Clinical Approach to the Anemic Reptile Miguel D

Topics in Medicine and Surgery Clinical Approach to the Anemic Reptile Miguel D. Saggese, DVM, MS, PhD

Abstract Anemia is a relatively common blood disorder but a seemingly underreported and investigated condition in reptiles. Better characterization of anemia in the sick reptile could help determine which available diagnostic tools could be best utilized to determine the cause of low red blood cell counts in these animals. There are, however, a number of limitations that exist when trying to interpret hematologic data for the large number of reptilian species presented to the exotic animal practitioner, including interspecific and intraspecific variation in hematological reference values, limited availability to specific reagents, and a lack of scientific- based studies that focus on anemic conditions in reptiles. Therefore, better documentation and consistency in the scientific-based studies of reptile anemia and rapid dissemination of this information are considered necessary to increase our knowledge in this area of reptile medicine. Collaborative research efforts between practitioners, owners, reptile collection curators, pathologists, immunologists, microbiologists, toxicologists, and laboratory scientists are urgently needed to augment our understanding of anemia and other hematological disorders in reptiles. Copyright 2009 Elsevier Inc. All rights reserved.

Key words: anemia; blood; erythrocytes; hemoglobin; reptiles; treatment

ematology is the medical science that stud- plication to other diseases. In the latter case, anemia ies blood in health and disease. As such, the rarely constitutes a diagnosis but is rather a clinical Hstudy of hematology has a deep impact on sign of some other pathological condition or illness. our understanding of multiple diseases. Without a Hb concentration and hematological indexes are doubt, one of the most useful interpretations of invaluable tools to investigate and diagnose reptile hematology in human and veterinary medicine is the hematologic maladies;1-8 however, thorough investi- change in the number and morphology of erythro- gation of the anemic patient appears to be rarely cytes that occur because of pathological conditions. conducted in reptile medicine. Identification of ane- Among these, one of the most commonly observed mia in a lizard, snake, crocodile, or chelonian should pathological conditions is anemia. challenge the veterinary practitioner to perform a The word anemia is derived from the Greek word comprehensive investigation with the aim to deter- anaemia, which means “without (an) blood (aemia).” It can be defined as a reduction in the number of red blood cells (RBC) or erythrocytes per volume of From the College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA USA. blood, with the subsequent reduction in oxygen de- Address correspondence to: Miguel D. Saggese, DVM, MS, PhD, livery to the tissues by hemoglobin (Hb). Ultimately, College of Veterinary Medicine, Western University of Health anemia should be defined as a qualitative or quanti- Sciences, Pomona, CA 91766. E-mail: [email protected]. tative deficiency in circulating Hb. Anemia can be © 2009 Elsevier Inc. All rights reserved. caused by a primary blood disorder (e.g., aplasia, 1557-5063/09/1802-$30.00 hypoplasia) or, more frequently, a secondary com- doi:10.1053/j.jepm.2009.04.003

98 Journal of Exotic Pet Medicine, Vol 18, No 2 (April), 2009: pp 98–111 Anemia in Reptiles 99 mine the underlying cause of the condition even in the absence of other clinical signs. Furthermore, early recognition of anemia in a reptile patient during annual or biannual examination helps to identify diseases before they progress into life-threatening disorders and allows the practitioner to implement a specific treatment regimen, thereby helping one establish a prognosis and monitor response to therapy.1-8 More than 400 causes of anemia have been reported in humans, with a smaller, although still significant, number of causes recognized in small and large animals.9,10 It is very likely that there are several causes for anemia in reptiles too, even though these remain unidentified or not clearly defined. Because there are more than 8000 species of reptiles described worldwide, it is unlikely that a detailed Figure 2. Collecting blood from the subcarapacial vein of an Argen- knowledge of the erythron and its response to dis- tine desert tortoise (Geochelone chilensis). Photo courtesy of Dr. Guillermo Perez Jimeno. ease for all these species will ever be achieved. How- ever, for the continued development of reptile medicine and hematology, a more exhaustive inves- Overview of Blood Collection in Reptiles tigation of the anemic patient, including its causes and associated changes in the erythron, is needed. Exotic The method of blood collection and the use of an animal veterinarians may contribute to this better un- appropriate sampling technique are critical steps to derstanding by reporting systematic investigation and the proper interpretation of the reptile hemogram, test results from their anemic reptile patients. especially during the investigation of an anemic pa- The goal of this article is to review and summarize tient. A basic understanding of reptile anatomy, with general aspects of reptilian hematology, sampling emphasis on the topographic anatomy of venipunc- techniques, and the clinical approach to the diagno- ture sites, is necessary for collecting blood and re- sis of anemia in reptiles. The intention is to generate ducing the discomfort that the procedure may cause more interest from the veterinary practitioner in in the patient. Excellent and detailed reviews on contributing to the study and understanding of the these topics have been recently published.4,5,8,11-14 causes and diagnosis of anemia in reptiles. Given Blood samples can be collected from multiple that excellent detailed reviews about reptile hema- locations on a reptile patient. (Figs 1-4).4,5,8,11-14 tology have been recently published, minimal de- Choosing the best site for blood collection will be scriptions of many of these basic principles are made determined by taxonomy, species anatomic charac- in this article.2-5,8

Figure 3. Blood collection from the ventral tail vein of a python Figure 1. Blood collection from the cervical sinus of a tortoise. (Python sp.). Photo courtesy of Drs. Paula Moreno and Angelica (Geochelone sp.) Photo courtesy of Dr. Carolina Torta. Rojas. 100 Saggese

ogy, plasma protein electrophoresis) is needed at a later date. Large volumes of blood, plasma, and/or serum can be saved at –80°C. In clinically ill patients or those suspected of being severely ill, a smaller volume of blood should be collected (e.g., 0.5 mL is usually enough to run a complete blood count including a plasma biochemistry panel). When investigating the progression of anemia using hematocrit (Ht) percentage and the RBC count, only small volumes of blood should be taken to avoid unnecessary and excessive blood loss in the patient. Veterinary clinicians must remember that reptiles have very well-developed lymphatic vessels that usually run in close proximity to the main vessels used to collect blood.3-5,12,14,15,19 As a result, contamination of Figure 4. Blood collection from the ventral tail vein of a spiny-tailed lizard (Uromastyx aegyptius). Photo courtesy Dr. Jaime Samour. the blood sample with lymph is a very common complication, especially in chelonians. Lymph contamination causes hemodilution, which can signifi- cantly alter the results and diagnostic value of a teristics, body condition, preference and experience blood