Prepared by Judith Handlinger

Systematic Fish Pathology Part 9. Pathology of the digestive system II. The liver, pancreas, peritoneum & swim bladder Section A: General aspects. Prepared by Judith Handlinger With the support of The Fish Health Unit, Animal Health Laboratory, Department Of Primary Industries, Parks, Water and Environment, Tasmania, for Animal Health Australia’s Australian Animal Pathology Standards program(AAPSP)

Acknowledgments & Introduction. This ninth module of the systematic examination of fish pathology (Gastrointestinal System Part B – liver, pancreas, swim bladder and peritoneum) is split into 4 sub-modules. Remember that the program aims to convey an approach to diagnosis and cover fish reactions of each organ system, rather than to cover all fish diseases, and is based largely on representative pathology found in the diagnostic laboratory of the Tasmanian Department of Primary Industries, Parks, Water & Environment (DPIPWE). It was funded by Animal Health Australia with in-kind support of DPIPWE as acknowledged previously Photos for this series, especially those of gross pathology, are generally also from DPIPWE archives and were generated by multiple contributors within DPIPWEFish Health Unit. Contributors of cases from other laboratories have been acknowledged wherever possible and specific material and photographs used with permission where the origin is known. Any inadvertent omissions in this regard are unintended. References quoted are listed at the end.

Course Outline A. Systematic Fish Pathology 1.Consider the Fish: An evolutionary perspective on comparative anatomy and physiology 2. Pathology of the kidney I – interstitial tissue Part A 3. Pathology of the kidney II – interstitial tissue Part B 4. Pathology of the kidney III – the nephron 5. Pathophysiology of the spleen 6. Fish haematology 7. Fish immunology – evolutionary & practical aspects 8. Pathology of the digestive system I – the oesophagus, stomach, & intestines. 9. Pathology of digestive system II – the liver and pancreas, swim bladder, peritoneum. (Thismodule - split into 4 sections: Section A (this presentation): General aspects Section B: Toxin related pathology and neoplasia. Section C: Pathology of microbial infections (viruses, bacteria & fungi) Section D: Parasitic diseases (protozoa & similar organism, metazoans). 10. Pathology of fish skin 11. Pathology and diseases of circulatory / respiratory system – heart, gills and vessels 12. Pathology of the musculoskeletal system and nervous systems 13. Pathology of gonads and fry

Anatomy & Microanatomy Major differences between species reflect: A) Whether the pancreas is separate or combined with the liver as a hepatopancreas b) Whether liver or pancreas are major fat storage organs

A Anatomy Salmonid (typical of many fish): A: Liverwith one major lobe lies to left of midline (though mirror-image reversal is not uncommon), with gall bladder towards the posterior margin (white arrow. Diffuse pancreas is located with the fat beds between pyloric caeca (yellow arrow). [Only residual tags of the ruptured swim bladder are visible, dorsal to arrows.] B: Thin swim bladder wall (blue arrow), visible in a dorsal position beneath the severed peritoneal lining of this gravid female salmonid. This is primarily an organ for buoyancy control, but also contributes to stability, and sometimes as a resonating chamber to produce or receive sound. B

Liver Microanatomy (salmonid) Salmonid liver is typical of fish with a diffuse pancreas and fat storage in the peri-caecal tissue. The organisation of the liver (like mammals) a dual-plated muralium, but the classical hexagonal lobular pattern round a central vein is not seen. The structure has been described as tubular, but this is disputed (or like animals, exact structure may depend on age). A general zonal structure can be distinguished but the distinct mammalian acinar pattern with visible portal and central vein regions for each functional acinus is not seen. (Evensen, 2006, quoted by Ferguson et al, 2006. Liver) . The double hepatocyte cords are separated by endothelial lined vascular spaces. Kupffer cells are notpresent*: the extensive surfaces of the fish heart, particularly the atria, provide an alternate site for specialized macrophages of the reticulo-endothelial system. Note the double cords, inconspicuous bile duct (top), and poorly defined lobulation. * Liver not a major site for RE macrophages in most fish, but this may not be universal.

