Prepared by Judith Handlinger

Systematic Fish PathologyPart 12. Pathology and diseases of the musculoskeletal and nervous systemsSection A: musculoskeletal systemPart I: anatomy and microanatomy of the musculoskeletal system 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

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

Acknowledgments & Introduction. • This is the twelfth module of the systematic examination of fish pathology, which 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). • As with previous modules, it was funded by the Australian Animal Pathology Standards program 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 DPIPWE Fish Health Unit, with many gross lesions taken by Kevin Ellard. • 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.

Anatomy & Microanatomy While fish differ little in their physiology of the musculoskeletal and nervous systems, there are some obvious anatomical differences: the appendages are not weight bearing, and the major muscle mass is trunk muscle.

Compare the bony vertebral column of a young teleost (above, white arrows), with the un-jointed notochord, with mineralized margins, of a larval lamprey (below, blue arrow). Mineralization of the notochord formed the first endoskeleton of fish, and occurs without cartilage formation. Teleosts still retain that first step in initial vertebral formation, but cartilage based bone is later involved in the growing skeleton of larger fish. As fish age, the notochord is reduced to the nucleus pulposus of the inter-vertebral discs, as in mammals. Key reviewreference for fish bones: Apschner et al, 2011.

Fish joints are simple, cartilage lined. Showing detail of cartilage, simple joint lining, and areas of slight peripheral mineralization (e. g. at arrow). (Fish bones do mineralize, but many routine sections that include bone are of young or very small fish, with little mineralization. Decalcification is necessary for other than very small fish.)

A more major joint (jaw articulation)…. .. Showing more bone mineralization. Bone of most fish are acellular, lacking osteocytes. They also lack the lacunar–canalicular (Haversian canal) system of mammals. Cellular bones do occur in a few families including the Salmonidae, Cyprinadae, and Clupeidae. Acellular bones are formed by osteoblast cells that show a polarized secretion of bone matrix, and which move away from the site of mineralization as bone deposition occurs so they are never incorporated into the matrix. They should be unable to undergo skeletal remodelling, but some remodelling still does occur, though how increased bone strain (indicating the need for remodelling) is detected still presents a conundrum (Currey & Shaharb, 2013). Although mineralization shown here only on the surface (perichondral), by osteoblasts derived from theh former perichondrium, is a pattern very typical for fish, other patterns of endochondrial bone formation do occur in larger teleosts. Joint

Joints in a tail fin of an Angel fish.

Showing ossified areas of the spinal column of a small salmon. A few cells (osteocytes) remain within the pink (decalcified) bone areas (arrows). The skeleton consists of two major subunits that evolved independently: the dermal skeleton and the endoskeleton. The dermal skeleton evolved first and is represented in mammals only as teeth, and in teleost fish as teeth, scales and scale-derived fin rays. More basal teleosts such as salmonids and cyprinids (which includes well studied fish such as the zebrafish), have cellular bone in the endoskeleton, but have dermal skeleton derived scales and fin rays that are acellular. Acellular bones of the endoskeleton (loss of entrapped ostecytes) is believed to have evolved over a long period in marine fish, and in freshwater fish are only found in those species that are regarded as "secondary freshwater" species, moving into this habitat only. One acellular mineralized tissue particular to all teleosts is the initial mineralized notochord sheath. The typical osteoclast associated with advanced teleosts (those with acellular bone) is mononuclear, though ultrastructural characteristics, including the ruffled borders, are similar to mammalian osteoclasts. "Primative" fish species (with cellular endoskeletons, containing osteocytes), show both mononuclear and multinuclear osteoclasts. Reference: Apschner et al, 2011.

The major propulsion force (and muscle mass) is usually trunk muscles, attached to the spine as parallel myomeresegments, not individual muscles with specific directional pull. • The myomere shape is accentuated by the muscle haemorrhage in the photo below.

