C O N F E R E N C E 25 17 May 2017

Joint Pathology Center

Veterinary Pathology Services

WEDNESDAY SLIDE CONFERENCE 2016-2017

C o n f e r e n c e 25 17 May 2017

Jeffrey C. Wolf, DVM, Diplomate ACVP Chief Scientific Officer, Experimental Pathology Laboratories, Inc. Sterling, VA

month and then the groups were combined CASE I: WSC 1617 Conf 25 Case 1 (JPC and maintained together in one large group 4065818). in an 85-gal (~ 322 L) tank. At 13 months of age, eight fish from this group were Signalment: 13-month-old male zebrafish selected at random for routine health (Danio rerio). assessment. These fish were euthanized by immersion in a solution of tricaine History: This fish was from a breeding methanesulfonate (MS-222) and then the colony of zebrafish. Offspring produced carcasses were fixed in toto in 10% neutral- from the breeding colony were either used in buffered formalin. The fixed carcasses were genomics research or were used to replace then submitted for histopathologic aged members of the breeding colony. examination. Because the breeding colony had been maintained as a “closed colony” for several The submitted slide contains whole-body years, there was concern about inbreeding. transverse sections from one of the eight Therefore, a group of adult zebrafish was fish. purchased from a commercial vendor to be used to breed with the established colony This fish was part of a research project and thus provide some genetic diversity. conducted under an IACUC approved protocol in compliance with the Animal After an uneventful 90-day quarantine Welfare Act, PHS Policy, and other federal period, one of the female fish from the statutes and regulations relating to animals recently-purchased group was bred with a and experiments involving animals. The male fish from the long-established colony. facility where this research was conducted is The resultant offspring were raised in accredited by the Association for several small groups up to the age of one Assessment and Accreditation of Laboratory

containing multiple basophilic nuclei with poorly-defined borders; and 3) 25-40 µm cyst-like structures containing multiple approximately 3 X 5 µm oval refractile spores with basophilic to clear cytoplasm. One or more spores are also occasionally present with macrophages.

Other than autolysis, there are no significant changes in other organs.

Contributor’s Morphologic Diagnosis: Skeletal muscle; multifocal to coalescing myodegeneration, moderate, with necrosis, lymphohistiocytic inflammation and Skeletal muscle, zebrafish. Four transverse sections of a numerous intracytoplasmic protozoa-like zebrafish are submitted for examination. At subgross organisms at various stages of development inspection, there is mild hypercellularity of the (including spores). musculature of the abdominal wall in multiple sections. (HE, 5X). Contributor’s Comment: The lesions in Animal Care, International and adheres to the muscles of this fish were unexpected principles stated in the Guide for the Care findings. These had not been seen and Use of Laboratory Animals, National previously in this fish colony and a review Research Council, 2011. of the available literature at that time did not reveal descriptions of similar lesions in Gross Pathology: NA zebrafish.

Laboratory results: NA Several special stains were done in an attempt to better characterize the organisms. Histopathologic Description: There are These stains revealed that the spores often four transverse sections of intact whole body had annular bands and a PAS-positive polar at different levels on this slide. Within two granule (Figure 6). The more mature spores of these sections, there are multifocal to were Gram-positive but immature spores coalescing degeneration and necrosis of were Gram-variable (Gram-negative or did not stain with Gram’s stain). These findings skeletal muscle fibers accompanied by 2,3,6 infiltrates of low to moderate numbers of are characteristics of microsporidia. macrophages and fewer lymphocytes. Numerous protozoa-like micro-organisms in Further studies conducted by personnel at various stages of development are present Oregon State University and the Zebrafish within the cytoplasm of affected myocytes. International Resource Center demonstrated that the species of microsporidia in this The different developmental stages of the 7 organisms include: 1) 5-15 µm diameter zebrafish is Pleistophora hyphessobryconis. round to oval basophilic uni-nucleate P. hyphessobryconis is a common pathogen of neon tetras (Paracheirodon innesi ) and organisms with an eosinophilic refractile 5,6 wall; 2) 15-30 µm round cyst-like structures other species of ornamental tropical fish. with an eosinophilc refractile wall and P. hyphessobryconis is so common in neon

Skeletal muscle, zebrafish. Left: Higher magnification of affected skeletal muscle demonstrates the presence of uninucleate sporoblasts as well as larger sporonts containing numerous mature spores in affected myocytes. Degenerate myocytes are fragmented and infiltrated by numerous histiocytes, which also expanded the perimysium. Right: Mature microsporidian spores exhibit birefringence.(HE. 256X) (Photo courtesy of: US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011)

tetras that the disease it causes is simply spp. (which at first are uninucleate and called “neon tetra disease”.5,6 Although eventually become multinucleate) are some older reports state that P. surrounded by a membrane derived from the hyphessobryconis has been seen in zebrafish host cell and are said to reside within a in the commercial pet trade, this case was parasitophorous vacuole.2,5 The meronts of the first time it was recognized in a Pleistophora spp. mature into sporogonial laboratory research colony.7 Subsequent to plasomodia that undergo fission to produce the discovery of this index case, other cases sporoblasts which ultimately develop into of P. hyphessobryconis infection in spores; multiple spores are contained within zebrafish have been seen in two other each thick-walled sporophorous vesicle.2,5 research colonies.7 Fish typically become infected by P. For many years, microsporidia were hyphessobryconis by ingesting spores taxonomically classified as protozoa. released into the environment from necrotic However, the results of more recent studies muscle fibers of other infected fish or by have caused them to be re-classified as scavenging dead, infected fish. It is not fungi.2,4 Microsporidial spores are the known how the fish of this report was infectious stage and each spore contains a exposed to P. hyphessobryconis spores. coiled structure called a polar filament or This animal had been raised in a laboratory polar tube.1 When a spore enters a suitable setting without any exposure to neon tetras host (usually by ingestion of the spore), the or other fish species. The water used to polar filament uncoils, emerges from the house this fish colony is a mixture of deep- spore, and penetrates a host cell. The well water and municipal water that has sporoplasm is then injected through the undergone reverse-osmosis filtration; thus, it polar filament into the host cell.2 The is highly unlikely that this water was the parasite proliferates asexually inside the host source of infection. The food used to feed cell, first through merogony and then the fish is a commercial flake-food that sporogony.3,6 The meronts of Pleistophora contains ground fish meal. However, the

processing procedures used to produce this to fry-raising tanks, and this bleaching food should have inactivated any micro- process should have inactivated any microsporidia spores that were accidentally sporidia spores present in the water. It is present. also worth noting that the affected fish was euthanized 13 months after conception and The mother of this fish had been purchased this would be a long time to harbor a from a commercial vendor and thus may subclinical infection. have been exposed to infected fish before her arrival at the research facility. Although When this infected fish was detected, all of the mother fish was clinically normal, it is the fish that had been purchased from the possible that she was harboring a low-grade commercial vendor had been removed from infection and thus may have shed spores into the colony months previously. Thus, it was the water during breeding. However, the not possible to examine them to determine if embryos produced from this breeding were they had been the source of infection. treated with bleach before being transferred

