Brittney Exarhos, LVT, RVT Toledo Zoo and Aquarium 2700 Broadway St. Toledo OH 43609 EXOTIC CLINICAL PATHOLOGY Veterinary technicians in a zoo setting often spend a lot of time in the lab. They must have an understanding of the cell morphology of multiple different species, and what is normal and abnormal for those species. Understanding common parasites, and how to recognize parasites on a blood smear or on a fecal slide is also very important. Hematology The first step in running blood work is to first collect the blood. Dealing with many different species means we must know where the best vessels are on all taxa. Not only do we need to know where to get the blood, but also how much blood we can safely take. In birds, 1% or less of the total weight of the bird can be removed in blood. This means that a 90 gram bird can safely have 0.9 ml of blood withdrawn from its body. The same rule applies to mammals. In reptiles and amphibians, we like to cut that number in half if possible. So a 90 gram snake would have 0.45 ml of blood withdrawn. Literature states that reptiles would most likely be okay with 1% of their body weight withdrawn in blood, but because of their slow metabolisms we like to stay on the cautious side. Fish are a little different because of the way they balance fluid within their body. The estimated total blood volume of a fish is 5% of their body weight and it is estimated that 30%-50% of the total blood volume can be collected from a healthy fish without negative side effects. No matter the species, we only require a minimum of 0.3 ml to run a full CBC and Chemistry in house. Having machines that can run samples at a small volume is important in a zoo setting. We have Abaxis equipment, the Vetscan being our chemistry machine and the HM5 being our CBC machine. The location of the blood draw always depends upon the actual species being dealt with and if the animal is handleable or sedated. In general, available vessels in a bird include the right jugular vein, wing vein (ulnar vein), and medial metatarsal. In snakes typically the tail is the only available vessel, but some practices still consider the heart as a blood sampling site. Chelonians are notoriously difficult to get blood from, but there are many sites that can be attempted. There is a sinus located directly under the carapace, called the subcarapacial venous sinus. Inserting a long needle midline where the neck meets the carapace and directing the needle upwards is typically where this sinus is located. A brachial, ventral coccygeal and dorsal coccygeal are all possible sites as well. Jugular samples are always the best when they can be collected (you will not get lymph contaminant using the jugular), but even the smallest of turtles are so strong it can be hard to withdraw their heads. The dorsal cervical sinus is the popular site for marine turtles. Blood is typically drawn from crocodilians via the lateral coccygeal vein or in smaller species, the ventral coccygeal vein. There is also an occipital sinus that blood can be collected from in crocodilians. Fish larger than 3 inches in length can have their blood collected from the ventral and lateral coccygeal vein, or the dorsal vascular sinus. Amphibians have a vein that runs along their ventral abdomen that can be sampled, also a lingual vein in larger species. Some larger
amphibians also have a sinus located behind their knee that can be sampled. On mammals big and small, the typical vessels are what we usually go for. The jugular vein is always the best pick for large sampling, and can be attempted on many species. Lateral saphenous, medial saphenous and cephalic veins are also available on most species. Tail blood draws work well for any animal with a large tail. Ear veins are good for elephants, rhinos and other animals with good auricle vessels. A femoral vein stick is typically used for primates but can also be attempted on other species. For bears, a good interdigital vessel can be sampled. Echidnas have a sinus at the end of their nose that can be sampled. If you are working with a new species for the first time, it is always good to research where the best sampling sites are prior to attempting to draw blood on the animal. For mammals, whole blood should be collected in lavender top tubes, and the rest of the blood should be placed in serum separator tubes unless plasma is needed for a certain test, then the blood should also be placed in a green top tube. For avians and reptiles we always place the whole sample in a green top tube (unless special testing requires serum). Since with these species we run manual CBCs, a lavender top tube is not necessary. The Avian/Reptilian CBC When running blood on any of our birds or reptiles, our goal is to be able to perform a PCV, total solids, WBC count (via hemacytometer), WBC differential and a chemistry. Using our equipment, we can perform all of the above using only 0.3mL of whole blood in a green top tube. Birds and reptiles have nucleated red blood cells and thrombocytes. This means that their blood cells confuse the automated machines we use for mammalian blood and give inaccurate values. Because of this, avian and reptilian blood must be ran manually. One of our first steps is to create two blood slides using the fresh blood. We prefer to use the squash technique rather than the push-pull technique for avian and reptilian blood slides, but either method is appropriate. The squash technique is accomplished by placing a drop of blood on the end of one slide and gently placing another slide on top of that drop of blood. Once the drop of blood has sufficiently been squished by