analysis, as well as blood or tissue samples for immunological,
serological or molecular studies. However, these procedures
will not be covered or discussed in detail in this book.
3.2
Sample Size and Euthanasia
The number of fish sampled for a health assessment will vary
according to the objectives of the study. For example, certification of freedom of a notifiable disease generally follows the
guidelines from the Office International des Epizooties (OIE).
Here the sample size is based upon an assumed prevalence
of the specific pathogen to an agreed level of confidence.
To obtain a 95–98 % probability of detecting at least one
infected fish in a clinically healthy population this translates
to a minimum of 30 individuals. Conversely, for disease
investigations 5–10 fish showing abnormal behaviour or the
characteristic signs of the condition will be adequate for necropsy. Fish removed for examination should, where practical,
be placed into a smaller container where further observations
can be made before any procedure or the removal of tissues or
body fluids. The fish should be euthanized by a humane
method, ideally through an overdose of anaesthetic and ideally
maintained at cool temperatures throughout the necropsy.
3.3
Necropsy Procedure
3.3.1 External Examination
Fish should be placed on a surface that prevents further
contamination to help the prosector to perform the work.
A steel tray is ideal due to containment, easiness for disinfection and durability. Normally fish are placed on the right
flank with head to the left, a convention based on fusiform
fish such as salmonids, where internal organs become readily
observable and easily accessed from the left flank, therefore
requiring minimal displacement of organs to observe or
access other structures. Different species and body forms
(e.g. Pleuronectiformes) will require a different approach
to achieve the same objective. The fish should be examined
in a cool environment and case notes taken throughout the
process of the post-mortem examination, and noting any
deviation from normality for the species. The provision of
a reference to the relative position of the abnormality or the
sample taken is an essential part of the report; a few useful
anatomical terms of location applicable to whole animals,
tissues or histological sections are outlined in Fig. 3.1.
For fish less than 2.0 cm in length they can be examined
under a dissecting microscope and when necessary, samples
of skin and mucous or gill scrapes can be taken for immediate analysis. However, for the purpose of histological examination fish of this small size can be preserved whole,
provided an abdominal flap has been cut open or removed,
a practice that allows the fixative to penetrate within the body
cavity and guarantee proper fixation. For larger fish, fresh
samples of tissues or body fluids can also be taken for initial
in situ analysis, but for histological examination tissues need
to be dissected using scalpel, scissors and forceps.
Gills are a delicate tissue that requires prompt examination
as once exposed to air, changes occur rapidly and they quickly
dry. The gills are protected by the opercula which should be
lifted, assisted by forceps and occasionally may need to be cut
to facilitate access to the gill arches. An inspection for evidence
of anaemia, increased mucous, blood clots or parasitic infection
of the gill filaments or the arch is carried out. A sample of the
whole first or second arch (from relative small fish), or a portion
(usually at the curved level) of gills (see Fig. 2.2) from larger
fish should be removed. For this, the arch can be held using
forceps by gripping the gill arch at a location that will not
become part of the sample, and carefully cutting the portion
to be fixed with scalpel or scissors. Special attention should be
paid to avoid any compression of the filaments. Tissue should
be promptly placed in the fixative. The pseudobranch (see
Fig. 2.6), located, on the inner surface of the operculum, is a
target tissue in salmon for the Myxozoa parasite Parvicapsula
spp. and should be checked and sampled if required.
A thorough check of the entire body external surface
should follow noting the integrity of the skin and fins,
changes of normal pigmentation for the species, excessive
mucus, raised or lost scales, erosion, ulcers, haemorrhage
(e.g. petechial), exophthalmia, grossly visible parasites, evidence of skeletal deformity or muscle atrophy. As fish skin is
in contact with the external environment, it is vulnerable to
damage from a variety of sources, including primary and
opportunistic pathogens. Moreover, factors such as handling
and net damage will also contribute to the health of the fish.
During the post-mortem examination some abnormalities
will become apparent, but others will only be recognised
through examination of tissues by light microscopy. A skin
sample should represent the affected area or lesion including
an edge of normal tissue. Both tegument and some of the
underlying musculature is usually included in the skin sample. A standard ‘normal’ skin sample that also includes red
and white muscle is also advisable and a default area is
usually at the level of the lateral line just below the dorsal
fin. Different parts of the fish surface have a particular
structure, e.g. head skin lack scales, and therefore samples
representing discrete areas of interest should be included.
Carefully remove a small piece approximately 1 cm
3 and
place immediately in fixative such as 10 % buffered formal
saline. The eye will normally be sampled next to prevent
excessive drying before analysis and if of interest for the
study. A gross examination of the eyes should include reference to corneal opacity, cataract or exophthalmia which,
although not necessarily pathognomonic, can indicate a
minor infection or a sign of a more serious condition. To
remove the eye carefully dissect the skin around the orbit
28
3 Post-mortem Examination and the Recognition of Tissue Abnormalities
serological or molecular studies. However, these procedures
will not be covered or discussed in detail in this book.