Skeletal muscle, zebrafish. Color plate showing various special stains. Top left: Spores stain strongly with Gram stains. (Lilly Twort, 400X). Top right: A Giemsa stain demonstrates annular bands (arrows). (Giemsa, 200X). Bottom left: A periodic Schiff stain demonstrates the polar granule. (PAS, 400X). Bottom right: A Luna mast cell stain is also an excellent choice for highlighting spores. (Luna, 100X) (Photo courtesy of: US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011)

After this fish was found to be infected, the mycobacteriosis and microsporidiosis.7,8 As remaining 135 fish in its sibling co-hort mentioned by the contributor, based on were euthanized and examined grossly (98 phylogenetic analysis and the presence of fish) and/or histologically (37); none of chitin within the spore, microsporidia are these were found to be infected and there classified as fungal-like organisms rather have not been any other cases in this colony than protozoans, as previously thought. since then. Microsporidia are a large group of obligate- intracellular eukaryotic parasites that infect Note: Opinions, interpretations, conclusions, a wide range of animal species.1,3 Their and recommendations are those of the author relatively simple life cycle consists of two and are not necessarily endorsed by the U.S. developmental stages: merogony and Army. sporogony.1,2,3,5,7,8 After infection, meronts multiply within the host cell, skeletal muscle JPC Diagnosis: Skeletal muscle: Myocyte in this case, eventually forming thick-shelled deneneration and necrosis, multifocal, mild spores. Spores are released from the with numerous intrasarcoplasmic meronts, ruptured host cell and reach the environment sporoblasts, and spores with histiocytic through various bodily secretions. The inflammation, zebrafish, Danio rerio. infectious spores have a thick, chitinous shell and are resistant to environmental Conference Comment: Zebrafish are of stress; thus allowing the infective spores to increasing importance as a research model remain viable in the aquatic environment for for the study of infectious diseases, a prolonged period of time. As mentioned by developmental biology, cancer, and the contributor, infective spores can also be toxicology. Their small size, ease of ingested through predation.7,8 husbandry, and ex-vivo transparent embryonic development make them a highly The most commonly identified micro- sought after laboratory species with a sporidian parasite affecting zebrafish is burgeoning transgenic and specific pathogen Pseudoloma neurophila.7,8 As is implied by free industry to support the increase in the name, P. neurophila have tropism for the laboratory use.8 This case represents central and peripheral nervous system and is infection by a microsporidian organism that associated with encephalomyelitis and may easily spread within a colony and could polyneuritis. Small groups and individual potentially confound experimental results; spores can be seen in a variety of extraneural thus making this and other zebrafish organs, including smooth and skeletal diseases of particular concern for muscle. Spores released from peripheral investigators and laboratory animal nerves and skeletal muscle typically cause pathologists. Conference participants severe inflammation.7,8 In contrast, discussed the importance of proper animal Pleistophora hyphessobryconis primarily acquisition, good husbandry, routine disease develops within skeletal muscle with and pathogen monitoring, and a robust multiple developmental stages (meronts, sentinel animal program as the cornerstones sporoblasts, and spores) present within the of laboratory animal disease prevention in sarcoplasm. The conference moderator any species. noted that the two Microsporidia species can be easily distinguished histologically due to The most commonly encountered infectious the presence of P.hyphessobryconis’ intra- diseases of laboratory zebrafish include sarcoplasmic developmental stages and

thick-walled sporophorous vacuoles laden 3. Gardiner CH, Fayer R, Dubey JP. An with spores.7,8 Additionally, spores of P. Atlas of Protozoan Parasites in hyphessobryconis are associated with only a Animal Tissues. 2nd ed. Washington, mild inflammatory response, as seen in this DC: Armed Forces Institute of case. In the areas of mild histiocytic Pathology, 1998. inflammation in the skeletal muscle of the 4. Lee SC, Corradi N, Byrnes EJ 3rd, et abdominal wall, birefringent spores are al. Microsporidia evolved from present within phagocytes. In photographs ancestral sexual fungi. Curr Biol. provided by the contributor, wet-mount 2008;18(21):1675-9. preparations of skeletal muscle from 5. Li K, Chang O, Wang F, Liu C, infected fish show individual spores as well Liang H, Wu S. Ultrastructure, as sporophorous vacuoles containing spores development, and molecular with a prominent posterior vacuole and phylogeny of Pleistophora coiled polar filament, typical of hyphessobryconis, a broad host Microsporidia spp.1,7,8 Another micro- microsporidian parasite of Puntius sporidium parasite of veterinary importance tetrazona. Parasitol Res. is Encephalitozoon cuniculi, which causes 2012;111:1715–1724. torticollis and phaecoclastic uveitis in the 6. Post G. Textbook of Fish Health. 2nd highly susceptible dwarf rabbit.1 Readers are ed. Neptune City, NJ: T.F.H encouraged to review WSC 2015 Publications, Inc., 1987. Conference 12 Case 4 for an outstanding 7. Sanders JL, Lawrence C, Nichols example of phaecoclastic uveitis caused by DK, et al. Pleistophora E. cuniculi in a double-maned lionhead hyphessobryconis (Microsporidia) rabbit. infecting zebrafish Danio rerio in research facilities. Dis Aquat Org. Contributing Institution: 2010;91:47-56. US Army Medical Research Institute of 8. Sanders JL, Watral V, Kent ML. Infectious Diseases Microsporidiosis in zebrafish Pathology Division research facilities. ILAR J. 2012; Fort Detrick, MD 53(2):106-113. http://www.usamriid.army.mil/

