Chapter 11. Microbiologial Quality Control for Laboratory Rodents & Lagomorphs Flashcards
Henry Foster
- Veterinarian who founded Charles River in 1948
- Pioneer in developing biosecurity systems to eliminate and exclude pathogens
Axenic & associated rodents mentioned in the Cesarean-originated barrier sustained (COBS) process are classified as?
- Gnotobiotic = completely known microbiota
- In contrast, barrier housed rodents are not gnotobiotic because acquire microbiota from environment, supplies, personnel - they are called SPF
Health monitoring for rodents
- Traditionally included direct gross and microscopic examinations of animal specimens, microbiology with culture, serology
- Now PCR common
- Commercial suppliers discontinued direct room-to-room transfers based on negative health monitoring results, as this surely contributed to inadvertant dissemination of unrecognized pathogens
Incidence
The rate of new contaminations or outbreaks
Prevalence
% positive within a time period
Genetically engineered mice and pathogens
- GE mice represent a significant source of adventitious agents not present in commercial colonies & capable of confounding experiments
- Import-export between institutions
Current Good Maufacturing Practices (CGMPs) for pharmaceuticals
- Strict control of production processes & comprehensive quality testing
- Similar to microbiological quality control of research animals and biological reagents
Principals or Principal Animals
The animal populations, or groups, being monitored, whether resident or in quarantine
-Residents include colony and study animals housed at a facility
Repeat testing
Containing & eradicating adventitious infections is costly = repeat testing is crucial, employing complementary methodologies if possible, to verify infection of resident animals before taking remedial measures
Exclusion lists for SPF mice and rats are more extensive than for rabbits and other common lab species
1) Mice and rats account for the vast majority of animals used in research - more is known about their indigenous pathogens
2) The diversity of inbred, and naturally and genetically engineered immunodeficient mutant rodent strains highly susceptible to infectious disease, in conjunction with sensitive immunoassay methods, and advances in molecular genetics, has contributed to the discovery and characterization of rodent pathogens found to be the cause of ubiquitous, inapparent infections of lab rodent colonies
3) Predominance of murine rodent research models has provided strong incentives for diagnostic laboratories and vendors to develop specific serology & PCR assays
4) Rederivation by hysterectomy or embryo transder (ET) to eliminate all exogenous pathogens is the standard practice for SPF mice and rats, but not other species
How are pathogenic bacteria and fungi distinguished from commensal & autochthonous (indigenous) organisms for SPF exclusion?
By their ability to establish themselves in niches devoid of other microorganisms such as the lower respiratory tract, urogenital tract, internal organs, & intracellularly
Primary pathogen
Can cause disease in an immunocompetent host
Examples: Salmonella, Mycoplasma pulmonis,
Helicobacter hepaticus, and Clostridium piliforme (the etiology of Tyzzer’s disease)
-For the most part, only primary pathogens are included on SPF exclusion lists for immunocompetent animals
Opportunistic pathogen
Cause disease mainly in immunocompromised hosts
Examples: Pseudomonas aeruginosa, β-hemolytic streptococci, Staphylococcus aureus, and Pneumocystis fungi
SOPF
Specific opportunistic pathogen-free
Lab animal biosecurity
Consists of all measure taken to eliminate, exclude, contain, and eradicate adventitious infections
Rederivation
- The most dependable approach for eliminating infections with
- Embryo transfer into pseudopregnant recipient females has supplanted nursing of C-section originated pups by gnotobiotic or SPF foster mothers as GOLD STANDARD
- C-section less reliable than embryo transfer d/t vertical transmission of viruses to fetuses & bacteria capable of colonizing uterus
- Neonatal transfer of pups after birth to SPF foster mothers after being immersed in disinfectant - cheaper, does not require euthanasia of breeders
Why is vertical transmission by embryo transfer unlikely?
- Zona pellucida that surrounds the embryos and oocytes excludes pathogens
- Risk of infecting recipient female minimized by extensive washing of embryos
Benefits of embryo transfer over C-section rederivation
- ET can also be combined with IVF & intracytoplasmic injection of sperm (ICSI) to overcome reproductive defects common in aged and GE mutant mice
- ET is more efficient because embryo donors are usually superovulated resulting in more offspring per female
- Embryos, ova, and sperm can be cryopreserved to reduce per diem costs, cage space
When is C-section rederivation still preferred over embryo transfer?
Certain GE rodents lines with fertility issues or whose embryos exhibit low viability in culture
Purpose of defined nonpathogenic bacterial cocktails given to ex-germfree animals
- Resist colonization by pathogenic microbes
- Normalize host physiology
- Stimulate the immune system
Cesarean-originated barrier-sustained (COBS)
Process for large-scale production of SPF rodents - gravid uterus pulled through disinfectant solution into sterile flexible film isolator where pups removed & suckled on axenic (germ free) foster dams - exposed to cocktail of nonpathogenic bacteria - transferred to barrier rooms
Alternatives to rederivation
Chemotherapy & Test-and-cull procedures
Chemotherapy as an alternative to rederivation
- Mainly used to cure or prophylactically treat pinworms & mites
- Avermectins (ivermectin, selamectin) & benzimidazoles (fenbendazole)
- Added to diet, drinking water, applied topically
Toxicity of ivermectin
When given to rodents with compromised BBB b/c of young age, genetic background, or GE mutations
Antibiotic treatments effective to eliminate which agents?
