Lecture 2 Flashcards
When submitting a live fish sample what is it important to consider
- Transport conditions - heat O2
- Allow 2L of water per 5-10 small fish
- Fill bags 1/4 full of water, leaving the remainder filled with air or oxygen
- Seal and place in second bag along with ice
- Place and seal in outer container - amount and layers of packing depend on the situation
- If you doubt that fish will arrive alive, select samples for individual tests, especially fixed material and smears and perhaps cultures of lesions
When submitting freshly dead samples what is it important to consider
- Submit freshly dead fish sealed in plastic bags, preferably individually wrapped and on ice
- Ensure the fish are not in direct contact with ice or water
- Anaesthetic overdose may cause some parasites to leave the fish so it is recommended smears for parasites be made as soon as possible
- For organic chemical analysis, percussion stunning with a sharp blow to the head is suitable unless the brain is needed for analysis
- Blunt force will also induce haemorrhage artefacts in the gills which may not be wanted
Australia post accepts biological substances under what conditions
- Australian addressee much be recognised by the lab
- The sender must
Qualified medical practitioner
Qualified vet surgeon
Public hospital, clinic or lab
A member of a commonwealth, state or territory police force - Triple packaging
How to set fish for histopathology
Formalin fixation - finfish are the same isotonicity as higher vertebrates, so standard vertebrates fixatives are suitable
What are the exceptions of histopathology
- Rapid penetration is required
- Tissue that tend to separate during processing
- Fixation of small parasites that would otherwise wash off epithelial surfaces during processing
What is Bouin’s fixative
Suitable for small specimens, especially very small fish that can be fixed and sectioned whole
What is Bouin’s fixative useful for
Skin and eyes, although eyes may be better fixed in neutral buffered formula after making a small slice in the back of the retina to aid penetration, as Bouin’s fixative may result in artefacts
What is Davidson’s fixative recommended for
Gils with suspected parasites
Within one week what must the tissue be trandered into from the Davidson’s fixative
70% ethanol within a week
External examination what to look at
- Corneal opacity may assessing the time of death
- (bi or uni) exophthalmia and/or hyphen is common finding with septicaemia but may also indicate gas disturbances
- open mouth as a common finding in a group of dead fish is likely of indicate respiratory distress
How to perform an external examination
- Lift and remove an operculum an examine gills and mouth
- Note gill colour the extent of mucous cover, and any gill or mouth lesion and parasite
- Gill colour is also a guide to the level of post mortem change as well as an indication of anaemia
- Focal lesions or increased mucus may indicate parasites or other irritants
What may parasitised fish show
Few gross signs, though surface parasites are common and often in sufficient numbers to be clinically significant
Where are protozoa and small flukes are best observed in
Wet smears
Where to prepare smears from
Gills and skin surfaces
For lab summations what must be done to the smears
air dried immediately
How are gills examined on microscope
Wet smear examination for microscopic external parasites
How to prepare wet smears of mucus from skin and gills
Lightly scrape the surface of the tissue with a scalpel blade, tranfer the mucus collected to a slide and dilute with a drop or two of water and place a cover slip o
What is important when doing wet smear
Use the same source of water for dilution as that of the fish
How to fix gills
Lift or cut the gill cover and fix one or more whole gill arches from small fish or at least 1.5 cm of an arch from larger fish
How to open the abdominal cavity
Using curved, blunt-ended scissors, open abdominal cavity along the mid-line to just in front of the vent (cut 1) and reflect dorsally (cut 2) or remove one abdominal wall (cut 2 and cut 3) to expose abdominal organs
For small fish, abdominal exposure is best made by one curved incision from in front of the anus curving towards the spine but remaining ventral to the kidney and extending forward to the gill arch (cut 2 and 3)
The abdominal flap can then be retracted ventrally to provide a sterile surface for handling tissues
What to look for in the abdominal cavity
Major organs and note size, colour, friability, haemorrhages, nodules, necrosis or parasites present in the organ
What is a good indicator of conditions
Level of abdominal or pancreatic fat
What may the presence of fluid in the abdominal cavity or swim bladder and/or serial congestion or petechiae indicate
Viral or bacterial diseases
What is the spleen
Major haematopoietic, lymphoid and phagocytic organ
If the spleen is typically enlarged what can it indicate
Infection or stress
How many chambers in the heart do fish have
2 chambers
How to expose the heart
Is exposed by cutting the diaphragm above the liver
What should the pericardial sac be like
Smooth
What fills much of the lumen
Myocardium trabeculae
What does poor heart morphology indicate
Developmental changes, nutritional imbalances or inactivity is relatively common and may result in rounder/more globular shape
What is the kidney
A dark organ closely apposed to the dorsal wall of the abdomen; retroperitoneal and covered but the swim bladder
What does the kidney contain
Haematopoietic tissue and tissue breakdown pigment as well as the nephrons
What is the Corpuscles of Stannius
A small place endocrine gland
How is the pancreas usually found
usually found as microscopic glandular clusters within the fat between the pyloric caeca in salmonids, through in some species e.g. Barra, pancreatic tissue is restricted to the liver in the hepatopancreas
How is the brain exposed
Expose the brain by slicing off the top of the skull
Alternatively, section through the head posterior to the eyes, grasping the fish firmly behind the head and cutting through the head transversely a short distance behind the eyes
Continue cutting until the oropharynx is reached
The incision for the for large fish is best made with culinary or butcher’s knife between 15-20cm ling that has finely serrated cutting edge to the clade or use as hack-saw
What is standard pathology practice
Fix samples for histopathology in 10x their volume of fixative
Samples should me no more than 0.5-1.0 cm thick in two dimensions
What is the preferred fixative to improve penetration of intact skin
Bouin’s fixative
What must be done before fixing whole
- The abdomen must be opened
- An abdominal flap and gill cover removed
- Preferably the swim bladder deflated
Fish should not be fixed unless
Internal organs are well exposed
How to sample for virology
Method of choice fro diagnosis of fish viral disease is virus isolation using fish cell lines
Other techniques, used aline or in combination with this, include, histopathology, direct visualisation by electron microscopy and PCR techniques
What is needed for testing for freedom of disease - OIE
A minimum number of 10 moribund fish or 10 fish exhibiting clinical signs of the disease in question
How to take samples for virology
5mm3 of liver, kidney and spleen, collected aseptically have been traditionally collected for virus isolation and are sufficient to detect most viruses, but heart and encephalon are now more generally included
Why don’t you pool organs such as skin, gut, gills with internal organs
They have high natural bacterial flora
What is the recommended sample number per pool
5 fish per pool
What is important when collecting sterile samples
Use separate sets of instruments for opening the fish and for collection of tissues
What must tool be cleaned of before soaking it in ethanol
Residual blood and tissue
What does the OIE recommend with small yolk sac fry
Be sampled whole if the yolk sac is removed
