Relevance to you? Immunodeficient mouse publications Pre Immunodeficient Options (J) & Background Effects Innate Back ground

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1 1 ImmunoDeficient Mice Options & Challenges 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 Relevance to you? Do you use? Nude? Scid? NOD/scid? NSG or NOG? Other immune deficient? Do you breed them? Do they live with or near competent mice? Do ID & competent mice share equipment? Imaging? Do you inoculate biological materials? Do you test them by MAP or PCR? How do you do microbial surveillance? Sentinels? Direct? 3 Brayton - Immunodeficient - P 1 AIMS 1. Select the best mice for your project. 2. Handle them to get best results with fewer animals. 3. Identify and eliminate threats before they damage your research & budget. 4 Discussion Plan Options 1. Mutations, Genetic backgrounds, Sources 2. Advantages & disadvantages RX: Irradiation Cytoxan etc - another lecture Challenges 1. Infections (manifesting as Enteric or Respiratory disease, death, sepsis abscesses, sepsis/death, or subclinical but patent infections of uncertain significance to your study) Biological Materials Viruses & Fungi Eukaryotes: Protozoa, Metazoan parasites Croy & al 2001 Immunodeficient mouse publications Pre 2001 Primary T cell deficient options Mutations T Cells B Cells Innate NK Leaky Foxn1 nu (nude) D N N yes Prkdc scid (scid) D D N- yes Prkdc scid Lyst bg (scid-bg) D D N Reduced by bg NOD-Prkdc scid (NOD-scid) D D D Minimal NOD, Prkdc scid, Il2rg tm (NSG, NOG) D D D No N = Normal D= Deficient 6 LEAKY mice B cells, T cells, immunoglobulin with age & microbial challenge (AKA conventional housing) Mice that survive infection & reject Xenografts. RX: CHECK CBC + IG levels BEFORE XENOGRAFT Tumors Thymic lymphoma Thymic lymphoma Thymic lymphoma B2m Foxn1 Nude Il2rg Prf1 perforin 1 Prkdc scid Rag1 NOD background 7 Functional (phenotypic effects) of some mutations in immunodeficient mouse models NK cell development/activity, CD8+ T cell activity nu/nu lack thymus T cell deficient unable to reject allogeneic & xenogeneic grafts nu/nu females are poor breeders, begin to ovulate late ~ 2.5 mo, & stop early, ~ 4mo NO thymic lymphomas on susceptible background, NOD.CB17 Prkdc scid /SzJ mice. NO NK cells, etc defects in innate immunity. NK cell activity, CD8+ T cell activity scid/scid no mature T & B cells, No cell mediated & humoral adaptive responses, BUT leaky some functional B and T cells as they age. Especially in non SPF conditions, leakiness C57BL/6J, BALB/cByJ backgrounds > C3H/HeSnJSmn > NOD/LtSzJ background ++ radiosensitive cannot be as thoroughly irradiated as other IDmodels before being engrafted Rag1 tm1mom /Rag1 tm1mom NO mature, functional T and B cells. On NOD background NO diabetes susceptible to immune mediated insulitis + diabetes NK, macrophage, APC, complement activity Immunodeficient Options (J) & Background Effects Innate Back ground Leaky H2 NAME Characteristics (Phenotypes) NK etc Normal B cells etc BALB/cBy Normal Yes T d CByJ.Cg-Foxn1 nu /J Extra thymic T cells with age POOR breeders, female fertility nu/+ F x nu/nu M NO functional B and T cells Normal High d CBySmn.CB17-Prkdc scid HIGH NK, complement activity (normal APC function) /J Thymic lymphomas but < NOD.CB17-Prkdcscid/SzJ RadioSensitive: C57BL/6J Normal NO b B6.129S7-Rag1 tm1mom NO functional B and T cells /J HIGH NK activity (normal APC, complement) Normal High b