sample.3-5,12,14,15,19 Recognition of lymph con- of the phlebotomist, size of the patient, and amount tamination is relatively simple if careful attention is of blood required. Selection of an adequate blood used when collecting the sample. When entering a sampling site is also essential if a significant volume 4,5,8,11-14 vessel with the needle, blood will slowly fill the sy- needs to be collected. Peripheral blood ves- ringe. However, when a lymphatic vessel is entered, sels should be the first option for reptile blood col- a clear, pale yellow or whitish liquid or a change in lection, given that these sites are relatively more the color of the previously collected blood (e.g., accessible and usually adverse to developing second- suddenly becoming more dilute) will be observed in ary complications. The brachial and jugular veins the sample when lymph is being collected instead of (e.g., lizards, chelonians), ventral tail vein (e.g., liz- blood. While the blood is being collected, careful ards, snakes, chelonians, crocodilians), and ventral observation of the syringe during blood draws will abdominal vein (e.g., lizards) are readily accessible 4,5,12,13,15 help to detect this contamination and consequent sites for most species of reptiles. In ophid- change in color.4,5 When lymph contamination is ians, given that these peripheral sites are not avail- detected, the sample should be discarded and a new able with the exception of the ventral tail vein, car- one obtained from a different anatomic location. diocentesis is usually recommended.4,5,12,16 It is a relatively simple and safe procedure that allows for the collection of a relatively large volume of blood. Preloading the Anticoagulant in Another potential venipuncture site in ophidians is the Syringe the palatine veins.4,5,12,16 In crocodilians, the ventral Anticoagulants are routinely used for holding and coccygeal veins and supravertebral vessels are safe storing reptile blood samples. Blood that has been and recommended when significant quantities of altered with an anticoagulant will allow for the eval- blood are needed.12,17 In chelonians, the jugular vein uation of blood cells or plasma components from is the preferred vessel because there is less risk of the same sample. Lithium heparin is the recom- collecting lymph-diluted blood as compared with the mended anticoagulant for preloading a syringe to other peripheral sites.4,5,18 Additional venipuncture collect a blood sample from a reptile.4,5,8,12,19,20 How- sites in chelonians are the postoccipital venous ever, other authors report the use of sodium ethyl- plexus, femoral and coccygeal veins, and the sub- enediamine tetraacetic acid (EDTA) as a better op- carapacial sinus.4,5,12,18 tion for preloading syringes when collecting blood For most reptiles, the total volume of blood col- from lizards and snakes.17,21 In chelonians, heparin lected should not be Ͼ1% (in grams) of body may cause hemolysis.20 As in birds, the use of EDTA weight.3-5,8,11 Usually, a blood volume equivalent to may impair blood cell staining and cause changes in 0.5% to 0.8% of the body weight is enough to pro- the coloration of erythrocytes and hemolysis.4,5,11 vide a diagnostically valuable sample. Ideally, the Nevertheless, blood smears are better prepared amount of blood obtained should allow the storage when blood is collected without the addition of of plasma or serum if additional testing (e.g., serol- anticoagulant in the collection syringe. Collecting a Anemia in Reptiles 101 viable blood sample without the use of an anticoag- nizing these possible variations in reptile hemato- ulant may be difficult to achieve in small reptiles, logic results will contribute to a judicious interpre- which offer limited blood volumes. tation of published reference values and, at the same When blood is collected without the use of an time, should motivate researchers and clinicians to anticoagulant, it should be quickly placed in tubes investigate these differences, obtain references val- containing anticoagulant to prevent clotting. The ues adjusted to these variations, and demonstrate use of 0.25 to 0.5 mL Microtainer blood tubes (BD their clinical significance.4-8,11,13,16-19 Ideally, at least Vacutainer Systems; Beckton, Dickinson and Co., 100 individuals from the same species, age, and sex, Franklin Lakes, NJ USA) is recommended.4 Micro- housed under similar conditions and fed a diet that tainer blood tubes allow for easy and fast mixing of is close in nutritional composition, should be used blood. Given that reptile erythrocytes are relatively when trying to determine normal blood reference fragile, it is advisable to remove the needle hub from values.24 Packed cell volume (PCV), RBC counts, and the syringe when transferring blood to the tube or hematological indices vary between species and making the blood film to avoid hemolysis. Once the within individuals, making the use of published ref- blood film has been made, they can be stained with erence values of limited value in most cases.4,5,8 Ul- the same methods used for birds, including Wright- timately, comparison of blood reference values in a Giemsa, May-Grünwald-Giemsa, or Diff Quick. Both patient with compiled data from multiple sources slide-to-slide and coverslide-to-coverslide techniques that either do not identify the methodology used to are recommended when preparing a blood smear.5,12 collect and test the blood sample or define the pop- The quality of the blood sample is always impor- ulation from which the sample is collected is not tant for precise examination and diagnosis. Presence recommended. In fact, a better approach to evaluat- of clots, hemolysis, excessive anticoagulant, and con- ing blood work values between sick and healthy pa- tamination with lymph will impede the collection of tients is to obtain in situ reference values for the blood samples and alter diagnostic values.3-5,8 It has individual through investigation of normal blood been stated that a laboratory test result is only as values for the same patient by collecting and analyz- good as the specimen collected. In another words, ing the samples at least two times in the year. This extreme caution should be used to prevent sending approach can provide more valuable information the laboratory a blood sample for analysis that will regarding what is within the normal range of healthy confer misleading results.11,19 individuals rather than a single value obtained from Special precautions should be taken when manip- a diseased patient. For example, a reptile patient ulating reptiles for venipuncture. They can harbor could experience a significant drop in its PCV (e.g., several infectious agents that are zoonotic or poten- from 45% to 25%), which suggests there is an ab- tially zoonotic.22 Blood samples should be treated as normality, but if the PCV values from the animal a potential source of blood-borne pathogens for the were captured by the reference range for that par- personnel as well as for other animals. Veterinarians ticular species it may be misclassified as being nor- are responsible for enforcing standard biosafety pre- mal. The scenario described above reinforces the cautions when working with reptiles, especially when need to compare blood reference values obtained collecting blood samples from the diseased patient. from the same patient or from a clearly defined Use of gloves, goggles, and/or a mask should always reptile population. In cases of severe anemia when be recommended when working with reptiles that the PCV values fall well below the published refer- present with clinical signs of disease. ence range, this comparison is not needed.