Pancreas micro-anatomy (salmonid) Pancreatic acinar cells are present as diffuse cords through the peri-caecal fat. Islets of Langerhans vary somewhat in size and distribution between species, and are rarely uniformly distributed, so several sections may need to be examined for assessment

A A: In this greenback flounder section, all the Islets are large and near the junction with the gut (white arrow), none in the more distal portions. Note that the pancreas tissue extends into the liver (blue arrow) B, C: detail. Note that this pancreas contains little fat (unlike the liver, though the latter may have a pathological component). B Peripheral parts of the pancreas, with few Islets. C Pancreas micro-anatomy (flounder)

Angel fish. This also has a few large Islets between liver and stomach wall. Such Islets may represent the total endocrine tissue of the pancreas, so the plane or number of sections may be critical for assessing Islet pathology.

Anatomic variations – liver as fat storage organ A different organisation of the pancreas is present in those fish without interstitial fat storage tissue between the caeca (many teleosts), or with no caeca at all (e.g. sharks). Note the liver is the fat storage organ in the this Port Jackson shark. Also that sharks have 2 major lobes (there is a third smaller ventral lobe). Sharks have a separate, discrete pancreas attached to the intestine. .

Highly vacuolated liver (and spleen) of a Dogfish shark (family Squalidae). Bile ducts but no pancreas tissue is present. Higher magnification shows the distinct rounded vacuole outline typical of fat vacuoles.

Anatomic variations - hepatopancreas Fish with without interstitial fat storage tissue between the caeca (as right) generally have a combined hepato-pancreas. The liver is also likely to store more fat in these species. Liver fat levels may also vary with maturity in some species - hepatocyte fat may be high prior to spawning, but not at other times.

Diffuse hepato-pancreas with the pancreas acinar cells clearly distributed round blood vessels. Some fish (such as the flounder shown above), may show both intra-and extra-hepatic pancreas tissue. Species also vary in the amount of fat and glycogenstored in normal livers: these two livers are of the same species – the top one is normal, the bottom is ill.

Variation in liver glycogen So far we have discussed vacuolation in terms of variations in the level of fat. Glycogenalso varies with nutritional status, and is also seen as pallor. Generally large distinct vacuoles are likely to be fat, indistinct foamy pallor without obvious vacuole boundaries is more likely to be glycogen (but could also be pathological). Right: two 28-day old striped trumpeter larva: Top: food in gut (upper left) and good liver glycogen levels Bottom: cohort with no food, gut slough, and little glycogen Note the indistinct glycogen vacuole outlines at higher magnification (top only).

Tuna – liver as a heat pump The abundant large vessels in this part of the liver of a Southern blue fin tuna are part of the heat-exchange mechanism allowing temperatures in the muscles and viscera to reach up to 14oC above that of the water. Veins draining the muscles and viscera are heated slightly by metabolic activity & digestion. Close apposition of these with arteries allows this heat to be retained in vessels supplying these organs, rather than equilibrated throughout the body and ultimately dissipated to the water. Slides courtesy of SBT industry project

Swim bladder anatomic variations Simple open swim bladder A A: Swim bladder of the relatively primitive salmonid is a thin-walled gas-filled tube derived from and connected to the oesophagus via the pneumatic ductthat remains patent throughout life . [Ignore the post-mortem imbibed water and solids.] Fish with a persistent pneumatic duct are called physostomes and regulate buoyancy via this duct. .

