Prepared by Judith Handlinger

1. Systematic Fish PathologyPart 12. Pathology and diseases of the musculoskeletal and nervous systemsSection A: musculoskeletal systemPart II: general pathology, infections and neoplasia of muscle Prepared by Judith Handlinger With the support of Animal Health Laboratory, Department Of Primary Industries, Parks, Water and Environment, Tasmania, for TheAustralian Animal Pathology Standards (AAPSP) program

2. 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. Section A (this presentation) – general & non-infectious pathology Section B (following presentation): Pathology of infectious causes. 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 A: Musculoskeletal system (this presentation) B: Nervous system 13. Pathology of gonads and fry

3. General pathology of muscles General patterns of muscle degeneration & repair Muscle atrophy due to cessation of eating. Four young Atlantic salmon that failed to adapt to the move to seawater & ceased eating, presumably to minimize osmotic challenge, plus their well adapted cohort, several weeks after movement.

4. More examples of “pin-head” salmon that failed to adapt and ceased feeding, plus their well adapted cohort. Not all Atlantic salmon undergo smoltification after their first year, though the proportion is so high under Tasmanian conditions that “parr” (fish that will smolt next year) are generally disregarded. Stress can also disrupt smoltification. These fish are basically still healthy, and will resume eating if returned to freshwater or offered moist food (such as fish) – both impractical. Note the head remains relatively large (unchanged), while fat & muscles have been metabolized but otherwise show little change.

5. Normal muscle: Well fixed Atlantic salmon trunk muscle in trans-section. Note the multiple peripheral nuclei.

6. Fixation artifact of muscle: Normal Atlantic salmon trunk muscle that has been fixed rapidly. Many individual fibres show pale lysed contents within the sarcolemma. Nuclei remain peripheral.

7. Fixation artifact of muscle cont: Note that the lytic artifacts are mostly adjacent to the fibrous raphae between myomeres that prevent contraction without membrane tearing. Affected muscle fibres are lysed, but show no other pathology. Chatter artifact, representing tearing of the fixed fibre mass, is also present (*) * * *

8. Muscle lysis due to hypokalemia: True pre-mortem muscle lysis may occur due to external chemicals or ion imbalances. Severe myopathy was seen in barramundi reared in potassium-deficient saline groundwater (Partridge and Creeper, 2004). Mineralization of degenerate fibres was seen, as well as swelling and fragmentation, readily differentiating this from lytic artifacts.

9. Hypokalemiacontinued:Other lesions included degenerative lesions in kidney. Marked hyperplasia of gill chloride cells occurred, as a response to hypernatraemia and hyperchloridaemia. Photos courtesy Fish Health Unit WA.

10. Haemorrhage in cranial muscles: Cranial muscles cut in longitudinal section, normal other than acute electrocution related haemorrhage. Some interstitial oedema, but surface location of nuclei still discernible.

11. Electrocution related haemorrhage in muscles: Another young fish, showing that an acute electrocution related haemorrhage is not all related directly to spinal cord injury.

12. Muscle repair – scar tissue. This fish also had a history of electrocution, resulting in spinal deformity. Affected muscles showing scar tissue (upper left), compared with more normal tissue (lower right). Scar tissue surrounding residual muscle fibres, some of which have internalized nuclei as they undergo regeneration. Detail of repairing fibres: these nuclei do not have the open vesicular form of invading macrophages, and the organization is more orderly.

13. Muscle repair – scar tissue & fibre regeneration. This Canadian fish is undergoing marked muscle repair (cause of muscle damage unknown). The damage is largely in the outer red layer, where there is a high level of fat, indicating that the fish was in excellent condition prior to the insult. Most of the muscle is pale, suggesting a loss of muscle fibres, and / or fibrous replacement. At higher magnification, many multinucleate cell can be seen. The tight nuclear cluster (white arrow) is clearly within a muscle fibre, that shows variable eosinophilic cytoplasm with loss of myofibril orientation, and at least one phagocyte in the lytic area at right. The nuclear cluster above (yellow arrow) also shows an eosinophilic cytoplasm indicative of fibre regeneration, though there appears to be fewer residual fibrils within the sarcolemma of this cell. At least some of these can be clearly seen within muscle fibres. All show the uniform moderately stained nuclei typical of muscle fibres.

