Hematology

Hematologic evaluation of fish is not routinely used in the diagnosis of fish diseases, but it can be useful in the detection of diseases that affect the cellular components of blood. Certain diseases of fish result in anemia, leukopenia, leukocytosis, thrombocytopenia, and other abnormal changes of blood cells. Evaluation of the hemogram may also be useful in following the progress of the disease or response to therapy.

The standard practices used for collecting, handling, and analyzing blood from mammals and birds can be misleading when applied to fish hematology. Emersion and handling of fish for venipuncture or cardiocentesis can have a marked effect on the hemogram resulting in a significant increase in the hematocrit (PCV) by as much as 25%. The magnitude of this effect is directly related to the handling and analytical time. Handling of fish (for as little as 20 seconds) results in the release of catecholamines which tend to cause hemoconcentration and swelling of the erythrocytes; therefore the hematocrit increases but the hemoglobin concentration remains the same, resulting in a decrease in the mean corpuscular hemoglobin concentration (MCHC).

Erythrocytes

In general, the packed cell volume (PCV) of fish is lower than that of mammals and birds. Hematocrit values vary between and within fish species and appear to correlate with the normal activity of the fish, where less active fish have lower hematocrit values than active, fast swimming fish. Hematocrit values also vary during the life cycle of fish. Age, sex, water temperature, photoperiod, and seasonal variation may also influence PCV of fish. The PCV of some species of male fish are larger enough so as to require two reference intervals.

Cartilaginous (sharks and rays) and bony fish appear to have different gas transport systems that affect their erythrocyte parameters. Bony fish exhibit a high cardiac work and blood pressure associated with a higher PCV and smaller erythrocytes. Sharks and rays, on the other hand, exhibit a relatively modest cardiac work, higher cardiac output, higher blood volumes and increased flow rates that are associated with lower concentrations of larger cells.

In general, fish with PCV greater than 45% are usually considered to be dehydrated, particularly when supported by increased serum osmolality or total protein. Anemic fish have low PCV values (i.e., less than 20%); however, PCV values for some species of normal fish may be as low as 20%.

Fish with regenerative anemia often have increased concentration of polychromatic and immature erythrocytes in their blood films. Anemic fish that exhibit little or no polychromasia have nonresponsive anemia. Because immature erythrocytes of fish are smaller than mature erythrocytes, a microcytosis is often associated with marked hemorrhagic or hemolytic anemias, where the regenerating immature erythrocytes represent the majority of the peripheral blood erythrocytes.

Leukocytes

There is a wide variation in the appearance of leukocytes, especially the granulocytes, among the various fish species. This has led to controversy and confusion when applying the nomenclature and classification of piscine leukocytes based upon descriptions of those found in avian and mammalian blood films stained with Romanowsky stains. Evaluation of the cellular ultrastructure, differential cytochemical staining, immunofluorescence, and function testing of fish leukocytes has helped alleviate some of this controversy in some species.

Piscine neutrophils and heterophils participate in inflammatory responses; however, they are not always phagocytic and little is known about their function including their methods of intracellular killing and digestion of phagocytized organisms. Because the function of fish granulocytes is not known, it would be inappropriate to view them as homologous to the granulocytes of higher vertebrates. Therefore, interpretation of changes in granulocyte concentrations in peripheral blood can be difficult. However, broad generalizations can be made until further studies indicate the function and responses of these cells to disease. For example, increases in concentration of fish neutrophils or heterophils are often associated with inflammatory diseases, especially those associated with infectious agents. A relative neutrophilia or heterophilia is often associated with lymphopenia, which can be interpreted as a stress response in fish.

Eosinophils are found in low concentrations in the peripheral blood of normal fish. An increase in eosinophil concentration in peripheral blood of fish is suggestive of an inflammatory response that is associated with parasitic infections or antigenic stimulation.

The functions of granulocytes of cartilaginous fish are not known; however, they do appear to participate in inflammatory responses. Because the granulocytes make up 20–30% of the leukocytes in sharks and rays, the normal granulocyte to lymphocyte (G:L) ratio is typically low (i.e., less than 0.5). An increase in granulocyte concentration is indicative of an inflammatory response. A decrease in lymphocyte concentration results from conditions that reduce the number of circulating lymphocytes, such as stress responses. Increases in granulocyte concentrations and decreases in lymphocyte concentrations of sharks can be associated with bacterial septicemias. The leukogram of cartilaginous fish can be used to follow the progress of the fish in the course of the disease or response to therapy. For example, an initial increase in granulocyte concentration or decrease in lymphocyte concentration that has returned to normal indicates a favorable response to therapy and prognosis.