References: CASE II: 15-3213 (JPC 4084010). 1. Cantile C, Youssef S. Nervous System. In: Maxie MG, ed. Jubb, Signalment: Adult female Northern puffer Kennedy, and Palmer's Pathology of fish (Sphoeroides maculatus). Domestic Animals. 6th ed. Vol 1. St. Louis, MO: Elsevier; History: This fish was presented to the 2016:385. CVMDL for euthanasia and necropsy after a 2. Curry A. Microsporidiosis. In: Cox one-week history of anorexia and labored FEG, Wakelin D, Gillespie SH, breathing. Reddish discoloration had been Despommler DD, ed. Topley & noted around multiple fins. Twelve Wilson’s Microbiology & Microbial individuals in two exhibits from this private Infections Parasitology. 10th ed. aquatic education center exhibited the same London, UK: Hodder Arnold, 2005: clinical signs, and several fish from affected 529-535. tanks had died over a one-month period. The

brown structures ranging in size from approximately 50 to 200 microns in diameter (presumptive trophonts of Amyloodinium ocellatum). Often smaller round forms were seen in organized aggregates of two to 16 organisms (dividing forms; tomonts).

Histopathologic Description: Gill: There is generalized mild to moderate hypertrophy and hyperplasia of lamellar epithelial cells Gill, pufferfish. Wet mount of gill clip gill reveals tomonts that often results in fusion along the lengths and trophonts (presumptive trophonts of Amyloodinium of some lamellae or at the lamellar tips ocellatum). (Photo courtesy of Department of (synechiae). Pseudocystic spaces formed by Pathobiology and Veterinary Science, Connecticut synechiae frequently contain sloughed Veterinary Medical Diagnostic Laboratory, Connecticut cellular debris, macrophages, numerous rod- http://www.pathobiology.uconn.edu/ shaped bacteria and occasional dinoflagellate organisms (consistent with tank was treated with copper and Amyloodinium sp.) in varying stages of metronidazole. Slight improvement was development. Present along the lamellar noted after the first treatment, but clinical surfaces are dinoflagellate trophonts that are signs then worsened with lethargy and lack circular to ovoid, range in size from 25 to of appetite. Additional treatment with 100 microns in diameter, and have a single, increased iodine and copper had no effect. round, deeply basophilic nucleus and wispy The submitted fish was euthanized with an eosinophilic cytoplasm containing several 3- overdose of MS-222 and necropsied 5 micron diameter, circular, brightly immediately. eosinophilic bodies and birefringent granules and spicules under polarized light. Gross Pathology: The fish was in poor Tomonts are occasionally trapped in body condition with scant coelomic adipose pseudocysts at varying stages of subdivision stores. External examination revealed slight into 2-20 dinospores, which have a thin reddening of the dorsal and pectoral fins that refractile and birefringent wall and are round was more severe at the proximal aspect, and and roughly 20-30 microns in diameter with the pectoral fins had some indistinct, cloudy granular eosinophilic cytoplasm and one to stippling. A skin scrape of the body was two irregularly shaped, stippled, deeply negative. The gills had disseminated, faint basophilic masses of chromatin. Lamellae dark-red stippling. The liver was diffusely are variably thickened by moderate to yellowish-tan and floated when placed in marked congestion, occasional hemorrhage formalin. The gallbladder was moderately and low to moderate numbers of dilated with thin, green, watery bile. The inflammatory cells consisting predominantly gastrointestinal tract was empty except for a of macrophages with fewer lymphocytes and small amount of mucus. rare granulocytes. There are a few, scattered areas of lamellar necrosis. The cytoplasm of Laboratory results: Cytology: Wet mount a few lamellar epithelial cells is markedly of the left pectoral fin and gill revealed few expanded by an intracytoplasmic, discrete, and numerous, respectively, round to ovoid,

round, finely granular basophilic inclusion This organism is considered an important (bacterial inclusion of epitheliocystis). parasite in aquarium fish and in tropical and subtropical brackish and marine fish culture, and it can infect both elasmobranch and teleost fish.5 Natural epidemics have also been documented.3 Amyloodinium ocellatum prefers warm water from 17 to 30 degrees Celsius (63-86 F) with potentially greater virulence in high Gill, pufferfish. There are multifocal synechia of secondary lamellae with formation of pseudocysts, as temperatures.2 well as secondary lamellae. Pseudocysts occasionally contain variable amounts of cellular debris and Isolates vary in multiple trophonts and tomonts of Amyloodinium. salinity tolerance with ranges from 3 to 45 ppt.5 Certain species of Contributor’s Morphologic Diagnosis: fish are resistant to infection; these fishes Gill: severe diffuse, proliferative and typically produce a thick mucus, possibly histiocytic branchitis with lamellar fusion, preventing attachment of trophonts, or synechiae, and intralesional dinoflagellate tolerate low oxygen levels.5 organisms, compatible with Amyloodinium ocellatum, gram-negative bacteria, and rare The life cycle of A. ocellatum is direct and epitheliocystis inclusions. triphasic. The parasitic, feeding stage, or trophont, attaches to the host’s epithelium by a root-like structured called a rhizoid.5 Contributor’s Comment: Although there After a period of feeding on the host, the were several etiologic agents seen trophont detaches from the host, retracts its microscopically in sections of gill that could rhizoid and becomes the encysted, dividing have contributed to the extensive pro- tomont stage in the substrate.5 The final liferative branchitis, the primary pathogen is stage is the free-swimming, infectious believed to be a heavy infestation with the dinospore that is released from the dividing obligate ectoparasitic marine dinoflagellate, tomont (up to 256 dinospores can come Amyloodinium ocellatum, the causative from a single tomont) and has flagella to agent of marine velvet disease or oodiniosis.

consequence of hypoxia, osmoregulatory imbalance and secondary bacterial infections.5 There are several modalities of treatment available, most of which only target the dinospore stage, which can make eradication of parasites difficult.5

