Atlas of Clinical Avian Hematology Disclaimer

The information on this publication comprises taken to verify the accuracy of the content at general statements based on scientifi c research. the time of publication, readers are advised to The reader is advised and needs to be aware confi rm that the infor mation complies with that such information may be incomplete or the latest knowledge and best standards of unable to be used in any specifi c situation. No practice and safety. To the extent permitted by reliance or actions should be made on infor- law, the publishers and authors exclude all mation contained in this publication without liability to any person for any consequences, seeking prior expert professional, scientifi c including but not limited to damages, costs, and technical advice. In addition, medical expenses and any other compensation, arising knowledge is constantly changing, particularly directly or indirectly from using this publica- with regard to procedures, equipment and tion (in part or whole) or any information or treatment. While all appropriate care has been material contained in it. Atlas of Clinical Avian Hematology

Phillip Clark Wayne S. J. Boardman Shane R. Raidal

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Library of Congress Cataloging-in-Publication Data Clark, Phillip, 1966– Atlas of clinical avian hematology / Phillip Clark, Wayne S.J. Boardman, Shane Raidal. p. cm. Includes bibliographical references and index. ISBN 978-1-4051-9248-4 (hardback : alk. paper) 1. Birds–Diseases–Diagnosis. 2. Veterinary hematology–Atlases. I. Boardman, Wayne. II. Raidal, Shane. III. Title.

SF994.C53 2009 636.5089’615–dc22 2008047432

A catalogue record for this book is available from the British Library.

Set in 10.5/12.5 pt Sabon by SNP Best-set Typesetter Ltd., Hong Kong Printed in Singapore

1 2009 Contents

Dedication vii Leukocytes 40 Authors viii Thrombocytes 49 Foreword ix Hematopoiesis 51 Preface xi Acknowledgements xiii 3 Particular hematological characteristics of birds 53 Introduction 53 1 Collection and handling of blood Anseriformes (ducks, geese samples 1 and swans) 54 Introduction 1 Apodiformes (swifts and Taxonomy 2 hummingbirds) 57 Safety aspects 5 Caprimulgiformes (frogmouths, Effects of anesthesia on the goatsuckers and owlet-nightjars) 57 hematological characteristics Charadriiformes (gulls and of birds 5 shorebirds) 58 Volume of blood 6 Ciconiiformes (herons, ibis and storks) 60 General aspects of collection of blood Coliiformes (mousebirds) 61 from birds 6 Columbiformes (pigeons and doves) 62 Sites for vascular access in birds 7 Coraciiformes (kingﬁ shers, Anticoagulants 15 bee-eaters, hornbills, motmots, Blood ﬁ lms 16 rollers and todies) 64 Post-collection artifacts 23 Cuculiformes (cuckoos, roadrunners Measurement of hematological cells 27 and turacos) 66 Falconiformes (falcons, hawks and 2 General hematological eagles) 66 characteristics of birds 33 Galliformes (grouse, megapodes, Introduction 33 curassows, pheasant, partridge, Erythrocytes 33 quail and turkey) 71 vi Contents

Gaviiformes (divers and loons) 74 4 Physiological and pathological Gruiformes (bustards, button quail, inﬂ uences on the hematological coots and cranes) 74 characteristics of birds 97 Opisthocomiformes (hoatzin) 77 Introduction 97 Passeriformes (song birds) 77 Physiological effects on the Pelecaniformes (cormorants, frigate hematological characteristics birds, gannets, pelicans and of birds 97 tropicbirds) 81 Pathological effects on the Phoenicopteriformes (ﬂ amingos) 82 hematological characteristics Piciformes (toucans and of birds 100 woodpeckers) 83 Podicipediformes (grebes) 85 5 Hemoparasites of birds 125 Procellariiformes (albatrosses, Introduction 125 fulmars, petrels and shearwaters) 85 Detection of hemoparasites 127 Psittaciformes (cockatoos, lories, Morphology of hemoparasites 129 macaws and parrots) 86 Sphenisciformes (penguins) 91 Strigiformes (barn owls and “typical” owls) 92 References 139 Struthioniformes (cassowaries, Species list: alphabetically by emus, kiwis, ostriches and rheas) 94 common name 163 Tinamiformes (tinamous) 96 Species list: by order 168 Trogoniformes (trogons and Glossary 173 quetzals) 96 Index 177 Dedication

This book is dedicated to our children:

Michael, Lachlan, Cameron & Lauren Clark Jamie Boardman Lachlan & Jackson Raidal Authors

Phillip Clark BVSc (Hons), PhD (Melb), DVSc Shane R. Raidal BVSc, PhD (Syd), FACVSc (Murd), MACVSc, Diplomate ACVP, Diplo- Associate Professor in Veterinary Pathobiol- mate ECVCP ogy, School of Agricultural and Veterinary Sci- P. O. Box 288, Daw Park, S. A. 5041, Australia. ences, Charles Sturt University, Wagga Wagga, N.S.W. 2678, Australia.

Wayne S. J. Boardman BVetMed (Lond), MRCVS, MACVSc Head of Veterinary Conservation Programs, Zoos South Australia, Adelaide, S.A. 5000, Australia. Foreword

This planet is home to a wide range of species scientists, zoologists, comparative hematolo- of birds, many of which are wild, others gists, ornithologists and evolutionary biolo- captive, and a smaller number domesticated. gists. Its excellent illustrations assist the reader Regardless of their habitat, there are occa- in expanding their understanding of the sions when, through illness or injury, they are complex processes involved in producing seen by veterinarians. In such circumstances, blood and maintaining its integrity and health. the analysis of their blood can assume a Given that species’ differences can affect the matter of great signifi cance. Until the avail- morphological appearance of blood cells, ability of this book, the information on avian which can also be perturbed by disease pro- hematology was fragmented and not easily cesses, the layout of the illustrations in this accessible, nor was it assembled in an orderly book is such that the reader is presented with manner. the relevant information to guide them through This volume, by three experts, provides these issues. such a logical compilation, which is both At a time when climatic changes are occur- extensive and yet organised in such a manner ring, causing stress and altered migratory pat- that it takes the reader from the collection of terns, and shifts in the niches occupied by a blood sample through a descriptive analysis species are emerging, an atlas covering the of the hematological characteristics of avian global avian species will be of value. species in general. It also assists the reader in The authors are to be congratulated on a attributing specifi c characteristics to some fi ne volume that will be of value to many who orders and explores the patho-physiological have dealings with “our feathered friends”. I characteristics observed in response to disease, commend the volume to you as a fi ne authori- including a chapter on hemoparasites. tative volume. While the atlas is of great value in the pursuit of diagnostic information, it will also be of Emeritus Professor David de Kretser benefi t to a wider readership, such as medical Governor of Victoria

Preface

Many species of birds are maintained by avi- these basic facts are not known for the major- culturalists, zoological institutions, Govern- ity of avian species, necessitating extrapola- ments and other organizations for captive tion from knowledge of the characteristics of breeding programs, research and to educate domestic species of birds and relatively few and inspire those that have the opportunity to “milestone” studies undertaken on “impor- view them. tant” or available species of birds. The task of maintaining the health of the This book aims to add to the available many species of birds requires constant atten- information by illustrating the general hema- tion and great effort. Many birds do not tological characteristics of birds, the hemato- exhibit clinical signs until late in a disease logical characteristics of selected species of process. Consequently, diagnostic procedures, birds in health and the hematological response such as hematology, may provide valuable to disease by birds, using clinical cases from insight into the health status of an individual many different species with a range of clinical before clinical signs become apparent, and disorders. allow early intervention. In this way, further fragments of informa- Interpretation of hematological results is tion are organized to increase our understand- most effective when the characteristics of a ing of the hematological response to disease particular species, both in health and in that may be encountered in these fascinating response to disease are known. Unfortunately, animals and further ensure their health.

Acknowledgements

We are grateful to the many people whom We would like to thank the many individu- have assisted us in our endeavors that led to als that have contributed material, knowledge, the production of this atlas. We would partic- encouragement, or constructive criticism ularly like to thank the staff of the clinical that have improved the quality of this book, pathology laboratories of Massey, Michigan including: Rachel Wicks, Pat Shearer, Nicolai State, Murdoch and Charles Sturt universities Bonne, Tim Hyndman, Margaret Sharp, Ken for their assistance in analysing blood samples Richardson, Peter Fallon, Trish Flemming, from clinical cases. We are also extremely Todd McWhorter, Kathryn Napier, Sue grateful to the staff of the Veterinary Depart- Jaensch, David Schulz, Peter Holz, Padraig ment of the Zoological Society London for Duignan, Richard Norman, Matthew collecting many blood samples to assist this Johnston, Jonathon Cracknell and Claire project and the faculty and staff of the clinical Cunningham. pathology laboratory of the Royal Veterinary We are extremely grateful to Carmel Clark College, University of London, for preparing for proof reading the manuscript. many blood ﬁ lms.

Collection and handling of blood1 samples

INTRODUCTION of physiological factors (such as age, sex, reproduction, season, nutrition, habitat and migration) and pathological factors (such as The class Aves contains more than 9,000 infectious disease) may have upon the hema- extant species of birds in 199 families. They tological characteristics of birds, has not been have evolved a wide range of morphologi- fully resolved. Hematology has an important cal, physiological and functional adaptations role in the assessment of the health of birds. that have allowed them to fi ll every ecological Many birds do not express clinical signs until niche imaginable. Hematology, the study of late in a disease process and the signs that they blood, has provided insight into some of do exhibit may be subtle and non-specifi c. the physiological characteristics of birds and Consequently, the use of hematological assays how they differ from mammals, the latter may aid in the early recognition of disease, being the subject of the overwhelming major- thus facilitating the best opportunity for man- ity of studies of “comparative” hematology. agement and therapy to resolve the process. Interestingly, the total number of species of Furthermore hematology may, by character- birds is approximately twice the number of izing the type of response, aid in the deter- species of mammals; consequently, many mination of the inciting etiology and allow hematological differences within the class institution of targeted therapy. Aves should be expected (as well as differences The hematological response to disease is in the hematological characteristics between complex and will depend on the inciting cause, birds and mammals and other classes of magnitude and duration of the disease process, vertebrates). the bird’s intrinsic hematological characteris- However, the hematological characteristics, tics and its ability to respond to a particular in health, have not been documented for the challenge. vast majority of bird species. Furthermore, in The accurate interpretation of hemato- most instances, the impact that the interaction logical data is facilitated by knowledge of 2 Atlas of Clinical Avian Hematology the hematological characteristics of healthy TAXONOMY members of the species and how those characteristics change in response to a particular disease. Unfortunately, for most species of For the purposes of this book, we have under- birds, neither their hematological characteris- taken to examine the hematological character- tics in health nor their hematological response istics of birds within each order of the Aves. As to disease have been documented. Conse- differing authorities have confl icting classifi ca- quently, the nuances in hematological charac- tion systems, we have adopted the classifi cation teristics exhibited in response to disease used in the “Handbook of Birds of the World” by a particular bird, may be diffi cult to (del Hoyo et al. 1992). This is outlined in Table recognize. 1.1. Consequently, the ensuing sections that This atlas of clinical avian hematology pro- describe the collection of blood and the mor- vides examples of the hematological response phological characteristics of hematological cells to disease, with an emphasis on the morphol- are ordered so that, wherever possible, material ogy of hematological cells, in a wide range from signifi cant and representative species of of non-domestic bird species. We consider each order has been included. Unfortunately, the assessment of the morphology of hemato- due to limitations in the material available it logical cells, both to ensure the accurate clas- was not possible to describe the hematological sifi cation of those cells and to identify the characteristics of birds within all of the orders. presence of any morphological atypia, an Furthermore, we recognize that there may be extremely important component of the ana- distinct differences in the hematological charac- lysis of avian blood samples. The latter is teristics of birds between species of birds placed particularly important because, as previously within the same order, family or even the same mentioned, of the uncertainty in the applica- genus. We hope that, in time, studies of indi- bility of available reference intervals to any vidual species will document the nuances in the specifi c bird. hematological characteristics of birds.

Table 1.1 Classiﬁ cations of orders with the Aves. Adapted from “Handbook of the Birds of the World” (1992–2009).

Order Family Common names

Anseriformes Anhimidae Screamers Anatidae Ducks, geese, swans Apodiformes Apodidae Swifts Hemiprocnidae Tree-swifts Trochilidae Hummingbirds Caprimulgiformes Steatornithidae Oilbird Podargidae Frogmouths Nyctibiidae Potoos Aegothelidae Owlet-nightjars Caprimulgidae Nightjars

(continued) Collection and handling of blood samples 3

Table 1.1 (continued)

Order Family Common names

Charadriiformes Jacanidae Jacanas Rostratulidae Painted-snipe Dromadidae Crab plover Haematopodidae Oystercatcher Ibidorhynchidae Ibisbill Recurvirostridae Avocets, stilts Burhinidae Thick-knees Glareolidae Coursers, pratincoles Charadriidae Plovers Scolopacidae Sandpipers, snipe Thinocoridae Seedsnipe Chionididae Sheathbills Stercorariidae Skuas Laridae Gulls, terns Rynchopidae Skimmers Alicidae Auks Ciconiiformes Ardeidae Herons Scopidae Hamerkop Ciconiidae Storks Balaencipitidae Shoebills Threskiornithidae Ibises, spoonbills Coliiformes Coliidae Mousebirds Columbiformes Pteroclididae Sandgrouse Columbidae Pigeons, doves Coraciiformes Alcenidae Kingﬁ shers Todidae Todies Momotidae Motmots Meropidae Bee-eaters Coraciidae Rollers Brachypteraciidae Ground-rollers Leptosomatidae Cuckoo-rollers Upupidie Hoopoe Phoeniculidae Woodhoopoes Bucerotidae Hornbills Cuculiformes Musophagidae Turacos Cuculidae Cuckoos Falconiformes Cathartidae New world vultures Pandionidae Osprey Accipitidrae Hawks, eagles Sagittariidae Secretary bird Falconidae Caracaras, falcons Galliformes Megapodiidae Megapodes Meleagridae Turkeys Odontophoridae New world quail Cracidae Guans, chachalacas, curassows Phasianidae Pheasants, grouse Numidae Guinea fowl

(continued) 4 Atlas of Clinical Avian Hematology

Table 1.1 (continued)

Order Family Common names

Gaviiformes Gaviidae Divers Gruiformes Mesitornithidae Mesites Turnicidae Button quail Pedionomidae Plains-wander Gruidae Cranes Aramidae Limpkin Psophiidae Trumpeters Rallidae Rails, coots Heliornithidae Finfoots Rhynochetidae Kagu Eurypigidae Sunbittern Cariamidae Seriemas Otididae Bustards Opisthocomiformes Opisthocomidae Hoatzin Passeriformes Six sub-orders (91 families) Song birds Pelecaniformes Phaethontidae Tropic birds Pelecanidae Pelicans Sulidae Gannets, boobies Phalacrocoracidae Cormorants Anhingidae Darters Fregatidae Frigatebirds Phoenicopteriformes Phoenicopteridae Flamingos Piciformes Galbulidae Jacamars Bucconidae Puffbirds Capitonidae Barbets Indicatoridae Honeyguides Rhamphastidae Toucans Picidae Woodpeckers Podicipediformes Podicipedidae Grebes Procellariiformes Diomedeididae Albatross Procellariidae Petrels, shearwaters Hydrobatidae Storm-petrels Pelecanoididae Diving-petrels Psittaciformes Loriidae Lories, lorikeets Cacatuidae Cockatoos Psittacidae Parrots Sphenisciformes Spheniscidae Penguins Strigiformes Tytonidae Barn owls Strigidae Typical owls Struthioniformes Struthionidae Ostrich Rheidae Rheas Casuaridae Cassowaries Dromaiidae Emu Apterygidae Kiwis Tinamiformes Tinamidae Tinamous Trogoniformes Trogonidae Trogons Collection and handling of blood samples 5

SAFETY ASPECTS that immobilizes their wings, legs and head in a way that presents the vein to be punctured. A towel or cloth to wrap a bird can be Handlers of birds need to be aware of the useful for physical immobilization of the wings defence mechanisms that an individual bird and legs and helps to prevent self-infl icted species may possess as well as the potential injuries such as wing fractures. The amount of zoonotic diseases that birds may be excreting. trans-coelomic pressure must be just suffi cient Sharp beaks, talons and claws have the poten- to hold and restrain the wings but not restrict tial to infl ict serious injury to anyone that respiratory movements. approaches or attempts to capture and restrain In most circumstances blood can be collected birds for blood collection. Members of the from superfi cial veins that can be readily seen Struthioniformes are large birds that can kick through the typically thin apterial avian skin. violently and cause signifi cant injury with their Only in the most obese of birds is there suffi - claws. Psittaciformes have powerful beaks that cient subcutaneous fat to obscure the view of can cause deep lacerations and crush injuries most of the superfi cial vasculature. Some of to fi ngers. Others with sharp straight beaks these veins, such as the right jugular vein in such as cranes, darters and bustards are long-necked birds, are relatively mobile owing renowned for selectively targeting the eyes of to a relative lack of fascial attachments to adja- their captors to cause injury. Face shields or cent anatomical structures and consequently protective eyewear must be worn by all people the birds need to be held in a way that stretches in the close vicinity of such birds when they the neck, thus stretching the vein and reducing are being captured, handled and restrained. its movement. In general, if a superfi cial vein Protective gloves should be worn by handlers cannot be visualized through the skin then of birds that are potentially excreting zoonotic venipuncture should not be attempted. pathogens such Chlamydophila psittaci. This should also be done to protect subsequent birds if more than one is to be handled, with gloves changed between birds each time. EFFECTS OF ANESTHESIA ON THE HEMATOLOGICAL Restraint of birds to facilitate CHARACTERISTICS OF BIRDS collection of blood samples The effects of anesthesia on the hematological The amount of restraint that is required to characteristics of birds have not been compre- safely collect a blood sample is dependent on hensively studied. A study of the hematologi- the size of the bird, its capacity to infl ict an cal effects of 10 minutes of isofl urane anesthesia injury, its familiarity with humans and the on American kestrels (Falco sparverius) skills of the phlebotomist. For well-trained, showed a mild decrease in packed cell volume humanized or human-imprinted birds it is (PCV) (0.40–0.45 to 0.35–0.42 L/L), basophil often possible to collect a blood sample from concentration (0.1–0.4 to 0–0.2 × 109/L) and the jugular vein with a minimal amount of plasma proteins (28–35 to 25–31 g/L) (Dressen restraint by using an approach that simulates et al. 1999). However, the hematocrits of red- allopreening of the head and neck region. tailed hawks (Buteo jamaicensis) administered Individual birds can be easily trained to not ketamine hydrochloride intramuscularly, when resist manipulation of the head and neck assessed 10, 20 and 40 minutes after adminis- region in a way that readily presents the jugu- tration, showed no signifi cant differences to lar vein for venipuncture. However, the vast the hematocrit values of conscious birds majority of birds require physical restraint (Kollias and McLeish 1978). 6 Atlas of Clinical Avian Hematology

VOLUME OF BLOOD It should be noted that all these studies were performed on clinically healthy birds. The amount of blood that can be safely harvested The blood volume of birds has been reported depends on the body size (and dependent to be 67 ± 3 mL/kg for common pheasants blood volume) and the health status of a bird. (Phasianus colchicus), 62 ± 5 mL/kg for red- Under most circumstances, the skilful removal tailed hawks and 111 ± 3 mL/kg for redhead of a single volume of blood less than 10% of (Aythya americana) and canvasback (Aythya the circulating blood volume (or approxi- valisineria) ducks (Bond & Gilbert 1958), and mately 1% or less of the bird’s body weight) 106 ± 3 mL/kg for galahs (Eolophus roseica- will not be detrimental to a healthy bird. When pillus) (Jaensch & Raidal 1998). The spleen dealing with ill birds, only the minimum does not provide a reservoir of erythrocytes in amount of blood necessary for analysis should birds (as it does in mammals) (Sturkie 1943). be removed to minimize any physiological However, birds have been shown to rapidly consequences. recover from experimentally induced blood loss. After the removal of about 30% of the estimated blood volume of Japanese quail GENERAL ASPECTS OF COLLECTION (Coturnix japonica), erythrocyte numbers OF BLOOD FROM BIRDS recovered to baseline values within 72 hours (Gildersleeve et al. 1985, 1987a, Schindler et al. 1987a). Furthermore, birds have been Collection of a high-quality sample of blood shown to restore blood volume after an episode is the most important part of any hematologi- of hemorrhage far more rapidly than mammals cal assessment. The integrity of the cellular (Djojosugito et al. 1968, Gildersleeve et al. and fl uid components will deteriorate once 1985, Schindler et al. 1987a,b). For example, removed from the circulating blood. Ulti- phlebotomized quail recovered their plasma mately, skill, experience and care are required volume by 1 hour after removal of blood to consistently obtain good quality samples of (Schindler et al. 1987a). However, multiple blood. blood samplings within a 24-hour period that Blood is usually collected from the venous resulted in the removal of an estimated component of the circulatory system. The 29% of total blood volume from ruffs (Philo- most accessible vein for venipuncture depends machus pugnax) and red knots (Calidris on the size and anatomy of the species. Con- canutus) resulted in a decreased hematocrit venient sites for vascular access in birds of that had resolved by the next sampling, 2 differing sizes and body shapes are described weeks later (Piersma et al. 2000). Similarly later in this chapter. However, some general removal of about 10% of estimated blood concepts apply to all venipuncture. volume, each day for 7 consecutive days, Notably, adequate restraint of the bird is decreased the PCV (0.30 L/L on day 1 to required to allow visualization of the vein and 0.24 L/L on day 8) in chickens (Christie 1978). subsequent venipuncture and withdrawal of Over a longer period, removal of 0.9 mL of blood. In some cases physical restraint may be blood (about 10% of estimated total blood adequate. However, subtle “reactive” move- volume) from American kestrels weekly, for ments, especially in response to the needle 20 weeks, did not alter the birds’ PCV (Rehder entering the skin may thwart the harvest of et al. 1982a). Stangel (1986) removed up to blood. 7% of estimated blood volume from small To aid visualization of the vein, the feathers birds (about 25–100 g) without ill effect on overlying the site may be dampened with an the health of the birds. alcohol solution (such has 70% ethanol), or a Collection and handling of blood samples 7 detergent then an alcohol solution. Contami- avoided as it may cause hemolysis and may nation of the blood with excess alcohol should cause the vein to “collapse”, which conse- be avoided as this may cause hemolysis (Tietz quently hinders withdrawal of blood. Failure 1994) and can exacerbate heat loss from the to apply any negative pressure typically results skin. In some species the overlying feathers in leakage of blood around the needle. may be plucked to aid in visualizing the vein Once blood has been collected into the but care must be taken when doing this because syringe, negative pressure within the syringe is many species have thin and fragile skin that is released, the needle is withdrawn and digital easily torn. pressure applied to the vein for a period of at In most species of bird, the veins do not least 30 seconds to prevent blood fl owing from need to be occluded to impede the fl ow of the damaged vein. Due to the relatively loose venous blood. Furthermore, such occlusion connective tissue that surrounds them, avian may result in perivascular egress of blood once veins are prone to develop hematomas and it the vein has been pierced and subsequent for- is possible for small birds to suffer signifi cant mation of a hematoma. Hematomas occur blood loss into the perivascular spaces or, in more frequently in birds (than in mammals) the case of the jugular vein, the clavicular air during venipuncture and may interfere with sacs. subsequent attempts at venipuncture, if a The needle should be removed from the sample is not initially obtained. Practically, syringe before blood is gently expelled into an when a hematoma forms, the phlebotomist appropriate container containing an anticoag- should collect blood from another vein. ulant. Blood should not be squirted through a In most cases a needle and syringe are needle as it will cause hemolysis, particularly employed to collect the sample of blood. with the small-gauge needles often used for The size and gauge of the needle and the birds. Blood samples should then be mixed, size and volume of the syringe should be thoroughly but not over-zealously (so as to appropriate for the size of the vessel and avoid hemolysis), to permit adequate diffusion the volume of blood to be collected. Both of the anticoagulant throughout the sample. the needle and syringe should be sterile and Needles and syringes should be disposed of needles should not be reused between animals appropriately in “sharps biohazard” contain- to avoid both contamination of samples and ers. Needles should not be recapped following the possible transmission of disease between use and care should be taken to avoid needle birds. injuries. The needle is placed on the syringe with bevel facing upwards, and the syringe aligned in the direction of the vein at an angle of SITES FOR VASCULAR ACCESS approximately 15º to horizontal. The syringe is advanced in one fl uid motion so that the IN BIRDS needle pierces the skin, subcutaneous tissue and the vessel. Avoid lateral movement of the The most appropriate site for vascular access tip of the needle as laceration of the vein may in birds will depend on the species of bird, result in subsequent hemorrhage and hema- the volume of blood required, the method of toma formation. restraint and the skill of the operator. A small amount of negative pressure is Typically blood is collected from the venous applied to the plunger of the syringe as the system, although under some circumstances vein is pierced and this is increased as the blood from capillary beds or arteries may be needle is aligned within the vein to facilitate collected. The nomenclature of veins of birds harvest of blood. Vigorous suction must be is confusing with many publications employ- 8 Atlas of Clinical Avian Hematology ing alternative names for the same vein. This border of the metacarpus and at the second is particularly evident for the pectoral limb, digit runs across the dorsal aspect of the meta- with veins described as “basilic”, “brachial”, carpus to the caudal aspect of the carpus where “cutaneous ulnar”, “median”, and “wing”. it becomes the ulnar vein. The ulnar vein Usually there is no anatomical description of passes along the caudal aspect of the ulna, the site provided with the name and the reader receiving several small tributaries. Approach- is uncertain of the actual site used. Some sites ing the elbow, it crosses to the medial surface that have been used to collect blood are of the forearm and, slightly proximal to the reported in Table 1.2. elbow, joins with the radial vein (the deepest of the forearm’s veins) to form the basilic vein. The basilic vein (also referred to as the Description of the anatomical median vein) is the largest vein of the bra- location of vessels that allow chium and passes along the medial surface of vascular access in birds the triceps brachii muscle. The confl uence of the basilic, brachial and deep brachial veins forms the axillary vein (Figure 1). Pectoral limb The ulnar vein provides convenient vascular The dorsal metacarpal vein is the continua- access in a wide range of bird species. It can tion of the dorsal digital vein of digit three. be visualized by wetting the area with alcohol. The former vein passes along the craniodorsal Using the appropriate size needle, blood

Table 1.2 Reported sites for the collection of blood from selected species of birds. Note that the terminology to describe the vein is as used in the original publication.

Species Site (Vein) Reference

Black duck Brachial Mulley 1979 Canvasback duck Jugular Kocan & Pitts 1976 Mallard Jugular Fairbrother et al. 1990 Snow goose Brachial Williams & Trainer 1971 Herring gull Brachial Grasman et al. 2000 Bald ibis Brachial Villegas et al. 2004 Puna ibis Jugular Coke et al. 2004 Rosy ﬂ amingo Brachial Hawkey et al. 1984b White stork (immature) Jugular Montesinos et al. 1997 Victoria crowned pigeon Brachial Peinado et al. 1992 White winged dove Medial metatarsal Small et al. 2005 American kestrel Jugular Dressen et al. 1999 Bald eagle Brachialis Bowerman et al. 2000 Eurasian kestrel Brachial Kirkwood et al. 1979 Harris’s hawk Basilic Parga et al. 2001 Peregrine falcon Ulnaris, jugular Lanzarot et al. 2001 Red-tailed hawk Brachial Rehder et al. 1982b Saker falcon Right jugular Samour et al. 1996 Sharp-shinned hawk Median metatarsal Phalen et al. 1995

(continued) Collection and handling of blood samples 9

Table 1.2 (continued)

Species Site (Vein) Reference

Spanish imperial eagle Cutaneous ulnar Garcia-Montijano et al. 2002 Attwater’s prairie chicken Jugular West & Haines 2002 Red-legged partridge Jugular, heart Lloyd & Gibson 2006 American coot Metatarsal, basilic, right jugular Newman et al. 2000 Rufous crested bustard Ulnaris, jugular Howlett et al. 2002 Sandhill crane Brachial Kennedy et al. 1977 Bar-tailed godwit Brachial Landys-Cianelli et al. 2002 Great tit Brachial, tarsal Ots et al. 1998 New Holland honey eater Right jugular Kleindorfer et al. 2006 Red knot Brachial Piersma et al. 2000 Swainson’s thrush Brachial Owen & Moore 2006 Superb fairy wren Alar Breuer et al. 1995 Brown pelican Medial metatarsal, cutaneous ulnar Zais et al. 2000 Giant petrel Jugular Uhart et al. 2003 Great frigate bird Cutaneous ulnar Work & Rameyer 1996 Great skua Tarsal Bearhop et al. 1999 Manx shearwater Brachial Kirkwood et al. 1995 Northern fulmar Medial metatarsal Edwards et al. 2006 Waved albatross Ulnar Padilla et al. 2003 African grey parrot Brachial Hawkey et al. 1982 Amazon parrot Cutaneous ulnar, jugular Tell & Citino 1992 Budgerigar Right jugular Scope et al. 2005 Hyacinth macaw Jugular, median metatarsal Calle & Stewart 1987 Red lori Right jugular Scope et al. 2000 Black-footed penguin Right jugular Grim et al. 2003 Humboldt penguin Brachial Villouta et al. 1997 Rock-hopper penguin Jugular Karesh et al. 1999 Great horned owl Brachiocephalic artery (arterial) Hawkins et al. 2003 Snowy owl Brachial Evans & Otter 1998 Lesser rhea Brachial Reissig et al. 2002

can be collected by aspiration into a syringe Pelvic limb or by allowing it to drip from the needle hub into a micro collection tube. Pressure is The medial metatarsal vein is recognizable required after removal of the needle for longer along the metatarsus in many species of birds. periods as this site is prone to hematoma It is formed by the conﬂ uence of plantar meta- formation. tarsal vein and dorsal metatarsal vein 1 and In larger species, the dorsal metacarpal vein subsequently forms the caudal tibial vein, may be pierced with a narrow gauge needle after passing over the tarsus. The main dorsal (25–27-gauge) and as the blood wells up in the metatarsal vein is formed from dorsal meta- hub of the needle it may be drawn away using tarsal veins 2 and 3. The medial and lateral capillary action of a glass tube. tarsal veins are formed proximal to the tarsus 10 Atlas of Clinical Avian Hematology

Patagial v. Brachial v. Dorsal metacarpal v. Axillary v.

Digital v. Cranial ext. thoracic v.

Coracoid v.

Subclavian v. Basilic v. Radial v.

Ulnar v. Caudal ext. thoracic v. Deep brachial v. Dorsal thoracic v.

Figure 1 Veins of the pectoral limb, ventrolateral view. Modifi ed from Fitzgerald, The Coturnix Quail, Anatomy And Histology. Copyright 1969, with permission of Blackwell Publishing. by branching of the dorsal metatarsal vein and veins, dorsal vein and lateral occipital vein. subsequently reform as the cranial tibial vein. It terminates by joining the subclavian vein to The cranial tibial vein traverses the dorsal form the cranial vena cava. aspect of the tibia and combines with the The jugular vein provides important vascu- caudal tibial vein to form the popliteal vein. lar access in many species of birds. It is usually The popliteal vein receives several smaller veins to form the femoral vein (Figure 2). The medial metatarsal vein provides conve- Cranial tibial v. Caudal tibial v. nient vascular access in species that have legs Lateral tarsal v. Medial tarsal v. of an adequate size and length (such as water- Anastomotic vv. fowl) and also in species that have a dense plumage that covers alternate sites of vascular Medial metatarsal v. Dorsal metatarsal v. access (such as the jugular vein). In smaller species, the proximity of the metatarsal veins, tarsal veins and tibial veins may make it diffi - Plantar metatarsal v. cult to discern the individual vessels. Dorsal metatarsal v.1 Dorsal metatarsal v. 2 Dorsal metatarsal v. 3 Head and neck Digital vv. The jugular vein arises from the confl uence of the cranial and caudal cephalic veins. The right jugular vein is typically larger and has a Digital vv. more ventral course (than the left jugular vein) through the cranial cervical region. Through- Figure 2 Veins of the pelvic limb, dorsal view. Modi- out its course, the jugular vein receives numer- fi ed from Fitzgerald, The Coturnix Quail, Anatomy and ous tributaries including: cutaneous veins, Histology. Copyright 1969, with permission of Black- ingluvies veins, esophageal veins, tracheal well Publishing. Collection and handling of blood samples 11

Dorsorostral cerebral v. Olfactory venous sinus Within the skull of birds, the occipital sinus is a vascular space containing venous blood, Dorsocaudal which lies within the dura mater and encircles cerebral v. the brain stem (Garrett et al. 1987). Collection Transverse venous sinus of blood from the occipital sinus has been demonstrated to be an effective non-lethal Occipital External occipital v. venous sinus method in ducks and geese (Vuillaume 1983, Garrett et al. 1987) and gallinaceous birds (Zimmermann & Dhillon 1985). When performed, the neck of the bird should be fl exed and an appropriately sized needle (for the size of the bird) attached to a syringe is directed perpendicular to the skin and aimed lateral to Internal the dorsal midline at the level of the foramen vertebral magnum (Figure 3). Penetration of this ana- Jugular v. sinus tomical location presents a risk of trauma to the brain stem or spinal cord and hemorrhage Figure 3 Access to the occipital venous sinus (modifi ed from Kaku, On the Vascular Supply in the Brain into the cerebrospinal fl uid. Consequently the of the Domestic Fowl. Copyright 1959, with permis- occipital sinus should not be recommended as sion of Fukuoka Acta Medica) can be achieved through a site for routine blood collection. When the the foramen magnum with the bird’s head held in a technique is used, the bird should be anesthe- fl exed position. The needle is directed (arrow) perpen- tized and it should be considered only when dicular to the skin and just lateral to the midline. blood collection is undertaken immediately Whilst the technique has been tolerated well in ducks prior to euthanasia. and geese there is a high risk of injury to the spinal cord and brain stem, particularly in small bird species whereby the technique should be learnt or performed only in anesthetized birds. Sites of vascular access for selected groups of birds

See Figures 4–11. easily visualized by parting feathers over the ventral part of the neck. However, in some Apodiformes and small passeriformes species, dense plumage may hinder visualization of the vein. Despite being able to readily In small bird species, such as ﬁ nches, hone- visualize the jugular vein in most birds, the yeaters and hummingbirds, the superﬁ cial “mobility” of the vein may frustrate the phle- veins may be too small to allow a hypodermic botomist’s attempts to pierce the vein. Holding needle to be inserted into a vein and blood the skin of the bird’s neck taut assists in withdrawn. In these species it is still possible restricting the lateral movement of the vein, to collect small volumes of blood (50–150 µL) and increases the likelihood of successful veni- using a 28- or 30-gauge needle to puncture the puncture. For most species of bird it is almost skin overlying a vein. Blood can be collected impossible to accidentally enter (or damage) directly from the hub of the needle or the skin the carotid artery while attempting jugular into a capillary tube. Depending on the species, venipuncture because the artery is normally the medial metatarsal, basilic, and external located deep within the epaxial muscles of the thoracic (which runs caudally and dorsally neck, below the ventral surfaces of the cervical from the axilla along the thorax) veins may vertebral bones. provide suitable vascular access. Topical 12 Atlas of Clinical Avian Hematology

Figure 4 A blue-breasted quail (Coturnix chinensis), weight about 50 g, showing the right jugular vein. In Figure 6 The left wing of a peregrine falcon (Falco small birds, the jugular vein provides the best site to peregrinus), weight about 700 g, showing the ulnar obtain a suffi cient volume of blood to allow quantita- vein. The feathers have been dampened by the appli- tive analysis. Care must be taken not to apply too cation of an alcohol solution which also disinfects the much negative pressure on the syringe, which acts to skin overlying the vein. Both the ulnar and basilic “collapse” the vein, prohibiting the fl ow of blood. veins provide convenient vascular access in a bird of Additionally, the typically slow fl ow of blood may this size. Diligent manual pressure must be applied result in the blood clotting in the syringe. after venipuncture to effect hemostasis.

Figure 7 The left wing of an Australian gannet (Morus serrator), weight about 2.0 kg, showing its prominent Figure 5 A galah (Eolophus roseicapillus), weight basilic vein. The feathers have been dampened by the about 500 g, showing the right jugular vein. In larger application of alcohol solution. Despite the large size birds, the jugular vein allows ready vascular access of the bird, the jugular vein was not able to be used and sufﬁ cient blood volume to be collected for for vascular access due to the dense plumage which analysis. precluded visualization of the vein. Collection and handling of blood samples 13

Figure 8 The wing of a grey teal (Anas gracilis) Figure 10 The leg of a galah (Eolophus roseicapillus) showing the leakage of blood from the ulnar vein, showing venipuncture of the cranial tibial vein with a which occurs during venipuncture when the phleboto- 23-gauge needle. The blood is allowed to ﬂ ow through mist fails to apply slight negative pressure to the the needle under hydrostatic pressure and is collected plunger of the syringe as the vein is pierced. into a container containing anticoagulant.

Figure 11 The leg of common eider (Somateria mollissima) illustrating venipuncture of the medial Figure 9 The wing of a little corella (Cacatua san- metatarsal vein and collection of blood into a syringe. guinea), weight about 650 g, showing the dorsal meta- (Courtesy of Claire Cunningham.) carpal vein. The vein has been pierced with a 27-gauge needle and as the blood “wells up” in the hub of the needle it is drawn away by the capillary action of the tube. 14 Atlas of Clinical Avian Hematology application of potassium permanganate or recommended the basilic vein for collection of ferric chloride solutions, using a cotton swab, blood from falcons, conscious and restrained can be used to achieve hemostasis. manually in dorsal recumbency. These authors noted the importance of hemostasis following venipuncture at this site. In smaller species of Anseriformes Strigiformes, blood can be obtained from the In larger species of ducks, geese and other right jugular vein (Aguilar 2003). birds that have webbed feet, it is possible to access the smaller superfi cial digital veins Galliformes that course through the inter-digital skin (“webbing”). This can often be done with In most species of galliformes, blood can be minimal restraint, with the bird in a standing collected from the right jugular vein or the position. Alternatively, the metatarsal, ulnar ulnar or basilic veins. The jugular vein has or basilic veins can be readily accessed in those been used successfully to obtain relatively species that have extensive cervical pterylae large volumes of blood from relatively small (that obscure the jugular vein). birds, such as quail (Gildersleeve et al. 1985, 1987a,b, Schindler et al. 1987a,b). Columbiformes Gruiformes Doves and pigeons have well developed cervical pterylae that surround the neck and the In cranes, blood can be collected from the cervical skin contains a well developed plexus right jugular vein or the medial metatarsal vein of veins (plexus venosus subcutaneous col- (Olsen & Carpenter 1997, Carpenter 2003). laris) that becomes engorged in both male and The birds should be held securely and the female adults that are reproductively active sample taken quickly. Blood is not often taken (Dalley et al. 1981). This plexus can make from the ulnar or basilic veins because of the visualization of the jugular vein diffi cult and diffi culties encountered in restraining all but for this reason alone it is usually easier to sedated birds. collect blood from the ulnar or basilic vein. Passerines Ciconiiformes Blood can be collected from larger species of In the Ciconiiformes, the long legs possessed passerine birds by venipuncture of the right by most species make the medial metatarsal jugular vein. For example, a 30-gauge needle vein the most accessible site for collection of and a tuberculin syringe can be used to take blood. The jugular and ulnar veins provide blood from the right jugular vein in starling alternative sites (Waters 2003). sized birds. Alternately, the ulnar vein can be used (Gentz 2003). Blood can be harvested from smaller species, as previously described, Falconiformes and strigiformes into a capillary tube after puncture of a super- Most species of raptors have long legs and in fi cial vein. most species the medial metatarsal vein is readily visualized and accessed for blood col- Pelecaniformes lection. Care must be taken to properly restrain the legs to avoid being injured by the talons. Pelicans and cormorants normally have exten- Alternatively, blood can also be collected from sive subcutaneous emphysema that can extend the ulnar or basilic veins. Werney et al. (2004) to the tips of the wings and the feet. This can Collection and handling of blood samples 15 interfere with visualization of the superfi cial recumbency or held in a vertical position, to veins of the wing and legs. Access to the jugular allow the leg to be held fi rmly and extended. vein is also impeded by extensive cervical pter- An external heat source applied to the feet can ylae. Branches of the dorsal metatarsal vein or help dilate the vein and facilitate the fl ow of the ulnar vein are the best sites for collection blood. of blood in these species. Struthioniformes Psittaciformes Blood can be collected from the median meta- Blood can be collected from the right jugular tarsal vein of large ratites, such as the ostrich vein in all species of psittacine birds. Larger and emu, with the bird in the standing posi- species, such as macaws, require restraint to tion. Restraint of the bird by an assistant is extend the neck and prevent injury from their effected by the person standing beside and beak. Alternative sites are the veins of the leaning into the bird with moderate down- wing, including the dorsal metacarpal vein. ward pressure to minimize movement of the Most parrots and cockatoos have short tarso- legs of the bird. The median metatarsal vein metatarsi, which makes access to the medial on the leg selected for venipuncture is then metatarsal vein diffi cult, but it is best accessed best approached from the caudomedial aspect. as it crosses over the hock. In neonatal psitta- Alternatively, the ulnar or brachial veins can cine birds (and the neonates of other altricial also be used for blood collection but care must species) that have sparse feathers, it can be be taken when restraining the wing, as they diffi cult to restrain the chicks unless their feet are easily fractured. and body are allowed to rest in a “neutral” sitting position. This then allows restraint of the head and neck, or extension of the wing, ANTICOAGULANTS to facilitate venipuncture.

