Transcript COMMON INFECTIOUS DISEASES IN LABORATORY RATS AND MICE Charles B. Clifford, DVM, PHD, DACVP Dir, Pathology and Technical Services Charles River Laboratories.

COMMON INFECTIOUS
DISEASES IN LABORATORY
RATS AND MICE
Charles B. Clifford, DVM, PHD, DACVP
Dir, Pathology and Technical Services
Charles River Laboratories
What’s common?
• MHV – 2%
• Parvoviruses
• Mouse – 2%
• Rat – 4%
• EDIM – 0.7%
• Norovirus ~30%
• RRV – 7%
Charles River Laboratories
•Helicobacter spp. – 15%
•C. bovis – 3%
•Pneumocystis carinii – 2%
•Pinworms –
Mouse – 0.3%
Rat – 1.3%
•Mites – 0.1% (mice only)
What’s common in mice?
Agent
Assay
# tested
# pos.
% pos.
Parv NS-1
ELISA
445,255
8,481
1.9048%
MPV
ELISA
457,062
8,974
1.9634%
MVM
ELISA
458,931
1,789
0.3898%
MHV
ELISA
441,098
7,949
1.8021%
EDIM
ELISA
364,793
2,459
0.6741%
GDVII
ELISA
342,312
991
0.2895%
MPUL
ELISA
352,563
32
0.0091%
REO
ELISA
338,054
43
0.0127%
SENDAI
ELISA
361,118
10
0.0028%
PVM
ELISA
353,043
12
0.0034%
Charles River Laboratories
What’s common in rats?
Agent
Assay
# tested
# pos.
% pos.
RPV
ELISA
73,289
1,324
1.8065%
H-1
ELISA
67,594
1,128
1.6688%
KRV
ELISA
73,400
1,136
1.5477%
RMV
ELISA
29,110
437
1.5012%
GDVII
ELISA
28,203
264
0.9361%
SDAV
ELISA
68,445
159
0.2323%
MPUL
ELISA
67,951
127
0.1869%
M pulmonis
Culture
3,558
2
0.0562%
PVM
ELISA
66,450
98
0.1475%
SENDAI
ELISA
67,193
16
0.0238%
REO
ELISA
61,016
5
0.0082%
Charles River Laboratories
Mouse Hepatitis Virus
(MHV)

Coronavirus, ss RNA, enveloped
– Very high evolutionary capacity (innumerable
strains)


Prevalence moderate
Virus types grouped as enterotropic (intestinal)
or polytropic (multiple tissue) – most field
strains are enterotropic
– Clinical signs very rare in immunocompetent mice
after weaning
– Wasting syndrome in many immunodeficient mice
Charles River Laboratories
MHV

As enveloped virus – does not persist in
environment. Probably not infective after 48
hrs.
– Short-term transfer by fomites (sleeves,
equipment, bedding)

Highly contagious and can spread rapidly
Charles River Laboratories
Enterotropic MHV




Strains: D, RI, Y, G, myriad others.
Most wild type strains are enterotropic
Clinical signs and gross lesions rare in immunocompetent
adult mice
Primary replication:
– GI tract, especially distal ileum, cecum, ascending colon


Secondary sites - uncommon
Clearance mediated by B cells
– Not cleared in μMT mice (anecdotally also in many GM lines)

Dissemination prevented by T cells
– Disseminates in TCR βδ- , IFN-γ - , RAG1, athymic nude mice
Charles River Laboratories
Research Impact of MHV
 Prolonged
immunologic effects:
– NK cells, T-cells, B-cells
– Infects monocytes, macrophages, bone marrow
dendritic cells
– Delayed allogeneic graft rejection

Alters course of concurrent infections, such as
Helicobacter hepaticus
Charles River Laboratories
MHV Detection

Serology
– Excellent cross-reaction among strains

MFIA or ELISA, with IFA for confirmation
– Seroconversion within 2 weeks (often one
week)

Histopathology
– Lesions should by confirmed by IHC, PCR
or serology
Charles River Laboratories
MHV Diagnosis

