2017 Brayton MICE INFECTIOUS

Jargon Mice Infectious Agents & SPF = Specific Pathogen Free – Defined by the Exclusion list Diseases (Phenotypes) Gnotobiotic = defined flora – ASF = altered Schaedler’s flora 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 Axenic = germ free Johns Hopkins University, School of Medicine Baltimore, MD 21205 Autochthonous flora (indigenous flora) [email protected] http://www.hopkinsmedicine.org/mcp/PHENOCORE/index.html – Microbiome/Microbiota Allochthonous flora (transient flora).

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Infectious Agents & Phenotypes FELASA 2014 recommendations Discussion Plan

1. FELASA recommendations –2014, 2015 updates 3 month testing 2. Competent Mice –by agent 1. More common  Annual testing : – Viral agents –Top few 2. More likely 1. Less likely – Bacteria –Top few 2. But concerning 3. Concerning – Eukaryotes –Top few 3. In a freezer near you 3. ‘Normal’ flora & the microbiome 4. In a pet store or wild 4. Immunodeficient mice ‐ by disease phenotype rodents near you… – Enteric / enterohepatic  USEFUL BUT Partial list of agents… – Respiratory  ‘other agents’ as ‘necessary’…. – OTHER  Some will be discussed…

125. Biological Materials … http://www.uni-heidelberg.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pdf

FELASA 2014 recommendations FELASA 2014 recommendations Mice Mice ‐ Viruses ‐ Bacteria

 3 month testing  3 month testing  Annual testing – add: 1. Helicobacter spp. x 1. MHV 1. LCMV  Annual testing – Add: +  H. hepaticus, H. bilis, 2. MAD1 (FL) 1. Citrobacter rodentium 2. Mouse rotavirus (EDIM) H. typhlonius 3. MAD2 (K87) 2. Clostridium piliforme 3. Murine norovirus 2. P pneumotropica 4. Mousepox 3. Corynebacterium kutscheri 4. Parvoviruses (NS1) (Ectromelia) virus 3. Strep b‐haemolytic (not 4. Mycoplasma pulmonis group D) 5. MVM 5. PVM 5. Salmonella spp 6. Reo3 4. Strep pneumoniae 6. Streptobacillus moniliformis 6. MPV 7. Sendai 7. TMEV

http://www.uni-heidelberg.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pdf http://www.uni-heidelberg.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pdf

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FELASA 2014 recommendations FELASA 2014 recommendations Mice – Other Agents Mice – Other Agents Test as appropriate, for example due to : Viruses: Bacteria & fungi: 1. Zoonotic concern  Hantaviruses  CAR bacillus 2. Historical concerns (in freezers near you!)  Herpesviruses  Klebsiella oxytoca, 3. Pet or wild rodent exposures (MCMV, MTV)  Klebsiella pneumoniae 4. Opportunists or too common to exclude  Lactate‐dehydrogenase  Other Pasteurellaceae practically from competent colonies & elevating virus (LDV)  Pneumocystis murina usually not a problem iin competent mice  Polyomaviruses (mouse 5. Experimental risk/exposure, e.g. polyomavirus, K virus)  Pseudomonas aeruginosa . Biological materials  Staphylococcus aureus . Experimental infections in the area . Gene therapy vectors http://www.uni-heidelberg.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pd6 f http://www.uni-heidelberg.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pdf

FELASA ‐ Mice –q 3m testing Infection phenotypes ‘endo/ectoparasites’ Ectoparasites  Protozoa/Protists 1. Fleas Immune modulation 1. Eimeria 2. Lice Polyplax Cancer 2. Entamoeba 3. Myobia Neurobehavioral 4. Myocoptes 3. Giardia – Hearing, vestibular 5. Radfordia 4. Spironucleus 6. Mesostigmatid mites – CNS – development/degeneration 5. Tritrichomonas muris Helminths Metabolic 1. Aspiculuris Respiratory 2. Syphacia Cardiovascular 3. 4. Rodentolepis 23

Nature: (mostly) genetics  Immune modulation Phenotypes in ‘Normal’? (+/+) Mice

129? Teratomas (Ter), lung tumors, acallosity, AMP, … Autochthonous flora (Indigenous) A/J Lung tumors, anomalies, amyloid, muscular dystrophy – Microbiome AKR Thymic Lymphoma… BALB/c Plasmacytoma etc tumors, heart dz, acallosity, kill each other – Opportunist C3H TUMORS - Mammary, Liver Microphthalmia, Hydrocephalus , MUD, Osteoporosis, Presbyacusis, Allochthonous flora (transient) C57BL/6 Amyloidosis, AMP, … – Most pathogens are allochthonous DBA Deaf, seizures, glaucoma, autoimmune • Commensal FVB/N? Blind, seizures, mammary/pituitary dz • Opportunist NOD Diabetes, immunoweird SJL/J Lymphoma, muscular dystrophy, kill each other • Subclinical DEAF C57BL/6, BALB, DBA, etc • Pathogen BLIND rd1 C3H, CBA, SJL, SWR, FVB + Different susceptibilities to infection and 24 26 disease !

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Examples of Some Strain Associated infectious disease phenotypes Immune Variations Agent DZ Susceptible Intermediate Resistant (More) Innate (More) Adaptive/Acquired MHV - Hc c5 hemolytic complement & TH1, TH2 biases B6, BALB/c SJL/J demyelination Sclc11a1 solute carrier family 11a MHC Parvo MPV1 member 1 (formerly Nramp1) C3H/HeNDISCLAIMERBALB/c, ICR, DBA B6 seroconversion Natural Killer Cell function - TMEV This is an over simplification of strain responses to Variation in NK complex Klra (Ly49) Tlr4 Toll like receptor 4 SJL/J, SWR, DBA/2 CBA, C3H A, B6, B10, DBA/1 demyelination these agents Klrb (Nkrp) etc Sendai NAIP neuronal Apoptosis inhibitor Il2m1 Il 2; mutation 1 = Hypo-active Emphasizes someDBA, 129 responses A, withBALB, SWRrelatively recentB6, SJL Pneumonia proteins & Nlrp- NOD like receptor variant of IL-2 with decreased T cell data protein polymorphisms activation DBA, BALB/c, C3H, Ectromelia B6, AKR immune def Sirpα in NOD phagocytosis Tcrbv8 T cell receptor beta variable 8 BALB/c, C3H, A/J Mycoplasma B6, B10 Slamf signaling lymphocyte activation DBA/2, AKR Cathepsin E function molecule family (CD48) polymorphisms A/J 3H/HeJ & N H hepaticus Nu scid B6, FVB/N Mx1, Mx2 variation 28 IL10- Rag2-

Some immune relevant B6 BALB/c DBA background strain genotypes Sclc11 MHC c5 Ahr ApoA2 Il3ra Rmcf OTHER (Nramp) oversimplified 129 H2 b ~~~~~~ Beware of substrain variations ~~~ Slc11a1R A H2 a Hc0 Ahrb-2 Apoa2c Il3ram1 Rmcfs Tnfrsf13c Bcmd1-A B6 BALB/c DBA A/J DBA/1 H2 q AKR H2 k Hc0 Ahrb-2 Apoa2a Il3ram1 Rmcfs H2 b H2 d DBA/2 H2 d BALB/c H2 d Ahrb-2 Apoa2b Slc11a1s BALB/cBy TH1 bias  IL12 TH2 bias  IL4 TH2 bias  IL4 C3H More cell mediated More humoral response H2b-2 Ahrb-2 Apoa2b Slc11a1R Tlr4Lps-d C3H/HeJ InT-Hi NK more Ly49 Int-less NK NK lack THy1.1 C57BL/6J Apoa2a H2 b Ahrb-1 Slc11a1s Specif Klr NK complex Klrd1DBA/2J C57BL/6N (Dock2Hsd) DBA/1 H2 q Slc11a1s More macrophage Less macrophage Ahrd Rmcfr DBA/2 H2 d Hc0 Slc11a1R activity , TNFa, IL12, activity, TNFa, IL12, FVB/N H2 q Hc0 Ahrb-2 Apoa2b bacterial killing ; more bacterial killing ; more local/less systemic systemic & acute MRLlpr H2 k Il2m1 Foxq1sa-J Faslpr response phase response, NOD H2 g7 Hc0 Il2m1 Sirpa B6J / B6N variation in Il2m1 SJL/J H2s Ahrd Apoa2c Rmcfs Ceacam1Hv2-r Th17response

FELASA 2014 recommendations Viral Agents Mice ‐ Viruses Top Few

 3 month testing 1998 2003 2009 2001 2009  Annual testing – add: Jacoby & Livingston Pritchett 1. MHV FELASA Mahler 1. LCMV Lindsay * & al Corning 1 MHV > 70% MHV MHV MNV MNV 2. Mouse rotavirus (EDIM) 2. MAD1 (FL) 2 Parvos ~ 40% MRV (EDIM) Parvoviruses MHV MHV 3. Murine norovirus 3. MAD2 (K87) 3 TMEV ~ 35% Parvoviruses MRV (EDIM) Parvoviruses Parvoviruses 4. Parvoviruses (NS1) 4. Mousepox 4 MRV(EDIM) ~30% PVM SEN MRV (EDIM) MRV (EDIM) 5. MVM (Ectromelia) virus 5 Sen ~20% SEN TMEV TMEV TMEV 5. PVM 6 PVM ~20% TMEV 6. MPV 6. Reo3 *% of positive non–spf facilities by survey 7. TMEV 7. Sendai  Norovirus (MNV) is fairly new to the list  Sendai & PVM are less common – Isolation by ventilated caging ? http://www.uni-heidelberg.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pdf 37

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Mouse Viruses Mouse viruses

 Similar findings & recommendations in different countries. Most common/likely (quarterly testing)  Most ‘Prevalences’ seem pretty low (< 5%) ‐ or are they? 1. MNV = Mouse Norovirus (Calicivirus)  Why test for an agent that’s not excluded? . NO disease expected in seropositive mice … 1. Less testing of positive areas  fewer + 2. More testing to confirm negative status more – 2. MHV = Mouse hepatitis virus (Coronavirus) . 3. Outbreak testing  more –(or more + ?) Disease unlikely in seropositive mice today .  Usually detected by serology now, LIVIM = lethal intestinal virus of infant mice – NOT by obvious clinical disease… 3. Parvoviruses ‐ NO disease expected  Surveillance (Sentinel & Quarantine testing) represent a lot of 4. MRV = Mouse Rotavirus (Type A rotavirus) time & $$£€ to test for agents that rarely kill, or even cause . EDIM = epizootic diarrhea of infant mice disease 5. TMEV (Gd7)= Theiler’s mouse encephalitis virus  All infections immunomodulate, potentially impact diverse . NO disease expected –famous for ‘mouse polio ‘ research …. (paralysis in susceptible strains) 38 39