In most species of birds, the use of ethylene- Sphenisciformes diamine-tetra acetic acid (EDTA) as an antico- Penguins have extensive pterylae and very agulant provides the best preservation of cell thick plumage. Consequently, it is very diffi - morphology and samples of blood should be cult to visualize or access the jugular vein routinely mixed with EDTA to prevent the or the superfi cial veins of the fl ipper. Tech- sample clotting (Jennings 1996, Wernery et al. niques have been described for blood collec- 2004). Hattingh and Smith (1976) found, for tion from the brachial vein (Samour et al. blood from pigeons (Columba livia), that sig- 1983), but the digital, pedal or dorsal meta- nifi cant hemolysis was evident in samples tarsal veins are more readily visualized in most mixed with heparin (30 hours) before it was species as they run over the dorsal aspect of evident in samples mixed with EDTA (70 the feet between the second and third digits hours). Interestingly, these authors found less (Cheney 1993). However, there is consider- hemolysis occurred in samples mixed with able variation in the anatomical location of EDTA, maintained at 20ºC than in samples these veins even within members of the same maintained at 4ºC. However, some birds may species. exhibit incomplete anticoagulation of the Dvorak et al. (2005) described techniques sample or hemolysis of the sample with EDTA for training individual birds to stand while (Campbell 2004). blood is collected. However, in most instances, Hemolysis may be evident when a sample the bird needs to be restrained in either dorsal of blood is centrifuged in a capillary tube, as 16 Atlas of Clinical Avian Hematology

(red) discolored plasma and a decreased Making blood fi lms proportion of erythrocytes. Microscopically, hemolysed samples are characterized by a lack See Figures 12–21. of intact, discernable erythrocytes and in their There are several methods that may be used stead are nuclei with indistinct erythrocytic to make blood fi lms. Prior to making any membranes and a distinctly eosinophilic back- blood fi lm the sample must be thoroughly (but ground due to the presence of a large amount gently) mixed to avoid any sedimentation of of “free” hemoglobin. cells. Hemolysis of blood samples mixed with EDTA has been reported in a number of species Two slide “wedge” method of birds including: ostriches (Struthio camelus) (Campbell 2004), black-crowned crane (Bale- A commonly used technique for making blood arica pavonina), laughing kookaburra (Dacelo fi lms from a wide range of species is the novaeguineae), many members of the Corvi- “wedge” method, which is suitable for many dae (Hawkey & Dennett 1989) and Megapo- situations. To produce a blood fi lm using this diidae (Vogelnest 1991). In species that exhibit method, place a slide on a fl at surface (such as EDTA-mediated hemolysis, heparin is the a bench top) then place a drop of blood (about anticoagulant of choice. However, lithium a generous “pin-head” size) towards the end heparin is not recommended as the routine of the slide. Stabilize this slide with a digit (of anticoagulant for birds as it results in exten- the operator’s non-preferred hand) then hold sive clumping of all hematological cell types a second (“spreader”) slide, between thumb (Robertson & Maxwell 1990). Notably, and forefi nger of the preferred hand, at about clumping of leukocytes and thrombocytes may 45 degrees to the (horizontal) fi rst slide. To result in inaccurate counts for these cell types spread the drop of blood; touch the “spreader” (Campbell 2004). slide to the fi rst slide in front of the drop of blood, “reverse” the “spreader” slide into the drop of blood, pause momentarily while the blood spreads laterally towards the edges of the slide and then rapidly and smoothly propel BLOOD FILMS the “spreader” slide forward. Blood fi lms pro-

To examine the morphology of hematological cells using light microscopy, a blood fi lm must be produced. These must leave cells intact and be thin enough to allow the transmission of light. Disruption of hematological cells is a signifi cant impediment to the analysis of avian blood and is promoted by the small volumes of blood commonly available from birds. Lysed cells are commonly encountered in blood fi lms and a spectrum of morphological effects may be encountered, from mild rupture of the cell’s membrane to comprehensive Figure 12 To make a blood fi lm using the “wedge” destruction of the cell, rendering it unrecogniz- method: a drop of blood is placed towards the end of able. The latter have been termed “smudge a slide (on a fl at surface) and the spreader slide is then cells”. “reversed” into the drop of blood. Collection and handling of blood samples 17

Figure 13 To make a blood fi lm using the “wedge” method: once the slide is in contact with the blood, Figure 15 To make a blood fi lm using the “cross” movement of the slide is paused to allow the blood to method: the blood is allowed to spread under the spread laterally, then the spreader slide is propelled weight of the “upper” slide and then the upper slide forward with a gentle, smooth motion to spread the is gently and smoothly advanced along the lower slide blood along the slide. to further spread the blood. duced by this method are typically thinnest at the leading edge of the blood fi lm; however, blood and the operator’s technique in making the cells are often distorted at this margin. the blood fi lm. Optimal cell morphology is usually found in the region of the “body” of the fi lm where the Two slide “cross” method cells are present as a monolayer. The size of this region is dependent on the PCV of the This method is typically less disruptive to cells than the “wedge” method and is useful in the

Figure 14 To make a blood fi lm using the “cross” method: a drop of blood is placed on a slide (held by Figure 16 To make a blood fi lm using the “slide and the operator), and then a second slide (held above and cover-slip” method: a drop of blood is placed on a at right angles to the fi rst slide to form a “cross”) is slide and then a cover-slip is placed on the drop of lowered to contact the drop of blood. blood. 18 Atlas of Clinical Avian Hematology

Figure 17 To make a blood fi lm using the “slide and Figure 19 To make a blood fi lm using the “two cover-slip” method: the blood is allowed to spread cover-slip” method: a drop of blood is placed on a under the weight of the cover-slip. cover-slip held between the thumb and forefi nger of the operator’s non-preferred hand. “fi eld” when no clean fl at surface is available. A drop of blood is placed on a slide (held by the at the poles and less dense in the center and operator), and then a second (“spreader”) slide sides. (held at right angles to the fi rst slide to form a “cross”) is fl atly touched to the drop of blood (with no downward pressure), the blood spreads Cover-slip and slide method under the weight of the slide and the slide is Similar to the above method, a cover-slip may gently and smoothly advanced along the fi rst be used instead of the second slide. A drop of slide. Blood fi lms produced by this method typically have an ovoid shape that is thicker

Figure 20 To make a blood ﬁ lm using the “two cover-slip” method: a second cover-slip is then gently Figure 18 To make a blood ﬁ lm using the “slide and placed on the drop of blood and blood is allowed to cover-slip” method: the cover-slip is then drawn along spread under its weight. The two cover slips are then the slide to further spread the blood. gently drawn apart to further spread the blood. Collection and handling of blood samples 19

ﬁ lms are mounted on a slide, either in a per- manent fashion using mounting medium or in a temporary fashion using immersion oil (with the side containing the blood ﬁ lm down).

All of the methods described may be used to produce blood fi lms of suffi cient quality to allow fruitful microscopic examination. The reader is encouraged to experiment with all of the methods to fi nd which one produces the best quality results under their particular circumstances.

Figure 21 Blood fi lms made from the blood of a lovebird (Agapornis rosiecollis) produced by the four Staining blood fi lms methods described above. (a) “Wedge” method, (b) “cross” method, (c) “cover-slip and slide” method (all See Figures 22–32. stained by an automated stainer Hematek, Bayer) and The staining of slides allows cellular detail (d) “two cover-slip” method, manually stained with to be visualized and the tinctorial characteris- Diff Quik (and subsequently mounted on a slide). tics of standardized staining methods have been used to classify hematological cells (viz, “eosinophils” and “basophils”). Poor quality blood is placed on a slide and then a cover-slip staining of a blood fi lm will decrease the value is placed on the drop of blood (with no digital of the fi lm. Inadequately stained samples do pressure). The blood spreads under the weight not allow fi ne cellular detail to be visualized. of the cover-slip and the cover-slip is drawn Similarly, “over-stained” samples often do along the long axis of the slide to further not allow fi ne cellular detail (particularly spread the blood. Any sized cover-slip may be nuclear detail) or tinctorial nuance to be used; however, greater dexterity is typically distinguished. achieved with longer cover-slips. The operator Avian blood fi lms are commonly stained must be careful not to apply too much pres- with Romanowsky stains, such as: Wright’s sure and fracture the cover-slip, as the resul- stain, May-Grünwald stain, Giemsa stain and tant shards of glass may cause an injury. Leishman’s stain, all of which contain varying proportions of methylene blue and eosin (Lynch et al. 1969), or “rapid stains” such as Two cover-slip method Diff Quik. All of these stains characteristically Finally, when very small volumes of blood are impart a basophilic color to nucleic acids and available the “two cover-slip” method is a the cytoplasmic granules of basophils and useful way to produce fi lms of blood. A drop impart an eosinophilic color to the hemoglo- of blood is placed on a cover-slip then a second bin-containing cytoplasm of erythrocytes and cover-slip placed on top of the fi rst. The blood the cytoplasmic granules of heterophils and spreads under the weight of the second cover- eosinophils. Prior to application of the stains, slip and when the two are drawn apart, two blood fi lms must be thoroughly dry. The fi lms are produced. This method is typically stains are generally applied by using either an more time consuming than the other methods automated stainer or by placing the slides in described, particularly as the fi lms must be Coplin jars containing the stain for a period stained by manual methods. After staining, the of time. Following application of the stain, 20 Atlas of Clinical Avian Hematology

Figure 22 Blood from an Australian raven (Corvus Figure 24 Blood from an ostrich (Struthio camelus) coronoides) showing the lysis of all erythrocytes due which has been mixed with heparin to act as an anti- to the effect of EDTA. The nuclei of erythrocytes are coagulant (as EDTA causes hemolysis in this species). evident but no distinct cell membranes can be dis- This slide has been stained with Diff Quik stain. cerned. The “free” hemoglobin gives a uniform eosin- Present are three heterophils that contain a high ophilic color to the background. Also present is a density of granules giving an overall brick-red color to heterophil. (Modiﬁ ed Wright’s stain.) the cytoplasm.

Figure 23 Blood from an Eurasian coot (Fulica atra) showing the aggregation of leukocytes that may occur Figure 25 Blood from the same ostrich as in Figure with the use of heparin as an anticoagulant. The 24. This slide has been stained with May-Grünwald uneven distribution of leukocytes is likely to affect the and Giemsa stains. Present are three heterophils, a accuracy of the determination of leukocyte monocyte and a lymphocyte. Notably, the heterophils’ concentration. granules are not as distinct with this stain (as they were with Diff Quik), thus illustrating the effect the staining process may have on the morphological appearance of the cells. This appearance of the heterophils’ granules must not be misinterpreted as a “toxic change”. Collection and handling of blood samples 21

Figure 28 Blood from a Stanley crane (Grus paradisea) showing stain precipitate, characterized by large Figure 26 Blood from an Australian kestrel (Falco aggregates of purple to basophilic, fl occulent material cenchroides). The blood fi lm has been stained with that overlies the erythrocytes and is present between modifi ed Wright’s stain. Present is a basophil showing cells. (Modifi ed Wright’s stain.) typical darkly basophilic cytoplasmic granules that obscure much of the cell’s nucleus and an adjacent, disrupted basophil with a lysed nucleus and dispersed basophilic granules.

Figure 27 Blood from the same Australian kestrel as Figure 29 Blood from a saker falcon (Falco cherrug) in Figure 26. The blood ﬁ lm has been stained with Diff showing small amounts of stain precipitate (arrows) Quik stain. Present is a basophil, in which most of the overlying cells and in between the hematological cells cytoplasmic granules have not stained. The cell is (a heterophil and a number of erythrocytes are also characterized by a round nucleus and pale cytoplasm present). (Modiﬁ ed Wright’s stain.) with many cytoplasmic “vacuoles” and a few basophilic granules. 22 Atlas of Clinical Avian Hematology

Figure 30 Blood from a masked lapwing (Vanellus miles) showing the remnant nuclear material of a Figure 32 Blood from a green pygmy goose (Net- number of markedly lysed cells amidst intact erythro- tapus pulchellus) showing mature erythrocytes that cytes. The extensive damage to the cells precludes have a refractile appearance (arrow) or pale “moth- identifi cation of the cell type. (Diff Quik stain.) eaten” appearance due to incomplete drying of the blood fi lm prior to being stained. Also present are two heterophils. (Diff Quik stain.) slides must be adequately washed (to remove excess stain) and dried. Slides should not be surface expedites drying. Slides that will be wiped dry as the sample is easily wiped from archived should have a cover-slip applied the slide. Contact with heated air or a heated using a suitable mounting medium and be stored away from direct sunlight to avoid fading of stain color. Alternative staining methods have been reported but have not been widely adopted (Rath et al. 1998, Kass et al. 2002). The tinctorial characteristics of the hematological cells may be affected by many factors, including: the type of anticoagulant used to prevent the blood from clotting, the ratio of anticoagulant to blood, the type of stain, the use of the stain, fi xation, and the time elapsed between the production of the blood fi lm and staining the blood fi lm. Robertson and Maxwell (1990) found the quality of May-Grünwald and Giemsa staining of fi lms of blood from domestic fowl was superior when EDTA, rather than lithium Figure 31 Blood from an orange-footed scrub fowl heparin, was used as an anticoagulant. These (Megapodius reinwardt) showing many erythrocytes latter resulted in extensive clumping of all with multiple, small, refractile “bodies” that occur as a result of incomplete drying of the blood fi lm (and hematological cell types, heterophils showed hence the presence of residual water) prior to being marked vacuolation of the cytoplasm, cells stained. This artifact is commonly encountered when had a blue color and small lymphocytes and atmospheric humidity is high. (Diff Quik stain.) thrombocytes were diffi cult to differentiate. Collection and handling of blood samples 23

Similarly, Hawkey and Dennett (1989) May-Grünwald and Giemsa stains were used. reported blood fi lms may stain with a “bluish” Recently, the effect of stain type on the appear- tinge with Romanowsky stains when heparin ance of eosinophils was demonstrated for the is used as an anticoagulant. However, addition gyr falcon (Falco rusticolus) (Samour et al. of less than 40% of the “correct” amount of 2005) with the “eosinophilic” granules most blood for the EDTA tube also compromised apparent with Wright’s and Giemsa stains and the quality of the staining of the blood fi lm less apparent with May-Grünwald and Giemsa (Robertson & Maxwell 1993). stains and a rapid stain. These authors also found unfi xed blood The method used to apply the stain may fi lms had superior quality staining than blood also affect the tinctorial characteristics. Typi- fi lms fi xed in methanol (for 6 minutes) when cally, blood fi lms stained with automated stained with May-Grünwald and Giemsa stain stainers are more uniform in appearance than (Robertson & Maxwell 1990). However, blood fi lms stained by manual methods. Bennett (1970) noted that, “Long delay in However, manual methods are less costly and fi xation results in deterioration of the smear facilitate greater fl exibility in staining and and poor stain contrast with Giemsa’s stain. allow the user to tailor the tinctorial charac- This is particularly noticeable in the cytoplasm teristics to the users’ preference. of erythrocytes which stain progressively more As the blood fi lms portrayed in this book blue as the time between making and fi xing were obtained from many sources and conse- smear increases.” This has also been the expe- quently were stained by different methods and rience of the authors. Hawkey & Samour types of stains, variation can be observed in (1988) recommended that when the staining the tinctorial characteristics of hematological of a blood fi lm was delayed for more than 48 cells between samples, sometimes even within hours, the fi lm be fi xed for 2 minutes in abso- individuals of the species. It is important to lute methanol prior to storage. distinguish these variations in staining from Signifi cant differences may be observed in pathological effects that result in morphologi- the appearance of hematological cells (even for cal changes to hematological cells. the same sample of blood) when rapid stains are compared to Romanowsky stains. Even within the Romanowsky stains, the colors of the cells and the fi ne cellular detail may be POST-COLLECTION ARTIFACTS quite different with different stains. Similarly, different commercial preparations and even See Figures 33–40. batches of the “same” preparation infl uence One of the most important decisions that a the appearance of cells. The greatest differ- hematologist, clinician or researcher will make ences are typically apparent in the cytoplasmic is whether the observed results are indicative granules of granulocytes. The hues of these of the hematological characteristics of the bird granules vary with the type of stain and in or whether they refl ect changes in the sample some instances the cytoplasmic granules may that have occurred since the blood was col- not be evident. Instead of granules, colorless lected. Failure to recognize the latter may “vacuoles” are evident in the cytoplasm of the result in incorrect interpretation of the data cell. In the authors’ experience, basophils are and consequently incorrect management of the the granulocyte most commonly affected, par- case. Post-collection changes in the sample due ticularly when rapid stains are employed. to handling or processing may result in changes However, we have also observed that hetero- in cell morphology that may be visualized in phil granules have had a greater intensity of blood fi lms. Effects on cells may also result in color when a rapid stain was used than when changes in some of the hematological values 24 Atlas of Clinical Avian Hematology

Figure 33 Blood from a lanner falcon (Falco biar- Figure 35 Blood from a cattle egret (Bubulcus ibis) micus) showing large, polyhedral, pale basophilic showing adipocytes mixed with hematological cells. anucleated squamous epithelial cells (arrow) amidst The adipocytes were most likely collected from peri- hematological cells. These are typically harvested vascular adipose tissue during venipuncture. (Modi- from the skin overlying the vein during the perfor- ﬁ ed Wright’s stain.) mance of venipuncture. (Modiﬁ ed Wright’s stain.)

Figure 34 An osteoclast (a) and osteoblasts (b) in the Figure 36 Blood from a peregrine falcon (Falco per- blood of a rainbow lorikeet (Trichoglossus haemato- egrinus). The sample of blood was grossly lipemic. dus) that had been collected by clipping a toenail Lipemia can be recognized on the blood fi lm as an (Clark & Tvedten 1999). The operator had clipped the overall “milky” appearance (in contrast to clear areas nail too far proximal, resulting in an inadvertent biopsy in the background, see arrow). The lipemia also results of the bone. (Modifi ed Wright’s stain.) in the cells present having a less distinct appearance. (Modifi ed Wright’s stain.) Collection and handling of blood samples 25

Figure 37 Blood from a whistling kite (Haliastur Figure 39 Blood from a yellow-eyed penguin (Mega- sphenurus). Several fungal hyphae (arrows) can be dyptes antipodes). Several refractile crystalline objects, observed and bacteria were recognized on the blood consistent with particles of sand, are present amidst fi lm. These represent contamination of the blood fi lm the erythrocytes. (Giemsa stain.) by extraneous organisms. The number and morphology of leukocytes was unremarkable, indicating no infl ammatory response. In contrast, Figures 267 and 269 illustrate phagocytosis of etiological agents and morphological atypia of leukocytes in response to infl ammatory challenge. (Diff Quik stain.)

Figure 40 Blood from a brolga (Grus rubicunda) Figure 38 Blood from a rusty-barred owl (Strix showing a large, linear, basophilic, clothing fi ber hylophila) showing a crystalline, angular, pale particle amidst hematological cells. (Modifi ed Wright’s stain.) of powder from the disposable gloves, worn by the phlebotomist. (Diff Quik stain.) 26 Atlas of Clinical Avian Hematology measured or calculated by automated hema- The morphology of cells is typically altered tology analysers. For example, hemolysis will according to their location in the blood fi lm. decrease the number of erythrocytes counted Towards the leading (“feathered”) edge of the but the total amount of hemoglobin will be the fi lm the cells may be distorted or lysed by the same, resulting in a spuriously increased mean forces generated in the making of the blood corpuscular hemoglobin concentration. fi lm. In the thicker regions of the fi lm, the cells The artifacts that may be encountered in may be “rounded up” because there is insuffi - blood fi lms may be broadly categorized as cient space for the cells to spread out, conse- altered cell morphology, staining artifacts, quently less light is able to penetrate the cell non-hematological cells, extraneous material and less cell detail is able to be visualized. The and contaminant etiological agents. Spurious fi ne detail of the morphology of cells is best hematological analytes may result from the able to be discerned in the monolayer of the disruption of cells, aggregation of cells or mis- fi lm. However, even in the monolayer, the classifi cation of cells. shape of some cells may be distorted by adja- A common problem leading to altered cell cent cells. In addition, the use of heparin as an morphology is the disruption (lysis) of cells. anticoagulant may result in clumping of leu- Lysis of cells is promoted by delayed process- kocytes and thrombocytes (Hawkey & Dennett ing, exposure of the sample to temperature 1989, Robertson & Maxwell 1990, Campbell extremes (both heat and cold), “rough han- 2004). dling” of the blood sample (such as squirting A commonly encountered issue with the blood through a narrow-bore needle into a quality of blood fi lms results from inadequately container or over-exuberant mixing of blood stained cells and consequently an inability to samples), or as previously mentioned the effect visualize fi ne cellular detail. This may be due of EDTA anticoagulant which effects almost to blood fi lms that are too thick and stain total hemolysis in some species of birds. penetration is decreased. Similarly, if the stain Marked hemolysis of avian blood is evident potency has decreased due to age or usage, the microscopically as nuclei suspended in a back- blood fi lm may not be adequately stained. ground of hemoglobin with no distinct cell Additionally, exposure of the blood fi lm to membranes evident. Less pronounced hemoly- formalin vapors reduces the cells’ permeability sis results the presence of “ghost cells”, that is to the stains. erythrocytes with their cell membrane still If the stain used becomes partially insoluble, evident but that contain decreased amounts of variably sized fl occulent aggregates of small, hemoglobin. round, basophilic granules will be evident For leukocytes, the magnitude of damage to throughout the slides (typically both on the cells may vary and several forms may be rec- surface of cells and in between cells). Large ognized including: the “stripping” of cyto- amounts of this precipitated stain may obscure plasm from the cell to leave a “naked” nucleus; cell detail. Small amounts, typically individual disruption of the cell membrane resulting in granules, may be misinterpreted as etiological an “exploded” cell with dispersal of granules agents (such as cocci bacteria or hematozoa) (if present) and less dense nucleus; almost by inexperienced hematologists. complete disruption of the cell making identi- Characteristic morphological artifacts may fi cation unreliable; and the remnant nuclear result if the slide is incompletely dried prior to material of disrupted cells, present as strands staining. When this occurs, the water on the in the blood fi lm. It is inappropriate to surface of the cells may interfere with the pen- interpret lysed cells and consequently the dif- etration of the stain into the cell and results in ferential leukocyte count may be biased if a inconsistent staining. Typically erythrocytes signifi cant number of cells are affected. are most affected and exhibit either a “moth- Collection and handling of blood samples 27 eaten” appearance, or small, irregularly “Numerical artifacts”, that is artifactual shaped, refractile “structures”. causes of a “change” in the concentration of In some instances, non-hematological cells cell(s), are only briefl y reviewed here as the may be encountered in a blood fi lm. Most focus of this book is the morphological commonly encountered are anucleated squa- characteristics of haematological cells. Post- mous epithelial cells from the skin of the bird, collection changes that disrupt cells or cause sampled during the venipuncture. Occasion- aggregation of cells will artifactually decrease ally other cell types that are anatomically asso- the concentration of those cells. As previously ciated with the sampling site may be harvested. mentioned, delayed processing, exposure to For example, cells from the bone may be environmental extremes and rough handling observed in blood collected from a clipped nail of the sample may result in disruption of (Clark & Tvedten 1999). Similarly adipocytes cells. Aggregation of thrombocytes occurs may be collected from subcutaneous adipose commonly in most species of birds and is pro- tissue adjacent to the vein selected for moted by tissue damage during the venipunc- venipuncture. ture or by stasis of blood during the collection Etiological agents, such as bacteria and (such as from small veins). As previously fungi, that are not part of a disease process, described, the type of anticoagulant used may may be observed in blood fi lms. These may affect the formation of the cells. Notably, the originate from contaminants in the sample use of lithium heparin as an anticoagulant may which subsequently replicate in the sample cause “clumping” of leukocytes. Such clump- (and therefore are most commonly encoun- ing of leukocytes results in uneven distribution tered with samples that have a delay in proof cells throughout the sample and conse- cessing) or from organisms that grow in the quently affects the measurement of concentra- staining solution. To prevent the latter, a tion of leukocytes. regular schedule of stain maintenance, either replacement or fi ltration, is recommended. In addition, airborne environmental contami- MEASUREMENT OF nants may alight on slides that are drying or waiting to be stained. These are usually a HEMATOLOGICAL CELLS single organism. In most cases there will be no phagocytosis of these organisms by leukocytes. See Figures 41–45. However, is some cases of “delayed process- As the measurement of hematological cell ing” leukocytes may phagocytose the prolifer- concentration is intertwined with the identifi - ating contaminant bacteria. cation of cell type by morphological features Extraneous substances may be observed in using light microscopy (especially for leuko- blood fi lms. Most commonly observed is glove cytes), the methods that may be used to provide powder, an irregularly round to polyhedral, a measurement of the erythrocytes, leukocytes pale colored structure of about 20 µm in diam- and thrombocytes of a bird are briefl y consid- eter, often with a refractile center. Also com- ered in the following text to allow the reader monly encountered are fi bers from clothing. to interpret the data provided in the descrip- These are often present towards the leading tion of the cases. edge of the fi lm and are typically linear with The characteristics of the erythron may a mildly basophilic color. A range of other be assessed using the PCV, to give an substances may be encountered in specifi c cir- overall estimate of erythrocyte “volume”, cumstances, such as granules of sand in blood erythrocyte concentration and hemoglobin samples that are collected from birds in coastal concentration. The concurrent interpreta- environments. tion of all of these values affords the best 28 Atlas of Clinical Avian Hematology

Figure 41 Blood from a Port Lincoln parrot (Barnar- Figure 42 Blood from an Australian kestrel (Falco dius zonarius) that has been diluted (1:200 with 0.85% cenchroides) that has been quantitatively mixed with saline) then applied to a disposable hemocytometer an acidophilic stain. Within the square of the hemo- (KOVA® Glasstic® slide 10, Hycor Biomedical LTD, cytometer central to the image, two acidophils can be UK), showing hematological cells, predominantly recognized. erythrocytes. The whole blood was diluted 1:40 (50 µL to 1.95 mL) with counting fl uid, containing stain that is taken up by the cytoplasmic granules of acidophils, then added to a disposable hemocytometer (KOVA® Glasstic® slide 10, Hycor Biomedical LTD, UK) which has a interpretation of the status of the erythrocytes volume of 0.9 µL within the grid. The concentration of the bird. of the acidophils (per microliter) is then calculated as: PCV is the proportion of the volume com- n (number of acidophils observed in the entire grid) × prised of erythrocytes in the whole blood. 1.1 (to convert the volume of the grid to one microliter) 40 (dilution factor). In the current example, It is typically measured after centrifugation of × n = 9, consequently the acidophil count was 0.396 × whole blood for 5 minutes at 10,000 G in a 103/µL (9 × 40 × 1.1) or 0.396 × 109/L. A differential glass capillary tube. It is expressed as the frac- leukocyte count from a Romanowsky stained blood tion that the erythrocyte volume comprises of fi lm identifi ed 26% heterophils, 66% lymphocytes, the whole blood volume, with the Système 2% monocytes, 1% eosinophils and 5% basophils. International d”Unités (SI units) of “litre of The absolute total leukocyte concentration = 9 erythrocytes per litre of blood” (L/L). “PCV” 0.396/(0.26 + 0.01) = 1.47 × 10 /L and consequently is often used interchangeably with “hemato- the component leukocyte concentrations were 0.38 × 109/L heterophils, 0.97 × 109/L lymphocytes, 0.03 × crit”; although in modern usage the latter 109/L monocytes, 0.01 × 109/L eosinophils and 0.07 × usually refers to a value calculated from the 109/L basophils. number and volume of erythrocytes in a blood Note that in both the current case and Figure 43 (Aus- sample using an automated hematology analy- tralian raven), the proportional values for the differen- ser. To complicate this interpretation, “hema- tial leukocyte count are the same, but as the total tocrit” has been used historically to refer to leukocyte concentration was different, the absolute the fraction of erythrocytes determined by the concentration for each type of leukocyte was different. This illustrates the necessity to interpret absolute leu- sedimentation of a sample of blood. kocyte values. Determination of the erythrocyte concentration of birds is typically not problematic and may be undertaken by counting an aliquot of blood in a hemocytometer or by an (appropri- Collection and handling of blood samples 29

results (Campbell & Dein 1984, Hawkey & Samour 1988). Recent reports have used a colorimetric method assessed at both 570 and 880 nm to compensate for turbidity of the sample (Samour et al. 2005). Using the measured values, additional characteristics of erythrocytes can be calculated. The average volume of erythrocytes may be used to give an indication of erythrocyte “size”. Mean corpuscular volume (MCV) may be determined by direct measurement of erythrocytes using an automated hematology analyser or, if the packed cell volume and Figure 43 Blood from an Australian raven (Corvus coronoides) that has been quantitatively mixed with erythrocyte concentration are known, may an acidophilic stain. Within the square of the hemo- be calculated according to the following cytometer central to the image, fi ve acidophils can be formula: recognized amidst numerous non-staining hematological cells (that can be distinguished because of the contrast achieved by a lowered condenser on the microscope). In this example, the Australian raven had 70 acidophilic leukocytes in the grid; consequently, the acidophil count was 3.08 × 103/µL (70 × 40 × 1.1) or 3.08 × 109/L. A differential leukocyte count from a Romanowsky stained blood fi lm identifi ed 26% heterophils, 66% lymphocytes, 2% monocytes, 1% eosinophils and 5% basophils. The absolute total leukocyte concentration = 3.08/(0.26 + 0.01) = 11.41 × 109/L and consequently the component leukocyte concentrations were 2.97 × 109/L heterophils, 7.53 × 109/L lymphocytes, 0.23 × 109/L monocytes, 0.11 × 109/L eosinophils and 0.57 × 109/L basophils. Note the effect of the proportion of acidophilic cells present can be illustrated by comparison with Figure 44 (an Australian black-shouldered kite) which has a similar Figure 44 Blood from an Australian black-shouldered acidophil concentration but a higher percentage of kite (Elanus axillaris) that has been quantitatively mixed acidophils and consequently a lesser absolute total with an acidophilic stain. Within the square of the leukocyte concentration. hemocytometer central to the image, four acidophils can be recognized. In the current case, the Australian black-shouldered ately calibrated) automated hematology analy- kite had 68 acidophilic leukocytes in the grid; conse- ser (Campbell & Dein 1984, Hawkey & quently, the acidophil count was 2.99 × 103/µL (68 × Samour 1988). 40 × 1.1) or 2.99 × 109/L. A differential leukocyte Colorimetric determination of hemoglobin count from a Romanowsky stained blood fi lm identi- concentration, by the cyanmethemoglobin fi ed 57% heterophils, 13% lymphocytes, 16% mono- method assessed at 540 nm, has been under- cytes, 7% eosinophils and 7% basophils. The absolute total leukocyte concentration = 2.99/(0.57 + 0.07) = taken on avian blood following the lysis of 4.67 × 109/L and consequently the component leuko- erythrocytes and centrifugation to remove cyte concentrations were 2.66 × 109/L heterophils, nuclei, which may contribute to turbidity of 0.61 × 109/L lymphocytes, 0.75 × 109/L monocytes, the samples and result in spuriously increased 0.33 × 109/L eosinophils and 0.33 × 109/L basophils. 30 Atlas of Clinical Avian Hematology

The MCH may be calculated from the hemoglobin and erythrocyte concentrations according to the formula:

MCH (pg) = Hemoglobin concentration (g/L)/erythrocyte concentration (× 1012/L)

For example, a blue-throated conure (Pyr- rhura cruentata) with a hemoglobin concentration of 128 g/L and an erythrocyte concentration of 3.9 × 1012/L had a MCH of 128/3.9 = 32.8 pg The MCHC may be calculated from hemo- Figure 45 A hemocytometer containing blood from globin concentration and packed cell volume an Australian gannet (Morus serrator) that has been according to the formula: quantitatively mixed with an acidophilic stain. Eight acidophils are present within the central square of the hemocytometer. In the current case, the Australian MCHC (g/L) = Hemoglobin concentration gannet had 157 acidophilic leukocytes in the grid; (g/L)/packed cell volume (L/L) consequently, the acidophil count was 6.91 × 103/µL 9 (157 × 40 × 1.1) or 6.91 × 10 /L. The differential leu- For example, a brown goshawk (Accipiter fas- kocyte count from a Romanowsky stained blood fi lm ciatus) with a hemoglobin concentration of identifi ed 85% heterophils, 10% lymphocytes, 4% monocytes, 0% eosinophils and 1% basophils. The 146 g/L, and a packed cell volume of 0.42 L/L absolute total leukocyte concentration = 6.91/(0.85) = had a MCHC of 146/0.42 = 346 g/L. 8.13 × 109/L and consequently the component leuko- Several methods have been used to deter- cytes concentrations were 6.91 × 109/L heterophils, mine the total leukocyte concentration of 0.81 × 109/L lymphocytes, 0.33 × 109/L monocytes and birds. These have included; automated counts 9 0.08 × 10 /L basophils. by hematology analysers, phase contrast microscopy, quantitative staining of leukocytes, quantitative staining of acidophils in MCV (fL) = packed cell volume (L/L)/ conjunction with a differential leukocyte count erythrocyte concentration (× 1012/L) from a blood fi lm, and fl ow cytometry. The presence of nucleated erythrocytes and For example, an eclectus parrot (Eclectus thrombocytes, as well as nucleated leukocytes, roratus) with a packed cell volume of 0.48 L/L complicates the measurement of the leukocyte and an erythrocyte concentration of 2.9 × concentration in avian blood samples using 1012/L had a MCV of 0.48/2.9 × 1012 = 166 × automated hematology analysers. Post et al. 10−15 L = 166 fL. (2003) used chicken blood mixed with EDTA The amount of hemoglobin per erythrocyte to compare manual differential counts (100 may be expressed by the calculated indices cells) with automated differential counts (Cell mean corpuscular hemoglobin (MCH) and Dyn 3500, Abbott Laboratories, IL, USA). mean corpuscular hemoglobin concentra- These authors found similar values for hetero- tion (MCHC). These may be used to deter- phils and lymphocytes for both methods, but mine the average amount of hemoglobin “per a signifi cantly greater proportion of mono- erythrocyte” which may aid the investigation cytes and a signifi cantly lesser proportion of of disorders of erythrocytes, particularly basophils with the automated method, com- anemia. pared to the manual method. A similar study Collection and handling of blood samples 31 that assessed chicken blood mixed with lithium (Unopette test 5877, Becton Dickinson, Ruth- heparin, that compared manual differential erford, NJ). counts (200 cells) with results from the Cell When the acidophil concentration is deter- Dyn 3500, found the total granulocyte counts mined in conjunction with a differential leu- had a good correlation (r = 0.80 to 0.93) kocyte count from a blood fi lm, the total (Lilliehook et al. 2004). However, monocytes leukocyte concentration and subsequently dif- were less well correlated (r = 0.70) and there ferential leukocyte concentrations may be cal- was poor correlation for lymphocyte counts. culated. For example, an aliquot of the blood Phase contrast microscopy has been used to collected from a masked owl (Tyto novaehol- distinguish the hematological cells in avian landiae) was diluted in a solution that con- blood. Janzarik (1981) described the morphol- tained phloxine B. The stained cells (acidophils) ogy of erythrocytes, leukocytes and thrombo- were counted in a hemocytometer and their cytes of chickens. Dilution of blood with 1% concentration determined to be 6.0 × 109/L. ammonium oxalate solution (1 : 20) and exam- A differential leukocyte count from a ination by phase contrast microscopy has been Romanowsky stained blood fi lm identifi ed used to identify leukocytes and thrombocytes 62% heterophils, 25% lymphocytes, 5% from numerous species of non-domestic birds monocytes, 2% eosinophils and 6% basophils. (Hawkey & Samour 1988, Samour et al. 1994, The absolute total leukocyte concentration = 1996, 2005). 6.0/(0.62 + 0.02) = 9.4 × 109/L and conse- Dein et al. (1994) reviewed “manual” quently the component leukocytes concentra- methods, which used a hemocytometer and tions were 5.8 × 109/L heterophils, 2.4 × 109/L light microscopy, for the quantitative determi- lymphocytes, 0.5 × 109/L monocytes, 0.2 × nation of the leukocyte concentration of avian 109/L eosinophils, and 0.6 × 109/L basophils. blood. The use of various diluting fl uids and This method may also be infl uenced by mea- stains has been employed to promote the surement (counting) variations in the differen- identifi cation of leukocytes. Nat and Herrick tial leukocyte count. Koepke (1980) presented (1952) developed a solution containing methyl 95% confi dence limits for the percentage of violet 2B that stains all types of leukocytes and leukocytes observed in differential leukocyte thus allows the total leukocyte concentration counts. For example, for an observed value of to be determined from quantitative counting 30% (for any cell type), the value may be of cells in a hemocytometer. Typically, leuko- between 21 and 40% when 100 cells are cytes have a dark blue color whereas erythro- counted and between 26 and 35% when 500 cytes have a palely basophilic nucleus and cells are counted. Furthermore, inaccurate colorless cytoplasm. Dein et al. (1994) reported identifi cation of cells may affect the calcula- an overall coeffi cient of variation (CV) of tion of total leukocyte concentration. For 14.2% for this method. example, if thrombocytes or rubricytes were Similarly stains, such as phloxine B, have misclassifi ed as lymphocytes, the proportion been used to identify acidophilic granulocytes of acidophils would be (erroneously) decreased and when counted in a hemocytometer to and consequently the total leukocyte concen- allow a quantitative determination of their tration (and subsequently the absolute concen- concentration to be made (Campbell & Dein tration of each of the types of leukocytes) 1984). The dilution of the sample and the would be increased. Consequently, users of volume of the particular hemocytometer this method must be aware of the sources of employed will affect the calculation to deter- error in the determination of leukocyte mine the concentration of acidophils. Dein et concentration. al. (1994) reported an overall CV of 6.8% Flow cytometry has been used for the mea- when using a prepackaged diluent system surement of avian leukocytes with populations 32 Atlas of Clinical Avian Hematology of erythrocytes, granulocytes, monocytes and 2003, Davis et al. 2004, Dunbar et al. 2005, lymphocytes able to be distinguished from the Owen & Moore 2006). However, there is little blood of Japanese quail, chickens and swan scientifi c data available to objectively compare geese (Anser cygnoides) (Morimoto et al. the results of such estimations with quantita- 2002, Uchiyama et al. 2005). However, it is tive methods for the determination of leuko- unlikely that this method will be routinely cyte concentration. applied for the analysis of clinical samples in A comparison of the leukocyte concentra- the foreseeable future. tions obtained by a quantitative hemocytome- In some instances, particularly with small ter method (Unopette eosinophil method) and birds, it is not possible to obtain a suffi cient estimated from a blood fi lm, showed that for volume of blood to perform a “quantitative leukocyte concentrations greater than 14 × measurement” of the concentration of leuko- 109/L, there was a signifi cant difference cytes. As an alternative, some authors have between the two methods with greater values suggested that “estimation” of the leukocyte recorded for the hemocytometer counts (Russo concentration from a blood fi lm to provide an et al. 1986). These authors also found greater indication of the leukocyte concentration variability in the estimated counts (mean CV (Campbell and Dein 1984, Woepel & Ross- of 28%) than for quantitative counts (mean kopf 1984). These methods have been CV of 12.5%). employed by a number of researchers to esti- Consequently, in the authors’ opinion, mate the leukocyte concentration of small “estimated counts” should be reserved for sit- birds (Tidemann et al. 1992, Ots & Horak uations where there is no facility to perform 1998, Acquarone et al. 2002, Mazerolle & quantitative measurement of leukocyte Hobson 2002, Ilmonen et al. 2003, Saks et al. concentration. General hematological characteristics2 of birds

INTRODUCTION This chapter describes the general morphological characteristics of the cells from the The blood of all birds contains erythrocytes, blood of healthy birds. leukocytes and thrombocytes. In contrast to mammals, the mature cells of each of these lines retain their nucleus throughout ERYTHROCYTES the life of the cell. The vast majority of avian species require effective circulation of See Figures 46–62. erythrocytes to provide adequate oxygen to The erythrocytes of birds, when examined the muscles of fl ight. Furthermore, given their in Romanowsky stained blood fi lms by light adaptation to extremely disparate environ- microscopy, are ovoid with a centrally located ments, from rainforest to desert and sea to ovoid nucleus and evenly colored eosinophilic mountain, it is no surprise to fi nd physiologi- cytoplasm. The “ovoid” shape varies between cal differences that may be refl ected in the species with some species having noticeably hematological characteristics of individual rounded cells whereas other species have a species. Table 2.1 illustrates some of the hema- narrower, elongated shape. The nucleus is tological characteristics reported for birds. ovoid and composed of darkly basophilic, However, the “species-specifi c” hematological coarsely clumped chromatin. When examined characteristics have not yet been described for by scanning electron microscopy, mature the majority of species of birds. Fewer studies erythrocytes are ovoid with a smooth, mostly still have assessed the hematological response fl at surface that may be distended in the central to disease in birds. The hematological idiosyn- region of the cell by the nucleus (Hawkey & crasies of any nominated species remain largely Samour 1988). This “bulge” is distinct in the unknown and have the potential to embarrass turkey (Meleagris gallopavo) (Hawkey & the best extrapolation of available scientifi c Dennett 1989) but was not distinct in the knowledge. majority of cells from representatives of several 34 Atlas of Clinical Avian Hematology et al. 1996 et al. 2006 wkey et al. 2002 1983 1994 1988 et al. 1999 1988 et al. 1982 1985 1988 eported) Jennings 1996 Jennings Jennings 1996 Jennings /L 9 × 10 0.00–1.45 Bearhop /L 9 × 10 0.00–1.12 0.00–0.88 et al. Hawkey 0.00–0.50 0.00 Ha /L 9 × 10 0.15–0.76 0.00–0.30 0.10–0.90 0.00–1.40 0.0–1.20 0.00–1.40 1992 Averbeck 0.10–0.60 0.00–0.10 0.10–1.10 Coke et al. 2004 0.20–1.49 0.00–0.75 0.00–1.55 et al. 2001 Parga 0.00–0.10 0.00–0.65 0.00–0.35 0.00–1.60 0.00–1.69 0.00–1.46 & Haines West 0.00–1.15 0.00–0.39 0.00–0.34 Samour et al. 0.00–0.20 0.00–0.30 0.10–1.60 & Samour Hawkey 0.00–0.30 0.00 0.00–0.20 & Samour Hawkey 0.00–0.21 0.00 0.00–0.80 Hawkey 0.00–1.42 0–0.540.07–1.68 0.00–4.63 0.09–5.72 Merritt 0.00–1.00 0.00–2.50 0.00–0.90 & Samour Hawkey 0.18–2.21 0.08–1.87 0.00 Uhart /L 9 × 10 /L 9 × 10 5.20–12.50 2.50–7.50 /L 9 × 10 4.70–9.40 2.70–5.60 0.65–4.00 4.30–31.00 0.50–14.0 0.30–14.1 4.00–11.00 3.50–6.97 1.38–1.93 5.50–27.70 1.81–15.51 1.32–14.23 5.70–11.60 4.59–9.243.20–7.85 0.53–2.83 1.70–5.70 0.00 0.80–3.30 4.50–15.40 2.30–8.00 0.90–3.30 3.30–10.30 1.85–7.31 0.78–2.11 1.53–15.90 0.59–12.45 0.93–14.50 3.55–23.8 1.41–11.6 0.89–11.9 3.20–10.20 2.20–6.10 0.60–2.90 0.00 11.50–22.30 4.22–17.70 2.59–11.93 0.48–4.56 136–145 1.40–7.60 0.10–2.20 0.70–2.50 147–163 4.80–10.0 2.30–6.71 0.60–2.36 152–182 5.00–15.00 4.20–9.30 2.70–4.50 g/L fL /L 12 × 10 0.46–0.57 2.60–3.48 122–181 163–177 0.38–0.50 1.40–3.10 103–165 142–270 0.43–0.53 2.60–3.80 121–177 141–156 0.35–0.63 NR NR NR 0.38–0.50 1.90–2.70 126–1680.43–0.55 180–204 2.09–3.36 133–163 153–228 0.41–0.51 2.70–3.50 148–189 132–157 0.43–0.51 3.00–3.60 142–170 137–155 0.38–0.58 1.12–1.85 92–177 234–419 0.34–0.49 1.40–1.90 133–192 238–302 L/L 0.44–0.54 NR NR NR wood duck wood backed gull backed goshawk chicken crane bustard macaw parrot fl amingo fl penguin SpeciesWhite-winged PCV RBC Hb MCV WBC Hets Lymphs Monos Eos Baso Reference Great black- Puna ibis 0.36–0.51 2.54–3.68 132–214 Harris’ hawk 0.40–0.55 2.63–3.50 121–171 Northern Greater prairie Greater prairie Red-crowned Red-crowned Houbara White pelican 0.33–0.45 1.90–2.70 98–166 Blue & gold African grey Great skua 0.39–0.51 2.08–3.52 NR 135–222 Greater Gentoo Barn owl 0.42–0.51 2.20–3.00 127–164 145–216 Units Greater rhea Table 2.1 Table : Lymphs Hets: heterophil concentration; species. (Hb: hemoglobin concentration; of selected avian Hematological characteristics NR: not r Baso: basophil concentration; Eos: eosinophil concentration; concentration; Monos: monocyte concentration; lymphocyte General hematological characteristics of birds 35

Figure 46 Typical avian erythrocytes in a thin blood fi lm, stained with a Romanowsky stain, and viewed by light microscopy. The erythrocytes have an ovoid Figure 48 Blood from a clinically healthy Australian shape, eosinophilic cytoplasm due to their hemoglo- kestrel (Falco cenchroides). The transmission electron bin content and a central, ovoid nucleus composed of micrograph of an erythrocyte (lead citrate and uranyl dense, coarsely clumped chromatin. Blood from a acetate stains) shows a moderately dense cytoplasm, black swan (Cygnus atratus). (Modifi ed Wright’s due to hemoglobin content, without notable organ- stain.) elles. The erythrocytes from this species have a narrower, more elongated, elliptical shape (compared with the rounded elliptical shape of the cell in the previous fi gure). Artifactual separation of the nucleus from the cytoplasm is evident. Figures 26 and 27 illustrate the light microscopic appearance of erythrocytes from this species.

