Section 4. ARAV Pre-conference Program

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1 Section 4 ARAV Pre-conference Program

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3 Reptile Clinical Pathology Vickie Joseph, DVM, DABVP (Avian) Session #121 Affiliation: From the Bird & Pet Clinic of Roseville, 3985 Foothills Blvd. Roseville, CA 95747, USA and IDEXX Laboratories, 2825 KOVR Drive, West Sacramento, CA 95605, USA. Abstract: Hematology and chemistry values of the reptile may be influenced by extrinsic and intrinsic factors. Proper processing of the blood sample is imperative to preserve cell morphology and limit sample artifacts. Identifying the abnormal changes in the hemogram and biochemistries associated with anemia, hemoparasites, septicemias and neoplastic disorders will aid in the prognostic and therapeutic decisions. Introduction Evaluating the reptile hemogram is challenging. Extrinsic factors (season, temperature, habitat, diet, disease, stress, venipuncture site) and intrinsic factors (species, gender, age, physiologic status) will affect the hemogram numbers, distribution of the leukocytes and the reptile s response to disease. Certain procedures should be adhered to when drawing and processing the blood sample to preserve cell morphology and limit sample artifact. The goal of this paper is to briefly review reptile red blood cell and white blood cell identification, normal cell morphology and terminology. A detailed explanation of abnormal changes seen in the hemogram and biochemistries in response to anemia, hemoparasites, septicemias and neoplasia will be addressed. Hematology and Chemistries Blood collection and preparation Although it is not the scope of this paper to address sites of blood collection and sample preparation, a few important points need to be explained. For best results to preserve cell morphology and decrease sample artifacts, hematologic testing should be performed as soon as possible following blood collection. Prolonged exposure to any anticoagulant will distort cell morphology and increase sample artifacts such as smudge cells. In general, EDTA is not recommended for use in reptiles because it may cause hemolysis, especially in chelonian blood samples. However, EDTA can be used in most of the lizard species without problems. Heparin has the advantage of being used as an anticoagulant for hematology and plasma for blood chemistry evaluation. However, heparinized samples may result in inadequate staining of the white blood cells, affect cell morphology, or stimulate clumping of the white blood cells and thrombocytes. 1,2 Regardless of the anticoagulant of choice, blood smears should be made immediately without exposure to anticoagulants. This preserves cell morphology and helps to prevent clumping of the white blood cells and sample artifact. Also important to remember is to air dry the blood smears with a rapid fanning of the slides. Smears allowed to dry too slowly may result in perinuclear ring artifacts. The technique used to make the blood smear is extremely important. Whether the choice is to use two slides, coverslip and slide, or coverslip to coverslip, if the technique is not perfected, lysis of the white blood cells occurs. ExoticsCon 2015 Pre-conference Proceedings 155

4 Cell identification Erythrocytes: Reptile erythrocytes are generally larger than avian and mammalian erythrocytes, but smaller than amphibian erythrocytes. The average life span of the reptile erythrocyte in the peripheral blood is days depending on the species. The slow metabolic rate of reptiles and age may play a role in the red blood cell life span. In general the packed cell volume (PCV) and the total erythrocyte count increases with age. The color of the adult cell is orange and contains a nucleus. Less than one percent of the circulating erythrocytes has a polychromatic color to the cytoplasm and is termed polychromatophilic erythrocytes. This reflects the slower turnover rate of reptilian erythrocytes and the long erythrocyte life span. They are similar in size and shape to the mature erythrocytes. These polychromatic erythrocytes reflect the final stage of maturation and are often termed reticulocytes. However a new methylene blue stain is required to verify the aggregate form of the reticulum encircling the erythrocyte nucleus and classifying these as reticulocytes. An increase of reticulocytes suggests a regenerative response to an anemia. Slight anisocytosis and poikilocytosis are considered normal in the peripheral blood. Immature erythrocytes are round to slightly irregular cells with large round nuclei and basophilic cytoplasm. They are occasionally seen in the peripheral blood of reptiles, especially very young reptiles or those going through ecdysis. Various terms used in the description of erythrocytes are used as a valuable diagnostic tool. 1-3 Hypochromasia reflects an erythrocyte with greater than half of the cytoplasm showing pallor. Microcytic erythrocytes reflect older aging cells, while macrocytic erythrocytes are cells recently released from hematopoietic tissue. Atypical erythrocytes, reflected as anisocytosis or poikilocytosis, may normally be seen in the peripheral blood in small numbers. Immature erythrocytes may vary in size, usually round, with a deeply basophilic cytoplasm and nuclear chromatin blue black in color. An important concept to remember is that all reticulocytes are polychromatic, but all polychromatic cells are not reticulocytes. Bone marrow is the primary site for erythropoiesis, granulopoiesis and thrombopoiesis, with the liver and spleen sites of extramedullary erythropoiesis. The thymus is considered the source of lymphoid cells Lymphocytes: Typically these cells are small with scant cytoplasm, a high nuclear to cytoplasm ratio, and nuclear chromatin clumping. Lymphocytes lack vacuoles but occasionally have azurophilic granules. Small lymphocytes can be confused with reptile thrombocytes. The function of the lymphocyte is similar to mammals and reflects immune stimulation. Plasma cells: This cell may occasionally be seen in the peripheral blood smear. They are larger than the normal lymphocyte, have a round nucleus that is eccentrically placed with chromatin clumping. The cytoplasm stains a deep blue and a perinuclear halo (Golgi) is present. Monocytes: A large cell with a round to amoeboid or lobed nucleus, they have less nuclear chromatin clumping as compared to the lymphocytes and the cytoplasm is abundant with a ground glass appearance. Monocytes have phagocytic capabilities and are active with chronic inflammatory conditions. The reptile monocytic cell has been referred to as monocytes, monocytoid azurophils, azurophilic monocytes or azurophils. Azurophils: An azurophilic leukocyte, the azurophil causes confusion in classification and identification. The granulopoietic origin of the azurophil has not been documented or confirmed. Many researchers believe this cell should be considered a monocyte and not a separate cell type. They may represent an immature form of the monocyte. The azurophil is slightly smaller than the monocyte, has an eccentrically placed round to oval to bilobed nucleus, basophilic cytoplasm and contains small numbers of cytoplasmic azurophilic granules. Similar to the monocyte, this cell has phagocytic capabilities. 156 Building Exotics Excellence: One City, One Conference

