The Elasmobranch Husbandry Manual: Captive Care of Sharks, Rays and their Relatives
Editors Mark Smith Doug Warmolts Dennis Thoney Robert Hueter
Published by Ohio Biological Survey, Inc. Columbus, Ohio 43221-0370
2004
Ohio Biological Survey Special Publication ISBN-13: 978-0-86727-152-3 ISBN-10: 0-86727-152-3 Library of Congress Number: 2004115835
Publication Director Brian J. Armitage Editorial Committee Barbara K. Andreas, Ph. D., Cuyahoga Community College & Kent State University Brian J. Armitage, Ph. D., Ohio Biological Survey Benjamin A. Foote, Ph. D., Kent State University (Emeritus) Jane L. Forsyth, Ph. D., Bowling Green State University (Emeritus) Eric H. Metzler, B.S., The Ohio Lepidopterists Scott M. Moody, Ph. D., Ohio University David H. Stansbery, Ph. D., The Ohio State University (Emeritus) Ronald L. Stuckey, Ph. D., The Ohio State University (Emeritus) Elliot J. Tramer, Ph. D., The University of Toledo
Literature Citation Smith, M., D. Warmolts, D. Thoney, and R. Hueter (editors). 2004. The Elasmobranch Husbandry Manual: Captive Care of Sharks, Rays and their Relatives. Special Publication of the Ohio Biological Survey. xv + 589 p. Cover and Title Page Illustration by Rolf Williams, The National Marine Aquarium, Rope Walk, Coxside, Plymouth, PL4 0LF United Kingdom Distributor Ohio Biological Survey, P.O. Box 21370, Columbus, Ohio 43221-0370 U.S.A. Copyright © 2004 by the Ohio Biological Survey All rights reserved. No part of this publication may be reproduced, stored in a computerized system, or published in any form or in any manner, including electronic, mechanical, reprographic, or photographic, without prior written permission from the publishers, Ohio Biological Survey, P.O. Box 21370, Columbus, Ohio 432210370 U.S.A. Layout and Design: Printing:
Brian J. Armitage, Ohio Biological Survey The Ohio State University, Printing Services, Columbus, Ohio Ohio Biological Survey P.O. Box 21370 Columbus, OH 43221-0370 www.ohiobiologicalsurvey.org 11-2004—1.5M ii
The Elasmobranch Husbandry Manual: Captive Care of Sharks, Rays and their Relatives, pages 467-472. © 2004 Ohio Biological Survey
Chapter 30 Necropsy Methods and Procedures for Elasmobranchs
GERALD L. CROW Waikiki Aquarium, University of Hawaii, 2777 Kalakaua Ave., Honolulu, HI 96815, USA. E-mail:
[email protected]
JAMES A. BROCK 95-430 Kamahana Place, Mililani, HI 96789, USA. E-mail:
[email protected]
Abstract: With declining populations of free-living elasmobranchs, collecting sharks and rays for public exhibition has become increasingly difficult. As a result, husbandry practices for captive elasmobranchs must be refined and improved continually to ensure the longevity of these animals. One critical aspect of husbandry is the understanding of life-threatening diseases through the use of detailed necropsy procedures. A skilled technician conducting a thorough necropsy gross examination can often provide a good diagnosis of the disease process or at least determine which organs were affected by the disease. When warranted, a complete necropsy with cultures, histology, tissue imprints, tissue and fluid stains, and SEM diagnostics can be used. The clinician should be familiar with experts in the various pathology fields and be prepared to ship tissue, parasites, and fluids to laboratories around the world for a full diagnostic workup. and evening, to note behavioral signs and physical responses to the environment that may provide clues to potential health problems within the elasmobranch collection. Detailed computerized records should be maintained on treated animals, and previous pathology case history information should be available, for ready access, in the event of a necropsy exam.
Necropsy examination is an essential part of elasmobranch husbandry and should be integrated thoroughly into the husbandry program (including collecting and handling procedures, exhibit design, daily observation, water chemistry, nutrition, and all aspects of veterinary care). Elasmobranchs are susceptible to a number of diseases (Stoskopf, 1993; Crow, 1996) and careful, detailed necropsy procedures will bring about better understanding of these processes. This chapter greatly benefited from the published work of Reimschuessel et al. (1993) and Noga (1996). For more detail on the examination of elasmobranchs and disease methodologies please refer to Chapters 20-29 of this manual.
