Matthew T. Hamilton

Evaluating the effect of sample type on American alligator (Alligator mississippiensis) analyte values in a point-of-care blood analyser

Point-of-care blood analyzers provide researchers with the opportunity to collect blood biochemistry data for a variety of non-domesticed organisms while in the field. We investigated the use of an i-STAT analyzer and CG4+ cartridge to analyze whole blood, fresh plasma, and previously frozen plasma samples collected from American alligators.

Blood and Plasma Biochemistry Reference Intervals for Wild Juvenile American Alligators (Alligator mississippiensis)

Abstract: American alligators (Alligator mississippiensis) are one of the most studied crocodilian species in the world, yet blood and plasma biochemistry information is limited for juvenile alligators in their northern range, where individuals may be exposed to extreme abiotic and biotic stressors. We collected blood samples over a 2-yr period from 37 juvenile alligators in May, June, and July to establish reference intervals for 22 blood and plasma analytes. We observed no effect of either sex or blood collection time on any analyte investigated. However, our results indicate a significant correlation between a calculated body condition index and aspartate aminotransferase and creatine kinase. Glucose, total protein, and potassium varied significantly between sampling sessions. In addition, glucose and potassium were highly correlated between the two point-of-care devices used, although they were significantly lower with the i-STAT 1 CG8+ cartridge than with the Vetscan VS2 Avian/Reptile Rotor. The reference intervals presented herein should provide baseline data for evaluating wild juvenile alligators in the northern portion of their range.

Influence of Collection Time on Hematologic and Immune Markers in the American Alligator (Alligator mississippiensis)

Crocodilians are important keystone species and indicators of environmental health. Much remains unknown, however regarding utility of field-collected crocodilian blood samples for ecologic assessments. Field sampling sites are also often distant to analysis centers, necessitating development of new techniques and panels of assays that will yield environmentally relevant data. Stability and viability of hematological and immunological indices have been of particular interest for linking ecosystem health to biomarkers in resident species. In this study, we investigated the effect of time at analysis post-blood sampling at 4 and 24 hr on a panel of potential biomarkers in alligator blood. Our results suggest alligator blood samples can be reliably evaluated for both hematologic and immunologic profile 24 hr after sampling.

First case of ranavirus and associated morbidity and mortality in an eastern mud turtle Kinosternon subrubrum in South Carolina

Ranaviruses are double-stranded DNA viruses that infect amphibians, fish, and reptiles, causing global epidemics in some amphibian populations. It is important to identify new species that may be susceptible to the disease, particularly if they reside in the same habitat as other at-risk species. On the Savannah River Site (SRS) in Aiken, South Carolina, USA, ranaviruses are present in several amphibian populations, but information is lacking on the presence, prevalence, and morbidity of the virus in reptile species. An eastern mud turtle Kinosternon subrubrum captured on the SRS in April 2014 exhibited clinical signs of a ranaviral infection, including oral plaque and conjunctivitis. Quantitative PCR analyses of DNA from liver tissue, ocular, oral, nasal, and cloacal swabs were all positive for ranavirus, and sequencing of the template confirmed infection with a FV3-like ranavirus. Histopathologic examination of postmortem tissue samples revealed ulceration of the oral and tracheal mucosa, intracytoplasmic epithelial inclusions in the oral mucosa and tongue sections, individualized and clusters of melanomacrophages in the liver, and bacterial rods located in the liver, kidney, heart, stomach, and small intestine. This is the first report of morbidity and mortality of a mud turtle with a systemic ranaviral infection.

Effects of selenium exposure on the hematology, innate immunity, and metabolic rate of yellow-bellied sliders ( Trachemys scripta scripta )