More viruses in mice MNV ‐ Murine Norovirus(es)

 Historical concerns that cause significant disease  Calicivirus (MNV1‐4…..) Henderson & al 2008 – Sendai – Single Stranded RNA virus – Ectromelia virus – Human Noroviruses = primary cause of non bacterial human gastroenteritis  Part of the genome (& Genetic engineers)  Seroprevalence: 30%‐(60%?!) in research colonies – Retroviruses – 0‐ low in vendor production colonies  Transmission: Fecal / oral  In freezers, pet stores, or wild mice near you – Like other Norwalk viruses – Arterivirus  – LCMV Competent mice – Seroconvert, no obvious disease  Common – ‘emerging’  Immune deficient mice – USUALLY NO disease – Astroviruses small round enteric viruse… – Pneumonia, hepatitis, encephalitis in severely innate immune deficient mice 40 41

MNV ‐ Murine Norovirus(es) MNV research effects Phenotypes

H&E IHC  Tropism for macrophages and dendritic cells  Usually no obvious phenotype / disease  Increased atherosclerotic lesion size ¯ophages in Ldlr(‐/‐) mice. – Paik & 2011, 2015  Subtle changes in lymphoid tissue in B6 model of diet‐induced obesity (DIO) and insulin resistance. – Did not alter weight gain, food intake, and glucose metabolism Paik & al 2010  Accelerated bacteria‐induced inflammatory bowel disease (IBD) progression in Mdr1a(‐/‐) mice.  – Did not alter survival, IBD scores, tumor incidence, or tumor H&E Typical mouse liver phenotype in Smad3(‐/‐) mice ‐ Lencioni & al 2011  IHC  MNV – Did not alter Helicobacter‐induced inflammatory bowel disease in Il10(‐/‐) mice ‐ Hsu & al 2014  Tac:SW sentinel mice = Competent Swiss mice – Perdue, & al. 2007. 42

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Stat1-/- Stat1-/- MNV- MNV+ Diagnosis/es? MHV • Liver necrosis, Mouse Hepatitis Virus(es) • Inflammation suppurative  Coronaviruses – mutable  many ‘strains’ Cause/s? • Immunodeficiencies  ‘seroprevalence’ ~ 5% • MNV 1 – Was > 50 % / US, Can, EU in 1980’s – 1990’s Rag1-/-/IfnγR-/- Rag1-/-/IfnγR-/- – Transmission: fecal oral ‐ Highly infectious (~ TGE, FIP) MNV-1 ProPol IHC MNV+  Competent mice – Usually seroconvert with no obvious disease – Used to have devastating epizootics LIVIM  Immune deficient mice F4/8 0 – Death, wasting with liver and enteric disease

Copyright © by American College of Veterinary Pathologists Hsu et al. Vet Pathol 2016;53:754-763 48

MHV MHV in Immune deficient

scid nu  Some  D Percy ‘wasting phenotype’ of Prkdc or Foxn1 etc competent immunodeficient … strains Liver necrosis  Subclinical ?   LET’s

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MHV MHV Liver Phenotypes Gut Phenotypes Necrosis LE’s (e.g. AST, ALT, LDH…..) Syncytia = histologic ‘hallmark’ Ascending colon target Distal SI too Syncytia, Necrosis

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MHV MHV enterotropic CNS Phenotypes (more common recently/currently) Inflammation  demyelination  many strains e.g. MHV Y Model for MS  In immune sufficient mice etc – restricted to intestine demyelinating Dz – Subclinical, self limiting, in juvenile‐adult – LIVIM enteritis mortality in neonates  In B cell‐deficient mice – restricted to intestine – chronic subclinical  In T cell‐deficient mice – Multisystem lethal infection e.g. progressive necrotizing hepatitis with syncytia….. 53 Coley et al. 2005. J Virol. 79(5):3097-106

MHV Polytropic/Respiratory Ceacam1 (Bgps) (more studied)

 Many strains e.g. MHV A59, JHM etc  CEACAM1 isoforms serve as receptors for several  Respiratory infection  then shows a primary viral and bacterial pathogens, e.g. tropism for liver, spleen, and brain – MHV in mice – Haemophilus influenzae, Neisseria gonorrhoeae, and  Syncytia in almost any tissue Neisseria meningitidis in humans.  May see eosinophilic cytoplasmic inclusions,  Most of the common inbred strains carry  Neurotropic strains  encephalitis, then Ceacam1a, & express an efficient MHV receptor that confers susceptibility – Death or  SJL/J mice carry the Ceacam1b allele, (encodes – Immune mediated demyelination (model of MS) defective receptor protein )  Ceacam 1 is an important receptor that confers resistance.

Ceacam1 & MHV MHV Research Relevance

 Susceptible Disease Models Ceacam1a binds ► Necrotizing MeningoEncephalitis + Demyelination MHV spike S ► Model for multiple sclerosis & immune mediate demyelinating protein disease Research interference ? ► Liver enzymes, liver, gut, CNS phenotypes ► Immune phenotypes ► Wasting in immunodeficient in chronic studies, or breeding ► Cell culture contaminant including ES cells

Bergmann et al. Nature Reviews Microbiology 4, 121–132 (February 2006) | doi:10.1038/nrmicro1343 59 60

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Mouse Parvoviruses Mouse Parvoviruses (MMV (MVM), MPV 1‐4) Disease/Phenotypes

Parvoviridae Tough & tiny (SS DNA) MMV (MVM) (c, i, m, p strains) MPV (MPV1-4) • Require S phase for infection / cytolysis In immune sufficient mice Subclinical, Immunomodulation . Lymphoid targets  immunomodulation Subclinical, Immunomodulation May not persist (Sero+/PCR-) . Oncolytic / oncosuppressive in vitro / in vivo (T tropic) MVMm  most prevalent, persistent Persistent/latent in mesenteric   ‘seroprevalence’ <5% But sneaky MVMi disrupt hematopoiesis in nodes C3H • Variable seroconversion so tough to detect In Immune deficient mice (scid, nude) • VERY persistent in environment MVMi exp  Lethal leucopenia in • Common biological contaminants scid In competent & immune deficient mice – usually no disease….

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Parvoviruses in developing Mouse parvovirus antigens (proliferating) tissues (important for detection)

In utero infections affect developing tissues NS1 – Non structural protein 1 E.g. cerebellar – Common to all the parvoviruses hypoplasia – Target for antibodies and serotesting – Rat  – Some mouse strains seroconvert late or poorly… – Hamster – More sensitive, less specific – Cat VP1 – Cow – Virus specific structural proteins – Mouse (experimental) – Less sensitive, more specific D Percy Image 63 64

Mouse Parvoviruses MRV (EDIM) Research Relevance  Experimental / model phenotypes Murine Rotavirus (Reoviridae) • EDIM = Epizootic Diarrhea of Infant Mice Neonate  multisystem infection  cerebellar hypoplasia, renal infarct anemia ‘seroprevalence’ <1% (from ~30%!) Oncotropic, oncolytic agents Transmission: fecal oral Gene therapy vectors • direct contact, fomites – pretty fragile Research interference ? • dirty bedding sentinels should detect well  Targets cells in S phase (developing embryo)  Competent mice Oncolytic in cancer models  Affect tumor growth ? – Seroconvert +/‐ neonatal yellow diarrhea Some are lymphotropic/lympholytic….  Immune deficient mice Competent sentinels may not seroconvert fast or reliably – Neonatal diarrhea Cell culture contaminant 65 66

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MRV (?)

 Transient Apical epithelial vacuolation Mouse diarrhea photo scoring system.  Looks like normal a. negative / normal stool ‘nursing’ pup gut b. Category 2 stool c. liquid yellow stool from with absorption of mouse inoculated with RRV hi fat milk…. d. similar stool expressed from mouse inoculated with PI‐RRV (d).

SHAW R & al. Rotavirus Diarrhea Is Caused by Nonreplicating Viral Particles. • AFIP WSC 14 J Virol, 1995, p. 5946–5950 Vol. 69, No. 10 1/14/98 67 68

MRV (EDIM) TMEV (GD7) Research Relevance

Theiler’s mouse encephalitis virus – PicoRNAvirus (related to poliovirus) Flaccid paralysis in susceptible Inflammatory Demyelination used to model

Experimental /model phenotypes multiple sclerosis  Neonatal diarrhea – model for rotavirus infection in human etc spp WAS a common infection Research Relevance In biomaterials near you  Neonatal diarrhea, in immune sufficient and deficient mice  Breeding colonies, runting – Baida & al 2008 (Tg) 69 Immunomodulation 70

TMEV demyelination TMEV susceptibility & MHC TMEV demyelination –MS model since 1970’s – Spectrum of strain susceptibility – Resistance – Why are B6, B10 so resistant ?

– 1985 Rodrigues & David – breeding study • on C57BL/10 background . Susceptible haplotypes: s, f, p, r, v, or q . Resistant haplotypes: b, k, or d

B6 SJL nice example of (experimental) strain – Confirmed with transgenics in after 2000 . Susceptible q haplotype (FVB/N) dependent susceptibility . Intermediate k, d, s haplotypes 71 – Lipton & Dal Canto (1979). 72 . Resistant b haplotype (B6, B10)

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TMEV (GD7) FELASA 2014 recommendations Research Relevance Mice ‐ Viruses

Experimental/model phenotypes  3 month testing  Annual testing – add:  Acute encephalitis with neuronolysis (virulent strains e.g. GD7, FA) - 1. MHV 1. LCMV  “Early Dz’ (less virulent strains e.g. TO DA) Encephalomyelitis, 2. Mouse rotavirus (EDIM) 2. MAD1 (FL) neuronolysis, meningitis, perivasculitis, poliomyelitis with 3. MAD2 (K87) neuronolysis, neuronophagia  paralysis 3. Murine norovirus 4. Mousepox  ‘Late Dz’ (less virulent strains e.g. TO DA) Demyelination (immune 4. Parvoviruses (NS1) (Ectromelia) virus mediated) – spastic 5. Reo3 5. MVM Research Interference ? 6. PVM 7. Sendai  Immune modulation 6. MPV  CNS phenotypes 7. TMEV  Cell culture contaminant

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(LCMV) Immunocompetent, LCMV-infected mice exhibit signs of uncal herniation.