Euthanasia If an elasmobranch is showing agonal signs and the decision is made to euthanize it, attempts should be made to obtain key diagnostic samples while the animal is still alive (i.e., blood samples and tissue scrapings). Euthanasia techniques should result in a rapid loss of consciousness, followed by cardiac and respiratory failure, and ultimate loss of brain function (Anon., 2001).
GENERAL METHODS It is recommended that the clinician and key aquarium staff inspect all exhibits, in the morning 467
CROW & BROCK An accurate diagnosis relies heavily on the experience of the clinician, supplies and media available, and the capability of the designated diagnostic laboratory. The examiner should be familiar with elasmobranch anatomy and, if needed, have a dissection manual available. A basic necropsy kit, support equipment, tissue sampling, and preserving fluids should be accessible at all times. A sample necropsy report form is provided in Chapter 36 of this manual.
Various methods can be employed to euthanize an elasmobranch (i.e., overdosing with anesthetics, severing of the spinal cord, etc.) and should take into consideration humane treatment and personal safety, as well as optimal sample collection. When using anesthetics as bath solutions, elasmobranchs should be left in solution for at least 10 minutes following cessation of gill movement (Anon., 2001). The decision to terminate life should only be taken when no veterinary procedures would improve the fish’s condition.
A complete necropsy can entail considerable time and expense; therefore, each case should be carefully evaluated to determine the extent of the necropsy procedure. It is important to note that results from cultures and histopathology may take several days to weeks before they are completed. A careful gross exam can provide immediate information.
Preparation Animals should not be frozen as it renders tissue unsuitable for diagnostics. It is critical that the necropsy procedure is conducted as soon as possible after the fish’s death. Elasmobranchs found dead for more than six hours, depending on the water temperature within the exhibit, are often autolyzed and will not provide useful cultures or usable tissues for histology. However, whenever possible, these animals should be subject to a gross exam which may still provide useful information.
Figure 30.1. organs.
The prosector (person conducting the necropsy) should be familiar with the general anatomy of elasmobranchs (Figures 30.1 and 30.2) and have an understanding of normal versus abnormal appearance (i.e., color, size, consistency, etc.) of tissues and organs. Wet tissue mounts, scrapes, smears, and tissue imprints often provide useful
Basic internal anatomy of the blacktip reef shark (Carcharhinus melanopterus) showing the location of principal
468
CHAPTER 30: NECROPSY METHODS AND PROCEDURES
Figure 30.2. Basic internal anatomy of the brown stingray (Dasyatis lata) showing the location of principal organs.
requested. Ideally, the necropsy supply kit should be ready for the evaluation of all potential disease etiologies: infectious (viral, bacterial, fungal, parasitic), trauma, tumors, toxins, metabolic, and nutritional. The contact details of some laboratories that routinely examine fish tissue have been provided in Table 30.2.
information, and the evaluation of most organs in this way is encouraged. Thin sections of tissue need to be taken for wet mounts, in order for adequate light penetration. Small tissue samples (i.e., 1.0 cm 3 or less) need to be taken for histology, in order to allow penetration of fixative. Fournie et al. (2000) review the fixation of tissues. The recommended tissue to fixative volume ratio is 1:10.
The following is a generalized procedure for organs to be examined during a necropsy. A regular sequence is recommended to ensure that each organ is examined. Attempts should be made to aseptically open any site that may require culture.
Some common mistakes made during necropsy include: not wearing gloves (even though there are no published reports of mycobacterium infections in elasmobranchs, many potential diseases are present); cutting tissues too thick, preventing penetration of fixative; not including all tissues; and not taking multiple samples of each tissue, which, if desired, can be placed in different fixatives. Tissue fixatives are typically considered hazardous material. Therefore, a certified shipper of hazardous material must always be used to ensure proper handling and labeling of shipped samples and fixatives.
External examination The external body should be closely inspected for any changes to normal body integrity. The specimen should be measured and weighed. The mouth should be inspected for any discoloration and blockage. The gills should be examined closely for signs of excessive bleeding and color changes. Gill clips should be taken and tissue samples examined under the microscope for evidence of parasites or gas super-saturation. Any skin lesions should be noted and samples of abnormal and normal tissue taken for histopathological examination. The cloaca should
THE NECROPSY The supplies needed for a thorough necropsy are listed in Table 30.1. Additional supplies and preservatives may be required if other tests are 469
CROW & BROCK
Table 30.1. List of items suggested for use during an elasmobranch necropsy procedure. BHI = Brain Heart Infusion agar; TCB = Thiosulfate Citrate Bile Salts agar.