14. Adjacent field, showing that as well as the obvious large clusters of internal nuclei, those of many other fibres are internalized, indicative of repair, sometime visible as single nuclei, sometimes as an internal string of nuclei (blue arrow).

15. Muscle repair – scar tissue and pigment formation. A single harvest salmon with melanotic muscle lesions, seen here at the margins of scar tissue, surrounded by healthy and degenerate myofibres. * The pigment is present in individual cells, apparently melanomacrophages (the pattern is not suggestive of an invasive melanoma). High magnification confirms the pigment pattern to be typical of non-aggregated melanomacrophages. Both degenerate muscle fibres (*) and other inflammatory cells are still present, despite what appears to be a well established reaction.

16. Solid Following myomere pattern Salmon fillets showing different patterns of melanisation. Focal, light Diffuse Melanisation is common following skin / muscle injury & repair. Even light residual pigment is a significant product quality issue. The differential diagnosis would include melanotictumours, which generally originate in skin, and in some species apparently idiopathic melanisation with a regular pattern, as occurs with the common flathead (Platycephalusbassensis). Such melanin is likely to be derived from melanomacrophages, which have been shown to contain a variety of metabolic end product pigments. Recent work (Haugarvoll et al, 2013) indicates they also produce their own melanin which has a direct role in defense against microbial infections.

17. Areas of diffuse reaction (right) and granuloma-like reactions, with melaninisation, round saponified foci. The latter contained fine spicules suggestive of cholesterol clefts. Pigment can also be derived from degeneration and reaction in the fat associated with trunk muscles. This shows melanisation (arrow) in fat adjacent to abdominal muscles of an Atlantic salmon which had received intraperitoneal vaccination (which contain adjuvant).

18. Gas bubble disease: Larvae in Striped Trumpeter (Latrislineata), 100 day post-hatch, with gas bubbles in superficial muscle layers (x4 objective magnification). These were usually in the tail (particularly the ventral tail) or the anterio-dorsal region behind the head. Other signs were lifting of scales (bottom of section), and overinflation of some swim-bladders. Junction of unaffected and affected muscle, showing larger bubbles between muscle fibres (x10 objective). .. And smaller bubbles within many fibres (x 20 objective) Detail showing disruption of striations by small bubbles within muscle fibres, and larger less well defined bubbles between (x40 objective). These fish were being reared in supersaturated water, which is well tolerated if levels are evenly maintained, with minimal turbulence, but result in gas bubble disease if excessive levels or less than ideal tank conditions used. (See also eye lesions, Section B this module.)

19. Infections of muscle Example 1: Muscle fibre splitting with Tasmanian aquabirnavirus. This incidental finding in the dorso-lateral skeletal muscle of the index case has not been seen in muscle examination of uninfected animals, and is presumed (but not proved) to be virus related (Crane et al, 2000). Apart from parasites, there are few infections that are specific to muscles though localisation of bacteria is common. Several viruses targeting heart & skeletal muscle (See Part 11) are exotic to Australia (no examples available). Bacteriallocalisation in muscle is common.

20. Example 2: Aeromonassalmonicida(atypical marine strain) infection in muscle of Atlantic salmon.

21. Same fish, before reflection of swollen and slightly inflamed skin.

22. Example 2b: Also Aeromonassalmonicida(atypical marine strain), external appearance... .. and on dissection. Clearly these are primarily muscle lesions, despite the secondary effects on skin.

23. Example 3: Vibrioanguillaruminfection may show a similar pattern. Note the elevated pale area of oedematous and partly denuded skin as well as the typical swollen vent reflecting internal infection. .. And on dissection.

24. Example 3b: Vibrioanguillarummuscleinfection of longer standing may be reflected by a surface depression, as lytic products are absorbed. .. on skin reflection .. and on deep dissection, when the extent of muscle lysis can be seen.

25. Example 3c: Histological appearance of an early similar Vibrioinfection. (Older lesions show too much lysis to be useful for sections, generally). The most obvious features at this stage are haemorrhage and muscle fibre disruption. Higher magnification confirms haemorrhage, sometimes within disrupted fibres, which clearly distinguishes these changes from fixation artifact.

