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Comparative Medicine Cystic Calculus in a Laboratory-housed Green Anole (Anolis carolinensis

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Comparative Medicine Cystic Calculus in a Laboratory-housed Green Anole (Anolis carolinensis
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  Comparative MedicineCopyright 2017 by the American Association for Laboratory Animal ScienceVol 67, No 2April 2017Pages 1–4 1 Green anoles (  Anolis carolinensis ) have a long history of use in ecological and evolutionary biologic research. 9  The anole’s small size, ready availability from the wild, and extensively studied ecology make this species a highly desirable animal model. 10  Reptiles in general and anoles specifically have been studied extensively and are easy to evaluate in terms of energetic and whole-organism performance traits, such as sprint speed. Sprint speed is a function of several factors, including stride length, stride frequency, muscle cross-sectional area, and muscle fiber type. 6  Sprint speed is an important locomotor trait and is neces-sary for prey capture and predator evasion. 9 The order Squamata contains more than 9400 species of lizards and snakes. 12  Cystic calculi in squamates have been reported in species with a urinary bladder. The presenting clinical signs for this condition range from nonspecific signs of inappetence and depression to signs indicating pelvic organ dysfunction, such as constipation, dystocia, cloacal prolapse, and rearlimb parapa-resis. 2  However, anoles are prey species and therefore will hide clinical signs of illness as long as possible. Treatment for cystic calculi in reptiles is usually surgical and carries a good prognosis as long as the stone can be removed successfully. 2 An adult, male green anole (  Anolis carolinensis ) was presented for anorexia. The anole was wild-caught in New Orleans, Louisi-ana, and housed in a laboratory setting at the University of New Orleans, where it was involved in a study evaluating the effects of diet restriction on locomotor performance. This case represents the first report of a cystic calculus in a wild-caught, laboratory-housed green anole .  Because the initial clinical signs of a cystic calculus in an anole may be subtle and nonspecific, we also pres-ent sprint-speed and morphologic data from the source study to further characterize the consequences of this lesion on the overall locomotor performance and clinical condition of the anole. Materials and Methods Information regarding source study.   Housing.  In the source study, anoles were housed singly in separate 30 ×  16 ×  16-cm cages containing 30 ×  0.5-cm perches. The room was maintained on a 12:12-h light: dark photoperiod. All shelves holding lizard cages were equipped with fluorescent bulbs to provide UV light. In addition, each pair of cages was provided with a 75-W incan-descent bulb directed toward the perch to allow opportunities for thermoregulation and basking. The cages were lined with a substrate of cypress mulch, which was cleaned and replaced with fresh mulch every 2 wk. To ensure appropriate hydration, the anoles were misted daily by using a misting bottle sprayed 2 to 3 times into each terrarium. The diet comprised live crickets dusted with calcium powder (Fluker, Port Allen, LA) and provided with-out restriction. Ambient temperature was maintained at 75 to 90 ° F (23.9 to 32.2 ° C), and the humidity was kept above 50%. The anoles’ care and usage was approved by University of New Or-leans IACUC and was consistent with the Guide for the Care and Use of Laboratory Animals . 7 Morphologic measurements.  To quantify growth, morphologic measurements including snout–vent length, tail-base thickness, and body weight were measured at the start and end of the ex-periment. Sprint-speed performance.  For each measurement of sprint-speed performance, each anole was removed from its enclosure and immediately chased down a 2-m racetrack into a black bag. Each anole was run 3 times per measurement; trials were sepa-rated by at least 1 h. The racetrack consisted of a wooden dowel (length, 2 m; diameter, 5 cm) that was covered in cork (for trac-tion) and equipped with vertically paired infrared photocells at 25-cm intervals so that a running anole broke the beams se-quentially; the elapsed time (in milliseconds) for each interval was computer-recorded. The track was placed at a 45 °  angle, to simulate natural conditions. 