Use of a Cardiac Access Port for Repeated Collec- tion of Blood Samples from Desert Tortoises (Gopherus agassizii)

J. WIMSATT, DVM, PHD, J. D. JOHNSON, DVM, AND B. A. MANGONE, DVM

Abstract ͉ Repeated collection of blood samples may be required during pharmacokinetic studies and can be challenging in tortoises because of size and location of the major vessels. The technique described here is relatively safe, inexpensive, and potentially reversible. A sterile rubber stopper is surgically inserted into a hole drilled in the plastron over the heart to allow collection of multiple blood samples over a period of several days or weeks, using a 27-gauge needle. During surgical placement of the port, pericardial excision was performed on some tortoises, but not others, to assess the risk of cardiac tampanade when collecting samples. In 13 of 14 tortoises, the technique appeared to be well tolerated. Pericardiectomy did not appear to be neces- sary. Ports were left in 6 tortoises for 6 months without evidence of adverse effects. Ports were removed from 5 other tortoises, and the defect in the plastron was repaired; these tortoises appeared healthy 8 months later. Four months after removal of the port, 2 other tortoises were euthanatized as a result of advanced respiratory tract disease. Postmortem examination revealed that the plastron defects were completely filled with bone; however, they also had evidence of mild fibrotic myocardial changes that were attributed to repeated cardiocentesis.

Repeated collection of blood samples in tortoises for the pur- considered to be carriers capable of spreading the Mycoplasma or- poses of research or clinical monitoring can be challenging. ganism. The U. S. Fish and Wildlife Service granted permission to Several blood collection methods can be used for turtles and use these seropositive tortoises for developing this technique as a tortoises. The jugular veins are potentially accessible for direct prelude to planned pharmacokinetic studies for the evaluation of venipuncture and catheterization (1–3). However, depending a new antimycoplasmal antibiotic. on the species, chemical immobilization or restraint is often Tortoises were housed in dirt substrate enclosures, using con- necessary. Therefore, repeated collection of samples from this trolled light conditions (12-h light: 12-h dark). Ambient site may be impractical. Other locations for blood collection in temperature was maintained at 29.4 to 33.3ЊC (85 to 92ЊF). Tor- chelonians include the brachial arteries and veins, scapular veins, toises had unlimited access to water and grass hay and were given occipital sinus, tail vein, orbital sinus, and toenails (4–9). It has leafy greens once weekly for 2 months before the study. All pro- also been reported that tortoises survive collection via repeated cedures described were approved by the Institutional Animal intracardiac puncture without adverse effects (7). Care and Use Committee of Colorado State University. The heart in chelonians is likewise less accessible than in other Tortoises were anesthetized, using 2 methods. In the first group, reptilian species, limiting the usefulness of direct cardiocentesis. 8 tortoises were induced with 5% isoflurane and 1.5 L of O2/min. In juvenile chelonians, a needle can be passed through the plas- After intubation with an appropriately sized endotracheal tube (e. tron into the heart before the shell calcifies (2, 3). A similar g., outer diameter of 2.0 to 3.5 mm), positive-pressure ventilation procedure has been described for adult tortoises, using an 18- was performed at 30-sec intervals. Anesthesia was maintained with gauge needle to configure a pilot hole at the same anatomic 1 to 3.5% isoflurane and 1.5 L of O2/min and by using intermit- location as described for juvenile tortoises (10). Alternatively, tent positive-pressure ventilation (10 cm of H2O, 1 to 2 times/ samples can be collected by drilling a hole in the plastron, col- min). In the second group, tortoises were given 25 ml of 0.9% lecting a blood sample from the heart, and repairing the plastron NaCl solution/kg of body weight epicoelomically 1 h before in- defect after collection of the sample is complete (2, 7). Blind duction. Tortoises were induced with propofol (4 to 7 mg/kg percutaneous approaches for intracardiac puncture have also diluted 1:2 in 0.9% NaCl solution) administered slowly into a jugu- been used, including directing a spinal needle through the axil- lar vein as a bolus injection. Anesthesia was maintained with 1 to lary or inguinal fossas; however, other organs sometimes were 3.5% isoflurane and 1.5 L of O2/min, with lower percentages of perforated and aspirated by use of this procedure (3, 11). In isoflurane administered via mask. other studies, a hole was made in the plastron by use of a saw, After induction of anesthesia, tortoises were positioned in trephine, or drill, and the hole then was left open to provide dorsal recumbency, the entire plastron was prepared for asep- investigators with an adequate view of structures and to allow tic surgery, and sterile drapes were applied. A sterile repeated cardiocentesis; or the site was closed with epoxy (12). stainless-steel drill bit (11/32-in in diameter, 0.8731 cm in di- ameter) and cordless drill were used to create a hole Materials and Methods perpendicularly through the plastron at a point on the midline Fourteen wild-caught subadult to adult desert tortoises (Gopherus at the junction of the humeral and pectoral scutes or just cau- agassizii) that weighed between 1.02 and 3.36 kg were used in the dal to that point, toward the junction of the pectoral and study. These tortoises were seropositive for Mycoplasma agassizii, abdominal scutes (Figure 1). The hole in the outer keratin scute the agent believed responsible for upper respiratory disease syn- and underlying dermal bone was slowly and carefully enlarged. drome. Tortoises were removed from the wild because they were Excessive downward pressure and high drill speeds were avoided, especially when the bit had almost passed through the plastron. Department of Clinical Sciences, College of Veterinary and Biomedical Sciences, Slow drilling speeds insured that thermal bone necrosis was mini- Colorado State University, Fort Collins CO 80523. mized. A sterile 0.9% NaCl solution was continually applied