26. Another area (same case, different fish), showing muscle fibres with pallor indicative of coagulative necrosis, as well as fibres with lysis of fibre membranes and fragmentation. Both V. anguillarum and A. Salmonicida produce severe exotoxins, which in other organs may produce death before this level of necrosis is reached.

27. Example 4: Mycobacteria in muscle. A reminder that other bacteria can either invade deep into muscles from the skin, such as this Mycobacteria infection (see Part 10, Skin). Others (for example, the exotic bacterial kidney disease, Renibacteriumsalmoninarum), may localise in muscle following septicaemia. Giemsa stain of this section. The muscle destruction seen with this mycobacterial infection appears to be related more to the extensive mononuclear infiltrate induced, than to bacterial exotoxins as bacteria are sufficiently rare to not be readily visible in many of the affected areas. .. Though they are present in other areas (Giemsa stain, same section).

28. Example 5: Fungal invasion of muscle, brook trout fry. The muscle of this fish is more open that normal, with increased spaces between fibres. In some of these spaces fungal hyphae are present (e.g arrow). In most fish fungal infection was restricted to the gut or adjacent peritoneum, but occasional deeper invasion into muscle was also seen.

29. Example 5b: Fungal invasion of muscle, rainbow trout fry. The muscle of this fish is also more open that normal, and anal obstruction (due to fungal invasion) well visualized. In this case the typical thin walled aquatic Saprolegnia fungi are not readily seen on H & E staining….. .. despite their abundance as demonstrated with silver staining. Both these cases were due to a mixture of poor tank hygiene and other stresses that allowed ingestion of fungi with reduced food intake, leading to long gut passage time & multiplication of fungi within the gut, followed by spread through adjacent tissues. [See also Part 10, Skin, for spread (or not) from skin infections.]

30. Example 6: Invasion with the fungus-like protistIchthyophonusin salmonid muscle. This is primarily a product quality issue, though this may be seen with lesions in other organs that may affect function. Infection in this fish is light (arrows). A light inflammatory infiltrate has surrounded one of the organisms, otherwise the muscle is unaffected.

31. Example 6b: A much more heavily infected fish with many Ichthyophonusthrough the smaller superficial fibres of red or “twitch” muscle… … extending into the adjacent larger and deeper red muscle. This was the only site of infection detected in this fish, though others in this group showed the organism in kidney, heart, gill, pancreatic fat, spleen, and / or brain. Large number of organisms can impart a bitter taste to flesh.

32. Example 6c: Another heavily infected fish (from the same group as 6b) of considerable duration, and with more obvious inflammatory reaction. Muscle fibrosis of this extent, especially the hard nodule formed round the organism at left (arrow), would be visible grossly, alter the texture of the muscle, and cause serious downgrading / discarding of the product.

33. Example 7: Protists that invade muscle as an extension of skin lesions are typified by Uronema (see also Part 10B, Skin), seen here in the muscle of big bellied seahorse (x10 objective magnification). No skin lesion is obvious in this section, indicating that the parasites have migrated a considerable distance through the adjacent muscle layers. Detail: At this stage there is no host reaction, and minimal evidence of tissue damage, although this can be considerable. (x20 objective magnification).

34. Example 8: Muscle is one of the most common sites for histiozoic (tissue-dwelling) myxosporea as muscle fibres are long-lived and large enough to support the growth of large pseudo-cysts in which large numbers of spores develop, to be released following the death of the fish. This example is also in a big bellied seahorse. The infection involves only a small cluster of muscle fibres, and has no clinical or economic significance at this level of infection. The pseudocyst is effectively a bag of spores & developing spores, wholely within the striated muscle fibre. There is no host reaction while the fibre remains intact.

35. Example 8b: Another example of big bellied seahorse muscle myxosporean infection, involving a single fibre in section. Oil immersion view showing developing spores in various levels of maturation and orientation. Several of the more mature spores marked (arrows).

