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Prevalent Infections of Laboratory Rats and Mice: Implications for their Use in Research Definitions Animal research model: An animal in which normative biology or behaviour can be studied, or in which a spontaneous or induced pathological process can be investigated, and in which the phenomenon in one or more respects resembles the same phenomenon in humans or other (target) species of animals [Wessler, 1976] Timing of discovery Upfront • During research • After research • Never……..hindsight • “Usual” Reasons Against Infected Rodents Some diseases are zoonotic Some may cause significant morbidity or mortality Accreditation/Regulatory pressure Complications in collaboration/sharing rodents strains and stocks Some subclinical infections reported to alter research results Prevalence (%) Agent (assay abbreviation) Method N NA Europe Total Ectromelia (ECTRO) Serology 246,857 0.02 0.00 0.02 Hantavirus (HANT) Serology 144,946 0.00 0.00 0.00 K virus (K) Serology 225,353 0.00 0.00 0.00 Lymphocytic choriomeningitis virus (LCMV) Serology 241,453 0.01 0.02 0.01 Mouse adenovirus 1 and 2 (MAV) Serology 230,351 0.02 0.22 0.02 Mouse cytomegalovirus (MCMV) Serology 146,511 0.04 0.00 0.04 Mouse hepatitis virus (MHV) Serology 558,673 1.57 3.25 1.59 Mouse norovirus (MNV) Serology 44,876 24.03 32.37 Parvovirus generic assay (NS-1) Serology 578,464 1.65 1.92 1.65 Mouse parvovirus Serology 594,539 1.83 3.64 1.86 Serology 595,903 0.33 0.46 0.33 Pneumonia virus of mice (PVM) Serology 447,656 0.01 0.01 0.01 Polyoma virus (POLY) Serology 225,868 0.02 0.20 0.02 Reovirus 3 (REO, REO-3) Serology 428,821 0.01 0.05 0.01 Rotavirus (EDIM) Serology 466,572 0.56 0.35 0.56 Sendai virus (SEND) Serology 462,209 0.00 0.00 0.00 Serology 435,772 0.26 0.27 0.26 1 and 2 (MPV) Mouse minute virus (MMV, MVM) Theiler's murine encephalomyelitis virus (TMEV, GD-VII) 32.64 Agent Citrobacter rodentium Prevale Disease* nce (%) 0 Transmissible murine colonic hyperplasia Clostridium piliforme** 4 Corynebacterium kutscheri 0 Mycoplasma spp.*** 0.01–2 Pasteurellaceae 13 Salmonella spp. 0 f3-Haemolytic streptococci (not group D)*** 0.2 Streptococcus pneumoniae^ 0 Helicobacter spp. 16 Streptobacillus moniliformis^^ 0 Tyzzer’s disease (classical trias enteritis, hepatitis, myocarditis), multifactorial Pseudotuberculosis,multifactorial Most relevant: M. pulmonis (murine respiratory mycoplasmosis), cofactorial disease P. pneumotropica causes opportunistic infections, e.g. Suppurative lesions Salmonellosis (mainly important as model organism) Rarely induce clinical disease; group B (Sc. agalactiae), systemic and suppurative lesions; a report of dermatitis of group G streptococci Usually subclinical H. bilis, H. hepaticus:enterohepatic lesions (dependent on strain and immune-status); H. muridarum: gastritis, H. typhlonius: typhlocolitis (Il10tm1Cgn mice) Septicemia,polyarthritis; zoonosis Bacteria to be monitored in mice according to the FELASA guidelines for health monitoring [1]. * For further reading concerning impact on research and pathology see [2,186]. ** Up to 65% in rats. *** Up to ∼4% in rats. ^ A recent outbreak of subclinical infection was reported at an European vendor in 200. ^^ Determined by culture, which unlikely detects latent infections of this bacterium [187]. A. Bleich, A.K. Hansen / Comparative Immunology, Microbiology and Infectious Diseases 35 (2012) 81– 92 Microbiological assessment of the health status of laboratory rats and mice The science of evaluating representative sample groups from given units of those species against a specific listing of etiological agents of disease to define the health status of the source colony. The purpose of this information is to prevent introduction of disease and to monitor the microbial status of resident colonies Principles of pathogen screening The current standard in health monitoring A first prerequisite for health monitoring is the definition of a hygienic unit, in which rodents of given colonies (e.g. at a vendor) are likely to display the same pathogen status, usually because they are maintained in the same barrier protected area without being exposed directly