Figure 47 Blood from a clinically healthy takahe Figure 49 Erythrocytes from a grey-faced petrel (Porphyrio mantelli). The transmission electron micro- (Pterodroma macroptera gouldi), examined by scan- graph of an erythrocyte (lead citrate and uranyl acetate ning electron microscopy. Illustrated is the ovoid stains) shows a moderately dense cytoplasm, due to shape and lack of a distinct central “bulge” and the hemoglobin content, without notable organelles. The relatively ﬂ at surface of erythrocytes in this species. light microscopic appearance of erythrocytes from this species is depicted in Figure 56. 36 Atlas of Clinical Avian Hematology

Figure 52 Scanning electron micrograph showing three erythrocytes from the blood of a blue-breasted Figure 50 Erythrocytes from a red-tailed black cock- quail (Coturnix chinensis). Note the rounded appear- atoo (Calyptorhynchus banksii) viewed by scanning ance of the erythrocyte, typical of Galliformes, and the electron microscopy. The erythrocytes are ovoid and lack of a distinct central bulge. The light microscopic lack a distinct distension of the central region of the appearance of erythrocytes from this species is illus- cell. The light microscopic appearance of erythrocytes trated in Figures 149, 150 and 244. from this species is shown in Figures 68, 194 and 195.

Figure 53 Blood from a New Zealand blue duck Figure 51 Erythrocytes from a barn owl (Tyto alba) (Hymenolaimus malacorhynchus) illustrating several viewed by scanning electron microscopy. The eryth- “types” of erythroid cells that may be distinguished by rocytes are ovoid and lack a distinct distension of the light microscopy including: mature erythrocytes, a central region of the cell. Mild anisocytosis can be polychromatophilic erythrocyte (arrow-head) and an discerned. The light microscopic appearance of eryth- anucleated erythrocyte or “erythroplastid” (arrow). rocytes from this species is depicted in Figures 57, (Diff Quik stain.) 73–78. General hematological characteristics of birds 37

Figure 56 Blood from a takahe (Porphyrio mantelli) Figure 54 Blood from a Mindanao bleeding heart showing the rounded erythrocytes encountered this dove (Gallicolumba criniger) illustrating mature eryth- species including three polychromatophilic erythro- rocytes, a polychromatophilic erythrocyte and an cytes, identiﬁ ed by their more basophilic cytoplasm. erythroplastid. (Modiﬁ ed Wright’s stain.) (Diff Quik stain.)

Figure 55 Blood from a helmeted honeyeater Figure 57 Blood from a barn owl (Tyto alba) illustrat- (Lichenostomus melanops) portraying mature erythro- ing a rubricyte (arrow) and a polychromatophilic cytes, a polychromatophilic erythrocyte, an erythro- erythrocyte amidst several mature erythrocytes. (Modi- plastid and an erythrocyte with a segmented nucleus ﬁ ed Wright’s stain.) Rubricytes are rounded cells dis- (arrow). (Modiﬁ ed Wright’s stain.) tinguished by their well delineated cell and nuclear membranes that contain, respectively, uniform agranulated basophilic cytoplasm and clumps of very dense chromatin interspersed with paler areas. A continuity of the nuclear structure from the rubricyte to polychromatophilic erythrocyte and mature erythrocytes can be seen. Lymphocytes from the same bird are illustrated in Figures 73–77. 38 Atlas of Clinical Avian Hematology

Figure 58 Blood from an anemic (PCV 0.20 L/L) Aus- Figure 60 Blood from an Australian black-shouldered tralian black-shouldered kite (Elanus axillaris) showing kite (Elanus axillaris) showing a Heinz body (arrow), an erythrocyte with basophilic stippling (arrow) amidst evident as a rounded protrusion, at the pole of an erythrocytes. (Modiﬁ ed Wright’s stain.) The basophilic erythrocyte. (Modiﬁ ed Wright’s stain.) Heinz bodies stippling represents aggregates of rRNA in the cyto- represent aggregates of oxidized hemoglobin. Also plasm of the erythrocytes. present is a heterophil.

Figure 61 A mitotic fi gure from the blood of a Figure 59 Blood from a black-necked aracari (Ptero- clinically healthy emu (Dromaius novaehollandiae). glossus aracari) showing a hemoglobin crystal (arrow) (Modifi ed Wright’s stain.) within an erythrocyte and several mature erythrocytes. (Modifi ed Wright’s stain.) The bird exhibited only very small numbers of these crystals and consequently they were unlikely to be clinically signifi cant. General hematological characteristics of birds 39

dimensions correlate to a mean corpuscular volume of about 150 fL. Some variation occurs between species, with the mean MCV reported to be: demoiselle crane (Grus virgo) 154– 162 fL (Hawkey et al. 1983); peregrine falcon (Falco pereginus) 118–146 fL (Jennings 1996); Victoria crowned pigeon (Goura victoria) 135–178 fL (Peinado et al. 1992); and great skua (Catharacta skua) 135–222 fL (Bearhop et al. 1999). In addition to typical “mature” erythrocytes that comprise the predominant type of cell in the blood of healthy birds, lesser numbers of cells representing the different stages of ery- Figure 62 A mitotic fi gure from the blood of a barn throid development may be encountered. owl (Tyto alba). (Modifi ed Wright’s stain.) Examination of the blood fi lms of most birds reveals a small proportion of polychromatophilic erythrocytes amidst mature erythrocytes. Polychromatophilic erythrocytes are the unrelated species including: barn owl (Tyto penultimate stage of the development of eryth- alba), red-tailed black cockatoo (Calyptorhyn- rocytes. Compared to mature erythrocytes, chus banksii), grey-faced petrel (Pterodroma polychromatophilic erythrocytes are charac- macroptera gouldi) and blue-breasted quail terized by cytoplasm which has a bluish col- (Coturnix chinensis). It is likely that some oration, due to increased amounts of ribosomal variation in the magnitude of distension RNA. In addition, the nucleus typically has occurs between species and consequently less dense chromatin than those of mature additional studies that encompass a wide erythrocytes. Polychromatophilic erythrocytes range of species are needed to determine the typically comprise 1–5% of all erythrocytes “typical” three-dimensional form of avian in healthy birds (Campbell & Dein 1984, erythrocytes. Campbell 1995). Increased numbers may be Avian erythrocytes are larger than those observed when increased erythropoiesis occurs encountered in mammals. Dimensions of in response to an anemia (a “regenerative erythrocytes from the marsh harrier (Circus response”). aeruginosus) were 13.78 ± 0.50 µm in length Reticulocytes are erythrocytes that when and 7.95 ± 0.35 µm in width with nuclear stained, by the incubation of living cells with dimensions of 6.46 ± 0.15 µm in length and a “supravital” stain, such as new methylene 2.42 ± 0.16 µm in width (Lavin et al. 1992). blue, exhibit granular aggregations of RNA The erythrocytes of another species of raptor, (“reticulum”). When stained in this manner, the gyr falcon (Falco rusticolus), were similar some residual cytoplasmic RNA will be evident in size: 14.82 ± 0.07 µm in length and 7.21 ± in most avian erythrocytes. As a consequence, 0.4 µm in width (Samour et al. 2005). Simi- the term “reticulocyte” should be reserved larly, for the rock dove (Columba livra), the for cells with aggregated “reticulum” that dimensions of erythrocytes were reported to forms a distinct (but incomplete) ring around be 12.20 ± 0.35 µm in length and 6.60 ± the nucleus. The number of these cells 0.00 µm in width with nuclear dimensions of typically correlates well with the number 6.80 ± 0.29 µm in length and 3.3 ± 0.00 µm of polychromatophilic erythrocytes in a in width (Gayathri & Hedge 1994). These Romanowsky stained blood fi lm. 40 Atlas of Clinical Avian Hematology

Less mature stages of erythroid develop- ophils, basophils, lymphocytes and mono- ment may be observed in the blood of birds. cytes. As heterophils, eosinophils and basophils Most commonly encountered are rubricytes. all possess distinct cytoplasmic granules they Rubricytes are typically smaller and more may be collectively referred to as granulo- rounded than mature erythrocytes. Their cytes. Furthermore, as the predominant cyto- nucleus is typically round and is composed of plasmic granules of heterophils and eosinophils coarsely clumped chromatin that is less dense both exhibit affi nity for acidic stains (such as than chromatin of mature erythrocytes. Rubri- eosin) they may be referred to as acidophils. cytes have a nuclear to cytoplasmic ratio that Lymphocytes and monocytes may be collec- is greater than the nuclear to cytoplasmic ratio tively referred to as mononuclear cells. of mature erythrocytes, with a small to moder- In many species of bird, heterophils are ate rim of markedly basophilic cytoplasm. typically the most commonly encountered Erythroid cells undergoing division in the granulocyte and often the most commonly peripheral blood, observed as mitotic fi gures, encountered leukocyte in the peripheral blood may occasionally be encountered in blood of birds. When examined in Romanowsky fi lms from clinically healthy birds. stained blood fi lms by light microscopy, het- A small number, typically less than 1%, of all erophils are typically irregularly round leuko- erythrocytes in the blood of birds are anucleated. cytes with a lobed nucleus, basophilic nucleus These are referred to as erythroplastids. There and prominent acidophilic cytoplasmic gran- have been no reports of the number of these cells ules. The nucleus commonly has two to three being specifi cally affected by a disease process. lobes, with the mean number of lobes reported Erythrocytes that exhibit a “variant” shape, to be 2.44 for chickens (Lucas & Jamroz that is not ovoid, are collectively known as poikilocytes and have been recognized in the blood of birds (Hawkey & Samour 1988, Campbell 1995, Fudge 2000, Campbell & Ellis 2007). These variant cells typically comprise a very small proportion of the total erythrocyte population in healthy birds. Example of variant shapes include: “drop-shaped” erythrocytes, fusiform erythrocytes and round erythrocytes. However, the association between increased proportions of these cells with variant shapes and a disease process is less well documented than it is for mammals. The presence of hemoparasites such as Hae- moproteus spp and Plasmodium spp may alter the morphology of erythrocytes. The presence Figure 63 Blood from a clinically healthy takahe (Por- of the organism(s) may distort the shape of the phyrio mantelli). Transmission electron microscopy cell, enlarge the size of the cell, or displace the illustrates the ultrastructure of a heterophil (lead citrate nucleus within the cell. and uranyl acetate stains). Notably, the structure of the large cytoplasmic granules can be seen to be fusiform in longitudinal section and “round” on cross section. LEUKOCYTES Other organelles including smaller round granules, endoplasmic reticulum, mitochondria and vacuoles may be observed in heterophils. Small pseudopodia See Figures 63–80. are commonly observed at the periphery of the cell. Five types of leukocytes are encountered in The light microscopic appearance of a heterophil from the blood of birds, namely: heterophils, eosin- this species is portrayed in Figure 152. General hematological characteristics of birds 41

Figure 64 The light microscopic appearance of a Figure 66 Blood from a clinically healthy Australian typical avian heterophil is characterized by many kestrel (Falco cenchroides). Transmission electron brick-red, fusiform granules present at high density microscopy illustrates the ultrastructure of an eosino- throughout the cytoplasm and a segmented nucleus phil (lead citrate and uranyl acetate stains). Notably, composed of coarsely clumped chromatin that is par- ovoid granules with homogeneous density can be tially obscured by the granules. Blood from a gang- observed in the cytoplasm. Eosinophils may also gang cockatoo (Callocephalon ﬁ mbriatum). (Modiﬁ ed contain other organelles such as endoplasmic reticu- Wright’s stain.) lum, mitochondria and vacuoles. Figure 126 illustrates the light microscopic appearance of eosinophils from this species.

Figure 67 Blood from a greater fl amingo (Phoenicop- Figure 65 A heterophil from a red-billed toucan terus ruber) demonstrating the large round, brightly (Ramphastos tucanus). The typical brick-red, fusiform eosinophilic granules within pale basophilic cyto- granules present at high density in the cytoplasm result plasm, characteristic of eosinophils of this species. in the nucleus being partially obscured with the result Also of note is the distinct segmentation of the nucleus that the cell appears to have three separate nuclei. in this cell. (Modifi ed Wright’s stain.) (Modifi ed Wright’s stain.) 42 Atlas of Clinical Avian Hematology

Figure 68 Blood from a red-tailed black cockatoo Figure 70 Blood from a sulphur-crested cockatoo (Calyptorhynchus banksii) illustrating an eosinophil (Cacatua galerita) showing a basophil with a high (left), with a high density of round, grey–mauve gran- density of dark basophilic cytoplasmic granules that ules, and a heterophil, with fusiform brick-red gran- obscure the characteristics of the nucleus. (Modiﬁ ed ules, amidst mature erythrocytes. (Modiﬁ ed Wright’s Wright’s stain.) stain.) In a number of avian species the “eosinophil” is a misnomer as the granules do not stain distinctly eosinophilic. This is further illustrated in Chapter 3.

Figure 69 Blood from a clinically healthy takahe Figure 71 Three basophils from a Stanley crane (Grus (Porphyrio mantelli). Transmission electron micros- paradisea) are illustrated. These have a low–moderate copy illustrates the ultrastructure of a basophil (lead density of basophilic cytoplasmic granules that allow citrate and uranyl acetate stains). Large, round gran- some nuclear and cytoplasmic characteristics to be ules with an internal “honey-comb” structure of vari- assessed. (Modiﬁ ed Wright’s stain.) able density can be observed in the cytoplasm, along with mitochondria, regions of endoplasmic reticulum and vacuoles. General hematological characteristics of birds 43

Figure 74 A medium sized lymphocyte, character- Figure 72 Blood from a clinically healthy takahe ized by a rounded nucleus, less dense chromatin and (Porphyrio mantelli). Transmission electron micros- a greater amount of basophilic agranulated cytoplasm copy illustrates the ultrastructure of a lymphocyte compared to the small lymphocyte, is shown. (Modi- (lead citrate and uranyl acetate stains). The cytoplasm ﬁ ed Wright’s stain.) typically exhibits relatively few organelles. In this cell only a small amount of endoplasmic reticulum and a few mitochondria are evident.

Figure 75 A large lymphocyte, with a large irregu- Figures 73–78 The mononuclear cells, comprising larly round nucleus with less dense chromatin and a lymphocytes and monocytes, may have a pleomor- greater amount of agranulated basophilic cytoplasm phic appearance and certain identifi cation of the cell’s compared to the medium lymphocyte, is depicted. lineage may not be possible using solely light micro- (Modifi ed Wright’s stain.) scopic examination of Romanowsky stained thin blood fi lms. The following six images all originated from the blood of a single bird (a barn owl, Tyto alba) and illustrate some of the morphologically identifi able categories of mononuclear cells. Figure 73 A typical small lymphocyte characterized by a rounded nucleus composed of coarse dense chromatin and a small rim of basophilic cytoplasm is illustrated. (Modifi ed Wright’s stain.) 44 Atlas of Clinical Avian Hematology

Figure 76 A “granular” lymphocyte, defi ned by the Figure 78 A monocyte, identifi ed by its large size, an presence of small azurophilic granules in the cyto- irregularly shaped nucleus composed of reticular plasm of the cell, is illustrated. (Modifi ed Wright’s chromatin and a moderate amount of basophilic cyto- stain.) These may be recognized in small numbers in plasm with several, variably sized pale vacuoles is the blood of clinically healthy birds of most species shown. (Modifi ed Wright’s stain.) and have been commonly observed in the blood of Gruiformes.

Figure 77 A stimulated (“reactive”) lymphocyte, identiﬁ ed by the increased basophilia of the cytoplasm (indicating increased amounts of RNA) and a pale perinuclear region (consistent with a Golgi zone) is portrayed. (Modiﬁ ed Wright’s stain.) The presence of Figure 79 Blood from a clinically healthy takahe these cells indicates a stimulated immune system. (Porphyrio mantelli). Transmission electron microscopy illustrates the ultrastructure of a monocyte (lead citrate and uranyl acetate stains). The cytoplasm typically exhibits a range of organelles, including endoplasmic reticulum, mitochondria, granules and glycogen particles. General hematological characteristics of birds 45

more prominent than the surrounding matrix of the granule. Few studies have assessed the ultrastructure of avian heterophils. Maxwell (1973) studied the ultrastructural characteristics of heterophils from six species of domestic birds and revealed variation between species in the size and length of granules and the morphology of the central granular body. Similarly a study of the roadside hawk (Buteo magnirostris) (Santos et al. 2003b), revealed a lobed nucleus with central euchromatin and peripheral heterochromatin, and cytoplasm that contained predominantly large, electron-dense granules Figure 80 Blood from a little penguin (Eudyptula with tapered extremities and lesser numbers of minor) that shows the light microscopic appearance of oval and “drop-shaped” granules. Also evident a monocyte. Moncytes are the most pleomorphic of were lesser numbers of small elliptical, rod or the leukocytes and the appearance of monocytes dumbbell-shaped granules and rare, very small within the same blood sample may be quite varied. electron-dense granules. Golgi, rough endo- (Modifi ed Wright’s stain.) plasmic reticulum (RER), mitochondria and small pinocytotic vesicles were also observed in the cytoplasm. 1961). In many cells, the lobes may not appear Eosinophils are the second type of acido- connected due to the presence of granules philic granulocytes that may be encountered overlying the nucleus. The nucleus, although in avian blood. They are typically less common basophilic, is often dark at the periphery and than heterophils and in many species are fades to a pale aqua-blue color centrally. defi ned by the expected brightly eosinophilic Characteristically, the nucleus of hetero- cytoplasmic granules when stained with phils is less basophilic than the nucleus of Romanowsky stains. However, in some eosinophils (Maxwell & Robertson 1998). species “eosinophil” is a misnomer as the The cytoplasm contains robust granules that cytoplasmic granules are not “eosinophilic”, are characterized in shape as fusiform (that is but may have a dull brown, aqua, grey or elongated, tapering to a point at each end) in pale blue color. longitudinal section and round in cross section Eosinophils are typically irregularly round and are “brick-red” to brown in color. The cells with a lobed nucleus composed of dark relative length and width of the granules varies basophilic chromatin. Most commonly the between species (Lucas & Jamroz 1961). The nucleus has two lobes, with a mean value of granules are typically evenly distributed 1.97 reported for the eosinophils of chickens at moderate to high density and often (Lucas & Jamroz 1961). Characteristically, obscure most of the cytoplasm (which is palely the chromatin of eosinophils stains more basophilic when apparent) and some of the darkly than that of heterophils in the same nucleus. In some heterophils, the granules may sample and, in contrast to heterophils, the exhibit a “central granular body” (Maxwell intensity of the color is similar throughout the & Robertson 1998). These are a round to entire nucleus. ovoid, pale or refractile structure located in Typically, the cytoplasm is pale to moder- the mid-section of the granule. In some ately basophilic and contains “eosinophilic” instances, the central granular body may be granules. The number, shape, size, hue and 46 Atlas of Clinical Avian Hematology density of granules vary between species. For electron-dense granules, RER, mitochondria example, species of fl amingos and cranes typi- and pinocytotic vesicles. cally have prominent, round, brightly eosino- The basophils of birds are most readily philic granules (Hawkey et al. 1983, 1984a,b, identifi ed by the darkly basophilic cytoplasmic 1985) whereas, species within the Anatidae granules they exhibit when stained with typically have many eosinophilic, rod-shaped Romanowsky stains. Basophils are typically granules. However, other species have less irregularly round cells that most commonly distinctive eosinophils. The appearance of have a nucleus with a single lobe; with a mean “eosinophils” is very variable between groups of 1.01 lobes reported for the basophils of of raptors (Lind et al. 1990). For example chickens (Lucas & Jamroz 1961). Classically, members of the genus Falco have eosinophils the cytoplasm contains darkly staining baso- with a few, “bluish pink to light blue” gran- philic granules at such high density that the ules in a pale blue cytoplasm (Lind et al. 1990) individual granules cannot be discerned. Com- or few small eosinophilic granules (Wernery et monly the nucleus appears pale (in compari- al. 2004, Samour et al. 2005). Samour et al. son to the overall color of granules in the (2005) noted the large effect the type of stain cytoplasm) and is partly obscured by the may have on the appearance of granules. In presence of the cytoplasmic granules, so that contrast to the falcons, birds within the genera nuclear detail is often diffi cult to discern. Buteo and Accipiter typically have round, Between species, there is some variation in the brightly eosinophilic granules (Lind et al. size, color, hue and density of granules. In 1990, Santos et al. 2003b). some case, “all” or the majority of cytoplas- Inexperienced hematologists may have dif- mic granules may fail to stain. In these cases, fi culty in distinguishing eosinophils from het- “basophils” exhibit regular, round vacuoles in erophils. When the color of the “eosinophil” a pale cytoplasm with occasional basophilic granules is eosinophilic, it is usually a brighter granules. In such cases, the nucleus is apparent “red–orange” color than the color of the gran- and is typically round to ovoid and composed ules of heterophils (typically a brown–red of moderately dense chromatin. The authors color). Additionally, the nucleus of eosinophils have noted this most commonly when Diff is typically less obscured by granules and Quik has been used to stain avian blood usually stains a darker basophilic color than fi lms. heterophils. Few studies have assessed the ultrastructure Few studies have assessed the ultrastructure of avian basophils. Maxwell (1973) reported of avian eosinophils. Maxwell and Siller (1972) the ultrastructural characteristics of basophils initially reported the ultrastructural character- from six species of domestic birds and a study istics of eosinophils from six species of domes- of the roadside hawk (Santos et al. 2003b) tic birds, then noted variation in the internal revealed that basophils had a central non- structure of eosinophil granules, with non- lobed nucleus with a nucleolus and cytoplasm crystalline (homogeneous) forms recognized in that contained granules with three distinct the European shag (Phalacrocorax aristotelis), appearances, RER, mitochondria and small crystalline in members of the Anseridae vacuoles. (Maxwell 1978), and “mixed” in the black- Lymphocytes are the most commonly crowned crane (Balearica pavonina) (Maxwell encountered mononuclear cell; in some species 1979). A study of eosinophils from the road- of birds, they are the most commonly encoun- side hawk (Santos et al. 2003b) revealed the tered leukocyte in the peripheral blood. Mor- cells had a lobed nucleus, with central euchro- phologically, lymphocytes vary in appearance. matin and peripheral heterochromatin and When stained with Romanowsky stains and cytoplasm that contained large, spherical viewed by light microscopy, these may be General hematological characteristics of birds 47 simply divided into small, medium and large tained up to six mitochondria, a Golgi appa- lymphocytes. ratus and centrioles, small amounts of RER Typical small lymphocytes are the smallest and occasional pinocytotic vesicles, lipid drop- of the leukocytes and have a round nucleus lets and small electron-dense membrane-bound composed of dense, coarsely clumped chroma- granules. Medium-sized lymphocytes were tin and a small, often incomplete “rim” of round to ovoid with large nuclear to cytoplas- cytoplasm that is moderately to deeply baso- mic ratio and small, thin pseudopodia. The philic. Medium-sized lymphocytes are larger nucleus was round or indented and was com- than small lymphocytes, often similar in size posed of peripheral heterochromatin and to granulocytes, and have an irregularly central euchromatin, with eccentric, variably round nucleus composed of moderately dense, sized nucleoli (some very large). The cytoplasm irregularly clumped chromatin with a moder- typically contained a paucity of organelles but ate amount of agranulated, moderately mitochondria, endoplasmic reticulum, Golgi basophilic cytoplasm. Large lymphocytes apparatus, centrioles, pinocytotic vesicles and are typically larger than granulocytes and membrane-bound granules with a whorl, stip- may be of similar size to monocytes. Large pled or homogeneous internal structure were lymphocytes typically have a round–ovoid observed in some cells. nucleus composed of moderately dense, irreg- Monocytes are large, pleomorphic leuko- ularly clumped chromatin with a moderate cytes. When observed by light microscopy in amount of agranulated, moderately basophilic Romanowsky stained blood fi lms, monocytes cytoplasm. have a nucleus that may be ovoid, indented Lymphocytes may exhibit several punctate, (reniform), “horse-shoe” shaped or irregularly azurophilic to basophilic granules in their shaped and is composed of fi ne to reticular cytoplasm. They are found regularly in the chromatin with a moderate to large amount of blood of most birds and have been commonly grey to basophilic cytoplasm. Granules are noted in some birds, such as Gruiformes, typically not evident in the cytoplasm; however, where they are believed to be a “normal” small eosinophilic granules may be observed feature (Hawkey et al. 1983). in some cells. One to several small vacuoles Although there is often variation in the mor- may be evident in the cytoplasm of some phology of lymphocytes within the blood of monocytes. an individual, there are no signifi cant distin- Although the morphology of monocytes guishing differences in the morphology of within the blood of an individual is often quite lymphocytes between the different species of varied, there are no signifi cant distinguishing birds. differences in the morphology of monocytes Few studies have assessed the ultrastruc- between species. As monocytes and large lym- ture of avian lymphocytes. Maxwell (1974) phocytes have a similar size and morphologi- reported the ultrastructural characteristics of cal appearance when viewed in Romanowsky lymphocytes from six species of domestic stained blood fi lms, it may be diffi cult to dis- birds, which he divided into small lympho- tinguish between these cell types by this cytes and medium-sized lymphocytes. Small technique. lymphocytes were round with a large nuclear Few studies have assessed the ultrastructure to cytoplasmic ratio and many pseudopodia. of avian monocytes. Maxwell (1974) reported The nucleus showed considerable pleomor- the ultrastructural characteristics of mono- phism being round, ovoid, reniform or indented cytes from six species of domestic birds. These and was composed of equal amounts of het- were characterized by a reniform nucleus, erochromatin and euchromatin, with one to composed of peripheral heterochromatin and two nucleoli. The cytoplasm typically con- central euchromatin with one to two nucleoli, 48 Atlas of Clinical Avian Hematology and extensive cytoplasm. The latter contained severe infl ammatory demand. The nuclear many organelles including a well defi ned structure is often diffi cult to assess when the Golgi apparatus and centrioles, microtubules, cell contains a typical number of cytoplasmic vesicles, mitochondria, RER, small dense granules, as the granules commonly obscure membrane-bound granules, lipid droplets and part of the nucleus. Consequently, changes in pinocytotic vacuoles. the complexity of the nucleus are most easily assessed when there is a concomitant decrease in the density of cytoplasmic granules. Direct Atypical morphology of effects of toxins on the nucleus of leukocytes avian leukocytes may result in karyolysis or karyorrhexis. These must be carefully distinguished from cells The morphology of “typical” leukocytes, as where the nucleus has been artifactually lysed described earlier in this chapter, may be altered during the production of the blood fi lm or due in response to infl ammation. These changes to ageing of the blood sample. In the authors’ may occur as a result of either the release of experience, artifactual causes of lysis are far leukocytes from sites of hematopoiesis before more commonly encountered than true patho- they are mature, by the direct actions of toxins logical effects on the cells in the peripheral upon the cells in the peripheral blood, or by blood. increased synthetic activity by the cell. Typi- As previously described, typical heterophils cally morphological atypia is most commonly contain many elongated, fusiform granules encountered in heterophils and lymphocytes, that typically obscure the cytoplasm and par- less commonly encountered in monocytes and tially obscure the nucleus. Atypical heterophils rarely recognized in eosinophils and basophils. may exhibit morphological abnormalities that The likelihood of morphological changes encompass changes in the cytoplasm and the occurring in response to infl ammation and the granules it contains. The cytoplasmic granules types and magnitude of the infl ammation of heterophils may change in shape, color and required to incite the changes, have not been number (density) in response to infl ammation. established for most species of birds. Typically, with mild infl ammation, the gran- Atypical heterophil morphology may be ules are less pointed and maintain typical observed in the nucleus, cytoplasm or both. color. In more signifi cant infl ammation, the Usually, atypia of the nucleus is indicated granules become round, larger and more by decreased segmentation, and consequently basophilic than typical granules. The typical decreased number of nuclear lobes. This de- number of granules is usually maintained with creased complexity of the nucleus refl ects mild infl ammation. In cases of more signifi cant decreased maturity of the heterophils and pre- infl ammation, the number of granules within mature release from the bone in response to cells is decreased and agranulated heterophils infl ammatory demand. “Band” form hetero- or those that possess only one to several gran- phils, metamyelocytes and myelocytes may all ules may be observed. be observed in response to signifi cant infl am- The decreased number of granules serves to matory demand. Band heterophils have a non- make the cytoplasm more visible and provides segmented nucleus, metamyelocytes have a greater opportunity to assess the characteris- reniform-shaped nucleus and myelocytes have tics of the cytoplasm. The cytoplasm may an ovoid (non-indented) nucleus. The chroma- exhibit an increased basophilia, due to the tin is typically less clumped (more diffuse) in presence of increased numbers of ribosomes. the less mature cells. Further indications of The cytoplasm may also exhibit a “foamy” or atypical myelopoiesis, such as an annular vacuolated appearance due to the direct action nucleus, may be observed in some cases of of toxins on the cells. However, these morpho- General hematological characteristics of birds 49 logical changes must be differentiated from a philic granules, was recorded in three species similar appearance that may occur due to from the genus Buteo with infl ammatory dis- delayed processing of a sample of blood. orders (Bienzle et al. 1997). A king shag (Phal- The morphological characteristics and the acrocorax albivenier) with aspergillosis had proportion of the cells that exhibit these char- heterophils that exhibited strongly basophilic acteristics vary with the severity of the infl am- spherical granules as well as oval eosinophilic mation. “Mild” changes typically include: granules (Hawkey et al. 1984c). Birds with mildly decreased numbers of granules, slightly mycobacteriosis, from several species of crane, rounded granules and increased basophilia of that demonstrated a heterophilia and monocy- the cytoplasm. “Moderate” changes include: a tosis, also had heterophils with a hypo-seg- greater decrease in the density of granules and mented nucleus, decreased concentration of rounded granules. Severe changes include: granules and extra, round basophilic granules. round granules, basophilic granules, large The monocytes of these birds lacked nuclear granules, “foamy”/vacuolated cytoplasm and indentation and exhibited cytoplasmic vacuo- karyolysis. The number of abnormal hetero- lation (Hawkey et al. 1990). Houbara bus- phils may be graded as “few” (5–10%), “mod- tards (Chlamydotis undulata) with chronic erate” (11–30%) and “marked” (greater than infl ammation exhibited heterophils with atypi- 30%) (Campbell 2004). cal morphology including: cytoplasmic baso- Lymphocytes may also exhibit variant philia, lack of granulation and decreased morphology, affecting both cytoplasmic and nuclear lobulation (D’Aloia et al. 1994). A nuclear characteristics; this refl ects that they lesser suphur-crested cockatoo (Cacatua sul- have responded to antigenic stimulation and phurea) with a traumatic foot injury exhibited have produced immunoglobulins. Typically, a heterophilia with atypical heterophil mor- these are medium-sized cells with increased phology including: decreased nuclear maturity amounts of deeply basophilic cytoplasm, due (“left-shift”), decreased granulation, round to an increased concentration of ribosomes, basophilic granules and “rice-shaped” eosino- and a prominent perinuclear halo that repre- philic granules (Bienzle & Smith 1999). sents the Golgi apparatus. Additionally, lym- Morphological atypia of leukocytes may phocytes that exhibit a prominent nucleolus not be present in all birds with infl ammation, may be observed. These “blast-transformed” however. For example, in a study of two cells usually represent a prelude to mitosis of species of black cockatoos with infl ammation stimulated lymphocytes. However, in large or traumatic injury, only 3/21 (14%) of birds numbers these may indicate a neoplastic pro- exhibited heterophils with morphological liferation of lymphocytes. changes (Jaensch & Clark 2004). The presence of hemoparasites, such as Leu- kocytozoon spp and Hepatozoon spp, may alter the morphology of their host cell, typically mononuclear leukocytes. The presence of THROMBOCYTES the organism(s) may distort the shape of the cell, enlarge the size of the cell, or displace the See Figures 81–83. nucleus within the cell. Thrombocytes are the nucleated hemostatic Examples of leukocytes exhibiting atypical cells of birds. Individual thrombocytes are morphology, in response to disease, have been typically smaller than all leukocytes and eryth- reported for a range of bird species. Morpho- rocytes. They have a very dense, darkly stain- logical atypia, including areas of cytoplasm ing, ovoid nucleus with a small to moderate without granules, cytoplasmic vacuolation, amount of “colorless”, pale grey or pale increased cytoplasmic basophilia and baso- basophilic cytoplasm. In some cases, fi ne 50 Atlas of Clinical Avian Hematology

Figure 81 Blood from a royal spoonbill (Platalea regia) portraying an aggregation of six thrombocytes Figure 83 Blood from a wattled crane (Grus carun- amidst mature erythrocytes. (Diff Quik stain.) Throm- culatus) illustrating a larger aggregate of thombocytes. bocytes typically have an ovoid nucleus composed of (Modiﬁ ed Wright’s stain.) In these thombocytes the uniformly dark, dense chromatin and a small amount cytoplasm is a distinct grey–basophilic color. Also of colorless to palely basophilic cytoplasm. Thrombo- present are an eosinophil and a basophil (in which the cytes are smaller than erythrocytes and most leuko- majority of granules have not taken up the basophilic cytes but may be similar in size to small stain). lymphocytes.

cytoplasmic projections (“pseudopodia”) may be evident. Several small, punctate, eosinophilic or azurophilic granules may be observed in the cytoplasm of some thrombocytes. In many cases, the thrombocytes will have aggregated into variably sized clumps throughout the blood fi lm. In these aggregates, distinct, individual cell outlines may be diffi cult to discern. When trying to distinguish thrombocytes from small lymphocytes, it is often useful to assess the morphology of thrombocytes within aggregates to determine the morphological characteristics of thrombocytes for the particular bird (or species), then use those features to identify non-aggregated thrombocytes, and fi nally distinguish the lymphocytes in contrast to the thrombocytes. Figure 82 Blood from a bald eagle (Haliaeetus leu- A study of the ultrastructure of thrombo- cocephalus) showing aggregated thrombocytes amidst cytes from roadside hawks revealed either a mature erythrocytes. (Modifi ed Wright’s stain.) Often predominantly elliptical shape or spherical thrombocytes are aggregated in blood fi lms and this shape, with organelles that included Golgi, characteristic may be used to aid the identifi cation of their cell type. However, if heparin is used as an anti- smooth endoplasmic reticulum, small mito- coagulant, leukocytes including lymphocytes may chondria, dense osmiophilic granules, periph- also occur in “clumps”. eral microtubules and canalicular system General hematological characteristics of birds 51 opened to the cell’s surface (Santos et al. of 75 clinically healthy quails. They found the 2003a). mean proportion of erythroid cells was 67.3%, the mean proportion of myeloid cells was 24.9% and the mean proportion of thrombo- HEMATOPOIESIS cyte line cells was 3.3%. The age of the bird had a signifi cant effect on the proportions of many of the cell types but no differences due Very few studies have assessed the hematologi- to the sex of the bird were observed. In the cal characteristics of bone marrow from birds, latter study, samples of bone marrow were other than chickens. These include studies of collected from the proximal tibiotarsus of 50 the Japanese quail (Coturnix coturnix japon- clinically healthy, adult gulls. The mean pro- ica) (Nazifi et al. 1999) and black-headed gull portion of erythroid cells was 39.9%, the (Larus ridibundus) (Tadjalli et al. 2002). The mean proportion of myeloid cells was 49.4% former study assessed samples of blood and and the mean proportion of thrombocytic cells bone marrow (from the proximal tibiotarsus) was 6.0%.

Particular hematological characteristics3 of birds

INTRODUCTION information presented in this chapter. It should be noted that the vast majority of bird species belong within the Passeriformes, which encom- The following chapter describes the hemato- passes six major sub-orders. To illustrate this logical characteristics of birds, where known, point, the number of species contained within within the orders of the Aves and highlights the 79 families of the Oscine sub-order, is any known nuances in those characteristics. In greater than the combined number of species the context of this book, a practical applica- from all other orders. As previously described tion of taxonomy has been used to structure in Table 1.1, the orders within the Aves the approach to the hematological character- include: istics of birds. Unfortunately, due to a dearth of published studies on species within some Order: Anseriformes orders and diffi culty in obtaining blood fi lms Order: Apodiformes and hematological data, it was not possible to Order: Caprimulgiformes include information about all the orders of the Order: Charadriiformes Aves. Further studies will no doubt elucidate Order: Ciconiiformes further nuances in the hematological charac- Order: Coliiformes teristics of particular species of birds. Until all Order: Columbiformes species have been described, extrapolation Order: Coraciiformes from the closest species for which the hemato- Order: Cuculiformes logical characteristics have been reported will Order: Falconiformes remain necessary, albeit with the caveat that Order: Galliformes even species within the same genus may exhibit Order: Gaviiformes signifi cant hematological differences. Order: Gruiformes There is considerable ongoing debate con- Order: Opisthocomiformes cerning the taxonomy of the class Aves and we Order: Passeriformes have used arguably the most recent and reli- Order: Pelecaniformes able source of information to summarize the Order: Phoenicopteriformes 54 Atlas of Clinical Avian Hematology

Order: Piciformes Order: Podicipediformes Order: Procellariformes Order: Psittaciformes Order: Sphenisciformes Order: Strigiformes Order: Struthioniformes Order: Tinamiformes Order: Trogoniformes

ANSERIFORMES (DUCKS, GEESE AND SWANS) Figure 85 Blood from an Australian shelduck (Tadorna tadornoides) showing a heterophil with See Figures 84–95. many uniform, fusiform brick-red granules and two Several studies have described selected basophils with variably sized round–ovoid basophilic hematological characteristics of non-domestic granules. Note that the nucleus of the basophils is Anseriformes, including: three species of palely stained in comparison to the cytoplasmic gran- geese (Williams & Trainer 1971), canvasback ules. The nucleus of the heterophil is a light blue color, duck (Aythya valisineria) (Kocan 1972, Kocan this is commonly observed and assists to distinguish heterophils from eosinophils (see Figure 86). (Modiﬁ ed & Pitts 1976), Paciﬁ c black duck (Anas Wright’s stain.)

Figure 84 Blood from a black swan (Cygnus atratus) showing two heterophils amidst mature erythrocytes. The granules of the heterophils are fusiform and brick- red color. Some granules exhibit a central, round, Figure 86 Blood from the same Australian shelduck refractile structure (“central granular body”). These are as in Figure 85 showing an eosinophil characterized regularly observed in birds from this Order and should by a segmented nucleus composed of dark basophilic, not be misinterpreted as a “toxic change”. (Modiﬁ ed coarse chromatin and many, brightly eosinophilic, Wright’s stain.) rod-shaped granules. (Modiﬁ ed Wright’s stain.) Particular hematological characteristics of birds 55

Figure 87 Blood from a pacifi c black duck (Anas Figure 89 Blood from the same bird as in Figure 88 superciliosa), showing a typical small lymphocyte and showing an eosinophil amidst mature erythrocytes. heterophil amidst mature erythrocytes. (Modifi ed The cytoplasmic granules are typically rod-shaped and Wright’s stain.) brightly eosinophilic. The nucleus can be seen to be a darker basophilic color than the nucleus of heterophils in Figure 88. (Modifi ed Wright’s stain.)

Figure 88 Blood from a Barrow’s golden eye (Buceph- Figure 90 Blood from the same bird as in Figure 88 ala islandica) showing a heterophil, with distinct gran- portraying a basophil and small lymphocyte amidst ules, amidst mature erythrocytes. (Modifi ed Wright’s mature erythrocytes. The illustrated “bleeding” of stain.) basophilic color from the basophil to outline adjacent cells is regularly observed in avian blood fi lms. (Modi- fi ed Wright’s stain.) 56 Atlas of Clinical Avian Hematology

Figure 93 Blood from the same goosander as in Figure 91 Blood from a goosander (Mergus mergan- Figure 91 showing a typical small lymphocyte amidst ser) showing a basophil (with some “bleeding” of the mature erythrocytes and a single polychromatophilic basophilic color to adjacent cells), heterophil (with erythrocyte. (Modiﬁ ed Wright’s stain.) palely staining nucleus) and monocyte amidst erythrocytes, including one polychromatophilic erythrocyte. (Modiﬁ ed Wright’s stain.)