PCR
– Sequencing of PCR product (nucleocapsid
gene) for epidemiology
– Fecal Shedding (quarantine,
immunodeficient mice)
– Environmental
– Confirmation of serology by PCR of
mesenteric lymph nodes
Charles River Laboratories
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
(test 1st)
 Infection persists in immunodeficient mice

Charles River Laboratories
Parvoviruses
Are you getting
mixed signals on
parvoviruses?
Parvoviruses in Mice

ssDNA, non-enveloped
– Virus remains active in environment

Resistant to desiccation and many (nonoxidizing) disinfectants
Fairly common
 Generally no clinical signs
 Cause persistent infection – no self-cure
 Need actively dividing cells to replicate

Charles River Laboratories
Parvoviruses of Mice

Mice Minute Virus (MMV or MVM)
– Multiple strains (i, p, c, m), MMVm is most prevalent
and is persistent. Others are culture-adapted strains.
MMVm reported to cause stunting, low reproduction and
early deaths in NOD μ-chain KO mice.
 Experimentally, caused hronic progressive infection in scid
mice.

Charles River Laboratories
Research Effects of MMV

Cell culture
– Can infect many mouse cell lines, as well as some rat embryo
lines and transformed human cells (324K, EL-4)

Immunity
– In vitro reduction of T-cell response by MMVi and in vivo late
reduction of cytotoxic memory cells by MMVp

Cytoskeleton
– In vitro (A9 cells) dysregulation of gelsolin (↑) and WASP (↓)
by MMVp

Tumor studies
– MMVp is oncotropic and oncolytic in some human tumors
(hemangiosarcoma) and mouse tumors
Charles River Laboratories
Parvoviruses of Mice

Mouse Parvovirus (MPV-1, MPV-2, MPV-3, MPV-4)
–
–
–
–

Prevalence higher than MMV
Causes persistent infection
No anatomic lesions, even in scid mice
Different strains not very cross-reactive by ELISA, MFIA
C57BL/6 mice and congenic strains partially
resistant to infection
– C57BL/6 mice require 10-100x infective dose
– DBA/2 only slightly better
Charles River Laboratories
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 in vivo

GEM expressing B19 NS1 have altered immune
system and high fetal mortality resembling
non-immune hydrops fetalis
Charles River Laboratories
Detection of Parvoviruses

Serology – Usually best for screening
– MFIA or ELISA - Traditional or recombinant antigens
– Use panel of antigens for each serotype, plus the
generic NS-1 antigen
Mice - MMV, MPV-1, MPV-2, and NS-1
 Rats - RV, H-1, RPV, RMV and NS-1

– IFA – Good follow-up assay for positive/equivocal
MFIA/ELISA
– Be careful with MPV serology of C57BL/6 mice!
Charles River Laboratories
Detection of Mouse
Parvoviruses

PCR
– Can be strain-specific (VP2) or generic (NS-1)
– Mesenteric LN stay positive indefinitely
– Pooled fecal samples to detect shedding (Beware of
fecal inhibitors of PCR)
– Biologicals and cell cultures
– Environmental swabs
Charles River Laboratories
Detection of Mouse
Parvoviruses
 Many
Challenges (sentinel parvovirus)
– Some strains partially resistant (C57BL/6, DBA/2)
– Not all mice may seroconvert to all antigens (NS-1)
– May have very low prevalence in IVC and filter-top
caging (hard to sort out from false positives)
– Seroconversion generally within 7 days, but may be
slow in adults exposed to low infectious dose
Charles River Laboratories
Control
of Parvoviruses


Can not “burn out” because infection is persistent
Can only eliminate by rederivation
– If caesarian section, must carefully test offspring and foster
dams. Primaparous dams more likely to be viremic.
– Reported as detected from sperm and pre-implantation
embryos

No envelope, so it stays active in environment
– Must thoroughly disinfect environment, materials and
equipment with oxidizing agent (Clidox, ozone, etc.)
Charles River Laboratories
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.)
Charles River Laboratories
Noroviruses