Arenavirus; envelopedhttps://www.askjpc.org/vspo/ RNA  Neonate infection  persistent, sublinical (’tolerant’) – Virus continually shed in urine, saliva, milk – immune complex GN  OR later infection (’non tolerant’) – Viremia death – Lymphocytic Choriomeningitis  Sources: Biologicals, wild rodents  Diagnosis/es?  Human risk: – Pet hamsters & feeder mice – ALimb extension, decerebrate posture – maternal infection assoc w hydrocephalus, – B D unilateral pupillary dilation (mydriasis) chorioretinitis, mental retardation – C E Uncal hermiation ? compression of brain matter  Marmoset hepatitis (from pinkies) against skull ?? Christine M. Matullo et al. J. Virol. 2010;84:312-320 Diseases of Research Animals –DORA http://dora.missouri.edu/ University of Missouri ‐ Comparative Medicine Program and IDEXX‐BioResearch

Mouse adenoviruses (MAdV‐1, MAdV‐2) Ectromelia Virus Mastadenovirus nonenveloped DNA Orthopoxvirus, causes mousepox – Species specific orthopoxvirus

MAdV‐1  Subclinical INIB Kidney MAdV‐2 tubules, endothelium  Subclinical INIB WAS a cause of devastating ‘outbreaks’  Neonatal infection  Intestine – Variation in mouse strain susceptibility necrosis + INIB liver, spleen, enterocytes In biological materials near you !! kidney, brain and adrenal  Shed in feces Diseases of Research Animals –DORA http://dora.missouri.edu/ – ‘outbreaks’ from commercial mouse serum…. University of Missouri ‐ Comparative Medicine Program and IDEXX‐BioResearch 78

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C57BL/6J  Diagnosis/es – Liver necrotizing multifocal – Splenomegaly  Cause/s? – Administered biologicals ? – Immunodeficiency X A. Ectromelia virus B. Mouse Hepatitis virus C. Murine Cytomegalovirus D. Murine Norovirus E. Clostridium piliforme F. Helicobacter hepaticus Ectromelia D Percy 79 Labelle etG. al. 2009.Salmonella Comp Med. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703149/e. typhimurium

C57BL/6J Ectromelia Virus  Diagnosis/es Research Relevance – Liver necrosis multifocal – Splenomegaly Experimental model phenotypes • necrosis multifocal  Model of orthopoxvirus infection  Cause/s?  Gene therapy vector  seroconversion – Administered biologicals A. Ectromelia virus Research interference ? B. Mouse Hepatitis virus  Biological materials C. Murine Cytomegalovirus  Sentinels do not reliably seroconvert D. Murine Norovirus  Seroconversion from gene therapy vectors can be confusing E. Clostridium piliforme F. Helicobacter hepaticus G. Salmonella e. typhimurium Labelle et al. 2009. Comp Med. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703149/ 82

Reovirus 3 (Reo3) Sendai Virus Research Relevance ?? Paramyxovirus ‐ respirovirus genus • ~ human parainfluenza 1 ‘seroprevalence’ <<1% (from much higher) Reoviridae Transmission: aerosol fecal oral Research Interference??? • Hily Infectious (mouse to mouse) ‐‐ But  Rare, unlikely unless exposed to pet or wildmice • Not so reliably detected by dirty bedding sentinels  Immunomodulation  Competent mice (significant strain variation)  Breeding colonies, historical runting, oily hair effect – Sublinical seroconversion  pneumonia/death  Immune deficient mice – Wasting with respiratory disease 83 84

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Pneumonia Virus of Mice (PVM) Sendai Research relevance ?? Paramyxovirus

Paramyxovirus Parainfluenza 1

Research interference ?? WAS a common cause of respiratory disease  Rare – eliminated? by microisolator caging in Susceptible Competent mice Pet store or wild mice? – E.g. DBA/2  Immunomodulation in immune sufficient mice  Immunodeficient mice are persistently infected, develop – Acute disease progressive bronchointerstitial pneumonia. Desquamation and hyperplasia of bronchiolar epithelium  Possible Cell culture contaminant – RADIL images

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Sendai Virus Sendai Virus

  Competent In nude, scid etc Susceptible mice – No T cell‐mediated  Immune mediated necrosis necrosis (cd8+T) – Not much  Acute Inflammation • Some neutrophils … – Necrotizing – Inflammmation – Proliferation – Syncytia – Syncytia – Inclusions – Intranuclear Inclusions

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Sendai Virus Sendai Virus Research relevance Competent mice Experimental/model phenotypes  Similar to clinical disease Chronic / repair phase  Model for human parainfluenza 1 – Proliferative Research interference – Syncytia  Not so likely today – contained by microisolator caging – Inclusions  Morbidity mortality in susceptible strains  Respiratory phenotypes, and Immunomodulation  Wasting dyspnea in Immune deficient in chronic studies, – D Percy breeding  Cell culture contaminant

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NOT so likely but Don’t WYD? forget/ignore them completely:  DNA viruses  Tissue? – Adenoviruses MAV1, MAV2 (~8% J&L 1998)  Lesion? – Herpesviruses (~5% J&L 1998)  Cause? • MCMV, MTV Thymic virus, gammaherpesviruses  INCLUSIONS – Papovaviridae PyV (MPV), K (polyomaviruses) – Poxviridae ECTV (Orthopoxvirus) Probably in a freezer, or in feral mice near you….  RNA viruses – Arteriviridae LDV ‐ chemistry test –not ‘serology’ – Arenaviridae LCMV (bunyavirus?) (~5% J&L 1998) – Astrovirus Common , small round enteric viruses, no disease? – Hantaviruses Wild mouse reservoirs  MuCMV in scid mouse – Paramyxoviridae Sendai, PVM, K – Respiratory …  In a freezer near you…. – Picornaviridae TMEV, EMCV , Ljungan? 94 – Reoviridae Reo3 (~5% J&L 1998)

Oncolytic virus THERAPY Mouse Polyomavirus (MPyV)  Amgen’s T‐VEC (now called Imlygic)  Immunodeficient & neonate  1st oncolytic virus to receive market approval. infections:  genetically engineered herpesvirus called talimogene  Cytolytic/cytopathic + laherparepvec (?) intranuclear inclusion bodies – KIDNEY tubule   treatment of melanoma in skin and lymph nodes (Salivary gland)  Dozens of oncolytic viruses in trial…  Cytopathic /proliferative:  many viruses will preferentially infect cancer cells  – Lung ureter etc immune reaction, oncolyssis  TUMORS  Physicians Noted cancer patients who went into – Mammary/salivary gland spontaneous remission after contracting a viral (myoepitheliomas) with infection more than a century ago… inflammation 96 – Sarcoma, osteosarcoma

Lactate dehydrogenase elevating virus (LDV) Astrovirus Arterivirus, enveloped SS RNA MuAstV

Subclinical persistent infections in monocytes  Recently identified macrophages  Widespread Elevated LDH (>2500) –also ALT AST SDH Etc  Shed in feces – (infected monocytes fail to clear these enzymes)  NSF • Significance: immunomodulation – No significant findings • Diagnosis: PCR is most useful (IFA, MFIA also) – ELISA is not useful Sources: Biologicals (mouse serum, cell lines) 102 Compton et al 2017 – Matrigel c 2010 Murine Astrovirus Infection and Transmission in Neonatal CD1 Mice

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Mouse Retroviruses EMCV MMTV’s EMV’s ERV’s IAP’s  sialodacryoadenitis in  Exogenous viruses  salivary, milk, semen mice. – eliminated from most commercially avail strains  Targets acinar cells of – MMTV Bittner agent historically important parotid and lachrymal  Endogenous viruses (ERV’s) glands, – ‘provirus’, ‘retroelements’ , loci, genes in mouse genome  submandibular, – Intracisternal A particles IAPs; transposable elements TEs  – named genes (Mtv1….; Mlv1…; Emv1… (Akv1..) sublingual and – d, rd1, hr etc Harderian glands are – 100% prevalence, strain variations –C3H very active free from lesions. – Many are not oncogenic  TEM Aggregation of EMCV particles. Bar,

0.2 μm 104

Mouse retrovirus – Mouse Retroviruses related phenotypes genetic engineers  Some Lymphomas e.g. Endogenous retroviral elements – AKR (C58 etc) thymic lymphoma – Moloney sarcoma, Friend Leukemia, Abelson virus – Aka ERVs Retroelements = Important parts of  Color ‐ Myo5ad  Dilute in DBA, etc mouse (genome  Hairless phenotype ‐ Hrhr – ~10% of reported spontaneous germ line  Vision ‐ Pde6brd1  rd blindness in C3H, SJL, FVB etc. mutations in laboratory strains. – Viral insertion (Xmv‐28) in intron 1 – extreme levels of polymorphism suggest ERV – + nonsense mutation (C  A) that truncates the protein insertions are important in genetic drift of mouse  i.e. Important part of mouse genomes lines. functional genes/alleles, markers – Emv’s, Mlv’s, Mtv’s, Xmv’s = loci Retroelements (transposons, IAP etc) ~ 30% of mouse – Intracisternal A particles IAPs genomes – transposable elements TEs 105

Genetic engineering by retroviruses: Retroviruses & Lymphomas

Hrhr ‐ hairless Hrrh - rhino  Retroviral integration  allele defined by NON – one or more MLV proviruses closely complementation with hr linked to hr allele.  similar to Hr/Hr except no hair  Normal coat up to ~ 10 do, then lose all regeneration & skin becomes hair, then waves of sparse fuzzy hair progressively thickened and growth… wrinkled More extensive  Cysts from hair canals, sheaths or hyperkeratosis in follicles  sebaceous glands  sebaceous large hair canal cysts with transformation, later keratinization plugs/balls of keratin  Abnormal mammae, nails …  Autoimmune ?  Leukemia  UV Irradiation Resistance?