5 Heterophils: The heterophil is functionally equivalent to the mammalian neutrophil. Often the most abundant cell in the peripheral blood, they have a lobed nucleus, primarily rod shaped granules that are bright orange (eosinophilic) and refractive in appearance. They are highly phagocytic with bactericidal activity. Heterophils also participate in viral and parasitic infections. Eosinophils: Similar in size to heterophils, this cell has a round to oval (lobed in some lizards) slightly eccentric nucleus with primarily round granules. Although their function is not entirely known, these cells may play a role in reflection of parasitic infections or hypersensitivity reactions. In general lizards have lower number of eosinophils versus turtles. Basophils: This round cell is often smaller conmpared with the eosinophil. The granules are round and deeply metachromic in color and often obscure the nucleus, which is nonlobed and eccentrically placed. The cytoplasmic granules contain histamine and their function is similar to the mammalian basophil and mast cell. Basophils produce, store and release histamine. The normal basophil percent ranges from 0-40% (desert tortoise) depending on the species. Thrombocytes: A small nucleated cell, the thrombocyte is the second most numerous cell in the peripheral blood. They are derived from a mononuclear precursor in the bone marrow, have a high nuclear to cytoplasm ratio and may be difficult to distinguish from the lymphocyte. The cytoplasm is colorless and may contain one or more azurophilic granules. The primary function of the thrombocyte is hemostasis. Normal thrombocyte numbers range between thrombocytes per 100 leukocytes in the peripheral blood smear. Clinical points for chemistry evaluation A detailed description of each biochemical enzyme evaluated in the reptile laboratory profile will not be presented. However, changes or trends of selected enzymes in response to disease will be discussed. Total protein will increase with dehydration, chronic disease and lipemia (Table 1). Decreases will be seen with starvation, liver dysfunction, kidney disease, gastrointestinal disorders resulting in maldigestion or malabsorption, and parasitism. 4 Protein electrophoresis for detailed protein evaluation is often recommended, especially when the globulin fractions are elevated. Physiologic elevations of calcium may be seen with dehydration, hyperalbuminemia, neoplasia and gravid females. Over supplementation of calcium or vitamin D, hyperparathyroidism, and osteolysis may also cause elevations of calcium. Hypocalcaemia is usually the result of a nutritional imbalance. Elevations of phosphorus may normally be present in the young growing reptile. However dietary excess, hypervitaminosis D, and renal disease may cause phosphorus elevations. Uric acid is a primary catabolic product of protein metabolism and tends to be higher in carnivores versus herbivores. Uric acid is often used to evaluate renal disease in the reptile, however 70% of kidney must be damaged for this value to elevate. Elevations may also be seen with gout, nephrocalcinosis and septicemia. An inverted Ca:P ratio is a reliable indication of renal disease. 4-6 Elevations of both phosphorus and potassium are often related to hemolysis of the sample, and may not reflect metabolic abnormalities. Similar to birds, reptile ALT and AST are not tissue specific. AST may be found in high concentrations in the liver and muscle, including the myocardium. Caution is used when evaluating AST in liver disease. Interpretation of reptile electrolyes follows the same guidelines as other animals. Elevations of sodium are seen with dehydration, while low values may reflect gastrointestinal losses. Elevations of potassium may be seen with dietary excess, acidosis or hemoylsis of the blood sample. Hypokalemia is seen with gastrointestinal losses or inadequate intake. Elevations of chloride may be seen with dehydration or renal disease. ExoticsCon 2015 Pre-conference Proceedings 157

6 Table 1. Reptile hematology guide. Value Increase Decrease PCV 1. Dehydration 1. Regenerative anemia, polychromasia 2. Abnormal myeloid production 2. Nonregenerative anemia, depression anemia 3. Hemolytic anemia 4. Hemorrhagic anemia TP 1. Dehydration (elevated alb) 1. Metabolic disorder (liver, kidney, GI) 2. Chronic inflammatory 2. Parasites disease (elevated globulins) 3. Nutrition WBC 1. Chronic inflammatory 1. Viral 2. Bacterial infections 2. Severe chronic infection 3. Parasites 3. Overwhelming septicemia 4. Fungal 4. Toxic chemicals Thrombocytes 1. Last cell line to become 1. Decrease production reactive 2. Increase peripheral utilization 2. Reactivity with any disease 3. Phagocytic properties Heterophils (+/- toxic) 1. Bacterial 1. Viral infections 2. Chronic inflammatory disease 2. Overwhelming septicemia 3. Parasitic infections 4. Stress 5. Neoplasia Lymphocytes 1. Viral 1. Bacterial (+/- reactive) 2. Parasites 2. Immunosuppressioin 3. Wound healing 3. Chronic stress 4. Ecdysis 4. Chronic malnutrition 5. Immune stimulation Monocytes/azurophils 1. Chronic bacterial infection 2. Granulomatous disease 3. Parasites 4. Chronic inflammatory disease Eosinophils 1. Parasites 1. Significance not known 2. Allergic reaction 3. Immune stimulation Basophils 1. Respiratory tract disease 1. Significance not known 2. Parasites 158 Building Exotics Excellence: One City, One Conference

