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Blood testing exotic animals: avian and reptile procedures

Author : Lesa Longley

Categories : Vets

Date : June 1, 2009

Owners of exotic pets frequently expect veterinary clinicians to take blood samples andinterpret results (Table 1) when dealing with ill animals, and understand normal values inhealthy individuals. This article will cover some haematology basics in exotic species,focusing on birds and reptiles.

Blood sampling

Before any analysis can be performed, a sample must be obtained. Depending on the species andpatient size, this may be the most difficult part of the procedure. Birds may require generalanaesthesia (with isoflurane, for example) for phlebotomy (Table 2). Smalldiameter needles should beutilised in birds to avoid damaging fragile avian veins. Gentle pressure should be applied aftervenepuncture, to reduce the risk of haemorrhage.

Long needles are required to access the ventral coccygeal vein in larger lizards (such as the greeniguana, Iguana iguana). In reptiles, sampling from several sites may produce a samplecontaminated with lymph (most notably in the dorsal coccygeal vein and subcarapacial sinus in Chelonia), with the dilution affecting the results obtained.

A maximum of 10 per cent of the total blood volume may be sampled safely from a healthyindividual. In birds, blood volume is approximately 10 per cent of bodyweight and, thus, a maximumof one per cent of bodyweight may be sampled (such as 3.5ml from a healthy 350g parrot). Theblood volume is five to eight per cent of bodyweight in reptiles. Therefore, a maximum of 0.5 percent bodyweight may be sampled (such as 0.5ml from a 100g snake). Smaller samples should betaken from ill animals that may be unable to cope with such large acute blood losses. It can beuseful to inject a similar volume of fluid intravenously after sampling to help maintain circulatoryvolume.

EDTA is used as the anticoagulant for blood from most species. Partial haemolysis may occur withsome species (consult other references for species lists), including some birds and Chelonia. If indoubt, use a heparin sample of whole blood for haematological assessment. In all cases, prepare afresh blood smear for morphological analysis and differential white cell count. In very smallpatients, a basic health check can be performed on a microhaematocrit tube and blood smear

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(estimated and differential white cell counts).

In-house haematology

While several UK laboratories offer excellent haematology services for exotics, there are timeswhen it is useful to perform in-house tests (such as when dealing with an emergency case at theweekend, or with an acutely ill bird). In these cases, it is advisable to run at least a screening panel(possibly before sending a sample to a commercial laboratory).

• Preparation of blood films

A small drop of fresh blood directly from the syringe (with the needle removed) is applied towardsone end of a clean microscope slide. A “spreader” slide is used to produce a bullet or flame-shaped film. Alternatively, two slides may be crossed and pulled apart to produce a blood smear ofeven thickness. The smear is air-dried by being shaken rapidly. Smears are stained in the samemanner as other species — with Romanowsky stains, for example.

• Whole cell counts

Automated counters used for mammals cannot be used for avian and reptilian haematology, due tothe similar cell sizes and the presence of nuclei in all blood cell types. Manual blood counts can beperformed using various techniques, such as a Neubauer-ruled haematocytometer chamber.

A rough estimate of the white cell count (x109/L) can be obtained from a smear. A monolayer ofcells is assessed (in bullet-shaped smears, this is near the feathered edge), counting the number ofwhite blood cells (WBCs) in 10 fields under medium (x400) magnification. The average iscalculated per field, and the result is multiplied by two.

To assess cell morphology and obtain a differential white cell count, the smear is examined underoil immersion (x1,000). Red blood cells (RBCs) and WBCs are assessed on the basis of size,shape and colour. Nuclei, cytoplasmic granules and cellular inclusions should also be described. Apacked cell volume (PCV) value can be obtained after centrifuging blood in a microhaematocrittube. Several factors affect PCV values, including RBC size and number, and plasma volume(dehydration). PCV is normally relatively high in birds, with anaemia present when it is less than 35per cent, and haemoconcentration when it is more than 55 per cent. The normal reptile PCV is 20to 40 per cent (Frye, 1991).

•Factors affecting haematology

Stress and disease processes will affect haematological values. Species, gender, age andenvironment (particularly in reptiles), diet and physiological variations are also seen.

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In reptiles that hibernate (including many tortoise species), haematological variations duringhibernation may include a fall in the total blood count, lower numbers of heterophils andlymphocytes, and higher numbers of eosinophils. There is usually little seasonal variation inbasophil and monocyte numbers.

