Chapter 11. Microbiologial Quality Control for Laboratory Rodents & Lagomorphs Flashcards
1
Q
Henry Foster
A
- Veterinarian who founded Charles River in 1948
- Pioneer in developing biosecurity systems to eliminate and exclude pathogens
2
Q
Axenic & associated rodents mentioned in the Cesarean-originated barrier sustained (COBS) process are classified as?
A
- Gnotobiotic = completely known microbiota
- In contrast, barrier housed rodents are not gnotobiotic because acquire microbiota from environment, supplies, personnel - they are called SPF
3
Q
Health monitoring for rodents
A
- 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
4
Q
Incidence
A
The rate of new contaminations or outbreaks
5
Q
Prevalence
A
% positive within a time period
6
Q
Genetically engineered mice and pathogens
A
- GE mice represent a significant source of adventitious agents not present in commercial colonies & capable of confounding experiments
- Import-export between institutions
7
Q
Current Good Maufacturing Practices (CGMPs) for pharmaceuticals
A
- Strict control of production processes & comprehensive quality testing
- Similar to microbiological quality control of research animals and biological reagents
8
Q
Principals or Principal Animals
A
The animal populations, or groups, being monitored, whether resident or in quarantine
-Residents include colony and study animals housed at a facility
9
Q
Repeat testing
A
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
10
Q
Exclusion lists for SPF mice and rats are more extensive than for rabbits and other common lab species
A
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
11
Q
How are pathogenic bacteria and fungi distinguished from commensal & autochthonous (indigenous) organisms for SPF exclusion?
A
By their ability to establish themselves in niches devoid of other microorganisms such as the lower respiratory tract, urogenital tract, internal organs, & intracellularly
12
Q
Primary pathogen
A
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
13
Q
Opportunistic pathogen
A
Cause disease mainly in immunocompromised hosts
Examples: Pseudomonas aeruginosa, β-hemolytic streptococci, Staphylococcus aureus, and Pneumocystis fungi
14
Q
SOPF
A
Specific opportunistic pathogen-free
15
Q
Lab animal biosecurity
A
Consists of all measure taken to eliminate, exclude, contain, and eradicate adventitious infections
16
Q
Rederivation
A
- 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
17
Q
Why is vertical transmission by embryo transfer unlikely?
A
- Zona pellucida that surrounds the embryos and oocytes excludes pathogens
- Risk of infecting recipient female minimized by extensive washing of embryos
18
Q
Benefits of embryo transfer over C-section rederivation
A
- 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
19
Q
When is C-section rederivation still preferred over embryo transfer?
A
Certain GE rodents lines with fertility issues or whose embryos exhibit low viability in culture
20
Q
Purpose of defined nonpathogenic bacterial cocktails given to ex-germfree animals
A
- Resist colonization by pathogenic microbes
- Normalize host physiology
- Stimulate the immune system
21
Q
Cesarean-originated barrier-sustained (COBS)
A
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
22
Q
Alternatives to rederivation
A
Chemotherapy & Test-and-cull procedures
23
Q
Chemotherapy as an alternative to rederivation
A
- Mainly used to cure or prophylactically treat pinworms & mites
- Avermectins (ivermectin, selamectin) & benzimidazoles (fenbendazole)
- Added to diet, drinking water, applied topically
24
Q
Toxicity of ivermectin
A
When given to rodents with compromised BBB b/c of young age, genetic background, or GE mutations
25
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
26
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
27
Test and cull procedures can be effective for which rodent pathogens?
```
Helicobacter spp.
Murine parvoviruses
MHV
MNV
Murine rotavirus
```
28
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
29
What is the most prevalent pathogen in mice?
MNV - shed indefinitely
30
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
31
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)
32
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
33
What is droplet nuclei?
The residue of dried droplets
34
What types of infections are not transmitted efficiently in soiled bedding?
- Host-adapted bacteria
- Enveloped viruses
- Other organisms that are unstable ex vivo
35
Most common source of MPV and mouse rotavirus infections in contemporary facilities?
