Signalment:  

Adult gravid female koi, (Cyprinus carpio koi).The adult female koi was submitted to UC Davis, VMTH Companion Avian and Exotic Animal Medicine and Surgery/Aquatic Animal Health service, dead on arrival from a non-commercial pond. Three other koi had died over the past 10-month period (August 2009 through May 2010), with similar clinical signs. The owner reported that this fish developed ulcerations on the dorsal head a few months ago and had been lethargic for the past three to four days. After being isolated from the pond, it developed generalized edema. The other affected fish similarly developed ulcerations prior to death. The owner offered antibiotic-containing feed to the pond after the third fish died. The pond is a converted swimming pool also being used by waterfowl. 

Upon clinical examination, ulceration on the lateral trunk and prominent edema of the gills were noted. Post mortem gill biopsy and skin scrapings were done using light microscopy immediately on arrival, and revealed gill flukes (Dactylogyrus sp.). Skin scraping did not reveal additional findings.

The fish was submitted for full necropsy to rule out viral, including koi herpes virus (KHV, Cyprinid herpesvirus 3), spring viremia of carp virus (SVCV), or bacterial infections. Fresh samples of gill and kidney were tested for KHV and carp edema virus (CEV). The molecular techniques used were PCR as described by Oyamatsu T. et al., for CEV and Taqman - PCR for KHV by Bercovier H. et al. A sample of water from the pond was submitted for toxicological and other water quality analyses. 

Gross Description:  

The submitted fish had irregular, coalescing, well demarcated areas of scale and skin loss with exposed underlying subcutis and muscles at the dorso-lateral trunk. The eroded areas were surrounded with hemorrhage. Diffusely, the scales fell out with little manipulation of the body. Clear fluid was exuding from the vesicles expanding the skin and elevating the scales producing a pinecone appearance. The skin on the head, around the vent, and diffusely throughout the body, had numerous pinpoint red discolorations (consistent with petechiation) and small vessel congestion. The mucosa of the upper palate of the mouth was diffusely swollen, partially occluding the pharyngeal region. The coelomic organs were intertwined and adhered to the parietal serosa with numerous thin fibrous attachments. The ovary, liver, spleen and kidneys were extremely friable. The gastrointestinal tract was surrounded with a large amount of adipose tissue.

Histopathologic Description:

Gill: The secondary gill lamellae are partially fused due to marked, diffuse epithelial cell hyperplasia and hypertrophy, mixed interstitial inflammation and edema. The interstitium of the primary lamellae is also expanded with marked edema and contains mixed inflammation. The inflammation consists of abundant mononuclear cells, such as lymphocytes and macrophages, as well as eosinophilic granular cells. Thickened and distorted secondary lamellae are lined with plump, sometimes vacuolated epithelium that piles up into confluent sheets with admixed inflammation, particularly at the base of the filaments. The gill epithelial cells frequently have rounded margins, hypereosinophilic cytoplasm, and karyorrhectic, pyknotic or indistinct nuclei. Sloughed necrotic cells mix in with cellular debris and erythrocytes at the gill surface. There is rare multifocal rodlet cell hyperplasia throughout the affected regions. Rare gill arch arterioles contain fibrin thrombi. Multifocally in the gill and mucosal epithelium, smudged basophilic aggregates and sometimes eosinophilic material disperses the chromatin (presumed cytoplasmic invagination, early chromatin changes in necrotic cells). Vessels throughout the gill are dilated.

Skin (slide not provided): Associated with superficial areas of ulceration are small, gram-negative bacterial aggregates (presumed Aeromonas sp.) mixed in with rare gram-positive larger rods. Adjacent to the ulcerated regions, on the intact epidermal surface, there were thin pyriform protozoa attached by thin stalks (flagella), approximately 6x5 μm (similar in size to a red blood cell), consistent with Ichthyobodo sp., (formerly known as Costia sp.). The scales are elevated above the dermis by clear spaces (edema). Skeletal muscles in the region of skin ulceration are inflamed and necrotic. The myocytes have fragmented, vacuolated or pale eosinophilic sarcoplasm with loss of distinct striations. Mononuclear inflammatory, often fragmented cells aggregate between myocytes, and along with erythrocytes spill onto the exposed surface.

Morphologic Diagnosis:  

1. Gill: Moderate diffuse subacute necrotizing branchitis with marked interstitial edema and multifocal branchial arteriole thrombosis (PCR positive for carp edema virus).
2. Integument, mid left dorso-lateral body wall: Severe multifocal subacute regionally extensive ulcerative and fibrinohemorrhagic dermatitis and necrotizing myositis with epidermal protozoal parasites (probable Ichthyobodo sp.).
3. Integument: Severe diffuse edema, petechiation, scale loss and lymphocytic and granulocytic dermatitis.