or indirectly to other rodents (a). Out of this unit, a predetermined number of animals has to be investigated for pathogens (b). This sampling size depends on the prevalence of a given agent in a colony of at least 100 animals and the confidence level at which one would like to detect a pathogen. A further prerequisite is a defined list of agents that are to be monitored (c) as well as the methods used to detect them. Finally, results need to be depicted in a formalized manner, usually a health report citing all details including current and historical results of health monitoring (d). A. Bleich, A.K. Hansen / Comparative Immunology, Microbiology and Infectious Diseases 35 (2012) 81– 92 Health surveillance programs detect by examination of 1or more representative sample groups the presence (even in a single individual) of any pathogen from a specific profile of infectious agents. Development of such data on a repetitive schedule forms the objective basis on which to establish and/or confirm the ongoing microbial status of commercial and institutional rodent production colonies develop institutional procurement standards for supplier eligibility based on animal health criteria continuously monitor the health status of institutional research animal residents, including recent arrivals undergoing equilibration or quarantine before use, those currently involved in research protocols, and those coming off study. Testing methods Clinical Evaluation, Gross and Microscopic Pathology Diagnostic Detection and Identification of Microorganisms ◦ Microscopy (ecto & endoparasites) ◦ Culture (resp. & enteric) ◦ Serology/PCR (tissue, feces) from animals or tissues (tumors and cell lines to be introduced) Factors that can influence the HM results Sampling method (colony animals, contact sentinels, bedding sentinels) The number of animals (sera or other samples) in the sample group (Prevalence and confidence) The periodicity of sample group submission The age and sex of animals in the group/room The testing method (serology, PCR, type of culture) Factors that can influence the HM results (cont.) The type of caging (isolators, IVC, static microiso., open cages) The immunological status of the animals (unknown in GM, stressed from research – can be used for dexamethasone provoked testing) The agent profile against which to evaluate the health status Examples of sample selection errors Result Methodology Falsenegative *Serology Falsepositive Error Acutely ill, serum antibodies not yet detectable Immunodeficient or immunosuppressed, weak or no antibody response Bacteriology/parasitolo Older and recovered from infection gy Site where organism is not normally resident All Small sample size Sentinels not adequately exposed via soiled bedding or contact to infectious agents carried by principals Serology Strain with autoimmune disease Immunized or inoculated with biological material (such as tumor cells) Maternal antibodies All Sentinels housed under less strict conditions than principals (such as principals kept in microisolation cages, but sentinels are in open cages) Comparison of typical and ideal serology test. Examples of sample selection errors Result Methodology Falsenegative Serology Falsepositive Error Acutely ill, serum antibodies not yet detectable Immunodeficient or immunosuppressed, weak or no antibody response Bacteriology/parasitolo Older and recovered from infection gy Site where organism is not normally resident All Small sample size Sentinels not adequately exposed via soiled bedding or contact to infectious agents carried by principals Serology Strain with autoimmune disease Immunized or inoculated with biological material (such as tumor cells) Maternal antibodies All Sentinels housed under less strict conditions than principals (such as principals kept in microisolation cages, but sentinels are in open cages) Subclinical infections are likely to increase variability Not all animals exposed on same day or to same dose of infectious agent Different strains, sexes, ages respond differently Co-infections, and maybe gut flora, can alter response Even in inbred animals of uniform signalment, there will be some variation Research