4  This procedure is a standard test for lizards and yields highly repeatable results. 4  Sprint speed was measured 1 wk before the beginning of the experiment and then at the end of the experiment. Case Report  Cystic Calculus in a Laboratory-housed Green Anole (  Anolis carolinensis ) Leslie L Birke, 1, * Ann M Cespedes, 2  Emma R Schachner, 3  and Simon P Lailvaux 2 An adult, male, wild-caught, laboratory-housed green anole (  Anolis carolinensis ) on a locomotor performance study was pre-sented for anorexia. The anole exhibited a 26% weight loss and a thin body condition but was otherwise alert and active. Despite supportive care, the anole’s clinical condition deteriorated, necessitating euthanasia. Postmortem examination revealed a 4.5 mm ×  2.5-mm cystic calculus, which consisted entirely of sodium urate. Here we describe the clinical findings and locomotor consequences of this disease in a green anole. Although urolithiasis has been reported clinically in reptiles, this report presents the first case of a cystic calculus in a laboratory-housed green anole. Received: 13 Jul 2016. Revision requested: 01 Sep 2016. Accepted: 18 Oct 2016. 1 Division of Animal Care and 3 Department of Cell Biology and Anatomy, Louisiana State University Health Sciences Center, New Orleans, Louisiana, and 2 Department of Biology, University of New Orleans, New Orleans, Louisiana*Corresponding author. Email: lbirke@lsuhsc.edu  Vol 67, No 2Comparative MedicineApril 2017 22 Clinical and diagnostic methods.   Urolith analysis.  The Minne-sota Urolith Center uses quantitative techniques including op-tical crystallography and infrared spectroscopy to identify the majority of uroliths submitted to it. For the current case, optical crystallography was performed by using a polarizing microscope. Trained technicians removed crystals from several areas within the sectioned urolith, with the aid of a stereomicroscope. Crys-tals, immersed in oils of known refractive index, were viewed through a polarizing microscope (BX50, Olympus, Tokyo, Japan, and Eclipse 50i, Nikon, Tokyo, Japan). On the basis of the refrac-tive index and microcrystalline properties, a determination of mineral composition was made. Infrared spectroscopy detects the vibrational characteristics of chemical functional groups. Diffuse reflectance infrared Fournier transform spectroscopy and attenu-ated total reflection analysis allow for determination of composi-tion with minimal sample preparation. The resulting spectrum was compared with purchased and lab-made spectral libraries of known compounds to determine the sample composition (Nico-let iS10 Infrared Spectroscope [ThermoScientific, Waltham, MA] with OMNIC and OMNIC Specta subtraction software). 13  Anatomic specimens.  To demonstrate the location of the urinary  bladder in an anole, anatomic specimens of  A. carolinensis  were obtained (Carolina Biological, Burlington, NC). In one specimen (Figure 1), a lateral incision was made to access the coelomic cav-ity, expose the urinary bladder, and inject the bladder with red latex solution (lab grade; catalog no. 470024-608, Ward’s Science, Rochester, NY). In another specimen, the cloaca was cannulated and injected with red latex dye, which filled the distal aspect of the colon (Figure 2). The specimens were then frozen overnight to solidify the latex and then dissected from a lateral approach to expose the pelvic viscera. The empty bladder lay dorsocranial to the fat pads. Case Report Physical exam, morphologic, and performance data.  The weight and tail-base thickness data for this anole are displayed in Figure 3. This anole was in thin body condition, and his weight had dropped from 5.25 g to 3.89 g in a 4-wk period, equivalent to a 26% weight loss and 1.54 SD below the initial mean body mass for all subject in the study. The BMI values at baseline through week 4 were 13.05 ×  10 –4 , 12.75 ×  10 –4 , 11.36 ×  10 –4 , 10.99 ×  10 –4 , and 9.67 ×  10 –4 , respectively. The anole was not interested in live prey. On physical exam, the oral cavity was negative for inflamma-tion or erythema. The eyes were slightly sunken, indicating mild dehydration. Despite his anorexia and weight loss, the anole was active and alert. The snout–vent length was 63.43 mm. His base-line sprinting