1
Q
- Name the two components of microbiological quality control.
A
Biosecurity and health surveillance
2
Q
- Define gnotobiotic, SPF, and conventional animals.
A
Gnotobiotic: animals are axenic (not contaminated by or associated with any foreign organisms) or have a defined microflora consisting of a few nonpathogenic bacteria
Specific pathogen free: animals tested negative for a limited list of exogenous viruses, bacteria, and parasites that may cause disease or otherwise interfere with research
Conventional: animals that are maintained with minimal micriobiological quality control and thus have a nominally defined microflora that often includes pathogens.
3
Q
- T/F Lactate dehydrogenase-elevating virus is easily transmitted from mouse to mouse.
A
F. LDV not easily transmitted from mouse to mouse although causes persistent viremia and excreted in large amounts; transmitted mainly by parenteral injection with contaminated biological materials.
4
Q
- Which common pathogens in mice and in guinea pigs cannot be eliminated by cesarean section or embryo transfer and why?
A
LCMV in mice and cytomegalovirus in guinea pigs because vertical transmitted
5
Q
- Fomite transmission with soiled bedding is the basis of most sentinel programs. Which pathogens won’t be at all or only inefficiently transmitted via soiled bedding?
A
Cilia-associated respiratory bacillus, Sendai virus
6
Q
- What parasites of mice and rats, respectively are transmitted by lice?
A
Eperythrozoon coccoides in mice
Hemobartonella muris in rats
7
Q
- Which pathogens/forms of pathogens are most resistant to inactivation by disinfection?
A
bacterial spores, free-living stages of parasites (e.g. pinworm eggs and protozoan cysts), nonenveloped viruses
8
Q
- All of the following are fomites, except:
a. food, bedding, supplies
b. contaminated water and/or air
c. biologics
d. arthropods
A
d
9
Q
- List 4 different forms of physical disinfection.
A
autoclaving, gamma radiation (usually emitted from 60Co source), UV irradiation, filtration
10
Q
- Autoclaving and electromagnetic irradiation are the treatments of choice for food and bedding. In comparison with gamma irradiation, autoclaving is ________ expensive but causes _________ reduction in the nutritional value of food. A drawback of autoclaving is _______________________________.
A
less, greater, the difficulty in achieving uniform steam penetration and temperature throughout a load
11
Q
- What is the main effect of ionizing irradiation as a disinfectant?
A
renders microorganisms nonviable by causing breakage in their nucleic acid
12
Q
- Does UV irradiation cause DNA breakage?
A
No; but damages DNA by production of thymine and other pyrimidine dimmers; this is a reversible process in contrast to DNA breakage
13
Q
- Filtration is the process most often employed to remove microbes from air and water. The filtration process is classified in ___, ____, and ___ according to the minimum size of particles retained.
A
microfiltration (range 0.1-10.0 um) ultrafiltration (range 1000-1,000,000 molecular weight) reverse osmosis (low-molecular-weight molecules, including salt)
14
Q
- Which filtration can exclude viruses?
A
ultrafiltration or reverse osmosis, microfiltration retains bacteria, fungi, and their spores but not necessarly viruses
15
Q
- Describe depth filters.
A
Depth filters entrap and adsorb.
They have a high dirt-handling capacity and are used for high-efficiency particulate air (HEPA) filtration and for clarification of particle-laden liquids. In contrast, membrane filters exclude particles according to pore size.
16
Q
- What murine viruses could be potentially transmitted via contaminated water, based on relatedness to waterborne human viruses?
A
Picornaviridae - TMEV
Reoviridae - reovirus, mouse rotavirus
Coronaviridae - MHV, SDAV
Adenoviridae - Mouse adenovirus
17
Q
- T/F Virus sensitivity to disinfectants is associated with viral solubility (lipophilic - lipid envelope and capsid; hydrophilic - naked capsid; intermediate - partially lipophilic capsid).
A
- T; phenolics and quaternary ammonium compounds more potent against lipophilic, enveloped viruses; oxidants inactivate hydrophilic as well as lipophilic viruses (see table IV, page 371 for examples)
18
Q
- What are factors that influence efficacy of a chemical disinfectant?
