Infectious Disease Management in Animal Shelters. Группа авторов

before testing Single‐well test devices (e.g. SNAP tests [IDEXX Laboratories, Westbrook, ME]) Anticoagulated whole blood, serum, plasma, or fecal samples^aSamples can be fresh, refrigerated up to 1 week, or frozen and thawed^aAllow samples and test kits to reach room temperature before testingStore test kits under refrigeration until expiration date OR at room temperature for 90 days (or until expired)Test kits removed from refrigeration for more than 24 hours should be used within 90 days or until expired Lateral flow assays (e.g., Solo Step^® tests [Heska Corporation, Loveland, CO], VetScan^® [Abaxis, Union City, CA], WITNESS^® tests [Zoetis, Parsippany‐Troy Hills, NJ]) Anticoagulated whole blood, serum, plasma, or fecal samples^aSamples can be fresh, refrigerated for 3–7 days (up to two weeks for feces), or frozen and thawed^aRun within 10 minutes of opening test pouch^aStore test kits at room temperature until the expiration date Fecal Examination Direct fecal smear Nematode larvae, protozoan trophozoites, heavyeggs, motile organisms Refrigerate if not examined within 1–2 hours of collectionExamine within 30 minutes if evaluating for protozoan trophozoitesBreak up a small piece of feces in a drop of saline on a glass slide; use a coverslipFeces remaining on rectal thermometer after use is sufficientUse of water instead of saline can result in lysis of fragile organismsDried fecal smears may be stained though motility will be lost Fecal flotation Nematode eggs, cestode eggs, protozoan cysts Approximately 1 teaspoon (4 grams) of fresh feces should be analyzedCentrifugation methods will recover more eggs, species with fewer eggs, and dense eggs; eggs will float in less time but additional steps are requiredGravitational methods are suitable for screeningSodium nitrate solution will float eggs in 10 minutes; immediate evaluation is necessary to reduce distortionSaturated sucrose solution will float eggs in 15–20 minutes Hematology Complete blood count Minimum metabolic database Use largest bore needle available to minimize hemolysisCollect directly in anticoagulant (e.g. EDTA) and promptly mixEnsure proper anticoagulant to sample ratio to minimize dilution of cell counts Chemistry Minimum metabolic database Use largest bore needle available to minimize hemolysisCollect without anticoagulant; allow to clot for 15–20 minutes (serum)For small volume samples (e.g. puppies, kittens, exotics), collect with lithium heparin and centrifuge immediately (plasma) Blood glucose Hypo or hyperglycemia Process whole blood sample within 30 minutes or separate serum and refrigerate until analysisStress, meals, anemia, sample handling and instrument variation can all lead to erroneous resultsPortable monitors are likely to slightly underestimate the concentration Urinalysis Specific gravity Minimum metabolic database, dehydration, evidence of urinary tract disease Use refractometer, reagent strip stick readings inaccurate Reagent strips Minimum metabolic database, dehydration, evidence of urinary tract disease Note collection method; cystocentesis and catheterization preferred; midstream voided sample acceptableUse refractometer to measure specific gravity, strip readings inaccurateDrop urine onto each pad, do not dip the strip in the sampleAnalyze immediately or refrigerate for up to 6 hoursAllow refrigerated samples to reach room temperature prior to analysisLeukocyte, nitrite, and specific gravity readings unreliableProtect strips from air, light and moisture; mind expiration dates Sediment analysis Minimum metabolic database, dehydration, evidence of urinary tract disease Note collection method; cystocentesis and catheterization preferred; midstream voided sample acceptableCollect 2–6 ml of urineAllow refrigerated samples to reach room temperature prior to analysisAnalyze within 4 hours or store under refrigeration up to 24 hoursCentrifuge sample for 5 minutes at 1,500 RPM, decant supernatant, resuspend sediment prior to analysisExamine stained and unstained sedimentExamine at 10x for crystals, casts, and epithelial cellsExamine at 40x for microorganisms, red blood cells, white blood cells Culture Evidence of urinary tract disease Note collection method; cystocentesis and catheterization preferred; midstream voided sample acceptableUse a sterile collection method and storage containerAerobic culture is indicated in most circumstancesStore under refrigeration and transport to the diagnostic laboratory within 12 hours

      ^a Vary widely based on specific tests and kit manufacturer, follow manufacturer's product insert guidelines.

      As the name implies, ELISAs are testing methods that rely on the detection of an enzyme‐linked protein within a variety of test samples to indicate either a qualitative or semi‐quantitative result. ELISAs can be direct or indirect, though the former are not routinely used outside of diagnostic laboratories. The basic operational principles of indirect ELISAs for antibody detection are as follows:

      1 Test substrates are pre‐coated with the antigen by the manufacturer.

      2 A biological sample containing the antibody to be detected is exposed to the substrate.

      3 Antibody in the sample binds to the pre‐coated antigen creating an antigen–antibody complex. Unbound antibody is washed away.

      4 An enzyme‐labeled antibody (i.e. conjugate) binds to the antigen–antibody complex. Unbound labeled antibody is washed away.

      5 An enzyme‐substrate reacts with the enzyme‐labeled antigen–antibody complex, resulting in a color change indicating the test result.

      In such tests, the intensity of color change is proportional to the amount of antibody present in the sample. Detection of antigen can be accomplished similarly, however, the well is pre‐coated with antibody allowing for binding and identification of antigen in the test sample.

A sandwich ELISA is a variation on the indirect ELISA commonly used to detect specific antigens. Many of the commercially‐available point‐of‐care test kits utilize this methodology in the form of manually activated bi‐directional flow devices (SNAP^® tests [IDEXX Laboratories, Westbrook, ME]) or lateral flow devices (also known as rapid immunochromatography). The former devices are largely automated and self‐contained; the benefits of which include minimization of background coloration, reduced washing time, and an increased opportunity for binding of the test sample to indicators (O'Connor 2015). Lateral flow assays generally offer more rapid analysis with fewer steps, lower operational costs, and greater environmental stability, however, they may be subject to decreased sensitivity and greater difficulty with interpretation of results, particularly in hemolyzed samples (O'Connor 2015; Sajid et al. 2015). Additional variations on the indirect ELISA include the labeled antigen ELISA and the competitive ELISA, see Table 4.4.

      4.3.2.2 Diagnostic Cytology

Examination and interpretation of cellular samples is a rapid,