Volume 37, No. 6 / November 1998 CONTEMPORARY TOPICS © 1998 by the American Association for Laboratory Animal Science 81 FIG. 2. Collection of a blood sample from a previously prepared cardiac access port, using a 27- gauge, 1.25-in needle. Repeated collection of large samples without lymph contamination are possible, and appear to cause less stress to the tortoises than collection of samples from the jugular veins, which requires that the head be repeatedly extended from the carapace.

FIG. 1. Photograph showing proper placement of the cardiac access port collection. Epoxy typically extended over the adjoining plas- on the midline just caudal to the intersection of the pectoral and hu- meral scutes of the plastron of a desert tortoise. Epoxy is visible in the tron to a distance approximately 1 cm from the edge of the area adjacent to the port and extends up over the port, protecting it stopper. Once the epoxy hardened, tortoises were returned to from abrasion during normal ambulation. their original enclosures. Prior to collection of each sample, the port was aseptically during drilling to prevent excessive heating of tissues. At this cleaned by use of a chlorhexidine-based surgical soap, which site, plastrons are approximately 5 to 6 mm in thickness in adult was allowed to remain in contact with the port for a minimum of tortoises and 2.5 to 4 mm in thickness in subadults. During the 4 minutes. The injection depression was cleaned with a sterile final phase of drilling through the plastron, intermittent posi- cotton-tipped swab prior to needle insertion. Tortoises were tive-pressure ventilation was briefly halted to prevent the heart manually suspended in ventral recumbency to enable collection from being elevated into the path of the drill. of samples. Alternatively, tortoises were positioned in dorsal re- After penetrating the underlying bone, the thick fibrous peri- cumbency and a rolled towel was placed transversely under their osteum and coelomic membrane were encountered. A curved body so that their cranial end was on an upward incline of ap- No. 12 scalpel blade (Becton-Dickinson, Rutherford, NJ) was used proximately 20Њ. Blood samples collected from tortoises in ventral to carefully excise the underlying periosteal and coelomic mem- recumbency usually required only a 27-gauge, 5/8-in needle, branes at the bottom of the hole. Occasionally, a small pectoral which was directed perpendicular to the plastron and inserted muscle attachment and intracoelomic fat were removed from to the needle hub. For tortoises in dorsal recumbency, a 27-gauge, the surgical path, using blunt dissection. Pericardiectomy was 1- or 1.5-in needle (depending on size of the tortoise) yielded performed in the initial 8 tortoises. Small rat-toothed cornea more consistent results, but rarely required that the needle be forceps (Sontec, Denver, CO) were used to elevate the pericar- fully inserted to the hub, even in larger tortoises (i. e., body weight dium from the heart, and a circular defect approximately 1.0 of more than 2 kg). Gentle steady suction with a 3-ml syringe was cm in diameter was created in the pericardium ventrally, using used to collect blood samples (Figure 2). tendonotomy scissors (Sontec, Denver, CO) or similar small, About once each month, additional epoxy was added to the narrow-bladed, blunt-tipped scissors. Care was taken to coordi- port site, because abrasion caused by housing the tortoises on a nate exteriorization of the pericardium with inhalation, because natural substrate tended to abrade the epoxy over time, exposing it is at that time that the heart could rise directly into the open- the port directly to wear. Port removal was easily accomplished. ing in the plastron. Large embedded vessels and nerves often The outer epoxy was removed, the body of the plug was grasped had to be avoided during incision of the pericardium. After peri- with a medium-sized hemostat, and the plug was rocked back and cardiectomy, the heart was easily visible through the hole and forth until it was freed. This technique was performed aseptically varied from bright red to a paler yellow-gray typical of many rep- and was used for replacement of ports when they were damaged tilian tissues. Pericardiectomy was not performed in the 7 or for port removal at completion of blood collection. Repair of tortoises in the second group. the plastron consisted of filling the plastron defect with bone wax Once the access hole was created, a sterile rubber stopper and then applying an epoxy patch externally over the site. from a 3-ml evacuated glass tube (Vacutainer®, Becton- Dickinson, Rutherford, NJ) was inserted (Figure 1). The stopper Results was trimmed to the thickness of the plastron, ensuring that it All tortoises resumed eating within 24 h after placement of was level with the visceral aspect of the dermal bone surface the port and did not have apparent adverse effects as a result of adjacent to the heart before placement. The rubber stopper fit the procedure. Blood collection was initiated 48 h after port snugly, but was not so tight as to cause pressure necrosis. Epoxy placement. Repeated collection of 9 to 23 blood samples (1 ml/ ® (Duro , Loctite Co., Cleveland, OH) was used to seal the rub- sample) was successfully performed (i. e., < 1% of body weight). ber stopper in position; the ventral surface of the stopper was Samples obtained from these tortoises during a 9-day period were coated with epoxy, but the injection depression in dorsal as- placed in syringes coated with 1,000 u/ml sodium heparin for pect of the rubber stopper was left uncovered to facilitate sample use in antibiotic pharmacokinetic studies.