Hematologic Reference Values Evaluation of the Reptilian Erythron Although published reference values are available for a number of reptile species, it is important to Evaluation of the reptilian erythron includes the remember that both intrinsic (e.g., species, gender, determination of the PCV or Ht, total RBC (TRBC) age, physiological status, breeding season, hiber- count, Hb concentration, and hematological indices nation) and extrinsic (e.g., season, temperature, such as mean corpuscular volume (MCV), mean cor- environmental conditions, husbandry, diet, living puscular hemoglobin (MCH), and mean corpuscu- conditions) factors may cause significant variations lar hemoglobin concentration (MCHC). Calculation in hematological parameters.4,5,8,11,23 Thus, when re- of these indices contributes to effectively measuring ferring to a set of references values, it is important to the size of erythrocytes and calculating the content determine the conditions under which the blood of Hb within cells. The detailed examination of these samples were obtained.4,5 Understanding and recog- parameters is necessary when diagnosing, investigat- 102 Saggese ing, and characterizing the anemic conditions that affect reptile species. The TRBC count can be determined by using a manual counter and the Natt Herrick or Unopette techniques. Both methods rely in the dilution of the blood sample and further manual count of cells through the use of a counting chamber.3-5,8,11,25,26 After subsequent adjustment to the dilution factor, the total number of cells/milliliter in blood are calculated. This can be easily performed in a veterinary clinic without the need for expensive equipment. Other methodologies used to obtain a reptile complete blood count include the use of flow cytometry or automatic counters. Although equipment to perform automated complete blood counts on reptile Figure 5. Blood film from a spiny-tailed lizard showing the normal species is expensive and can be purchased, it is not shape/size of mature erythrocytes. A single teardrop erythrocyte can available through most veterinary diagnostic labora- be observed in the upper left quadrant. Photo courtesy of Dr. Jaime tories.3-5 Samour. Reptiles usually have a lower number of erythrocytes per volume of blood than mammals or birds.27-29 An inverse relationship between size and 100/Hct (%). MCHC values reported for reptiles number of circulating erythrocytes apparently exists. range from 22% to 41%.3-5,8,13,14,16-19,28,30 Those species with smaller erythrocytes tend to have Detailed descriptions of the reptilian RBC have a larger number of this cell type in their blood.27-29 been published in other articles.4,5,29,31-35 The combi- The PCV or Ht measures the percentage of RBCs in nation of basic and acid dyes allows for the differen- the blood. Reference PCV values in reptiles range tial staining of blood cells and the study of their from 20% to 45%, whereas some authors report a morphology under the light microscope (Fig 5). 3-5,8,16-19,28,30 wider range of values (e.g., 15%-50%). With practice, clinicians and technicians can be- Hb concentration is measured with a solution of come proficient in evaluating reptile erythrocytes. potassium cyanide and potassium ferricyanide. A specific volume of blood is combined with cyanide forming cyanmethahemoglobin, which is read using Anemia light photometry.4,8,9 Reported Hb concentration values for reptiles range from 5.5 to 12 g/dL.3-5,8,16-19,28,30 Reptile patients with anemia have a decreased oxy- The above-mentioned parameters, together with the gen-carrying capacity. This may occur because of a hematological indices described below, are neces- reduction in the number of circulating erythrocytes sary to effectively investigate RBC function. Hemato- or a decrease in the concentration of Hb within the logical indices allow accurate measurements of erythrocytes. Anemias may be classified as: 1) regen- erythrocyte size and Hb concentration. When com- erative, when the bone marrow maintains its eryth- bined, these parameters and indices provide impor- ropoietic function (e.g., hemorrhage, hemolysis) tant information that contributes to a better diagnosis, and the patient is capable of producing and releas- identification of the etiology, and characterization of ing newly formed erythrocytes, and 2) nonregenera- anemia. The hematological indices are: tive, where no new RBCs are formed or released from the hematopoietic sites. MCV (fL) correlates to the average volume of erythrocytes. It can be calculated with the following for- Regenerative Anemia mula: Ht (%) ϫ 10/RBC count (ϫ 1012/L). MCV Regenerative anemias are usually caused by hemor- values range from 200 to 1200 fL.3-5,8,13,14,16-19,28,30 rhage and hemolysis. Hemorrhagic anemia in rep- MCH measures the average weight of Hb in the eryth- tiles occurs because of blood loss after trauma and rocyte. The MCH can be calculated as follows: Hb surgery, hemathophagous external parasites, coagu- (g/dL) ϫ 10/RBC count (ϫ 1012/L). MCH values lation disorders, gastrointestinal ulcers, and neopla- ranges from 6 to 10 g/dL.3-5,8,13,14,16-19,28,30 sia.4,5,8,36-39 MCHC expresses the average concentration of Hb in Anemia is a common finding in reptiles with the erythrocyte. It is calculated as: Hb (g/dL) ϫ heavy ectoparasite infestations. Ticks from the fami- Anemia in Reptiles 103