107 108

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Viral phenotypes Viral phenotypes oversimplified oversimplified Phenotype Viruses Also consider Helicobacters Seroconversion is probably the most common MNV C piliforme Enteric / MHV Salmonella finding in contemporary research colonies…. MRV Enterohepatic Giardia? Spironucleus? (mCMV) Pinworms  rectal prolapse Disease may not be obvious but Pneumocystis murina Respiratory Sendai, PVM Pasteurella pneumotropica immunomodulation and other effects can primarily MPV (pneumotropic virus) Klebsiella oxytoca etc Bordetella avium, hinzii impact diverse research… Ectromelia MHV Death & necrosis CMV C piliforme; Salmonella Subclinical Parvoviruses Many possibilities immunomodulation etc Adenoviruses, Subclinical + Herpesviruses, inclusion bodies Polyomaviruses 110 111 Most common agents in orange

FELASA 2014 recommendations Mice Likely bacterial phenotypes ‐ Bacteria (usually worse in immunodeficient) Phenotype Likely Bacteria Also consider  3 month testing Helicobacters 1. Helicobacter spp.  Annual testing – Add: Enteric Clostridium piliforme MNV? MHV, MRV, mCMV  Citrobacter rodentium Giardia? Spironucleus? + H. hepaticus, H. bilis, 1. Citrobacter rodentium /enterohepatic Salmonella Pinworms  rectal prolapse H. typhlonius 2. Clostridium piliforme Enterococcus? Brachyspira? 2. P pneumotropica Pasteurella pneumotropica Pneumocystis murina 3. Corynebacterium kutscheri Respiratory Klebsiella oxytoca etc M pulmonis, Filobacterium 3. Strep b‐haemolytic 4. Mycoplasma pulmonis primarily Bordetella avium, hinzii rodentium (CARbacillus) pseudohinzii? Sendai, MPV (not group D) 5. Salmonella spp Staphylococcus spp Streptococcus Abscesses primarily 4. Strep pneumoniae 6. Streptobacillus P pneumotropica? Gram negatives Corynebacterium bovis Check for mites! moniliformis Other Skin disease MUD + opportunists, Staph etc Ringworm ? Death & Sepsis Klebsiella oxytoca Strep/enterococcus spp Not Staphylococci Discussed with RATS (usually Proteus mirabilis Pseudomonas immunodeficient) Endo/enterotoxemia http://www.uni-heidelberg.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pdf

Helicobacter, Pasteurella, Rectal prolapse Staph aureus MOST likely cause today? What can they do. Helicobacters Mouse strain susceptibilities. Research impact.  Pinworms?  Citrobacter rodentium ?  Tumors?  Other phenotype ?

115 116

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Rectal prolapse

Perineal skin

Perianal glands Colitis – Proctitis Inflammatory Bowel Disease phenotypes

117 118

Silver stains lots Helicobacters in mice of bacteria Liver  Chronic (lymphocytic) and active (neutrophilic) inflammation  Cholangiolar proliferation  Anisocytosis, anisokaryosis, aneuploidy, hepatocytomegaly,  intranuclear ‘inclusions’ of invaginated cytoplasmic material can be common in older mice.  relation ship with helicobacter or other infections is not clear

119 120

H hepaticus hepatitis Helicobacters in mice Strain & Sex influence Liver AxB RI mice  Modified Steiner’s Silver stain 14 mpi  LOW sensitivity B = Resistant compared to PCR – WNL  Careful examination is time consuming A = Susceptible  Silver stains are expensive – Inflammation  Rodent helicobacters are – Biliary hyperplasia small Males more susceptible  Liver Histology is terminal – to liver dz for the mouse 121 122 Ihrig & al. 1999

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H hepaticus & tumors Helicobacters in humans… ?  Stomach Hepatocellular – H Pylori adenoma, carcinoma  Hepatobiliary– Zhou et al 2013 Meta analysis – H pylori, H bilis, H hepaticus, H ganmani studied. – discovered in A/J dt – Higher pooled infection rate of Helicobacter spp. in the liver tumors biliary cancer group compared with the normal group (P=0.0001) and the benign biliary disease group, Lymphoma respectively (P=0.0001). – Studies from East Asia and South Asia show higher Hemangiosarcoma prevalence of Helicobacter spp. in the malignant group. – Evidence supporting higher presence of Helicobacter spp. in the cancer group was obtained using PCR and IHC from bile and biliary tissues.

123 Ihrig & al. 1999

Helicobacter ganmani ? Helicobacter pullorum ?

 Enterohepatic Helicobacter species(EHS) isolated from  urease‐negative anaerobe in mouse intestine – Humans (healthy or with enteritis) – Common in rats? Humans? – Commercial broiler and layer chickens with hepatitis and enteritis attributed to H. pullorum.  Pathogenicity of Helicobacter ganmani in Mice • Cecal lesions in experimentally infected chickens Susceptible and Resistant to Infection with H. hepaticus. Alvarado & Al 2015 CM  C57BL/6NTac and C3H/HeNTac mice spontaneously infected – in this study failed to induce typhlitis that is the with H. pullorum hallmark of H. hepaticus infection, –  seropositive by ELISA, – A/J, C57BL/6, IL10‐deficient mice – H. pullorum‐specific IgG levels decreased as colonization was lost over time in selected mice.  Previous reports of Typhlocolitis in naturally – C57BL/6 mice did not develop gross or histological lesions of the infected IL10 deficient mice liver or . – New mouse model for an emerging zoonosis? – Turk, M. L., et al. (2012).

Helicobacters implicated in mouse Helicobacter mastomyrinus disease

1. H hepaticus  etc "Ulcerative typhlocolitis associated with 2. H bilis Helicobacter mastomyrinus in telomerase‐ 3. H typhlonius deficient mice.“ 4. H trogontum  Eaton et al. (2011). VP 5. H mastomyrinus 6. H ganmani 7. H muridarum 8. H rodentium

127

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Helicobacter spp. & Helicobacters Rectal Prolapses Research Relevance  Miller ...Fox 2014. CM.  Enterohepatic Helicobacter species (EHS) often are associated Experimental model phenotypes with typhlocolitis and rectal prolapse in mice.  Model helicobacter infection  Mild to severe typhlocolitis, often with hyperplastic and  IBD inflammatory bowel disease dysplastic foci.  Helicobacter influence on enterohepatic tumors – Neoplastic foci tended to occur at the ileocecal‐colic junction.  Strain and sex influences on disease – most severe in strains that had lower‐bowel inflammatory disease, notably IL10, Rag1, and Rag2 knockout strains;  Prolapses also occurred IL10, Rag1, and Rag2 knockouts in Research interference ? Helicobacter‐free barrier facility.  Immunomodulation  Most mice with prolapses were immunocompromised GEM  Enterohepatic inflammation – Lamellipodin knockout was noteworthy for prolapse, distal lesions, and  Enterohepatic and other tumors lack of known immunodeficiency.  Zoonotic concern? H bilis, ganmani, hepaticus?  Most colons tested PCR‐positive for EHS, often coinfections. 130

P pneumotropica Respiratory infection: Rhinitis

 Gram-negative, short pleomorphic rod Mice are obligate nose breathers  bipolar staining but stains poorly with gram or silver ….  Very common opportunist This could kill them Who dunnit ? Research interference ?  Import/export – P pneumotropica  Immunomodulation – B hinzii, avium ?  Respiratory disease and death in susceptible/compromised – Klebsiella (oxytoca) ? animals • Immunodeficient ?  Asthma studies  Auditory studies – Mycoplasma ?  Sub Fertility in susceptible/compromised animals • Not so common  Abscesses in susceptible/compromised animals – Streptococci ? 131 132

Respiratory infection: Tracheitis Respiratory infection: Otitis

Who dunnit ? Pretty common in our mouse submissions – P pneumotropica Effects on – B hinzii, avium ? – Hearing? – Klebsiella (oxytoca) ? – Behavior? • Immunodeficient ? – Immune – Mycoplasma ? responses? • Not so common – Streptococci ?

133 134

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Otitis Aural Cholesteatoma Not just humans & gerbils … Opportunists ? Or Pathogens ? – P pneumotropica? – K oxytoca ? – B hinzii/avium ? – Mycoplasma ? • Historical ? – Pseudomonas ? • Historical ? • Only? in immunodeficient ? • Neutrophil deficient

135 136

Bronchopneumonia (+ AMP) Infertility Pyometra

 Likely Causes? – P pneumotropica? – M pulmonis? – Imperforate vagina • Vaginal septa

 Opportunists in immuno‐deficient immunoweird

137 139 P pneumotropica pyometra D Percy

Conjunctivitis P pneumotropica Blepharoconjunctivitis biovar Heyl Likely causes? Bom:NMRI‐Foxn1nu • Inbred or outbred?  Strain‐related? e.g. Microphthalmia, entropion, KCS? • Diagnosis/es?  P pneumotropica ? • Orbital/periorbital abscesses  Bordetella spp ? C57BL/6JBom – May isolate a lot of things • Inbred or outbred? • Diagnosis/es? NOT so likely: • Subcutis abscess  Ectromelia virus ? • Heyl (vs JAwetz) is usually – Common finding in recent outbreaks implicated in clinical disease

Adhikary et al. Copyright © 2017 Laboratory Animals Limited 140 http://journals.sagepub.com/doi/figure/10.1177/0023677217698503?

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Fig 4 Pasteurella pneumotropica Streptococci • Outbreak strains = SAME STRAIN  On the list: B hemolytic + S Pneumoniae • Clonal outbreaks imply – Beta‐hemolytic species primary role in infection. • cause complete rupture of red blood cells • E.g. S pyogenes, S pneumoniae, S agalactiae, S equi • Low grade Opportunists in immune sufficient • Undesirable in immune deficient Table 1 Affected mouse strains – Streptococcus pneumoniae • Immune • Secondary pathogen –human risk ? To or from rats? sufficient + • Fibrinosuppurative poly serositis pleuropneumonia • Immune – Enterococci E durans, E faecalis (formerly group D) deficient • Gamma hemolytic (incomplete hemolysis) • Historical ‘outbreaks’ in weanling rats: diarrhea death • Gram positive cocci adhered to villus surfaces Adhikary et al. Copyright © 2017 Laboratory Animals Limited

FELASA 2014 recommendations Abscesses Mice ‐ Bacteria

 3 month testing Often one end or the other  Annual testing – Add: 1. Helicobacter spp. 1. Citrobacter rodentium +  H. hepaticus, H. bilis, H. typhlonius 2. Clostridium piliforme 2. P pneumotropica 3. Corynebacterium kutscheri 4. Mycoplasma pulmonis 3. Strep b‐haemolytic 5. Salmonella spp (not group D) 6. Streptobacillus moniliformis 4. Strep pneumoniae Discussed with RATS Not Staphylococci http://www.uni-heidelberg.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pdf 145

S aureus & Citrobacter rodentium opportunistic bacteria (C freundii 4280) Research Relevance  Transmissible murine colonic hyperplasia in susceptible mice – GROSS: Short thick colon Experimental model phenotypes – HISTO: Crypt elongation  Model burn wound infections & immune responses  MRSA model (B6 resistant, A/J susceptible)  What mouse strain has mutant Dock2? – C57BL/6NHsd (some)  What infection is this Research interference ?  Usually secondary invaders, but can elicit profound immune used to model? responses  Can cause death, or warrant euthanasia for humane reasons  Difficult to identify origin and eliminate from colonies or environment 146 Liu et al 2016 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4904218/

