Protecting Animal Health. Serban Morosan, DVM, PhD, Dip. Vet LAS UMS 28 phenotype du petit animal Faculty of Medicine Pierre et Marie Curie, Paris

Transcription

1 Protecting Animal Health Serban Morosan, DVM, PhD, Dip. Vet LAS UMS 28 phenotype du petit animal Faculty of Medicine Pierre et Marie Curie, Paris 1

2 Topics Health, health status & exclusion list Developing an exclusion list Health Monitoring Protecting the health status of your mice Introducing mice into the colony Barriers Animal care Researchers Precaution during research 2

3 Healthy Mouse What is a Healthy Mouse? 3

4 Mouse Health vs. Health Statuts Healthy mouse : No signs of disease; exhibiting normal behavior Many pathogens do not cause clinical disease Health status : microbial status, usually of the colony Defined in terms of specific : Pathogenic viruses, bacteria, fungi, etc. Parasites Opportunistic pathogens 4

5 Definitions Pathogen : an infectious agent that can cause overt disease and/or alter biologic response(s) during experimentation An organism need not cause disease to be undesirable Opportunist : en infectious agent that is not normally pathogenic but that may cause disease or alter biologic responses under certain circumstances (e.g;, sick/injured or immunodeficient mouse) Commensal : an organism that inhabits and derives benefit from a host without either benefiting or harming the host Zoonotic : transmissible from animals to human Reverse zoonotic : transmissible from human to animals 5

6 A Spectrum of Harmful Potential 6

7 Exclusion list A list of organisms that you will not tolerate in your animal colony facility Defines the desired health status of your mice Basis for making good decisions regarding : Handling of news additions to the colony, e.g., quarantine procedures Standard operating procedures Animal care Research What should be on it? Depends on the needs of your program 7

8 Developing An Exclusion List for Your Program or Facility 8

9 Animal facilities and Budget Program Needs Motivation Exclusion List 9

10 Health Status What do you want? What do you need? What can you maintain? 10

11 Possible Impact on Research Infectious agents may affect breeding performance Disruption in physiology may cause anomalous results even if mice appear healthy: MPV : alteration of CD4+ and CD8 T cells mediated responses; accelerated rejection of tumor and skin allografts; altered tumor development; Inhibition of diabetic phenotype in NOD by many agents, inclusive MHV, LCMV, pinworms, mycobacterium. Pinworm : stimulate mucosal immunity; may induce or accelerate lymphoma development in nude mice 11

12 Host factors may Be Critical Age, stress, immune status Strain and specific genetic modifications: Mice lacking intact interferon signaling pathways due to a lack of type I and/or type II IFN receptors or STAT-1 molecule lethal MNV infection C3H/HeJ (homozygous for the defective LPS response allele Tlr4 Lps-d & Tlr4 knockouts Gram negative infections 12

13 Other considerations Zoonotic potential Collaborations Your health monitoring capabilities/budget Your ability to : Adhere to exclusionary measures Act in the event of a break 13

14 Commonly Excluded Pathogenic viruses, Mycoplasma/pathogenic bacteria: All alter normal physiology Most can affect breeding productivity Some cause illness +/- death Parasites : Stressors that alter normal physiology May affect clinical health and/or breeding All may limit ability to share mice with collaborators 14

15 Some Opportunist to Consider Mouse adapted organism : Mouse norovirus Pneumocystis murina Helicobacter Pasteurella Corynebacterium Human commensals/environmental contaminants : Staphylococcus Pseudomonas B hemolytic Streptococcus Klebsiella Escherichia coli 15

16 Health monitoring program A health monitoring program is designed to : Assure the health and well-being of our animal colonies by routine and extensive monitoring for undesirable agents Prevent the entry of infectious agents by rederivation and strict health monitoring of all incoming mice Minimize the opportunity for transmission of infectious agents between cages (specific housing equipment and rigorous animal health practices) 16