Red muscle (& fat) Fish swim by contracting muscles on either side of their body to generate waves of flexion that generally get larger as they move caudally. In long slender fish such as eels there is no increase in amplitude caudally. There are two types of striated skeletal muscle. The thin outer “red” muscle is aerobic and used for sustained low speed swimming. The bulky inner “white” muscle is largely anaerobic and used for high speed or bursts of swimming. It is this that will lead to fatigue. Note that the “white” muscle of larger salmonids is usually salmon-pink due to natural or added pigments (above), which are not part of the juvenile diet (below). White muscle White muscle

Epithelium covering all surfaces – both above and beneath the projecting portions of the scale. Bony scale Melanophores Scale pocket Dense connective tissue layer Smaller fibres of superficial (twitch) or red muscle Fat layer Larger fibred of the deep (white) muscle Sk-1C

Skeletal fractures & deformity A Deformities occurring during early development will be covered by Part 13. This section deals with deformities occurring during the growing phases. Example 1 : Deformity following disruption to the spinal cord. Young Atlantic salmon, with disruption of the cord at the site indicated (arrow). There is no indication of overt vertebral dislocation sufficient to injure the spinal cord, but there is sharply demarcated muscle atrophy caudal to the point of injury, as well as flexion. Possibly post-electrocution, which resulted in severe spasm, causing vertebral & cord rupture, with subsequent vertebral healing.

Example 1 B of Deformity following disruption to the spinal cord. Salmon B shows marked focal unilateral muscle loss, leading to lateral flexion (scoliosis) (arrow). N B B This 1990 example is almost certainly due to previous bird attack (typical cages being smaller, shallower, and unprotected from birds at that time). Both spinal cord & muscle damage were common consequences in fish that survived. They remained unproductive / unmarketable.

Example 2: Acute spinal injury likely to lead to deformity. Young salmon with spinal damage from spasms resulting from electrocution. Sudden deaths are common, but survivors likely to be deformed, as above. Without deformity as an indicator, it is very easy to miss spinal fractures, as there is likely to be little resulting haemorrhage to suggest a lesion & routine post-mortem dissection is usually focused on internal organs, and may not involve deep dissection to the spine. [The same can be said for post-mortems of many mammals!] Radiography would work well with these spinal vertebral lesions.

Another acute spinal injury from electrocution, showing lateral displacement of the spine.

Histological appearance of a similar spinal fracture, also from elctrocution, with displacement of bone into the spinal cord (arrow). Note the disrupted and vacuolated appearance of the spinal cord, indicating demyelination. Be aware that fish neurological tissue can appear much more open and apparently vacuolated in section than mammalian tissues, but without the obvious disruption or degeneration.

Another example. While it may be difficult at first glance to determine if the bone area arrowed does transect the cord, or reflects lateral flexion, the disruption to the vertebral bodies / notochord remnant is marked and obvious. .. So, too, is the recent haemorrhage. Higher magnification also indicates that the material in question has the appearance of vertebral disc pulp (notochord like cells), which should not be in this location. These several examples suggest electrocution of fish is common? Probably not, but a number of fish may be involved, and repeat episodes may occur until the source of current is determined. The particular vulnerability is their aquatic environment, with transmission along wet surfaces. A typical scenario is leakage along old wiring, especially if underground, where problems only occur during or after wet weather, when ground water is high. Note this can also pose significant human risk. Under these circumstances, low level leakages can also occur, resulting in fish with sub-lethal muscle lesions rather than overt spasm with spinal fractures.

Example 3: chronic low level myositis due to electrical leakage. Archive slide (J Langdon) of phagocytosis of a single degenerate white muscle fibre due to low level leakage. This should always be considered in the differential diagnosis of such lesions.

Example 4: spinal injury in large Atlantic salmon following lightning strike. Two adult Atlantic salmon from a freshwater group presenting (live) with floating tails. The adjacent / interspersed ponds of adult rainbow trout were not affected. No injury was evident on external or internal examination – dissection down to the spinal column was necessary to detect the spinal fractures & determine that the symptoms were due to distal paresis & inability to control tail muscles & swim bladder inflation levels. Further history confirmed recent severe thunder-storm above the farm. Reports of the storm confirmed both groups experienced lightning strike through the ponds, resulting in severe transient spasm. This resulted in spinal fracture only in the Atlantic salmon, which have a higher muscle: spinal mass ratio.