Food & bedding
| -Gamma-irradiate or autoclave to reduce risk
36
Lice are biological vectors for which rodent pathogens
Eperythrozoon coccoides
| Mycoplasma haemofelis
37
What is the most likely animal reservoir of infection for SPF rodent colonies?
Other rodents - wild or feral (escaped)
38
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
39
GE mutant mice at academic institutions have a high prevalence of infection with which agents?
MNV, Helicobacter spp, P. pneumotropica, parasites
40
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
41
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)
42
Temperature for heating of food during pelleting
75-80 C
| -Substantially reduces bacterial count but not sufficient to inactivate thermostable pathogens
43
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
44
Gamma irradiation
- Emitted from a cobalt-60 source
- More expensive than autoclaving
- Breaks nucleic acids in microorganisms
- Passes through solid objects
45
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
46
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
47
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
48
Microfiltration
Range 0.1–10.0 μm
- Retains bacteria, fungi, spores
- Cannot be relied upon to exclude viruses
49
Ultrafiltration
Range 1000–1,000,000
molecular weight
-Can remove viruses from water
50
Reverse osmosis
Low-molecular weight
molecules, including salts
-Can remove viruses from water
51
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)
52
Denaturants
Quaternary ammonium compounds (benzalkonium chloride)
Phenolics
Alcohols
53
Reactants
Aldehydes (formaldehyde, glutaraldehyde)
| Ethylene oxide
54
Oxidants
Halogens (chlorine bleach, chlorine dioxide, povidone-iodine)
Peroxygens (vapor-phase H2O2, Virkon® S)
55
Klein-DeForest Scheme for Viral Sensitivity
-Associates sensitivity to disinfectants with viral solubility
-Phenolics and quaternary
ammonium compounds, which disrupt lipid membranes, are more potent against lipophilic, enveloped viruses than against hydrophilic, nonenveloped viruses
-Oxidants attack all organic compounds and thus inactivate hydrophilic as well as lipophilic viruses
56
Klein-DeForest Category A
Lipophilic; lipid envelope + capsid
Paramyxo (Sendai, PVM)
Corona (MHV, SDAV)
Arena (LCMV)
57
Klein-DeForest Category B
Hydrophilic; naked capsid
Picorna (TMEV)
Parvo (MVM, MPV, KRV,
RPV)
58
Klein-DeForest Category C
Intermediate; partially lipophilic capsid
Adeno (MAV-1,2)
Reo (Reo-3)
Rota (EDIM, IDIR)
59
Scale for Susceptibility of Laboratory Animal Pathogens to Disinfectants
A: Enveloped viruses, non-spore-forming bacteria
B: Partially lipophilic, nonenveloped viruses
C: Hydrophilic, nonenveloped viruses
D: Bacterial endospores and parasite ova and cysts
60
Why does increasing pH or temperature of water render chlorine less effective?
-Reduces of concentration of hypochlorous acid in favor of hypochlorite (OCl-) ion, which is less biocidal
61
Why should surfaces be cleaned prior to disinfection?
Inorganic substances exert a chemical demand that reduces effectiveness of disinfectant agents
62
Which agents can be used to remove biofilms in water?
- H2O2
| - Alkaline peroxide
63
Why is it important to test biologics for bioburden?