82 CONTEMPORARY TOPICS © 1998 by the American Association for Laboratory Animal Science Volume 37, No. 6 / November 1998 Blood gas analysis of blood samples collected via this tech- venipuncture may be difficult to perform. Sample collection via a nique indicated that left ventricular or mixed left and right port can be used in chelonian species for which it is difficult to ventricular samples were obtained depending on the position obtain access to the jugular veins without chemical restraint; how- of the needle in the ventricle at the time of sample collection. ever, the landmarks for placement of a port in those species may Mean cardiac blood pressures could also be obtained, using a differ slightly from those described here. Sample collection with 20-gauge intravenous catheter inserted into the heart through this technique is possible under natural or semi-natural labora- the port. tory conditions, and we believe it will greatly reduce restraint-related Of the 14 tortoises reported here, 13 appeared to tolerate the stress caused by extending the neck during collection of blood technique well. The port was maintained in these 13 tortoises samples. On the basis of results obtained for the series of tortoises for 3 to 6 months, apparently without adverse effects. The re- reported here, ports apparently can be successfully removed. Con- maining tortoise died 2 days after initiation of the repeated tinued clinical evaluation of the surviving tortoises indicated that collection period, and a postmortem examination was per- long-term complications are rare. formed. Grossly, important lesions (e. g., tampanade) were not evident. Histologic examination revealed minimal myocardial Acknowledgments changes resulting from collection of samples; only small self-con- The authors thank Heather Branvold and Alyssa Tothill for tained hemorrhages on the myocardium at the puncture site providing care for the tortoises. Support was provided, in part, were seen. Evidence of advanced upper respiratory disease syn- by the Zoological Medicine Service and Department of Clinical drome as a result of infection with Mycoplasma agassizii was Sciences, College of Veterinary Medicine and Biomedical Sci- observed and may have caused the death of this tortoise. Alter- ences, Colorado State University. natively, cardiac arrest could have been the cause of death. Two additional tortoises were euthanatized 4 months after their ports were removed. These tortoises were euthanatized because References of progression of upper respiratory disease syndrome and weight 1. Jacobson, E. R. 1987. Reptiles. In: Harkness, J. E., (ed.), loss. Both of these tortoises were in the first group. In these 2 Vet. Clin. North Am. Small Anim. Pract. 17:1203–1225. tortoises, complete self-repair of the plastron had occurred dur- 2. Jenkins, J. R. 1996. Diagnostic and clinical techniques. pp. ing the 4-month interval since port removal. Histopathologically, 264-276, In: Mader, D. R., (ed.), Reptile medicine and sur- the myocardium had mild fibrotic changes that were probably gery. W. B. Saunders Co., Philadelphia, Pa. associated with the repeated collection procedure performed 3. Jacobson, E. R. 1992. Laboratory investigations. pp 49-62, previously. Other changes were not detected, and the pericar- In: Beynon, P. H., (ed.), Manual of reptiles, J. Looker Print- dium had completely regenerated. ers, Shurdington, England. None of the other tortoises had adverse effects during blood 4. Avery, H. W. and L. J. Vitt. 1984. How to get blood from a collection or for 8 months after completion of this study. In this turtle. Copeia 1:209–210. small series, cardiac problems