as tumor cells)
Maternal antibodies
All
Sentinels housed under less strict
conditions than principals (such as
principals kept in microisolation cages, but
sentinels are in open cages)
Comparison of typical and ideal
serology test.
Examples of sample selection errors
Result
Methodology
Falsenegative
Serology
Falsepositive
Error
Acutely ill, serum antibodies not yet
detectable
Immunodeficient or immunosuppressed,
weak or no antibody response
Bacteriology/parasitolo Older and recovered from infection
gy
Site where organism is not normally
resident
All
Small sample size
Sentinels not adequately exposed via
soiled bedding or contact to infectious
agents carried by principals
Serology
Strain with autoimmune disease
Immunized or inoculated with biological
material (such as tumor cells)
Maternal antibodies
All
Sentinels housed under less strict
conditions than principals (such as
principals kept in microisolation cages, but
sentinels are in open cages)
Subclinical infections are likely to
increase variability
Not all animals exposed on same day or to
same dose of infectious agent
 Different strains, sexes, ages respond
differently
 Co-infections, and maybe gut flora, can alter
response
 Even in inbred animals of uniform
signalment, there will be some variation

Research Effects of Subclinical
Infections

In addition to currently documented
effects, potential interactions can be
suspected through cell types infected,
intracellular components and functions
involved, and systemic host response
mechanisms
Presenter bias

How clean/pure do you want
your glassware and reagents?

Would you buy reagents if
vendor said they are
contaminated, and that the
contamination might affect
some, but not all, research?
What’s common?
• MHV – 2%
• Parvoviruses
• Helicobacter spp. – 15%
• P. pneumotropica
• Rotavirus – 0.7%
• Norovirus - 30%
• RRA – 7%
• Theilovirus
• C. bovis – 3%
• Pneumocystis carinii – 2%
• Pinworms –
• Mouse – 2%
• Rat – 4%
• Mouse – 0.3%
• Rat – 1.4%
• Mouse – 15%
• Rat – 5%
• Mouse – 0.3%
• Rat – 1.3%
• Mites – 0.1%
Rats and Mice

Mycoplasmosis (CRD, MRM*):
◦
◦
◦
◦
◦
◦
Mycoplasma pulmonis: causative agent
major problem in rats; stress worsens condition
primarily affects the respiratory tract
may cause inner or middle ear infection (head tilt)
can become endemic and difficult to eradicate
causes severe pathologic lesions in the lungs:
abscesses, red to gray consolidation
* Murine respiratory mycoplasmosis
Rats and Mice

CAR (Cilia associated respiratory)
bacillus:
◦ gram negative bacteria attached to cilia
◦ respiratory disease or lesions in rats, mice,
rabbits, swine
◦ require special staining techniques on
histopathology (Warthin-Starry silver stain)
◦ dirty bedding inadequate for detection
Mouse Hepatitis Virus (MHV)

Coronavirus, ssRNA, enveloped
◦ Innumerable strains
Prevalence: ~2% of serum samples, so
present in most universities
 Strains grouped as enterotropic or
polytropic

◦ Infection generally asymptomatic in postweaning immunocompetent mice
◦ Wasting syndrome in many immunodeficient
mice
◦ BALB/c mice more susceptible than C57BL/6
mice
MHV - polytropic

Virus attaches to CEACAM1a glycoproteins
(members of the immunoglobulin - Ig
superfamily) via spike protein

Primary tropism for upper respiratory tract
mucosa

Secondary sites - lung, liver, CNS, intestines,
ovaries, epididymis
Research Impact of MHV
 Prolonged immunologic effects:
◦ MHV depletes NK cells through apoptosis
and syncytia formation
◦ T-cells, B-cells
 > 1,000 CD8 T cell clonotypes estimated to
respond to dominant MHV S protein epitope
(JHM in C57BL/6)
◦ Infects monocytes, macrophages, bone
marrow dendritic cells
◦ Delayed allogeneic graft rejection

