Systematic Fish Pathology: Part 3. Kidney II: Interstitial Tissue Focal Reactions

1. Systematic Fish Pathology: Part 3. Kidney II: Interstitial TissueFocal Reactions Prepared by Judith Handlinger Fish Health Unit, Animal Health Laboratory, Department of Primary Industries & Water, Tasmania for The Australian Animal Pathology Standards (AAPSP) Program

2. Before Entering Training Program READ ME • This series of training modules has been prepared from a teaching slide set used for short courses on fish histopathology aimed at students with a range of prior knowledge of either pathology or fish. • Slides are representative of the pathology found in Australian fish in this (Tasmanian) fish laboratory, rather than a comprehensive record of all fish diseases, or diseases of all fish. There are some examples from other laboratories, including exotic diseases. • The major aim is to convey an approach to diagnosis, not to cover all fish diseases. The systematic approach is relevant, regardless of species (indeed this is just applying mammalian pathology training to fish). Nevertheless the pathology of any species can only be interpreted in comparison with the known normal. As there are more fish species than all the other vertebrates together, it is necessary to overcome the temptation to consider fish just a new “species” for study, and appreciate the diversity of fish species. • That this course does not cover all Australian fish, then, is within the context of using available material to impart a general knowledge of fish pathology including general patterns of fish diseases and an introduction to types of pathogen common in fish.

3. Acknowledgments. • Most material used has largely been generated within the Tasmanian Department of Primary Industries & Environment Fish Health Unit, and represents contribution of cases and photographs from many other contributors including Jeremy Carson, David Taylor, Stephen Pyecroft, Richmond Loh, Kevin Ellard, Paul Hardy-Smith, and Barry Munday. • Contributors of cases from other laboratories have been acknowledged wherever possible and specific material and photographs used with permission. Any inadvertent omissions in this regard are unintended. • Material exotic to Australia includes slides distributed for general teaching purposes and slides contributed specifically for the DPIW Fish Teaching set, and are acknowledged as such.

4. Introduction • This is the second of three Powerpoint presentations of fish kidney that are being used to demonstrate major fish response patterns, and to introduce major groups of fish pathogens. • Please refer to Part I “Consider the Fish” for a general introduction to fish anatomy, histology and immunology, and to Part 2 for generalised responses of the kidney interstitial tissue. • This Powerpoint presents a range of responses to pathogens localised to the kidney interstitium. However, similar responses are likely to occur in other organs.

5. Kidney Interstitial Tissue:Inflammation & Degeneration B. Focal reactions 1. Chronic reactions - granuloma formation

6. Head kidney of Goldfish with a granuloma. ( ) What are the other eosinophilic structures? ( ) (Yes – more thyroid follicles)

7. Granuloma shows a well developed fibrous wall, and macrophage outlines are still visible.

8. A very common inciting cause for granuloma formation, especially in aquarium fish, is mycobacterial infection, shown here in a gourami (Ziel–Neelsen stain). Several marine & freshwater mycobacterial species have been implicated. (a moderate zoonosis: “fish-fancier’s fingers”).

9. Many agents incite a granulomatous reaction & not all acid fast objects are bacterial - the contents of this old granuloma suggest fragments of a capsule, although the organism has been killed.

10. Parasitic granuloma with whole & clearly nucleated spores Inter-renal tissue Granulomas Goldfish kidney showing similar granulomas at an earlier stage. (Note thyroid follicles.) Note more macrophages migrating round older granuoloma

11. Introducing fish parasites Pathogen groups typical of fish will be introduced as they are encountered. The range of pathogens affecting fish is in general similar to that of higher vertebrates (viruses, bacteria, fungi, parasites etc), but the water environment does favour some types of bacteria, and the range of parasites in land animals has become defined to some extent by the restrictions of that environment. Two types of microscopic parasites are very common in fish: Myxosporea Microsporea are much more common in fish than mammals, though a few mammalian examples such as Nosema do occur.