Piscine monocytes are actively phagocytic cells and participate in acute inflammatory responses in fish. Monocytes occur in low numbers (i.e., less than five percent of the leukocyte differential) in the peripheral blood of normal fish. Therefore, a monocytosis would be suggestive of an inflammatory response in fish, perhaps associated with an infectious agent.

Lymphocytes are the most commonly observed leukocytes in the peripheral blood of most normal fish, where they typically represent greater than 60% (as high as 85% in some species) of the leukocyte differential. Lymphocytes play a major role in the humoral and cell-mediated immunity of fish. Therefore, a lymphocytosis is suggestive of immunogenic stimulation, whereas a lymphopenia is suggestive of immunosuppressive conditions, such as stress or excessive exogenous glucocorticosteroids. Bacterial septicemias commonly affect fish and result in marked leukopenia and lymphopenia.

Thrombocytes and Hemostasis

Fish blood clots in response to injury as with other vertebrates. However, the speed and effectiveness is variable. The clotting of blood is much more rapid in bony fish compared to sharks and rays. Sharks and rays appear to rely primarily on extrinsic pathways of coagulation; the addition of skin, high calcium solutions, sea water, or other extrinsic factors enhances clotting. Clot formation in bony fish usually occurs within five minutes, whereas clotting of blood samples taken from sharks and rays can take 20 minutes or longer.

Blood Biochemistry

Blood biochemical evaluation is not routinely part of the clinical assessment of piscine patients, most likely owing to the expense involved and lack of meaningful reference intervals. As a result, much of the blood biochemical studies have focused on economically important species, such as salmonids (salmon and trout), catfish, and cyprinids (carp, goldfish, and koi). Routine assay methods for the biochemical evaluation of mammalian blood appear to be useful for fish blood; however, interpretation of the results can be difficult. Many endogenous (i.e., species, age, nutritional status, gender, reproductive status) and exogenous factors (i.e., environmental conditions, population density, time of day, and method of capture) influence the plasma biochemistry results of fish. These factors should be taken into consideration when establishing reference intervals for fish.

Wet Mount Microscopy in Fish

Gill Biopsies

Each gill arch has rows of macroscopic fingerlike primary lamellae. Each primary lamella has rows of microscopic secondary lamellae. Each secondary lamella contains blood vessels that move blood countercurrent to the water flow to facilitate gas and nitrogenous waste exchange. Normal gill filaments as viewed in a wet-mount preparation under the microscope have slender, triangular, smooth-surfaced lamellae, and interlamellar water channels are approximately equal in size.

Abnormal gill filaments are thick, have a ragged surface, and are coated with a layer of hyperplastic epithelium and mucus. Hyperplasia and hypertrophy of epithelial cells indicate gill damage and can lead to fusion of adjacent secondary lamellae. Hyperplastic lesions lead to death of the fish owing to a reduction or blockage of the respiratory water flow over the lamellar epithelium, thus reducing the exchange of gases and ions across the lamellar epithelium.

Severe gill damage results in telangiectasis or necrosis of the gill filaments. Telangiectasis occurs with gill infections or environmental toxins and is indicated by dilatation of groups of small blood vessels in the secondary lamellae. Severe necrosis of gill tissue is characterized by the destruction of secondary lamellae with the loss of the epithelium and exposure of the underlying cartilaginous skeleton of the primary lamellae.

Mucus Smears and Fin Biopsies

Normal wet-mount preparations from mucus smears and fin biopsies reveal mucus strands, a few superficial squamous epithelial cells, and an occasional scale that may contain normal pigment. Bacteria and ectoparasites can occur in low numbers in healthy fish. Many of the protozoan parasites are commensals that utilize the integument and gills as a substrate and only become harmful (usually in immunocompromised fish or amphibians) when their increased numbers interfere with skin or gill function. Others are obligate parasites of skin and gill epithelium that cause disease and death. High numbers of ectoparasites, such as numbers greater than two per low-power (40x to 100x magnifications) field and numerous bacteria and fungi are considered abnormal.

The significance of the amount of bacteria present on a wet-mount preparation is usually the subjective opinion of the cytologist as no guideline is available for the assessment of bacterial numbers. However, the presence of inflammatory cells associated with increased numbers of bacteria is suggestive of bacterial involvement. Stained, air-dried smears made by allowing the wet-mount preparation to dry following the removal of the coverslip are often helpful in the assessment of bacterial involvement. Septic lesions are identified by the presence of bacterial phagocytosis by leukocytes.

