We have encountered several diseases of unknown cause (idiopathic etiology) and a few that are related to non-infectious causes. These include egg-associated inflammation and fibroplasia, hepatic megalocytosis, severe dilation of the heart or pericardium, and nephrocalcinosis. In addition, we have seen many neoplasms in zebrafish research facilities. They are discussed in the following section.

This condition occurs in many species of fish held in captivity, and we occasionally observe it in our diagnostic cases. Nephrocalcinosis is the accumulation of calcium deposits in kidney tubules and collecting ducts. Causes of nephrocalcinosis include high CO₂ (e.g., > 12 mg/L) in water or excessive levels of calcium and magnesium in the diet. The use of calcium carbonate (rather than sodium bicarbonate) to buffer water in recirculating systems has been associated with the condition (Chen et al. 2001). Only when the lesions are extensive is the condition associated with overt clinical disease. In other words, we see many fish with the condition (diagnosed by histology) in which it was probably not a significant cause of the disease.

We have not observed macroscopic changes due to nephrocalcinosis in zebrafish. However, with larger fish, kidneys with severe nephrocalcinosis may exhibit distinct, white, opaque deposits in the kidney. The ureters may be filled with a chalk-like material, and vermiform deposits may occur in collecting ducts and tubules.

Histological sections reveal basophilic, crystalline deposits (uroliths), in renal tubules and collecting ducts. The deposits often cause severe dilation of the affected structures.

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Histological section showing masses of basophilic crystalloid material (arrows) in kidney tubules and collecting ducts

Observation of calcium (basophilic) accumulations in the kidney by histology is generally sufficient for confirmatory diagnosis.

With zebrafish, high CO₂ associated with crowding or the use of calcium carbonate (e.g., crushed coral) are two factors that have the potential to cause nephrocalcinosis. Chen et al. (2001) recommended buffering with sodium bicarbonate. In addition, proper CO₂ concentrations should be maintained by proper water exchange and avoiding crowded conditions. Proper atmospheric CO₂ levels should be maintained by appropriate ventilation.

The heart region (ventral, posterior to the head) is very swollen. Dissection of the affected area may hemorrhage severely or may reveal large blood clots.

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Zebrafish with enlarge heart or pericardial effusion, resulting in protrusion in heart region (arrow)

Histological sections revealed that the pericardial sac in either severely congested with blood or a proteinaceous exudate. Fish may also present with a ventricle that is extremely dilated and congested.

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Pericardial effusion. Pericardial sac is enlarged congested with an eosinophilic exudate (E) (suggesting proteinaous fluid). V = ventricle of heart

Hemorrhage of the heart at necropsy is strong presumptive evidence. Histology will confirm the diagnosis.

The cause of this condition in zebrafish is unknown, and thus at this date we have no recommendations for control.

We frequently observe severe, chronic inflammation in the visceral cavity associated with degenerating eggs in diagnostic cases. Rarely, in the most severe cases, aggressive fibroplasia leading to development of fibromas and fibrosarcomas may be observed. We have named the condition “Egg Associated Inflammation” or EAI. Anecdotal information indicates that it is caused by egg retention, and infectious agents are not usually found in the lesions. Occasionally we observe Mycobacterium spp. within granulomas in these lesions, but these infections are probably not the primary cause.

Female zebrafish typically present with an enlarged or distended abdomen. The ovaries grossly appear as a solid, tumor-like mass in the visceral cavity. In several cases the mass will cause adhesion to the wall of the visceral cavity, and ultimately a free raft of scar tissue will be extruded through the body wall and skin of the fish. This results in a large ulcer with a whitish center on the lateral flank of the fish.

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Ulcer on flank of zebrafish associated with EAI. Note whitish center corresponding to fibrotic tissue and debris.

Histological examination revels severe, chronic inflammatory changes that originate in the ovaries and may extend throughout the peritoneal cavity. Eggs of varying states (from intact to completely degenerated) are found within the lesion. Prominent fibroplasia occasionally occurs, and in some cases appears to lead to the formation of fibrosarcomas.

The second histological figure (B) shows a mass of degenerating eggs and fibrotic tissue that has extended through the body wall. This same phenomenon occurs in wild striped bass with chronic pertionitis associated with larval tapeworms of Lacistorhynchus tenuis (Moser et al. 1984).

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Histological section of zebrafish with EAI. Note extensive, chronic inflammation in ovaries (demarcated by arrows). E = egg

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Figure B Histological section of zebrafish with EAI in which a raft of chronic inflammation and eosinophilic debris (R) is extruded through the body wall. Arrows demark edge of ulcer.

Identification of the condition is based on observing histological changes described above.

The precise cause of EAI is unknown. Because abnormal egg retention is a suspected cause, we recommend timely spawning of females. Holding males and females together may also aid in preventing abnormal egg retention. Striping egg bound females has been tried, but apparently is not reliable for curing the problem.

Hepatic megalocytosis is characterized by tremendous enlargement of the cytoplasm and nuclei of hepatocytes. It is generally thought to be caused by hepatotoxicants, either anthropogenic (Myers et al. 1987) and natural (e.g., algatoxins such as microcystin) (Kent 1990; Andersen et al. 1994). The affected cells are polyploid, resulting from failure of cell division. Exposure to hepatic carcinogens often induces the condition, but the affected cells probably do not go on to develop into neoplasms. We have found that the affected hepatocytes will be present many months after exposure to the agent (Kent 1990). The source of the agent(s) causing hepatic megalocytosis in zebrafish colonies is unknown. As it can be either natural or anthropogenic causes, all possibilities should be considered, including diet

We have not related direct clinical disease to hepatic megalocytosis, and it is only observed during histological examinations.

Histological sections of the liver reveal massive hypertrophy of the cell and nucleus of individual hepatocytes, at times 10 to 20X, or greater, than normal size. The abnormal hepatocytes are usually found randomly dispersed within the normal liver parenchyma.

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Hepatic megalocytosis. Note severe enlargement of hepatocyte nuclei and cytoplasm

Diagnosis is based on observation of extremely enlarged hepatocyte nuclei within enlarged cells by histology.

Identification of the agents and their sources are required before we can recommend treatment or avoidance strategies.