Vinyl or latex gloves Calipers Dissection scope Binocular microscope with 800x Two sizes of scissors Forceps Bone cutters Scalpel blades Slides/cover slips Syringes 1cc and 5cc with needles Scale and measuring tape Labeled tissue containers for preserved tissues Sterile loop or culturette with transport media 10% buffered formalin, Bouin's, or Davidson's solutions Absolute methanol Bacterial growth media (BHI and TCBS with 2% salts) Fungal growth media (Sabouraud dextrose agar 2% salts) Stains for slides (Geimsa, Diff-Quick, and Acid Fast) SEM fixative (Millonig's buffer and glutaraldehyde) Digital, slide, and video cameras
cartilage without hitting the brain. Then either cut along the side of the chondrocranium with bone cutters or slice over the top of the chondrocranium to expose the brain. Cerebral spinal fluid should be checked for discoloration and a culture taken if excessive, or discolored, fluid is present. Fluid should be removed with a syringe and needle, and placed on a slide for examination. The fluid can be placed on a mini-tip culturette and inoculated onto media. The brain should be removed intact and, depending on size, placed directly in, or sectioned before placing in, fixative.
be examined for the presence of parasites, and any exudate or discharge should be collected for evaluation.
Internal examination The brain should be the first internal organ sampled, as brain tissue deteriorates rapidly. The brain of elasmobranchs is surrounded by a cartilaginous case called the chondrocranium. The elasmobranch brain consists of five parts, the telencephalon (olfactory), diencephalon (pineal organ), mesencephalon (vision), metencephalon (cerebellum), and myelencephalon (hearing). Expose the brain by removing the skin just posterior to the eyes. The chondrocranium will be exposed as whitish cartilage. As the skin is peeled away at the posterior end of the chondrocranium there is an endolymphatic foramen. Just posterior to the foramen gently slice down to cut this
Eyes are often overlooked in the necropsy procedure. The eyes should be removed intact and placed in fixative for histological examination. Eyes should be slit for fixative penetration. The thyroid gland is commonly ignored during necropsy. It is, however, critical to body function, affected by environmental stressors, and should
470
471
Marine Pathology Laboratory University of Rhode Island Kingston, RI 02882 (401) 792-2334
Zoo/Exotic Pathology Services Dr. Drury Reavill 2825 KOVR Drive West Sacramento, CA 95605 (916) 725-5100
Fish Pathology Laboratory Department of Pathology Ontario Veterinary College Guelph, Ontario N1G 2W1 Canada (519) 824-4120
Utrecht University Department of Veterinary Pathology Section Pet Avian, Exotic An. and Wildlife Yalelaan 1 3584 CL Utrecht The Netherlands (313) 025-34357
Central Institute for Animal Disease Control Fish Pathology P O Box 65 8200 AB Lelystad The Netherlands (313) 202-38238
Animal Health Laboratory Food, Agriculture, and Fisheries Division DPIWE P O Box 46 Kings Meadows TAS 7249 Australia
Joseph M. Groff, VMD, PhD Department of Pathology, Microbiology and Immunology Room 1149, Haring Hall One Shields Avenue School of Veterinary Medicine University of California Davis, California 95616 (530) 753-8739 e-mail:
[email protected] (e-mail contact for correspondence preferred)
Fish Diagnostic Medicine College of Veterinary Medicine Drawer V Mississippi State, MS 39762 (601) 325-3432
Canada
Fisheries Western Australia Locked Bag 39 Cloisters Square WA 6850 Australia (61) 363-365216
Northwest ZooPath 18210 Waverly Drive Snohomish, WA 98296 (360) 668-6003
Fish Diagnostic Laboratory Department of Avian and Aquatic Animal Medicine College of Veterinary Medicine Cornell University Ithaca, NY 14853 (607) 253-3365
North Georgia Diagnostic Ass. Lab. College of Veterinary Medicine University of Georgia Athens, GA 30602 (404) 542-5260
Netherlands
CSIRO Livestock Industries Australian Animal Health Laboratory Private Bag 24 Geelong VIC 3220 Australia (61) 352-275426
Yeerongpilly Veterinary Laboratory Animal research Institute 665 Fairfield Road Yeerongpilly QLD 4105 Australia (61) 733-629440
Pathology Laboratory Osborn Laboratories of Marine Science New York Aquarium, NY Zoological Soc. Boardwalk & West 8th Street Brooklyn, NY 11224 (718) 265-3417
Aquatic Toxicology and Pathology Laboratory Department of Pathology, 711 MSTF University of Maryland School of Medicine 10 S. Pine St. Baltimore, MD 21201 (410) 328-7230
Australia
Department of Fisheries and Aquaculture College of Veterinary Medicine 7922 NW 71 St. Gainesville, FL 32606 (904) 392-9617
University of California at Davis Department of Medicine School of Veterinary Medicine Davis, CA 95616 (916) 752-3411
United States
Table 30.2. A sample of diagnostic laboratories from around the world that specialize in the examination of fish tissue.
