DEVELOPMENT OF A HEMOLYMPH COLLECTION TECHNIQUE AND REPORT OF BIOCHEMICAL PARAMETERS OF HEMOLYMPH IN A MANAGED POPULATION OF FEMALE THORNY DEVIL STICK INSECTS (EURYCANTHA CALCARATA).

Insects are increasingly common in households, zoological collections, research, and food industries. Increased knowledge of insect health parameters is necessary to ensure proper welfare. The study goal was to develop a hemolymph collection technique and report hemolymph serum biochemical parameters for the thorny devil stick insect (Eurycantha calcarata). Clinically healthy adult stick insects (5 males and 14 females, 15.1-24.7 g) were enrolled. Four collection techniques were evaluated. Hemolymph collection was unsuccessful in males, but was successful in females by using a single technique. The insect was manually restrained in an elevated position and an 18-ga × 2.54-cm needle was used to puncture the membrane just caudal to the third pair of legs. With the puncture site directed ventrally, ≤1 ml of hemolymph was collected via gravity and allowed to visibly clot. The sample was then centrifuged, and the serum was separated. Serum samples were individually analyzed (Avian/Reptilian Profile Plus, VetScan VS2, Abaxis, Inc, Union City, CA 94587, USA). Fourteen samples (0.2-1.0 ml) were collected from 14 females. Median (minimum-maximum) parameters included the following: aspartate aminotransferase 12 (0-45) U/L, creatinine kinase 25 (0-76) U/L, uric acid 7.5 (3.1-13.7) mg/dl, glucose 12 (8-22) mg/dl, calcium 18.6 (17.2-19.4) mg/dl, phosphorus 15.0 (n = 1) or >30.0 (n = 13) mg/dL, total protein 2.7 (1.6-2.9) g/dL, albumin 0.9 (0.2-1.2) g/dL, globulin 1.7 (1.6-1.8) g/dL (n = 6) or not quantified (n = 8), potassium 10.6 (9.0-11.8) mmol/L, sodium < 100 mmol/L, and bile acids 0 lmol/L. This is the first report of biochemistry parameters in clinically healthy female stick insects. Larger sample sizes are needed to establish statistically valid reference ranges. Hemolymph collection techniques for male stick insects warrant further investigation.

Dirofilaria immitis Identified in Galapagos Sea Lions (Zalophus wollebaeki): A Wildlife Health and Conservation Concern.

The Galapagos sea lion (Zalophus wollebaeki), an endemic and endangered pinniped, faces an increasing threat due to infectious diseases related to domestic animals. Dirofilaria immitis, the parasite responsible for canine heartworm disease, is one such threat, as canine infections on the archipelago have been documented. We used a canine heartworm antigen test kit to analyze the blood from 25 juvenile Galapagos sea lions for D. immitis. Two (8%) sea lions tested positive for D. immitis antigen. Using morphologic and genetic assessments, we evaluated 20 filarial-like worms collected from within the heart of an adult male Galapagos sea lion during a previous routine postmortem examination. The intracardiac worms were morphologically consistent with adult D. immitis, and sequence analysis of targeted PCR amplicons confirmed their identity. This is the first report of D. immitis infection in Galapagos sea lions, which could become a major health problem for these pinnipeds. Further studies are necessary to confirm the level of threat from this parasite; however, widespread adoption of routine heartworm testing, prevention, and treatment in the canine population, and the control of mosquitos, could potentially reduce the disease impact on this endangered pinniped species.

Evaluating the Possibility of Transfusion Medicine, Through Crossmatching in Juvenile Galapagos Sea Lions (Zalophus wollebaeki).

The Galapagos sea lion (Zalophus wollebaeki) is an endemic pinniped to the Galapagos archipelago, and like most wild mammals, is at risk for anemia due to trauma, infectious disease, and poor nutrition. This study evaluated the health status of 26 juvenile Galapagos sea lions on the island of San Cristobal prior to evaluating 100 crossmatch combinations. On evaluation, all but one sea lion had no major systemic abnormalities. Of the 100 crossmatches performed, 23% had minor reactions. The most significant reaction was weak macroscopic agglutination found in 4% of samples. The small percentage of agglutination reactions suggests a small proportion of naturally occurring alloantibodies in this species and may be consistent with a low risk of acute immune-mediated hemolytic transfusion reaction.

Development and Evaluation of a Serum Transfusion Process in the Thorny Devil Stick Insect (Eurycantha calcarata).