B6.CB17-Prkdc scid NO functional B and T cells /SzJ HIGH NK, complement activity (normal APC function) NO functional B and T cells NOD/LtSzJ Low NO g7 NOD.129S7(B6)-Rag1 tm1mom /J Pre-B cell > Thymic lymphomas ( 10.5mo) Somewhat RadioResistant NO functional B &T cells; NO NK cell activity Low NO g7 NOD.Cg-Rag1 tm1mom Prf1 tm1sdz /Sz Thymic lymphomas (short lifespan ~8.5mo) RadioResistant: survives up to 8 Gy NO functional B and T cells; NOD background low NK activity, NO Low Low g7 NOD.CB17-Prkdc scid hemolytic complement activity, defects in myeloid development, poor APC /SzJ functions -- RadioSensitive: tolerates up to 4 Gy Thymic lymphomas (short lifespan ~8.5mo) NO functional B and T cells Low Low g7 NOD.Cg-Prkdc scid B2m tm1unc No MHC 1 expression ~NO NK activity /J Thymic lymphomas (short lifespan ~6.3mo) Hemachromatosis dt? NO functional B and T cells Low NO g7 NOD.Cg-Prkdc scid Il2rg tm1wjl /SzJ NO NK cell activity Lymphoma-resistant and long-lived > 16m adapted from Jax notes #501, 2006 NIH stock Normal Yes T q NU/J (Foxn1nu) NIH outbred nude stock inbred at TJL 8 More Foxn1 nu options Source Background Name INbred AKA Tac Bom B6N/Tac B6.Cg/NTac-Foxn1 nu + B6 nude BALB/cAnN C.Cg/AnNTac-Foxn1 nu + BALB/c nude BALB/cBom C.Cg/AnBomTac-Foxn1 nu + BALB/cA nude BALB x NIH(S) CrTac:NCr-Foxn1 nu NIH Nude NMRI (S) BomTac:NMRI-Foxn1 nu Swiss nude NIH(S) NTac:NIHS-Foxn1 nu Swiss Nude Crl BALB/cAnN CAnN.Cg-Foxn1 nu /Crl + BALB/c Nude CD1 (S) Crl:CD1-Foxn1 nu CD1 nude NIH Nu Crl:NU-Foxn1 nu Nu/nu NIH nude Hsd Ola NIH Nu Hsd:Athymic Nude-Foxn1 nu NIH nude NMRI (S) HsdCpb:NMRI-Foxn1 nu Swiss Nude CD1 (S) HsdHli:CD1-Foxn1 nu CD1 nude ICR (S) HsdOla:ICRF-Foxn1 nu Swiss Nude MF1 HsdOla:MF1-Foxn1 nu MF1 nude 9 BALB/cOla BALB/OlaHsd-Foxn1 nu + BALB nude

2 More Prkdc scid options Source Background Name INbred AKA Tac Bom C.B17 C.B-Igh-1 b /IcrTac-Prkdc scid + CB17 scid IcrTac IcrTac:ICR-Prkdc scid Scid - Swiss C.B-17 C.B-Igh-1b/GbmsTac-Prkdc scid -Lyst bg + scid-beige NOD + NOD/MrkBomTac-Prkdc scid + NODscid NOD NOD.Cg-Prkdc scid Il2rg tm1sug /JicTac + CIEA NOG Crl C.B17 CB17/Icr-Prkdc scid /IcrCrl + CB17 scid Crl:SHO-Prkdc scid Hr hr Scid hairless outbred Hsd Ola BALB/cJ BALB/cJHan Hsd-Prkdc scid + BALB/c Scid 10 C.B-17 C.B-17/IcrHsd-Prkdc scid + CB17 scid C3H C3H.C-Prkdc scid /IcrSmnHsd + C3H scid ICR (S) HsdIcr:Ha(ICR)-Prkdc scid Scid NOD CB17 NOD.CB17/JHliHsd-Prkdc scid + NODscid IF the name begins with the SOURCE, it is OUTbred (or not inbred) May not be as homogeneous as inbred BUT May be more robust & easier to breed / maintain 11 More other options Source Background Name INbred AKA Tac C.B17 C.B-Igh-1b/GbmsTac-Prkdc scid -Lyst bg + Scid beige NIH(S) Tac:NIHS-Lyst bg Foxn1 nu Btk xid NIHIII nude NOD NOD.Cg-Prkdc scid Il2rg tm1sug /JicTac + CIEA NOG Crl C.B-17 CB17.Cg-Prkdc scid Lyst bg /Crl + Scid beige NIH(S) Crl:NIH-Lyst bg Foxn1 nu Btk xid NIHIII nude HSD CBA CBA/HNOlaHsd-Btk xid + XID NIH(S) Hsd:NIHS-Lyst bg Foxn1 nu Btk xid NIHIII nude B6 C57BL/6OlaHsd-Lyst bg + Beige C.B-17 C.B-17/IcrHsd-Prkdc scid Lyst bg + Scid beige NSG or NOG? NOD.Cg-Prkdc scid Il2rg tm1wjl /SzJ NSG -Il2rg null Schultz at J - (Shultz & al. 2005). NOD.Cg-Prkdc scid Il2rg tm1sug /JicTac NOG - Il2rg deficient CIEA (Jic) Japan (Ohbo & al Brayton - Immunodeficient - P 2 Immunodeficient options Conclusion: LOTS OF OPTIONS. QUIZ: Do you order from the same vendor? Do you order exactly the same mice? Do you check the health reports? Do you