Chelonians Flashcards

Question

What parameters were best to predict BCS in EBTs evaluated with CT?

Answer 1

BODY CONDITION OF EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) EVALUATED BY COMPUTED TOMOGRAPHY. dePersio S, Allender MC, Dreslik MJ, Adamovicz L, Phillips CA, Willeford B, Kane L, Joslyn S, O'Brien RT. J Zoo Wildl Med. 2019 Jun 13;50(2):295-302. Key Points: Mass alone or mass and carapace width were the best variables to predict body condition score Previous unvalidated models for chelonian BCS are likely inaccurate In sea turtles, carapace length and mass are the best indicators of body condition index Conclusions: Mass and carapace width are the best predictors of body condition index.

Answer 2

CHARACTERIZING THE 25-HYDROXYVITAMIN D STATUS OF TWO POPULATIONS OF FREE-RANGING EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) Watson MK, Byrd J, Phillips CA, Allender MC. Journal of Zoo and Wildlife Medicine. 2017 Sep;48(3):742-7. Omnivorous reptiles obtain vit D from diet or secondary exposure to UVB radiation. Vitamin D3 in skin has a longer half-life than dietary vitamin D3 because it skin D3 is 100% protein bound Recommended method of measuring Vit D is 25-OHD3. Key Points: Vitamin D3 was positively correlated with UVB at time of capture. No correlation with latitude, calcium, Ca:P, age, or sex Conclusions: 25-OHD3 levels in wild eastern box turtles were correlated with UVB levels at the time of capture.

Answer 3

ASSESSMENT OF A VISUAL SCORING SYSTEM FOR IDENTIFYING AND QUANTIFYING ANEMIA IN MALE EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA). Cerreta AJ, Mehalick ML, Stoskopf MK, Dombrowski DS, Lewbart GA. J Zoo Wildl Med. 2018 Dec;49(4):977-982 FAffa MAlan CHArt (FAMACHA) is used in small ruminant species to estimate anemia based on conjunctival color Male eastern box turtles have an non-pigmented iris, which causes it to have a red hue Key Points: There was a strong correlation between eye-color score and PCV. (Lighter = more anemic) Using 5 or 6 color options resulted in less variable and more specific assessments than 9 color options. 5 or 6 options were highly specific (100%) but not sensitive (30-44%) for anemic EBT

Answer 4

SURGICAL ANATOMY OF CELIOTOMY APPROACHES TO THE STOMACH IN THE COMMON SNAPPING TURTLE (CHELYDRA SERPENTINA). Jimenez IA, Hermanson JW, Childs-Sanford SE. J Zoo Wildl Med. 2019 Mar 1;50(1):82-88. Options for surgical removal of GI foreign bodies in chelonians: Plastron osteotomy; may not have good access to the stomach because of limited organ mobility Prefemoral celiotomy Axillary celiotomy Key Points: Left axillary approach: 4 cm adjacent to plastron edge Required transection of superficial muscle bellies to see coelomic membrane Greater curvature stomach visualized but not easily exteriorized Left prefemoral: 4 cm adjacent to plastron edge Fascia excised and coelom penetrated easily Able to exteriorize duodenum and exteriorize stomach Simple and atraumatic Conclusion: Celiotomy to access stomach for surgery in snapping turtles is recommended as prefemoral approach in snapping turtles.

Answer 5

Long-term Oral Clarithromycin Administration in Chelonians with Subclinical Mycoplasma spp. Infection Rettenmund CL, Boyer DM, Orrico WJ, Parker SG, Wilkes RP, Seimon TA, Paré JA. Journal of Herpetological Medicine and Surgery. 2017;27(1-2):58-61. Mycoplasma agassizii and Mycoplasma testudineum = agents of contagious upper respiratory tract disease (UTRD) in tortoises Mycoplasma testudinis = asymptomatic infection M. agassizzi and M. testudienum affect Gopher tortoises Can find mycoplasma in conjunctival swabs of normal chelonians Intermittent shedding of Mycoplasma, so PCR can give false negatives Clarithromycin is macrolide- used to successfully treat M. agassizii in desert tortoises in another paper (took 6 mo) Good intracellular penetration because it travels in phagocytes to sites of infection Accumulates in respiratory lining cells and secretions Conclusions: Long-term clarithromycin PO is safe but does not stop shedding of Mycoplasma in Forsten’s tortoises or Sulawesi forest turtles.

Answer 6

A PRELIMINARY ANALYSIS OF PROLONGED ABSORPTION RATE OF PONAZURIL IN RED-FOOTED TORTOISES, CHELONOIDIS CARBONARIA Benge SL, Heinrichs MT, Crevasse SE, Mahjoub B, Peloquin CA, Wellehan JF. Journal of Zoo and Wildlife Medicine. 2018 Sep;49(3):802-5. TINC (Testudine intranuclear coccidiosis)- first found in radiated tort in 1990 Systemic disease, affects multiple organ systems: GI, resp, endocrine, lymphoid Coccidia associated with inflammation and necrosis Similar to Sarcocystis neurona (equine protozoal myeloencephalitis) High mortality Ponazuril inhibits all developmental stages of coccidia Key Points: Prolonged and variable oral absorption (continual absorption over an entire week!) 20mg/kg did not reach Cmax consistent with effect in mammals Suspect inadequate dosing of ponazuril reason no documented cases of tortoise clearing TINC Highest concentration of the drug was at 168 hours Take Home Message: Red footed tortoises did not reach mammalian Cmax necessary to treat coccidial disease at a dose of 20mg/kg. The hope was that ponazuril could be used to treat TINC

Answer 7

Efficacy of a Topical Formulation Containing Emodepside and Praziquantel (Profender®, Bayer) against Nematodes in Captive Tortoises Tang PK, Pellett S, Blake D, Hedley J. Journal of Herpetological Medicine and Surgery. 2017 Dec;27(3-4):116-22. Oxyurids have a direct life cycle and are host specific Inhabit lower intestinal tract of tortoises High levels have been associated with fatal post-hibernation anorexia Most common ascarid in herbivorous tortoises = Angusticaecum holopterum Direct life cycle after ingestion of embryonated eggs Low levels are likely insignificant, but heavy burdens can cause intestinal obstruction and perforation Fenbendazole is a common oral antiparasitic in tortoises, but can have negative clinicopathologic effects Heteropenia, transient hypoglycemia, hyperuricemia, and hyperphosphatemia (source below) Potential for reduced fenbendazole efficacy in chelonians Emodepside is a cyclooctadepsipeptide with activity against gastrointestinal nematodes Triggets inhibitory neurotransmitter release from presynaptic membrane → flaccid paralysis in nematodes Displayed efficacy against a variety of nematodes in a wide variety of species Praziquantel has anthelmintic activity against trematodes and cestodes Increases permeability of calcium channels to cause an influx of calcium across muscle/tegumental membranes of parasites Ivermectin is a parenteral anthelmintic which in chelonians can cause paresis, flaccid paralysis, hepatic lipidosis, nephrotoxicity, and death Key Points: Oxyurid were not changed at day 14, but decreased by ~60% at day 33 Delayed, moderate anthelmintic activity Suboptimal temperatures may have inhibited drug absorption Ascarids reduced by 67% at day 14 (Not enough samples for day 33) Conclusion: Profender produced delayed, moderate anthelmintic activity in tortoises and may be an option to reduce but not eliminate oxyurid burden in tortoises when oral medications not a treatment option.

Answer 8

Buscaglia, N. A., Inman, D. N., Chen, S., Arheart, K. L., & Cray, C. (2021). Partial hematology, biochemistry, and protein electrophoresis reference intervals for captive spotted turtles (clemmys guttata). Journal of zoo and wildlife medicine, 52(2), 704-709. Protein electrophoresis - semiautomated agarose gel electrophoresis traditionally used. Capillary zone electrophoresis - Increased precision of fraction quantitation, better resolution of new protein fractions, easier to use, completely automated, faster results, smaller sample can be run.

Answer 9

Buscaglia, N. A., Inman, D. N., Chen, S., Arheart, K. L., & Cray, C. (2021). Partial hematology, biochemistry, and protein electrophoresis reference intervals for captive spotted turtles (clemmys guttata). Journal of zoo and wildlife medicine, 52(2), 704-709. Protein electrophoresis - semiautomated agarose gel electrophoresis traditionally used. Capillary zone electrophoresis - Increased precision of fraction quantitation, better resolution of new protein fractions, easier to use, completely automated, faster results, smaller sample can be run. It is possible to compare results of CZE using serum and plasma.

Answer 10

COMPARISON OF SUBCARAPACIAL SINUS AND BRACHIAL VEIN PHLEBOTOMY SITES FOR BLOOD COLLECTION IN FREERANGING GOPHER TORTOISES (GOPHERUS POLYPHEMUS) Journal of Zoo and Wildlife Medicine 52(3): 966–974, 2021 - Neiffer, Dipl ACZM, et al. Higher brachial vein mean values for most parameters. Attributed to greater hemodilution of subcarapacial samples with lymph. Subcarapacial prone to lymph dilution. Can collect larger samples, allows for more rapid processing and does not require chemical restraint. Assoc with serious complications i.e. paralysis. All CBC parameters were higher in brachial vein samples except for lymphocytes (high in lymph, SC). All plasma chem, EPH, and vitamin parameters higher in brachial vein samples except NaCl and BUN.

Answer 11

INTRAVENOUS LIPID EMULSION TREATMENT REDUCES SYMPTOMS OF BREVETOXICOSIS IN TURTLES (TRACHEMYS SCRIPTA). JZWM 50(1): 2019 Cocilova, Flewelling, Granholm, Manire and Milton Karenia brevis - Brevitoxicosis Animals treated with IL 30 min after brevetoxin exposure had greatly reduced symptoms within first 2 hrs. Fully recovered within 24 hrs. Animals receiving ILE first showed little to no clinical signs post exposure. Increase observed in brevetoxin in bile and feces 24h post ILE tx vs non treatment exposure control.

Answer 12

F8 Chelonians; Mader 2019 - Ch. 7 Chelonian Taxonomy, Anatomy, and Physiology * Chelonians (turtles, tortoises, terrapins) * Diapsids – have two temporal fenestra * Sister clade to Archosaurs (crocodilians, birds, extinct dinosaurs) * Less closely related to Lepidosaurs (lizards, snakes, amphisbaenids, tuataras) * Most highly endangered of any of the major vertebrate groups. * Threats – hunting, traditional medicine, pets, intro of invasive spp, disease, anthropogenic trauma, fisheries, habitat degradation, fragmentation. * Taxonomy: Chelonia * Suborder Cryptodira (11 families, 250+ spp) - Hidden-neck turtles * Can retract head and neck straight back into shell, hiding neck * Includes sea turtles although unable to retract * Family Carettochelyidae: Pig-nosed turtle * Family Cheloniidae: Hard-shelled sea turtles * Family Chelydridae: Snapping turtles * Family Dermatemydidae: Central American river turtles * Family Dermochelyidae: Leatherback sea turtle * Family Emydidae: Box turtles, pond turtles, map turtles, wood turtles, terrapins, sliders, cooters * Family Geoemydidae: Asian river, lead, roofed, or Asian box turtles * Family Kinosternidae: Mud and musk turtles * Family Platysternidae: Big-headed turtle * Family Testudinidae: Land tortoises * Family Trionychidae: Softshell turtles * Suborder Pleurodira (3 families, 80+ spp) - Side-necked turtles * Not fully able to retract neck, fold it sideways * Family Chelidae: Austro-American side-necked and snake-necked turtles * i.e. Comon snake-neck turtle, mata mata * Family Pelomedusidae: Afro-American side-neck turtles * i.e. African helmeted turtle * Family Podocnemididae: Madagascar big-headed turtles, big-headed Amazon river turtles, South American side-neck river turtles * Common names vary: * Tortoise – typically terrestrial i.e. family Testudinidae; Australians will refer to turtles as tortoises, UK terrapin refers to FW chelonians, turtle refers to marine, tortoise refers to terrestrial. * North America – terrapin used for single spp (diamondback terrapin), all other aquatic turtles called turtles.

Answer 13

* Musculoskeletal system * Shell – upper carapace, lower plastron, joined laterally by bony bridges * Carapace - ~50 bones derived from ribs, vertebrae, dermal elements * Plastron – 9 bones from clavicles, coracoids, interclavicles, gastralia (ribs) * Most spp have keratinized epithelium covering a thin dermis layer consisting of collagen fibers, melanophores, vessels, and nerves, beneath which is dermal bone. * Scutes – superficial layer of keratin * Staggered, seams between not directly over bone * New scutes produced with each major growth period * Terrestrial chelonians retain scutes * Aquatic/semiaquatic chelonians shed scutes * Grow outward from central nucleus (areola) * Each year, new scute forms beneath previous year’s * Outer edge will form annulus (growth ring) * Nutritional deficiencies – inversion of annuli * Counting annuli not accurate for determination of age * Scute nomenclature – Fig. 7.23 * Shell modifications depending on spp * Leatherback sea turtles, softshell turtles, Fly River turtles – scutes replaced by leathery skin (alpha keratin) * All other turtles both alpha and beta keratin * Hatchling tortoises have fontanelles b/t carapacial bones, fuse with age * Some spp i.e. pancake tortoises retain fenestrae * Plastronal hinges i.e. box turtles, spider tortoises, mud turtles * Caudal carapacial hinge – hinged-back tortoises * Chelonians = only extant vertebrates with pectoral and pelvic girdles within ribcage * Tripartite rectilinear pectoral girdle * Dorsoventral scapula * Ventromedial acromium process * Ventrocaudal corcoid (procoracoid) * Vertebrae are incorporated into the carapace from first thoracic vertebra caudally to coccygeal vertebra. * Marine spp and the Fly River turtle have elongated metacarpals and phalanges (flippers)

Answer 14

* Respiratory system * Obligate nasal breathers – open-mouth breathing is pathologic * Complete tracheal rings * Cryptodiran turtles – short trachea, bifurcation is mid-cervical * Cranial bifurcation and complete tracheal rings enable breathing unimpeded when neck is withdrawn. May be a hazard for intubation. * Lungs adherent to carapace, dorsal * Ventrally separated by septum horizontale – attached to liver and GIT * Pleuroperitoneal/coelomic cavity * Multicameral (partitioned), saccular, 3-11 chambers dep on family * Reticular surface, smooth muscle and connective tissue * Large lung volume, advantage as hydrostatic organ for aquatic turtles * Three-toed box turtles, relatively small tidal volumes coupled with high respiratory rates. * Peak airway pressures during anesthesia recommended \< 10 cm H20 * Antagonistic muscles decrease or increase lung and visceral volume * Gular pumping to assist olfaction, not typically ventilation (except some aquatic spp) * In submerged snapping turtle – active inspiration, expiration passive. * On land, passive inspiration, active expiration. * Some aquatic turtles – supplemental cloacal, buccopharyngeal, or cutaneous respiration for prolonged submergence. * Long periods of apnea make induction of gas anesthesia difficult. * Open fractures of shell with lung exposure do not typically result in obvious respiratory distress. * Many factors make removing secretions or FB from lungs difficult: * Termination of mucociliary elevator outside the glottis. * Poor drainage through dorsally located bronchi. * Compartmentalization of lungs. * Large potential space within the lungs. * Lack of a complete muscular diaphragm to cough. * Pneumonia can be difficult to manage and life threatening Zoo Path * ● Nares open into paired vestibules or nasal passages (no sinuses, turbinates) * o Mucosa - keratinized epithelium that transitions to multilayered olfactory epithelium dorsally & ciliated epithelium w/ mucus glands ventrally * ● Trachea short, bifurcates early into bronchi 🡪 open into singular saccular lung lobes (R,L) 🡪 subdivided into multiple variably sized compartments by smooth muscle trabeculae and smaller faveoli * o Lungs firmly attached dorsally to carapace & separated from coelomic cavity by fibrous membrane (**septum horizontale** or **pleuroperitoneal membrane**) * o No diaphragm - can’t cough; clearance of lower resp. infection/inflammation nearly impossible * o Some aquatic species use air in lungs for buoyancy control * o Evidence (controversial) some aquatic spp. have marginal extrapulmonary gas exchange (moving water through oral and cloacal cavities)

Answer 15

* Digestive system * Most terrestrial spp herbivorous * Aquatic species carnivorous, omnivorous - Numerous exceptions * No teeth, swallow bite-sized pieces, saliva is not enzymatic * Esophagus is ciliated * Sea turtles – large esophageal papillae * SI relatively short vs mammals * LI – primary fermentation site in tortoises, includes cecum, colon * Cecum lacks mesenteric attachments * Transverse colon has wider mesenteric attachment to stomach, gives dorsoventral mobility * Heavy ingested FB sink ventrally in the transverse colon, become entrapped, and accumulate at the descrnding colon. * FB removal facilitated by milking material anterograde into cecum, easiest to exteriorize. * Stomach, SI, pancreas produce digestive enzymes * Liver – two major lobes, envelops GB on right * May have normal melanomacrophages * Pale yellow to tan may indicate hepatic lipidosis or normal vitellogenesis in females. * One study did not detect postprandial increase in bile acids in RES. * Pancreas directly contacts spleen (cryptodirans) or separate in mesentery with duodenum (pleurodirans) * Factors affecting GI transit time: * Temperature, spp, feed frequency, food size, water or fiber content of food. * Transit time omnivores \< carnivores \< herbivores. * Captive diets generally move faster, especially in tortoises. * Loggerhead sea turtles, GI contrast 2-3 weeks * Metoclopramide, cisapride, and erythromycin did not significantly reduce GI transit time compared with water in desert tortoises. Zoo Path ● No teeth - keratinized horny ridges line the upper (rhamphotheca) and lower (dentary) beak ● Green sea turtles - esophagus lacks mucus glands, Cd-directed conical papillae help retain ingested prey ● Several marine turtle species have a crop to retain food ● GI tract simple, suspended from dorsal midline by a single mesentery ● Liver has 2 main lobes w/ gallbladder w/in or along caudal border of R lobe o Histo – hepatocytes in discrete trabeculae, but *distinct lobular architecture absent* o Melanomacrophages considered normal cellular population in liver

Answer 16

* Kidneys – retrocoelomic, deep to caudodorsal carapace, posterior to acetabulum. * Except marine turtles – anterior to acetabulum. * Metanephric, no loop of Henle. * Cannot concentrate urine above that of plasma. * Urine not sterile. * Ammonia and urea require large amounts of water for excretion. * Renal portal system – PK studies have shown no significant difference in drug metabolism if injections are given in caudal region vs cranial limbs. * Differences from mammals markers (BUN, Cr) making detection of kidney disease difficult: * Amino-ureotelic: Marine and highly aquatic FW turtles excrete more ammonia and urea than uric acid. * Ureo-uricotelic: Semiaquatic turtles excrete mostly urea. * Ureo-uricotelic to uricotelic: Terrestrial chelonians produce more insoluble uric acid and urates passed in semisolid state. * Healthy sea turtles very high BUN vs other chelonians (\> 100 mg/dL). * Healthy herbivorous tortoises have basic urine. * Tortoises in catabolic state acidic urine, not specific to disease. * Urinary bladder * Some spp accessory urinary bladders. * Terrestrial chelonians – largest, bilobed, thin, membranous distensible, ciliated, mucus secreting wall. * Used for water storage and K/Na exchange during drought. * Care should be taken to prevent urination when handling wild tortoises, could result in significant fluid losses for that individual. * Aquatic chelonians – small bladders, thicker walls. * Cloaca, colon, UB can reabsorb urinary water. * Bladder prolapse may occur with uroliths or colonic FB. Zoo Path: ● Paired lobular kidneys in retrocoelomic space; *no distinct cortex and medulla grossly* o Fewer nephrons, no loops of Henle 🡪 chelonians cannot concentrate urine o Proportion of N waste excreted as NH₃, urea, & uric acid varies by spp., habitat, season, diet, age ▪ FW turtles excrete equal amts of ammonia and urea ▪ Sea turtles also excrete uric acid ▪ Tortoises (esp. desert spp.) excrete far more uric acid & K⁺-urate salts in semisolid state o Ureters enter dorsal urodeum 🡪 excreted or diverted into urinary bladder ▪ Urinary bladder - ciliated epithelium w/ mucus glands ▪ In some spp. - 2 additional small accessory bladders lateral to urodeum ▪ Bladder - water reservoir, resorptive capacity in some spp. o BUN has limited utility as marker of renal fxn, except perhaps in aquatic spp. ▪ BUN also ↑ during hibernation in desert tortoises (↓ H2O intake, protein catabolism) ▪ Urea - better for hydration status, water availability than kidney function

Answer 17

* Gonads. * Paired, anterior to kidney. * Fertilization is internal. * Seasonal hormone patterns. * Spermatogenesis is temp and testosterone dependent. * Rising in spring/summer, falling in fall/winter. * Spermatozoa may be retained through winter. * Sperm may be stored months to years by female in albumin gland within isthmus of oviduct. * Following fertilization, produces membranes/albumin around ovum. * Shell gland produces shell membranes and eggshell. * Ovary/follicles. * Previtellogenic follicles – secrete estradiol in response to pituitary gonadotropin. * Estradiol stimulates liver to secrete vitellogenic protein. * Taken up by maturing follicles. * Nesting F sea turtles have higher TP, alb, glob, Ca, P, TG, and cholesterol. * Testosterone regulates seasonal reproduction in males and females. * Follicular testonsterone increases with maturation of follicle. * Biphasic increase associated with spring and fall mating. * Falls after ovulation, females become nonreceptive. * Ovulation occurs after courtship and mating, then within days of nesting. * Associated with LH and progesterone surges. * Single or multiple clutches of eggs dep on spp. * Arginine vasotocin peaks during first oviposition, baseline within an hour. * Follicles that have not ovulated undergo atresia. * Reduces in size, becomes a corpora albicans. * Repeated folliculogenesis and atresia without production of eggs may lead to coelomitis. * Thyroxine (T4) peaks in both sexes following hibernation. * Males undergo second peak in late summer as male combat and spermatogenesis return. * Temperature dependent sex determination * Pattern 1a: Typical, produces females at higher temperatures, males at lower temps * Pattern 2: Females at both high and low temps and males at IM temp. * Smaller gender typically occurs at cooler temps. * Some turtles have genetic sex determination: * Common snake-necked turtle, Wood turtle * Phallus * Spade-shaped. * Ventral floor of the proctodeum, no urethra. * Seminal groove for transport of sperm. * No inversion occurs as it does in squamates. * Males often vocalize during copulation. * Typically last part of the turtle to be desensitized under general anesthesia. * Sexual dimorphism. * Coloration, tail or claw length, size, shell shape. * Males have longer, thicker tails, distal vent, curved or concave plastron, anal notch on plastron deeper than female. * Cryptodiran turtles, most significantly larger - F. * Males typically larger in larger-sized spp. * Male aquatic turtles tend to have elongated foreclaws for courting. * Males of some marine spp have hooked claw on front flipper. * Female leopard tortoises have elongated rear claws. * Box turtle males have red iris vs yellow/brown of females.

Answer 18

* Reproduction (F8): * All chelonian lay shelled eggs on land. * Clutch size may range from 1-100+. * Size and number of clutches per season may be influenced by nutritional status. * Incubation times vary. * Most spp have temp-dependent sex det. * Generally females produced at higher temps, males at lower temps. * Plasma Ca may exceed 25 mg/dL during folliculogenesis. * Globs may \> 8 and chol 200+. * Can place in shallow water and administer oxytocin IM or ICe to try to stimulate laying. * Retention of shelled eggs in otherwise healthy animal not a concern. * Anorexic or lethargic, otherwise systemically affected, can administer fluids, Ca gluc, repeat oxytocin with prostaglandin. * Can assess repro status of wild females with US. * Don’t have to expose the ovary to multiple radiographic events. * Presence of mature ovarian follicle does not ensure ovulation will occur. * Some females are capable of resorbing preovulatory follicles. * Primary repro issue in males is phallus prolapse. * Cleanse, manually replace and purse string. * May require amputation.

Answer 19

* Cardiovascular system * Four-chambered heart: one sinus venosus (pacemaker), two atria, one ventricle with three subchambers (cavas). * Segregate oxygenated and deoxygenated blood. * Deoxygenated blood returns to sinus venosus (receives blood from right and left cranial vena cavas, caudal vena cava, left hepatic vein). * Right atrium \>\> left atrium, no auricles. * Great vessels (from ventricle) – two aortas, common pulmonary artery or trunk. * Left trunk is pulmonary artery, splits into each lung. * Middle trunk (left aorta) – supplies viscera * Right trunk (right aorta) has brachiocephalic trunk arising from base, bifurcates into subclavian arteries. * Landmarks for thyroid gland. * Left and right aortae join caudal to heart to form dorsal aorta. * Located on midline except in Trionychidae – displaced to right. * Bordered laterally by acromium and coracoid processes. * More pericardial fluid vs mammals. * Ligamentous gubernaculum cordis attaches ventricular apex to pericardial sac. * Ventral midline coelomic mebrane incision can inadvertently enter pericardial sac. * Apnea and diving * HR and pulmonary blood flow decreases by 50% and 80%, 150% increase in pulmonary resistance. * Results in more intracardiac R-L shunting. * When breathing, HR and pulmonary blood flow increase two and three fold, results in net L to R shunt. * Renal portal system of unknown clinical significance. Zoo Path ● Heart - pericardial sac connected to apex of heart via gubernaculum cordis; 4 chambers (sinus venosus, 2 atria, ventricle) o Ventricle - 3 interconnected chambers (cavum venosum, cavum arteriosum, cavum pulmonale) o _Purkinje fibers_ absent o Contractions initiated via sinus venosus, propagated by arrangement of myofibers o Muscular sphincters in pulmonary arteries of some marine spp.

Answer 20

* Nervous system * CNS – brain and SC * Brain – olfactory bulb, cerebral cortex, thalamus, hypothalamus, pituitary, optic lobes, cerebellum, medulla. * Most reptiles lack neocortex, some evidence dorsal cortex homologous in chelonians. * Traumatic midline carapace injuries may affect the spinal cord. * Bran and SC surrounded by two meningeal layers – inner leptomeninx and outer dura mater. CSF in between (site of intrathecal injections). * Reptiles lack subarachnoid space. * Epidural space is rich in vascular supply, does not contain CSF. * Extreme tolerance for hypoxia and anoxia. * 12 cranial nerves. * Nervous terminalis considered nerve 0. * PNS

Answer 21

* Nonreproductive endocrine system * Thyroid gland – unpaired, anterior to heart * Thymus – anterior to thyroid gland * Not part of endocrine system * Two pairs of parathyroid glands – one within thymus, one near aortic arch. * Parathyroid hormones regulate calcium and phosphorus metabolism. * Within PG are paired ultimobranchial bodies. * Homologous with thyroid parafollicular cells of mammals that secrete calcitonin. * Adrenal glands - Retrocoelomic, cranial to kidneys * Catecholamines, glucocorticoids, mineralicorticoids * Increased corticosterone documented under physical or physiological stress. * Pituitary gland - Below optic chiasma in sella tursica of sphenoid bone. * Pancreas typical endocrine functions – insulin/glucagon.

Answer 22

* Special senses and salt glands. * Lack parietal eye. * Pineal gland may affect behavior, gonadal activity, thermoregulation. * Smell - Large olfactory bulbs, modified jacobson’s organ. * Excellent hearing - Low tones from 100-700 Hz. * Large middle and inner ear beneath tympanic membrane. * Sound reception: tympanic membrane -\> extracolumella cartilaginous footplate -\> osseous columella -\> inner ear. * Eustacian tube between inner ear and pharynx. * Sight. * FW turtles far-sighted. * Cones predominant photoreceptors. * Color vision good for red, yellow, orange. * Retina avascular, conus papillaris is a vascular projection from optic nerve. * Upper and lower eyelids, nictitating membrane, scleral ossicles. * Iris composed of skeletal muscle. * PLRs difficult to observe. * IOP measured in several spp. * Harderian glands and lacrimal glands produce tear film. * Glands may obstruct, become cystic with hypovitaminosis A. * Herbivorous chelonians can synthesize vitamin A endogenously. * No nasolacrimal ducts, so tears spill over lid margins. * Tears also lost by conjunctival tissue absorption and evaporation. * Sea turtles and diamondback terrapins – lacrimal gland has evolved into salt-excreting gland. * Regulate plasma electrolytes in hypertonic environment. * Plasma osmolality and Na, Cl, K, Mg can be influenced by salt gland function.

Answer 23

* Special physiology (F8): * All chelonians considered ectothermic except leatherback sea turtle. * Leatherback can regulate body temperature through series of vascular plexuses deep within musculature of limbs. * Chelonians may demonstrate episodic breathing and may shunt blood to or away from the lungs via vascular and IC shunting mechanisms. * High tolerance of hypoxia, large lung volume, rapid and extensive air exchange during ventilation, and physiologic buffering by bone, blood and pericardial fluid of lactic acid and hydrogen ions built up during anaerobic metabolism. * Gas exchange may also occur through the integument, pharynx, or cloacal tissues in some spp. * Soft-shelled turtles may obtain 70%+ of oxygen during submergence through leathery shell.

Answer 24

**Ch 23. Tortoises, Freshwater Turtles, and Terrapins (Mader 2019)** Lighting: * Chelonians require UV light – natural is best * Tropical species – 12 hr light cycle year round * No light at night * Temperate species – annual photoperiod should be followed Temperature: * Heliotherms – obtain heat by basking * Nonlethal temp range (all chelonians) - 8°C (46°F) to 45°C (113°F) * Terrestrial species optimal temp - 28°C (84°F) w/ range: 22° to 30°C (72°–86°F) * \<15°C (59°F) - inactive and anorexic * \<10°C (50°F) – hibernating * \>35°C (95°F) – seek cooler areas, may aestivate * 39° to 43°C (102°–109°F) – critical or lethal * \>45°C (113°F) – rapidly lethal, all species * Box turtles – largest freeze tolerant taxa * Sudden freezes still major cause of mass mortality box turtles * Drop in temp at night – beneficial, may help lessen pyramidal shell growth * Captivity - temp gradient within the POTZ is best * Drop in temp at night preserves natural circadian rhythms * In general: * Captive animals should not be subjected to constant temps but should be provided with a diurnal fluctuation and a thermal gradient. * Aquatic spp – maintaining appropriate water temp ranges critical. Hibernation/Brumation: * Brumation - precedes reproduction, utilizes stored glycogen and fat, can be important husbandry component * Colder temp – thyroid hormones decreases, often anorexic * All temperate chelonians should hibernate if healthy, do not hibernate if unhealthy * Species hibernation: * most Gopherus and Testudo tortoises hibernate, with the exception of African Testudo tortoises * none of the tropical tortoises hibernate * most temperate zone terrestrial and freshwater turtles hibernate * all hibernate north American box turtles hibernate except for the Gulf Coast and Florida box turtles * wood turtles, spotted turtles, common snapping turtles, northernly distributed eastern mud turtles, stinkpots, red-eared sliders, and painted turtles should hibernate * Falling temps stimulate hibernation, decreased appetite, slowed metabolism * Hibernaculi - areas that are slightly warmer than surrounding environment to avoid freezing and provide moisture to protect against dessication * Glycogen and fat stores in the liver and body – main source of energy during hibernation * Emergence from hibernation stimulated by increasing temperature * Jackson’s ratio - compares body weight with midline, straight carapace length * partial estimation of healthy body condition for Testudo graeca and T. hermanni (not applicable to other Testudo) * presence of uroliths, coelomic exudates, or intestinal gravel can elevate body weight of ill tortoises * Maximum carapace length, width, and height (volume) against weight - another estimation of the health status of desert tortoises * Prior to hibernation, discontinue supplemental food * Indoor humidity can be low during hibernation – recommend soaking intermittently * Disturbing during hibernation not harmful * Box turtles and Testudo tortoises can tolerate much colder hibernation temperatures * Avoid freezing – can cause blindness, damage to extremities, and death * Postemergence turtles often dehydrated, immunocompromised, vulnerable to disease * Tortoises – should lose no more than 6-7% BW over hibernation * Ideally Testudo spp lose \<1% BW * Aquatic turtles can hibernate under water or on land General Nutrition * Tortoises – Herbivores, graze on grasses and annual forbs, very little natural exposure to fruits. * FW turtles – Primarily omnivorous, consume fish, crustaceans, snails, aquatic grasses, fruits, other items. * Green sea turtles – Sea grass, algae. * Loggerhead and Ridleys – Diet of molluscs and crustaceans. * Hawksbills – Specialized diet of sponges. * Leatherback – Primarily jellyfish. * Captive sea turtles will eat fish, crustaceans, and mollusks and may be acclimated to balanced diets in pellet or gelatin form. * Hatchling chelonians of all spp should be fed daily. * Most spp should be fed at least twice a week as adults.

Answer 25

Analgesia * Opioids * Morphine has been shown to provide analgesia, butorphanol did not have any effect on thermal threshold * Meperidine at 20 mg/kg – equivalent analgesia to morphine * Buprenorphine (0.075-0.1 mg/kg) maintains concentrations considered analgesic in humans * Tramadol has also increased the thermal threshold * NSAIDS have not shown pharmaodynamic evidence of analgesia but are commonly used * Meloxicam pharmacokinetics vary widely * Local Anesthetics * Prevent transmission of nerve impulses by interrupting sodium influx into the neurons * Bupivacaine is more potent and longer acting than lidocaine * Lidocaine injected intrathecally at 1mL(20 mg)/25 kg Galapagos tortoises was sufficient to perform phallectomy

Answer 26

Anesthesia * Pectoral muscles are preferred site for injections * Dorsal coccygeal vein is a reliable IV site for dangerous unsedated turtles * Death has been associated with subcarapacial injections that were accidentally intrathecal * Propofol – 4-8 mg/kg as a single agent * Ketamine + alpha 2s * Alfaxalone * Softshell turtles can absorb MS-222 through their skin * Tidal volumes vary – sea turtles have much higher tidal volumes than terrestrial tortoises Specific Drug Info * Alpha 2 Agonists * Cardiopulmonary effects similar to mammals * Bradycardia & hypoxemia described * IV administration of antagonists can produce severe hypotension * Ketamine * 30-50 mg/kg required to reach anesthesia alone, has long recovery * Benzodiazepine & alpha-2s enhance muscle relaxation and reduce doses * Propofol * Large species appear to require smaller doses than small chelonians * Fast induction and recovery time make it a preferred drug for short procedures * Neuromuscular Blocking Agents * Rocuronium – allowed for endotracheal intubation in box turtles and amazon turtles * Can be antagonized with neostigmine & glycopyrrolate (to reduce neostigmines parasympathetic effects) Recovery * Catechomalines modulate cardiac shunting and may shorten recovery

Answer 27

*Hematology* ● PCV and most other hematologic/biochemical parameters vary sig. by spp., sex, age, nutrition, reproductive status, hibernation, season ● All circulating blood cells retain their nuclei ● Chelonian RBCs large compared to other vertebrates (except amphibians) o Length approx. same as heterophils o May contain basophilic cytoplasmic inclusions, thought to be degenerating organelles or hemoglobin ppts (no pathologic sig.) ● Assessing 1º hemostasis challenging - thrombocytes tend to clump and appear similar to lymphocytes ● Thrombocytes - small amounts of cytoplasm, round to ovoid nuclei o Activated cytoplasm - vacuolated, +/- magenta granules ● Heterophils - largest WBC w/ numerous linear, pink-orange, refractile granules that “melt” together o Toxic Δ - increased cytoplasmic basophilia, retention of 1º granules - severe inflamm/infection ● Mature lymphocytes - thin rim of basophilic cytoplasm and round nuclei ● Monocytes - round to amoeboid, blue-gray cytoplasm, ovoid to reniform nuclei w/ loosely clumped to coarsely granular chromatin o Monocytosis - acute & chronic infections ● Eosinophils - round to ovoid, eccentric nuclei, round, pink-orange granules ● Basophils - smallest granulocyte, fine purple/deeply basophilic granules

Answer 28

*Clinical Chemistry* ● Ca and P important indicators of renal function in reptiles ● ↓ in Ca:P can indicate renal dysfunction ● ↑ in solubility index (Ca-P product) ↑ risk of dystrophic mineralization o Females - typically ↑ Ca, iCa, solubility indices, ↓ Ca:P ● ALT, AST, and ALP - wide tissue distribution, spp. dependent organ conc. ● ALT - kidney, liver, cardiac; not useful for renal, normally released in urine ● AST - ↑ w/ hepatic injury but cardiac/skeletal mm. damage also ↑ o Sig. ↑ in male (vs female) desert tortoises ● ALP - kidney, intestine, bones, lungs, repro tract - limited ability as useful hepatic indicator o ↑ may reflect bone remodeling more reliably than hepatic enzyme induction ● GLDH - holds promise as indicator of hepatocellular necrosis o Localized to mitochondrial matrix, involved in urea production and amino acid metabolism o Though present in CNS & kidney, damage leads to excretion in CSF/urine o Therefore, _↑ in plasma GLDH relatively liver specific_ ● Biliverdin - main hemoglobin breakdown product o Green-tinged plasma may indicate liver dz, no commercial assay available ● CK - predominates in skeletal/cardiac mm. w/ smaller amounts in CNS and GI tract ● GLU – varies w/ season, age; hyper- d/t stress or hypo- d/t prolonged anorexia, severe liver dz, septicemia ● ALB – hypo- 2º to renal or intestinal losses, as a neg. acute phase protein, or d/t ↓ protein intake o Adult nesting seat turtles - ↑ TP, Alb, Glob compared to juv. of same spp. o Desert tortoises - dehydration has minimal effect of TP, Alb conc. ● TRIG/CHOL - vary widely, esp. in reproductively active females

Answer 29

● Metabolic bone disease (MBD) - nutritional or renal in origin o **Nutritional MBD** more common; almost exclusively in captivity ▪ D/t diets deficient in Ca or Vit D, imbalances in Ca:P, lower ambient temps, inadequate UV light thought to play a role ▪ Common in aquatic turtles housed indoors; w/o UV, can’t synthesize calcitriol (active form of D3) necessary for intestinal Ca absorption ▪ **Hypocalcemia** **🡪** ↑ **PTH** **🡪** **enhanced bone Ca resorption** **🡪** **osteopenia** (rickets - young animals; osteomalacia - older animals) ▪ Serum Ca low normal, P can be normal to slightly low d/t ↑ renal excretion o **Renal MBD** - sequela to 1º chronic renal dz ▪ **Decreased GFR** **🡪** **hyperphosphatemia (hallmark of RMBD)** **🡪** **hypocalcemia** **🡪** **soft tissue mineralization & osteodystrophy** o Overlap in appearance of lesions of osteoporosis, osteomalacia, rickets, and fibrous osteodystrophy (not commonly reported in chelonians like in other reptiles) o Clinical signs 🡪 generalized mm. weakness, cloacal/rectal prolapse, tremors, ataxia, repro. stasis, growth retardation, pathologic fx (uncommon in chelonians), softening of shell, pyramiding, separation of scutes, thickening of rhamphotheca, bowing of the limbs o Aquatic/semi-aquatic - often erosive shell lesions w/ 2º infections o Histo – main (but not pathognomonic) 🡪 osteoclastic bone resorption & ineffective mineralization of osteoid