Figure 6. Ticks (Amblyoma argentina) on an Argentinean desert Figure 8. Blood film from a spiny-tailed lizard. An intracellular tortoise. Photo courtesy of Dr. Guillermo Perez Jimeno. hemoparasite (Haemoproteus sp.) can be observed inside some of the erythrocytes. Photo courtesy of Dr. Jaime Samour. lies Ixodidae and Argasidae are commonly identified as the underlying cause of anemia in reptile cases in ingestion of a foreign body (e.g., ingesting a fish- 39,40 which the arthropods are present (Fig 6). The hook) are other possible causes of internal hemor- introduction of new or recently acquired reptiles in rhage in reptiles.42 Hemorrhagic conditions are rare an otherwise well-managed reptiliary could cause in ophidians, although internal hemorrhage caused outbreaks of tick infestations and anemia in pre- by the ingestion of rodents exposed to anticoagulant viously healthy individuals. Ophionyssus natricis, the rodenticides may occur in free-ranging snakes.43 Co- snake mite, is a common cause of anemia in captive agulation disorders caused by thrombocytopenia snakes. Juvenile animals are especially susceptible to have been described in reptile species and are often developing anemia from these mite infestations. the result of decreased production, accelerated use, Leeches may cause significant blood loss in aquatic or destruction of thrombocytes.4,5,8 41 reptiles and are often a difficult problem to diag- Hemolytic anemia occurs when erythrocytes are nose if the parasites are not observed while attached destroyed within the blood vessels (e.g., intravascular to their victims. hemolysis) or outside the lumen of the vessel (e.g., In chelonians, traumatic wounds caused by fight- extravascular hemolysis). Hemolytic anemia can be ing, dog bites, and free falling from balconies will congenital (e.g., inherited hemolytic anemia) or ac- result in anemia due to internal or external blood quired. An anemia can also be classified based on the loss (Fig 7). Gastric ulcers or trauma related to the mechanism of damage to the plasmatic membrane of the erythrocyte. This can be an immune-mediated response (e.g., antibodies) or the result of an infectious agent, drug, or toxin on the erythrocyte membrane. Inherited hemolytic anemia has not been reported in reptiles to the author’s knowledge. The most common cause of acquired hemolytic anemia in reptiles is associated with severe infestation by the malarial hemoparasites (Plasmodium).5,39,40,44,45 Haemopro- teus and Saurocytozoon are two additional genera of hemoparasites that can also cause anemia in reptiles (Fig 8).5,45 Special considerations should be taken to identify and differentiate these parasites from other nonpathogenic hemoparasites found in reptile blood. Idiosyncratic drug-induced hemolysis can arise theoretically through the administration of any med- Figure 7. Traumatic hemorrhage in an Argentinean desert tortoise. ication prescribed for reptiles. Drug-induced hemo- Photo courtesy of Dr. Guillermo Perez Jimeno. lysis is more likely to occur with drugs well recog- 104 Saggese nized for causing anemic conditions (e.g., nonsteroidal antiinflammatory drugs, antifungal agents, sulfon- amide antibiotics).46-48 Heavy-metal toxicity, especially lead and zinc toxicosis, can induce a hemolytic anemia, in addition to other gastrointestinal and neurological clinical signs.4,5,8,43,49-51 Lead toxicosis may result from the ingestion of tainted paint, fishing sinkers, spent lead ammunition, or from other environmental sources. Zinc toxicosis has been associated with overdosing zinc supplements, ingestion of pennies coined after 1982, thermometer weights (Fig 9), and zinc-containing ointments. Use of anticoagulants containing calcium EDTA has been reported to cause hemolysis in some species Figure 10. Severe fungal hepatitis (caused by Schizangiella serpen- of chelonians.20 Inappropriate venipuncture tech- tis, a zygomycete of the order Entomophthorales, family Basidiobo- niques, such as excessive negative pressure, pro- laceae) associated with a nonregenerative anemia in a snake. Photo longed storage, and manipulation of the sample, are courtesy of Dr. Tracey McNamara. common causes of iatrogenic hemolysis.