Effects of Subclinical Infections In addition to currently documented effects, potential interactions can be suspected through cell types infected, intracellular components and functions involved, and systemic host response mechanisms Presenter bias How clean/pure do you want your glassware and reagents? Would you buy reagents if vendor said they are contaminated, and that the contamination might affect some, but not all, research? What’s common? • MHV – 2% • Parvoviruses • Helicobacter spp. – 15% • P. pneumotropica • Rotavirus – 0.7% • Norovirus - 30% • RRA – 7% • Theilovirus • C. bovis – 3% • Pneumocystis carinii – 2% • Pinworms – • Mouse – 2% • Rat – 4% • Mouse – 0.3% • Rat – 1.4% • Mouse – 15% • Rat – 5% • Mouse – 0.3% • Rat – 1.3% • Mites – 0.1% Rats and Mice Mycoplasmosis (CRD, MRM*): ◦ ◦ ◦ ◦ ◦ ◦ Mycoplasma pulmonis: causative agent major problem in rats; stress worsens condition primarily affects the respiratory tract may cause inner or middle ear infection (head tilt) can become endemic and difficult to eradicate causes severe pathologic lesions in the lungs: abscesses, red to gray consolidation * Murine respiratory mycoplasmosis Rats and Mice CAR (Cilia associated respiratory) bacillus: ◦ gram negative bacteria attached to cilia ◦ respiratory disease or lesions in rats, mice, rabbits, swine ◦ require special staining techniques on histopathology (Warthin-Starry silver stain) ◦ dirty bedding inadequate for detection Mouse Hepatitis Virus (MHV) Coronavirus, ssRNA, enveloped ◦ Innumerable strains Prevalence: ~2% of serum samples, so present in most universities Strains grouped as enterotropic or polytropic ◦ Infection generally asymptomatic in postweaning immunocompetent mice ◦ Wasting syndrome in many immunodeficient mice ◦ BALB/c mice more susceptible than C57BL/6 mice MHV - polytropic Virus attaches to CEACAM1a glycoproteins (members of the immunoglobulin - Ig superfamily) via spike protein Primary tropism for upper respiratory tract mucosa Secondary sites - lung, liver, CNS, intestines, ovaries, epididymis Research Impact of MHV Prolonged immunologic effects: ◦ MHV depletes NK cells through apoptosis and syncytia formation ◦ T-cells, B-cells > 1,000 CD8 T cell clonotypes estimated to respond to dominant MHV S protein epitope (JHM in C57BL/6) ◦ Infects monocytes, macrophages, bone marrow dendritic cells ◦ Delayed allogeneic graft rejection Polytropic MHV Brain and spinal cord ◦ Meningitis and necrotizing encephalitis NOS2 KO mice (amyloid beta deposits - Alzheimer's disease-like pathology accompanied by behavioral changes) have less mortality in experimental infection, associated with decreased neuronal apoptosis ◦ Demyelination (brain stem) Apoptosis of oligodendroglia Immune-mediated (antibody, CD8 bystander, MBP autoreactive T cell clones, macrophages) ◦ Intracerebral MHV (JHM – a neurotropic variant of MHV) in C57BL/6 altered expression of 80 genes, at least 27 of which relate to innate or acquired immunity Enterotropic MHV - common Most wild type strains are enterotropic Highly contagious, shed in large quantities in feces Transmitted by fomites (gloves, cages, bedding, etc.) Clinical signs and gross lesions rare in immunocompetent adult mice Primary replication: ◦ GI tract, especially distal ileum, cecum, ascending colon Secondary sites - uncommon MHV Detection Serology ◦ Excellent cross-reaction among strains ◦ Seroconversion within 2 weeks (often one week) PCR ◦ Shedding?, Environment ◦ Mesenteric lymph nodes to confirm serology ◦ Epidemiology Histopathology ◦ Lesions should by confirmed by IHC, PCR or serology CONTROL OF MHV Immunocompetent mice self-cure Enveloped virus: not stable in environment, easy to disinfect Can eliminate from immunocompetent colonies by not breeding and no new mice for 6-8 weeks (always test to confirm) Infection persists in immunodeficient mice Parvoviruses Compared to MHV, parvoviruses are harder to detect, harder to eliminate, and tend to have lower prevalence. This leads to confusion and frustration. Parvoviruses ssDNA, (5.4 kb genome), non-enveloped ◦ Virus remains active in environment Resistant to desiccation, non-oxidizing disinfectants Most are fairly common Very