appeared to be rare, and tortoises 5. Dessauer, H. 1970. Blood chemistry of reptiles: physiologi- with and without pericardiectomy tolerated frequent sample cal and evolutionary aspects. pp. 1–72. In: Gans, C. and T. collection equally well. Moreover evidence of pressure necrosis S. Parsons, (eds). Biology of the reptilia. Vol. 3, Morphol- was not found at the plug-plastron interface in any tortoises at ogy C, Academic Press, New York. the time of port removal. For 5 tortoises, removal of the port 6. McDonald, H. S.1976. Chapter 2. Methods for the physi- and repair of the plastron as long as 8 months after insertion of ological study of reptiles. pp 19–125, In: Gans, C., (ed.), the port did not appear to cause adverse effects. Biology of the reptilia. Vol. 5 (Physiology A), Academic Press, London. Discussion 7. Maxwell, J. H. 1979. Turtles: perspectives and research. pp The materials needed for port placement are readily available 127–152, In: Harles, M. and H. Morlock, (eds.), John Wiley and inexpensive. It was advantageous to dull a sharp drill bit & Sons, New York. before attempting to penetrate the plastron. Dulling of the bit 8. Nagy, K. A. and P. A. Medica. 1986. Physiological ecology could be accomplished with a metal file or by repeated drilling of desert tortoises in southern Nevada. Herpetologica into wood before use on the plastron. Sharper drill bits had a 42:73–92. tendency to cut and rip through the periosteum and coelomic 9. Rosskopf, W. J. 1982. Normal hemogram and blood chem- membrane on penetration of the bone, whereas duller drill bits istry values for California desert tortoises. Vet. Med. Small would push the periosteum away and not perforate further when Anim. Clin. 77:85–87. the internal aspect of the dermal bone was reached. 10. Gandal, C. P. 1958. A practical method of obtaining blood Pericardiectomy appeared to be unnecessary. In small domes- from anesthetized turtles by means of cardiac puncture. tic animals, the pericardium appears to be nonessential and can Zoologica 43:93–94. be fenestrated or removed without consequence (13). In theory, 11. Stephens, G. A and J. S. Creekmore. 1983. Blood collec- the risk of tampanade might be reduced when sample collection tion by cardiac puncture in conscious turtles. Copeia: is performed with a tortoise in a sternal position after the fenestra- 2:522–523. tion is made. However, signs indicative of cardiac tampanade were 12. Crawshaw, G. J. and P. Holz. 1996. Comparison of plasma not observed in any tortoises in this small series. biochemical values in blood and blood lymph mixtures from Although invasive, collection of blood samples from the heart red-eared sliders, Trachemys scripta elegans. Bull. Assoc. Rept. via a surgically placed port has the potential advantage of provid- Amphib. Vet 6:7–9. ing blood samples uncontaminated with lymph (12). The 13. Orton, E. C. 1995. Chapter 17. The pericardium. pp 177- technique described here greatly facilitated repeated collection 184, In: Orton, E. C., (ed.), Small animal thoracic surgery. of blood samples and decreased the number of tortoises needed Williams and Wilkins, Malvern, Pa. to complete pharmacokinetic studies. These are potential advan- 14. Raphael, B. L., M. Papich, and R. A. Cook. 1994. Pharma- tages over studies that use conventional blood collection techniques cokinetics of enrofloxacin after a single intramuscular (14). Collection of blood samples via a port is often preferable to injection in Indian star tortoises (Geochelone elegans). J. Zoo conventional methods when studying smaller tortoises in which Wildl. Med. 25:88–94.

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