Columnaris is a bacterial disease of freshwater fish, caused by Flexibacter columnaris or related bacteria, and a disease of marine fish, caused by Flexibacter maritimus. Lesions associated with these bacteria include cutaneous ulcers, fin necrosis (fin rot), and gill necrosis. Wet-mount preparations of the lesions reveal long, thin, bacterial rods (0.5–1.0 µm wide and 4–10 µm long) that form moving mounds or columns that resemble haystacks. Bacteria on the outer edges of the specimen exhibit a characteristic gliding or flexing motion.

Fungal infections are another major cause of disease in fish and are usually the result of immunosuppression associated with poor water quality, chronic stress, and coexisting diseases. Saprolegniasis is a catch-all term for white, fuzzy mold growth on the skin of fish. Gross lesions caused by these fungi appear as white, cotton-like growths on the skin and gills. Wet-mount preparations reveal wide (7–30 µm) aseptate hyphae that are often associated with other pathogens, such as bacteria and protozoa.

Lymphocystis is a common viral disease of freshwater and marine fish. It is caused by a DNA iridovirus that causes a marked hypertrophy of infected cells (dermal fibroblasts). These cells can be up to 100,000 times their normal size, therefore they are visible without magnification. Advanced lesions exhibit large wart-like tumorous growths on the skin and fins. Microscopic examination of wet-mount preparations from lymphocystis lesions reveal extremely enlarged dermal fibroblasts.

Ciliate Protozoa

The majority of the ciliate protozoa found on fish are commensals and harmless. However, a few are notoriously pathogenic. For example, Ichthyophthirius of freshwater fish and Cryptocaryon of marine fish are highly pathogenic ectoparasites that feed on host cells. Because ciliate protozoans have a direct life cycle, they can occur in high numbers and create heavy infestations. Ichthyophthirius multifiliis, a common parasite of freshwater fish, is easily recognized in mucus scrapings or gill or fin biopsies by its size (up to 1 mm in diameter), characteristic horseshoe-shaped macronucleus, and slow rolling movement. Cryptocaryon irritans is the marine counterpart of the freshwater Ichthyophthirius multifiliis.

Chilodonella spp. are important holotrich ciliate protozoan parasites of tropical and subtropical freshwater fish. These parasites appear as flattened, ovoid- shaped protozoa that measure up to 80 µm in length and are covered by rows of cilia. They are identified by their shape and slow steady gliding movements in wet-mount preparations. Brooklynella spp. are the marine counterpart to the freshwater Chilodonella spp. and are significant pathogens of the skin and gills of marine aquarium fish.

Trichodina spp. of freshwater and marine fish are another pathogenic ciliate protozoan that can damage the gills and skin, especially when they occur in large numbers. Trichodina are peritrich ciliate protozoa that are easily recognized by a ring of internal denticles, which have a skeletal function.

Epistylis spp. are colonial stalked peritrich protozoal parasites that are commonly found on the surface of freshwater fish in ponds with high organic material. The surface of affected fish is used as a substrate for attachment, creating white tuft-like lesions on the surface of the skin or fins. They measure up to 100 µm in length and they have buccal cilia at one end of a bell-shaped structure called a zooid, which can recoil and extend.

Tetrahymena spp. are free-living, ciliated, protozoan parasites that become secondary pathogens capable of becoming highly invasive and resulting in systemic infestations in freshwater fish. Wet-mount preparations of mucus smears reveal numerous actively motile, pear-shaped ciliate protozoa that measure 60 µm by 100 µm. The appearance and movement of this protozoan is like that of a spiraling football. Uronema marinum is the marine counterpart to Tetrahymena.

Flagellate Protozoa

Flagellate protozoa are another common type of ectoparasite of fish. They often have direct life cycles and some have resistant cyst stages. The hemoflagellates have indirect life cycles.

The best known flagellate protozoan parasite of fish is Ichthyobodo necator (formerly Costia necatrix). This parasite affects practically any freshwater fish and is cosmopolitan in distribution. Although it is considered to be highly pathogenic to freshwater fish (especially young fish and those with immunosuppression), Ichthyobodo necator can cause disease and mortality in marine fish. It is detected in its free living stage, which is a small oval to kidney-bean-shaped protozoan, measuring 10–15 µm in length, about the size of many fish erythrocytes. It has two pairs of flagella of unequal length that are held in a groove over most of the length of the body. This causes the parasite to swim in a jerky spiral that exhibits a flickering or flashing image as it turns its crescent-shaped body in the light path of the microscope. When attached, it swims in a circular motion.