Answer 30

● **Secondary nutritional hypothyroidism** (& subsequent goiter) o In captive animals o Assoc. w/ diets ↓ in iodine or ↑ in goitrogenic plants (kale, broccoli, cabbage) o Iodine toxicity can also lead to goiter o Galapagos, Aldabra tortoises more susceptible o Exact pathogenesis unclear, but common in sea turtles in artificial salt water aquaria for prolonged periods o Grossly - gland enlarged, follicles engorged o Selenium def. in other spp., not described in chelonians

Answer 31

● **Gout -** frequent in captive terrestrial chelonians o Hyperuricemia - most common cause ▪ Assoc. w/ renal dz, dehydration, inappropriate diet, reduced renal perfusion o Uric acid circulates in blood as insoluble monosodium urate, excreted directly by renal tubules o Hyperuricemia 🡪 precipitation of urates and salts as insoluble crystals ▪ Common in kidneys and joints, can also be visceral surfaces o Grossly - kidneys swollen/mottled w/ disseminated pinpoint to linear, white, chalky deposits, soft tissues around joints/joint spaces swollen - can hinder mobility o Renal tubular damage via mechanical and occlusion of tubules o Tophi form when crystals bind w/ cellular/inflamm, & organic debris, form stellate aggregates o Urate crystals dissolve readily in alcohol; unfixed, unstained cytology 🡪 polarized crystals ● **Pseudogout** o Crystal deposition than urate salts (Ca hydroxyapatite in Red-bellied turtles) - typically in joints ● **Renal oxalosis** o Scattered granulomatous inflamm. assoc. w/ oxalate crystals o Not uncommon in some herbivorous marine and terrestrial turtles ● **Uroliths** o Relatively common in terrestrial chelonians, esp. desert tortoises o Majority in captive, but reported in free-ranging as well o Uric acid is most common component, usually complexed w/ Ca, K, and ammonium salts o Radiodense - Rads or CT o Dehydration most common predisposing factor; others - vit A, D or Ca deficiency, excess dietary protein or oxolates, bacterial infection, complication related to FBs o Most common in urinary bladder - single, smooth lamellated concretion, but can have multiple o Can be in ureters, cloaca - not reported in reptile kidneys o Serum UA not typically elevated unless concurrent renal disease, does not correlate w/ uroliths o Small stones - mucosal irritation, mild hematuria o Large stones - mucosal erosion/ulceration, thickening/hypertrophy of wall, transmural inflamm., necrosis, coelomic/visceral adhesions o Retropulsed eggs occasionally trapped in urinary bladder

Answer 32

● **Obesity** o Common in captivity - excessive calories, poorly balanced diet, sedentary lifestyle o Can predispose to fatty liver syndrome in face of starvation o Interpret w/ caution - fat content of liver can vary dramatically w/ metabolic state (hibernation, reproductive season) o Obese turtles may have reduced mobility d/t excessive SQ fat around neck, limbs ● **Anorexia, prolonged** - severe protein malnutrition/cachexia o Signs - loss of mm. mass, weakness, lethargy, reduced SQ fat, loss of coelomic fat bodies o Gallbladder ectasia w/ normal bile consistency may indicate recent anorexia; distended gallbladder w/ thick, mucoid, inspissated bile can be assoc. w/ prolonged anorexia

Answer 33

*Metabolic* ● **Hypothermic (cold) stunning** o Free-ranging; abrupt drops in water temps o Juv. - cool down faster 🡪 smaller bodies, higher surface:volume o Reduced activity, prone to predation: GI and resp. Dz common o Sequelae 🡪 fungal/bacterial pneumonia, ulcerative enteritis/gastritis, parasites, related to immune system dysfunction 2º to debilitation ● **Hepatic lipidosis** o Physiologic (hibernation, reproduction, etc.) or pathologic o Cytoplasmic accumulation of triglycerides w/in hepatocytes o Grossly - enlarged w/ rounded edges, light brown to pale tan o Early stages of pathologic HL - anorexia, protein malnutrition, hepatic injury, systemic disease; grossly indistinguishable o Liver may float in fixative, water ● Disseminated **hepatic fibrosis** and **hepatocellular nodular hyperplasia cirrhosis)** o Can be 2º to several chronic disease processes ▪ Hepatic inflamm. or severe lipidosis, heart failure, hepatic/post-hep. biliary obstruction o Grossly - SQ edema/ascites, small liver w/ pale yellow/tan hyperplastic nodules surrounded and separated by thick fibrous connective tissue septae, often w/ pigmented macrophages

Answer 34

● **Aural abscesses** o Common w/ suboptimal conditions; uni- or bilateral, well documented in E. box turtles o Etiology multifactorial - improper housing/unsanitary conditions, poor nutrition o Usually mixed Gram-neg., aerobic bacteria that are part of normal flora of the oral cavity & gain access to tympanic cavity via the Eustachian tube o **Squamous metaplasia assoc. w/ chronic hypovitaminosis A** ▪ Not essential for development of aural abscesses, but predisposes to accumulation of keratinized debris and facilitates bacterial colonization ▪ Hypovit A also assoc. w/ metaplasia of lacrimal & Harderian gland ducts 🡪 can lead to development of cysts in 3^rd eyelid, interference w/ closing of eyelids

Answer 35

*Viruses* ● **Herpesviruses** o All reptile **herpesviruses** belong to subfamily *alphaherpesviridae* o 1^st herpesvirus isolated in terrestrial chelonians named **tortoise herpesvirus 1 (THV-1),** has since been ID’d in other spp. of chelonians ▪ Causes necrotizing stomatitis, glossitis, diphtheritic plaques in oral cavity, Cr. esophagus, & trachea (some coinfected w/ *Mycoplasma*) ▪ Rhinitis, hepatomegaly, enteritis, conjunctivitis also reported ▪ **Eosinophilic to amphophilic glassy intranuclear inclusions** in mucosal epithelium of oral cavity, tongue, GI tract, resp tract, urinary/genital tract; only during **acute phase** o **THV-2 -** isolated from a CA desert tortoise ▪ Similar clinical and pathologic findings as THV-1 ▪ **Endotheliotropism** 🡪 vasculitis, edema, thrombosis in several cases (unlike THV-1) **Ch. 169 Testudinid Herpesvirus (Mader 2019)** * Definition: * Herpesviridae; Alphaherpesvirinae. * Testudinid herpesviruses (TeHVs): * TeHV3 – Widespread, most pathogenic, any tortoise. * Group B assoc with worse lesions vs group A. * TeHV 1 – Russian tortoises. * TeHV2 – Agassiz’s desert tortoises. * TeHV4 – African tortoises, Leopard tortoises. * Transmission y contact +/- vertical. IN and IM inoculation causes dz. * Clinical presentation: * Stomatitis, glossitis, oral/nasal discharge, ocular discharge, conjunctivitis, dyspnea, neuro signs, cervical edema, anorexia, wt loss. * Oral diphtheronecrotic plaques are classic. * TeHV3 is neurotropic, but inclusions present systemically. * Virus reactivation unpredictable, shedding common after hibernation. * Diagnostic confirmation: * Ddx – Ranavirus, virus X (picornavirus), Mycoplasma. * Serology – SN and ELISA. * ELISA validated in Greek and Hermann’s tortoises for TeHV3. * Minimum 2 ELISAs at least 8 wks apart for definitive TeHV3 infection, and 10 wks apart for SN. Both equally reliable. * Helpful in chronic infection without clinical disease. * PCR. * Specific and pan-herpesvirus protocols. * Test of choice during acute infection. * VI, EM, Histo. * VI definitive proof. * Tx: * Acyclovir, ganciclovir reduce viral replication in vitro. * Effective concentrations could not be reached in vivo. * Supportive care – fluid therapy, tx secondary infections. * Vx has been unsuccessful. * Prognosis and prevention: * Lifelong infection. * Quarantine 6+ mos, repeated serology and PCR. * Separate animals with clinical signs. * Avoid overcrowding and mixing spp.

Answer 36

* **Fibropapillomatosis (FP)** * Emerging, panzootic infectious dz affecting **green sea turtles,** documented in all sea turtle spp.; recognized in 1938; pathogenesis and epidemiology not yet elucidated * Putative agent currently - **chelonian herpesvirus 5 (ChHV-5)**, previously chelonid fibropapilloma-assoc. herpesvirus * *Alphaherpesvirus,* genus *scutavirus*; at least 4 genetic variants * Has been identified in non-affected animals (suggests not strict viral cause) * Common in post-benthic juv. migrating to coastal shallow feeding areas * Both genders; suggests horizontal transmission (aggression, mating) * Has not been identified in deep ocean-dwelling turtles * Stressors, new environments, social dynamics may play a role * Water temp, anthropogenic activity, algal blooms implicated * Spontaneous regression noted in some location * External tumors in natural & experimentally induced - identical, pathognomonic * Tumors - vary, flat, plaque-like lesions to raised exophytic gray to black, verrucous masses over 30 cm in diameter * Arise from skin around eyes, mouth, limbs; also on cornea, surface of shell * Localized fibroblast proliferation w/in superficial dermis 🡪 hyperplastic epidermis 🡪 exophytic nodules composed of papilliferous fronds * Histo – acanthosis, basal cell necrosis, cleft formation along dermo-epidermal jxn * **Eosinophilic intranuclear inclusions are rare/transient** * Internal, well-demarcated, smooth tumors (firm, white, nodular) described in oral cavity, glottis, lungs, kidney, heart, liver, spleen, GI tract; tend to develop late in dz process * Foreign body granulomas assoc. w/ sprirochiid trematode ova or epidermal inclusion cysts not uncommon in FP lesions of free-ranging turtles * Ulcerated regions often assoc. w/ bacterial or fungal colonization

Answer 37

* **Adenoviruses** - until recently unrecognized in chelonians * **Sulawesi tortoise adenovirus-1 (STAdv-1)** * Mortality event in 105 confiscated Sulawesi tortoises 2006 * Only reptilian adenovirus w/in *Siadenovirus* genus * Interspecies transmission is likely possible * Clinical signs - poor BCS, anorexia, lethargy, ulcerative stomatitis, nasal/ocular discharge, diarrhea * **Intranuclear, glassy, amphophilic to basophilic inclusions** that displace nuclear chromatin * Inclusions in many sites - epithelial, hepatocytes, Kupffer cells, cardiac & vascular endothelium, bone marrow myeloid and stromal cells, spleen, pancreas, testes, ovaries, kidneys, resp. epithelium, cerebral glia, choroid plexus * Adenovirus-related dz most common in young & immunocompromised animals, or naïve * Novel adenovirus from n=1 ornate box turtle in Europe - similar gross/histo findings to STAdv-1 * Pet trade may be at higher risk d/t mixing of geographic species, inappropriate husbandry and nutrition, other stressors

Answer 38

● **Papillomaviruses** o ***Caretta caretta*** **papillomavirus (CcPV)** – ID’d in a free-ranging loggerhead o ***Chelonia mydas*** **papillomavirus (CmPV)** – ID’d in in free-ranging green sea turtles ▪ Both developed few to many soft, white raised smooth nodules over head and flippers ▪ Histo – **eosinophilic intranuclear inclusions** surrounded by clear halo

Answer 39

* ***Iridoviridae*** – diverse group of viruses that infect vertebrates and invertebrates * Contains **Iridovirus** and **Ranavirus** genus (all OIE reportable); implicated in cause of disease and mortality in chelonian populations worldwide * Mediterranean and gopher tortoises, Eastern box turtles (EBT), red-eared sliders * **Turtle virus -3 (TV-3)** (nearly identical to **Frog virus-3 (FV-3)**) - recovered from EBT * Enviro. temps may have sig. effect on virus-assoc. morbidity/mortality * Transmission unknown, but because iridoviral particles can be observed in circulating WBCs; may be by blood-feeding parasites or biting insects * Other possible transmission modes - consumption of infected animals, enviro. contamination, fomites, amphibian/fish reservoirs * Lesions (vary some by spp.) 🡪 oral plaques/ulcers, necrotizing glossitis/tracheitis, pneumonia, multifocal gray foci, hemorrhage in liver, hepatic lipidosis, splenomegaly * Necrotizing splenitis w/ hemorrhage - consistent in red-eared sliders * raised, often ulcerated abscesses in EBT skin fairly common * Fibrinoid vasculitis - consistent finding * **Intracytoplasmic basophilic inclusions in epithelial cells** (renal, hepatic), although _uncommon and difficult to ID_

Answer 40

● Ulcerative lesions of skin and shell, 2º infection w bacteria primarily in captive FW chelonians o Often assoc. w/ suboptimal housing, nutrition, management o Often start as abrasions or result of conspecific aggression o If becomes systemic - **SCUD (septicemic cutaneous ulcerative disease)** ▪ Signs - Lethargy, weakness, limb paralysis, cutaneous abscesses, necrosis of digits or shell ▪ Reported in N. American, Europe; identical presentation in African terrapins ▪ ***Citrobacter freundii*** most common; also ***Serratia* sp.** & ***Beneckea* sp.** ▪ Grossly - cutaneous erosions/ulcers, loosening of scutes; w/ disseminated 🡪 necrotic foci in liver, spleen, kidney, heart, caseous yellow mass in coelomic cavity ▪ Histo - Intense heterophilic and histiocytic inflammation w/ necrosis o Recent ID of fungi consistent w/ *Nannizziopsiaceae* in some lesions complicates pathogenesis; not all should be attributed to bacterial infection

Answer 41

● **Upper respiratory tract disease (URTD)** – rhinitis and oculonasal discharge o 1^st described free-ranging CA desert tortoises, assoc. w/ sig. declines in free-ranging pop o 5 spp. of *Mycoplasma* have been ID’d in tortoises ▪ ***Mycoplasma agassizii*** (desert torts) & ***M. testudineum*** (gopher torts) assoc. w/ URTD o Mycoplasmas thought to be commensal, opportunistic pathogens ▪ Interplay of factors - strain virulence, # of organisms, external stressors, immunocompetency, coinfections o Acute high morality rare but reported in exp. study w/ gopher tort hatchlings o Spontaneous clearance is rare; usually chronic, cyclic infections o Transmission - likely horizontal, close proximity; latent infections reported o Clinical signs - palpebral edema, conjunctivitis, rhinitis, oculonasal discharge ▪ Erosions/vertical grooves ventral to the nares in chronic cases ▪ Often cachectic, lethargic w/ systemic disease (2º infections) when die ▪ Nasal cavity lesions can vary dramatically b/w individuals o Cytoadherence of *Mycoplasmas* (at least *M. pneumonia*) to resp. epithelium is by an attachment structure coated w/ adhesins; once adhered accessory proteins allow intracellular translocation

Answer 42

● ***Salmonella -*** recognized as most important zoonotic dz of reptile origin o Turtle-assoc. human salmonellosis 1^st rep. 1943 🡪 legislation on aquatic turtle sale in pet trade o Routinely isolated from clinically asymptomatic turtles, generally considered normal GI microbes o Up to 85% may be infected by up to 5 diff. serotypes o ***S. durham*** may be most common isolate (one institution) o Intermittent shedding reported in healthy hosts o Debilitated animals can develop pathogenic infections - _clinical disease in chelonians far less common than other reptiles_ o Transmission - fecal-oral, urine, vertical documented (eggs pass through cloaca) o Virulence factors vary greatly b/w *Salmonella* spp. and serovars

Answer 43

● ***Mycobacteria*** **spp.** o Slender, aerobic, acid-fast, and Gram-positive bacilli o Infections most common in aquatic chelonians o ***M. marinum*** & ***M. chelonae*** common; *M. kansasii* some, few rep. of *M. avium -* green sea turtles o More common in free-ranging (can be captive), and can be acute or chronic in nature ▪ Acute – lethargy, limb petechial hemorrhages, bloody nasal d/c, internal hemorrhages ▪ Chronic – more common; granulomatous dz - lungs, coelomic viscera, skin; bones, joints o Bacterial emboli and disseminated IV coagulopathy reported o Histo - heterophil aggregates w/ or w/o central liquefied core surrounded by histiocytes, macrophages, multinucleated giant cells o Typically stain positive w/ acid fast stains such as Ziehl Neelson and Fites method **but atypical mycobacteria may not** o Diagnosis - a/f bacteria in exudates, but positive culture and molecular more definitive

Answer 44

*Fungi* * Largely considered opportunistic, typically under managed care * Managed care - suboptimal housing, overcrowding, inappropriate temps, poor hygiene; allow organic debris to build up, promote fungal growth * Free-ranging - adverse enviro. conditions * Hyalohyphomycoses and phaeohyphomycoses reported in chelonians * **Superficial dermatomycoses** * Typically lesions on shell, less commonly on skin * More common in marine and fresh water turtles than terrestrial * White to lightly colored blemishes on carapace 🡪 scute necrosis, pitting, can ulcerate * Fatal infections rare, but outbreaks of ***Mucor*** (FL softshell turtles) and ***Paecilomyces*** (Fly River turtles) reported * In terrestrial - 2º fungal pododermatitis can develop (d/t substrate putatively) * **Necrotizing scute dz (NSD) –** caused by ***Fusarium incarnatum*** * Isolated from free-ranging and managed Texas tortoises * Blemishes on shell confined to superficial epidermal keratin layers * Differs from SCUD – lesions *_do not_* result in systemic dz/death * ***Microsporidiosis*** * Reported in Hermann’s tortoises w/ granulomatous/necrotizing hepatitis, pneumonia w/ intralesional basophilic spores * Stain positive w/ Grocott-Gomori’s methenamine Silver & PAS * ***Phaeohyphomycoses*** * ***Ochroconis*** and ***Exophiala*** (dematiaceous fungi) * Granulomatous to ulcerative skin lesions on feet and tail of box turtles * *Exophiala* - reported oral granuloma (radiated tort), deep ulcerative shell lesions (Aldabra tort), unilateral ocular infection/systemic disease (Galapagos tort) * ***Veronea*** sp. - obstructive tracheitis in stranded green sea turtles * ***Nannizziopsiaceae*** * Unique presentation of mycotic shell disease * Reported in variety of managed and free-ranging aquatic/semi-aquatic chelonians * Grossly - multifocal to coalescing scute ulceration, keratin discoloration, deformation, necrotizing osteomyelitis (radiographically nodular lytic lesions) * Cystic - central accumulation of tan/gray caseous material, large lesions may compress coelomic viscera * Hyphae readily visible w/ GMS stain but may be inapparent w/ routine HE staining * Phylogenetic analysis 🡪 distinct from related reptile pathogens (*Ophidiomyces, CaNV)* * **Systemic mycoses** * Respiratory tract is primary route of infection (usually the lungs), dissemination possible * ***Fusarium*** spp. - assoc. w/ cold-stunning events, causing fungal pneumonias * Fungal pneumonia assoc. w/ sporulating fungi (***Aspergillus, Paecilomyces, Penicillium*** spp.) relatively common in giant tortoises * *Aspergillus* spp. fungal pneumonias usually involve lower resp tract * Caseous, firm, nodular masses, fungal plaques on mucosal or coelomic surfaces * Dissemination to liver, spleen, kidneys, other viscera possible * **Yeasts**, (esp. ***Candida albicans***) - widespread in GI tract; pathologic infections are rare * Reports include oronasal ulcers (radiated tortoise), pneumonia (Greek tortoise), enteritis and pneumonia (stranded loggerhead sea turtle)

Answer 45

*Metazoa* * **Spirorchiid trematodes** – at least 10 genera documented in sea turtles * One of most common/widespread CV parasites in world * Thought to play a role in sea turtle stranding and mortality * Life cycle unknown; thought to involve a gastropod and a motile cercarial stage * ***Amphiorchis*** sp. identified as key intermediate host for infection in loggerheads * Infection extremely common in free-ranging sea turtles, but trematode burden does not always correlate w/ clinical disease or death, however ↑ tissue ova burden may correlate w/ declining BCS * **Spirorchiid infections often colocalize anatomically w/ other dz processes,** like FP-assoc. skin & internal tumors, coccidian-assoc. enteritis, systemic Gram-neg bacterial inf. * Adult trematodes 🡪 cardiac chambers, lumina of great vessels * Gross - cardiac and vascular lesions 🡪 endocarditis, arteritis, tunica muscularis thickening, mural mineralization, intimal nodular proliferation, thrombosis, aneurysms * Ova - round to elongated (500 um x 100 um) deposited in vascular lumen, wall, or @ perivascular sites (most commonly spleen), often assoc. w/ granulomatous inflammation

Answer 46

* **Intranuclear coccidian** * Reported in confiscated/pet-trade radiated, giant, flat-tailed, spider, bowsprit, and impressed tortoises, E. box turtles, and Arakan forest turtles * Clinical presentation variable 🡪 clear mucoid nasal/ocular discharge, lethargy, weakness, anorexia, anemia, leukocytosis, hypoNa, hyperuricemia, hyperglycemia * Life cycle and mode of infection/transmission not been elucidated * Generally systemic and widespread infections; concurrent mycoplasmosis, amoebiasis, bacterial septicemia possible * Grossly - severity and distribution vary; mucous membrane pallor, rhinitis w/ oronasal fistulas, generalized SQ edema, coelomic effusion, pseudomembranous enteritis, epicardial and intestinal petechiae, tan liver discoloration, splenic congestion, gray pallor to red mottling in kidneys, urinary bladder distension, pulmonary congestion, skin ulceration * Intranuclear stages more commonly in renal tubular epi cells, hepatocytes, enterocytes, pancreatic ductular epi, acinar cells, u bladder, ureteral epi, nasal mucosa, tracheal epi, and pneumocytes * Most common histo lesion 🡪 single cell necrosis assoc. w/ intranuclear (or less commonly intracytoplasmic) stages of organism * Organisms not ID’d in CNS but inflamm. infiltrates in brain/spinal cord of some affected animals * Can be difficult to ID on HE stain, do not selectively stain w/ Fite’s acid-fast of PAS methods * Cytology of nasal exudate w/ Wright-Giemsa may highlight organisms * qPCR on swabs from conjunctiva, oral, choanal, or cloaca for confirmation in suspect cases

Answer 47

* **GI protozoal parasites** * ***Eimeria*** **spp.** most common in chelonians * ***Eimeria caretta*****,** * ***Caryospora cheloniae*** - assoc. w/ mortality in mariculture-raised green sea turtles and mass mortality events in green sea turtles in Australia * Main lesion - heterophilic to granulomatous meningoencephalitis and fibrinous or fibrinonecrotizing enteritis * Meronts packed w/ numerous basophilic merozoites - cytoplasm of intestinal epi cells and w/in WBCs in intestinal mucosa & lamina propria, thyroid, brain; also renal tubular epi. * ***Cryptosporidium*** **spp. -** at least 2 novel spp. ID’d in GI tract of tortoises (Testudo) * Signs not always obvious 🡪 diarrhea, pica, weight loss, undigested food in feces, anorexia * **Gastric cryptosporidiosis** – stomach dilated and filled w/ fluid, organisms along apical surface of gastric mucosal epithelial cells * **Intestinal cryptosporidiosis** – fibrinonecrotic lesions, ulceration, hemorrhage w/ intracellular, extracytoplasmic organisms assoc. w/ apical border of enterocytes * **Amoebiasis** (usually ***Entamoeba invadens***) * Pathogenesis similar to other reptiles * Lesions – fibrinonecrotic enteritis/colitis, +/- necrotizing hepatitis via dissemination through portal vasculature or common bile duct * Amoeba readily apparent on HE stain

Answer 48

*Ectoparasites* * **Epibiont load** – # of barnacles, leeches, algae, & other marine life adhered to shell of sea turtles * Can be indicator of weakness/disease, esp. if adhered to skin as well as shell * Excessive algae suggests consistently floating near the surface * Excessive barnacles suggests inability to scrape barnacles off on rocks * Interpret w/ caution as epibiont loads vary with species * **Ixodid** and **argasid ticks** * Typically in free-ranging; usually skin of limbs/neck, some spp. localize in seams b/w scutes * Anemia w/ severe burdens, can cause skin wounds that become 2º infected * No reports of tick-borne disease in chelonians, very rare in reptiles in general * ***Amblyomma*** ticks are vectors for mammal pathogens such as *Ehrlichia ruminatium*, the cause of heartwater dz in domestic ruminants in Africa - have been found on leopard tortoises * Careful exam and removal of ticks necessary to prevent inadvertent pathogen introduction

Answer 49

Acute Mortalities * Multiple deaths- infectious, environmental failure, or intoxication. * Herpesvirus, intranuculear coccidiosis, adenovirus, entamebiasis, ranavirus, chlamydiosis, reovirus, and paramyxovirus. * Indoors or in a green house, overheating lethal - above 38 to 43 C (100-110 F), all spp.

Answer 50

Dermatological Signs * Cutaneous or SQ swelling * Box turtles, torts, or aquatic turts - myiasis caused by Cistudinomyia cistudinis. * Maggots encyst SQ, breathe through small black-rimmed pores on base of neck, base of legs, axilla, prefrm fossa, pericloaca. * Joints or feet - gout, pseudogout or calc circumscripta, infection, FB, parasites, puncture wounds, fracture, luxations. * Aural abscess * Tumors- epidural squamous papillomas (aquatic turtles). * Bacterial (i.e. mycobacteria) or sub bacterial or fungal granulomas. * Fibropapilomas in green sea turtles. * Gas bubble disease. * Spirochid blood fluke eggs - granulomatous tissue reactions. * Can occlude blood vessels and manifest as ulcerative ecchymyotic shell lesions * Erythema, petechiae, eccymoses, purpura, foul odor of shell or skin – unsanitary husbandry. * Bacterial or fungal shell infections. * Focal areas, white to yellow to serosanguinous under scutes – osteomyelitis. * Diffuse shell erythema also seen with septicemia, starvation. * Erythema * Septicemia (endotoxins, exotoxins, hemoprotozoans), bacterial dermatitis, burns, or hypervitaminosis A * Juvenile freshwater turtles - reddish aposematic coloration is normal, fades with age. * Aural Abscess – Common in Emydid turtles. * Whitish Growth on Skin - shed skin piecemeal (normal), scar tissue, fungal, viral, parasitic infection. * Whitish areas on shell - healed shell lesions (normal), exposed healed bone missing scutes, mineral deposits, ongoing shell infection – bacterial or fungal. * White areas may be seen in annuli between skutes in rapidly growing turtles, normal, darkens with age. * Texas tortoises - superficial whitish scute necrosis that spares the new growth annuli, caused by Fusarium semitectum. * Sloughing Skin * Full thickness is abnormal- hypervit A, bacterial infections i.e. anaerobes, trauma, starvation, chemical or thermal burns. * Desert tortoises - too damp during hibernation - ulcerative dermatitis. * Aeromonas hydrophila * Sloughing Scutes * Bacterial or fungal infections - moist environment or from chronic renal failure. * RF – Bone plates may loosen and ooze azotemic fluid. * Ascites may also be present. * Tortoises - nutritional deficiencies may cause flaking and sloughing of scutes, secondary infections. * Aquatic species normally flake off portions and shed scutes annually. * Shell Ulceration and Necrosis * Trauma * Terrestrial chelonians – Wet shell necrosis (bacterial), dry shell necrosis (fungal). * Ulcerative shell disease or shell necrosis - chronic contagious disease (Beneckea chitonovora - bacterial from shellfish may cause pitting of shell of aquatic turtles). * Speticemic cutaneous ulcerative disease (SCUD)- caseated craterform ulcers, ventral surfaces of aquatic turtles. * Untreated - hepatic necrosis, paralysis, and death * Free-ranging desert tortoises - cutaneous dyskeratosis – lesions commence at seams and spread towards middle of scutes, high mortality. * Overgrown Rhamphotheca and Toenails * Nutritional disease like NSHP, protein deficiency or excess, lack of wear (less likely). * Elongated nails (but not rhampotheca) on front or rear feet (not both) is secondary sex characteristics of some males. * Soft Shell or Distorted shell * Usually NSHP (rarely fractures, just bends/folds). * Normal hatchlings should have firm shells within first few years (solid bone, most spp). * NSHP can cause scoliosis of plastron, lordosis, upturned marginal, increased vertical growth esp at bridge, narrowed or increased shell openings, lack of growth. * Shell appears too small for the chelonian, often assoc with penile prolapse. * Scute abnormalities may be congenital or metabolic bone dz. * Pyramidal Shell Growth * Juveniles most affected, etiology remains unknown, multifactorial. * NSHP, too rapid growth, low humidity, continuous temp maintenance. Not protein excess. * Dermal Fistulas – Trauma or myiasis, abscesses. * Edema or Ascites * Liver (hepatic lipidosis), kidney, cardio-pulm dz, vascular or lymphatic obstruction or hypoproteinemia. * Fat chelonias have excessive lipid deposition in axial and prefemoral areas - don't confuse with edema. * Myxedema has been observed in chelonians with thyroid/thymus dysfunction.

Answer 51

Gastrointestinal Signs * Anorexia – Nonspecific. Winter anorexia common in wild-caught spp that normally hibernate. * Pale Tongue and Mucous Membranes * Anemia of chronic disease (poor nutrition), pore peripheral circulation, blood loss. * Many chelonians normally have lower PCVs than other reptiles or vertebrates. * Infectious Stomatitis * Herpes – Necrotic caseous oropharyngeal stomatitis, glossitis, pharyngitis, tracheitis. * Russian tortoises remain latently infected. * All terrestrial chelonians considered susceptible. * Ranavirus, mycobacterial infections, infectious stomatitis are otherwise rare. * Moisture or Dried Mucus on Rhamphotheca * Oral infection i.e. herpes, pneumonia, regurgitation, ulceration, jaw fracture, rhampotheca abnormalities. * Constipation * Chelonians typically defecate daily to every few days. * Constipation most commonly from colonic impaction from FB i.e. sand, gravel, rocks, cage litter, yard debris. * Fine sand, crushed gravel particular concern. * Few stones may pass uneventfully. * Cystic calculi, low environmental temps, parasites less common causes. * Emesis/Regurgitation * Parasites, FB, septicemia * Adverse drug reactions * 30 mins post admin of enrofloxacin or Vit A at normal dose * Ptyalism, oronasal fluid regurgitation, vomiting common with intestinal/colonic impactions. * Toadstools * Bloating * Fermentation of food in GI tract * Diet change to protein-rich or ccarb-rich foods, force-feeding sick chelonians inappropriate foods, excessive volumes to cachexic animals at suboptimal temps. * Intestinal/colonic obstruction or hypomotility. * Diarrhea * Food with high water concents i.e. fruits. Infectious causes. * Tortoises – Lack of roughage, often when housed indoors. Semiformed feces may not be abnormal. * Mass protruding from cloaca * Phallus is most common, should retract within hours. * Large pink, purple, tan mass with spade-shaped tip. * Ddx prolapse of urinary bladder cloaca, colon, or oviduct. * Bleeding from cloaca * Phallus laceration is common. * Parasites. * Urocystoliths - can lodge in proctodeum. * Cloacaliths - seen in several tortoise species. * Dilated Cloaca - may take several months to reduce, usually from urocystoliths

Answer 52

Respiratory Signs * Nasal Discharge * Pressure applied on gular area should not elicit any nasal discharge nor should forcing the head posteriorly into the shell. * Mycoplasma – most common upper tract disease (usually no mass mortalities). * Herpes is the next most common, almost always has whitish oral plaques. * All terrestrial chelonians susceptible. * White discharge or bubbling from nares, rhinitis, ocular discharge, conjunctivitis, mild palpebral edema, decreased to no appetite, lethargy and weight loss, without lesions in mouth. * Nasal dc may accumulate on forelimbs from wiping. * Herpesvirus (white plaques in oral cavity), ranavirus, adenovirus, intranuclear coccidiosis, chlamydiosis, reovirus, paramyxovirus, FB, and oronasal fistulas. * Multiple deaths common with viruses and TINC. * Nasal Abnormalities * Chronic discharge - erode or depigment nares. * NSHP in growing torts or mycobacteriosis may cause area around nose to bulge asymmetrically (like atrophic rhinitis of swine). * Dyspnea and Abnormal breath sounds. * Stretch neck and gape or pump head and legs. * Gular pumping assoc with olfaction and not a dyspneic sign. * Whistling or click - lower tract dz or glottal/tracheal obstruction. * Gram negative bacteria most common cause. Other infectious diseases or FB or neoplasia i.e. tracheal chondromas. * Uneven Floating * Listing of aquatic turtles – Abscesses, tumors, cystouroliths, GBs, asymmetrical gas or bloating, pneumonia. * Inability to submerge * Common in sea turtles – Emaciation, pneumonia, tumors, GI or coelomic gas (infection, trauma, obstruction).

Answer 53

Reproductive Signs * Failure to Lay Eggs * NSHP, hypovit A, hypcalcemia, dehydration, lack of nesting area. * Need deep substrate (twice length of carapace). * Ruptured, large, or anomalous eggs, oviductal rupture, infection, mass impinging on the oviduct i.e. urolith, fecalith, colonic FB can lead to egg retention. * Eggs may become ectopically located in UB, coelom, colon, or lodge in pelvis.

Answer 54

Musculoskeletal Signs ● Lameness ○ Abscesses, bite wounds, FB, fractures, osteomyelitis, luxation, trauma, gout, pseudogout, sepsis. Females – short-term sequela to egg laying and imbalances in Ca homeostasis. ■ Egg retention, cystic calculi, cloacaliths, constipation may contribute to lameness. ■ N or RSHP. ■ Large breeding males can traumatize limbs when dismounting females. ○ Asymmetrical toenail wear ■ Sign of lameness in terrestrial species, but don’t mistake for secondary sex characteristics. ○ Swollen joints ○ Swollen long bones ■ Fractures, neoplasia, osteomyelitis ■ Fibrous osteodystrophy - common in lizards with NSHP, RARE in chelonians

Answer 55

Neurological Signs ● Ataxia/Hypermetric Gait ○ Tortoises - hyperthermia, near-drowning, or joint luxations ○ Cerebellar injury – hypoxia, cerebral edema, encephalitis. ○ Toxin exposure – Ivermectin, Ops in mite and tick sprays, heavy metals. ● Circling - freezing during hibernation (blindness), toxemia, encephalitis, central nervous system damage, or hepatic encephalopathy. ● Flaccid Paresis, Paralysis or Coma ○ Severe systemic illness – septicemia, organ failure, starvation, death. ○ Toxicity to ivermectin, Rhododendrons, shock, drowning, asphyxiation, hyperthermia, bloat. ○ Check heart beat with doppler.

Answer 56

Ocular ● Blindness ○ Testudo spp after freezing, may be assoc with hyphema, vitreal haze, lenticular opacities, retinal damage. ■ May be anorexic, have head tilt, circl, hold head high. ● Palpebral edema ○ Hypovitaminosis A, herpes infections (more keeping eyes shut). ○ Trauma, FB, bacterial or viral infection, parasites, metabolic disturbances. ○ Box turtles may hold one eye shut due to aural abscess secondary to swelling. ○ Ulcers, panophthalmitis, fire ant stings may cause blepharospasm. ● Expopthalmia - Retrobulbar abscess, tumor, or injury. ● Enophthalmia – Microphthalmia (if OU), thiamine deficiency (frozen fish diet). Herpes, circulatory collapse or hypotension in sepsis or debilitated chelonians. ○ Eyes often sink before death. ○ Unilateral – orbital injury i.e. HBC, phthisis bulbi. ● Palpebral Edema ○ Hypovit A ○ Emaciated or dehydrated turts with herpesvirus ● Conjunctivitis and ocular discharge ○ Sea turtles, red foot and yellow foot tortoises have clear ocular discharge b/c they lack lacrimal ducts.

Answer 57

**Ch. 99 – Prefemoral Coeliotomy (Mader 2019)** * Two major approaches to coelom of chelonians: transplastron and prefemoral. * Prefemoral approach – Through prefemoral fossa. * Less invasive. * Can provide unilateral surgical access to caudal lung, liver, small and large intestine, reproductive tract, kidney, and bladder. * Positioned in lateral recumbency, head down helpful. * Left prefemoral approach often preferred by right-handed surgeons (with a right approach easier for those who are left-handed). * Ipsilateral pelvic limb maximally extended and secured caudal. * Skin incision cranial to extended limb and in a cranial-to-caudal direction, in middle of prefemoral fossa, starting close to skin insertion at plastrocarapacial (bridge) junction and ending just cranial to musculature of limb. * Blunt dissection cranial to sartorius and ventral to iliacus muscles exposes pale coelomic aponeurosis formed by external abdominal oblique muscle * Coelomic membrane, musculature, subcutaneous tissues closed with simple continuous pattern using absorbable sutures. * Skin closed using nylon or polydioxanone and simple, horizontal mattress sutures * Removed (if still present) 8 weeks postop.

Answer 58

**Ch. 100 – Chelonian Transplastron Coeliotomy (Mader 2019)** * Disadvantages: * Increased surgical trauma, pain and discomfort, and prolonged bone healing. * Indications: * Extensive exposure (for large cystic calculus, foreign body, or mass removal) or exploration required. * Patients with small prefemoral fossae. * Locations of the heart (usually at midline intersection of pectoral and abdominal scutes), paired abdominal veins (running caudocranially within ventral abdominal musculature), hinges (between abdominal and femoral scutes in Testudo, and between pectoral and abdominal scutes in Terrapene), and plastron thickness (including its expansion toward plastrocarapacial bridge) have to be considered before surgery. * Access cannot be easily enlarged - ensure initial cuts are sufficient. * Plastron incision: * In neonates and young juveniles - scalpel blade can be used. * Oscillating sagittal saw couples accuracy with reduced collateral damage and minimal soft-tissue trauma needed in most cases. * Thinner incisions can often be obtained using a fine blade rotary saw but rotating saws are less forgiving on soft tissues, and over-scoring at corners occurs with larger-blade diameters. * Saw held at 45 to 60 degrees to bevel lateral shell incisions - made first. * Fine hypodermic needle or scalpel blade can be used to gauge depth. * Caudal cut and final cranial cut made in a similar fashion, but bevelling not necessary. * Caudal or cranial cut can be incomplete, with a few millimeters bone thickness left. * Soft tissues are bluntly dissected free while elevating cranial and caudal margins, staying as close to the shell as possible. * Paired abdominal veins - closely associated with plastron. * Soft-tissue attachments at fractured bone line can be left intact, because plastron flap is reflected caudal and covered with moistened, sterile gauze. * Entry into coelom options: * Midline incision, between abdominal veins, through poorly defined linea alba. * Unilateral or bilateral paramedian incision(s) lateral to abdominal veins. * Even ligation and transection of one abdominal vein to permit an L-shaped flap for removing large structures. * Ligation of both abdominal veins not recommended, because severe circulatory disturbance would likely ensue. * Coelomic membrane closed in simple continuous pattern using absorbable suture material. * Poliglecaprone 25 or polyglyconate can be used for closure of paramedian incisions, but polydioxanone preferred for linea alba. * 3-4 stabilizing sutures (usually polydioxanone, but wire can be used for large species) placed through drilled holes to anchor bony section to plastron. * Closure - epoxy resin or acrylic (e.g., polymethylmethacrylate), either at 4 points or around the entire bony incision. * Common for plastron flap to become a sequestrum that provides temporary protection for new bone growth beneath. * Plastron typically heals within 12 weeks but can take longer, and therefore the covering is usually left in place for 6 to 12 months. * Rads seldom helpful in assessing healing - radiolucent lines persist long after healing is complete but, osteolysis may suggest osteomyelitis.

Answer 59

* Venipuncture * Sodium or lithium heparin. * EDTA causes hemolysis. * Sites – Jugular most reliable (minimizing lymph), dorsal coccygeal vein, axillary branch of the brachial vein in forelimb, subcarapacial sinus. Occipital sinus. * Sea turtles – external jugular vein/dorsal cervical sinus aka supravertebral.