Physostome swim bladder micro anatomy Simple thin-walled swim bladder of a small salmon fry (a physostome). Simple epithelium height varies with level of inflation. The partially collapsed state of this bladder probably reflects the patent duct, allowing loss of some air with relaxation of the sphincter. Swim bladder

Closed (glandular) swim bladder B: In many more advanced fish, such as thisGoldfish,the swim bladder is two-lobed, aiding the secretion and resorption of gas by a gas-gland. Fish relying on gas secretion (following initial inflation via the pneumatic duct that then closes over) are termed physoclists. A lining of guanine crystals on a fully developed swim bladder reduces permeability to gas

Physoclist swim bladder micro-anatomy Gambusia sp. swim bladder sectioned through the gas-gland (between the arrows). The gland covers only a small proportion of the surface – this is a fortuitous section. Higher magnification shows the glandular nature of this part of the gland.

Swim bladder of a Tasmanian whitebait Lovettia sealii, a small physoclist of the Galaxiidae family, sectioned through the swim bladder gland. Note the two parts of the gland: the glandular or secretory portion, and the vascular retemirabile (counter-current blood flow / gas exchange complex). Intestine Glandular component Swimbladder (SB) SB gland Rete mirabile component Spine

Swim bladder gland from a much larger fish (a 3 kg male striped trumpeter), showing detail of the well developed gland, and the associated vascular retemirabile. This gland excretes lactic acid and produced carbon dioxide, resulting in blood acidity, loss of oxygen from haemoglobin (via the Root effect). Some of the oxygen diffused directly into the bladder, the rest plus the CO2 is removed by the rete, allowing very high levels of gas (mainly 02) to be maintained in deep water fish. Gasses may return to the blood on exposure of another blood rich area, the oval window.

Another gas gland, from an agedoreo dory(possibly a deepwater species), showing relationship of the gland to rete. (Nature of large dark nuclei not established.)

Other swim bladder variations. Only the ray-finned fish have a swim bladder. Sharks and rays separation pre-date evolution of this organ. The precursor organ differentiated into the lung in lobe-finned fish (and their descendents such as ourselves). Thus lobe-finned fish have a lung, but no swim-bladder. Fish without swim bladders control their depth by active swimming, and / or store oils to aid buoyancy. Some deep water fish use low buoyancy solids instead of gas in the swim bladder. An example is the Orange Hoplostethus atlanticus (depth 700-1800 m) which has a wax-ester swim bladder (right). This is utilized in population surveys as acoustic methods can differentiate this from gas filled bladders.

Physiological variations – liver & pancreas As well as major species differences in the organs that store fat, the level of fat & glycogen storage within hepatocytes also varies with food intake, and some physiological states (such as sexual maturation & approaching spawning). Note though that food intake itself may change with maturation – salmonids, for example, cease feeding during freshwater spawning runs. Such changes are usually uniform: pathological fatty change may appear similar but is often less regular. In some cases (especially aquarium fish, but also during early attempts at species cultivation), regular and common fatty change may appear normal but be due to inappropriate food composition. Thus “normal” hepatocyte appearance is poorly defined for many species. A comparison of “good” & “bad” fish is always desirable if available.

This is the liver of a normal, healthy 1 year old farmed Atlantic salmon. The fat level is much higher than a healthy wild caught fish is likely to be, reflecting the high plain of nutrition for maximum growth. (Remember that high fat content is also an aim for such farmed fish, as a desirable source of Omega-3 acids.). A bile duct is clearly visible even at low magnification but smaller ducts are inconspicuous, and not associated with a clearly defined portal triad. Small lymphoid-rich perivascular aggregates are common (fish, like birds, lack lymph nodes). L-801

Another healthy young salmon, with less vacuolation 802

The liver of this clinically healthy two-year old farmed Rainbow trout shows high diet-related fat in typical large single vacuoles. Note the inconspicuous bile duct (arrow). 020623 trout N

A lower level of vacuolation in a larger sea-run rainbow trout with thin heart muscle (possibly indicating a slight nutritional imbalance, or reduced food intake). These hepatocytes are also normal.

