Fish & Invertebrate Diagnostics & Clinical Pathology

Question 1

Ideally, reference intervals should meet what criteria to be valid?

Why is this so difficult in fish medicine?

Answer 1

Reference Intervals & Material in Fish Medicine

• Ideally, RI = derived by the laboratory at which testing is done, from a pop of at least 120 of the same species, under the same husbandry conditions, using the same instrumentation.
• This = virtually impossible due to the low number of clinically normal fish specimens available for testing and the small blood volumes associated with some species.
• When RI are unavailable, online databases (IVIS) or primary or 2ndary lit can be used
  
  • Use caution with these sources = diff labs, health status of animals, diff methodologies,
    
    • etc

Question 2

Describe the preparation of wet mount for diagnostics in fish.

What diseases can be diagnosed with skin scrapes and gill clips?

What is the appearance of eosinophilic granulocytes on cytology?

How is enteritis confirmed on fecal wet mounts?

Answer 2

Wet Mount Exams

• Most common diagnostic used in fish med
• Water used on slide should be from system of origin of fish/parasite (external samples), or
• physiologic saline (internal samples)
• For most accurate results, wet mounts should be evaluated immediately (to prevent false -)
• Certain parasites (ie. monogeneans) inhabit specific locations, and can be missed unless multiple sites sampled

Skin Scrapes

• A few scales + mucus should be collected & spread out beneath coverslip
• Esp useful in identifying ectoparasites (Ciliates, Dinoflagellates, Monogeneans, Leeches, Copepods, & Branchiurans)
• Other uses
  
  • ID of columnaris (Flavobacterium columnare), oomycetes (saprolegnia), and hyperplastic cells from lymphocystis disease

Gill Biopsies/Gill Clips

• Distal portions of filaments should be clipped, far enough from arch to see filaments separately via microscope
  
  • Don’t attempt to view entire gill arches w/ filaments attached, or have filaments on top of each other
• Normal appearance
  
  • Interlamellar spaces should be free of tissue debris & excess mucus
  • Should see a central cartilage rod bordered by marginal arterioles and an epithelial layer
• Common abnormal/pathologic changes
  
  • Swelling & hyperplasia of lamellae resulting in clubbing of entire filaments.
  • Congestion, hemorrhage, telangiectasia (lamellar aneurysms), excess mucus, necrosis, parasites
  • Gill tissues deteriorate rapidly after death, and can falsely look like path lesions

EGCs (Eos Gran Cells) or CEG (Coarse Eosin granulocytes) can be mistaken for parasites, but are equivalent to a mammalian mast cell, and are no protozoa or eosinophils

• Tissue-associated EGCs = abundant, large, round cytoplasmic granules and eccentric ovoid nuclei granules are intensely eosinophilic on cytologic and histologic stains.
• In contrast, EOSINOPHILS = smaller cells, with smaller, less intensely staining
• granules.

Fin Bx/Fin Clips

• Place on flat slides & manually spread to eliminate folds & tissue overlap
• Fin Bx samples have dendritic pigment cells w/ branched tree-like projections
• Pay special attention to free-margin of fin clip = often tattered or scalloped due to necrosis or tissue loss

Fecal Wet Mount

• Teleost intestines have relatively few bacteria compared to mammals (Lumsden 2006).
• While high bacterial # may indicate enteritis, it’s more commonly d/t postmortem autolysis.
• If enteritis is suspected, look for inflammatory cells and phagocytized bacteria on stained
• cytologic samples

Tissue Wet Mounts/Squash Preparations

• Samples of organs/tissue should be small (~1 mm3) & significant pressure may be needed to produce a thin sample.
• Tissue wet mounts can also be useful for sex determination & assessment of gonadal maturity.

Question 3

Describe the use of cytology in fish medicine.

What are some issues that can cause some issues with the quality of the cytologies?

What stains are used in fish cytology and what are their benefits?

Answer 3

Cytologic Examination

• Most commonly used cytologic stains = Romanowsky stains (e.g. Dif-Quik).
• Gram & AF stains can be used in addition to further categorize findings.

Factors that Affect the Diagnostic Quality of Stained Cytologies

• Typically, tissues (aspirates & impression smears) of epithelial origin (skin, gills) exfoliate better than mesenchymal tissues, which often yield too few cells to evaluate adequately
• While the staining techniques listed on the package inserts typically provide adequate cellular detail, prolonged staining times may be needed for thick samples (fecal swabs, gill clips).
• Slides exposed to formalin, including fumes = typically deeply basophilic and lack adequate detail for interpretation.
• Formalin fumes permeate most things, so unstained slides should not be stored or shipped in packages containing biopsy or other specimens fixed in formalin
• Pre-staining slides prior to shipping prevents this dilemma.