12. Polar capsule with with coiled polar filament (upper left) & 3 shapes of bi-capsular spores. Spores can have 2-7 polar capsules. Introducing Myxosporea The previous example was a granulomatous response to myxosporea. • Myxosporea were long considered as a Class of protozoa, then as a separate phylum (Myxozoa), but are now known to be a derivative of jelly-fish stinging cells (nematocyts). (Siddal et al, 1995) • The defining stage is the bi- to multi-valved spore. Each capsid (polar capsule) within the spore contains a coiled filament (the polar filament) that resembles that of the nematocyte and is extruded during penetration of the host cell on primary infection of the host, allowing the sporoplasm to enter and infect the cell. • The complete life cycle is known for very few myxosporea, but from those few it is known that an intermediate host is required (typically an oligochaete worm), in which a spore with different appearance is produced. This spore type was previously considered as a separate class of the phylum Myxozoa (the Actinosporea, now abandoned, Kent et al, 1994). This has long caudal appendages to the main stalk (e.g 3 for the Triactinomyon species), but the same coil structure in miniature in the capsides at the tip of the stalk. • There are many fish myxosporean parasites - either within the tissues (histiozoic) or outside the reach of the host immune system in coeliozoic locations such as the gall bladder or kidney tubules. We will see many examples. Two Hoferellus carassii with polar filaments extruded. Note brush of fine posterior filaments & 2 dark capsules Pentacapsulate spores, stained smears and wet prep

13. References • The Demise of a Phylum of Protists: Phylogeny of Myxozoa and Other Parasitic Cnidaria. Mark E. Siddall, Donald S. Martin, Diane Bridge, Sherwin S. Desser, David K. Cone. The Journal of Parasitology, Vol. 81, No. 6 (Dec., 1995), pp. 961-967 • The Demise of a Class of Protists - Taxonomic and Nomenclatural Revisions Proposed for the Protist Phylum Myxozoa Grasse, 1970. Kent M. L. ; Margolis l. ; Corliss J. O. Canadian Journal of Zoology,1994, vol. 72 (5), pp. 932-937

14. 2 1 4 5 3 Oil immersion (x100 obj.) of previous granuloma. Bi-valve spore in frontal view (1), side view (2), tangential (3). And in cross section through the capsules (4), and through the nucleus of the sporoplasm (5)

15. The above example shows myxosporea within a host reaction, where many may be destroyed over time (as in the first case shown), but the strategy of these parasites is to avoid host reactions if possible. The spores of histiozoic forms will therefore usually develop within a host cell (thereby avoiding host recognition). The host cell becomes very much enlarged, forming a zenoma or “cyst”, as in this River Blackfish (Gadopsis marmoratus ) kidney. Under the influence of mild autolysis & fixation, the normally tightly packed polar filament in many polar capsule has partially uncoiled and become visible.

16. Two more examples of spores from this fish.

17. Myxosporea can occasionally be picked up in smears - in this case methylene blue smear from the above goldfish with myxosporean granulomas.

18. Sometimes a hint can be found in the outlines of a granuloma in a smear: well established granuloma walls can be hard to disrupt. Look within these for pathogens - in this case myxosporean spores.

19. Back to other agents inciting granuloma formation

20. Granuloma in a Goldfish kidney - necrotic tissue only in centre, so the only place to look for pathogens is at the macrophage zone of the margin. These tiny parasites (previous slide) have long been regarded as amoeba-like, the condition being known as systemic amoebiasis. Although there has been considerable doubt as to their exact nature recent genetic analysis suggests they are closely related to the more primitive amoebae (Stephen Pyecroft, pers. comm.). Whether they are opportunists or not, this condition may occur in outbreaks within a pond system, sometimes repeated with an annual pattern. Look particularly at the interface of dead and live macrophages. Under oil (x100), tiny nucleated parasites are present free & in macrophages – many parasites!