the upper slide, the upper slide is gently pulled towards the end of the lower slide and a blood smear is formed. Using the squash technique allows for a larger reading field than the push-pull technique. In the push-pull technique the only readable portion is within the monolayer, whereas using the squash technique the whole slide can be used. This is beneficial if a patient has a low white blood cell count (like many reptiles) and finding 100 white blood cells in the monolayer may prove to be difficult. Running a PCV and TS on an avian or reptilian patient is the same as a mammalian patient. The normal values for each species varies greatly, and all values should be compared to a reference range made for the actual species you are dealing with. Finding the white blood cell count of reptilian and avian blood can be done in a direct (Nat and Herrick s) and semi direct manual method. We will be discussing the semi direct method which uses phyoxine B stain to find the total leukocyte concentration. This is a specific stain that only stains the granules of heterophils and eosinophils. Pre-made kits (leukopet system) can be ordered and come with vials with the stain already measured out. The kits also provide a 25 ul
pipette. Using the provided pipette, 25 ul of blood is inserted into the vial of stain, providing a perfect 1:32 diluted ratio. A Neubauer-ruled hemacytometer is also needed, and must be charged with the stained sample on both sides. The Neubauer-ruled hemacytometer is then read under the microscope at 100x magnification. All 9 large squares on both sides of the hemacytometer should be counted systematically. Record each side s number. These two numbers should be within 10% of each other, if not a new hemacytometer should be charged and recounted. If the numbers are within 10% of each other, add those two numbers together and you have found your raw count. The raw count is not your white blood cell count, and is only a number that is used in an equation once the differential has been read and the percentages of heterophils and eosinophils has been found. Once the differential is done, the following equation will be how the white blood count is calculated: (Raw count from hemacytometer x 1.1 x 16 x 100) %heterophils + %eosinophils on differential For example: On one side of the hemacytometer I have counted 120 stained WBC, on the other side of the hemacytometer I have counted 109 stained WBC. That gives me a raw count of 229. On the differential I counted 36 heterophils, 52 lymphocytes, 5 eosinophils and 7 monocytes. So I multiply my raw count, 229 by 1.1 x 16 x 100 and then divide that number by 41 (the number of heterophils and eosinophils) and get 9,830.24, which is your white blood cell count. A disadvantage to this method is when a blood sample is very high in lymphocytes, the accuracy of the number gets skewed. When a sample is high in lymphocytes, an estimated WBC count should also be performed. This is done the same way in birds and reptiles as it is in mammals. Under 400x, count the WBCs seen on 10 fields, times that number by 2,000 and you get your estimated WBC count. Leukocytes Bird leukocytes include lymphocytes, monocytes, heterophils, eosinophils and basophils. An avian lymphocyte resembles a mammalian lymphocyte. They are round cells, typically with a large nucleus and a small amount of blue cytoplasm showing. A typical avian lymphocyte looks very similar to an avian thrombocyte, but the color of the cytoplasm is different. A thrombocyte will have colorless cytoplasm, whereas the lymphocyte cytoplasm is blue. Large lymphocytes can also be seen in an avian blood smear and can be confused with monocytes. The nucleus in these large lymphocytes should still be rather round and condensed, and the cytoplasm should still be a blue color without any vacuoles. Reactive lymphocytes do occur in avian blood and may be a sign that some sort of infectious disease process is happening. The reactive lymphocytes can be small to medium in size, and will appear darker in color than your average lymphocyte. Avian monocytes also resemble mammalian monocytes. They can be round or amoeboid, and the nucleus also varies in shape. The nucleus is typically more pale than a lymphocyte nucleus, and the cytoplasm is abundant with a blue-gray color. Heterophils are the avian equivalent to the mammalian neutrophil. It is also the most abundant granulocyte of most avian species. The morphology of the heterophil varies by species, but generally they are round with a multi-lobed nucleus. The granules often hide the nucleus, and the size and shape of these eosinophilic granules depends upon the species, but they are typically rod-shaped or round. Heterophils often get confused for eosinophils. Immature and toxic heterophils can be seen in a