Figure 94 Blood from a Baer’s pochard (Aythya baeri) showing a heterophil amidst mature erythrocytes. (Modiﬁ ed Wright’s stain.) Figure 92 Blood from the same goosander as in Figure 91 showing a typical eosinophil; with a high density of brightly eosinophilic granules and segmented basophilic nucleus (darker than the nucleus of the heterophils), amidst mature erythrocytes. (Modi- ﬁ ed Wright’s stain.) Particular hematological characteristics of birds 57

APODIFORMES (SWIFTS AND HUMMINGBIRDS)

To the authors’ knowledge few studies have described the hematological characteristics of species within the Apodiformes, and these did not describe the morphological characteristics of hematological cells.

CAPRIMULGIFORMES (FROGMOUTHS, GOATSUCKERS AND OWLET-NIGHTJARS) Figure 95 Blood from a Baer’s pochard showing a basophil, monocyte and eosinophil amidst mature See Figure 96. erythrocytes (Modifi ed Wright’s stain.) Few studies have described the hematological characteristics of members of the Caprimul- giformes. McCracken (2003) reported selected hematological values for the tawny frogmouth (Podargus strigoides) and described eosinophils to have pale mauve granules. superciliosa) (Mulley 1979), Australian wood duck (Chenonetta jubata) (Mulley 1980) and mallard (Anas platyrhynchos) (Driver 1981, Fairbrother et al. 1990, Fairbrother & O’Loughlin 1990, Levengood et al. 2000). Few of these have described the morphological characteristics of hematological cells. Erythrocytes were ovoid cells with an ovoid nucleus composed of dense chromatin and homogeneous eosinophilic cytoplasm. Small numbers of polychromatophilic erythrocytes, exhibiting palely basophilic cytoplasm were evident. Heterophils had many fusiform, brick-red colored granules that were present at high density throughout the cytoplasm. Figure 96 Blood from a tawny frogmouth (Podargus Eosinophils had many small, rod-shaped, strigoides) showing: a heterophil, with brick-red brightly eosinophilic granules present at granules and a basophilic nucleus (top middle); an high density throughout the cytoplasm. eosinophil with a segmented basophilic nucleus and Basophils had many round, deeply basophilic basophilic cytoplasm that contains sparse, pale eosin- granules that obscured both nucleus and ophilic granules (left); a basophil with many dark basophilic granules that preclude observation of the cytoplasm. Lymphocytes and monocytes had fi ne detail of the cell’s structures (top right); a lympho- morphology consistent with the general cyte and a thrombocyte with a dark ovoid nucleus and characteristics displayed by birds, as previ- pale basophilic cytoplasm (top left), amidst mature ously described. erythrocytes. (Modifi ed Wright’s stain.) 58 Atlas of Clinical Avian Hematology

Erythrocytes from the tawny frogmouth were ovoid cells with an ovoid nucleus composed of dense chromatin and even eosinophilic cytoplasm. Heterophils were irregularly round cells with fi ne, fusiform, brick-red colored granules present at moderate–high density throughout the cytoplasm. Eosinophils had a palely basophilic cytoplasm with few, small pale blue–grey to eosinophilic granules (depending on the stain), consequently the nucleus is distinct and characterized by segmented coarse chromatin. Basophils had fi ne round, deeply basophilic granules present at high density that obscured both nucleus and cytoplasm. Lymphocytes and monocytes had Figure 98 Blood from a fairy tern (Sterna nereis) morphology consistent with the general char- showing a heterophil amidst mature erythrocytes. (Modifi ed Wright’s stain.) acteristics displayed by birds, as previously described. include the pigeon guillemot (Cepphus columba) (Seiser et al. 2000), black-headed CHARADRIIFORMES (GULLS gull (Larus ridibundus) (Munoz & De la AND SHOREBIRDS) Fuente 2003, Mostaghni et al. 2005), the herring gull (Larus argentatus) (Threlfall 1966, Averbeck 1992, Grasman et al. 2000), great See Figures 97–104. black-backed gull (Larus marinus) and several There have been few studies that have species of wading birds (Ball 2003). The mor- described the hematological characteristics of phological characteristics of hematological species within the Charadriiformes; these cells were not described. Work (1996) described

Figure 97 Blood from a great knot (Calidris tenuiros- Figure 99 Blood from a fairy tern showing an eosino- tris). A heterophil and a thrombocyte are present phil amidst mature erythrocytes. (Modiﬁ ed Wright’s amidst mature erythrocytes. (Diff Quik stain.) stain.) Particular hematological characteristics of birds 59

Figure 100 Blood from a stone curlew (Burhinus oedicnemus) showing two heterophils and an aggrega- Figure 102 Blood from a silver gull (Larus novaehol- tion of thrombocytes amidst mature erythrocytes. landiae) showing two heterophils amidst mature eryth- Amongst the thombocytes, several cells with a round rocytes. Note that the nucleus of each of the heterophils nucleus and a cell with an elongated nucleus are has not taken up an adequate amount of stain and evident. (Modiﬁ ed Wright’s stain.) appears as pale blue, rather than basophilic color. (Modiﬁ ed Wright’s stain.)

Figure 101 Blood from the same stone curlew as in Figure 103 Blood from a silver gull showing an Figure 100, showing an eosinophil, monocyte and eosinophil with a high density of small brown–grey aggregation of thrombocytes. The eosinophil contains cytoplasmic granules. A thrombocyte and mature many small round dull brown–eosinophilic granules erythrocytes are also present. (Modiﬁ ed Wright’s within a basophilic cytoplasm. (Modiﬁ ed Wright’s stain.) stain.) 60 Atlas of Clinical Avian Hematology

tered within this order. Basophils had ﬁ ne round, deeply basophilic granules present at high density that obscured both nucleus and cytoplasm. Lymphocytes and monocytes had morphology consistent with the general characteristics displayed by birds, as previously described.

CICONIIFORMES (HERONS, IBIS AND STORKS)

See Figures 105–107. Reports of the hematological characteristics Figure 104 Blood from a silver gull showing a baso- have been published for a number of species phil amidst mature erythrocytes. (Modifi ed Wright’s belonging to this order including: bald ibis stain.) (Geronticus calvus) (Dutton et al. 2002, Vil- legas et al. 2004), puna ibis (Plegadis ridgwayi) (Coke et al. 2004), white stork (Ciconia the morphology of hematological cells from ciconia) (Alonso et al. 1991, Montesinos et al. the sooty tern (Sterna fuscata); heterophils, basophils, monocytes and lymphocytes had morphology similar to that previously described for birds. Eosinophils had “densely packed orange, round, plump granules”. The morphology of haematological cells of stone curlews (Burhinus oedicnemus) has been described (Samour et al. 1998). These authors reported that eosinophils had a bi-lobed nucleus and pale blue cytoplasm that contained “very small round-oval shaped pink- red/orange” granules. The remaining leukocytes had an appearance similar to that previously described for birds. In the species examined, heterophils were irregularly round cells with fi ne, fusiform, brick-red colored granules present at moder- Figure 105 Blood from a royal spoonbill (Platalea ate–high density throughout the cytoplasm. regia) illustrating: an eosinophil (left) with four lobes Eosinophils of the silver gull (Larus novaehol- to the nucleus evident and a moderate density of landiae) had many small, grey–blue granules round, pale eosinophilic granules in the cytoplasm; a and, in contrast to a previously published heterophil with a segmented nucleus and a high density of fusiform, brick-red granules; a lymphocyte report, the eosinophils of the stone curlew had with coarse clumped chromatin and basophilic small ovoid to rod-shaped, brown–eosino- cytoplasm, amidst mature erythrocytes and a single philic granules. Consequently, variation in the polychromatophilic erythrocyte. (Modifi ed Wright’s morphology of eosinophils may be encoun- stain.) Particular hematological characteristics of birds 61

1997), black stork (Ciconia nigra) (Puerta et al. 1989), night heron (Nycticorax nycticorax), cattle egret (Bubulcus ibis) and little egret (Egretta garzetta) (Celdran et al. 1994). However, these studies did not report the morphology of hematological cells. Waters and Hart (2002) assessed the morphology of hematological cells from the scarlet ibis (Eudocimus ruber), American wood stork (Mycteria americana) and marabou stork (Leptoptilos crumeniferus) and described variation in the morphology of eosinophils, notably in the size and hue of the cytoplasmic granules. Erythrocytes were ovoid cells with an ovoid Figure 106 Blood from a cattle egret (Bubulcus ibis) nucleus composed of dense chromatin and showing an eosinophil with a segmented basophilic eosinophilic cytoplasm. Heterophils were nucleus and a high density of small, indistinct eosino- irregularly round cells with prominent fusi- philic granules in the cytoplasm. (Modiﬁ ed Wright’s form, brick-red to dull brown colored stain.) granules that were present at high density and obscured the nucleus in many cells. Eosino- phils typically had small round, brightly eosinophilic granules present at a moderate to high density in a palely basophilic cytoplasm. The size of the granules varied between species. Basophils were smaller, round cells with round, deeply basophilic granules present at high density that obscured both nucleus and cytoplasm. In some degranulated cells, the nucleus was round and the cytoplasm “colorless”. Lymphocytes and monocytes had morphology consistent with the general characteristics displayed by birds, as previously described.

COLIIFORMES (MOUSEBIRDS) Figure 107 Blood from a black-faced ibis (Theristicus melanopis) showing a heterophil (left) with thin, fusiform brick-red cytoplasmic granules and an eosinophil To the authors’ knowledge few studies have with brightly eosinophilic round to rod-shaped cyto- described the hematological characteristics of plasmic granules amidst erythrocytes. (Modiﬁ ed members of the Coliiformes. Pye (2003) pre- Wright’s stain.) sented selected hematological data for four species of mousebird but did not describe the morphological characteristics of hematological cells. 62 Atlas of Clinical Avian Hematology

COLUMBIFORMES (PIGEONS AND DOVES)

See Figures 108–114. The hematological characteristics were reported for fi ve species of pigeons (Peinado et al. 1992), rock pigeons (Columba livia) (Gayathri & Hegde 1994) and three species of pigeons (Schulz 2003). These studies did not report the morphological characteristics of hematological cells. Erythrocytes were ovoid cells with an ovoid nucleus composed of dense chromatin and even eosinophilic cytoplasm. Heterophils were Figure 109 Blood from the same bird as in Figure irregularly round cells with prominent fusi- 108 showing an eosinophil with a bi-lobed basophilic form, brick-red to dull brown colored granules nucleus and many small round pale eosinophilic gran- that were present at high density and fi lled ules in a pale basophilic cytoplasm, amidst erythro- most of the cytoplasm. Eosinophils had many cytes. (Modifi ed Wright’s stain.) small, round, eosinophilic granules. The color of the granules varied notably with the stain. Basophils had many, deeply basophilic granules that obscured both nucleus and cytoplasm. Lymphocytes and monocytes had morphology consistent with the general characteristics displayed by birds, as previously described.

Figure 110 Blood from the same bird as in Figure 108 showing a classically shaped monocyte (left) with a “horse-shoe” shaped nucleus, reticular chromatin and a moderate amount of basophilic cytoplasm and a lymphocyte amidst mature erythrocytes. (Modiﬁ ed Wright’s stain.)

Figure 108 Blood from a Mindanao bleeding heart dove (Gallicolumba criniger) showing a heterophil and a lymphocyte amidst erythrocytes, including a single polychromatophilic erythrocyte. (Modiﬁ ed Wright’s stain.) Particular hematological characteristics of birds 63

Figure 111 Blood from a chestnut-naped imperial pigeon (Ducula aenea paulina) illustrating two hetero- Figure 113 Blood from a spotted dove (Streptopelia phils, each with many fusiform, brick-red granules. chinensis) portraying a heterophil (top) with many fusi- (Modiﬁ ed Wright’s stain.) form, brick-red granules and an eosinophil with a bi- lobed basophilic nucleus and many small round brightly eosinophilic granules, amidst mature erythrocytes. (Modiﬁ ed Wright’s stain.)

Figure 112 Blood from a chestnut-naped imperial Figure 114 Blood from a spotted dove showing a pigeon showing an eosinophil with a lobulated baso- basophil with many basophilic cytoplasmic granules philic nucleus and many small round pale eosino- that obscure the nucleus, and two thrombocytes amidst philic granules in a pale basophilic cytoplasm, amidst mature erythrocytes. (Modiﬁ ed Wright’s stain.) erythrocytes. (Modiﬁ ed Wright’s stain.) 64 Atlas of Clinical Avian Hematology

CORACIIFORMES (KINGFISHERS, BEE-EATERS, HORNBILLS, MOTMOTS, ROLLERS AND TODIES)

See Figures 115–124. To the authors’ knowledge few studies have described the hematological characteristics of members of the Coraciiformes. Selected hematological data was presented for eight species of Coraciiformes (Dutton 2003) but did not describe the morphological characteristics of hematological cells. Several species of kingfi shers, including the azure kingfi sher (Alcedo azurea), sacred kingfi sher (Todirhamphus sanctus) and laughing Figure 116 Blood from the same bird as in Figure kookaburra, had erythrocytes that were ovoid 115 illustrating an eosinophil. Note the segmented cells with an ovoid nucleus composed of dense nucleus and variably sized ovoid eosinophilic chromatin and even eosinophilic cytoplasm. granules within a pale basophilic cytoplasm. (May- Grünwald and Giemsa stains.) Heterophils were irregularly round cells with many, prominent fusiform, brick-red to dull brown colored granules that partially obscured at high density that obscured both nucleus and the nucleus in many cells. Eosinophils had cytoplasm. Lymphocytes and monocytes had many robust, round, brightly eosinophilic morphology consistent with the general char- granules present at a moderate to high density acteristics displayed by birds, as previously in a palely basophilic cytoplasm. Basophils described. had many deeply basophilic granules present

Figure 115 Blood from a laughing kookaburra Figure 117 Blood from a blue-winged kookaburra (Dacelo novaeguineae) showing a heterophil with fi ne (Dacelo leachii) showing a heterophil amidst erythro- fusiform, brick-red granules in a pale basophilic cyto- cytes. The density of cytoplasmic granules makes plasm and a monocyte amidst erythrocytes. (May- assessment of the fi ne detail of the cell diffi cult. (Diff Grünwald and Giemsa stains.) Quik stain.) Figure 118 Blood from the same bird as in Figure Figure 121 Blood from a sacred kingfi sher (Todirh- 117 illustrating an eosinophil. In this sample the amphus sanctus) illustrating three heterophils and a brownish color of the granules likely refl ects the stain basophil. A pale center can be distinguished in the employed. (Diff Quik stain.) some of the heterophils’ granules. (Modifi ed Wright’s stain.)

Figure 119 Blood from an azure kingﬁ sher (Alcedo azurea) depicting a typical heterophil, amidst mature erythrocytes. (Diff Quik stain.)

Figure 122 Blood from a Von der Decken’s Hornbill (Tockus deckeni) showing a heterophil with “brick- red” cytoplasmic granules and palely-staining basophilic nucleus. Two polychromatophilic erythrocytes are also present amongst numerous mature erythrocytes. (Modiﬁ ed Wright’s stain.)

Figure 120 Blood from the same bird as in Figure 119 showing a lymphocyte. (Diff Quik stain.) 66 Atlas of Clinical Avian Hematology

The hematocrit has been reported for birds from three genera of Bucerotidae (hornbills) (Villegas et al. 2006). For the Von der Deck- en’s hornbill (Tockus deckeni), erythrocytes were ovoid cells with an ovoid nucleus composed of dense chromatin and even eosinophilic cytoplasm. Heterophils were irregularly round cells with many, prominent fusiform, brick-red to dull brown colored granules that partially obscured the nucleus in many cells. Eosinophils had many robust, round, brightly eosinophilic granules present at a moderate to high density in a palely basophilic cytoplasm. Basophils had many deeply basophilic granules present at high density that obscured both nucleus and cytoplasm. Lymphocytes and Figure 123 Blood from the same bird as in Figure monocytes had morphology consistent with 122 portraying an eosinophil identiﬁ ed by a bi-lobed the general characteristics displayed by birds, basophilic nucleus and ovoid eosinophilic granules in as previously described. a palely basophilic cytoplasm. (Modiﬁ ed Wright’s stain.)

CUCULIFORMES (CUCKOOS, ROADRUNNERS AND TURACOS)

To the authors’ knowledge few studies have described the hematological characteristics of species within the Cuculiformes. Selected hematological values were presented from the Guira cuckoo (Guira guira) and greater roadrunner (Geococcyx californianus) (Abou-Madi 2003). These did not describe the morphological characteristics of hematological cells.

FALCONIFORMES (FALCONS, HAWKS AND EAGLES)

Figure 124 Blood from the same bird as in Figure See Figures 125–140. 122 depicting a basophil with a relatively palely staining basophilic nucleus and ovoid, darkly basophilic Numerous studies have reported selected cytoplasmic granules. (Modiﬁ ed Wright’s stain.) hematological characteristics of the Falconi- formes. The hematological characteristics of a number of species within the genus Falco, including the Eurasian kestrel (Falco tinnunculus) (Kirkwood et al. 1979), Mauritius kestel (Falco punctatus) (Cooper et al. 1986), saker Particular hematological characteristics of birds 67

Figure 125 Blood from a clinically healthy Austra- Figure 127 Blood from a Lanner falcon (Falco biar- lian kestrel (Falco cenchroides) illustrating two hetero- micus) showing a heterophil (right), with a segmented phils, each with a segmented nucleus and fusiform, nucleus and fusiform, brick-red cytoplasmic granules brick-red colored granules. (Diff Quik stain.) and an “eosinophil” with a bi-lobed nucleus and pale basophilic–grey cytoplasm with no distinct eosinophilic granules but small pale “vacuoles”. (Modiﬁ ed Wright’s stain.)

Figure 126 Blood from a clinically healthy Austra- Figure 128 Blood from the same Lanner falcon as in lian kestrel representing an eosinophil. The small Figure 127, showing a typical small lymphocyte. eosinophilic granules evident within basophilic cyto- (Modiﬁ ed Wright’s stain.) plasm with modiﬁ ed Wright’s stain were not apparent in a samples stained with Diff Quik stain. This staining characteristic of eosinophil granules has been observed in other species of falcons (Samour et al. 2005). 68 Atlas of Clinical Avian Hematology

Figure 129 Blood from the same Lanner falcon as in Figure 131 Blood from a peregrine falcon showing a Figure 127 showing two monocytes with reticular basophil (top left), lymphocyte (mid right) and throm- chromatin, reniform to horse-shoe shaped nucleus and bocyte (bottom right) amidst erythrocytes. These moderate amounts of basophilic cytoplasm. (Modifi ed “darkly staining cells” often need to be viewed at Wright’s stain.) higher magnifi cations to be accurately distinguished. (Modifi ed Wright’s stain.) falcon (Falco cherrug) (Samour et al. 1996), peregrine falcon (Jennings 1996, Lanzarot et al. 2001) and gyr falcon (Samour et al. 2005) et al. 1995), red-tailed hawk (Rehder et al. have been published. Other species also studied 1982b), white-backed vulture (Gyps africa- include the marsh harrier (Lavin et al. 1992), nus) (van Wyk et al. 1998), bald eagle (Hali- sharp-shinned hawk (Accipiter striatus) and aeetus leucocephalus) (Bowerman et al. 2000) Cooper’s hawk (Accipiter cooperii) (Phalen and imperial eagle (Aquila adalberti) (Garcia-

Figure 130 Blood from a peregrine falcon (Falco per- Figure 132 Blood from a brown goshawk (Accipiter egrinus) showing a heterophil with a high density of fasciatus) showing two heterophils, each with a high brick-red cytoplasmic granules, amidst erythrocytes. density of brick-red fusiform granules, amidst erythro- (Modiﬁ ed Wright’s stain.) cytes. (Modiﬁ ed Wright’s stain.) Particular hematological characteristics of birds 69

Figure 133 Blood from a brown goshawk showing an eosinophil amidst erythrocytes. Birds in this genus typically have round, brightly eosinophilic granules. Figure 135 Blood from a red kite illustrating three (Modifi ed Wright’s stain.) lymphocytes. Mild anisocytosis is evident. These cells are typical of avian lymphocytes. (Modifi ed Wright’s stain.) Montijano et al. 2002). However, few of these reported the morphology of the hematological cells. granules that were present at high density Erythrocytes were ovoid cells with an ovoid throughout the cytoplasm. Consequently the nucleus composed of dense chromatin and nucleus was (partially) obscured in many cells even eosinophilic cytoplasm. Heterophils were and the number of nuclear lobes could not be irregularly round cells with fi ne fusiform to accurately determined. The appearance of elongated, brick-red to dull brown colored “eosinophils” is very variable between groups

Figure 134 Blood from a red kite (Milvus milvus). A Figure 136 Blood from a black kite (Milvus migrans) heterophil (left), with a pale blue nucleus and brick- illustrating an eosinophil with a segmented basophilic red fusiform granules and an eosinophil, with baso- nucleus and many round brightly eosinophilic gran- philic segmented nucleus and many round, eosinophilic ules within basophilic cytoplasm. (Modiﬁ ed Wright’s granules, are shown. (Modiﬁ ed Wright’s stain.) stain.) 70 Atlas of Clinical Avian Hematology

Figure 137 Blood from a tawny eagle (Aquila rapax) portraying an eosinophil with a bi-lobed basophilic nucleus and many round brightly eosinophilic gran- Figure 139 Blood from a black vulture illustrating a ules in basophilic cytoplasm. Note the similarity thrombocyte (center), a small lymphocyte and a large between this cell and the eosinophil in Figure 136. lymphocyte amidst erythrocytes. The thrombocyte is (Modiﬁ ed Wright’s stain.) distinguished by its dense, dark-staining nucleus. The cytoplasm of all three cells has a similar appearance. (Modiﬁ ed Wright’s stain.)

Figure 140 Blood from a king vulture (Sarcoramphus papa) showing a heterophil (right), an eosinophil with Figure 138 Blood from a black vulture (Coragyps cytoplasmic granules that are a dull brown–grey color atratus) showing an eosinophil (left) with a basophilic and a lymphocyte (distorted by surrounding erythro- nucleus and rod-shaped eosinophilic granules within cytes). (Modifi ed Wright’s stain.) a basophilic cytoplasm and a heterophil with a pale blue nucleus and brick-red granules in which the central granular body is prominent and the granules are indistinct. (Modifi ed Wright’s stain.) Particular hematological characteristics of birds 71 of raptors. Species within the genus Falco have eosinophils with a few, “bluish pink to light blue” granules in a pale blue cytoplasm (Lind et al. 1990) or few small eosinophilic granules (Wernery et al. 2004, Samour et al. 2005). Samour et al. (2001, 2005) noted the large effect the type of stain may have on the appearance of eosinophils’ granules, with granules most prominent when stained with modifi ed Wright’s stain and least prominent when stained with Diff Quik stain. Birds within the genera Accipiter, Aquila, Buteo and Haliaee- tus typically have prominent round, brightly eosinophilic granules (Lind et al. 1990, Santos et al. 2003b), and the northern harrier (Circus Figure 141 Blood from a Congo peafowl (Afropavo cyaneus) had round, lightly eosinophilic gran- congensis) showing two typical heterophils amidst ules. Basophils were typically round cells with erythrocytes. (Modifi ed Wright’s stain.) small round, intensely basophilic granules present at high density that obscured both nucleus and cytoplasm. Lymphocytes and (Dunbar et al. 2005) and common pheasant monocytes had morphology consistent with (Hauptmanova et al. 2006). These studies did the general characteristics displayed by birds, not describe the morphology of hematological as previously described. cells. The morphology of hematological cells from Japanese quail has been described (Tadjalli et al. 2003). Erythrocytes were typically rounded ovoid GALLIFORMES (GROUSE, cells with an ovoid nucleus composed of dense MEGAPODES, CURASSOWS, chromatin and even eosinophilic cytoplasm. PHEASANT, PARTRIDGE, QUAIL AND TURKEY)

See Figures 141–151. Numerous studies have described the hematological characteristics of domestic fowl. The morphology of hematological cells from chickens was comprehensively described and beau- tifully illustrated by Lucas and Jamroz in their 1961 monograph, “Atlas of Avian Hematology”. Species within the Galliformes for which hematological characteristics have been studied, include red grouse (Lagopus lagopus scoticus) (Wilson & Wilson 1978), turkey (Bounous et al. 2000), greater prairie-chicken Figure 142 Blood from a Congo peafowl showing an (Tympanuchus cupido) (West & Haines 2002), eosinophil amidst erythrocytes. (Modiﬁ ed Wright’s sage grouse (Centrocercus urophasianus) stain.) 72 Atlas of Clinical Avian Hematology

Figure 145 Blood from a crested wood partridge Figure 143 Blood from a Congo peafowl showing (Rollulus roulroul) showing an eosinophil amidst eryth- a basophil amidst erythrocytes. (Modiﬁ ed Wright’s rocytes. Note the rounded shape of the erythrocytes. stain.) (Modiﬁ ed Wright’s stain.)

Heterophils were irregularly round cells eosinophilic granules present at moderate with prominent fusiform, brick-red to dull to high density in a palely basophilic cyto- brown colored granules that were present plasm. The size and number of the eosino- at high density and consequently (partially) philic granules varied between species. obscured the nucleus in many cells. Eosino- Basophils were round cells with round, phils had many discrete, round, brightly intensely basophilic granules present at moderate to high density that obscured both nucleus and cytoplasm. Lymphocytes and

Figure 144 Blood from a green peafowl (Pavo muticus) showing an eosinophil with round eosinophilic granules and some colorless vacuoles in a pale Figure 146 Blood from a brown quail (Coturnix ypsi- basophilic cytoplasm, amidst erythrocytes. (Modiﬁ ed lophora) showing a heterophil amidst mature erythro- Wright’s stain.) cytes. (Diff Quik stain.) Figure 147 Blood from a brown quail showing a Figure 150 Blood from a blue-breasted quail showing lymphocyte amidst erythrocytes, including a single a basophil with a moderate density of pleomorphic polychromatophilic erythrocyte. (Diff Quik stain.) basophilic granules and several vacuoles within a pale basophilic cytoplasm, amidst erythrocytes. (Modiﬁ ed Wright’s stain.)

Figure 148 Blood from a brown quail showing two monocytes amidst mature erythrocytes. (Diff Quik stain.)

Figure 151 Blood from an orange-footed scrub fowl (Megapodius reinwardt) showing an intact heterophil and a lysed heterophil, the latter allowing individual fusiform granules to be visualized. (Diff Quik stain.)

Figure 149 Blood from a blue-breasted quail (Cotur- nix chinensis) showing an eosinophil with large round- ovoid cytoplasmic granules, amidst erythrocytes. A heterophil from this species is shown in Figure 244. (Modiﬁ ed Wright’s stain.) 74 Atlas of Clinical Avian Hematology monocytes had morphology consistent with the general characteristics displayed by birds, as previously described.

GAVIIFORMES (DIVERS AND LOONS)

Hematological data has been reported for common loons (Gavia immer) (Haefele et al. 2005). This study did not report the morphology of hematological cells.

Figure 153 Blood from a brolga (Grus rubicunda) GRUIFORMES (BUSTARDS, BUTTON showing a typical heterophil amidst erythrocytes. QUAIL, COOTS AND CRANES) (Modiﬁ ed Wright’s stain.)

See Figures 152–160. (Grus grus) (Abelenda et al. 1993) and several Selected hematological values have been species of bustard (Samour et al. 1994, Flach reported for a number of species of Gru- 1995, D’Aloia et al. 1995, 1996, Howlett iformes, including: demoiselle crane (Grus et al. 1995, 2002). virgo) (Conetta et al. 1974), whooping crane The morphology of hematological cells has (Grus americana), sandhill crane (Grus been described for ten species of crane (Hawkey canadensis) (Gee et al. 1981), common crane et al. 1983), the houbara bustard (Samour

Figure 152 Blood from a clinically healthy takahe (Porphyrio mantelli) illustrating a heterophil, a small lymphocyte and two medium-sized lymphocytes Figure 154 Blood from a brolga (Grus rubicunda) amidst mature erythrocytes. Pleomorphism of lympho- showing a basophil with a moderate density of cyto- cytes is commonly observed in the blood of clinically plasmic granules and a medium-sized lymphocyte, healthy birds. (Diff Quik stain.) amidst erythrocytes. (Modiﬁ ed Wright’s stain.) Particular hematological characteristics of birds 75

Figure 155 Blood from the same brolga as in Figure 154, showing two monocytes amidst erythrocytes. Figure 157 Blood from a wattled crane (Grus carun- (Modiﬁ ed Wright’s stain.) culatus) showing a heterophil, lymphocyte (bottom right) and a disrupted basophil, which allows the individual basophilic granules to be observed. (Modiﬁ ed Wright’s stain.)

Figure 158 Blood from the same wattled crane as in Figure 156 Blood from a Stanley crane (Grus para- Figure 157, portraying a granular lymphocyte and an disea) illustrating: a heterophil (top right) with pale eosinophil amidst erythrocytes. Hawkey et al. (1983) blue nucleus and fusiform granules, some with a noted that lymphocytes from cranes commonly con- prominent central granular body; and an eosinophil tained basophilic granules. (Modiﬁ ed Wright’s stain.) with a basophilic nucleus and a high density of brightly eosinophilic granules that precludes recognition of individual granule shape, amongst mature erythrocytes. Basophils from this species are portrayed in Figure 71. (Modiﬁ ed Wright’s stain.) 76 Atlas of Clinical Avian Hematology

used as an anticoagulant in other species of cranes (Conetta et al. 1974, Hawkey et al. 1983) and bustards (Samour et al. 1994, Howlett et al. 1995) without untoward consequences. Erythrocytes were ovoid cells with an ovoid nucleus composed of dense chromatin and even eosinophilic cytoplasm. Occasional polychromatophilic erythrocytes were evident. Heterophils were irregularly round cells with prominent fusiform, brick-red to dull brown colored granules that were present at high density throughout the cytoplasm. Hawkey et al. (1983) reported the nucleus of heterophils from cranes was typically bi-lobed. Eosino- Figure 159 Blood from an Eurasian coot (Fulica atra) showing a monocyte and an eosinophil with brightly phils had many prominent, round, brightly eosinophilic cytoplasmic granules, amidst mature eosinophilic granules present at moderate to erythrocytes. (Modifi ed Wright’s stain.) high density in a palely basophilic cytoplasm. Samour et al. (1994) reported that eosinophils from the houbara bustard had a “bi-lobed et al. 1994) and kori bustard (Ardeotis kori) nucleus with a slightly basophilic cytoplasm (Howlett et al. 1995). almost completely obscured by overlying large Notably, the use of EDTA as an anticoagu- disc-like and slightly basophilic (blue–grey) lant causes progressive hemolysis in crowned granules”. Howlett et al. (1995) reported that cranes (Balearica spp) (Hawkey et al. 1983) for the kori bustard, “the eosinophil had a bi- and should not be employed as an anticoagu- lobed nucleus, enclosed by a slightly basophilic lant for that species. However, EDTA was cytoplasm that contained distinct bright-red round granules”. Similarly, cranes were reported to have “brightly eosinophilic, round or oval granules” (Hawkey et al. 1983). Basophils were typically round cells with round, intensely basophilic granules present at varied density. Some basophils contained only a few granules that allowed the fi ne detail of the cell to be discerned, whereas other basophils had a greater density of granules that obscured both nucleus and cytoplasm. Lym- phocytes and monocytes had morphology consistent with the general characteristics displayed by birds, as previously described. Lympho- cytes that contained one to several, azurophilic cytoplasmic granules were commonly observed in blood from cranes (Hawkey et al. 1983) and Figure 160 Blood from an Eurasian coot (Fulica atra) have been observed regularly, by the authors, showing a basophil and thrombocyte amidst mature in the blood from other species of erythrocytes. (Modifi ed Wright’s stain.) Gruiformes. Particular hematological characteristics of birds 77

OPISTHOCOMIFORMES (HOATZIN)

To the authors’ knowledge no studies have described the hematological characteristics of the hoatzin (Opisthocomus hoazin) which is the sole extant representative of this order.

PASSERIFORMES (SONG BIRDS)

See Figures 161–175. A substantial number of studies have employed hematological methods as a component of the study of effects of various physio- Figure 161 Blood from a superb lyrebird (Menura logical or pathological infl uences on species of novaehollandiae) illustrating: a heterophil with a high Passeriformes. Notably, the use of EDTA as density of brick-red fusiform granules that obscure an anticoagulant causes hemolysis in samples part of the nucleus (top left); an eosinophil with from many species of Corvidae (Hawkey & a segmented basophilic nucleus and round to ovoid eosinophilic granules in basophilic cytoplasm Dennett 1989). Species for which some hema- (bottom right); and a typical small lymphocyte tological data has been published include: (bottom left), amidst mature erythrocytes. (Modifi ed house martin (Delichon urbica) (Kostelecka- Wright’s stain.) Myrcha & Jaroszewicz 1993), superb blue wren (Malurus cyaneus) (Breuer et al. 1995), great tit (Parus major) (Ots & Horak 1998, Ots et al. 1998), rufous-collared sparrows (Zonotrichia capensis) (Ruiz et al. 2002), hooded crow (Corvus corone) (Acquorone et al. 2002), house fi nches (Carpodacus mexicanus) (Davis et al. 2004), welcome swallow (Hirundo neoxena), fairy martin (Hirundo ariel) (Simmons & Lill 2006) and three species of thrushes (Owen & Moore 2006). However, few of these have been primarily a study of the “hematological characteristics” of the species of bird and consequently very few have described the morphological characteristics of hematological cells. Furthermore, the small mass of many species of Passeriformes often precludes the collection of a suffi cient volume of blood to allow a “complete” hematological Figure 162 Blood from a New-Holland honeyeater assessment to be performed. However, the rig- (Phylidonyris novaehollandiae) showing a heterophil orous assessment of a blood fi lm to estimate with a pale blue nucleus and brick-red fusiform gran- the concentration of hematological cells and ules, a lymphocyte and a thrombocyte (top) amidst particularly interpretation of the morphology erythrocytes. (Modifi ed Wright’s stain.) Figure 166 Blood from a red wattlebird (Anthochaera Figure 163 Blood from a New-Holland honeyeater carunculata) showing a heterophil (right) and an eosin- showing an eosinophil and a lymphocyte amidst ophil (left) amidst erythrocytes including several poly- erythrocytes. (Modifi ed Wright’s stain.) chromatophilic erythrocytes. Note the similarity in the morphology of acidophils for the honeyeaters illustrated, representing three different genera. (Modifi ed Wright’s stain.)

Figure 164 Blood from a New-Holland honeyeater depicting a basophil. The density of dark basophilic granules prevents visualization of the nucleus. (Modi- Figure 167 Blood from an Australian raven, with ﬁ ed Wright’s stain.) lithium heparin used as an anticoagulant, illustrating a heterophil, a monocyte and two lymphocytes. (Mod- iﬁ ed Wright’s stain.) The use of heparin as an anticoagulant typically results in paler blue color of basophilic structures (than would occur with blood mixed with EDTA) and may result in clumping of leukocytes. However, heparin must be used in this species as EDTA will result in hemolysis (see Figure 22).

Figure 165 Blood from a singing honeyeater (Lichenostomus virescens) showing intact acidophils; a heterophil with a high density of brick-red fusiform granules (left) and an eosinophil with ovoid eosinophilic granules within a basophilic cytoplasm (right) as well as a disrupted basophil (arrow) amidst erythrocytes. (Modifi ed Wright’s stain.) Figure 168 Blood from an Australian raven (Corvus coronoides) showing an eosinophil. (Modifi ed Wright’s stain.) Figure 171 Blood from a hooded pitta exhibiting a monocyte (left), a lymphocyte and two thrombocytes (adjacent to the monocyte), amidst mature erythrocytes. The thombocytes have a darker nucleus than the mononuclear leukocytes. (Modifi ed Wright’s stain.)

Figure 169 Blood from a hooded pitta (Pitta sordida) showing a typical avian heterophil amidst mature erythrocytes. (Modiﬁ ed Wright’s stain.)

Figure 172 Blood from a superb starling (Spreo superbus) showing a heterophil amidst erythrocytes, including two polychromatophilic cells. (Modiﬁ ed Wright’s stain.)

Figure 170 Blood from a hooded pitta portraying an eosinophil with a segmented basophilic nucleus and large, ovoid, brightly eosinophilic granules within a basophilic cytoplasm; and a basophil with such a density of basophilic granules that cellular detail cannot be discerned, amidst mature erythrocytes. (Modiﬁ ed Wright’s stain.) 80 Atlas of Clinical Avian Hematology

Figure 175 Blood from a violet-backed starling Figure 173 Blood from a superb starling showing an showing an eosinophil amidst mature erythrocytes. eosinophil (left) with a segmented basophilic nucleus (Modifi ed Wright’s stain.) and many eosinophilic granules within a basophilic cytoplasm and a basophil. The basophils of this bird exhibited variable numbers of granules. (Modifi ed Wright’s stain.) had colorless cytoplasm with “eosinophilic rod-shaped granules” and a bi-lobed nucleus, whereas eosinophils had a darker nucleus, of hematological cells can yield important pale blue cytoplasm and round eosinophilic information about the health of the bird. granules. Basophils had a central round The morphology of haematological cells of nucleus often hidden by deeply basophilic the hill mynah (Gracula religiosa) has been granules. described (Archawaranon 2005). Heterophils For the species of passerine birds examined, erythrocytes were ovoid cells with an ovoid nucleus composed of dense chromatin and even eosinophilic cytoplasm. Variable numbers of polychromatophilic erythrocytes were evident. Heterophils were irregularly round cells with prominent fusiform, brick-red to dull brown colored granules that were present at high density and fi lled most of the cytoplasm and consequently (partially) obscured the nucleus in many cells. Eosinophils had many small, irregularly round, dull eosinophilic granules present at moderate density in a pale basophilic cytoplasm. Basophils had many small round, deeply basophilic granules present at high density that often obscured both nucleus and cytoplasm. Lymphocytes and Figure 174 Blood from a violet-backed starling (Cin- nyricinclus leucogaster) showing a heterophil with monocytes had morphology consistent with typical morphology amidst mature erythrocytes. (Mod- the general characteristics displayed by birds, ifi ed Wright’s stain.) previously described in this chapter. Particular hematological characteristics of birds 81

PELECANIFORMES (CORMORANTS, FRIGATE BIRDS, GANNETS, PELICANS AND TROPICBIRDS)

See Figures 176–179. The hematological characteristics of several species belonging to the Pelecaniformes have been reported, these include the white pelican (Pelecanus onocrotalus) (Puerta et al. 1991), brown pelican (Pelecanus occidentalis) (Weber 2003, Zais et al. 2000), pink-backed pelican (Pelecanus rufescens), guanay cormorant (Phal- acrocorax bougainvillii) (Weber 2003), ﬂ ightless cormorant (Phalacrocorax harrisi), black-faced cormorant (Phalacrocorax fuscescens) (Melrose Figure 177 Blood from the same bird as in Figure & Nicol 1992), double-crested cormorants 176 illustrating an eosinophil with a segmented baso- (Phalacrocorax auritus) (Kuiken & Danesik philic nucleus and many round to ovoid eosinophilic 1999) and brown boobies (Sula leucogaster) granules within basophilic cytoplasm, amidst erythro- (Work 1999). These studies did not describe the cytes. (Diff Quik stain.) morphological characteristics of hematological cells. Work (1996) described the morphology of species, heterophils, basophils, monocytes and hematological cells from three species of Pele- lymphocytes had morphology similar to that caniformes; red-footed boobies (Sula sula), red- previously described for birds. Eosinophils from tailed tropic birds (Phaethon rubricauda) and the boobies had round large and brightly orange great frigate birds (Fregata minor). In these granules whereas the eosinophils from frigate

Figure 176 Blood from a brown booby (Sula leucogaster) portraying two heterophils each with a high Figure 178 Blood from an Australian gannet (Morus density of brown-red fusiform cytoplasmic granules serrator) depicting a heterophil with a high density of that partially obscure the nucleus of the cell, amidst brick-red fusiform granules and a pale basophilic cyto- erythrocytes including polychromatophilic erythro- plasm amidst mature erythrocytes. (Modiﬁ ed Wright’s cytes. (Diff Quik stain.) stain.) 82 Atlas of Clinical Avian Hematology

Chilean fl amingos (Phoenicopterus chilensis) (Hawkey et al. 1984a), greater fl amingos (Phoenicopterus ruber) (Hawkey et al. 1984b, 1985, Merritt et al. 1996, Mostaghni et al. 2005) and lesser fl amingos (Phoenicopterus minor) (Hawkey et al. 1985). The morphological characteristics of hematological cells were described in a number of these studies (Hawkey et al. 1984a, b, 1985). Erythrocytes were ovoid cells with an ovoid nucleus composed of dense chromatin and eosinophilic cytoplasm. Heterophils were irregularly round cells with prominent fusiform, brick-red to dull brown colored granules Figure 179 Blood from an Australian gannet showing that were present at high density that obscured a basophil and monocyte amidst mature erythrocytes. the nucleus in many cells. Eosinophils typically (Modifi ed Wright’s stain.) had robust round, brightly eosinophilic granules present at a moderate density in a pale birds had “loosely packed, plump, elliptical basophilic cytoplasm. Basophils were smaller, granules distributed among variably sized clear round cells with round, deeply basophilic granules” and tropicbirds’ eosinophils had granules present at high density that obscured “sparse numbers of amorphous to round, large, dull-orange granules interspersed with numerous small, clear, well defi ned vacuoles”. Erythrocytes were ovoid cells with an ovoid nucleus composed of dense chromatin and eosinophilic cytoplasm. Heterophils had prominent fusiform, brick-red to eosinophilic colored granules. Eosinophils observed in blood from the brown booby had small round eosinophilic granules that were present at moderate density in a palely basophilic cytoplasm. Basophils had many small round, deeply basophilic granules present at high density that often obscured both nucleus and cytoplasm. Lymphocytes and monocytes had morphology consistent with the general characteristics displayed by birds, as previously Figure 180 Blood from a greater fl amingo (Phoe- described. nicopterus ruber), illustrating: a heterophil (left) with a pale basophilic nucleus and brick-red fusiform cytoplasmic granules; an eosinophil (right) with a seg- PHOENICOPTERIFORMES mented basophilic nucleus and variably sized, brightly (FLAMINGOS) eosinophilic, round to ovoid cytoplasmic granules; and a monocyte (top) with constricted nucleus composed of reticular chromatin and agranulated baso- See Figure 180. philic cytoplasm, amidst erythrocytes. Note that the Hematological values have been reported shape of the cells has been distorted by the adjacent for several species of fl amingos including: cells. (Modifi ed Wright’s stain.) Particular hematological characteristics of birds 83 both nucleus and cytoplasm. Lymphocytes and monocytes had morphology consistent with the general characteristics displayed by birds, as previously described.