A
temperature, pH, chemical demand of the medium being treated, dirt and organic matter, biofilms
19
Q
- What is the classical test for rodent viral contaminant surveillance?
A
mouse and rat antibody production tests (MAP and RAP), lately also PCR
20
Q
- People and insects can be mechanical vectors. List practices that should be instituted to reduce the risk.
A
1) effective pest control
2) animal care technicians should not have pet rodents
3) limit access to facilities and breeding rooms: dedicated staff for each room if possible, if not flow of people and supplies from clean to dirty, no visitors who had recent contact to other rodent colonies
4) PPE
5) limitation of animal-human contact by housing in microisolation cages
6) manipulation of rodents in laminar flow hoods and handling them with disinfected forceps or disinfected gloves
21
Q
- Gross and microscopic examination of animal specimens may reveal disease during the active phase of an infection, prior to __________. It is sometimes the most reliable diagnostic methodology when a specific in vitro test is unavailable or unsatisfactory.
A
seroconversion
22
Q
- Which etiologic agent was discovered by examination, which can cause hepatitis and hepatocellular carcinoma in mice?
A
Helicobacter hepaticus
23
Q
- In laboratory animal pathology tissues and organs are inspected for gross abnormalities and selected specimens for histopathological changes. The most routinely employed tissue stain is with hematoxylin and eosin whereas microbial antigens or nucleic acid in tissue sections can be specifically stained by ______ or _____.
A
immunohistochemistry or in situ hybridization
24
Q
What is the most effective method of detecting coccidia in rabbits?
A
Fecal floatation
Found to be superior intestinal wet mounts and histology.
25
41. Why is viral isolation not practical for routine animal monitoring in contrast to bacterial isolation?
Because immunocompetent animals usually clear viral infections rapidly; different viral species and strains have diverse host ranges in culture and tissue tropisms in vivo, making host and specimen selection problematic; some fastidious viruses such as mouse thymic virus (MTLV) will not grow in any cell-culture system
26
Describe selectivity of MacConkey's agar.
| What is the best choice of specimen collected from a rat to isolate Corynebacterium kutscheri?
crystal violet and bile salts - inhibit Gram +
Lactose fermenters produce pink-red colonies; non-lactose fermenters produce colorless colonies
submaxillary lymph node
27
MacConkey's agar
Contains crystal violet and bile salts
Inhibits Gram +, allows Gram -
Differentiates lactose or non-lactose fermenters
Lactose fermenters: pink-red
Non-lactose fermenters: colorless
28
How to culture Helicobacter from feces or intestinal contents?
media that has a mixture of of antibiotics
29
Tracheal cultures for Bordetella brochiseptica?
Selective media usually not necessary since they are typically few extraneous bacteria species
30
What tissue best for isolation of Corynebacterium kutcheri?
lymph nodes
31
Selenite broth
Salmonella spp. from feces or intestinal tract
32
How culture Mycoplasmas?
media supplemented with serum as a source of cholesterol, and an atmosphere with additional CO2 because they're capnophilic
33
What species require a microaerophilic environment?
Campylobacter and Helicobacter
34
Culture of Clostridium piliforme?
embryonated chicken eggs or mammalian cell culture
35
43. What does viral tropism mean? Give an example for a pneumotropic, neurotropic, and pantropic virus.
Sendai virus - pneumotropic
MHV, TMEV - neurotropic
Reoviruses - pantropic
36
44. Name the three host systems used to isolate viruses in diagnostic laboratories, in order of decreasing importance.
1. cell culture (immortalized cell lines, primary cell cultures enzymatically dispersed directly from animal tissues, explant cultures consisting of tissue fragments)
2. laboratory animals, particularly neonatal mice, but also immunodeficient hosts such as nude mice, or laboratory animal strains for which a particular viral infection is more pathogenic
3. embryonated eggs
37
45. If a virus does not induce a specific cytopathic effect, evidence of virus replication can be obtained by alternative methods such as ______, _______ or _________.
hemadsorption (e.g. Sendai agglutinates erythrocytes), immunofluorescence with virus-specific antibodies, or electron microscopy
38
46. T/F Animal hosts are more susceptible than cell culture to nonspecific specimen toxicity and bacterial or fungal contamination.
False. Animal hosts are less susceptible.