low or no morbidity All can cause persistent infection Require mitotically active cells for replication ◦ S phase for activation of P4 promoter Parvoviruses of Mice Mice Minute Virus (MMV or MVM) ◦ Multiple strains (i, p, c, m), MMVm is most prevalent and is persistent. Others are cultureadapted strains. MMVm causes growth retardation, reduced fecundity and premature deaths in NOD μ-chain KO mice. Chronic progressive infection in scid mice. MMVi Intranasal inoculation causes lethal leukopenia in scid mice, infects CFU-GM, BFU-E, CFU-MK and HSC Research Effects of MMV Can infect many mouse cell lines, as well as some rat embryo lines and transformed human cells (324K, EL-4) In vitro (A9 cells) dysregulation of gelsolin (↑) and WASP (↓) by MMVp In vitro reduction of T-cell response by MMVi and in vivo late reduction of cytotoxic memory cells by MMVp MMVp is oncotropic and oncolytic in some human tumors (hemangiosarcoma) and mouse tumors Parvoviruses of Mice Mouse Parvovirus (MPV-1, -2, -3, -4, -?) ◦ ◦ ◦ ◦ Prevalence higher than MMV Cause persistent infection No anatomic lesions, even in scid mice Strains not reliably cross-reactive by serology Research Effects of MPV MPV-1a (cell culture adapted) modulates immune response (McKisic et al, 1996) ◦ Suppression of T cell response in vitro CD8+ T lymphocyte clones lose function and viability Cytokine- and antigen-induced T cell proliferation in vitro suppressed after exposure to MPV-1a ◦ Potentiates allograft rejection ◦ Induces isograft rejection Parvoviruses of Rats (RPV-1, RPV-2, RMV, RV [KRV] I H-1) RV - Rat Virus (previously KRV, Kilham Rat Virus) ◦ Natural infections usually asymptomatic, but persistent, with prolonged shedding ◦ Infects rapidly growing cells: Endothelium, lymphoid and hematopoietic tissues, developing cerebellum and liver ◦ Rare epizootic disease in fetal/neonatal rats: Cerebellar hypoplasia, anemia, thrombocytopenia ◦ Very rare disease in older rats: Hemorrhagic disease Rat Virus (RV) formely Kilham’s rat virus - KRV Rat Virus (RV) – Research Effects Research Effects: RV induced diabetes in DR BB rats (Guberski et al., 1991) ◦ Possibly due to imbalance in Th1 and Th2 responses (Jun and Yoon, 2001) RPV NS protein induced epigenetic modification in thymic lymphoma line, causing reversion to benignancy (Iseki H , 2005) Parvoviruses of Rats Rat Parvovirus (RPV) Few studies in literature, very difficult to isolate ◦ Multiple strains exist ◦ No clinical disease reported ◦ Research effects: Suppression of lymphoid tumor growth in vivo: RPV-1a Parvoviruses of Rats (Toolan’s) H-1 - no natural disease ◦ Significance through research interference: liver ◦ Historic interest as cancer therapy Rat Minute Virus (RMV) ◦ Almost nothing in literature ◦ May be most common parvovirus of rats ◦ Serologically and genetically more similar to RV than to RPV Exclusion of Parvoviruses Consider sources of research animals: ◦ Vendors, GM animals, immunodeficient Wild rodents Biological materials Risk from personnel handling infected rodents (pets, snake food) Fomites (Feed, bedding, water, used/shared equipment etc.) Detection of Parvoviruses Serology – Best for screening ◦ Use panel of antigens for each serotype, plus the generic NS-1 antigen PCR - strain-specific (VP2) or generic (NS1) ◦ ◦ ◦ ◦ Mesenteric LN stay positive indefinitely PCR of feces to detect shedding Valuable for testing biologicals and cell cultures Environmental swabs Parvovirus detection problems Mice on C57BL/6 background and congenic strains are partially resistant to infection and infrequently seroconvert after exposure ◦ 10-100x increase in ID50, cf. BALB/c or CD-1 ◦ DBA/2 also, not quite as bad If small amount of virus in environment, sentinels will be positive, but only an occasional C57 will receive an ID DBA/2 only slightly better Parvovirus diagnostic problems Older sentinels less susceptible, seroconversion does not necessarily mean shedding enough to transmit infection ◦ May have one positive, with negative cagemates Prevalence also kept low by filter-top cages ◦ Low prevalence means low predictive value of positive result Control of Parvoviruses Can only eliminate by rederivation ◦ Multiple