Although not considered the primary disease in this case, epitheliocystis was also noted in this fish. Characterized histologically by large, granular, basophilic, intracytoplasmic, bacterial inclusions within enlarged branchial lamellar epithelial cells, this type Gill, pufferfish. Amyloodinium trophont with of infection is caused by a group of obligate, stomatopode . (HE, 400X). intracellular, gram-negative, chlamydia-like organisms.5 Recent advances in molecular facilitate swimming to find a suitable host.5 techniques have identified various Amyloodinium ocellatum causes severe physical damage to the host cells through attachment of the trophont to epithelium, with the gill typically being the primary site of infestation.5,6 In cases with heavy parasite burdens, the skin and eyes can also be infected, sometimes producing the dusty appearance of the skin that accounts for the name “velvet disease.”5 In some reports infestation of larval fish affected only the skin rather than the gill.6 The gill was the tissue most severely affected in this puffer fish, although a few of the organisms were seen on a wet mount of the fin; cutaneous Gill, pufferfish. Amyloodinium tomont with four changes were mild in histologic sections. divisions. There is a synechia between adjacent secondary lamellae. (HE, 400X). Trophonts may also be seen in the pseudobranch, branchial cavity, nasal passages, and gastrointestinal tract (if epitheliocystis agents as belonging to the swallowed).5 order Chlamydiales, the first of which was Candidatus Piscichlamydia salmonis in the Microscopic lesions of severely affected gill Atlantic salmon.1,7 Infections of gills, include epithelial hyperplasia, often with pseudobranch, and rarely the skin by these lamellar fusion and distortion, mild organisms have been described in many inflammatory infiltrates, hemorrhage and freshwater and marine species of fish.7 epithelial degeneration and necrosis,3,5,6 as Pathogenicity is dependent upon the host seen in this case. The feeding activity and response and the chlamydiales bacterium, the detachment of large numbers of and microscopic lesions can range from organisms can damage epithelium, and none to severe epithelial hyperplasia with potentially result in mortality as a associated inflammation.7

JPC Diagnoses: 1. Gill: Branchitis, proliferative and necrotizing, multifocal, moderate with marked lamellar adhesions (synechiae) and fusion, and numerous dinoflagellate ectoparasites, Northern puffer fish, Sphoeroides maculatus. 2. Gill, lamellar epithelial cells: Chlamydial inclusions, multiple.

Conference Comment: Amylodinuym ocellatum is one of the most important pathogenic parasites of both marine and Gill, pufferfish. Bifringence of multiple life stages of estuarine fish. It is one of the few organisms Amyloodinium. (HE, 100X) that can infect the gills and skin of both teleost fish (bony ray-finned fish) and increased respiratory rate (indicated by rapid elasmobranchs (cartilaginous fish, ex. movement of the gill opercula) and sharks).2,5 This dinoflagellate parasite has gathering at the surface or in other areas of the capacity for rapid reproduction and increased oxygen concentration. The skin is severe infections, such as in this case, can also infected and fish can develop white or cause devastating disease and high mortality brown “velvety” appearance.2 This case also in a wide range of fish species.2 As nicely contains excellent examples of summarized by the contributor, the life cycle epitheliocystis with large inclusions is simple and requires no intermediate hosts. composed of colonies of Chlamydia-like The parasite has three different organisms within the cytoplasm of lamellar developmental stages. The trophont stage is epithelial cells. Epitheliocystis is often the adult stage that feeds directly on the host considered an incidental finding, and most and attaches via the rhizoid; the tomont conference participants agreed that its stage is detached from the fish and divides presence in this case is unrelated to the to the third dinospore stage. Dinospores are significant gill pathology caused by the free swimming infectious flagellated Amylodinuym ocellatum.7 phase.2,5 The simple life cycle and free Prior to the conference, the moderator swimming infectious dinospore stage briefly reviewed the normal anatomy and contribute to severe epizootics in both wild histology of the gill. The gill arch is a series and cultured fish, especially when crowded. of bony or cartilaginous curved structures A. ocellatum is a major concern to the that support double rows of paired filaments, aquaculture industry because outbreaks have also called the primary lamellae. Each an acute onset, spread rapidly, and have a filament is composed of numerous high mortality. There are many nice perpendicularly-arranged secondary examples of all three life stages in this case, lamellae.4 The gill arch is covered by including several trophonts with prominent epidermis and at the origin of the primary rhizoids.2,5 This case is an excellent example lamellae the epidermis is thicker and of the gill as the primary site of infection for contains many mucous cells and subjacent this parasite. Predominant antemortem lymphoid tissue. The primary lamellae are clinical signs will most often be disturbances covered by a mucoid epidermis which

in the respiratory system, including contains a pale-staining, eosinophilic, salt-

lamellar adhesions are present in this case.

Multifocally, within the secondary lamellae of this case, there are dilated capillaries that contain organizing fibrin thrombi, in- terpreted by conference participants as examples of telangiectasia. The moderator noted that this vascular change can be a perimortem artifact secondary to capture of the fish or an antemortem change secondary to the disease process. Distinguishing between peri- and antemortem telangiectasis Gill, pufferfish. Occasional epithelial cells contain large is often difficult, even for those experienced bacterial inclusions characteristic of Epitheliocystis. in gill histopathology; however, in this case, (HE, 400X) the presence of organizing fibrin thrombi is characteristic of antemortem telangiectasia secreting chloride cells that function in ionic rather than a perimortem capture artifact. transport and detoxification. The surface of the secondary lamellae consists of a single Contributing Institution: layer of interdigitating squamous epithelial Department of Pathobiology and Veterinary cells supported by pillar cells and lamellar Science blood channels.4 The surface of the lamellar Connecticut Veterinary Medical Diagnostic epithelium has numerous microvilli which Laboratory serve as a substrate for cuticular mucus and College of Agriculture, Health and Natural aid in gas exchange and defense against Resources infection and trauma. Thus, gills are University of Connecticut important for gas exchange, ionic balance, http://www.pathobiology.uconn.edu/ and the excretion of the nitrogenous wastes. As a result, damage or infection of the gill References: often results in serious systemic con- 1. Draghi A, Popov VL, Kahl MM, et al. sequences to the fish.4 Characterization of “Candidatus Pischichlamydia salmonis” (Order Conference participants also discussed the Chlamydiales), a Chlamydia-like difference between lamellar fusion and bacterium associated with epitheliocystis lamellar adhesion, both prominent features in farmed Atlantic Salmon (Salmo in this case. Lamellar fusion is most often salar). J Clin Microbiol. 2004; associated with inflammation and is a 42(11):5286-5297. consequence of marked epithelial 2. Francis-Floyd R, Floyd MR. proliferation. In contrast, lamellar adhesions Amyloodinium ocellatum, an important (also known as lamellar clubbing or parasite of cultured marine fish. SRAC. synechiae) occur when there is adherence of 2011; 4705:1-12. two or more secondary lamellae forming a 3. Kuperman, BI, Matey VE. Massive pseudocyst, usually in the absence of infestation by Amyloodinium ocellatum epithelial proliferation or (Dinoflagellida) in a highly saline lake, inflammation. Both lamellar fusion and Salton Sea, California, USA. Dis Aquat Org. 1999; 39:65-73.