request Klebsiella negative? Do you order from the same room/isolator? Every time? Discussion Plan 1. Options 1. Mutations, Genetic backgrounds, Sources 2. Advantages & disadvantages RX: Irradiation Cytoxan etc - another lecture Competent vs Immunodeficient Infectious Phenotypes Really Oversimplified Competent Immunodeficient Immune response Innate + acquired Antibody response seroconversion immune mediated lesions, necrosis Control/eliminate infection. Less inflammation Cytopathic effect /necrosis Failure to seroconvert Failure to clear heavy burden GREAT RESERVOIR & risk to others Competent vs Immunodeficient Infectious Phenotypes Really Oversimplified Competent Immunodeficient Immune response Innate + acquired Antibody response seroconversion immune mediated lesions, necrosis Control/eliminate infection.. Less inflammation Cytopathic effect / necrosis Failure to seroconvert Failure to clear heavy burden GREAT RESERVOIR & risk to others Challenges 1. Infections (manifesting as Enteric or Respiratory disease, death, sepsis abscesses, sepsis/death, or subclinical but patent infections of uncertain significance to your study) Biological Materials Viruses & Fungi Eukaryotes: Protozoa, Metazoan parasites 14 IMPLICATIONS Immunodeficient don t seroconvert reliably hard to detect by serology seroconversion ID don t clear many infections - if they survive: Heavier burdens Easier to detect agents Harder to eliminate agents Source of agents that may be hazard to others 15 Implications for Microbial surveillance Competent sentinels Should seroconvert but may not (e.g. Parvoviruses) May clear opportunist infections to undetectable levels May pose a risk to immunodeficient neighbors Direct testing (e.g. fecal PCR) of immunodeficient residents, may be a useful adjunct Likely Infectious Phenotypes & Agents in Enteric enterohepatic Respiratory Other MNV Sendai Parvoviruses MHV PVM (paramyxo-) Ectromelia viruses MRV mcmv Helicobacters Pasteurella pneumotropica Staph spp Clostridium piliforme Bordetella spp Klebsiella oxytoca Salmonella Klebsiella o or p? Strep/enterococcus Pseudomonas Giardia? Pneumocystis murina Proteus mirabilis Spironucleus? C bovis Pinworms Etc. 16 More Common and or Problematic agents in red NOT primarily enteric or respiratory (Oversimplified) Phenotype Agents Death & Necrosis Ectromelia virus - orthopoxvirus Cytomegalovirus mcmv Gram Positive: Strep/enterococcus spp etc. Death & Sepsis Gram negative: Pseudomonas Klebsiella Proteus etc Endotoxemia, enterotoxemia Abscesses Staphylococccus spp, etc. Skin disease Corynebacterium bovis MUD + opportunists Subclinical immunomodulation etc Parvoviruses Subclinical + inclusion bodies Adenoviruses, Herpesviruses, Polyomaviruses 17 Likely viral phenotypes in Phenotype Viruses Also consider Enteric / Enterohepatic Respiratory primarily MNV MHV MRV (mcmv) Sendai, PVM MPV (pneumotropic virus) Death & necrosis Ectromelia CMV Subclinical immunomodulation etc Parvoviruses Subclinical + inclusion bodies 18 Adenoviruses, Herpesviruses, Polyomaviruses Helicobacters C piliforme Salmonella Giardia? Spironucleus? Pinworms rectal prolapse Pneumocystis murina Pasteurella pneumotropica Klebsiella oxytoca etc Bordetella avium, hinzii MHV C piliforme;, Salmonella Many possibilities