7 Abnormal Clinical Pathology Evaluating the reptile hemogram is challenging. Extrinsic factors (season, temperature, habitat, diet, disease, stress, venipuncture site) and intrinsic factors (species, gender, age, physiologic status) will affect the hemogram numbers, distribution of the leukocytes and the reptile s response to disease. Low environmental temperatures may suppress and inhibit the immune response of the reptile. Lymphocyte counts in general are lower during the winter months and higher during the summer. Variation of the heterophil numbers is similar to the lymphocyte seasonal variation. Reptiles have the ability to stimulate a marked leukamoid response to an inflammatory disease. This can be confused with an emerging neoplastic disorder, but will resolve itself with treatment of the underlying disease process. Blood parasites Blood parasites are common in the reptile species and are often considered an incidental finding. However, some blood parasites have the potential to cause disease and hemolytic anemia. 2 Hemoproteus species: is a protozoan parasite found lizards, turtles and snakes, and resembles the Hemoproteus species found in birds. Hemoproteus is transmitted by blood sucking vectors such as midges, mosquitoes and hippoboscid fly. Clinical signs associated with an infection may include anorexia and lethargy. This parasite will cause dehemoglobinization of the infected erythrocyte. The gametocyte may appear as a developing ring or as an elongated crescent-shaped mature gametocyte encircling the erythrocyte nucleus. The granules in the gametocyte contain refractive yellow to brown pigments representing iron pigment deposition. It is also possible to see extraerythrocytic gametocytes in the peripheral blood. Plasmodium species: is a protozoan parasite that has the potential to cause severe hemolytic anemia and illness in the reptile. The plasmodium life cycle does require the sporogony stage to develop in the mosquito, while the schizogony and gametogony stages develop in the reptile host. This parasite may also distort the erythrocyte and push the nucleus to one end. The presence of schizonts (round to oval inclusions containing deeply staining merozoites) is diagnostic for the identification of Plasmodium sp. Trypanosome species: is occasionally found in the peripheral blood of reptiles. Transmission is usually a result of a biting insect such as the mosquito, hippoboscid fly, black flies and mites. The leech appears to be the invertebrate host transmitting Trypanosome in the aquatic and semi aquatic reptiles. These large parasites have an undulating membrane, a slender tapering posterior end and a short anterior directed flagellum. They are considered to be an incidental finding and not considered pathogenic. Microfilaria: is not an uncommon finding in the peripheral blood. For the most part, microfilaria is not considered pathogenic. Reptiles can survive years with this parasite. Piroplasmids: of reptiles include Sauroplasma (lizards), Serpentoplasma (snakes), Aegyptianella and Babesia. Transmission of this parasite occurs by biting insects or arthropods. The parasite appears as a small round to piriform, nonpigmented signet-ring like vacuole in the erythrocyte cytoplasm. Lainsonia and Schellackia spcies: are coccidian parasites, resembling avian Atoxoplasma, and are found in lizards and snakes. This parasite is a round to oval, pale staining, nonpigmented inclusion that deforms the host cell s nucleus (erythrocyte and lymphocyte) into a crescent shape. ExoticsCon 2015 Pre-conference Proceedings 159

8 Hemogregarine: parasites are the most common hemoparasite affecting reptiles, except for sea turtles and tortoises (considered rare). Hemogregarine, Hepatozoon and Karyolysus species are the three genera commonly found in the reptiles. However classification of this parasite cannot be accomplished based on the gross appearance of the gametocyte within the erythrocyte cytoplasm, so the general term hemogregarine is used when reporting the presence of this parasite. Mites, ticks, mosquitoes and flies transmit this parasite to terrestrial reptiles, while leeches are the primary intermediate host for transmission to aquatic reptiles. The sausage shaped intracytoplasmic gametocyte in the reptile erythrocyte identifies Hemogregarine, and often distorts the erythrocyte nucleus. Erythrocytes Erythrocytic values will be affected by species, gender, age and physiologic status of the reptile, season, temperature, habitat, diet, disease, stress, and venipuncture site. Lymphatic contamination of the blood sample draw often occurs in the reptile and will affect the hemogram by decreasing the hematocrit, hemoglobin concentration, total red and white blood cell count. In general, the erythrocytic indices are at their highest prior to hibernation and lowest immediately following hibernation. Hypochromasia: greater than 2+ may be a result of nutritional deficiencies leading to iron deficiencies, lead toxicity or chronic inflammatory disease Anisocytosis: greater than 1+ with an increase of polychromasia suggest a regenerative anemia Poikilocytosis: greater than 1+ is associated with erythrocytic regeneration (anisocytosis and poikilocytosis may also be found in the post hibernation reptile or those with severe inflammatory disease) Basophilic stippling suggests a regenerative response, but may also occur in reptiles with iron deficiency or less commonly lead toxicity. Peri-nuclear rings or clear irregular refractile spaces in the cytoplasm are artifacts in the erythrocyte as a result of the blood smear drying too slowly. 2,5 Do not confuse these with gametocytes of Hemoproteus or Plasmodium. Round to irregular basophilic inclusions (clumping endoplasmic reticulum) are frequently seen in the erythrocyte cytoplasm and are considered artifact of slide preparation. Anemia The normal packed cell volume (PCV) ranges from 15-55%. Values above this range with an elevated total protein suggest dehydration or erythrocytosis (polycythemia) with a normal or low total protein. Values below this range suggest anemia if hemodilution with lymph is not a factor. Anemia is classified according to pathophysiology and termed hemolytic, hemorrhagic or hypoplastic (depression anemia). Polychromasia is usually not graded as it is in the avian species, due to the fact that polychromasia is less than1% of the total erythrocyte population. The presence of increased polychromatic erythrocytes and reticulocytes is evidence of active erythropoiesis. The presence of rubriblasts indicates a marked erythropoietic response to anemia. Hemorrhagic anemia: is typically a result of traumatic injuries. Blood sucking parasites and conditions such as coagulopathies or ulcerative lesions should also be considered. Hemolytic anemia: is often due to systemic or hematogenous bacterial infections (sepsis), toxins (lead, zinc, aflatoxins, petroleum products and certain plants), hemoparasites (plasmodium), or infectious diseases. This usu- 160 Building Exotics Excellence: One City, One Conference