PICIFORMES (TOUCANS AND WOODPECKERS)

See Figures 181–188. To the authors’ knowledge, very few studies have described the hematological characteristics of Piciformes. Cubas (2003) provided selected hematological values for species of Figure 182 Blood from the same black-necked toucans, but did not describe the morphologi- aracari as in Figure 181 showing an eosinophil, with cal characteristics of hematological cells. a segmented basophilic nucleus and many prominent Erythrocytes were ovoid cells with an ovoid round faintly eosinophilic cytoplasmic granules that nucleus composed of dense chromatin and are outlined by the surrounding basophilic cytoplasm, amidst erythrocytes. (Modiﬁ ed Wright’s stain.) eosinophilic cytoplasm. Heterophils had prominent fusiform, brick-red to eosinophilic colored granules. Eosinophils exhibited mor- obscuring the nucleus) but varied in size and phological variation in the cytoplasmic gran- hue. For example, eosinophils from red-billed ules between species. These were typically toucan (Ramphastos tucanus) had grey gran- round and present at high density (partially ules with a faint eosinophilic hue, whereas

Figure 181 Blood from a black-necked aracari (Ptero- Figure 183 Blood from a toco toucan (Ramphasto glossus aracari) showing a heterophil with a basophilic toco) portraying a typical heterophil with a segmented nucleus and prominent robust fusiform brick-red cyto- basophilic nucleus and prominent robust fusiform plasmic granules. Also present is a lymphocyte and brick-red cytoplasmic granules. (Modifi ed Wright’s erythrocytes. (Modifi ed Wright’s stain.) stain.) Figure 186 Blood from a red-billed toucan (Rampha- stos tucanus) illustrating a typical heterophil and two Figure 184 Blood from a toco toucan illustrating an lymphocytes, both of which contain several azuro- “eosinophil” that has round granules that have a blue– philic granules. (Modifi ed Wright’s stain.) grey color with a faint eosinophilic hue. Several individual granules can be discerned overlying the nucleus. (Modifi ed Wright’s stain.)

Figure 187 Blood from a red-billed toucan illustrating an “eosinophil”. The granules are mostly a blue– grey color with occasional eosinophilic granules observed. (Modiﬁ ed Wright’s stain.) Figure 185 Blood from a toco toucan showing two lymphocytes; one (right) has about 20 small amphophilic cytoplasmic granules whereas the other (left) exhibits only two small granules. (Modiﬁ ed Wright’s stain.)

Figure 188 Blood from the same bird as in Figure 187, showing a monocyte, characterized by its large size, reticular chromatin and agranulated basophilic cytoplasm. Also present is a thrombocyte exhibiting a single large cytoplasmic vacuole. (Modifi ed Wright’s stain.) Particular hematological characteristics of birds 85 eosinophils from black-necked aracari (Ptero- PROCELLARIIFORMES glossus aracari) had brightly eosinophilic (ALBATROSSES, FULMARS, granules. Basophils were not observed in the PETRELS AND SHEARWATERS) samples examined. Lymphocytes and monocytes had morphology consistent with the general characteristics displayed by birds, as See Figures 190–191. previously described. The hematological characteristics of several species belonging to the Procellariiformes have been reported, these include manx shearwaters (Puffi nus puffi nus) (Kirkwood et al. 1995), PODICIPEDIFORMES (GREBES) seven species of pelagic sea birds (Work 1996), great skua (Bearhop et al. 1999), waved alba- See Figure 189. tross (Diomedea irrorata) (Padilla et al. 2003), To the authors’ knowledge, no studies have southern giant petrels (Macronectes giganteus) described the hematological characteristics of (Uhart et al. 2003) and northern fulmar (Ful- the Podicipediformes. marus glacialis) (Edwards et al. 2006). Most Blood from an Australasian grebe (Tachy- of these studies did not describe the morpho- baptus novaehollandiae) contained erythro- logical characteristics of hematological cells. cytes that were ovoid cells with an ovoid Work (1996) described the morphology of nucleus composed of dense chromatin and hematological cells from three species of Pro- eosinophilic cytoplasm. Heterophils had fi ne, cellariiformes, namely Hawaiian petrel (Ptero- fusiform, brick-red to eosinophilic colored droma phaeopygia), wedge-tailed shearwater granules. Eosinophils and basophils were not (Puffi nus pacifi cus) and Layasan albatross recognized. Lymphocytes and monocytes had (Diomedea immutabilis). The heterophils, morphology consistent with the general char- basophils, monocytes and lymphocytes acteristics displayed by birds, previously had morphology similar to that previously described in this chapter. described for birds. Eosinophils from petrels

Figure 190 Blood from a ﬂ uttering shearwater (Pufﬁ - Figure 189 Blood from an Australasian grebe (Tachy- nus gavia). Illustrated is a heterophil amidst mature baptus novaehollandiae). A heterophil and a large erythrocytes. Overall, the basophilic stain is very pale, lymphocyte, amidst mature erythrocytes, are illus- as can be determined by the pale color of the eryth- trated. (Diff Quik stain.) rocytes’ nuclei. (May-Grünwald and Giemsa stains.) 86 Atlas of Clinical Avian Hematology

Figure 191 Blood from a short-tailed shearwater (Puffi nus tenuirostris) showing a heterophil and a lym- Figure 192 Blood from an eclectus parrot (Eclectus phocyte amidst mature erythrocytes. Some precipi- roratus) showing a typical heterophil (left) with a baso- tated stain is also present around the lymphocyte. philic nucleus partially obscured by many fusiform (May-Grünwald and Giemsa stains.) brick-red cytoplasmic granules and a monocyte (right) with reticular chromatin and agranulated basophilic cytoplasm. (Modifi ed Wright’s stain.) and shearwaters had “densely packed orange, round, plump granules” whereas eosinophils from albatross had “loosely packed tiny round formes have been reported, including: 19 bright red-orange granules”. species of captive psittacine birds (Polo et al. Blood from fl uttering shearwater (Puffi nus 1998), hyacinth macaws (Anodorhynchus gavia), short-tailed shearwater (Puffi nus tenui- hyacinthinus) (Calle & Stewart 1987), red- rostris) and providence petrel (Pterodroma fronted macaws (Ara rubrogenys) (Garcia del solandri) contained erythrocytes that were Campo et al. 1991), Cuban Amazon parrots ovoid cells with an ovoid nucleus composed of (Amazona leucocephala) (Tell & Citino 1992), dense chromatin and eosinophilic cytoplasm. Heterophils had fi ne, fusiform, brick-red to eosinophilic colored granules. Eosinophils from the shearwaters exhibited many, small rounded, eosinophilic granules that were present at high density. Lymphocytes and monocytes had morphology consistent with the general characteristics displayed by birds, as previously described.

PSITTACIFORMES (COCKATOOS, LORIES, MACAWS AND PARROTS)

Figure 193 Blood from the same eclectus parrot as See Figures 192–210. in Figure 192, showing a basophil with a moderate The hematological characteristics of a density of robust round to ovoid dark basophilic gran- number of species belonging to the Psittaci- ules, amidst erythrocytes. (Modiﬁ ed Wright’s stain.) Particular hematological characteristics of birds 87

Figure 194 Blood from a red-tailed black cockatoo Figure 196 Blood from a white-tailed black cockatoo (Calyptorhynchus banksii) showing a heterophil and (Calyptorhynchus baudinii) portraying two typical het- two monocytes each with vacuoles evident in their erophils and a small lymphocyte amidst erythrocytes. cytoplasm. The shape of the latter has been distorted (Modiﬁ ed Wright’s stain.) by surrounding cells. (Modiﬁ ed Wright’s stain.)

describe the morphology of hematological blue-fronted Amazon parrots (Amazona cells. Hawkey et al. (1982) described the mor- aestiva) (Deem et al. 2005), orange-bellied phology of hematological cells from African parrot (Neophema chrysogaster) (Melrose et grey parrots (Psittacus erithacus) but did not al. 1995), budgerigars (Melopsittacus undulatus) (Harper & Lowe 1998, Fischer et al. 2006) and red lories (Eos spp) (Scope et al. 2000). Most of these studies did not did not

Figure 197 Blood from a Major Mitchell’s cockatoo (Cacatua leadbeateri) depicting: a heterophil with a pale blue nucleus and many brick-red, fusiform cytoplasmic granules; a basophil with variably sized, unevenly distributed, dark basophilic cytoplasmic Figure 195 Blood from the same red-tailed black granules; and a monocyte with an irregularly shaped cockatoo as in Figure 194, showing a basophil with a nucleus and basophilic cytoplasm containing several moderate density of small basophilic granules. (Modi- vacuoles, amidst erythrocytes. (Modiﬁ ed Wright’s ﬁ ed Wright’s stain.) stain.) 88 Atlas of Clinical Avian Hematology

Figure 198 Blood from a Major Mitchell’s cockatoo Figure 200 Blood from a Port Lincoln parrot (Barnar- showing an “eosinophil” with a basophilic nucleus dius zonarius) illustrating a heterophil (left) with a and ovoid, pale blue–grey with a faint eosinophilic basophilic nucleus and many brick-red, fusiform cyto- hue, granules, amidst erythrocytes. (Modiﬁ ed Wright’s plasmic granules; and an “eosinophil”, which in this stain.) species, has many blue–grey cytoplasmic granules. (Modiﬁ ed Wright’s stain.) recognize eosinophils in clinically healthy birds. Campbell (2004) described the general morphological features of hematological cells irregularly round cells with prominent fusi- from psittacine birds. form, brick-red to dull brown colored granules Erythrocytes were ovoid cells with an ovoid that were present at high density and conse- nucleus composed of dense chromatin and quently (partially) obscured the nucleus in even eosinophilic cytoplasm. Heterophils were many cells. The term “eosinophil” is a misno-

Figure 199 Blood from a galah (Eolophus roseicapil- Figure 201 Blood from a northern rosella (Platycer- lus) showing an “eosinophil” with a bi-lobed baso- cus venustus) showing a heterophil, with a basophilic philic nucleus and many small ovoid, pale blue–grey, nucleus and many brick-red, fusiform cytoplasmic granules, amidst erythrocytes. (Modiﬁ ed Wright’s granules, amidst mature erythrocytes. (Diff Quik stain.) stain.) Particular hematological characteristics of birds 89

Figure 204 Blood from a princess parrot showing a basophil with a moderate density of round–ovoid Figure 202 Blood from a northern rosella showing basophilic cytoplasmic granules that permits some an “eosinophil” with a bi-lobed basophilic nucleus nuclear and cytoplasmic characteristics to be dis- and many small ovoid, pale blue–grey granules, amidst cerned. (Modiﬁ ed Wright’s stain.) erythrocytes. (Diff Quik stain.)

philic, aqua, pale blue or grey in color and are mer for many species of psittacine birds, for typically round to ovoid (with size varying examples the cockatoos, as eosinophils lack between species) and present at high density. classically eosinophilic cytoplasmic granules. In some birds, the color of granules within the Encompassing species and staining variation, same cell may vary with palely eosinophilic the granules of “eosinophils” may be eosino- granules, palely basophilic granules and colorless (degranulated) spaces all apparent.

Figure 203 Blood from a princess parrot (Polytelis alexandrae) showing a disrupted “eosinophil” that allows the individual pale blue–grey cytoplasmic Figure 205 Blood from a sun conure (Aratinga solsti- granules to be visualized. Also present are two lym- tialis) showing an eosinophil with a basophilic nucleus phocytes amidst erythrocytes. (Modiﬁ ed Wright’s and ovoid, variably sized, pale eosinophilic granules, stain.) amidst erythrocytes. (Modiﬁ ed Wright’s stain.) 90 Atlas of Clinical Avian Hematology

Figure 206 Blood from the same sun conure as in Figure 208 Blood from a military macaw (Ara milita- Figure 205 depicting a basophil with a moderate ris) illustrating a heterophil. The central granular body density of pleomorphic basophilic cytoplasmic gran- appears refractile and prominent in many of the cyto- ules that obscure nuclear and cytoplasmic character- plasmic granules. Also present is a thrombocyte and istics. (Modiﬁ ed Wright’s stain.) several erythrocytes. (Modiﬁ ed Wright’s stain.)

Basophils were round cells with ﬁ ne to robust round, deeply basophilic granules present at moderate high to density that Lymphocytes and monocytes had mor- often obscured both nucleus and cytoplasm. phology consistent with the general characteristics displayed by birds, as previously described.

Figure 207 Blood from a blue-throated conure (Pyr- rhura cruentata) showing a basophil with a moderate density of round–ovoid basophilic cytoplasmic gran- Figure 209 Blood from a military macaw showing an ules, with a largely polar distribution, that permits eosinophil with a basophilic nucleus and large, ovoid, some nuclear characteristics to be discerned. (Modi- variably colored (pale eosinophilic to amphophilic) ﬁ ed Wright’s stain.) cytoplasmic granules. (Modiﬁ ed Wright’s stain.) Particular hematological characteristics of birds 91

Figure 210 Blood from a green-winged macaw (Ara Figure 211 Blood from a little penguin (Eudyptula chloropterus) showing a heterophil amidst mature minor) depicting: a heterophil (left) with a pale baso- erythrocytes. The central granular body appears refrac- philic nucleus and many brick-red fusiform cyto- tile in many of the cytoplasmic granules and its promi- plasmic granules; and an eosinophil (right) with a nence makes overall structure of the granule diffi cult segmented basophilic nucleus and many eosinophilic, to discern. (Modifi ed Wright’s stain.) round to ovoid granules within a basophilic cytoplasm, amidst erythrocytes. (Modifi ed Wright’s stain.)

SPHENISCIFORMES (PENGUINS) Erythrocytes were “rounded ovoid” cells with an ovoid nucleus composed of dense See Figures 211–214. chromatin and eosinophilic cytoplasm. Het- Selected hematological characteristics have erophils exhibited a high density of fusiform, been reported for the little penguin (Eudyptula brick-red to eosinophilic colored granules that minor) (Nicol et al. 1988, Sergent et al. 2004), partially obscured the nucleus in many cells. rock-hopper penguin (Eudyptes chrysocomes) (Hawkey et al. 1989, Karesh et al. 1999), gentoo penguin (Pygoscelis papua) and magellanic penguin (Spheniscus magellanicus) (Hawkey et al. 1989), Humboldt penguins (Spheniscus humboldti) (Wallace et al. 1995, Villouta et al. 1997, Cranfi eld 2003), king penguins (Aptenodytes patagonicus) (Cran- fi eld 2003) and Galapagos penguin (Sphenis- cus mendiculus) (Travis et al. 2006). These studies did not describe the morphological characteristics of hematological cells. Nicol et al. (1988) noted the relatively large size of erythrocytes (MCV 226 ± 26 fL, length 18.8 ± 1.6 and width 9.2 ± 0.8 µm) and low concentration of erythrocytes (1.66 ± 0.31 × 12 10 /L) in the little penguin. Similar values Figure 212 Blood from a little penguin exhibiting a were subsequently observed for other species basophil amidst erythrocytes. (Modifi ed Wright’s of penguins (Hawkey et al. 1989). stain.) 92 Atlas of Clinical Avian Hematology

ovoid eosinophilic granules. Basophils had many round, deeply basophilic granules present at high density that obscured both nucleus and cytoplasm. Lymphocytes and monocytes had morphology consistent with the general characteristics displayed by birds, as described in the previous chapter.

STRIGIFORMES (BARN OWLS AND “TYPICAL” OWLS)

See Figures 215–221. Several studies have investigated the hemato- Figure 213 Blood from a black-footed penguin logical characteristics of the Strigiformes and (Spheniscus demersus) portraying: a heterophil (left) with a segmented basophilic nucleus and many brick- selected hematological characteristics have been red, short fusiform cytoplasmic granules; a small lym- reported for several species of owls (Cooper phocyte; a “round” polychromatophilic erythrocyte 1975, Elliot et al. 1974, Smith & Bush 1978, and several mature erythrocytes. (Modiﬁ ed Wright’s Hawkey & Samour 1988, Evans & Otter stain.) 1998). More recently, Aguilar (2003) presented hematological values for nine species of owl. Eosinophils typically had a segmented nucleus that was more basophilic and less obscured by granules than heterophils. The cytoplasm was moderately basophilic and contained many

Figure 215 Blood from a southern boobook (Ninnox boobook) exhibiting: two heterophils (right) with paler basophilic nucleus and many brick-red cytoplasmic granules; an eosinophil (left) with a segmented nucleus Figure 214 Blood from a black-footed penguin and many rod-shaped eosinophilic cytoplasmic gran- (Spheniscus demersus) showing a basophil with an ules; and a basophil with a number of prominent ovoid (non-segmented nucleus) and sparse, fi ne baso- basophilic granules in an overall basophilic cytoplasm, philic cytoplasmic granules, and a small lymphocyte, amidst a number of erythrocytes including two amidst mature erythrocytes. (Modifi ed Wright’s polychromatophilic erythrocytes. (Modifi ed Wright’s stain.) stain.) Particular hematological characteristics of birds 93

Figure 216 Blood from a masked owl (Tyto novaehollandiae) showing two heterophils, an eosinophil Figure 218 Blood from the same rusty-barred owl as (arrow) and a monocyte. (Modiﬁ ed Wright’s stain.) in Figure 217 illustrating a basophil with a round– Note the subtle differences between the eosinophil, ovoid nucleus and moderate density of pleomorphic which has smaller rod-shaped more brightly eosino- basophilic granules within a basophilic cytoplasm. philic granules, and the heterophils which have larger, (Modiﬁ ed Wright’s stain.) more fusiform and brown–red colored granules. The shape of the leukocytes has been distorted by the surrounding cells.

Figure 219 Blood from the same rusty-barred owl as Figure 217 Blood from a rusty-barred owl (Strix in Figure 217 showing a lymphocyte (left) and a (larger) hylophila) showing a heterophil (left) and an “eosino- monocyte (right) amidst erythrocytes. (Modiﬁ ed phil”. The granules of the latter have a blue–grey color Wright’s stain.) with a faint eosinophilic hue. (Modiﬁ ed Wright’s stain.) 94 Atlas of Clinical Avian Hematology

colored granules. The morphology of eosinophils from Strigiformes has received careful description and marked variation between species has been noted (Lind et al. 1990). Species within the genus Tyto have long thin, rod-shaped eosinophilic granules, whereas species within the genus Strix have round, bluish to dully eosinophilic granules and species within the genera Asio and Otus have robust, round, brightly eosinophilic granules. Basophils had a high density of variably sized, round to ovoid, deeply basophilic granules that often obscured the nucleus. Lymphocytes and monocytes had morphology consistent with the general characteristics displayed by Figure 220 Blood from a spectacled owl (Pulsatrix birds, as previously described. perspicillata) portraying a heterophil (left) and an eosinophil. The latter has large round–ovoid brightly eosinophilic granules. (Modiﬁ ed Wright’s stain.) STRUTHIONIFORMES (CASSOWARIES, EMUS, KIWIS, However, these studies did not report the mor- OSTRICHES AND RHEAS) phological description of hematological cells. Erythrocytes were ovoid cells with an ovoid See Figures 222–228. nucleus composed of dense chromatin and The hematological characteristics of ostrich eosinophilic cytoplasm. Heterophils had (Palomeque et al. 1991, Fudge 2000), emu prominent fusiform, brick-red to eosinophilic

Figure 222 Heparinized blood from an ostrich (Struthio camelus) showing an eosinophil (left) with a Figure 221 Blood from a white-faced scops owl segmented basophilic nucleus and many small round (Otus leucotis) showing a heterophil (left) and an eosinophilic granules within a basophilic cytoplasm eosinophil, with small round eosinophilic granules and a heterophil (right) with a segmented basophilic within a basophilic cytoplasm. (Modiﬁ ed Wright’s nucleus and indistinct brown–red fusiform cytoplas- stain.) mic granules. (Diff Quik stain.) Particular hematological characteristics of birds 95

Figure 223 Blood from a southern cassowary (Casu- arius casuarius) portraying an eosinophil (left) with a Figure 225 Blood from the same emu as Figure 224, segmented basophilic nucleus and many small pleo- portraying an eosinophil with a bi-lobed basophilic morphic brightly eosinophilic granules within a nucleus and many small round eosinophilic granules basophilic cytoplasm and a heterophil (right) with a within a basophilic cytoplasm. (Modiﬁ ed Wright’s segmented basophilic nucleus and brick-red fusiform stain.) cytoplasmic granules, amidst mature erythrocytes. (Diff Quik stain.)

Figure 226 Blood from a brown kiwi (Apteryx australis) showing a heterophil with a basophilic nucleus Figure 224 Blood from an emu (Dromaius novaehol- that is largely obscured by the presence of many brick- landiae) exhibiting: a heterophil (left) with a segmented red fusiform cytoplasmic granules. Also present are a pale blue nucleus and brick-red fusiform cytoplasmic number of erythrocytes and two thrombocytes (top granules; a basophil many dark basophilic cytoplas- right); one of which exhibits a “round” shape and the mic granules that obscure the nucleus; and a mono- other a fusiform shape. (Modiﬁ ed Wright’s stain.) cyte with its nucleus folded, reticular chromatin and basophilic agranulated cytoplasm. (Modiﬁ ed Wright’s stain.) Note the shape of some leukocytes has been distorted by adjacent cells. 96 Atlas of Clinical Avian Hematology

EDTA has been reported to cause hemolysis of ostrich blood and consequently is not recommended as an anticoagulant (Hawkey & Dennett 1989). Erythrocytes were ovoid cells with an ovoid nucleus composed of dense chromatin and even eosinophilic cytoplasm. Heterophils had prominent fusiform, brick-red to dull brown colored granules that were present at high density. Eosinophils typically had a basophilic nucleus and ovoid, dull to brightly eosinophilic granules present at high density throughout the cytoplasm. Basophils typically had Figure 227 Blood from a brown kiwi illustrating: an ovoid, deeply basophilic granules present at eosinophil with a segmented basophilic nucleus and high density that obscured both nucleus and many small ovoid to rod-shaped eosinophilic granules cytoplasm. However, in some cells, the major- within a basophilic cytoplasm; and a small lympho- ity of granules did not stain. Lymphocytes and cyte, amidst erythrocytes. (Modiﬁ ed Wright’s stain.) monocytes had morphology consistent with the general characteristics displayed by birds, as previously described. (Fudge 2000), lesser rhea (Rhea pennata) (Reissig et al. 2002) and greater rhea (Rhea americana) (Uhart et al. 2006) have been TINAMIFORMES (TINAMOUS) reported. These studies did not describe the morphology of hematological cells. To the authors’ knowledge, there have been few studies that have described the hematological characteristics of Tinamiformes. Smith (2003) presented selected hematological values for the red-winged tinamou (Rhynchotus rufescens); however, the morphological characteristics of hematological cells were not described.

TROGONIFORMES (TROGONS AND QUETZALS)

To the authors’ knowledge, there have been few studies that have described the hematological characteristics of Trogoniformes. Figure 228 Blood from a brown kiwi showing a Neiffer (2003) reported selected hematological basophil in which many of the granules have not taken values for two species of trogon and two up the basophilic stain leaving negatively staining species of quetzal. The morphology of hema- spaces in the cytoplasm. (Modiﬁ ed Wright’s stain.) tological cells was not reported. Physiological and pathological inﬂ uences on the hematological characteristics4 of birds

INTRODUCTION PHYSIOLOGICAL EFFECTS ON THE HEMATOLOGICAL The analysis of blood provides a minimally CHARACTERISTICS OF BIRDS invasive way to gain an insight into the health status of an individual. To be able to recognize A number of physiological infl uences, including the hematological response to disease, the age, sex and season, may affect the hematologi- hematologist must be aware of the many cal characteristics of a bird and a number of factors, artifactual, physiological and patho- studies have reported these effects. However, it logical, that may affect the measured hemato- is usually diffi cult to isolate the effect of a single logical values and morphology of hematological physiological factor, as many such effects may cells. In the following sections a number of be interrelated. For example, the age of a bird is physiological and pathological infl uences, that often related to the season (such as fl edged birds may affect hematological characteristics, are entering their fi rst winter) which may affect the considered. In many cases, the physiological availability of food (and consequently the nutri- infl uences result in a change to the concentration of the bird). These infl uences may affect the tion of hematological cells but do not affect concentration of hematological analytes but cell morphology and will only be briefl y usually do not affect the morphology of hema- described here. Pathological infl uences may tological cells. Consequently, physiological affect the concentration of hematological cells, infl uences will be only briefl y discussed. the morphology of hematological cells or both the concentration and morphology of cells. As previously stated, our focus is the morphologi- Age cal appearance of cells and how changes in the morphology may be recognized and used to Age has been shown to affect the hematologi- detect response to disease. cal characteristics of a number of species of 98 Atlas of Clinical Avian Hematology birds. A study of house martin nestlings No differences between sexes was observed showed a rapid increase in erythrocyte concen- in the measured hematological values of tration and hematocrit, and a decrease in Eurasian kestrels (Kirkwood et al. 1979), MCV with age (Kostelecka-Myrcha & Jaro- Cooper’s and sharp-shinned hawks (Gessaman szewicz 1993). A number of species have et al. 1986), marsh harrier (Lavin et al. 1992), shown an increasing PCV, erythrocyte concen- American kestrel (Rehder & Bird 1983, tration and hemoglobin concentration with Dawson & Bortolotti 1997), great tits age (to maturity). That is, younger birds typi- (Hauptmanova et al. 2002), houbara bustards cally have lesser values than adults. This char- (Samour et al. 1994) or common loons (Haefele acteristic has been observed in a number of et al. 2005). species including; Chilean fl amingos (Phoe- Differences in the erythrocyte concentration nicopterus chilensis) (Hawkey et al. 1984a), and hemoglobin concentration were observed houbara bustard (Jimenez et al. 1991, Samour between males and females, during a non- et al. 1994), rufous-crested bustard (D’Aloia breeding period, for a range of captive species et al. 1995), ostrich (Palomeque et al. 1991), of birds including cranes, geese, raptors, and white storks (Montesinos et al. 1997) and quail (Gee et al. 1981). Mature male common common pheasant (Dos Santos Schmidt et al. pheasants had a greater PCV than mature 2007). A study of common loons revealed that females (Hauptmanova et al. 2006, Dos Santos chicks had lesser mean PCV than adults but Schmidt et al. 2007). Rehder et al. (1982a) adults had a wider range (Haefele et al. noted that female American kestrels, while 2005). laying, had a lower PCV than males. In contrast, erythrocytic values did not Hematological values may vary depending differ between adult (greater than 1 year old) on the reproductive status of the bird. For and young (less than 1 year old) common example a higher PCV was observed in non- cranes (Abelenda et al. 1993). reproductive female pigeons (0.54 L/L) com- Some of these studies also showed differ- pared to those undertaking reproductive ences in the concentrations of leukocytes activities such courtship, mating, incubation between juveniles and adults. Decreasing total and brooding (0.41–0.45 L/L) (Gayathri & leukocyte and lymphocyte concentrations were Hegde 2006). Similarly, female great skuas observed with increasing age in white storks that produced a greater number of eggs (six) (Montesinos et al. 1997); juvenile rufous- had lesser erythrocytic values than females crested bustards had greater total leukocyte producing fewer eggs (two) (Kalmbach et al. and lymphocyte concentrations, but a lesser 2004). basophil concentration, than adults (D’Aloia et al. 1995). The chicks of Chilean fl amingos had greater and more variable concentrations Season of leukocytes than adults (Hawkey et al. 1984a). Season has been reported to affect the hematological characteristics of some species of birds. Captive American kestrels exhibited Sex and reproductive status maximum hematocrit in winter (mean 0.48 L/ L for females) and minimum hematocrit in The effect of the sex of the bird and its repro- summer (0.29 L/L for females) (Rehder & Bird ductive status has been investigated in a 1983). In contrast, no signifi cant difference number of studies. They have been shown to was observed in the PCV or erythrocyte con- affect the hematological characteristics of centration of captive red-tailed hawks between some species of birds but not in others. November and February (Rehder et al. 1982b). Physiological and pathological infl uences 99

A study of four passerine species of birds in Effect of migration south-eastern Australia revealed that an increased erythrocyte concentration and The effect of long-distance fl ight on the hema- decreased MCV (but no signifi cant change in tological characteristics of migratory birds has hematocrit) occurred during winter (Breuer been studied. A decreased hematocrit, associ- et al. 1995). A lesser hematocrit and erythro- ated with decreased amounts of subcutaneous cyte concentration was seen in common cranes fat, has been observed during migration in during early autumn than at other times of goldcrests (Regulus regulus) and blue tits the year (Abelenda et al. 1993). Seasonal (Merila & Svensson 1995, Svensson & Merila (and geographical) variation was observed 1996). Similarly, in bar-tailed godwits (Limosa in the hematological characteristics of lapponica) assessed during a stop-over in several passerine birds (Booth & Elliot 2003). migration, the arriving birds had a lesser Hematocrit, erythrocyte concentration and hematocrit and hemoglobin concentration hemoglobin concentration were greatest in than “refueling” birds, however no signifi cant winter and early spring and least in summer differences in reticulocytes were observed (erythrocyte concentration) and autumn (Landys-Cianelli et al. 2002). (hematocrit and hemoglobin concentration) A study of the hematological characteristics for snow geese (Anser caerulescens) and of four species of passerine birds and red knots Canada geese (Branta canadensis) (Williams (Calidris canutus) undertaking endurance & Trainer 1971). fl ight revealed that hematocrit was dependent In contrast to the above reports for eryth- on body mass (Jenni et al. 2006). For red rocytes, no changes in the concentration of knots, typically a small decrease in hematocrit any leukocyte have been reported to be due to (0.52 to 0.49 L/L) occurred within 1 hour the season. after the commencement of the fl ight. Simi- larly, the hematocrit of pigeons after a 2–4- hour fl ight was, on average, 1.1% less than Time of day their pre-fl ight values (Adams et al. 1997). In contrast, no signifi cant differences were Variation in the hematological values of observed between pre-migration and post- birds with the time of day has been observed. migration hematocrit values of adult black Rose-ringed parakeets (Psittacula krameri) headed gulls. However, juvenile females had exhibited a circadian rhythm in the dif- lower values in the post-migratory period ferential leukocyte count, with heterophil (Munoz & De la Fuente 2003). and eosinophil proportions greater at 0600 h than 2400 h and lymphocyte proportions less at 0600 h than 2400 h (Choudhury Geographic location and population et al. 1982). Rehder et al. (1982b) noted the PCV and erythrocyte concentration of captive Geographic location and population (that is red-tailed hawks was greater in the morning the community of its species to which the bird and decreased throughout the day. A similar belongs) may affect the hematological values effect was noted for the PCV of American of a bird. This is typically an overall indication kestrels (Dawson & Bortolotti 1997). The of the complex interactions of many factors mechanism underlying this observation was that include habitat, competition, nutrition, not elucidated. In contrast, the PCV of Ameri- age, sex, social structure, “stress” and health can kestrels was not signifi cantly different status. between 0900 h and 1200 h (Rehder et al. Reports of the effect of geographic location 1982b). and population on hematological characteris- 100 Atlas of Clinical Avian Hematology tic have been published. For example, the cal characteristics make it necessary to critically hematological reference intervals of common appraise the applicability of any reference loons differed with geographic location intervals employed for comparative purposes. (Haefele et al. 2005), and Dutton et al. (2002) The greater the dissimilarity between the phys- reported differences in the hematological value iological state of the bird under study and that between two captive populations of bald ibis. of the birds for which the reference intervals The latter authors suggested the differences were established, the greater the uncertainty in may be due to diet and state of hydration. the comparison and interpretation as to the cause of any recognized difference. As previ- Beyond the described physiological infl uences, ously stated, physiological infl uences rarely many additional factors have been studied to manifest as morphological changes of hemato- determine their effects on the hematological logical cells. Consequently, the presence of characteristics of birds. It is beyond the scope morphological atypia is strong evidence of of this book to consider the many studies that response to a disease process. have investigated the effects of physiological infl uences on the hematological characteristics of birds that include inactivity, molt, nutrition Characteristics of the concentration and territory. Furthermore, how the interac- and morphology of erythrocytes in tion of two or more of these factors may affect response to disease the hematological characteristics of the bird is complex and diffi cult to predict. See Figures 229–243. The clinical assessment of the erythrocytic characteristics of a bird is best achieved by considering the measurement of a bird’s PCV, PATHOLOGICAL EFFECTS ON THE HEMATOLOGICAL CHARACTERISTICS OF BIRDS

In the following section, the hematological response(s) exhibited by birds in response to “disease” is considered. These may be evident as changes in the concentration of hematological cells or alterations in the morphology of hematological cells (or both). While dramatic changes in hematological values may be identifi ed as a response to disease without reference to the hematological values of a particular type of bird in health, these represent a small Figure 229 Gross agglutination of blood from an minority of cases. In the vast majority of cases, eclectus parrot (Eclectus roratus) with an agglutinating the detection of a disease process is greatly immune-mediated hemolytic anemia, resulting in a assisted by the recognition of deviation from PCV of 0.17 L/L The “particles” visible represent hematological values exhibited by comparable immune-mediated aggregates of erythrocytes. Repro- duced from Johnston et al., Immune-mediated hemo- birds lacking the disease process. lytic anemia in an eclectus parrot, in Journal of Consequently, even without the infl uence of American Veterinary Medical Association, copyright any disease process, the multitude of physio- 2007 with permission of the American Veterinary logical factors that may affect the hematologi- Medical Association. Physiological and pathological infl uences 101

Figure 230 Blood from a black-footed penguin Figure 232 Blood from an Australian black-shoul- (Spheniscus demersus) with increased erythropoiesis, dered kite (Elanus axillaris) that had suffered trauma evidenced by 9% polychromatophilic erythrocytes in and blood loss after being hit by a motor vehicle. The the peripheral blood. Three polychromatophilic eryth- bird had a PCV of 0.20 L/L and 19% polychromato- rocytes are present near the center of the fi eld. Figure philic erythrocytes and 2% rubricytes indicated 214 illustrates erythrocytes from this species, with increased erythropoiesis in response to the anemia. typical morphology. (Modifi ed Wright’s stain.) Several polychromatophilic erythrocytes are shown. (Modifi ed Wright’s stain.)

Figure 231 Blood from an injured and clinically Figure 233 Blood from a white-tailed black cockatoo dehydrated barn owl (Tyto alba) that had a PCV of (Calyptorhynchus baudinii) with a PCV of 0.28 L/L. 0.32 L/L. The hemoconcentration (relative polycythe- Polychromatophilic erythrocytes comprised 21% of mia) due to dehydration may “mask” the magnitude erythrocytes which indicated increased erythropoiesis of the anemia. Increased erythropoiesis was indicated in response to the anemia. Illustrated are several poly- by 10% polychromatophilic erythrocytes and 1% chromatophilic erythrocytes (one indicated by an rubricytes. Illustrated are several polychromatophilic arrow) amongst mature erythrocytes. (Modifi ed erythrocytes and two rubricytes (one identifi ed by an Wright’s stain.) arrow). (Modifi ed Wright’s stain.) 102 Atlas of Clinical Avian Hematology

Figure 234 Blood from an eclectus parrot (Eclectus Figure 236 Blood from a severely anemic muscovy roratus) with a PCV of 0.30 L/L, erythrocyte concentra- duck (Cairina moschata) (PCV = 0.09 L/L). The 12 tion of 2.0 × 10 /L, hemoglobin concentration of 59 g/ decreased density of erythroid cells may be observed L, MCV of 150 fL and MCHC of 197 g/L. A regenera- at this magnifi cation. A few mature erythrocytes and tive response was indicated by the presence of 12% a rubricyte are present (arrow), as well as two hetero- polychromatophilic erythrocytes but the microcytosis phils and a lymphocyte. (Modifi ed Wright’s stain.) and hypochromasia indicate decreased hemoglobin production. Illustrated are several polychromatophilic cells (one identifi ed by an arrow). These are diffi cult to distinguish as most of the cells present are signifi - cantly hypochromatic. (Modifi ed Wright’s stain.)

Figure 237 Blood from the same bird as in Figure Figure 235 Blood from the same eclectus parrot as 236 stained to illustrate reticulocytes. Reticulocytes in Figure 234. The concentration of reticulocytes was comprised 37% of all erythrocytes, which correlated 216 × 109/L (10.8%, similar to the observed proportion to a concentration of 185 × 109/L. (New methylene of polychromatophilic cells). A reticulocyte is identi- blue stain.) Note the importance of calculating the fi ed by the arrow. Note that most avian erythrocytes absolute values of reticulocytes, as the duck has a have some “reticulum” but are not classifi ed as reticu- greater proportion but lesser absolute concentration of locytes unless they contain multiple aggregates distrib- reticulocytes than the eclectus parrot in Figures 234 uted around the nucleus. (New methylene blue and 235. stain.) Physiological and pathological infl uences 103

Figure 238 Blood from a black-footed penguin Figure 240 Blood from an anemic white-tailed black (Spheniscus demersus) with severe anemia and a cockatoo (Calyptorhynchus baudinii) with a PCV of marked regenerative response. A large number of 0.24 L/L. No polychromatophilic erythrocytes are immature erythroid cells are present, including poly- evident in the fi eld, indicating the absence of a “regen- chromatophilic erythrocytes and rubricytes, with few erative response”. (Modifi ed Wright’s stain.) This lack mature erythrocytes. Also illustrated are a heterophil of response can be contrasted with the increased and a monocyte (center). In contrast, Figures 213 and erythropoiesis in a different bird of the same species 214 illustrate erythrocytes from this species with portrayed in Figure 233. typical morphology, and Figure 230 shows an increased proportion of polychromatophilic erythrocytes. (Modifi ed Wright’s stain.)

Figure 241 Blood from a depressed and lame Eur- Figure 239 Blood from the same black-footed asian coot (Fulica atra) with a PCV of 0.32 L/L, which penguin as in Figure 238. Many rubricytes (a) and had less than 1% polychromatophilic erythrocytes, several polychromatophilic erythrocytes (b) are present indicating a lack of response to the anemia. (Modifi ed amidst mature erythrocytes. Also present is a lympho- Wright’s stain.) Two heterophils are also present, cyte (c), identifi ed by its fi ner chromatin. (Modifi ed one of which (right) exhibits atypical cytoplasmic Wright’s stain.) granules. 104 Atlas of Clinical Avian Hematology

hemoglobin concentration and erythrocyte concentration and the assessment of the morphology of the erythrocytes. The morphological characteristics that may be encountered are summarized in Table 4.1.

Polycythemia The mechanisms resulting in polycythemia, the increase in the circulating mass of erythrocytes, and erythrocytosis, the increase in the circulating concentration of erythrocytes, are not as well described for birds as they are for mammals. These may occur due to relative changes in the blood that occur as consequence Figure 242 Tissue section of liver from a Port Lincoln of the redistribution of the erythrocytic or parrot (Barnardius zonarius). (Hematoxylin and eosin stains.) A macrophage exhibiting erythrophagocytosis fl uid components; or due to an increased pro- is illustrated; these were distributed throughout the duction of erythrocytes (erythropoiesis) that liver but were not evident in other tissues. Peripheral results in an absolute increase in the number blood was not available from this bird. (mass) of erythrocytes. Birds do not store a reserve of erythrocytes in their spleen (Sturkie 1943). Consequently, relative polycythemia due to redistribution of erythrocytes (in response to exercise, excitement, fear or pain) is not encountered in birds as it is in mammals. Relative polycythemia due to hemoconcentration of the blood as a result of water loss may occur in birds. The magnitude of such hemoconcentration is diffi cult to predict and may be complicated by variation in plasma volume between individuals and between species (Jaensch & Raidal 1998). Experimen- tal deprivation of water from pigeons for 48 hours did not change the PCV but did result in signifi cant changes in the total solids and serum osmolality (Martin & Kollias 1989). However, dehydration due to decreased water Figure 243 Blood from an Australian kestrel (Falco intake may incite differing hematological cenchroides) with heterophilic meningoencephalitis. effects on different species of birds, even Linear arrangement of erythrocytes, equivalent to rou- species within the same genus. For example, a leaux in mammals, is evident. Rouleaux are a viscosity comparison of blue-breasted quail, a wet mediated property, not usually encountered in the blood of birds. The addition of physiological saline to grassland species, and stubble quail (Coturnix the sample typically will result in dissolution of the pectoralis), a dryland species, found notable phenomenon. (Modifi ed Wright’s stain.) differences in the measured hematological analytes following restriction of water intake (Roberts & Baudinette 1984). After 10 days Physiological and pathological infl uences 105

Table 4.1 Morphological characteristics of avian erythrocytes that may indicate a disease process.

