Zebra fish techinques and diseases

Most common areas for blood collection in ZF

Dorsal aorta or posterior cardinal vein (survival), decapitation or dorsal aorta through transverse incision of anal and caudal fins (terminal)

Monitoring fish under anesthesia

operculum movement tail fin pinch

Stage 1 anesthesia

teens disoriented, sedated

Stage 2 anesthesia

excited in your 20s

Stage 3 anesthesia

Stage 3 – loss muscle tone in your 30’s

Stage 4 anesthesia

Stage 4 – down hill in your 40’s – no muscle tone

Only FDA approved anesthetic for fish:

MS222

Important notes of MS 222 regarding solubility, acidity and heat

Important notes of MS 222: soluble in water, acidic so requires buffering, potentancy increases with heat

Can you give isoflurane with MS222 in fish?

 Yes, provides a nice surgical plane of anesthesia in 90 minutes, fast recovery,

What is the dose for MS222 for surgical anesthesia?

140 – 164 mg/ L

What is the dose for isoflurane for anesthesia in fish?

325 mg/L

What is the safety margin of isoflurane in fish?

Narrow safety margin, 30% mortality at 350 mg/L.

Eugenol, cost, safety, induction time, and solubility

Important components of eugenol: low cost, wide safety margin, fast induction, metabolism. Require ethanol to become soluble.

Side effects of eugenol

Side effects: long recovery time and can cause adverse effects

Does Metatomidate provide analgesia?

no

side effects of metatomidate?

Side effects: long recovery, can see fasiculations, decrease cortisol and glucose, increase pigmentation

Can you cool fish for anesthesia?

Yes, get water to 10  - 12 C, for quick nonpainful procedures.

Does quinaldine have any analgesic properties?

Yes, but minimal

Immersion techniques for acceptable euthanasia?

Immersion: MS222, benzocaine, lidocaine, quinaldine sulfate, 2-phenoxyenthanol

Can chilling be an acceptable form of euthanasia?

Rapid Chilling (2-4°C)

What are the acceptable with conditions of euthanasia for fish?

a.      Immersion: CO_2, eugenol/clove oil

b.      Rapid Chilling (2-4°C): small-bodied tropical/sub-tropical

c.      Maceration

d.      Decapitation + pithing

e.      Manual blunt force trauma + pithing/exsanguination

Examples of unacceptable methods of euthanasia?

a.      Flushing

b.      Slow chilling or freezer

c.      Anoxia / dessication by removal from water

d.      Caustic chemicals

e.      Prolonged trauma

f.        Metomidate (illegal for extralabel euthanasia

Rapid chilling components based on age: adults

10 minutes

Rapid chilling components based on age: fry (4-14 dpf)

20 minutes

Rapid chilling components based on age: <3dpf

<3dpf: add-in sodium/calcium hypochlorite

What is being shown here?

pericardial effusion

What is being shown here?

Dropsy, edema

Most common mycobacterium in ZF:

M. chelonae (nonpathogenic)

Most common pathogenic mycobacterium in ZF:

M. marinum, and M. haemophilum

Common clinical signs of Mycobacterium haemophilum

Poor growth, wasting, emaciation

Most common clinical signs of M. marinum:

dropsy, edema

Most common generic clinical signs of mycobacteriosis in fish:

Granulomas (external and internal), Spinal curvature, muscle deformities

What likely pathogen causes this gross pathology?

mycobacteriosis

Which mycobacterium species cause the gross pathology in A, B, and C?

A / B: M. marinum C. M. haemophilum

Stains used for staining acid fact mycobacterium?

Fite-Faraco, Ziehl-Neelsen, Kinyoun

What organs of the fish are best for diagnostic staining for mycobacterium?

spleen and kidney

How long does it typically take to grow M. marinum and M. haemphilum:

a.      M. marinum (10 – 14 days)

b.      M. haemophilum (6-8 wks)

What type of bacteria and what is the stain?

Mycobacterium and Ziehl-neelsen

Control of mycobacterium

eradicate completely and bleach. M. marinum is zoonotic and cause fish handler granulomas.

Which mycobacterium species is zoonotic for fish? And what does it cause in humans?

M. marinum, cause fish handler granulomas on the fingers, where broken skin is. Usually in immunocompromised people

Edwardsellosis gram stain and what is the name of the disease in catfish (there are two)?

Gram negative. hole in head disease / enteric septicemia in cat fish

Which species of edwardsellosis is most common in ZF and what is the severity of the disease?

E. ichtaluri can cause severe mortality and morbididty in ZF.

CS of E. ichtaluri

CS of edwardsellosis: acute presentation w/septicemia=> lethargy, raised scales, dropsy, skin ulceration, hemorrhage in skin near eyes/opercula, fins, ventral abdomen

What pathogen is likely the cause of this presentation? There was a rapid and almost 100% mortality in the tank of this fish.