9 ally presents with a strong response to the anemia with small round erythrocytes (spherocytes) and biliverdinuria due to extravascular hemolysis. Hypoplastic anemia: is a non-regenerative, normocytic, normochromic anemia. Often termed depression anemia, this is the most common anemia seen in the reptilian species. There is a decrease of erythropoiesis due to chronic inflammation or infection, drug reaction, renal or hepatic disease and neoplasia. Leukocytes Leukocyte parameters are affected by many factors and at times may be difficult to properly interpret. As mentioned previously, intrinsic and extrinsic factors affect the reptile s response to disease. Heterophils become active and toxic with systemic illness. The severity of the systemic illness affects the degree of toxic changes present and the number of immature heterophils released into the peripheral blood. The toxic changes seen in the heterophil include an increase of cytoplasmic basophilia, degranulation, abnormal granulation (rounding), and vacuolization. The immature heterophils consist of bands, metamyelocytes and myelocytes. Significant increases of the heterophils are associated with inflammatory diseases, stress and neoplasia. Lymphocytes show reactivity by developing a basophilic cytoplasm or forming scalloped edges. Inclusion Body Disease (IBD) viral inclusions can be seen in the lymphocyte and confirmed with H&E stain. Plasma cells distended with clear to light blue round structures are termed Mott cells. The round structures are termed Russell bodies, which contain immunoglobulins. Monocytes/ azurophils become larger with exaggerated foamy cytoplasm. Cytoplasmic vacuoles and blebbing may occur with reactivity. A monocytosis suggests chronic or granulomatous inflammation. Monocytes have phagocytic capabilities and often engulf leukocytes and erythrocytes in response to anemia and infectious disease. They also actively engulf bacteria in a sepsis condition. Eosinophils increase with parasitic infections and stimulation of the immune system. Basophils increase with parasitic and viral infections, wounds and respiratory disease. Thrombocytes play an active role in the response to disease. The presence of an increased number of immature thrombocytes is a regenerative response to excessive utilization. A decrease of thrombocytes suggests decrease bone marrow production or excessive peripheral utilization. This may be due to septicemia or disseminated intravascular coagulation (DIC). Activated thrombocytes will appear as aggregated clusters of cells. Total protein Most plasma proteins are synthesized in the liver. Exceptions are the immunoglobulins produced by the lymphocytes and plasma cells. Their biological activity is dependent upon their primary and secondary structure (enzymes, hormones, transport and carrier compounds). Hypoproteinemia may be a result of chronic hepatopathies, malabsorption, protein-losing enteropathies, renal disease, blood loss, malignant tumors, starvation, and malnutrition. Hyperproteinemia may result from chronic infectious disease and overproduction of gamma globulins, lymphoproliferative disease or dehydration. ExoticsCon 2015 Pre-conference Proceedings 161

10 Leukocyte response to disease Reptiles with a severe inflammatory disease (usually bacterial etiology) develop a marked leukocytosis with the presence of immature heterophils (bands, metaheterophils, myeloheterophils, progranulocytes). 1-3,5 Lymphocytosis may occur with wound healing, parasitic infections, viral infections and inflammatory disease. A lymphocytosis may also be present during ecdysis. The presence of reactive lymphocytes and plasma cells reflects stimulation of the immune system. Leukopenia reflects the consumption or decreased production of the peripheral leukocytes. The presence of an absolute heteropenia in conjunction with immature heterophils and toxicity represents overwhelming demand for heterophils in the periphery due to bacterial sepsis or viral disease. This is a degenerative response and carries a grave prognosis. Leukopenia with heteropenia, anemia and thrombocytopenia suggest injury to the bone marrow. Heteropenia may suggest acute inflammatory disease, degenerative response to disease, stem cell injury or bone marrow toxicity (chemicals, drugs, viral). A heteropenia with no left shift, no anemia and adequate thrombocytes may suggest an acute viral infection or acute marrow toxicity. Lymphopenia may occur secondary to diseases that cause immunosuppression, chronic malnutrition and chronic stress. References 1. Nardinin G, Leopardi S, Bielli M. Clinical hematology in reptilian species. Vet Clin Exot Anim. 2013;16: Campbell TW, Ellis CK. Hematology of reptiles. In: Campbell TW, Ellis CK, ed. Avian and Exotic Animal Hematology and Cytology. 3 rd ed. Ames, IA: Blackwell Publishing; 2007: Campbell TW. Clinical pathology. In: Mader DR, Divers SJ, ed. Current Therapy in Reptile Medicine and Surgery. 1 st ed. St Louis, MO: Elsevier Saunders;2014: Griswold WG. Basic reptilian clinical pathology. Paper presented at: Western Veterinary Conference; February 20, 2005; Las Vegas, NV. 5. Campbell TW. Comparative clinical chemistries in exotic animal medicine. Paper presented at: Western Veterinary Conference; February 23, 2011; Las Vegas, NV. 6. Campbell TW, Grant K. Herptile hematology case studies. In: Campbell TW, Grant K, ed. Clinical Cases in Avian and Exotic Animal Hematology and Cytology. 1 st ed. Ames, IA: Wiley-Blackwell Publishing; 2010: Building Exotics Excellence: One City, One Conference

