Viral diagnostics for animal diseases


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Viral diagnostics for animal diseases

  1. 1. Viral diagnostics for animal diseases I.Sophia P-1699 VBM
  2. 2.  substances or devices which aid in the diagnosis of viral infections  Classification of viral diagnostic tests  (i) direct virus detection  (ii) viral antigen detection  (iii) virus isolation  (iv) viral antibody detection. Viral diagnostics
  3. 3.  Electron microscopy, immunoelectron microscopy  Histopathology  Fluorescent antibody technique – direct, indirect  Immunohistochemistry  ELISA  Immunochromatography  Latex agglutination test Direct identification of viruses
  4. 4.  ELECTRON MICROSCOPY  Rapid diagnosis  No need of additional probes  Disadvantage  Procurement and maintenance of the instrument – expensive  Require skilled & well trained personnel for operation.  Sensitivity is comparatively low – require at least 10 6 viral particles for + ve diagnosis.  Single specimen examined at a time.  Applications:  Rotavirus from faecal samples, rabies virus from brain, norwalk virus from faecal samples
  5. 5.  Employs viral antibody  Increases the sensitivity & specificity of EM  Two methods (i) classical IEM (ii) solidphase IEM  classical IEM: Requires prior incubation of the clinical sample with virus specific antibody .  Solidphase IEM: solid support (copper grid) is coated with specific antibody which captures viruses from the clinical sample. Immunoelectron microscopy (IEM)
  6. 6.  Helps in diagnosis of viral inclusion bodies  Viral inclusion bodies: Aggregates of viral nucleocapsids in the cytoplasm or nucleus.  Can be detected by H&E staining Applications  Negri bodies in rabies virus  Guarnieri bodies – vaccinia virus  Bollinger bodies - fowlpox Histopathology
  7. 7.  Use of two specific antibodies – nanoparticle conjugated antibody, antibody fixed in chromatography paper.  The sample dropped on sample pad – forms complex with the antibody. When moves along the membrane pad- sandwiched between two antibodies- production of colour.  diagnosis of avian influenza, dengue etc. IMMUNOCHROMATOGRAPHY
  8. 8.  Particulate antigen +virus specific antibody - crosslinking of polyvalent antigens – agglutination.  Visibility can be improved by coating the latex beads with virus specific antibodies (sensitized latex beads).  Sensitized latex beads + clinical sample (containing viruses) visible aggregates.  Applications:  Avian influenza  Rota virus  FMD (Sugimura et al.,2000) Latex agglutination test
  9. 9. Fluorescence antibody technique (FAT)  Fluorochromes like fluorescent isothiocyanate and rhodamine isothiocyanate. Are used.  Viral antigens can be detected from smears from clinical samples, frozen tissue sections and also from formalin fixed tissue samples. Direct Fluorescence antibody technique The virus specific antibodies are directly tagged with fluorescent dyes. Indirect fluorescence antibody technique (IFAT) second antibody known as anti-species immunoglobulin (Igs) are used. Advantage:  Available easily commercially than the virus specific antibodies  More sensitivity than direct FAT.  Better signal amplification
  10. 10. Immunohistochemistry Principle  Employs antibodies conjugated with enzymes like horseradish peroxidase or alkaline phosphatase.  When suitable substrates are used, colour reaction takes place in case of positive sample which can be viewed through light microscope.  Both direct and indirect immunoperoxidase tests are used.  Advantages  The procedure can be applied for formalin fixed tissues as well.  Comparative study of site of localization of viruses and the tissue damage is possible
  11. 11. Sandwich ELISA The viral antigen is sandwiched between two antibodies namely capture and detection antibodies. For the assay both the antibodies should target different epitopes of the antigen. This is followed by addition of enzyme conjugated anti- species immunoglobulin. On adding suitable chromogen-substrate colour reaction develops. Advantages Assay is quantitative, amount of viral antigen can be detected Assay has high sensitivity and specificity More samples can be tested at the same time Disadvantages Need ELISA reader for result interpretation; not possible under field conditions The method is time consuming and labourious. Applications: Used in diagnosis of PPR, Bluetongue, FMD etc
  12. 12. Immunoblotting Applications: useful in differentiation of infected and vaccinated animals in FMD using recombinant proteins (Fu et al., 2011).
  13. 13. Complement fixation test Principle  The ability of the complement system to fix the antigen- antibody complex forms the basis of complement fixation test.  Sheep RBCs and its corresponding antibody acts as indicator system.  When the antigen reacts with specific antisera, antigen antibody complex will be formed and the complement will be unavailable for the indicator system. Then there will be no haemolysis and the test is positive.  If the antiserum is not specific, then the complement is free to fix the indicator system resulting in haemolysis.  Sensitive assay like ELISA have replaced it.  Simple; easy to perform. Applications  Complement fixation test is prescribed by the OIE for diagnosis of equine diseases like African horse sickness, equine encephalomyelitis (eastern, western, Venezuelan), vesicular stomatitis.