17 Health monitoring program A health monitoring program provide information about: Prevalence rates of infectious agents Microbiological status of laboratory animals Continuous reporting of prevalence rates on regular basis is important : For the design of a cost effective health monitoring program For the interpretation of diagnostic test results For an update of standard health monitoring recommendation (such as FELASA) 17

18 Health monitoring program Purpose is to find at least one infected animal if an organism from your exclusion list is present in the colony Elements of a health monitoring strategy Frequency of monitoring Number and distribution of animal for testing Age, sex and immunological status of animals Colony animals vs sentinels Testing methods 18

19 Health monitoring program Purpose is to find at least one infected animals if a organism from your exclusion list is present in the colony Elements of a health monitoring strategy Frequency of monitoring Number and distribution of animals for testing Age, sex and immunologic statute of animals Colony animals vs. sentinels Testing methods 19

20 Which Animals to Test Colony animals random selection, but all strains Sentinels : 6 wk generally best Younger animals best for parasitology Test animals from all strains/investigators Sentinels strain can make the difference Choose CD1, Swiss, NMRI, Balb/c, C 3 H or DBA over C 57 BL/6 Immunodeficient good for direct test (PCR, culture); useless for serology 20

21 Sentinels Dirty bedding Poor transmission of some organisms: Organism not spread by fecal oral route, e.g. Sendai virus Labile organism, e.g., MHV, Pasteurella Suboptimal exposure to infectious agent : delayed detection, e.g. MPV, MNV Sentinels don t share history with colony animals (shipping, research manipulation) Contaminated sentinel False indicator of colony health May introduce contaminants 21

22 Sentinels Recommendations Don t rely exclusively on dirty bedding sentinels (DB) Supplement with colony animals and/or contact sentinels DB sentinels can improve coverage at lower cost Improve results with DB sentinels Small ratio of colony cages per sentinel Allow at least 1 month exposure, preferably 2-3 Make sure that sentinels are clean prior to placement 22

23 Sample Size Inverse ratio between prevalence and # tested Prevalence = percentage of animals in the colony that are infected Most commonly aim for detection at 50% prevalence Can calculate mathematically Formulas assume random spread and random sampling When assumptions don t hold 1 cage of DB sentinels per colony cages Lower ratio better if relying exclusively on sentinels 5-16 colony animals/contact sentinels per room 23

24 Factors Affecting Prevalence Latency to reach a certain prevalence after introduction of the contaminant will vary with: The organism, especially in epizootic contaminations Shorter with rapidly spreading organisms (e.g., MHV) than those that spread more slowly (e.g., Helicobacter) Caging system microisolater/ivc caging will slow spread Animal handling SOPs Strict microisolator technique can prevent spread 24

25 Monitoring Frequency Test more frequently for the organisms of greatest concern, e.g., MHV, MPV Test less frequently for organisms of less concern, e.g., K virus, Polyoma virus Highest frequency for special cases, e.g., Organism present in one part of the facility, excluded from another Recovering from a break Highly protected colony, e.g., sentinel or foster mother colony 25

26 Testing Methods Serology Sensitive, specific, cost effective methods for detection of most viruses, Mycoplasma, some bacteria False positives can occur, esp. with tests for complex organism and with tests of lower specificity Nonspecific tests can occur, esp. with older animals or animals prone to autoimmune disease Retest suspicious MFI positives with ELISA or IFA 26

27 Testing Methods PCR Advantages Extraordinary sensitivity Potentially applicable for detection of viruses, parasites, bacteria, fungi Disadvantages False negatives Wrong tissue sampled Infected animal sampled at wrong time Reaction inhibitors in some tissues False positives Easily contaminated Easily misinterpreted 27

28 Serology vs. PCR Virus Antibodies Virus Titer Anti-body Titer Latent Period Virus Exposure Seroconversion 28

29 Pathogen Time after onset of infection in weeks >12 MVM MPV MHV MNV PVM Sendai TEMV intermittent spread possible EDIM consistent viral shedding possible Reo3 Ectromelia Clostridium piliforme Mycoplasma pulmonis Adenovirus-2 LCMV KRV Toolan H1 RPV RCV / SDA Pathogens as listed above Presence of antibodies in immune competent animals: serology Pathogen Time after onset of infection in weeks >12 Helicobacter spp. Pasteurella pneumotropica Syphacia spp. Aspiculuris tetraptera