11 Reptile Sedation, Anesthesia and Analgesia Christoph Mans, Dr med vet, Dipl ACZM Session #284 Affiliation: From the School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA. Abstract: Utilizing chemical restraint methods in reptile patients is commonly needed for diagnostic and therapeutic procedures. However, the significant differences in anatomy and physiology amongst different groups of reptiles as well as several other factors such as body temperature, injection site and route as well as underlying disease processes make sedation and anesthesia in reptiles challenging at times. Reducing environmental temperatures below the preferred temperature range of reptile patients will lead to a delay in onset of action of anesthetic drugs, longer action and delayed recovery. If temperatures are increased, onset of sedation or anesthesia is more rapid, but the duration of effect can be shorter. Therefore it is critical to ensure that reptile patients are maintained at an appropriate temperature prior to and during sedation and anesthesia. Sedation, Anesthesia and Analgesia Injection of sedative and anesthetic drugs in the caudal body-half should be avoided if possible, since some drugs may undergo a hepatic first pass effect, which may be more important for most anesthetic drugs, compared to the more often discussed renal first-pass effect. 1,3 Plasma levels and efficacy of anesthetic drugs can be greatly reduced and may require repeated drug administration. 3 Historically the intramuscular route has been recommended as the route of choice for non-vascular administration of anesthetic and sedative drugs in reptiles (Tables 1 and 2). However, the subcutaneous route provides a suitable alternative, in particular in animals with reduced muscle mass or if larger volumes of drugs need to be administered. While the onset of effect is often slower following subcutaneous administration, the depth of sedation or anesthesia reached is ultimately similar following intramuscular administration. 1 By considering short acting drugs and partial or completely reversible drug protocols for sedation and anesthesia, prolonged recoveries or fatalities can be avoided. Sedation often is sufficient for most diagnostic procedures as well as certain therapeutic procedures, particularly if combined with local anesthetics (Table 1). 2 Lidocaine and bupivacaine can be used for local infiltration, nerve block as well as for spinal anesthesia in reptiles. 2,4 Our knowledge on reptile analgesia continues to grow, and increasing scientific evidence is available to guide the selection of drugs which are likely to provide analgesia in reptiles. 1,5 However, the side effects of opioid analgesics, such as sedation and respiratory depression and well as differences in response amongst groups of reptiles should be carefully considered. In general most mu-opioid receptor agonists have been shown to provide analgesia in turtles and tortoises as well as lizards and crocodilians. Drugs such as morphine, hydromorphone, fentanyl and tramadol are most commonly used (Table 3). 5-7 However, limited information is available in regards to drug side effects across different reptile species. ExoticsCon 2015 Pre-conference Proceedings 163

12 Table 1. Sedation protocols commonly used in reptiles. a Protocol Dose [mg/kg] Route b Species Comments Chelonians Midazolam 2 SC/IM Red-eared slider Mild sedation, inconsistent effects, completely reversible Mild to moderate sedation, Dexmedetomidine Red-eared slider SC/IM completely reversible; rapid + midazolam NA box turtles recovery after reversal Dexmedetomidine Moderate sedation, partially midazolam + SC/IM Red-eared slider reversible; rapid recovery after ketamine reversal Medetomidine + Deep sedation, suitable for SC/IM Red-eared slider ketamine intubation Deep sedation - light anesthesia, Medetomidine SC/IM Tortoises partially reversible; rapid recovery ketamine + morphine after reversal Deep sedation - light anesthesia, Medetomidine SC/IM Tortoises completely reversible; rapid midazolam + morphine 1 recovery after reversal. Alfaxalone 10 IM Red-eared slider Moderate sedation, suitable for intubation Propofol 2-5 IV Moderate sedation - light anesthesia Lizards Moderate sedation, completely Dexmedetomidine 0.1 Bearded dragon, SC/IM reversible; rapid recovery after + midazolam + 1 green iguana reversal Dexmedetomidine + midazolam + ketamine SC-IM Bearded dragon, green iguana Alfaxalone 10 IM, IV Green iguana Moderate - deep sedation, partially reversible; rapid recovery after reversal Light-moderate sedation, maximum effect after ~ 5 min, duration of effect ~ 10 min Propofol 3-5 IV, IO Deep sedation - light anesthesia Snakes Midazolam 1-2 IM Mild sedation, inconsistent effects Telazol 2-5 SC, IM Large snakes Ketamine 5-10 SC, IM Mild-moderate sedation, endotracheal intubation Mild-moderate sedation, endotracheal intubation Propofol 3-5 IV Moderate sedation - light anesthesia a Atipamezole is used to antagonize medetomidine/dexmedetomidine in 1:1 volume, SC or IM. Flumazenil is used to antagonize midazolam: 0.05mg/kg SC, IM or IV. Nalaxone is used to antagonize morphine or hydromorphone: 0.04mg/kg SC, IM, or IV. b Abbreviations: SC=subcutaneous; IM=intramuscular; IV=intravenous; IO=intraosseous. 164 Building Exotics Excellence: One City, One Conference

13 Table 2. Anesthetic protocols commonly used in reptiles. a Protocol Medetomidine + midazolam + ketamine + hydromorphone Dose [mg/kg] Route b Species Comments Chelonians SC/IM Tortoises Medetomidine + ketamine SC/IM Red-eared slider Medetomidine + ketamine + morphine Dexmedetomidine + ketamine + hydromorphone Propofol 2-10 IV SC/M SC/IM Map turtle Propofol IV Red-eared slider Alfaxalone 20 IM Red-eared slider Surgical anesthesia, maintain with gaseous anesthetic if necessary Surgical anesthesia partially reversible Surgical anesthesia, Administration of nalaxone recommended if recovery is slow or to prevent renarcotization Surgical anesthesia, supplement with gaseous anesthesia if necessary Induction agent; use lower dose in large tortoises Light anesthesia for ~ 60 min (10mg/kg) or ~ 90 min (20mg/kg) Induction agent; light anesthesia for 28 ± 13 min at 20 C Lizards Alfaxalone IM Green iguana Induction agent, effect after 5 minutes, duration of effect min Alfaxalone 10 IV Green iguana Induction agent; maintain with gaseous anesthetic Propofol 5-10 IV, IO Green iguana Induction agent, apnea common, requires intubation and IPPV Snakes Propofol 3-10 IV, IC Induction agent; maintain with gaseous anesthetic Alfaxalone 9 IV Australian Light anesthesia, endotracheal snakes intubation Telazol 2-6 IM Induction agent; maintain with gaseous anesthetic All reptiles Isoflurane 2-5% Induction 5%, maintenance 2-3% Sevoflurane 2.5-8% Induction 7-8%, maintenance % a Atipamezole is used to antagonize medetomidine/dexmedetomidine in 1:1 volume, SC or IM. Flumazenil is used to antagonize midazolam: 0.05 mg/kg SC, IM or IV. Nalaxone is used to antagonize morphine or hydromorphone: 0.04 mg/kg SC, IM, or IV. b Abbreviations: SC=subcutaneous; IM=intramuscular; IV=intravenous; IO=intraosseous. ExoticsCon 2015 Pre-conference Proceedings 165

