Health Monitoring

Question 1

Why is PCR so sensitive?

Answer 1

Daddy issues
Due to exponential amplification

Question 2

Generally, what pathogens are poorly transmitted via SBS?

Answer 2

Respiratory viruses, hist-adapted bacteria, and parasites

Question 3

What is the gold standard for diagnosing helminth infestations?

Answer 3

Direct exam of macerated gastrointestinal tract.

Question 4

What is the sensitivity of direct exam?

Answer 4

Low analytical sensitivity - Lesions/organisms must be present at high concentrations

Question 5

Describe the selective growth on MacConkey’s agar.

Answer 5

Contains crystal violet and bile salts that selectively inhibit the growth of gram positive bacteria, supports gram negative bacterial growth. Lactose fermenters produce pink to red colonies, lactose non-fermenters remain colorless.

Question 6

How is selenite broth used?

Answer 6

Enrichment media to recover Salmonella from feces or intestinal tract.

Question 6

What organisms are fastidious and noncultivable? (5)

Answer 6

M. pulmonis (Slow)
Helicobacter (Slow)
C. piliforme (NC)
Filobacter rodentium (NC)
Pneumocystis (NC)

Question 7

For which organisms is serotyping common?

Answer 7

Salmonella - Serovars based on somatic O and flagellar H antigens
B-Hemolytic streptococci - Group-specific, cell-wall carbohydrate (C) antigens

Question 8

What type of samples does MALDI-TOF test? What are advantages of this modality?

Answer 8

Actively growing cultures or extracts from them. Based on unique peptidic spectra primarily of ribosomal and other housekeeping proteins.
Rapid (minutes) and lower cost per identification than biochemical testing.

Question 9

Question 10

Question 11

How are samples prepared prior to IFA so that antibodies can enter the tissue?

Answer 11

Fixative with cold acetone, which permeabilizes the cell membrane

Question 12

How are ELISAs and IFA generally used?

Answer 12

ELISAs for high-throughput testing, IFA for confirmatory testing as pattern of fluorescence in cell can distinguish specific from nonspecific reactions. IFA also more inclusive - able to detect seroconversion to heterologous viral strains and serotypes.

Question 13

What does virus neutralization and HAI test for? How does this impact exclusivity?

Answer 13

Only recognize antibodies to viral surface protein antigens that are unique to the serotype or strain of virus used in this test. Highly exclusive. Used for confirmatory testing.

Question 14

Define xMAP and MFI/MFIA.

Answer 14

xMAP = Luminex’s Multi-Analyte Profile - Assay multiplexing, where an array of tests are performed simultaneously in a single well, tube, or chip location
MFI(A) = Multiplexed fluorescence immunoassay

Question 15

Question 16

What does a Southern and Northern blot test for?

Answer 16

Southern - DNA
Northern - RNA

Question 17

How can altering the temperature of PCR impact specificity?

Answer 17

Increases the temp during hybridization enhances specificity by increasing the degree of complementarity needed for stable probe-target hybrids to form.

Question 18

Question 19

What do PCR primers for bacteria and parasites chiefly target?

Answer 19

Ribosomal genes

Question 20

Can PCR copy DNA? How are RNA agents identified?

Answer 20

Can only copy DNA. RNA templates can be detected provided they are first transcribed by a reverse transcriptase to a complementary DNA template. This is RT-PCR.

Question 21

Question 22

Question 23

Describe the real-time fluorogenic nuclease qPCR (real-time PCR).

Answer 23

AKA quantitative PCR (qPCR) or TaqMan PCR

Question 24

Question 25

What is the limit of detection (LOD)? How does this impact sensitivity?

Answer 25

Limit of detection - Lowest concentration of a target analyte in a specific matrix that can consistently yield a positive result. Sensitivity is measured as the limit of detection.

Question 26

Define analytical specificity? What does it include?

Answer 26

The ability of an assay to distinguish target from nontarget analytes, including matrix components. Comprises selectivity, inclusivity, and exclusivity.

Question 27

Define selectivity, inclusivity, and exclusivity as it pertains to specificity.

Answer 27

Selectivity - Restricts matrix-related effects Inclusivity - Ability of a single test to detect related organisms of interest Exclusivity - Test able to differentiate an agent from others that are closely related Inclusive assays for initial test, exclusive for confirmatory and to speciate.

Question 28

Why does PCR have higher specificity than traditional tests that rely on phenotypic tests?

Answer 28

Can be located in regions of the microbial genome that do not encode proteins. Sequence of a gene often varies more than the aa sequence of the protein. Specificity of genetic targets are unaffected by environmental and host-related factors that can alter the phenotype of an organism or humoral immune response to infection.

Question 29

Define predictive value. What is it influenced by?

Answer 29

The probability that a positive or negative result is correct. Influenced by the assay itself and prevalence of infection.

Question 30

Describe positive predictive value.

Answer 30

Percentage of all positive results that are true positives. PPV = TP/(TP + FP)Decreases along with prevalence. If something is very rare, a substantial percentage of positive results are expected to be false positives, even with high specificity.

Question 31

Which age of animals are most likely to be serologically positive? Positive for active infection?

Answer 31

Serology - Aged animals, as they are most likely to have encountered and seroconverted. Young animals post weaning are more likely to be actively infected, as maternal antibodies wane.

Question 32

What number of animals would need to be tested to achieve a 95% probability of detecting a pathogen it the prevalence was approx. 30%, 10%, and 1%?

Answer 32

30% - 8 animals 10% - 30 1% - 300

Question 33

Should inbred or outbred sentinels be used? Why? Age?

Answer 33

Outbred - Good serologic responders, cheaper. Some inbred strains have been shown to be comparatively resistant to infection or mount a delayed antibody response. Avoid older animals, as they may be more resistant to infection

Question 34

How much bedding is transferred from dirty to sentinel cages? How long are animals exposed?

Answer 34

5-15 mL, should not be diluted with clean bedding but can add new nesting material. Exposed for minimum of 3 months based on quarterly testing schedule.

Question 35

Describe SBS for MPV.

Answer 35

Occasionally all animals in a cage may not seroconvert, so testing only a subset of sentinel animals may decrease sensitivity. Could submit mesenteric ln for PCR or test the second sentinel only for parvovirus.