Cytologic Sample Evaluation

Romanowsky Stains

• Stain proteins pink, nuclear material purple, and most bacterial organisms blue (Figure
• A7.12).
• Mycobacteria do not take up Romanowsky stains.
• Stains include Wright’s stain (used in large diagnostic labs), and the rapid Diff-Quik and Camco stains.
• It is important to note that the vast majority of bacterial pathogens stain blue with Romanowsky stains and should not be equated with a positive Gram stain reaction.

Gram Stains

• Should be used in conjunction with Romanowsky-stained smears for concurrent evaluation of inflammatory cells to minimize over-interpreting bacterial contaminants.
• Bacterial presence does not = pathology
• Heat fixation of the slides is typically recommended.

Acid Fast Stains

• e.g. Ziehl–Neelsen, Kinyoun’s, Fite’s
• 🖉 The Kinyoun’s stain (known as a “cold method” or as a modified Ziehl–Neelsen stain) is particularly user friendly = since no heating step is required.
• Mycobacteria (and other Acid Fast bacteria & cellular structures) stain pink/red
• With Romanowsky stains, mycobacteria = “negatively” stained
• Nocardia spp. bacteria stain weakly or not at all with Ziehl–Neelsen stains, but are strongly AF (+) with the Fite’s modification of the AF
• Microsporidia, the shell valves of myxozoans, and coccidian sporozoites also stain acid-fast positive

Question 4

Describe fish hematology.

What anticoagulants should be used?

What methods should be used for leukogram evaluation?

What is the most numerous leukocyte in teleosts?

What do the nuclei of teleost neutrophils look like? Are there any exceptions to this?

What are the most abundant leukocytes in stingray versus shark blood?

What are the differences between coarse and fine eosinophilic granulocytes?

Answer 4

Hematology

• Sample Processing
  
  • EDTA works well for teleosts & most elasmos, but can cause lysis in some spp.
    
    • Powdered (or spray-on) EDTA tubes will help avoid any dilution affect from inaprop tube fill
  • Stingray blood should be placed in HEPARIN. EDTA causes their blood to become viscous
    
    • However, Heparin causes thrombocyte clumping, making quant difficult
      
      • Recommend Erythrogram from heparin, and thrombogram/Leukogram from EDTA ifenough blood available.
  • Elasmobranch blood may be adjusted for its high osmolality by adding urea & Na-Cl to preventcell lysis
• Sample Evaluation
  
  • Only PCV can be performed on fish blood (not HCT, which requiresautomated hematology analyzers)
  • Low PCV can be due to pathology, as well as decreased water temperature
    
    • May be d/t decreased metabolic demand for O2 or presenceof increased DO at lower temps
  • High PCV seen in more active spp., handling stress, and hemoconcentration
• Leukogram
  
  • Teleosts = use Natt & Herrick’s solution, and a hemocytometer
    
    • With Natt & Herrick’s solutions, RBCs = light pink/red,Thrombocytes = slightly darker purple than RBCs w/ indistinct margins, and Leukocytes = dark purple.
  • Elasmobranchs = use Modified Natt & Herrick’s, w/ addition ofNaCl to match osmolality of elasmo blood, performed on a Neubauer counting chamber
    
    • Hard to differentiate thrombocytes from lymphocytes
  • Leukocyte estimation from blood smears (non-hemocytometer ornon-Neubauer) should be limited to qualitative (low/normal/high) assessments.
• Erythrogram
  
  • Elasmobranch RBCs = typically larger than teleosts, w/ lower N:C ratio.
  • In most spp., low # (up to 3%) of circulating RBCs are immature = NORMAL
• Thrombogram
  
  • Fish thrombos = nucleated w/ pale, basophilic cytoplasm = easily confised with lymphocytes
    
    • ID of thrombo clumps is helpful in differentiating these
  • In elasmos, a subset of thrombos contains rod-shaped pinkgranules, and when active, rods can become elongated & easier to distinguish
• Teleost Leukocytes
  
  • Lymphs = most numerous LEUKOCYTE in teleosts
    
    • Occasional granular lymphocytes can be seen (azurophilic granules)
  • Neutrophils = most numerous GRANULOCYTE in most telesots
    
    • Nuclei = usu. eccentrically placed & round or lobed, oval, or slightly reniform.
      