- Eliminate pathogens
- A high bioburden is problematic even when free of pathogens b/c commensal bacteria are more likely to cause disease by circumventing natural host defenses when parenterally injected along with tumor cells into immunodeficient recipients
64
Direct gross & microscopic examination of animal specimens
Techniques include:
- Pathology: gross & histopathology
- Special stains, IHC, in situ hybridization
- Parasitology with dissecting microscopy - gold standard for helminth infx
- Phase-contrast microscopy: helminths & mites, protozoan cysts
65
Fur mite microscopy for mice
- Fur mites can be found in a higher percentage of mice by microscopic exam of adhesive tape applied to the dorsal fur than by checking skin or skin scrapings
- Fur plucks also give reasonable accurate results (between scapulae, base of tail)
- Pelt digestion
66
Limitations of gross & microscopic examination
- Lesion are seldom diagnostic
- Low sensitivity: lesions & organisms must be present in high concentrations to be observed
- Sample pools control costs, but have low sensitivity
- Postmortem exams usually need to be restricted to a small number of sentinel animals & sentinels may not be positive for agents in the resident rodents
67
Minimum concentration of virus that can be detected by transmission electron microscopy
10^5-10^6 particles per milliliter
68
Microbiology techniques for health monitoring
- Cultural isolation: sites most often sampled (upper resp & large intestine) possess a complex microbiome that can overgrow cultures & obscure colonies of interest - use selective media
- MacConkey's agar: contains crystal violet and bile salts that selectively inhibit growth of G(+) bacteria; lactose fermenting will be pink to red, non lactose fermenting will be colorless
- Helicobacter media contains an abx mixture to inhibit growth of intestinal microbiome
- Selenite broth: salmonella in feces or intestinal tract
- Cultures usually incubated aerobically at 35-37 C b/c clinically impt bacteria are usually facultative anaerobes, will strict anaerobes of autochthonous microbiome will not grow
69
Most reliable sample to isolate Corynbacterium kutscheri in rats
Submaxillary lymph nodes
70
Fastidious and noncultivable microbial pathogens
- M. pulmonis
- Helicobacter spp.
- C. piliforme
- CAR bacillus
- Pneumocystis spp.
- Use PCR instead for these
71
Serotypes for salmonella
Based on somatic O & flagellar H antigens
72
Lancefield classiciation system for beta-hemolytic streptococi
Based on group-specific cell-wall carbohydrate (C) antigens
73
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)
- Run on actively growing cultures or extracts made from them
- Based on unique peptidic spectra primarily of ribosomal & other house-keeping proteins that are expressed at high levels
- Peak intensity and position of the spectra are compared to a database
74
Limitations of microbiology
- Limited to microorganisms that can be cultivated in cell-free media
- Usually need postmortem samples = limited to indirect surveillance of sentinels
- Bacteria of interest need to be viable & present in high numbers to detect among the normal microbiome
75
When are serum antibody responses detectable?
Usually by 1-2 weeks post-infection and can last for long periods (at least months or sometimes life of rodent)
76
Homogenous serology techniques
- Homogenous = indicates sample & assay reagents are mixed and incubated together in solution
- Hemagglutination inhibition (HAI)
- Complement fixation
- Virus neutralization
77
Indirect Enzyme-linked immunosorbent assay (ELISA)
Antigen (purified virus particles, microbial cell extracts, recombinant proteins) immobilized on surface of wells in microtiter plates ("solid phase")
- Some well coated with extract of noninfected host cells ('tissue control') to detect nonspecific binding of immunoglobin (Ig)
- Test serum added (with Ab to antigen)
- Enzyme-labeled anti-Ig added - binds to test Abs
- Enzyme substrate added - create color changes on bond enzyme-labeled anti-Ig
- Measure color change (optical density w/ spectrophotometer, plate reader) = amount of enzyme-labeled anti-Ig = amount of serum antibodies
- ELISA is primary screening test (over IFA)
78
Immunofluorescence assay (IFA)
- Infected & uninfected cells fixed to wells with cold acetone - makes cell membranes permeable so intracellular viral antigens are accessible to Abs in serum samples
- Examined manually by fluorescence microscope = ELISA better than IFA for high throughput
- IFA is excellent confirmatory test; can tell if infection in host cell nucleus or cytoplasm or both
- Can be more 'inclusive' (able to detect heterologous viral strain and serotypes) than ELISA
79
Heterogenous serology techniques
- Each incubation period followed by a wash step
- ELISA
- IFA
80
Indirect serology techniques
-Indirect = detection of serum Abs bound to the solid phase by labeled anti-Ig or Ig-binding bacterial proteins (Proteins A & G)
81
Common radioisotope labels
- ELISA: horseradish peroxidase, alkaline phosphatase
| - IFA: fluorescent dye fluorescein
82
Direct serology techniques
- Direct = a label is coupled to the microbial antibodies rather than anti-Ig or Ig-binding bacterial protein
- More common for research on time course and distribution of infections in situ
83
Cons of solid-phase antigen capture immunoassays
- Active infection and shedding often short-lived
- Even when infx is active, the concentration of microbial antigens in animal specimens may be below the assay's detection limit
- Obtaining microbial antibodies may be a long & expensive process
84
Human group A rotavirus kits can be used to detect which rodent virus?