Selenium (Se) is a naturally occurring essential element that can be toxic to vertebrates at high concentrations. Despite studies that have documented that wild reptile species can accumulate copious amounts of Se, little is known regarding specific toxicologic effects of Se. In this study, 70 juvenile yellow-bellied sliders (Trachemys scripta scripta) were exposed to one of three seleno-l-methionine (SetMet) treatments (control, n = 24; 15 mg/kg, n = 23; and 30 mg/kg, n = 23) via weekly oral gavage for 5 weeks. At the conclusion of the experiment, kidney, liver, muscle, and blood samples were collected for quantitative Se analysis. Turtles in the SeMet treatment groups accumulated significantly higher amounts of Se in all tissue types relative to controls (all p < 0.001). Turtles in the 30 mg/kg SeMet group also accumulated significantly higher amounts of Se compared to the 15 mg/kg group (all p < 0.001). Although toxicity thresholds for reptiles have not been established, Se concentrations in liver tissue from both SeMet treatment groups exceeded reported avian toxicity thresholds for liver tissue. Neither oxygen consumption nor innate bactericidal capacity were impacted by SeMet exposure. However, turtles in the 30 mg/kg SeMet group exhibited anemia, which has been reported in other vertebrates exposed to Se. Furthermore, juvenile T. s. scripta in the 30 mg/kg SeMet group experienced 17% mortality compared to 0% in the 15 mg/kg treatment and control groups. To our knowledge, this study is the first to report dose-dependent Se-associated anemia and mortality in a chelonian species.

Corticosterone in American alligator (Alligator mississippiensis) tail scutes: Evaluating the feasibility of using unconventional samples for investigating environmental stressors

Baseline plasma corticosterone (CORT) concentrations have been widely used to investigate the effects of stressors in wild and captive crocodilians. However, collecting baseline plasma CORT samples from wild crocodilians may be particularly difficult due to the capture and handling protocols used for large individuals. Thus, it may prove beneficial to use recently modified techniques for extracting CORT deposited in keratinized and non-keratinized tissues to better quantify the effects of long-term stress in crocodilians. In this study, we investigated the feasibility of using American alligator (Alligator mississippiensis) tail scute tissues to quantify CORT by collecting blood and tail scutes from 40 alligators before and after a short-term handling stressor. The objective of the current study was to better understand CORT deposition in crocodilian scutes and whether short-term increases in CORT could be detected. We found that CORT can be reliably extracted from alligator scute tissue and quantified using a commercially available enzyme immunoassay. However, there was a significant increase in scute CORT concentrations following an alligator being exposed to a short-term stressor (p = 0.017), although the magnitude of change was less than observed in plasma samples from the same individuals (p = 0.002). Furthermore, our results indicate that there was a significant effect of body condition on an alligators post-stressor CORT concentration (p = 0.02). While our study is among the first to experimentally examine the usefulness of tissue CORT in crocodilians, a combination of field and laboratory experiments are needed to better understand deposition rates of CORT in scute tissues and to further validate the usefulness of tissue glucocorticoids for evaluating the effects of stress.

Coal combustion residues and their effects on trace element accumulation and health indices of eastern mud turtles (Kinosternon subrubrum)

Coal combustion is a major energy source in the US. The solid waste product of coal combustion, coal combustion residue (CCR), contains potentially toxic trace elements. Before 1980, the US primarily disposed of CCR in aquatic settling basins. Animals use these basins as habitat and can be exposed to CCR, potentially affecting their physiology. To investigate the effects of CCR on eastern mud turtles (Kinosternon subrubrum), we sampled 30 turtles exposed to CCRs and 17 unexposed turtles captured in 2015-2016 from the Savannah River Site (Aiken, SC, USA). For captured turtles, we (1) quantified accumulation of CCR in claw and blood samples, (2) used bacterial killing assays to assess influences of CCR on immune responses, (3) compared hemogregarine parasite loads, and (4) compared metabolic rates via flow-through respirometry between CCR-exposed and unexposed turtles when increased temperature was introduced as an added stressor. Turtles exposed to CCR accumulated CCR-associated trace elements, corroborating previous studies. Blood Se and Sr levels and claw As, Se, and Sr levels were significantly higher in turtles from contaminated sites. Average bacterial killing efficiency was not significantly different between groups. Neither prevalence nor average parasite load significantly differed between CCR-exposed and reference turtles, although parasite load increased with turtle size. Regardless of site, temperature had a significant impact on turtle metabolic rates; as temperature increased, turtle metabolic rates increased. The effect of temperature on turtle metabolic rates was less pronounced for CCR-exposed turtles, which resulted in CCR-exposed turtles having lower metabolic rates than reference turtles at 30 and 35 °C. Our results demonstrate that turtles accumulate CCR from their environment and that accumulation of CCR is associated with changes in turtle physiological functions when additional stressors are present.