Diagnostics Final Flashcards
What are our detection goals for viruses?
-Prevent introduction
-Target care (treatment of secondary infections)
-Limit spread (to both animals and humans)
How can we detect viruses?
-Host antibodies or antigen
Antigen -> viral proteins, nucleic acid, live virus
What are tests we use to detect anibodies?
ELISAS, lateral flow assays, agar gel immunodiffusion, other agglutination assays
How do antibody detecting tests work?
-test contains the antigen
-patient antibodies react with test antigen
-reaction is visualized
-some tetss for iGM, others IgG
-lag time from infection
Direct Elisa?
The primary antibody conjugate has the antigen and enzyme
Indirect ELISA?
Has a secondary antibody conjugate
Competitive ELISA?
There is an inhibitor antigen
Describe a lateral flow assay?
-labeled antigen
-binds patient antibodies
-easy and cheap
Example :FIV
Agar gel immunodiffusion test
-equine infectious anemia (coggins test)
avian influenza
-postive will have a band of precipiate
Hemoagglutination inhibiton test
Figure on slide 12
Detection of viral proteins
Lateral flow assay
labeled antibodies
-bind antigen
easy and cheap
Example : parvo
Viral proteins
ELISA
-capture or sandwhich assay
Viral proteins
fluorescent antibody testing
-direct method
-fluroescent labeled antibodies (bind target antigens)
-rabies testing
Viral proteins
Immunohistochemistry
-fixed tissue is treated to expose antigen
-sections exposed to antibody
-example: FIP, Mareks disease
What are the pros and cons of ELISAs and lateral flow assays?
Pros -> quick, less expensive, readily avaiable, ELISAS have titers
Cons ->less specific, risk of false positives, not as likely to be accepted for regulatory purposes, cross reactivity
Pros and cons of AGID and IH
Pros -> specific, gold standard for some tests, often accepted for import/export
Cons -> harder to find, longer incubation, labor intensive, often more expensive
Pros and cons of FA and IHCs
Pros ->usually quite specific
FAs: fast, visualize pathogen location
IHC: option for fixed tissue
visualize pathogen location
Cons -> rarely an antemortem test, can be expensive, may be species specific, limited assays
Why would we pick antigen vs antibody test?
-antigen -> detecting current infection , detects in immunotolerant animals
Antibody ->detecting previous/chronic infection
Detects vaccination that elicits IgG
PCR
-real time PCR quantifies viral load
DNA virsues
-reverse transcriptase PCR for RNA viruses
Sequencing
Sequencing -> whole genome, metagenomic
-In development/limited use
What are some examples of sequencing?
-sequencing mutated FMD
-testing samples for canine respiratory outbreak
-tracking vaccines related ILT outbreak
What are the pros and cons for PCR?
Pros -> fast, sensitive, reliable
Cons ->no distinction live vs inactive
may miss if virus mutated
contamination can be an issue
need to know what you are looking for
What are pros and cons of sequencing?
Pros -> large amount of data
ID unknown pathogens
track mutations
Cons ->expensive, lots of analyaiss, best on pure sample
How do we detect viruses?
viral isolation, electron microspy, histopathology,
Describe viral isolation
Goal : grow virus from diagnostic sample then identify
usually tissue samples, body swabs as well
Gold standard -:only test to prove viable virus is present
What cell lines can be used for viral isolation?
embryonic or adult
kidney
liver
others
How do we identify that we isolated a virus?
cyotopathic effects
electron microsopy
PCR
sequencing
aggluination
What are some examples of why we try to isolate viruses?
Detect low viral load: BVD in milk, IBR in semen
(grow enough virus to detect)
-unknown pathogens
What is Electron Microscopy?
transmission electron microscope
stained with heavy metal salts
visualize surface structures
used on diseases samples or purified virus
can search blindly
can detect primary, secdonary antigens
Histopathology?
Visualize viral inclusions inside cells
standard H and E stain
usually pathopnomonic
usually present at a certain stage of infection
What are the pros and cons of virus isolation?
Pros -> detecting live virus
detect and identify unknown virus
Sensitive
amplify and collect live virus
Cons -> Diffcult, long wait time, expensive, sensitive to contamination
What are pros and cons of electron microscopy?
Pros -> visualize pathogen
will work even if mutated
false positives are rare
Cons -> expsensive, less common, longer turnaround time
Pros and cons of histopathology?
Pros -> false positives are unlikely
inclusions are often distinct and pathopneumonic
-Virus is inactivated
Cons ->Absences does not equal negative
Longer turn around time
What swabs are safe to use?
Polyester, dacron, nylon, ryon,
Flocked = catch more virus
What swabs should we aboid?
wooden handles
Calcium alginate ->inactivate virus and interfere with PCR
Does a FCoV neagative ELISA mean this cat doesnt have FIP
No, in late stage FIP antibodies drop and may not be detected
What are more defintiive options than PCR on biopsy tissues?
Histopathology and immunohistochemistry
How do we ship samples for virus isolation?
ship whole fish overnight on icepacks
ship overnight on dry ice
Formalin inactivates virus
