Ornamental fish may present to the veterinary practitioner for a variety of different diseases, including poor water quality, inappropriate husbandry, nutritional deficiencies, infectious and parasitic diseases, and neoplasia. To determine which of these etiologies is responsible for a disease in a particular fish (or fishes), diagnostic testing is required. Although the concept of performing these tests may appear overwhelming, with practice, diagnosing disease can become second nature.

There are two ways to approach a disease issue in fish: 1) antemortem tests and 2) postmortem tests. Antemortem tests are done when the aquarist is interested in saving a particular fish, while postmortem tests are pursued when the aquarist is interested in saving a group of fish. A necropsy can provide a great deal of insight into the disease condition of a particular fish, and therefore the population of animals from which it originates. The purpose of this presentation is to review the common diagnostic tests used to assess the disease status of a fish.

The most common antemortem tests performed on fish are gill biopsies, skin scrapes, fin biopsies, complete blood counts, cultures, fecal direct smears, and diagnostic imaging (e.g., radiographs, ultrasound, endoscopy).¹ Selecting which test to perform should be based on the clinical signs of the fish. Dyspnea in fish is suggestive of gill disease, and a gill biopsy would be appropriate. Lesions found on the skin (e.g., excessive mucus production) or fins (e.g., erosions) may be suggestive of infectious or parasitic disease, and a skin scrape or fin biopsy would be appropriate. Fish that are depressed, anorectic (not eating), or thin (muscle wasting) may have an internal disease (e.g., infectious or parasitic disease). A bacterial culture can be done to identify a specific bacterial pathogen. An antibiotic sensitivity profile can also be done to determine which antibiotic is best suited for eliminating the infection. A complete blood count can be used to interpret the animal's overall well-being or a fecal exam can be used to assess the potential for internal parasites. Diagnostic imaging can be used to characterize a disease or collect samples (e.g., ultrasound-guided biopsy). To minimize discomfort for the fish and decrease the likelihood of collecting substandard samples, it is recommended to anesthetize the fish for these different diagnostic procedures. Tricaine methane sulfonate (MS-222; Argent Laboratories, Redmond, WA 98052) (100–200 mg/L) is the preferred anesthetic for anesthetizing fish.

Gill biopsies are an excellent method for assessing the quality of the gills.¹ Teleosts, or bony fish, have 4 pairs of gills. The gills reside in the protective buccopharyngeal chamber under the operculum (gill cover). At the microscopic level, the gills can be divided into the primary and secondary lamellae. The primary lamellae represent the individual gill filaments that can be observed with the naked eye, while the secondary lamellae are comprised of a single layer of epithelial and endothelial cells and line the primary lamellae. The secondary lamellae are the site for gas exchange (e.g., oxygen absorption and carbon dioxide off-loading) and the excretion of wastes (e.g., ammonia). The surface area of the gills is vast, and allows for the rapid movement of water across the gill surface. Any damage to the gills can decrease the surface area associated with the secondary lamellae, and lead to dyspnea and death. Elevated levels of chlorine, ammonia, and nitrite, along with infectious and parasitic diseases, are the most common causes of gill disease in ornamental fish.¹ To confirm which of these problems is associated with a specific case, diagnostic tests, such as a gill biopsy, should be done. If ammonia, nitrite or chlorine toxicity is suspected, then a water test should be done too. Elevated levels of any of these toxins, in combination with microscopic changes in the gills (e.g., excessive mucus production and a loss of respiratory surface area), are diagnostic. The presence of infectious (e.g., bacterial or fungal) or parasitic diseases with abnormal gills is also diagnostic. Once a diagnosis is made, an appropriate treatment plan can be devised. For example, water changes can be made to reduce the toxicity associated with ammonia or nitrite, sodium thiosulfate used to dechlorinate water, or an appropriate antibiotic or antiparasitic given to treat infectious or parasitic agents.

When performing a gill biopsy, the thumb of your non-dominant hand should be inserted under the operculum, and the operculum lifted. Once elevated, a fine pair of scissors can be inserted under the operculum to collect the gill biopsy. A small amount of bleeding may occur, but generally ceases within seconds. The gill sample should be placed onto a glass microscope slide, a drop of water from the animal's aquarium placed on the sample, and a coverslip added to protect the sample. Water from the aquarium is preferred because it is isotonic (balanced) for any pathogens found on the gill. Adding water from another source that is not balanced can lead to the death of the organism and an inability to make a diagnosis. The sample should be reviewed immediately after collection to ensure best results.

A skin scrape should be done in cases where a fish has lesions on the skin.¹ The skin scrape can be used to identify infectious or parasitic organisms. A glass microscope slide can be used to collect the sample. The slide should be held at a 45° angle and drawn in a cranial to caudal direction (e.g., from head to tail). The sample should be placed on a second microscope slide, mixed with a drop of water from the aquarium, and covered with a coverslip. Again, the sample should be read immediately for best results. If bacteria are a concern, then a Gram stain or Diff-Quik stain can be done to evaluate the types of bacteria present. To prepare these slides, the sample and drop of water are mixed, the sample heat fixed using a match or lighter, and the sample stained according to the manufacturer's recommendation.

