Lecture 18: Diagnostic Immunology Case Studies Flashcards
Neurological Symptoms in wild raccoons and several <1 y/o dogs with oculonasal discharge and 1 with seizures.
Differential diagnosis: rabies, toxin, canine distemper
What tests can be run
Rabies direct fluorescent antibody (FA) testing, submit ocular/nasal swabs for distemper FA testing, and submit blood for serology and ethylene glycol testing
Describe the steps in rabies direct FA testing
Requires a fresh Brain, take impression stains of tissue from brain stem, cerebellum and cerebrum
Add fluorescently- labeled rabies-specific antibodies
Describe the steps in submitting ocular/nasal swabs for distemper direct FA antibody testing
Swipe swab on slide and add fluorescently labeled distemper specific antibodies
What are direct fluorescent antibody tests looking for
Using patient samples and fluorescently labeled antibodies specific for a virus, we are looking for a viral antigen
Describe the steps involved in submitting serology for virus/serum neutralization
Obtain patient serum, perform dilutions, and run tests to determine the lowest effective dilution where antibodies can fight off infection. Will determine normal healthy cells that have antigen bound to antibody and neutralized vs virus infected cells (cytopathic effect) not enough antibodies present in dilution to neutralize antigen
Analyzing results of neighborhood dogs tested using FA and VN:
VN: threshold > or equal to 1:80
Astro, 2yr, clinical signs include ocular discharge DFA for distemper= + and VN< 1:40
What stage is Astro at in disease
Astro is probably in early stages of infection so CDV antigen is present, specific immunity has not developed yet so antibody titer is low
analyzing results of neighborhood dogs tested using FA and VN:
VN: threshold > or equal to 1:80
Barley, 4 month old, clinical signs include seizures, DFA for CDV= - and VN titer =1:320
What stage of disease is Barkley at
Barkley is in the very late stages of infection so viral antigens are long gone, however an immune response is present as indicated by the elevated titers
analyzing results of neighborhood dogs tested using FA and VN:
VN: threshold > or equal to 1:80
Carlie is a 1yr, showing no clinical signs, DFA= -, VN titer= 1:640
What stage of disease is Carlie at
Carlie has high titers either from previous vaccination or she has recovered from an infection at young age
Tibetan Terrier in UK: 2yr/old never outside UK with 1 month progressive lethargy, pancytopenia and non-regenerative anemia, has received 2x packed RBC transfusions and steroid and antibiotic treatment
Differential diagnosis are IMHA/IMTP or tick borne disease (many agents not reported in UK)
What are some tests that can be run
Blood work: anti-nuclear antigen testing, Coombs/blood typing/agglutination testing
Test for tick borne disease- 4Dx SNAP and ehrlichia indirect fluorescent antibody testing
Describe the steps in Anti-nuclear antibody (ANA) testing for systemic lupus erythematosus
Identifies the presence of antibodies targeting dsDNa/ssDNA and histones
Take diluted patient serum add anti-dog/cat/ etc antibodies that are fluorescently labeled
What does a saline agglutination test, test for and how would histology appear
Identifies autoantibodies in patient serum directed against RBC’s
RBC clumped together, misshaped
What does an indirect Coombs test, test for
Identifies antibodies exogenous RBC’s in patient serum
Describe how IDEXX SNAP 4Dx for tick/mosquito borne disease test works
Obtain patient blood add enzyme linked antibody conjugate
Antigen in blood will bind to conjugate then will bind to antibody bound matrix that is pre coated on snap device
Add wash to remove any non-specific unbound conjugate (RBC’s) and then substrate will move across the matrix, react with color changing substrate and appear blue on SNAP device
What does an indirect FA test for
Antibody against antigen is the target
Describe the steps in ehrlichia indirect FA test
Take diluted patient serum and slide with ehrlichia culture, add wash and then anti-dog/cat antibodies that are fluorescently labeled
The diagnostic results for Tibetan terrier:
Saline Agglutination (+)
SNAP 4Dx (+) for Ehrlicha
Ehrlichia IFA (+)
Ehrlichia PCR (+)
Patient sent home on antibiotic and steroid then returned days later with gastrointestinal hemorrhage and hepatomgaly
What could have happened?
Why is saline agglutination (+)
If UK is Ehrlicha free how did dog become infected
Saline agglutination could have been positive from either improper matching with transfusions (exogenous RBC’s) or Ehrlichia antigens present on RBC’s targeting them for lysis
Ehrlichia displays similar symptoms to systemic Lupus- bone marrow suppression which can lead to non-regenerative anemia, thrombocytopenia and leukopenia (pancytopenia)
Respiratory disease in Feedlot Cattle TX:
Hx: chronic respiratory disease in group of cattle on feedlot, ~40 deaths in 6 months
Differentials: shipping fever, BRD, Mannheimia/pasteurella, BVDV, IBR
What are some tests that can be run
Culture for mannheimia/pasteurella
Bovine respiratory disease panel- VN and FA
BVDV ear notch testing- ELISA, ACE
Describe the steps in BVDV ear notch testing
Test with antigen capture ELISA, ACE
Need labeled anti-BVDV antibodies, wash reagents, color reagent and control
Requires FRESH tissue
Determine ELISA threshold for positive samples through graph of optical density
Describe the steps in BVDV ear notch testing with immunohistochemistry
Requires fixed tissue, add labeled antibodies
Analyze the following results for cattle
IBR VN threshold < or equal to 1:80
BVDV VN threshold < or equal to 1:80
Blue 451 IBR VN 1:1600 and BVDV 1:800, BVDV ACE test is (-)
Red 207 IBR VN 1:3200, BVDV VN: 1:1600, BVDV ACE test is (-)
Green 769 IBR VN 1:1600, BVDV VN 1:40, BVDV ACE test is (+)
What conclusions can be drawn
All three cattle have been sick for > 2 weeks and likely infected with both IBR and BVD
Green 769 has no antibodies against BVDV despite having positive ACE result which is because it is PERSISTENTLY INFECTED
Remove Green 769 to reduce transmission
What samples can be used for Fluorescent assays
Serum, tissue, ocular/nasal swabs
What does DFA detect
Antigen
What does IFA detect
Antibody
What samples can be used for virus neutralization
Serum
What does virus neutralization detect and what does it indicate about disease stage (above or below threshold)
Lowest effective level of AB’s to neutralize antigen
If VN is below threshold and patient is experiencing symptoms, with a positive antigen test then likely in early stages of disease
If VN is above threshold could be multiple things
- above threshold with clinical signs, and negative antigen test- late stages of disease with circulating antibodies but no antigen present
- above threshold without clinical signs and negative antigen test then patient either has been recovered from disease for a while or has been vaccinated previously
What does anti-nuclear antigen test for
DsDNA, ssDNA, and histones in SLE
What does saline agglutination test for
Autoantibodies to RBC’s
what does SNAP detect
Antigen- binds antibody and conjugate
What samples can be used in ELISA tests
Fresh tissueh
What does an ELISA (ACE) detect
Antigens
What are two signs of persistent infection
ELISA (ACE) test is positive but BVDV is low
What are 3 tests that target self antigens
- Saline agglutination
- Anti-nuclear antigen testing (ANA)
- Misfolding protein assays-prions