14 Table 3. Analgesic drugs commonly used in reptiles. Drug Dose [mg/kg] Route Species Comments Hydromorphone SC/IM Chelonians q24h Morphine 1-2 SC/IM Chelonians q24h Tramadol 5-10 PO, SC/IM Chelonians q48-72h Buprenorphine SC/IM Chelonians No analgesia demonstrated Butorphanol 1-8 SC/IM Chelonians No analgesia demonstrated Meloxicam 0.2 PO, SC/IM Green iguanas q24h, PK data only Ketoprofen 2 SC/IM Green iguanas > q24h, PK data only Drugs previously believed to have analgesic properties in reptiles, such as butorphanol and buprenorphine, have so far not shown to be effective in providing analgesia in reptiles. 7,8 Non-steroidal anti-inflammatory drugs are frequently administered to reptiles. However, to date, no evidence for analgesic efficacy of these drugs in reptiles exists. Meloxicam appears to be safe and even at high doses no adverse effects were found. 9 Future research is necessary in order to determine safe and effective analgesic drug protocols for the reptile species most commonly kept in captivity. References 1. Sladky KK, Mans C. Clinical anesthesia in reptiles. J Exot Pet Med. 2012;21: Schumacher J, Mans C. Anesthesia. In: Mader D, Divers, SJ, eds. Current Therapy in Reptile Medicine and Surgery. St. Louis, MO:WB Saunders; Kummrow MS, Tseng F, Hesse L, et al. Pharmacokinetics of buprenorphine after single-dose subcutaneous administration in red-eared sliders (Trachemys scripta elegans). J Zoo Wildl Med. 2008;39: Mans C. Clinical technique: Intrathecal drug administration in turtles and tortoises. J Exot Pet Med. 2014;23: Sladky KK. Analgesia In: Mader D, Divers SJ, eds. Current Therapy in Reptile Medicine and Surgery. St. Louis. MO: WB Saunders; Giorgi M, Salvadori M, De Vito V, et al. Pharmacokinetic/pharmacodynamic assessments of 10 mg/kg tramadol intramuscular injection in yellow-bellied slider turtles (Trachemys scripta scripta). J Vet Pharmacol Ther In press. 7. Mans C, Lahner LL, Baker BB, et al. Antinociceptive efficacy of buprenorphine and hydromorphone in red-eared slider turtles (Trachemys scripta elegans). J Zoo Wildl Med. 2012;43: Sladky KK, Miletic V, Paul-Murphy J, et al. Analgesic efficacy and respiratory effects of butorphanol and morphine in turtles. J Am Vet Med Assoc. 2007;230: Divers SJ, Papich M, McBride M, et al. Pharmacokinetics of meloxicam following intravenous and oral administration in green iguanas (Iguana iguana). Am J Vet Res. 2010;71: Building Exotics Excellence: One City, One Conference

15 Reptile Radiology Stephen J Divers, BVetMed, DZooMed, Dipl ACZM, Dipl ECZM (Herpetology), Dipl ECZM (Zoo Health Management), FRCVS, and Scott Stahl, DVM, DABVP (Avian) Session #082 Affiliation: From the Department of Small Animal Medicine and Surgery (Zoological Medicine), College of Veterinary Medicine, University of Georgia, 2200 College Station Road, Athens, GA 30602, USA. Abstract: Diagnosing disease in reptiles can be a challenge for even the most experienced veterinarian. The clinical signs exhibited by these ectotherms are often subtle and physical findings are seldom pathognomonic. Diagnostic imaging should be part of the clinical evaluation of any sick reptile. Radiology and ultrasonography are common diagnostic tools used in reptiles and can be instrumental in the identification of disease. Although less readily available to practitioners, computed topography and magnetic resonance imaging can provide excellent images. This review will focus on radiology. Introduction Diagnosing disease in reptiles can be a challenge for even the most experienced veterinarian. The clinical signs exhibited by these ectotherms are often subtle and physical findings are seldom pathognomonic. Diagnostic imaging should be part of the clinical evaluation of any sick reptile. Radiology is a common diagnostic tool used in reptile practice and can be instrumental in the identification of disease. There are several anatomical differences that make it difficult to obtain quality radiographs of good contrast and detail. The relatively small size of most pet reptiles, coupled with the lack of diffuse body fat often produces images of poor contrast. The presence of thick, highly keratinized scales, osteoderms or shells can severely hinder the x-ray beam, necessitating greater power and a subsequent loss of fine soft tissue detail. Space limitations prevent the inclusion of detailed descriptions or the addition of advanced modalities including ultrasonography, CT and MRI; however these will be presented in the lecture. 1-4 Radiographic Equipment Despite the aforementioned difficulties, most high-capacity radiographic units can be set to produce quality radiographs of reptilian patients. High-detailed screen-film combinations (eg, mammography film) are essential to obtain sufficient detail and contrast, especially in smaller animals. Many practices have moved to digital radiography which offers improvements in contrast and data storage. Various agents can be used to improve contrast. Barium sulphate (30%) can be used for gastrointestinal studies, although the authors prefer water soluble iodine compounds such as iohexol for upper gastrointestinal, urogenital and intravenous techniques. It should not be forgotten that the simple injection of air into the coelom of a lizard can greatly improve the appreciation of pre-ovulatory follicles. ExoticsCon 2015 Pre-conference Proceedings 167