P. pneumotopica, H. hepaticus
-These agents do not survive for long ex vivo & therefore unlikely to reinfect hosts after treatment stopped
Test and cull as alternative to rederivation
- Viral infections cannot be treated, but can be eradicated with test and cull
- Stop breeding - test 100% of cages at regular intervals by serology, PCR, or both - positive cages culled - repeat until remaining cages repeatedly negative or the racks depopulated & resident animals euthanized, relocated, or rederived
Test and cull procedures can be effective for which rodent pathogens?
Helicobacter spp. Murine parvoviruses MHV MNV Murine rotavirus
Breeding moratorium (Burnout)
- Historical alternative to rederivation for nonpersistent infections of immunocompetent hosts w/ enveloped viruses (Sendai, SDAV)
- 6-8 wk moratorium on breeding & introduction of new animals
- Expected that colony animals would recover from infx, stop shedding virus & excreted virus would quickly become noninfectious
- NOT recommended in contemporary rodent colonies - if tried despite this, confirm eradication by PCR or serology of sentinels rather than colony offspring that will likely have maternal antibodies
What is the most prevalent pathogen in mice?
MNV - shed indefinitely
Barrier systems
- Opportunistic pathogens not reliably excluded - production of rodents that need to by SOPF involves transfer from barrier rooms to isolators & filter-covered microisolationg cages
- IVC cages can be exhausted directly to facility HVAC to improve room environment & run under negative pressure for pathogen containment
- Microisolation cages should only be opened in a HEPA-filtered air laminar flow change station or biological safety cabinet
For lab animal, is a pathogen’s source or reservoir more practical to control?
-Much more practical to control source (may be feed, bedding, water, caging, biologics) rather than reservoir (may be wild animal, the environment)
Which murine virus is mainly spread by parenteral injection of transplantable mouse cell lines?
Lactate dehydrogenase-elevating virus (LDV)
-Contaminates a basement membrane matrix used by tumor biologists and the cell line from which it was derived
What is droplet nuclei?
The residue of dried droplets
What types of infections are not transmitted efficiently in soiled bedding?
- Host-adapted bacteria
- Enveloped viruses
- Other organisms that are unstable ex vivo
Most common source of MPV and mouse rotavirus infections in contemporary facilities?
Food & bedding
-Gamma-irradiate or autoclave to reduce risk
Lice are biological vectors for which rodent pathogens
Eperythrozoon coccoides
Mycoplasma haemofelis
What is the most likely animal reservoir of infection for SPF rodent colonies?
Other rodents - wild or feral (escaped)
Recommended procedures for captured wild rodent
- Identify to species
- Handle as if infected
- Anesthetize & collect blood for serology, examine for internal and external parasites
- Submit tissues, feces, swabs for PCR
GE mutant mice at academic institutions have a high prevalence of infection with which agents?
MNV, Helicobacter spp, P. pneumotropica, parasites
Quarantine protocols for incoming rodents
- 4-8 week quarantine with cohousing with sentinels
- 2 wk quarantine with PCR testing of feces, fur, oral cavity
- Direct PCR testing of animals is more sensitive than indirect sentinel screening
In general, which pathogen stages are resistant to inactivation?
Bacterial spores, free-living stages of parasites (pinworm eggs, protozoan cysts), and hydrophilic nonenveloped viruses (MPV)
Temperature for heating of food during pelleting
75-80 C
-Substantially reduces bacterial count but not sufficient to inactivate thermostable pathogens
Drawbacks of autoclaving
- Greater reduction in nutritional value of food vs. gamma irradiation
- Difficulty in achieving uniform steam penetration and temperature throughout a load
- Presterilization vaccuum cycles help preserve nutritional value of food by promoting rapid and uniform steam penetration
Gamma irradiation
- Emitted from a cobalt-60 source
- More expensive than autoclaving
- Breaks nucleic acids in microorganisms
- Passes through solid objects
UV radiation
- 210-328 nm
- Does not possess sufficient energy to cause ionization
- Also inactivates microorganisms by damaging their DNA, but does NOT cause DNA breakage
- Produces thymine and other pyrimidine dimers
- Bactericisal activity is maximal near peak of DNA absorption (260 nm)
- Does not pass through solid objects - only for surfaces or drinking water
- Attractive for water compared to chlorination b/c does not convert organic precursors into potentially carcinogenic trihalomethanes
Radiosensitivity of organisms corresponds with what properties
- Genome volume
- Ability to repair DNA damage
- Why small viruses (parvoviruses) highly resistant to UV & gamma irradiation, as are bacterial spores, protozoan cysts, and vegetative bacteria with highly efficient DNA repair capabilities
Filtration
- Depth filters entrap and adsorb - no pore size so do not have ratings
- Membrane filters exclude particles according to pore size
- 99.97% rating given to HEPA filters based on 0.3-μm particle retention size
Microfiltration
Range 0.1–10.0 μm
- Retains bacteria, fungi, spores
- Cannot be relied upon to exclude viruses
Ultrafiltration
Range 1000–1,000,000
molecular weight
-Can remove viruses from water
Reverse osmosis
Low-molecular weight
molecules, including salts
-Can remove viruses from water
Types of chemical disinfection
- Denaturants that disrupt protein or lipid structures
- Reactants that form or break covalent bonds
- Oxidants (chlorine dioxide, bleach, vapor phase H2O2, peroxygen Virkon S) are most frequently used b/ considered more effective for inactivating resistant pathogens (spore-forming bacteria, nonenveloped viruses, free-living parasites form)
Denaturants
Quaternary ammonium compounds (benzalkonium chloride)
Phenolics
Alcohols
Reactants
Aldehydes (formaldehyde, glutaraldehyde)
Ethylene oxide