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Citrobacter rodentium C rodentium model for (C freundii 4280) Attaching effacing E coli Diagnosis/es?  Diagnosis/es? – Intestine, colon – GROSS: – Adherent bacilli via pedestal Proliferative colitis like structures; – HISTO: – Loss/effacement of microvilli Hyperplasia, ‘Crypt’  Cause/s Interpretation? – C rodentium induces elongation; not much attaching and effacing (A/E) inflammation lesions similar to Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC)

http://dora.missouri.edu/mouse/ Liu et al 2016 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4904218/

C rodentium model for Salmonella enterica typhimurium Attaching effacing E coli  A, C control   Gram negative invasive toxin B, Dinfected producing enteric  B, D – Historical cause of devastating Attaching/ effacing outbreaks in mouse colonies lesions with effacement – Necrosis, bacteria in liver spleen cecum of the brush border – Human risk (food poisoning) microvilli and actin accumulation  IBD colitis model ? – Role of Nramp1  Gram negative – Permissive mutation in adherent coliforms in C57BL/6 colon – (Attenuated salmonella spp)

Crepin et al 2016. Nature Protocols Månsson et al. ©2012 by American Physiological Society 150 http://www.nature.com.proxy1.library.jhu.edu/nprot/journal/v11/n10/full/nprot.2016.100.html

153 154 Experimental S typhimurium Experimental S typhimurium ‘cleared the infection’……  Hepatitis with necrosis  Typhlitis (necrotizing) (necrotizing) necrosis ulceration  Usually see bacteria  Usually see bacteria

153 154

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Likely bacterial phenotypes Lethal Liver necrosis (usually worse in immunodeficient) in immunodeficient mice… Phenotype Likely Bacteria Also consider Helicobacters Clostridum piliforme – Enteric Clostridium piliforme MNV? MHV, MRV, mCMV Citrobacter rodentium Giardia? Spironucleus? also /enterohepatic Salmonella Pinworms  rectal prolapse Enterococcus? Brachyspira? – MHV Pasteurella pneumotropica Pneumocystis murina – (MCMV) Cytomegalovirus Respiratory Klebsiella oxytoca etc M pulmonis, Filobacterium primarily Bordetella avium, hinzii rodentium (CARbacillus) – ECT Ectromelia virus pseudohinzii? Sendai, MPV • (Spleen necrosis, inclusions, ectromelia) Staphylococcus spp Streptococcus Abscesses primarily P pneumotropica? Gram negatives – Helicobacters? Corynebacterium bovis Check for mites! Other Skin disease MUD + opportunists, Staph etc Ringworm ? – Salmonella enterica typhimurium Death & Sepsis Klebsiella oxytoca Strep/enterococcus spp (usually Proteus mirabilis Pseudomonas immunodeficient) Endo/enterotoxemia 157

Morphologic diagnosis: ______Etiology: ______

 Liver – Necrosis + – INTRACELLULAR filamentous bacteria  Giemsa  Warthin Starry  UCD CCM images 158 159

Brachyspira  Intestinal spirochetes  Mouse models of porcine (& human) infections – Brachyspira hyodysenteriae (Serpulina hyodysenteriae) B pilosicoli  Isolated from wild mice on pig farms  C3H/HeJ + C3H/HeOuJ similar cecal lesions but more severe on defined diet – Hutto & al 1998.  End on attachment to cecal enterocytes  Sacco & al 1997. S Pilosicoli in C3H Sacco & al 1997. S Pilosicoli in C3H  – Weakly hemolytic avian isolate – Cf strongly hemolytic, pathogenic S hyodysenteria in swine

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Likely bacterial phenotypes Upper Respiratory Disease (URI) (usually worse in immunodeficient) Phenotype Likely Bacteria Also consider Otitis Helicobacters Enteric Clostridium piliforme MNV? MHV, MRV, mCMV – Common Citrobacter rodentium Giardia? Spironucleus? /enterohepatic Salmonella Pinworms  rectal prolapse Enterococcus? Brachyspira? Gram+ Pasteurella pneumotropica Pneumocystis murina – Strep/Enterococcus Respiratory Klebsiella oxytoca etc M pulmonis, Filobacterium Gram‐ primarily Bordetella avium, hinzii rodentium (CARbacillus) pseudohinzii? Sendai, MPV – Pasteurella Bordetella Staphylococcus spp Streptococcus Klebsiella (C3H/HeJ etc) Abscesses primarily P pneumotropica? Gram negatives – Mycoplasma Corynebacterium bovis Check for mites! Other Skin disease MUD + opportunists, Staph etc Ringworm ? Death & Sepsis Klebsiella oxytoca Strep/enterococcus spp (usually Proteus mirabilis Pseudomonas immunodeficient) Endo/enterotoxemia 163

Upper Respiratory Disease (URI) Bronchopneumonia (+ AMP) Lobar consolidation with bronchitis, broncho‐ Otitis pneumonia Common Also acidophilic macrophage pneumonia – P pneumotropica? Myd88 – B hinzii, avium? Gram+ – Klebsiella oxytoca ? – Strep/Enterococcus K pneumoniae ?

164 165

Likely bacterial phenotypes Bordetella spp (usually worse in immunodeficient) Phenotype Likely Bacteria Also consider Gram negative Bacilli adhere to cilia Helicobacters Enteric Clostridium piliforme MNV? MHV, MRV, mCMV Hard to identify on histo Citrobacter rodentium Giardia? Spironucleus? /enterohepatic Salmonella Pinworms  rectal prolapse Enterococcus? Brachyspira? Pasteurella pneumotropica Pneumocystis murina SEM of B bronchiseptica Respiratory Klebsiella oxytoca etc M pulmonis, Filobacterium on cultured rabbit primarily Bordetella avium, hinzii rodentium (CARbacillus) pseudohinzii? Sendai, MPV tracheal epithelium Abscesses Staphylococcus spp Streptococcus primarily P pneumotropica? Gram negatives Corynebacterium bovis Check for mites! Other Skin disease Edwards & al 2005 MUD + opportunists, Staph etc Ringworm ? Death & Sepsis Klebsiella oxytoca Strep/enterococcus spp (usually Proteus mirabilis Pseudomonas 166 immunodeficient) Endo/enterotoxemia

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Staphylococcus spp. Facial Abscesses

 S aureus (1 of few coagulase POSITIVE species) Folliculitis  Furunculosis ? – Lymph node abscesses, botryomycosis, ulcerative dermatitis in competent mice – Furunculosis, abscesses in immunodeficient

 S xylosus, sciureus, hominis, hyicus etc ‘commensal’ (coagulase Negative) isolated from abscesses + Botryomycosis lesions in immunodeficient & GEM

Myd88 deficient

168 169

Facial Abscesses in ABSCESSES! immunodeficient mice  Lawson 2010 CM  Staphylococci "Etiopathogenesis of mandibulofacial and – S aureus maxillofacial abscesses in – S hyicus, hominis, mice." sciureus, xylosus, etc  S aureus from all cultures  Embedded hairs in all  Periodontal disease  All the other  Maxillofacial (63) in opportunists various strains  Mandibulofacial (9) in 1 group of 129& 129xB6

P pneumotropica  Fig 4 Pasteurella pneumotropica • Outbreak strains = • Bom:NMRI‐Foxn1nu SAME STRAIN • Inbred or outbred? • • Diagnosis/es? Clonal outbreaks imply • Orbital/periorbital abscesses primary role in infection.

• C57BL/6JBom • Inbred or outbred? • Diagnosis/es? Table 1 Affected mouse strains • Subcutis abscess • Immune sufficient +

• Immune deficient Adhikary et al. Copyright © 2017 Laboratory Animals Limited http://journals.sagepub.com/doi/figure/10.1177/0023677217698503? Adhikary et al. Copyright © 2017 Laboratory Animals Limited

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Scurfy skin + pup mortality in Mouse: Nude, Rag1, NODscid, NSG etc  Scurfy skin Corynebacterium bovis  Etiology?  Morphologic (Similar lesions from S xylosus ?) Gram diagnosis/es?  Histology findings?  Etiology?  Special Stain?

– RADIL

175175 176

 Nude mouse  S xylosus  1 report  Scaley skin (hyperkeratosis in a GEM may be genotype driven…. – Do a Gram stain & Check for C bovis, S xylosus anyway  Macroscopic phenotype of ML.VS in the absence of endogenous loricrin (ML.VS Lor−/−).

Yasushi Suga et al. J Cell Biol 2000;151:401-412

A. Russo et al. Vet Pathol 2013 Copyright © by American College of Veterinary Pathologists 177 © 2000 The Rockefeller University Press

Likely bacterial phenotypes  Mortality NSG Post Xenograft  (usually worse in immunodeficient) Phenotype Likely Bacteria Also consider Helicobacters Liver Enteric Clostridium piliforme MNV? MHV, MRV, mCMV Citrobacter rodentium Giardia? Spironucleus? Gram stain /enterohepatic Salmonella Pinworms  rectal prolapse Enterococcus? Brachyspira? Usually not so Pasteurella pneumotropica Pneumocystis murina Respiratory Klebsiella oxytoca etc M pulmonis, Filobacterium obvious primarily Bordetella avium, hinzii rodentium (CARbacillus) pseudohinzii? Sendai, MPV Bacteremia Staphylococcus spp Streptococcus Abscesses primarily sepsis P pneumotropica? Gram negatives Corynebacterium bovis Check for mites! Other Skin disease MUD + opportunists, Staph etc Ringworm ? Death & Sepsis Klebsiella oxytoca Strep/enterococcus spp (usually Proteus mirabilis Pseudomonas immunodeficient) Endo/enterotoxemia 180

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 Mortality in NSG   Mortality in Myd88 

Brain Kidney Gram stain Intravascular bacteria Overwhelming gram positive bacteremia and bacterial colonization E faecalis isolated from some 181 182

Mortality in LAD Streptococci / Enterococci cd11/18 deficient

Meningoencephalitis Morphology Gram positive cocci – Gram positive cocci Experimental IP streptococcus infection – Pairs (diplococci) – Unexpected mortality – Chains (streptococci)

183 184

Klebsiella oxytoca Klebsiella oxytoca ?