16 Snakes Restraint and positioning Snakes can be difficult to position and restrain for radiographic examinations unless anesthetized. If the purpose of the examination is simply to rule out radiodense foreign bodies, the snake may be allowed to coil in its natural position while the radiograph is taken. If detailed examination of the skeletal, respiratory and digestive system is desired, the snake must be extended. A plastic restraint tube can be utilized for this purpose; however, this may produce some radiographic artifact. In larger snakes, several films will be needed to radiograph the entire length of the body. It is important to properly label each exposure in order to keep track of all the different views. Lateral views are best taken using horizontal beams to avoid displacement artifact of the viscera. However, standard laterals with the snake taped in lateral recumbency can be useful especially where horizontal beams are not possible or safe to undertake. The interpretation of dorsoventral views is hindered by the spine and ribs, but can still be useful when dealing with obvious lesions including eggs and mineralized masses. Musculoskeletal system Traumatic fractures, metabolic bone diseases, spondylitis/spondylosis, osteomyelitis, and congenital abnormalities are common indications for examining the skeletal system of snakes. Fractured ribs with periosteal bone formation are a common finding in snakes. Another common finding is exuberant vertebral periosteal bone formation. On radiographs, this appears as several fused vertebrae. Digestive system Common indications for radiographically evaluating the digestive system include hypertrophic gastritis, foreign body ingestion/impaction, constipation, hepatomegaly and hepatic masses. Contrast studies are useful in diagnosing intestinal obstruction and constipation. In addition, contrast material in the gastrointestinal tract can often outline and help determine the origin of a non-specific intracoelomic masses; intraluminal or extraluminal. Cardiopulmonary system Cardiomyopathy has been reported in snakes, which can be indicated by cardiomegaly on radiographs. Metastatic mineralization of large blood vessels is often apparent around the heart due to the negative contrast afforded by the adjacent lung(s). The superimposition of other organs such as liver and stomach over the lung fields can make the radiographic interpretation of respiratory disease challenging. Common indications for evaluating the respiratory system are rhinitis, suspected neoplastic and infectious disorders of the trachea and lung, as well as abscesses or granulomas. Urogenital system The kidneys are not always radiographically evident, unless enlarged or mineralized. Disease processes that can cause renomegaly include renal gout and neoplasia. Eggs of oviparous species are leathery and poorly calcified, but can often be appreciated on plain radiographs. In viviparous species fetal skeletons become visible as they mineralize late in gestation. The hemipenes of some species may appear mineralized and can be detected radiographically. Common indications for evaluating the reproductive system include dystocia, apparent infertility and reduced fecundity. 168 Building Exotics Excellence: One City, One Conference

17 Miscellaneous The presence of any swelling is an indication for radiography. Abscesses, which can either be extracoelomic or intracoelomic and associated with a specific organ or the coelomic wall are common findings in snakes. Lizards Restraint and positioning Small lizards can often be restrained by taping them to the radiography film or table for a dorsoventral view. Placing cotton balls over the eyes, and wrapping them with self-adhesive tape (VetWrap, 3M Products) will often produce a calm, motionless lizard. Placing a mirror in front of a lizard can distract the animal long enough to take a radiograph. A dorsoventral view can be helpful to identify foreign bodies, intestinal impaction, or coelomic masses. A horizontal x-ray beam provides the best lateral imaging in lizards, especially when evaluating the respiratory system. The positioning for this view involves rotating the x-ray tube 90 degrees and placing the film vertically behind the lizard. Elevating the body of the lizard on rolled towels or foam pads helps to prevent superimposition of the limbs with the coelomic cavity. The positioning for, and interpretation of, crocodilian radiographs are similar to those employed for lizards. Skeletal system Radiography is very useful to identify skeletal abnormalities in lizards such as metabolic bone diseases, osteomyelitis, gout, fractures and septic arthritis. Of all the metabolic bone diseases, nutritional secondary hyperparathyroidism is most common. Radiographically, this condition can be manifested as generalized lack of density of all bones, cortical bone thinning and/or pathologic fractures. In addition, fibrous osteodystrophy is manifested by soft tissue swelling of the limbs, in conjunction with lack of cortical bone density of the long bones and spine. Other common radiographic signs of bone disease can include folding or incomplete fractures, chronic luxation of toes and ribs, hypertrophic osteopathy, scoliosis, kyphosis, and/or vertebral fusion. Osteomyelitis is a lytic process in reptiles. Acutely, localized bone lysis will be evident on radiographs. Digestive system The anatomy of the different species of lizards must be taken into account when interpreting radiographs of the digestive tract. For example, Green iguanas (Iguana iguana) and Chuckwallas (Sauromalus obesus) are primarily herbivorous, hind-gut fermenters, and as such, a large, gas-filled, distended sacculated colon can be expected on radiographs. Common indications for evaluating the digestive system of lizards include foreign body ingestion, intestinal impaction, hepatopathy and enteritis. Foreign bodies are common in lizards, especially if housed on substrate they can easily consume. Enteritis is often marked by gas distension of the intestines and/or gastrointestinal ileus. Contrast radiography is helpful in identifying foreign bodies that are not radio-opaque, foreign body impaction and some cases of enteritis. Transit time for the contrast material can be days to weeks, depending on the amount of contrast administered, the species of lizard, the type of digestion, environmental temperature and activity of the lizard. An enlarged liver will often displace the intestines and lungs dorsally, or present as a large soft tissue structure obstructing the visualization of the other organs in the mid to caudal coelom. ExoticsCon 2015 Pre-conference Proceedings 169