Dinoflagellates are found on the epithelial surfaces of marine and freshwater fish, usually in tropical or subtropical waters. They are the cause of the disease commonly known as "velvet disease," owing to the dust-like sheen they create on the skin of infected fish. Amyloodinium ocellatum is considered by many to be the most important parasite of marine fishes cultured in warm waters. It is one of the few fish parasites that can infest both teleost fish and elasmobranchs. Amyloodinium is detected by identifying the trophonts in biopsy samples, which are relatively large (50–350 µm), irregularly shaped organisms attached to the skin or scales. Piscinoodinium, the freshwater counterpart to Amyloodinium, contains chlorophyll, creating the "velvet disease" or "rust disease" of tropical pet fish.

Diplomonad flagellate protozoa (Spironucleus spp. and Hexamita spp.) are commonly encountered in the digestive tract of a wide variety of hosts, including fish, amphibians, rodents, and birds. These are small, pleomorphic, very active flagellates that are typically 10–20 µm long, which makes their identification difficult using light microscopy alone. Hexamita and Spironucleus are flagellate protozoans in the gastrointestinal tract of freshwater and marine fish that can cause anorexia, lethargy, and death. Massive systemic infections, especially with Spironucleus, are lethal.

Trypanosome infections are usually asymptomatic and the pathogenesis is unknown. They are incidental findings in blood films or imprints of tissues (i.e., kidneys).

Myxozoa

Members of the phylum Myxozoa that infect fish are in the class Myxosporea, often known as myxosporeans or myxozoan parasites. These organisms are characterized by their multicellular spores (usually measuring between 8–25 µm) which contain two polar capsules. Henneguya infection is an example of a disease caused by a myxosporean parasite that causes serious gill damage in freshwater fish with the formation of interlamellar cysts.

Microsporidians

Microsporidians are parasites of the order Microsporidia that have smaller spores (usually less than 7 mm) than the myxosporeans, and their spores contain only one polar capsule. Microsporidians, such as Glugea, can affect the skin and gills of fish where they create masses that resemble those caused by myxozoan parasites.

Monogeneans

Monogenean (skin or gill fluke) infestation occurs in both freshwater and marine fish. They are easily identified on wet-mount preparations of the gills, skin, or fins by their morphology and stretching and recoiling (caterpillar-like) movements. Monogeneans contain hooks or suckers. Most measure around 4 cm or less. The most important groups are gyrodactylid and dactylogyrid monopisthocotyleans. These monogeneans possess a haptor (attachment organ), have a direct life cycle, and are ectoparasites that live on skin, gills, and fins.

Digenean Trematodes

Digenean trematodes are endoparasites with an indirect life cycle. Digenean infestations are common in wild freshwater and marine fish. Diagnosis is based on the detection of the encysted metacercariae in wet-mount preparations of squash preparations, such as the skin, gill, fins, and internal organs. The encysted metacercariae have many of the characteristics of the adult digenean, such as suckers. Melanin pigment associated with the encysted digenean parasite results in "blackspot disease."

Crustaceans

Crustacean parasites include the branchiurans and copepods. Branchiurans are ectoparasites with a dorsoventrally flattened body, measure over 1 cm in length, and possess prehensile suckers that attach to the body of freshwater fish. Argulus spp., often called the fish louse, is the best known parasitic crustacean of the branchiurans group. Argulus is large enough (5–8 mm in length) to be seen with the naked eye. Copepods have a diversity of body forms with variable appendages. Lernaeid copepods are common copepod species of freshwater and marine fish. Lernaea spp., called the anchor worm, is a parasite of freshwater fish and the best known example of a parasitic copepod. This parasite is large enough to be seen with the unaided eye. The head of Lernaea spp. is a stellate anchor that embeds into the body musculature of the host fish. Females appear as Y- shaped parasites composed of an elongated trunk and two egg sacs hanging from the skin or fins of affected fish.

Nematodes

Parasitic nematodes are typically found as internal parasites of the gastrointestinal tract of fish. Fish are often intermediate hosts for nematodes; therefore, larval forms can be found in other tissues including the subcutis.

References

1.  Campbell TW, ed. Exotic Animal Hematology and Cytology. 4th ed. Ames, IA: Wiley Blackwell; 2015.

2.  Thrall MA, Weiser G, Allison RW, Campbell TW, eds. Veterinary Hematology and Clinical Chemistry. 2nd ed. Ames, IA: Wiley Blackwell; 2012.