36. Example 9: Myxosporean infection in tuna muscle (wild caught albacore tuna, Thunnusalalunga). Typical appearance of Kudoa pseudocysts in tunas (several Kudoa species across a range of tuna species). This condition is also primarily a product quality issue, and not primarily because of the unsightly appearance but because pseudocysts break down after death rapidly causes liquefaction of muscle (within 1-3 days in fish on ice).

37. Kudoa in tuna muscle continued (same case). Histological appearance of histiozoic (tissue dwelling) Kudoa pseudocysts in albacore tuna muscle. Note variable host reaction. Both pseudocysts are producing spores, though these are reduced in the cyst on the left with more host reaction. The host reaction extends into the cyst at left, while the cyst at right appears contained within the muscle fibre, and does not appear to have been exposed to the host immune system. Details of host reaction: note the eosinophilic granules in the dominant tuna granulocyte. One developing spore (arrow, right) has assumed the typical folded 6-star shape (cross section, showing 2 polar capsules)

38. Detail of the cyst (right, above) showing that inflammation does not extend into the muscle fibre of cyst wall. Inset: Wet prep of K crumena spores from Southern Bluefin Tuna, Thunnusmaccoyii, which shows the shape of this group of myxosporea. Photo courtesy Nowak et al, 2006. Another area with deeply staining polar capsules in cross section (white arrow, 2 visible), and in flat section (blue arrow, 3 deeply staining, one off centre and / or less well developed capsule. Evidence suggests this is a 4-polar capsule Kudoa species (possibly K crumena or K. nova, both known in tuna), rather than the 6-shell valve / 6-polar capule(K. (formerly Hexacapsula) neothunni, is also known in albacore (Munday et al, 2003). Detail of developing spores, showing some with the crown of 4 developing polar capsules (3 visible) around the still abundant sporoplasm (white arrow), and 1 with at least 3 deeply staining polar capsules near maturity (blue arrow).

39. Another field, showing many more spores with fully developed and refractile capsules. Kudoa species (having 4 or more spores arranged radially in a drooping disc shape), generally have a broad species range. Genetic analysis is clarifying the number of species, so nomenclature of this group is evolving. Almost all those affecting tuna, plus others in other commercially important fish species, induce marked and rapid liquefaction after harvest making this one of the commercially most costly myxosporean infections. For example, there has been similar concern about high levels of Kudoa thyrstites(also a cause of liquifaction) in salmonids in several countries. Fortunately the incidence of this parasite in Tasmanian Atlantic salmon is very low (Munday et al, 1998 and on-going health monitoring). This probably reflects the lack of native wild sea-going salmonids, as well as management factors minimizing the ingestion of wild food.

40. Example 10: Encysted metazoans (trematodes) in galaxids. Two fish with bulges and skin discolouration from encysted digeneanmetacercaria. One intact & 1 ruptured cyst (above).

41. Example 10b: Encysted metazoans (trematodes & nematodes) in galaxids. Three more galaxids, two with metazoans encysted in muscle: “redworms” (Eustrongyloides) nematodes, upper fish, and “black spot” (encysted metacercaria), bottom fish. [Parasite encystment can be seen in a wide range of species, but galaxids to appear to be prone to these.]. Metacercariae encysted in fish are often the intermediate stages of bird flukes.

42. Example 11: Encysted metazoans (nematodes) in large abscess-like swelling in flounder muscle. Incidental finding, nematode not identified further.

43. Example 12: Larval trypanorhynchcestodes between muscles of barracouta (Thyrsitesatun). These are the intermediate larval stages (plerocercoids). Some species can also be found in the body cavity. The adults typically occur in the intestines of elasmobranchs (sharks & rays), and the invertebrate (procercoid) stage in copepods. There may be little associated pathology.

44. Neoplasia of muscle Example 1: Neurofibroma-like mass in muscle of a wild caught brown trout. This single example showed softening and apparent liquifaction of abdominal muscle. The abdominal cavity was filled with “creamy viscous fluid”. Some autolysis was also present. Primary muscle neoplasia appears to be rare in fish, though some are reported. Secondary neoplasia in muscle is commonly either an extension from skin neoplasms, or (in Atlantic salmon in Tasmania), a secondary lymphoid tumour.