Lab Results:  

-+ Molecular analysis of the fresh tissues (gill, kidney) were positive for Carp Edema Virus (CEV) or Koi Sleepy Disease, a pox - like virus, and negative for KHV.  -+ Toxicological analysis of the water did not demonstrate any elevated heavy metals except for copper, measuring 0.02ppm (reference at 0.01ppm).  -+ Elevated nitrite (0.085mg/L NO₂-N) and ammonia nitrogen (0.11mg/L NH₃-N) were detected using spectrophotometer.

Condition:  

Carp edema virus (CEV)

Contributor Comment:  

Carp edema virus (CEV) is the causative agent of sleepy disease of koi (SDK) with devastating outbreaks in koi or ornamental variety of carp (Cyprinus carpio koi, also known as Japanese color carp). This disease occurs epizootically in the fall and spring in commercially cultured young fish. Occurrence of outbreaks is associated with mild water temperature ranges of 15 to 25 degrees Celsius. Affected fish are lethargic, found at the bottom/surface of the tank or pond with sunken eyes and skin erosions/ulcerations. The most distinct gross finding is diffuse edema, particularly affecting the gills. Microscopic findings include clubbing and swelling of gill filaments with interlamellar fusion, and hypertrophy, hyperplasia and necrosis of the gill epithelium. CEV, a pox like virus, particles can be visualized with transmission electron microscopy in the infected gill epithelium.(6) Electron microscopy demonstrates cytoplasmic viral particles in the gill epithelium with immature virus measuring up to 450nm in diameter and mature, roughly oval, virions measuring about 400x413nm. Mature virions are decorated with surface globules and have a dense core enclosed by a prominent membrane.(6) Cytoplasmic inclusions characteristic of pox virus infections were not observed in the gills of this fish, neither they were described by T. Miyazaki et al. The CEV PCR assay can detect the virus in multiple different tissues from affected fish including skin, liver and kidney.(6)

In this case, the diagnosis of carp edema virus was made on the characteristic gross and histologic findings with confirmation by PCR. No virions were identified in gill tissue by electron microscopy; however, the tissue was not optimally preserved. Combination of CEV, protozoal, and bacterial infections may have played a role in the skin ulceration. The latter two could potentially be opportunistic pathogens infecting the compromised host. 

Based on the history (mild water temperature, season, age of affected fish) and gross lesions (skin ulcerations and petechiae), differentials for this case would include KHV and SVC. The pronounced branchial edema distinguishes the CEV infection, grossly. As a brief review, KHV is characterized grossly by necrotizing branchitis and is a reportable disease of wild and cultured common carp. Histological findings include epithelial proliferation, fusion of the gill lamellae, and epithelial necrosis (cytopathic effect of the virus). Typical of KHV, there are intranuclear inclusions in many cell types, including but not limited to respiratory epithelial cells, macrophages, hematopoietic cells in the kidney, and cardiac myocytes. Electron microscopy demonstrates enveloped herpes virus with mature nucleocapsids measuring up to 117nm and mature enveloped nucleocapsids up to 180nm in the affected cells.(7) KHV virus can be isolated from multiple organs and confirmed with PCR and immunohistochemistry.(2,5) Spring viremia of carp virus (SVCV) from the family Rhabdoviridae, genus Vesiculovirus, is the causative agent of another reportable, contagious, fatal disease of farmed carp and related species. The virus causes petechial hemorrhages in the gill and skin, as well as internal hemorrhage in the kidneys, spleen and liver, and exophthalmia. SVCV targets the swim bladder, resulting in edema and inflammation, as well as ascites.(1,3) Skin ulceration can also be caused by parasitic infection such as Ichthyobodo sp. (formerly known as Costia sp.) that may also affect the gills.(4,9) The latter agent can be seen on scrapings from gills and skin lesions, and on histological examination. Koi ulcer disease (also known as summer ulcer disease and carp erythrodermatitis), associated with bacterial pathogens such as Aeromonas spp., can also present similarly.(4,9)

JPC Diagnosis:  

1. Gill: Branchitis, proliferative, diffuse, severe, with marked epithelial hypertrophy and hyperplasia, lamellar fusion, arteriolar fibrin thrombi and mild goblet cell hyperplasia.
2. Oral mucosa: Stomatitis, proliferative and lymphocytic, diffuse, mild, with numerous intraepithelial intranuclear inclusions.