Polytropic MHV
Brain and spinal cord
◦ Meningitis and necrotizing encephalitis
 NOS2 KO mice (amyloid beta deposits - Alzheimer's
disease-like pathology accompanied by behavioral changes)
have less mortality in experimental infection, associated with
decreased neuronal apoptosis
◦ Demyelination (brain stem)
 Apoptosis of oligodendroglia
 Immune-mediated (antibody, CD8 bystander, MBP
autoreactive T cell clones, macrophages)
◦ Intracerebral MHV (JHM – a neurotropic variant of MHV) in
C57BL/6 altered expression of 80 genes, at least 27 of which
relate to innate or acquired immunity
Enterotropic MHV - common
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Most wild type strains are enterotropic
Highly contagious, shed in large quantities in feces
Transmitted by fomites (gloves, cages, bedding, etc.)
Clinical signs and gross lesions rare in
immunocompetent adult mice
Primary replication:
◦ GI tract, especially distal ileum, cecum, ascending colon

Secondary sites - uncommon
MHV Detection

Serology
◦ Excellent cross-reaction among strains
◦ Seroconversion within 2 weeks (often one
week)

PCR
◦ Shedding?, Environment
◦ Mesenteric lymph nodes to confirm serology
◦ Epidemiology

Histopathology
◦ Lesions should by confirmed by IHC, PCR or serology
CONTROL OF MHV
Immunocompetent mice self-cure
 Enveloped virus: not stable in
environment, easy to disinfect
 Can eliminate from immunocompetent
colonies by not breeding and no new
mice for 6-8 weeks (always test to
confirm)
 Infection persists in immunodeficient mice

Parvoviruses
Compared to MHV,
parvoviruses are
harder to detect,
harder to eliminate,
and tend to have
lower prevalence.
This leads to
confusion and
frustration.
Parvoviruses

ssDNA, (5.4 kb genome), non-enveloped
◦ Virus remains active in environment
 Resistant to desiccation, non-oxidizing disinfectants
Most are fairly common
 Very low or no morbidity
 All can cause persistent infection
 Require mitotically active cells for
replication

◦ S phase for activation of P4 promoter
Parvoviruses of Mice

Mice Minute Virus (MMV or MVM)
◦ Multiple strains (i, p, c, m), MMVm is most
prevalent and is persistent. Others are cultureadapted strains.
 MMVm causes growth retardation, reduced fecundity
and premature deaths in NOD μ-chain KO mice.
Chronic progressive infection in scid mice.
 MMVi
 Intranasal inoculation causes lethal
leukopenia in scid mice, infects CFU-GM,
BFU-E, CFU-MK and HSC
Research Effects of MMV

Can infect many mouse cell lines, as well as some
rat embryo lines and transformed human cells
(324K, EL-4)

In vitro (A9 cells) dysregulation of gelsolin (↑) and
WASP (↓) by MMVp

In vitro reduction of T-cell response by MMVi and in
vivo late reduction of cytotoxic memory cells by
MMVp

MMVp is oncotropic and oncolytic in some human
tumors (hemangiosarcoma) and mouse tumors
Parvoviruses of Mice

Mouse Parvovirus (MPV-1, -2, -3, -4, -?)
◦
◦
◦
◦
Prevalence higher than MMV
Cause persistent infection
No anatomic lesions, even in scid mice
Strains not reliably cross-reactive by serology
Research Effects of MPV

MPV-1a (cell culture adapted) modulates
immune response (McKisic et al, 1996)
◦ Suppression of T cell response in vitro
 CD8+ T lymphocyte clones lose function and viability
 Cytokine- and antigen-induced T cell proliferation in
vitro suppressed after exposure to MPV-1a
◦ Potentiates allograft rejection
◦ Induces isograft rejection
Parvoviruses of Rats
(RPV-1, RPV-2, RMV, RV [KRV] I H-1)

RV - Rat Virus (previously KRV, Kilham Rat Virus)
◦ Natural infections usually asymptomatic, but persistent,
with prolonged shedding
◦ Infects rapidly growing cells: Endothelium, lymphoid and
hematopoietic tissues, developing cerebellum and liver
◦ Rare epizootic disease in fetal/neonatal rats: Cerebellar
hypoplasia, anemia, thrombocytopenia
◦ Very rare disease in older rats: Hemorrhagic disease
Rat Virus (RV)
formely Kilham’s rat virus - KRV
Rat Virus (RV) – Research Effects

Research Effects: RV induced diabetes in DR
BB rats (Guberski et al., 1991)
◦ Possibly due to imbalance in Th1 and Th2 responses
(Jun and Yoon, 2001)

