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Hemolymph Chemistry Reference Ranges of the Chilean Rose Tarantula

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Hemolymph Chemistry Reference Ranges of the Chilean Rose Tarantula HEMOLYMPH CHEMISTRY REFERENCE RANGES OF THE CHILEAN ROSE TARANTULA GRAMMOSTOLA ROSEA (WALKENAER, 1837) USING THE VETSCAN BIOCHEMISTRY ANALYZER BASED ON IFCC-CLSI C28- A3 Author(s): Mark A. Eichelmann, D.V.M., and Gregory A. Lewbart, M.S., V.M.D., Dipl. A.C.Z.M., Dipl. E.C.Z.M. (ZHM) Source: Journal of Zoo and Wildlife Medicine, 49(3):528-534. Published By: American Association of Zoo Veterinarians https://doi.org/10.1638/2015-0145.1 URL: http://www.bioone.org/doi/full/10.1638/2015-0145.1 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Journal of Zoo and Wildlife Medicine 49(3): 528–534, 2018 Copyright 2018 by American Association of Zoo Veterinarians HEMOLYMPH CHEMISTRY REFERENCE RANGES OF THE CHILEAN ROSE TARANTULA GRAMMOSTOLA ROSEA (WALKENAER, 1837) USING THE VETSCAN BIOCHEMISTRY ANALYZER BASED ON IFCC-CLSI C28-A3 Mark A. Eichelmann, D.V.M., and Gregory A. Lewbart, M.S., V.M.D., Dipl. A.C.Z.M., Dipl. E.C.Z.M. (ZHM) Abstract: The use of invertebrate hemolymph chemistry analysis has the potential to become a major diagnostic tool. The goal of this study was to generate statistically sound hemolymph reference ranges from healthy tarantulas. Hemolymph was drawn from wild caught, acclimatized, and apparently healthy female Chilean rose tarantulas Grammostola rosea (Walkenaer, 1837) (n ¼ 43) using a modified technique. Hemolymph samples were separately analyzed using the Avian-Reptilian Profile Plus reagent rotor for VetScant for the following chemistries: aspartate aminotransferase, bile acids, creatine kinase, uric acid, glucose, total calcium, phosphorus, total protein, albumin, potassium, and sodium. With this method the authors were able to establish statistically sound reference ranges for aspartate aminotransferase, creatine kinase, glucose, phosphorus, and total protein. Further in situ studies will determine the practical usability of these values in the evaluation of tarantula health. Key words: Arachnida, Grammostola rosea, hemolymph chemistry, spider, Theraphosidae, VetScan. INTRODUCTION captured in Chile and shipped to the author’s laboratory in Germany (Ho¨ hle M, pers. comm.) Like many species of exotic pets, invertebrates from March to April. On the day of the arrival can provide the clinician with diagnostic challeng- each tarantula appeared healthy based on gross es. Utilizing tested and proven clinical pathology examination. Quarantine for the spiders lasted for methods provides for consistent and reliable data thatcanbeusedtoassistinclinicalcase 2 wk under the following husbandry parameters: management. Due to the small size and fragility temperature: 22 6 28C (71.6 6 3.68F) during the of many arachnid patients, the volume of hemo- day and 20 6 28C (68 6 3.68F) at night; humidity: lymph that can be obtained is necessarily small. 80%; feeding: after 2 wk house crickets (Acheta Thus, in some cases dilution is necessary in order domesticus) once and then weekly with adult dubia to meet the requirements of different chemistry roaches (Blaptica dubia) from the same source; analyzers. One advantage of the method used in water: cotton buds, which were replenished every this study is the low required sample size of 0.09– 4days. 0.12 ml, making it possible to extract hemolymph The spiders were restrained with a modified from animals with a very low body weight without technique. For this inverted pinching method, the dilution. This study was designed to investigate animals were approached cranially. Thumb and the development of a practical method of taran- middle finger were placed in the intercoxal spaces tula hemolymph collection and examination. between legs II and III while applying gentle pressure with the index finger on the carapace. MATERIALS AND METHODS Hemolymph withdrawal was achieved by intra- Forty-three female adult Chilean rose tarantu- cardiac puncture in which the needle was posi- las Grammostola rosea (Walkenaer, 1837) were tioned with the tip pointing upwards and the bevel towards the puncture site (Figs. 1, 2). When indicated, hemolymph stasis was achieved with From Tierarztpraxis Maja Firle´, Emser Strasse 40, 60486 sterile cotton swabs and n-Butyl cyanoacrylate Frankfurt, Germany and the University of Veterinary tissue glue (Surgibond Gewebekleber, SMI AG, Medicine Hannover, Foundation, Bu¨nteweg 9, 30559 St. Vith, 4780, Belgium). Hannover, Germany (Eichelmann); and the North Caro- The body weight of the animals ranged from 5 lina State University, College of Veterinary Medicine, Department of Clinical Sciences, 1060 William Moore to 13 g (mean 7.6, median 7.0, standard deviation Drive, Raleigh, NC 