45. Example 1 continued: Histological appearance of the margin of the above brown trout tumor. Demarcation is poor, with extension into surrounding muscle fibres, some of which show vacuolar change, especially those close to the tumor. Tumor cells are relatively uniform, pale, spindle shaped, and arranged in loose whorls that extended into muscle. Showing arrangement of tumor cells, in a pattern judged to be consistent with neurofibroma. Some of which are clearly invading normal muscle cells.

46. Example 2: Lymphoid tumor in salmon muscle. These tumors commonly have kidney as the likely primary site, metastases to internal organs and / or muscle being relatively common. (See Part 2, Kidney Interstitia re the primary tumors). Lesions in muscle may be moderately well demarcated, though in this case there is extensive lateral spread, especially through the red muscle & fat layer. Showing the typical uniform population of lymphoid cells.

47. References – musculoskeletal system. Re anatomy & bone development: • Apschner, A, Stefan Schulte-Merker, S, and Witten, P.E. 2011. Not All Bones are Created Equal - Using Zebrafish and Other Teleost Species in Osteogenesis Research. Methods In Cell Biology, Vol 105, p. 239-256. DOI 10.1016/B978-0-12-381320-6.00010-2 • Currey, JD and Shaharb, R. 2013. Cavities in the compact bone in tetrapods and fish and their effect on mechanical properties. Journal of Structural Biology, Volume 183, Issue 2, August 2013, Pages 107–122 • Roberts, R.J, Hardy, R.W., Sigiura, S.H., 2001. Screamer disease in Atlantic salmon, Salmosalar L., in Chile. J. Fish Dis. 24, 543–549. • Lall, S P, Lewis-McCrea, L M. 2007. Role of nutrients in skeletal metabolism and pathology in fish — An overview. Aquaculture 267, 3–19. • Fernández, I and Gisbert, E. 2011. The effect of vitamin A on flatfish development and skeletogenesis: A review. Aquaculture 315, 34–48. • M.J. Darias, MJ, Mazurais, D, Koumoundouros, G, Cahu, CL and Zambonino-Infante, JL. 2011. Overview of vitamin D and C requirements in fish and their influence on the skeletal system. Aquaculture 315. 49–60. • McGrouther, M. 2013. Australian Museum. http://australianmuseum.net.au/Hyperostosis-Swollen-Bones Re muscular pathology: • Partridge, G J and Creeper J. 2004. Skeletal myopathy in juvenile barramundi, Latescalcarifer(Bloch), cultured in potassium-deficient saline groundwater. Journal of Fish Diseases 27, 523–530 • Haugarvoll, E et al. 2013. Norwegian School of Veterinary Science (2009, February 2). Secretive Immune System Of Salmon. ScienceDaily. Retrieved October 28, 2013, from http://www.sciencedaily.com­ /releases/2009/01/090127123117.htm In press as: Vaccine-assosiatedgranulomatous inflammation and melanin accumulation in Atlantic salmon white muscule • Crane, M. St.J, Hardy-Smith, P., Williams, L. M., Hyatt, A. D., Eaton, L. M., Gould, A., Handlinger, J., Kattenbelt, J. and Gudkovs, N. 2000. First isolation of an aquatic birnavirus from farmed and wild fish species in Australia. Diseases of Aquatic Organisms 43:1-14. • Munday, B L, Sawada, Y, Cribb T and Hayward, C J. 2003. Review: Diseases of tunas, Thunnus spp. Journal of Fish Diseases, 26, 187–206 • Nowak, B., Johnston, C., Hayward, C., Aiken, H., Adams, M., Evans, D., Deveney, M., Carson, C., Jones, B., Evans, R., Dyková, I., Porter, M., Naeem, S., Kruesmann, M., Bayly, T. & Pitney, C. 2006. Southern Bluefin Tuna Health. (AquafinCRC, on disc) B Nowak (Ed). University of Tasmania. ISBN 978-1-86295-374-1 • Munday, B L, Su, X-q, and Harshbarger, J C. 1998. A survey of product defects in Tasmanian Atlantic salmon (Salmosalar). Aquaculture 169 Ž1998. 297–302.