Many male reptiles have higher erythrocytic values compared to females – for example, PCV andhaemoglobin concentrations in African hingeback turtles (Kinixys erosa). Conversely, female greeniguanas have higher haemoglobin, PCV and mean corpuscular haemoglobin concentration countsthan males.

Blood cells

Peripheral blood cells comprise erythrocytes (RBCs), leukocytes (WBCs) and haemostatic cells(these are called thrombocytes in birds and reptiles, rather than platelets as in mammals). Bloodsmears are important, as cell morphology (including size) varies greatly between taxonomicgroups, species and disease.

• Red blood cells

These cells are elliptical and, unlike their mammalian counterparts, are nucleated. The nucleus iscentrally situated, oval or round, and contains densely clumped chromatin. The nucleus may havean irregular outline in reptiles. Avian RBCs are larger than in mammals, and reptilian cells largeragain – size also varies between species.

RBC lifespan varies between species. It is much shorter in birds (28 to 35 days in chickens– Sturkie, 1976) compared to mammals, and longer in reptiles (600 to 800 days – Sypek andBorysenko, 1988; Frye, 1991).

Basophilic inclusions are commonly seen within the cytoplasm of reptilian RBCs. These may beinfectious agents (haemoparasites or viral inclusion bodies) or degenerate organelles.

Immature RBCs are often seen in the peripheral circulation of birds and reptiles. These cells havemarked polychromasia and round nuclei. Reticulocytes have a ring of aggregated reticulum at theperiphery of the nucleus. Large numbers may be associated with regenerative anaemia.

Regenerative responses may also be seen in reptiles waking from hibernation, and those withinflammatory disease or malnutrition. Changes include moderate to marked anisocytosis andpoikilocytosis. Increased polychromasia and immature RBCs may be seen in young reptiles orthose undergoing ecdysis.

• White blood cells

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The aim of blood smear analysis in veterinary practice is often to identify changes in WBCs thatreflect disease processes. However, it can be difficult for the first-opinion veterinary practitioner tobecome familiar with WBCs from birds and reptiles, as there are great inter-species variations inthe appearance of normal cells. Relative and absolute numbers of WBCs also vary betweenspecies (the clinician is referred to other texts for normal values).

– Heterophils. In most species, these are the predominant WBCs. In normal mature heterophils,the cytoplasm is clear and contains eosinophilic granules. The appearance of these granules variesbetween species, and also with disease processes. In most reptiles, the heterophil nucleus isspherical or oval, but is lobed in birds and some lizards – all contain coarse, clumped chromatin.

Immature heterophils have increased cytoplasmic basophilia, non-segmented nuclei and granulesoccupying less than half the cytoplasmic volume, and they display pleomorphism. The presence ofsuch immature heterophils in the peripheral blood (comparable to the “left shift” in mammals)suggests inflammatory disease. An overwhelming inflammatory response (often with infection) mayresult in these immature cells and heteropaenia.

Heterophils are functionally equivalent to mammalian neutrophils. They participate in inflammatorylesions and have phagocytic activity. Heterophils respond to inflammation by undergoing changesreferred to as “toxic change” – the presence of these changes is typically associated with aworsening prognosis. Changes seen may include altered granulation (such as degranulation anddeeply basophilic and/or coalescing granules), cytoplasmic basophilia, vacuolation in the cytoplasmand/or the presence of a band nucleus.

– Eosinophils. Granules within the cytoplasm of eosinophils are typically round and stronglyeosinophilic. However, the granules may be oval or elongate, and in some species (such asiguanas and African grey parrots) are basophilic. The cytoplasm shows some basophilia. Nucleiare usually lobed in birds, and elongated to lobed in reptiles. There is great species variation ineosinophil numbers.

– Basophils. These small, spherical cells have a non-lobed nucleus. The granules are large, roundand basophilic (sometimes obscuring the nucleus). Normal cell numbers and size vary greatlybetween species.

– Lymphocytes. These cells are round, as is the (occasionally indented) nucleus, which issurrounded by a rim of weakly basophilic cytoplasm. The cytoplasm does not normally contain anyvacuoles or granules. On blood smears, lymphocytes are often seen moulded around or adhered toneighbouring RBCs. As with other WBCs, morphological changes are seen when lymphocytesbecome activated in disease (such as antigenic stimulation in infectious disease or lymphocyticleukaemia). These “reactive” changes may include clumping of nuclear chromatin and a deeplybasophilic cytoplasm.