RPV NS protein induced epigenetic modification
in thymic lymphoma line, causing reversion to
benignancy (Iseki H , 2005)
Parvoviruses of Rats

Rat Parvovirus (RPV)

Few studies in literature, very difficult to
isolate
◦ Multiple strains exist
◦ No clinical disease reported
◦ Research effects: Suppression of lymphoid tumor
growth in vivo: RPV-1a
Parvoviruses of Rats

(Toolan’s) H-1 - no natural disease
◦ Significance through research interference: liver
◦ Historic interest as cancer therapy

Rat Minute Virus (RMV)
◦ Almost nothing in literature
◦ May be most common parvovirus of rats
◦ Serologically and genetically more similar to RV than to RPV
Exclusion of Parvoviruses

Consider sources of research animals:
◦ Vendors, GM animals, immunodeficient

Wild rodents

Biological materials

Risk from personnel handling infected
rodents (pets, snake food)

Fomites (Feed, bedding, water, used/shared
equipment etc.)
Detection of Parvoviruses

Serology – Best for screening
◦ Use panel of antigens for each serotype, plus
the generic NS-1 antigen

PCR - strain-specific (VP2) or generic (NS1)
◦
◦
◦
◦
Mesenteric LN stay positive indefinitely
PCR of feces to detect shedding
Valuable for testing biologicals and cell cultures
Environmental swabs
Parvovirus detection problems

Mice on C57BL/6 background and congenic
strains are partially resistant to infection and
infrequently seroconvert after exposure
◦ 10-100x increase in ID50, cf. BALB/c or CD-1
◦ DBA/2 also, not quite as bad
If small amount of virus in environment,
sentinels will be positive, but only an
occasional C57 will receive an ID
 DBA/2 only slightly better

Parvovirus diagnostic problems

Older sentinels less susceptible, seroconversion
does not necessarily mean shedding enough to
transmit infection
◦ May have one positive, with negative cagemates

Prevalence also kept low by filter-top cages
◦ Low prevalence means low predictive value of positive
result
Control of Parvoviruses

Can only eliminate by rederivation
◦ Multiple anecdotal reports of success with early
cross-fostering

No envelope, so it stays active in
environment
◦ Must thoroughly disinfect environment, materials
and equipment with oxidizing agent (Clidox,
ozone, etc.)
Human Norovirus Infection
(good model for mouse disease)

Genus named for Norwalk virus, discovered
in 1972
◦ Typical signs in humans - vomiting and diarrhea
for ~ two days.

Noroviruses cause most nonbacterial
epidemic gastroenteritis worldwide.
◦ In US, CDC estimates 23 million cases of
noroviral diarrhea each year
◦ If similar rate in rest of world, > 500 million cases
each year.
Human Norovirus Infection

Norovirus infamous on
cruise ships, high
morbidity of passengers
and crew

“Vomiting Bug Sickens
Hundreds of Thousands in
the UK”: By Anna Boyd
17:07, January 3rd 2008, eFluxMedia
Human Norovirus Infection

Lessons from human outbreaks
1. As a nonenveloped virus, disinfection is
difficult
2. Infectious amounts of virus shed for
weeks after symptoms abate
3. Immunity not cross-protective
Murine Norovirus (MNV)
Small, nonenveloped, ssRNA viruses in
Caliciviridae
 Capsid is single protein, with cup-like
projections, or calices
 Myriad MNV variants, as with humans

◦ CRL Molecular Diagnostics has identified more
than 50 different variants - All in Genogroup V
MNV Epizootiology

Fecal-oral transmission

As a nonenveloped virus, MNV can remain
infectious in the environment, possibly weeks

Seroconversion slow

Infectious dose for mice is not known
◦ Infectious dose for humans may be only a few
virions
MNV Epizootiology

Infected mice, as infected humans, shed:
◦ Massive amounts of virus for a few days after
infection
◦ Shed small but probably infectious amounts of
virus for weeks
Prevalence appears very high, estimated at
~30% of mice
 Production colonies of major vendors are
MNV negative