Type virus is Norwalk
virus, “cruise ship virus”
– Non-enveloped, RNA

Cause >90% nonbacterial
epidemic gastroenteritis
worldwide, 23M cases/yr
in US (per CDC)
– Cruise ships, institutions,
military
Noroviruses

MNV
– Genetically distinct (genogroup V) from human
noroviruses (I, II, IV), zoonotic spread unlikely
– No evidence of clinical disease or lesions in
immunocompetent mice

No noroviruses yet reported in other lab
rodents
Charles River Laboratories
MNV-1
No disease in immunocompetent mice
 High mortality in RAG (-/-) STAT (-/-)double
KO mice, with disseminated infection and
encephalitis and pneumonia

– Encephalitis only with IC inoculation
Charles River Laboratories
MNV
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
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Many variants isolated at this point, > 50 at CRL
MNV widespread in lab mouse research facilities
No clinical disease reported in natural infections
Most major vendors (including CRL) reporting all
colonies negative for MNV by serology and/or PCR
Charles River Laboratories
MNV

Research interference unknown, but:
– MNV-1 was detected in macrophage-like cells in
vivo and grew in vitro in dendritic cells and
macrophages. Growth was inhibited by the
interferon αβ receptor and by STAT-1 (Wobus et al.,
2004)
– Possible macrophage aggregates in RAG livers
Charles River Laboratories
MNV

Diagnosis:
– MFIA/ELISA – recombinant capsid protein selfassembles into VLP. Good cross-reaction among
variants
– PCR – Virus shed in feces for long periods, should
persist in environment. PCR must be properly
designed to be able to detect multiple strains.
Charles River Laboratories
MNV

Management
– Virus probably present in mice for a long time (so
no hurry)
Nonpathogenic
 Widely distributed
 Numerous strains

– Noroviruses should not cross placenta, so c-section
or ET rederivation should be successful
– Must consider environmental decontamination
Charles River Laboratories
Rat Respiratory Virus (RRV)
a.k.a. Idiopathic pneumonitis

Biology
– non-classified virus (apparently). Apparently enveloped.


Prevalence: Common
Epidemiology
– Host range - rats are the only known host, all strains
susceptible
– Transmitted by aerosol and/or dirty bedding
– Additional fomites transmission likely
Charles River Laboratories
Rat Respiratory Virus
(RRV)

Pathogenesis
– If no previous exposure
Lesions first seen about 4-5 weeks post-exposure
 Lesions reach peak severity at 7 weeks, then decline
 Lesions present for at least 13 weeks post-exposure

– In chronically infected colony (young have
maternal antibodies)

Best time to screen is 8 - 12 weeks of age
Charles River Laboratories
Rat Respiratory Virus
(RRV)

Diagnosis
– Gross Lesions: Scattered brown to grey areas on
pleural surface
– Histopathology
Dense perivascular lymphoid cuffs distributed in lungs
 Interstitial pneumonia (lymphohistiocytic)
 Syncytial cells
 Lesions graded minimal to mild, rarely moderate

– Serology: None available
Charles River Laboratories
RRV


Control
– Eliminate by rederivation
– Persistent infection? -No definite answer
If enveloped - Should be readily deactivated by
disinfectants, drying
Charles River Laboratories
Helicobacter Infection in
Laboratory Rodents

Biology and Epidemiology
 Microaerophilic (H. ganmani is anaerobic)
 Does not persist in environment – sensitive to
drying
 Transmission fecal-oral
 Many can infect multiple species
Charles River Laboratories
H. hepaticus




Host range: Mice, rats (mostly experimental)
Prevalence: High (12.7% in mice, 0.6% in rats by specific PCR)
Infection acquired early, persistent in mice
Chronic hepatitis in aging immunocompetent mice of some
strains.
– A/J inbred mice - hepatitis, increased hepatocellular carcinomas
– C57BL/6 mice resistant to disease, but still get infected
– Typhlocolitis in some strains

Immunodeficient mice
– Typhlocolitis and prolapsed rectum
– Hepatitis, may be necrotizing
Charles River Laboratories
Helicobacter hepaticus



Prolapsed rectum in
immunodeficient
mice
Proliferative
typhlocolitis
Suggestive, but not
diagnostic
Helicobacter hepaticus
Background Lesions

