Chapter 4 - Diagnostics Flashcards
what is the purpose of ‘Observation’?
recognize abnormal behaviour, physiology as indicators of possible pathogen or disease
what would you look for in your observations?
behavior -spinning, flashing color external lesions internal pathology bleeding
can you use observation as sole method for diagnosis?
nope
What is the purpose of Microscopy?
-magnify image of specimen or host tissues for examination and identification
what are the two types of electron microscopy?
SEM: scanning
TEM: transmission
how do you manipulate the following for microscopy observation?
- Viruses
- Bacteria
- Fungi
- Protists
- Histology
V: need EM B: Gram stain F: Wet mount + Stain P: wet mount + Stain H: stain fish tissues to detect cell and tissue damage.
what is the purpose of Culture Techniques?
demonstrate pathogen is present and viable by growing it.
four media types for culture techniques
- general
- selective
- enrichment
- diferencial
for Culture Techniques, what is API 20E?
20 biochemical tests condensed on one strip (differential media)
what sort of media would you use for viruses?
Live media (cell culture): use living cells as growth medium.
CPE. name, what does it do
Cytopathic effect.
Concept: pathogen destroys cells if present.
Use: Viruses
give some example types of live media
- CHSE: chinook salmon embryo
- SHK: salmon head kidney
- RTG: rainbow trout gonad
- FHM: fathead minnow
- SSN-1: striped snakehead
- EPC: epithelioma papillosum cyprini
CHSE:
Chinook salmon embryo. LIVE MEDIA
SHK:
Salmon head kidney. LIVE MEDIA
RTG:
Rainbow trout gonad. LIVE MEDIA
FHM:
fathead minnow: LIVE MEDIA
SSN-1:
striped snakehead. LIVE MEDIA
EPC:
epithelioma papillosum cyprini: LIVE MEDIA
What is the purpose of Serological Techniques?
detect presence or part of pathogen by using commercial antibodies to attach to pathogen
what do serological tests do?
detect antigens in sample or detect specific antibodies in the fish
FAT: acronym
fluorescent antibody technique. SEROLOGICAL TECHNIQUE
IFAT: acronym
Indirect Florescent Antibody Technique.
SEROLOGICAL TECHNIQUE
ELISA: acronym
Enzyme Linked Immunosorbent Assay
SEROLOGICAL TECHNIQUE
describe FAT (5 points)
fluorescent antibody technique
1: commerical antibodies (Ab) prepared by injecting lab rabbit with fish pathogen
2: rabbit produces Ab to fish pathogen, then a fluorescent dye is added.
3: Obtain sample of fish tissue suspect with pathogen, smear on slide
4: Add commercial labeled rabbit Ab to slide, then rinse
5: If pathogen present, stick to Ab and reacts with Ab. see glow, using flourescent microscope.
describe IFAT (7 points)
indirect fluorescent antibody technique
1: rabbit blood injected into goat, goat produces Ab against rabbit blood, a florescent dye is added
2: rabbit injected w fish pathogen, rabbit produces Ab to fish pathogen (commercially prepared)
3: Obtain sample of fish tissue suspect w pathogen, smear on slide
4: Add rabbit Ab to slide, then rinse
5: Add labelled goat Ab to slide, then rinse
6: If pathogen present, sticks to rabbit Ab and reacts with rabbit Ab, then goat Ab attach to rabbit Ab- see glow
7: With indirect method, get enhanced signal and sometimes more cost effective.
describe ELISA (5 points)
enzyme linked immnosorbent assay
1) Plate coated with a capture antibody, rinse
2) sample added, any antigen present binds to capture antibody, rinse
3) Detecting antibody added and binds to antigen, rinse
4) Enzyme- linked secondary antibody added, binds to detecting antibody, rinse
5) Substrate added, converted by enzyme to detectable form = glow or colour
3 types of Commercial Antibody Kits. Why use these? How do you tell if they’re working?
- Serum Agglutination
- Latex Agglutination
- Co-agglutination Test (antibody-sensitized staphylococci)
- > real simple kits. good for a ‘quick test’ then you would do more samples or send off to lab.
- > if the target organism is there, you get clumping. If not, smooth. Can be variable
What is the purpose of Genetic Techniques?
detect a specific genetic code of the pathogen or its antigenic component.
What does Genetic Techniques DO?
provides genetic signal, but not necessarily mean pathogen was viable and virulent.
what sort of technique is PCR?
genetic technique
PCR: acronym
Polymerase Chain Reaction
what does PCR do?
amplifies target genome (via thermal cycling) to produce a copy large enough to be detected. 20 cycles= 10^6 copies of target DNA
what are the three steps of PCR?
1) DENATURING @ 94-96C 20-30s DNA is split via melting
2) ANNEALING @ ~ 65C (20-40s) DNA target binds to original DNA strand
3) ELONGATION at 72*C (~ 1000 bp min^-1) DNA target is copied.
((heating, cooling, replication))
name two types of PCR
RT-PCR: reverse transcription polymerase chain reaction (for RNA viruses)
Q-PCR: quantitative, real-time PCR measures amount of DNA amplified over times
what does Sensitivity mean? (interpreting diagnosis tests)
% true positive.
reflects how well tests perform among group of DISEASED fish
what does Specificity mean? (interpreting diagnosis tests)
% true negatives
reflects how well tests performs among a group of NON-DISEASED fish
what are both sensitivity and specificity dependant on?
prevalence and number of fish sampled.
what happens to specificity in moribund (almost dead, close to dead) fish?
secondary invaders can mask primary pathogen. specificity DECREASES
what are the implications for ELISA when measuring specificity vs sensitivity?
It measures ANTIBODY response.
false positive: prior exposure.
false negative: no measurable antibody response
what are the implications for PCR or RT-PCR when measuring specificity vs sensitivity?
It measures GENETIC code
- false positive: genome present, but inactive
- false negative: genome present, but insufficient quantity
What is the implication for Culture Media when measuring specificity vs sensitivity?
Growth
-false negative. no growth, expired reagents, temperature, fastidious species, contamination.
how would you test for covert (hidden) infections?
you could stress your fish and see if disease occurs. separate the stock, crank up the heat, wait and see.
what do all diagnoses start with?
Observation
when beginning your diagnosis, how should you get to know your fish?
check records
what is adequate for diagnoses of many parasites, but not sufficient for viruses and most bacteria?
microscopy
what method of diagnoses is very important for viruses and bacteria?
Culture
how is culture important for viruses and bacteria?
demonstrates viability
what sort of techniques are great for commercially important pathogens?
Serological techniques
Genetic techniques