      • Salmonids = exception ➔ segmented nuclei similar to mammalian granulocytes
    • Nuclei surrounded by a moderate amount of pale blue cytoplasm containing non-staining (“neutral”)granules
      
      • Goldfish = exception ➔ heterophils w/ pink, rod-shaped intracytoplasmic granules
  • Monocytes = largest fish leukocytes in low #s
    
    • Resemble those of other animals
  • Eosinophils & Basophils = Existence in teleosts is controversial, but reported in some spp.
• Elasmo Leukocytes
  
  • Basophils are abundant in stingrays but present in low numbers in other elasmobranchs.
    
    • They have blue-black granules which often obscure the nucleus.
  • Sharks, unlike skates and rays, have neutrophils in peripheral blood.
    
    • However, there is much discordant information regarding the classificationof other elasmobranch granulocytes
      
      • Descriptive System:
        
        • Coarse eosinophilic granulocytes (CEGs) = contain large round pink/red/orangegranules
          
          • have been likened to eosinophils
            
            • These should not be confused with the eosinophilic granular cells (EGCs)described under gill biopsies of teleosts.
        • Fine eosinophilic granulocytes (FEGs) = contain round or lobed nuclei surrounded by cytoplasm w/ slender, rod-shaped pink granules ➔ have been likened to heterophils.
• Special Stains for Hematology:
  
  • New methylene blue (NMB) = stain used to identify immature erythrocytes in circulation
    
    • It’s a vital stain = requires live cells to take up dye ➔ should not be used on premade bloodsmear
      
      • Needs a 10 min incubation of a drop of anticoagulated blood with a drop ofstain, then make blood smear

Question 5

Describe biochemistry analysis in fish.

What tubes are usually used?

How do changes in sodium and chloride occur as a result of stress or gill disease in freshwater as opposed to marine fish?

Changes in potassium concentrations may be associated with what diseases?

Hypermagnesemia is associated with what sample error?

Increased clacium and phosphorus is likely indicative of what?

Increases and decreases in urea in elasmobranchs may be indicative of what?

Answer 5

Blood Biochemistry

• Sample Processing & Evaluation
  
  • RTT or GTT
    
    • Heparin most common in fish
      
      • If analysis is delayed beyond 24 hours, the plasma or serum should be frozen.
    • Stress-induced catecholamine release increases gill permeability of teleosts, leadingto changes in sodium (Na) and chloride (Cl) ➔ freshwater (decrease d/t loss) and marine (increase d/t influx)
  • Sodium and chloride levels are expected to decrease in freshwater teleosts with gill disease and increase in marine teleosts with the same clinical condition
  • Lactate release from white mm can induce lactic acidosis w/in 5 min of capture/restraint, affectingelectrolytes
  • MS-222 reportedly induces Hyper-glycemia & -kalemia & K+ and increases urinary electrolyte excretion inteleosts
  • Blood gas parameters that are temp-dependent (pH, pCO2, and pO2)
• Water temp (equivalent to patient temp in poikilotherms) needs to be entered into the analyzer
• Marine elasmos = plasma osmolality is slightly hyperosmotic to seawater (800–1100 mOsmol/kg), due to high levels of urea, trimethylamine oxides (TMAO), Na, and Cl.
• Na and Cl = substantially higher in elasmos than teleosts ➔ often exceed linearity of mostanalyzers (error messages or erroneous values)
• Specific Electrolyte Changes
  
  • Decreased K = associated with loss through the GI and skin, & nitrite tox.
    
    • Less affected by changes in gill permeability and renal blood flow than Na & CL
  • Increased K = often artifactual but has been associated with muscle damage, handling stress, andhemolysis.
  • Increased Mg = can also increase up to 10-fold in hemolyzed samples ➔ fish RBCs are rich in Mg.
  • Increased Ca & P = can be due to vitellogenesis via mobilization from scales
  • Increased Urea (in teleosts) = gill damage or dysfunction
    
    • In teleosts, urea, UA, creatinine are of minimal use in renal evaluation
  • Increased Urea (in elasmos) = renal disease (since resorbed by the kidney)
  • Decreased Urea (in elasmobranchs) = hepatic disease (since made by kidney), and prolonged anorexia
  • Fish can maintain glucose concentrations during prolonged starvation (up to 150 days), so liverglycogen stores (hepatic assessment) may not correlate well with BG