Section 1

Question 1

When is cell culture indicated

Answer 1

Detection of organisms dependent on living host cells for replication

Question 2

What are the disadvantages of cell culture?

Answer 2

Slow
Costly
Technically difficult to perform and interpret results

Question 3

What are the main applications of cell culture?

Answer 3

Discovery of new pathogens
Pathogen identification when results of molecular testing/serology equivocal
Research
Vaccination
Establishment of efficacy of novel antimicrobial drugs

Question 4

What is the significance of timing of collection of samples for cell culture?

Answer 4

Must be collected early (within first week), when viral shedding occurring

Question 5

What are the different cell lines for cell culture? When might one be preferred?

Answer 5

Primary - directly derived from animal
Immortalised/continuous cell line - derived from cancer cells or from introduction of cellular mutations

Primary are needed for isolation of some viruses

Question 6

List examples of immunoassays

Answer 6

IFA (direct/indirect), IHC, ELISA, Western immunoblotting, agglutination, gel immunodiffusion, serum neutralisation

Question 7

Describe direct IFA

Answer 7

Used to detect antigen
Uses antibody tagged with fluorescent label, or an enzyme

Question 8

What is IHC/ICC?

Answer 8

ICC - antibody vs antigen tagged with enzyme, substrate added, enzyme causes substrate to change into insoluble precipitate
IHC - same method applied to tissue

Question 9

How does indirect IFA differ from direct IFA

Answer 9

Normally (not always) used to detect antibody
When detecting antigen more sensitive than direct
Patient serum diluted and reacted with known antigen on glass slide. Second antibody designed to react to dog/cat antibody added, conjugated with marker

Question 10

Describe ELISA testing

Answer 10

Can detect antigen or antibody
Antigen detection - antigen immobilised in wells of plate (direct binding or bound using capture antibody - sandwich ELISA). Antigen detected using antibody bound to enzyme.

Question 11

Describe Western immunoblotting

Answer 11

Used for antibody detection
After physical/chemical disruption of an organism, proteins separated based on molecular mass using gel electrophoresis. Protein bands then blotted onto a membrane using an electrical current.
Membrane cut into vertical strips, if antibodies in serum to antigens in organism, antibodies bind to antigens in the strip. Visualised using immunoperoxidase reaction

Question 12

Describe agglutination testing
When might a false negative be seen

Answer 12

Detects antibody or antigen
Serial dilutions of serum tested for ability to cause or inhibit agglutination

When excess antibody present (prozone effect)

Question 13

How should anaerobes be sample for culture and how should samples be stored?

Answer 13

Aspirates/tissue preferred, not refrigerated

Question 14

Which organisms are detected with acid-fast stains?
Give an example of a acid fast stain

Answer 14

Nocardia, Mycobacterium
Ziehl-Neelsen, Fite’s, Kinyoun

Question 15

What are the advantages of dark field and phase contact microscopy?

Answer 15

Allow visualisation of organism without fixation or staining, allows motility to be assessed.

Question 16

Common blood culture contaminants

Answer 16

Coagulase-negative staph, Bacillus, corynebacteria, propionibacteria

Question 17

What is the significance of Mycoplasma in the nasal cavity of a cat?

Answer 17

Uncertain - can be isolated from healthy cats and cats with rhinosinusitis

Question 18

What level of bacterial growth from a cystocentesis sample is significant?

Answer 18

Any level significant, most >10^3 CFU

Question 19

When is anaerobic urine culture indicated?

Answer 19

If there is evidence of emphysematous cystitis

Question 20

In an animal with a urinary catheter and suspected UTI; what should be cultured?

Answer 20

Urine (obtained by cysto or freshly placed catheter) - NOT catheter tip (likely contaminated with urethral commensals

Question 21

What is the structure of mold?

Answer 21

Molds exist as filaments - hyphae
Mass of filaments = mycelium
Granule formed by compacted mat of fungal filaments = actinomycotic mycetoma

Question 22

What is the structure of yeast?

Answer 22

Single-celled organisms, reproduce by budding
Sometimes buds remain attached and form a chain - pseudohyphae

Question 23

What is a dimorphic fungi?

Answer 23

Exist as mold in the environment and convert to yeast form in the body

Question 24

How to fungi reproduce?

Answer 24

By forming spores - sexual or asexual reproduction possible

Question 25

What are the terms for the a) asexual and b ) sexual stage of a fungus?

Answer 25

a) Anamorph
b) Teleomorph

Question 26

What are the two types of nucleic detection assays - give an example of each

Answer 26

Probe-based assays - ISH
DNA amplification - PCR

Question 27

Describe DNA/RNA probes

Answer 27

Short single-stranded nucleic acids, complementary to a specific segment of DNA. Labelled with fluorescent or chemical tag

Question 28

Describe how PCR works

Answer 28

2 short DNA primers used in conjunction with a thermocycler to amplify DNA segment

Question 29

What is the CT number of a PCR and how does it relate to the quantity of target DNA present?

Answer 29

The number of cycles that elapse in a RT-PCR before fluorescence appears
Lower the CT number = more target DNA

Question 30

What is RT-PCR used for?

Answer 30

RNA detection

Question 31

What is nested PCR? What is an advantage and disadvantage of the technique?

Answer 31

Small amount of amplified PCR product subjected to another set of primers
Increases specificity and greatly increases sensitivity
More affected by contamination