39
48. LDV does/does not elicit an easily measured antibody response. LDV can be diagnosed by _____ and measurement of increased level (10-to 20-fold) of ______in serum or plasma.
does not; PCR ; LDH activity
40
50. A common usage of antigen assays is to serotype isolates of bacteria. Salmonella have been delineated by agglutination with antisera to ________O and ________H antigens and ¥â-Hemolytic streptococci usually have group-specific, ______________C antigens (basis of Lancefield classification system), demonstrated by precipitation or by the agglutination of antibody-coated latex particles.
somatic, flagellar, cell-wall carbohydrate
41
51. T/F Neutralization, complement fixation, and hemagglutination inhibition (HAI)
tests are traditional serologic methods that discriminate among related viral
strains as antigen assays only.
F. this tests can be applied as antigen assays and as antibody assays.
42
52. Among the antigen assays, labeled antibody methods have been preferred for direct microorganism identification because it is simple, sensitive and highly specific through the possibility of the incorporation of monoclonal antibodies. List the three groups of standard labels!
fluorescent dyes (fluorescein isothiocyanate [FITC] most popular)
enzymes (horseradish peroxidase [HRP], alkaline phosphatase [AP])
radioisotopes
43
54. Immunofluorescence assay results are read with a ________ or ________ directly following the final labeled-antibody incubation and wash steps, whereas an additional ____________ is required to develop the results of an enzyme immunoassay.
Fluorescence microscope or fluorometer, incubation with substrate
44
57. T/F Immunochemical staining is not possible when tissue is formalin-fixed and paraffin-embedded.
F. Immunochemical staining of formalin-fixed tissue can be performed, provided that tissue sections are first digested with trypsin to unmask microbial antigen.
45
63. What can be labels of reporter probes (same as antibodies)?
Reporter probes can be labeled directly or indirectly with radioisotopes, enzymes that act on chromogenic or chemiluminescent substrates, or fluorophores.
46
64. What are the limitations of labeled antibody probes and nucleic acid probes for direct detection of microorganisms in clinical specimen?
fixed target quantities in specimens and background due to nonspecific binding of the labeled probes
47
66. Although PCR is characterized by simplicity, speed, sensitivity, and specificity, why is it unlikely that it will replace serology in routine viral surveillance?
Many viral infections are short-lived and convalescence of the host is complete, making attempts at virus detection futile regardless of the assay sensitivity.
48
71. It is common to obtain readily detectable quantities of DNA from just a single template copy by PCR. In contrast, how many copies of a target nucleic acid sequence are required for detection by blot hybridization?
approximately 100,000 copies
49
78. List traditional serologic tests.
Complement fixation (CF); hemagglutination inhibition (HAI), neutralization test (NT); enzyme-linked immunosorbent assay (ELISA); indirect immunoflourescence assay (FIA)
50
79. Why is serology not as often employed for bacterial surveillance in contrast to viral and mycoplasmal monitoring?
Because bacteria are genetically and antigenically complex and many antigens are shared across species, bacterial serology with whole-cell antigen is likely to detect cross-reacting antibodies to clinically irrelevant bacterial species or strain. False negative results can occur when antigen purified from one bacterial strain does not cross-react with antibodies to others or when noninvasive bacteria do not stimulate detectable antibody production.
51
80. CF and HAI are considered traditional serologic methods. CF is no longer in routine use because it is _________ and _________, whereas HAI is still commonly employed as a confirmatory test, because it is _________ and _____.
CF is time-consuming and not very sensitive, whereas HAI is simple to perform and can distinguish among antibodies to different species or strains of viruses.
52
81. Explain how the Hemagglutination inhibition test works.
Serial dilutions of a serum specimen are incubated with viral antigen in V-bottom microtiter plate wells and a suspension of erythrocytes is then added. If the serum specimen contains antibodies to the viral hemagglutination, these will coat the virus and prevent it from agglutinating the red blood cells. Conversely, if the sample is HAI antibody-negative hemagglutination occurs.
53
82. Why is the indirect ELISA, a solid-phase immunoassay, the method most often used to screen serum samples for antibodies to infectious agents?
Because it is highly sensitive, amenable to automation, and can be read with a spectrophotometer and sent to a computer to be compiled into reports.