30 PCR on feces What is the chance of detecting pathogens by PCR? Sample of choice: Feces Pathogen: 1. Helicobacter 2. MNV (Murine Norovirus) 3. Pasteurellaceae 4. MHV (Mouse Hepatitis Virus) 5. Sendai / PVM (Pneumonia Virus of Mice)

31 Chance of success depends on: 1. Prevalence (occurrence) of infection 2. Prevalence (spread) within animal colony 3. Period of shedding pathogen 4. Optimal sampling site

32 PCR on feces What is the chance of detecting Helicobacter by PCR? Absent Present

33 PCR on feces What is the chance of detecting Helicobacter by PCR? Absent 20% Present 80%

34 Chance of success for Helicobacter: 1. Prevalence (occurrence) of infection High 2. Prevalence (spread) within animal colony High 3. Period of shedding pathogen Long term 4. Optimal sampling site Feces / coecum contents Other diagnostic techniques?

35 PCR on feces What is the chance of detecting Pasteurellaceae by PCR? Absent Present

36 PCR on feces What is the chance of detecting Pasteurellaceae by PCR? Absent 70% Present 30%

37 Chance of success for Pasteurellaceae: 1. Prevalence (occurrence) of infection High 2. Prevalence (spread) within animal colony High 3. Period of shedding pathogen No shedding of viable bacteria in feces 4. Optimal sampling site Oral swab Other diagnostic techniques?

38 PCR on feces What is the chance of detecting MNV by PCR? Absent Present

39 PCR on feces What is the chance of detecting MNV by PCR? Absent 40% Present 60%

40 Chance of success for MNV: 1. Prevalence (occurrence) of infection High 2. Prevalence (spread) within animal colony High 3. Period of shedding pathogen Long term 4. Optimal sampling site Feces / coecum contents Other diagnostic techniques?

41 PCR on feces What is the chance of detecting MHV by PCR? Absent Present

42 PCR on feces What is the chance of detecting MHV by PCR? Absent 50% - 100% Present 0% - 50%

43 Chance of success for MHV: 1. Prevalence (occurrence) of infection High 2. Prevalence (spread) within animal colony High 3. Period of shedding pathogen Short term! 4. Optimal sampling site Feces only during shedding Blood; antibody detection Other diagnostic techniques?

44 Chance of success for MHV: 3. Period of shedding pathogen Short term! 4. Optimal sampling site Feces only during shedding. Blood; antibody detection MHV infected mouse animal 1 Time after onset of infection in weeks >12 animal 2 animal 3 animal 4 animal 5 animal 6 animal 7 animal 8 animal 9 animal 10 chance in % Spread of infection through feces Presence of antibodies in immune competent animals

45 PCR on feces What is the chance of detecting Sendai / PVM by PCR? Absent Present

46 PCR on feces What is the chance of detecting Sendai / PVM by PCR? Absent 100% Present 0%

47 Chance of success for Sendai / PVM: 1. Prevalence (occurrence) of infection Low 2. Prevalence (spread) within animal colony High 3. Period of shedding pathogen Not through feces 4. Optimal sampling site Blood; antibody detection Other diagnostic techniques?

48 Is PCR the solution? Advantages Non invasive (feces) Sensitive Fast (compared to waiting time antibody response) Pooling of samples possible Identification of new pathogens (general PCR s) Disadvantages Non invasive sample site not always the best sample site Time window (spread of infection) No discrimination between viable / non viable pathogen More expensive than serology / culture Species specific PCR s narrows the window of screening Complete FELASA panel not possible

49 Is PCR the solution? PCR Yes PCR No Live saving sampling (Vivum) Import check Pathogens which are difficult to culture Follow up after proven infection When complete FELASA panel is needed Active infection (no discrimination viable / nonviable agents) Short term shedding of pathogens When sample required for PCR is suboptimal Biological samples Environmental check (?)

