Clinical Medicine

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Clinical Medicine Necropsy Techniques

• Claudia Harper, DVM, Dipl. ACLAM

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Clinical Evaluation of Zebrafish Presentation
Outline

• Sample Submission
• Comprehensive Evaluation
• Clinical Evaluation
• Physical Examination
• Anesthesia Euthanasia
• Gill, Skin, Fin Wet Mount Preparation
• Microbiology and Fungal Evaluation
• Histology

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Steps In Working-Up a Clinical Case

• History
• Clinical presentation
• Number of animals affected
• Systems affected
• Duration of clinical presentation
• Water quality parameters
• Peracute, acute, chronic
• Water
• Water quality log
• Parameters within normal range
• Fluctuations
• Collect a water sample
• Oxygen, temperature
• Ammonia, nitrite, pH, hardness, salinity
• Evaluating the appearance of fish, tank and
  facility
• Biological scaffolding
• Chemicals in the room

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Water Quality and Housing System Evaluation
Most fish health problems are due to poor water
quality and husbandry.

• System Evaluation
• Quarantine program / Source of fish
• Acclimation and prevent fluctuations
• Evaluate tank appearance
• Biological scaffolding
• Water Parameters
• Frequency of testing
• Monitoring systems
• Water sample submission

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Sample Submission and Techniques

• Samples Submitted
• Live Fish - preferable
• Tissue culture containers or sealed water tight
  plastic bags (double bag). Plastic bag should
  contain 1/3 water and 2/3 air
• Ship in styrofoam lined cardboard shipping box
• Overnight shipping
• Heat pads added to shipping box
• Fixed Fish
• Bouins
• 10 Buffered Formalin
• Water Sample
• Common Diagnostic Procedures Available
• Necropsy
• Parasitology
• Bacterial Fungal Culture
• Hematology
• Histology
• PCR

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Comprehensive System Evaluation (MGH)

• Fish Necropsy
• Fish Parasitology
• Baffle or Trough Evaluation
• Water Parameters
• Histology
• Microbiology
• PCR?
• Minimum of fish requested is 3

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Physical Examination
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Clinical Evaluation of Zebrafish

• Behavior
• Buoyancy
• Flashing
• Location in water column
• Body Condition
• Abdominal distention
• Emaciation
• Eyes
• Exophthalmia
• Gill / Operculum
• Coloration
• Anatomy missing operculum
• Flaring
• Musculoskeletal System
• Deformities

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Clinical Evaluation of Zebrafish (contd)

• Fins / Skin
• Frayed fins
• Nodule
• Ulceration
• Erythema, Hyperemia
• Excess mucus
• Discoloration
• Urogenital pore
• Fecal casts
• Ulceration

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This table can be used as a guideline
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• Clinical Signs Differential Diagnosis
• Opercular flaring respiratory distress,
  parasites, bacterial
• Sloughed mucus, skin chemical irritation,
  parasites
• Clamped fins parasites
• Hemorrhage bacterial infection, parasites
• Scale loss parasites, trauma
• Improper buoyancy swim bladder failure,
  parasites
• Lethargy bacterial infection, virus, stress,
  starvation
• Surface breathing hypoxia
• Acute mortality chemical toxicity, poor water
  quality
• Wasting, emaciation bacterial infection, virus,
  fungal, stress
• Rough, thickened skin parasites, fungal
  infection

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Zebrafish Euthanasia Anesthesia
2000 Report of the AVMA Panel on Euthanasia
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2000 Report of the AVMA Panel of Euthanasia
External or Topical Agents

• Tricaine Methane Sulfonate (TMS, MS-222)
• Water bath
• Should be buffered with sodium bicarbonate pH of
  7.07.5
• Benzocaine Hydrochloride
• Water bath
• 2-phenoxyethanol
• Water bath
• Carbon Dioxide
• Water bath
• Cooling
• Does not reduce pain in ectothermic species
• Freezing not recommended unless animal deeply
  anesthetized as formation of ice crystals on skin
  and in tissues of animal may cause pain or
  distress
• Clove Oil
• Water baths are not acceptable in fish

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Euthanasia of Zebrafish Tricaine Methane
Sulfonate (TMS, MS-222)
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Euthanasia of Zebrafish Tricaine Methane
Sulfonate (TMS, MS-222)

• Prevent inhalation, ingestion, eye or skin
  contact
• Proper PPE should be worn
• Work under hood when handling the powder
• Mask, eye protection, gloves are recommended
• Stock solution is good for a couple of days.
  Stock solution will loose efficacy after a week
  or so.
• MS-222 is light sensitive

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External Anatomy of a Zebrafish
Caudal fin
Dorsal fin
Operculum
Eye
Vent
Anal fin
Pectoral fin
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Cutaneous Wet Mount Preparations