performance rank was in the 10th percentile among the 60 adult male anoles in the study. Tape tests for external para-sites were negative as was a direct fecal examination. The anole was given fresh crickets to stimulate food consumption and extra mistings with tap water for hydration.The next day, the anole had still not eaten, so 0.1 mL liquid diet (Emeraid Carnivore, Emeraid, Cornell, IL) was adminis-tered by gavage. Extra crickets were provided the next day, but the patient was still not interested in live prey. Given the lack of response to supportive care and because continued gavage treat-ment would comprise experimental data, it was determined that a humane endpoint had been reached. The anole was euthanized  by a 2-step procedure involving intracoelomic injection with 1% (v/v) buffered MS222 (0.025 mL per gram of body mass) for Figure 1.  Anatomic specimen of  A. carolinensis . The arrow indicates the latex-injected urinary bladder. Figure 2.  Anatomic specimen of  A. carolinensis . The white line circles the unstained urinary bladder. The distal colon is injected with red latex. anesthesia, followed after loss of consciousness by intracoelomic injection of 50% (v/v) MS222 (0.1 mL per gram of body mass) for euthanasia. 3 Necropsy.  On necropsy, the anole was in thin body condition with no discernible subcutaneous fat and scant intracoelomic fat  bodies. The lungs showed evidence of pulmonary congestion and hemorrhage. The gastrointestinal tract was fluid-filled. The en-tire intestinal tract was examined under a dissecting microscope (model M50, Leica, Wetzlar, Germany) for endoparasites, and none were found. An approximately 4.5 ×  2.5-mm white, hard, irregularly shaped object (Figure 4) was discovered in the urinary  bladder and submitted for analysis.  Cystic calculus in a green anole 3 reptilian species is a proposed cause of urolithiasis. 2  Another pos-sible cause of urolith formation in reptiles is lack of access to wa-ter or dehydration, which was more likely than inappropriate protein intake in the current case. The large size of the urolith in this anole indicates that the stone probably was present at cap-ture. Clinically this patient was alert and active; however, prey species typically hide signs of illness as a protective mechanism. Morphologic and sprint data from the source study provided data that helped to unmask this protective mechanism in this anole. Sprint speed is a performance measure used in the source study of life-history theory to determine how allocation of re-sources from one trait affect the energy available to another trait. 8  In this anole, the sprint performance data were important clinical-ly because they demonstrated that although the anole displayed normal behavior, his sprint speed was adversely affected. This anole’s low baseline sprinting performance rank among his con-specifics in this experiment ( n  = 60) suggests that the large stone was painful and might have interfered with his ability to evade predation in the wild.As a urolith increases in size, it compresses the pelvic organs, vasculature, and nerves, potentially causing rearlimb paresis, inappetence, intestinal ileus, and fecal impaction. Inappetence, intestinal compression, and ileus but not rearlimb paresis were observed in this case. Although the anole’s weight dropped con-tinuously over the 4-wk period, the weight declined more dra-matically between weeks 1 to 2 and 3 to 4, suggesting reduced and sporadic food intake during this time rather than complete anorexia (Figure 3). Tail-base thickness is used as a morphologic indicator of body condition, especially for lizards in a dietary restriction study in which weight fluctuations are expected. In this anole, tail-base thickness was not a sensitive measure of  body condition because whereas body weight and BMI showed dramatic reductions over time, the tail base thickness did not (Figure 3).Although the case we present is the first report of urolithiasis in a laboratory anole, urolithiasis frequently affects other rep-tiles that possess a urinary bladder. Compared with larger rep-tiles, 10  very little information is known about the veterinary care of anoles. Urolithiasis may simply be underdiagnosed in this species due to a low incidence of suspicion. However, one of the authors has performed more than 120 necropsies of wild-caught anoles from the same geographic area, and this was the first uri-nary stone found. Although sodium urate stones are radiolucent, the calculus in this anole was large enough to be palpable in the area of the prefemoral fossa. Herpetologic veterinarians advise palpation of the prefemoral fossa during physical examination of chelonians and iguanas to help rule in a cystic calculus in some patients. 