MNV infection

Immunocompetent mice – No clinical signs
◦ Studies in 129 (129S6SvEvTac) mice, inoculated
PO with 107 PFU MNV-1.CW3
 Minimal change ( 13 vs. 8 per high power field) in
inflammatory cells in lamina propria of small intestine at
24 hours post infection
 Increased nuclear staining in red pulp of spleen, but no
change cell number
 Viral nucleic acid found in small intestine, spleen,
mesenteric lymph nodes, liver
 Possible decreased “stool contents” at 3 days P.I.
MNV Disease

Mice deficient in acquired immunity
(RAG, SCID)
◦ MNV antigen and nucleic acid detected in
mesenteric lymph nodes – possibly in
dendritic cells
MNV Disease

Mice deficient in innate immunity
◦ Lethal infection in STAT1 -/- (with or without
RAG2 and PKR), and IFN Rαβγ -/◦ hepatitis, interstitial pneumonia
◦ Encephalitis only with intracerebral inoculation
◦ Virus present in dendritic cells
Homozygous disruption of the Stat1 gene eliminates the intracellular mechanism by which
cells respond to interferons, resulting in a mouse model with extreme susceptibility to bacterial
and viral infections, and with increased tumor formation
Applications for the Stat1 Targeted Mutation Mouse Model
MNV Research Effects
Unknown, other than disease in some
strains
 Potential interference with studies of
innate immunity and/or dendritic cells

MNV Effects on Research [after Hsu, 2012]
•
•
•



•
•
MNV infects dendritic cells and macrophages (effect on
immunological and infectious disease research?) [Wobus et
al., 2004]
Elevated type I interferon levels (intestines: 12 and 24h PI;
Serum: 24h PI) [Mumphrey, et al., 2007]
MNV exacerbates bacterial-induced IBD
- Mdr1a-/- mice + Helicobacter + MNV = ↑ wt. loss and
colitis @ 2wks PI
- MNV enhances antigen presentation by dendritic cells
- ↑ IFN-γ production by T cells 2d PI (but not at later
time points) [Lencioni et al., 2008]
MNV increases the duration of MPV fecal shedding and MPV
DNA levels in tissues of BALB/c mice [Compton et al., 2010]
MNV infection + susceptibility gene interactions determines
the phenotype for a mouse model of Crohn’s Disease
[Cadwell et al., 2010]
MNV (Lack of ) Effects on Research [after Hsu, 2012]
But….
• MNV has no significant effect on C57BL/6 mouse models of
vaccinia virus or influenza A virus models [Hensley et al., 2009]
• MNV has a mild impact on CD8 T cell responses in a mouse
model of murine cytomegalovirus (MCMV) infection, but
“not likely to affect most experimental outcomes in
immunocompetent mice in the MCMV model” [Doom et al.,
2009]
• MNV infection in a mouse model of bacterial induced colon
cancer (SMAD3-/- mice + Helicobacter) did not impact
survival, IBD scores, tumor incidence or tumor phenotype
[Lencioni et al., 2011]
MNV Diagnosis

Colony screening by Serology
◦ Good cross-reaction among strains
◦ Seroconversion often slow, so sentinels should have 8
weeks of exposure

PCR
◦ Immunodeficient mice
◦ Pooled fecal samples
◦ Release from quarantine
 Avoids long exposure period
◦ Environmental monitoring
◦ Epidemiology
MNV Management

Virus apparently present for a long time
◦ Very well adapted to host
◦ Many strains
◦ Wide geographic distribution
Current situation does not recent development,
and does not require urgent action – do not panic
if positive results received
 Consensus seems to be to survey

◦ Many facilities waiting to see how attitudes toward MNV
evolve in the near future, before taking action
◦ Many trying to start new facilities with MNV-free mice
MNV Management

Rederivation by embryo transfer or
caesarian section should be successful
◦ Possible early cross-foster

Environmental decontamination requires
use of oxidizing disinfectants or heat > 56oC
Helicobacter

Originally included with Campylobacter
◦ Helicobacter created in 1989

H. pylori probably discovered in 1875,
◦ linked to human gastric disease in 1899 (Polish text)

“Re”discovered in 1979 by Warren
◦ 1984 - linked to most gastric ulcers and gastritis
(Warren and Marshall, Nobel Prize awarded in 2005)
Helicobacter in rats and mice - Discovery
H. muridarum in mice: 1992
 H. hepaticus: 1994