Mouse rotavirus in feces
85
Inclusivitity of IFA
Its ability to detect antibodies to the constructural proteins
conserved among rodent parvoviruses in mouse and rat populations that were largely MVM and rat virus (RV) seronegative by serotype-specific HAI and by ELISA with virus particle antigen) provided the initial evidence for the existence of then-novel rodent parvoviruses later
identified as MPV, rat parvovirus, and rat minute virus
86
Inclusivity of an assay enhances diagnostic sensitivity or specificity?
Sensitivity - more inclusive, better as a primary screening assay
-Risk of false negatives if the antibody response to the conserved antigen is delayed, weak, absent (ex: antibody response of rodents to the parvovirus nonstructural protein NS1)
87
Why do solid-phase immunoassays tend to be more inclusive than corresponding traditional test?
Can detect antibodies to any of the epitopes (i.e., antigenic sites) presented by the microbial antigens attached to the solid phase and at much lower levels
88
Why are virus neutralization and HAI highly exclusive?
Only recognize antibodies to viral surface protein antigens that are unique to the serotype or strain of virus being used in the test
- Not good for screening
- Good for confirmatory testing
89
Recombinant DNA technology
- Made it possible to produce large quantities of recombinant protein (important for sold-phase immunoassays) rapidly from a cloned gene of interest inserted into microbial, mammalian, or baculovirus (an insect virus) expression vector systems
- Can produce pure, potent, noninfectious antigen
- Recombinant viral capsid proteins, such as parvovirus VP2, self-assemble into virus-like particles, or VLPs, than can be purified by gradient centrifugation & other techniques
- Incorporating recombinant protein antigens representing different viral strains and proteins makes serosurveillance panels more inclusive
90
Luminex Multi-Analyte Profile (xMAP) platform
- Suspension microarray
- Tests are colorcoded with beads that are covalently bonded to antigens or controls - beads flow through modified flow cytometer where bead color set (test) & emitted phycoerythrin fluorescence (median fluorescence intensity; MFI - across all beads for a specific test) are measured
- Indirect, heterogenous solid-phase tests
- Abbreviated MFI or MFIA
- Diagnostic performance comparable to ELISA
- Include bead set coated with Ig of the test species to detect procedural errors (e.g., failure to add anti-Ig to a well) & set coated with anti-test species Ig to identify samples with inadequate levels of Ig (failures of this usually from animal that is immunocompromised or different from the test species)
91
Dried blood spot (DBS)
New method to collect small volume samples for multiplexing tests
92
Limitations of serologic tests
- Applicable mainly to viruses & seldom bacteria (except M. pulmonis, CAR bacillus, E. cuniculi) or fungi d/t poor specificity compared to viruses
- Bacteria and fungi induce weak antibody responses unless invasive
- Require immunocompetent host
- Period of 7-10 days at least btwn infx and seroconversion
93
Hybridization assays
- Labeled reported probes are directly or indirectly labeled with a radioisotope an enzyme that acts on a chromogenic or chemiluminescent substrate, or a fluorescent dye
- Immobilize the sample nucleic acid by blotting onto nitrocellulose or nylon membrane, OR target sequences captured by unlabeled probe attached to a solid phase
- Denature double stranded sample and probe DNA by heating (90-100 C) or alkaline conditions
- Reaction mixture then cooled (55-65 C) to permit formation of stable probe-target hybrids (RNA to RNA, DNA to DNA, or DNA to RNA)
- Degree of hybridization determined by measuring the signal emitted by the probe label or the enzyme-substrate product
94
Southern blot
DNA
95
Northern blot
RNA
96
Effect of raising temperature of incubation during hybridization?
Enhances assay specificity by increasing the degree of complementarity necessary for stable probe-target hybrids to form
97
Limitations of hybridization
- Constrained by quantity of organisms found in specimen
| - Nonspecific binding of the labeled probe
98
PCR main components
(1) Synthetic oligonucleotide
primers (15–25 bases long) that anneal in opposite
directions to complementary strands of the DNA template
at sites separated by up to 500 base pairs (for surveillance
assays)
(2) A heat-stable DNA polymerase (such as the Taq DNA polymerase originally isolated from the thermophilic bacterium Thermus aquaticus) with
the unique ability to tolerate the 95°C denaturation
99
PCR DNA polymerase originally isolated from what bacterium?