Edwardselli ichtaluri

Aeromonas hydrophila and sobria gram stain and what is another name of this disease in fish (reflects the clinical signs)?

Aeromonas hydrophila and sobria: gram negative, ulcer disease

What is the pathogen that could cause these clinical signs of ulcerations?

Aeromonas spp.

Clinical signs of Aeromonas in ZF

Clinical signs in ZF: petechial hemorrhages of skin, fins, oral cavity, muscle

Treatment and prevention of aeromonas in ZF

Treatment: remove stressors, antibiotics (oxytetracycline, nifurpirinol)

Is A. hydrophila zoonotic?

yes low zoonotic risk.

Flavibacterium branchiophilum (BGD) and F. columnare (Columnaris) gram stain and common names

gram negative, bacterial gill disease, cotton wool, fin rot

Which pathogen is opportunistic and is commonly seen with shipping stress in zf?

Flavibacterium branchiophilum (BGD) and F. columnare (Columnaris)

CS of BGD (Flavibacterium bronchiophilum)

labored breathing, surface swimming

CS of Flavibacterium columnare

Saddleback, loss of pigment and scales.

What pathogen is the picture describing?

F. bronchiophilum

Diagnosis of BGD?

Diagnosis: Wet mounts of gills: filmentous bacteria, fused secondary lamella

What pathogen causes this disease in fish:

F. columnare

What pathogen causes this disease in fish:

F. bronchiophilum

What is the most common pathogen of ZF?

Microsporidiosis

What are the most common microsporidia of ZF:

Pseudoloma neurofilia, Pleistophora hyphessobryconis

Are pseudoloma and pleistophora intracellular or extracellular?

intracellular

How is microsporidia transmitted?

Both vertically and horizontally.

Where do pseudoloma and pleistophora preference in the body?

Pseudoloma (neuro), Pleistophora (skeletal muscle).

What filamentous pathogen causes this grosely?

microsporidiosis

Clinical signs of microsporidiosis:

Degenerative muscle atrophy, scoliosis, lordosis, reduced growth

What pathogens present with this?

Pseudoloma neurophila and Pleistophora.

How long should you have sentinels to ensure SPF populations don’t have microsporidia?

3 months

Sanitation for microsporidia?

UV sterilization, Chlorine 100 ppm (>95% of spores) = useful for equipment disinfection. Can aerosolize

Are classic egg surface sterilization techniques sufficient for preventing microsporidiosis?

Egg surface sterilization practices are insufficient in inactivating spores on outside/inside embryo

What are saprolegnia

Water molds, are pseudo-fungi. Opportunistic, ubiquitous in environment.

What pathogen causes this?

Saprolegnia

What is the type of pathogen and common disease name of Piscinoodinium:

motile protozoa, velvet disease, gold dust disease.

What are the common species of Piscinoodinium?

P.pillulare and P.limneticum

Describe the transmission lifecycle of piscinoodinium

Horizontal transmission, Trophont feeds on skin / gills via rhizocyst projections skin / gills segments slough.

Trophont drops off sessile tomont produces infective dinospores

What is the infective stage of piscinoodinium?

dinospore, produced by sessile tomont

Clinical signs of piscinoodiumium in fish?

excess mucus, skin darkening, anorexia, depression, surface swimming, labored breathing, flashing/rubbing side of tank due to epithelial irritation/pruritis, yellow/rusty color (heavy infestation)

What pathogen is this in fish?

piscinoodiumium

Treatment of piscinoodinium:

Saltwater baths, Copper sulfate (dangerous), Raise water temp 33-34°C (ZF can tolerate)

Ichthyophythirius multifilis, what type of pathogen, and what is a common disease name for this species?

ciliated protozoa, causes white spot disease

Lifecycle of I. multifilis?

a.      Trophonts – feed on fin, skin, gills

b.      Tomonts – substrate, sticky

c.      Theronts – infectious/motile

What pathogen causes this?

Ichthyophythirius multifilis

Roundworm of ZF?

Pseudocapillaria tormentosa

What species is this? Whats a unique characterisitic on the eggs?

Pseudocapillaria tormentosa, Bipolar plugs on eggs

Clinical signs of pseudocapillaria:

a.      Wasting, ↓ fecundity, Appearing darker in color, Proliferative enteritis, Neoplasms at intestinal-esophageal junction, intestinal cell carcinomas or mixed malignant

Treatment of pseudocapillaria?

TX: Fenbendazole in Artemia

Most common neoplasm of ZF:

1.      seminoma

a.      Older males

b.      Rarely invade locally/metastasize