• Three Cutaneous Sites
• 1) Gill
• 2) Skin / mucus / scales
• 3) Fins
• Materials Needed
• 1) Glass slide
• 2) Coverslip
• 3) Scalpel blade, scissors, forceps
• 4) Dissecting microscope
• 5) MS-222

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Gill Biopsy

• The Gill Epithelium is The Major Site of
• Gas exchange
• Acid-base balance
• Ionic regulation
• Excretion of nitrogenous waste
• Gills are Evaluated for The Presence of
• Parasites
• Bacteria
• Hyperplasia
• Fused lamellae
• Telangiectases
• Mucus

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Gill Wet Mount Preparation

• Remove the operculum
• Lift up segment of the gill lamellae and biopsy a
  small portion using scissors
• Transfer gill biopsy to cover slip
• Add a drop of water onto slide and place the
  covers lip

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Melanocytes
Secondary lamellae
Telangiectases
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Normal Zebrafish Gill Wet Mount
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Fin Wet Mount Preparation

• Zebrafish paired fins
• Dorsal, pectoral, ventral, anal, caudal
• Extend and examine the fins
• Parasitic and fungal organisms
• Frayed tissue
• Superficial lesions

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Fin Biopsy
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Fin margin
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Skin Wet Mount Preparation

• The epithelium provides
• Mucus production
• Immunoglobulins
• Assist in maintaining osmotic regulation
• Skin are evaluated for the presence of
• Parasites
• Erosion
• Ulceration

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Skin Wet Mount Preparation
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Skin Wet Mount
Cycloid scales
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Skin Wet Mount
Epistylis
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Necropsy

• Petri dish
• Dissecting microscope
• Slides
• Cover slip
• Scissors
• Forceps
• Pipette

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Female Zebrafish
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Anatomy / Squash Preps
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Drawing from Spike Cover 2003. www.akca.org/kht/an
atomy.pdf
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Adult Female Zebrafish Anatomy
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Diagnostic Procedures

• Necropsy
• Wet Mount Preparations
• Parasitology
• Bacterial Fungal Culture
• Hematology
• Histology
• PCR

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Sample Collection for Bacteria Fungi

• Sampling for bacteria and fungi should be done on
  live zebrafish.
• For cutaneous samples the fish is alive or
  euthananized. The area to be cultured should not
  be handled prior to culturing. Use sterile loop
  or culturette.
• Beckon Dickinson Mini-Culturette Culture Swab
  Plus Amies Gel without Charcoal / Flexible
  twisted wire

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Sample Collection for Bacteria Fungi(contd)

• Culture Samples
• Cutaneous
• A mixed bacterial culture should be expected.
• Gills
• Euthanize the fish
• Gently rub the sterile culturette through the
  gill arches.
• The culturette should pick up mucus.
• A mixed bacterial culture should be expected. If
  a pure culture of a potential pathologic bacteria
  is obtained, this should be considered a possible
  cause of disease in this fish.
• Blood
• Euthanize the fish (?)
•  

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Sample Collection for Bacteria Fungi(contd)

• Kidneys
• Most important internal organs to culture.
• There are two methods of culturing the kidney.
• The first method is to cut the dorsal fin off
• Sterilize the open area with heat or dip the fish
  in alcohol
• Cut the vertebra with a sterile scissors or
  scalpel
• Bring the head and tail together
• Exposes the kidney for culturing.
• The second method is collecting the samples
  internally
• Possible contamination by internal organs.
• Dipped into 70 alcohol
• Open the abdominal cavity aseptically to allow
  all organs to be exposed.
• Sterilize with heat, the desired internal organs
  to be cultured, cut open the organ with a sterile
  scalpel and culture.

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Blood Collection

• No survival technique described so far.
• Collection from the dorsal aorta.
• Yield range from 1-10 µl per fish.
• Usually samples are pooled post mortem.
• Micropipette tip used for blood smears
• EDTA coated microtube used for erythrocyte count.
• Lymphocyte 82.95
• Total RBC 3.02 X 106 cells/ microl

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Histology

• Fixatives
• Bouins
• 10 Formalin
• Others
• Technique
• Perfuse gills with a 25G needle
• For optimal fixative penetration tissue should
  be no thicker than 3 mm for any fixative.
• Fix tissue 12-20 hours, if tissue is fixed in
  formalin.
• Place tissue in 70 ethanol for storage

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Zebrafish Histology

• Fixatives
• Bouins
• 10 Buffered Formalin
• Dietrich's
• Technique with Bouins
• Perfuse gills with a 25G needle
• For optimal fixative penetration, tissue should
  be no thicker than 3 mm for any fixative
• Fix tissue 12-20 hours
• Place tissue in 70 ethanol for storage

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Conclusions

• Working with live zebrafish provides far superior
  diagnostic value
• Comprehensive system evaluations provide a global
  perspective of diagnostic cases or sentinel
  evaluation
• Ask for a minimum of 3-5 fish for a system
  evaluation
• Work with other well establish fish medicine
  groups from other industries