Answer 60

* Hematology * RBCs long life spans i.e. 600-800 days in box turtles. * Limitations of blood collection 5-8% of total i.e. 0.5-0.8 mL for 100 g animal. * Heparin preferred anticoagulant. * Total WBC counts – direct via Natt-Herricks or indirect via phloxin B sol or estimation from smear. * Chelonian leukocyte response less predictable vs mammals or birds. * Varies by spp, season, nutritional status, type of stain, venipuncture site, handling of sample, age, sex, anticoag used. * Twofold changes in a parameter constitute a significant change. * Het counts incren ase during summer and decrease during brumation. * Toxic hets display cytoplasmic basophilia, abnormal granulation, lobed nuclei, are present in cases of inflammation. * Basophilia may be present with parasites or viral dz. * Lymph counts low to absent in winter and low in cases of malnutrition and dz secondary to stress and immunosuppression. * Lymphocytosis seen with wound healing, parasites, viral infections. * Monocytes increase with granulomatous disease.

Answer 61

* Biochemistry * EDTA -\> changes in K and Ca directly and other parameters due to hemolysis. * Biochemical assays routinely run on plasma. * Glucose, Ca, P, Na, urea, enzymes in lymph are comparable with those in plasma. * Lymph lower in TP and K vs plasma. * BUN, Cr poor indicators of renal disease. * Low BUN in marine spp may indicate chronic anorexia. * Plasma UA in chelonians generally \< 5 mg/dL. * Elevations may be seen in sepsis, nephrocalcinosis, nephrotoxicity, gout, recent high-protein diet. * Hyponatremia may result from GI or renal dz, oversupplementation of fluids low in sodium, dz of salt gland, maintenance of SW spp in FW. * Hypernatremia may occur in dehydration or excessive dietary intake. * Potassium elevated with hemolysis, reduced renal secretion, reduced intake or excess GI loss. * Ca may increase 2-4x with follicular development. * Hypocalcemia may be caused by anorexia, dietary deficiencies of calcium or vit D3, hypoalbuminemia, alkalosis, hypoparathyroidism. * Phosphorus can decrease with starvation, nutritional deficiency. * Hyperphosphatemia from excessive dietary phosphorous, hypervitaminosis D, renal dz, severe tissue trauma or be falsely elevated with hemolysis. * AST, LDH high in liver tissue but not specific. CK muscle specific. * Increased LDH, AST in absence of high CK indicates liver disease. * Low TP seen with chronic malnutrition, PLE, maldigestion, chronic liver and kidney dz. * High TP seen during folliculogenesis and dehydration. Increased globulins. * Hypoglycemia may be caused by starvation, malnutrition, hepatobiliary dz, septicemia. * Hyperglycemia likely iatrogenic.

Answer 62

**Ch. 56 – Radiography of Chelonians (Mader 2019); Review images from this chapter.** * Restraint and positioning o Elevation of chelonian by placement of plastron on an appropriately narrow lucent structure (central plastron stand) encourages head and limb extension. o Chemical restraint helpful for evaluation of head or limbs (depending on patient) – tape can also be used for limb arrangement. o Position as close to cassette as possible. o Shell curvature – structures further away from cassette will be magnified or blurred – right and left laterals recommended for larger chelonians. o Orthogonal views: Dorsoventral, horizontal beam lateral(s), cranial caudal images. o Technique adjustments for shell may be needed – increase mAs.

Answer 63

* Cardiovascular system * Radiography not commonly used to assess for heart disease – heart poorly visualized with indistinct borders -\> echocardiography. * Heart located as soft-tissue opaque structure ventral to the tracheal termination. * Loggerhead – triangle formed by scapula and coracoids is landmark. Respiratory system * Lung is arborized – small airways terminating in spongy (edicular) air spaces; dorsal lung adheres to carapace parietal serosa, interspersed muscular fibrous bands. * Pulmonary vasculature should be visible. * Limb and head extension are important for lung expansion. * PPV – less risk of misinterpreting overinflation compared to snakes and lizards. * Diseases – increased pulmonary soft tissue opacity- focal, multifocal or diffuse. * Fibrosis is differential for diffuse or multifocal pattern. * Focal – focal pneumonia, granuloma, abscess, neoplasia. * Cold-stunned Kemp’s ridleys distribution and pattern of pulmonary infiltrates have not been reported with disease/condition specificity (honeycomb pattern). * CT optimal

Answer 64

* Gastrointestinal * Temperature, nutrition, time since last meal must be considered. * DV and HBL, best visualized on DV. * Stomach in left mid portion, minimal gas should be present (aerophagia, fermentation, obstruction). * Large intestinal contents – mixed soft tissue and gas appearance or more granular. * GIT is uncommonly empty in chelonians. * Barium contrast studies reported for a variety of species – EBT, red-eared slider, leopard tortoise, Geoffroy’s side necked turtle, yellow-spotted river turtle, and Arrau river turtle (dosages vary by species and temperature). * RES transit time: 72-192 hours. * Leopard tortoise: 144-166 hours. * Iodinated contrast studies – red-eared slider, Greek tortoise, faster transit times. * GT had faster transit times between 27 and 34 deg C. * Avoid respiratory administration. * Generalized ileus –progressive accumulation of ingesta or abnormal motility. * Severe gas distension – obstruction. * Fish hooks are common.

Answer 65

Reproductive system * Reproductive/dystocia monitoring – evaluate uniformity of shell and size, thickness. * Ultrasound and CT recommended as second line. * Dystocia – enlarged eggs, fractured shells, follicular stasis, normal eggs outside of reproductive season. * Increased but even mineralization and smooth – usually prolonged retention in lower repro. * Thickened and rough shells can indicate uric acid and mineral deposition of shells that have been retained within urinary bladder. * Soft tissue mass effect – follicular stasis is differential, soft tissue accumulation can also indicate yolk coelomitis.

Answer 66

Urinary system * Expansive in volume, bilobed, can extend to mid cranial coelom close to stomach. * Commonly evaluated for urinary calculi – urate is most common in chelonians (radiolucent, but can mix with other minerals); more common in left lobe of bladder; irregularly rounded shape; obstruction uncommon. * Severe urinary bladder distension due to spinal fracture reported. * Kidneys hard to see and renal calcification is not commonly seen.

Answer 67

Musculoskeletal system * Radiography most useful for this in chelonians. * Long bones have distinction between cortical and medullary regions, but less distinct than mammals. * The pectoral girdle bones should be clearly visualized with sharp margins, small medullary canal and homogenous cortical radiopacity. * Shell fractures – help document the degree of displacement and extensiveness of shell trauma and investigate concurrent soft-tissue abnormalities associated with injuries (pulmonary); CT helpful. * Healing of shell – fibrous callus -\> fracture lucent line can persist for years; recommended to repeat rads 10-20 weeks after stabilization, complete healing/remodeling may take 12-18 months. * If carapace fracture crosses midline, spinal fracture should be expected – further imaging modalities helpful to evaluate spinal cord. * Orthogonal views of limbs – lateral and craniocaudal/dorsopalmar(plantar) images. * CTs better define abnormalities- good for surgical planning. * Osteomyelitis and septic arthritis common – lytic (greater degree than mammals, accompanied by caseous abscessation) and osteoproliferative changes, soft tissue enlargement. * Gout – soft tissue mineralization, gout tophi, pseudogout. * Degenerative joint disease common – they live a long time, trauma, resolved septic arthritis. * Periarticular mineral proliferation. * Osteophytes. * Early stages – difficult to distinguish active septic arthritis from degenerative changes. * **Advanced disease – soft tissue mineralization around joint (not common in mammals) – limit range of motion.** Nutritional secondary hyperparathyroidism common. * Know expected degree of mineralization for species and life stage. * Juveniles have reduced mineralization for a year; assess pectoral or pelvic girdles. * Diffusely decreased opacity of bones is primary abnormality. * Cortical bone thinning, shell and rarely limb thickening, pathologic fractures.

Answer 68

**Ch. 61 – Medical Aspects of Giant Tortoise Relocation in the Galapagos Islands (F9)** * Giant tortoise population decimated by human influence with 3 species that has gone extinct. * Keystone species Rebuilding tortoise populations involves 4 major program activities: 1. Captive breeding and release 2. Head-starting and release of hatchlings from wild nests 3. Release of sterilized hybrid adult animals 4. Repopulating islands with breeding populations of genetically selected surrogate species * Galapagos islands discovered in 1535 - as many as 250000 tortoises at the time * Tortoises harvested from 17th to 20th century * Population estimate in 1970s to be as low as 8000 individuals 15 species described with two general morphologies: 1. Dome shaped (carapace has smooth and rounded contour) 2. Saddleback (front of carapace raised and looks like a spanish saddle from the side) 3. Dome shaped animals generally larger and are found on islands with higher elevation where vegetation is denser 4. Saddleback smaller (males up to 100kg) and are found on more arid islands. Elevation of the front of the carapace allows animals to reach higher when foraging in dry vegetation 5. Few populations are intermediate between dome and saddleback shapes * Population impacts: Human predation, introduction of livestock, introduction of dogs, cats, norway rats, black rats, house mice, invasive plants \>\> destruction of food sources, destruction of canopy of vegetation that naturally captures fog and mist precipitation, predation of eggs and hatchlings, damage to nesting areas **Restoration of Giant Tortoise Populations** * Restoration of tortoise populations dependent on eradication of introduced pest species and removal of hunting pressure through public education * No successful recruitment of hatchlings occurred on Pinzon island for nearly 100 years due to predation by black rats. Hatchling eggs and hatchlings still in the nest were collected and transported to Santa Cruz island for rearing until 5yo and then returned. * Other populations from Santiago island and santa cruz island were identified for head starting. **Head starting** * Hatchlings and eggs are collected from nesting areas on Pinzon, Santiago and Santa Cruz islands * Nests opened with hand digging, movement only during late incubation, egg orientation kept the same as it was in the nest. * Eggs and hatchlings transported in small plastic containers with lightly moistened vermiculite to maintain humidity * Embryonic mortality encountered when incubation temperature too high or low and eggs incubated at lower temperatures have longer incubation times. * Hatchlings are reared in rodent proof containers, outdoors on a substrate of crushed lava. * Diet consists of leaves of two plant species: Arrowleaf elephant ear and coral bean which are offered 3 times a week * Moved to preadaptation enclosure at 1.5 years of age where they have a substrate of lava rocks and natural vegetation up to 5 years of age * Transponders placed in left hind leg which will ID them permanently. Prior to release, each individual is branded on the carapace. * Released when curved carapace length of 20cm is reached, at approximately 3-4 years of age. * Release occurs in suitable habitat, near nesting zone where hatchlings were collected. **Ecosystem Restoration** * GNPD intends to reintroduce tortoises where they have been driven to extinction, with the goal of restoring ecosystems * Woody vegetation obstructs tortoise movement, prevents the growth of cactus spp, and shades the ground - reducing opportunity for tortoises to feed and thermoregulate. * Feral goats have controlled woody vegetation, but at the same time threatened native endemic vegetation. Islands need tortoises to restore natural balance upon goat removal. * Genetically sound (as pure as possible) animals were collected for re-introduction in 2015 **Surrogate Species Use as Ecologic Engineers** * Pinta Island NP chose to use surrogate sterilized mixed heritage tortoises to fulfill the role of ecological engineers until a breeding population can be re-introduced. Surrogate females were oophorectomied and males phallectomized.

Answer 69

**Fowler 6 Ch 18 - Raising Giant Tortoises** Basics: * Order Testudines, families Testudinae and Emydidae * Adults readily differentiated externally: Males have distinctly longer tails than females and the plastron is concave * *Geochelone nigra* = Galapagos tortoise * Current numbers est 12,000-15,000 * Head wider than neck, short nose, small prefrontal scales, absent nuchal scute, single caudal scute * Diet more that of browser * *Geochelone gigantea* = Seychelles tortoise * Current numbers est 100,000 * Approx 20% smaller than G. nigra * Head with similar diameter as neck, rounded ridge of nose, pointed nose, nuchal scute present in 98%, caudal scute mostly double, large prefrontal scales * Diet more that of grazer Captive Management * Challenging to keep outside of natural climate due to large size and requirements * Indoor considerations * UV light, heated environment (24-27 C, hot spot of 38 C), 60% humidity, shallow pool for soaking, mud wallow if able Nutrition * Little known about exact nutritional needs of these animals * Possible nutritional disorders include excessive geophagy with subsequent constipation, loose stools, bloat, metabolic bone disease * Natural diet: grass, leaves, flowers, and fruits * Carnivory and coprophagia have been observed but do not make up more than 0.5% of total diet * 90% chopped high-quality grass hay, other 10% includes variety of herbs, dried leaves, and occasionally produce * Fiber content very important (crude fiber content of 30-40% on dry-matter basis) * Recommend calcium supplementation * Ca:P ratio of 4:1 to 6:1 recommended * Oversupplementation of calcium may result in Ca concretions in UB or urolithiasis * Water at all times Breeding Giant Tortoises * Spatial and social variability important * One male should be significantly larger than others, social groups should not be heavily male biased * Females must be able to avoid males * Large groups of at least 12 animals appears to increase breeding activity * Most mating in summer (temps \>73.5 F) in the morning and late afternoon * Northern hemisphere: mating June-Oct, oviposition Nov-Mar * Southern hemisphere: mating Feb-May, oviposition June-Sept * Galapagos tortoises: follicles became preovulatory at a diameter of 40-42 mm and eggs laid 34-84 days after thin-shelled eggs detected in oviduct * Eggs with shells can be retained until next breeding season without adverse effects * Seychelles artificial incubation of eggs * At least 80% humidity, temperatures 82.4-87.8 F * Temps \>84.2 F result in females * Incubation lasts 125-136 days at temps btwn 28-30 C (90 d at temps btwn 30-32 C) * Galapagos tortoise egg incubation * 29-32 C with 65% humidity, incubation lasts 105-164 days * Takes 2 days from pipping to hatching, best not to “assist” * Recommended to soak juvenile giant tortoises at least twice daily * Captive Galapagos tortoises at age 4 yrs were 2x as long and weighed 10x more than animals under natural conditions * FAST GROWTH * May lead to sexual maturity at younger age and shorter life expectancy * Biting * Bigger incidence if food is reduced, boredom, hunger, overcrowding Restraint and handling * Dorsal positioning (automobile tire is useful); positioning on a flowerpot * Anesthesia: Medetomidine (0.1 mg/kg IM) and Ketamine (10 mg/kg IM) or Propofol (5 mg/kg IV) Noninfectious Diseases in Juvenile Giant Tortoises * Metabolic developmental disease * Metabolic bone disease (differentiate btwn renal and nutritional origin) * Hypomineralization, fibrous osteodystrophy of bones, pyramiding of the carapace * Major predisposing factor is probably the phenotypic plasticity that enables giant tortoises to grow at an increased rate when food is available in large amounts * High protein levels in diet should be avoided because of a repeated link to pyramiding * Lower levels of humidity resulted in significantly increased pyramidal growth of the carapace * Other factors include endoparasites, nephropathy, and typhlohepatitis are possible causes for pyramidal shell development * Recommend UVB light to help prevent, enteral and parenteral doses of calcium and vitamin D should be given if developmental disorders are recognized * Recommend Annual radiographic exams Infections Diseases in Juvenile Giant Tortoises * Respiratory tract disease * Secondary to stress, anatomy (poor mucociliary apparatus), weather (humid and cold), parasitism, deficiencies in husbandry/sanitation/nutrition * Most cases caused by bacteria (also viral, fungal, and parasitic agents) * Clinical signs usually appear late in process * Extended neck, inspiratory and expiratory Dyspnea, anorexia, lehtargy * DX: rads (especially lateral and craniocaudal views) * Aggressive antibiotic treatment necessary * Parenteral and nebulization * Use enrofloxacin cautiously * Increased humidity also proves beneficial by promoting proper hydration and breakup of necrotic and inflammatory debris * Endoparasites * More likely to occur in captivity secondary to stress, inadequate diet, low temperature, and overcrowding * *Entamoeba invadens* * CS: bloody diarrhea and subsequent dehydration, DX: fecal, TX: Metronidazole (50 mg/kg PO x 10 d) * *Coccidia* * CS: diarrhea or unthriftiness, tx only if clinical * Normal flora: *Balantidium and Nyctotherus* * Unknown clinical significance * Oxyurids and strongylids

Answer 70

JAVMA 84(1): 2023 **Bioencapsulation is a feasible method of terbinafine administration in Emydomyces testavorans-infected western pond turtles (Actinemys marmorata)** Anthony J. Cerreta, DVM, MS1; Jennifer M. Reinhart, DVM, PhD, DACVIM, DACVCP1; Lauren R. Forsythe, PharmD, DICVP1; Matthew R. O’Connor, DVM, MPVM2; Karisa N. Tang, DVM, MS, DACZM2; Sherry Cox, PhD3; Krista A. Keller, DVM, DACZM1 Key Points: - Emydomyces testavorans (ET) = emerging onygenalean fungus associated with chronic lesions in FW chelonians - Effective mycosis treatment in reptiles involves long tx course (usually oral) -- Terbinafine = high margin of safety, rare reports of toxicity, drug accumulation in keratinized tissue, published MIC data for ET. Clears through liver & kidney - Oral meds can be challenging in chelonians. -- Alternative = bioencapsulation (drug accumulated into biological substrate) - Terbinafine 30mg/kg SID to EOD via encapsulation in earthworm may be useful tx for ET in WPTs - Oral gavage of same dose followed by small meal may be useful, but steady-state predications were less favorable for this ROA

Answer 71

**Relationship, difference, and diagnostic discordance between blood ionized and total calcium concentrations in client-owned chelonians**. Di Girolamo N. Journal of the American Veterinary Medical Association. 2022;260(S2):S101-110 Key Points: - Lower calcium in reptiles with limited UVB, or low Ca diets - Higher total calcium in females during repro activity and dependent on circulating estrogens - Plasma calcium: ionized/free, protein bound, bound to other anions (phosphate, lactate, bicarb) - iCa and tCa in chelonians do not necessarily reflect each other, their relationship is complex and affected by multiple factors - Recommend measuring both and calculating bound Ca to evaluate overall calcium status and for repro disease - tCa was higher in females and with repro disease but iCa was not affected by either suggesting bound Ca may be more useful

Answer 72

JHMS 2022 32(2):130-5 **Feasibility, Gross Postmortem Results and Participant Perception of Oblique Prefemoral Ovariectomy in Red Eared Sliders (Trachemys scripta elegans) During a Teaching Workshop** Di Girolamo N, Studer K, Maranville R, Caron M, Murciano G, Sellers M, Cococcetta C Key Points: - Chelonians have paired ovaries and oviducts - Surgical access to the reproductive tract in chelonians can be achieved through: -- Incision on the plastron (i.e., plastronotomy) -- Plastronotomy is suspected to be more painful -- Prolonged healing times associated with it can be detrimental for aquatic and semiaquatic chelonians who must be dry-docked post-op -- Incision through the prefemoral fossa (i.e., prefemoral celiotomy) - Modified prefemoral technique = positioning the chelonian in oblique dorsolateral recumbency - Main advantage is that ovaries and oviducts bearing eggs tend to be heavier - Gravity contributes in the exteriorization of the reproductive tract for its excision TLDR: Oblique prefemoral ovariectomy may be an effective technique. In juvenile chelonians a fully endoscopic approach should be considered

Answer 73

**Molecular and Serological Detection of Leptospira spp. in Italian Tortoises (Testudo spp.)** Journal of Herpetological Medicine and Surgery, 32(2) : 136- 143 Key Points: - Lepto DNA and antibodies seen in captive Hermanns and marginated tortoises (20% PCR prevalence, low seroprevalence) - Need for standardization in direct and indirect diagnoses and harmonization of testing procedures - Leptospira able to infect wide range of species – humans, mammals, reptiles, amphibians, birds and inverts -- Seasonal – dependent on availability of water in the environment -- Isolation of lepto from kidney and cloaca of turtles has been reported along with seroprevalence in multiple species in freshwater turtles raising the possibility of a potential zoonotic risk

Answer 74

JHMS 2022 32(3):162-70 **Mycobacterial Cloacitis with Systemic Dissemination in a Hermann's Tortoise (Testudo hermanni)** Ollé RD, Sevilla IA, Juan-Sallés C, Garrido JM, Santamaría JJ Case: - 21yo F Hermann’s tortoise presented for a 15-day history of apathy and tail swelling -- PE: BAR, swollen tail/cloaca, necrotic area at muco-cutaneous junction of the cloaca -- Cloacal lesion was debrided/disinfected, started on ceftazidime -- Switched to enrofloxacin/marbofloxacin after cloacitis worsened -- Ultimately, euthanized due to chronic weight loss, cloacal lesion progression Key Points: - Clinical signs consistent with chronic mycobacteriosis (non-healing external lesions, weight loss) -- No tail/distal hind limb edema like a previously reported group of Hermann’s tortoises - Necropsy consistent w/ mycobacteriosis: granulomatous/necrosuppurative inflammation -- Presence of intralesional acid-fast bacilli confirmed the diagnosis - Mycobacteriosis is seldom reported in reptiles -- Majority of reported cases involved aquatic and semiaquatic chelonian TLDR: First case of cloacitis secondary to mycobacteriosis in a tortoise caused by an atypical mycobacterial agent from the M. terrae complex

Answer 75

JHMS 2022 32(3) 225-229 **Effects of Artificial Ultraviolet B Radiation on Plasma 25-Hydroxyvitamin D3 Concentrations in Juvenile Blanding's Turtles (Emydoidea blandingii)** Key Points: - In RES, leopard geckos, corn snakes, and burmese pythons exposure to artificial UVB increased plasma 25-hydroxyvitamin D3. In ball pythons and hermann’s tortoises it did not - This finding affirms that Blanding’s turtles can synthesize vitamin D via photochemical conversion - Not measured in adults, further research needed as some studies in humans show that young adults can have more circulating vitamin D precursors - Based on the results herein, we recommend exposing juvenile Blanding’s turtles to daily artificial UVB (290– 320 nm) as part of their standard care.

Answer 76

JHMS 2022 32(4):262-70 **Successful Treatment of Severe Ulcerative Dermatitis in an Aubry's Flapshell Turtle (Cycloderma aubryi)** DiRuzzo S, Praschag P, Miller L, Brodsky M Key Points: - Softshell turtles (Trionychidae) have unique shell anatomy: -- Bony shell is greatly reduced -- Flat bony elements made of compact layers w/ an inner cancellous core -- Shell surface is covered by leathery skin rather than horny scutes -- Dermatitis can progress quickly and invade the thin bony structures and coelom - Benefits of photobiomodulation (PBM) are well described in the literature -- MOA not fully known but effects mediated via biologically active chromophore in the mitochondria and light-sensitive ion channels within cell membranes -- PBM reduces pain, positively influences all phases of wound healing, & increases wound tensile strength via acceleration of fibroblasts, collagen synthesis, & neovascularization - PBM in reptiles have found varying results -> subjective improvement in quality/rate of healing TLDR: PBM should be considered for treating ulcerative dermatitis in softshell turtles, especially in patients with delayed wound healing or chronic dermatologic lesions

Answer 77

**Mycoplasma sp. Detection in the State Endangered Illinois Alligator Snapping Turtle (Macrochelys temminckii)**. Johnson SJ, Adamovicz L, Kessler EJ, Allender MC. Journal of Herpetological Medicine and Surgery. 2022;32(4):277-280 Background - Alligator snapping turtles, family Chelydridae - Mycoplasma spp. Thought to be commensal organism that may opportunistically cause disease - CS in chelonians: blepharedema, oculonasal discharge, conjunctivitis, anorexia, lethargy; may recrudesce Key Points - ONE out of 156 samples from an Alligator snapping turtle headstart program in Illinois tested positive for Mycoplasma spp. And was sequenced for GenBank -- This positive turtle was in prerelease and wasn’t tagged so couldn’t compare to postrelease - None of the turtles had clinical signs in pre or post-release Conclusion: clinical significance of Mycoplasma spp. In common snapping turtles is unknown at this time but it is considered unlikely to be a threat to the endangered Illinois population at this time

Answer 78

The Journal of Herpetological Medicine and Surgery, Volume 32, No. 4, 2022 pp. 281–290 **Corticosterone, Lactate, and Injury Severity as Short- Term and Long-Term Prognostic Indicators in Eastern Box Turtles (Terrapene carolina carolina) with Traumatic Injuries Admitted to a Wildlife Rehabilitation Clinic** Emily K. Tucker-Retter, Gregory A. Lewbart Key Points: - Corticosterone is the primary glucocorticoid of reptiles -- Markedly elevated in cold stun kemps ridleys but no diff btwn survivor and nonsurvivors -- Elevated lactate at intake or rise in lactate in first 24h when initial lactate > 5 assoc with poorer px - Lactate concentrations at intake significantly associated with 24h survival - Turtles whose corticosterone increased in the first 24h were more likely to die in the first week that those that decreased or remained the same - Survival at 1 month predicted only by triage score -- Lactate concentration signif assoc with triage score - Lactate, H:L ratio and PCV all decreased over the course of hospitalization - Healthy EBT had static mild elevations in corticosterone - no observable effect of handling Take home: - Mortality in the first 24hr of EBT admitted for traumatic injuries was assoc with elevated lactate and higher triage scores. - Mortality after 24h but within first week was loosely associated with an increase in corticosterone. - Mortality after first week but within 1 month was associated with higher triage scores. - Tissue damage and acute severe stress likely contribute to mortality in first 24h to 1 wk

Answer 79

**PREVALENCE OF MULTIPLE REPTILIAN PATHOGENS IN THE OROPHARYNGEAL MUCOSA, CLOACAL MUCOSA, AND BLOOD OF DIAMONDBACK TERRAPIN (MALACLEMYS TERRAPIN) POPULATIONS FROM MARYLAND AND GEORGIA, USA** Bryan S. Vorbach, Leigh A. Clayton, Willem M. Roosenburg, Terry M. Norton, Laura Adamovicz, Catherine A. Hadfield, and Matthew C. Allender Journal of Wildlife Diseases, 58(4), 2022, pp. 782–790 Key Points: - Diamondback terrapin (Malaclemys terrapin) – only exclusively brackish water turtle in NA -- Current threats – habitat destruction, drowning in crab pot, boat strikes - Sampled terrapins from Maryland (free-ranging) and GA (in rehab) - M. testudineum detected in both populations 🡪Maryland 87% (48 of 55) positive and Georgia 86% (6 of 7) positive from oropharyngeal-cloacal swabs -- No clinical signs seen in any turtle -- Juveniles had lower infection rates and copy number than adults 🡪 suggest horizontal transmission. Copy number greater in males then females Take Home: High prevalence of mycoplasma testudineum in diamondback terrapins, though no clinical signs seen. Be careful not to comingle multiple turtles species in zoos.

Answer 80

**PREVALENCE AND PARASITE INTENSITY OF HAEMOGREGARINES IN AFRICAN BELL HINGE-BACK (KINIXYS BELLIANA) AND AFRICAN HOME'S HINGE-BACK (KINIXYS HOMEANA) TORTOISES IN IBADAN, NIGERIA**. Adetunji VE, Ogundipe GA, Adeyemo OK. The Journal of Wildlife Diseases. 2022;58(4):825-835 Background - Genus Kinixys: hinge-back tortoises, native to west/central Africa, omnivores, IUCN vulnerable - Haemogregarines: apicomplexan intracellular parasites; global distribution of chelonian infx -- Four genera: Hepatozoon, Haemogregarina, Karyolysus, Hemolivia -- Transmitted by blood-sucking inverts (ticks, leeches, tsetse flies, sandflies, mosquitos) -- Tortoises likely ingest infected ticks vs freshwater turtles are bitten by leeches -- CS: anemia, low Hg, basophilia, eosinophilia, heterophilia, azurophilia Key Points - Intraerythrocytic sausage- or banana-shaped, slender or elongated gamonts, concave site facing nucleus, lying eccentrically or centrally in RBC cytoplasm - Infected RBCs show hypertrophy with atrophied and marginalized nuclei - Overall high prevalence, low intensity - PCR more sensitive (75%) than light microscopy (58%) - Parasite prevalence and intensity was higher in males and during the wet season - Only ectoparasite identified: Amblyomma hard ticks - Tortoises with ticks were more likely to be infected with haemogregarines

Answer 81

JZWM 2023 54(1):164-174 **DEGENERATIVE CARDIAC DISEASE IN TWO SPECIES OF TORTOISE (CHELONOIDIS NIGRA COMPLEX, CENTROCHELYS SULCATA)** Walker B, Eustace R, Thompson KA, Schiller CA, Leone A, Garner M Key Points: - Case series describes 9 Galápagos and 2 sulcata tortoises with degenerative cardiac disease -- All individuals were considered young for their species (x̄ = 19 yo) -- All individuals displayed clinical signs of illness prior to death -- Variable clinical signs included lethargy, anorexia, and peripheral tissue edema -- Tissue edema = common sign of severe cardiac disease in dogs, cats, and reptiles -- Five tortoises had peripheral edema in the limbs and cervical region - Extensive antemortem diagnostic testing was not routinely performed - Diagnostic imaging used in these cases included ultrasound and CT -- Pericardial effusion was the most common finding associated with cardiac disease -- Minimal pericardial effusion anecdotally reported as an incidental finding in tortoises -- Epicardial fibrosis/adhesions postmortem were likely due to chronic pericardial effusion -- Ultrasound-guided pericardiocentesis was performed using a dorsal midline approach - Cardiomegaly was not obvious in any tortoises with degenerative cardiac disease - However, age-based heart measurements for these species have not been established - Myocardial fibrosis in tortoises ranged from mild to marked -- In reptiles associated w/ improper nutrition/husbandry, parasites, and infectious diseases - Hepatic fibrosis/lipidosis and pulmonary edema commonly observed -- Hepatic lipidosis is a common, often reversible metabolic condition in anorexic tortoises -- Cardiogenic pulmonary edema occurs in CHF due to pulmonary venous congestion -- Coelomic effusion was reported in two cases TLDR: * Degenerative cardiac disease may be more prevalent in Galápagos and sulcata tortoises in human care than previously reported. * Severe peripheral edema = most common clinical presentation. * Pericardial effusion was the most common finding via imaging

Answer 82

JZWM 2022 53(2):402-411 **Extravasation Of Contrast Media After Subcarapacial Vessel Injection In Three Chelonian Species** Rockwell K, Rademacher N, Osborn ML, Nevarez JG Background: - Subcarapacial sinus/vessel -- Anastomosis of caudal branch of external jugular & common intercostal veins -- Along midline just cranial to the eighth cervical vertebrae -- Lymphatic structures run in close relationship, creating a high risk of lymph dilution - Repeated sampling and IV injections have resulted in temporary to permanent paresis of the tail or hindlimbs, unilateral or bilateral forelimb paresis, and contamination of CSF - Chelonians lack an epidural space; well-developed intrathecal or subarachnoid (or subdural) space surrounding the spinal cord filled with CSF Key Points: - All species had contrast extravasation in the subarachnoid space, with red-footed tortoises having the largest percentage (70% compared to 50% and 33% of box turtles and red-eared sliders, respectively). - Extravasation of contrast in the trachea or bronchi (70%) and lungs (80%) was seen in the red-footed tortoises only. - Majority of red-footed tortoises had contrast extravasation into unintended sites -- Incidence of extravasation was higher compared to box turtles and red-eared sliders - Lungs of terrestrial chelonians occupy approximately 2/3 of the body cavity, whereas in loggerhead sea turtles their lungs only occupy the dorsal 1/3 of the body cavity -- The lungs of tortoises may take up a larger space in the cranial coelom compared to aquatic turtle species, providing a greater risk of accidental injury or injection - Successful administration was seen in 100% of sliders vs. 50% of tortoises -- A higher success rate may be attributed to the shape of their carapace - Iohexol is indicated for intrathecal use -- The dose of iohexol contrast used in this study was 300 mg/kg -- This resulted in a substantial volume to be administered to each individua -- Given the small relative vasculature anatomy, possible leakage was probable TLDR: Caution is advised with phlebotomy and IV administration in chelonian species with more dome-shaped carapaces

Answer 83

**COMPUTED TOMOGRAPHY FOR MEASURING BODY FAT RESERVES IN THE THREATENED MOJAVE DESERT TORTOISE (GOPHERUS AGASSIZII)** Margarete A.Walden,MS, Rachel Jania, DVM, Dipl ACVR,Matthew E. Kinney, DVM, Dipl ACZM, Anne Devan-Song, PhD, K. Kristina Drake, PhD, Todd C. Esque, PhD, and Kevin T. Shoemaker, PhD Journal of Zoo and Wildlife Medicine 53(2): 412–423, 2022 Take-Home Message: - CT effective noninvasive technique to measure body fat location and volume. - Fat volume calculated by CT did not correlate with visual assessment of BCS nor quantitative BCI. - Relative temporalis depth calculated from CT was not related to BCS.

Answer 84

JZWM 2022 53(2) 424-432 **BACTERIAL IDENTIFICATION AND ANTIBIOTIC SENSITIVITY FROM THE ABSCESSES OF CAPTIVE TORTOISES—CLINICAL ANTIBIOTIC RECOMMENDATIONS** Key Points: - We observed that over 75% of the identified bacteria from tortoise abscesses were anaerobic bacteria - Based on our antibiotic-sensitivity testing results, amikacin, ceftazidime, and ceftiofur were recommended to treat anaerobic gram negative bacterial infections - amoxicillin, ampicillin, carbenicillin, and penicillin are proposed to treat abscesses caused by anaerobic gram-positive bacteria - The three most frequently identified bacteria in shells were Proteus mirabilis, Enterococcus spp., and Escherichia coli - The three most common bacteria found in skin wounds were Pseudomonas aeruginosa, Klebsiella pneumonia, and Aeromonas hydrophila

Answer 85

**Leatherback Sea Turtle (Dermochelys coriacea) Nesting and Health Parameters in St. Kitts, 2003– 2016**. JHMS Volume 33, No. 1, 2023. Kimberly M. Stewart1,2, Anne Conan2,4, Terry M. Norton1,3, Brayton A. Hill2 , Maria Smeriglio2 , Kathleen Clements2 , Darryn L. Knobel2 Key Points: - Majority of nesting females presented with external lesions -- External lesions most frequently documented on the front flippers (38.1%) > carapace (21.6%) -- The most frequently observed lesion types were notches (41.2%) and linear lesions (34.1%) -- Lesions were most frequently found in the remodeling stage (76.6%) of healing -- Barnacle loads were most commonly mild > moderate > severe - increases were seen in total white blood cell counts and number of monocytes in individual females from the first sample acquired during the nesting season to the last sample -- Negative correlation between monocyte counts and hatch success was documented in a nesting population of loggerheads in previous study (Flower et al 2018) -- Decreases TS, TP, Ca, and ALB between first sample and the last sample of the season -- Note – they used bromocresol green method which can overestimate albumin – rec. EPH -- No significant changes in hematological and biochem values observed between nesting seasons. - hatch success for relocated nests was significantly lower than non-relocated nests, but w/o relocation the nests would have been lost to tidal inundation, erosion, depredation, poaching, vehicular or pedestrian traffic

Answer 86

ZB 2023 42(1):86-97 **Dietary protein requirement for captive juvenile green turtles (Chelonia mydas)** Jualaong S, Thongprajukaew K, Kanghae H, et al Key Points: - Juvenile green turtles are carnivorous but turn omnivorous or herbivorous with age - Green turtles fed with 40% protein had significantly higher performance -- Also improved calcium and phosphorus deposition in carapace - Estimated optimal protein requirement for green turtles as 40.6% -- This dietary protein level is similar to freshwater turtles -- No negative effects on the green turtles at this dietary protein level - Green turtles fed with carnivorous fish feed containing 44.8% protein had superior growth and feed utilization vs, those fed with shrimp (42.5% protein) or omnivorous fish feed (18.5% protein) -- Supports artificial diets to rear captive green turtles for head-starting before release to the natural environments TLDR: The estimated optimal dietary protein level for juvenile green turtles is about 40.6%

Answer 87

1. **What tramadol dose achieved plasma concentrations above 100 ng/ml for 24 hours in giant tortoises?** *5 mg/kg orally.* 2. **What is the approximate half-life of oral tramadol in giant tortoises?** *12–13 hours.* 3. **Why is O-desmethyltramadol (M1) important in tramadol pharmacology?** *M1 has stronger μ-opioid receptor affinity than tramadol and contributes significantly to analgesic effects.* 4. **Was M1 a major metabolite in giant tortoises?** *No, low levels suggest M1 is a minor metabolite in this species.* 5. **What side effect was observed in tortoises receiving tramadol?** *Transient sedation, especially at 5 mg/kg.* 6. **How does tramadol pharmacokinetics in giant tortoises compare to mammals?** *Longer half-life than dogs, cats, horses, goats, and macaques.* 7. **How does the tramadol Cmax in giant tortoises compare to loggerhead sea turtles at the same dose?** *Higher Cmax in giant tortoises (518 ng/ml vs 373 ng/ml in sea turtles).* 8. **What considerations should clinicians have when using tramadol in giant tortoises?** *Monitor for sedation, adjust dosing carefully, and recognize species-specific metabolic differences.* ## Footnote *J. Zoo Wildl. Med.* 55(1): 86–91, 2024 **PHARMACOKINETICS OF TRAMADOL AND O-DESMETHYLTRAMADOL IN GIANT TORTOISES (CHELONOIDIS VANDENBURGHI, CHELONOIDIS VICINA)** Daniela Yuschenkoff, Gretchen A. Cole, Jennifer D’Agostino, Brad Lock, Sherry Cox, Kurt K. Sladky * * * **Key Points** - Oral tramadol at 5 mg/kg achieves plasma concentrations >100 ng/ml (human therapeutic threshold) for 24 hours in giant tortoises. - Tramadol has a long half-life in giant tortoises (~12–13 hours), longer than in many mammals (dogs, cats, horses, goats, macaques). - O-desmethyltramadol (M1), the active metabolite, was detected at low levels, suggesting it may not be a major metabolite in giant tortoises. - Mild transient sedation was observed post-tramadol administration, particularly at 5 mg/kg, notably in juvenile tortoises. - Comparison to other chelonians shows interspecies variation: higher tramadol concentrations but shorter half-life than in loggerhead sea turtles. - Species-specific differences in metabolism and bioavailability may influence tramadol effectiveness across chelonian taxa. - Oral tramadol dissolved from powder (rather than tablets) was used to ensure accurate dosing. - No pharmacodynamic correlation (plasma levels vs analgesia) established for tortoises; therapeutic thresholds in reptiles remain unknown. * * * **Introduction** - Pain management in reptiles is challenging due to limited pharmacological data and difficulty in assessing pain. - Previous opioid studies (butorphanol, morphine, buprenorphine) in reptiles showed inconsistent efficacy. - Tramadol offers oral administration advantages with fewer respiratory depressive effects compared to morphine. - Prior studies in turtles (red-eared sliders, yellow-bellied sliders, loggerhead sea turtles) demonstrated tramadol's efficacy but no data existed for tortoises. - A clinical case prompted this pharmacokinetic study following observed sedation in a giant tortoise on tramadol therapy. * * * **Materials and Methods** - Eleven giant tortoises (3 adults, 8 juveniles) received oral tramadol at 1 mg/kg or 5 mg/kg in a crossover design (3-week washout). - Blood sampling occurred at 0, 2, 6, 24, 48, 72, and 96 hours post-administration. - Plasma concentrations of tramadol and M1 were measured using HPLC. - Noncompartmental pharmacokinetic analysis was performed to determine t½, Cmax, Tmax, AUC, and MRT. * * * **Results** - Tramadol half-life: 11.9 ± 4.6 h (1 mg/kg) and 13.2 ± 6.1 h (5 mg/kg). - Maximum plasma concentrations (Cmax): 125 ± 69 ng/ml (1 mg/kg) and 518 ± 411 ng/ml (5 mg/kg). - Therapeutic levels (>100 ng/ml) maintained for 24 hours at 5 mg/kg. - M1 concentrations were low and inconsistent, precluding pharmacokinetic parameter calculation. - Mild transient sedation and decreased appetite observed, more pronounced at higher doses. * * * **Discussion** - Giant tortoises metabolize tramadol slowly compared to mammals but faster than loggerhead sea turtles. - M1 seems to be a minor metabolite, similar to findings in dogs, goats, and horses. - Differences in tramadol metabolism and effect between chelonians may be influenced by formulation, metabolism pathways, and administration methods. - Clinical significance of low M1 levels in tortoises is unknown; may impact analgesic effectiveness. - Sedation should be monitored closely during tramadol therapy in giant tortoises. - Further pharmacodynamic studies are needed to correlate plasma concentrations with clinical analgesia.