4. Mumford S, Heidel J, Smith C, Morrison nodular surface that bulged into the J, MacConnell B, Blazer V. Fish coelomic cavity. Histology and Histopathology. US Fish and Wildlife Service, National Conservation Training Center. http://nctc.fws.gov/resources/cou rse-resources/fish- histology/index.html. Accessed May 25, 2017. 5. Noga EJ. Problem 27 Marine Velvet Disease (Amyloodiniosis, Marine Oodinium Disease, Oodiniosis). IN Fish Disease: Diagnosis and Treatment, 2nd ed, Ames, IA: Wiley-Blackwell; 2010:143-147. 6. Paperna I, Steinitz AH. Amyloodinium ocellatum (Brown, 1931) (Dinoflagellida) infestations in cultured marine fish at Eilat, Red Sea: epizootiology and pathology. J Fish Dis. 1980; 3:363-372. 7. Stride MC, Polkinghorne A, Nowak BF. Chlamydial infections of fish: Diverse pathogens and emerging causes of disease in aquaculture species. Vet Microbiol. 2014; 171:258–266.

CASE III: A15-25281 (JPC 4084652).

Signalment: Adult male long snout Kidney, seahorse: Renal parenchyma is expanded and seahorse (Hippocampus reidi). architecture is diffusely effaced by granulomatous inflammation. (HE, 5X) History: This animal was one of 20 seahorses that experienced an epizootic of ulcerative dermatitis shortly after arrival into Laboratory results: Cultures of kidney and quarantine facilities of a public aquarium. liver yielded a Nocardia sp. PCR The 15.4 cm, snout to tail tip, the male amplification of the 16S rRNA gene became moribund and was euthanized 14 produced a 524 bp sequence with 100% days after shipment. similarity to Nocardia nova (GenBank KP025810.1). Mycobacterial and fungal Gross Pathology: Unlike other animals cultures were negative. from the group, there was no evidence of dermatitis. The caudal third of the kidney Histopathologic Description: was enlarged and pale, with an irregularly Approximately 75% of the normal renal

12 parenchyma is severely altered by large of elongate, slender, branching filaments. numbers of variably-sized, multifocal to coalescing, haphazardly organized sheets Contributor’s Morphologic Diagnosis: and nodular collections of macrophages, Kidney: Granulomatous nephritis, multifocal with scattered lymphocytes and variable to coalescing, chronic, severe, with multiple degrees of fibrous encapsulation. Similar granulomas, multiple foci of necrosis and streams of macrophages, fibrous tissue and modified acid-fast branching bacterial scattered fibroblasts extensively displace filaments. and isolate normal hematopoietic tissue and groups of tubules. Discrete, well-organized Contributor’s Comment: Microscopic granulomas are uncommon. Also present are findings are typical of piscine nocardiosis. multiple, often large, encapsulated foci with Changes vary between groups of slides, central areas of hemorrhage, necrotic particularly the extent of extrarenal cellular debris and individualized macro- involvement, which includes connective phages. On H&E and Ziehl-Neelsen stained tissues, muscle, intestine and the coelomic sections, rod-shaped and filamentous cavity in some sections. Additional findings bacteria can be poorly visualized, in some sections include renal calculi, particularly in areas of necrosis. With Lillie- intestinal nematodes, and parasitic Twort stains, bacteria appeared as Gram- granulomas. Smaller lesions were widely positive, coccobacillary forms and beaded distributed in the gills, skeletal muscle, one filaments. Fite’s stain reveals large numbers ocular choroid, and dermal and hypodermal connective tissues. Similar bacteria were present in all lesions. Three additional animals from the group were examined microscopically, but none showed evidence of infection.

Nocardiosis in fish has been summarized in multiple sources.3,7 Nocardia asteroides was Kidney, seahorse: Higher magnification of the kidney, demonstrating poorly demarcated, coalescing first reported granulomas with a necrotic core. (HE, 100X) from neon tetra in 1962 and has

demonstrated experimentally by injection, dermal abrasion, immersion, feeding and cohabitation.6 Losses of 15-17% from N. seriolae have been reported in cultured sea bass and yellow croaker, respectively.2,9 While microscopic lesions are frequently described, many reports only include bacterial identification to the genus level.5 Identification of Nocardia nova in this case suggests greater species diversity of Nocardia could be involved in fish disease.1

Kidney, seahorse: Higher magnification of affected JPC Diagnosis: Kidney: Nephritis, necro- kidney with marked loss of tubules. Remaining tubules granulomatous, diffuse, severe, with are ectatic and contain numerous sloughed epithelial cells numerous extracellular filamentous bacilli, and cellular debris. (HE, 400X) long snout seahorse, Hippocampus reidi.

caused epizootics primarily in freshwater fish. N. seriolae is a major pathogen of marine fish worldwide, particularly in Asian mariculture.2,9 Nocardia salmonicida has received little attention since its first isolation from sockeye salmon.1 The systemic granulomatous disease is characterized by progressive lethargy and emaciation. Infections can be confused both clinically and grossly with those of mycobacteriosis, which is extremely common in captive seahorses. Gross lesions can include skin ulcers, muscle necrosis, and organomegaly, with small nodules in Kidney, seahorse: Necrotic core of one of the granulomas. (HE, 400X) parenchymal organs and the gills. Microscopically, nodules are variously Conference Comment: Nocardia spp. are described as granulomas and abscesses, ubiquitous, saprophytic, gram-positive, frequently with necrotic centers, peripheral higher order bacteria that are associated with macrophages, lymphocytes, possibly giant 7 both opportunistic and primary infections in cells, and variable fibrous encapsulation. a variety of terrestrial and aquatic species The filamentous branching bacteria, stain worldwide. Morphologically, the bacteria weakly and irregularly gram-positive, poorly are long, thin, beaded filaments with or not at all with the Ziehl-Neelsen acid-fast frequent right-angle branching resembling stain, and are best visualized with modified Chinese letters.8 They can be seen on acid-fast stains, such as Fite’s. standard H&E stained sections; however,

they are best visualized by the histochemical Although contaminated feed has been stains Gomori methenamine silver (GMS), suggested as a source of infection, the gram stains, and modified acid-fast stains, pathogenesis of natural disease is poorly such as Fite-Faraco, as demonstrated by the understood. Transmission has been