Conference Comment:  

The moderator began with a brief review of the normal anatomy, histology and physiology of the gill. The gill arch is a curved bony structure with double rows of paired primary lamellae (filaments). Each primary lamella, in turn, encompasses an array of perpendicularly oriented secondary lamellae. The entire gill arch is covered by epidermis; the epidermis overlying the origin of the primary lamellae is thicker and often contains numerous mucous cells, with a subepidermal array of lymphoid tissue. The primary lamellae are covered by a mucoid epidermis which may contain round, pale, eosinophilic, salt-secreting chloride cells (especially at the basal/proximal part of the lamellae). These chloride cells function in ionic transport and may also play a role in detoxification.(8)

Gas exchange occurs via countercurrent exchange at the surface of the secondary lamellae, which are lined by overlapping squamous epithelial cells, usually one layer thick, surrounding numerous capillaries that are supported by rows of pillar cells. Where the pillar cells encroach on the basement membrane, they spread to coalesce with neighboring pillar cells to complete the lining of lamellar blood channels. Pillar cells contain contractile protein elements that resist distension and support the lamellar blood spaces. The surface of the lamellar epithelium gives rise to microvilli that aid in attachment of the epidermal (cuticular) mucus. This mucus, in addition to providing protection against abrasion and infection, is important in the exchange of gas, water and ions. The combined thickness of the cuticle, respiratory epithelium and flanges of the pillar cells (which is the total diffusion distance for gas exchange) ranges from 0.5 to 4 μm. Low to moderate numbers of goblet cells are scattered among lamellar squamous epithelial cells of both primary and secondary lamellae.(8)

Much like mammalian lungs, the gill epithelium is thin with a large surface area in order to maximize the exposure of gill capillaries to water. While this is an important factor for efficient gas exchange, it is a fairly ineffective physical barrier and results in increased branchial vulnerability to inflammation and infection. Gills also play an essential role in regulating the exchange of salt and water, as well as the excretion of the nitrogenous wastes (primarily ammonia). Thus, even minimal damage can result in significant osmoregulatory and respiratory difficulties.(8)

As noted by the contributor, the proliferative nature of these microscopic lesions is striking, with marked epithelial cell hypertrophy and hyperplasia leading to lamellar fusion; however, many conference participants also identified moderate numbers of fairly prominent, eosinophilic, intracytoplasmic inclusions within the epithelium of the (presumed) oral mucosa, which appear to peripheralize the chromatin. After scrupulous examination of H&E sections, and consideration of the laboratory results reported by the contributor (molecular analysis for koi herpes virus was negative), we are unable to elucidate the nature of these inclusions. There is also some slide variation - not all sections contain fibrin thrombi within arterioles of the gill arch, as reflected in the JPC morphologic diagnosis. Furthermore, the moderator observed that cartilage of the gill arch appears somewhat irregular and deformed, which may suggest a previous nutritional deficiency, but is probably unrelated to the current disease process.

References:

1. Ahne W, Bjorklund HV, Essbauer S, et al. Spring viremia of carp (SVC). Dis of Aquat Org. 2002;52:261272. 

2. Bercovier H, Fishman Y, Nahary R, et al. Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis. BMC Microbiol. 2005;5:13.

3. Dikkeboom AL, Radi C, Toohey-Kurth K, et al. First report of spring viremia of carp virus (SVCV) in wild common carp in North America. Journal of Aquatic Animal Health. 2004;16:169178.

4. Ferguson HW. Systemic Pathology of Fish. 2nd ed. London, UK: Scotian Press; 2006:55, 72-73.

5. Ilouze M, Dishon A, Kotler M. Characterization of a novel virus causing a lethal disease in carp and koi. Microbiology and Molecular Biology Reviews. 2006;70:147156.

6. Miyazaki T, Isshiki T, Katsuyuki H. Histopathological and electron microscopy studies on sleepy disease of koi (Cyprinus carpio koi) in Japan. Dis of Aquat Org. 2005;65:197207.

7. Miyazaki T, Kuzuya Y, Yasumoto S, et al. Histopathological and ultrastructural features of koi herpesvirus (KHV)-infected carp Cyprinus carpio, and the morphology and morphogenesis of KHV. Dis of Aquat Org. 2008;80:111.

8. Mumford S, Heidel J, Smith C, Morrison J, MacConnell B, Blazer V. Fish Histology and Histopathology. US Fish and Wildlife Service, National Conservation Training Center. http://nctc.fws.gov/resources/course-resources/fish-histology/index.html. Accessed April 26, 2014.

9. Noga EJ. Fish Disease: Diagnosis and Treatment. St. Louis, MO: Wiley-Blackwell; 1996:108-110, 141-146.

Click the slide to view.

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1-1. Skin

1-2. Skin around vent

1-3. Gill

1-4. Gill

1-5. Gill

1-6. Gill

1-7. Gill

1-8. Skin

1-9. Skin

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