◦ Liver tumors and hepatitis in A/J mice on
carcinogenicity study
◦ Lesions resembled aflatoxicosis
◦ Spiral organisms discovered in bile canaliculi with
Steiner stain

Currently >40 species of Helicobacter
described, with many in rodents
Helicobacter

Gram-negative bacteria colonizing intestinal tract
of warm-blooded animals
◦ Gastric
◦ Large intestine - Some of these reach liver
(enterohepatic)





Microaerophilic (H. ganmani is anaerobic)
Highly sensitive to desiccation
Highly adapted to hosts, although many are capable
of colonizing multiple host species
Mechanisms of disease similar among helicobacters
Animal models useful in studying human disease
Helicobacter
Epizootiology
Fecal-oral transmission
Short-term fomites (soiled
bedding) possible
 Colonization by weaning,
persist for lifetime


◦ Short-term colonization
noted in a few experiments,
significance unknown

Prevalence: high (~15% in
mice, ~8% in rats),
especially high in GM mice
Helicobacter Disease
Disease varies with Helicobacter species, rodent
strain, immune status, sex, possibly age
 General – Outcome depends on interaction with
gut flora and on immune response, with most
disease being a by-product of the host response

◦ Important components of host response include IL-10,
TNF, TH1:TH2 balance, CD4+CD45RB(lo)CD25+ T
regulatory cells, and TGF-beta

Infection with multiple Helicobacter species or with
other pathogens, e.g., MHV, can be synergistic
H. hepaticus Disease
Most common Helicobacter sp. in mice
 Proliferative colitis in A/J, C3H/HeN, athymic nude,
SCID and many other immunodeficient strains, e.g.,
IL-10 -/ Currently, only common infectious cause of rectal
prolapse, especially in immunodeficient mice*
 Chronic hepatitis (necrosis, hepatocytomegaly,
biliary proliferation, nonsuppurative inflammation)
in A/J, C3H/HeN, and some other
immunocompetent strains

◦ Hepatitis may be necrotizing in immunodeficient strains
Helicobacter hepaticus
H. hepaticus Disease
Most common Helicobacter sp. in mice
 Proliferative colitis in A/J, C3H/HeN, athymic nude,
SCID and many other immunodeficient strains, e.g.,
IL-10 -/ Currently, only common infectious cause of rectal
prolapse, especially in immunodeficient mice*
 Chronic hepatitis (necrosis, hepatocytomegaly,
biliary proliferation, nonsuppurative inflammation)
in A/J, C3H/HeN, and some other
immunocompetent strains

◦ Hepatitis may be necrotizing in immunodeficient strains
H. hepaticus Disease
Hepatocellular carcinomas in
A/J, C3H/HeN, SCID mice
 Colon carcinoma in SMAD-3
deficient mice
 Increased incidence of
mammary carcinoma in RAG2/- Apc(min/+)mice (secondary
to inflammation)
 C57 resistant to disease,
but can carry high level of
colonization

6-week old AL-ras x AL-myc dual Tg mouse
H. bilis Disease
Prevalence about 1/4 that of H. hepaticus
 Proliferative typhlocolitis in athymic mice
and rats

◦ Occasional rectal prolapse

Mild chronic hepatitis in
immunocompetent mice (low incidence)
Helicobacter spp. - Research Effects
Direct effects, depending on variables above, on
large bowel and liver, with broad activation of
specific and non-specific aspects of host defense
system including development of tertiary
lymphoid follicles
 Indirect effects of infection without lesion
production are not as well described but may
influence remainder of gut flora

Diagnosis of Helicobacter
infection
Similar for all Helicobacter spp.
 PCR – best. Can be generic or specific.

