Avian blood may contain various disease agents including viruses, bacteria, rickettsiae, protozoa, microfilariae, and rarely fungi. Except for viruses, these organisms often can be identified by microscopic examination of wet mounts, buffy coat, or blood smears; appropriate culturing techniques; or subinoculation of blood into susceptible birds. Microscopically, some are within blood cells (Plasmodium, Haemoproteus, Leucocytozoon, Atoxoplasma, Hepatozoon, Babesia, Aegyptianella), while others are free in the plasma (Trypanosoma, microfilariae, bacteria, spirochetes). None live exclusively in the blood; most are found in tissues but are present in blood during part of their life cycle. Some, such as microfilariae and Plasmodium, may have a periodi-city when numbers or stages of parasites are present at different times. In such cases, examining multiple smears at intervals will increase the likelihood of obtaining a diagnosis. Seasonal variations in infection rates relate to the activity of arthropod vectors. When possible, tissue cytology is also a useful adjunct to examination of blood. Most bloodborne organisms are either uncommonly or not associated with clinical disease. However, weakened or injured raptors infected with hemoprotozoa had higher mortality and delayed recovery compared with uninfected birds. Routine examination for bloodborne organisms should be included in the clinical and diagnostic procedures for any ill bird.

Thin blood smears should be made with blood directly from the bird if possible. Anticoagulants, storage, and cooling of the blood can distort protozoal morphology and introduce artifacts. A small drop of blood can be collected using a syringe and needle. The drop should be spread on a clean glass slide to make a thin smear. A good quality Romanowsky-type stain that gives good polychromatic coloration (eg, Giemsa stain) should be used. At least 200 oil-immersion fields (~20,000 RBC) for single smears or 100 for multiple smears from the same bird should be examined. Leucocytozoon and microfilariae are found around the periphery of smears and can be easily seen on low-power magnification.

Bloodborne organisms in plasma or WBC are concentrated in the buffy coat. The microhematocrit tube is cut just below the buffy coat above the packed RBC. The buffy coat should be expressed from the cut end with a small amount of plasma to make a suspension, and a thin smear prepared. Stained buffy coat smears are recommended for detecting bacteremia, spirochetes, and chronic Leucocytozoon, Trypanosoma, or Atoxoplasma infections. An excellent technique for identifying low numbers of motile organisms such as spirochetes and microfilariae is direct examination of the buffy coat by darkfield or phase contrast microscopy. The buffy coat and all of the plasma should be expressed onto a glass slide, and covered with a coverglass, which is depressed slightly to spread the buffy coat. The buffy coat/plasma interface should be examined with darkfield or reduced light microscopy to detect motile organisms.

Plasmodium infections can be determined by subinoculation. Ideally, birds of the same or a known susceptible avian species should be used, but this is often not practical. In general, canaries are used for detecting passerine infections, and turkeys are susceptible to most plasmodia that infect gallinaceous birds. Parasites remain viable in blood stored at 32°F (in ice) for at least 7 days. Inoculation IV is preferred and will result in earlier parasitemia, but any parenteral route can be used. Recipients should be examined twice weekly for a minimum of 4 wk if exposed IV; longer times are needed if other routes of inoculation are used. Spirochete, Aegyptianella, and bacterial infections can also be detected by subinoculation of infectious blood; bacteria can usually be identified by blood culture.

To make a diagnosis of infection with an intracellular blood protozoan on a thin blood film, it first should be determined that the “parasites” in question are neither normal nor artifact. The following should then be determined: the host cell and whether it is normal or deformed beyond identification, whether pigment granules (hemozoin) are present or absent, and whether merogony is occurring (see Bloodborne Organisms: Characteristics of Protozoa Encountered in Avian Blood). Identification of an organism beyond genus (or subgenus in the case of Plasmodium spp) is difficult and usually unnecessary for clinical purposes.

Table 1
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Characteristics of Protozoa Encountered in Avian Blood Protozoan Host Cell Pigment Present Merogony in Blood Plasmodium RBC Yes Yes Haemoproteus RBC Yes No Leucocytozoon RBC or WBC; distorted and enlarged often beyond recognition No No Atoxoplasma WBC; in nuclear indentation No Noa Hepatozoon b WBC, large, elongated oval shape No No Babesia b RBC No Noc a Multiple intracellular parasites may be seen in acute infections. b Uncommon to rare. c Babesia are pyriform and may be in a V, X, or fan pattern.

Last full review/revision March 2012 by Arnaud J. Van Wettere, DVM, MS, DACVP