Theromophilic bacterium: Thermus aquaticus
100
Conserved vs. Differentiating PCR targets for parvoviruses
Conserved = non-structural NS-1 gene
| Differentiating capsid gene
101
Common gene targets for PCR
- Bacterial & parasitic ribosomal genes
| - Bacterial RNA polymerase rpoB gene
102
Reverse transcriptase (RT)-PCR priming options
- Sequence-specific primers: the most efficient
- Nonspecific primers: oligo-dT (anneals to the polyA sequence appended to RNA transcripts) & random hemaxers
- Nonspecific priming preferred when testing for multiple RNA viruses
103
Steps of PCR
Denaturation (95 C)
Annealing (50-70 C)
Elongation (72 C)
-PCR has 30-50 of these cycles done automatically by a thermocycler
104
Gel-based PCR
PCR product (amplicon) is identified in an ethidium bromide-stained gel electrophoretogram exposed to UV light as a visible fluorescent band of an expected size
105
PCR primer binding
Bind at 3' end
106
Labeled probe hybridization
More specific, sensitive, and amenable to automation and computer data processing than the gel-based method
107
TaqMan assay
- Amplification and hybridization occur concurrently
- The TaqMan probe is an oligonucleotide that anneals to a DNA template sequence between the forward and reverse primers & is tagged on opposite ends with a fluorescent reporter dye and a quencher dye
- When extending a primer, the Taq DNA polymerase uses its 5'-3' exonuclease activity to digest annealed probe - resultant separation of reporter from its quencher generates a sequence-specific fluorescent signal that can be read after each amplification cycle ('real time')
- Number of cycles required to reach a threshold signal (Ct) is inversely related to the copies of DNA template added to the reaction
108
Real-time PCR assays
Called quantitative PCR: qPCR
109
Advantages of TaqMan qPCR technique
- Estimating the copy number is helpful for identifying and discounting low-copy positive results due to contamination with template from other samples, controls, environment
- Better analytical specificity and sensitivity than gel-based d/t internal probe
- Less risk of contamination because reaction tubes stay closed post amplification
- Higher throughput because no post-PCR processing steps
110
Limitations of PCR
- Detection of microbial nucleic acid template can occur in absence of infection (false positives) - contamination of samples at even minute levels
- May miss agents that are shed transiently (although has been shown to detect MHV & MPV in fees for weeks to months after infected mice are no longer contagious)
- Will continue to detect template in EAD swabs even after animals have stopped shedding
- Can have false negatives due to sample-mediated inhibition (detect by using internal control assay)
- Labor-intensive & costly
111
Sample classified as indeterminate
- Equivocal (+/-) compared to positive and negative cutoff values
- Sample reacted nonspecifically or was inhibitory
- Quantity or target concentration was insufficient
112
Assay optimization
The goal of assay optimization is to reduce the percentages of samples that yield false-negative (FN), false-positive (FP), or +/− results by increasing the specific signal given by KP samples and decreasing
the background ‘noise’ level and variation for KN samples. Following optimization, cutoffs can be adjusted to favor DSe or DSp depending on whether FN or FP determinations are more problematic
-Assay cutoff values might
be increased to favor DSp and avoid FP results in tests
for rare pathogens; conversely, they may be decreased to enhance DSe when testing for common agents to reduce the likelihood that adventitious infections will be missed
due to FN findings
113
Assay sensitivity
- Measured as the limit of detection (LOD): the lowest concentration of a target analyte in a specific matrix that can consistently yield a positive result
- Performing serology on acutely infected or PCR on convaslescent hosts can reduce the concentration of the targeted analyte below the test LOD
114
Assay specificity
- Comprises selectivity, inclusivity, and exclusivity
- Inclusivity: ability of a single test to detect related organisms or interest; preferred for primary screening tests
- Exclusivity: describes a test able to differentiate an infectious agent from other that are closely related; preferred for confirmatory testing
115
Example of inclusive viral antigens
- NS1 protein in parvoviruses
- VP6 capsid of group A rotaviruses
- Nucleocapsid NP protein of LCMV
116
T/F. IFAs for viral antibodies are intrinsically inclusive
True. Because they typically use as antigen-infected cells containing all virally encoded proteins
117
Raising the positive cutoff to compensate for higher background is likely to reduce...sensitivity or specificity?
