Systemic Pathology of Fish A Text and Atlas of Normal Tissues in Teleosts and their Responses in Disease
Second Edition edited by
Hugh W. Ferguson, BVM&S, PhD, DipACVP, MRCVS, FRCPath
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Introduction
chapter 1 by Hugh W. Ferguson
Post-mortem techniques Sample submission and processing Unlike mammals which tend to cool down after death, thereby slowing autolysis, fish out of water often warm up. Tissues must, therefore, be placed into fixative as quickly as possible or artifacts can start to hinder interpretation. We have found this to be especially true with gills, and a time lag of even five minutes (not long if one is doing a necropsy!) may produce lamellar epithelial swelling and lifting from the basement membrane, partly due to the lack of compensatory water pressure within the branchial cavity. Sick but live fish are probably the most useful samples to receive for diagnostic purposes, therefore, followed by freshly dead animals, kept cool, but not frozen. Keeping marine species cool by placing them directly on top of a bed of freshwater ice inevitably leads to osmotic destruction of skin and most associated pathogens. Ten per cent neutral buffered formalin is the fixative of choice for most situations and 24 hours on a shaker is the optimum, prior to trimming. If keen to get tissues into the processor overnight so that slides can be read the next day, even 3-4 hours fixation can be sufficient in an emergency. Using seawater instead of saline is also a good substitute, as it is quite well buffered. Use of unbuffered formalin often leads to oxidation of haem and the presence of haematin in sections, presenting as an annoying brown pigment. Bouin’s fixative is especially useful for eyes and gills, or for skin, or small fish, where its demineralizing properties minimize distortion and tearing artifacts from scales and other bony structures, and in the case of small fish, permits whole-body sections without the need to dissect out (and often destroy!) the various organs. Fixation for 24-48 hours, followed by storage in 70% alcohol until processing, is usually adequate. Decal is also used as a routine, as is decalcification of the block face (hard tissues) for an hour immediately prior to section cutting. One of the many advantages of working with fish over mammals is the ability to get a section of the entire animal on a single slide; the relative proportions and inter-relationships of the organs are thereby easily examined (Fig 1.1A).
Fig 1.1A. Saggital section through young seahorse
For ultrastructural work, 2-2.5% glutaraldehyde in a cacodylate or phosphate buffer with a total osmolality of roughly 320 mOsm, and pH 7.2 has been found acceptable for primary fixation under most circumstances. This is usually followed by secondary fixation with 1 per cent osmium tetroxide in a phosphate buffer, and then dehydration in graded ethanols prior to resin embedding. So much of our diagnostic material is mailed to us, however, that when we feel the need for some ultrastructural work, we usually have nothing except formalin fixed tissues with which to work, and getting new material appropriately fixed is either not possible, or it would take too long. Accordingly, we use a post-formalin fixation strategy of 30 minutes in glutaraldehyde, followed by buffer rinse and then osmium staining. The results are more than acceptable for diagnostic purposes. The routine processing and staining methods used in any pathology laboratory are employed for the preparation of paraffin wax sections from fish tissues. Any discussion of fixative at this stage is possibly premature because the importance cannot be over-emphasized of routinely employing tissue scrapes, smears and squashes from live and anaesthetized, or freshly dead fish. This is especially true for skin and gill diseases and where protozoan or other parasitic diseases are suspect: some of the protozoa do not survive processing very well, and there is little substitute for seeing the cell move when trying to decide if it is of host origin or not! And even then, you
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Chapter 3 – Skin
Fig 3.2. SEM of normal rainbow trout skin showing fingerprint-like microridged pattern of the outer epidermal cells and 3 mucus cell pores. The outline of individual cells can be seen. This material was fixed in aldehydes which rapidly remove the mucus covering normally present.
of a variety of pigment-cell types are present at different levels within the dermis: these include melanophores, xanthophores and iridophores. These latter contain reflective plates of guanine which thereby render a silvery appearance to the skin when orientated at 90º to the oncoming light. In transparent fish (like glass catfish) the arrangement of dermal collagen fibres resembles that seen in the cornea. Scales are a major feature of most species of teleosts (Fig 3.3), and it is important to appreciate that, where present, they originate in scale-pockets in the dermis and become covered by a layer of epidermis as they emerge, often to overlap one another, thereby providing significant structural support and physical protection. Thus, scale-loss represents a very significant breach in
the osmotic and/or physical defences of the fish. Scales comprise an outer reticulated osseous part with sawtooth-like ridges, and an inner fibrillar layer that is uncalcified in some species and partially calcified in others. This latter consists of parallel collagen fibres embedded in an organic matrix. Scale modifications provide protection for the lateral line. This canal runs along both sides of the fish and joins up with a series of sensory canals on the head, including the infra- and supraoptic canals. They contain neuromasts, and connect to the outside water through pores in the scales. They can be a common site for lesions including improper closure, ulceration and even parasites (Fig 3.4). A dermal component of the skeleton, scales represent a ready source of calcium, and
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Chapter 3 – Skin
Fig 3.4B. Acute inflammation within and surrounding supra-optic canal.
E
Fig 3.4C. Trichodinids within sensory canal, apparently having little effect.
Fig 3.3. Normal trout skin showing overlapping distribution of scales: note the ridges on their outer surface. The epidermis (E) contains goblet cells but no club cells in this species.
during periods of starvation or pre-spawning, they may be resorbed in preference to the skeletal reserves. Beneath the deep compact dermal layer, the loose relatively well-vascularized hypodermis provides a frequent avenue for lateral movement of pathogens and inflammatory processes.
Disease
Fig 3.4A. Sensory canal from salmonid within encasing bone. Neuromasts can also be seen (arrow).