The thorny devil stick insect (Eurycantha calcarata) is a common invertebrate maintained under human care. Blood (hemolymph) transfusions are a widely used therapeutic tool in other species, but investigation in terrestrial arthropods remains scarce. Study objectives were development and evaluation of a serum transfusion process in the thorny devil stick insect. Twenty-five clinically healthy adult insects (9.9-23.0 g) were enrolled and baseline body weights were collected. Hemolymph collection was not successful in males, thus, all were recipient only (n = 12, MR). Females were divided into donor (n = 4, FD) or donor and recipient (n = 9, FDR) groups. Females were manually restrained and up to 1 mL of hemolymph was collected from the abdominal segment caudal to the proximal hindlimb using an 18 g hypodermic needle and passive collection via gravity. Hemolymph was quantified, centrifuged, and the serum separated. Insects were then injected superficially at the lateral aspect of the abdomen with 0.01 mL/g FD serum (MR), lactated Ringer's solution (LRS) equal to collected hemolymph volume (FD), or a combination of FDR serum and LRS equal to collected hemolymph volume (FDR). Response to stimulation, surface temperature, and righting reflex and mortality were serially assessed for up to 24 h and 7 days, respectively, following injection. In FD, median (range) injected LRS dose was 0.04 (0.03-0.06) mL/g. In FDR, median (range) injected serum, LRS, and combined serum and LRS dose was 0.03 (0.02-0.04), 0.01 (0-0.04), and 0.04 (0.02-0.06) mL/g, respectively. A mild temperature increase (maximum +2.9°C) (MR n = 10, FD n = 3, FDR n = 8) and delayed righting reflex (MR n = 4, FD n = 3, FDR n = 7) occurred in a subset of insects following injection. Two deaths occurred at 2 min (n = 1, FDR) and 96 h (n = 1, FD) post-injection. This is the first report of serum transfusions in thorny devil stick insects, and while largely successful, minor to severe transfusion reactions may occur.

EVALUATION OF PROGNOSTIC INDICATORS FOR INJURED TURTLES PRESENTING TO A WILDLIFE CLINIC.

The Turtle Rescue Team (TRT) at North Carolina State University College of Veterinary Medicine currently sees over 500 chelonian cases a year. Each turtle receives a thorough physical examination as the main evaluation of its health because further diagnostics are cost prohibitive in a donation-based facility. Thus, there is a need to further understand how a few economical diagnostics can evaluate the prognosis of chelonians. The objectives were to evaluate lactate, PCV, total solids, glucose, temperature, and HR as prognostic indicators in multiple chelonian species and to determine how the change in lactate concentrations over a 24 h period affects prognosis. Eastern box turtles (Terrapene carolina carolina), yellow-bellied sliders (Trachemys scripta scripta), river cooters (Pseudemys concinna), painted turtles (Chrysemys picta), and common snapping turtles (Chelydra serpentina) were evaluated because of prevalence of presentation to the TRT, differences in habitat, and differences in physiology. Hyperlactatemia, an increased lactate over 24 h when the initial lactate concentration was greater than 5 mM/L, and anemia were determined to be significant prognostic indicators of mortality for all turtles, and hypoglycemia was a significant prognostic indicator of mortality for male turtles (P < 0.05).

Pharmacokinetics of ketorolac in juvenile loggerhead sea turtles (Caretta caretta) after a single intramuscular injection.

Ketorolac is a non-steroidal anti-inflammatory drug administered as an analgesic in humans. It has analgesic effects comparable to opioids but without adverse effects such as respiratory depression or restrictions because of controlled drug status. We designed this study to examine the potential of ketorolac as an analgesic for sea turtle rehabilitative medicine. Our objective was to determine the pharmacokinetics of a single 0.25 mg/kg intramuscular dose of ketorolac in a population of 16 captive-raised juvenile loggerhead sea turtles (Caretta caretta). A sparse sampling protocol was utilized, and blood samples were collected for 12 hours after administration of ketorolac. Samples were analyzed with high-pressure liquid chromatography (HPLC), and a nonlinear mixed effects model (NLME) was used to determine parameters for the population. With these methods, we identified a long elimination half-life (ßT1/2  = 11.867 hr) but a low maximum concentration (CMAX  = 0.508 µg/mL) and concentrations were below the level proposed to be therapeutic in humans (EC50  = 0.1-0.3 µg/mL) for most of the collection period. We conclude that ketorolac may not be an appropriate long-term analgesic for use in loggerhead sea turtles at this dose; however, it may have some benefit as a short-term analgesic.