3.2
Sample Size and Euthanasia
The number of fish sampled for a health assessment will vary
according to the objectives of the study. For example, certification of freedom of a notifiable disease generally follows the
guidelines from the Office International des Epizooties (OIE).
Here the sample size is based upon an assumed prevalence
of the specific pathogen to an agreed level of confidence.
To obtain a 95–98 % probability of detecting at least one
infected fish in a clinically healthy population this translates
to a minimum of 30 individuals. Conversely, for disease
investigations 5–10 fish showing abnormal behaviour or the
characteristic signs of the condition will be adequate for necropsy. Fish removed for examination should, where practical,
be placed into a smaller container where further observations
can be made before any procedure or the removal of tissues or
body fluids. The fish should be euthanized by a humane
method, ideally through an overdose of anaesthetic and ideally
maintained at cool temperatures throughout the necropsy.
3.3
Necropsy Procedure
3.3.1 External Examination
Fish should be placed on a surface that prevents further
contamination to help the prosector to perform the work.
A steel tray is ideal due to containment, easiness for disinfection and durability. Normally fish are placed on the right
flank with head to the left, a convention based on fusiform
fish such as salmonids, where internal organs become readily
observable and easily accessed from the left flank, therefore
requiring minimal displacement of organs to observe or
access other structures. Different species and body forms
(e.g. Pleuronectiformes) will require a different approach
to achieve the same objective. The fish should be examined
in a cool environment and case notes taken throughout the
process of the post-mortem examination, and noting any
deviation from normality for the species. The provision of
a reference to the relative position of the abnormality or the
sample taken is an essential part of the report; a few useful
anatomical terms of location applicable to whole animals,
tissues or histological sections are outlined in Fig. 3.1.
For fish less than 2.0 cm in length they can be examined
under a dissecting microscope and when necessary, samples
of skin and mucous or gill scrapes can be taken for immediate analysis. However, for the purpose of histological examination fish of this small size can be preserved whole,
provided an abdominal flap has been cut open or removed,
a practice that allows the fixative to penetrate within the body
cavity and guarantee proper fixation. For larger fish, fresh
samples of tissues or body fluids can also be taken for initial
in situ analysis, but for histological examination tissues need
to be dissected using scalpel, scissors and forceps.
Gills are a delicate tissue that requires prompt examination
as once exposed to air, changes occur rapidly and they quickly
dry. The gills are protected by the opercula which should be
lifted, assisted by forceps and occasionally may need to be cut
to facilitate access to the gill arches. An inspection for evidence
of anaemia, increased mucous, blood clots or parasitic infection
of the gill filaments or the arch is carried out. A sample of the
whole first or second arch (from relative small fish), or a portion
(usually at the curved level) of gills (see Fig. 2.2) from larger
fish should be removed. For this, the arch can be held using
forceps by gripping the gill arch at a location that will not
become part of the sample, and carefully cutting the portion
to be fixed with scalpel or scissors. Special attention should be
paid to avoid any compression of the filaments. Tissue should
be promptly placed in the fixative. The pseudobranch (see
Fig. 2.6), located, on the inner surface of the operculum, is a
target tissue in salmon for the Myxozoa parasite Parvicapsula
spp. and should be checked and sampled if required.
A thorough check of the entire body external surface
should follow noting the integrity of the skin and fins,
changes of normal pigmentation for the species, excessive
mucus, raised or lost scales, erosion, ulcers, haemorrhage
(e.g. petechial), exophthalmia, grossly visible parasites, evidence of skeletal deformity or muscle atrophy. As fish skin is
in contact with the external environment, it is vulnerable to
damage from a variety of sources, including primary and
opportunistic pathogens. Moreover, factors such as handling
and net damage will also contribute to the health of the fish.
During the post-mortem examination some abnormalities
will become apparent, but others will only be recognised
through examination of tissues by light microscopy. A skin
sample should represent the affected area or lesion including
an edge of normal tissue. Both tegument and some of the
underlying musculature is usually included in the skin sample. A standard ‘normal’ skin sample that also includes red
and white muscle is also advisable and a default area is
usually at the level of the lateral line just below the dorsal
fin. Different parts of the fish surface have a particular
structure, e.g. head skin lack scales, and therefore samples
representing discrete areas of interest should be included.
Carefully remove a small piece approximately 1 cm
3 and
place immediately in fixative such as 10 % buffered formal
saline. The eye will normally be sampled next to prevent
excessive drying before analysis and if of interest for the
study. A gross examination of the eyes should include reference to corneal opacity, cataract or exophthalmia which,
although not necessarily pathognomonic, can indicate a
minor infection or a sign of a more serious condition. To
remove the eye carefully dissect the skin around the orbit
28
3 Post-mortem Examination and the Recognition of Tissue Abnormalities