3 Likely bacterial phenotypes in Phenotype Also consider Enteric /enterohepatic Respiratory primarily Death & Sepsis Abscesses primarily Skin disease 19 Helicobacters Citrobacter rodentium Clostridium piliforme Salmonella Enterococcus? Pasteurella pneumotropica Klebsiella oxytoca etc Bordetella avium, hinzii Pseudomonas Strep/enterococcus spp Staphylococccus spp Corynebacterium bovis MUD + opportunists MNV? MHV, MRV, mcmv Giardia? Spironucleus? Pinworms rectal prolapse Pneumocystis murina Sendai, MPV Klebsiella oxytoca Proteus mirabilis Endo/enterotoxemia Streptococcus Gram negatives Ringworm? 20 Infectious Agents in Biological Materials Tumor xenografts, cell lines, sera, etc. In a freezer, in specimens, or in mice near you Risk to projects & personnel. ATCC does NOT test for most of these. MAP Test or PCR test to detect them 21 Brayton - Immunodeficient - P 3 Biological Materials Recent (also historical) contaminants Agents found recently Some Phenotypes in immunodeficient Mycoplasma sp. Respiratory dz + shedding LDV Immunomodulate LE s + shedding Human herpesviruses BIOHAZARD + shedding HIV BIOHAZARD + shedding Hepatitis B BIOHAZARD + shedding Parvoviruses Immunomodulate, Tumor growth? + shedding Polyoma virus Immunomodulate, Tumors + shedding LCMV Immunomodulate, BIOHAZARD + shedding Mouse adenovirus Immunomodulate INIB? Kid, Gut + shedding Mouse hepatitis virus Wasting + enterohepatic DZ + shedding mcmv) Death hepatic etc necrosis + shedding Ectromelia virus Death & shedding Pneumonia virus of mice (PVM) Respiratory DZ + shedding Murine norovirus Enteritis encephalitis n some + shedding Sendai virus Wasting + respiratory Dz + shedding E. cuniculi Kid + CNS? + shedding from CBW RADIL & CC CRL AALAS Rodent Agents in Biomaterials Agent % Positive Mycoplasma sp. ~8.0% Lactic dehydrogenase elevating virus (LDV) > 1% Parvoviruses < 1% Polyoma virus < 1% Lymphocytic choriomeningitis virus (LCMV) < 1% Mouse adenovirus < 1% Mouse hepatitis virus < 1% Mouse cytomegalovirus (mcmv) < 1% Ectromelia virus < 1% Pneumonia virus of mice (PVM) < 1% Murine norovirus < 1% Sendai virus < 1% E. cuniculi <1% from CBW RADIL & CC CRL AALAS Human & other viruses found in human biomaterial submissions ( ) Infectious agent Percent positive HIV <1% Hepatitis B <1% Human herpesvirus 6 >1% Human herpesvirus 8 <1% Epstein Barr virus >1% Reo, LDV, LCMV, parvo <1% from CBW LR RADIL & CC CRL AALAS Compmed May 16, 2011 Inoculated mice became ill, had ascites and splenomegaly The (commercial mouse) serum was positive with high titer to mouse hepatitis virus, Theiler's meningoencephalitis virus, mouse rotavirus, mouse norovirus and Sendai virus. Comparative Medicine List [[email protected]] Monday May 16, 2011 Viruses Enteric +/- hepatic disease in MNV - Hepatitis etc in stat deficient MRV neonatal diarrhea like competent mice MHV Hepatitis enterocolitis wasting CMV mouse cytomegalovirus death with liver, necrosis etc Helicobacters Clostridium piliforme Salmonella typhimurium Protozoa Giardia, Spironucleus? proliferative enteritis? Bigger 25 Pinworms heavy burdens rectal prolapse NOT associated with disease Segmented Filamentous Large intestine protozoa MNV - Murine Norovirus(es) in Subclinical in most or Pneumonia, hepatitis, vasculitis, (encephalitis) in severely innate immune deficient mice e.g. Rag2 / /Stat1 / Rag1 / /Stat1 / Rag1 / /IFNγR / Ward & al.; Wobus & al Innate immunity? Macrophage function? Paik & al IBD, enterohepatic phenotypes? Paneth cells Cadwell & al Cell culture contaminant 26 Sick Nude mouse Small intestine Necrosis + crypt abscesses elsewhere Paneth cell degeneration Similar to Cadwell 2010? 27 MNV?