Clinical signs of skin disease include hyperactivity, and obvious evidence of irritation such as rubbing against the sides or bottom of tanks or ponds; in so doing, the sides of the fish become visible from above and the behaviour is known as “flashing”. In some cases the fish may even jump out of the water. As with gill disease, there is usually excess mucus production and this can result in foam accumulating on top of the water. Severe epidermal hyperplasia may produce a grossly visible white
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Kidney
chapter 4 Renate Reimschuessel and Hugh W. Ferguson
Head kidney
Posterior Cardinal Trunk kidney Corpuscles of Stannius
Archinephric duct
❶
❷
❸
❹
❺
Fig 4.1. Gross morphology of the teleost kidney (after Ogawa, in Hickman and Trump). Most freshwater fish kidneys are in groups 1-3, while marine species also include groups 4 & 5 (with permission).
General anatomy This varies greatly between different species of fish, both grossly and histologically. Grossly, five main shapes are described (Fig 4.1). Often fused, it lies in a retroperitoneal location just ventral to the vertebral column, and can extend from the head region to the posterior abdomen as one organ, or it can have distinct head and trunk regions (Fig 4.2A). In general, the kidney in the cranial region (also called anterior or head kidney) contains primarily haematopoietic and lymphoid tissue with few if any renal tubules, while the kidney located along the trunk (also called posterior kidney) contains more renal tubules with a lesser amount of interstitial haematopoietic and lymphoid tissue. Renal arteries supply blood to the glomeruli via arterioles. The efferent arterioles then contribute blood to the peritubular capillaries. In many species, the other major source of blood to these capillaries is the renal portal system, which receives blood and lymph draining from the tail region of the fish. Thus both anterior and posterior regions of the kidney possess a sinusoidal system of blood vessels that are lined by highly phagocytic cells (Fig 4.2B). This dual blood supply imbues the kidney with a certain degree of resistance to the consequences of anoxic insults that target one particular supply. But the high trapping abilities also ensure that microbiological diseases target the organ.
Fig 4.2A. Gross appearance of normal kidney in a brook trout. Ureters (green arrows) and urinary bladder (blue arrow) are easily seen.
Fish kidney also contains endocrine elements including thyroid follicles, corpuscles of Stannius, chromaffin cells and inter-renal tissue, the latter being located around major blood vessels, and which represent the equivalent of the mammalian adrenal medulla and cortex respectively. In elasmobranchs these can be visible with the unaided eye, while in most fish these tissues are microscopic. Corpuscles of Stannius may be seen as small white nodules located in the posterior kidney or at the junction of the anterior and posterior kidney. They should not be mistaken for parasites or granulomas! The grossly visible ureters fuse and may form a urinary bladder prior to ducting urine to the outside, posterior to the anus (Fig 4.2A).
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The Eye
chapter 10 by Erling Olaf Koppang and Ellen Bjerkås
Normal structure and function The purpose of the eye (bulbus oculi) is to project optical images of the surroundings onto the retina where they are recorded and transmitted to the brain for recognition and interpretation. To fulfil this aim, the construction of the eye must establish an optical axis, which is composed of the cornea, anterior chamber, lens, and vitreous body (Fig 10.1). The axis must be transparent and it must remain so, as any reduction will impair its function. Minor distortions of the optical axis cause major disruption of the projected optical image. To an animal, this may be life-threatening, and nature has invested heavily in constructing the eye in a manner that maintains and secures its precise function. Further, the optical axis must be able to regulate the light scattering, or focus the light that is projected onto the retina. Some deep-water species possess asymmetric, tubular eyes, while others possess no eyes at all, but these are the exceptions. In general, the globular construction of the eye, as found in most fish species, is ideally suited to minimising changes in shape due to alterations in water pressure. The construction and function of the organ are aimed solely at one single task: to maintain a functional optical axis, and this must be achieved despite the demands of life in an aqueous environment under sometimes extreme conditions. While these conditions are not experienced by mammals, the camera-type architecture of the eye in the two groups is remarkably similar, making it one of the most sophisticated and specialized organs throughout the animal kingdom. The globe of fish is attached to the orbit by three pairs of striated muscles. These are the dorsal, lateral, ventral and medial rectus muscles and the dorsal and ventral oblique muscles. These muscles allow the eye to be turned in any direction. The ability for voluntary eye movement varies between species, and therefore some species track objects mainly by changing their body position. Three major arteries enter the eye. These are the ophthalmic artery (arteria ophthalmica), which receives its blood from
Fig 10.1. Internal structures of the Atlantic salmon eye, lateral view. A dotted line indicates the optical axis. Following structures are indicated by letters: SC: scleral cartilage. Li: limbus. C: cornea. CR: choroid rete. C: choroid. IR: iris. R: retina. L: lens. AC: anterior chamber. V: ventral ciliary cleft. M: external muscles. N: optic nerve. VB: vitreous body.
the pseudobranch and enters the eye caudodorsally to the optic nerve (nervus opticus) (Fig 10.2). It branches out in the capillaries of the choroid rete (see under the uveal tract). The second vessel is the retinal artery (a. retinalis) which is a branch of the internal carotid artery (a. carotis interna). The retinal artery enters the eye ventral to the optic nerve, but it also branches out to supply this nerve, the eye muscles and the periocular tissues. After entering the eye, this artery, like the ophthalmic artery, forms a structure similar to the choroid rete, but much smaller. A branch enters the falciform process (processus falciforme) and nourishes the retractor lentis muscle. Blood from both of the above mentioned arteries feeds the choroid. Finally, the iris is supplied by the iris artery which derives from the external carotid artery (a. carotis externa).
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