Answer 88

Which anticoagulant is best for snapping and painted turtles? **Lithium heparin (LH)** Which turtle species tolerated both LH and dEDTA? **Blanding’s turtle** Major problem seen with dEDTA in snapping turtles? **Hemolysis and clotting** Why does EDTA cause hemolysis? **Calcium chelation → osmotic swelling → RBC lysis ** PCV effect in dEDTA samples from snapping turtles? **Falsely increased PCV** Should anticoagulant recommendations be extrapolated across turtles? **No, species-specific evaluation is necessary ** ## Footnote *J. Zoo Wildl. Med.* 55(1): 92–101, 2024 **COMPARING THE EFFECTS OF DIPOTASSIUM EDTA AND LITHIUM HEPARIN ON HEMATOLOGIC VALUES IN BLANDING’S, PAINTED, AND SNAPPING TURTLES** Alexis Davidson, Maura Ryan, Amy N. Schnelle, William Graser, Laura Adamovicz, Matthew C. Allender * * * **Quick Key Points for ACZM Board Review** - **Preferred anticoagulant**: - Painted turtles & snapping turtles → **Lithium heparin (LH)** - Blanding’s turtles → **Either LH or dEDTA**, but use anticoagulant-specific reference intervals. - **dEDTA issues**: - **Snapping turtles**: Severe hemolysis and clotting in dEDTA tubes. - **Painted turtles**: Moderate hemolysis observed in dEDTA samples. - **Hemolysis Mechanism**: - EDTA chelates calcium → increases erythrocyte membrane permeability → osmotic swelling → cell lysis. - **Species-specific differences**: - Blanding’s turtles had **no gross hemolysis** with either anticoagulant. - **Bias introduced**: - Anticoagulant choice causes **constant and proportional errors** in hematologic parameters across species. - **Important hematologic findings**: - **Snapping turtles**: dEDTA raised PCV, TS, monocyte, and eosinophil counts. - **Painted turtles**: dEDTA raised PCV and heterophil:lymphocyte ratio; ESR higher with LH. - **Clinical recommendation**: - **Always validate anticoagulant selection per species**; do not assume consistency across chelonians.

Answer 89

What were the primary clinical signs associated with intestinal tympany in D. mawii?

**Abnormal buoyancy (9/10 cases) and anorexia (2/10 cases).**

What factors were suspected to contribute to intestinal tympany and volvulus?	Abrupt dietary changes (especially high sugar fruits), ingestion of indigestible material, suboptimal temperatures.
How rapidly did fatal cases progress from clinical signs to death or euthanasia?	1–2 days.
Why might cultivated fruits pose a risk to D. mawii?	Higher sugar and lower fiber content compared to wild fruits, promoting excessive fermentation.
How was volvulus diagnosed in these cases?	Mostly postmortem; difficult to confirm radiographically.
What necropsy findings were common in fatal cases?	Small intestinal volvulus, colonic volvulus (some), hepatic lipidosis, vascular thrombosis.
What preventive husbandry measures are recommended?	Maintain stable diets (low in fermentable fruits), ensure optimal temperature, monitor and address buoyancy changes early.
Is tympany alone fatal in D. mawii?	Not necessarily; mild cases resolved with supportive care, but tympany may predispose to fatal volvulus.

## Footnote *. Zoo Wildl. Med.* 55(1): 224–234, 2024 **INTESTINAL TYMPANY AND VOLVULUS IN CENTRAL AMERICAN RIVER TURTLES (DERMATEMYS MAWII)** Tabitha B. Gionfriddo, Tim Georoff, Kendra Baker, Donna Ialeggio, Keith Hinshaw, Barbara Toddes, John G. Trupkiewicz * * * **Quick Key Points for ACZM Board Review** - **Main Clinical Signs**: Abnormal buoyancy (9/10 cases) and anorexia (2/10 cases) were the primary clinical presentations. - **Mortality Risk**: Intestinal volvulus was fatal in 5/10 cases, with rapid progression (1–2 days) from clinical signs to death/euthanasia. - **Dietary Risks**: Abrupt dietary changes (e.g., excessive fruit or mulberry browse) were associated with 6/10 cases. - **Temperature Influence**: Suspected suboptimal environmental temperatures were noted in 4/10 cases prior to clinical signs. - **Pathology Findings**: Volvulus involved the small intestine, often with concurrent colonic involvement; common necropsy findings included hepatic lipidosis and thrombosis. - **Radiographic Findings**: Tympany was consistently evident on radiographs, but volvulus was difficult to diagnose antemortem. - **Prevention Recommendations**: Maintain stable, appropriate diets (low fermentable fruit), optimize environmental temperatures, and recognize buoyancy abnormalities early. - **Comparative Species Information**: Similar volvulus and tympany risks reported in other chelonians like Aldabra tortoises and green sea turtles. - **Husbandry Adjustments**: Reducing water depth, dietary corrections, and avoiding abrupt diet changes may prevent progression from tympany to volvulus. * * * **Section-by-Section Bullet Summary** **Introduction** - *Dermatemys mawii* is critically endangered and largely herbivorous. - No prior reports of intestinal volvulus in this species exist; however, it has been reported in other chelonians. - Study documents 10 cases under managed care. **Case Series** - 10 turtles developed intestinal tympany; 5 survived, 5 died or were euthanized. - Clinical progression was often rapid (1–2 days). - Associated risk factors included diet changes (especially fruit/mulberry) and possible suboptimal temperatures. - Some cases involved foreign body ingestion. - Housing conditions (indoor vs outdoor) varied; ambient indoor temp ~23–25°C. **Discussion** - Intestinal tympany suspected as a risk factor for volvulus. - Volvulus could not be reliably diagnosed radiographically; diagnosis confirmed postmortem. - Wild vs cultivated fruit composition may predispose captive animals to fermentation problems (cultivated fruits are higher in sugar, lower in fiber). - Suboptimal temperatures may delay GI transit and increase gas buildup. - Early identification of buoyancy issues critical to prevention. - Analogous volvulus cases in other chelonians suggest multifactorial causes. **Conclusions** - Avoid abrupt dietary changes, especially high-fruit intake. - Maintain environmental temperatures around 21–23°C for *D. mawii*. - Reduce water depth and correct diet immediately if abnormal buoyancy is observed. - Tympany might predispose to volvulus; prevention and early intervention critical.

Answer 90

What major physical abnormality was common in spotted turtles? **Carapace and plastron lesions**. What novel virus was detected in spotted turtles? **Clemmys adenovirus-1.** Were any herpesviruses or ranaviruses detected in the study? **No**. Which metal was found at higher concentrations in female painted turtles compared to males? **Strontium (Sr).** What environmental factor prompted heavy metal testing in this turtle population? **Camp Edwards is a former Superfund site with known contamination**. How were pathogen screenings performed for painted turtles? **qPCR for 21 pathogens on a Fluidigm platform.** Did turtles positive for the novel adenovirus show clinical signs? **No, they appeared healthy**. Why are turtles useful for environmental monitoring? **Their longevity, habitat use, and trophic level make them sentinel species for contaminants and pathogens.** What sampling method was used for pathogen detection from shell lesions? **Aggressive shell swabbing.** ## Footnote *J. Zoo Wildl. Med.* 55(3): 743–749, 2024 **HEALTH ASSESSMENT OF SPOTTED (CLEMMYS GUTTATA) AND PAINTED (CHRYSEMYS PICTA) TURTLES IN CAPE COD, MASSACHUSETTS, U.S.A, WITH DETECTION OF A NOVEL ADENOVIRUS** Lauren M. Vincent, Matthew C. Allender, Annie E. Curtis, John C. Garrison, Stacey Lance, Adam McFall, Amber Simmons, Kaitlin Moorhead, Laura Adamovicz * * * **Quick Key Points for ACZM Board Review** - **Primary Findings**: High prevalence of carapace (90%) and plastron (46.7%) lesions in spotted turtles; shell abnormalities also common in painted turtles. - **Novel Pathogen**: Detection of a previously undescribed adenovirus (proposed Clemmys adenovirus-1) in three spotted turtles. - **Pathogen Screening**: No detection of herpesviruses, ranavirus, Mycoplasmopsis spp., or Emydomyces testavorans in either species. - **Heavy Metal Burden**: Painted turtles had measurable but relatively low plasma concentrations of metals; females had lower copper and higher strontium than males. - **Clinical Pathology**: Baseline hematologic data established for both species; no major differences found related to sex, age, or physical examination findings. - **Conservation Implication**: Continued surveillance is critical due to the detection of novel adenovirus and high prevalence of shell lesions. - **Environmental Concerns**: Camp Edwards is a Superfund site with a history of contamination; turtles serve as sentinels for environmental health. - **Methodology Notes**: Shell swabs for pathogen detection, aggressive sampling attempted but may have missed deeper fungal infections. * * * **Section-by-Section Bullet Summary** **Introduction** - Health assessments are essential for conservation; turtles are important environmental sentinels. - Spotted turtles (Clemmys guttata) are endangered and at risk for disease outbreaks. - Painted turtles (Chrysemys picta) are more common and may serve as a health model for spotted turtles. - Historic contamination (e.g., lead) at Camp Edwards warranted investigation of health and toxicant burdens. **Materials and Methods** - Turtles were captured by hand or baited traps; strict biosecurity protocols followed. - Physical exams, blood collection, hematology, heavy metal analysis, and pathogen PCR screening performed. - Painted turtles tested for 21 pathogens by qPCR; spotted turtles evaluated by consensus PCR protocols. - Plasma metals assessed included Co, Ni, Cu, Zn, Sr, Mo, Ag, Cd, Pb, As, and Se. **Results** - 30 spotted turtles and 24 painted turtles sampled. - Spotted turtles: high prevalence of carapace/plastron lesions; some gravid females. - Painted turtles: shell abnormalities common; ectoparasites in 25%. - Heavy metals: low levels detected; females had lower Cu, higher Sr. - Pathogens: Novel adenovirus detected in spotted turtles; no other pathogens detected. - No clinical signs associated with adenovirus-positive individuals. **Discussion** - High shell lesion prevalence warrants future monitoring; lesions appeared minor but may mask deeper issues. - Heavy metals present but at relatively low levels; blood may underestimate body burden. - First detection of a novel adenovirus in spotted turtles; currently no signs of associated disease. - Need for continued health monitoring to detect emerging threats and better inform conservation actions. **Conclusions** - Painted turtles appear healthy compared to other populations; spotted turtles had high shell lesion rates. - Detection of Clemmys adenovirus-1 calls for ongoing pathogen surveillance. - Integrating health assessments with conservation efforts is vital to early identification of health threats.

Answer 91

What disease does Emydomyces testavorans cause? **Chronic ulcerative shell disease in freshwater turtles**. What is the advantage of bioencapsulation for drug delivery in turtles?**Improved drug exposure (AUC), easier administration, and better maintenance of therapeutic plasma levels**. What was the dose of terbinafine used in this study? **30 mg/kg.** How was terbinafine administered in the BEC group? **Injected into earthworms, which were then fed to the turtles.** Did BEC or OG administration lead to higher plasma terbinafine concentrations? **BEC (bioencapsulation) led to higher plasma exposure (AUC)**. What was the approximate half-life of terbinafine in western pond turtles? **26–27 hours**. Was the plasma concentration after terbinafine administration sufficient to exceed the MIC for ET? **Yes, for both OG and BEC, but more reliably for BEC**. What were the limitations of this study? **Small sample size, male-only turtles, single-dose study, ET-infected population**. Why is feeding status important to consider in future studies? **Feeding may alter drug absorption and pharmacokinetics**. ## Footnote *Am. J. Vet. Res.* 85(1): 1–8, 2024 **Bioencapsulation is a feasible method of terbinafine administration in *Emydomyces testavorans*\-infected western pond turtles (Actinemys marmorata)** Anthony J. Cerreta, Jennifer M. Reinhart, Lauren R. Forsythe, Matthew R. O’Connor, Karisa N. Tang, Sherry Cox, Krista A. Keller * * * **Quick Key Points for ACZM Board Review** - **Disease Focus**: *Emydomyces testavorans* (ET) causes chronic shell lesions in freshwater chelonians, notably western pond turtles (*Actinemys marmorata*). - **Study Purpose**: Evaluate pharmacokinetics of terbinafine administered via oral gavage (OG) and bioencapsulation (BEC) in earthworms. - **Bioencapsulation Benefits**: BEC significantly increased drug exposure (AUC) compared to OG and achieved better predicted therapeutic plasma levels. - **Therapeutic Findings**: Plasma terbinafine exceeded MIC (15–60 ng/mL) in all turtles using BEC every 24 or 48 hours. - **Pharmacokinetics**: Terbinafine half-life was approximately 26–27 hours; Tmax longer with BEC (~14 hours vs. 1.8 hours OG). - **Species Comparison**: Terbinafine absorption and exposure were relatively high in western pond turtles compared to other species (e.g., horses, dogs, penguins). - **Clinical Relevance**: BEC is a promising method for delivering long-term antifungal therapy to chelonians with ET infections. - **Limitations**: Small sample size (n=7 males), all animals clinically infected with ET; results may vary with disease status or between sexes. - **Future Needs**: Multidose pharmacokinetics, feeding effect studies, and clinical trials are needed to refine treatment recommendations. * * * **Section-by-Section Bullet Summary** **Introduction** - *Emydomyces testavorans* is an emerging shell pathogen causing morbidity/mortality in turtles. - Terbinafine is promising due to its high safety margin, tissue accumulation, and efficacy. - Oral dosing in turtles is challenging; bioencapsulation into prey (earthworms) offers a novel solution. **Materials and Methods** - Seven male western pond turtles were enrolled; all had confirmed ET infection. - A crossover pharmacokinetic study compared 30 mg/kg terbinafine via oral gavage (OG) or bioencapsulated (BEC) in earthworms. - Blood sampling occurred up to 120 hours post-administration; plasma analyzed by HPLC. **Results** - Terbinafine plasma levels exceeded MIC (>60 ng/mL) quickly in all turtles for both methods. - BEC significantly increased AUC0-∞ compared to OG (P = 0.031). - Steady-state predictions indicated better maintenance of therapeutic levels with BEC, both with 24- and 48-hour dosing intervals. - No adverse effects observed. **Discussion** - Bioencapsulation led to greater drug exposure (higher AUC) and more consistent therapeutic plasma concentrations. - Pharmacokinetic profile (half-life ~26-27h) similar to other species like bearded dragons and African penguins. - Interspecies and interindividual variation in pharmacokinetics noted. - Bioencapsulation may improve compliance and treatment efficacy in reptile fungal infections. - Need for studies to assess effects of feeding, chronic dosing, and disease state on pharmacokinetics. **Conclusions** - BEC terbinafine (30 mg/kg q24–48h) may provide an effective method for ET treatment in western pond turtles. - Oral gavage is less reliable for maintaining therapeutic drug levels. - Controlled clinical studies are required to validate findings.

Answer 92

What dose of metronidazole was studied in sea turtles? **20 mg/kg.** How long after oral dosing did plasma concentrations exceed 8 µg/ml? **About 8 hours.** How quickly did plasma concentrations exceed 8 µg/ml after IV dosing? **Within 5 minutes.** For how long did plasma levels remain above 8 µg/ml after a single dose? **About 24 hours.** What is the recommended dosing interval for most infections based on this study? **20 mg/kg every 48 hours**. What adjustment to dosing interval is suggested for treating higher MIC pathogens like some Clostridium spp.? **20 mg/kg every 24 hours.** Were there major species differences in pharmacokinetics among the sea turtles studied? **No, pharmacokinetics were very similar across species.** What minor side effect was noted during IV administration? **Mild, transient pectoral flipper tremors in two loggerhead turtles.** Why is IV administration sometimes preferred initially in critical sea turtle patients? **It rapidly achieves therapeutic plasma levels.** Why is further research needed despite the positive findings of this study? **To assess multi-dose kinetics, accumulation risk, and clinical efficacy.** ## Footnote *J. Zoo Wildl. Med.* 55(4): 1049–1055, 2024 **PHARMACOKINETICS OF METRONIDAZOLE IN GREEN (CHELONIA MYDAS), LOGGERHEAD (CARETTA CARETTA), AND KEMP'S RIDLEY (LEPIDOCHELYS KEMPII) SEA TURTLES AFTER SINGLE ORAL AND INTRAVENOUS DOSES** Terry M. Norton, Sherry Cox, Charles A. Manire, Bette Zirkelbach, Ian Morrison, Rachel Overmeyer, Stephanie Stowell, Leah Adelman, Sarah Buttrey, Taylor Marcialis, Maximillian M.R. Polyak, Samantha Clark, Jamie Gamby, Allison Hardman, Isabelle Gilbert, Krista Breshears, Maria Chadam, Whitney Crowder, Emily Mirowski, Shelby Hoover, Annie Page * * * **Quick Key Points for ACZM Board Review** - **Drug Focus**: Metronidazole (20 mg/kg) evaluated in green, loggerhead, and Kemp’s ridley sea turtles. - **Pharmacokinetics**: Plasma levels ≥8 µg/ml reached by 8 hours (oral) and within 5 minutes (IV); maintained ≥8 µg/ml for ~24 hours and ≥4 µg/ml for ~48 hours. - **Species Comparison**: Pharmacokinetic parameters were very similar across all three turtle species. - **Bioavailability**: Oral bioavailability estimated at ~85–112% depending on species, indicating good absorption. - **Administration Observations**: IV infusion over 30–40 minutes minimized adverse effects (mild transient tremors seen with faster infusions). - **Clinical Application**: A dosing regimen of 20 mg/kg every 48 hours is appropriate for most anaerobic/protozoal infections; every 24 hours recommended for higher MIC pathogens (e.g., some *Clostridium* spp.). - **Metabolism and Excretion**: Metronidazole extensively metabolized in liver, excreted mainly via feces. - **Safety**: No major adverse effects at 20 mg/kg; caution needed at higher doses (>40 mg/kg reported toxic in reptiles). - **Comparative Notes**: Pharmacokinetics similar to prior studies in reptiles (e.g., red-eared sliders, snakes, iguanas). * * * **Section-by-Section Bullet Summary** **Introduction** - Anaerobic bacterial and protozoal infections occur in sea turtles. - Limited antibiotic studies exist for sea turtles; metronidazole is effective in other reptiles and is commonly used. - MICs range: 0.25–8 µg/ml for bacteria; 1–50 µg/ml for protozoa. **Materials and Methods** - 18 sea turtles sampled (6 of each species). - Single oral (20 mg/kg) or IV (20 mg/kg) administration of metronidazole. - Blood collected at multiple intervals up to 144 hours post-administration. - Plasma drug concentrations determined by HPLC. **Results** - Plasma concentrations after oral dosing achieved ≥8 µg/ml by ~8 hours, maintained ≥8 µg/ml for ~24 hours, ≥4 µg/ml for ~48 hours. - Plasma concentrations after IV dosing achieved ≥8 µg/ml within 5 minutes, similar maintenance durations. - No severe adverse effects; minor transient tremors noted in two loggerheads during faster IV infusion. - Oral bioavailability was high; steady pharmacokinetics across species. **Discussion** - Metronidazole at 20 mg/kg is effective for anaerobic and protozoal infections in sea turtles. - Oral route convenient and effective; IV route beneficial for critical patients. - Dosing every 48 hours generally sufficient; every 24 hours for infections with higher MIC targets. - Minimal differences between species suggest a unified dosing approach. - Further studies needed to assess accumulation with multiple dosing. **Conclusion** - 20 mg/kg metronidazole q48h (oral or IV) is appropriate for green, loggerhead, and Kemp’s ridley sea turtles. - Clinical efficacy still needs validation via multi-dose and outcome-based studies.

Answer 93

What anesthetic and oxygen fractions were studied in green sea turtles? **Sevoflurane with 21% or 100% inspired oxygen (FIO₂).** Did 100% O₂ prolong time to extubation compared to 21% O₂? **No, extubation times were similar.** What difference was observed in bite block response between groups? **Turtles ventilated with 21% O₂ bit the bite block earlier.** What major acid-base disturbance was observed during anesthesia?**Respiratory alkalosis (higher pH, lower PCO₂).** Were there significant differences in blood lactate between 21% and 100% O₂ groups? **No, no difference in lactate levels** Why might sea turtles remain apneic even after anesthesia discontinuation? **Their physiology includes natural breath-holding and diving adaptations.** How did heart rate change during anesthesia? **HR decreased by about 25% during sevoflurane anesthesia.** Is high SpO₂ always necessary for sea turtles under anesthesia? **Not necessarily; they can physiologically tolerate low arterial oxygen levels.** Should 100% oxygen be avoided during anesthesia in sea turtles? **No, 100% oxygen is safe and does not prolong recovery under mechanical ventilation.** ## Footnote *Am. J. Vet. Res.* 85(4): 1–8, 2024 **The fraction of inspired oxygen does not affect the time to extubation in mechanically ventilated, sevoflurane-anesthetized green sea turtles (Chelonia mydas)** André A. Justo, Gustavo H. P. Dutra, Adriano B. Carregaro, Silvia R. G. Cortopassi * * * **Quick Key Points for ACZM Board Review** - **Study Objective**: Evaluate whether inspired oxygen fraction (FIO₂) (21% vs 100%) affects recovery time in green sea turtles under sevoflurane anesthesia with mechanical ventilation. - **Key Finding**: No meaningful difference in time to extubation between turtles ventilated with 21% O₂ vs 100% O₂. - **Biting Response**: Turtles under 21% O₂ bit the bite block sooner than those under 100% O₂, suggesting faster early lightening of anesthesia. - **Blood Gases**: Respiratory alkalosis (high pH, low PCO₂) was present during anesthesia with both FIO₂ levels, but no metabolic acidosis or hyperlactatemia observed. - **SpO₂ Trends**: SpO₂ values were consistently higher in 100% O₂ turtles but clinical significance is unclear because sea turtles physiologically tolerate low oxygen tensions. - **Mechanical Ventilation**: All turtles remained apneic under mechanical ventilation regardless of oxygen fraction, suggesting spontaneous ventilation may not return until full recovery. - **Heart Rate Changes**: HR dropped ~25% during anesthesia, attributed to a “diving reflex” (parasympathetic predominance), not an anesthetic complication. - **Clinical Implication**: Use of 100% O₂ or 21% O₂ during anesthesia does not clinically impact overall recovery or acid-base balance under studied ventilation settings. - **Research Importance**: Supports using mechanical ventilation in sea turtles without concerns about prolonged recovery linked to oxygen supplementation. * * * **Section-by-Section Bullet Summary** **Introduction** - Inhalation anesthesia is preferred in sea turtles for major procedures. - Concerns exist that 100% O₂ could prolong recovery by suppressing breathing. - No controlled clinical studies previously confirmed this theory. **Materials and Methods** - Eleven juvenile green sea turtles studied in a randomized, blinded, crossover design. - Each turtle received anesthesia twice (once with 100% O₂ and once with 21% O₂) with a 1-week washout. - Monitored HR, PetCO₂, SpO₂, cloacal temperature, venous blood gases, and lactate. **Results** - HR decreased by ~25% during anesthesia in both treatments; PetCO₂ declined over time. - SpO₂ higher in 100% O₂ group but without clinical consequences. - Venous blood gases showed mild respiratory alkalosis during anesthesia (higher pH, lower PCO₂) for both groups. - No significant lactate increase or evidence of anaerobic metabolism. - Time to bite block shorter with 21% O₂, but no difference in time to extubation or spontaneous ventilation. - All turtles tolerated procedures well and fully recovered. **Discussion** - Biting the bite block earlier with 21% O₂ might indicate earlier lightening of anesthesia, but clinical significance minor. - Hypotheses about oxygen concentration affecting recovery times not supported. - Apnea persisted post-anesthesia under both treatments, suggesting sea turtles' respiratory physiology differs fundamentally from mammals. - Mechanical ventilator settings may mask subtle differences; sea turtle-specific ventilators recommended for future studies. **Conclusion** - In green sea turtles, providing 100% oxygen during sevoflurane anesthesia does not meaningfully affect anesthetic recovery or time to extubation compared to 21% oxygen. - Both oxygen levels are safe to use during mechanical ventilation.

Answer 94

What euthanasia method was tested in this study? **Transmucosal administration of pentobarbital (oral or cloacal).** Which transmucosal route was more reliable in turtles? **Oral administration.** What was the median time to confirmed death (asystole) after administration? **18 hours (range 6–26 hours).** How soon after administration did turtles typically lose pain responses? **Within 15 minutes.** What percentage of cloacal-administered turtles had leakage of euthanasia solution? **75%.** What cardiac changes were observed during euthanasia? **Bradyarrhythmias in 61.5% of turtles.** Why is maintaining turtles within their POTZ important during euthanasia? **To optimize drug absorption and distribution.** What complication was observed in turtles that received incomplete cloacal dosing? **Regaining of movement and reflexes (incomplete euthanasia).** How was death confirmed in this study? **Absence of reflexes, heartbeat, and electrical activity on ECG.** What potential future improvements were suggested? **Evaluating higher doses, alternative temperatures, and postmortem histology effects.** ## Footnote *Am. J. Vet. Res.* 85(4): 1–8, 2024 **Transmucosal pentobarbital is a successful euthanasia method in pond slider turtles (Trachemys scripta)** Zachary C. Ready, Krista Keller * * * **Quick Key Points for ACZM Board Review** - **Study Objective**: Assess transmucosal administration (oral and cloacal) of pentobarbital as a method of euthanasia in pond slider turtles. - **Success Rates**: Oral transmucosal administration was successful in 100% of cases; cloacal administration was successful in 75% (2/8 cloacal turtles required secondary euthanasia). - **Timing to Death**: Median time to death (confirmed via asystole) was 18 hours (range 6–26 hours). - **Speed of Anesthesia Onset**: Loss of pain response and voluntary movement occurred in ≤15 minutes for most turtles, achieving deep anesthesia rapidly. - **Leakage Issues**: 75% of cloacal administrations had leakage; no leakage occurred after oral administration. - **Clinical Implication**: Oral transmucosal pentobarbital provides a humane, non-invasive euthanasia method without requiring IV access. - **Cardiac Effects**: Bradyarrhythmias occurred in 61.5% of turtles, consistent with expected effects of pentobarbital. - **Post-mortem Considerations**: Prolonged time (up to 24 hours) under optimal temperature was needed to ensure complete euthanasia. - **Future Directions**: Higher doses or alternative protocols may reduce euthanasia time further; histologic effects of transmucosal pentobarbital in turtles remain unknown. * * * **Section-by-Section Bullet Summary** **Introduction** - Reptile euthanasia is challenging due to breath-holding, temperature-dependent metabolism, and limited IV access. - The AVMA recommends barbiturates, but transmucosal methods have not been validated in chelonians. - Study aimed to evaluate if oral or cloacal transmucosal pentobarbital could reliably induce humane euthanasia within 24–48 hours. **Materials and Methods** - 16 pond sliders (Trachemys scripta) randomized into oral (n=8) or cloacal (n=8) groups. - 100 mg/kg pentobarbital administered either orally (esophageal mucosa) or cloacally (vent area). - Parameters recorded: movement, heart rate, respiratory rate, palpebral/corneal reflexes, and responses to noxious stimuli. - Death confirmed via absence of reflexes, movement, heartbeat, and ECG asystole. **Results** - 13/16 turtles successfully euthanized via transmucosal route (7 oral, 6 cloacal). - 2 cloacal turtles failed euthanasia (regained movement); 1 oral turtle excluded due to overdose. - Leakage was common with cloacal delivery (75%), absent with oral delivery. - Loss of respiration and voluntary movement occurred within 15 minutes for most turtles. - Median time to loss of corneal reflex: 45 minutes. - Median time to cardiac arrest: 18 hours. **Discussion** - Oral transmucosal delivery is more reliable than cloacal due to reduced leakage. - 15-minute time to loss of pain responses indicates rapid induction of unconsciousness. - Bradyarrhythmias were common but expected due to pentobarbital action. - Maintaining turtles within POTZ (~28°C) likely critical for drug absorption and efficacy. - Suggests oral transmucosal pentobarbital can be a humane first-step method in a 2-step euthanasia protocol if necessary. - Further research needed on cerebral death timing, impact on postmortem histology, and efficacy of higher doses. **Key Figure:** - **Figure 1**: Scatter plots of times to loss of movement, heart rate, reflexes, and death after oral or cloacal administration. - No significant differences between oral and cloacal groups for loss of any parameter (P = 0.17–0.99).

Answer 95

What species accounted for the majority of fishhook injury cases **Yellow-bellied slider (Trachemys scripta scripta).** What was the most common anatomical site for fishhook injuries? **Oral cavity (55%).** What was the most commonly used removal technique? **Retrograde removal.** Which anatomical location was associated with the highest complication risk? **Esophagus.** What was the overall release rate after treatment? **89.2%.** Was endoscopy commonly used for removal in this study? **No, it was rarely used (1 successful case).** What removal method involved using a tube to cover the hook barb?**Modified disgorger technique. This involved using 10 Fr and 13 Fr red rubber catheters to thread over the hook with the goal of covering the barb embedded in the soft tissue and slide it out.** What was a common postoperative complication associated with fishhook injuries? **Infection and abscess formation.** How did esophageal injuries impact the risk of complications? **Increased odds of complications (OR 3.49).** Why is successful treatment of fishhook injuries important for conservation? **It reduces adult mortality, critical for slow-reproducing turtle populations**. ## Footnote *JAVMA* 261(12): 1829–1837, 2023 **The clinical features and treatment of fishhook injuries in freshwater turtles: 126 cases from 1997–2022** Lily M. Hale, Sabrina L. Kapp, James B. Robertson, Gregory A. Lewbart, Sarah M. Ozawa * * * **Quick Key Points for ACZM Board Review** - **Species Affected**: Yellow-bellied sliders (Trachemys scripta scripta) were the most common species (54.8%) with fishhook injuries. - **Most Common Injury Location**: Oral cavity (55% of hook locations); esophagus second (18.5%). - **Treatment Approaches**: Retrograde removal (63.3%) was the most common and successful removal method; esophagostomy used for esophageal hooks. - **Outcomes**: High release rate (89.2%); mortality or euthanasia occurred in 10.8% of turtles. - **Complication Risk**: Fishhooks embedded in the esophagus significantly increased complication rates (OR 3.49). - **Sedation/Analgesia**: Analgesics used in 86.5% of cases; ketamine and dexmedetomidine were common sedatives. - **Alternative Techniques**: Endoscopic and modified disgorger techniques were successful for specific cases. - **Clinical Significance**: Early, minimally invasive removal and managing complications are key to improving survival. - **Conservation Relevance**: Successful treatment of fishhook injuries can aid freshwater turtle conservation by reducing adult mortality. * * * **Section-by-Section Bullet Summary** **Introduction** - Fishing activities pose significant risks to freshwater turtles, often resulting in fishhook injuries. - Chelonians' slow metabolism and long lifespans make them vulnerable to population-level impacts. - Study objective: describe clinical features, treatments, and outcomes of freshwater turtles with fishhook injuries. **Methods** - Retrospective review of 126 turtles presented to North Carolina State University's Turtle Rescue Team (1997–2022). - Data collected: species, injury location, treatment methods, complications, and outcomes. - Various statistical analyses performed to evaluate risk factors for complications and mortality. **Results** - **Species**: Yellow-bellied sliders most common, followed by Pseudemys spp. and common snapping turtles. - **Locations of Fishhooks**: Oral cavity (55%), esophagus (18.5%), eye (18.5%), lower GI tract, limbs. - **Removal Techniques**: - Retrograde removal: 63.3%. - Esophagostomy: 12 cases. - Endoscopy: 1 successful retrieval, 2 diagnostic uses. - Celiotomy (subaxillary approach) for gastric foreign body. - Modified disgorger technique successful in one case. - **Outcomes**: - 89.2% released, 10.8% died or euthanized. - Median time in care: 16 days. - Esophageal injuries increased complication risk but not mortality directly. - Complications significantly associated with mortality (P < 0.001). **Discussion** - Retrograde removal effective and associated with shorter care times. - Esophageal hook injuries complicated by infection and necrosis; careful monitoring needed. - Endoscopy offers a minimally invasive option but was rarely used. - Fishhook injuries can be managed successfully with appropriate analgesia, sedation, and wound care. - Limitations: retrospective design, incomplete follow-up after release, possible underreporting of complications. **Key Tables and Figures** - **Table 1**: Species breakdown — Yellow-bellied sliders most common (54.8%). - **Table 2**: Injury locations — Oral cavity most frequent. - **Table 3**: Removal methods per anatomic site. - **Figure 1**: Turtle with fishhooks in oral cavity and foot. - **Figure 2**: Radiographic progression of internalized fishhooks. - **Figure 3**: Complications by hook location.

Answer 96

What protozoan was identified as causing systemic infection in the pancake tortoise? Entamoeba invadens What was the primary organ affected in this case?Liver (severe necrotizing granulomatous hepatitis). How was the diagnosis of Entamoeba invadens confirmed? PCR testing on colon tissue. What were the characteristic histological features of the trophozoites?Round 10–15 µm organisms with peripherally displaced nucleus, chromatin plaques, and clear endosome. What vascular pathology was associated with amoebic infection in this tortoise? Fibrinoid vasculitis and thrombosis. What are common stressors that predispose reptiles to amoebiasis outbreaks? Dietary changes, transport, mixing of animals, husbandry changes. How does E. invadens spread among reptiles? Fecal-oral transmission via contaminated food, water, or fomites. What histologic stains were useful for highlighting trophozoites?Periodic acid–Schiff (PAS) and Giemsa stains. Besides the liver, which other organs were notably affected? Colon (ulcerative colitis) and spleen (infarction). Why is molecular testing important in diagnosing reptile amoebiasis?Morphology alone is nonspecific; PCR provides definitive species-level diagnosis. ## Footnote *JAVMA* 261(12): 1829–1837, 2023 **Sudden death in a 5-year-old female African pancake tortoise (Malacocherusus tornieri)** Tatiane Terumi Negrão Watanabe, Daniel Felipe Barrantes Murillo, Anne White, Ashley Talley, Larry J. Minter, Brigid V. Troan * * * **Quick Key Points for ACZM Board Review** - **Etiology**: Death attributed to systemic *Entamoeba invadens* infection causing necrotizing hepatitis, vasculitis, splenic infarction, and colitis. - **Clinical History**: No prior clinical signs; found dead in exhibit. - **Pathologic Findings**: Severe hepatic granulomatous inflammation, vascular invasion by amoebic trophozoites, gastrointestinal fibrinoid vasculitis, and splenic infarction. - **Diagnosis**: Confirmed by histopathology and *E. invadens*\-specific PCR on formalin-fixed tissues. - **Characteristic Histology**: Trophozoites (10–15 µm) with peripherally displaced nucleus, chromatin plaques, and a clear endosome. - **Predisposing Factors**: Stressors like dietary changes, mixing of animals, or asymptomatic carriers can trigger outbreaks. - **Infection Mechanism**: Fecal-oral transmission; trophozoites invade gastrointestinal mucosa and vasculature leading to systemic dissemination. - **Clinical Signs**: In reptiles, can vary from asymptomatic to sudden death, with signs like anorexia, hematochezia, and wasting in affected animals. - **Conservation Concern**: *E. invadens* outbreaks in captive tortoise collections can cause high morbidity and mortality. * * * **Section-by-Section Bullet Summary** **History** - 5-year-old female African pancake tortoise found dead with no observed prior clinical signs. **Clinical and Gross Findings** - Liver extensively replaced by firm yellow nodules (~90% of parenchyma). - Intestinal nematodes and protozoal organisms detected on fecal exam. - Cytology of liver showed abundant heterophils; no acid-fast organisms. **Histopathological and Microbiologic Findings** - Liver: Multifocal to coalescing heterophilic granulomas with necrosis. - Numerous amoebic trophozoites highlighted by PAS and Giemsa staining. - Portal vein thrombi containing trophozoites; vascular wall invasion (vasculitis). - Colon: Ulcerative colitis with intralesional amoebae. - Spleen: Fibrinoid vasculitis and infarction. - PCR on colon tissue positive for *Entamoeba invadens*. **Morphologic Diagnosis and Case Summary** - Severe amoebic hepatitis, colitis, splenic infarction, and gastrointestinal vasculitis due to *E. invadens*. **Comments** - *E. invadens* typically asymptomatic in reptiles but can cause fatal systemic infections under stress. - Reptilian amoebiasis primarily affects colon but can disseminate. - Histologic ID supported by special stains; definitive diagnosis requires PCR. - Stress, mixing populations, or environmental changes often precede outbreaks. **Figures and Tables** - **Figure 1**: Gross image of nodular, yellow liver lesions (bar = 1 cm). - **Figure 2**: Histologic images of liver granulomas and colonic ulceration showing amoebic trophozoites.