A fin biopsy should be considered in cases where lesions are found on the fins.¹ Many times these lesions are associated with a bacterial, fungal or parasitic infection. A fine pair of scissors should be used to collect the sample. If the sample can be collected between fin rays, that is preferred; however, this is not always possible, and the fin will regenerate. The sample should be handled in a similar fashion to the skin scrape, and either is placed on a slide with a coverslip or stained.

Fecal exams for parasites can be done on free-catch samples (e.g., found in the tank) or via enema. The samples should be placed on a slide with a drop of water and a coverslip and reviewed.

Blood can be collected from the ventral tail vein or heart. Fish blood does tend to clot quickly, so an anticoagulant may be required. Because fish have nucleated erythrocytes and thrombocytes, manual counts are necessary.

Postmortem examinations should always be performed immediately after the fish has expired.² Autolysis can occur rapidly in fish, and can severely limit the value of a necropsy. Fish that have been dead in the water for even a couple hours, depending on the water conditions and temperature, may have limited value. Therefore, it is important to perform the procedure as soon as possible after death. In cases where this is not possible, the animal should be stored in a refrigerator in an airtight bag. Freezing a fish can lead to tissue crystallization and eventual autolysis with thawing and is not recommended. Storing a fish in water is also not recommended, again, because of the potential for autolysis.

A fish postmortem can be divided into two major parts: the gross examination and the microscopic examination.² Veterinarians interested in submitting samples can find individuals capable of reviewing a case by searching the internet or local/state diagnostic laboratory. When performing a necropsy on a fish, it is important to protect yourself against potential zoonotic diseases. The author highly recommends wearing exam gloves when performing a necropsy. There are a number of bacterial and fungal fish diseases that can cause localized or even systemic diseases in humans. The cuts and scrapes we have on our hands can serve as excellent sites of entry for these pathogens, and thus the reason gloves are important.

The postmortem examination should start with an external examination of the fish. The general appearance of the fish should be closely inspected. How is the muscling? Is the animal thin? This can usually be determined by evaluating the epaxial muscles along the spine. Animals with chronic disease typically lose muscle in an attempt to generate energy to defend against an infectious disease (e.g., mycobacteriosis). Are there erosions or ulcers on the skin? How large are they? Are they full thickness (e.g., can you see the underlying muscles)? These types of lesions may be indicative of aggressive bacterial infections that may be contagious to other fishes (e.g., Aeromonas spp.). A close external examination can provide a significant amount of insight into the health status of the animal. Not fully evaluating the fish can result in misdiagnosis. Once the external examination is completed, a thorough internal examination should be done.

Prior to opening the coelomic cavity (abdomen), it is important to evaluate the oral cavity and gills. The operculum should be removed and the gross appearance of the gills recorded. If the fish is only recently expired, they should remain moist and red. If the fish has been expired for an extended period of time, then they may appear deteriorated. Excessive mucus production or a loss of color is suggestive of disease. A clip of the gills can be taken and reviewed (unstained) under a light microscope to identify potential pathogens.

The author prefers to open the fish on the left side for the internal examination, as it provides better access to the spleen. The initial incision should be made on the ventral surface of the fish, cranial (in front of) to the anus. The incision should then be extended cranially to the level of the operculum. The incision should then be extended dorsally towards the spine. At this point, the incision can be extended caudally towards the tail, parallel to the spine. Finally, the incision can be extended ventrally back to the level of the initial incision. Once the incision is completed, the entire lateral aspect of the body wall can be removed. With the body wall removed, it will be possible to visualize the internal organs. With over 20,000 different teleosts, it is impossible to describe the variation in organ position, size, color, and texture in a single article. For the most part, these things are similar, but you can expect to be stumped on occasion. With time and practice, a veterinarian can become quite adept at identifying organs and knowing what looks normal and what looks abnormal. The gross examination of the organs can certainly provide some insight into the health status of the animal, but is generally limited without histopathology.

To truly characterize a specific cause of disease in a fish or a group of fish, diagnostic tests must be performed. For many veterinarians, the idea of performing these tests may be daunting; however, with practice any veterinarian can become proficient at performing and interpreting these tests.

References

1.  Noga EJ. The clinical workup. In: Noga EJ, ed. Fish Disease: Diagnosis and Treatment. 2nd ed. Ames, IA: Wiley-Blackwell; 2010:13–48.

2.  Noga EJ. The clinical workup. In: Noga EJ, ed. Fish Disease: Diagnosis and Treatment. 2nd ed. Ames, IA: Wiley-Blackwell; 2010:49–64.