Another animal from this group. As well as the spinal fracture, there is congestion of the gut, apparently also a consequence of loss of caudal innervation. Electrical current is transmitted weakly through freshwater. Transmission is dependent on the impurities and the low but necessary ion levels. It did not kill outright as the fish did not make contact with the ground and therefore did not act as a conduit for concentrated current to the ground. Electrical current is transmitted much more readily in seawater, lightning strikes generally being dispersed around the fish (which have a lower ion concentration that the seawater). Despite this, adverse effects of lightning strike were reported from one sea farm. Neither acute deaths nor spinal rupture were seen (no fish floating tail up due to paralysis round the swim bladder), but a number of fish were found on the cage floor following the event. These fish failed to swim the several meters to the surface, and therefore obtained little food, but mostly remained healthy otherwise until culled. Culled fish showed a collapsed empty swim bladder suggesting that the lightning had generated sufficient spasm for a cough-like reaction that expressed the air from the swim bladder. As a physostome (fish with patent pneumatic duct in adult life), they should have been able to gulp air if they swam to the surface, though it is possible the bladder was emptied too completely to allow this.

Example 5. Nutrition related spinal fracture - Lovettiasealii(Johnston). Archive slide of L. seallii, Tasmanian whitebait, in experimental culture, showing focal disruption of the spine with extensive areas of lysis and narrowing of the cord. Bones were thin, with poor mineralization. This was suspected to be related to vitamin C deficiency. Nutritional deficiencies are more likely to occur during initial attempts at culture, before nutritional requirements of the species are established.

Example 6: nutrition related jaw deformity, Atlantic salmon. Jaw deformity of varying severity, which, contrary to the previous statement, occurred in rapidly growing salmon some years after nutritional requirements for this species had been established & several years after marine culture of this species occurred in Tasmania. Triploid fish were most affected, but this was also seen in diploid fish. A nutritional cause was suspected but difficult to prove as one feed type was being used across the industry. Normal jaw

Jaw deformity, Atlantic salmon cont. Two Tasmanian salmon showing that deficits of the opercular bones were seen in this condition. (The operculum core is formed by 4 fused bones.) Though jaw deformities were usually the dominant lesion, a malformed operculum was sometimes the major lesion, leading to deformed twisted gills. Gill distortions are likely to be both a direct effect of the nutritional deficits on gill cartilage / bones, and secondary deformity due to turbulence as a result of uneven gill cover contribute to “raggy” gills.

Example 6: nutrition related jaw deformity, Atlantic salmon cont. Proof of a nutritional link came when the same syndrome was seen in the larger and more diverse Chilean industry, though this was only one of the factors (Robert et al, 2001). The Tasmanian fish (right) shows the facial appearance the Chileans associated with Edvard Munch’s paintings entitled “The Scream” leading to the term “screamers” for such fish. In Chile this condition was restricted to out-of-season smolt (imported as eggs from the northern hemisphere), which were released into sea water at 20oC or greater in December, and were fed particular diets (The in-country derived smolt are transferred earlier, June to November). Investigation confirmed that the condition was primarily due to phosphorus deficiency in very rapidly growing fish. Improved fish strains, triploidy, newer high energy diets, and high water temperatures all increased growth rate – which already receives a physiological boost following transfer to a seawater environment. Feeds met previously published standards, but these required revision. Pathology: These gross abnormalities reflect a serious deficiency in calcification of most skeletal structures (Roberts et al found spinal & rib distortions also, that are not always obvious on gross examination). Chondocytes were disordered, ground substance pale, often deficient perichondrial or periostealtisissue, and often osteoclastic activity at centres of ossification. Jaws showed exuberant cartilage growth & excess highly cellular collagen tissue. Deformity followed increased flexibility of poorly calcified bones bending to the jaw action involved with respiration (pumping water over the gills).

Example 7: Similar deformities in freshwater, not primarily food related. This and the following slide are from a hatchery where such lesion occurred in the freshwater phase, and were markedly exacerbated on transfer to sea, despite adjusted diets. Note poor operculae, and the start of jaw deformity.