Nonregenerative Anemia Infectious diseases should always be considered as one of the top differential diagnoses and are highly Nonregenerative anemia is the most common man- prevalent in captive reptiles.56-62 Systemic chronic ifestation of anemia in reptiles, although it is rarely infectious diseases such as mycobacteriosis, myco- fully investigated and, as such, rarely reported. In plasmosis, chlamydiosis, salmonellosis, herpesvirosis, reptiles, the development of a nonregenerative ane- iridovirosis, coccidiodomycosis, and aspergillosis are mia is a slow process, which is likely because of the commonly associated with a nonregenerative anemia idiosyncrasy of many chronic diseases and the long when disseminated disease is present, or when the half-life of the reptilian erythrocyte. liver, kidney, spleen, bone marrow, and/or lungs are The most common causes of nonregenerative ane- seriously compromised.56-62 mia described in domestic animals and other exotic Renal disorders, including nephritis, nephrosis, 1-5,8,11 pets have been identified in reptiles. Neoplasia, amyloidosis, and nephrocalcinosis, have been re- systemic infectious diseases, and any moderate to se- ported in reptiles.53,63 All of the above diseases can vere chronic degenerative or inflammatory, systemic, cause a nonregenerative form of anemia. Either in- or localized disorder that affects the liver, kidney, flammatory or degenerative disease conditions can spleen, or lungs may be accompanied by a nonregen- adversely affect liver and renal health, ultimately 4,5,8,52-55 erative anemia typical of chronic diseases. resulting in the development of nonregenerative anemia (Fig 10).63 Hepatic lipidosis is a common liver disorder in captive lizards, and many infectious agents have been reported as underlying causes of hepatitis.64,65 Tumors, toxins, and other liver disorders are rare but can also cause anemia (Fig 11). Internal hemorrhage caused by the rupture of blood vessels in highly vascularized or ulcerative tumors may cause a regenerative anemia, but most neoplastic conditions are accompanied by nonregenerative forms of anemia.52 Chronic pneumonia caused by bacterial, viral, or mycotic infections are common in lizards, snakes, and chelonians, and should be investigated in the anemic patient.56-62 Gastrointestinal disorders associated with parasitic infestation, chronic diarrhea, and inflammation can result in a nonregenerative anemic condition.40 Figure 9. Ingestion of zinc pieces from a broken aquarium ther- Other causes of nonregenerative anemia observed in mometer weight by a red-eared slider (Trachemys scripta). Photo birds and other domestic animals may apply to the courtesy of Dr. Jennifer Burr. differential list of the anemic reptile, including hy- Anemia in Reptiles 105

Figure 11. Hepatic lymphoma. Tumors are often associated with nonregenerative anemia in reptiles. Photo courtesy of Dr. Tracey McNamara. Figure 12. Blood film from a spiny-tailed lizard with erythrocytes showing moderate hypochromasia (post-hibernation anemia). Photo courtesy of Dr. Jaime Samour. perestrogenism, leukemia, nutritional deficiencies, stomatitis, chronic gastritis, starvation, hypovitamin- osis A, urinary calculi, amyloidosis, gout, metabolic tion in the delivery of oxygen to the tissues are bone disease, chronic stress, and hypothyroidism.66 briefly discussed below. A complete description of Chemotherapy for cancer and the use of certain the clinical signs typically associated with most antibiotics (e.g., chloramphenicol) may induce my- reptilian diseases that result in secondary anemia elosuppression and nonregenerative anemia.52 Pri- is beyond the scope of this review. The reader is mary causes of bone marrow suppression that have encouraged to look at other sources for more been reported in mammals and birds may also occur details.1,66 4,5,8 in reptiles. Typical clinical signs of anemia in vertebrates Most nonregenerative anemias will be of the nor- are weakness, exercise intolerance, reluctance to mocytic normochromic type, indicating that hema- walk, tachycardia, and dyspnea. However, reptiles’ tological indices will be within reference ranges for physiological idiosyncrasies and captive conditions the species being considered. Hypochromic anemia could contribute to subclinical disease signs that in reptiles is rare and is classically associated with are hard for even the most dedicated keeper, ferropenic anemia. The development of ferropenic owner, or veterinarian to recognize. Moreover, anemia has been associated with excessive iron loss mild to moderate decreases in the number of cir- due to sequestration by bacteria, iron-deficient diets, culating RBCs and Hb concentration are not nec- or incomplete iron absorption as seen in malabsorp- essarily accompanied by clinical signs. Given their tion syndromes and chronic diarrhea.3 Rarely, a lo- restricted activity, reptiles kept in small enclosures calized infection will manifest with hypochromic rarely show respiratory distress, even when suffer- anemia. The primary disorders in iron metabolism ing from the most severe cases of anemia. It is only that are associated with nonregenerative hypochro- when the nonregenerative anemia is moderate to mic anemia in other classes of vertebrates have not severe, and when other clinical signs associated been reported in reptiles. Mild anemia with hypo- with the primary cause become evident, that the chromia is also observed in reptiles coming out of anemic condition will be clinically diagnosed. brumation (Fig 12). Moderate to severe paleness of oral and cloacal mucosa is usually an indication of anemia in a reptile Clinical Presentation of Anemia patient. An observant owner should be able to detect in Reptiles this change in the mucosal color. However, this examination parameter is difficult to observe in reptile Clinical signs of anemia can be divided into two species with a pigmented oral mucosa. Other com- main groups: those associated with a reduction in mon signs of chronic, nonregenerative anemia in the delivery of oxygen to the tissues and those that reptiles, although nonspecific, are prolonged hiber- are associated with the primary cause of the ane- nation or brumation times, anorexia, lethargy, and mic condition. Only those associated with a reduc- depression. 106 Saggese

