Signalment:  

Adult male long snout seahorse (Hippocampus reidi).This animal was one of 20 seahorses that experienced an epizootic of ulcerative dermatitis shortly after arrival into quarantine facilities of a public aquarium. The 15.4 cm, snout to tail tip, the male became moribund and was euthanized 14 days after shipment.

Gross Description:  

Unlike other animals from the group, there was no evidence of dermatitis. The caudal third of the kidney was enlarged and pale, with an irregularly nodular surface that bulged into the coelomic cavity.

Histopathologic Description:

Approximately 75% of the normal renal parenchyma is severely altered by large numbers of variably-sized, multifocal to coalescing, haphazardly organized sheets and nodular collections of macrophages, with scattered lymphocytes and variable degrees of fibrous encapsulation. Similar streams of macrophages, fibrous tissue and scattered fibroblasts extensively displace and isolate normal hematopoietic tissue and groups of tubules. Discrete, well-organized granulomas are uncommon. Also present are multiple, often large, encapsulated foci with central areas of hemorrhage, necrotic cellular debris and individualized macro-phages. On H&E and Ziehl-Neelsen stained sections, rod-shaped and filamentous bacteria can be poorly visualized, particularly in areas of necrosis. With Lillie-Twort stains, bacteria appeared as Gram-positive, coccobacillary forms and beaded filaments. Fite�s stain reveals large numbers of elongate, slender, branching filaments.

Morphologic Diagnosis:  

Kidney: Granulomatous nephritis, multifocal to coalescing, chronic, severe, with multiple granulomas, multiple foci of necrosis and modified acid-fast branching bacterial filaments.

Lab Results:  

Cultures of kidney and liver yielded a Nocardia sp. PCR amplification of the 16S rRNA gene produced a 524 bp sequence with 100% similarity to Nocardia nova (GenBank KP025810.1). Mycobacterial and fungal cultures were negative.

Condition:  

Nocardia asteroides

Contributor Comment:  

Microscopic findings are typical of piscine nocardiosis. Changes vary between groups of slides, particularly the extent of extrarenal involvement, which includes connective tissues, muscle, intestine and the coelomic cavity in some sections. Additional findings in some sections include renal calculi, intestinal nematodes, and parasitic granulomas. Smaller lesions were widely distributed in the gills, skeletal muscle, one 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 first reported from neon tetra in 1962 and has 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 parenchymal organs and the gills. Microscopically, nodules are variously described as granulomas and abscesses, frequently with necrotic centers, peripheral macrophages, lymphocytes, possibly giant cells, and variable fibrous encapsulation.⁷ The filamentous branching bacteria, stain weakly and irregularly gram-positive, poorly or not at all with the Ziehl-Neelsen acid-fast stain, and are best visualized with modified acid-fast stains, such as Fite�s.

Although contaminated feed has been suggested as a source of infection, the pathogenesis of natural disease is poorly understood. Transmission has been 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

JPC Diagnosis:  

Kidney: Nephritis, necro-granulomatous, diffuse, severe, with numerous extracellular filamentous bacilli, long snout seahorse, Hippocampus reidi.

Conference Comment:  

Nocardia spp. are ubiquitous, saprophytic, gram-positive, higher order bacteria that are associated with both opportunistic and primary infections in a variety of terrestrial and aquatic species worldwide. Morphologically, the bacteria are long, thin, beaded filaments with frequent right-angle branching resembling Chinese letters.⁸ They can be seen on standard H&E stained sections; however, they are best visualized by the histochemical stains Gomori methenamine silver (GMS), gram stains, and modified acid-fast stains, such as Fite-Faraco, as demonstrated by the outstanding photographs provided by the contributor.⁸