18 Respiratory system Most lizards have sponge-like lungs. However, in some species, the lungs are more sac-like and thus, no reticulations are visible on radiographs. Pneumonia is not uncommon in lizards and can be distinguished by an increased soft tissue opacity of the lungs or distinct fluid lines. Cardiovascular system The heart cannot always be fully appreciated as the thoracic girdle and forelimbs may overlap the cardiac silhouette. The descending aorta can best be visualized on a horizontal lateral view coursing from the caudal aspect of the cardiac shadow through the cranial coelom. Given the low incidence of reported cardiac disease in lizards, there is little information on the standards for size and shape for different species. Urogenital system The kidneys of iguanid lizards are mainly located within the pelvic cavity. However, when enlarged they protrude into the caudal coelom. In some lizards like the monitors (Varanus species) the gender can be determined by visualizing the calcified hemipenes. One of the primary reasons for radiographing lizards is to evaluate the reproductive system. Dystocia, egg binding and other related reproductive disorders are common in this group of reptiles. Lizard eggs may be calcified or leathery. Several round soft tissue objects of approximately equal size with very thin mineralized walls may represent a normal gravid female. If the eggs are over- or under-mineralized, too large or of irregular size, disease should be suspected. Miscellaneous Radiography should be part of the standard investigation for the presence of any soft tissue swelling. Abscesses are common in lizards. It is important to evaluate an abscess radiographically prior to surgical resection to determine the presence of osteomyelitis frequently associated with abscesses as well as the involvement of other major structures. Chelonians Restraint and positioning Fortunately, most chelonia are fairly easy to position and restrain. For vertical beam dorsoventral radiographs, most conscious individuals will remain motionless long enough to permit exposure. Ideally, the head and limbs should be extended from the shell in order to reduce superimposition of the limb musculature on the coelomic viscera. More active animals can be restrained by taping them to the cassette or by placing them in a radiolucent container although this should be avoided particularly with smaller specimens (and lower exposures) as material artifacts may appear on the films. For lateral horizontal beam radiographs the chelonian is best placed on a central plastron stand. By lifting the animal clear of the ground the limbs and head will be encouraged to extend but the tortoise will remain immobile. Both left and right lateral projections should be taken with the lateral edge of the shell touching (or as close as possible to) the cassette. The third basic coelomic view is the horizontal craniocaudal (or anterior-posterior) view. Again the chelonian is positioned on a central plastron stand, with the caudal edge of the carapace touching (or as close as possible to) the cassette, with the head facing the x-ray tube and the beam centered on the midline of the cranial rim of the carapace. Radiology of the head and limbs will require their exteriorization from the shell and this will usually require general anesthesia. The use of sandbags, foam and tape will aid positioning. Standard interpretation requires that both true lateral and dorsoventral views should be taken even slight rotation makes interpretation very difficult. 170 Building Exotics Excellence: One City, One Conference

19 Musculoskeletal system The common musculoskeletal problems that can be detected on radiographs include metabolic bone diseases (often nutritional secondary hyperparathyroidism), shell and limb fractures, dislocations, joint disease and osteomyelitis. Nutritional metabolic bone disease (nutritional secondary hyperparathyroidism) produces characteristic radiographic lesions including; reduced bone density, cortical thinning, and shell (and less commonly limb) thickening as a result of fibrous proliferation. The opacity of the pelvic and pectoral girdles provides the best assessment of ossification and mineral depletion. Conversely, soft tissue mineralization, mineralized gout tophi, or pseudogout may be observed as areas of increased opacity in the periosteal or periarticular areas, or within any visceral tissue. The diagnosis of limb fractures is usually straightforward. Fractures of the shell are more common and may be obvious or subtle and difficult to appreciate on radiographs unless displaced or appropriately sky-lined. The situation is further complicated by the lack of periosteal new bone growth and obvious callus formation, such that a radiolucent line may persist in an asymptomatic individual with acquired fibrous fracture stabilization for years. Following diagnosis and any fracture repair, follow up radiographs should be taken at 10 and 20 weeks to assess the repair, although remodeling is likely to take 6-18 months or more. Rarely, traumatic joint dislocations can occur as a result of mishandling or predator attack. Dislocations of the stifle and elbow are easily appreciated but coxofemoral and scapulohumeral dislocations may be more difficult to detect without lateral, dorsoventral and craniocaudal views. Any joint surgery should also be followed up after 3, 6, 12 and 24 and 36 months for evidence of degenerative joint disease and arthritis. Chelonia are amongst the longest lived of animals and age related orthopedic disease, namely degenerative joint diseases, are becoming increasingly common. Other causes of degenerative joint disease include articular gout, pseudogout and trauma. Evidence of bone lysis at the articular surfaces and joint swelling are most noticeable. Osteomyelitis is a common presentation in reptiles and tends to be lytic rather than proliferative as it is in mammals. There is often gross enlargement and distortion of the local anatomy with loss of corticomedullary definition. Digestive system The radiographic appearance of the alimentary tract will depend upon the temperature, nutritional status of the animal, nature of the ingesta, and the period of time elapsed between feeding and radiographic examination. The gastro-intestinal tract of chelonians is shorter than that of mammals but the transit time is also slower. For an overview, a dorsoventral radiograph is most helpful, however the ancillary digestive organs, namely, pancreas and liver are not usually discernible. Radiodense objects, typically stones or gravel, are often encountered and may be considered significant only if concurrent with clinical signs. Metallic objects are less commonly seen but lead intoxication has been reported. Gastrointestinal ileus due to improper husbandry and diet is common in chelonians. The use of gastrointestinal contrast studies can help to differentiate between intestinal and extra-intestinal diseases. These techniques can also help to distinguish between intraluminal (eg, radiolucent foreign bodies), intestinal (eg, abscessation, neoplasia) and extraluminal diseases, and aid in the diagnosis of gastrointestinal perforation. Cardiopulmonary system The heart is located in the cranial third of the coelomic cavity, resting above the plastron, but is often not radiographically discernible. Radiographic assessment of the lung fields in cases of suspected pulmonary disease is very valuable and effective as, despite the shell, the air provides excellent contrast. The horizontal beam lateral and craniocaudal views are most informative, although severe consolidation can be appreciated on a dorsoventral image. ExoticsCon 2015 Pre-conference Proceedings 171

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