14 outstanding photographs provided by the infectious organism and examination of contributor.8 histologic sections of infected organs. Histologically, Mycobacteria sp. are non- Although generally thought to cause low filamentous, non-branching, gram-positive, mortality in fish, Nocardia spp. can induce strongly acid-fast bacteria and are easily severe chronic granulomatous systemic differentiated from Nocardia with the disease.1,7 In this case, the normal renal special histochemical stains previously architecture is almost completely effaced by mentioned.1,7,8 Additionally, virulent multifocal to coalescing necrotizing and Mycobacteria spp. are obligate intracellular granulomatous inflammation. Although pathogens that replicate within host

Lung, penguin: Special stain panel demonstrating numerous gram-positive and acid-fast bacilli within the necrotic cores of the granulomas. (Photo courtesy of: University of Georgia College of Veterinary Medicine, Department of Pathology, 501 DW Brooks Drive, Athens, GA 30602, http://www.vet.uga.edu/VPP) there is some slide variability, in several macrophages. In contrast, pathogenic examined tissue sections, inflammation Nocardia spp. are facultative intracellular extends into the adjacent coelomic cavity bacteria that have complex cell wall lipids and skeletal muscle. As mentioned by the that allow for resistance to phagocytosis by contributor, the primary differential host macrophages.7,8 In this case, the vast diagnosis for piscine nocardiosis includes majority of bacteria are extracellular. infection with the much more common Mycobacteria sp., which produces nearly In a small number of tissue sections, identical gross and histological lesions in conference participants noted a focal fish.7 Gross lesions include cachexia, granuloma within the intestinal wall ascites, dermal ulceration, multifocal centered on a degenerate larval cestode skeletal muscle necrosis, and small white characterized by a 2 um well-circumscribed granulomas in the eosinophilic tegument, a kidney, spleen, heart, and liver.7 As a result, lacy, fibrillar eosinophilic parenchymatous nocardiosis can be easily misdiagnosed as body cavity, scattered 5 um diameter, mycobacteriosis. Positive differentiation basophilic, calcareous corpuscles, and requires isolation and identification of the birefringent hooks in some sections.4 The

15 pathologic significance of the parasitic 6. Itano T, Kawakami H, Kono T, granuloma in the intestinal wall, in this case, Sakai M. Experimental induction of is unclear; however, it may indicate that this nocardiosis in yellowtail, Seriola seahorse was immunocompromised, quinqueradiata Temminck & allowing for the proliferation of various Schlegel by artificial challenge. J concurrent opportunistic pathogens. Fish Dis. 2006; 29:529–534. 7. Lewis S, Chinabut S. Contributing Institution: Mycobacteriosis and Nocardiosis. In: University of Georgia Woo PTK, Bruno DW eds, Fish College of Veterinary Medicine Diseases and Disorders, Volume 3: Department of Pathology Viral, Bacterial and Fungal Athens, GA Infections 2nd ed. CABI Publishing, http://www.vet.uga.edu/VPP Oxfordshire; 2011:397-423. 8. Mauldin E, Peters-Kennedy J. References: Integumentary system. In: Maxie MG, ed. Jubb, Kennedy, and 1. Brown-Elliott BA, Brown JM, Palmer’s Pathology of Domestic Conville PS, Wallace RJ. Clinical Animals. Vol 1. 6th ed. Philadelphia, and laboratory features of the PA:Elsevier; 2016:637-638. Nocardia spp. based on current 9. Wang G-L, Yuan S-P, Jin S. molecular taxonomy. Clin Microbiol Nocardiosis in large yellow croaker, Rev. 2006; 19:259-282. Larimichthys crocea (Richardson). J 2. Chen SC, Lee JL, Lai JC, Gu YW, Fish Dis. 2008; 28:339-345. Wang CT, Chang HY, Tsai KH. Nocardiosis in sea bass, Lateolabrax japonicus, in Taiwan. J Fish Dis. 2000; 23:299-307. CASE IV: R1 or R2 (JPC 4085971). 3. Cornwell ER, Cinelli MJ, McIntosh DM, Blank GS, Wooster GA, Signalment: Post-smolt, unknown gender, Groocock GH, Getchell RG, Bowser Atlantic salmon (Salmo salar). PR. Epizootic Nocardia infection in cultured weakfish, Cynoscion regalis History: This case was submitted as part of (Bloch and Schneider). J Fish Dis. a sample of five fish from a seawater tank in 2011; 34:567–571. which fish were presenting with loss of 4. Gardiner, CH, Poynton, SL. An Atlas appetite and lethargy. of Metazoan Parasites in Animal Tissues. Washington, D.C: Armed Gross Pathology: There was no Forces Institute of Pathology, digesta/ingesta in the gastrointestinal tract American Registry of Pathology, with small amounts of soft yellow material 1999:35–38. (fecal casts) in the distal intestine. 5. Elkesh A, Kantham KPL, Shinn AP, Crumlish M, Richards RH. Systemic Laboratory results: Positive for salmonid nocardiosis in a Mediterranean alphavirus (SAV) by RT-PCR population of cultured meagre, Argyrosomas regius Asso Histopathologic Description: Pyloric (Perciformes; Sciaenidae). J Fish caeca/pancreas: one section is examined Dis. 2013; 36:141-149. consisting of multiple cross sections of

cells. In the white muscle, there are scattered necrotic and degenerating myofibres, with swollen fragmented, eosinophilic sarcoplasm, central migration of nuclei, and sarcoplasmic infiltration by macrophages. Affected myofibres are surrounded by low numbers of lymphocytes and macrophages.

