Prepared by Judith Handlinger

Systematic Fish PathologyPart 9. Pathology of the digestive system II. The liver, pancreas, peritoneum & swim bladder SectionD: pathology of parasitic infections. 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)

Introduction to Section D. For acknowledgements of funding and photographs see Module 9, Section A. 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: General aspects Section B: Toxin related pathology and neoplasia. Section C : Pathology of microbial infections (viruses, bacteria & fungi) Section D (this presentation): Parasitic diseases (protozoa & similar organisms, 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

Parasitic Diseases of liver (protozoa & similar organism, metazoans) This section will follow-on from section C (microbial diseases) that finished with examples of granulomatous liver disease due to bacteria and fungi. A granulomatous response is also typical of host responses to many parasites, though as the more successful parasites avoid significant interaction with the host immune system, the most florid responses are often in aberrant hosts. Note that “parasitic” is being used in its wider context, and microsporea (now grouped with the fungi) are included as these are traditionally considered with the protozoa. So too are the myxosporea, now regarded as a phylum within the metazoan (Phylum Myxozoa), but still often discussed with protozoa.

Parasitic granulomas Large number of parasitic granulomas containing degenerate metazoan parasites in liver and other abdominal tissues of a flathead (one of the Platycephalusspecies). This was an incidental finding in an apparently clinically normal wild-caught fish, demonstrating considerable replacement of hepatic tissue, without obvious clinical effect. (Histology very poor as tissue had been frozen, so histopathology is not presented.) 901654

Parasitic granulomas 2: Wild caught Red cod (Pseudophycis brachus) with multiple granulomas through liver, kidney and heart. An incidental finding - the fish submitted because of a large ulcer (2 cm x 1 cm deep) near the head (probably traumatic in origin – hook or predator). Some granulomas show a degenerate core. Most hepatocytes are normal though some are compressed by fibrosis. Cod, Tamar L863

At higher magnification small single-celled organisms are visible at the periphery of the active zone of the granulomas. These small parasites have a similar appearance to the goldfish amoeba (see PPT 3). Similar amoeboid-like granulomas are also seen occasionally in other species (pers com R Bowater).

Note the residual pigment in some of the macrophages, sometimes retaining the size & shape of these organisms, indicating some host killing of the parasites.

Stains such as Giemsa (right) did not enhance the appearance – consistent with the simple cell structure of amoeba. Aside - other findings in this fish: these were not the only granulomatous lesions in this fish. A granulomatous reaction not containing these cells, but containing fungal hyphae and bacteria, was seen in the ulcerous lesion near the head. Mixed bacteria including Vibrio anguillarum (not one of the recognised pathogenic strains); Shewanella putrefaciens were isolated from this ulcer / wound, but not from kidney. The fungus was cultured and was confirmed as Beauveria bassiana (i.e. the one discussed in Part C). Because the infection was local, almost certainly secondary and incidental, it was not considered that this single finding represented a risk to fish from this fungus, which is a normal inhabitant of soil and vegetable matter.

Case exercise: Similar section – see if you can work out what is going on here.

Well did you get it? What made you think this was liver anyway? The lobular structure based on circulation is not unique to the vertebrate liver. The keys are the lack of erythrocytes, and this These are sperm These are developing eggs. And this is a spawning sponge! (from an abalone shell)(OK got me too at first – quick look through a new tray of slides for anything particularly interesting.)

Parasites causing minimal reaction:Well adapted parasites may be walled-off by a thin fibrous capsule, avoiding further reaction until either the parasite dies or disrupts the capsule to facilitate further spread. The effect on host health is minimal. This coccidian (probably a Goussia sp.) from liver of greenback flounder (Rhombosolea tapirina) is a good example.

There is slight enhancement of the coccidial staining but no metachromasia with Giemsa stain (A) The capsules of mature spores are acid fast (Ziehl-Neelson stain) (B, C). This clearly shows the tetrasporocystic nature (4 spores / cyst), though detail of the longitudinal suture joining the 2 valves of the spores can’t readily be seen by light microscopy. B C A

These microsporea, also from greenback flounder, show similar pathology (or lack of it). The zenomas formed are considerably smaller than those that may constrict the gut (see PPT8) 951886-9

In the previous example the heavy hepatocyte vacuolation obscured hepatocyte details. The lower level of vacuolation in this poorly fed flounder (with a thin gut) clearly shows nearby hepatocytes are unaffected by the microsporidian zenomas.

Another example of microsporean zenomas in flounder liver, showing the individual nucleated spores

Different levels of maturation of spores are shown by spore staining: mature spores are slightly refractile. L860 965258-2

Any breech of the zenoma wall will lead to inflammation: clinical effects (if any) relate to such reactions.