4 Mouse Rotavirus (MRV - EDIM) in Neonatal diarrhea as in competent mice Breeding colonies, runting Immunomodulation 28 MHV Mouse Hepatitis Virus in Adult wasting with necrosis/syncytia liver, spleen, lymph nodes, GALT, marrow, brain Don t clear virus i.e. persistent infection (granulomatous peritonitis/ pleuritis in Ifn Ko similar to Feline Infectious Peritonitis? ) Is it a problem for you? Wasting in old mice in chronic studies, or breeding Liver enzymes, liver, gut phenotypes Immune phenotypes 29 Cell culture contaminant including ES cells 30 MHV in wasting phenotype of Prkdc scid or Foxn1 nu etc immunodeficient Liver necrosis progressive severe Brayton - Immunodeficient - P 4 MHV Liver Phenotypes Necrosis extensive in immunodeficient +/- LE s AST, ALT, LDH, GGT Not much inflammation in immunodeficient Syncytia = histologic hallmark MHV Gut Phenotypes Ascending colon target Distal SI too Syncytia, Necrosis Syncytia in other tissues too Morphologic Diagnosis: Cause (most likely Today): Helicobacters Likely sex? Also consider: Citrobacter? Pinworms? Tumors? IBD Phenotype? 33 Colitis Proctitis Inflammatory + Proliferative Bowel Disease phenotypes Lymphocyte deficient mice more neutrophils + macrophages Helicobacters often subclinical Usually typhlocolitis, often proliferative (IBD) H hepaticus, mastomyrinus, typhlonius, etc Females more susceptible? to bowel disease Immunomodulate Gut, Liver phenotypes, Liver enzymes Liver, Gut, Mammary tumors Morphologic diagnosis: Etiology:

5 Liver Necrosis + INTRACELLULAR filamentous bacteria Giemsa Warthin Starry UCD CCM images Clostridium piliforme Tyzzer s disease (Bacillus piliformis) Kills hamsters + susceptible mice, rats, horses, etc Gram negative filamentous bacilli Warthin Starry silver stains it best Obligate intracellular parasite Cultured in eggs or cell culture Historically significant agent in research rodents low prevalence now. Segmented Filamentous bacteria Florid growth suggests immunoweird? Competent mice typically clear to very low levels by ~ 1mo Good for your mice? Th17 etc development 39 Brayton - Immunodeficient - P 5 Giardia muris Spironucleus muris (Hexamita muris) Large Intestine Flagellates Nude sentinel Abundant in nu/nu but NOT nu/+ cagemates nu/+ cleared to undetectable levels 0-minimal inflammation Expect these in hamsters 40 Nude sentinel Abundant in nu/nu but NOT nu/+ cagemates nu/+ cleared to undetectable levels 0-minimal inflammation 41 Expect these in hamsters In Crypts 42 Entamoeba muris Pinworms in Expect this mouse to be immunodeficient because of heavy load Nematodes Pinworms (oxyurids) Adults, larvae in cecum, colon Detect by (terminal) direct examination 43 Amoebae ~>10 u probably has helicobacters too easy to detect shedding lots of infectious eggs in environment, on equipment Syphacia obvelata (muris) LOTS OF asymmetric ) eggs on perineum Detect by tape test Aspiculuris tetraptera Sneakier FEWER symmetric () eggs in feces Detect by fecal flotation RADIL images

6 Viruses Fungi 46 Respiratory disease in Sendai virus Paramyxovirus Pneumonia virus of mice Paramyxovirus Mouse pneumotropic virus Polyomavirus Pasteurella pneumotropica etc opportunists Mycoplasma CARbacillus Bordetella avium, hinzii etc Klebsiella oxytoca or pneumoniae? Pneumocystis murina Clinical Phenotypes Dyspnea maybe chattering Sick- dead Wasting in chronic disease Sendai Virus in Progressive wasting, dyspnea, death Proliferative (tumor-like) lesions instead of necrotizing lung lesions; syncytia, inclusions Not much inflammation Wasting dyspnea in chronic studies, breeding Open cages contained by microisolator caging Immunomodulate Cell culture contaminant 47 Sendai Virus In nude, scid etc No T cell-mediated