  14. 14. Haemagglutination inhibition tests Principle Certain viruses namely paramyxoviruses and orthomyxoviruse have the ability to bind to sialic acid residues in RBCs. This property is called haemagglutination. If specific antibody and viruses are mixed prior to the addition of RBCs haemagglutination is inhibited. HI test is used for serotyping and also for measuring antibody titre. Applications  OIE recommends this test for diagnosis of viral infections like blue tongue, avian laryngeo tracheitis, avian influenza, marek’s disease, infectious bursal disease, enzootic bovine leucosis, equine infectious anaemia (Coggin’s test), myxomatosis and caprine arthritis encephalitis disease.
  15. 15. Complement fixation test -ve +ve
  16. 16. Virus neutralization test Principle  When a serum sample to be tested is specific for the virus, there will be antigen-antibody complex formation. The virus particles are unavailable to cause cytopathic effect in cell culture which can be visualized microscopically or by staining with vital dyes. Applications  Prescribed test by OIE for diagnosis of pseudorabies, rabies, Riftvalley fever, vesicular disease and IBRT-IPV.
  17. 17. Plaque reduction neutralization test Principle The ability of virus specific antibody to inhibit the plaque forming property of viruses by 50% when plated in semisolid media is the basic principle involved. Applications PRNT is recommended by the OIE for diagnosis of eastern equine encephalitis and Venezulean equine encephalitis disease. Disadvantages: The interpretation of results requires at least 3-5 days which is very slow when compared to EIA and RIA. Both the tests involve use of cell culture where false results due to contamination of cell culture are possible.
  18. 18. Agar gel immunodiffusion test Principle  It is basically a precipitation test in which soluble antigen reacts with its homologous antibody in the presence of electrolytes. At the optimum proportion, an insoluble antigen-antibody precipitate will be formed. Applications  OIE recommends this test for diagnosis of viral infections like blue tongue, avian laryngeo tracheitis, avian influenza, marek’s disease, infectious bursal disease, enzootic bovine leucosis, equine infectious anaemia (Coggin’s test), myxomatosis and caprine arthritis encephalitis disease.
  19. 19. Counter immunoelectrophoresis PRINCIPLE Counter immunoelectrophoresis is the immunodiffusion modified by electrophoresis to drive antigen and antibody towards each other. The specimen to be tested is placed in the cathode side, and the antiserum is placed in the anode. At neutral or alkaline pH, antigens are negatively charged and hence migrate towards anode in barbital or veronal buffer. Applications: Counter immunoelectrophoresis is used in diagnosis of blue tongue virus, orf virus and pox viral diseases
  20. 20. Radio immunoassay Principle It is the competitive binding assay which binding of known amount of radiolabeled substrate and antibody. When the test sample is added, the radiolabeled substrate gets displaced by it resulting in free radiolabelled substrate in the solution. Finally, the radioactivity of free substrate in the solution is measured which is proportional to the amount of unknown antigen bound to antibody. The assay is more sensitive and specific compared to the above methods. Disadvantage: Assay needs sophisticated instruments like gamma counter. Radioactive compounds are hazardous to human health ie. Carcinogenic. Therefore, it is not routinely used for viral diagnosis.
  21. 21. Indirect ELISA PRINCIPLE  The viral antigen is immobilized in a solid support followed by addition of serially diluted antibodies. The antibodies in turn are captured by enzyme conjugated anti-species immunoglobulin.  Addition of chromogen / substrate facilitates the colour reaction which can be measured calorimetrically.  Currently, use of recombinant viral proteins as antigens reduce the risk of handling of dangerous organisms and made the assay sensitive and specific. Advantages Simplicity; easy to perform Rapid than neutralization assays Safer: Use of enzymes instead of radiolabels Require ELISA reader for result interpretation
  22. 22. Nucleic acid based diagnostics  PCR  RFLP  Microarray  Hybridization techniques
  23. 23. RFLP  The nucleotide signatures of each species are unique. This fact is explored in the technique where endonucleases recognizing restriction sites are used to cut the genome at various sites for each organism. The genome fragments are resolved by running in agarose gel electrophoresis. The characteristic nucleotide base pair length corresponds to each organism.
  24. 24. PCR
  25. 25. Real time PCR
  26. 26. MICROARRAY
  27. 27. Nanobiosensor  There are various types of nanobiosensors based on various principles namely, electrochemical biosensors, voltametric and amperometric sensors, impedance sensors, optical fiber based sensors, surface plasmon resonance based biosensors, quartz crystal microbalance and atomic force microscopy based nanobiosensors.  Nanobiosensors have been designed for diagnosis of dreadful human viral diseases like HIV, hepatitis B, Hepatitis C, Ebola virus etc. Nanobiosensors have also been designed for diagnosis of animal diseases like avian influenza virus, infectious bovine rhinotracheitis, rabies, bovine leukemia virus.