Answer 97

What venipuncture site is recommended for blood sampling in eastern box turtles? Jugular vein. What artifact is associated with subcarapacial venous sinus sampling?Dilution with lymphatic fluid and/or cerebrospinal fluid. How did head-started turtles differ from free-ranging turtles in blood analytes? Lower TS, lower H:L ratio, higher lymphocyte counts. What factors might confound interpretation of blood work in this study? Age, sex, seasonal differences, stress response. Why are free-ranging turtles likely to have a higher H:L ratio? Increased stress from human handling leading to lymphopenia. What common table-side measurements are affected by venipuncture site choice? Packed cell volume (PCV) and total solids (TS). How were turtles screened for health before release? CBC, biochemistry panel, and PCR for Mycoplasma, herpesvirus, adenovirus, and ranavirus. What seasonal behavior in box turtles could influence blood analytes?Brumation causing lymphoid atrophy and lymphopenia. What was the age makeup of head-started turtles at release? Mostly juveniles (~73%). How does stress physiologically alter reptile bloodwork? Stress typically decreases lymphocytes and increases heterophil counts (higher H:L ratio). ## Footnote *JAVMA* 261(12): 1829–1837, 2023 **Venipuncture site and population affect blood analytes in head-started and free-ranging eastern box turtles (Terrapene carolina)** Sara J. Wint, Terry M. Norton, Kimberly M. Andrews, Rachel K. M. Overmeyer, Nicole I. Stacy, Stephen J. Divers * * * **Quick Key Points for ACZM Board Review** - **Study Focus**: Compared blood analytes between head-started (captive-reared) and free-ranging eastern box turtles and between venipuncture sites (jugular vein vs subcarapacial sinus). - **Jugular Vein Preferred**: Blood collected from the jugular vein had less evidence of lymphatic/CSF contamination compared to subcarapacial venous sinus. - **Blood Analyte Differences**: Head-started turtles had lower total solids (TS), lower heterophil-to-lymphocyte (H:L) ratios, and higher lymphocyte counts compared to free-ranging turtles. - **Subcarapacial Samples**: Showed significant dilution (lower PCV, TS, WBC counts, and heterophils) compared to jugular samples. - **Confounding Factors**: Differences likely influenced by age (juveniles vs adults), sex, seasonal effects, and stress from handling. - **Clinical Implication**: Blood sampling methodology (site and population) must be standardized when interpreting hemograms. - **Conservation Relevance**: Highlights the need to accurately assess health in head-started vs free-ranging turtles for conservation programs. - **Recommendation**: Prefer jugular sampling when possible; exercise caution with subcarapacial samples due to dilution artifacts. * * * **Section-by-Section Bullet Summary** **Introduction** - Eastern box turtles are vulnerable species impacted by anthropogenic threats. - Head-start programs aim to increase survival rates of young turtles. - Blood work critical for health assessments; venipuncture site effects not well studied in this species. **Methods** - Study conducted on Jekyll Island, Georgia (2011–2015). - Head-started turtles reared for 2–3 years before release. - Blood collected from jugular vein and/or subcarapacial sinus; CBC and plasma TS measured. - PCR screening done for adenovirus, herpesvirus, ranavirus, Mycoplasma. **Results** - 15 head-started and 24 free-ranging turtles included (after exclusions). - **Head-started vs Free-ranging**: - Head-started had lower TS, lower H:L ratio, higher lymphocytes. - No differences in PCV or total WBC count. - **Jugular vs Subcarapacial** (Head-started turtles): - Subcarapacial samples had lower PCV, TS, WBCs, and heterophils (indicating dilution). - **Figures/Tables**: - **Table 1**: Morphometrics of head-started turtles at release. - **Table 2**: Age/sex breakdown of study groups. - **Table 3**: Infectious disease testing results. - **Table 4**: Detailed blood analyte comparisons by group and venipuncture site. **Discussion** - Lower TS in head-started turtles could reflect life stage, hydration, or minor husbandry effects. - Stress response differences likely contributed to H:L and lymphocyte variation. - Sampling site critical for data consistency; subcarapacial sampling risks significant dilution artifacts. - Confounding variables included age, sex, and seasonality. - Emphasizes need for standardized serial sampling for meaningful comparisons.

Answer 98

What was the overall complication rate for surgical incisions in sea turtles in this study? 20%. Which type of surgery had a higher complication rate, orthopedic or soft tissue? Orthopedic (37.5% vs 6.5%). On average, when were complications first detected postoperatively?~21 days. How did healing time differ between uneventful and complicated cases? 41 days vs 74 days, respectively. Was there a correlation between body weight and healing time? Yes, heavier turtles had longer healing times. Which skin closure method had fewer complications, staples or sutures? Staples, though confounded by surgery type. Did preoperative antibiotic use affect complication rates? No significant impact found. Why is early return to seawater recommended after surgery? To reduce stress, maintain feeding, and support physiologic homeostasis. What factors were NOT associated with healing duration or complications? Species, surgery duration, hospitalization time, timing of diagnostics, antibiotic use. What surgical site observations were commonly seen with complications? Swelling, erythema, dehiscence, purulent effusion, eschar formation, granulation tissue. ## Footnote *JAVMA* 265(1): 1–8, 2025 **Surgical incisions in sea turtles have a moderately high complication occurrence but heal well with appropriate management: 70 incisions for 52 turtles (2008–2024)** Florence Coderre, Kelly Rockwell, Kathryn A. Tuxbury, Melissa J. Joblon, Julie M. Cavin, Robert J. McCarthy, Constance Merigo, Adam Kennedy, Charles J. Innis * * * **Quick Key Points for ACZM Board Review** - **Incision Healing**: 80% of sea turtle surgical incisions healed uneventfully; 20% developed complications. - **Orthopedic Surgeries Higher Risk**: Orthopedic procedures had a significantly higher complication rate (37.5%) than soft tissue procedures (6.5%). - **Healing Times**: Mean healing time was ~6 weeks for uneventful incisions and ~10 weeks for complicated cases. - **Complication Onset**: Complications typically noted ~21 days postoperatively (range 12–31 days). - **Body Weight Impact**: Heavier turtles had longer healing times, though weight was not associated with complication rate. - **Staples vs Sutures**: Skin closure with staples had a lower complication rate compared to sutures, but this may relate to the type of surgery (more orthopedic incisions closed with sutures). - **Antibiotic Use**: Most turtles received antibiotics perioperatively; no significant impact of antibiotic use on healing outcomes. - **Species Focus**: 92% Kemp’s ridley turtles (Lepidochelys kempii), 8% loggerhead turtles (Caretta caretta). - **Clinical Implication**: Sea turtle surgical wounds require extended monitoring (weeks to months) due to delayed and sometimes complicated healing. * * * **Section-by-Section Bullet Summary** **Introduction** - Sea turtles are highly threatened; surgical intervention often needed for rehabilitation. - Sparse data existed regarding expected healing timeframes or complication rates. - Aim: document surgical incision healing and complications over 16 years at one institution. **Methods** - Retrospective review (2008–2024) at New England Aquarium. - Included non-traumatic, non-ophthalmic, non-transmitter surgeries. - Documented species, surgical procedure, closure method, use of antibiotics, and major comorbidities. - Healing outcomes categorized as "uneventful" or "complicated." **Results** - 70 incisions in 52 turtles (mostly Kemp’s ridley). - **Complication Rate**: 20% overall; 37.5% orthopedic vs 6.5% soft tissue surgeries. - **Healing Time**: - Uneventful: Mean 41 days (range 20–81). - Complicated: Mean 74 days (range 40–113). - **Closure Method**: Staples associated with fewer complications than sutures, though confounded by surgery type. - **Figures**: - **Figure 1-2**: Examples of uneventful healing. - **Figure 3-4**: Examples of complicated healing (dehiscence, purulence, granulation). - **Tables**: - **Table 1**: Healing outcome vs surgery type, closure method, and species. - **Table 2**: Healing durations and body weights by outcome. - **Important Trends**: - No association found between healing time/complications and duration of hospitalization, surgery time, pre-op diagnostics, or antibiotic use. - Body weight positively correlated with longer healing time. **Discussion** - Healing generally successful even if complicated, but requires patience and ongoing wound care. - Orthopedic surgeries inherently more prone to complications, likely due to joint movement and pre-existing infection. - Staples may be better for soft tissue closures, but more research needed. - Sea turtles should be rapidly returned to clean seawater to reduce stress postoperatively. - Highlights importance of prolonged monitoring post-surgery. - Limitations included retrospective design, small sample size, missing data, and surgeon variability.

Answer 99

What dose of tiletamine-zolazepam was used in this study for sedation? 10 mg/kg SC. Was there a significant difference between forelimb and hindlimb injection sites for sedation quality? No significant difference. What was the average onset time for sedation? ~11–14 minutes. How successful was simulated jugular venipuncture after sedation?88% success in both groups. Did turtles completely lose their withdrawal reflexes after sedation?No, only decreased reflex observed. What drug was used to attempt sedation reversal? Flumazenil at 0.05 mg/kg SC What was the average recovery time following TZ sedation? ~135–165 minutes. How did forelimb injection compare to hindlimb injection regarding ease of jugular venipuncture? Slightly easier after forelimb injection, but not statistically significant. Why might hindlimb injections be theoretically less effective? Potential hepatic portal first-pass effect; though not evident with TZ in this study. Should sedation protocols for reptiles be species- and drug-specific?Yes, due to varying pharmacokinetics and physiology across taxa. ## Footnote *JAVMA* 265(1): 1–8, 2025 **Subcutaneous tiletamine-zolazepam produces moderate sedation in red-eared sliders (Trachemys scripta elegans) regardless of injection site** Katharine Hausmann Farris, Elijah J. Collins, Christoph Mans, Grayson A. Doss * * * **Quick Key Points for ACZM Board Review** - **Drug Evaluated**: Subcutaneous (SC) tiletamine-zolazepam (TZ) at 10 mg/kg produces moderate sedation in red-eared sliders. - **Injection Site**: No significant difference in sedation efficacy whether injected into the forelimb or hindlimb SC regions. - **Clinical Effectiveness**: Moderate sedation achieved in most turtles (~88% successful simulated jugular venipuncture). - **Onset and Recovery**: Onset of sedation ~11–14 minutes; recovery time ~135–165 minutes. - **Venipuncture Ease**: Slightly easier in forelimb injection group, but not statistically significant. - **Sedation Depth**: Good reduction in muscle tone (neck, limbs, jaw, cloaca) in both groups, but no complete loss of withdrawal reflexes. - **Physiologic Impact**: No significant changes in heart rate; respiratory rates difficult to assess. - **Clinical Use**: Suitable for physical exams, blood collection, or minor procedures; recovery times may limit use for very short procedures. - **Species and Drug-Specific Considerations**: Highlights need for species-, drug-, and site-specific sedation protocols in chelonians. * * * **Relevant ACZM Job Task Analysis Section** - **Category II.C.5.a.2 (Clinical Medicine > Diagnostic Procedures > Chemical restraint, sedation, and anesthesia)** - **Category II.A.2 (Species-Specific Management > Reptiles > Anatomy, physiology, husbandry, and clinical procedures)** * * * **Section-by-Section Bullet Summary** **Introduction** - Few standardized injectable sedation protocols exist for semiaquatic chelonians. - Tiletamine-zolazepam widely used but mostly evaluated for anesthesia rather than moderate sedation. - Injection site (cranial vs caudal) has mixed effects across reptile species on drug efficacy. **Methods** - Eight adult red-eared sliders studied in a randomized, blinded, crossover design. - 10 mg/kg TZ administered SC into either the forelimb (FL) or hindlimb (HL). - Sedation parameters assessed at 5-minute intervals; flumazenil administered at 45 min post-TZ for recovery. **Results** - **Onset**: 11 ± 4 min (FL) vs 14 ± 6 min (HL); no significant difference. - **Muscle Tone Reduction**: 75% FL vs 62.5% HL showed decreased tone. - **Jugular Venipuncture Simulation**: Successful in 7/8 turtles for both groups; HL group needed more restraint. - **Heart Rate**: No significant differences between groups. - **Recovery Times**: 135 ± 55 min (FL) vs 165 ± 45 min (HL); no statistical difference but longer in HL group. - **Figures and Tables**: - **Figure 1**: Turtle demonstrating sedation with head resting on ground. - **Figure 2**: Boxplot of time to decreased tone in various body parts. - **Figure 3**: Sedation score curves over time. - **Table 1**: Scoring rubric for muscle tone, jaw tone, cloacal tone, withdrawal reflex. **Discussion** - Injection site (cranial vs caudal) did not significantly affect sedation quality or recovery. - Longer recovery times than ideal for short procedures, despite flumazenil reversal. - Previous reports showed site-dependent drug efficacy in reptiles; differences appear drug- and species-specific. - Clinical consideration: FL injection may offer slightly easier handling but not significantly better sedation. - Caution: Results in healthy turtles; effects could differ in ill individuals. **Conclusion** - 10 mg/kg SC tiletamine-zolazepam provides moderate sedation in red-eared sliders suitable for clinical procedures. - Either forelimb or hindlimb SC injection site acceptable, with careful planning due to prolonged recovery.

Answer 100

What was the mean IOP measured in Sonoran Desert tortoises? 15.8 mm Hg (OD) and 16.8 mm Hg (OS) What method and setting were used to measure IOP in this study?Rebound tonometry, "dog" setting on iCare Tonovet Plus. Was there a significant difference in IOP between male and female tortoises? No. How did the IOP in Sonoran Desert tortoises compare to aquatic species like loggerhead turtles? It was higher in Sonoran Desert tortoises. What restraint method was used during tonometry? Placement on a circular container to encourage head extension without force. Was topical anesthesia needed for rebound tonometry in tortoises? No. Why should IOP values not be extrapolated across reptile species? Differences in anatomy and physiology (e.g., ocular structures) can significantly affect IOP. What anatomical structure is hypothesized to influence reptilian IOP variation? Conus papillaris (analogous to avian pecten oculi) What is the conservation status of Gopherus morafkai? Vulnerable (IUCN Red List). What types of eye complications were observed after the tonometry procedures? None ## Footnote *J. Herpetol. Med. Surg.* 35(1): 22–25, 2025 **Intra-ocular Pressure in the Sonoran Desert Tortoise (Gopherus morafkai) Using Rebound Tonometry** Julio Alfonso Mercado Rodríguez, Itza Denisse Vizcarra Alcantar, Cesar Augusto Flores Dueñas, Alberto Barreras Serrano * * * **Quick Key Points for ACZM Board Review** - **Study Focus**: First report measuring intra-ocular pressure (IOP) in Sonoran Desert tortoises (Gopherus morafkai) using rebound tonometry. - **IOP Results**: Mean IOP was 15.8 mm Hg (OD) and 16.8 mm Hg (OS), higher than in aquatic and some terrestrial chelonian species. - **Clinical Methodology**: Rebound tonometry (iCare Tonovet Plus, "dog" setting) was reliable without the need for local anesthetics. - **Sex and Eye Differences**: No significant difference in IOP between sexes or between right and left eyes. - **Species Comparisons**: Sonoran Desert tortoise IOP values were higher than loggerhead sea turtles, red-eared sliders, and Texas tortoises. - **Anatomical Hypothesis**: Differences in ocular anatomy (e.g., presence or absence of the conus papillaris) may explain variations in IOP among reptile species. - **Clinical Implication**: IOP values should not be extrapolated between reptile species; species-specific baselines are critical. - **No Adverse Effects**: No discomfort or ophthalmic complications were observed after tonometry. - **Conservation Context**: Gopherus morafkai is a vulnerable species per IUCN, although its legal protection status in Mexico is under debate. * * * **Relevant ACZM Job Task Analysis Section** - **Category II.C.3.c.1 (Clinical Medicine > Diagnostic Procedures > Ophthalmologic examination including intra-ocular pressure measurement)** - **Category II.A.2 (Species-Specific Management > Reptiles > Anatomy, physiology, husbandry, and clinical procedures)** * * * **Section-by-Section Bullet Summary** **Introduction** - *Gopherus morafkai* is a distinct species separated from G. agassizii (Mojave Desert tortoise). - IOP measurement is an essential part of ophthalmic exams in reptiles. - Prior IOP studies exist for various chelonians, but none for the Sonoran Desert tortoise. **Methods** - Twenty captive-born Sonoran Desert tortoises were evaluated (13 females, 7 males). - Restraint involved placing tortoises on circular containers to promote head extension without force. - Rebound tonometry performed using the "dog" setting; six measurements per eye collected. - Statistical analysis tested effects of eye, sex, and their interaction on IOP. **Results** - Mean IOP: - Right Eye (OD): 15.8 ± 2.66 mm Hg - Left Eye (OS): 16.8 ± 2.75 mm Hg - No significant difference based on eye or sex. - No need for topical anesthetic; no complications observed. **Discussion** - Sonoran Desert tortoise IOP higher than most aquatic species and slightly higher than other tortoises like *Chelonoidis chathamensis* (San Cristobal giant tortoise). - No correlation found between tortoise body size and IOP across species. - Potential anatomical influences on IOP proposed (e.g., conus papillaris structure differences). - Emphasized the importance of species-specific IOP references for clinical assessment. - Rebound tonometry proven effective and safe for chelonians. * * * **Key Figures and Tables** - **Table 1**: Mean IOP values for right and left eyes by sex (no significant differences). - **Table 2**: IOP values for marine, semi-aquatic, and terrestrial chelonians using rebound tonometry or applanation tonometry (important for exam comparison!).

Answer 101

What biological sample was used to measure trace elements in this study? Heparinized blood plasma. Which sex had lower trace element concentrations overall? Males. Which trace elements were significantly lower in males compared to females? Ag, Au, Ba, Hg, Mg, Mn, Mo, Ni, Sb, Se, Zn. What method was used to analyze the plasma trace elements? Inductively coupled plasma mass spectrometry (ICP-MS). Name two trace elements found in higher concentrations in turtles from southern Germany. Barium (Ba) and Magnesium (Mg). Which elements showed a positive correlation with turtle body weight? Ba, Hg, Mg, Mn, Mo, Ni, Se, Tl, Zn. Why are turtles good bioindicators for environmental pollution?< Long lifespan, aquatic habitat, and consistent exposure to environmental contaminants. What major limitation affects the lower bound of some reference intervals? Some trace elements were below the lower limit of quantification (LOQ). What sample contamination was avoided by excluding visibly lymph-contaminated samples? Lymphatic contamination. Why should extrapolation across species and habitats be done cautiously? Different species and environments significantly affect trace element absorption and metabolism. ## Footnote *J. Herpetol. Med. Surg.* 35(1): 31–39, 2025 **Trace Elements in Pond Sliders (Trachemys scripta)—Variations between Sex and Region of Sample Collection in Germany** Christoph Leineweber, Gregor Geisler, Sabine Öfner, Rachel E. Marschang * * * **Quick Key Points for ACZM Board Review** - **Study Focus**: Measured plasma concentrations of 17 trace elements in 167 pond sliders (Trachemys scripta) from northern and southern Germany. - **Sex Differences**: Males had significantly lower levels of Ag, Au, Ba, Hg, Mg, Mn, Mo, Ni, Sb, Se, and Zn compared to females. - **Regional Differences**: Significant differences based on collection region; e.g., Hg and Cu levels were higher in southern Germany turtles, Tl and Zn higher in northern Germany. - **Body Weight Correlations**: Positive correlations for Ba, Hg, Mg, Mn, Mo, Ni, Se, Tl, and Zn with turtle body weight. - **Environmental Implication**: Local environmental exposure, sex, and possibly body mass influence blood trace element levels. - **Sampling Methodology**: Heparinized plasma analyzed via inductively coupled plasma mass spectrometry (ICP-MS); rebound tonometry avoided lymph-contaminated samples. - **Comparison to Other Species**: Levels compared to published data in other freshwater turtle species, showing species and habitat differences. - **Clinical/Conservation Relevance**: Established preliminary reference intervals for turtles under human care, useful for environmental monitoring and health assessment. - **Important Limitation**: Lower quantification limit for several elements; plasma values may underestimate tissue accumulation. * * * **Relevant ACZM Job Task Analysis Section** - **Category II.B.4.a.1 (Preventive Medicine > Environmental monitoring, ecotoxicology, toxicologic disease surveillance in zoological species)** - **Category II.A.2 (Species-Specific Management > Reptiles > Anatomy, physiology, husbandry, and clinical procedures)** * * * **Section-by-Section Bullet Summary** **Introduction** - Trace elements can be both essential and toxic depending on concentration. - Turtles, due to long lifespans and aquatic habits, serve as excellent bioindicators. - Few studies exist assessing blood trace element concentrations in freshwater turtles. **Materials and Methods** - 167 pond sliders (49 males, 118 females) sampled during clinical exams (July–September 2022). - Blood collected from dorsal coccygeal vein or subcarapacial sinus; plasma analyzed. - 17 elements measured by ICP-MS: Ag, As, Au, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Mo, Ni, Sb, Se, Tl, Zn. - Statistical tests included t-tests, Wilcoxon, and Spearman correlation; P < 0.05 considered significant. **Results** - **Sex Differences**: Males had significantly lower levels of 11 trace elements compared to females. - **Regional Differences**: - Higher Au, Ba, Mg, Tl, Zn in southern turtles. - Higher Cu, Hg, Sb in northern turtles. - **Body Weight Correlations**: Positive for Ba, Hg, Mg, Mn, Mo, Ni, Se, Tl, Zn. - **Tables/Figures**: - **Figure 1**: Map showing sampling regions. - **Figure 2**: Boxplots of sex-based differences. - **Figure 3**: Boxplots of region-based differences. - **Table 1**: Reference intervals for each element. - **Table 2**: Comparison with published data from other turtle species. **Discussion** - Differences influenced by sex, weight, region, possibly diet and reproductive excretion (e.g., females shedding elements via eggs). - Regional differences may reflect geological soil variation and local pollution. - Results emphasize need for species- and site-specific baselines for environmental assessments. - Blood sampling is minimally invasive and suitable for ongoing monitoring, though tissue concentrations may differ. - Study limitations include sample type, LOQ challenges, and lack of seasonality assessment.

Answer 102

**1\. What was the main pathogen targeted by the nasal flush protocol in this study?** *Mycoplasma agassizii.* **2\. What clinical sign was used to assess treatment success in tortoises?** *Resolution of nasal discharge.* **3\. How many nasal flush treatments were performed per tortoise before evaluation?** *At least five treatments.* **4\. What systemic antibiotic was administered during the nasal flush protocol?** *Enrofloxacin at 10 mg/kg IM.* **5\. What adjunct anti-inflammatory was included in the nasal medicated solution?** *Dexamethasone.* **6\. What percentage of tortoises tested negative for *Mycoplasma* one month after treatment?** *80%.* **7\. What diagnostic tests were used to detect *Mycoplasma* infections?** *PCR and next-generation sequencing (NGS).* **8\. What agreement statistic indicated good consistency between PCR and NGS results?** *Kappa = 0.64.* **9\. What factors may contribute to *Mycoplasma* recurrence even after initial negative testing?** *Stress, environmental issues, immunosuppression, or latent infection.* **10\. Is a negative PCR or NGS result definitive proof of *Mycoplasma* eradication?** *No, due to detection thresholds and possible latency of infection.* ## Footnote *J. Herpetol. Med. Surg.* 35(1): 40–44, 2025 **Effectiveness of Nasal Flush Treatments in Mycoplasma PCR- or DNA Sequencing–Positive Tortoises** Marianne Caron, Ian Kanda, Mark Mitchell, Thomas Boyer * * * **Key Points for ACZM Board Review** - **Study Purpose**: Evaluated nasal flush protocol combined with systemic enrofloxacin for treating *Mycoplasma*\-positive tortoises. - **Clinical Resolution**: 100% of tortoises had resolution of acute nasal discharge at the end of treatment. - **PCR/NGS Results**: 80% (12/15) tested negative for *Mycoplasma* one month after treatment; statistically significant improvement. - **Nasal Flush Protocol**: Involved five saline flushes plus medicated enrofloxacin/dexamethasone solution every 48–72 hours under sedation. - **PCR vs NGS Agreement**: Good agreement between PCR and next-generation sequencing (kappa = 0.64), though PCR appeared more sensitive. - **Recurrence**: 3/6 tortoises re-tested between 4–10 months post-treatment had recrudescence; stress and environmental factors were likely contributors. - **Treatment Limitations**: Despite acute resolution, complete long-term elimination of *Mycoplasma* remains uncertain. - **Diagnostic Insight**: Negative tests may not guarantee absence of infection due to organism latency or detection limits. - **Conservation Relevance**: Improved management of mycoplasmosis could enhance captive and wild tortoise conservation efforts. * * * **Relevant ACZM Job Task Analysis Section** - **Category II.C.6.a.3 (Clinical Medicine > Infectious disease diagnosis and management > Diagnosis and treatment of bacterial diseases)** - **Category II.B.4.a.1 (Preventive Medicine > Environmental monitoring, ecotoxicology, and infectious disease surveillance in zoological species)** * * * **Section-by-Section Bullet Summary** **Introduction** - *Mycoplasma agassizii* causes chronic upper respiratory tract disease (URTD) in tortoises. - Leads to mucosal destruction, decreased olfaction, reduced appetite, and impaired reproduction. - Current treatments are limited; prior nasal flush protocols showed promise but lacked confirmatory testing. **Materials and Methods** - Inclusion: Captive Testudinidae with nasal discharge and positive *Mycoplasma* detection by PCR or NGS. - Protocol: - Nasal flushes with saline under sedation every 48–72 hours (≥5 treatments). - Medicated nasal instillation (enrofloxacin + dexamethasone). - Systemic enrofloxacin (10 mg/kg IM) after each flush. - Diagnostics: PCR and NGS at initial flush, final flush, and 1-month post-treatment. - Statistical tests: Cochran’s Q test and kappa agreement statistics. **Results** - 15 *Mycoplasma*\-positive tortoises treated (desert, leopard, and sulcata species). - At final flush: - 26.7% remained positive. - One month post-flush: - 20% remained positive. - Resolution of nasal discharge occurred in all treated tortoises. - Recurrence of nasal discharge in 2 tortoises >4 months post-treatment. - Table 1: Detailed PCR and NGS results at different time points for each patient. **Discussion** - Nasal flush + systemic enrofloxacin resulted in acute clinical resolution. - Treatment improved clinical signs but may not fully eradicate infection. - NGS was slightly less sensitive than PCR for *Mycoplasma* detection. - Stressors, husbandry issues, and possible re-infections are critical post-treatment variables. - Repeat flushing recommended for persistent positives at fifth treatment. * * * **Key Diagrams, Figures, and Tables** - **Table 1**: Sequential PCR and NGS mycoplasma test results for each tortoise (before treatment, after fifth flush, 1 month post-flush).

Answer 103

**1\. What conservation intervention was studied in this project?** *Headstarting of Blanding’s turtles (Emydoidea blandingii).* **2\. What group of turtles showed the poorest health indices in this study?** *1-year post-release headstarts.* **3\. What pathogen was newly identified in post-release Blanding’s turtles?** *Emydoidea adenovirus 1.* **4\. Which hematologic finding was higher in pre-release headstarted turtles compared to wild-reared juveniles?** *Total solids, albumin, and beta globulins.* **5\. What physical abnormalities were more common in pre-release headstarted turtles?** *Shell and musculoskeletal deformities.* **6\. What trend was seen in ectoparasite and hemoparasite prevalence after release?** *Increased prevalence in post-release turtles.* **7\. What environmental factors might contribute to musculoskeletal abnormalities in headstarts?** *High stocking density, UVB deficiency, dietary imbalances.* **8\. What are two pathogens besides adenovirus detected in these turtles?** *Mycoplasmopsis sp. and Emydomyces testavorans.* **9\. What clinical pathology value indicated increased physiological stress in pre-release headstarts?** *Higher heterophil:lymphocyte ratio.* **10\. Why is proactive pathogen surveillance critical in headstart programs?** *To detect emergent infections early and prevent pathogen release into wild populations.* ## Footnote *J. Wildl. Dis.* 61(1): 30–45, 2025 **Hematology, Plasma Biochemistry, Protein Electrophoresis, and Pathogen Surveillance in Headstarted and Wild-Reared Populations of Blanding’s Turtles (Emydoidea blandingii) in Three Northern Illinois, USA, Counties** Alexis Davidson, Michelle W. Kendall, Maura Ryan, Kayla Ladez, Samantha Bradley, Carley Lionetto, William Graser, Gary Glowacki, Daniel Thompson, Richard B. King, Callie K. Golba, Kaitlin Moorhead, Laura Adamovicz, Matthew C. Allender * * * **Key Points for ACZM Board Review** - **Study Focus**: Assessed health parameters (physical exams, hematology, biochemistry, protein electrophoresis, pathogen surveillance) in headstarted vs wild-reared Blanding’s turtles. - **Health Shifts After Release**: Significant health parameter shifts occur after release; 1-year post-release turtles had the poorest overall health indices before partial rebound by 2 years. - **Clinical Pathology Trends**: Pre-release headstarts had higher total protein, albumin, and alpha globulins but lower heterophil:lymphocyte ratios compared to wild-reared turtles. - **Pathogen Findings**: Adenoviruses (tentatively named Emydoidea adenovirus 1), Mycoplasmopsis sp., and Emydomyces testavorans detected primarily in post-release individuals. - **Shell and Musculoskeletal Abnormalities**: Higher rates in pre-release headstarts, possibly linked to high-density rearing and nutritional/metabolic issues. - **Environmental Exposure**: Increased ectoparasite and hemoparasite prevalence post-release reflected natural exposure risks. - **Infectious Disease Surveillance**: Demonstrated critical importance of proactive pathogen monitoring in headstart programs. - **Management Implications**: Adjustments to husbandry (UV light, diet, rearing density) and careful disease surveillance are vital to improving headstart success. - **Conservation Relevance**: Findings directly inform evidence-based conservation practices for Blanding’s turtles. * * * **Relevant ACZM Job Task Analysis Section** - **Category II.B.4.a.1 (Preventive Medicine > Environmental monitoring, ecotoxicology, infectious disease surveillance)** - **Category II.C.2.a.1 and II.C.5.a.2 (Clinical Medicine > Diagnostic procedures > Physical examination, chemical restraint, clinical pathology including hematology and biochemistry)** * * * **Section-by-Section Bullet Summary** **Introduction** - Blanding’s turtles are endangered; headstarting is a conservation strategy, but health impacts are poorly understood. - Concerns exist regarding pathogen introduction during headstart release. **Materials and Methods** - Compared headstarted turtles (pre- and post-release) to wild-reared juveniles across three Illinois counties. - Data collected: physical exam findings, hematology, plasma biochemistry, protein electrophoresis, pathogen surveillance (PCR, qPCR, NGS). **Results** - 767 assessments from 561 turtles. - Pre-release headstarts: Higher rates of shell/musculoskeletal abnormalities; higher albumin, prealbumin, total protein, bile acids. - Post-release headstarts: Increased ectoparasites, hemoparasites, asymmetrical nares; decreased plasma proteins and increased heterophil:lymphocyte ratios. - Pathogens: - Emydoidea adenovirus 1 detected post-release (up to 11% prevalence). - Mycoplasmopsis sp. and Emydomyces testavorans also detected sporadically. - Figures and Tables: - **Figure 1-3**: Hematology, biochemistry, and protein electrophoresis differences by rearing history. - **Tables 2-6**: Physical exam abnormalities, hematology, plasma biochemistry, and electrophoresis results across groups. **Discussion** - Health parameters shifted significantly after release but rebounded over time. - Musculoskeletal abnormalities linked to husbandry practices. - Infectious disease surveillance critical; novel adenovirus identified. - Recommendations include rearing modifications (UV exposure, diet optimization) and rigorous health monitoring. **Conclusions** - Headstarting success can be enhanced by minimizing health disruptions through improved husbandry and surveillance. - Long-term studies linking health indices to survival needed.

Answer 104

**1\. What was the target organism in this disinfectant efficacy study?** *Emydomyces testavorans, an emerging fungal pathogen in aquatic turtles.* **2\. Which common disinfectants were shown to be completely effective against E. testavorans?** *Bleach, Betadine, chlorhexidine, Rescue™, F10 SC, Roccal-D Plus, Lysol, and heat (60°C).* **3\. What concentration and duration of heat exposure was fungicidal?** *60°C for 5 minutes.* **4\. Which disinfectant unexpectedly showed complete efficacy in this study despite past reports of ineffectiveness against similar fungi?** *Chlorhexidine (2%).* **5\. Why is benzalkonium chloride not recommended for disinfection of E. testavorans?** *It showed delayed, incomplete inhibition suggesting fungistatic but not fungicidal activity.* **6\. What was the efficacy of desiccation in eliminating E. testavorans?** *Desiccation for 24 hours did not prevent fungal regrowth.* **7\. What disinfectant commonly used in aquaculture did not eliminate E. testavorans in this study?** *Virkon S (1%).* **8\. What type of test was used to confirm fungal colony identity post-treatment?** *Quantitative PCR.* **9\. What implication does this study have for surgical site preparation in turtles?** *Betadine is effective and should be preserved for sterile surgical prep to avoid resistance.* **10\. Why must Roccal-D Plus be used cautiously in field or aquatic environments?** *It is toxic to aquatic organisms and may cause environmental harm if not rinsed thoroughly.* ## Footnote *J. Wildl. Dis.* 61(1): 234–240, 2025 **Evaluating the Efficacy of Disinfectant Methods Against Emydomyces testavorans, a Fungus Associated with Shell Disease in Freshwater Aquatic Turtles** Nicholas C. Liszka, Laura Adamovicz, Kaitlin A. Moorhead, Maris J. Daleo, Kamila Grochowski, Matthew C. Allender * * * **Key Points for ACZM Board Review** - **Study Focus**: First study to test efficacy of 13 disinfectants against *Emydomyces testavorans*, an emerging fungal pathogen in aquatic turtles. - **Effective Disinfectants**: Bleach (3–10%), chlorhexidine, Rescue™ (accelerated hydrogen peroxide), Betadine, Roccal-D Plus, F10 SC, F10 SCXD, Lysol, and heat (60°C for 5 min) completely inhibited fungal growth. - **Ineffective Methods**: Ethanol (100%), Virkon S (1%), benzalkonium chloride, and desiccation were not consistently fungicidal. - **Chlorhexidine Findings**: Contrary to prior findings in related fungi, 2% chlorhexidine was effective against *E. testavorans* in this study. - **Heat and Desiccation**: Drying for 24 hours was insufficient; heat at 60°C for 5 minutes effectively eliminated fungal growth. - **Virkon S**: Showed delayed growth inhibition, indicating fungistatic rather than fungicidal activity at 1%. - **Field vs Clinical Use**: Recommendations vary by application; bleach and chlorhexidine for field use, povidone-iodine preserved for surgical prep. - **Biosecurity Significance**: Data provide essential guidance for disease prevention protocols in conservation and captive turtle programs. - **Environmental Caution**: Some disinfectants (e.g., Roccal-D Plus) are toxic to aquatic organisms and require careful handling in field use. * * * **Relevant ACZM Job Task Analysis Section** - **Category II.B.4.a.1** – Preventive Medicine → Environmental monitoring, ecotoxicology, infectious disease surveillance in zoological species - **Category II.C.6.a.3** – Clinical Medicine → Infectious disease diagnosis and management → Management of fungal diseases - **Category II.D.1.a.1** – Facility Biosecurity and Husbandry → Disinfection, sanitation, and hygiene protocols * * * **Section-by-Section Bullet Summary** **Introduction** - *E. testavorans* is an emerging fungal pathogen linked to shell disease in freshwater turtles, especially in managed care. - Effective disinfectant protocols are lacking; prior recommendations borrowed from protocols for *Ophidiomyces* and *Nannizziopsis* spp. - Study goal: test 13 commonly used disinfectants against *E. testavorans* in vitro. **Methods** - In vitro assay using fungal strain ATCC TSD-145. - Disinfectants tested at manufacturer-recommended concentrations and contact times. - Exposures included chemical agents, heat (60°C), and desiccation. - Growth monitored over 6 weeks; fungal colonies counted and confirmed by qPCR. **Results** - Disinfectants that completely inhibited growth: - Bleach (3%, 10%) - Chlorhexidine (2%) - Accelerated hydrogen peroxide (Rescue™) - Povidone-iodine (Betadine) - Roccal-D Plus - F10 SC, F10 SCXD - Lysol All-Purpose Cleaner - Heat (60°C for 5 min) - Ineffective: - Ethanol (1 min and 5 min) - Virkon S (1% for 2–10 min) - Benzalkonium chloride - Desiccation for 24 hrs - **Table 1**: Disinfectants, dilutions, contact times. - **Table 2**: Colony growth by dilution and treatment. **Discussion** - Chlorhexidine effective despite prior reports of ineffectiveness for related fungi. - Benzalkonium chloride may be fungistatic but not fungicidal. - Virkon S delayed fungal growth but didn’t eliminate it. - Desiccation alone not recommended due to fungal resilience. - Practical guidance: use bleach or chlorhexidine for field decontamination, and reserve Betadine for surgical preparation. - Limitations: Morphologic ID used on most plates, contact time variability, no host organism testing. **Conclusion** - Disinfectant choice should be tailored to the setting. - Multiple effective disinfection strategies validated for use in controlling *E. testavorans*. - Findings support enhanced biosecurity in conservation, rehabilitation, and research programs.

Answer 105

**1\. What pathogen was the focus of this epidemiologic study in Florida turtles?** *Turtle fraservirus 1 (TFV1), a novel negative-sense RNA virus, family Tosoviridae* **2\. Which tissue and sample types were most reliable for PCR detection of TFV1?** *Kidney and urine, especially in decomposed carcasses.* **3\. What clinical sign was most strongly associated with TFV1 infection in softshell turtles?** *Presence of oral and/or cloacal plaques.* **4\. How did ambient temperature influence the probability of TFV1 infection?** *Lower maximum temperatures (especially <25°C) were associated with higher infection risk.* **5\. How was a “clustered mortality event” defined in this study?** *Two or more sick/dead turtles within the same waterbody over a 7-day period.* **6\. What statistical outcome showed the strongest predictor of TFV1 positivity?** *18-fold increased odds in turtles from clustered mortality events (CL7).* **7\. Was the probability of TFV1 infection influenced by turtle sex or maturity?** *No significant association was found for sex or maturity.* **8\. What seasonal trend was observed in TFV1 case frequency?** *Most positive cases were collected between January and March.* **9\. Why are softshell turtles a conservation concern in TFV1 outbreaks?** *High frequency of infection in gravid/vitellogenic females may reduce reproductive output.* **10\. What management recommendation was emphasized to prevent TFV1 spread?** *Strict biosecurity and spatial buffer zones (≥5 km) around positive detection sites.* ## Footnote *PLOS ONE* 20(4): e0320097, 2025 **Epidemiological Factors Associated with Turtle Fraservirus 1 (TFV1) in Freshwater Turtles in Florida, USA** Lisa A. Shender, Andrea Sylvia, Brian A. Stacy, Veronica Guzman-Vargas, Thais C.S. Rodrigues, Pedro Viadanna, et al. * * * **Key Points for ACZM Board Review** - **Emerging Virus**: TFV1 is a newly recognized negative-sense RNA virus from the *Tosoviridae* family, first detected in 2018 in Florida softshell turtles. - **Infection Dynamics**: >50% of tested turtles were TFV1-positive. Infection strongly associated with oral/cloacal plaques (especially in softshells) and spatial-temporal clustering. - **Host Range**: TFV1 affects softshells and hardshell turtles (Pseudemys, Trachemys), with softshells more severely affected. - **Transmission Hypotheses**: Data support waterborne and potentially direct transmission. Virus detected in urine; environmental spread is likely. - **Diagnostic Recommendations**: RT-qPCR is more sensitive than RT-cPCR; kidney and urine are preferred sample types in decomposed carcasses. - **Environmental Risk**: Colder temperatures increased infection probability; urbanization metrics were not strong predictors, but site clustering was. - **Biosecurity Guidance**: Findings emphasize biosecurity for rehabilitation, translocations, and waterbody management. - **Surveillance Strategies**: Cluster detection (≥2 turtles at the same site within 7 days) and plaque observation in softshells are strong indicators for TFV1 testing prioritization. - **Population Impacts**: Female reproductive softshell turtles were frequently affected, raising concern for long-term population stability. * * * **Relevant ACZM Job Task Analysis Sections** - **II.B.4.a.1** – Preventive Medicine → Environmental monitoring, ecotoxicology, infectious disease surveillance - **II.C.6.a.3** – Clinical Medicine → Infectious disease diagnosis and management - **II.C.2.a.1** – Clinical Medicine → Physical examination, diagnostic planning in free-ranging reptiles * * * **Section-by-Section Summary** **Introduction** - Chelonians are globally imperiled; emerging diseases like TFV1 pose new threats. - TFV1 was first discovered in 2018; much about its epidemiology remains unknown. - Study goals: assess risk factors for TFV1, inform management guidelines, and propose future research directions. **Methods** - 117 necropsied freshwater turtles from 2018–2021 evaluated for TFV1 via RT-cPCR and RT-qPCR. - Datasets created: Field (e.g., species, season, urbanicity), Necropsy (e.g., plaques, sex), Weather (e.g., temperature), Spatial (e.g., proximity to infected turtles). - Statistical modeling used to determine associations with TFV1 infection. **Results** - 93 turtles tested: 47 (50.5%) positive (57% softshells, 37% hardshells). - Strongest predictors: - Clustered mortality events (18x higher odds of infection). - Oral/cloacal plaques in softshells (31x increased odds). - Lower maximum temperatures associated with higher infection risk. - Urine and kidney were reliable diagnostic samples. - Spatial clustering within 1–5 km of other positive turtles predicted infection likelihood. **Discussion** - TFV1 likely spreads through water, secretions, or direct contact—suggested by cluster and temperature data. - Cold weather may impair turtle immunity, increasing disease susceptibility. - In softshells, the disease may be acute; in hardshells, possibly more chronic or opportunistic. - Urban metrics did not predict infection, but spatial patterns strongly did. **Conclusion** - TFV1 is highly transmissible and poses significant conservation risks. - Cluster-based mortality detection and plaque observation in softshells are strong diagnostic cues. - Biosecurity practices are essential to prevent disease spread between water bodies.