necrosis Not much Inflammation Some neutrophils Proliferation Syncytia Intranuclear Inclusions 48 Brayton - Immunodeficient - P 6 Proliferative tumor like Adenomatous change in immunodeficient mice 49 Sendai Virus Upper Respiratory Disease (URI) Mice are obligate nose breathers Rhinitis can kill. P pneumotropica? B hinzii, avium? Klebsiella oxytoca? K pneumoniae? Mycoplasma? Not so common Streptococci? Not as likely 50 Upper Respiratory Disease (URI) Otitis Common Gram 0???? Mycoplasma Gram+ Strep/Enterococcus Gram- Pasteurella Bordetella Klebsiella 51 Upper Respiratory Disease (URI) Otitis Common Gram+ Strep/Enterococcus Gram- Pasteurella Bordetella Klebsiella Gram 0? Mycoplasma Upper Respiratory Disease (URI) Otitis Common Myd88 Gram+ Strep/Enterococcus Bronchopneumonia (+ AMP) Lobar consolidation with bronchitis, broncho-pneumonia Also acidophilic macrophage pneumonia P pneumotropica? B hinzii, avium? Klebsiella oxytoca? K pneumoniae?

7 55 Pasteurella pneumotropica Gram negative bacillus Usually considered Commensal opportunist Normal? gut microflora, isolated from healthy nasopharynx 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 Bordetella spp Gram negative bacillus B avium, B hinzii B bronchiseptica historic? in mice Rabbit & Guinea pig problem Isolated from implicated in respiratory disease with upper respiratory lesions (rhinitis tracheitis) in mice Short gram negative rods Look among cilia in respiratory epithelium Warthin starry silver stain cf B bronchiseptica, Kennel cough Bordetella spp Gram negative Bacilli adhere to cilia Hard to identify on histo SEM of B bronchiseptica on cultured rabbit tracheal epithelium Edwards & al 2005 Brayton - Immunodeficient - P 7 Respiratory disease in Sudden Death Diffuse consolidation interstitial pneumonia Respiratory disease in Consolidations Intra-alveolar eosinophilic foamy material 0-Minimal inflammation Special stain? Other diagnostic tests? Respiratory disease in Pneumocystis murina (P carinii) GMS silver stains Cysts Fungus NOT primarily enteric or respiratory Phenotypes - Oversimplified Death with Necrosis - Viral Death with Necrosis bacterial (sepsis) Abscesses bacterial Skin disease No obvious disease Viruses Fungi Death, Necrosis, Sepsis in Ectromelia virus - orthopoxvirus Cytomegalovirus mcmv (also MHV with necrosis in liver) Gram Positive: Strep/enterococcus spp etc. Gram negative: Pseudomonas Klebsiella Proteus etc (Also c piliforme, Salmonella with necrosis in liver) Opportunists?.g. Paecilomyces Trichosporon, zygomycetes Ectromelia Virus DEATH Many immunodeficient Hily susceptible to acute lethal dz Few Gross lesions (die too quickly) +/- liver spleen necrosis with inclusions Biological materials Sentinels do not reliably seroconvert

8 l sepsis in Mortality NSG Post Xenograft Brayton - Immunodeficient - P 8 Mortality in NSG Gram Positive Streptococcus Enterococcus Gram Negative Klebsiella oxytoca Proteus mirabilis Pseudomonas Enterotoxemias post antibiotics Any? bacteria that are not cleared can bacteremia Liver Gram stain Usually not so obvious Bacteremia sepsis Kidney Intravascular bacteria 64 Clinical Phenotypes Death Ill-moribund mice Brain Gram stain Mortality in Myd88 Overwhelming gram positive bacteremia and bacterial colonization E faecalis isolated from some 67 Mortality in LAD cd11/18 deficient Meningoencephalitis Gram positive cocci Experimental IP streptococcus infection Unexpected mortality Streptococci / Enterococci Morphology Gram positive cocci Pairs (diplococci) Chains (streptococci) Streptococci / Enterococci S pneumoniae (beta hemolytic, non A, non B) Many mice relatively resistant but otitis / meningoencephalitis / mortality in LAD Pneumonia/mortality in C3H/HeJ (Tlr4 deficient) Group B (S agalactiae) may rhinitis, meningitis, encephalomyelitis in nude Alpha hemolytics septicemia in scid Non-hemolytic (= gamma hemolytic) isolates from septic immunodeficient S zooepidemicus dermatitis (Lancefield type C considered to be non-pathogen) E faecalis emerging problem in immunodeficient? Especially NSG? Foreman & al Gram Negative Sepsis bacteremia in Klebsiella oxytoca emerging? NSG etc Pseudomonas aeruginosa most famous Proteus mirabilis can have these and do fine until stressed. Do you check vendor health reports? Do you request mice that are negative for these agents? Gram negative rod capsule Opportunist in NSG etc Esp in UTI urinary tract infections Foreman & al Klebsiella oxytoca

9 Gram negative rod capsule Opportunist in NSG etc Esp in UTI urinary tract infections Foreman & al 2010 Rule out E coli Klebsiella oxytoca? Note capsules K oxytoca otitis in C3H/HeJ Macarthur & al 2007 Brayton - Immunodeficient - P 9 Pseudomonas aeruginosa Common isolate from healthy mice P aeruginosa etc pseudomonads are common in tapwater control with acidification, hyperchlorination Opportunist bacteremia, mortality in neutropenic (esp irradiated) mice A lot of are fine - until irradiated, then succumb (no neutrophils). Also urolithiasis, otitis etc Proteus mirabilis Bone marrow Abscesses in 76 Common isolate from healthy mouse GI tract Grows rapidly & Overgrows other species in culture may mask a real cause of disease Dx: Proteus overgrowth septicemia, fibrinosuppurative peritonitis, pyelonephritis, hepatitis, pneumonitis, mortality 77 MDA 468 Happy hematopoietic marrow MCF7 (+ estrogen) Too much bone Not much marrow 1. Staphylococcus spp 78 Also consider Klebsiella oxytoca E coli Streptococcus etc Corynebacterium kutscheri pseudotuberculosis historical? Etc Gram Neg Fungal Opportunists abscesses, granulomas, mycosis Paecilomyces variotii Trichosporon beigelii Zygomycetes Abscesses Often one end or the other Facial S. sciureus isolated Abscesses Head & Neck tumor? Ulcerated = SCC? Abscesses S aureus or Coagulase NEG Staphylococcus spp e.g. S xylosus, S hyicus etc. in immunodeficient or immunoweird Botryomycosis

10 Staphylococcus spp. S aureus (1 of few coagulase POSITIVE species) Lymph node abscesses, botryomycosis, ulcerative dermatitisin competent mice Furunculosis, abscesses in immunodeficient S xylosus, sciureus, hominis, hyicus etc commensal (coagulase Negative) isolated from abscesses + Botryomycosis lesions in immunodeficient & GEM Skin disease in Corynebacterium bovis Opportunists in ulcerative dermatitis, - likely? to progress to sepsis Fungi Ringworm? T mentagrophytes? Historic? Bigger Brayton - Immunodeficient - P 10 Demodex muris in follicles Skin Disease Nude mouse Etiology? Histology findings? Special Stain? Scurfy skin + pup mortality in Nude, Rag1, NODscid, NSG etc Gram Corynebacterium c/o RADIL DORA at C. diphtheriae Gram POSITIVE irregular short rods Chinese letter configuration Very small Corynebacterium bovis HAC = Hyperkeratosis Associated Coryneform bacteria Orthokeratitic epidermitis, dermatitis looks like corn meal in Nude mice Mortality in suckling mice Also isolated from hyperkeratotic, haired immunodeficient (and competent?) mice Tough to eradicate. Mouse Ulcerative Dermatitis Isolate anything from chronic MUD MUD likely to progress to sepsis in immunodeficient Staphylococcus spp are common Epidermolytic toxins can staphylococcal scalded skin syndrome (SSSS) Subclinical Infectious concerns in Subclinical immunomodulation etc Subclinical + inclusion bodies Sublinical until stressed, further suppressed. Parvoviruses Adenoviruses, Herpesviruses, Polyomaviruses Gram positives Gram negatives Almost anything?