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– Monocytes. These are large, round or amoeboid in shape, with a speckled or basophiliccytoplasm that contains vacuoles or fine eosinophilic granules. The nucleus is centrally placed andvaries in shape. Compared to lymphocytes, monocyte nuclei have less chromatin clumping andthere is more cytoplasm.

In a similar fashion to mammalian monocytes, in birds and reptiles these cells are phagocytic andprocess antigens.

Reptile monocytes with azurophilic staining (commonly called “azurophils”) are functionally thesame as those without this staining.

Reactive monocytes have phagocytic activity and migrate into tissues to become macrophages.They have an important role in antigen processing.

• Thrombocytes

These cells are functionally similar to mammalian platelets. They are ellipsoid in shape, with acentrally located dark nucleus and nonstaining or weakly basophilic cytoplasm. Thrombocytesusually contain less cytoplasm than RBCs, and may clump on smears. Activated thrombocytesmay show changes, such as irregular cytoplasmic margins and cytoplasmic pseudopodia. Nuclearand cytoplasmic changes may occur, and vacuoles and degranulation may be present.

Haemoparasites

Protozoan parasites are commonly seen on fresh blood smears from birds and reptiles. Many areasymptomatic infections. In cases where clinical signs are seen, they usually relate to anaemia,and include lethargy and anorexia.

• Birds

Haemoproteus are common in many wild birds. The mature gametocytes wrap around the RBCnucleus with a characteristic “halter shape”.

Plasmodium infection may result in clinical malaria in some species, including canaries, raptors andpigeons. Several life stages are seen in RBCs, WBCs and thrombocytes. Trophozoite stages resultin a “signet ring” appearance in the host cell.

Gametocytes from Leukocytozoon parasites are often seen pushing the host cells’ nuclei to theedge of the cell in many wild birds. Some species, such as young waterfowl, are highly susceptibleto disease. Microfilaria may be seen extracellularly in peripheral blood.

Atoxoplasma is uncommon but highly pathogenic. This coccidian parasite affects passerine birds.

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Sporozoites are seen mostly within lymphocytes as intracytoplasmic inclusions indenting the hostcell nucleus.

• Reptiles

Haemogregarine gametocytes are seen as intracytoplasmic sausage-shaped structures in RBCs.Trypanosomes are large extracellular protozoa. Microfilaria are a common incidental finding,particularly in wild-caught chameleons.

Gametocytes of Plasmodium have refractile pigment granules. Schizogony may be seen, andtrophozoites are signet ring structures in the cytoplasm of RBCs. Infection may result in severehaemolytic anaemia.

References and further reading

Frye F L (1991). Hematology as applied to clinical reptile medicine. In Frye F and Malabar FL, Biomedical and Surgical Aspects of Captive Reptile Husbandry, Krieger Publishing, 1:209-277.Fudge A M (1999). Laboratory Medicine: Avian and Exotic Pets, Saunders. Harcourt-BrownN and Chitty J (2005). Manual of Psittacine Birds (2nd edn), BSAVA, Quedgeley,Gloucester.McArthur S, Wilkinson R and Meyer J (2004). Medicine and Surgery of Tortoises andTurtles, Wiley Blackwell. Sturkie P D (1976). Blood: physical characteristics, formedelements, hemoglobin, and coagulation. In Sturkie P D, Avian physiology, New York,Springer Verlag: 53-75.Sypek J and Borysenko M (1988). Reptiles. In Rowley A and Ratcliffe N, Vertebrate BloodCells, Cambridge, Cambridge University Press: 211-256.Thrall M A, Campbell R W, DeNicol D et al (2006). Veterinary Hematology and ClinicalChemistry, Blackwell Publishing, Ames, Iowa.

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Far left: a blood smear from an Amazon parrot, showing red blood cells and a heterophil.

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Near left: blood smear from an African spurred tortoise, showing red blood cells and abasophil.

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Right: a blood smear from a Fischer’s chameleon, showing a microfilarial parasite amongred blood cells.

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Most haematology profiles can be run on a blood sample stored in EDTA. In some species,EDTA causes haemolysis and heparin should be used as the anticoagulant.

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TABLE 1. Examples of haematology results for common problems in birds and reptiles

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TABLE 2. Phlebotomy sites in birds and reptiles

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