Diagnosing Anemia in a Reptile 300,000 to 2,500,000 erythrocytes/uL. More specific range values have been provided for lizards When compared with nonreptilian species, the princi- (1,000,000 to 1,500,000 erythrocytes/␮L), snakes ples and approaches used for the diagnosis of anemia (700,000 to 1,600,000 erythrocytes/␮L), and chelo- in reptiles are the same. Based on the limited knowl- nians (300,000 to 500,000 erythrocytes/␮L).2-5,8,28-30 edge existing for reptiles, anemia rarely appears to It becomes self-evident that for each group, when constitute a single or primary problem. Often, anemia total RBC counts are well below the low reference is just another sign or manifestation of an associated range, the RBC count can be useful in identifying medical condition or disease. Therefore, an exhaustive anemia. However, only a limited number of cases will examination of the whole patient is required to identify be severe enough to fall under the lower ranges. the etiology for the underlying disease that may be Mild to moderate physiologic changes may occur contributing to the anemic condition. with the total RBC count falling within the reported Most anemic conditions identified in reptiles will overall reference range, even if anemia is present. have a traumatic, degenerative, inflammatory, neo- Therefore, cautious interpretation of published data plastic, toxic, metabolic, nutritional, or infectious and evaluation of other hematological values, indi- etiology.1-5,8,11,13,14,19,36,38 In cases where one of these ces, and cell morphology is required when evaluat- etiologies is not self-evident, a complete anamnesis, ing erythrocytic parameters of reptile patients. evaluation of the husbandry, thorough physical ex- The same considerations mentioned for RBC amination, and judicious use of appropriate comple- counts may apply to the PCV and Hb concentration. mentary diagnostic tests (e.g., fecal tests, radio- Subsequent calculation of the hematological indices graphs, ultrasound, urinalysis, complete blood could help to further characterize the anemia. For count, plasma protein electrophoresis, plasma bio- example, by measuring the average size of the eryth- chemistry panel, bacterial cultures, fungal cultures, rocytes (MCV), it would be possible to determine testing using polymerase chain reaction technology, whether the patient has a microcytic, normocytic, or fine-needle aspirates, biopsies, coeliotomy, laparos- macrocytic anemia. Additional characterization of copy, blood cultures, lung washes, bronchial washes) the anemia may be achieved through measuring the can be done to determine the underlying cause of MCH to determine whether a reptile is hypochromic anemia in a reptile patient.42,53,63,64,67-70 or normochromic. Overall, anemia in reptiles is usu- Given the idiosyncratic physiological and behav- ally of the normocytic and normochromic type.1-5,8 ioral characteristics that tend to mask signs of disease Hypochromic anemias are less commonly observed in reptile patients, overt signs of sickness may not be and usually associated with iron deficiency, heavy evident for many owners. Without the observed pres- metal toxicosis, starvation, and malnutrition.4,5 ence of other clinical signs, anemia is easily over- Other types of anemias (e.g., microcytic, macrocytic) looked in its early stages. Annual or biannual physical have not been widely reported in reptile species. examinations and diagnostic testing are recom- To determine the etiologic origin of an anemic mended for early diagnosis of many diseases in rep- condition requires the examination of the plasma tiles, including anemia. color, investigation of blood films for RBC morphol- A complete investigation of the reptile hemogram ogy and the presence of hemoparasites, and mea- is one of the most valuable, simple, and least expen- surement of the reticulocyte count.2,4,5,8,9 The reticu- sive diagnostic tools available to the practitioner in- locyte count reflects the ability of the bone marrow terested in identifying the cause of anemia or disease to produce new blood cells. Newly produced eryth- condition in reptile patients. A complete hematolog- rocytes can be easily identified and counted manu- ical investigation should include a review of the RBC ally when stained with vital stains (e.g., new methyl- numbers, morphology and size, and the accompany- ene blue). Given that reticulocytes are part of the ing white blood cell response.3-5,8 regenerative response to anemia in birds and mam- The first specific step needed when investigating mals, they are usually a good quality indicator as to the cause of a reptile anemia is to estimate the total whether the anemic condition is regenerative or RBC count, PCV, and Hb concentration. Unfortu- nonregenerative in reptiles, and can also provide nately, reference values for these parameters are information about bone marrow function. However, only reported for a small percentage of reptiles, and it is important to note that reptiles have a very long certain parameters, such as the total RBC count, may erythrocyte half-life (up to 600 days) and a very slow vary because of intrinsic and extrinsic factors, limit- turnover time for these cells.27 For most reptiles, the ing the value of the reference. Nevertheless, the total normal percentage of reticulocytes is less than RBC counts for most reptiles usually range from 2.5%.4,5,8 Therefore, an increase in the number of Anemia in Reptiles 107 reticulocytes may not be present in reptile patients Nevertheless, although a low PCV and reddish col- during the early stages of hemolytic or hemorrhagic oration of plasma suggest hemolytic anemia, the test anemia. Failing to understand the difference be- should be repeated to rule out technical problems tween reptiles and other domestic and exotic pet that could result in a misdiagnosis. animals can result in the inability to diagnose a Because malaria is a relatively common cause of nonregenerative cause in the early regenerative ane- hemolytic anemia in reptiles, blood films should mic patient. In reptiles, it has been reported that it always be examined for the presence of pathogenic can take up to 2 months to detect an increase in hemoparasites within the RBCs.1-5,39,40,45 The pres- newly formed erythrocytes to rule out a nonregen- ence of malarial organisms will often confirm a di- erative anemia without an obvious cause.5 An obvi- agnosis of a parasite-induced hemolytic anemia. ous increase in the number of reticulocytes is a clear Care should be taken not to assign a parasitic cause indicator of regenerative anemia.2,4,5,8 