Sensitivity
118
Degeneracy of the genetic code
Multiple codons are translated into the same amino acid = sequence of a gene often varies more than the amino acid sequence of the protein it encodes
-Specificity of PCR exceeds that achievable by traditional tests; also unaffected by environment or host-related factors that can alter phenotype looked at by traditional tests
119
Positive predictive value (PPV)
- PPV = TP / (TP+FP)
- Percentage of all positive results that are true positive
- More false positives are expected with lower prevalence
120
True positive (TP)
Known positive (KP) x Sensitivity
121
False positive (FP)
Known negative (KN) x (1-Specificity)
122
Known Positive (KP)
Tested x Prevalence
123
Known Negative (KN)
Tested x (1-Prevalence)
124
Immune competence of animals and health monitoring
- Immunocompetent: needed to produce antibodies to viral or microbial agents for serology
- Immunodeficient: may enhance sensitivity of surveillance relying on direct detection of infectious agents (PCR, bacteriology, parasitology)
125
If testing resident animals, which age group is most likely to test positive?
Recently weaned to young adult
- Newborns may be protected by maternal antibodies
- Older animals may have been infected a while ago and already cleared infection
126
Why should sentinel animals be outbred stock?
- Generally good serologic responders & less expensive
- Inbred strains have been shown to be comparatively resistant to infection with or to mount a delayed antibody response to certain pathogens
127
Soiled-bedding sentinels
- Transfer as much soiled bedding as possible to minimize diluting of infectious material (5-15 mL from each principal cage)
- Sentinels should be exposed for a minimum of 3 mth before being tested
- Typical recommendation is 1 cage of 2-3 sentinels per 50-80 cage rack or side of rack
- Testing usually performed quarterly
- Sentinels should not be kept for more than 6 mths as sensitivity to certain pathogens may decrease with age
128
Sentinel testing for MPV
- Occasionally not all animals in a cage will seroconvert to MPV (may be missed is only a subset tested)
- Test a second sentinel or collect mesenteric LNs for PCR as a supplement to serology
129
First step when health monitoring finds an unexpected positive result?
Contact testing laboratory & confirm positive results with confirmatory testing!
130
Shedding patterns of example murine pathogens
Rotavirus: Shedding stops quickly & infection cleared, antibodies persist
MPV: Shedding continues for a little longer but eventually stops even though infection continues, antibodies persist
MNV: Infection and shedding persist indefinitely, antibodies also persist
131
Outbreak response
1) Confirmatory testing
2) 100% colony screen
3) Two additional screens at 21- to 28-day intervals
4) Track any relocations or exports that occurred within the last 90 days prior to detection of the excluded agent
132
What testing is required to life a room quarantine?
Two consecutive negative screens of 100% of cages in the room are required to lift quarantine
133
Examples of sterilants
Vapor-phase hydrogen peroxide, chlorine dioxide gas, formaldehyde
134
General plan for an outbreak room
Depopulate the room.
● Discard any nonessential or easily replaceable
equipment.
● Place any materials leaving the room in a bag; spray
the outer surface of the bag with a disinfectant and
then place it into a second bag outside the room.
The bagged caging may then be autoclaved, cleaned,
and then autoclaved again before reentering a clean
room.
● Clean the floors, walls, and room surfaces
thoroughly with a detergent solution and then
rinse.
● After rinsing, apply an aqueous-based disinfectant
as per the manufacturer’s recommendations
(concentration, time, temperature, humidity, etc.).
● Rinse again.
● Apply at least one other aqueous-based disinfectant
(with a different mode of action than the first).
Many facilities will go further and use a sterilant such as vapor-phase hydrogen peroxide or chlorine dioxide.