Cell characteristic Description Signiﬁ cance

Altered erythrocyte content Ghost erythrocytes Pale erythrocytes due to disrupted cell Indicative of hemolysis; may occur membrane that permits loss of with contact with EDTA in some hemoglobin species; may also occur in aged samples. Hypochromatic Erythrocytes that have increased region of Indicates decreased hemoglobin erythrocytes central pallor due to decreased production, e.g. iron defi ciency hemoglobin content Polychromatophilic Contain ribosomes and resultant basophilic Increased numbers indicate increased erythrocytes hue with Romanowsky stains due to erythropoiesis, such as in young rRNA content birds or response to anemia Erythrocyte formation Agglutination Clumping of erythrocytes usually mediated Indicates immune-mediated by antibodies “bridging” cells hemolytic anemia Anisocytosis Variation in the size of cells, typically May be encountered with a range of applied to erythrocytes disorders affecting production or destruction of erythrocytes Decreased density Increased distance between cells, paucity May be indicative of anemia of erythrocytes of cells Increased density Blood fi lm may lack monolayer, crowding May be indicative of polycythemia of erythrocytes of cells Rouleaux Linear arrangement of erythrocytes Indicates increased protein mediated by plasma viscosity concentration, such as acute phase proteins. Not commonly encountered in avian blood samples Structures within erythrocytes Basophilic stippling The presence of small, dark blue staining Indicative of increased erythropoiesis, “granules” within erythrocytes, usually occurs with a “regenerative due to the presence of residual response” to anemia aggregations of RNA. (Also occasionally due to the presence of iron aggregations) Heinz bodies Rounded projections from the surface of Indicative of oxidative injury erythrocytes that represent denatured hemoglobin that are eosinophilic with Romanowsky stains and blue when stained with new methylene blue stain Intracellular E.g. Babesia spp, Haemoproteus spp, Variable depending on species of hemoparasites Hepatozoon sp. Variable appearance hemoparasite, host, immune depending on the type of hemoparasite. function etc., insignifi cant to May increase the size, distort the shape pathogenic or displace the nucleus of the erythrocyte 106 Atlas of Clinical Avian Hematology without water the weight of blue-breasted to hypoxia, whereas other species may exhibit quail had decreased to 80.5% of their original an erythrocytosis in response to the hypoxia weight but the PCV was not signifi cantly of high altitude. For example, Pekin duck increased (102% of the original value); (Anas platyrhynchus forma domestica) devel- however, plasma osmolarity had increased oped erythrocytosis in response to acclimation (119.9% of the original value). In comparison, to altitude (5640 m) but this did not occur in after 20 days without water, the stubble quails’ bar-headed geese (Black & Tenney 1980). A weight had decreased to 85.6% of their origi- study of many species showed that highland nal with an increased PCV (115.4% of the birds had a mean hematocrit and mean eryth- original value) but unchanged osmolarity rocyte concentration that were 7% and 23% (101.2% of the original value). greater than the values of the same species Clinical disease causing dehydration has from lower altitudes (Carey & Morton 1976). also been reported to increase the PCV of Similarly, the embryos of American coots birds. Increased erythrocytic values were (Fulica americana) from montane regions had observed in common pheasants and red-legged signifi cantly greater hematocrit than embryos partridges (Alectus rufa) with severe spironu- from lowland regions (Carey et al. 1993). The cleolisis due to relative polycythemia (hemo- hematocrit of pigeons exposed to an experi- concentration) as a result of diarrhea causing mental recreation of high altitude was increased dehydration (Lloyd & Gibson 2006). Increased (0.48 ± 0.02 L/L compared to 0.34 ± 0.6 L/L) PCV in stranded wedge-tailed shearwater when the birds were acclimated to high-alti- (Puffi nus pacifi cus) chicks, compared to tude conditions but showed no signifi cance healthy chicks, was consistent with hemocon- difference when birds were acutely exposed centration (Work & Rameyer 1999). A study (Weinstein et al. 1985). Japanese quail accli- of American kestrels incubating eggs, revealed mated to a simulated altitude of 6100 m for 6 the hematocrit was increased with ambient weeks had a 31% increase in mean hematocrit temperature, likely refl ecting hemoconcentra- and 37% increase in mean hemoglobin con- tion (Dawson & Bortolotti 1997). centration (Weathers & Snyder 1974). Absolute polycythemia resulting in the Bond and Gilbert (1958) found that both increased production of erythrocytes may be “diving” and “dabbling” species of ducks had due to autonomous production of erythrocytes a greater blood volume than, but similar ery- (primary polycythemia), or be stimulated by throid characteristics to, non-aquatic birds. an increased production of erythropoietin in More recent studies of the physiological char- response to hypoxia (secondary polycythe- acteristics of diving birds, including oxygen mia). The latter may result from disease of the utilization, have not considered hematological cardiovascular, pulmonary or renal systems or values (Green et al. 2005, Halsey & Butler physiological response to high altitude. 2006). A study of burrowing owls (Speotyto Species of birds that have evolved to under- cunicularia), which routinely become hypoxic take high-altitude fl ight, such as the bar-headed below ground, had erythrocytic values similar goose (Anser indicus), Rueppell’s griffon (Gyps to non-burrowing birds (Boggs et al. 1983). rueppellii) and white-headed vulture (Trigono- Maxwell et al. (1987) found that PCV, eryth- ceps occipitalis), posses hemoglobin with an rocyte concentration and haemoglobin con- increased affi nity for oxygen, compared to centration were greater in young broiler chicks species of birds that do not undertake such with experimentally induced hypoxia (com- fl ight (Weber et al. 1988, Hiebl et al. 1989, pared to control chicks). Zhang et al. 1996). Consequently, these Most commonly, when the concentration of adapted species typically do not exhibit an erythrocytes is increased, the erythrocytes typ- increased erythrocyte concentration in response ically have a “normal” morphological appear- Physiological and pathological infl uences 107 ance. However, if the polycythemia is due to A number of experimental and clinical increased erythropoiesis then an increased examples of hemolytic anemia affecting birds proportion of polychromatophilic erythrocytes have been reported. Few cases of suspected (Romanowsky stains) or reticulocytes (new primary immune-mediated hemolytic anemia methylene blue stain) may be observed. have been reported. Presumed immune-mediated hemolytic anemia was reported in a blue- crowned conure (Aratinga acuticaudata) with Anemia a PCV of 0.28 L/L (Jones et al. 2002). Simi- Anemia, the decrease in the circulating mass larly, immune-mediated hemolytic anemia was of erythrocytes, may result from causes that reported in an eclectus parrot, that had gross may be broadly classed as “hemorrhagic” (loss agglutination of erythrocytes, a PCV of 0.17 L/ of erythrocytes), “hemolytic” (destruction of L and 19.6% reticulocytes (indicating a erythrocytes) or “hypoproliferative” (decreased marked increase in erythropoiesis) (Johnston erythropoiesis). Erythrocytes with variant et al. 2007). morphology may be evident, depending on the A number of cases of hemolytic anemia cause of the anemia and the hematological incited by hemoparasites have been reported. response. Anemia may be due to a lack of Natural infection of snowy owls (Nyctea scan- erythrocytes but those present exhibit typical diaca) with Haemoproteus noctuae resulted in size (normocytic) and hemoglobin content severe anemia (PCV of 0.13 L/L) with mor- (normochromatic). Alternately, if erythropoi- phological evidence of regeneration (Evans & esis is increased in response to the anemia, an Otter 1998, Mutlow & Forbes 2000). Simi- increased proportion of (morphologically dis- larly, a sandhill crane (Grus canadensis) chick tinct) immature erythroid cells may be evident infected with Haemoproteus balearicae had in the peripheral blood. a severe anemia (0.13 L/L) (Dusek et al. A number of experimental and clinical 2004). Natural infection with Babesia shortii examples of hemorrhagic anemia affecting resulted in anemia (PCV of 0.22 L/L) in a Eur- birds have been reported. Experimental asian kestrel (Munoz et al. 1999) and a saker removal of 30% of estimated blood volume in falcon (PCV of 0.29 L/L) (Samour & Pierce Japanese quail induced peak reticulocytes at 1996). 48 hours and recovery of erythrocyte numbers Extra-vascular hemolytic anemia due to by 72 hours after phlebotomy (Gildersleeve oxidative damage to erythrocytes that results et al. 1985, Schindler et al. 1987a). Similarly, in the formation of Heinz bodies has been for pigeons with experimental blood loss (15– produced under experimental conditions and 30% of estimated total blood volume), the has been observed in naturally occurring birds recovered their PCV to about 90% of disease. Heinz body anemia was experimen- initial values within 168 hours (Finnegan et al. tally induced in chickens by treatment with 1997). dimethyl disulphide. Birds maintained eryth- Clinically evident hemorrhage, that resulted rocyte concentration with increased numbers from brodifacoum toxicity, caused severe of polychromatophilic erythrocytes and rubri- anemia in a captive white-winged wood duck cytes (Maxwell 1981). Erythrocytes stained (Cairina scutulata) (James et al. 1998). The with methyl violet exhibited an average of six duck had ongoing bleeding and was severely Heinz bodies 0.3–2.0 µm in diameter. anemic (PCV of 0.16 L/L) 10 days after initial Experimental zinc toxicosis in mallards clinical signs. The PCV increased after treat- induced variable hematological effects (Chris- ment, including a blood transfusion, to 0.37 L/ topher et al. 2004). Birds that died had severe L at 16 days and 0.57 L/L at 45 days after anemia (0.19 ± 0.07 L/L) with many hypo- initial clinical signs. chromatic erythrocytes, fusiform erythrocytes 108 Atlas of Clinical Avian Hematology and erythrocytes with nuclear abnormalities. ropoiesis. A number of reports of birds with Birds that survived had PCV (0.48 ± 0.075 L/ mild anemia associated with infl ammatory L) similar to the PCV of control birds (0.50 ± disease have been published. Red grouse with 0.04 L/L). Clinical cases of zinc intoxication an infl ammatory response to Trichostrongylus have been reported for a number of species of tenuis also developed a mild anemia (Wilson birds, both captive and free-living, including a and Wilson 1978). African grey parrots that hyacinth macaw (Romagnano et al. 1995), a had an infl ammatory leukogram in response gray-headed chachalaca (Ortalis cinereiceps) to a range of clinical disorders also exhibited (Droual et al. 1991) and a trumpeter swan anemia, microcytosis and hypochromasia (Cygnus buccinator) (Carpenter et al. 2004). (Hawkey et al. 1982). The majority of indi- Heinz body formation and subsequent viduals (88%) from several species of cranes extra-vascular hemolytic anemia has been with naturally occurring mycobacteriosis observed in herring gulls and Atlantic puffi ns exhibited an anemia (Hawkey et al. 1990). (Fratercula arctica) administered crude oil Mild anemia, consistent with anemia of infl am- (Leighton et al. 1983, Leighton 1985, 1986). matory disease was reported in two species of Similarly, anemia with increased numbers of black cockatoo with infl ammatory disorders polychromatophilic erythrocytes was observed (Jaensch & Clark 2004). in wild white-winged scoters (Melanitta fusca) Many other factors may also affect the pro- following contamination with fuel (Yamato duction of erythroid cells. For example, thy- et al. 1996). In contrast, rhinoceros auklets roidectomy of redhead bunting (Emberiza (Cerorhinca monocerata) experimentally bruniceps) reduced erythroid values (PCV, administered crude oil did not develop Heinz erythrocyte concentration and hemoglobin bodies and had hematological values similar concentration) which was restored by admin- to control birds (Newman et al. 1999). istration of exogenous thyroxine, illustrating A number of experimental and clinical the necessity of thyroid hormones (Thapliyal examples of hypoproliferative anemia affect- et al. 1983). Similarly a defi ciency of one or ing birds have been reported. A non-regenera- more nutrients may adversely impact on effi - tive anemia was observed in wild herring gulls cient production of hemoglobin and erythro- brought into captivity with the nadir in PCV cytes. However comprehensive discussion of occurring 12–20 days after capture (from 0.43 these many factors is beyond the scope of this ± 0.04 L/L to 0.32 ± 0.05 L/L) (Hoffman & book. Leighton 1985). Similarly, the PCV of rhinoceros auklets maintained in captivity decreased (from 0.56 ± 0.02 L/L to 0.46 ± 0.01 L/L) Characteristics of the concentrations within 3 weeks that persisted throughout the and morphology of leukocytes in duration of the study (Newman et al. 1999). response to disease A commonly encountered cause of hypoproliferative anemia is “anemia of infl amma- See Figures 244–286. tory disease” that is typically mild, normocytic, Interpretation of the avian leukogram is normochromatic and non-regenerative (Means best served by consideration of the absolute 2000, Waner & Harrus 2000). Anemia of differential concentration of each type of leu- infl ammatory disease is mediated by increased kocyte and the morphology of the leukocytes, secretion of cytokines during infl ammation particularly the heterophils. The hematologi- that results in decreased availability of iron cal indications of an infl ammatory response (due to sequestration within macrophages), exhibited by a particular bird is a complex decreased erythrocyte lifespan, impaired pro- interaction of many factors that include: the duction of erythropoietin and decreased eryth- inherent hematological characteristics of the Physiological and pathological infl uences 109

Figure 244 Blood from a blue-breasted quail (Cotur- Figure 246 Blood from a wild Eurasian coot (Fulica nix chinensis) that had healing cutaneous wounds atra) that presented with wounds infl ected by a preda- (infl icted by other quails). A band heterophil with tor. Illustrated is a heterophil showing mild morpho- notable cytoplasmic basophilia but typical granule logical atypia, including rounded granules, large morphology, representing a mild atypia, is shown. granules (arrow) and mild cytoplasmic basophilia. (Modifi ed Wright’s stain.) (Modifi ed Wright’s stain.)

Figure 245 Blood from a wild galah (Eolophus roseicapillus) injured by a motor vehicle. Two heterophils Figure 247 Blood from an injured barn owl (Tyto are illustrated; one (upper) has distinctly basophilic alba) that had a heterophil concentration of 6.1 × cytoplasm and a decreased density of granules while 109/L. Illustrated is a heterophil with rounded cyto- the other (lower) heterophil has typical cytoplasmic plasmic granules and basophilic cytoplasm (arrow). granule morphology. (Modifi ed Wright’s stain.) Other Also present is an eosinophil, amidst erythrocytes. heterophils (not shown) from this bird exhibited addi- (Modifi ed Wright’s stain.) Many other heterophils from tional morphological atypia including: rounded gran- this bird also had a decrease in the overall number of ules, basophilic granules, hyposegmentation of the cytoplasmic granules. nucleus and decreased chromatin density. Figure 248 Blood from a wedge-tailed eagle (Aquila audax) that presented with blindness in both eyes. Figure 250 Blood from the same Australian black- Hematological assessment revealed a heterophil con- shouldered kite as in Figure 249, 2 days later. It had 9 centration of 10.67 × 109/L, lymphocyte concentration a heterophil concentration of 9.10 × 10 /L, lympho- 9 of 0.58 × 109/L and monocyte concentration of 1.30 cyte concentration of 0.89 × 10 /L and a monocyte 9 × 109/L. Illustrated is a heterophil representative of the concentration of 1.11 × 10 /L. Morphological atypia mild morphological atypia exhibited by most hetero- of heterophils that included basophilic cytoplasm, phils, which included rounded and basophilic gran- decreased granulation and rounded granules were ules amidst typical fusiform, brick-red granules. observed. Fifty-two percent of heterophils exhibited some atypia. Illustrated is a heterophil with large and rounded granules, but a typical density of granules. (Modifi ed Wright’s stain.)

Figure 249 Blood from an Australian black-shouldered kite (Elanus axillaris) that was likely hit by a motor vehicle, that had a concentration of creatine kinase of 17,560 IU/L and a heterophil concentration of 2.67 × 109/L, lymphocyte concentration of 0.61 × Figure 251 Blood from a brown falcon (Falco berig- 109/L, monocyte concentration of 0.75 × 109/L, eosin- ora) with a talon injury, which had a heterophil con- ophil concentration of 0.33 × 109/L and basophil concentration of 5.94 × 109/L and a lymphocyte centration of 0.33 × 109/L. “All” of the heterophils concentration of 1.1 × 109/L. Many heterophils exhib- exhibited some morphological atypia that included ited atypical morphology. A mature heterophil with decreased segmentation of the cell’s nucleus, an fusiform granules (left) and an atypical heterophil with annular form nucleus, basophilic cytoplasm, decreased shorter more rounded granules, a decreased number granulation, large granules and rounded granules. of granules and basophilic cytoplasm are illustrated. Illustrated are two heterophils, each with a “band” (Modifi ed Wright’s stain.) nucleus, decreased granulation and basophilic cytoplasm. (Modifi ed Wright’s stain.) Figure 252 Blood from a red wattlebird (Anthochaera carunculata) with meningoencephalitis. Two hetero- Figure 254 Blood from a clinically dehydrated Aus- phils with basophilic cytoplasm and rounded granules tralian magpie (Gymnorhina tibicen) that had a PCV are shown, amidst erythrocytes. For comparison, of 0.70 L/L (a marked polycythemia) and a heterophil 9 Figure 166 illustrates typical heterophils from this concentration of 8.2 × 10 /L with atypical morphol- 9 species. (Modifi ed Wright’s stain.) ogy, and a monocyte concentration of 2.0 × 10 /L. The heterophils illustrated have large and rounded granules amidst more typical granules. This atypia indicated a response to infl ammation by the bird. The erythrocytes have been partially lysed, as evidenced by the paler eosinophilic color of the cytoplasm and the distinctly eosinophilic background, most likely due to contact with the EDTA used as an anticoagulant.

Figure 253 Blood from a yellow-throated laughing thrush (Garrulax galbanus). The bird’s plasma is evident due its milky, opaque appearance that indicates lipemia. Three heterophils, that exhibit distinctly basophilic cytoplasm and a decreased density of granules Figure 255 Blood from an Australian king parrot (Ali- with some rounded granules, are portrayed. The lym- sterus scapularis) that had a heterophil concentration phocyte present has intensely basophilic cytoplasm of 34.0 × 109/L, a lymphocyte concentration of 4.8 × and a small perinuclear pale zone (arrow). These mor- 109/L and a monocyte concentration of 1.2 × 109/L. phological characteristics indicate an infl ammatory Illustrated are four heterophils that have rounded gran- response. (Modifi ed Wright’s stain.) ules and a decreased density of granules as well as increased basophilic cytoplasm. Both the increased concentration of heterophils and heterophil morphology indicate signifi cant infl ammatory demand. (Modi- fi ed Wright’s stain.) Figure 256 Blood from an Australian king parrot (Ali- sterus scapularis) with a notable heterophilia (47.1 × Figure 258 Blood from a princess parrot (Polytelis 109/L) and atypical heterophil morphology. Lympho- alexandrae) with a heterophil concentration of 19.2 × 9 9 cyte concentration was 6.8 × 109/L and monocyte 10 /L, lymphocyte concentration of 3.4 × 10 /L and 9 concentration was 7.4 × 109/L. Two atypical hetero- monocyte concentration of 3.4 × 10 /L, that indicated phils are represented, one has small round granules infl ammation. Illustrated are three heterophils with (left); the other has larger granules that are slightly decreased number of granules and rounded granules. rounded. Both these cells have increased basophilic (Modifi ed Wright’s stain.) cytoplasm. (Modifi ed Wright’s stain.)

Figure 259 Blood from a sulphur-crested cockatoo (Cacatua galerita) with a PCV of 0.27 L/L, heterophil Figure 257 Blood from a Port Lincoln parrot (Barnar- concentration of 58.8 × 109/L, lymphocyte concentra- dius zonarius) with a PCV of 0.36 L/L, heterophil con- tion of 8.9 × 109/L and monocyte concentration of 0.7 centration of 19.1 × 109/L, lymphocyte concentration × 109/L. Illustrated are a granulated heterophil (right), of 6.2 × 109/L and monocyte concentration of 1.6 × a large mononuclear cell (center) and a heterophil that 109/L. Atypical heterophil morphology, including is markedly hypo-granulated, with only two needle- decreased granularity and rounded granules, was shaped granules evident (left). The heterophilia and observed. Illustrated is a heterophil with decreased atypical heterophil morphology indicate infl amma- granule density, rounded granules (including one tion. In this case, concurrently increased amylase con- large, prominent, round granule) and increased baso- centration indicated pancreatic disease. (Modifi ed philia of the cytoplasm. For comparison, Figure 200 Wright’s stain.) illustrates typical heterophil morphology for this species. (Modifi ed Wright’s stain.) Figure 260 Blood from a Major Mitchell cockatoo Figure 262 Blood from a rusty-barred owl (Strix (Cacatua leadbeateri) that had been injured by another hylophila) showing two heterophils with atypical 9 bird, with a heterophil concentration of 24.1 × 10 /L. appearance. One is characterized by indistinct, Illustrated are two heterophils that exhibit atypical rounded eosinophilic granules and a few round granule morphology, including rounded granules and strongly basophilic granules. The other bears little basophilic granules, and decreased segmentation of resemblance to a typical heterophil with a predomi- the nucleus. For comparison, Figure 197 illustrates nance of these strongly basophilic, round granules. typical heterophil morphology for this species (Modi- These represent marked atypia in response to infl am- fi ed Wright’s stain.) matory demand. Comparison with the granulocytes from a healthy bird of the same species may be used to distinguish this as a “toxic change” (and not, for example a basophil). For comparison, Figures 217– 219 illustrate the typical morphology of leukocytes from this species. (Modifi ed Wright’s stain.)

Figure 261 Blood from a sun conure (Aratinga solstitialis) that contained few heterophils (0.89 × 109/L), Figure 263 Blood from a blue bonnet (Northiella “all” with atypical morphology, indicating a signifi - haematogaster) that presented in a weak state and with cant infl ammatory demand. Represented is a hetero- poor body condition. The bird had a PCV of 0.35 L/L, phil with rounded cytoplasmic granules that have an a heterophil concentration of 2.03 × 109/L, a lympho- increased basophilic color. (Modifi ed Wright’s stain.) cyte concentration of 0.20 × 109/L and a monocyte concentration of 0.86 × 109/L. The heterophils exhibited marked morphological atypia, notably rounded basophilic granules, which indicated signifi cant infl ammatory demand. (Modifi ed Wright’s stain.) Figure 264 Blood from a wild spotted dove (Strepto- Figure 266 Blood from an Australian kestrel (Falco pelia chinensis) that had extensive cutaneous wounds cenchroides) with heterophilic meningo-encephalitis that appeared to have been present for some time. and bacteremia. An immature heterophil exhibiting “All” of the heterophilic lineage cells exhibited mor- decreased granulation and basophilic granules and a phological atypia including: decreased granulation, hyposegmented nucleus is shown. For comparison, rounded granules, basophilic granules, notable cyto- Figure 125 shows typical heterophil morphology from plasmic basophilia, hyposegmentation of the nucleus, this species. (Modifi ed Wright’s stain.) and decreased chromatin density. Illustrated is an example of such a heterophil. For comparison, Figure 113 illustrates typical heterophil morphology from this species. (Modifi ed Wright’s stain.)

Figure 265 Blood from the same wild spotted dove as in Figure 264, 6 days after the initial blood sample was collected, during which time the wounds had Figure 267 Blood from the same Australian kestrel been debrided and antimicrobial therapy adminis- as in Figure 266, with heterophilic meningoencepha- tered. Two representative heterophils that exhibit mild litis and bacteremia. A macrophage that has phagocy- morphological atypia, including decreased density of tosed several bacteria and some cellular material is granules, mildly rounded granules and mildly cyto- shown. (Modifi ed Wright’s stain.) plasmic basophilia, are illustrated. (Modifi ed Wright’s stain.) The morphology of the bird’s heterophils indicates it is now better able to produce heterophils in response to the infl ammation. A subsequent blood sample from the same bird, 13 days after the initial sample, revealed heterophils with typical morphology. Figure 268 Blood from an Australian hobby (Falco Figure 270 Blood from an injured southern boobook longipennis) which had a heterophil concentration of owl (Ninox novaeseelandiae). Present are a cell of 5.1 × 109/L, a lymphocyte concentration of 10.0 × heterophilic lineage that has decreased granules, 109/L and a monocyte concentration of 1.15 × 109. which were round and basophilic, within basophilic The heterophils exhibited marked morphological cytoplasm. Also present is a mononuclear cell and atypia. Illustrated is a heterophil with decreased several polychromatophilic erythrocytes. The latter numbers of cytoplasmic granules, which are rounded indicate increased erythropoiesis, in this case, most and have increased basophilia, in a cytoplasm that has likely in response to hemorrhage. For comparison, increased basophilia. These indicate severe infl amma- Figure 215 illustrates typical heterophil morphology tion and marked demand for heterophils. Bacteria for species. (Diff Quik stain.) were evident in the sample (see Figure 269) and the bacteremia accounts for the severe infl ammatory challenge indicated by heterophil morphology. (Modifi ed Wright’s stain.)

Figure 271 Blood from a Mallee fowl (Leipoa ocellata) with a fractured tibiotarsus, which had a heterophil concentration of 9.8 × 109/L (all stages) and a monocyte concentration of 3.56 × 109/L. Numerous immature heterophils were observed, these exhibited a hypo-segmented nucleus and decreased cytoplasmic granularity. The erythrocytes had lysed due to contact Figure 269 Blood from the same Australian hobby as with the EDTA anticoagulant. Illustrated are two in Figure 268. Two bacteria are evident within the immature heterophils, with no segmentation or inden- monocyte. Bacteria were also evident throughout the tation of the nucleus but some cytoplasmic granules fi lm (arrowhead). (Modifi ed Wright’s stain.) evident, and two monocytes. (Modifi ed Wright’s stain.) 116 Atlas of Clinical Avian Hematology

Figure 274 Blood from a black-footed penguin Figure 272 Blood from a pied butcher bird (Cracticus (Spheniscus demersus) with air sacculitis had a hetero- nigrogularis) showing a heterophil with an annular philia (32.0 × 109/L), lymphocytosis (14.5 × 109/L) and nucleus, which refl ects atypical myelopoiesis. Also monocytosis (3.5 × 109/L). Illustrated are four hetero- present is a monocyte. (Modifi ed Wright’s stain.) phils, with typical morphology, amidst erythrocytes (including three polychromatophilic erythrocytes). The “mature” heterophilia indicates the bone marrow’s production of heterophils is adequate for the infl ammatory demand. Also present are aggregated thrombocytes (bottom left). (Modifi ed Wright’s stain.)

Figure 273 Blood from a little penguin (Eudyptula minor) with aspergillosis exhibiting a heterophilia and a monocytosis. Illustrated are three heterophils, two of which have a band form nucleus, and a mononuclear cell. Some amphophilic, rounded cytoplasmic gran- Figure 275 Blood from a white-tailed black cockatoo ules are evident in the segmented heterophil. However, (Calyptorhynchus baudinii) with an ulnar fracture that the central granular bodies of the granules are also had a PCV of 0.42 L/L, heterophil concentration of prominent, making the rounded granules more diffi - 34.6 × 109/L, lymphocyte concentration of 4.1 × 109/L cult to discern. Note also that some heterophil gran- and monocyte concentration of 3.9 × 109/L, indicating ules can also be discerned “free” in the background. an infl ammatory response. Illustrated are six hetero- For comparison, Figure 211 illustrates the typical het- phils amidst mature erythrocytes. Most of the hetero- erophil morphology for this species. (Diff Quik phils exhibit typical density of granules; however, stain.) rounded granules can be observed in some cells. (Modifi ed Wright’s stain.) For comparison, Figure 196 illustrates the typical heterophil morphology for this species. Physiological and pathological infl uences 117

Figure 276 Blood from a woolly-necked stork Figure 278 Blood from a red wattlebird (Anthochaera (Ciconia episcopus) with bacterial pododermatitis that carunculata) with meningoencephalitis. A heterophilia exhibited increased numbers of heterophils and mono- was evident and six heterophils are illustrated. These cytes. Illustrated are three heterophils, a monocyte and show varied granule morphology within a basophilic a lymphocyte amidst erythrocytes, including several cytoplasm. (Modiﬁ ed Wright’s stain.) polychromatophilic erythrocytes. The heterophils illustrated exhibited typical granule morphology whereas other heterophils had rounded granules. (Modiﬁ ed Wright’s stain.)

Figure 279 Blood from a male Brolga (Grus rubicunda) with diarrhea that had a heterophil concentra- Figure 277 Blood from a Stanley crane (Grus para- tion of 2.8 × 109/L, a lymphocyte concentration of disea) with aspergillosis; the four heterophils illustrated 0.48 × 109/L, a monocyte concentration of 0.65 × have mostly indistinct brick-red cytoplasmic granules 109/L and a basophil concentration of 0.08 × 109/L. with a few small, round basophilic granules. These The heteropenia is consistent with infl ammatory cytoplasmic characteristics represent cytoplasmic demand and the lymphopenia is consistent with con- immaturity and increased demand for cells in response current “stress”. The decreased concentration of het- to infl ammation. For comparison, Figure 156 illus- erophils with mostly typical morphology suggests a trates typical heterophils morphology for this species. rapid consumption of circulating cells that have not (Modifi ed Wright’s stain.) yet been replaced from the bone marrow. Illustrated is a heterophil with mostly fusiform granules and a few rounded granules. (Modifi ed Wright’s stain.) Figure 280 Blood from a tawny frogmouth (Podargus strigoides) with a fractured wing, which had a hetero- Figure 282 Blood from an Edwards’ pheasant phil concentration of 1.1 × 109/L, lymphocyte concen- (Lophura edwardsi) with aspergillosis exhibiting a tration of 0.1 × 109/L and monocyte concentration of monocytosis. The clumping of cells and vacuolation 0.2 × 109/L. Despite the heteropenia, the heterophils of cells is an artifact due to the use of lithium heparin exhibited mostly typical morphology, suggesting rapid, as an anticoagulant and delayed processing of the recent depletion of circulating heterophils. Illustrated sample. (Modifi ed Wright’s stain.) is a heterophil with typical granule morphology with slight basophilia of the cytoplasm. For comparison, Figure 96 illustrates the typical heterophil morphology for this species. Also present are a lymphocyte and a number of erythrocytes. (Modifi ed Wright’s stain.)

Figure 281 Blood from a white-tailed black cockatoo Figure 283 Blood from a crested wood-partridge (Calyptorhynchus baudinii) with weight loss, vomiting (Rollulus roulroul) with dyspnea that exhibited a het- and oral lesions, that had a PCV of 0.32 L/L, a hetero- erophilia (25.8 × 109/L) and a monocytosis (32.4 × phil concentration of 17.2 × 109/L, a lymphocyte con- 109/L). Depicted are a monocyte with typical morphol- centration of 4.6 × 109/L, a monocyte concentration ogy and two heterophils, one of which has atypical of 23.9 × 109/L and a basophil concentration of 0.2 × granulation (arrow). (Modifi ed Wright’s stain.) 109/L. Illustrated are three monocytes amidst erythrocytes. (Also present is an erythroplastid). (Modifi ed Wright’s stain.) Monocytosis occurs more rapidly in birds than in mammals (Branton et al. 1997). Physiological and pathological infl uences 119

Figure 284 Blood from the same crested wood-par- Figure 286 Blood from a varied lorikeet (Psitteuteles tridge as in Figure 283. Illustrated is a monocyte with versicolor) with weight loss. The bird had a lympho- atypical morphology, namely a prominent nucleolus penia (lymphocyte concentration of 0.5 × 109/L). Illus- (arrow). (Modifi ed Wright’s stain.) trated is a fi eld of erythrocytes with a single heterophil evident. Notably lymphocytes were absent from this and many of the fi elds observed. Those lymphocytes observed had a typical morphology. (Modifi ed Wright’s stain.)

species; the characteristics of the individual bird; the etiological agent (or the cause of infl ammation); the “magnitude” of infl ammation; and the duration of the infl ammation. The wide range of avian species and the dearth of information regarding specifi c response to defi ned etiological agents in a nominated species typically necessitate extrapolation from an unrelated species. In the following section selected studies are used to illustrate trends in the avian hematological response to disease. These characteristics are also summarized in Figure 285 Blood from a cockatiel (Nymphicus hol- Tables 4.2 and 4.3. landicus) with renal insuffi ciency, which had a lym- 9 Amongst the Anseriformes, hematological phopenia (0.14 × 10 /L). Lymphocytes were absent response to an infl ammatory challenge has from most of the fi elds examined. Hematological cells had typical morphology. Pictured are two heterophils been reported for individuals of several species amidst mature erythrocytes. (Modifi ed Wright’s (Bienzle et al. 1997). A captive teal (Anas spp) stain.) with a Staphlococcus aureus septicemia had a heteropenia (0.6 × 109/L) with a left shift (1.4 × 109/L) and “toxic changes”. The same authors reported a juvenile black-bellied whistling duck (Dendrocygna autumnalis) 120 Atlas of Clinical Avian Hematology

Table 4.2 Disease processes indicated by changes in the leukocyte concentration of birds.

Leukocyte concentration Description Signiﬁ cance

Heterophilia A concentration of heterophils that is Commonly indicative of greater than the upper limit of an infl ammation appropriate reference interval. Increased numbers of heterophils may be evident on a blood fi lm Heteropenia A concentration of heterophils that is Indicative of marked infl ammation less than the lower limit of an resulting in depletion of bone appropriate reference interval. marrow granulocyte reserve; Decreased numbers of heterophils may also occur with decreased may be evident on a blood fi lm granulopoiesis Lymphocytosis A concentration of lymphocytes that Indicative of immune stimulation is greater than the upper limit of an appropriate reference interval. Increased numbers of lymphocytes may be evident on a blood fi lm Lymphopenia A concentration of lymphocytes that Commonly indicative of “stress” (lymphocytopenia) is less than the lower limit of an appropriate reference interval. Decreased numbers of lymphocytes may be evident on a blood fi lm Monocytosis A concentration of monocytes that is Indicative of infl ammation greater than the upper limit of an appropriate reference interval. Increased numbers of monocytes may be evident on a blood fi lm Eosinophilia A concentration of eosinophils that is Indicative of infl ammation, often greater than the upper limit of an of parasitic or allergic etiology appropriate reference interval. Increased numbers of eosinophils may be evident on a blood fi lm

with osteoarthritis, and multifocal necrotizing and toxic changes evident had a maximum tissue lesions caused by S. aureus, that had heterophil concentration of 12.2 × 109/L with marked heterophilia with a left shift (130.7 × 0.7 × 109/L immature heterophils, before the 109/L heterophils and 105.6 × 109/L immature infl ammation resolved with antimicrobial heterophils) and “toxic changes” evident as therapy. well as a lymphocytosis (22.2 × 109/L), mono- Examples of changes in leukocyte con- cytosis (11.1 × 109/L) and eosinophilia (8.3 × centration and morphology in response to 109/L). An Aleutian goose (Branta canadensis naturally occurring disease have been docu- leucopareia) with multifocal, cutaneous infl am- mented for a number of species of Falconi- matory lesions that initially had 4.6 × 109/L formes. Bienzle et al. (1997) reported the heterophils, 0.8 × 109/L immature heterophils hematological characteristics of representa- Physiological and pathological infl uences 121

Table 4.3 Morphological characteristics of avian leukocytes that may indicate a disease process.

Leukocyte morphology Description Signiﬁ cance

Heterophil Nuclear immaturity Band The penultimate stage of granulocyte Increased numbers are development that is characterized by a nucleus indicative of infl ammation that has no constriction greater than half the width of the nucleus and less coarsely clumped chromatin and a smoother nuclear membrane than mature (segmented) granulocytes. Note granules may obscure nucleus and prevent classifi cation as a band/segmented Metamyelocyte/ Less mature stages of heterophil development Indicative of infl ammation myelocyte with no constrictions of the nucleus. Typically not observed in the peripheral blood unless depletion of reserve of mature heterophils Cytoplasmic immaturity Basophilia Increased bluish coloration of the cytoplasm, due Indicative of infl ammation to increased number of ribosomes, when stained with a Romanowsky stain Decreased density of Fewer granules. Decreased granulation allows Indicative of infl ammation granules better visualization of the fi ne detail of the nucleus and cytoplasm Changes in the Larger granules, rounded granules, variably sized Indicative of infl ammation morphology of granules; indicative of atypical granulopoiesis granules Changes in the color Granules are amphophilic or basophilic, instead Indicative of infl ammation of granules of “brick-red” color. Often concurrent changes in the morphology of the granules. Indicative of atypical granulopoiesis Lymphocytes Nucleolus Complex of nuclear proteins and RNA where Indicates cellular activity. May RNA transcripts are produced be encountered with both antigenic/mitogenic stimulation and neoplasia Increased cytoplasmic Represent increased amounts of cytoplasmic RNA Indicates cellular activity basophilia Perinuclear pale region Represents the Golgi apparatus Indicates cellular activity

tives from several birds of prey, namely red- had heteropenia (1.2 × 109/L) with a left shift tailed hawk, ferruginous hawk (Buteo regalis) (3.1 × 109/L) and marked toxic changes. A and rough-legged hawk (Buteo lagopus) with second red-tailed hawk with aspergillosis and naturally occurring bacterial and mycotic Staphylococcus aureus abscess, when initially disease. A red-tailed hawk with aspergillosis assessed, had a heterophilia (23.2 × 109/L) 122 Atlas of Clinical Avian Hematology with a left shift (31.2 × 109/L) and mild toxic lymphocytes, monocytes and basophils, 1 hour changes. The heterophil concentration had post-injection (Wang et al. 2003). These values increased markedly by day 7 (230.5 × 109/L) had returned to pre-injection values (in surviv- with a left shift (7.8 × 109/L). Similarly, a ing birds) by 48 hours after injection. Similarly, captive ferruginous hawk with a mycotic infec- injection of lipopolysaccharide into domestic tion had a heterophilia (63.9 × 109/L) with a turkeys incited a transient leukopenia due to left shift (20.7 × 109/L) and toxic changes. A lymphopenia (2–4 hours after injection) and rough-legged hawk with mycotic disease had then a leukocytosis due to heterophilia (8–24 heterophilia (19.6 × 109/L) with a left shift (3.5 hours after injection) (Carmichael in Harmon × 109/L) and monocytosis (6.3 × 109/L) at pre- 1998). Japanese quail experimentally infected sentation and by day 28 had a greater hetero- with Aspergillus fl avus exhibited an increased philia (49.2 × 109/L) with a left shift (5.8 × total leukocyte concentration 3–7 days after 109/L) and monocytosis (13.0 × 109/L). All infection that was characterized by an increased these birds died or were euthanased. Cellular percentage of heterophils and a decreased per- morphology was a better predictor of outcome centage of lymphocytes (Pandita et al. 1991). than the magnitude of the heterophilia. Red grouse experimentally infected with cecal Wernery et al. (2004) reported that falcons threadworm (Trichostrongylus tenuis) had rarely exhibited leukocytosis of greater than increased concentrations of both heterophils 17.0 × 109/L in response to infl ammation and eosinophils (Wilson & Wilson 1978). and the interpretation of the leukogram was Leukocyte response to naturally occurring complicated by response to handling which disease has been reported for several species of could increase leukocyte concentration to Gruiformes. Seventeen birds, representing 13 × 109/L. These authors also reported the several species of crane, naturally infected hematological changes in falcons in response with Mycobacterium avium, all exhibited a to various etiological agents, such as viral leukocytosis due to a heterophilia and mono- diseases (leukocytes 6.34 ± 2.21 × 109/L), cytosis and many (71%) had a concurrent bacterial diseases (leukocytes 13.76 ± 2.91 × lymphocytosis (Hawkey et al. 1990). Affected 109/L) and aspergillosis (leukocytes 13.97 ± cranes exhibited heterophil concentrations up 2.59 × 109/L). to 150 × 109/L and morphological atypia of Many studies have investigated and reported heterophils that included decreased segmenta- the leukocyte characteristics of commercial tion of the nucleus, decreased concentration poultry and these studies provide an important of granules, and atypical shape of granules, reservoir of knowledge that may be extrapo- notably rounded granules were observed. lated to non-domestic birds. It is beyond the Additionally, monocytes without indentation scope of this book to comprehensively review of the nucleus and with cytoplasmic vacuola- the reservoir of studies of domestic poultry; tion were observed. however, selected, illustrative studies are pre- Houbara bustards with chronic infl amma- sented. Acute infl ammation, experimentally tory disorders had leukocytosis, heterophilia, induced in chickens, caused a heterophilia that monocytosis and basophilia and morphologi- peaked at 12 hours (18.3 × 109/L), with a “left cally atypical heterophils characterized by shift” between 12 and 24 hours, and leukocy- cytoplasmic basophilia, degranulation and tosis that persisted for 7 days (Latimer et al. decreased nuclear lobulation (D’Aloia et al. 1988). Intravenous injection of lipopolysac- 1994). Reactive lymphocytes were also charide from Salmonella typhimurium into observed. Kori bustards infected with Pseudo- commercial broiler chickens resulted in monas aeruginosa had leukocytosis due to decreased concentrations of total leukocytes heterophilia and monocytosis (Bailey et al. (by about 66% of initial values), heterophils, 2000). Similarly, kori bustards with ventricular Physiological and pathological infl uences 123 foreign bodies exhibited a heterophilia (Bailey majority of captive gentoo penguins with et al. 2001). bumble foot (12/14) had a heterophilia (8.9 ± For Phoenicopteriformes, eight captive 3.4 × 109/L) compared to the heterophil con- greater fl amingos with a variety of disorders centrations of healthy birds (Hawkey et al. exhibited a heterophilia (Hawkey et al. 1984b). 1985) and black-footed penguins infected with The concentration of heterophils was greater eastern equine encephalitis virus exhibited a than 40 × 109/L in two birds and one of these leukocytosis due to a heterophilia (27.4 ± 10.0 had heterophils with atypical morphology × 109/L) (Tuttle et al. 2005). (round, strongly basophilic granules). One Overall, for a range of species and disor- bird with biliary carcinoma and terminal peri- ders, a hematological response to disease has tonitis had a heteropenia (0.6 × 109/L). been documented. Although these studies Hematological response to disease has been reveal a heterogeneous response, as would be documented for some species of Procellari- expected for the broad range of species and iformes. Monocytosis (maximum observed the varied disorders, some general characteris- concentration of 2.38 × 109/L) was noted in tics can be identifi ed. Commonly a hetero- some diseased manx shearwaters (Kirkwood philia is present, with variation in the et al. 1995) and 24% of stranded wedge-tailed magnitude (that is the concentration of hetero- shearwater chicks had heterophils with atypi- phils obtained) observed. A “left shift” is regu- cal morphology (Work & Rameyer 1999). larly observed with signifi cant infl ammatory The hematological response to disease has challenges, as are morphological atypia of het- been reported for a number of species of psit- erophils. Monocytosis may be evident at levels tacine birds. An infl ammatory leukogram with greater than would be observed in mammals. leukocytosis and heterophilia was encountered in 11 African grey parrots with a range of Effects of “stress” and excitement on clinical disorders with the maximum hetero- hematological characteristics phil concentration of 29.6 × 109/L recorded for a bird with dyspnea (Hawkey et al. 1982). The hematological effects of glucocorticoid Heterophilia with a left shift was recorded for mediated “stress” in birds due to increased psittacine birds, namely, yellow-collared concentrations of corticosterone have been macaw (Ara aricollis), blue and gold macaw reported both in experimental and clinical sit- (Ara ararauna) and yellow-crowned Amazon uations. Three-week-old chickens injected (Amazona ochrocephala) (Tangredi 1981). with ACTH initially showed a leukopenia, due Heterophilia (45.4 × 109/L) with a left shift to a lymphopenia, at 1 hour post-injection; (6.1 × 109/L) and morphological atypia then exhibited a leukocytosis, due to a hetero- (rounded and basophilic granules) was docu- philia, at 4 and 12 hours post-injection mented in a lesser sulphur-crested cockatoo (Davison & Flack 1981). There were no con- after surgery to debride a traumatic leg injury sistent changes in the basophils, eosinophils or (Bienzle & Smith 1999). Heterophilia monocytes. Similarly, administration of corti- (maximum concentration of 25.28 × 109/L) costerone to chickens via drinking water and monocytosis (maximum concentration of resulted in an increased heterophil concentra- 23.87 × 109/L) were observed in two species tion at 24 hours post-administration (Post et of black cockatoo with a range of disorders al. 2003). No signifi cant effects were noted for (Jaensch & Clark 2004). Morphological other leukocytes. changes were infrequently encountered (3/28 Birds placed in “stressful” situations may be cases). expected to exhibit similar hematological Hematological response to infl ammation characteristics to the effects of increased cor- has been reported for Sphenisciformes. The ticosterone concentration determined experi- 124 Atlas of Clinical Avian Hematology mentally. Transported, untrained Harris’ showed density dependent effects on the hema- hawks (Parabuteo unicinctus) had a lympho- tological characteristics (Voslarova et al. penia and eosinopenia (Parga et al. 2001). 2006). At the greatest density, total leukocyte However, similarly handled peregrine falcons and heterophil concentration was decreased did not exhibit similar effects. In trained birds, and basophil concentration increased. a monocytosis was exhibited by the Harris’ Overall, the wide variation in the hemato- hawks but not the peregrine falcons. Wernery logical characteristics exhibited in clinically et al. (2004) stated that, in response to han- “stressful” situations likely refl ects a complex dling, the leukocyte concentration of falcons interaction of the effect, magnitude and dura- could increase to 13.0 × 109/L. tion of the “stresses” on the intrinsic hemato- Humboldt’s penguins captured from the logical characteristics of the individual and the wild and maintained in captivity exhibited typical responses of its species. mild changes in absolute heterophil concentration (increased from day 3 to week 3 then Decreased leukopoiesis decreased between week 3 and 7) and lymphocyte concentration (decreased to week 7 then Leukopenia due to bone marrow hypoplasia increased to a value similar to at capture by in birds treated with either fenbendazole or week 15) (Villouta et al. 1997). Racing pigeons albendazole has been reported (Howard et al. showed an increased heterophil concentration 2002). Similarly, painted storks (Mycteria and a decreased lymphocyte concentration, 3 leucocephala) treated with fenbendazole hours after handling and transport (Scope developed severe heteropenia (Weber et al. et al. 2002). Transport of common pheasant 2002). Hemoparasites5 of birds

INTRODUCTION Trichomonads and Histomonads may develop suffi cient parasitaemia to be detected in blood (Stabler 1954, McKeon et al. 1997) but such There have been many hemoparasites described events are rare. and reported that have been discovered in the The signifi cance of infection with a particu- blood of birds, and it is well beyond the scope lar species of hemoparasite varies with the of this atlas to comprehensively consider the parasite, the magnitude of the parasitemia, the great body of knowledge that has been accu- species of host, and the physiological state of mulated. We aim to provide a “working” the host. In many instances infection appears knowledge of the morphological characteris- to have no signifi cant detrimental effect on the tics that distinguish the various “types” of health of the host. A review of 5640 articles hemoparasites that may be encountered by a published on avian hemoparasites concluded hematologist when examining avian blood “There are remarkably few reports of mortal- fi lms. Following recognition, investigation ity caused by blood parasites in wild birds” employing appropriate morphological and (Bennett et al. 1993). However, some hemo- molecular biology methods may be undertaken parasites may impact on the health or fi tness to further classify the organism. of a particular host. Consequently, it is impor- Hemoparasites that have been recognized in tant for the hematologist to recognize hemo- the blood of birds include: Atoxoplasma spp, parasites during examination of blood fi lms Babesia spp, Haemoproteus spp, Hepatozoon that are part of an investigation of avian spp, Leukocytozoon spp, Trypanosoma spp, health. Plasmodium spp, and the microfi laria(e) Some organisms are pathogenic and infec- of fi laroid nematodes. Selected examples of tion of a particular host results in clinical species reported from each of these groups of disease of that host. For example, natural hemoparasites and their avian hosts are given infection of bobwhite quail (Colinus virginia- in Table 5.1. Occasionally the motile interme- nus) with Haemoproteus lophortyx resulted in diate or free-living stages of protozoal para- a cumulative mortality of 20% of birds within sites from the alimentary tract such as the affected fl ocks (Cardona et al. 2002). 126 Atlas of Clinical Avian Hematology

Table 5.1 Selected reports of hemoparasites and their hosts.The references refer to the study in which the parasite was reported, not necessarily the original description of the organism.