11 Parvoviruses in Usually No Evidence of Disease Persistent infection, shedding Runting, death, INIB in NOD.Cg-H2H4-Igh-6 / MVMi exp Lethal leucopenia in scid Targets cells in S phase Oncolytic in cancer models Poor tumor growth? Competent sentinels may not seroconvert fast or reliably Cell culture contaminant MVM (MMV) no lesions? NOD.Cg-H2H4-Igh-6 / mouse Runting & death - Naugler et al Spleen - intranuclear inclusion bodies IHC positive for MVM VP2 Frankin 2006 ILAR J 93 Brayton - Immunodeficient - P 11 Immunodeficient Challenges Conclusion: LOTS OF CHALLENGES. QUIZ: DO you MAP or PCR test biological materials that you use in? Do you check the health reports? Do you request Klebsiella negative? Do you assess ill mice for agents that could be a risk to other mice or experiments? Is CLEANEST BEST? for the mice? for the project? YES for that are expected to survive, and accept grafts from other animals. Maybe not necessarily?? for GEM where Environmental/Microbial challenge may expose a phenotype & Rederivation may disappear it 94 SUMMARY & Conclusions Options Lots 1. Mutations, Genetic backgrounds, Sources 2. Advantages & disadvantages Challenges Lots 1. Beware of Biological Materials! 2. Enteric, Respiratory & OTHER diseases - What is likeliest in your situation? 3. Consider direct testing (PCR) as well as sentinels 95 Infectious REFERENCES Baker, D. G. (2007). FLYNN S PARASITES OF LABORATORY ANIMALS. 2 nd ed. ACLAM, Blackwell Publishing. Hedrich, THE HANDBOOK OF EXPERIMENTAL ANIMALS SERIES: THE LABORATORY MOUSE. Elsevier. Percy, D. H. and S. W. Barthold (2007). PATHOLOGY OF LABORATORY RODENTS AND RABBITS. Ames, IA, Iowa State University Press. 3 rd ed Sellers, R; Clifford; Treuting; Brayton Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice. Vet Path. 49(1): p Treuting, P; Clifford; Sellers; Brayton Of mice and microflora: considerations for genetically engineered mice. Vet Path Various authors, specific diseases and agents In Fox & al. Ed s. THE MOUSE IN BIOMEDICAL RESEARCH 2nd Ed. ACLAM series. Elsevier. Various Authors ILAR Journal Volume 49(3) Detection and Management of Microbial Contamination in Laboratory Rodents Various authors, University of Missouri RADIL Diseases of Research Animals (DORA) site 97 Sources of mice and surveillance information Charles River (Crl) /research_models/mice_a_b.html Harlan (Hsd, Ola) Jax (J) NCI Frederick (N, Cr) ogram/strain_information/ Taconic (Tac) Research & Reporting Guidelines Workman, P., E. O. Aboagye, et al. (2010). "Guidelines for the welfare and use of animals in cancer research." Br J Cancer 102(11): Kilkenny, C., W. J. Browne, et al. (2010). "Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research." PLoS Biol 8(6): e National Academy of Sciences: Guidance for the Description of Animal Research in Scientific Publications (2011) Animal/