 Gram negative rod  Gram negative rod – capsule – capsule  Opportunist in NSG etc  Opportunist in NSG etc  Esp in UTI urinary tract  Esp in UTI urinary tract infections –Foreman & infections –Foreman & al 2010 al 2010  Rule out E coli

185 186

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K oxytoca otitis in C3H/HeJ Macarthur & al 2007 Pseudomonads

P aeruginosa is usually implicated Source: water Note ‘capsules’ – Eliminated by acidified or hyperchlorinated water – So we don’t see it so much anymore Usually not a problem UNTIL/UNLESS granulocytes are knocked out – Nude or scid mice are fine until they are irradiated

187 188

Proteus

P mirabilis is usually implicated What  Common enteric resident happened to – Immunodeficient mice are fine until surgery or Protozoa??? other significant intervention  peritonitis sepsis Beware of aerobic culture: ‘4+ Proteus’ Six – Proteus overgrows everything, rapidly, and may overgrow the real culprit Kingdoms of living things tree Maulucioni y Doridí 189

Respiratory disease in Eukaryotes: Fungi immunodeficient mice Usually Commensals, Opportunists – Or experimental infections Sudden Death Pneumocystis murina (P carinii) • Pathogen in immunodeficient mice Trichophyton mentagrophytes Diffuse consolidation • skin ‐ ringworm interstitial Aspergillus, Paecilomyces • Opportunists –see hyphae pneumonia Blastomyces, Histoplasma etc • Opportunists, Dimorphic, see budding yeasts Candida spp (C albicans etc) • Opportunists, trimorphic, see yeasts hyphae, pseudohyphae Torulopsis / Kazachstania ? 193 • Yeasts ON gastric mucosa (PAS positive Easter eggs) 194

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Lethal Respiratory disease Pneumocystis murina in In immunodeficient mice immunodeficient mice

1. Pneumocystis murina ‐ then consider  Consolidations  Intra‐alveolar eosinophilic – All of the bacterial opportunists foamy material • P pneumotropica?  0‐Minimal inflammation • B hinzii, avium? • Klebsiella oxytoca ? K pneumoniae ?  Special stain ? • Streptococci  Other diagnostic tests ? – Sendai virus, PVM, K virus ? • Should not see these today…

195 196

Pneumocystis murina in immunodeficient mice

GMS (silver) stains Cysts Fungus ‘trophozoites’ do not stain PCR probably is most practical test IHC is frustrating

197 198

Acidophilic Macrophage Pneumonia Mouse Stomach: WYD? Eosinophilic Crystalline Pneumonia

1990 (Murray & Luz)  Granulocyte / ‘Charcot Leyden’ like material granulocyte breakdown.

1990 (Guo & al.) YM1 Chitinase like Protein Chitinase 3 like 3 protein

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Figure 5. Candida albicans pseudohyphae (A&B, Wright GMS stain). Hyphae of Aspergillus fumigatus (C&D, GMS stain).

Sudbery, P. E. (2011). "Growth of Candida albicans hyphae." Mouse stomach

 A striking feature of its biology is its ability to grow in yeast, pseudohyphal and hyphal forms  The hyphal form has an important role in causing disease by invading epithelial cells and causing tissue damage. 211

Kazachstania species Gastric Yeasts (Candida pintolopesii) Savage & Dubos. 1967. Localization of (Torulopsis pintolopesii) indigenous yeast in the murine stomach. Journal of bacteriology. 94(6): p. 1811‐6.  In mice probably K pintolopesii – Torulopsis pintolopesii ‐ ON surface  Kazachstania (Arxiozyma) telluris species complex  A telluris ‐ first described as a Saccharomyces – Candida bovina, C pintolopesii, C slooffiae are phenotypically similar to A. telluris – ALL phylogenetically in Kazachstania genus –for now…  A good yeast? In some probiotics …

213 215

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Kazachstania spp. e.g. Degu stomach (Torulopsis, Candida)

Surface of gastric mucosa Non pathogen Find in hamsters, rats etc. rodents….

PAS positive Easter eggs

219 220

FELASA ‐ Mice ‐ annual testing from Mouse Eukaryota: Results ‐ 2009 Parasites /’protozoa’ Pritchett‐Corning, Cosentino & Clifford 2009 (Charles River –PRE PRIA) Ectoparasites ‘Protozoa’ 1. Fleas PREVALENCE % 1. Eimeria Site Agent Method N NA Europe Total 2. Lice Polyplax Protozoa 2. Entamoeba 3. Myobia Duodenum Giardia sp. Wet mount 102,093 0.00 0.00 0.00 3. Giardia Duodenum Spironucleus sp. Wet mount102,093 0.08 0.00 0.08 4. Myocoptes Cec/colon Trichomonads Wet mount 94,890 8.88 nr 8.88 4. Spironucleus 5. Radfordia Cec/colon Entamoeba sp. Wet mount 94,890 8.08 nr 8.08 5. Tritrichomonas muris 6. Mesostigmatid mites Cec/colon Hexamastix sp. Wet mount 94,890 4.45 nr 4.45 Cec/colon Chilomastix sp. Wet mount94,890 3.74 nr 3.74 Helminths Cec/colon Monocercomonas sp. Wet mount 94,890 0.04 nr 0.04 Cec/colon Retortamonas sp. Wet mount94,890 0.03 nr 0.03 1. Aspiculuris Cec/colon Octomitus ? 2. Syphacia 3. Hymenolepis 4. Rodentolepis >1% Pathogenic ?

EUKARYOTES NO longer in protozoa … (or protists) Kingdom Protista (formerly Protozoa)  NOT ‘proto animals’ … In 2005 Society of Protozoologists changed their name to the – Microsporidia (now in Kingdom Fungi) International Society of Protistologists. • E cuniculi, enterocytozoon bieneusii  unicellular eukaryotes that do not fit into other categories: protozoa, unicellular algae, slime molds – Ascetosporea (now in Kingdom Rhizaria) – Ciliates: Ciliophora – Myxosporidia (now in Kingdom Cnidaria) – Flagellates: Zoomastigophora • Diplomonads Giardia, Spironucleus • Trichomonads Pneumocystis (now in Fungi) – Amoebas: Sarcodina • Entamoeba muris – Freshwater radiolarans: Heliozoa (have axopods) – Alveolata Apicomplexans (sporozoa) etc

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Medical Parasitology Taxonomy Update: Giardia muris, maybe G duodenalis (G lamblia) Species Major host(s) January 2012 to December 2015 G. agilis Kunstler, 1882 Amphibians G. ardeae Noller, 1920 Birds  ‘Protozoa’ G. microti Benson, 1908 Muskrats and voles G. muris Benson, 1908 Rodents – Giardia duodenalis from Giardia intestinalis or Giardia lamblia G. psittaci Erlandsen & Bemrick, 1987 Birds – G duodenalis ONLY species found in humans and many other mammals, G. . varani Lavier, 1923a Lizards including pets and livestock G. duodenalis Davaine, 1875 Mammals • (not the only species in rodents) Assemblage A Humans, nonhuman primates, domestic and wild ruminants, (=G. duodenalis sensu stricto?b) alpacas, pigs, horses, domestic and wild canines, cats, ferrets, rodents, marsupials, other mammals  and Ascaris suum may be identical  Parastrongylus (Angiostrongylus, (Rattostrongylus)) cantonensis Assemblage B (=G. enterica?b) Humans, nonhuman primates, cattle, dogs, horses, rabbits, beavers, muskrats and Parastrongylus mackerrasae may be identical Assemblage C (=G. canis?b) Domestic and wild canines  eosinophilic meningitis in humans etc accidental hosts Assemblage D (=G. canis?b) Domestic and wild canines Assemblage E (=G. bovis?b) Domestic ruminants, pigs  PJ Simner 2017. Assemblage F (=G. cati?b) Cats Assemblage G (=G. simondi?b) Mice, rats https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5228259/ Assemblage H Seals

Feng & Xiao 2011 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3021202/

Small intestine nu/nu

Historically considered to be pathogenic… May be 0-minimal inflammation…

 Giardia muris  Spironucleus muris – (G lamblia) – (Hexamita muris) – Flying saucers – Torpedoes • ‘On’ mucosa • In crypts – Enteritis? – Enteritis?

– Hamsters – Hamsters Diagnosis? – Wild mice – Wild mice • Peromyscus • Peromyscus 228 227 Significance ?

Flagellates 65286 6 in large intestine Commensal –always? Cecum Pyriform trophozoites Colon Can fill lumen of cecum, prox colon Some in SI Characteristic movements – Trichomonads ( T muris, T diminuta..) – Hexamastix muris… – Chilomastix bettencourti.. – Cercomonas, Monocercomonoides – Retortomonas, Octomitus etc 230 229

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Cecum, Enteric Flagellates Colon  Eukaryota › Diplomonadida › Hexamitidae › Giardiinae › Octomitus  Eukaryota › Diplomonadida › Hexamitidae › Hexamitinae › Spironucleus  Eukaryota › Diplomonadida › Hexamitidae › Giardiinae › Giardia

 Eukaryota › Parabasalia › Tritrichomonadida › Tritrichomonadidae › Tritrichomonas  Eukaryota › Parabasalia › Honigbergiellida › Hexamastigidae › Hexamastix

 Eukaryota › Rhizaria › Cercozoa › Cercomonadida › Cercomonadidae > Cercomonas

 Eukaryota › Retortamonadidae › Chilomastix  Eukaryota › Retortamonadidae › Retortamonas 232 231 Entamoebae ~>10 u – probably has helicobacters too

Apicomplexa Mouse Stomach: WYD?

Not likely today (but fun for pathologists) – Eimeria spp – not likely – wild mice – Cryptosporidium spp • C muris –stomach • C tyzzeri (C parvum) ‐ Ileum – Klossiella muris –Kidney • Wild/feral mice near us… – Sarcocystis – Toxoplasma gondii • Cats!

233 From an anonymous source via J Ward

Cryptosporidia in mice Stomach (glands) Ileum C muris

Tyzzer. 1910. – An extracellular Coccidium, Cryptosporidium muris (Gen. Et Sp. Nov.), of the gastric glands of the Ren 2012 Common Mouse.

– Merozoites 5‐8u – Macrogametocyte 5x4u – Oocysts 5‐8u

235 236 Tyzzer 1910 Slapeta

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Ileum: C tyzzeri (C parvum)

Fig. 5 Phylogenetic relationship of Cryptosporidium tyzzeri to other Cryptosporidium species and genotypes inferred by a neighbor‐joining analysis of the partial SSU rRNA gene sequence, based on genetic dis...  not all C parvum anymore  C tyzzeri or C parvum in mice Fig. 4 Cryptosporidium tyzzeri in jejunum and ileum of mice under transmission intestine electron microscopy. (A) A trophozoite in the jejunum. (B) A trophozoite in the ileum. (C) A type I meront containing eight merozoites in the jejunum  C muris (gastric glands) is in a  NOTE SIZE = TINY – oocysts ~ 4u different group Ren, X., & al, Cryptosporidium tyzzeri n. sp. (Apicomplexa: Cryptosporidiidae) in domestic mice (Mus musculus). Experimental parasitology, 2012. 130(3): p. 274‐81 http://dx.doi.org/10.1016/j.exppara.2011.07.012 (Tyzzer, E.E., 1912.C ryptosporidium parvum (sp. nov.), a coccidium found in the small intestine of the common Ren, X., & al. 2012. , Cryptosporidium tyzzeri n. sp. (Apicomplexa: Cryptosporidiidae) in mouse. Arch. Protisenkd., 26: p. 394–412.) domestic mice (Mus musculus). Experimental parasitology, 130(3): p. 274‐81 http://dx.doi.org/10.1016/j.exppara.2011.07.012

WYD? WYD?