21. Florid granulomatous disease in a Canadian salmon, virtually obliterating the kidney. With many very small bodies within the reactions …..of relatively even size and shape, but variable pigment Heavily pigmented forms may be dead (residual pigment, retaining shape), but occasional tiny nucleated forms can be seen (arrow).

22. These tiny organism are the Rosette agent (now called Sphaerothecium destruens) is now know to belong to a small clade of unusual organisms known as the DRIP clade. (This is the “R” member of the name. We will meet some others.) • A disseminated form with little host reaction is also known. • The disease affects salmonids in the Pacific North West of North america. It is florid and nearly uniformly fatal – but the prevalence of affected animals is low, implying good resistance in most members of adapted populations, and / or low numbers in the environment and an indirect life cycle. (Same section, H&E). The organisms are readily seen with a PAS (Periodic acid-Schiff reaction stain for carbohydrates). • Apparently similar organisms may occasionally seen in other fish species (see Liver PPT) • The pathology shown demonstrates that much of the kidney can be destroyed without immediate risk to life - nitrogenous wastes are excreted across the gill, not the kidney.

23. Another florid exotic granulomatous disease Macrophage contents are vaguely granular with H&E stain

24. Same slide: Gram stain is rewarding! Showing the large number of Gram positive bacteria, and their intracellular location (within macrophages). This is a pathognomonic finding: Gram positive rods surviving in macrophages within granulomas indicates infection with Renibacterium salmonarum,causing the internationally significant Bacterial Kidney Disease (BKD). The infection spreads throughout the kidney, and to the rest of the body during periods of stress such as late winter when the impaired immune system fails to maintain the active granulomatous reaction.

25. Kidney swelling due to Bacterial kidney disease (BKD) – infection by Renibacterium salmonarum – in a Chilean post-smolt (Chile, 2002). Same cohort: Note the lesions in the liver, also due to BKD.

26. More about granulomatous reactions.

27. This slide shows 2 hallmarks of granulomas: A wall of macrophages partially joined to form an “epithelioid” appearance (white arrows) • A giant cell, formed by more complete fusion - a syncytium (blue arrow)

28. This shows the multinucleate nature of the giant cells more clearly. But what is the nature of the inciting agent? Any guesses?

29. All the section of these bodies appear round, suggesting a spherical shape, with a marked capsule or wall. The other major characteristic is the multiple small nuclei with no apparent cytoplasmic division. Additional information: on culture medium the agent forms a thallus. These characteristics suggest a very large fungus. This is a relatively common finding in marine fish, leading to the name Ichthyophonus (literally fish-fungus). Actually it is not a fungus. It does not fit any of the major categories, but is now know to belong to a small clade of unusual organisms known as the DRIP clade. (This is the “I” part of the name).

30. Appearance of tissue form of Ichthyophonus in wet smear.

31. Ichthyophonus hoferi in salmonids (continued): Sometimes this pathogen is killed…

32. .. but the infection may become overwhelming, as in this kidney, where despite an obvious host response, the organisms are not contained. In such heavy infections the organisms are distributed throughout the body.

33. This is another salmon kidney showing giant cells and extensive destruction and replacement with inflammatory reaction. At higher magnification this material clearly has a basophilic core. A large focus of necrosis is present, outlined by eosinophilic material (arrows). Similar material is scattered through the remaining kidney interstitium.

34. Another area from the same kidney clearly shows the granulomatous nature of the reaction. These are Gram-positive filamentous rods, but were not identified. (No cultures undertaken. Nocardia is suspected on clinical history, and has been associated with Splendore-Hoppli reaction in mammals.) Splendore-Hoeppli protein is a response to continued exposure to certain types of antigen. It is relatively rare in fish, with the possible exception of a response to vaccines containing adjuvant. More examples can be expected here as adjuvant vaccines become more common in Australia. Oil emersion shows this reaction to be round bacteria, in this case. The eosinophilic material is Splendore-Hoeppli material (also known as club-colonies). This is comprised largely of antigen-antibody complex. The similarity to such material in mammals demonstrates the similarity of the immunology of all vertebrate classes.