Parasites with minimal reactions 2: coelozoic existence. The gall bladder (like renal tubules) provides a coelozoic site for myxosporea to avoid the host immune system. Note “hairy” appearance of this greenback flounder gall bladder, allowing detection of heavy infection at very low magnification, despite rapid autolysis typical of gall bladders. Most are pre-spore plasmodia, but developing spore are visible (white arrows) at higher magnification. Minimal impact on gall bladder epithelium or host health. L856

Another area (same slide). Although the majority of developing spores are close to the epithelium, the adhered parasite cell processes extend well into the lumen, with some spores developing in more distal locations. L856

The various stages of spore development are shown in this well preserved partially collapsed gall bladder (cohort of the above). Higher magnifications are of the partially developed bicapsular pores (white arrows), and a nearby multinucleate stage that precedes this (black arrow). L856

High power view of the disrupted surface of another flounder (same parasite), showing the shape of the developing bicapsular spores (curved and flattened boomerang shaped). The shape is typical of Ceratomyxaspecies – a number of species are common in various flounder and other flat fish. Reference: Lom & Dykova, 1992. L860

Ceratomyxacontinued: Only spores with well developed capsules are selectively stained with Giemsa stain. L852-3-5

Left: another cohort of above flounder showing very elongated myxosporean sporangia (but no mature spores). Other diagnostic tools: a) Myxosporean spores are readily detected in wet preparations from gall bladder wall (right).

L852-3-5 The shape of these cells means they can often be detected in stained smears, even with considerable shrinkage or other artefacts. (Top) Smear of flounder gall bladder showing elongated Ceratomyxa sp. cells. (Bottom) Smear of Yellowtail Kingfish gall bladder showing multinucleate plasmodial stages of another myxosporean.

Myxosporea in extra-coelic locations:Liver from an adult Chinook salmon, showing multifocal pallor, seen at higher magnification to be necrosis, not reflecting a lobular pattern. Large cells are present within the necrotic areas. From slide donated by the Pacific Biological Station, Fisheries & Oceans, Canada.

(Continued): These large cells are often multinucleate (arrows).

(Continued): Others (black arrow) show a bicapsular boomerang shape (similar to the flounder gall bladder myxosporea). One is cut at an angle showing the coiled spiral polar filament (blue arrow). Also present in this field are the multinucleate forms (white arrow),

In some areas a few spores show the capsular basophilia and exclusion of dye penetration to the cytoplasm typical of mature myxosporean spores. This is obviously another myxosporean – in fact another Ceratomyxa species (Ceratomyxa shasta). This is unusual in that while gall bladder infection (as for other Ceratomyxa species) does occur, the developmental stages are not restricted to this coelozoic site. The entire digestive tract, liver, spleen, kidney, gills and muscle tissue may also be involved (lesions varying somewhat between salmonid species). Gut perforation may occur. It infects salmonids on the Pacific coast of North America. Its restriction to this location appears due to geographic restriction of it’s intermediate (alternate) host, the freshwater polychaete, Manayunkiaspeciosa(Stocking, 2006). Despite high mortality (e.g. up to 100% in some age groups of rainbow trout – see Stocking), the lack of demonstrated spread has seen international concern for this parasite gradually reduced, but translocation with stock movements is an issue within the potential range.

C. Shasta continued: Section of liver and gut of juvenile Chinook salmon with focal liver necrosis seen as pallor (arrows) From slide donated by the Pacific Biological Station, Fisheries & Oceans, Canada.

This is the same parasite (C. shasta). but in this young animal has not progressed to spore production, and only the single or multinucleate stages (arrows) are present.

In foci of parasite abundance there are areas of degenerating hepatocytes (right). Part of the effect could be vascular, as parasite cells are abundant in vessels (arrow).

Continued: The parasites are also abundant over serosa (A), and in the gut lumen (B). Once spores are present, those that do develop in the gall bladder do enter the gut lumen with bile, and can be detected with gut smears which have been used as a screening test for this parasite. However this will not detect animals (such as this one), with early (pre-spore) infections. A B

More parasites inciting florid reactions: Widespread reactions (here including liver & serosa) to circulating myxosporean stages is not common, but does reflect the number of circulating forms in this aberrant florid infection (See kidney PPT - guppy with many blood forms prior to establishment in the kidney tubules). The number of circulating forms reflects the duration of the dispersal stage, which is usually short, with few parasites moving from entry point (gut, skin etc) to the maturation site at any one time. This is longer in Sphaerospora infections which multiply in the blood stream, but in this case parasites apparently ‘losing their way” contributes to greater interaction with the immune system and widespread reactions.