Answer 106

**1\. What virus was genetically detected in turtles and leeches in this study?** *Chelonid herpesvirus 5 (ChHV5).* **2\. Which gene was targeted by PCR to detect ChHV5 in this study?** *UL30 (DNA polymerase gene).* **3\. What host species tested positive for ChHV5 in this study?** *One olive ridley turtle (tumor), one black turtle (skin), and one leech (Ozobranchus branchiatus).* **4\. What phylogenetic group did the ChHV5 sequences cluster with?** *Eastern Pacific group.* **5\. What concern does low PCR positivity raise in the context of ChHV5 surveillance?** *PCR targeting UL30 may miss latent infections, underestimating true prevalence.* **6\. Why are UL22 and UL27 suggested as better diagnostic targets for ChHV5?** *They detect viral glycoproteins and are not constrained to active replication, improving latent infection detection.* **7\. What was notable about the leech that tested positive for ChHV5?** *It came from a clinically healthy turtle that tested negative, suggesting possible vector transmission.* **8\. How might turtle ecology contribute to ChHV5 spread in the region?** *Turtles use multiple foraging areas, facilitating viral transmission across the Eastern Pacific.* **9\. What was the main limitation of this study’s molecular detection method?** *Conventional PCR using only one gene (UL30) may miss ChHV5 in latency.* **10\. What does this study emphasize for future marine turtle disease research?** *Expanded genetic surveillance and investigation of host–parasite–pathogen dynamics.* ## Footnote *EcoHealth* 17: 258–263, 2020 **Genetic Analysis of Chelonid Herpesvirus 5 in Marine Turtles from Baja California Peninsula** Joelly Espinoza, Elsa Hernández, María Mónica Lara-Uc, Eduardo Reséndiz, Alonzo Alfaro-Núñez, Sawako Hori-Oshima, Gerardo Medina-Basulto * * * **Key Points for ACZM Board Review** - **Study Focus**: First molecular detection and genetic characterization of Chelonid herpesvirus 5 (ChHV5) in marine turtles and leeches from the Baja California Peninsula, Mexico. - **Host Species Affected**: ChHV5 DNA detected in one tumor from an olive ridley turtle (Lepidochelys olivacea), skin of one black turtle (Chelonia mydas), and one leech (Ozobranchus branchiatus). - **Genetic Findings**: Two ChHV5 variants identified; sequences clustered within the Eastern Pacific phylogenetic group, suggesting regional viral circulation. - **Vector Insight**: Leech collected from a clinically healthy turtle carried a ChHV5 strain, supporting its potential vector role. - **Diagnostic Limitations**: Only 3 of 87 samples (3.4%) were ChHV5-positive, likely underestimating prevalence due to limited PCR sensitivity for latent infections. - **Molecular Tools**: Conventional PCR targeting the UL30 gene may miss latent infections; alternate targets like UL22 or UL27 are recommended for future studies. - **Ecological Relevance**: Turtles in the region exhibit foraging behavior across multiple sites, possibly promoting viral spread via host movement. - **Conservation Implication**: Ongoing ChHV5 surveillance is needed due to FP’s link with environmental degradation and potential vector involvement. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.6.a.3** – Clinical Medicine → Infectious disease diagnosis and management → Herpesvirus detection and fibropapillomatosis surveillance - **II.B.4.a.1** – Preventive Medicine → Infectious disease surveillance, wildlife disease ecology - **II.A.2** – Species-Specific Management → Reptiles → Virology and conservation medicine in marine turtles * * * **Section-by-Section Summary** **Introduction** - Fibropapillomatosis (FP), a tumor disease linked to ChHV5, affects all sea turtle species and has a circumtropical distribution. - ChHV5 transmission may occur via direct contact or potential vectors like Ozobranchus spp. leeches. - Prior to this study, ChHV5 had not been confirmed in turtles from Baja California via molecular techniques. **Materials and Methods** - Samples from 87 turtles (black, loggerhead, olive ridley) and 5 leeches were collected (2016–2017) across five coastal feeding sites. - PCR targeting the UL30 gene of ChHV5 was used for detection. - Positive amplicons were sequenced and compared with GenBank entries; phylogenetic analyses conducted using Bayesian methods. **Results** - ChHV5 detected in: - Tumor from one olive ridley turtle. - Skin sample from one black turtle. - One leech (O. branchiatus) from a clinically healthy black turtle. - Phylogenetic tree showed all sequences belonged to the Eastern Pacific group. - Leech and olive ridley sequences were identical; black turtle sequence had a unique 4-nucleotide insertion. - Table 1 and Figure 2: Sampling site overview and phylogenetic tree showing Eastern Pacific group clustering. **Discussion** - Results support leeches as possible mechanical vectors. - Low detection rate could reflect latent infections undetectable by UL30-based PCR. - Need for more sensitive markers and expanded surveillance is highlighted. - Ecological behaviors (e.g., shared foraging areas) may facilitate virus dispersal across the Eastern Pacific. - FP etiology likely multifactorial—ChHV5 presence, environmental stressors, and host factors all contribute. **Conclusion** - First molecular evidence of ChHV5 in turtles and leeches in Baja California Peninsula. - Study contributes to understanding regional ChHV5 dynamics and supports further surveillance using broader genetic markers. * * * **Key Figures and Tables** - **Figure 1**: Map of five turtle sampling locations in Baja California Peninsula. - **Figure 2**: Bayesian phylogenetic tree of UL30 sequences showing clustering with Eastern Pacific ChHV5 variants. - **Table 1**: Sample origin, turtle species, ChHV5 detection status, and leech host data.

Answer 107

**1\. What virus is associated with fibropapillomatosis in green turtles?** *Chelonid alphaherpesvirus 5 (ChAHV5).* **2\. What percentage of green turtles sampled in this study showed visible FP lesions?** *30% overall (33% in Guinea-Bissau, 28% in Mauritania).* **3\. What tissues had the highest viral loads of ChAHV5 DNA?** *Tumor tissues.* **4\. What percentage of asymptomatic turtles tested positive for ChAHV5 DNA?** *Approximately 45%.* **5\. Which turtle life stage showed the highest FP prevalence?** *Juveniles.* **6\. What was the general body condition of turtles with even severe FP lesions?** *Good; they did not appear emaciated or impaired.* **7\. How was tumor severity scored in this study?** *On a scale of 0–3, based on number and size of tumors.* **8\. Why might smaller turtles have been under-sampled?** *Large mesh size in entanglement nets allowed escape of smaller individuals.* **9\. What finding suggests ChAHV5 is enzootic in these populations?** *High prevalence of ChAHV5 DNA in FP-free turtles.* **10\. What is recommended for future studies on FP in West Africa?** *Long-term monitoring, flipper tagging, and inclusion of blood health parameters.* ## Footnote *EcoHealth* 18: 229–240, 2021 **Fibropapillomatosis and the Chelonid Alphaherpesvirus 5 in Green Turtles from West Africa** Jessica Monteiro, Margarida Duarte, Kidé Amadou, Castro Barbosa, Nahi El Bar, Fernando M. Madeira, Aissa Regalla, Ana Duarte, Luís Tavares, Ana Rita Patrício * * * **Key Points for ACZM Board Review** - **First FP Baseline in Region**: This is the first study reporting the prevalence of fibropapillomatosis (FP) and Chelonid alphaherpesvirus 5 (ChAHV5) in foraging green turtles in Guinea-Bissau and Mauritania. - **Moderate FP Prevalence**: FP was observed in 30% of turtles overall (33% in Guinea-Bissau, 28% in Mauritania); most were mildly affected. - **ChAHV5 Detection**: ChAHV5 DNA was detected in 83% of tumor samples and 45% of asymptomatic turtles, supporting viral latency and multifactorial disease expression. - **FP and Age**: Juveniles had a higher FP prevalence compared to subadults, consistent with age-related immunity development. - **Tumor Scoring**: Most FP-afflicted turtles had mild lesions (score 1); a minority (12.5%) were heavily afflicted but maintained good body condition. - **Viral Load Findings**: Tumor tissue carried significantly higher ChAHV5 DNA loads than normal skin; viral DNA was also present in clinically normal skin. - **Site Differences**: Turtles from Mauritania had higher ChAHV5 prevalence in both lesions and normal skin than those from Guinea-Bissau. - **One Health Implications**: Despite low anthropogenic disturbance, FP prevalence remains moderate, suggesting environmental and host-pathogen factors influence disease dynamics. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.6.a.3** – Clinical Medicine → Infectious disease diagnosis and management → Viral diseases and tumorigenesis - **II.B.4.a.1** – Preventive Medicine → Environmental monitoring and infectious disease surveillance - **II.A.2** – Species-Specific Management → Reptiles (marine turtles) → Clinical assessment and diagnostic testing * * * **Section-by-Section Summary** **Introduction** - FP is a tumorigenic disease affecting all sea turtle species but primarily green turtles. - Etiology involves ChAHV5, but disease expression depends on host-pathogen-environment interactions. - There were no previous data on FP or ChAHV5 prevalence in green turtles in West African foraging areas. **Materials and Methods** - Study sites: Bijagós Archipelago (Guinea-Bissau) and Banc d’Arguin National Park (Mauritania). - 108 green turtles were captured; 76 tested for ChAHV5 via qPCR. - Tumor severity scored (0–3); DNA extracted from tumor and normal skin biopsies; viral load quantified. **Results** - **FP Prevalence**: 30% overall; 33% in Guinea-Bissau, 28% in Mauritania. Most turtles had mild tumors. - **ChAHV5 Detection**: - 83.3% of tumor samples positive. - 26% of normal skin from FP turtles and 45% of FP-free turtles also positive. - **Viral Load**: Highest in tumors, lower in normal skin; significant differences between sites (higher in Mauritania). - **Age Trends**: Juveniles more commonly afflicted; FP risk peaked at ~50–60 cm curved carapace length. - **Table Highlights**: - **Table 1**: FP and ChAHV5 prevalence by site and tissue type. - **Table 2**: FP severity by life stage. - **Figures**: - **Figure 2**: Life stage distribution. - **Figure 3**: FP risk vs turtle size. - **Figure 4**: Viral DNA load comparisons. **Discussion** - High viral DNA in tumors supports ChAHV5 as the causative agent. - Presence of viral DNA in asymptomatic turtles indicates latency or early infection. - Mild FP severity and good body condition suggest subclinical or non-impairing disease in most turtles. - Potential under-detection in smaller turtles due to net mesh size. - Disease progression, immunity, or regression remains unknown due to the study’s snapshot nature. - Seasonal or environmental changes may influence FP outbreaks. **Final Considerations** - Establishes a critical baseline for FP and ChAHV5 in a major Atlantic green turtle rookery and foraging site. - Future long-term studies needed to assess FP dynamics and potential environmental drivers. - Recommends use of flipper tags and bloodwork to monitor health trends over time.

Answer 108

**1\. What disease affecting green turtles was the focus of this study?** *Fibropapillomatosis (FP).* **2\. What virus is associated with fibropapillomatosis in sea turtles?** *Chelonid alphaherpesvirus 5 (ChHV5).* **3\. Which bay in Martinique showed the highest FP prevalence increase?** *Anse du Bourg/Chaudière.* **4\. What body parts were most frequently affected by FP tumors?** *Eyes, fore flippers, and neck.* **5\. What environmental factor showed a slight positive association with FP prevalence?** *Net primary productivity (NPP).* **6\. Which water quality indicators were elevated in high-FP prevalence sites?** *Nitrites and E. coli (plus enterococci in Anse du Bourg/Chaudière).* **7\. What spatial pattern was associated with increased FP prevalence?** *High turtle density in small, localized areas.* **8\. What mode of disease transmission is supported by the findings?** *Horizontal transmission via close physical contact and contaminated water.* **9\. What ecological feature makes Martinique important for green turtles?** *It is a critical developmental foraging area for immature green turtles.* **10\. Why might FP prevalence be underestimated in this study?** *Diagnosis relied on external tumors; asymptomatic turtles may carry latent ChHV5.* ## Footnote *EcoHealth* 19: 190–202, 2022 **Fibropapillomatosis Prevalence and Distribution in Immature Green Turtles (Chelonia mydas) in Martinique Island (Lesser Antilles)** Thibaut Roost, Jo-Ann Schies, Marc Girondot, Jean-Patrice Robin, Pierre Lelong, et al. * * * **Key Points for ACZM Board Review** - **Emergence in Lesser Antilles**: First study documenting fibropapillomatosis (FP) prevalence in immature green turtles in Martinique. - **Prevalence Trend**: FP prevalence increased from 0% in 2011 to 16.8% in 2019, driven mainly by rising cases at Anse du Bourg/Chaudière. - **Tumor Distribution**: Tumors primarily affected the eyes (41.4%), fore flippers (21.9%), and neck (9.4%), suggesting contact-mediated transmission. - **Spatial Clustering**: High-density turtle aggregations occurred in small, defined areas of bays, potentially facilitating FP transmission. - **Environmental Correlates**: No effect of sea surface temperature (SST) was found; net primary productivity (NPP) had a slight positive association with FP prevalence. - **Water Quality Indicators**: Elevated nitrites and presence of enterococci (indicative of fecal pollution) were found at high-FP sites. - **Transmission Hypothesis**: Results support horizontal transmission of FP, likely enhanced by turtle interactions and environmental factors. - **Conservation Concern**: Martinique is a critical developmental habitat for green turtles in the Atlantic, and FP may impact long-term survival and recruitment. * * * **Relevant ACZM Job Task Analysis Sections** - **II.B.4.a.1** – Preventive Medicine → Environmental monitoring and infectious disease surveillance - **II.C.6.a.3** – Clinical Medicine → Infectious disease diagnosis and management - **II.A.2** – Species-Specific Management → Reptiles (marine turtles) → Clinical assessment and disease ecology * * * **Section-by-Section Summary** **Introduction** - FP is a panzootic disease in green turtles linked to Chelonid herpesvirus 5 (ChHV5). - Horizontal transmission is suspected, particularly in dense foraging grounds. - This study provides baseline FP data for Martinique’s critical green turtle habitats. **Materials and Methods** - Study along 60 km of Martinique’s Caribbean coast (2010–2019); 405 individual turtles monitored. - Data collected via capture-mark-recapture (CMR), visual tumor scoring, environmental sampling (SST, NPP, chemical and microbial water parameters), and turtle density mapping. **Results** **Fibropapillomatosis Prevalence and Body Distribution** - 12.8% overall FP prevalence; site-specific differences: - Anse du Bourg/Chaudière: increase to 50% by 2019. - Grande Anse: consistently low prevalence. - Tumor location: most frequently the eyes, fore flippers, and neck. **Fibropapillomatosis and Environmental Cofactors** - Best-fit model: FP prevalence driven by site and year. - Slight positive association with NPP; SST not significant. - Elevated nitrites and E. coli levels at Anse du Bourg/Chaudière. - Enterococci detected only at this high-FP site. **Turtle Density Distribution** - Highest densities observed at Anse du Bourg and Petite Anse. - Turtles aggregated in small areas; FP clustered in high-density zones. **Discussion** - FP increase appears localized, not uniform across Martinique. - High turtle densities and poor water quality likely contribute to FP dynamics. - Tumor location patterns and behavioral observations (e.g., head rubbing) support horizontal transmission. - Environmental degradation (e.g., wastewater discharge) may exacerbate disease risk. **Conclusion and Perspectives** - FP prevalence rose significantly in Martinique, with site-specific dynamics. - Continued CMR and health monitoring with qPCR and serology are needed. - Understanding disease impact on survival and broader Atlantic populations is essential for conservation planning. * * * **Figures and Tables to Know** - **Figure 2**: FP prevalence trend by year and site (2010–2019). - **Figure 3**: Tumor distribution by body part. - **Figure 4**: Water quality by site (ammonium, nitrites, nitrates, chlorophyll a, E. coli, enterococci). - **Figure 5**: Green turtle density maps (2018–2019). - **Table 1**: Model selection results for FP predictors. - **Table 2**: Statistical significance of water quality parameters. - **Table 3**: Post hoc water quality comparisons by site.

Answer 109

**1\. What disease affecting green turtles was the focus of this study?** *Fibropapillomatosis (FP).* **2\. What virus is associated with fibropapillomatosis in sea turtles?** *Chelonid alphaherpesvirus 5 (ChHV5).* **3\. Which bay in Martinique showed the highest FP prevalence increase?** *Anse du Bourg/Chaudière.* **4\. What body parts were most frequently affected by FP tumors?** *Eyes, fore flippers, and neck.* **5\. What environmental factor showed a slight positive association with FP prevalence?** *Net primary productivity (NPP).* **6\. Which water quality indicators were elevated in high-FP prevalence sites?** *Nitrites and E. coli (plus enterococci in Anse du Bourg/Chaudière).* **7\. What spatial pattern was associated with increased FP prevalence?** *High turtle density in small, localized areas.* **8\. What mode of disease transmission is supported by the findings?** *Horizontal transmission via close physical contact and contaminated water.* **9\. What ecological feature makes Martinique important for green turtles?** *It is a critical developmental foraging area for immature green turtles.* **10\. Why might FP prevalence be underestimated in this study?** *Diagnosis relied on external tumors; asymptomatic turtles may carry latent ChHV5.* ## Footnote *EcoHealth* 19: 190–202, 2022 **Fibropapillomatosis Prevalence and Distribution in Immature Green Turtles (Chelonia mydas) in Martinique Island (Lesser Antilles)** Thibaut Roost, Jo-Ann Schies, Marc Girondot, Jean-Patrice Robin, Pierre Lelong, et al. * * * **Key Points for ACZM Board Review** - **Emergence in Lesser Antilles**: First study documenting fibropapillomatosis (FP) prevalence in immature green turtles in Martinique. - **Prevalence Trend**: FP prevalence increased from 0% in 2011 to 16.8% in 2019, driven mainly by rising cases at Anse du Bourg/Chaudière. - **Tumor Distribution**: Tumors primarily affected the eyes (41.4%), fore flippers (21.9%), and neck (9.4%), suggesting contact-mediated transmission. - **Spatial Clustering**: High-density turtle aggregations occurred in small, defined areas of bays, potentially facilitating FP transmission. - **Environmental Correlates**: No effect of sea surface temperature (SST) was found; net primary productivity (NPP) had a slight positive association with FP prevalence. - **Water Quality Indicators**: Elevated nitrites and presence of enterococci (indicative of fecal pollution) were found at high-FP sites. - **Transmission Hypothesis**: Results support horizontal transmission of FP, likely enhanced by turtle interactions and environmental factors. - **Conservation Concern**: Martinique is a critical developmental habitat for green turtles in the Atlantic, and FP may impact long-term survival and recruitment. * * * **Relevant ACZM Job Task Analysis Sections** - **II.B.4.a.1** – Preventive Medicine → Environmental monitoring and infectious disease surveillance - **II.C.6.a.3** – Clinical Medicine → Infectious disease diagnosis and management - **II.A.2** – Species-Specific Management → Reptiles (marine turtles) → Clinical assessment and disease ecology * * * **Section-by-Section Summary** **Introduction** - FP is a panzootic disease in green turtles linked to Chelonid herpesvirus 5 (ChHV5). - Horizontal transmission is suspected, particularly in dense foraging grounds. - This study provides baseline FP data for Martinique’s critical green turtle habitats. **Materials and Methods** - Study along 60 km of Martinique’s Caribbean coast (2010–2019); 405 individual turtles monitored. - Data collected via capture-mark-recapture (CMR), visual tumor scoring, environmental sampling (SST, NPP, chemical and microbial water parameters), and turtle density mapping. **Results** **Fibropapillomatosis Prevalence and Body Distribution** - 12.8% overall FP prevalence; site-specific differences: - Anse du Bourg/Chaudière: increase to 50% by 2019. - Grande Anse: consistently low prevalence. - Tumor location: most frequently the eyes, fore flippers, and neck. **Fibropapillomatosis and Environmental Cofactors** - Best-fit model: FP prevalence driven by site and year. - Slight positive association with NPP; SST not significant. - Elevated nitrites and E. coli levels at Anse du Bourg/Chaudière. - Enterococci detected only at this high-FP site. **Turtle Density Distribution** - Highest densities observed at Anse du Bourg and Petite Anse. - Turtles aggregated in small areas; FP clustered in high-density zones. **Discussion** - FP increase appears localized, not uniform across Martinique. - High turtle densities and poor water quality likely contribute to FP dynamics. - Tumor location patterns and behavioral observations (e.g., head rubbing) support horizontal transmission. - Environmental degradation (e.g., wastewater discharge) may exacerbate disease risk. **Conclusion and Perspectives** - FP prevalence rose significantly in Martinique, with site-specific dynamics. - Continued CMR and health monitoring with qPCR and serology are needed. - Understanding disease impact on survival and broader Atlantic populations is essential for conservation planning. * * * **Figures and Tables to Know** - **Figure 2**: FP prevalence trend by year and site (2010–2019). - **Figure 3**: Tumor distribution by body part. - **Figure 4**: Water quality by site (ammonium, nitrites, nitrates, chlorophyll a, E. coli, enterococci). - **Figure 5**: Green turtle density maps (2018–2019). - **Table 1**: Model selection results for FP predictors. - **Table 2**: Statistical significance of water quality parameters. - **Table 3**: Post hoc water quality comparisons by site.

Answer 110

**Flashcard Questions** **1\. What species was the focus of this 14-year nesting and health study in St. Kitts?** *Leatherback sea turtle (Dermochelys coriacea).* **2\. What was the overall hatch success rate observed in this population?** *16.75%.* **3\. How did hatch success compare between relocated and in situ nests?** *Relocated nests = 2.0%; in situ nests = 24.5%.* **4\. What was the median remigration interval for tagged females?** *2 years (range 1–5 years).* **5\. What lesion types were most commonly observed in nesting females?** *Notches (41.2%) and linear lesions (34.1%).* **6\. What hematologic trend was seen between first and last samples of the nesting season?** *Increased WBCs and monocytes; decreased TP, ALB, and Ca.* **7\. What is a potential cause of decreasing emergence events over time?** *Extended remigration intervals due to poor nutrition or environmental stress.* **8\. How did the study influence conservation policy in St. Kitts?** *Data supported the removal of leatherbacks from the legal harvest list.* **9\. What beach in St. Kitts had the highest nesting activity?** *Keys Beach.* **10\. Why is long-term monitoring important in leatherback conservation?** *To detect shifting baselines and inform adaptive management strategies.* ## Footnote *J. Herpetol. Med. Surg.* 33(1): 18–34, 2023 **Leatherback Sea Turtle (Dermochelys coriacea) Nesting and Health Parameters in St. Kitts, 2003–2016** Kimberly M. Stewart, Anne Conan, Terry M. Norton, Brayton A. Hill, Maria Smeriglio, Kathleen Clements, Darryn L. Knobel * * * **Key Points for ACZM Board Review** - **Nesting Trend**: Documented decline in female leatherback emergence from 369 (2003) to 30 (2016); nesting began as early as February, peaking April–May. - **Reproductive Metrics**: Mean clutch size = 113 eggs; mean yolked = 83.5; yolkless = 29.3; mean clutch frequency = 2.2 per season; incubation = ~60 days. - **Low Hatch Success**: Overall hatch success = 16.75%; lower in relocated nests (2%) vs. in situ nests (24.5%), reinforcing need for relocation only when necessary. - **Health Profiles Established**: Baseline hematologic and plasma biochemical values established over 191 samples; consistent with other leatherback populations. - **Physical Exam Findings**: High prevalence of external lesions, especially notches and linear wounds on flippers and carapace; most lesions were remodeling stage. - **Remigration Trends**: Median remigration interval = 2 years; range 1–5 years; increasing interval over time suggests nutritional/environmental stress. - **Population Concerns**: Declining emergence and low hatch success imply conservation urgency; St. Kitts leatherbacks exhibit high site fidelity, contributing to local vulnerability. - **Management Impact**: Study informed regulatory changes (e.g., pending removal of leatherbacks from legal harvest list in St. Kitts). * * * **Relevant ACZM Job Task Analysis Sections** - **II.B.4.a.1** – Preventive Medicine → Population monitoring, reproductive metrics, environmental risk assessment - **II.C.5.a.2** – Clinical Medicine → Physical examination and clinical pathology of reptiles - **II.C.6.a.3** – Infectious disease diagnosis and wound characterization - **II.A.2** – Species-Specific Management → Marine turtles → Nesting biology and field diagnostics * * * **Section-by-Section Summary** **Introduction** - Leatherbacks are globally distributed but face major anthropogenic threats. - St. Kitts had historic harvesting; this study initiated to monitor reproductive and health parameters and assess recovery. **Methods** - 2003–2016: Night and morning beach patrols at major nesting beaches. - Data collection included emergence events, tagging, physical exams, blood sampling, nest metrics. - Health assessment: hematology, biochemistry, lesion characterization. **Results** - **Nesting Trends**: - Total emergence events = 2,419. - Peak nesting: April–May; shift toward earlier season over time. - **Tagging and Remigration**: - 259 individual females identified. - High site fidelity; few inter-beach or inter-island remigrants. - Median remigration = 2 yrs; increasing to 5 yrs by 2016. - **Nest Metrics**: - Clutch size = 113; yolked = 83.5; yolkless = 29.3. - Hatch success: in situ = 24.5%; relocated = 2.0%. - **Physical Examination**: - Lesions in 2,447 cases; common types = notches (41.2%), linear lesions (34.1%); front flippers most affected. - Most lesions in remodeling phase; possible causes include predation, mating, boat strikes, or debris. - **Clinical Pathology**: - Total of 191 samples from 118 turtles. - Significant seasonal changes: increased WBCs and monocytes; decreased TP, ALB, Ca—consistent with fasting and egg production. - **Poaching and Damage**: - Poaching decreased over time; most incidents early in the study. - Poaching, erosion, and sandmining more prevalent at Keys Beach. **Discussion** - Decline in nesting likely multifactorial: shifting remigration, poor foraging resources, delayed maturity. - Hatch success far below global average (~50%); low success in relocated nests emphasizes minimal intervention. - Lesions likely due to a combination of trauma, predation, and anthropogenic impact. - Health parameter trends support understanding of leatherback physiology during nesting fast. - Conservation implications: study data informed policy changes, such as pending protection from legal harvest. * * * **Key Figures and Tables** - **Figure 3**: Nesting seasonality trend (monthly emergence). - **Figure 4**: Remigration interval trend. - **Figure 5–7**: Lesion location, type, and healing stage distributions. - **Tables 1–5**: Annual turtle identification, nest threats, hematology and biochemistry reference intervals, paired seasonal comparison of blood values.

Answer 111

**1\. What was the main objective of this study?** *To establish fall-specific hematologic reference intervals for adult red-eared sliders in Germany.* **2\. Which hematologic parameter differed significantly between male and female turtles?** *Absolute monocyte count (higher in females).* **3\. What was the median packed cell volume (PCV) in the study population?** *22.5%.* **4\. What anticoagulant was used for preserved blood samples?** *Lithium heparin.* **5\. Which blood collection sites were used in this study?** *Dorsal coccygeal vein and subcarapacial sinus.* **6\. Why were samples with PCV <10% excluded?** *To avoid influence from lymphatic dilution or poor sample quality.* **7\. How did leukocyte and thrombocyte counts compare to yellow-bellied slider data?** *Generally lower in red-eared sliders in this study.* **8\. What season were the samples collected?** *Fall (September).* **9\. Why were blood smears made from non-anticoagulated blood?** *To avoid thrombocyte clumping and improve smear quality.* **10\. What is a potential source of variation in turtle hematology besides sex and season?** *Geography, subspecies, infection, and venipuncture site.* ## Footnote *J. Herpetol. Med. Surg.* 33(1): 35–39, 2023 **Hematological Values of Red-eared Sliders (Trachemys scripta elegans) in Fall** Christoph Leineweber, Anke C. Stöhr, Sabine Öfner, Karina Mathes, Rachel E. Marschang * * * **Key Points for ACZM Board Review** - **Species-Specific Data**: This study establishes fall-specific hematologic reference intervals for adult red-eared sliders sampled from a wild population in Southern Germany. - **Sex Independence**: Most parameters showed no statistically significant differences between sexes, except for absolute monocyte counts (higher in females). - **PCV and Hemoglobin**: PCV (median 22.5%) and hemoglobin (median 58.0 g/L) were lower than prior published values in captive red-eared sliders and yellow-bellied sliders. - **Differential Counts**: Red-eared sliders had lower heterophil, basophil, and monocyte percentages but higher lymphocyte and eosinophil percentages than yellow-bellied sliders. - **Venipuncture Consideration**: Blood collected from dorsal coccygeal vein or subcarapacial sinus; visible lymph contamination or PCV <10% were exclusion criteria. - **Anticoagulant & Smear Handling**: Smears were prepared from non-anticoagulated blood to avoid thrombocyte clumping; lithium heparin used for preserved samples. - **Environmental and Seasonal Influence**: Hematologic values may vary with geography, subspecies, season, and environmental factors—underscoring the need for localized reference intervals. - **Health Status**: All turtles were clinically healthy with no parasites on fecal examination, supporting validity of data for reference intervals. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.5.a.2** – Clinical Medicine → Diagnostic procedures → Hematology and blood sample interpretation in reptiles - **II.A.2** – Species-Specific Management → Reptiles (Freshwater turtles) → Clinical pathology and physiology - **II.C.2.a.1** – Physical examination and diagnostic data collection * * * **Section-by-Section Summary** **Introduction** - Red-eared sliders are invasive in Europe but frequently encountered in veterinary practice. - Hematology is important for health evaluation, but reference values specific to species, season, and geography are limited. **Materials and Methods** - 42 clinically healthy adult red-eared sliders (11 males, 31 females) captured in Munich in September. - Blood collected from coccygeal vein or subcarapacial sinus; smears made immediately and stained with Diff-Quick. - PCV measured; hemoglobin and differential counts evaluated. - Statistical tests: t-test, Wilcoxon, and Shapiro-Wilk for normality; nonparametric reference intervals calculated. **Results** - Only absolute monocyte count significantly higher in females (P = 0.042); all other parameters did not differ by sex. - Reference intervals (all sexes combined): - PCV: median 22.5% - Hemoglobin: 58.0 g/L - Leukocytes: 2.4 G/L - Thrombocytes: 12.16 G/L - Differential counts included heterophils, eosinophils, basophils, lymphocytes, and monocytes. - No fecal parasites or signs of illness were found. **Discussion** - PCV and hemoglobin lower than other published red-eared slider data—may reflect subspecies, geography, or season (fall). - Differential counts differed from yellow-bellied sliders, possibly due to climatic or environmental differences. - Emphasizes the importance of localized, season-specific reference intervals. - Venipuncture site, anticoagulant type, and sample handling all influence results. - Calls for further studies to evaluate effects of age, infection, and seasonal variation in this species.

Answer 112

**1\. What is the estimated pivotal temperature range for most sea turtle species and what is a pivotal temperatrue?** *28.6–31°C (83.5–87.8°F). Incubation temperatures below the pivotal temperature will produce predominately male offspring, while those above the pivotal temperature will result in a majority of female hatchlings.* **2\. Which species of sea turtle is believed to be the most ancient lineage?** *The leatherback turtle (Dermochelys coriacea).* **3\. What physiological changes occur in nesting turtles due to reproductive fasting?** *Decreased PCV, electrolytes, proteins, and lipid stores.* **4\. What are the three main neonatal life history strategies in sea turtles?** *Type I: Neritic only; Type II: Oceanic to neritic; Type III: Fully oceanic.* **5\. Which species are known to participate in arribadas?** *Olive ridley and Kemp’s ridley turtles.* **6\. How do sea turtles likely navigate back to their natal beaches?** *Through magnetic imprinting and detection of inclination angle and magnetic intensity.* **7\. What hormone stimulates vitellogenesis in female sea turtles?** *Estrogen.* **8\. How does embryonic arrest benefit egg transport in sea turtles?** *It allows safe transfer by pausing development until post-oviposition oxygen exposure resumes it.* **9\. What trend is observed in hatchling sex ratios due to climate change?** *Feminization—more females due to higher nest temperatures.* **10\. Why is operational sex ratio often balanced despite skewed hatchling ratios?** *Males breed more frequently, offsetting lower primary sex ratio male numbers.*

Answer 113

**1\. What bacterial species caused the corneal perforation in this turtle case?** *Providencia rettgeri.* **2\. What diagnostic imaging modality confirmed corneal perforation in this turtle?** *Transpalpebral ocular ultrasonography.* **3\. What surgical procedure was performed due to the poor prognosis of the affected eye?** *Transconjunctival enucleation.* **4\. What systemic antibiotic was effective against P. rettgeri in this case?** *Trimethoprim-sulfadoxine.* **5\. What environmental factor contributed to the corneal ulcer in the contralateral eye?** *Ocular surface drying under a heat lamp during dry recovery.* **6\. What histological findings supported a diagnosis of corneal perforation?** *Stromal edema, heterophilic infiltration, fibrin exudation, hemorrhagic debris.* **7\. What was the likely source of immunosuppression or vulnerability in this turtle?** *Poor diet, lack of UVB exposure, and inadequate thermal/humidity regulation.* **8\. What is the typical habitat of Providencia rettgeri in the environment?** *Soil, sewage, water, and intestinal tracts of humans and animals.* **9\. What is an important surgical consideration for enucleation in chelonians?** *Eyelid closure should leave a medial gap for drainage in infected cases.* **10\. How can corneal drying be prevented in turtles during out-of-water recovery?** *Apply eye lubrication every 8 hours and avoid heat lamps that dry the air.* ## Footnote *J. Herpetol. Med. Surg.* 33(3): 134–138, 2023 **Corneal Perforation Associated with Providencia rettgeri in an African Helmeted Turtle (Pelomedusa subrufa)** Sabrina Vieu, Coline Jondeau, Matthieu Bott, Edouard Reyes-Gomez, Thomas M. Donnelly, Sabine Chahory * * * **Key Points for ACZM Board Review** - **Novel Case**: First report of corneal perforation due to Providencia rettgeri in a reptile. - **Presentation**: A 4-year-old captive African helmeted turtle presented with blepharedema, a melting corneal ulcer, and limb edema. - **Diagnostics**: Ocular ultrasound confirmed corneal perforation with anterior uveitis. Histopathology confirmed stromal destruction and heterophilic inflammation. - **Surgical Treatment**: Transconjunctival enucleation was performed under general anesthesia due to poor prognosis and sepsis risk. - **Culture Results**: Bacterial culture identified P. rettgeri; fungal culture was negative. P. rettgeri was sensitive to trimethoprim-sulfadoxine. - **Contralateral Eye**: Developed a superficial ulcer after surgery, likely due to environmental drying—resolved with topical and systemic treatment. - **Pathogen Background**: P. rettgeri is a rare but emerging opportunistic Gram-negative bacterium in animals and humans; causes ocular disease in immunosuppressed hosts. - **Contributing Factors**: Poor diet (chicken, fish), lack of UVB, and improper environmental control likely predisposed the turtle to infection and poor healing. - **Management Lessons**: Emphasizes the importance of optimal husbandry, pain management, and environmental humidity control in ocular disease cases. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.5.a.2** – Clinical Medicine → Diagnostic procedures → Ophthalmology in reptiles - **II.C.6.a.3** – Clinical Medicine → Infectious disease diagnosis and management (Gram-negative infections in reptiles) - **II.D.1.a.1** – Facility Biosecurity and Husbandry → Environmental, nutritional, and thermal management * * * **Section-by-Section Summary** **Case Presentation** - Male Pelomedusa subrufa with 3-day history of left-eye swelling and systemic edema. - History of poor husbandry: diet limited to fish/chicken; no temperature/humidity control. **Diagnostics** - Slit-lamp exam: corneal ulcer with stromal melting and edema. - Ultrasound: collapsed anterior chamber, uveitis, and lens changes. - Cultures: bacterial (P. rettgeri); fungal negative. - Histopathology: corneal perforation, heterophilic infiltrates, hemorrhagic debris. **Treatment and Surgery** - Initial topical antibiotics and supportive care failed. - Enucleation performed under general anesthesia using microscope-assisted transconjunctival technique. - Postoperative care: systemic antibiotics, anti-inflammatories, and warm-water soaks. - Contralateral eye developed superficial ulcer likely due to drying under heat lamp—resolved with treatment. **Outcome** - Full healing of surgical site in 3 weeks; complete healing of contralateral eye by 6 weeks. - Limb edema resolved after husbandry improvements. **Discussion** - P. rettgeri is rare but increasingly reported in exotic species, including reptiles. - Contributing factors included immunosuppression and inadequate husbandry. - Preventing ocular complications requires adequate hydration, thermal support, and eye lubrication. - Enucleation in turtles follows mammalian protocols; eyelid closure must allow for drainage if infection is suspected. * * * **Key Figures** - **Figure 1**: Corneal abscess and blepharedema on presentation. - **Figure 2**: Ultrasound image showing hyperechoic material in anterior chamber and lens cortex changes. - **Figure 3**: Histology showing fibrin, hemorrhage, and heterophilic infiltration at corneal perforation site. - **Figure 4**: Healing superficial ulcer in contralateral eye one week after enucleation.