Contributor’s Morphologic Diagnoses: Exocrine pancreas: Severe, diffuse, pancreatic degeneration and loss

Viscera, Atlantic salmon. Left-right: Skeletal muscle, Heart, myocardium: Marked, multifocal to heart, and pyloric ceca and mesentery are presented for coalescing, cardiomyocyte degeneration and evaluation. (HE, 5X) necrosis, with endocardial hyperplasia and pyloric caeca and adjacent adipose tissue. hypertrophy, and mild to moderate, There is extensive loss of exocrine multifocal, lymphohistiocytic epicarditis pancreatic tissue. Multifocally, there are small to moderate numbers of mononuclear Skeletal muscle (red myofibres): Moderate inflammatory cells (lymphocytes and to marked, chronic, multifocal to coalescing, macrophages). Remaining exocrine necrotizing myositis epithelial cells have reduced numbers of zymogen granules, and the islets of Skeletal muscle (white myofibres): Mild to Langerhans remain intact. moderate, chronic, multifocal, necrotizing myositis Heart: There are degenerative changes of the cardiomyocytes in the spongy and compact layers of the ventricle, characterized by marked cytoplasmic hypereosinophilia, with variable vacuolation and occasional shrunken nuclei. In these areas, there are low to moderate numbers of lymphocytes and macrophages. Multifocally, there is increased cellularity, prominence and occasional karyomegaly of endocardial endothelium (hyperplasia). Multifocally, the epicardium is expanded by mild to moderate numbers of lymphocytes and macrophages. Skeletal muscle, lateral body wall, Atlantic salmon. Skeletal muscle: Multifocally, there is There is marked degeneration of the overlying red muscle marked loss of myofibres in the red muscle. (arrows). There is milder degeneration and atrophy of the exterior portion of the underlying white muscle. (HE, The majority of the remaining myofibres are 40X) shrunken with densely eosinophilic sarcoplasm, surrounded by moderate numbers of mononuclear inflammatory

Contributor’s Comment: The microscopic detected in freshwater systems, causing features of this case are consistent with sleeping disease in freshwater trout. Current previously published findings for pancreas research also suggests there may be disease (PD) in farmed Atlantic salmon, and differences between strains in the infection the histopathological diagnosis was dynamics7 as well as minor differences in confirmed by RT-PCR. PD is caused by prevalence and severity of the tissue salmonid alphavirus (SAV), which was first damage8. Naturally occurring outbreaks of described in farmed Atlantic salmon from PD in farmed Atlantic salmon have only Scotland in the mid-seventies.16 It has been reported in the seawater phase of subsequently been responsible for major production.15 To date, there has been no economic losses to the Atlantic salmon evidence of vertical transmission of the farming industries in Scotland, Ireland, and disease11 with horizontal transmission being Norway. At present, six closely related by far the most important means of subtypes have been identified for SAV4,5, spreading the virus1,19, with shedding of which differ in host specificity, geographical mucus and feces described as transmission location7,10 and aquatic environments (Table routes for SAV. 1). SAV-2 is the only subtype commonly

Table 1. Summary of Salmonid alphavirus subtypes, their geographical distribution and species susceptibility SAV Production Species Country Subtype Phase SAV-1 Atlantic salmon (Salmo salar) Seawater Ireland and Scotland Rainbow trout France, England, Scotland, SAV-2 Freshwater (Oncorhynchus mykiss) Spain, Croatia and Germany SAV-2 Atlantic salmon (Salmo salar) Seawater Scotland and Norway (Marine) Atlantic salmon (Salmo salar) SAV-3 Rainbow trout Seawater Norway (Oncorhynchus mykiss) SAV-4 Atlantic salmon (Salmo salar) Seawater Ireland and Scotland SAV-5 Atlantic salmon (Salmo salar) Seawater Scotland SAV-6 Atlantic salmon (Salmo salar) Seawater Ireland

Gross findings at postmortem examination The acute phase of the disease is relatively during the early stages of the disease may short-lived with rapid destruction of the include the absence of food in the gut and exocrine pancreatic tissue and a variable the presence of fecal casts in the hindgut.15 inflammatory response ranging from no Occasionally, petechial hemorrhages can be inflammation to moderate mononuclear cell detected over the surface of the pyloric infiltration and/or fibrosis of the periacinar tissue.15 In recovering fish, regeneration of exocrine tissues can take place as early as four weeks post infection. Acute heart lesions can be observed concurrently or slightly lagging behind the acute pancreatic changes and includes multifocal cardiomyocyte necrosis with affected cells having a Skeletal muscle, lateral body wall, Atlantic salmon. Higher magnification of red (left) and white shrunken, deeply muscle(right.) There is marked muscle atrophy and numerous degenerate and necrotic fibers. eosinophilic There is prominent hypertrophy of satellite nuclei and expansion of the perimysium with histiocytes cytoplasm and and lymphocytes. (HE, 100X) pyknotic nuclei. caeca and surrounding visceral fat. During Both the compact the chronic stages of the disease, non- and spongy layer of the ventricular feeding fish with low body condition and myocardium and the atrial myocardium can minimal internal body fat (runts) tend to be be affected to varying degrees, ranging from mild focal changes to involvement of the more common due to the failure of the 15 pancreas to recover20; these fish are more entire heart musculature. Hypertrophy of susceptible to parasitic and secondary myocardial nuclei, particularly at the bacterial infections, which may present junction of the spongy and compact concurrently. Apparently healthy fish may ventricular myocardium can be evident in also die suddenly due to cardiac and skeletal the recovery phase of the disease. Skeletal muscle damage and exhaustion; this muscle pathology tends to first appear 3–4 manifestation of the disease appears to be weeks after the appearance of pancreatic and more common when older grower fish are heart lesions, and fish sampled in late-phase infected during their second year at sea. disease may only have skeletal muscle lesions. The skeletal muscle lesions consist

19 of hyaline degeneration with swollen suggested to be associated with HSMI fragmented eosinophilic sarcoplasm, central infection17,21. migration of myocytic nuclei and subsequent invasion of the sarcoplasm by Cardiomyopathy syndrome (CMS) affects phagocytic macrophages.15 harvest-sized Atlantic salmon, 12–18 months after sea-transfer, and mainly affects The differential diagnoses for these cardiac the heart without involvement of skeletal lesions should at least include the two muscle. Histopathological changes include following diseases: necrosis and inflammation of the spongy layer of ventricle and atrium, epicarditis, and Heart and skeletal muscle inflammation infiltration of lymphocytes and macro- (HSMI) has been reported in farmed phages, with rupture of atrium or sinus Atlantic salmon in Norway, Scotland, and venosus visible macroscopically.3,18 A Chile. It typically affects fish 5 to 9 months totivirus (piscine myocarditis virus after sea-transfer. Mortality in affected cages (PMCV)) is proposed as the causative agent may be negligible but in some cases can for this syndrome14,18,19,21. Both HSMI and reach up to 20%. Histologically, HSMI CMS are not typically associated with any mainly targets cardiomyocytes and pancreatic pathology. myofibres of red skeletal muscle. Affected myocytes show signs of degeneration, Differential diagnosis for pancreatic necrosis including loss of striation, sarcoplasmic should include infectious pancreatic necrosis eosinophilia, vacuolation, centralized nuclei (IPN), a viral disease that primarily targets (in skeletal muscle), and karyorrhexis. In the pancreatic tissue and is caused by an HSMI inflammation is more prominent than aquatic birnavirus (known as IPN virus). myocyte necrosis in most cases.11,13,18 However, IPN does not typically cause Recently piscine reovirus (PRV) has been cardiac and skeletal muscle pathology.