Although generally thought to cause low mortality in fish, Nocardia spp. can induce severe chronic granulomatous systemic disease.^1,7 In this case, the normal renal architecture is almost completely effaced by multifocal to coalescing necrotizing and granulomatous inflammation. Although there is some slide variability, in several examined tissue sections, inflammation extends into the adjacent coelomic cavity and skeletal muscle. As mentioned by the contributor, the primary differential diagnosis for piscine nocardiosis includes infection with the much more common Mycobacteria sp., which produces nearly identical gross and histological lesions in fish.⁷ Gross lesions include cachexia, ascites, dermal ulceration, multifocal skeletal muscle necrosis, and small white well-circumscribed granulomas in the kidney, spleen, heart, and liver.⁷ As a result, nocardiosis can be easily misdiagnosed as mycobacteriosis. Positive differentiation requires isolation and identification of the infectious organism and examination of histologic sections of infected organs. Histologically, Mycobacteria sp. are non-filamentous, non-branching, gram-positive, strongly acid-fast bacteria and are easily differentiated from Nocardia with the special histochemical stains previously mentioned.^1,7,8� Additionally, virulent Mycobacteria spp. are obligate intracellular pathogens that replicate within host macrophages. In contrast, pathogenic Nocardia spp. are facultative intracellular bacteria that have complex cell wall lipids that allow for resistance to phagocytosis by host macrophages.^7,8 In this case, the vast majority of bacteria are extracellular.

In a small number of tissue sections, conference participants noted a focal granuloma within the intestinal wall centered on a degenerate larval cestode characterized by a 2 um eosinophilic tegument, a lacy, fibrillar eosinophilic parenchymatous body cavity, scattered 5 um diameter, basophilic, calcareous corpuscles, and birefringent hooks in some sections.⁴ The pathologic significance of the parasitic granuloma in the intestinal wall, in this case, is unclear; however, it may indicate that this seahorse was immunocompromised, allowing for the proliferation of various concurrent opportunistic pathogens.

References:

1.      Brown-Elliott BA, Brown JM, Conville PS, Wallace RJ. Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy. Clin Microbiol Rev. 2006; 19:259-282.

2.      Chen SC, Lee JL, Lai JC, Gu YW, Wang CT, Chang HY, Tsai KH. Nocardiosis in sea bass, Lateolabrax japonicus, in Taiwan. J Fish Dis. 2000; 23:299-307.

3.      Cornwell ER, Cinelli MJ, McIntosh DM, Blank GS, Wooster GA, Groocock GH, Getchell RG, Bowser PR. Epizootic Nocardia infection in cultured weakfish, Cynoscion regalis (Bloch and Schneider). J Fish Dis. 2011; 34:567�571.

4.      Gardiner, CH, Poynton, SL. An Atlas of Metazoan Parasites in Animal Tissues. Washington, D.C: Armed Forces Institute of Pathology, American Registry of Pathology, 1999:35�38.

5.      Elkesh A, Kantham KPL, Shinn AP, Crumlish M, Richards RH. Systemic nocardiosis in a Mediterranean population of cultured meagre, Argyrosomas regius Asso (Perciformes; Sciaenidae). J Fish Dis. 2013; 36:141-149.

6.      Itano T, Kawakami H, Kono T, Sakai M. Experimental induction of nocardiosis in yellowtail, Seriola quinqueradiata Temminck & Schlegel by artificial challenge. J Fish Dis. 2006; 29:529�534.

7.      Lewis S, Chinabut S. Mycobacteriosis and Nocardiosis. In: Woo PTK, Bruno DW eds, Fish Diseases and Disorders, Volume 3: Viral, Bacterial and Fungal Infections 2^nd ed. CABI Publishing, Oxfordshire; 2011:397-423.

8.      Mauldin E, Peters-Kennedy J. Integumentary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer�s Pathology of Domestic Animals. Vol 1. 6th ed. Philadelphia, PA:Elsevier; 2016:637-638.

9.      Wang G-L, Yuan S-P, Jin S. Nocardiosis in large yellow croaker, Larimichthys crocea (Richardson). J Fish Dis. 2008; 28:339-345.

Click the slide to view.

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3-1. Kidney, seahorse:

3-2. Kidney, seahorse:

3-3. Kidney, seahorse:

3-4. Kidney, seahorse:

3-5. Lung, penguin:

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