The easiest way to arrive at a diagnosis is when a specific disease-causing agent such as a bacterium or parasite can be identified associated with pathological changes. However this is not always the case and it can be very difficult to identify what are described as pathognomonic changes ie unique changes that can be ascribed to a specific cause. Fish tissues have limited range of reactions in any particular disease process. So it is imperative that as full a clinical history is provided with samples. This clinical information is essential in allowing the histopathologist to arrive at a diagnosis. The use of a histopathology service by fish farmers and others can allow an investigation to proceed without the requirement for an initial visit. This can be especially important for farms situated in remote areas. The only requirement is that the person taking the samples is properly trained in this critical area.
In the initial stages of a disease problem it is easy to plump for the most obvious as the primary responsible factor eg:
severe lesions on skin of fish were caused by physical abrasion
one might also decide, not unreasonably, that fish with bite marks had been simply attacked by a predator.
What simple observation does not allow is to identify whether in fact there was an underlying reason for the obvious damage. The fish may have developed skin lesions because they were not properly smolted, had experienced a plankton insult, suffering from sunburn or had an infectious disease such as Infectious Pancreatic Necrosis or Salmon Pancreas Disease. All these reasons will make fish unable to maintain their position and swim properly and very likely to be swept onto nets causing severe loss of skin.
Histological examination will often confirm an experienced farmers observation but may also identify other primary factors that can then be addressed.
Fish that appear to be swimming oddly may be suffering a virus infection, a bacterial infection of even a parasite infecting its brain. It is only by examining appropriate tissues under high magnification that these problems can be identified.
An awareness of the range of appearance of "normal" tissues is essential before a competent histopathologist can identify genuine pathological change. Tissues sampled from different ages and species of fish will have a different appearance and these must be recognised in order to interpret sections correctly.
Recognition of these differences and a degree of commonsense is paramount when it comes to interpretation and drawing any conclusions for any specific case examination.
Histopathology should never be used in isolation but as an adjunct along with any number of other considerations including gross observations of fish behaviour, pattern of mortality, identification of potential factors such as recent history eg handling, vaccination, grading, net changing, feed and feeding, water quality, presence of predators, post-mortem findings,
Histology can be usefully used in a number of ways including:
- Most commonly in the differential diagnosis of disease by simple direct microscopical examination of stained tissues
- In conjunction with specific immunological reagents histology will allow identification of pathogenic bacteria and viruses
- Confirmation of vaccination
- Assessment of any pathological reactions in novel feed and feed supplement trials
- Can be used to indicate recovery following a disease outbreak and ensure that further infections, the same or new opportunists, are not missed
1. Sampling
Sampling of suitable fish - the right fish must be sampled. Ideally moribund fish should be sampled or very fresh dead. We all know how difficult it can be to catch that one "moribund" fish but time spent catching the right fish is essential. Sampling the correct tissues is also necessary in allowing the correct, best diagnosis. Standard tissues for sampling include skin, muscle, gills, heart, liver, caecae, pancreas, intestine, spleen and kidney. However if fish are seen to be swimming abnormally then should consider sampling brain. If fish show cloudy lenses then eyes should be sampled. If fish are showing buoyancy problems then swimbladder should be sampled.
2. Fixation
Fixation preserves the tissues in the same condition as when they were sampled, fish tissues deteriorate very quickly and must be preserved as quickly as possible. Many types of fixatives are available but formaldehyde bases are the commonest in current use. A number of problems can occur at this stage leading to difficulties later in the process:
Too much tissue for the amount of fixative used or tissue pieces too large for effective penetration by the fixative. The ideal ratio of tissue to fixative 1 part of tissue to 20 parts of fixative with individual tissue pieces around 0.5x0.5x0.5 cu cm.
When small fish are being processed they are often handle whole but if the abdominal cavity is not open to allow fixative to enter then the internal tissues will breakdown making any interpretation very difficult.
3. Tissue processing
In order to be able to cut the very thin sections required, usually 5 microns, 5 thousands of a millimetre, the tissues must be passed through a series of solvents before finally being impregnated fully with paraffin wax. Only once the wax has been allowed to solidify can sections be cut using a specialised piece of equipment called a microtome.
4. Sectioning
The use of microtome produces a ribbon of wax containing the tissue sections. These ribbons are floated on warm water to flatten out the section before carefully collecting the section onto a glass slide. The slides now containing the tissue sections are allowed to dry fully before proceeding to the next step in the process.
5. Staining
The final stage is the staining of the tissue to highlight the individual cells and tissue components. Before staining can proceed the wax has to be removed and this is achieved by again using a range of solvents ending up with the tissue in water. Now staining can take place and the most commonly used method is the Haematoxylin and Eosin which is a general staining procedure allowing effective recognition of the majority of tissues.
Other staining methods are available that allow specific recognition of disease causing agents and disease produced factors. These include stains for Mycobacteria, fungi or parasites and stains for factors associated with disease effects such as haemosiderin, which is found in haemolytic anaemias or ceroid which is associated with extreme cell membrane destruction eg where low tissue antioxidants or rancid diets are present. Once staining is completed section finally must pass through another sequence of solvents ending up in xylene.
6. Microscopical examination
Before slides can be examined they need to be removed from the xylene solvent and mounted in a special fluid covered with a very fine coverslip and left to dry.
The whole process from tissue sampling to examination can be completed in as short as 24 hours but usually takes longer if slides are allowed to dry fully before examination.
So in conclusion, I would advise that farmers do not rely solely on the evidence of their eyes for the answers in any potential disease problem. Rather they should consider the role that histological examination can serve in the investigation by providing a clearer, more accurate and deeper insight into the problem.
For remote and difficult to reach sites histology can provide a much-needed lifeline in expediting any disease investigation and arriving at an early diagnosis allowing prompt remedial action.
Source: Fish Vet Group - April 2002
