Charles B. Clifford, DVM, PHD, DACVP
Dir, Pathology and Technical Services
Charles River Laboratories

• MHV – 2%
• Parvoviruses
• Mouse – 2%
• Rat – 4%
• EDIM – 0.7%
• Norovirus ~30%
• RRV – 7%
• Helicobacter spp. – 15%
• C. bovis – 3%
• Pneumocystis carinii – 2%
• Pinworms –
Mouse – 0.3%
Rat – 1.3%
• Mites – 0.1% (mice only)
Charles River Laboratories

Agent Assay # tested # pos.
% pos.
Parv NS-1
MPV
MVM
MHV
EDIM
GDVII
MPUL
REO
SENDAI
PVM
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA
445,255
457,062
458,931
441,098
364,793
342,312
352,563
338,054
361,118
353,043
8,481
8,974
1,789
7,949
2,459
991
32
43
10
12
1.9048%
1.9634%
0.3898%
1.8021%
0.6741%
0.2895%
0.0091%
0.0127%
0.0028%
0.0034%
Charles River Laboratories

RPV
Agent
H-1
KRV
RMV
GDVII
SDAV
Assay # tested # pos.
% pos.
ELISA 73,289 1,324 1.8065%
ELISA 67,594 1,128 1.6688%
ELISA
ELISA
ELISA
ELISA
73,400 1,136 1.5477%
29,110
28,203
68,445
437
264
159
1.5012%
0.9361%
0.2323%
MPUL ELISA
M pulmonis Culture
PVM
SENDAI
ELISA
ELISA
REO
Charles River Laboratories
ELISA
67,951
3,558
66,450
67,193
61,016
127 0.1869%
2 0.0562%
98 0.1475%
16 0.0238%
5 0.0082%




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



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








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


– 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



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


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





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





 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


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




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



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


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


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


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


– 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




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


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



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



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



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





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


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


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


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




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


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


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



Control
– Eliminate by rederivation
– Persistent infection? -No definite answer
If enveloped -
Should be readily deactivated by disinfectants, drying
Charles River Laboratories


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






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




Prolapsed rectum in immunodeficient mice
Proliferative typhlocolitis
Suggestive, but not diagnostic


Prolapsed rectum in immunodeficient mice can also be non-infectious (sporadic)






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

– 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?




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







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





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


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



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


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


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