CHAPTER 30: NECROPSY METHODS AND PROCEDURES
CROW & BROCK be included in the exam. The thyroid gland in healthy elasmobranchs is a flattened organ located in loose connective tissue between the ventral side of the coracohydral and the medial side of the coracomandibular muscles. A general description of the location of the thyroid gland has been given in Figure 30.1.
its natural flora (Grimes et al., 1985). Bacterial cultures should be taken if these organs are suspected in the disease process. The stomach and valvular intestine should be opened and examined for abrasions, obstructions, lesions, and parasites. Stomach contents should be collected, rinsed, and placed in a petri dish or bowl for metazoan parasite identification using a dissection scope.
To enter the body cavity, a midline incision is recommended. Place the elasmobranch on its back. Gently pull up a piece of skin, posterior to the pectoral girdle, with a pair of forceps and make a small incision with a scalpel blade. Keeping the skin elevated, cut toward the tail stopping just short of the cloacal area. It is important not to touch any internal organs with the scalpel or gloves when doing the cutting. A quick inspection of the organs prior to any manipulation of body contents should be done to observe any abnormalities. Cultures and fluid samples should be taken.
REFERENCES Anon. 2001. 2000 report of the AVMA panel on euthanasia. Journal of the American Veterinary Medical Association 218: 669-696. Crow, G. L. 1996. A review of the diseases and pathology of captive elasmobranchs. In: AZA Annual Conference Proceedings, September 17-21, Waikiki, Hawaii, p. 7681. American Zoo and Aquarium Association, Silver Spring, Maryland, USA. Fournie, J. W., R. M. Krol, and W. E. Hawkins. 2000. Fixation of fish tissues. In: The Laboratory Fish, p. 569-578. G. Ostrander (ed.). Academic Press, San Diego, California, USA. Grimes, D. J., P. Brayton, R. R. Colwell, and S. H. Gruber. 1985. Vibrios as autochthonous flora of neritic sharks. Systematic Applied Microbiology 6: 221-226. Noga, E. J. 1996. Fish Disease Diagnosis and Treatment. Mosby, St. Louis, Missouri, USA. 367p. Reimschuessel, R. 1993. Postmortem examination. In: Fish Medicine, p. 160-165. M. K. Stoskopf (ed.). W. B. Saunders Co., Philadelphia, Pennsylvania. USA. Stoskopf, M. K. (ed.). 1993. Fish Medicine. W. B. Saunders Co., Philadelphia, Pennsylvania, USA. 882 p.
The liver is typically the most prominent organ in the body cavity of elasmobranchs. The color should be reddish/beige and the edges should be sharp and well-demarcated. Vitamin E deficiency typically creates rounded edges and a mushy texture that easily comes apart in your hand. If a liver infection is suspected, the external surface can be sterilized and the tissue sliced open for culture samples. Care must be taken during interpretation because Vibrio spp. are a normal part of the liver flora in elasmobranchs (Grimes et al., 1985). Liver tissue imprints can be made on slides and fixed in 100% methanol. The gallbladder is a thin-walled greenish sac located at the junction of the left and right lobes of the liver. Parasites have been discovered in this organ, and fluid stains may be useful for disease diagnosis. The spleen and pancreas are located alongside the pyloric stomach. The spleen should be bright red or maroon and the pancreas beige. Both organs should be inspected for any abnormalities. If disease is suspected, cultures and tissue imprints should be taken. The reproductive tract should be examined for egg or sperm development and traced from the testis (male) or ovary (female) to the sperm sac (male) or uterus (female). The epigonal glands and kidneys are located on both sides of the vertebral column. The kidney has been reported to contain bacteria as part of 472