Answer 114

**1\. What hormone was the focus of this ELISA validation study in Texas tortoises?** *Androstenedione.* **2\. What was the established reference interval for serum androstenedione in adult male Gopherus berlandieri?** *0.57–18.58 ng/ml.* **3\. Which physical measurements positively correlated with androstenedione concentration?** *Weight, carapace length, and shell height.* **4\. Which male morphological feature did not correlate with androstenedione levels?** *Gular horn length.* **5\. Why is androstenedione a promising hormone for reproductive assessment in tortoises?** *It is a precursor to testosterone and may be more stable or accessible to measure; useful in assessing reproductive status or disease.* **6\. What were the intra- and inter-assay coefficients of variation for the ELISA used?** *Intra-assay CV = 5.69%; inter-assay CV = 10.23%.* **7\. What method was used to validate the ELISA performance in reptile serum?** *Spiking charcoal-stripped serum with known androstenedione standards.* **8\. What is the significance of a wide reference interval in this species?** *Reflects natural variation in hormone levels and the need for individualized interpretation or longitudinal data.* **9\. What is one potential clinical application of androstenedione monitoring in tortoises?** *Assessment of reproductive maturity or screening for adrenal/reproductive disorders.* **10\. What future research did the authors recommend to improve the utility of this hormone as a diagnostic tool?** *Include females, juveniles, serial sampling for seasonal trends, and comparison to disease states.* ## Footnote *J. Herpetol. Med. Surg.* 33(3): 155–161, 2023 **Validation of an Enzyme-Linked Immunosorbent Assay and Establishment of a Reference Interval for Androstenedione in Texas Tortoises (Gopherus berlandieri)** Garrett C. Crooks, Jacquelyn K. Grace, Jianhua Guo, Alice Blue-McLendon, J. Jill Heatley * * * **Key Points for ACZM Board Review** - **New Tool for Reproductive Assessment**: This study validated an ELISA for measuring serum androstenedione and established the first reference interval for this hormone in Texas tortoises. - **Reference Interval Established**: Serum androstenedione ranged from 0.57 to 18.58 ng/ml in adult male tortoises sampled during the breeding season. - **Correlation with Morphometrics**: Androstenedione levels positively correlated with tortoise weight, carapace length, and height—but not with gular horn length. - **Assay Validation**: The ELISA was validated with charcoal-stripped serum and spiking; R² values >0.99 and intra-assay CV = 5.69%, inter-assay CV = 10.23%. - **Novelty**: First reptilian study measuring androstenedione, a precursor to testosterone, which may be useful in noninvasively assessing reproductive status or disorders. - **Potential Applications**: Useful in sexing, breeding program selection, health assessments, and possible diagnosis of reproductive or adrenal pathology. - **Limitations**: Small sample size (n=25), only adult males sampled; parallelism testing not performed due to limited sample volume. - **Further Research Needed**: To evaluate females, juveniles, reproductive seasonality, and disease correlations across populations. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.5.a.2** – Clinical Medicine → Diagnostic Procedures → Clinical pathology (hormone testing) - **II.A.2** – Species-Specific Management → Reptiles (Chelonians) → Reproductive endocrinology - **II.C.6.a.3** – Clinical Medicine → Diagnosis and management of reproductive diseases - **II.B.4.a.1** – Preventive Medicine → Population health assessment and conservation diagnostics * * * **Section-by-Section Summary** **Introduction** - Androstenedione is a steroid hormone precursor to testosterone and estrone, with reproductive roles in other taxa but poorly studied in reptiles. - A4 may serve as a biomarker for maturity, aggression, reproductive capability, or disease. - Texas tortoises are threatened in Texas; improving reproductive monitoring is important for captive management and conservation. **Materials and Methods** - 25 adult male Texas tortoises sampled from a managed colony. - Weight, carapace dimensions, and gular horn length recorded. - Jugular blood drawn, serum separated, and stored at −80°C. - Commercial ELISA validated using hormone-spiked, charcoal-stripped serum; optical density measured at 450 nm. - Reference interval calculated per ASVCP guidelines using bootstrap quantile methods. **Results** - Mean androstenedione = 5.40 ± 5.16 ng/ml; reference interval = 0.57–18.58 ng/ml (90% CI). - Positive correlations: - Carapace length (P=0.0128) - Shell height (P=0.0138) - Weight (P=0.0155) - No correlation with: - Carapace width - Gular horn length (P=0.6134) - Assay had excellent linearity and precision (R²=0.99); recovery = 90.61%. **Discussion** - Validated ELISA can be used to assess reproductive condition in tortoises; correlations support body size as a predictor of androgen levels. - Gular horn length, despite its behavioral role in combat, was not associated with hormone levels. - Values were comparable to other reptiles but higher than typical human male values, with wide natural variation. - Suggested uses: screening for reproductive maturity, health assessments, and monitoring response to husbandry changes or clinical conditions. - Recommends expanding work to include females, juveniles, seasonal sampling, and field-collected tortoises.

Answer 115

**1\. What is the scientific name of the critically endangered species studied in this article?** *Batagur trivittata (Burmese roofed turtle).* **2\. Which blood parameters were significantly higher in female turtles?** *Lymphocyte count, glucose, and calcium.* **3\. What was the median heterophil:lymphocyte ratio in this study?** *0.59.* **4\. What white blood cell type was most predominant in Burmese roofed turtles?** *Lymphocytes (42%).* **5\. Why might CK and AST levels be elevated in some individuals despite short handling times?** *Capture stress, muscle exertion due to pond draining, or individual variability.* **6\. What methodology was used for hematologic cell counting in this study?** *Hemocytometry with Natt and Herrick solution; manual differential from stained smears.* **7\. What site was used for blood collection in this study?** *Lateral saphenous vein.* **8\. What electrolyte was significantly higher in male turtles?** *Potassium.* **9\. How did B. trivittata's PCV compare to that of B. baska and B. borneoensis?** *Higher in B. trivittata.* **10\. Why are species- and institution-specific reference intervals critical in turtle conservation medicine?** *Because analyte values vary significantly with species, environment, season, and husbandry practices.* ## Footnote *J. Herpetol. Med. Surg.* 33(3): 162–168, 2023 **Hematologic and Biochemical Reference Intervals of a Single Population of Captive Burmese Roofed Turtles (Batagur trivittata)** Guillaume Douay, Wenjing Clara Yeo, Pei Yee Oh, Delia Chua, Shangari Sekar * * * **Key Points for ACZM Board Review** - **First Reference Ranges**: This study established the first hematologic and biochemical reference intervals (RIs) for captive Burmese roofed turtles (Batagur trivittata), a critically endangered species. - **Sex Differences**: Females had higher lymphocyte counts, glucose, and calcium; males had higher potassium. - **Heterophil:Lymphocyte Ratio**: Median H:L ratio was 0.59 (range 0.21–3.69); useful for assessing chronic stress. - **Low Monocyte Counts**: Monocytes were nearly absent in both sexes; combined monocyte-azurophil counts were within expected turtle ranges. - **Species-Specific Traits**: Lymphocytes were the most abundant WBC (42%), followed by heterophils, similar to other chelonians. - **Stress Indicators**: Handling, pond draining, and sampling may have influenced AST, CK, and glucose despite rapid collection. - **Environmental Consistency**: All animals were in a uniform, well-controlled captive environment; limiting abiotic variation. - **Comparison to Related Species**: PCV and RBC counts were higher in B. trivittata than in B. baska and B. borneoensis; useful for interspecies comparisons in Batagur. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.5.a.2** – Clinical Medicine → Diagnostic Procedures → Hematology and biochemistry in reptiles - **II.A.2** – Species-Specific Management → Reptiles → Physiology and diagnostics in endangered chelonians - **II.B.4.a.1** – Preventive Medicine → Conservation diagnostics and health baselining for ex situ populations * * * **Section-by-Section Summary** **Introduction** - Burmese roofed turtle (Batagur trivittata) is among the top 25 most endangered turtles. - No published hematologic or biochemical baselines exist; establishing RIs is essential for conservation management. **Materials and Methods** - 23 clinically healthy captive turtles (11 females, 12 males) aged 8–9 years sampled over 4 months. - Samples taken via lateral saphenous vein during monthly health assessments; smears stained with Wright’s stain. - Hematology via hemocytometer; biochemistry via IDEXX Catalyst One. - RIs calculated using robust or parametric statistics; sex comparisons done with t-tests or Mann-Whitney U-tests. **Results** - **Sex Differences**: - Females: ↑ lymphocytes (P=0.004), glucose (P=0.006), calcium (P=0.001) - Males: ↑ potassium (P=0.001) - H:L ratio median = 0.59; not significantly different by sex. - AST and CK showed wide ranges, possibly due to handling or pond draining stress. - Table 1: Full RIs for 23 analytes (PCV, WBC, RBC, differential counts, glucose, electrolytes, enzymes). - Table 2: Analytes significantly different between sexes. - Table 3: Comparisons with B. baska and B. borneoensis from ZIMS database. **Discussion** - Lymphocytes were the predominant WBC (42%)—consistent with other chelonians. - Low monocyte counts may reflect species-specific norm or stress physiology. - Elevated CK and AST may reflect capture stress or activity. - Biochemical differences between sexes not fully explained by reproductive physiology given off-season sampling. - Comparison with ZIMS data shows variation between Batagur species and highlights value of species- and institution-specific RIs.

Answer 116

**1\. What fungal species caused systemic disease in this African side-neck turtle?** *Nannizziopsis arthrosporioides.* **2\. What type of fungal lesion was found on histopathology in the lungs and liver?** *Granulomatous inflammation with fungal hyphae.* **3\. What diagnostic method was used to confirm the fungal species?** *Multi-gene PCR sequencing (ITS, D1/D2, actin, β-tubulin).* **4\. What was unusual about this case compared to typical Nannizziopsis infections?** *No fungal elements were found in the skin despite systemic infection.* **5\. What staining technique confirmed the presence of fungal organisms?** *Grocott’s methenamine silver (GMS) stain.* **6\. What organism did the phylogenetic analysis group the fungal isolate with?** *Nannizziopsis arthrosporioides, with 99% bootstrap support.* **7\. What clinical signs developed prior to euthanasia?** *Anorexia, lethargy, skin sloughing, bleeding beak, open-mouth breathing.* **8\. What husbandry factor may have contributed to infection?** *Enclosure was directly below that of bearded dragons (possible cross-species contamination).* **9\. Which co-isolated bacteria were found, but of uncertain significance?** *Elizabethkingia meningoseptica and Chryseobacterium spp.* **10\. Why is this case significant for chelonian medicine?** *It demonstrates that systemic fungal infections can occur in turtles even without cutaneous lesions.* ## Footnote *J. Herpetol. Med. Surg.* 33(4): 223–228, 2023 **Systemic Nannizziopsis arthrosporioides in an African Side-neck Turtle (Pelomedusa subrufa)** Kennymac Durante, Julie D. Sheldon, Laura Adamovicz, Patrick J. Roady, Krista A. Keller * * * **Key Points for ACZM Board Review** - **First Report**: This is the first documented case of systemic Nannizziopsis arthrosporioides infection in an aquatic turtle (Pelomedusa subrufa). - **Clinical Signs**: Initial presentation included nasal discharge and a skin ulcer; later progression included skin sloughing, anorexia, weight loss, and multisystemic decline. - **Histopathologic Findings**: Fungal granulomas were present in the lungs, liver, small intestine, and coelom; skin was not affected by fungal invasion despite visible ulceration. - **Molecular Diagnostics**: Culture and sequencing (ITS, D1/D2, actin, β-tubulin) confirmed >98% similarity to N. arthrosporioides; phylogenetics confirmed monophyletic grouping. - **Unusual Host & Pathogenesis**: This species is usually associated with squamates; the case demonstrates a broader host range and capacity for systemic infection without cutaneous involvement. - **Environmental and Husbandry Factors**: Co-housing with bearded dragons, suboptimal water quality, and recent antibiotic administration may have contributed to susceptibility and infection. - **Differential Diagnosis**: Mycoplasma, herpesvirus, and ranavirus were ruled out via PCR; cytology failed to detect fungi, highlighting diagnostic limitations. - **Clinical Implications**: Onygenalean fungi can cause severe systemic infections in chelonians and should be considered even in the absence of cutaneous signs. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.6.a.3** – Clinical Medicine → Infectious disease diagnosis and management → Systemic mycoses and opportunistic fungal pathogens - **II.C.5.a.2** – Clinical Medicine → Diagnostic procedures → Histopathology, cytology, and molecular diagnostics in reptiles - **II.D.1.a.1** – Facility Biosecurity and Husbandry → Disease transmission risk from cross-species exposure * * * **Section-by-Section Summary** **Introduction** - Onygenalean fungi like Nannizziopsis spp. are increasingly recognized in reptiles, typically causing cutaneous disease. - This case expands host range and tissue tropism for N. arthrosporioides, showing systemic disease without skin fungal involvement in a semiaquatic turtle. **Case Presentation** - A 2-year-old captive African side-neck turtle presented with nasal discharge and a foot ulcer. - Bloodwork showed monocytosis/azurophilia and hyperuricemia; cytology of the ulcer was non-septic. - Treated with ceftazidime and supportive care; husbandry changes advised. **Progression and Outcome** - One month later: anorexia, lethargy, skin sloughing, weight loss, beak bleeding, and open-mouth breathing observed. - Humane euthanasia elected due to poor prognosis. - Necropsy: fungal granulomas in multiple organs, but no fungal elements found in skin. **Diagnostics and Mycology** - Histopathology showed granulomas with fungal hyphae; Grocott’s methenamine silver stain confirmed fungi in lungs and liver. - Aerobic bacterial culture yielded Elizabethkingia and Chryseobacterium spp. (unknown clinical relevance). - Fungal culture grew white colonies with pyriform conidia; four-gene sequencing confirmed N. arthrosporioides. - Phylogenetic trees (concatenated and individual genes) showed 99% bootstrap support for N. arthrosporioides grouping. **Discussion** - Rare presentation of systemic mycosis without skin involvement; cutaneous ulcer may have been the entry point. - Environmental factors (co-housing with bearded dragons, temperature/humidity conducive to fungal growth) likely enabled cross-species transmission. - Yeast forms seen in tissue are unusual for N. arthrosporioides; typically filamentous. - Highlights importance of multi-gene sequencing for definitive identification. - Suggests need for consideration of Nannizziopsis in chelonians with systemic illness even if classic skin lesions are absent.

Answer 117

**1\. What two sea turtle species were studied in this clinical pathology reference interval study in St. Kitts?** *Green (Chelonia mydas) and hawksbill (Eretmochelys imbricata) sea turtles.* **2\. Which species had higher packed cell volume (PCV)?** *Hawksbill sea turtles.* **3\. Which analytes were significantly higher in green sea turtles compared to hawksbills?** *Total protein, monocytes, heterophils, and phosphorus.* **4\. What was a novel PIT tag placement site tested in this study?** *Subcutaneously in the prescapular region.* **5\. What was the suspected cause of elevated CPK and AST levels in some turtles?** *Stress from capture, handling, and venipuncture.* **6\. What hematologic finding in green turtles raised concern for possible subclinical fibropapillomatosis (FP)?** *Elevated heterophils and monocytes.* **7\. What analyte differed between species and was also positively correlated with carapace length?** *Packed cell volume (PCV).* **8\. How were leukocyte counts performed in this study?** *Manual estimation from blood smears under microscopy.* **9\. What analytical equipment was used for plasma chemistry in this study?** *Abaxis VetScan analyzer with avian/reptile rotors.* **10\. What were the criteria for inclusion in the reference interval analysis?** *Clinically healthy appearance, good BCI, absence of FP or acute lesions, and sample quality without hemolysis or lipemia.* ## Footnote *J. Herpetol. Med. Surg.* 33(4): 236–250, 2023 **Clinical Pathology Reference Intervals for Juvenile Green (Chelonia mydas) and Hawksbill (Eretmochelys imbricata) Sea Turtles Captured in St. Kitts, 2004–2017** Kimberly M. Stewart, Terry M. Norton, Kathleen Clements, Mark A. Mitchell, Darryn L. Knobel * * * **Key Points for ACZM Board Review** - **First RIs for St. Kitts Juveniles**: This study established the first hematology and biochemistry reference intervals (RIs) for juvenile green and hawksbill turtles in St. Kitts, crucial for health monitoring. - **Species Differences**: Green turtles had significantly higher WBC, heterophils, monocytes, phosphorus, and total protein; hawksbills had higher PCV, glucose, eosinophils, and uric acid. - **Body Size Correlations**: Several analytes (e.g., PCV, TP, Ca) were positively correlated with carapace length, independent of species. - **Hawksbill Dive Activity Hypothesis**: Elevated PCV and glucose in hawksbills may reflect higher diving activity, supported by similar findings in diving reptiles. - **Subclinical FP Concerns**: Elevated heterophils and monocytes in green turtles suggest potential subclinical fibropapillomatosis (FP); follow-up qPCR or serology warranted. - **Methodology Rigor**: Blood samples processed within 6 hours and analyzed using standardized protocols; appropriate statistical methods used for RI determination (robust, parametric, nonparametric). - **New PIT Tag Site**: Subcutaneous prescapular PIT tagging showed no migration across 7 years, suggesting a safe and effective site for juvenile sea turtles. - **Stress Effects on CPK/AST**: Elevated CPK and AST likely due to capture and restraint stress; values remained within reference ranges and consistent with other studies. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.5.a.2** – Clinical Medicine → Diagnostic procedures → Hematology and clinical pathology in reptiles - **II.A.2** – Species-Specific Management → Reptiles (Cheloniidae) → Population health monitoring - **II.B.4.a.1** – Preventive Medicine → Population health assessments and baseline data for conservation * * * **Section-by-Section Summary** **Introduction** - Juvenile hawksbill and green turtles are present year-round around St. Kitts. - No prior health baselines existed for these cohorts; the study addresses this gap using 13 years of data. **Methods** - 211 turtles captured via snorkel; 124 samples included for analysis after exclusions. - Morphometrics, lesion scoring, PIT/flipper tagging, blood collection, and processing followed established protocols. - Biochemistry analyzed with VetScan analyzer; WBC counts via manual differential and estimation from smears. **Results** - 103 juvenile turtles included in final RI dataset. - Significant species differences in many analytes; CCL-NT used to assess body size correlation. - Hawksbills had higher PCV and glucose; greens had higher TP and monocytes. - Reference intervals presented separately for each species (Tables 2 & 3). **Discussion** - PCV positively correlated with carapace length; hawksbills had higher values, possibly due to greater diving. - Green turtles had higher inflammatory cell counts—possibly reflecting subclinical FP. - Biochemistry differences likely reflect dietary and physiological differences. - First report of successful prescapular PIT tag use without migration. - Data support health baseline establishment and inform conservation policy in a region where juvenile turtles remain at risk.

Answer 118

**1\. What were the most common lesions in hawksbill hatchlings that died in rehabilitation?** *Dermatitis (34%), skeletal malformations (23%), and pneumonia (23%).* **2\. What percentage of sexed turtles in this study were female?** *93%.* **3\. What kinds of organisms were associated with dermatitis in these turtles?** *Opportunistic bacteria (Gram-positive and Gram-negative) and fungi (Penicillium, Aspergillus, Paecilomyces).* **4\. What pattern of pneumonia was most common in these hatchlings?** *Bronchopneumonia, suggestive of inhalation.* **5\. What lesion was suggestive of temperature-related developmental issues or mechanical compression?** *Carapacial compression (a form of skeletal malformation).* **6\. How did caregivers identify dermatitis in most cases involving the eyes?** *Eye lesions like blepharitis were consistently noted clinically.* **7\. What kidney lesion was found in 13% of hatchlings and unrelated to drug administration?** *Acute renal tubular necrosis.* **8\. What preventive strategy was suggested to reduce opportunistic fungal infections?** *Washing hatchlings upon admission and during rehabilitation.* **9\. What systemic lesion was observed in 15% of cases and was often associated with renal or respiratory disease?** *Thrombosis.* **10\. What gastrointestinal lesion occurred in turtles in care for >1 month and was linked to buoyancy issues?** *Enteritis (ulcerative or necrohemorrhagic).* ## Footnote *J. Wildl. Dis.* 59(1): 109–120, 2023 **Pathology of Hatchling Hawksbill Sea Turtle (Eretmochelys imbricata) Mortalities Occurring While Under Rehabilitative Care, 2015–21** Paula Sotomayor Rodríguez, Kimberly M. Stewart, Angela Picknell, Lemuel Pemberton, Alissa Tepedino, Dominique Capaldo, Michelle M. Dennis * * * **Key Points for ACZM Board Review** - **Widespread Lesions**: 95% of 44 hawksbill hatchlings dying in rehabilitation had one or more lesions; most frequent were dermatitis (34%), skeletal malformations (23%), and pneumonia (23%). - **Opportunistic Infections**: Both fungal (23%) and bacterial (36%) infections were prevalent; lesions included dermatitis, pneumonia, rhinitis, esophagitis, and yolk sacculitis. - **Dermatitis Observations**: Skin lesions were often subtle or missed on visual exam; periocular dermatitis was consistently recognized; pathogens included Penicillium, Aspergillus, and Paecilomyces spp. - **Respiratory Disease**: Pneumonia had varied etiologies (bacterial, fungal, aspiration), with bronchopneumonia being most common. - **Skeletal Malformations**: Affected 23% of turtles, including carapacial compression and dysmelias, often co-occurring with other systemic pathology. - **Female Bias**: 93% of sexed turtles were female, suggesting strong temperature-dependent sex determination and potential climate-related sex skewing. - **Rare Findings**: Additional lesions included thrombosis (15%), skeletal muscle necrosis (13%), acute renal tubular necrosis (13%), and bacterial yolk sacculitis (15%). - **Implications for Rehab**: Highlights diagnostic challenges in live hatchlings and the need for improved prophylactic and diagnostic strategies. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.6.a.3** – Clinical Medicine → Infectious disease diagnosis and management (fungal and bacterial infections) - **II.B.4.a.1** – Preventive Medicine → Conservation diagnostics, pathology of early life-stage mortalities - **II.C.5.a.2** – Clinical Medicine → Diagnostic procedures → Postmortem and histopathology - **II.A.2** – Species-Specific Management → Sea turtles → Reproductive ecology and developmental health * * * **Section-by-Section Summary** **Introduction** - Hawksbill turtles are critically endangered; hatchling survival is essential for species recovery. - Study goal: Describe pathology in hatchlings dying during rehabilitation in St. Kitts and Nevis over a 7-year period. **Materials and Methods** - Forty-four hatchlings underwent postmortem exams with histopathology, Gram and fungal staining, and occasional culture. - Animals were admitted for dehydration, weakness, or gross lesions; care included saltwater tanks, feeding, and supportive therapy. **Results** - 96% had at least one lesion; most common: - **Dermatitis** (34%): pustular, hyperkeratotic, ulcerative; fungal and bacterial etiologies common. - **Pneumonia** (23%): bronchopneumonia most frequent; aspiration and hematogenous spread suspected in some cases. - **Skeletal Malformations** (23%): carapace compression, dysmelias, scute anomalies; often had concurrent disease. - **Thrombosis** (15%) and **Muscle Necrosis** (13%): associated with systemic illness. - **Renal Tubular Necrosis** (13%): not linked to treatment toxicity. - **Yolk Sacculitis** (15%): Gram-negative bacterial infections, often concurrent with pneumonia. - Additional findings included enteritis, esophagitis, hydrocoelom, phytobezoars, and single cases of traumatic injury or proptosis. - Sex determination (n=27): 93% female. **Discussion** - Opportunistic infections are common; fungal involvement higher than other sea turtle hatchling studies. - Subtle dermatitis may be overlooked; improved visual diagnostics needed. - Skeletal malformations likely multifactorial (temperature, mechanical compression, genetic); linked to lower fitness. - Female-biased sex ratio likely climate driven; concern for future sex ratio imbalance. - Need for preventative hygiene (e.g., turtle washing), and attention to environmental conditions, nutrition, and pathogen exposure.

Answer 119

**1\. What genus of ciliate was responsible for the infestations described in this study?** *Epistylis spp.* **2\. What species of turtles were affected by the infestations?** *Sonoran mud turtle (Kinosternon sonoriense) and rough-footed mud turtle (Kinosternon hirtipes).* **3\. What environmental conditions were associated with the infestations?** *Shallow, warm, eutrophic livestock ponds with no outflow, especially during drought.* **4\. What clinical signs were observed in the affected turtles?** *Impaired swimming and diving, emaciation, ocular occlusion, and lethargy.* **5\. How was the Epistylis spp. confirmed in this case?** *Microscopy and 18S rRNA sequencing.* **6\. What treatments were effective in resolving the infestations?** *Tap water soaking and daily 10% saltwater baths.* **7\. What behavior or physiological traits may increase susceptibility in Kinosternon spp.?** *Low basking behavior and slow scute shedding.* **8\. What was the suspected impact of the infestation on the turtles’ ability to feed?** *Obstructed vision and lethargy likely impaired foraging, leading to poor body condition.* **9\. Which turtle species sharing the same ponds were unaffected by the infestation?** *Yellow mud turtles (Kinosternon flavescens).* **10\. Why is further investigation into Epistylis infestations in turtles important?** *Because heavy infestations may contribute to morbidity, especially in degraded aquatic habitats.*

Answer 120

**1\. What was the prevalence of Mycoplasmopsis spp. in usable oral cultures from northern diamond-backed terrapins?** *33% (10/18).* **2\. Which two major reptile pathogens were not detected in any turtles in this study?** *Herpesvirus and ranavirus.* **3\. What percentage of turtles were found to have intestinal parasites?** *43%.* **4\. What blood parasite was observed in 7% of the sampled turtles?** *Haemogregarina spp.* **5\. What hematologic ratio was elevated in four turtles, suggesting possible inflammation?** *Heterophil to lymphocyte (H:L) ratio >4.5.* **6\. Was there a significant difference in blood values between gravid and nongravid females?** *No.* **7\. What technique was used to detect Mycoplasmopsis spp.?** *Culture on in-house agar with identification by colony morphology.* **8\. What was the likely source of variability in creatine kinase (CK) levels among turtles?** *Muscle damage during handling or PIT tag implantation.* **9\. What diagnostic samples were collected to test for herpesvirus and ranavirus?** *Oral swabs.* **10\. Why is this health assessment important for terrapin conservation in New Jersey?** *It provides baseline health data to inform reintroduction of confiscated turtles.* ## Footnote *J. Wildl. Dis.* 59(2): 234–244, 2023 **Health Assessment of Wild Northern Diamond-Backed Terrapins (Malaclemys terrapin terrapin) in New Jersey, USA** Nicole Lewis, Brian Zarate, John Wnek, and Benjamin Wurst * * * **Key Points for ACZM Board Review** - **Baseline Data Established**: Study provides hematologic, biochemical, parasitologic, and pathogen screening data for 30 wild female northern diamond-backed terrapins during nesting season. - **Mycoplasmopsis Prevalence**: 33% of interpretable samples were positive for Mycoplasmopsis spp.; no signs of upper respiratory disease observed. - **No Herpesvirus or Ranavirus**: All turtles were negative for herpesvirus and ranavirus via PCR, though wood-handled swabs may have reduced detection sensitivity. - **Parasite Findings**: 43% had intestinal parasites (trematodes, nematodes); 7% had blood parasites (Haemogregarina spp.), consistent with previous studies. - **Gravid vs. Nongravid**: No statistically significant differences in hematology or biochemistry values between gravid and nongravid females. - **Elevated H:L Ratios**: Four terrapins had H:L ratios >4.5, suggesting possible subclinical inflammation or stress; not statistically linked to Mycoplasmopsis. - **No Pathogen Coinfection Observed**: No cases of coinfection with Mycoplasmopsis, herpesvirus, and ranavirus found. - **Sample Limitations**: All turtles sampled were females in a single season; small sample size precluded formal reference interval calculation. * * * **Relevant ACZM Job Task Analysis Sections** - **II.B.4.a.1** – Preventive Medicine → Population health surveillance, pathogen monitoring - **II.C.5.a.2** – Clinical Medicine → Diagnostic procedures → Hematology, clinical chemistry, parasitology - **II.C.6.a.3** – Clinical Medicine → Infectious disease management → Mycoplasmosis, herpesvirus, ranavirus - **II.A.2** – Species-Specific Management → Reptiles (Emydidae) → Health assessments, reintroduction planning * * * **Section-by-Section Summary** **Introduction** - Terrapin populations have declined due to overharvest, habitat loss, road mortality, and crab traps. - Increasing confiscations due to illegal wildlife trade necessitate informed reintroduction practices. - This study aimed to provide health baseline data to guide such efforts in New Jersey. **Materials and Methods** - 30 wild females sampled in June 2020 while crossing roads to nest. - Each turtle underwent physical measurements, tagging, fecal collection, blood sampling, and pathogen testing. - Tests included: - WBC counts, differentials, and H:L ratio - Serum chemistry - Oral swabs for Mycoplasmopsis spp., ranavirus, and herpesvirus - Fecal floatation for parasites **Results** - Average age = 10 years; 70% gravid. - **Mycoplasmopsis spp.**: 10/18 usable cultures positive (33%); no clinical respiratory signs. - **Herpesvirus & Ranavirus**: All negative via PCR. - **Intestinal Parasites**: 43% had low burdens of trematodes (Cymatocarpus spp.) and nematodes (Strongyloidea). - **Blood Parasites**: 2 turtles had Haemogregarina spp. - **No associations**: Gravid status, parasite presence, and Mycoplasmopsis infection not associated with clinical parameters or chemistry differences. **Discussion** - Findings support the presence of Mycoplasmopsis in healthy turtles, aligning with other studies in emydids. - No signs of ranavirus or herpesvirus; possibly due to low prevalence or sampling limitations. - Blood parameters were consistent with published ranges for similar species. - No evidence of disease impact on health status, but elevated H:L ratios in some turtles warrant future investigation. - Study supports the utility of bloodwork and pathogen screening in reintroduction evaluations.

Answer 121

**1\. What are the two methods used to measure ESR in this study?** *Microhematocrit tubes (MHT) and Winpette commercial kits.* **2\. Which method tended to overestimate ESR values?** *The MHT method.* **3\. What physiologic or demographic factors were positively associated with ESR?** *Adult age class, gravidity, and site (SBCP).* **4\. What was the relationship between ESR and packed cell volume (PCV)?** *Negative correlation—higher ESR with lower PCV.* **5\. Why is the MHT method considered advantageous in the field?** *It uses less blood, is less expensive, and easily implemented under field conditions.* **6\. What biologic change likely explains elevated ESR in gravid turtles?** *Increased plasma lipids, proteins, and fibrinogen due to reproductive physiology.* **7\. What is the significance of Kendall’s τ = 0.64 in this study?** *It shows a moderate-to-strong correlation between ESR methods.* **8\. What key insight does this study provide for chelonian health assessment?** *ESR is a promising, underutilized biomarker for inflammation in turtle conservation medicine.* **9\. What future directions did the authors recommend for ESR research in turtles?** *Larger, seasonal studies including sick turtles and co-occurring species to generate broader reference data.* **10\. What statistical tool confirmed that the two ESR methods are not directly interchangeable?** *Passing-Bablok regression and Bland-Altman analysis.* ## Footnote *J. Wildl. Dis.* 59(4): 743–747, 2023 **Evaluating Different Methods for Determining Erythrocyte Sedimentation Rate in Free-Living Blanding’s Turtles (Emydoidea blandingii)** Samantha E. Bradley, Laura Adamovicz, Kirsten Andersson, Lauren Mumm, Gary Glowacki, Matthew C. Allender * * * **Key Points for ACZM Board Review** - **Objective**: This study compared two erythrocyte sedimentation rate (ESR) measurement methods—microhematocrit tubes (MHT) and a commercial Winpette kit—in free-ranging Blanding’s turtles. - **Method Agreement**: MHT ESR values were highly correlated with Winpette ESR but systematically overestimated them, indicating results are not interchangeable. - **Biologic Associations**: ESR was higher in adults vs. subadults/juveniles and in gravid vs. non-gravid females, and negatively correlated with PCV and total solids. - **Clinical Utility**: Both ESR methods correlated similarly with health and demographic variables, supporting their use for inflammation monitoring with method-specific reference intervals. - **Field Practicality**: The MHT method required less blood (<0.1 mL), was more economical, and easier to perform under field conditions. - **Physiologic Insights**: Elevated ESR in gravid turtles likely reflects reproductive changes (e.g., increased plasma lipids/proteins); age-related increases may reflect accumulated inflammatory exposure. - **Implications for Conservation**: ESR offers a minimally invasive, cost-effective tool to support health assessments in threatened chelonians like Emydoidea blandingii. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.5.a.2** – Clinical Medicine → Diagnostic Procedures → Hematology and inflammation biomarkers - **II.B.4.a.1** – Preventive Medicine → Population health assessment and conservation diagnostics - **II.A.2** – Species-Specific Management → Reptiles (freshwater turtles) → Physiology and field diagnostics * * * **Section-by-Section Summary** **Introduction** - ESR measures red cell sedimentation and is used in many vertebrates to assess inflammation. - Its role in reptile diagnostics is emerging; this study explores its utility in Blanding’s turtles, a threatened species. **Materials and Methods** - 60 turtles were captured in Illinois over two summers and underwent physical exams. - Blood was drawn from the subcarapacial sinus and tested for PCV, total solids, and ESR via: - Winpette commercial system - Microhematocrit tubes (MHT) - Statistical comparisons included regression modeling and method agreement (Passing-Bablok and Bland-Altman). **Results** - MHT values were consistently higher than Winpette values but correlated (Kendall’s τ = 0.64). - ESR was: - Higher in turtles from Spring Bluff Chiwaukee Prairie (SBCP) - Higher in adults vs. subadults/juveniles - Higher in gravid vs. non-gravid females - Negatively associated with PCV and total solids - Bland-Altman and regression analysis confirmed proportional bias (MHT > Winpette). **Discussion** - ESR values were influenced by demographic and physiological variables. - The MHT method is valid for field use, especially in small-bodied reptiles. - Gravid-related ESR increases are consistent with mammalian findings and reflect hormonal changes (e.g., fibrinogen). - Age-related ESR elevation parallels observations in other taxa (humans, swans, box turtles). - ESR should be further explored in broader turtle health assessments and conservation strategies.

Answer 122

**1\. What was the most commonly isolated bacterial species from nesting olive ridley turtles?** *Pseudomonas aeruginosa (22%).* **2\. What diarrheagenic E. coli pathotypes were identified in this study?** *Enteroaggregative (EAEC) and enteropathogenic (EPEC).* **3\. Which serotype of V. parahaemolyticus was pathogenic?** *OUT:KUT with the tdh+ gene.* **4\. What percentage of bacterial isolates were resistant to at least one antibiotic?** *100%.* **5\. What proportion of isolates were classified as MDR and XDR, respectively?** *30.9% MDR, 29% XDR.* **6\. What methods were used for bacterial identification and confirmation?** *Culture, biochemical tests, PCR, and serotyping with antisera.* **7\. Why is bacterial colonization in nesting turtles a concern for hatch success?** *Pathogenic bacteria can infect eggs during oviposition, impacting embryo survival.* **8\. What anthropogenic factors may contribute to bacterial contamination at nesting beaches?** *Agriculture runoff, aquaculture, human settlements, and fecal pollution.* **9\. What does the presence of these bacteria suggest about sea turtles' ecological role?** *They serve as sentinel species for coastal ecosystem health and pollution.* **10\. Which bacteria in this study showed the highest rates of drug resistance?** *P. aeruginosa, K. pneumoniae, and P. mirabilis.* ## Footnote *EcoHealth* 20: 390–401, 2023 **Potentially Pathogenic Bacteria in Nesting Olive Ridley Turtles in Northwestern Mexico** Jorge Valdés-Flores, Uriel Angulo-Zamudio, Nidia León-Sicairos, Héctor Flores-Villaseñor, C. Paul Ley-Quiñonez, Jorge Velázquez-Román, Ingmar Sosa-Cornejo, Alan Zavala-Norzagaray, A. Alonso Aguirre, Vicente Olimón-Andalón, and Adrián Canizalez-Román * * * **Key Points for ACZM Board Review** - **High Bacterial Diversity**: 100 bacterial isolates representing 21 species were identified from oral and cloacal swabs of 47 nesting olive ridley turtles at Ceuta Beach Sanctuary. - **Common Pathogens**: The most frequently isolated species were *Pseudomonas aeruginosa*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, *Vibrio parahaemolyticus*, *Klebsiella pneumoniae*, and *E. coli*. - **E. coli Pathotypes**: Diarrheagenic strains including enteroaggregative (EAEC) and enteropathogenic (EPEC) E. coli were identified; phylogroups B1, A, F, and unknown were present. - **Vibrio parahaemolyticus Serovars**: Multiple serotypes including O1, O4, K8, K12, and untypeable (OUT/KUT) were detected; one isolate carried the tdh virulence gene (pathogenic strain). - **Cocolonization**: 18 cases of dual-species and 2 cases of triple-species bacterial cocolonization were observed, suggesting complex microbial ecology. - **Antibiotic Resistance**: All bacterial isolates exhibited resistance to ≥1 antibiotic; 30.9% were multidrug-resistant (MDR) and 29% were extensively drug-resistant (XDR). - **Public and Wildlife Health Implications**: The presence of pathogenic and drug-resistant bacteria poses health risks to turtles, hatchlings, and potentially humans via illegal turtle meat/egg consumption. - **Sentinel Role of Turtles**: Findings support the utility of sea turtles as sentinels for coastal ecosystem pollution and antimicrobial resistance surveillance. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.6.a.3** – Clinical Medicine → Infectious disease diagnosis and management → Bacterial and antimicrobial resistance - **II.B.4.a.1** – Preventive Medicine → Environmental monitoring, microbial exposure, and conservation health - **II.A.2** – Species-Specific Management → Reptiles (marine turtles) → Reproductive ecology, microbial threats, and nest success * * * **Section-by-Section Summary** **Introduction** - Olive ridley turtles (Lepidochelys olivacea) are ecologically important but face anthropogenic threats. - Ceuta Beach is a key nesting site, but little is known about the pathogenic bacteria these turtles carry. **Methods** **Study Site and Specimen Collection** - Samples from 52 turtles were collected post-nesting at Ceuta Beach Sanctuary (Sinaloa, Mexico) during Aug–Nov 2018. - Oral and cloacal swabs were collected for microbial culture and molecular analysis. **Isolation and Identification** - Swabs cultured on selective media for Vibrio and Enterobacteriaceae; isolates identified biochemically and via PCR. **PCR and Serotyping** - Pathotype-specific PCR assays were used for E. coli and V. parahaemolyticus. - Serotyping was conducted for V. parahaemolyticus using commercial antisera kits. **Antimicrobial Testing** - 55 isolates tested using the Kirby-Bauer disk diffusion method against 9 antibiotics; CLSI standards used to classify MDR and XDR profiles. **Results** **Isolated Bacterial Species** - 100 isolates from 47 turtles (53 oral, 47 cloacal); most frequent species were *P. aeruginosa* (22%), *A. hydrophila* (20%), and *V. alginolyticus* (8%). **E. coli Phylogroups and Pathotypes** - 5 isolates; 2 EAEC, 1 EPEC, and 1 non-DEC; phylogroups B1, A, F, and unknown detected. **V. parahaemolyticus Serovars** - 6 isolates; 5 non-pathogenic and 1 pathogenic strain (OUT:KUT, tdh+); various O and K serogroups including untypeable combinations. **Cocolonization** - 18 turtles had mixed infections: 16 with two bacterial species and 2 with three species, e.g., *E. coli* + *V. alginolyticus*, *K. pneumoniae* + *V. mimicus*. **Antibiotic Resistance** - 100% of isolates were resistant to ≥1 drug. - Most common resistances: ampicillin (85.4%), cefotaxime (67.2%), nalidixic acid (54.5%). - 30.9% of isolates were MDR; 29% XDR. - *P. aeruginosa*, *K. pneumoniae*, and *P. mirabilis* were the most drug-resistant genera. **Discussion** - Bacteria likely reflect environmental exposure from polluted nesting beaches. - DECs and V. parahaemolyticus may negatively affect hatching success and represent public health risks. - Mixed infections may influence turtle immune health and represent zoonotic concerns. - Antibiotic resistance may arise from environmental contamination, illegal consumption, or rehab practices. **Conclusion** - Nesting olive ridley turtles harbor diverse and drug-resistant bacteria, some with human and turtle pathogenic potential. - Sea turtles serve as sentinels of environmental health, and their microbiota should be monitored in conservation strategies.