◦ Can use pooled fecal pellets
 Culture – difficult, lacks sensitivity and rarely done
 Histopathology with silver stains (in tissue only)
 Serology
Sentinels in Helicobacter
screening

Transmission to sentinels has been
questioned
◦ Effective in as little as 2 weeks for H. hepaticus
(Livingston, et al, Comp Med, 48:219 1998)
◦ Less effective for H. bilis, H. rodentium (Whary, et
al, Comp Med 50:436 2000)
◦ May consider direct monitoring of principal
animals

Based on epizootiology, frequent in-house
monitoring is unnecessary if incoming animals are
negative
Helicobacter Management

Relatively easy to contain within a research facility
◦ Anecdotal reports of cages side-by-side without
transmission
◦ Note reports of difficulty in transferring by soiled
bedding

Elimination often successful by cross-fostering
before 24 hrs of age (Singletary KB, Kloster CA, Baker
DG, Comp Med 2003 Jun;53(3):259-64 )
Mixed reports on success of medicated feed or
antibiotic administration by gavage
 Rederivation seems uniformly successful

Interference by opportunists in rodents

Opportunists: A usually harmless organism that can cause
disease under favourable conditions
•
Beta haemolytic streptococci
Klebsiella oxytoca
Klebsiella pneumoniae
Pseudomonas aeruginosa
Proteus spp
Staphylococcus aureus
•
•
•
•
•
Interference by opportunists in rodents
Nude Mice-Various and Numerous Examples

- Immune Modulation Effects

- Clinical Signs - Abscesses, Unthriftiness

- Interference with Breeding Colonies
• CB 17 SCIDs - It Gets Worse!
• Conventional Rodents

- Post Operative Infections in Surgical Models

- Safety Assessment Studies – Survival Rates, Clinical
Issues
•
Opportunists example: Staphylococcus aureus
• C57Bl/6 mice most resistant: innate immune mechanisms
• A/J, DBA/2, BALB/c mice highly susceptible
• For A/J mice 3 chromosomes influence susceptibility
• Findings suggest 2 genes, Tnfaip8 and Seh1l, may contribute
to susceptibility to S. aureus in A/J mice, and represent
promising candidates for human genetic susceptibility
studies.


[Kockritz et al.,2008; Ahn et al., 2010]
Mycobacteria in NHP
Mycobacteria (NHP): M.Tuberculosis; M. kansasii

- Personnel safety concerns if M.Tb, M. avium

- Regulatory obligations and documentation

- Animal Welfare Concerns

- Increased spending for equipment and supplies

- “Work around” costs

- Delayed research start

- M. kansassii Interferes with Gene Expression
•
Primate Diseases

Tuberculosis
◦ Draining lymph
nodes
◦ Tuberculin testing
◦ Tissue changes
◦ Method of tb testing
in monkeys
Mycobacteriosis

Mycobacterium spp.
◦
◦
◦
◦
◦
M. tuberculosis* -human to animal
M. avium*
M. marinum*
M. bovis*
Etc………
* Zoonosis
Morbidity+ Mortality+
 Transmission

◦ inhalation, direct contact
Tuberculosis: Diagnosis
TB skin test
 Culture

◦ Very slow growing
Acid-fast staining
 PCR
 Radiographs
 QuantiFERON-TB test (Humans only)

Herpes B
◦ Alphaherpesvirus
◦ In macaques:
 Nodules on lips or mouth
 Carriers for life
◦ Fatal for humans (and
vervets)
◦ Trans: bite, broken skin,
mucosa
Rickettsial Diseases
Q-fever: Reservoir Species

Goats, sheep and cattle:
◦ No obvious illness in animals
◦ Can cause abortions
Cats
 Rabbits
 Birds
 Rodents ????

Q-fever: Transmission to Humans
Organism is excreted in urine, feces, milk,
and especially in birth fluids
 Humans are usually infected by inhalation of
the organism from contaminated
environments
 Occasionally raw milk

Q-fever: Risk Factors
Direct contact with infected animals
 Farmers
 Veterinarians
 Slaughterhouse workers
 Sheep researchers

Rabbits

Pasteurellosis:
◦
◦
◦
◦
Pasteurella multocida: bacterial cause
very common in rabbits and difficult to treat
spread by aerosol or direct contact
diseases: snuffles (sneezing, nasal and
ocular discharge), otitis media or interna,
localized abscesses, genital infections,
pneumonia
Frogs

Redleg
◦ caused by Aeromonas hydrophila
◦ more common in leopard frogs (Rana
pipiens)
◦ factors: stress, population density, dietary
change, injury to skin or slime coat
◦ signs: cutaneous hemorrhages on legs, but
can occur anywhere on the body.
Rats and Mice