54
83. IFA, another solid-phase immunoassay is rarely used as primary screening assay since its results have to be read manually. With IFA, virus-infected cells and uninfected cells are fixed to wells on a glass slide, using cold acetone. The binding of primary antibodies to the solid phase in the IFA is demonstrated with an FITC-labeled antispecies immunoglobulin. Slides are covered with buffered mounting medium and examined with fluorescence microscope. How are antibody-virus reactions differentiated from nonspecific reactions?
Bright, granular fluorescence is typical of an antibody-virus reaction, whereas diffuse fluorescence is characteristic of nonspecific reactions.
55
90. The microorganisms selected for monitoring in mice and ras have been compiled, and the basis for their selection has been categorized, in the ________ published by the National Institute of Health.
Manual of Microbiologic Monitoring of Laboratory Animals
56
93. There are occasions when it is helpful to use sentinels of one species to monitor principals of a second. Explain such a situation.
One such occasion is when little is known about the viruses that infect a species, which is the case for gerbils (better to do serology on sentinel mice or rats to determine whether gerbils shedding murine viruses). Another example would be when the different species is more likely to become ill following infection than the principal species (e.g. gerbils uniquely susceptible to Tyzzer¡¯s disease).
57
94. Match the following types of sentinels with the purpose of testing:
a) retired breeder
b) immunocompetent animal
c) sentinels of multiple ages
d) immunocompromised or immunosuppressed animals
retired breeder - production colonies, because they've had time to become infected and seroconvert
immunocompetent animal - serology
sentinels of multiple ages - pathology, bacteriology, and parasitology because prevalence of infection is often age-dependent
immunocompromised - diagnosis of certain latent infections (e.g. C. piliforme)
58
97. T/F Sample size is also related to the number of animals in a colony. The number of animals that must be sampled to achieve a certain level of confidence increases as the colony size decreases.
True
59
98. Define prevalence and incidence.
Prevalence is the percentage of positive animals at a point in time in a designated area. Incidence is the percentage of new positives over a designated period.
60
99. In discussions of laboratory animals health surveillance the __________ formula is often cited for determining sample size. What does the correctness of sample size depend on when using this formula? How is the accuracy influenced by the trend away from open cages toward filter-top microisolation cages?
binomial distribution formula; The correctness of sample size depends on the assumptions that there is a random spread of infection and that the colony includes at least 100 animals. It also depends on the accuracy of the prevalence estimate. The use of filter-top on cages may result in smaller effective population sizes and adventitious infections that spread more slowly and have a lower prevalence.
61
100. T/F The relevance of the sampling formulas to sentinel animals kept on pooled, soiled bedding has not been addressed.
True
62
101. The frequency of testing should be adjusted based on historical contamination rates. Interpret the importance/frequency of bacteriology, serology, and parasitology in regards of Gnotobiotic colonies, immunodeficient and immunocompetent rodents, and barrier rooms.
In gnotobiotic colonies bacteriology should be more frequently performed than serology and parasitology. Bacteriology should also be performed often on immunodeficient rodents for which many opportunistic bacteria are pathogenic, but can be done less regularly on immunocompetent SPF colonies. In barrier rooms/facilities serology is most important whereas bacteriology and parasitology can be performed less often.
63
103. What do sensitivity and specificity mean in regards to a diagnostic assay?
103. What do sensitivity and specificity mean in regards to a diagnostic assay?
64
105. Name some of the most common sample selection errors.
Sample selection errors include sampling of acute ill animals when antibodies
are not yet detectable, faulty sample site for bacteriology specimen, small sample size, sentinels not adequately exposed via soiled bedding or contact to infectious agents carried by principals, maternal antibodies when young animals used for serology, weak or no antibody response in immunodeficient or immunosuppressed animals.
65
Give examples of denaturant disinfectants.
Quaternary ammonium compounds
Phenolics
Alcohols
66
Give examples of reactant disinfectants.
Aldehydes
| Ethylene oxide
67
Give examples of oxidant disinfectants.
Halogens - chlorine bleach, chlorine dioxide, povidone-iodine
Peroxygens - hydrogen peroxide, peracetic acid
Ozone
Microbiological Quality Control
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