COMMON INFECTIOUS DISEASES IN LABORATORY RATS AND MICE

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

2. What’s common? • 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)

3. What’s common in mice?

4. What’s common in rats?

5. 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

6. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. Parvoviruses Are you getting mixed signals on parvoviruses?

15. Parvoviruses in Mice • ssDNA, non-enveloped • Virus remains active in environment • Resistant to desiccation and many (non-oxidizing) disinfectants • Fairly common • Generally no clinical signs • Cause persistent infection – no self-cure • Need actively dividing cells to replicate

16. 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.

17. 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

18. 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

19. 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

20. 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!

21. 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

22. 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

23. 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.)

24. 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.)

25. 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

26. 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

27. 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

28. MNV • 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

29. 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

30. MNV • Diagnosis: • MFIA/ELISA – recombinant capsid protein self-assembles 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.

31. 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

32. 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

33. 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

34. 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

39. RRV • Control • Eliminate by rederivation • Persistent infection? -No definite answer • If enveloped - Should be readily deactivated by disinfectants, drying

40. 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

41. 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

42. Helicobacter hepaticus • Prolapsed rectum in immunodeficient mice • Proliferative typhlocolitis • Suggestive, but not diagnostic

44. Helicobacter hepaticus

45. Background Lesions • Prolapsed rectum in immunodeficient mice can also be non-infectious (sporadic)

46. 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

47. 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 H. cinaedi - hamster, human, dog, macaque H. cholecystus - hamster H. aurati - hamster H. mesocricetorum - hamster OTHERS? Additional lab rodent Helicobacter spp.

48. 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

49. 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

50. 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