Example 7 cont. This fish, from the same hatchery, following year, shows in addition a caudal deformity following fracture of the soft vertebral bones. These fish were not undergoing very rapid growth, and were on the same feed as other unaffected hatcheries. The pathology was the same but the phosphorus deficiency was due to excessively high calcium levels in the spring water supplying the hatchery (now disbanded). Generally calcium is absorbed from water, but levels of phosphorus are low in both seawater & freshwater, & this is absorbed from food. The excess levels of Ca blocked this absorption. [Other food factors such as phytates also render P unavailable, but availability is generally high from fish meal, which remains a significant component of young fish diets.] Vitamin C, also essential for normal bone growth, is protective against pathological fractures with mild mineral deficiencies.

Example 8: Poor growth and fin deformities in Angel fish. A range of fin lesions, and other health problems, were seen in Angel fish grown at slightly increased temperatures. These Angel fish had been “pushed” for fast growth with slightly higher temperatures. The previously adequate high quality salmon starter diet was found to be inadequate under these circumstances. Problems resolved by doubling the vitamin-mineral premix, to better match their normal high metabolic rate, as well as lowering the temperature. (The normal temperature for Angel fish is already much higher than that of young salmonids, so higher levels of vitamins are required.) • Other vitamins, especially vitamin C, are also important for normal bone growth (reviewed by Lall & Lewis-McCrea, 2007; Fernández and Gisbert, 2011; Darias et al, 2011). • Vitamin E decreases bone resorption through suppression of cytokines and stimulates bone matrix production; ascorbic acid regenerates the α-tocopherol reduced form after it has been oxidized (i.e protects against Vitamin E deficiency), and is also essential for bone matrix and collagen synthesis. Vitamin K, zinc, manganese and copper are also required. Fish bones contain high levels of lipids: the fatty acid balance & antioxidant availability are also essential. The physiological role of vitamin D in fish is unclear, but fish (especially marine fish), may have high stores of vitamin D. • Care must be taken as toxicities (mineral and vitamin A) can occur if too much is added.

Example 9: Whirling disease. The best studied infectious cause of bone destruction and deformity leading to spinal compression is infection by the myxosporeanMyxobolus(formerly Myxosoma)cerebralis, known as “whirling disease”. This disease of salmonids was first described in Germany and is now known throughout most of Europe, with well documented spread to the United States and to other countries including South Africa. It has been seen in New Zealand, but not Australia. Below: resulting skeletal deformity in mature brook trout. Inset above: black tail due to loss of innervation (see Part 10A). Photo Dr Thomas L Wellborn, Jr, collection US Fish & Wildlife service, Wikimedia commons From http://en.wikipedia.org/wiki/Myxobolus_cerebralis

Whirling disease life cycle: It had long been inferred that myxosporeans required an intermediate host, but tubificid worms as the intermediate host of M. cerebraliswas the first to be found. Animals named as Triactinomyxons(morphology below), were found to be intermediate stages of myxosporeans. Note that the spores contain polar capsules, as do those of the myxosporean stage. From http://en.wikipedia.org/wiki/Myxobolus_cerebralis

* * * * * Spinal cord of a rainbow trout with whirling disease (Aquatic Slide of the Quarter, circulated by AAHL Fish Diseases laboratory) . The cord appears interrupted due to lateral flexion. M. cerebralis is a parasite of cartilage, affecting growth & bone formation. Several sites of infection are marked (*). Affected spinal processes are starting to impinge on the spinal cord (arrows), and more marked parasite invasion is present in the lower section.