 Tissue?  Renal coccidiosis  Lesion?  Klossiella muris  Cause? – sporozoites in sporocysts  In wild mice near you ….  Feral mouse Caught on campus

 1st described in 1889

Mouse Eukaryota: Results ‐ 2009 Pritchett‐Corning, Cosentino & Clifford 2009

PREVALENCE %  Diagnosis/es? Agent Method N NA Europe Total – Brain rprotozoal cyst Metazoa Helminths (Oxyurids)*  Cause/s? Aspiculuris tetraptera Direct 135,860 0.19 – T gondii Syphacia muris Direct 128,657 0.01 1.31? 0.25 – Usually expermintal Syphacia obvelata Direct 128,657 0.11 infection Mites Direct 130,976 0.11 0.43 0.12 Lice Direct 126,482 0.00 nr 0.00  S muris (rat ) more common in EU??  How often do you test known positive colonies ?  In house testing is frequent

243 http://noahsarkive.cldavis.org/cgi-bin/show_image_info_detail.cgi?image=F07266

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Metazoan parasites Nematodes: Helminths = worms Pinworms (oxyurids) Nematodes (Pinworms) Adults, larvae in cecum, colon  Syphacia spp Easier to Detect –tape test – Detect by (terminal) direct examination…  Aspiculuris spp Sneakier … Cestodes (tapeworms) ‐‐ Syphacia obvelata (muris)  Requires cat / carnivore exposure – asymmetric |) eggs on perineum – Cysticercus fasciolaris  Taenia taeniaeformis  – Detect by tape test Asymptomatic with liver cysts RADIL images  Wild mouse exposure – Rodentolepis nana (smallest) ‐ Small intestine –dwarf tapeworm Aspiculuris tetraptera  intermediate host (found in humans too) – symmetric () eggs in feces – ‐‐ Small intestine –‘rat tapeworm’ – R microstoma ‐‐ bile pancreatic ducts, duodenum – Detect by fecal flotation 245 246

Mouse Pinworms Mouse Pinworms

 Syphacia obvelata (muris) – asymmetric eggs ‐ tape test – > 400 egg/d !!; 12d cycle ! – Cecum, colon, retroinfection/anus? ? Human pinworm is Enterobius vermicularis – Old report of possible human infection by Syphacia…

 Aspiculuris tetraptera RADIL images – symmetric eggs in feces – <20 egg/d; 24d cycle • Sneakier  low level infestation is hard to detect – Cecum, colon, larvae in crypts

247 248

NOT how you want to diagnose NOT how you want to diagnose pinworms in a clean barrier ….. pinworms in a clean barrier …..

Do you expect this burden in competent mice?

Larvae deep in glands, with eosinophilic inflammation 249 250

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Not a research problem …. ? Cecum

 Infection with S. obvelata induced a transient Th2‐ type immune response with elevated IL‐4, IL‐5, IL‐13 & parasite‐specific IgG1….  BALB/c mice deficient in IL‐13, IL‐4/13, or IL‐4 Pinworms receptor alpha chain showed chronic disease, with >100x parasite burden, increased Ifn gamma Protozoa production, parasite‐specific IgG2b, and default Th2 Flagellates response… – Michels & al 2006

251 252

Pinworms + protozoa Helminth infections Immunodeficient? or dirty ? Research Relevance

Gravid Pinworm Experimental/model phenotypes Gut  Helminth infections Eggs  Asthma, Allergies (beneficial effects ?)

Protozoa Research interference ? Flagellates  Immunomodulation Entamoeba  Minimal interference?  Often found in conventional housing with other agents.

253 254

Hymenolepid tapeworms More Helminths – Rodent definitive hosts (various Mus spp, rats, hamsters, gerbils) – Arthropod Intermediate hosts (or direct cycle); found in human feces….  Hymenolepis diminuta – Adults: 10 ‐60 mm!!! & 3‐4 mm wide – UNARMED ‐ NO hooks on scolex Not likely today (but fun for pathologists) in small intestine Cestodes – Eggs: 54 –85 µm, yellowish and spherical.  Rodentolepis nana dwarf tapeworm – Hymenolepis, Rodentolepis – – Adults: 25‐40 mm long, <1mm wide; armed rostellum • Small intestine, bile ducts – Eggs: 40‐60 µm oval, 2 membranes w 3 pair polar filaments from inner membrane – Excyst in duodenum – in lamina propria –adult attaches in ileum • Where are the wild rodents? – Beetle intermediate host or DIRECT life cycle – Taenia taeniaeformis,  Rodentolepis microstoma tapeworm –mouse bile duct tapeworm – Adults: similar to R nana; armed rostellum Cysticercus (Strobilocercus) fasciolaris ‐Eggs: similar to but larger than Rnana with polar filaments • Liver & Serosal cysts – Excyst in duodenum – cysticercoid in lamina propria – attaches in bile duct (& SI?)) – Beetle intermediate host or DIRECT cycle in nude, NODscid, NOG (Andreassen2007) • Where are the cats ?  DIAGNOSIS: Histo, Eggs on fecal float – speciate by PCR  Rodentolepis vs Vampirolepis 255 – armed hymenolepid controversy of 1954

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H nana, R nana, V nana

a) Eosinophil infiltration ‐ cysticercoid of H. nana in  Two generations of H. nana villus of competent mouse in small intestine of mouse b) no infiltration in nude inoculated with mouse at Day 4 of oral egg (indirect inoculation. cycle). http://www.cdc.gov/parasites/hymenolepis/biology.html Ito – 2015 http://onlinelibrary.wiley.com/doi/10.1111/pim.12167/pdf

Radiographic and Pathological Features of Rodentolepis Malignant .  unstained Rodentolepis nana and Rodentolepis microstoma eggs. a) R. nana laboratory reference isolate (rodent); b) R. microstoma laboratory reference isolate HIV pt (rodent); c) R. nana field isolate (human);  A, B ‐ CT lung nodules d) R. microstoma field isolate (rodent); e) R. nana showing polar filaments;  f) R. microstoma showing polar filaments; C cervical L node g) R. nana egg showing elliptical shape  D cytology  Macnish, M. G., et al. (2003). "Detection of the rodent tapeworm Rodentolepis (=Hymenolepis)  E‐F – histology proliferating microstoma in humans. A new zoonosis?" Int J Parasitol 33(10): 1079‐1085. http://dx.doi.org/10.1016/S0020- atypical cells 7519(03)00137-1  Cunningham, L. J. and P. D. Olson (2010).  H TEM "Description of Hymenolepis microstoma (Nottingham strain): a classical tapeworm model for research in the genomic era." Parasit Vectors 3: 123. http://www.parasitesandvectors.com/content/3/1 /123 http://www.nejm.org/doi/full/10.1056/NEJMoa1505892#t=article Muehlenbachs A et al. N Engl J Med 2015;373:1845-1852.

262 Confirmation of H. nana Infection Mouse: Cause/s? What is/are the morphologic diagnosis/es? Liver, larval cestode cysts A. IHC T Solium GP50 What is the etiology? Taenia taeniaeformis – cross-reactive (Hydatigera taeniaeformis), Cysticercus polyclonal fasciolaris (Strobilocercus fasciolaris) B. ISH Cestode 18S What is the etiologic diagnosis? Hepatic RNA larval cestodiasis, hepatic C. ISH Human Alu D. H nana Co1 sequence

http://www.nejm.org/doi/full/10.1056/NEJMoa1505892#t=article Muehlenbachs A et al. N Engl J Med 2015;373:1845-1852. 262

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MOUSE: Cysticercus fasciolaris Arthropod Parasites (T. taeniaeformis) Where is the cat? Mites Lice  FUR  Polyplax serrata –fur –nits – Myobia musculi – Radfordia affinis Fleas – Myocoptes musculinus  Xenopsylla, Leptopsylla  Follicles – Demodex musculi – Psorergates simplex Psocids, Psocoptera  book lice, dust mites, in  mouse rooms, storage Mesostigmatid areas (Biting/sucking)  NOT parasites on mice – Ornithonyssus bacoti  Cellulose & humidity – Laelaps echidnina  Allergenic? 263 264

NOT how you want to diagnose mites in Fur mites  itchy mice a clean barrier ….. hypersensitivity  dermatitis

Don’t like nude or hairless mice

Myocoptes musculinus (A.), Myobia musculi (B.), Radfordia affinis (C.)

265 266

Mesostigmatid mites Mesostigmatid mites

Disease Diagnosis transmission  NOT on mice – Eat & leave  1mm moving spot on a filter top

0.5 mm Laelaps echidnina Ornithonyssus bacoti  Or rash on • Spiny rat mite • Tropical rat mite investigator 267 268 Whatsthatbug.com

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68716 ‐ WYD 68716 not a problem any more?

Demodex in Mice How to Diagnose Parasites  D musculi head, neck, abdomen, back, limbs, perigenital /anal, tail  S. Marculus Abdomen, back, limbs, peri anal  Enteric – Most similar to D musculi slightly smaller   D fusiformis Abdomen, back, limbs – Direct microscopic PCR  Flagellurus Perigenital – Fecal flotation – Also identify viruses, – Long with with whip like tail bacteria –in patent – Tape test infections – Histo – Immunodeficient  Izdebska, J. N. and L. Rolbiecki (2015). "Two New Species of Demodex (Acari: Demodecidae) with a Redescription of  Fur mites – Improve? Accelerate? Demodex musculi and Data on in Mus musculus (Rodentia: Muridae)." J Med Entomol 52(4): 604‐613. – Direct microscopic Quarantine (Poland) – Fur plucks  Demodecid mites (scaled) from M. musculus. – Pelt digestion – (A and B) D. musculi, ♂, ♀. – (C and D) D. marculus sp. nov., ♂, ♀. – Histo – (E and F) D. fusiformis sp. nov., ♂, ♀. – (G and H) D. flagellurus, ♂, ♀. 272

Lice Psocids Pests NOT Parasites Human Head lice • Book (Bark) lice Psocids (on tape) – Pediculus humanus − 6 legs + antennae capitis − Wings in some adults

273 http://entoplp.okstate.edu/labs/go/psocid/p_key. 274 hl

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Pests NOT Parasites Infectious Agents & Phenotypes Psocids Discussion Plan

Book lice ‘Dust mites’ 1. FELASA recommendations 2. Competent Mice –by agent Moving critters in room – Viral agents –Top few Not on mice – Bacteria –Top few Like Cellulose & Humidity – Eukaryotes –Top few 3. ‘Normal’ flora & the microbiome Allergenic ? 4. Immunodeficient mice ‐ by disease phenotype  hypersensitivity ? – Enteric / enterohepatic – Respiratory – OTHER

275 2775. Biological Materials …

What is/are ‘normal’ flora ? FYI MICROBIOME

What is in your GEM ? What is in your control mice? Does it matter ?