“Pinhead” salmon (smolt that has not adapted to sea-water and virtually stopped eating). Remember that the increased kidney pigment indicates catabolism. Lack of liver vacuolation reflects a low recent plane of nutrition. These fish remain otherwise healthy until catabolic reserves are exhausted, and will resume feeding if returned to freshwater or offered wet food. (Neither of these options are generally economically viable.) L-810

Another “Pinhead” salmon showing negligible pancreatic fat & hepatocyte vacuolation, prominent bile duct and interstitial lymphoid aggregates. Despite the lack of visible fat there is no pathology of pancreas tissue.

G19 Adult salmon (with some nutritional imbalances & a low overall plane of nutrition, therefore less fat), showing major typical bile duct structure (similar to other vertebrates).

Liver of two areas of the liver of a greenback flounder (Rhombosolea tapirina) showing variation in the level of vacuolation that may be present within the same liver. L-808

L-805 Variations continued: Adult lamprey liver: what is different?

Lamprey liver continuedDid you pick it? No bile ducts! Bile ducts are present in larval lampreys (called ammocoetes) which live in fresh water and may make up the majority of the life-time of a lamprey, but the entire biliary system degenerates during metamorphosis into the adult (marine) stage. Larval liver consists of blind-ending single cell tubules of hepatocytes lined by microvilli (the equivalent of canalicular lumens of mammals), and a convoluted bile duct system surrounded intimately by sinusoids enabling a large surface interface with vessels. In the adults, the biliary system has disappeared, and hepatocytes lose their tubular arrangement. Despite early reports to the contrary, bile pigment does not accumulate in the adult liver, but there is marked non-haem iron and ferritin iron accumulation in the liver (and in the renal tubules as the pronephros also degenerates during metamorphosis). Non-haem iron levels in Geotria australia increase also during larval life, rising from 0.15-0.55 µg.mg wet wt-1 in larvae to 8.8 µg.mg wet wt-1 in recently metamophosed migrants (Harris et al, 1990). Ferritin iron (same units, same time-frame) rose from 0.06-0.26 in larvae to 5.3 in the migrants. Non-haem iron levels are relatively high even during larval life, with high plasma iron (>19,000 µg 100ml-1 and very high haemoglobin concentration and red cell numbers. (Macey et al, 1985) Superoxide dismutase (SOD) fell during metamorphosis, apparently to reduce the potential toxic effects of iron accumulation. Despite this, lampreys are considered a useful model for studying iron accumulation. During biliary regression, many hepatic enzymes are relocated from the canalicular-like surface to the external surface .

Physiological variations and degeneration of the pancreas complex: 1. Simple starvation. The following cases are from Atlantic salmon “pinheads” – fish that did not adapt well to recent transfer from freshwater to sea as smolt (either because they had not completed smoltification or were stressed during this process) and ceased eating. Normal feeding salmon (top) and “pinheads” with various levels of muscle catabolism & lack of growth The pancreas tissue is normal (though generally inactive), the major change being a decrease in fat.

Pancreas from another ‘pinhead’ with almost completely metabolised fat, and moderately heavy resultant kidney pigment.

Same fish, detail. Note that Islets of Langerhans, acinar cells and fat cells are all intact, despite the reduction in fat.

General aspects of liver pathology (non-infectious insults) For those who have followed this Power-point series it should be obvious by now that damage to fish cells and fish responses vary little from those of higher vertebrates. The major differences are likely to be the circumstances whereby they encounter toxins (especially for the liver), and some difference in the toxins and infectious pathogens encountered.

Liver cysts Cysts as a developmental abnormality are not common in our experience, but do occur. They are usually of little clinical significance, but need to be differentiated from cysts of infectious origin.