Area 2: * Multi-cellular pre-spore stages of the parasite. After the triactinomyxons stage enters the fish (facilitated by firing of the polar filaments within the polar capsules to gain attachment), the sporoplasm enters epithelial cells (typically of gut) and multiplies to produce amoeboid stages which migrate and find cartilages. * * * Infected cartilage sites marked (*). Note disruption of bone formation, with fibrous reaction & multinucleate giant cells (green arrows) – presumably osteoclasts as they are in an area of bone disruption, not directly associated with the parasites. * Poorly decalcified bone * Normal cartilage

Another section of the same section, showing some head cartilage, near the pharynx. Note the heavily infected cartilage (between arrows), and the rupture through malformed and resorbed bone (black arrow). The cartilage cells (left) near the infected area are degenerating. The parasites in this area are a mixture of the multi-cellular stage as seen above, and the more consolidated ovate shapes of developing spores (arrows). M. cerebralismyxospores (which are infective to the Tubifextubifexoligochaetes), are formed from six cells: two forming the polar capsules, two merge to form a binuclear sporoplasm, and two form protective valves. More examples. Another view (X 100 objective), showing well developed spores with pale refractile capsules, photographed at several angles.

ACUTE LESION RESIDUAL HEALED LESIONS Example 10: Reminder. Fin and tail fin loss is also a common bone deformity, usually the result of flavobacterial infection. This can also result from chemical injury such as low pH, especially in very small fish.

Tail fin from an ~800g Atlantic salmon from a group with ongoing skin lesions of at least 2 weeks duration, related to fluctuating temperatures and high algal load. Tenacibaculummaritimum (formerly Flexibactermaritimus) was amoung the bacteria present. In this section the epithelium is largely intact, except for the area marked, but deep congestion & / or haemhorrhage is extensive. Note that fin bones are fragmenting. … and osteoclasts (multinucleate syncytia associated with focal bone erosion) are abundant. Thus the erosion of fin bones that occurs with “fin-rot” and “tail-rot”, is not just due to the erosive and devitalizing effect of the necrotising surface bacteria, but is partly mediated by osteoclastic activity following the stimulus of deep inflammation.

Example 11: Idiopathic hyperostosis. Hyperostosis has been recorded in about 80 species across at least 20 families, at particular sites or with particular patterns that are fairly consistent within a species. A well known example is the hump on the head of an adult Snapper. The cause (s) is unknown: though pollution and disease have been suspected the evidence suggests these have a genetic cause (McGrouther, 2013). The hyperostoses in the leatherjacket (below, sites arrowed), are typical of this species. They are not regarded as affecting eating quality. (It is less clear if the more anterior swellings are the same process of post-fracture, but the gross disruption is a harvest artifact.)

Example 12: Osteoma in a triploid Rainbow trout (1990). True osteomas and osteo-sarcomas are rare. This is the only DPIW laboratory example of this lesion in salmonids, and one of only two in total.

Osteoma in a triploid Rainbow trout continued.Section from this trout tumor. An area from the margin of the tumor, showing bone growth within a highly vascular fibrous tissue stroma. Bone spicules (probably both osteoblastic and osteoclasticactivty), and areas of imperfectly decalcified bone (*) An area from the margin of the tumor, showing bone growth within a highly vascular fibrous tissue stroma. The cellular nature of this bone is still obvious. Many of the large activated marginal cells appear to be mononuclear osteoclast. Remember both multinuclear osteoclasts (shown remodelling scales in Part 10A) and mononuclear osteoclasts are found in fish with cellular bones, with mononuclear forms the typical osteoclast in those with acelluarl bones. *

Same section, different area of tumor. Areas of necrosis are also present within the tumor.

Example 12b: This goldfish tumour was classed as an osteoma. It was one of several sub-cutaneous tumors in this fish, one with the appearance of a lipoma, another resembling a fibroma at the margins and large pale cells plus collagen deeper in the nodule.

Example 12b cont: Detail of the osteoma-like growth, showing areas of dense bone which in the interior of the lesion appear as bone spicules within a fat matrix. Either a fibrovascular or lipid support tissue is usually associated with true osteomas and osteosarcomas in fish, as they are developing in bones without either a medullary cavity or Haversian blood supply. The lesion is more cellular at the outer margins, which appear fibrous in some areas… With foci of active growing cartilage, both on the surface and deep within the lesion (arrow). Given the relationship of fish bone tumours blood supply and support tissues, it is possible that these tumours are all expressions of the same neoplastic process.

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.

Prepared by Judith Handlinger