Table 2. Comparison of histopathological lesions in farmed Atlantic salmon with Pancreas disease (PD), Heart Skeletal Muscle Inflammation (HSMI), Cardiomyopathy Syndrome (CMS) and Infectious Pancreatic necrosis (IPN) PD Lesions Sub- HSMI CMS IPN Acute Chronic Recovery acute Myocardial + + + - + + - necrosis Endocardial + + + + + + +/- proliferation

Cardiomyocyte - +/- + + + + - hypertrophy Epicarditis + + + + - Exocrine + + + +/- - - + acinar necrosis Myositis - - + + + -

is to recognize the complete absence of a normal structure in a tissue section, especially when there is little to no inflammation associated with that loss. Within the multiple cross sections of the pyloric cecae, in this case, conference participants astutely noted a near diffuse loss of exocrine pancreas with only small clusters of remaining exocrine pancreatic cells containing brightly eosinophilic zymogen granules surrounded by mild lymphocytic inflammation that extends into the peripancreatic fat. The islets of Heart, Atlantic salmon. At subgross magnification, there Langerhans are mostly unaffected. This is a cellular infiltrate within the epicardium and at the junction between the compact and spongy layers of the diffuse loss of exocrine pancreas with ventricular myocardium (arrows). (HE, 20X). relatively mild inflammation is typical for the chronic phase of this disease. As mentioned by the contributor, the acute JPC Diagnosis: 1. Pancreas: Loss, diffuse, phase is associated with a short-lived with severe with mild lymphocytic pancreatitis, rapid necrosis of the exocrine pancreatic Atlantic salmon, Salmo salar. tissue and an inflammatory response ranging 2. Heart, ventricle: Epicarditis and from no inflammation to moderate myocarditis, lymphocytic, diffuse, moderate mononuclear cell infiltration. The with multifocal mild myocardiocyte contributor provides an outstanding and necrosis. thorough review to SAV and includes 3. Skeletal muscle, red: Degeneration and helpful tables to rule out potential necrosis, multifocal to coalescing, severe differential diagnoses based on the lesions with histiocytic myositis. present in this case. 4. Skeletal muscle, white: Degeneration and necrosis, multifocal to coalescing, mild to moderate with histiocytic myositis.

Conference Comment: This excellent case demonstrates the prototypical constellation of lesions associated with chronic salmonid alphavirus (SAV) infection. Histological examination of infected fish typically reveals a near complete loss of exocrine pancreatic tissue, epicarditis and myocarditis centered on the ventricle, and skeletal muscle degeneration and necrosis of the white and red muscle.9,15 Each subtype of SAV listed in Table 1 produces similar Heart, Atlantic Salmon. Higher magnification morphologic changes and are unable to be demonstrating the lymphocytic infiltrate in the epicardium (left) and within the myocardium (center). distinguished histologically. One of the most (HE, 200X) difficult tasks for the novice histopathologist

Both pancreas disease (PD) of salmon, seen University of Edinburgh in this case, and sleeping disease (SD) of Easter Bush Campus rainbow trout is caused by related SAV Midlothian infection. This virus is in the genus United Kingdom Alphavirus and family Togaviridae, a group of important enveloped ssRNA viruses.9,15 References: Other members of the Togaviridae family include eastern, western, and Venezuelan 1. Bratland A, Nylund A. Studies on equine encephalitis viruses. Generally, this the possibility of vertical family of viruses is associated with transmission of Norwegian salmonid transmission by insects, usually mosquitoes. alphavirus in production of Atlantic An aquatic arthropod vector has not yet been salmon in Norway. J Aquat Anim identified for SAV; however, direct Health. 2009; 21:173–178. horizontal transmission has been well 2. Christie KE, Graham DA, documented.9,15 The virus can survive for McLoughlin MF, Villoing S, Todd extended periods of time in the water D, Knappskog D. Experimental outside of the host with a marked increase in infection of Atlantic salmon Salmo survival in sea water compared to fresh salar pre-smolts by IP injection with water. The virus can also survive for long new Irish and Norwegian salmonid periods within the fat of dead fish and alphavirus (SAV) isolates: a leaked fat droplets floating on the surface comparative study. Dis Aquat Organ. may contribute to long distance spread of 2007; 75:13–22. the virus. Vertical transmission has not been 3. Ferguson HW, Poppe TT, and shown to be a significant route of Speare DJ. Cardiomyopathy in infection.9,15 farmed Norwegian salmon. Dis Aquat Organ. 1990; 8:225–231. Contributing Institution: 4. Fringuelli E, Rowley HM, Wilson The Royal (Dick) School of Veterinary JC, Hunter R, Rodger H, Graham Studies DA. Phylogenetic analyses and molecular epidemiology of European salmonid alphaviruses (SAV) based on partial E2 and nsP3 gene nucleotide sequences. J Fish Dis. 2008; 31:811–23. 5. Graham DA, Fringuelli E, Wilson C, Rowley HM, Brown A, Rodger H, et al. Prospective longitudinal studies of salmonid alphavirus infections on two Atlantic salmon farms in Ireland; evidence for viral persistence. J Fish Dis. 2010; 33(2):123–35. 6. Graham DA, Fringuelli E, Rowley Mesentery and pyloric ceca, Atlantic salmon: The HM, Cockerill D, Cox DI, Turnbull mesentery is devoid of pancreatic tissue. Few pancreatic

islets remain. (HE, 30X) T, et al. Geographical distribution of salmonid alphavirus subtypes in marine farmed Atlantic salmon,

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