35. Which brings us to..Inflammation & degeneration B. Focal reactions 2. Acute reactions - & more patterns of bacterial infection.

36. This slide shows large clumps of bacteria free within the interstitial kidney interstitial tissue of a Canadian salmon (arrows), with little host response apparent. The clumped nature implies non-motile bacteria (indeed quite “sticky” , a characteristic obvious in cultures). These characteristics are hallmarks of Aeromonas salmonicida. One strain of this bacterium, Aeromonassalmonicida salmonicida, isthe cause of “Furunculosis” - one of one of the oldest and most significant bacterial diseases of fish (particularly salmonid fish). The toxins from these large clumps of bacteria may cause death before an effective host response can be mounted. Other “Atypical” strains are known, particularly in marine fish but also Goldfish. The pathology is similar, though the course of the disease may be less dramatic, with more host response evident.

37. Classic or “typical” strain (Aeromonas salmonicida salmonicida) in a Gram stained section. From slide donated to EAFP teaching set by the Marine Laboratory, Aberdeen, of a moribund Scottish (farmed) Atlantic salmon (x 40 objective)

38. Tasmanian greenback flounder kidney with large focal lesion with faint basophilic masses at the margins. Higher magnification shows these to be similar to previous A. salmonocida slides: This is infection with an atypical A. salmonicida strain (of which there are increasing numbers being recognised). These tend to be overall less pathogenic than the typical salmonid strain, in that fewer animals are affected, but the pathology is similar, although animal may survive longer and mount more of a response.

39. More examples of flounder A. salmonicida showing the bacterial clumps and fibrin at the margin of expanding necrotic zone

40. Flounder isolate of atypical A. salmonicida in Atlantic salmon in interstitium and blood vessel (experimental infection). Similar natural infections have been seen in marine environment. Demonstrates that the various strains are not species-specific, though the susceptibility may vary. (Goldfish strains are also pathogenic to salmon.)

41. This is the kidney of a sea-horse (Hippocampus species). Although clumps of non-motile bacteria evoking little host response are a hall-mark of A. salmonicida infection, other bacteria must be considered, especially as fish responses can be inhibited by many factors such as temperatures outside the normal range for the fish species. However these organisms are acid-fast (ZN stain, x 40 objective). The basophilic mass clearly consists of bacteria free within the tissues, with little evidence of a local host response They are mycobacteria. Because mycobacteria can frequently survive within host macrophages (e.g within a granuloma), there can be an assumption that they are obligate intracellular organisms, but this is not the case. They are capable of replication outside (e.g in soil) and inside the host. If the host is unable to mount effective containment through macrophage phagocytosis they may continue to multiply, as shown here.

42. Another sea-horse from this group, showing massive mycobacterial colonies.

43. Detecting infections where the pathogen is less obvious.

44. Sometimes changing patterns visible at low magnification indicate areas of change that are not otherwise obvious when scanning a slide. Here an area devoid of the normal pigment pattern (arrows) indicates an area of increased interstitial reaction, although the cell overall density of the tissue is little altered.

45. Another example, more obvious at lower power.

46. Another less defined example, as outlined to be examined in more detail for the pathogen (Atlantic salmon kidney). There is clearly both inflammation and disruption of tubules Large focal lesion in Atlantic salmon kidney (outlined) Higher magnification (x 40 objective) shows degenerate inflammatory cells, many suggestive of polymorphonuclear granulocytes (blue arrows) & cells within macrophages (white arrows), containing small organisms. These are rickettsia-like organisms (RLO).

47. Another field (same section)

49. Another fish with RLO (rickettsia-like organisms) in the kidney... Large irregular areas of pallor. A B

50. And another example of RLO in the kidney.