Cystic liver - histological appearance (less affected area amenable to sectioning). Note the presence of reaction to collapsed cysts (arrow, 2rd image) and the mild fibrosis near reactions. The healthy appearance of uncompressed liver cells (see last slide) is consistent with the usual lack of clinical significance of this lesion. 890683

Liver Atrophy The liver (arrowed) of the goldfish shown above is markedly reduced. Long term history was available (it belonged to a staff member), indicating great old-age, which was regarded as a major factor. It should also be remembered that few aquarium fish are fed a totally suitable diet (few diets sold for many species cultured, little research, sometimes prolonged storage). Foraging may be important to correct some imbalances.

General pathology & toxicity of liver– degeneration, necrosis & repair • Liver vacuolation changes that are widespread but non-uniform are more likely to be due to pathology, or a sudden change in nutrition, than the variations shown above. • This may be due to toxins • Or secondary to pathology in other tissues (including endogenous toxins, circulatory compromise etc) • Changes involving altered fat metabolism may involve both liver and pancreatic fat (less commonly also body / muscle fat). • Localised degeneration is likely to be due to toxins or infections. • [For examples see next presentation, Section B: • nitrite toxicity • Copper toxicity • Jelly fish toxin • Algal toxins - Nodularia; net-pen disease] • Sequelae to degeneration include ceroid formation. • Other cell changes may also reflect toxins.

Widespread but uneven vacuolation (in this case loss of vacuolation), in Angel fish B with widespread iridovirus infection. Occasional virus-affected cells were present in the liver (arrow), but the major liver change reflects the general ill-health (probable anorexia, possibly also cessation of breeding?) Note the zonal distribution A: Liver of a healthy Angel fish from this population & diet B:Angel fish with widespread iridovirus infection. Note vacuolation level and a single large dark infected cell (arrow, HP). • 022142-F4 021571-2

Yellowtail kingfish Seriola lalandi with a pale liver. Gill flukes (blood sucking) were present though PCV was within normal range. Even without knowledge of the normal vacuolation level in the species, the uneven pattern suggests pathology, although it is unclear whether this is primarily damage to hepatocytes or a secondary response.

This is also a lesion of chronic anoxia (due to a constrictive cardiomyopathy), that was probably exacerbated by stress, leading to increased necrosis in the compromised cells. Note that lesions are zone-related, but the pattern is less regular. (Old peritoneal tags are also present.) 020625

The focal vacuolation of the liver may an early sign of degeneration. In this sea-caged salmon this is likely to be due to an external toxin as the kidney also showed signs of tubule degeneration and regeneration. History suggested an algal toxin. The gut shows eosinophilic casts, suggesting the mild liver change is secondary to current anorexia, whatever the nature of the primary insult. K2 061558-1

Diet-related pathology: Excess fat may be good for the fish farmer, not necessarily for the fish. Trout in particular will develop fatty livers with high carbohydrate levels, particularly if protein is low (<25%) or vitamin E levels are low. This is accompanied by marked reduction in liver glycogen and increased susceptibility to stress. Yellow-bronze liver of overfed (?) trout of similar age. [Carbohydrate levels not given] Normal liver colour – sea-run Atlantic salmon. Reference: http://www.fao.org/docrep/field/003/t8389e/T8389E05.htm

Secondary liver change: at first inspection this Chilean salmon post-smolt suggests a liver problem (pale icteric liver). Further examination suggests this is secondary to a major bacterial gill erosion: you need to examine the whole fish – a problem if only single organs are submitted

Liver repair and fibrosis: repair withbasophilia and mild biliary fibrosis following an insult such as widespread single cell necrosis is usually not common in fish, but was reported as common in some species of fish with mild Netpen liver disease (related to microcystin-LRtoxicity – see Section B). Below is a ~1.5 kg Atlantic salmon collected for routine surveillance, showing basophilia, typically round bile-ducts, that is characteristic of liver repair. (Basophilia is due to increased RNA of actively dividing cells).

Same fish: in some areas only basophilia is present. The cause of liver damage in this fish was not established. Reovirus was present and is therefore a possible cause, but this is uncertain as this virus is often persistent and widespread liver damage is rare (at least in surviving fish).

Prepared by Judith Handlinger