Hemoparasite Host Reference

Atoxoplasma sp Greenfi nch Ball et al. 1998 Atoxoplasma sp Bullfi nch McNamee et al. 1995 Atoxoplasma sp Tanagers Adkesson et al. 2005 Babesia bennetti Yellow-legged gull Merino 1998 Babesia poelea Brown booby Work & Rameyer 1997 Babesia shortti Saker falcon Samour & Peirce 1996 Babesia kiwiensis Brown kiwi Peirce et al. 2003 Haemoproteus balearicae Sandhill crane Dusek et al. 2004 Haemoproteus danilewskyi Blue jay Garvin et al. 2003 Haemoproteus iwa Great frigatebird Work & Rameyer 1996 Haemoproteus lophortyx Bobwhite quail Cardona et al. 2002 Haemoproteus nisi Eurasian sparrow hawk Peirce et al. 1990 Haemoproteus tinnunculi American kestrel Apanius & Kirkpatrick 1988 Hepatozoon kabeenin Sedge warbler Kruszewicz & Dyrcz 2000 Hepatozoon kiwii Brown kiwi Peirce et al. 2003 Leucocytozoon atkinsoni Common jery Savage et al. 2006 Leucocytozoon ibisi White ibis Adlard et al. 2002 Leucocytozoon marchouxi Pink pigeon Peirce et al. 1997 Leucocytozoon toddi Eurasian sparrow hawk Ashford et al. 1990 Plasmodium forresteri Barred owl Telford et al. 1997 Plasmodium juxtanucleare Black-footed penguin Grim et al. 2003 Plasmodium relictum Lesser fl amingo Peirce 2005b Plasmodium rouxi Mountain thrush-babbler Valkiunas et al. 2005b Plasmodium subpraecox Eastern screech owl Tavernier et al. 2005 Trypanosoma avium Common buzzard Votypka et al. 2002 Trypanosoma bennetti American kestrel Kirkpatrick et al. 1986 Trypanosoma everetti Common redstart Rintamaki et al.1999 Microfi lariae Red-billed blue magpies Simpson et al. 1996 Microfi lariae Australian magpie Reppas et al. 1995 Microfi lariae Willow ptarmigan Holmstad et al. 2006 Microfi lariae Little owl Bedin et al. 2007

Natural infection of a saker falcon and Eur- acutely before erythrocytic stages are evident asian kestrel with Babesia shortii resulted in in peripheral blood (Peirce 2005b). Similarly, signifi cant anemia (Samour and Peirce 1996, extensive retinal and central nervous system Munoz et al. 1999). Deaths of black-footed lesions have been described associated with a penguins have been attributed to infection fatal Leucocytozoon infection in Australian with Plasmodium juxtanucleare; both in wild Falco species (Raidal & Jaensch 2000). birds admitted to a rehabilitation center (Grim The pathogenic sequelae of infection may be et al. 2003) and captive birds, which may die greater in immature birds, which may be due Hemoparasites of birds 127 to a lack of immunity because of no previous Decreased defence of the nest was exhibited exposure to the organism (Sol et al. 2003). An by Tengmalm’s owls (Aegolius funereus) infection with both Haemoproteus noctuae infected with Trypanosoma avium (Hakkara- and Leucocytozoon ziemanni was fatal in inen et al. 1998). A study of great tits showed juvenile snowy owls (Evans & Otter 1998). that urban birds infected with Haemoproteus Similarly, natural infection of pink pigeons spp had lower body weight than uninfected (Columba mayeri) with Leucocytozoon mar- birds. However, rural birds did not exhibit a chouxi had the greatest pathological effect on difference in body weight (Ots & Horak squabs and juvenile birds (Peirce et al. 1997). 1998). Female blue tits treated to reduce the However, infection of a young bird with a level of infection with Haemoproteus majoris hemoparasite does not necessarily result in and Leucocytozoon majoris had better body clinical disease; for example, a study of Leu- condition and their offspring had a greater cocytozoon toddi in Eurasian sparrowhawks fl edging success, than non-treated birds (Accipiter nisus) showed no signifi cant evi- (Merino et al. 2000). dence of initial mortality or decreased longev- ity (Ashford et al. 1991). Under some circumstances, hemoparasites DETECTION OF HEMOPARASITES that typically do not incite clinical consequences to the host may assume greater signifi cance when the host has decreased As this text is intended primarily for hematol- immunity. For example, stress and increased ogists, hemoparasites will typically be encoun- photoperiod were shown to induce recrudes- tered during examination of a blood fi lm to cence of latent, natural infections with Hae- assess the hematological characteristics of the moproteus belopolskyi and Trypanosoma spp host. Commonly, this will be a thin blood fi lm in blackcaps (Sylvia atricapilla) (Valkiunas & of adequate quality to permit reliable assess- Iezhova 2004). Similarly, the greater burden ment of the host cells and typically stained of Leucocytozoon dubreuili (compared to with a Romanowsky stain and examined by healthy birds) observed in an injured great tit light microscopy. was attributed to the debilitation of the host Initial examination of the blood fi lm at (Hautmanova et al. 2002). The increased lower power magnifi cation (×4 or ×10 objec- numbers of Leucocytozoon toddi observed in tive lens) promotes the detection of larger the blood of Eurasian sparrow hawks during organisms, such a microfi laria(e) and Try- “spring relapse”, which coincides with nesting panosoma spp, that may be present in low season, was believed to refl ect the “stress” numbers (and may not be recognized if the associated with reproduction (Ashford et al. microscopist omits this step). Distorted leuko- 1990). Similarly, increased numbers of hemo- cytes containing Leucocytozoon spp and dis- parasites were observed during the breeding torted erythrocytes containing Haemoproteus season in captive yellowhammer (Emberiza spp may also be recognized during the lower citronella) (Allander & Sundberg 1997) and power examination of the fi lm. Often, the wild common redstarts (Phoenicurus phoeni- larger organisms are found near the leading curus) (Rintamaki et al. 1999). edge of the fi lm and distributed throughout the There is an increasing body of knowledge thin (“monolayer”) region located behind the that reveals that while infection with hemo- leading edge. After the initial examination of parasites may not incite clinical disease in the the blood, closer examination using ×20, ×40 host, it may have an effect on the complex, and ×100 objectives is employed to recognize multi-factorial interactions that infl uence the smaller hemoparasites, such as Plasmodium fi tness and reproductive success of the host. spp and Babesia spp and to allow examination 128 Atlas of Clinical Avian Hematology of the fi ne detail of the structure of all organ- and advised that additional methods, such as isms. Hemoparasites are more diffi cult to rec- restriction fragment length polymorphism ognize in thick blood fi lms and poorly stained (RFLP) analysis or direct sequencing, were blood fi lms (particularly those that are insuf- necessary to identify organisms. Similarly, the fi ciently stained). The presence of large use of nested PCR for cytochrome b did not amounts of stain precipitate may complicate recognize mixed infections of haemosporidian detection of small hemoparasites, such as parasites in naturally infected birds (Valkiuˇnas Babesia spp. et al. 2006). Light microscopy, by an experienced and The relative ability of light microscopic diligent operator, provides a simple and effec- examination of blood fi lms and PCR to detect tive method to recognize hemoparasites in the the presence of hemoparasites has been com- blood of birds. The detection limit of light pared in a number of studies with varied microscopic examination, specifi cally the ability outcome. Ribeiro et al. (2005) found that for to recognize the presence of small numbers of Plasmodium spp, PCR detected organisms in organisms, has been the subject of scientifi c dis- 39.6% of samples tested whereas light micro- cussion (Sehgal et al. 2001, Riberio et al. 2005). scopic examination detected organisms in Typically, an infection of intra-erythrocytic 16.5% of samples. In contrast, in a study of organisms is considered “detectable” using Plasmodium and Haemoproteus, Beadell et al. light microscopic examination of thin blood (2004) found the same detection rate (35/40) fi lms when the parasites are present in greater for samples analyzed by both light microscopy than 0.1% of erythrocytes (Gaunt 2000). and PCR and noted that the two primer sets Muñoz et al. (1999) detected parasitemias of had failed to detect infection in 17% and 30% 0.1% for heucocytozoon spp. and Haemopro- of cases respectively. Similarly, a study of Try- teus spp. in blood from a range of hosts. panosoma spp initially detected organisms in Diagnosis of hemoparasite infections by 37/193 (19%) of samples by light microscopy light microscopy can also be complicated by and 53/193 (28%) of samples by PCR (Sehgal the ability of some organisms to incite patho- et al. 2001). Of the samples that had discor- genic consequences without appearing in the dant results between the two methods, 6/193 peripheral blood; for example, Plasmodium (3%) samples were initially negative by PCR spp in penguins (Pierce 2005b, Pierce et al. but had organisms observed by microscopy 2005). and when the PCR was repeated had a positive Increasingly, molecular biology methods, result. In the same study, 6/193 (3%) of blood most notably the polymerase chain reaction fi lms were initially classed as negative but, (PCR), have been applied to the detection of when re-examined after a positive PCR result, avian hemoparasites (Sehgal et al. 2001, were found to contain organisms. Votypka et al. 2002, Beadell et al. 2004, Hell- Concentration methods for the detection of gren et al. 2004, Ribeiro et al. 2005, Wiersch hemoparasites may be applied to aid in the et al. 2007). PCR provides an effective way detection of certain avian hemoparasites when detect hemoparasites when only small volumes suffi cient volume of blood is available. Bennett of blood are available and few hemoparasites (1962) reported that the detection of hemo- are present. Use of “multiplexed PCR” for the parasites could be increased by centrifugation concurrent detection of different hemopara- of blood in a capillary tube and subsequent sites promotes convenient and expedient anal- light microscopic examination of i) the serum ysis of samples (Hellgren et al. 2004). However, immediately above “buffy coat” layer for Try- Cosgrove et al. (2006) reported that primers panosoma spp and microfi laria(e) and ii) a designed to amplify DNA from Plasmodium stained, thin fi lm prepared from the “buffy spp and Haemoproteus spp also (unintention- coat” for hemoparasites such as Haemopro- ally) amplifi ed DNA from Leucocytozoon spp teus spp and Leucocytozoon sp. The author Hemoparasites of birds 129 noted these organisms’ shape may be distorted but it is limited in what it can tell us about by the process and consequently they may be the basic life history strategies of these diffi cult to speciate. Similarly concentration organisms.” methods, such as Knott’s test, may be employed From the hematologists’ perspective, micros- to detect microfi laria(e) that may be present in copy also provides important information on circulation in small numbers (Zajac & Conboy the parasites’ effect on the health of its host; 2006). such as, if the bird is anemic, if there is In the context of this book, the recognition increased erythropoiesis or if there is an infl am- of hemoparasites using morphological charac- matory response. These effects are not cur- teristics during examination of blood fi lms by rently able to be determined by molecular light microscopy is the most likely method methods. used by the hematologist to initially detect hemoparasites. These characteristics are described in the following section. Additional Plasmodium methods may then be employed to further classify the organism, quantify infection or See Figures 287–288. undertake hemoparasitological surveys of Many species of Plasmodium have been additional birds. reported from birds (Soulsby 1982, Laird 1988). Plasmodium infections are characterized by the concomitant presence of several stages of the life-cycle of the organism within MORPHOLOGY OF the erythrocytes of the host. Notably, gameto- HEMOPARASITES cytes and schizonts (containing merozoites) may be recognized (Soulsby 1982). The morphology of the organism varies The recognition of hemoparasites in avian between species of Plasmodium and it is blood fi lms using light microscopy provides an important method for the detection of hemoparasites. The classifi cation of hemoparasites has traditionally employed morphological characteristics (Peirce et al. 1990, 2003, Peirce 2000, Adlard et al. 2002) in conjunction with host specifi city and life cycle information. Increasingly molecular methods are being uti- lized; however, Peirce et al. (2005a) stated “The expanding use of molecular techniques in the study of avian hematozoa should even- tually clarify the status of many species which have a similar morphology but occur in different host families or subfamilies. However, molecular studies should only be considered a useful adjunct in taxonomy; morphology and life cycle will remain the core attributes in defi ning a new species”. Similarly, Valkunias Figure 287 Blood from a pied currawong (Strepera graculina) with gametocytes of Plasmodium sp. evident et al. (2005a) stated “Certainly, molecular within the cytoplasm of several mature erythrocytes genetics provides inexhaustible opportunities and a schizont within one polychromatophilic eryth- for investigations into the phylogenetic rela- rocyte (bottom left). Also present is a lymphocyte. tionships of haemosporidian parasites (1–4), (Modifi ed Wright’s stain.) 130 Atlas of Clinical Avian Hematology

or polar to the nucleus. Both mature erythrocytes and polychromatophilic erythrocytes may contain schizonts. Typically the shape of the host cell is not distorted by the presence of the schizont but the nucleus may be slightly displaced. Each schizont is composed of merozoites. The number of merozoites in a schizont varies between species of Plasmodium and within schizonts of the same species of Plas- modium. For example, Plasmodium forresteri had small schizonts with two to six merozoites (Telford et al. 1997). Merozoites have a pleomorphic appearance and may be round, ovoid, elongate in shape Figure 288 Blood from a sacred kingﬁ sher (Todirh- amphus sanctus) showing organisms consistent with with a small dark basophilic nucleus and baso- gametocytes of Plasmodium sp. within the cytoplasm philic cytoplasm. Merozoites within a schizont of erythrocytes. However, the morphological similar- commonly exhibit a “fan” formation and may ity of the large gametocytes to Haemoproteus sp also exhibit a rosette, cruciform or “bow-tie” demands that a dual infection with Plasmodium sp and formation, depending on the number present Haemoproteus sp cannot be discounted solely by in the schizont. morphological assessment. Remarkably, the bird had 93% of its erythrocytes parasitized. (Diff Quik stain.) Babesia beyond the scope of this book to detail the See Figure 289. morphological characteristics of the many The taxonomy of the organisms within the species. In general, the gametocytes of species genus Babesia, that may be found within the of Plasmodium may be irregularly round, blood of birds, has been reviewed and some elongate, “U” or “V” shape, with a round amphophilic central nucleus, and moderately basophilic cytoplasm that contains several brown–black pigment granules. Plasmodium juxtanucleare has relative small round gametocytes and P. relictum has round to irregular gametocytes which, in both species, may displace the nucleus of the host erythrocyte (Soulsby 1982). In contrast, Plasmodium forresteri had elongate, slender gametocytes that rarely distorted the shape of the host erythrocyte but regularly displaced the nucleus (Telford et al. 1997) and P. fallax has large, elongate gametocytes that resemble Haemo- proteus spp that tend to surround the nucleus of the host cell without displacing it (Soulsby, Figure 289 Blood from a brown kiwi (Apteryx aus- 1982). tralis) with a hemoparasite morphologically consistent Schizonts are round to ovoid structures with Babesia sp. (arrow) within the cytoplasm of an within the cytoplasm of erythrocytes, lateral erythrocyte. (May Grünwald and Giemsa stains.) Hemoparasites of birds 131

13 valid species have been recognized (Peirce 2000). Typically, species of Babesia are small (1–3 µm in diameter), intra-erythrocytic organisms. Classically Babesia spp have a round “ring form” appearance with a single eccentric darkly basophilic, chromatin mass, and a fi ne limiting membrane that encloses pale basophilic cytoplasm (which often contrasts with the eosinophilic cytoplasm of the host erythrocyte). Other forms encountered include ovoid, elongate and pyriform. Schizonts are typically composed of several merozoites. For example, B. shortii is commonly observed to have four merozoites in a cruciform or fan- shaped confi guration (Pierce 2000). Figure 291 Blood from a blue-winged kookaburra (Dacelo leachii) showing intra-erythrocytic Haemo- proteus sp. A basophilic macrogametocyte (within the erythrocyte adjacent to the heterophil) and a “pink” Haemoproteus microgametocyte can be discerned. (Diff Quik stain.) See Figures 290–293. Organisms classifi ed within the genus Hae- families (Bennett & Peirce 1988, 1990, Peirce moproteus have a cosmopolitan distribution et al. 1990, Peirce 2005a). Typically, gametog- with greater than 100 species identifi ed from ony of Haemoproteus spp occurs within eryth- host birds representing many of the avian rocytes, whereas schizogony occurs within endothelial cells. Consequently, only gametocytes are observed within erythrocytes (in con-

Figure 290 Blood from a grey teal (Anas gracilis) showing lower power magnifi cation of many mature and several polychromatophilic erythrocytes. Haemo- Figure 292 Blood from a southern boobook (Ninox proteus sp. organisms can be identifi ed in two of the boobook) with three gametocytes of Haemoproteus erythrocytes. Observation at lower magnifi cation sp. evident within the cytoplasm of erythrocytes. In allows detection of the organisms and an impression addition, two extracellular organisms are observed. of the proportion of erythrocytes infected to be gained, The punctate, dark pigment often present in Haemo- but does not allow assessment of the fi ne morphologi- proteus spp is clearly visible in all the organisms. (May cal detail of the organism. (Modifi ed Wright’s stain.) Grünwald and Giemsa stains.) 132 Atlas of Clinical Avian Hematology

yellow to black–brown granular pigment and punctate purple granules, and a centrally located nucleus. In some species, the morphological difference between macrogametocytes and microgametocytes may be identifi ed. Mac- rogametocytes, in comparison with microgametocytes, typically have cytoplasm that stains a darker basophilic color with greater numbers of pigment granules distributed throughout the cytoplasm and a dense nucleus that stains an amphophilic to basophilic color. In contrast, microgametocytes typically have cytoplasm that stains a pale blue to “rose” color with fewer granules, that are often polar in Figure 293 Blood from a brown falcon (Falco berig- distribution, and a nucleus that is more diffuse ora) illustrating a “circumnuclear” form of a gameto- (less dense) and stains an amphophilic to red cyte of Haemoproteus sp. within the cytoplasm of an erythrocyte. (Modifi ed Wright’s stain.) color. trast to Plasmodium spp). Multiple gametocytes Leukocytozoon within a single erythrocyte are commonly observed with some species, such as H. tin- See Figures 294–300. nunculi (Peirce et al. 1990). Occasional extra- Numerous species of Leucocytozoon have cellular organisms may be observed. been identifi ed from many families of avian Bennett & Peirce (1988) divided the macro- host (Bennett et al. 1991, Bennett & Peirce gametocytes of Haemoproteus spp into fi ve 1992, Adlard et al. 2002, Peirce et al. 2005, morphological categories according to body shape, namely: microhalteridial, halteridial, circumnuclear, rhabdosomal and discosomal. Classically, the gametocytes of Haemoproteus spp are halteridial; that is elongated and curved, often around the host erythrocyte’s nucleus. However, the predominant form of the gametocytes varies between species. For example, considering the haemoproteids of the order Falcoformidae, H. tinnuniculi is typically halteridial whereas H. janovyi is highly pleomorphic (Peirce et al. 1990). Similarly, H. sacharovi is typically a large and pleomorphic organism that may exhibit a leukocytozoid appearance (Bennett & Peirce 1990). The pro- pensity of gametocytes to displace the nucleus of and cause distension of the host erythrocyte Figure 294 Blood from an Australian magpie (Gym- norhina tibicen) with the gametocyte of a Leucocyto- varies with the species of Haemoproteus. zoon sp. evident within a mononuclear leukocyte, In general, the gametocytes of Haemopro- which has been distorted by the organism. Also present teus spp have a distinct peripheral outline, are two lymphocytes and several erythrocytes. (Modi- cytoplasm that contains variable amounts of a fi ed Wright’s stain.) Hemoparasites of birds 133

Figure 297 Blood from an Australian kestrel (Falco cenchroides) showing the gametocyte of a Leucocyto- Figure 295 Blood from a singing honeyeater zoon sp. within the cytoplasm of a mononuclear leu- (Lichenostomus virescens) showing the gametocyte of kocyte. Also present are a basophil, two lysed cells a Leucocytozoon sp. within a leukocyte. (Modiﬁ ed and mature erythrocytes. (Modiﬁ ed Wright’s stain.) Wright’s stain.)

Peirce 2005b, Savage et al. 2006). The game- others exhibit only fusiform or only round togony of species of Leucocytozoon occurs forms. For example, both fusiform and round within hematological cells, whereas the schi- forms of L. maclaeni are recognized (Bennett zogony occurs in various parenchymatous and et al. 1991). However, the leucocytozoids endothelial cells. described from Passeriformes all exhibited The gametocytes of leucocytozoids are round forms (Bennett & Peirce 1992), as did highly pleomorphic with some species exhibit- L. podargii, L. ibisi and L. otidis (Adlard ing both fusiform and round forms whereas et al. 2002).

Figure 296 Blood from a tawny frogmouth (Podargus Figure 298 Tissue section of the brain from an Aus- strigoides) showing the gametocyte of a Leucocyto- tralian kestrel showing the schizont of a Leucocyto- zoon sp. within a leukocyte. (Modiﬁ ed Wright’s zoon sp. within the wall of a vessel. (Haematoxylin stain.) and eosin stains.) 134 Atlas of Clinical Avian Hematology

fusiform forms have an ovoid to elongated shape with the host cell’s nucleus evident as a crescent along the long axis of the parasite and the host cell’s cytoplasm tapering to a point at each pole of the long axis of the cell. Morpho- logical differences between macrogametocytes and microgametocytes may be evident in some preparations for some species of Leucocyto- zoon. In general, macrogametocytes are larger and have a darker basophilic nucleus. In contrast microgametocytes are often more pleomorphic, slightly smaller (allowing more of the host cell to be visualized), and have a pale nucleus with less dense, amphophilic Figure 299 Transmission electron micrograph of a chromatin. schizont from the brain of an Australian kestrel showing individual organisms. (Lead citrate and uranyl acetate stains.) Atoxoplasma

Typically, the round forms have a ﬁ nely See Figure 301. granular amphophilic to basophilic nucleus Atoxoplasma spp (formerly Lankesterella) that may contain small vacuoles. The presence are coccidian parasites of birds. Reclassiﬁ ca- of the parasite may enlarge the host cell. The tion of these organisms as Isospora spp has nucleus of the infected host cell is typically been considered (Upton et al. 2001). Game- distorted to form a long dark homogeneous togony typically occurs within intestinal crescent along one edge of the parasite and enterocytes cells and merogony occurs within displaced to the margin of the host cell. The

Figure 300 Blood from an Australian hobby (Falco Figure 301 Blood from a Bali myna (Leucopsar roth- longipennis) portraying the gametocyte of a Leucocy- schildi) exhibiting an organism morphologically con- tozoon sp. within the cytoplasm of a mononuclear sistent with Atoxoplasma sp. within a mononuclear leukocyte. Also present is a heterophil exhibiting mor- cell. Also present are a small lymphocyte and two phological atypia due to an infl ammatory challenge. thrombocytes amidst erythrocytes. (Modifi ed Wright’s (Modifi ed Wright’s stain.) stain.) (Courtesy of Jonathon Cracknell.) Hemoparasites of birds 135 mononuclear leukocytes. Merozoites may be nucleus, in a centric or eccentric sub-terminal present at low concentration within leukocytes location, and pale basophilic cytoplasm with and detection of the organisms may be assisted occasional punctate azurophilic granules. A by the concentration of leukocytes in a buffy capsule, seen as a white halo around the para- coat preparation. Merozoites are diffi cult to site, is present in some species; for example, defi nitively identify using solely light micros- H. kiwii (Peirce et al. 2003). copy (Adkesson et al. 2005). The merozoites The organism may displace the host’s of Atoxoplasma spp are small round to ovoid nucleus which becomes compressed at the structures with agranulated pale basophilic periphery of the cell. The host cell may be cytoplasm and a central to sub-central baso- distended as the parasite matures. philic to amphophilic nucleus. Organisms are Macrogametocytes and microgametocytes located within the cytoplasm of the host cell cannot be distinguished morphologically. and may mildly displace the host cell’s nucleus. Trypanosomes

Hepatozoon See Figures 303–306. Organisms classiﬁ ed within the genus Try- See Figure 302. panosoma have life-cycle stages within the There have been some 15 species of Hepa- blood of a vertebrate host, which may be tozoon recorded from avian hosts (Bennett encountered by hematologists, as well as stages et al. 1992, Peirce et al. 2003). The gameto- within an invertebrate host. Although about cytes of Hepatozoon spp are usually encoun- 100 species of avian trypanosomes have been tered within the cytoplasm of mononuclear described, the validity of many of these species leukocytes, both monocytes and lymphocytes. has been questioned (Votypka et al. 2002). The organisms are typically ovoid or elongate Trypanosoma avium has been reported in a with rounded ends, with a round basophilic wide range of birds and a number of “species”

Figure 302 Blood from a brown kiwi (Apteryx australis) exhibiting a hemoparasite morphologically con- Figure 303 Blood from a red-collared lorikeet sistent with Hepatozoon sp. within the cytoplasm of a (Trichoglossus haematodus rubritorquis) depicting an mononuclear leukocyte. (May Grünwald and Giemsa extracellular Trypanosoma sp. amidst erythrocytes. stains.) (Diff Quik stain.) 136 Atlas of Clinical Avian Hematology

Figure 304 Blood from a whistling kite (Haliastur Figure 306 Blood from a New Holland honeyeater sphenurus) exhibiting a Trypanosoma sp. and a het- (Phylidonyris novaehollandiae) portraying a Trypano- erophil amidst erythrocytes. The morphological fea- soma sp. and a nucleus amidst erythrocytes. (Modiﬁ ed tures of the organism, namely the kinetoplast, nucleus, Wright’s stain.) undulating membrane and ﬂ agellum, can all be clearly discerned. (Diff Quik stain.)

amidst the host’s hematological cells. Detec- of avian trypanosomes are considered to be tion of organisms is further aided by centrifu- part of the “T. avium complex”. gation of a sample of blood in a capillary tube Typically, Trypanosoma spp have a low and examination of the buffy coat preparation parasitemia. Cosequently, blood smears need (Bennett 1962). to be thoroughly examined at low magniﬁ ca- The different life-cycle stages of Trypanosoma tion to facilitate the detection of parasites. spp exhibit varied morphology. The most recog- Fortunately the size and distinctive morphol- nizable stage is the trypomastigote. In general, ogy of the parasites assists their recognition trypomastigotes have a “blade” like, elongated shape that tapers to a posterior ﬂ agellum and a pointed anterior. An undulating membrane extends along much of the organism’s length, a dark, basophilic focal kinetoplast is located in the anterior of the organism, and a round, basophilic to amphophilic nucleus is located around the mid-section of the organism. The cytoplasm is typically a pale basophilic color. The morphology and the relative dimension of the described structures vary with the species and the stage of development of the organism.

Microﬁ lariae

Figure 305 Blood from a brown honeyeater (Lich- See Figures 307–311. mera indistincta) illustrating a Trypanosoma sp. organ- The microfi laria(e) of fi laroid nematodes are ism amidst erythrocytes. (Modifi ed Wright’s stain.) large (in comparison to all the host cells), Hemoparasites of birds 137

Figure 307 Blood from a brown falcon (Falco berig- Figure 309 Blood from a northern rosella (Platycer- ora) illustrating a serpentine microfi laria amidst mature cus venustus) depicting a microfi laria amidst mature erythrocytes. (Diff Quik stain.) erythrocytes. Note the difference in shape between this organism and the microfi lariae in Figures 307 and 308. (Diff Quik stain.) extracellular organisms that typically have a serpentine appearance and a basophilic color. Variation in the relative length and width Fortunately the large size and distinctive occurs between species and results in a spec- morphology of the parasites assists their rec- trum of shapes from relatively broad, short ognition amidst the host’s haematological organisms to thin, elongated organisms. cells. Detection of organisms is further aided Typically birds have a low microfi laremia by centrifugation of a sample of blood in a and only a few organisms are present in the capillary tube and examination of the buffy volume of blood used to make a blood fi lm. Cosequently, blood smears need to be thoroughly examined at low magnifi cation to facilitate the detection of microfi laria(e).

Figure 310 Blood from an Australian raven (Corvus coronoides) showing a microfi laria amidst mature erythrocytes, a single heterophil and several lysed Figure 308 Blood from a tawny frogmouth (Podargus cells. (Modifi ed Wright’s stain.) The lesser magnifi ca- strigoides) showing a microfi laria amidst mature eryth- tion needed to include the entire organism illustrates rocytes. (Diff Quik stain.) it greater size. 138 Atlas of Clinical Avian Hematology

coat preparation (Bennett 1962) or concentration methods, such as a Knott’s test (Zajac & Conboy 2006). The adults of nematodes may be found in a range of anatomical locations, including air sacs, thoracic cavity, abdominal cavity and subcutaneous tissues (Mackerras 1962, Reppas et al. 1995).

Figure 311 Histological section of subcutaneous tissue from an Australian magpie (Gymnorhina tibicen) showing many microﬁ lariae within a subcutaneous vessel. (Hematoxylin and eosin stain.) References

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Species list: alphabetically by common name

A Bali myna (Leucopsar rothschildi) Bar-headed goose (Anser indicus) African grey parrot (Psittacus erithacus) Barn owl (Tyto alba) Aleutian goose (Branta canadensis Barrow’s golden eye (Bucephala islandica) leucopareia) Bar-tailed godwit (Limosa lapponica) American coot (Fulica americana) Black kite (Milvus migrans) American kestrel (Falco sparverius) Black stork (Ciconia nigra) American wood stork (Mycteria americana) Black swan (Cygnus atratus) Atlantic pufﬁ n (Fratercula arctica) Black vulture (Coragyps atratus) Australasian grebe (Tachybaptus Black-bellied whistling duck (Dendrocygna novaehollandiae) autumnalis) Australian black-shouldered kite (Elanus Blackcap (Sylvia atricapilla) axillaris) Black-crowned crane (Balearica pavonina) Australian gannet (Morus serrator) Black-faced cormorant (Phalacrocorax Australian hobby (Falco longipennis) fuscescens) Australian king parrot (Alisterus scapularis) Black-faced ibis (Theristicus melanopis) Australian magpie (Gymnorhina tibicen) Black-footed penguin (Spheniscus demersus) Australian raven (Corvus coronoides) Black-headed gull (Larus ridibundus) Australian shelduck (Tadorna tadornoides) Black-necked aracari (Pteroglossus aracari) Australian wood duck (Chenonetta jubata) Blue and gold macaw (Ara ararauna) Azure kingﬁ sher (Alcedo azurea) Blue bonnet (Northiella haematogaster) Blue-breasted quail (Coturnix chinensis) Blue-crowned conure (Aratinga acuticaudata) B Blue-fronted Amazon parrot (Amazona aestiva) Baer’s pochard (Aythya baeri) Blue-throated conure (Pyrrhura cruentata) Bald eagle (Haliaeetus leucocephalus) Blue-winged kookaburra (Dacelo leachii) Bald ibis (Geronticus calvus) Bobwhite quail (Colinus virginianus) 164 Species list: alphabetically by common name

Brolga (Grus rubicunda) Eurasian kestrel (Falco tinnunculus) Brown booby (Sula leucogaster) Eurasian sparrowhawk (Accipiter nisus) Brown falcon (Falco berigora) Brown goshawk (Accipiter fasciatus) Brown honey-eater (Lichmera indistincta) F Brown kiwi (Apteryx australis) Brown pelican (Pelecanus occidentalis) Fairy martin (Hirundo ariel) Brown quail (Coturnix ypsilophora) Fairy tern (Sterna nereis) Budgerigar (Melopsittacus undulatus) Ferruginous hawk (Buteo regalis) Burrowing owl (Speotyto cunicularia) Flightless cormorant (Phalacrocorax harrisi) Fluttering shearwater (Pufﬁ nus gavia) C

Canada geese (Branta canadensis) G Canvasback (Aythya valisineria) Cattle egret (Bubulcus ibis) Galah (Eolophus roseicapillus) Chestnut-naped imperial pigeon (Ducula Galapagos penguin (Spheniscus aenea paulina) mendiculus) Chilean fl amingo (Phoenicopterus chilensis) Gang-gang cockatoo (Callocephalon Cockatiel (Nymphicus hollandicus) fi mbriatum) Common crane (Grus grus) Gentoo penguin (Pygoscelis papua) Common eider (Somateria mollissima) Goldcrest (Regulus regulus) Common loon (Gavia immer) Goosander (Mergus merganser) Common pheasant (Phasianus colchicus) Gray-headed chachalaca (Ortalis Common redstart (Phoenicurus phoenicurus) cinereiceps) Congo peafowl (Afropavo congensis) Great black-backed gull (Larus marinus) Cooper’s hawk (Accipiter cooperii) Great frigate bird (Fregata minor) Crested wood partridge (Rollulus roulroul) Great knot (Calidris tenuirostris) Cuban Amazon parrot (Amazona Great skua (Catharacta skua) leucocephala) Great tit (Parus major) Greater fl amingo (Phoenicopterus ruber) Greater prairie-chicken (Tympanuchus D cupido) Greater rhea (Rhea americana) Demoiselle crane (Grus virgo) Greater roadrunner (Geococcyx Double-crested cormorant (Phalacrocorax californianus) auritus) Green peafowl (Pavo muticus) Green-winged macaw (Ara chloropterus) Grey teal (Anas gracilis) E Grey-faced petrel (Pterodroma macroptera gouldi) Eclectus parrot (Eclectus roratus) Guanay cormorant (Phalacrocorax Edwards’ pheasant (Lophura edwardsi) bougainvillii) Emu (Dromaius novaehollandiae) Guira cuckoo (Guira guira) Eurasian coot (Fulica atra) Gyr falcon (Falco rusticolus) Species list: alphabetically by common name 165

H Little corella (Cacatua sanguinea) Little egret (Egretta garzetta) Harris’ hawk (Parabuteo unicinctus) Little penguin (Eudyptula minor) Hawaiian petrel (Pterodroma phaeopygia) Lovebird (Agapornis rosiecollis) Helmeted honeyeater (Lichenostomus melanops) Herring gull (Larus argentatus) M Hill mynah (Gracula religiosa) Hoatzin (Opisthocomus hoazin) Magellanic penguin (Spheniscus Hooded crow (Corvus corone) magellanicus) Hooded pitta (Pitta sordida) Major Mitchell’s cockatoo (Cacatua Houbara bustard (Chlamydotis undulata) leadbeateri) House fi nch (Carpodacus mexicanus) Mallard (Anas platyrhynchos) House martin (Delichon urbica) Mallee fowl (Leipoa ocellata) Humboldt penguin (Spheniscus humboldti) Manx shearwater (Puffi nus puffi nus) Hyacinth macaw (Anodorhynchus Marabou stork (Leptoptilos hyacinthinus) crumeniferus) Marsh harrier (Circus aeruginosus) Masked lapwing (Vanellus miles) I Masked owl (Tyto novaehollandiae) Mauritius kestel (Falco punctatus) Imperial eagle (Aquila adalberti) Military macaw (Ara militaris) Mindanao bleeding heart dove (Gallicolumba criniger) J Muscovy duck (Cairina moschata)

Japanese quail (Coturnix japonica) N K New Zealand blue duck (Hymenolaimus malacorhynchus) King penguin (Aptenodytes patagonicus) New-Holland honeyeater (Phylidonyris King shag (Phalacocorax albivenier) novaehollandiae) King vulture (Sarcoramphus papa) Night heron (Nycticorax nycticorax) Kori bustard (Ardeotis kori) Northern fulmar (Fulmarus glacialis) Northern harrier (Circus cyaneus) Northern rosella (Platycercus venustus) L

Lanner falcon (Falco biarmicus) O Laughing kookaburra (Dacelo novaeguineae) Layasan albatross (Diomedea immutabilis) Orange-bellied parrot (Neophema Lesser ﬂ amingo (Phoenicopterus minor) chrysogaster) Lesser rhea (Rhea pennata) Orange-footed scrub fowl (Megapodius Lesser suphur-crested cockatoo (Cacatua reinwardt) sulphurea) Ostrich (Struthio camelus) 166 Species list: alphabetically by common name

P Rough-legged hawk (Buteo lagopus) Royal spoonbill (Platalea regia) Pacifi c black duck (Anas superciliosa) Rueppell’s griffon (Gyps rueppellii) Painted stork (Mycteria leucocephala) Ruff (Philomachus pugnax) Pekin duck (Anas platyrhynchos forma Rufous-collared sparrow (Zonotrichia domestica) capensis) Peregrine falcon (Falco peregrinus) Rufous-crested bustard (Eupodotis rufi crista) Pied butcher bird (Cracticus nigrogularis) Rusty-barred owl (Strix hylophila) Pied currawong (Strepera graculina) Pigeon guillemot (Cepphus columba) Pink pigeon (Columba mayeri) S Pink-backed pelican (Pelecanus rufescens) Port Lincoln parrot (Barnardius zonarius) Sacred kingfi sher (Todirhamphus sanctus) Princess parrot (Polytelis alexandrae) Sage grouse (Centrocercus urophasianus) Providence petrel (Pterodroma solandri) Saker falcon (Falco cherrug) Puna ibis (Plegadis ridgwayi) Sandhill crane (Grus canadensis) Pygmy-goose (Nettapus pulchellus) Scarlet ibis (Eudocimus ruber) Sharp-shinned hawk (Accipiter striatus) Short-tailed shearwater (Puffi nus tenuirostris) R Silver gull (Larus novaehollandiae) Singing honeyeater (Lichenostomus Rainbow lorikeet (Trichoglossus virescens) haematodus) Snow geese (Anser caerulescens) Red grouse (Lagopus lagopus scoticus) Snowy owl (Nyctea scandiaca) Red kite (Milvus milvus) Southern boobook (Ninnox boobook) Red knot (Calidris canutus) Southern cassowary (Casuarius casuarius) Red lory (Eos bornea) Southern giant petrel (Macronectes Red wattlebird (Anthochaera carunculata) giganteus) Red-billed toucan (Ramphastos tucanus) Spectacled owl (Pulsatrix perspicillata) Red-collared lorikeet (Trichoglossus haema- Spotted dove (Streptopelia chinensis) todus rubritorquis) Stanley crane (Grus paradisea) Red-footed booby (Sula sula) Stone curlew (Burhinus oedicnemus) Red-fronted macaw (Ara rubrogenys) Stubble quail (Coturnix pectoralis) Redhead (Aythya americana) Sulphur-crested cockatoo (Cacatua galerita) Redhead bunting (Emberiza bruniceps) Sun conure (Aratinga solstitialis) Red-legged partridge (Alectus rufa) Superb blue wren (Malurus cyaneus) Red-tailed black cockatoo (Calyptorhynchus Superb lyrebird (Menura novaehollandiae) banksii) Superb starling (Spreo superbus) Red-tailed hawk (Buteo jamaicensis) Swan geese (Anser cygnoides) Red-tailed tropic bird (Phaethon rubricauda) Red-winged tinamou (Rhynchotus rufescens) Rhinoceros auklet (Cerorhinca monocerata) T Roadside hawk (Buteo magnirostris) Rock dove (Columba livia) Takahe (Porphyrio mantelli) Rock-hopper penguin (Eudyptes Tawny eagle (Aquila rapax) chrysocomes) Tawny frogmouth (Podargus strigoides) Rose-ringed parakeet (Psittacula krameri) Tengmalm’s owl (Aegolius funereus) Species list: alphabetically by common name 167