of anemia when nonpathogenic hemoparasites are When evaluating erythrocyte polychromasia, it is identified (e.g., hemogregarines).4,39,40,45 Furthermore, it important to recognize that up to 1% of circulating is important to remember that a low prevalence of polychromatic cells is a normal finding in rep- malarial parasites is not necessarily associated with tiles.4,5,8 Furthermore, a polychromatic erythrocytic clinical signs and hemolysis. Other causes of hemo- percentage of more than 1% is usually considered a lytic anemia in reptiles may include drug-induced good indicator of RBC regeneration. Conversely, a toxicity and heavy metal toxicosis, and these should lack of polychromasia suggests a nonregenerative be investigated following the same diagnostic proce- anemia. Repeated evaluation of more than 500 RBCs dures used in small animal and avian medicine. may be required to estimate the true status of poly- Diagnosing internal hemorrhage in reptiles can chromasia in the reptile patient. Presence of mitotic be challenging. In most cases, comprehensive phys- figures within the erythrocytes, other immature cells, ical examination and judicious use of complimen- and basophilic stippling of erythrocytes are good tary imaging techniques should be effective enough indicators, although not exclusive, of regeneration.3-5,8 to identify this type of anemia. Recovery of blood by When investigating the anemic reptile, practitio- gentle flushing of the coelomic cavity will be highly ners should keep in mind that in certain physiolog- supportive of a diagnosis of internal hemorrhage. If ical stages, variations in RBC morphology may occur. these other diagnostic tests are insufficient to con- Growing reptiles and those undergoing ecdysis may firm active internal hemorrhage, an exploratory coe- have increased numbers of polychromatic erythro- liotomy or coelioscopy can be performed to confirm cytes. In brumating reptiles, the number of cells with the diagnosis. mitotic figures may be more prevalent. The condi- Secondary poisoning with the newer coumarinic tion described above should be considered when rodenticides is a possible cause of hemorrhage in trying to establish a diagnosis of anemia.5,8 On the reptiles fed contaminated rodents.43 In many cases, contrary, anemia is easily masked if blood samples melena and epistaxis will be present, and it is rare for are taken shortly after brumation. The PCV and Hb internal hemorrhage to be the only overt clinical concentrations will be elevated because of the natu- manifestation. A definitive diagnosis for rodenticide ral dehydration process that occurs during the time toxicity can be made in reptiles with the same tech- of brumation. A new blood sample should be taken niques recommended for birds and mammals. Mea- 2 to 4 weeks after the initial test to verify the results, suring vitamin K–dependent clotting factors is pos- thus taking into account normalization of the bru- sible in reptiles and may contribute to the diagnosis mation-affected blood values if anemia is suspected. of a rodenticide poisoning. Investigation of the pro- Most regenerative causes of anemia become obvi- thrombin, partial thromboplastine, and coagulation ous after completing the anamnesis, physical exam- times may also have some diagnostic value if the ination, hematological investigation, and ancillary results are compared with those of unaffected indi- diagnostic tests. For example, sudden changes in viduals from the same species, although the signifi- PCV are suggestive of a hemolytic or hemorrhagic cance of these tests in reptiles needs further investi- disorder. Usually, a sudden drop in PCV is accom- gation. Primary hemostatic disorders are very rare in panied by a reddish color of the plasma or serum if reptiles and constitute an area with little scientific a hemolytic etiology is present. The plasma color of investigation. reptiles is generally a yellow/clear to orange color, Nonregenerative causes of anemia present as a but may have a greenish tinge to it. Therefore, a completely opposite picture to the regenerative re- sudden drop in PCV without changes in plasma sponse. The absence of reticulocytes, polychroma- color is more suggestive of a hemorrhagic problem. sia, and mitotic figures is usually a good indicator 108 Saggese of nonregenerative anemia. However, given that a that is part of some intravenous catheter sets when small percentage of reticulocytes (Ͻ 1%) and poly- performing this procedure. chromatic erythrocytes (Ͻ 2.5%) are usually found General anesthesia is highly recommended when in the blood of healthy reptiles, a clinician may not performing a bone marrow biopsy, except in the be able to use the absence of these cells as confir- critical patient in which the risk of anesthesia could mation early in the anemic process. Again, these are compromise the patient’s condition. In this last case, general reference values and may not apply to in- the use of a local anesthetic is recommended. Once dividual cases; therefore, careful interpretation of the area is aseptically prepared, a small incision these parameters is required. should be made on the skin and the needle inserted The diagnosis of nonregenerative causes of anemia into the bone with continuous and steady pressure requires the combination of collecting a complete and a slight left-right rotational movement. Once the patient database, thorough physical examination, needle is within the medullary cavity, the stylet and the investigation of most common secondary should be removed and a syringe attached for sam- causes of nonregenerative anemia. If a diagnosis ple collection. Bone marrow can be collected by cannot be made based on this information, a bone aspirating the syringe with a slight but steady nega- marrow aspirate is required. tive pressure. No anticoagulant is needed. Once the sample is collected, multiple films can be prepared for evaluating cell morphology and numbers. Sam- Evaluating the Bone Marrow ples can be stained with Romanowsky or hematoxy- lin/eosin stains to evaluate the cells, and Gram’s A bone marrow biopsy is required to determine a and/or Ziehl-Neelsen stains to screen for microor- reptile’s status in a case of nonregenerative anemia. ganisms. If a bone marrow culture is desired, the hub An examination of the cell types from the bone of the needle can be swabbed and submitted for marrow will allow the clinician to determine why the bacteriology, mycology, or