Commensal or Opportunist ? – No problem in competent or control… – Problem ? in immuno weird or other mutant – Some may enhance immunity & survival (SFB)

278 279

ASF 356a ASF 360a ASF 361a ASF 457 ASF 492 ASF 500 ASF 502 ASF 519a

Mouse Nelson b Collins Swiss NCS NCS NCS CD-1 CD-1 CD-1 NCS What is/are ‘defined’ flora ? strain (NCS) Date Early 1960s Early 1960s Early 1960s Early 1960s Early 1970s Early 1970s Early 1970s Early 1960s

Rockefeller Rockefeller Rockefeller Rockefeller Charles River Charles River Charles River Rockefeller Location University University University University Laboratories Laboratories Laboratories University Russell Russell Russell Russell Russell Scientist Roger Orcutt Roger Orcutt Roger Orcutt Schaedler’s flora Schaedler Schaedler Schaedler Schaedler Schaedler No fungi Intestine/ Intestine/ Intestine/ Intestine/ Intestine/ Intestine/ Source Stomach Stomach various recipes Cecum Cecum cecum cecum Cecum Cecum Lactobacill Eubacteriu Mucispirill Pseudoflav Parabacte No protozoa Taxonomy Clostridiu us Lactobacill m Clostridiu um onifractor roides ~ 8 bacterial spp e.g. (Gram) m species intestinali us murinus plexicauda m species schaedleri species goldsteinii s tum • 2 lactobacilli Genome No SFB 2.91 Mb 2.01 2.17 2.33 6.51 3.70 6.48 6.87 size • 2 clostridia THUS you should NOT see AQFQ00000000 AQFR00000000 AQFs00000000 AYGZ00000000 AQFT00000000 AYJP00000000 AQFU00000000 AQFV00000000 GenBank # .1 .1 .1 .1 .1 .1 .1 .1 • 1 Bacteroides these in gnotobiotic, or in Morphology • 1 Eubacterium clean immunodeficient rodents from isolators… • 2 ASF#’s You should not see protozoa in clean rederived rodents…

280 Wymore et al 2015 ILARJ

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Common findings ??? significance Esophagus ‐ Forestomach

 Lactobacillus sp  Streptococcus / Stomach ‐ yeast – In ASF recipes enterococcus – like Small Intestine ‐ Segmented Filamentous Bacteria – Chains of cocci Large intestine – Flagellates + Entameobae – NOT in ASF

You should NOT see these in gnotobiotic, or in clean immunodeficient mice from isolators… You should not see protozoa in clean rederived mice.

Most Vendors test for & exclude the protozoa.

282 283

Gastric Yeasts Ileum SFB Savage & Dubos. 1967. Localization of  Most potent microbial indigenous yeast in the murine stomach. stimulus for induction of Journal of bacteriology. 94(6): p. 1811‐6. intestinal IgA‐producing – Torulopsis pintolopesii ‐ ON surface cells  Also important for TH17 responses  Normally ‘disappear’ from weanling mice after IgA induced  persist in IgA deficient mice  This is a NSG mouse

284 286

Segmented Filamentous Bacterium (SFB) of the Small Bowel of mammals and birds SFB ‐ Ileum After the initial “bite” (A),  Described by Savage, Dubos etc 1970’s & earlier Holdfast segments move  Gram Positive away from remaining part  Spore former of the organisms (B and C),  Attach to brush border Holdfast segments penetrate  Role in Mucosal immunity? more deeply while  Distal SI –esp in immuno‐ microvillar borders and deficient out of isolators – RP Orcutt cytoplasmic membranes  Could not be grown in pure culture return to an apparent integrity (D–F). not in Schaedler’s flora Grown on cells + complex media –Schnupf& al 2015 288 287 Caselli, & Al 2010.

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SFB & Th17 & B6 sources SFB & Th17 & B6 sources Ivanov & al. 2009. (Tac vs J B6) Denning & al. 2011. (Crl vs J B6)

Figure 2. SFB in Th17 Cell‐Sufficient and  Ability of DC and macrophage subsets to induce Foxp3(+) T(reg) cells Th17 Cell‐Deficient Mice versus Th17 cells was strikingly dependent on Mouse source: (A)qPCR for SFB &total – DCs from C57BL/6 mice from CRL (+ SFB which robustly induce bacterial (EUB) 16S rRNA Th17 cells in situ) were more efficient at inducing Th17 cells in Tac & Jax B6 feces. and less efficient at inducing Foxp3(+) T(reg) cells than DCs (B) SEM&TEM ileum 8wo from B6 from TJL (‐ SFB). Jax & Tac B6 after cohousing  Only CD11c(+)CD11b(+)CD103(+) DCs efficiently induced Th17 cells (C)qPCR 14d post – Distribution correlates with Th17 cells: duodenum > jejunum > ileum > colon. cohousing w Tac B6 (Jax‐Coh)  Conversely, CD11c(+)CD11b(‐)CD103(+) DCs, macrophages, and (D)SFB colonization of Jax Foxp3(+) T(reg) cells were most abundant in colon & scarce in B6 14 d postcohousing duodenum. Ivanov & al. 2009. Induction of intestinal Th17 cells by segmented289 290 filamentous bacteria. Cell. 2009;139(3):485‐98.

Cecum, colon Further down … Large intestine B6,129,FVB TmTg Flagellates + Entamoebae

Common Don’t seem to cause a problem Some agents might ‘invade’ damaged mucosa ? NOT in ASF. Pyriform, undulating membrane Compatible with Trichomonad Diagnosis?

291Significance ? 292

Friends in our mouse guts Beware of Biological Materials Recent (also historical) contaminants

Agents found recently… Some Phenotypes in immunodeficient Stomach Mycoplasma sp. Respiratory dz + shedding LDV Immunomodulate LE’s + shedding – Yeasts cw Kazachstania (Torulopsis pintolopesii) Human herpesviruses BIOHAZARD + shedding HIV BIOHAZARD + shedding – [Lactobacillus, Enterococcus forestomach mucosa] Hepatitis B BIOHAZARD + shedding Parvoviruses Immunomodulate, Tumor growth ? + shedding Small Intestine Polyoma virus Immunomodulate, Tumors + shedding LCMV Immunomodulate, BIOHAZARD + shedding – Segmented filamentous bacterium of the small Mouse adenovirus Immunomodulate – INIB? Kid, Gut + shedding Mouse hepatitis virus Wasting + enterohepatic DZ + shedding intestine of mammals & birds (Ileum) mCMV) Death – hepatic etc necrosis + shedding • Gram POS, spore forming, anchored in ileum enterocytes Ectromelia virus Death & shedding Pneumonia virus of mice (PVM) Respiratory DZ + shedding Murine norovirus Enteritis encephalitis n some + shedding Cecum/colon Sendai virus Wasting + respiratory Dz + shedding – Trichomonads etc protozoa? E. cuniculi Kid + CNS ? + shedding  from CBW RADIL & CC CRL – AALAS 2010 – Important /useful (TH17 etc) 295

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Is CLEANEST BEST? Immunodeficient mice & for the mice? for the project? Infectious Agents Over simplified ‘Barrier’ defined by the agents excluded, and the exclusion practices – More severe disease ‘SPF’ defined by the agents excluded, and – Higher mortality the exclusion practices – Persistent infections (not cleared) ‘Clean’ moving target, as new issues/pathogens – Don’t seroconvert emerge or are rediscovered – Reservoirs of infectious agent • Risk to humans & other animals Environmental/Microbial ‘challenge’ may expose an important phenotype. Rederivation may lose it…. 296 297

Is CLEANEST BEST? Sources of mice for immunodeficient mice? and surveillance information …

‘Barrier’ defined by the agents excluded, and Charles River (Crl) http://www.criver.com/find‐a‐model?loc=US the exclusion practices ‘SPF’ defined by the agents excluded, and Envigo (Harlan) (Hsd, Ola) http://www.envigo.com/products‐ the exclusion practices services/research‐models‐services/resources/health‐ monitoring‐reports/ ‘Clean’ moving target, as new issues/pathogens emerge or are rediscovered Jax (J) https://www.jax.org/

Probably, usually, YES. Taconic (Tac) https://www.taconic.com/quality/health‐reports/

298 299

SPF NSG

 Diagnosis/es: NOT a 1. Lung • Cause? Super Power. 2. Mammary gl Must be 3. Uterus tumor 4. Uterus +PAS defined in 5. Uterus tumor reporting… • Chromogranin A 6. Lymph Node

Santagostino et al. 2017 http://journals.sagepub.com/doi/full/10.1177/0300985817698210

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MOUSE: 306 NSG Etiology?

 Diagnosis/es:  What is/are the morphologic diagnosis/es? Necrosis and loss, 7. Skin + PAS distal limbs (+tail) • Cause?  What is/are the Etiology(ies)? Ectromelia virus (Pox viridae) 8. Kidney  What is the name of the disease? Mousepox 9. Kidney EM  What is a possible NON infectious cause? Flooded cage  • Cause? gangrene 10. Head (decal)  (+/‐ tail ‘tipping’ of tg / tm) 11. Spine  King Likely mouse strain ? 12. Kidney  Looks Like DBA –which are susceptible to Ectromelia

Santagostino et al. 2017 306 http://journals.sagepub.com/doi/full/10.1177/0300985817698210

Diagnosis/es?

Cause/s?

Don’t panic yet… Are these BALB/c or SJL/J males? 307 308 http://dora.missouri.edu/mouse/

309 Mouse: Morphologic Diagnosis (es)? Common ? Which is:  B. Ornithonyssus Bacoti Likely A blood sucking mite ? C. musculi etiologies?  A fur mite ?  A non parasitic pest ? A. Psocid (bark or book louse)

309 A 0.5 mm C 310 B

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