anecdotal reports of success with early cross-fostering No envelope, so it stays active in environment ◦ Must thoroughly disinfect environment, materials and equipment with oxidizing agent (Clidox, ozone, etc.) Human Norovirus Infection (good model for mouse disease) Genus named for Norwalk virus, discovered in 1972 ◦ Typical signs in humans - vomiting and diarrhea for ~ two days. Noroviruses cause most nonbacterial epidemic gastroenteritis worldwide. ◦ In US, CDC estimates 23 million cases of noroviral diarrhea each year ◦ If similar rate in rest of world, > 500 million cases each year. Human Norovirus Infection Norovirus infamous on cruise ships, high morbidity of passengers and crew “Vomiting Bug Sickens Hundreds of Thousands in the UK”: By Anna Boyd 17:07, January 3rd 2008, eFluxMedia Human Norovirus Infection Lessons from human outbreaks 1. As a nonenveloped virus, disinfection is difficult 2. Infectious amounts of virus shed for weeks after symptoms abate 3. Immunity not cross-protective Murine Norovirus (MNV) Small, nonenveloped, ssRNA viruses in Caliciviridae Capsid is single protein, with cup-like projections, or calices Myriad MNV variants, as with humans ◦ CRL Molecular Diagnostics has identified more than 50 different variants - All in Genogroup V MNV Epizootiology Fecal-oral transmission As a nonenveloped virus, MNV can remain infectious in the environment, possibly weeks Seroconversion slow Infectious dose for mice is not known ◦ Infectious dose for humans may be only a few virions MNV Epizootiology Infected mice, as infected humans, shed: ◦ Massive amounts of virus for a few days after infection ◦ Shed small but probably infectious amounts of virus for weeks Prevalence appears very high, estimated at ~30% of mice Production colonies of major vendors are MNV negative MNV infection Immunocompetent mice – No clinical signs ◦ Studies in 129 (129S6SvEvTac) mice, inoculated PO with 107 PFU MNV-1.CW3 Minimal change ( 13 vs. 8 per high power field) in inflammatory cells in lamina propria of small intestine at 24 hours post infection Increased nuclear staining in red pulp of spleen, but no change cell number Viral nucleic acid found in small intestine, spleen, mesenteric lymph nodes, liver Possible decreased “stool contents” at 3 days P.I. MNV Disease Mice deficient in acquired immunity (RAG, SCID) ◦ MNV antigen and nucleic acid detected in mesenteric lymph nodes – possibly in dendritic cells MNV Disease Mice deficient in innate immunity ◦ Lethal infection in STAT1 -/- (with or without RAG2 and PKR), and IFN Rαβγ -/◦ hepatitis, interstitial pneumonia ◦ Encephalitis only with intracerebral inoculation ◦ Virus present in dendritic cells Homozygous disruption of the Stat1 gene eliminates the intracellular mechanism by which cells respond to interferons, resulting in a mouse model with extreme susceptibility to bacterial and viral infections, and with increased tumor formation Applications for the Stat1 Targeted Mutation Mouse Model MNV Research Effects Unknown, other than disease in some strains Potential interference with studies of innate immunity and/or dendritic cells MNV Effects on Research [after Hsu, 2012] • • • • • MNV infects dendritic cells and macrophages (effect on immunological and infectious disease research?) [Wobus et al., 2004] Elevated type I interferon levels (intestines: 12 and 24h PI; Serum: 24h PI) [Mumphrey, et al., 2007] MNV exacerbates bacterial-induced IBD - Mdr1a-/- mice + Helicobacter + MNV = ↑ wt. loss and colitis @ 2wks PI - MNV enhances antigen presentation by dendritic cells - ↑ IFN-γ production by T cells 2d PI (but not at later time points) [Lencioni et al., 2008] MNV increases the duration of MPV fecal shedding and MPV DNA levels in tissues of BALB/c mice [Compton et al., 2010] MNV infection + susceptibility gene interactions determines the phenotype for a mouse model of Crohn’s Disease [Cadwell et al., 2010] MNV (Lack of ) Effects on Research [after Hsu, 2012] But…. • MNV has no significant effect on C57BL/6 mouse models of vaccinia virus or influenza A virus models [Hensley et al., 2009] • MNV has a mild impact on CD8 T cell responses in a mouse model of murine cytomegalovirus (MCMV) infection, but “not likely to affect most experimental outcomes in immunocompetent mice in the MCMV model” [Doom et al., 2009] • MNV infection in a mouse model of bacterial induced colon cancer (SMAD3-/- mice + Helicobacter) did not impact survival, IBD scores, tumor incidence or tumor phenotype [Lencioni et al., 2011] MNV Diagnosis Colony screening by Serology ◦ Good cross-reaction among strains ◦ Seroconversion often slow, so sentinels should have 8 weeks of exposure PCR ◦ Immunodeficient mice ◦ Pooled fecal samples ◦ Release from quarantine Avoids long exposure period ◦ Environmental monitoring ◦ Epidemiology MNV Management Virus apparently present for a long time ◦ Very well adapted to host ◦ Many strains ◦ Wide geographic distribution Current situation does not recent development, and does not require urgent action – do not panic if positive results received Consensus seems to be to survey ◦ Many facilities waiting to see how attitudes toward MNV evolve in the near future, before taking action ◦ Many trying to start new facilities with MNV-free mice MNV Management Rederivation by embryo transfer or caesarian section should be successful ◦ Possible early cross-foster Environmental decontamination requires use of oxidizing disinfectants or heat > 56oC Helicobacter Originally included with Campylobacter ◦ Helicobacter created in 1989 H. pylori probably discovered in 1875, ◦ linked to human gastric disease in 1899 (Polish text) “Re”discovered in 1979 by Warren ◦ 1984 - linked to most gastric ulcers and gastritis (Warren and Marshall, Nobel Prize awarded in 2005) Helicobacter in rats and mice - Discovery H. muridarum in mice: 1992 H. hepaticus: 1994 ◦ Liver tumors and hepatitis in A/J mice on carcinogenicity study ◦ Lesions resembled aflatoxicosis ◦ Spiral organisms discovered in bile canaliculi with Steiner stain Currently >40 species of Helicobacter described, with many in rodents Helicobacter Gram-negative bacteria colonizing intestinal tract of warm-blooded animals ◦ Gastric ◦ Large intestine - Some of these reach liver (enterohepatic) Microaerophilic (H. ganmani is anaerobic) Highly sensitive to desiccation Highly adapted to hosts, although many are capable of colonizing multiple host species Mechanisms of disease similar among helicobacters Animal models useful in studying human disease Helicobacter Epizootiology Fecal-oral transmission Short-term fomites (soiled bedding) possible Colonization by weaning, persist for lifetime ◦ Short-term colonization noted in a few experiments, significance unknown Prevalence: high (~15% in mice, ~8% in rats), especially high in GM mice Helicobacter Disease Disease varies with Helicobacter species, rodent strain, immune status, sex, possibly age General – Outcome depends on interaction with gut flora and on immune response, with most disease being a by-product of the host response ◦ Important components of host response include IL-10, TNF, TH1:TH2 balance, CD4+CD45RB(lo)CD25+ T regulatory cells, and TGF-beta Infection with multiple Helicobacter species or with other pathogens, e.g., MHV, can be synergistic H. hepaticus Disease Most common Helicobacter sp. in mice Proliferative colitis in A/J, C3H/HeN, athymic nude, SCID and many other immunodeficient strains, e.g., IL-10 -/ Currently, only common infectious cause of rectal prolapse, especially in immunodeficient mice* Chronic hepatitis (necrosis, hepatocytomegaly, biliary proliferation, nonsuppurative inflammation) in A/J, C3H/HeN, and some other immunocompetent strains ◦ Hepatitis may be necrotizing in immunodeficient strains Helicobacter hepaticus H. hepaticus Disease Most common Helicobacter sp. in mice Proliferative colitis in A/J, C3H/HeN, athymic nude, SCID and many other immunodeficient strains, e.g., IL-10 -/ Currently, only common infectious cause of rectal prolapse, especially in immunodeficient mice* Chronic hepatitis (necrosis, hepatocytomegaly, biliary proliferation, nonsuppurative inflammation) in A/J, C3H/HeN, and some other immunocompetent strains ◦ Hepatitis may be necrotizing in immunodeficient strains H. hepaticus Disease Hepatocellular carcinomas in A/J, C3H/HeN, SCID mice Colon carcinoma in SMAD-3 deficient