12  Although the bladder may be difficult to palpate in the prefemoral fossa of very small or large chelonians and in large iguanas, the anatomic specimens demonstrate that the bladder is easily palpable at the prefemoral fossa in an anole. Therefore, pal-pation of the prefemoral fossa during the physical examination of anoles, especially those presenting with inappetence or rearlimb paresis could be a useful clinical tool to guide further diagnostic evaluation. Acknowledgments We thank Lisa Ulrich at the Minnesota Urolith Center for analyzing the stone and Robert Romance for assisting with the anatomical specimens. Urolith analysis.  Analysis at the Minnesota Urolith Center (St Paul, MN) revealed the stone to be composed entirely of sodium urate. Discussion We have described the case of a wild-caught, laboratory housed anole that presented with anorexia. Antemortem diagnostics and examination failed to reveal an etiology. Necropsy revealed a cys-tic calculus. Cystic calculi present commonly in reptilian species that have a urinary bladder, such as chelonians, iguanas, 1  and reportedly anoles. 5  Because few anatomic diagrams describe the urinary bladder of anoles and given that this organ is not easily apparent on dissection, we have illustrated the location of the  bladder on an anatomic specimen (Figures 1 and 2). The bladder is located dorsal to the fat pads and is thin and transparent when empty.Interestingly, green anoles are insectivores, whereas most of the reptilian species in which cystic calculi have been found were herbivores. 11  Inappropriate intake of dietary protein in herbivorous Figure 3.  Changes in the anole’s body weight and tail-base thickness over time. Figure 4.  Sodium urate cystic calculus found in a laboratory-housed anole .  Vol 67, No 2Comparative MedicineApril 2017 44 References  1. Alworth   LC,   Hernandez   SM,   Divers   SJ.  2011. Laboratory reptile surgery: principles and techniques. J Am Assoc Lab Anim Sci 50:  11–26. 2. Bojrab JM, Waldron DR, Toombs JP.  2014. Current techniques in small animal surgery. Jackson (WY): CRC Press. 3. Conroy   CJ,   Papenfuss   T,   Parker    J,   Hahn   NE.  2009. Use of tricaine methanesulfonate (MS222) for euthanasia of reptiles. J Am Assoc Lab Anim Sci 48: 28–32. 4. Cox   RM,   Calsbeek   R.  2010. Severe costs of reproduction persist in  Anolis  lizards despite the evolution of a single-egg clutch. Evolution 64: 1321–1330. 5. Gredler   ML,   Sanger   TJ,   Cohn   MJ.  2014. Development of the cloaca, hemipenes, and hemiclitores in the green anole,  Anolis carolinensis . Sex Dev 9: 21–33. 6. Higham   TE,   Korchari   PG,   McBrayer   LD.  2011. How muscles define maximum running performance in lizards: an analy-sis using swing- and stance-phase muscles. J Exp Biol 214: 1685–1691. 7. Institute of Laboratory Animal Resources.  2011. Guide for the care and use of laboratory animals. Washington (DC): National Acad-emies Press. 8. Lailvaux SP, Husak JF . 2014. The life history of whole-organism performance. Q Rev Biol 89: 285–318. doi:10.1086/678567 9. Losos    JB.  2009. Lizards in an evolutionary tree; ecology and adap-tive radiation of anoles (organisms and environments). Los Angeles (CA): University of California Press. 10. Lovern   MB,   Holmes   MM,   Wade    J.  2004. The green anole (  Anolis carolinensis ): a reptilian model for laboratory studies of reproductive morphology and behavior. ILAR J 45: 54–64. 11. Mans   C,   Sladky   KK.  2012. Endoscopically guided removal of cloacal calculi in 3 African spurred tortoises ( Geochelone sulcata ). J Am Vet Med Assoc 240: 869–875. 12. Pyron   RA,   Burbrink   FT,   Wiens    JJ.  2013. A phylogeny and revised classification of Squamata, including 4161 species of lizards and snakes. BMC Evol Biol 13: 93. 13. Ulrich   LK,   Bird   KA,   Koehler   LA,   Swanson   L.  1996. Urolith analysis. Submission, methods, and interpretation. Vet Clin North Am Small Anim Pract 26: 393–400.
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