50 Other Methods Microbiologic culture - method of choice for most bacteria and fungi Difficult or impossible to cultivate some organisms Suppression of growth by competing organisms Parasitology fecal flotation, direct microscopy, examination of skin or pelage, cellophane tape Gross and histopathology can suggest most likely etiology of disease for further investigation; can be used to confirm diagnosis 50

51 Positive Finding? Single positive result may not mean anything! Retest by same and other test methods If first animal confirmed positive test additional animals Interpret based on all test results and experience If results are confirmed and make sense... Time to implement your action plan! 51

52 Protecting Mouse Health Introduction of new animals Housing and husbandry Use in research 52

53 Receipt Check shipping box for integrity Thoroughly disinfect the outside of the box Briefly check each mouse as it s unpacked 53

54 Introduction News animals (Three Options) Send to colony room Quarantine Rederive Greatest risk Least risk Least expensive Most expensive 54

55 Pros Cons Send to room Immediate access Greatest risk to colony; no built in acclimatation Quarantaine Reduced risk for colony Delayed access; space &cost Rederive Least risk to colony eliminate future pathogens Delayed access; space &cost Recommend 3-5 days Benefit depends on procedures & testing 55

56 Quarantaine Principles Physically separate from established colonies Separate animals by source (& shipment if possible) Dedicated caretakers, caretakers who care 1º for nonrodents, or end of day Handle latest arrivals last At least 3 wk; longer better Test before release 56

57 Rederivation Newborn fostering most risky Hysterectomy derivation long history of proven effectiveness with most pathogens Embryo transfer / in vitro fertilization faster; effectiveness depends on thorough washing Ovarian transplantation : Unlikely to be effective with all organisms 57

58 Levels of Protection Conventional Low barrier High barrier Isolator Risk Cost/incovenience Risk Cost/incovenience 58

59 Barriers and animal Health Barrier separates/isolates the animal from the surrounding environment Prevents introduction +/- spread of unwanted infectious agents Levels (and effectiveness) vary from maximum to minimum Barrier may be at the cage or at the room/facility Both physical and operational elements Adherence to well-designed procedures is crucial to success 59

60 Facility/Room Level Barrier 1 focus on preventing introduction of contaminants Exclude wild rodents Air pressure differentials (usually + vs. corridor) HEPA-filtered air Animals in open caging, changed on open tables May also utilize cage-level barrier to prevent spread 60

61 Barrier Procedures Maximun Barrier Rederive mice Shower (wet or air) Sterilize water, e.g. RO, UV, filtered, acidified, chlorinated Low Barrier Quarantine mice Lab coat or gown + shoe covers Tap water Cap, mask, gloves, restricted entery 61

62 Cage Level Barrier Isolates individual cages (microisolater, IVC) Prevents introduction and spread of contaminants Emphasis on procedures 62

63 IVC Technique Sterilize all materials contacting mice No cages opened outside of change station All materials entering change station sterilized or cleaned with high-level disinfectant Gloved hands frequently disinfected Sleeve guards increase protection Handle mice with disinfected forceps Avoid touching materials that will come into direct contact with mice, e.g., inside of cage 63

64 Isolators Can be viewed as very small maximum barrier rooms Sterilize all materials entering isolator Disinfection of ports before introduction or removal of materials is critical 64

65 What Level of Barrier Is Needed? Exclude only rodent-adapted pathogens or opportunists? 1 focus on preventing: introduction of contaminants via mice or tissues spread of contaminants from one colony to another Exclude opportunists that are widespread in environment or carried by humans? Additional focus on materials, people, air, water At least high barrier maximum or isolator better for long term success 65

66 Animal Care Dedicated caretakers if possible Handle higher health status before lower Caretaker training critical Your operation is only as good as your caretakers! 66