mice Increased incidence of mammary carcinoma in RAG2/- Apc(min/+)mice (secondary to inflammation) C57 resistant to disease, but can carry high level of colonization 6-week old AL-ras x AL-myc dual Tg mouse H. bilis Disease Prevalence about 1/4 that of H. hepaticus Proliferative typhlocolitis in athymic mice and rats ◦ Occasional rectal prolapse Mild chronic hepatitis in immunocompetent mice (low incidence) Helicobacter spp. - Research Effects Direct effects, depending on variables above, on large bowel and liver, with broad activation of specific and non-specific aspects of host defense system including development of tertiary lymphoid follicles Indirect effects of infection without lesion production are not as well described but may influence remainder of gut flora Diagnosis of Helicobacter infection Similar for all Helicobacter spp. PCR – best. Can be generic or specific. ◦ Can use pooled fecal pellets Culture – difficult, lacks sensitivity and rarely done Histopathology with silver stains (in tissue only) Serology Sentinels in Helicobacter screening Transmission to sentinels has been questioned ◦ Effective in as little as 2 weeks for H. hepaticus (Livingston, et al, Comp Med, 48:219 1998) ◦ Less effective for H. bilis, H. rodentium (Whary, et al, Comp Med 50:436 2000) ◦ May consider direct monitoring of principal animals Based on epizootiology, frequent in-house monitoring is unnecessary if incoming animals are negative Helicobacter Management Relatively easy to contain within a research facility ◦ Anecdotal reports of cages side-by-side without transmission ◦ Note reports of difficulty in transferring by soiled bedding Elimination often successful by cross-fostering before 24 hrs of age (Singletary KB, Kloster CA, Baker DG, Comp Med 2003 Jun;53(3):259-64 ) Mixed reports on success of medicated feed or antibiotic administration by gavage Rederivation seems uniformly successful Interference by opportunists in rodents Opportunists: A usually harmless organism that can cause disease under favourable conditions • Beta haemolytic streptococci Klebsiella oxytoca Klebsiella pneumoniae Pseudomonas aeruginosa Proteus spp Staphylococcus aureus • • • • • Interference by opportunists in rodents Nude Mice-Various and Numerous Examples - Immune Modulation Effects - Clinical Signs - Abscesses, Unthriftiness - Interference with Breeding Colonies • CB 17 SCIDs - It Gets Worse! • Conventional Rodents - Post Operative Infections in Surgical Models - Safety Assessment Studies – Survival Rates, Clinical Issues • Opportunists example: Staphylococcus aureus • C57Bl/6 mice most resistant: innate immune mechanisms • A/J, DBA/2, BALB/c mice highly susceptible • For A/J mice 3 chromosomes influence susceptibility • Findings suggest 2 genes, Tnfaip8 and Seh1l, may contribute to susceptibility to S. aureus in A/J mice, and represent promising candidates for human genetic susceptibility studies. [Kockritz et al.,2008; Ahn et al., 2010] Mycobacteria in NHP Mycobacteria (NHP): M.Tuberculosis; M. kansasii - Personnel safety concerns if M.Tb, M. avium - Regulatory obligations and documentation - Animal Welfare Concerns - Increased spending for equipment and supplies - “Work around” costs - Delayed research start - M. kansassii Interferes with Gene Expression • Primate Diseases Tuberculosis ◦ Draining lymph nodes ◦ Tuberculin testing ◦ Tissue changes ◦ Method of tb testing in monkeys Mycobacteriosis Mycobacterium spp. ◦ ◦ ◦ ◦ ◦ M. tuberculosis* -human to animal M. avium* M. marinum* M. bovis* Etc……… * Zoonosis Morbidity+ Mortality+ Transmission ◦ inhalation, direct contact Tuberculosis: Diagnosis TB skin test Culture ◦ Very slow growing Acid-fast staining PCR Radiographs QuantiFERON-TB test (Humans only) Herpes B ◦ Alphaherpesvirus ◦ In macaques: Nodules on lips or mouth Carriers for life ◦ Fatal for humans (and vervets) ◦ Trans: bite, broken skin, mucosa Rickettsial Diseases Q-fever: Reservoir Species Goats, sheep and cattle: ◦ No obvious illness in animals ◦ Can cause abortions Cats Rabbits Birds Rodents ???? Q-fever: Transmission to Humans Organism is excreted in urine, feces, milk, and especially in birth fluids Humans are usually infected by