Toco toucan (Ramphasto toco) White pelican (Pelecanus onocrotalus) Trumpeter swan (Cygnus buccinator) White stork (Ciconia ciconia) Turkey (Meleagris gallopavo) White-backed vulture (Gyps africanus) White-faced scops owl (Otus leucotis) White-headed vulture (Trigonoceps occipitalis) V White-tailed black cockatoo (Calyptorhyn- chus baudinii) Varied lorikeet (Psitteuteles versicolor) White-winged scoter (Melanitta fusca) Victoria crowned pigeon (Goura victoria) White-winged wood duck (Cairina scutulata) Violet-backed starling (Cinnyricinclus Whooping crane (Grus americana) leucogaster) Woolly-necked stork (Ciconia episcopus) Von der Decken’s Hornbill (Tockus deckeni)

Y W Yellow collared macaw (Ara aricollis) Wattled crane (Grus carunculatus) Yellow crowned amazon (Amazona Waved albatross (Diomedea irrorata) ochrocephala) Wedge-tailed eagle (Aquila audax) Yellow-eyed penguin (Megadyptes antipodes) Wedge-tailed shearwater (Pufﬁ nus paciﬁ cus) Yellowhammer (Emberiza citronella) Welcome swallow (Hirundo neoxena) Yellow-throated laughing thrush (Garrulax Whistling kite (Haliastur sphenurus) galbanus) Species list: by order

Order: Anseriformes Swan geese (Anser cygnoides) Trumpeter swan (Cygnus buccinator) Aleutian goose (Branta canadensis White-winged scoter (Melanitta fusca) leucopareia) White-winged wood duck (Cairina scutulata) Australian shelduck (Tadorna tadornoides) Australian wood duck (Chenonetta jubata) Order: Caprimulgiformes Baer’s pochard (Aythya baeri) Bar-headed goose (Anser indicus) Tawny frogmouth (Podargus strigoides) Barrow’s golden eye (Bucephala islandica) Black swan (Cygnus atratus) Black-bellied whistling duck (Dendrocygna Order: Charadriiformes autumnalis) Canada geese (Branta canadensis) Atlantic pufﬁ n (Fratercula arctica) Canvasback (Aythya valisineria) Black-headed gull (Larus ridibundus) Common eider (Somateria mollissima) Fairy tern (Sterna nereis) Goosander (Mergus merganser) Great black-backed gull (Larus marinus) Grey teal (Anas gracilis) Great knot (Calidris tenuirostris) Mallard (Anas platyrhynchos) Great skua (Catharacta skua) Muscovy duck (Cairina moschata) Herring gull (Larus argentatus) New Zealand blue duck (Hymenolaimus Masked lapwing (Vanellus miles) malacorhynchus) Pigeon guillemot (Cepphus columba) Paciﬁ c black duck (Anas superciliosa) Red knot (Calidris canutus) Pekin duck (Anas platyrhynchos forma Rhinoceros auklet (Cerorhinca domestica) monocerata) Pygmy-goose (Nettapus pulchellus) Ruff (Philomachus pugnax) Redhead (Aythya americana) Silver gull (Larus novaehollandiae) Snow geese (Anser caerulescens) Stone curlew (Burhinus oedicnemus) Species list: by order 169

Order: Ciconiiformes Bald eagle (Haliaeetus leucocephalus) Black kite (Milvus migrans) American wood stork (Mycteria americana) Black vulture (Coragyps atratus) Bald ibis (Geronticus calvus) Brown falcon (Falco berigora) Black stork (Ciconia nigra) Brown goshawk (Accipiter fasciatus) Black-faced ibis (Theristicus melanopis) Common kestrel (Falco tinnunculus) Cattle egret (Bubulcus ibis) Cooper’s hawk (Accipiter cooperii) Little egret (Egretta garzetta) Eurasian kestrel (Falco tinnunculus) Marabou stork (Leptoptilos crumeniferus) Eurasian sparrowhawk (Accipiter nisus) Night heron (Nycticorax nycticorax) Ferruginous hawk (Buteo regalis) Painted stork (Mycteria leucocephala) Gray-headed chachalaca (Ortalis Puna ibis (Plegadis ridgwayi) cinereiceps) Royal spoonbill (Platalea regia) Gyr falcon (Falco rusticolus) Scarlet ibis (Eudocimus ruber) Harris’ hawk (Parabuteo unicinctus) White stork (Ciconia ciconia) Imperial eagle (Aquila adalberti) Woolly-necked stork (Ciconia episcopus) King vulture (Sarcoramphus papa) Lanner falcon (Falco biarmicus) Order: Columbiformes Marsh harrier (Circus aeruginosus) Mauritius kestel (Falco punctatus) Chestnut-naped imperial pigeon (Ducula Northern harrier (Circus cyaneus) aenea paulina) Peregrine falcon (Falco peregrinus) Mindanao bleeding heart dove (Gallicolumba Red kite (Milvus milvus) criniger) Roadside hawk (Buteo magnirostris) Pink pigeon (Columba mayeri) Rough-legged hawk (Buteo lagopus) Rock dove (Columba livia) Rueppell’s griffon (Gyps rueppellii) Spotted dove (Streptopelia chinensis) Saker falcon (Falco cherrug) Victoria crowned pigeon (Goura victoria) Sharp-shinned hawk (Accipiter striatus) Tawny eagle (Aquila rapax) Order: Coraciiformes Wedge-tailed eagle (Aquila audax) Whistling kite (Haliastur sphenurus) Azure kingﬁ sher (Alcedo azurea) White-backed vulture (Gyps africanus) Blue-winged kookaburra (Dacelo leachii) White-headed vulture (Trigonoceps occipitalis) Laughing kookaburra (Dacelo novaeguineae) Sacred kingﬁ sher (Todirhamphus sanctus) Order: Galliformes Von der Decken’s hornbill (Tockus deckeni) Blue-breasted quail (Coturnix chinensis) Order: Cuculiformes Bobwhite quail (Colinus virginianus) Brown quail (Coturnix ypsilophora) Greater roadrunner (Geococcyx californianus) Common pheasant (Phasianus colchicus) Guira cuckoo (Guira guira) Congo peafowl (Afropavo congensis) Crested wood partridge (Rollulus roulroul) Order: Falconiformes Edwards’ pheasant (Lophura edwardsi) Greater prairie-chicken (Tympanuchus American kestrel (Falco sparverius) cupido) Australian black-shouldered kite (Elanus Green peafowl (Pavo muticus) axillaris) Japanese quail (Coturnix japonica) Australian hobby (Falco longipennis) Mallee fowl (Leipoa ocellata) 170 Species list: by order

Orange-footed scrub fowl (Megapodius Hooded pitta (Pitta sordida) reinwardt) House fi nch (Carpodacus mexicanus) Red grouse (Lagopus lagopus scoticus) House martin (Delichon urbica) Red-legged partridge (Alectus rufa) New-Holland honeyeater (Phylidonyris Sage grouse (Centrocercus urophasianus) novaehollandiae) Stubble quail (Coturnix pectoralis) Pied butcher bird (Cracticus nigrogularis) Turkey (Meleagris gallopavo) Pied currawong (Strepera graculina) Red wattlebird (Anthochaera carunculata) Order: Gaviiformes Redhead bunting (Emberiza bruniceps) Rufous-collared sparrow (Zonotrichia Common loon (Gavia immer) capensis) Hoatzin (Opisthocomus hoazin) Singing honeyeater (Lichenostomus virescens) Order: Gruiformes Superb blue wren (Malurus cyaneus) Superb lyrebird (Menura novaehollandiae) American coot (Fulica americana) Superb starling (Spreo superbus) Black-crowned crane (Balearica pavonina) Violet-backed starling (Cinnyricinclus Brolga (Grus rubicunda) leucogaster) Common crane (Grus grus) Welcome swallow (Hirundo neoxena) Demoiselle crane (Grus virgo) Yellowhammer (Emberiza citronella) Eurasian coot (Fulica atra) Yellow-throated laughing thrush (Garrulax Houbara bustard (Chlamydotis undulata) galbanus) Kori bustard (Ardeotis kori) Rufous-crested bustard (Eupodotis rufi crista) Order: Pelecaniformes Sandhill crane (Grus canadensis) Stanley crane (Grus paradisea) Australasian gannet (Morus serrator) Takahe (Porphyrio mantelli) Black-faced cormorant (Phalacrocorax Wattled crane (Grus carunculatus) fuscescens) Whooping crane (Grus americana) Brown booby (Sula leucogaster) Brown pelican (Pelecanus occidentalis) Order: Passeriformes Double-crested cormorant (Phalacrocorax auritus) Australian magpie (Gymnorhina tibicen) Flightless cormorant (Phalacrocorax harrisi) Australian raven (Corvus coronoides) Great frigate bird (Fregata minor) Bali myna (Leucopsar rothschildi) Guanay cormorant (Phalacrocorax Bar-tailed godwit (Limosa lapponica) bougainvillii) Blackcap (Sylvia atricapilla) King shag (Phalacrocorax albivenier) Brown honeyeater (Lichmera indistincta) Pink-backed pelican (Pelecanus rufescens) Common redstart (Phoenicurus Red-footed booby (Sula sula) phoenicurus) Red-tailed tropic bird (Phaethon rubricauda) Fairy martin (Hirundo ariel) White pelican (Pelecanus onocrotalus) Goldcrest (Regulus regulus) Great tit (Parus major) Order: Phoenicopteriformes Helmeted honeyeater (Lichenostomus melanops) Chilean fl amingo (Phoenicopterus chilensis) Hill mynah (Gracula religiosa) Greater fl amingo (Phoenicopterus ruber) Hooded crow (Corvus corone) Lesser fl amingo (Phoenicopterus minor) Species list: by order 171

Order: Piciformes Hyacinth macaw (Anodorhynchus hyacinthinus) Black-necked aracari (Pteroglossus aracari) Lesser suphur-crested cockatoo (Cacatua Red-billed toucan (Ramphastos tucanus) sulphurea) Toco toucan (Ramphasto toco) Little corella (Cacatua sanguinea) Lovebird (Agapornis sp) Order: Podicipediformes Major Mitchell’s cockatoo (Cacatua leadbeateri) Australasian grebe (Tachybaptus Military macaw (Ara militaris) novaehollandiae) Northern rosella (Platycercus venustus) Orange-bellied parrot (Neophema Order: Procellariiformes chrysogaster) Port Lincoln parrot (Barnardius zonarius) Fluttering shearwater (Puffi nus gavia) Princess parrot (Polytelis alexandrae) Grey-faced petrel (Pterodroma macroptera Rainbow lorikeet (Trichoglossus gouldi) haematodus) Hawaiian petrel (Pterodroma phaeopygia) Red lory (Eos bornea) Layasan albatross (Diomedea immutabilis) Red-collared lorikeet (Trichoglossus Manx shearwater (Puffi nus puffi nus) haematodus rubritorquis) Northern fulmar (Fulmarus glacialis) Red-fronted macaw (Ara rubrogenys) Providence petrel (Pterodroma solandri) Red-tailed black cockatoo (Calyptorhynchus Short-tailed shearwater (Puffi nus banksii) tenuirostris) Rose-ringed parakeet (Psittacula krameri) Southern giant petrel (Macronectes Sulphur-crested cockatoo (Cacatua galerita) giganteus) Sun conure (Aratinga solstitialis) Waved albatross (Diomedea irrorata) Varied lorikeet (Psitteuteles versicolor) Wedge-tailed shearwater (Puffi nus pacifi cus) White-tailed black cockatoo (Calyptorhynchus baudinii) Order: Psittaciformes Yellow collared macaw (Ara aricollis) Yellow crowned amazon (Amazona African grey parrot (Psittacus erithacus) ochrocephala) Australian king parrot (Alisterus scapularis) Blue and gold macaw (Ara ararauna) Blue bonnet (Northiella haematogaster) Order: Sphenisciformes Blue-crowned conure (Aratinga acuticaudata) Blue-fronted Amazon parrot (Amazona Black-footed penguin (Spheniscus demersus) aestiva) Galapagos penguin (Spheniscus mendiculus) Blue-throated conure (Pyrrhura cruentata) Gentoo penguin (Pygoscelis papua) Budgerigar (Melopsittacus undulatus) Humboldt penguin (Spheniscus humboldti) Cockatiel (Nymphicus hollandicus) King penguin (Aptenodytes patagonicus) Cuban Amazon parrot (Amazona Little penguin (Eudyptula minor) leucocephala) Magellanic penguin (Spheniscus Eclectus parrot (Eclectus roratus) magellanicus) Galah (Eolophus roseicapillus) Rock-hopper penguin (Eudyptes Gang-gang cockatoo (Callocephalon chrysocomes) fi mbriatum) Yellow-eyed penguin (Megadyptes Green-winged macaw (Ara chloropterus) antipodes) 172 Species list: by order

Order: Strigiformes Order: Struthioniformes

Barn owl (Tyto alba) Brown kiwi (Apteryx australis) Burrowing owl (Speotyto cunicularia) Emu (Dromaius novaehollandiae) Masked owl (Tyto novaehollandiae) Greater rhea (Rhea americana) Rusty-barred owl (Strix hylophila) Lesser rhea (Rhea pennata) Snowy owl (Nyctea scandiaca) Ostrich (Struthio camelus) Southern boobook (Ninnox boobook) Southern cassowary (Casuarius casuarius) Spectacled owl (Pulsatrix perspicillata) Order: Tinamiformes Tengmalm’s owl (Aegolius funereus) White-faced scops owl (Otus leucotis) Red-winged tinamou (Rhynchotus rufescens) Glossary

Acidophil: a classiﬁ cation of granulocytes, that has no constriction greater than half that includes heterophils and eosinophils, based the width of the nucleus and less coarsely on the characteristic of cytoplasmic granules to clumped chromatin and a smother nuclear imbibe acidophilic stains (such as eosin) membrane than in mature (segmented) granulocytes Adipocyte: a cell that has a specialized role to store fat Basophil: a leukocyte (granulocyte) that contains basophilic cytoplasmic granules Agglutination: clumping of erythrocytes mediated by antibodies “bridging” between cells Basophilia: 1. increased bluish color of the cytoplasm when stained with a Romanowsky Amphophilic: simultaneous staining with stain. 2. a concentration of basophils that is both basophilic and eosinophilic dyes greater than the upper limit of an appropriate reference range (basophilic leukocytosis) Anemia: a decreased mass of erythrocytes characterized by decreased hematocrit (packed Basophilic: staining readily with “basic” dyes cell volume), decreased erythrocyte concentra- that impart a blue–purple color tion or decreased hemoglobin concentration Basophilic stippling: the presence of small, Anisocytosis: variation in the size of cells of dark blue staining foci within erythrocytes, the same type, commonly used to describe usually due to the presence of residual aggre- erythrocytes gations of RNA but also occasionally may be due to the presence of aggregations of iron- Azurophilic granules: the granules that have containing substances an azure to basophilic color Bone marrow: the tissue typically found in Band: the penultimate stage of granulocyte the medullary cavity of bones that is the prin- development that is characterized by a nucleus ciple site of hematopoiesis in adult birds 174 Glossary

Buffy coat: the layer of leukocytes and Ghost cell: erythrocyte that has lost hemoglo- thrombocytes that collects immediately above bin content through a deﬁ cit in the cell the erythrocytes when whole blood is membrane centrifuged Golgi apparatus (complex): an organelle Chromatin: a complex of DNA and nuclear composed of cisternae that are involved in the proteins production of proteins that may be seen as a pale perinuclear region by light microscopy Cytoplasm: the portion of a cell that excludes the nucleus Granulocyte: a leukocyte that contains prominent granules in the cytoplasm of the cell, e. Dacrocyte: a “drop-shaped” erythrocyte g. heterophils, eosinophils and basophils

Diff Quik: a commercially available rapid Granulopoiesis: the production of stain that is commonly used to stain blood granulocytes ﬁ lms Hematocrit: the volume of erythrocytes rela- EDTA: ethylene-diamine-tetra-acetic acid, a tive to whole blood; typically calculated from compound commonly used as an the number and size of the erythrocytes mea- anticoagulant sured by a hematology analyser but has also been determined by sedimentation of cells (see Endothelial cell: the cell type that composes also packed cell volume) the inner lining of blood vessels Hematology: the study of blood Eosinophil: a leukocyte (granulocyte) that typically contains eosinophilic cytoplasmic Hematopoiesis (hemopoiesis): the produc- granules tion of the cells present in the blood

Eosinophilia: a concentration of eosinophils Hemoglobin: the oxygen-carrying protein that is greater than the upper limit of an contained within erythrocytes appropriate reference range Hemolysis: lysis of erythrocytes Eosinophilic: characteristic of exhibiting an afﬁ nity for eosin that consequently imparts an Hemolysate: the product of hemolysis orange to red color Hemosiderin: an insoluble form of iron that Eosinopenia: a concentration of eosinophils appears as a golden brown to black colored, that is less than the lower limit of an appropri- granular cytoplasmic pigment with ate reference range Romanowsky stain and has a blue color with Perl’s Prussian blue stain Erythrocyte: a red blood cell Heinz bodies: rounded projections from the Erythroplastid: an anucleated erythrocyte in surface of erythrocytes that represent dena- the peripheral blood of a bird tured hemoglobin following oxidative damage; they typically appear eosinophilic with Erythropoiesis: the production of Romanowsky stains and blue when stained erythrocytes with new methylene blue stain Glossary 175

Heterophil: a leukocyte with a segmented Lymphopenia: (lymphocytopenia) a concen- nucleus and cytoplasm that contains typically tration lymphocytes that is less than the lower many prominent, brick-red to eosinophilic, limit of an appropriate reference range fusiform cytoplasmic granules (which often partially obscure the nucleus) Lymphopoiesis: the production of lymphocytes Heterophilia: a concentration of heterophils that is greater than the upper limit of an Macrocyte: a large erythrocyte (increased appropriate reference range volume)

Heteropenia: a concentration of heterophils Macrophage: a differentiated monocytic cell, that is less than the lower limit of an appropri- present in tissues, that has roles in phagocyto- ate reference range sis, antigen presentation and cytokine production Hypochromasia: erythrocytes that lack hemoglobin and consequently have a pale color Microcyte: a small erythrocyte (decreased volume) Karyolysis: the lysis of a cell’s nucleus Monocyte: a large, pleomorphic, agranulated Left shift: an increased concentration of leukocyte characterized (using Romanowsky immature heterophils, commonly band form stain) by an irregularly shaped nucleus com- cells, in the peripheral blood posed of reticular chromatin, and a blue to grey cytoplasm that frequently exhibits one to Leukemia: a neoplastic proliferation of hema- several small vacuoles topoietic cells Monocytosis: a concentration of monocytes Leukocyte: a white blood cell that is greater than the upper limit of an appropriate reference range Leukocytosis: a concentration of total leukocytes that is greater than the upper limit of an Monocytopenia: a concentration of mono- appropriate reference range cytes that is less than the lower limit of an appropriate reference range Leukopenia: a concentration of total leukocytes that is less than the lower limit of an Monocytopoiesis: the production of appropriate reference range monocytes

Leukopoiesis: the production of leukocytes Myeloid: 1. pertaining to the bone marrow 2. pertaining to leukocyte precursor cells (e.g. Lymphocyte: a non-granulocytic white blood myeloid:erythroid ratio) cell characterized (using Romanowsky stain) by a round nucleus composed of dense chro- New methylene blue stain: an aqueous stain matin and a thin peripheral rim of basophilic used to identify reticulocytes and Heinz cytoplasm bodies

Lymphocytosis: a concentration of lympho- Non-regenerative anemia: an anemia that cytes that is greater than the upper limit of an does not exhibit morphological features of appropriate reference range increased erythropoiesis 176 Glossary

Nuclei: more than one nucleus releasing increased numbers of immature cells following increased erythropoiesis Nucleus: the part of the cell that contains the nuclear material and typically stains basophilic Reticulocyte: the penultimate stage of eryth- with Romanowsky stains rocyte development, that when stained with new methylene blue, shows aggregations of Nucleolus: a typically round structure in the RNA; corresponds to the presence of poly- nucleus that is the site of RNA synthesis chromatophilic erythrocytes in Romanowsky stained blood ﬁ lms Osteoblast: a cell that produces osteoid in bone Romanowsky stain: a class of stains that contain a combination of methylene blue and Osteoclast: a large multinucleated cell eosin, including: Wright’s stain, Leishman’s involved in the remodelling of bone stain and Giemsa stain

Packed cell volume (PCV): the proportion of Rouleau (pl. rouleaux): the viscosity medi- erythrocytes relative to centrifuged whole ated property of erythrocytes that results in blood (see also hematocrit) overlapping linear or branching arrays of erythrocytes Plasma: the non-cellular component of whole blood (in which coagulation has been Rubricyte: a stage of erythroid develop- prevented) ment immediately preceding polychromatophilic erythrocytes that can be identifi ed Plasma cell: a differentiated B-lymphocyte in Romanowsky stained blood fi lms as that has an eccentric nucleus with coarse chro- smaller cells with dense chromatin and matin and basophilic cytoplasm that may more basophilic cytoplasm than mature contain a pale, perinuclear Golgi zone or erythrocytes eosinophilic granules (Mott cell) or diffuse cytoplasmic eosinophilia (fl ame cell) Serum: the fl uid component of blood after clotting has occurred Poikilocyte: an abnormally shaped erythrocyte Smudge cell: a cell that has been lysed and Polychromasia: presence of increased numbers cannot reliably be identifi ed of polychromatophilic erythrocytes in a blood fi lm Thrombopoiesis: the production of thrombocytes Polychromatophilic erythrocyte: the penultimate stage of erythrocyte production that has Toxic change: a number of morphological a bluish color with Romanowsky stain due to characteristics (such as: cytoplasmic baso- the presence of residual, diffuse RNA philia, foamy/vacuolated cytoplasm, decreased density of granules, rounded granules, baso- Pyknotic cell: a cell with a shrunken nucleus philic granules or karyolysis) that represents comprised of condensed chromatin abnormal maturation of the heterophil in response to infl ammatory demand or direct Regenerative anemia: an anemia in which action of toxic substances upon the heterophil; the bone marrow is attempting to correct by also referred to as “morphological atypia” Index

Note: page numbers in italics refer to fi gures, those in bold refer to tables acidophils 171 reticulocytes 102 concentration measurement 31 rubricytes 103 adipocytes 24, 27, 171 zinc toxicosis 107–8 age effects on hematological characteristics anisocytosis 36, 171 97–8 disease process 105 agglutination 171 Falconiformes 69 erythrocytes 105 Anseriformes 2, 167 air sacculitis 116 hematological characteristics 54, 54 –7, 57 albendazole 124 vascular access sites 14 altitude, high 106 anticoagulants 15–16, 22, 23 anesthesia 5 artifacts 27, 118 anemia 171 erythrocyte lysis 115 erythrocytes 38, 101, 102, 103, 107–8 Gruiformes 76 erythropoiesis 101 Passeriformes 77, 78 Heinz bodies 107, 108 Struthioniformes 96 hemolytic 107 see also ethylenediaminetetraacetic acid erythrocytes 100 (EDTA); heparin; lithium heparin extravascular 107, 108 Apodiformes 2, 57 hemoparasites 107 vascular access sites 11, 14 PCV effects 100 aspergillosis hemorrhagic 107 heterophil changes 49, 116, 117, 120–2 heterophils 103 monocytosis 118 hypoproliferative 107, 108 Atoxoplasma 125, 126, 134–5 infl ammatory disease 108 Aves 1, 2 mycobacteriosis 108 axillary vein 8, 10 PCV 101, 102 regenerative 174 Babesia 125, 126, 130–1 regenerative response 103 Babesia shortii 107, 126 178 Index bacteria brachial vein 8, 10, 15 blood fi lm contamination 25, 27 deep 8, 10 pododermatitis 117 brodifacoum toxicity 107 severe infl ammation 115 buffy coat 172 basilic vein 8, 10, 11, 12, 14 methods 128 basophils 19, 21, 24, 40, 42, 46, 171 anesthesia effects 5 Caprimulgiformes 2, 167 Anseriformes 54, 55–6, 57 hematological characteristics 57–8 artifacts 26 carotid artery 11 Caprimulgiformes 57, 58 cell morphology in disease 105 Ciconiiformes 61 cephalic vein, caudal 10, 11 Columbiformes 62, 63 Charadriiformes 3, 167 Coraciiformes 64, 66 hematological characteristics 58, 59, 60 cytoplasmic granules 46 chromatin 48, 172 disease-induced changes 118 Ciconiiformes 3, 167–8 Falconiformes 68, 71 hematological characteristics 60–1 Galliformes 72, 73 monocytes 61 Gruiformes 74, 75, 76 vascular access sites 14 Passeriformes 78, 79, 80 circadian effects on hematological characteristics 99 Pelecaniformes 81, 82 Coliiformes 3 Phoenicopteriformes 82–3 hematological characteristics 61 Podicipediformes 85 Columbiformes 3, 168 Procellariiformes 85 hematological characteristics 62, 62 –3 Psittaciformes 86, 87, 89, 90 vascular access sites 14 Sphenisciformes 91, 92 Coraciiformes 3, 168 stippling 105, 171 hematological characteristics 64, 64 –6, 66 stress response 124 cranial vein 10, 11 Strigiformes 93, 94 Cuculiformes 3, 168 Struthioniformes 95, 96 hematological characteristics 66 ultrastructure 42, 46 cutaneous vein 10, 11 blindness, heterophil changes 110 blood collection 6–7 dehydration 104, 106, 111 equipment 7 diarrhea restraint of birds 5 dehydration 106 blood fi lms 16–19, 20 –1, 22–3 heterophil concentration 117 artifacts 23, 24 –5, 26–7 Diff Quick stain 19, 20, 21, 22, 172 cover-slip and slide method for 18–19, digital veins 14, 15 19 diving birds, blood volume 106 cross method 17–18, 19 dorsal vein 10, 11 fi xed 23 dyspnea 118, 119 hemoparasites 127–8 incomplete drying 22, 26–7 EDTA see ethylenediaminetetraacetic acid (EDTA) non-hematological cells 24, 25, 27 eosinophils 19, 40, 41–2, 43, 45–6, 172 staining 19, 20 –1, 22–3 Anseriformes 54, 55, 56, 57, 57 two cover-slip method for 18, 19 Caprimulgiformes 57, 58 unfi xed 23 Charadriiformes 58, 59, 60 wedge method for 16–17, 19 Ciconiiformes 60, 61 blood volume 6 Columbiformes 62, 63 diving birds 106 Coraciiformes 64, 65, 66 Passeriformes 77 cytoplasm 45–6 bone marrow 51, 171 cytoplasmic granules 55, 71 hypoplasia 124 Falconiformes 67, 69, 70, 71 Index 179

Galliformes 71, 72, 73 Anseriformes 55, 56, 57 granules 46 Charadriiformes 58, 59 Gruiformes 75, 76 Ciconiiformes 60, 61 Passeriformes 77, 78, 79, 80 Columbiformes 62, 63 Pelecaniformes 81–2 Gruiformes 74, 75, 76 Phoenicopteriformes 82 Passeriformes 77, 79 Piciformes 83, 84, 85 Pelecaniformes 81 Podicipediformes 85 Sphenisciformes 92 Procellariiformes 85–6 Struthioniformes 95 Psittaciformes 88–9, 90 trauma 116 Sphenisciformes 91, 92 meningoencephalitis 104, 111 Strigiformes 92, 93, 94 nucleated 30 Struthioniformes 94, 95, 96 nucleus 33, 37 time of day effects 99 Anseriformes 57 ultrastructure 41, 46 oxidative damage 107 erythrocytes 22, 33, 35–9, 39–40, 172 Passeriformes 77, 78, 79, 80 age effects 98 Pelecaniformes 81, 82 agglutination 105 Phoenicopteriformes 82 aggregation 100 Piciformes 83 anemia 38, 101, 102, 103, 107–8 Plasmodium schizonts 130 Anseriformes 55, 56, 57 Podicipediformes 85 shape 57 polychromatophilic 36, 37, 38, 39, 174 anucleated 36 anemia 101, 103 artifacts 26–7 Anseriformes 56 basophilic stippling 38 bacterial infection 117 Caprimulgiformes 58 Ciconiiformes 61 Ciconiiformes 60, 61 Coraciiformes 65 Columbiformes 62, 63 disease process 105 concentration 27, 28–9 erythropoiesis 101 increase 106–7 hemorrhage response 115 Coraciiformes 64, 65, 66 Passeriformes 78, 79, 80 cytoplasm 35, 37 Pelecaniformes 81 dehydration 111 Sphenisciformes 92 density 105 Strigiformes 92 development stages 39–40 trauma 101 dimensions 39 polycythemia 104, 106–7 disease response 100, 100 –4, 104, 105, 106–8, Procellariiformes 85, 86 111 Psittaciformes 86, 87, 88, 89, 90 Falconiformes 69 reproductive status effects 98 Galliformes 71, 72, 73 rouleaux 104, 105 ghost 105 seasonal effects 99 Gruiformes 74, 75, 76 sex effects 98 Haemoproteus 131–2 shape 33, 35, 36, 39 hemoglobin crystal 38 variant 40 hemolytic anemia 100 size 39 hemoparasites 40 Sphenisciformes 91, 92 heterophilic meningoencephalitis 104 staining 20 hypochromatic 105 Strigiformes 92, 93, 94 increased production 106 Struthioniformes 95, 96 lysis 115 time of day effects 99 mature 36, 37, 38 trauma 101 anemia 103 volume 29–30 180 Index erythrocytosis 104 head and neck, vascular access 10–11 hypoxia 106 Heinz body 38, 105, 107, 172 erythroid cells 51 extra-vascular hemolytic anemia 107, 108 anemia 103 hematocrit disease effects on production 108 age effects 98 erythrophagocytosis, macrophages 104 dehydration 106 erythroplastids 36, 37, 40, 172 migration effects 99 erythropoiesis 101, 172 seasonal effects 98, 99 anemia 101 hematomas 7 hemorrhage 115 hematopoiesis 51, 172 polycythemia 107 hemocytometer 31, 32 trauma 101 hemoglobin 34, 172 esophageal vein 10, 11 age effects 98 ethylenediaminetetraacetic acid (EDTA) 15, 20, colorimetric determination 29 22, 172 decreased production 102 erythrocyte lysis 115 high-altitude fl ight birds 106 Gruiformes 76 measurement 29–30 Passeriformes 77, 78 migration effects 99 Struthioniformes 96 seasonal effects 99 sex effects 98 Falconiformes 3, 168 hemolysis 15–16, 20, 26, 172 hematological characteristics 66, 67, 68–9, 70, 71 Gruiformes 76 vascular access sites 14 Passeriformes 77, 78 femoral vein 10 Struthioniformes 96 fenbendazole 124 hemoparasites 49, 125–38 ferric chloride 14 blood fi lms 127–8 fi tness of hemoparasite host 127 clinical disease 125–7 fl ow cytometry 31–2 concentration methods 128–9 fungal hyphae 24 detection 127–9 blood fi lm contamination 27 diagnosis 127–9 erythrocyte shape 40 Galliformes 3, 168 fi tness of host 127 hematological characteristics 71–2, 73, 74 hemolytic anemia 107 vascular access sites 14 immature birds 126–7 Gaviiformes 4, 168 immunity 127 hematological characteristics 74 intracellular 105 geographic location, effects on hematological light microscopy 128 characteristics 99–100 morphology 129–38 ghost cells 26, 172 polymerase chain reaction 128 Giemsa stain 19, 20, 22–3 reproductive success of host 127 glove powder particles, blood fi lm contamination hemorrhage 115 25, 27 hemostasis, blood collection 14 glucocorticoids, stress 123 heparin 16, 23 granulocytes 172 leukocyte clumping 50 staining 31 Passeriformes 78 Gruiformes 4, 169 see also lithium heparin hematological characteristics 74, 75, 76 Hepatozoon 49, 125, 126, 135 vascular access sites 14 heterophilia 112, 173 air sacculitis 116 Haemoproteus 40, 125, 126, 131–2 aspergillosis 116 handling of birds 5, 14, 15 dyspnea 118 Index 181

lymphocytes 112 infl ammation/infl ammatory disease 108, 119–23 meningoencephalitis 117 anemia 108 pancreatic disease 112 bacterial pododermatitis 117 heterophils 20, 21, 34, 40, 41, 45, 173 heterophil response 48–9, 112, 113, 115, 120, anemia 103 122–3 Anseriformes 54, 55, 56, 57 lipemia 111 aspergillosis 49, 116, 117, 120–2 lymphocytes 112, 115 atypical morphology 48, 112, 113, 114, 115 monocytes 112, 115 atypical myelopoiesis 116 toxic changes 119–20 band 48, 109 ingluvies vein 10, 11 basophilic granules 49 injury see trauma Caprimulgiformes 57, 58 central granular body 45, 54 jugular vein 10–11, 12, 14, 15 Charadriiformes 58, 59, 60 Columbiformes 62, 63 karyolysis 48, 173 concentration with diarrhea 117 karyorrhexis 48 Coraciiformes 64, 65, 66 Knott’s test 129 cytoplasmic granules 45, 48–9 dehydration 111 Leishman’s stain 19 disease-induced changes 111, 112, 116, 117, Leucocytozoon 126, 132–4 118 leukocytes 20, 34, 40, 41–5, 45–9, 173 distinction from eosinophils 46 age effects 98 eosinophilic granules 49 artifacts 26, 27 Falconiformes 67, 68, 69, 70 atypical morphology 48–9 Galliformes 73 basophilic granules 49 Gruiformes 74, 75, 76 clumping 27 hemorrhage response 115 concentration measurement 30–2 infl ammation 48–9, 112, 113, 115, 120, 122–3 estimation 32 lipemia 111 count 28, 29 meningoencephalitis 104, 111, 114, 117 cytoplasmic changes with disease 49 mycobacteriosis 49 differential counts 31 Passeriformes 77, 78, 79, 80 disease response 49, 108, 109 –19, 119–24 Pelecaniformes 81, 82 concentration 120 Phoenicopteriformes 82 morphological characteristics 121 Piciformes 83, 84 hemoparasites 49 Podicipediformes 85 infl ammation response 48, 108, 119–23 pododermatitis 117 measurement 30–2 Procellariiformes 85, 86 nucleated 30 Psittaciformes 86, 87, 88, 90, 91 nucleus atypia 48 Sphenisciformes 91, 92 nucleus toxin effects 48 stress response 124 staining 31 Strigiformes 92, 93, 94 stress response 123–4 Struthioniformes 94, 95, 96 time of day effects 99 time of day effects 99 total concentration 31 trauma response 49, 109, 110, 113–16, 118 see also basophils; eosinophils; heterophils; ultrastructure 38, 45 lymphocytes; monocytes hypochromasia 102, 173 Leukocytozoon 49, 125 hypoxia, burrowing birds/high altitude 106 leukopenia 124, 173 light microscopy, hemoparasite detection 128 immature birds, hemoparasites 126–7 lipemia 24, 111 immunity, hemoparasites 127 lithium heparin 16, 22, 27, 118 182 Index lymphocytes 20, 40, 43, 44, 46–7, 173 meningoencephalitis age effects 98 erythrocytes 111 Anseriformes 55, 56, 57 heterophil changes 111, 117 atypical morphology 49 heterophilic 104, 114, 117 basophilic granules 47 metacarpal vein, dorsal 9, 10, 13 blast-transformed cells 49 metamyelocytes 48 Caprimulgiformes 57, 58 metatarsal vein 14 Ciconiiformes 60, 61 dorsal 9, 10, 15 Columbiformes 62, 63 medial 9, 10, 11, 13, 14, 15 Coraciiformes 64, 66 plantar 9, 10 disease-induced changes 111, 117, 118, methyl violet 2B 31 119 microcytosis 102 Falconiformes 67, 69, 70, 71 microfi lariae 126, 136–8 Galliformes 72, 73, 74 microscopy Gruiformes 74, 75, 76 light heterophilia 112 hemoparasite detection 128 infl ammation 112, 115 leucocyte concentration determination lipemia 111 31 nucleolus 49 phase contrast 31 Passeriformes 77, 78, 79, 80 migration effects on hematological characteristics Pelecaniformes 81, 82 99 Phoenicopteriformes 83 mitotic fi gure 39, 40 Piciformes 83, 84, 85 monocytes 20, 40, 43, 44 –5, 47–8, 173 Podicipediformes 85 Anseriformes 56, 57, 57 Procellariiformes 85, 86 Caprimulgiformes 58 Psittaciformes 87, 89, 90 Charadriiformes 59, 60 size 47 Columbiformes 62 Sphenisciformes 92 Coraciiformes 64, 66 stress response 124 disease-induced changes 112, 117, 118, 119 Strigiformes 93, 94 Falconiformes 68 Struthioniformes 96 Galliformes 73, 74 time of day effects 99 Gruiformes 75, 76 trauma response 110, 116 infl ammation 112, 115 ultrastructure 43, 47 mycobacteriosis 49 lymphocytosis 173 Passeriformes 78, 79, 80 air sacculitis 116 Pelecaniformes 81, 82 lymphopenia 119, 173 Phoenicopteriformes 83 lysis of cells 26 Piciformes 84, 85 erythrocytes 115 Podicipediformes 85 see also hemolysis Procellariiformes 85, 86 Psittaciformes 86, 87, 90 macrophages 173 Sphenisciformes 92 erythrophagocytosis 104 Strigiformes 93, 94 heterophilic meningoencephalitis 114 Struthioniformes 96 May–Grünwald stain 19, 20, 22–3 trauma response 115, 116 mean corpuscular hemoglobin (MCH) 30 ultrastructure 44, 47–8 mean corpuscular hemoglobin concentration monocytosis 173 (MCHC) 30 air sacculitis 116 mean corpuscular volume (MCV) 29–30, 34 aspergillosis 116, 118 age effects 98 disease-induced changes 118 measurement of hematological cells 27–32 dyspnea 118 Index 183 mononuclear cells 40, 43 Phoenicopteriformes 4, 169 hemoparasites 49 hematological characteristics 82–3 mycobacteriosis physiological effects on hematological anemia 108 characteristics 97–100 leukocyte changes 49 Piciformes 4, 169 myelocytes 48 hematological characteristics 83, 83–4, 85 myeloid cells 51 plasma proteins, anesthesia effects 5 myelopoiesis, atypical 48, 116 Plasmodium 40, 125, 126, 129–30 plexus venosus subcutaneous collaris 14 neck, vascular access 10–11 Podicipediformes 4, 169 nematodes hematological characteristics 85 adults 138 poikilocytes 40, 174 microfi lariae 126, 136–8 polycythemia nucleolus 174 erythrocytes 104, 106–7 lymphocyte 49 erythropoiesis 107 polymerase chain reaction (PCR), hemoparasite occipital sinus, access 11 detection 128 occipital vein, lateral 10, 11 popliteal vein 10 Opisthocomiformes 4 population effects on hematological characteristics hematological characteristics 77 99–100 osteoblasts 24, 174 Procellariiformes 4, 169–70 osteoclasts 24, 174 hematological characteristics 85–6 Psittaciformes 4, 5, 170 packed cell volume (PCV) 27–8, 34, 174 hematological characteristics 86–90, 91 age effects 98 vascular access sites 15 anesthesia effects 5 anemia 102 radial vein 8, 10 hypoproliferative 108 rapid stains 19, 23 dehydration effects 106, 111 see also Diff Quick stain disease response 100, 100–4, 104 red blood cells (RBCs) 34 hemolytic anemia 100 renal insuffi ciency, lymphocytes 119 reproductive status effects 98 reproductive status, effects on hematological sex effects 98 characteristics 98 time of day effects 99 reproductive success of hemoparasite host trauma 101 127 pancreatic disease, heterophilia 112 restraint of birds 5, 14, 15 parasites see hemoparasites reticulocytes 39–40, 174 Passeriformes 4, 169 anemia 102 hematological characteristics 77, 77–80, migration effects 99 80 Romanowsky stains 19, 23, 31, 174 vascular access sites 11, 14 see also Giemsa stain; Leishman’s stain; pathological effects on hematological May–Grünwald stain; Wright’s stain characteristics 100–24 rouleaux 104, 105, 174 pectoral limb, vascular access 8–9, 10 rubricytes 37, 40, 174 pedal veins 15 anemia 103 Pelecaniformes 4, 169 trauma 101 hematological characteristics 81–2 vascular access sites 14–15 sand particles, blood fi lm contamination 25, 27 pelvic limb, vascular access 9–10 seasonal effects on hematological characteristics phase contrast microscopy 31 98–9 phloxine B 31 sex effects on hematological characteristics 98 184 Index species thrombocytic cells 51 common names 163–6 tibial vein orders 167–70 caudal 9, 10 Sphenisciformes 4, 170 cranial 10, 13 hematological characteristics 91–2 time of day, effects on hematological vascular access sites 15 characteristics 99 spironucleosis 106 Tinamiformes 4, 170 staining of blood fi lms 19, 20 –1, 22–3 hematological characteristics 96 artifacts 26 toxic change 174 granulocytes 31 infl ammation 119–20 leukocytes 31 tracheal vein 10, 11 Romanowsky stains 19, 23, 31, 174 trauma stress, hematological effects 123–4 erythrocyte changes 101 Strigiformes 4, 170 erythropoiesis 101 hematological characteristics 92, 92–4, 94 heterophil changes 49, 109, 110, 113–16, vascular access sites 14 118 Struthioniformes 4, 5, 170 lymphocyte changes 110, 116 hematological characteristics 94, 94 –6, 96 monocytes 115, 116 vascular access sites 15 PCV 101 subclavian vein 10, 11 Trichostrongylus tenuis 108 Trogoniformes 4 tarsal vein, lateral/medial 9–10 hematological characteristics 96 taxonomy 2, 2–4, 53–4 Trypanosoma 125, 126, 135–6 see also species thoracic vein, external 11 ulnar vein 8–9, 10, 12, 13, 14, 15 thrombocytes 49–51 aggregation 27, 116 vascular access sites 7–11, 12–13, 14–15 Caprimulgiformes 57 bird groups 11, 12–13, 14–15 Charadriiformes 58, 59 veins, access to 5, 6–7 clumping 50 vena cava, cranial 10 Columbiformes 63 venipuncture 5, 6–7 Falconiformes 68, 70 Passeriformes 77, 79 white blood cells see leukocytes Piciformes 84 Wright’s stain 19, 20, 21, 23 Psittaciformes 90 Struthioniformes 95 zinc toxicosis, anemia 107–8 ultrastructure 50–1 zoonotic diseases 5