blood smear. Immature heterophils are also called band cells, and can be identified by a nonsegmented nucleus. These can be hard to identify because the eosinophilic granules of the heterophil usually hide the nucleus so to truly get a good band cell count a nuclear stain, such as hematoxin, should be used. Heterophils can exhibit toxic changes just like a neutrophil does, and this can signify severe systemic illness. A toxic heterophil typically has cytoplasmic basophilia, cytoplasm vacuolation, abnormal granulation or degeneration of the nucleus. The avian eosinophil looks very similar to the heterophil. They are similar in size and both have eosinophilic granules that can be either rod-shaped, or round depending on the species. Eosinophils do tend to have more swollen looking granules, and also tend to stain more intensely than heterophils do. The avian basophil is much more common to see in a peripheral blood smear than in a mammal blood smear. The basophil contains very dark staining granules that often obscure the non-lobed nucleus. The avian basophil looks very much like a mammalian mast cell. A stress leukogram is commonly seen in avian patients and is associated with leukocytosis, moderate mature heterophilia and lymphopenia. Birds can get very stressed when being caught up and restrained so it is advised that blood is collected as soon as possible during a procedure to decrease the likelihood of creating a stress leukogram. Reptilian leukocytes also include heterophils, eosinophils, basophils, lymphocytes and monocytes (also azurophilic monocytes). Reptilian heterophils are considered large, and have colorless cytoplasm with eosinophilic rod shaped granules. The nucleus of the heterophil is not typically centered and is round or oval in shape. Toxic changes also occur in the reptilian heterophil and is characterized by blue cytoplasm with purple-colored granules and vacuoles. The reptilian eosinophil is also considered a large white blood cell. Snakes have the largest eosinophils whereas lizards have the smallest. The cytoplasm in these cells is light blue and contains a large number of sphere shaped pink granules. Basophils in reptiles are small and round. They contain dark colored granules that obscure the nucleus. Turtles and crocodiles often have larger basophils than lizards. The reptilian lymphocyte closely resembles the mammalian and avian lymphocyte, a round cell with blue cytoplasm and a large, darkly staining nucleus. Monocytes are typically the largest leukocyte in circulation. They vary in shape, and have a either round, oval or lobed nucleus that stains lighter than a lymphocyte nucleus. The cytoplasm stains pale blue-grey and may be foamy in appearance. There is a type of monocyte that is often found in low numbers in lizards, chelonians and crocodilians and in high numbers in snakes. This monocyte is referred to as an azurophil and is typically irregularly shaped and smaller than an average monocyte. The cytoplasm is basophilic and lavender-blue in color, and contains small pink staining granules. Vacuolation can be seen in azurophils and is considered normal and not a toxic change. Many people do not categorize azurophils as separate cells, and consider them just a monocyte. Their purpose is not fully understood, but it is thought that these may be an immature form of the monocyte. Erythrocytes Mature avian erythrocytes are larger than mammal erythrocytes and are elliptical with an elliptical, purple staining nucleus centrally located. Although avian erythrocytes are larger than mammalian erythrocytes, they are smaller than reptilian ones, which are also elliptical with elliptical nuclei. Evaluation of erythrocyte morphology is an important part of reading a blood
smear. Special attention should be paid to the size and color of erythrocytes. Microcytosis, macrocytosis and anisocytosis can all be seen in exotic erythrocytes. A slight variation in size is considered normal, but a greater degree of anisocytosis is indicative of regenerative anemia and is also associated with polychromasia. Reticulocytes also circulate in avian and reptilian blood and can occur in low numbers in a healthy animal. A reticulocyte is a young red blood cell that has made it into circulation early. They tend to be smaller in size and less elongated than the mature erythrocytes. If an erythrocyte lacks color and has a larger area of cytoplasmic pallor it is considered hypochromatic and may be indicative of an iron deficiency when seen in higher numbers. The shape of the nucleus should also be paid attention to. Micronuclei and nuclear budding may be a sign of environmental genotoxic exposure. If a blood smear has a large number of binucleated red blood cells it may be a sign of neoplasia, genetic or viral disease. Also, occasionally an erythrocyte may be missing the nucleus and is called an erythoplastid. Anucleated erythrocytes can be seen in a normal avian or reptilian blood smear. Parasitology Parasites are a big concern in a zoo setting, and each animal is on a fecal analysis schedule. Each animal gets both a direct smear and a centrifuged fecal sample. We look for protozoan organisms on a direct smear, evaluating the organism s movement on the slide and identifying them. A centrifuged fecal sample is considered gold standard for finding parasitic ova, and exotic animals are no exception. Occasionally we perform quantitive fecal counts, in which the exact number of parasitic ova are counted per gram of feces. Quantitative fecal counts are useful on species of animals that already have a known parasitic population that cannot be fully dewormed. A monthly quantitative can ensure that the parasitic population stays beneath an acceptable number, and if the number creeps up the animal is then dewormed. Our exotic mammals get many of the same parasites as domestic animals. The most commonly seen parasites in birds are giardia, capillaria, coccidia and occasionally ascarids. In reptiles, ascarids, coccidia (specifically cryptosporidium is of concern and can be identified with an acid fast stain), pinworms and strongyloides can be found among other parasites. Conclusion Many of the same in house diagnostics that are performed on dogs and cats can be performed on birds, reptiles, and other exotic pets. With the proper equipment and knowledge, veterinary technicians can provide these helpful diagnostics to their veterinarians so better care can be provided to even the oddest of animals.