Table 5.1.
Principles and Objectives of Diagnostic Methods
| Principle | Method | Specimens/Findings | Characteristics |
|---|---|---|---|
| Visual Information Leading to a Presumptive Diagnosis | |||
| Review of the disease history, clinical examination, chemistry, hematology, etc. | Subject animal and its body fluids/Abnormal values | Essence of differential and rule out diagnoses; presumptive diagnosis determines the specimens and methods for further testing | |
| Pathology, histopathology, ultrastructural pathology | Animals, organs, tissues, cells/Characteristic lesions, inclusion bodies | Although slow and expensive, still important in veterinary diagnostics | |
| Detection of viruses by electron microscopy | Tissues, cells, secretions, excretions, vesicular contents/Particles of uniform, characteristic morphology | Rapid; sensitive enough with many diseases, especially diarrheas; expensive; technically demanding, expertise unavailable in many settings | |
| Detection and Identification of Viral Antigens | |||
| Enzyme immunoassay methods (e.g., antigen-capture enzyme immunoassay) | Tissues, cells, secretions, excretions/Reaction of viral antigen with antibody of known specificity | Rapid, sensitive and specific. Most common methods in use today | |
| Immunochromatography, immunogold-binding assays (the equivalent of the home pregnancy test) | Blood, secretions, excretions/Viral antigen identified by reaction with antibody of known specificity | Rapid, sensitive, specific, suitable for testing of individual specimens in the clinical setting | |
| Immunofluorescence | Tissues and cells/Viral antigen identified in situ by reaction with antibody of known specificity | Rapid, sensitive and specific. Localization of antigen in specific cells adds to confidence in diagnosis; technically demanding | |
| Immunohistochemistry (immunoperoxidase staining) | Tissues and cells/Viral antigen identified in situ by reaction with antibody of known specificity | Slow, but sensitive and specific. Localization of antigen in specific cells adds to confidence in diagnosis; technical expertise involved is more like an extension of histopathology | |
| Immunoelectron microscopy | Tissues, cells, secretions, excretions/Character and aggregation of virus by specific antibody of known specificity | Extension of diagnostic electron microscopy. Rapid, sensitive and specific. Expensive and technically demanding; expertise unavailable in many settings | |
| Direct Detection and Identification of Viral Nucleic Acids | |||
| Hybridization methods, including in-situ hybridization, Southern blot hybridization and dot-blot filter hybridization methods | Extracts from tissues, cells, secretions, excretions/Viral nucleic acid identified by reaction with specific DNA probe | Dot-blot methods are rapid, simple to carry out, very sensitive, and with suitable reagents very specific. Largely being replaced with polymerase chain reaction (PCR) procedures | |
| PCR, reverse transcriptase-PCR, real-time PCR, and amplification by isothermal amplification | Extracts from tissues, cells, secretions, excretions/Viral nucleic acid specifically amplified using primer sets and then identified by various methods such as fragment size analysis, labeled DNA probes, probe hydrolysis, and partial sequencing | Some methods can be subject to contamination, causing false-positive results. Nevertheless, because of incredible sensitivity and specificity, becoming used very widely in circumstances where the “state of the art” is required. Automation and new methods for identifying amplified products are leading to quicker, more reliable, and less expensive tests | |
| Viral genomic sequencing and partial sequencing | Extracts from tissues, cells, secretions, excretions/Viral nucleic acid specifically amplified, usually via PCR and then subjected to automated sequencing, usually of only 100–300 bases in selected genomic regions | When combined with automated genome amplification methods and computer-based analyses of results, this becomes the new “gold standard” in identifying a virus | |
| Oligonucleotide fingerprinting and restriction endonuclease mapping | Extracts from tissues, cells, secretions, excretions/Viral nucleic acid amplified, usually via PCR or growing the virus in cell culture, then restriction enzyme digestion and gel electrophoresis to determine characteristic banding patterns (“viral bar-coding”) | Very slow, expensive, difficult to automate, and complex to analyze. Methods largely being replaced with PCR and sequencing | |
| Virus Isolation and Identification | |||
| Virus isolation in cultured cells | Tissues, cells, secretions, excretions/Specimens inoculated into suitable cell cultures and presence of virus detected by various methods, usually immunological methods | Relatively slow, expensive, and technically demanding. However, this is the only method that provides a virus isolate for further testing (e.g., strain typing) and is therefore widely used in reference centers | |
| Virus isolation in animals | Tissues, cells, secretions, excretions/Specimens inoculated into animals, usually newborn or 3-week-old mice, usually by the intracerebral or intraperitoneal routes, with sickness or death as indication of viral growth. Identification of virus by various methods, usually immunological methods | Even slower, more expensive, and technically demanding than virus isolation in cell culture. However, for viruses that do not grow well in cell culture, this is the only method that provides a virus isolate for further testing (e.g., strain typing) and is therefore still used in reference centers in special circumstances | |
| Detection and Quantitation of Antiviral Antibodies (Serologic Diagnosis) | |||
| Enzyme immunoassay (EIA)–enzyme-linked immunosorbent assay (ELISA) | Serum/Specimens tested for presence of specific antibodies indicating recent or past infection | Rapid, sensitive, and specific; the pillar of retrospective diagnosis for many clinical and epidemiological purposes. In many cases, paired sera are needed to confirm infection or recent vaccination | |
| IgM class-specific antibody EIA–ELISA | Serum/Specimens tested for presence of specific IgM antibodies indicating recent infection | Rapid, sensitive, and specific; becoming the pillar of serologic diagnosis of recent infection in human medicine, with limited development in veterinary medicine. In many cases a single serum suffices | |
| Serum (virus) neutralization assay | Serum/Specimens tested for presence of specific antibodies indicating recent or past infection | Cell culture-based method; slow, expensive, and technically demanding. However, this is the “gold standard” of serology, as neutralizing antibodies correlate best with immune protection | |
| Immunoblotting (Western blotting) | Serum/Specimens tested for presence of specific antibodies indicating recent or past infection | Slow, expensive, and technically demanding, mostly used as confirmatory test | |
| Indirect immunofluorescence assay | Serum/Specimens tested for presence of specific antibodies indicating recent or past infection | Rapid, sensitive, but subject to uncontrollable, non-specific reactions | |
| Hemagglutination-inhibition assay | Serum/Specimens tested for presence of specific antibodies indicating recent or past infection | Rapid, sensitive, and specific; widely used for retrospective diagnosis for epidemiological and regulatory purposes. Still a pillar in avian virus diagnostics and for many mammalian virus diseases | |
| Immunodiffusion | Serum/Specimens tested for presence of specific antibodies indicating recent or past infection | Rapid, but can lack sensitivity and subject to specificity problems. There are very good tests available for some diseases | |