67 Care during experimental use as important as husbandry! 67

68 Research In Barrier Facilities Folow all barrier procedures Ventilated changing station/hoods whenever possible One-way trips best Need to use shared resources (e.g. irradiateurs, phenotyping equipment) or remove animals from barrier ( e.g. lab)? 68

69 News Technological Developments in Health Monitoring of rodent colonies Serban Morosan, DVM, PhD, Dip. Vet LAS UMS 28 phenotype du petit animal Faculty of Medicine Pierre et Marie Curie, Paris 69

70 Maintaining an SPF Facility Depend on Realiable screening of incoming biological materials and animals Maintain o good biesecurity program Reliable health monitoring of resident animal to detect problems and to certify SPF status Monitor key prosesses such as autoclaving, e.g 70

71 Biological Microbial contamination Mycoplasma Viruses Bacteria &fungi Biological : - Cell lines - Serum - Antibody - Material archived in freezers 8-10 % are contaminated with Mycoplasma 2% are contaninated with a human or rodent pathogen 6% of cell lines contaminated with slow-growing bacteria or fungi ATCC: The Global Bioresource Center 71

72 Questions to determine if incoming animals threaten the facility Are you positive? Are you infective? 72

73 Incoming animals (if quarantine) Time : 6-8 weeks after arrival Sample(s) : live mice (1-2 per shipment) Protocol(s) : HM complete ( results in 1-2 we) Final time : 8-10 weeks 73

74 Incoming animals (if quarantine) Test by mouse line few days post-arrival 1. Faecal Pellets : a faecal pellet from each rodent 2. Oral Swabs : respiratory agents 3. Skin Swabs : C. bovis and ectoparasites Time : 2-3 day Sample(s) : samples (pooled) from up to 10 mice Protocol(s) : PCR ( results in 1-2 we) Final time : 2-3 weeks 74

75 Incoming animals (if quarantine) Bact Fungi Respiratory Skin Enteric Oral Swab Skin swab Feces Viruses Enteric Feces Parasites All Pinworms and Protozoa Ectoparasites Serum Feces Pelt/cage Swab 75

76 Optimal quarantine Criteria for Testing Program (example) 1. Exclusion list ( identifies all pathogens on your exclusion list) Detect organism that are being shed, e.g. pinworms Detects antibodies to current and past infections, e.g. MPV Detects organism that are not readily transmitted to sentinels, e.g. Pasteurella, Helicobacter 2. Minimizes quarantine time Animals are tested directly No need to wait for sentinels to become infected or seroconvert Time : 2-3 weeks 3. Minimizes costs No sentinels 76

77 Questions associated with sentinel screening 1. The infection is transmitted to the sentinel cage? 1. Biological materiel is enough to infect the sentinel? 1. Is the sentinel receptive ( line, age..) 1. Is the exposure long enough? 1. What do you do if you have a positive result? 77

78 Variables associated with sentinel screening 1. Infected animal may not be shedding substantial doses of the agent 2. Frequency and amount of bedding transferred to the sentinel cages 3. Some agents may not survive well outside of the host e.g. enveloped virus, helicobacter 4. The age and strain of sentinel animal can impact susceptibility 78

79 Infectious agents and IVC 1. Agent that are likely to reach the sentinel cage 2. Agent that are likely to reach the sentinel cage but lose infectivity 3. Agents that are less likely to reach the sentinel cage 79

80 Sentinel testing 1. Proven to detect many prevalent pathogens 2. Soiled bedding transfer easy to implement 3. Survey many cages for pathogens 4. Misses pathogens not transmitted in bedding 5. Positive results = disease present for many weeks 1. Best accuracy Sentinel testing 2. May not have access to colony animals 3. Run more test to screen all colonies : high cost Environmental monitoring 1. Survey many cages for pathogens 2. Accuracy dependent on sample location ( cage vs rack vs room) 3. Cannot detect pathogens that are not shed 80

81 Optimize your health monitoring program Optimal quarterly health monitoring requires a combination of samples from sentinels, colony animals and environment? 81

82 «Success is the sum of small efforts repeated day in and day out» Robert Collier Questions? 82