virology.60,67,70 hematopoietic organ is not producing cells in the A different approach and technique are recom- wake of needed production.4,5,11 Primary failure of mended in snakes and other reptiles lacking hind and erythropoiesis (i.e., the presence of bone marrow forelimbs. The rib is the preferred site for a bone aplasia, hypoplasia, or both) is the most common marrow biopsy in these animals, and collecting the cause of nonregenerative anemia due to bone mar- sample requires a surgical approach. This procedure row origin. Possible reasons for a primary failure of should be done with the patient under general anes- bone marrow erythropoiesis include immune-medi- thesia. Once the site is aseptically prepared, a section of ated, genetic, radiation, and drug-mediated causes. a rib can be removed and submitted for testing.4,5,8 Infiltration of bone marrow with fat, inflammatory When evaluating the bone marrow biopsy, it is im- cells, or fibrosis may also result in a reduction in RBC portant to review the myeloid:erythroid relationship, production. A careful anamnesis will help to rule out quantitative evaluation of different RBC precursors, possible radiation or drug-induced causes for de- morphological abnormalities, presence of inflamma- creased RBC production; however, diagnostics for tory cells and fatty deposits, and presence of aplasia or immune-mediated and genetic causes are not widely hypoplasia. In some cases, hyperplasia of bone marrow available for reptiles. A general lack of basic research RBC precursors is observed and indicates regeneration limits our ability to diagnose some of these etiolo- and normal bone marrow function. However, ineffec- gies, and little information about bone marrow in- tive erythropoiesis may occur if RBC precursors are terpretation has been published for reptiles.4,5 observed but not their circulating forms. Seven basic A bone marrow biopsy can easily be collected from stages of erythrocyte maturation have been reported: lizards, chelonians, and crocodilians. The preferred rubriblasts, prorubricytes, basophilic rubricytes, early biopsy sites are the femur and tibia. The landmarks for polychromatic rubricytes, late polychromatic rubri- inserting the biopsy needle are the trochanteric fossae cytes, polychromatic erythrocytes, and mature erythro- (e.g., lizards and crocodilians) and tibial crest (e.g., cytes.4,5,27,71,72 Relative percentages of these different lizards, chelonians, and crocodilians). The technique is cell populations in healthy or anemic reptile patients conceptually similar to the biopsy technique per- are not available. formed in birds. A spinal needle or a regular hypoder- When suspected, identification of infectious cau- mic needle of adequate size and length can be used to ses of osteomyelitis should also be investigated. collect the sample. When using a regular needle, it is An increased number of inflammatory cells in the best to use a stylet to avoid blocking the needle channel biopsy sample may be suggestive of osteomyelitis. with bone and/or cartilage. The author uses the stylet Polymerase chain reaction, culture, and specific Anemia in Reptiles 109 stains are necessary to identify fungi as well as various References bacteria in the bone marrow. Additional staining with Periodic acid Schiff, Congo red, and Prussian 1. Frye FL: Hematology as applied to clinical reptile blue are useful to detect amyloid, fat deposits, and medicine, in Frye FL (ed): Biomedical and Surgical iron in the bone marrow. Aspects of Captive Reptile Husbandry. Melbourne, FL, Krieger Publishing Co, pp 209-277, 1991 2. Rosskopf WJ: Disorders of reptilian leukocytes and erythrocytes, in Fudge AM (ed): Laboratory Medicine. Therapeutics Philadelphia, PA, Avian and Exotic Pets, pp 198-204, 2000 Very little information is available regarding treatment 3. Campbell TW: Clinical pathology of reptiles, in Mader DR (ed): Reptile Medicine and Surgery (ed of anemia in reptiles; therefore, specific therapeutic 2). St Louis, MO, Elsevier/Saunders, pp 453-470, recommendations cannot be provided. Anecdotal and 2006 clinical evidence suggests that the basic therapeutic 4. Campbell TW, Ellis CK: Hematology of reptiles, in principles applied to the anemic nonreptile patient Campbell TW, Ellis CK (eds): Avian and Exotic He- may also be of benefit to the anemic reptile. Detailed matology and Cytology. Ames, IA, Blackwell Publish- ing, pp 51-81, 2007 information about general husbandry, nutrition, and 5. 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Hematology of the Pascagoula map turtle (Graptemys gibbonsi) and the southeast Asian box turtle (Cuora amboinensis). J Zoo Wildl Med 39:460-463, 2008 8. Sykes JM, Klaphake E: Reptile hematology. Vet Clin Conclusion North Am (Exotic Anim Pract) 11:481-500, 2008 9. Rodak BF, Fritsma GA, Doig K: Hematology: Clinical Principles and Applications, New York, NY, Saun- Anemia is a very common presentation in veterinary ders, 2007 medicine, but it is underreported in the scientific 10. Feldman BF, Zinkl JG, Jain NC: Schalm’s Veterinary literature as it relates to reptile species. Therefore, Hematology. Philadelphia, PA, Lippincott Williams better case documentation, consistency in the inves- & Wilkins, 2000 tigation of anemia, and rapid dissemination of the 11. Murray MJ: Reptilian blood sampling and artifact considerations, in Fudge AM (ed): Laboratory Med- available information are needed to increase our icine. Avian and Exotic Pets. Philadelphia, PA, WB knowledge in this area of veterinary medicine. Col- Saunders, pp 185-192, 2000 laborative research efforts between practitioners, 12. Dyer SM, Cervasio EL: An overview of restraint and herpetologists, reptile curators, pathologists, immu- blood collection techniques in exotic pet practice. nologists, microbiologists, toxicologists, and clinical Vet Clin North Am (Exotic Anim Pract) 11:423-443, 2008 pathologists are urgently needed to augment our 13. Hernandez-Divers SJ: Diagnostic techniques, in Mader understanding of anemia in reptiles. DR (ed): Reptile Medicine and Surgery (ed 2). St Louis, MO, Elsevier/Saunders, pp 490-532, 2006 14. Heard D, Harr K, Wellehan J: Diagnostic sampling and laboratory tests, in Girling SJ, Raiti P (eds): Acknowledgments BSAVA Manual of Reptiles. London, Blackwell, pp 78-79, 2004 15. Lopez-Olvera JR, Montane J, Marco I, et al: Effect of The author is deeply grateful to Drs. 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