Pinworms (Oxyurids)
◦ Syphacia oblevata, S. muris, Aspicularis tetraptera
◦ affects rats, mice, hamsters, gerbils, rabbits,
horses, NHP
◦ probably # 1 contaminant in rat & mouse
colonies in the US
◦ no clinical signs (may cause rectal prolapse in
young animals)
◦ easily spread and survive in environment forever
◦ fecal floatation, anal tape, or direct exam
Rats and Mice

Ectoparasites:
◦ Mites (Myobia sp.) and Lice (Polyplax sp.)
◦ detection by examining pelt under
microscope
◦ pruritis, dermatitis, greasy coat, alopecia
The gut microbiota of rodents
Influence of gut microbiota on caecum size. The caecum size
decreases with increasing number of bacteria present in the microflora,
although degree of normalization also depends on the composition of the
intestinal microbiota. Germfree mouse (a), mouse associated with the 8
species of the altered Schaedler flora (b), and conventionalized mouse (c).
A. Bleich, A.K. Hansen / Comparative Immunology, Microbiology and
Infectious Diseases 35 (2012) 81– 92
Examples of the impact of the gut microbiota on rodent models of human diseases.
↑, increased incidence in germ free animals and ↓, decreased incidence in germ free animals.
A. Bleich, A.K. Hansen / Comparative Immunology, Microbiology and Infectious Diseases 35 (2012) 81– 92
Disease
Organ
Species
Model
Type 1
diabetes
β-Cells
Mouse
Non-obese diabetic (NOD) ↑[65]
Mouse
Rat
MyD88 KO NOD
Bio-breeding
Rat
Alloxan
Rat
Rat
Collagen induced
HLA-B27 Tg
↓ [151]
↓ [60]
Mouse
Mouse
IL-2 KO
IL-10 KO
↓ [61,152]
↓ [62,63]
Mouse
Mouse
TCRα KO
Dextran sulphate sodium
↓ [155]
↔ [156,157]
↑ [158,159]
Mouse
Mouse
SAMP1/Yit
T-cell transfer
↓ [168]
↓ [169]
Rat
HLA-B27 Tg
↓ [60]
Arthritis
IBD
Joints
Intestine
Incidence/sympt
oms in germ free
animals
↑ [146]
Examples of the impact of the gut microbiota on rodent models of human diseases.
↑, increased incidence in germ free animals and ↓, decreased incidence in germ free animals.
A. Bleich, A.K. Hansen / Comparative Immunology, Microbiology and Infectious Diseases 35 (2012) 81– 92
Disease
Organ
Specie
s
Contact
hypersensitivity
Dermis
Mouse
2, 4-dinitrofluorobenzene
Oral Lactobacillus casei as
well as oligofructose (and
associated increase in
Bifidobacteria spp.)
treatment are protective
[178,179]
Allergy
Systemic
Rat
Mouse
Cow milk
Ovalbumin-specific T
cell receptor TG
↓ [181]
beta-lactoglobulin
↑ [182]
C57BL/6
Lepob
↓ [67]
Pristane
↔ [183]
Lepob
Streptozotocin
Lactobacillus spp. delays
progression [74,185]
Mouse
Obesity
Lupus
erythematosus
Type 2
diabetes
Systemic
Mouse
Mouse
Systemic Mouse
Systemic
Mouse
Rat
Model Incidence/symptoms in germ
free animals
Type of Contact
Skin
 Animal bites
 Inhalation

11 (39%)
5 (18%)
4 (14%)
Animal Species
Rodent/rabbit
 Dogs
 Cats
 NHP

5 (17%)
4 (14%)
4 (14%)
4 (14%)
LAB ANIMAL ALLERGIES
Prevalence ranges from 11-44% depending
on the study.
 Prevalence estimates vary primarily because
of differing ways of defining the disease:

◦ Objective tests vs. subjective symptom
reporting: the lowest estimates were from
studies which relied on employer reports; the
highest estimates simply use self-reporting of
symptoms (i.e. “do you ever get a stuffy nose at
work”).
Specific Animal Allergens
Urine is the major source of rodent allergen
exposure.
 Mouse:

◦ Mus m 1 (prealbumin): Previously known as major
urinary protein. Found primarily in urine, but also
in dander and hair
◦ Mus m 2: Found mostly in hair and dander
◦ Albumin: Found in serum