Intra-nuclear parasite from Dwarf Gourami (Colisa lalia) This parasite was a surprise finding during detailed examination of an iridovirus outbreak. A number of nuclei in the liver appeared to show intranuclear bodies. You can go a bit crazy looking for these, running the risk of concerns regarding nucleoli, but these did seem to have structure with a defined wall. Too small to be certain (or photograph) on H&E, but confirmed with Gram stain. Why Gram stain? Recall this stains mature spores of intranuclear microsporean (see kidney I), and may also stain other spores. Gram stain showing apparent intra-nuclear parasites (arrows) in liver of Dwarf Gourami Colisa lalia with iridovirus infection. As intranuclear microsporean Enterocytozoon salmonis is known to be immuno-suppressive, there was concern that the this might be a similar intranuclear microsporean and may have contributed to emergence of the virus disease. Sp-15

Higher magnification (oil emersion, Gram stain), showing apparent intranuclear location within a hepatocyte and oval shape (white arrow). Compare with other hepatocyte nuclei (blue arrow) Iridovirus cells at lower right (*) * * Sp-15

Another oil emersion field, also showing the shape. Note that the form is that of a nucleated spore. Sp-15

Gram stained liver from another fish with a relatively high number of infected cells, with parasites in various planes of section. Sp-16

Another field from the same liver photographed at different depth of field, showing the highly refractile nature of the spore wall (arrows) Sp-16

Same fish showing similar cells in kidney, including glomerular tuft (arrows). Sp-16

.. And glomerular space (arrow). Same glomerulus, different focus. .. and tubules. The parasites seemed most common in the liver and appear to have been filtered from the blood through glomeruli to tubules, but it is possible both types of epithelia do support growth.

Same kidney showing cells within a putative macrophage in the interstitial tissue (arrow). A few cells were also occasionally seen in the spleen, also suggestive of macrophage removal of circulation parasites or host cells.

Intranuclear location of at least some parasites was more obvious on resin sections (arrow, toluidine blue stained section, deparaffinised liver from same block). Sp-16

Electron microscopy from same block, showing these bodies within the nucleus (N) of a hepatocyte. N N N

Another field from this liver showing parasites within the cytoplasm. Little internal detail can be seen as electron microscopy was only available on de-paraffinised formalin fixed tissue, and the spores walls appear also to have reduced fixative penetration. However a suggestion of coiled contents, such as with a polar filament, could occasionally be seen (arrows).

Discussion: dwarf gourami microsporean: • The size, shape, and appearance including the thick exospore and endospore walls, osmophilic contents, and staining characteristics provide enough information to confirm this is a microsporidian. • The characteristic refractile cell wall - one of the features that alerted us to their presence - provides the strong staining of microsporidia with Gram and Giemsa stains - (see PPT 2, kidney 1, for comparison with staining of other microsporidian including Enterocytozoan salmonis, the best known intranuclear example). This wall also prevents the penetration of fixatives, compromising electron microscopy. • Thus there is insufficient detail to determine the genus of microsporidian, but sufficient to differentiate this from the possible alternative of an intra-nuclear coccidian. This is important as this group of dwarf gouramis (and some at least of the same animals) were also infected with gut coccidia (see PPT 8 GIT-I), and that some coccidia of cichlids (the best studied being Eimeria vanasi) can have intracellular, epicellular, and intranuclear forms (Paperna, 1995). However: • E. vanasi IN forms are seen in the gut, and only as merozoites or meronts, which are larger and do not have thick cell walls. (Note though that reptilian IN coccidia can complete their entire endogenous development within the host-cell nucleus - Paperna & Vilenkin, 1999). • Are they likely to have caused immune suppression? • IN forms generally evade the immune response until the cell breaks down & they are released (Sitja-Bodadill, 2008): their presence in the glomerular tuft, renal tubules and spleen ellipsoids suggests this may have occurred, so their effect on host cells is also unresolved.

Parasitic Diseases (protozoa & metazoa) of peritoneum & swim bladder Metazoan parasites include some of the most important zoonoses obtained from eating fish, (such as anisakosis). Although the main tissue eaten is muscle, many of these parasites inhabit the abdominal cavity and associated serosa. Some are simply attached to the muscle wall, but others may migrate into adjacent muscles after death (apparently away from the autolysing gut). Thus freshness is a major factor in safe harvesting for raw or poorly cooked fish product.

Metazoan parasites, especially third stage nematode larvae, are a common finding in the abdominal cavity of fish. Pathology is usually minimal, but reactions to dying or migrating parasites may lead to adhesions. Rainbow trout serosa with Eustrongyloides sp. Nematodes in the abdominal cavity and partially embedded in organs such as liver. Note adhesions (white arrow), and a partially resorbed ova from the previous spawning season (yellow arrow)

Another example, showing physical damage to liver, plus minor adhesions.

More severe adhesions – possible consequence of either bacterial or nematode Peritonitis.

Prepared by Judith Handlinger