Answer 123

**1\. What was the primary histopathologic lesion in all tortoises in this case series?** *Ventricular myocardial fibrosis.* **2\. What clinical signs were most commonly observed in tortoises with cardiac disease?** *Peripheral edema, lethargy, and inappetence.* **3\. What novel ultrasonographic approach was described in this study?** *A dorsal cervical approach for cardiac imaging and pericardiocentesis.* **4\. What other organ systems commonly showed pathology in affected tortoises?** *Liver (lipidosis, fibrosis), lungs (edema), kidneys (nephritis).* **5\. What were some common gross necropsy findings in these cases?** *Pericardial effusion, epicardial adhesions, generalized edema.* **6\. What imaging modalities were useful for diagnosing cardiac disease in these tortoises?** *Ultrasound and non-contrast CT.* **7\. What were the proposed non-infectious contributors to cardiac disease in this study?** *Poor diet, inappropriate environmental parameters, and chronic stress.* **8\. What diagnostic challenges exist in detecting cardiac disease in tortoises?** *Nonspecific clinical signs and limited validated diagnostic tools.* **9\. In what location was fibrosis most commonly seen histologically in the heart?** *At the junction of the inner and outer myocardial tunics.* **10\. What is the clinical relevance of pericardial effusion in tortoises?** *May reflect underlying cardiac dysfunction; functional significance remains unclear.* ## Footnote *J. Zoo Wildl. Med.* 54(1): 164–174, 2023 **Degenerative Cardiac Disease in Two Species of Tortoise (Chelonoidis nigra Complex, Centrochelys sulcata)** Bridget Walker, Ronan Eustace, Kimberly A. Thompson, Chris A. Schiller, Angelique Leone, and Michael Garner * * * **Key Points for ACZM Board Review** - **Young Tortoises Affected**: Eleven cases of degenerative cardiac disease were identified in young captive Galápagos (Chelonoidis nigra complex) and sulcata tortoises (Centrochelys sulcata), with a mean age of 19 years. - **Consistent Histologic Lesions**: All cases exhibited ventricular myocardial fibrosis; many also showed epicardial adhesions, pericardial effusion, and myocardial edema. - **Common Clinical Signs**: Peripheral edema, lethargy, and inappetence were the most frequent clinical signs prior to death. - **Diagnostic Imaging**: Ultrasonography and CT revealed pericardial effusion and decreased cardiac function in two cases, supporting their utility in tortoise cardiology. - **Contributing Hepatic and Pulmonary Disease**: Frequent concurrent hepatic lipidosis, fibrosis, and pulmonary edema suggest multifactorial disease or systemic compromise. - **Etiology Unknown**: No infectious or toxic causes were identified; husbandry, diet, and environmental parameters are suspected contributors. - **Novel Diagnostic Technique**: A dorsal cervical ultrasound approach was successfully used for pericardiocentesis, offering a new method for tortoise cardiac evaluation. - **Differential Diagnosis Challenge**: Cardiac disease may be underdiagnosed in reptiles due to nonspecific signs and limited diagnostics. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.6.a.3** – Clinical Medicine → Diagnosis and management of cardiovascular disease in reptiles - **II.C.5.a.2** – Diagnostic Procedures → Histopathology, imaging (ultrasound, CT), and clinical pathology - **II.A.2** – Species-Specific Management → Chelonian medicine → Husbandry, nutrition, and chronic disease prevention * * * **Section-by-Section Summary** **Introduction** - Degenerative cardiac disease is poorly documented in tortoises. This study retrospectively reviews cases of myocardial fibrosis in Galápagos and sulcata tortoises, both in zoological and private care. **Methods** - Eleven cases met criteria of myocardial fibrosis in tortoises ≤50 years. Data collected included necropsy reports, histology, clinical signs, diet, and limited bloodwork and imaging. **Results** **Clinical Signs and Diagnostics** - Common signs: peripheral edema (6/11), lethargy (6/11), inappetence (3/11). - Imaging: pericardial effusion and reduced ejection fraction (ultrasound), enlarged cardiac silhouette (CT). - Pericardiocentesis performed using a dorsal cervical approach. **Gross Necropsy Findings** - Frequent: pericardial effusion (4/11), epicardial adhesions (4/11), myocardial mottling (2/11), pericardial thickening (2/11), generalized edema (7/11). **Histopathologic Findings** - Myocardial fibrosis (all cases), most severe at the junction of myocardial tunics. - Epicardial adhesions (5/11), myocardial edema (2/11), epicarditis (2/11). - Hepatic disease: lipidosis (7/11), fibrosis (6/11), hepatitis (5/11), biliary hyperplasia (4/11). - Pulmonary edema (4/11), pneumocytic hypertrophy (3/11), interstitial fibrosis (1/11). - Renal lesions: nephritis and tubular necrosis in 3/11 cases. **Discussion** - Tortoises showed signs consistent with mammalian congestive heart failure. - Many had extracardiac disease, suggesting systemic illness or shared pathophysiology. - No infectious etiology was identified; diet, environmental conditions, and poor husbandry are possible contributors. - Importance of early diagnosis emphasized; ultrasound and CT are promising tools. - First report of a dorsal ultrasound approach for pericardiocentesis in chelonians. **Conclusion** - Degenerative cardiac disease may be underrecognized in tortoises. - Appears to be more prevalent than expected in young captive tortoises. - Suggests need for better nutritional, environmental, and diagnostic practices in tortoise management. * * * **Key Figures and Tables** - **Figure 1**: Novel dorsal ultrasound technique and cardiac imaging results. - **Figure 2**: Gross and histologic images of cardiac fibrosis and pericardial adhesions. - **Table 1**: Clinical signs per case. - **Table 2**: Gross necropsy findings. - **Table 3**: Histologic findings across organ systems.

Answer 124

**1\. What anatomic window was used for echocardiographic imaging in giant tortoises?** *The cervicobrachial window (left or right).* **2\. What proportion of tortoises had physiologic pericardial effusion?** *77%.* **3\. What were the reference intervals established for EF% and AVo MAX?** *EF%: 43.94–76.12%; AVo MAX: 0.55–1.58 m/s.* **4\. Which species had a higher rate of successful echocardiographic measurements?** *Aldabra tortoises.* **5\. What restraint techniques were used for echocardiography?** *Behavioral restraint, elevation using tires, and food motivation.* **6\. Why was modified Simpson’s method used in this study?** *To calculate ventricular volumes from 2D echocardiographic images.* **7\. Which cardiac features were consistently visualized in all tortoises?** *Right and left atria, single ventricle, AV, aortic, and pulmonic valves.* **8\. What Doppler measurements were used to assess cardiac function?** *A-Vvo (AV inflow), AVo and PVo (aortic and pulmonary peak velocity), AoVTI and PVTI.* **9\. What challenges limited measurement collection in some Galapagos tortoises?** *Lower tolerance to handling and reduced socialization compared to Aldabras.* **10\. What is the clinical significance of this study for zoo medicine?** *Establishes a practical, non-invasive method for cardiac evaluation in large tortoises.* ## Footnote *J. Zoo Wildl. Med.* 54(2): 310–318, 2023 **Determination of Echocardiographic Techniques for Clinical Assessment of Galapagos (Chelonoidis nigra Complex) and Aldabra (Aldabrachelys gigantea) Tortoises** Camden Rouben, Simon Swift, Cassidy Soehnlein, Amy B. Alexander, Michael Aherne, Jane Christman, and Darryl Heard * * * **Key Points for ACZM Board Review** - **Non-anesthetized Imaging Feasible**: Echocardiographic evaluation was successfully performed in awake Galapagos and Aldabra tortoises using manual or behavioral restraint. - **Reliable Acoustic Window Identified**: The cervicobrachial window provided consistent access to all cardiac chambers and great vessels using long-axis imaging. - **Physiologic Pericardial Effusion Common**: Anechoic pericardial effusion was present in 77% of tortoises and considered physiologic in the absence of tamponade signs. - **Functional Parameters Assessed**: Ejection fraction (EF%), aortic and pulmonary valve flow velocities, and AV inflow velocities were measurable in all subjects using Doppler imaging. - **Structural Visualization Achieved**: All cardiac valves and chambers were consistently visualized; AV valve morphology was reliably assessed, while pulmonic/aortic valves were more challenging. - **EF% and Aortic Velocity Norms Proposed**: Reference intervals for EF% and AVo MAX were calculated (EF%: mean 60.04%, RI 43.94–76.12; AVo MAX: mean 1.07 m/s, RI 0.55–1.58). - **Aldabra Tortoises More Compliant**: More echocardiographic measurements were successfully obtained from Aldabra than Galapagos tortoises, possibly due to greater socialization. - **Foundation for Cardiac Disease Diagnosis**: Provides a baseline for evaluating suspected cardiac disease, especially given reports of myocardial and pericardial fibrosis in these species. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.5.a.2** – Diagnostic Procedures → Ultrasonography, echocardiography in reptiles - **II.C.6.a.3** – Clinical Medicine → Cardiovascular disease diagnosis and monitoring in chelonians - **II.A.2** – Species-Specific Management → Chelonian physiology, diagnostics, and health management * * * **Section-by-Section Summary** **Introduction** - Giant tortoises are vulnerable/endangered species requiring effective health monitoring. - Cardiac disease has been reported, but echocardiographic techniques and reference values are lacking for live, non-anesthetized individuals. **Materials and Methods** - 51 tortoises (Galapagos = 23, Aldabra = 28) from zoological or private collections were studied. - Echocardiography was performed in ventral recumbency using a 5-MHz phased array probe. - Left or right cervicobrachial window used; images acquired in dorsal and ventral long-axis views. - No sedation used; food motivation or elevation used to reduce movement. - Parameters measured included EF%, AV inflow/outflow velocities, ventricular dimensions, and flow integrals. **Results** - Full cardiac visualization achieved in all tortoises. - EF% and AVo MAX were normally distributed and reference intervals calculated. - Physiologic pericardial effusion detected in 77% of tortoises. - Aldabra tortoises had higher compliance, yielding more complete datasets. - Three Galapagos tortoises with systemic disease (pneumonia, osteomyelitis) did not show abnormal echocardiographic parameters. **Discussion** - Echocardiography is a feasible and useful diagnostic tool in awake giant tortoises. - Provides first large dataset on echocardiographic anatomy and function for these species. - Functional and structural parameters were consistently measurable, especially in socialized animals. - Physiologic pericardial effusion appears normal in these species, supporting previous observations. - Reference intervals for EF% and AVo MAX help guide future clinical interpretation. - Future work should explore associations between echocardiographic changes and pathology. **Conclusion** - Echocardiographic assessment of cardiac health is achievable in non-anesthetized giant tortoises. - Establishes clinically useful protocols and reference values for cardiac evaluation in Chelonoidis and Aldabrachelys species.

Answer 125

**1\. Which hematologic values were significantly higher in Phrynops geoffroanus during the rainy season?** *Hemoglobin (Hb) and mean corpuscular hemoglobin concentration (MCHC).* **2\. Which white blood cell types were elevated during the dry season?** *Heterophils, eosinophils, lymphocytes, and monocytes.* **3\. What hematologic differences were found between captive and urban turtles?** *Captive turtles had higher RBC, PCV, basophils, and monocytes; urban turtles had higher lymphocytes.* **4\. What hematologic parameter was significantly higher in females?** *Eosinophils.* **5\. Was straight carapace length (SCL) correlated with any blood parameters?** *No, there was no significant correlation.* **6\. What was the most abundant leukocyte found in all turtles?** *Heterophils.* **7\. Did the heterophil:lymphocyte ratio vary by season, environment, or sex?** *No, it remained consistent across all variables.* **8\. What might explain the elevated eosinophil counts in urban turtles?** *Increased exposure to parasites and environmental stressors.* **9\. Why might hematologic values be higher in captive turtles?** *Possibly due to better nutrition and reduced environmental stress.* **10\. What is the clinical relevance of this study for chelonian medicine?** *It establishes that hematologic interpretation must consider season, environment, and sex to be diagnostically useful.* ## Footnote *J. Zoo Wildl. Med.* 54(3): 484–497, 2023 **Influence of Season, Environment, and Sex on Selected Blood Parameters of Geoffroy’s Side-Necked Turtles, Phrynops geoffroanus (Schweigger, 1812)** Rachel S.L. Pulcherio, Marcela N.A. Rocha, Rachel V.P. de Barros, Elizângela S. Brito, Denize P. dos Santos, Adriane J. Mendonça, Christine Strüssmann, Rosa H. S. Ferraz * * * **Key Points for ACZM Board Review** - **Seasonal Variation Documented**: Hemoglobin (Hb) and mean corpuscular hemoglobin concentration (MCHC) were significantly higher in the rainy season; WBC, heterophils, eosinophils, lymphocytes, and monocytes were elevated in the dry season. - **Environmental Impact**: Turtles from captivity had significantly higher RBC, PCV, basophils, and monocytes, while urban turtles had higher lymphocytes. - **Sex-Based Differences**: Females had significantly higher eosinophil counts than males, possibly related to reproductive physiology or parasitism. - **No Size Effect**: Straight carapace length (SCL) showed no significant correlation with any hematologic parameter. - **No H:L Ratio Differences**: The heterophil:lymphocyte (H:L) ratio did not differ significantly by season, environment, or sex. - **Leukocyte Dominance**: Heterophils were the most frequent leukocytes, followed by lymphocytes, basophils, monocytes, and eosinophils. - **High Diagnostic Value**: This study supports the need to interpret hematologic values in chelonians in context, considering environmental and seasonal influences. - **Field-Relevant Methodology**: Blood sampling, smear preparation, and hematologic analysis methods are feasible and applicable to both wild and captive freshwater chelonians. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.5.a.2** – Clinical Medicine → Diagnostic Procedures → Hematology and leukocyte differential in reptiles - **II.B.4.a.1** – Preventive Medicine → Population health surveillance and physiological baselines - **II.A.2** – Species-Specific Management → Freshwater turtles → Physiology, stress response, and seasonal biology * * * **Section-by-Section Summary** **Introduction** - Reptilian hematologic parameters vary with environmental and individual factors. - Study aimed to evaluate how season, environment (captive vs. urban), sex, and body size influence blood values in Phrynops geoffroanus. **Materials and Methods** - 38 turtles sampled from captivity and an urban stream in Cuiabá, Brazil, during rainy and dry seasons. - Blood collected from the post-occipital sinus; smears made; counts and biochemistry performed within 2 hours. - Hematology included RBC, WBC, PCV, Hb, MCV, MCHC, leukocyte differentials, and TS. - Statistical models (GLM) evaluated effects of all variables. **Results** - **Seasonal Influence**: - Rainy season: ↑ Hb, ↑ MCHC - Dry season: ↑ WBC, heterophils, eosinophils, lymphocytes, monocytes - **Environmental Influence**: - Captive turtles: ↑ RBC, ↑ PCV, ↑ basophils, ↑ monocytes - Urban turtles: ↑ lymphocytes - **Sex Influence**: - Females: ↑ eosinophils - No significant differences in MCV, TS, or H:L ratio due to any variable. - No hematologic parameters correlated with SCL. **Discussion** - Hematologic shifts likely reflect climate, nutritional status, reproductive activity, and parasite exposure. - Higher RBC and PCV in captivity may reflect better nutrition or reduced parasite burden. - Eosinophilia in females and urban turtles may relate to greater parasite exposure. - Seasonal leukocytosis corresponds with reproductive and immune modulation. - Importance of multi-variable analysis to refine diagnostic interpretation in field and clinical settings.

Answer 126

**1\. What was the goal of this study on praziquantel in European pond turtles?** *To assess pharmacokinetics and safety of praziquantel administered via three routes (PO, IM, SC).* **2\. Which administration route resulted in the highest plasma praziquantel concentration?** *Intramuscular (IM).* **3\. Which administration route had the fewest adverse effects in EPT?** *Subcutaneous (SC).* **4\. What dosing protocol was used in this study for all groups?** *25 mg/kg praziquantel given three times at 3-hour intervals.* **5\. What were the main side effects observed in the IM group?** *Local swelling, lameness, and decreased food intake.* **6\. What was the mean Cmax of R-praziquantel in the PO group?** *1,082 ng/ml.* **7\. What is a hypothesized mechanism for IM injection site reactions to praziquantel?** *Activation of TRPM ion channels leading to local muscle irritation.* **8\. Why is PO administration of praziquantel challenging in EPT?** *Risk of regurgitation and difficulty with esophageal delivery in small turtles.* **9\. What disease prompted the need for this pharmacokinetic study?** *Spirorchiidosis caused by Spirorchis spp.* **10\. Which PZQ administration route is currently recommended for future treatment in EPT based on this study?** *Subcutaneous (SC) injection.* ## Footnote *J. Zoo Wildl. Med.* 54(4): 728–737, 2023 **Safety and Pharmacokinetics of Praziquantel in European Pond Turtles (Emys orbicularis)** Marion Stettler, Christian Lotz, Alexandra Probst, Nelson Marreros, Britta Lundström-Stadelmann, Caroline F. Frey, Jennifer Keiser, Andreas Hofer, and Stefan Hoby * * * **Key Points for ACZM Board Review** - **First PK Data in EPT**: This is the first study to describe pharmacokinetics and safety of praziquantel (PZQ) in European pond turtles (EPT), addressing the need for treatment protocols against spirorchiid blood flukes. - **Three Administration Routes Tested**: 12 turtles were treated with 25 mg/kg PZQ three times at 3-hour intervals via oral (PO), intramuscular (IM), or subcutaneous (SC) routes. - **SC Recommended**: SC administration achieved high plasma concentrations (Cmax 5,643 ng/ml), was well tolerated, and avoided the side effects seen with IM administration (lameness, local swelling). - **PO Less Effective, Variable**: Oral dosing produced lower and more variable plasma levels (Cmax 1,082 ng/ml), with some regurgitation, making it less practical for EPT. - **IM Side Effects Noted**: IM administration resulted in significant local adverse reactions and decreased food intake; although plasma concentrations were highest, side effects reduce its clinical desirability. - **Pharmacokinetic Findings**: - Tmax at 6 h in most turtles - AUC significantly higher in SC and IM than PO groups - T1/2 varied widely between individuals - **Spirorchiidosis Concern in EPT**: Disease threatens conservation efforts in Switzerland; treatment protocols must be safe, effective, and practical for reintroduction programs. - **TRPM Channels Hypothesized in Side Effects**: Muscle pain and swelling after IM may be linked to TRPM channel activation, as in schistosome PZQ activity. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.6.a.3** – Clinical Medicine → Treatment of parasitic disease in reptiles (anthelmintics, spirorchiids) - **II.C.5.a.2** – Diagnostic and pharmacologic procedures → Pharmacokinetics and drug safety - **II.A.2** – Species-Specific Management → European pond turtles → Conservation medicine, pharmacotherapy, and reintroduction health planning * * * **Section-by-Section Summary** **Introduction** - Spirorchiidosis poses a risk to captive and wild EPT populations in Switzerland. - Treatment protocols for spirorchids are lacking in this species. - This study evaluated pharmacokinetics and safety of PZQ administered via PO, IM, and SC routes in adult EPT. **Materials and Methods** - 12 adult turtles randomly assigned to receive 25 mg/kg PZQ q3h × 3 via PO (n=6), IM (n=3), or SC (n=3). - Plasma PZQ measured at 3, 6, 24, and 48 hours post first dose via LC-MS/MS. - Clinical signs, body weight, and adverse reactions recorded. - Non-compartmental analysis used to calculate Cmax, Tmax, AUC, and T1/2. **Results** - **IM Group**: Highest Cmax (7,532 ng/ml), but all turtles developed swelling, lameness, and decreased food intake. Required meloxicam therapy. - **SC Group**: High Cmax (5,643 ng/ml) with no adverse effects. Preferred route. - **PO Group**: Lowest and most variable Cmax (1,082 ng/ml); two turtles regurgitated PZQ. - AUC significantly higher in SC and IM groups than PO. - T1/2 varied but was not significantly different among groups. - One PO turtle (ID 8) had delayed Tmax at 48 h (excluded from AUC/T1/2 analysis). - No mortality occurred; all side effects were transient and resolved. **Discussion** - SC administration is the most practical and safest method with good bioavailability. - IM administration results in discomfort, likely due to local muscle irritation or TRPM channel activation. - Oral dosing is feasible but technically challenging and less reliable. - Results support 25 mg/kg q3h × 3 SC as a preferred treatment for spirorchids in EPT. - Further research is needed on therapeutic efficacy and in vivo parasitological outcomes. **Conclusion** - SC administration of PZQ at 25 mg/kg q3h × 3 is a promising protocol for use in conservation programs targeting spirorchiid infections in EPT.

Answer 127

**1\. What percentage of green turtles had positive blood cultures at intake in this study?** *24%.* **2\. Which bacterial species was the most frequently isolated from positive blood cultures?** *Vibrio alginolyticus.* **3\. What clinical finding was significantly associated with a positive blood culture?** *External injury.* **4\. Name two blood parameters that were significantly lower in culture-positive turtles compared to culture-negative turtles.** *Total WBC and total protein.* **5\. What outcome was significantly more likely in culture-positive turtles?** *Death during rehabilitation.* **6\. Was there a significant association between blood culture positivity and presence of fibropapillomatosis?** *No.* **7\. What is one recommended improvement for blood culture technique based on this study?** *Standardized and more rigorous aseptic skin preparation before venipuncture.* **8\. What challenge exists in differentiating bacteremia from septicemia in reptiles?** *Lack of defined systemic inflammatory response criteria in reptiles.* **9\. What limitation related to blood volume collection was noted in this study?** *Small volumes may reduce sensitivity for detecting bacteremia.* **10\. Why are blood cultures at intake important in sea turtle rehabilitation medicine?** *They help confirm septicemia early, guide treatment, and predict prognosis.* ## Footnote *. Zoo Wildl. Med.* 54(4): 766–775, 2023 **Retrospective Analysis of Blood Cultures and Their Association with Clinical Findings and Outcome in Green Sea Turtles (Chelonia mydas) at a Florida Sea Turtle Rehabilitation Facility, 2017–2020** Alan R. Glassman, DVM, and Trevor T. Zachariah, DVM, MS, DACZM * * * **Key Points for ACZM Board Review** - **24% Positive Blood Culture Rate**: 24% of green turtles had positive aerobic blood cultures at intake; common isolates included *Vibrio alginolyticus*, *Shewanella algae*, *Achromobacter xylosoxidans*, and *Photobacterium damselae*. - **Association with External Injuries**: External injuries significantly increased the odds (OR 2.23) of a positive blood culture. - **Laboratory Changes**: Culture-positive turtles had significantly lower total WBCs, lymphocytes, monocytes, total protein, albumin, and globulin compared to culture-negative turtles. - **Poorer Prognosis**: Culture-positive turtles were significantly more likely to die in rehabilitation (OR 2.47). - **Microbial Flora**: Many isolates were typical of marine and turtle cloacal flora but acted opportunistically in compromised hosts. - **No Significant Association**: No link found between blood culture status and fibropapillomatosis or Caryospora-like infections. - **Procedural Considerations**: Nonstandardized venipuncture asepsis and small blood volumes could influence contamination rates or detection sensitivity. - **Recommendations**: Emphasizes early blood culture collection at intake, better aseptic protocols, and cautious interpretation of culture results in debilitated sea turtles. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.6.a.3** – Clinical Medicine → Diagnosis and management of infectious diseases in reptiles - **II.C.5.a.2** – Diagnostic Procedures → Clinical pathology, microbiology, and interpretation of blood culture results - **II.A.2** – Species-Specific Management → Marine turtles → Clinical care and rehabilitation protocols * * * **Section-by-Section Summary** **Introduction** - Sea turtles are often debilitated by trauma and disease; septicemia is a common cause of death. - Blood cultures are underutilized at intake despite their diagnostic value. - This study aims to correlate blood culture results with clinical findings and rehabilitation outcomes in green turtles. **Materials and Methods** - 167 juvenile to subadult green turtles from 2017–2020. - Blood cultures obtained at intake before antibiotics; cultures processed using aerobic methods. - External injuries, fibropapillomatosis, Caryospora-like infections, and CBC/biochemistries were evaluated. - Outcomes classified as death, euthanasia, or release. **Results** - **Culture Positivity**: 24% (40/167) were blood culture positive. - **Bacterial Isolates**: Vibrio spp., Shewanella spp., Achromobacter spp., and Photobacterium spp. predominated. - **Injury Correlation**: Positive cultures significantly associated with external injuries. - **Laboratory Differences**: - Lower WBC, lymphocyte, monocyte counts, and lower protein levels in culture-positive turtles. - **Outcome**: Culture-positive turtles were more likely to die naturally in rehabilitation. - **No Association**: Between culture results and Caryospora-like coccidia, fibropapillomatosis, or evidence of WBC toxicity. - **Asepsis Validation**: Occasional low-level bacterial contamination detected in skin swabs despite disinfection. **Discussion** - Bacteremia in green turtles frequently involves marine opportunists. - External injuries likely serve as portals of entry. - Differences in hematology support the clinical significance of positive cultures (suggesting septicemia). - Aseptic technique inconsistencies and small blood volumes are potential study limitations. - Positive cultures and associated laboratory findings (hypoproteinemia, cytopenias) inform prognosis. - Blood cultures should become standard at sea turtle rehabilitation intake. **Conclusion** - Blood cultures are valuable diagnostic and prognostic tools in rehabilitating green turtles. - External injury increases the risk of bacteremia. - Positive blood culture is associated with poorer outcomes and important clinicopathologic changes.

Answer 128

**1\. What percentage of eastern box turtles sampled showed evidence of cutaneous myiasis?** *30.5%.* **2\. Which habitats were most associated with myiasis in turtles?** *Bog habitats.* **3\. Name three clinical pathology parameters that were significantly lower in turtles with myiasis.** *WBC count, PCV, and total solids.* **4\. What hematologic marker was elevated in turtles with myiasis, suggesting inflammation?** *Erythrocyte sedimentation rate (ESR).* **5\. Was there an association between heavy metal concentrations and presence of myiasis?** *No.* **6\. Which two heavy metals were elevated in turtles with plastron lesions?** *Zinc and selenium.* **7\. What pathogen was detected in a small number of turtles, but not associated with myiasis?** *Terrapene herpesvirus 1.* **8\. How long did it generally take for myiasis infections to resolve naturally in surviving turtles?** *Approximately 4 to 8 weeks.* **9\. Did the presence of myiasis statistically predict mortality in this study?** *No, although heavily infested individuals were among those that died.* **10\. Why is continued health monitoring of this population important despite the ability of turtles to recover from myiasis?** *Environmental stressors could compound health impacts, affecting conservation outcomes.* ## Footnote *J. Zoo Wildl. Med.* 54(4): 785–795, 2023 **Cutaneous Myiasis and Its Relationship to Wellness in Eastern Box Turtles (Terrapene carolina carolina) in Cape Cod, Massachusetts** Lauren M. Vincent, Matthew C. Allender, Annie E. Curtis, Nicole E. Madden, Carolyn Cray, Stacey Lance, Adam McFall, Laura Adamovicz * * * **Key Points for ACZM Board Review** - **High Prevalence of Myiasis**: 30.5% of turtles were affected by cutaneous or subcutaneous fly larvae (myiasis), primarily in protected anatomical locations (prescapular and prefemoral fossae). - **Health Impact**: Turtles with myiasis had significantly lower body condition index (BCI), WBC, PCV, total solids, albumin, calcium, and phosphorus, and higher erythrocyte sedimentation rate (ESR). - **Habitat Association**: All turtles found in bog habitats had evidence of fly larvae infection, indicating environmental risk factors. - **No Pathogen Link**: No association between myiasis and terrapene herpesvirus 1 or other pathogens tested. - **Heavy Metals**: No association between myiasis and plasma heavy metal concentrations, although plastron lesions correlated with higher zinc and selenium levels. - **Myiasis Resolution**: Many myiasis cases resolved naturally within 4–8 weeks, suggesting individual recovery possible without intervention. - **Mortality Observations**: Six turtles died during the study; severe myiasis appeared to contribute in some cases but was not statistically associated with death overall. - **Conservation Implications**: Although not immediately population-limiting, myiasis can affect individual wellness and could become more significant under compounding environmental stresses. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.6.a.3** – Clinical Medicine → Diagnosis and management of ectoparasites and wound infections in reptiles - **II.C.5.a.2** – Diagnostic Procedures → Clinical pathology, toxicology, and disease surveillance - **II.A.2** – Species-Specific Management → Terrestrial turtles → Disease ecology and health surveillance for conservation * * * **Section-by-Section Summary** **Introduction** - Eastern box turtles (EBT) in Cape Cod are a conservation concern. - Emerging reports of high rates of myiasis (fly larvae infection) prompted investigation into health impacts and associations with other factors (pathogens, metals). **Materials and Methods** - 59 turtles sampled from May–August 2021 at Camp Edwards and nearby habitats. - Full physical exams, blood collection, environmental data recording, qPCR for pathogens, plasma protein electrophoresis, heavy metal analysis, and bone marrow pathogen testing were performed. **Results** - **Myiasis**: - Present in 30.5% of turtles. - Higher prevalence in bog habitats. - Lesions predominantly in fossae near limbs or head. - **Clinical Pathology**: - Myiasis associated with reduced BCI, WBC, PCV, total solids, calcium, and phosphorus; increased ESR. - Females had higher calcium and globulins than males overall. - **Pathogen Screening**: - Only terrapene herpesvirus 1 detected (5.1%); no association with myiasis. - **Toxicology**: - No differences in heavy metals between myiasis-positive and -negative turtles. - Higher zinc and selenium levels found in turtles with plastron lesions. - **Mortality**: - Six turtles died; myiasis appeared to contribute in some cases, but no statistical association. **Discussion** - Myiasis negatively affects several health parameters. - Recovery without intervention is possible in many cases, but heavy infestations can be fatal. - Wetland habitats increase myiasis risk; mosquito and fly interactions may facilitate larval deposition. - Heavy metals may predispose turtles to shell lesions but were not linked directly to myiasis. - Continued health monitoring is essential for long-term conservation management.

Answer 129

**1\. What species is described in this case of esophageal stricture?** *Green sea turtle (Chelonia mydas).* **2\. What clinical signs suggested an esophageal stricture in this turtle?** *Progressive dysphagia and regurgitation of water and food from the nares.* **3\. What diagnostic imaging modalities were used to evaluate this case?** *Computed tomography (CT), ultrasound, and endoscopy (esophagoscopy).* **4\. What histologic findings characterized the esophageal stricture?** *Marked fibrosis, edema, and mild perivascular lymphoplasmacytic inflammation.* **5\. What were the two treatment methods attempted for this turtle's esophageal stricture?** *Endoscopic bouginage and surgical esophagotomy.* **6\. What is the likely cause of esophageal stricture in sea turtles based on this case?** *Prior unknown trauma, likely anthropogenic (e.g., fishing gear, boat strike).* **7\. Why was surgical resection not successful in this case?** *Anatomical location and insufficient viable esophageal tissue for anastomosis.* **8\. What other signs of chronic illness were found on necropsy?** *Serous atrophy of fat, diffuse muscle atrophy, and emaciation.* **9\. In sea turtle rehabilitation, what clinical signs should prompt investigation for esophageal stricture?** *Progressive weight loss, dysphagia, and regurgitation.* **10\. What is the prognosis for sea turtles diagnosed with esophageal strictures?** *Guarded for survival and poor for release.* ## Footnote *J. Herpetol. Med. Surg.* 34(1): 6–10, 2024 **Esophageal Stricture and Occlusion in a Green Sea Turtle (Chelonia mydas)** Nicholas G. Dannemiller, Ashley R. Souza, Taylor Sherrod, Mandy Womble, Sarah M. Ozawa, Gregory A. Lewbart, Kathryn A. Zagzebski, Craig A. Harms * * * **Key Points for ACZM Board Review** - **Rare Case in Sea Turtles**: This report describes a rare, severe esophageal stricture and complete occlusion in a juvenile green sea turtle, likely secondary to prior trauma. - **Clinical Signs**: Progressive dysphagia, regurgitation of water and food from the nares, emaciation, and poor body condition developed during rehabilitation. - **Diagnosis**: Endoscopy (esophagoscopy) confirmed a cranial esophageal stricture; CT and ultrasound ruled out mass lesions or foreign bodies. - **Treatment Attempts**: Endoscopic bouginage and surgical esophagotomy failed due to complete occlusion and insufficient esophageal tissue for anastomosis. - **Histopathology**: Revealed marked fibrosis, edema, and mild perivascular lymphoplasmacytic inflammation; surrounding soft tissue showed edema and discoloration. - **Potential Causes**: Suspected secondary to prior traumatic injury rather than congenital stenosis; environmental or anthropogenic trauma are possible etiologies. - **Clinical Implication**: Esophageal strictures should be considered in turtles showing progressive weight loss, dysphagia, or regurgitation during rehabilitation. - **Guarded Prognosis**: Even with intervention, the prognosis for functional recovery and release is poor. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.6.a.3** – Clinical Medicine → Diagnosis and management of gastrointestinal disorders and trauma in reptiles - **II.C.5.a.2** – Diagnostic Procedures → Advanced imaging (CT, endoscopy) and surgical diagnostics in reptiles - **II.A.2** – Species-Specific Management → Marine turtles → Clinical care, rehabilitation, and release assessment * * * **Section-by-Section Summary** **Introduction** - Esophageal strictures are rare in reptiles but occur due to esophageal injury and fibrosis. - Causes include trauma, ingestion of foreign bodies, or surgical/iatrogenic injury. - Sea turtle esophageal anatomy (keratinized papillae) makes them susceptible to trauma-induced strictures. **Case Report** - Juvenile green sea turtle stranded during cold-stunning event, presenting with severe emaciation, abnormal buoyancy, and a healing cervical wound. - Despite initial supportive care, turtle exhibited worsening dysphagia and regurgitation. - Imaging (CT, ultrasound) excluded masses; endoscopy revealed a complete esophageal occlusion. - Attempts at bouginage and surgical esophagotomy were unsuccessful due to fibrosis and lack of viable tissue. - Euthanasia elected based on poor prognosis; necropsy confirmed a 5-mm-thick fibrous occlusion 3 cm caudal to oropharynx. **Discussion** - Likely acquired rather than congenital; potentially secondary to prior traumatic injury (e.g., boat strike, fishing gear). - Regurgitation and dysphagia are hallmark clinical signs of esophageal stricture in both domestic animals and turtles. - Treatment challenges include anatomical limitations for surgical correction. - Diagnostic approach: radiography with contrast, CT, and esophagoscopy. - Prognosis remains guarded even with advanced interventions; permanent managed care may be necessary if survival is achieved. - Highlights the need for early identification of esophageal injury signs during rehabilitation.

Answer 130

**1\. What new radiographic view was described for evaluating coxofemoral injuries in tortoises?** *A craniodorsal–caudoventral 45° oblique (45CC) view.* **2\. Why is the 45CC view superior to standard views for pelvic injuries in tortoises?** *It reduces superimposition of shell, pelvis, and soft tissues.* **3\. What orthopedic condition was diagnosed in both tortoises in this report?** *Coxofemoral luxation.* **4\. What surgical procedure was used to treat the luxations?** *Femoral head ostectomy (FHO).* **5\. How did the presence of telemetry equipment affect imaging?** *Minimal interference; 45CC view still diagnostic.* **6\. What potential complications were seen in the tortoises postoperatively?** *One had minor residual lameness; one was later found deceased (cause unknown).* **7\. What anesthesia protocols were used for surgical procedures?** *Tiletamine-zolazepam sedation, propofol induction, isoflurane maintenance.* **8\. What are other potential uses for the 45CC view besides coxofemoral luxation?** *Diagnosis of pelvic fractures, thoracic girdle injuries, and pelvic canal diseases.* **9\. What supportive care measures were used during rehabilitation?** *Esophagostomy tube feeding, UVB exposure, and outdoor enrichment.* **10\. How does this study suggest adapting radiography across species?** *Techniques developed in avian radiology can improve chelonian imaging and diagnosis.* ## Footnote *J. Herpetol. Med. Surg.* 34(1): 16–21, 2024 **Use of Craniodorsal–Caudoventral 45° Oblique Radiographs for Diagnosis and Treatment of Coxofemoral Luxation in Two Mohave Desert Tortoises, Gopherus agassizii** Ian Kanda and Thomas Boyer * * * **Key Points for ACZM Board Review** - **New Radiographic Technique**: A craniodorsal–caudoventral 45° oblique (45CC) radiographic view provided superior visualization of the coxofemoral joint and pelvic girdle in tortoises compared to standard views. - **Enhanced Diagnosis**: The 45CC view clearly identified coxofemoral luxations and pelvic fractures obscured by shell and soft tissue overlap in dorsoventral and lateral views. - **Successful Surgical Interventions**: Femoral head ostectomies (FHO) were performed in both cases to manage luxations; tortoises recovered with minimal impairment. - **Case Specifics**: One tortoise had a contaminated open wound with luxation, and the other had a closed luxation with an associated pelvic fracture. - **Use Despite Telemetry Devices**: Radiotelemetry equipment on the carapace did not interfere with diagnostic quality in the 45CC view. - **Minimal Sedation Needed**: Tortoises were imaged without sedation, although sedation may improve diagnostic quality if positioning is difficult. - **Broader Application Potential**: Authors suggest the 45CC view may help detect additional disorders, including thoracic girdle fractures and pelvic canal disease. * * * **Relevant ACZM Job Task Analysis Sections** - **II.C.5.a.2** – Diagnostic Procedures → Imaging (radiography) of reptiles - **II.C.6.a.3** – Clinical Medicine → Diagnosis and management of orthopedic injuries in reptiles - **II.A.2** – Species-Specific Management → Terrestrial turtles → Rehabilitation and orthopedic surgery * * * **Section-by-Section Summary** **Introduction** - Coxofemoral luxations and pelvic fractures are challenging to diagnose in chelonians due to superimposition in standard radiographic views. - Advances in avian radiography (oblique views) have inspired similar techniques in turtles. - Study evaluated the use of a 45° craniodorsal–caudoventral view in Mohave desert tortoises with orthopedic injuries. **Case Reports** **Tortoise 1** - Adult male with a severe, infected wound exposing the left coxofemoral joint. - 45CC radiograph confirmed luxation; no fractures. - Surgical FHO performed; recovery involved passive range of motion exercises, esophagostomy tube feeding, and UVB exposure. - Outcome: Gained weight, good recovery with minor residual lameness, released under biologist supervision. **Tortoise 2** - Subadult with right hindlimb paresis and missing pedal scales. - 45CC radiograph revealed a right coxofemoral luxation and a puboischiatic fracture involving the acetabulum. - Surgical FHO performed with maintenance of the pelvic fracture. - Outcome: Good postoperative recovery, normal behavior initially, found deceased months later (no necropsy performed). **Discussion** - 45CC imaging provides critical visualization of pelvic and femoral structures otherwise obscured. - Minimal radiographic distortion was acceptable and did not affect clinical interpretation. - Sedation and limb positioning may improve imaging further. - Suggests potential broader application for diagnosing pelvic and thoracic injuries or pelvic canal diseases. - Emphasizes adapting techniques from avian to reptilian species where applicable.