27606, USA (Lewbart). Correspon- 2.4). Physiologic hemolymph volume is estimated dence should be directed to Dr. Eichelmann (mark. at 20% of the body weight in one species of [email protected]). tarantula.35 For the purpose of this study, the 528 EICHELMANN AND LEWBART—HEMOLYMPH REFERENCE RANGES IN GRAMMOSTOLA 529 Figure 1. Intracardiac hemolymph withdrawal technique from an adult female Grammostola rosea, macroscopically. maximum hemolymph volume in ml was chosen as 10% of the physiologic hemolymph volume. The amount of hemolymph required by the analyzer was 0.09 ml, just below the 0.10 ml maximum for the smallest animal. Hemolymph samples were obtained using heparinized (Hepa- rin-Natrium-25000-ratiopharmt, 5000 UI/ml) 1- ml syringes fitted with 23-ga needles (BD Micro- lanceTM 23 G 1-inch No. 16 0.6–25 mm, Henry Schein, Langen, Hessen, 63225, Germany). These samples were directly transferred to a 1-ml Figure 2. Comparison between the classical hemo- lymph withdrawal technique (a)andthemodified lithium heparin tube (Multivettet 600 LH, Sars- method (b) from an adult female Grammostola rosea, tedt AG & Co., Nu¨ mbrecht, Nordrhein-West- microscopically (340). Urticating hair were removed falen, 51588, Germany) and centrifuged with an from the cutis with a cotton bud and the area was EBA 20 centrifuge (Andreas Hettich GmbH & lucubrated with 75% isopropanol prior to the proce- Co. KG, Tuttlingen Baden-Wu¨ rttemberg, 78532, dure to visualize the puncture site for imaging. The Germany) at 3.46 x g for 10 min. Hemolymph curved rigid cutis and hemolymph pressure leads to a supernatant was extracted and examined by a rapid ejection of fluid when the classical punction method is performed. With the modified method, the VetScant Point-of-Care Blood Analyzer, Model lesion is sealed much more efficiently with no observed No. 200-1000 (Abaxis Inc, Union City, CA 94587, disadvantages. USA) using Avian-Reptilian Profile Plus reagent rotors. Advisor v2.1 (Microsoft Office Professional Plus International recommendations for determina- 2010 Version 14.0.4760.1000t, Microsoft Corpo- tion of reference intervals continuously updated ration, Redmond, WA 98052, USA) and the by the International Federation of Clinical Chem- istry (IFCC) and the Clinical and Laboratory computed reference intervals placed in a spread- 13,19 Standards Institute (CLSI) confirm that a non- sheet according to CLSI guidelines. parametric method is preferred if the number of Following the hemolymph collection, the ani- reference individuals is between 1 and 120. If the mals were maintained as during the quarantine number of specimens is low, the CLSI guideline period and monitored daily for 6 mo. provides a calculation of reference values based on a robust method. A distribution as close as RESULTS possible to the Gaussian distribution is preferred. Therefore, a Box-Cox transformation is often Aspartate aminotransferase (AST): After Box- used to transform data to normality. The data of Cox transformation (k1 ¼ 0.087, k2 ¼ 0.371), the this study was processed by Reference Value Anderson-Darling test indicated a Gaussian dis- 530 JOURNAL OF ZOO AND WILDLIFE MEDICINE tribution (P ¼ 0.855). The standard method for Total protein (TP): The results indicated a high Box-Cox transformed values lead to the reference possibility (P ¼ 0.788 and visualization via Q-Q interval for AST ranging between 1.9 (90% plot) for a Gaussian distribution of untrans- confidence interval ¼ 0.8–3.6 U/L) and 54.7 U/L formed data. Computed with the standard meth- (90% confidence interval ¼ 44.0–65.7 U/L). One od, the reference interval ranged between 1.7 and outlier was detected by the Tuckey method (57.9 9.0 g/dl (90% confidence interval, 1.0–2.5 g/dl U/L) and was retained. and 8.2–9.8 g/dl). Bile acids (BA): The results for BA in this Albumin (ALB), Globulin (GLOB): Precision of study were 35 lmol/L for each specimen, which is the results for ALB was low because values are the lower limit for the dynamic range of the rotor. concentrated in the area of the rotors lower Computing reference intervals was not possible. detection limit (1 g/dl). The amount of GLOB is Creatine kinase (CK): The results for CK were directly dependent on ALB because it is calculat- quite similar to those for AST. Values trans- ed from ALB and TP. Therefore, it was not formed via Box-Cox (k1 ¼ 2.33, k2 ¼ 0.431) showed possible to compute the reference interval for a suspected Gaussian distribution (P ¼ 0.843). ALB and GLOB. Values calculated lead to a reference interval from Potassium (Kþ): Data distribution of Kþ was 1.2 (90% confidence interval ¼0.5 to 3.4 U/L) to very compact around the lower limit of the rotors 50.3 U/L (90% confidence interval ¼ 41.6–59.1 U/ dynamic range (37% of the values: 25.5% at 1.5 L).
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