inhalation of the organism from contaminated environments Occasionally raw milk Q-fever: Risk Factors Direct contact with infected animals Farmers Veterinarians Slaughterhouse workers Sheep researchers Rabbits Pasteurellosis: ◦ ◦ ◦ ◦ Pasteurella multocida: bacterial cause very common in rabbits and difficult to treat spread by aerosol or direct contact diseases: snuffles (sneezing, nasal and ocular discharge), otitis media or interna, localized abscesses, genital infections, pneumonia Frogs Redleg ◦ caused by Aeromonas hydrophila ◦ more common in leopard frogs (Rana pipiens) ◦ factors: stress, population density, dietary change, injury to skin or slime coat ◦ signs: cutaneous hemorrhages on legs, but can occur anywhere on the body. Rats and Mice Pinworms (Oxyurids) ◦ Syphacia oblevata, S. muris, Aspicularis tetraptera ◦ affects rats, mice, hamsters, gerbils, rabbits, horses, NHP ◦ probably # 1 contaminant in rat & mouse colonies in the US ◦ no clinical signs (may cause rectal prolapse in young animals) ◦ easily spread and survive in environment forever ◦ fecal floatation, anal tape, or direct exam Rats and Mice Ectoparasites: ◦ Mites (Myobia sp.) and Lice (Polyplax sp.) ◦ detection by examining pelt under microscope ◦ pruritis, dermatitis, greasy coat, alopecia The gut microbiota of rodents Influence of gut microbiota on caecum size. The caecum size decreases with increasing number of bacteria present in the microflora, although degree of normalization also depends on the composition of the intestinal microbiota. Germfree mouse (a), mouse associated with the 8 species of the altered Schaedler flora (b), and conventionalized mouse (c). A. Bleich, A.K. Hansen / Comparative Immunology, Microbiology and Infectious Diseases 35 (2012) 81– 92 Examples of the impact of the gut microbiota on rodent models of human diseases. ↑, increased incidence in germ free animals and ↓, decreased incidence in germ free animals. A. Bleich, A.K. Hansen / Comparative Immunology, Microbiology and Infectious Diseases 35 (2012) 81– 92 Disease Organ Species Model Type 1 diabetes β-Cells Mouse Non-obese diabetic (NOD) ↑[65] Mouse Rat MyD88 KO NOD Bio-breeding Rat Alloxan Rat Rat Collagen induced HLA-B27 Tg ↓ [151] ↓ [60] Mouse Mouse IL-2 KO IL-10 KO ↓ [61,152] ↓ [62,63] Mouse Mouse TCRα KO Dextran sulphate sodium ↓ [155] ↔ [156,157] ↑ [158,159] Mouse Mouse SAMP1/Yit T-cell transfer ↓ [168] ↓ [169] Rat HLA-B27 Tg ↓ [60] Arthritis IBD Joints Intestine Incidence/sympt oms in germ free animals ↑ [146] Examples of the impact of the gut microbiota on rodent models of human diseases. ↑, increased incidence in germ free animals and ↓, decreased incidence in germ free animals. A. Bleich, A.K. Hansen / Comparative Immunology, Microbiology and Infectious Diseases 35 (2012) 81– 92 Disease Organ Specie s Contact hypersensitivity Dermis Mouse 2, 4-dinitrofluorobenzene Oral Lactobacillus casei as well as oligofructose (and associated increase in Bifidobacteria spp.) treatment are protective [178,179] Allergy Systemic Rat Mouse Cow milk Ovalbumin-specific T cell receptor TG ↓ [181] beta-lactoglobulin ↑ [182] C57BL/6 Lepob ↓ [67] Pristane ↔ [183] Lepob Streptozotocin Lactobacillus spp. delays progression [74,185] Mouse Obesity Lupus erythematosus Type 2 diabetes Systemic Mouse Mouse Systemic Mouse Systemic Mouse Rat Model Incidence/symptoms in germ free animals Type of Contact Skin Animal bites Inhalation 11 (39%) 5 (18%) 4 (14%) Animal Species Rodent/rabbit Dogs Cats NHP 5 (17%) 4 (14%) 4 (14%) 4 (14%) LAB ANIMAL ALLERGIES Prevalence ranges from 11-44% depending on the study. Prevalence estimates vary primarily because of differing ways of defining the disease: ◦ Objective tests vs. subjective symptom reporting: the lowest estimates were from studies which relied on employer reports; the highest estimates simply use self-reporting of symptoms (i.e. “do you ever get a stuffy nose at work”). Specific Animal Allergens Urine is the major source of rodent allergen exposure. Mouse: ◦ Mus m 1 (prealbumin): Previously known as major urinary protein. Found primarily in urine, but also in dander and hair ◦ Mus m 2: Found mostly in hair and dander ◦ Albumin: Found in serum