Prolapsed rectum in immunodeficient mice
can also be non-infectious (sporadic)
H. bilis





Host range: mice, rats, gerbils, dogs, cats, humans,
others?
Prevalence: high (3.5% in mice, 0.1% in rats)
Overall, similar to H. hepaticus in immunodeficient
mice
Similar to, but less severe, in immunocompetent mice
No lesions confirmed in immunocompetent rats
– Typhlocolitis in immunodeficient rats
Charles River Laboratories
Additional lab rodent
Helicobacter spp.
– H. rodentium – mouse, rat
– H. typhlonius – mouse, rat
– H. ganmani - mouse
– H. muridarum - mouse, rat
– F. (H.) rappini - mouse,
pig, sheep, dog, cat,
human
– H. trogontum – rat, mouse
Charles River Laboratories
– H. cinaedi - hamster,
human, dog, macaque
– H. cholecystus - hamster
– H. aurati - hamster
– H. mesocricetorum hamster
– OTHERS?
Research Interference



H. hepaticus - causes inflammation of liver and large intestine.
Increases inflammatory mediators (IP-10, MIP-1α, IL-10, IFN-γ,
and MIG mRNA, Livingston et al., 2004), in A/JCr mice, with
greater increases in females than in males.
Coinfection of H. hepaticus and H. rodentium, exacerbated the
inflammation and expression of inflammatory mediators, but
infection with H. rodentium alone did not cause hepatitis or
enteritis in A/JCr or SCID mice (Myles et al., 2004).
H. hepaticus infection in A/J mice caused upregulation of
putative tumor markers correlated temporally with increasing
hepatocellular dysplasia (Boutin et al., 2004). Also leads to
increased liver tumors
Charles River Laboratories
Helicobacter Detection



Similar for all Helicobacter
PCR – best. Generic or specific
Sentinels on dirty bedding 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)

Culture (microaerophilic or anaerobic, Brucella agar,
filtration)
– Different species require different filter pore size, culture
conditions


Histopathology with silver stains (in tissue only)
Serology
Charles River Laboratories
Control of Helicobacter


Probably similar for all Helicobacter spp.
Control (Elimination)
– Oral antibiotics – perhaps for small groups of mice. Questionable
efficacy.
– Rederivation by caesarian section or embryo transfer
– Cross-fostering pups onto “clean” dams

Before 24 hrs of age (Singletary KB, Kloster CA, Baker DG, Comp Med 2003
Jun;53(3):259-64 )

Control (Containment)
– Isolators
– Microisolators

Good review by Whary and Fox, Comp Med 54:128 2004
Charles River Laboratories
Oxyuriasis
(pinworms)

Biology:
– Oxyurid nematodes (Aspiculuris tetraptera, Syphacia
obvelata, S. muris)
– Direct life cycle
A. tetraptera eggs shed in feces, embryonate in 6-7 days
 Syphacia eggs attached to perianal hairs, embryonate in a
few hours
 Prepatent period 12-15 d for Syphacia, 23-25 d for
Aspiculuris

Charles River Laboratories
Oxyuriasis

Epidemiology:
– Transmission by contact with infective materials
– Eggs remain infective for months

Significance
– Old reports attributed colitis and rectal prolapse to
heavy infestation
– Newer reports describe changes in behavior and
immune response
Charles River Laboratories
Syphacia sp.
Aspiculuris tetraptera
Tape test
Fecal Exam
Oxyuriasis

Control
– Rederivation
– Exclusion Theoretically could be introduced by bedding, feed,
other supplies.
 Practically, introduced by other rodents or shared
equipment, then spread by fomites

Charles River Laboratories
Oxyuriasis

Control (cont.)
– Treatments
Fenbendazole (150 ppm in feed for at least three 7 day
periods over at least 5 weeks, combined with
environmental decontamination)
 Excellent review by Pritchett and Johnston,
Contemporary Topics, 2002, 41(2):36-46

Charles River Laboratories
Gambian Giant Pouched Rat
Cricetomys gambianus