necropsy techniques in reptiles part 1: general introduction and lizards and crocodilians

6

Click here to load reader

Upload: christopher-j

Post on 04-Apr-2017

214 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Necropsy techniques in reptiles Part 1: General introduction and Lizards and Crocodilians

waiting for the post-mortem examination. Freezingshould be always be avoided in order to preventartefacts resulting from the freezing procedure.Freezing should only be used if there is no othermeans of refrigeration or if ancillary procedures(virus isolation, PCR, etc.) will be hampered by aprolonged post-mortem interval. Small carcasses willcool down faster than larger ones and adequatehistological preparations can be obtained even afterseveral days of refrigeration.

Carcasses in advanced stages of decomposition can bestill examined and prominent macroscopic lesionscan be still detected. Tissues from decomposedanimals can be collected for toxicologicalexamination, although histological evaluation ofmost tissues may be compromised and in some casesnon diagnostic.

If the body needs to be sent to a pathologylaboratory for examination ensure that the body iskept cold during shipping and is well insulated toslow down autolysis. If the necropsy is done by theclinician and only selected formalin fixed tissuesamples are to be sent out for examination, ensurethat the container is water tight and sealed to preventformalin spillage. In addition, all informationregarding the submitted case should be shipped withthe carcass or formalin fixed tissues in a separate,sealed, waterproof bag. Packaging must conform topostal regulations and remaining tissues/carcass mustbe disposed of appropriately.

EQUIPMENT

For reptiles it is important to have specificknowledge about the anatomy and physiology of theanimal that will be necropsied post-mortem. Anecropsy usually requires a small set of easilyobtainable equipment: scissors (different sizes), scalpelblades, forceps (different sizes), a ruler, a bone cutter,a cast cutting saw (i.e. Stryker®) and/or a rotary tool(i.e. Dremel®), microbial culturettes, sterilecontainers, labelling tags and or plastic embeddingcassettes, latex gloves, face masks, microscope glassslides (for smears and impression smears) andformalin containers (Fig. 1).

INTRODUCTION

Historically, reptiles have been viewed as unusual andunpleasant animals. Despite these mistaken beliefs,the last four decades have shown that both thescientific community including herpetologists,veterinarians, and biologists, and the people adoptingreptiles as pets or objects of collections have steadilygrown. Along with the increased interest in theseanimals as pets, the number of specialisedveterinarians working predominantly or exclusivelywith reptiles has also flourished. Although greatstrides have been taken in the understanding ofreptile husbandry, physiology, medicine and surgery,many diseases are still not well understood, easilytreated, or easily diagnosed. In some cases the onlyway to obtain a definitive diagnosis is with athorough necropsy, particularly when animals arefound dead with no previous clinical signs.

A necropsy is the examination of a cadaver todetermine or confirm the cause of death and is alsocalled a post-mortem examination. A necropsy is anunfortunate event, but is an extremely valuablesource of information and opportunity for learning.There are several reasons for performing a necropsy,other than just as a diagnostic procedure. In aveterinary teaching hospital necropsies can also beused for teaching normal reptile anatomy,comparative anatomy, and even reptile physiology.Practitioners, students, herpetologists and pathologistcan also learn about disease processes from a reptilenecropsy. By observing the final result of a disease,more can be learned about ante-mortem diagnosisand potential treatment for the next clinical case.Because so much valuable information can be lostfrom a poorly performed reptile necropsy, it isimportant to understand how to preserve and be ableto systematically examine a reptile cadaver.

PRESERVATION OF THE CARCASS

The ideal necropsy should be performed on a freshcarcass soon after the death of the animal, in order toavoid post-mortem artefacts related to the decayprocess (autolysis). More often, however, many hoursto days will pass before the body can be examined. Inthese cases the animal should be refrigerated while

SMALL ANIMAL � EXOTICS ���UK Vet - Vol 15 No 5 June 2010 1

Raffaele Melidone DVM, Christopher J. Gibson VMDTUFTS CUMMINGS SCHOOL OF VETERINARY MEDICINE, 200 WESTBORO ROAD,NORTH GRAFTON, MASSACHUSETTS.

Necropsy techniques in reptiles Part 1: General introduction and Lizards and Crocodilians

Page 2: Necropsy techniques in reptiles Part 1: General introduction and Lizards and Crocodilians

WHAT MAKES A GOOD NECROPSY?

A complete, accurate necropsy starts with thecollation of all available information for the animalto be examined, including radiographs, blood work,surgical reports, an accurate medical history,biological and environmental information (especiallyfor reptiles with specific environmentalrequirements), medical treatments if any, and aclinical diagnosis or suspected cause of illness ifpossible.

In addition, the clinician submitting the animalshould have in mind and ask a few specific questionsthat he/she would like addressed during the postmortem examination.

Every complete necropsy starts with a thoroughexternal examination including examination of theskin, shell, oral cavity, cloacal opening, ears, eyes andpalpation of bones (Figs. 2a and 2b). All biologicalmeasurements should be accurately taken andrecorded including body weight, organ weights, fluidvolumes, and the sizes and locations of all lesions.Careful dissection and gentle tissue handling isextremely important for smaller animals. Accuratedescription of the macroscopic findings, withpictures, will help recall the post-mortem findingslater and facilitate easy communication with othersthat were not present at the original necropsy.Representative samples of all tissue should be placed

SMALL ANIMAL � EXOTICS ��� UK Vet - Vol 15 No 5 June 20102

immediately in 10% buffered formalin and/or frozenfor toxicological or other ancillary tests. Blood,urine, gastrointestinal contents, and exudates can alsobe frozen if required for further investigations.Bacterial cultures should be taken aseptically andsubmitted as soon as possible for accurate results.

A complete, well performed necropsy can provide anenormous amount of information and lead to anaccurate diagnosis. In contrast, a poorly performednecropsy will be likely to result in frustration and amissed diagnosis.

NECROPSY TECHNIQUES AND INTERNAL

EXAMINATION

With the anatomical differences between reptilespecies, the post-mortem examination of reptiles canbe divided into three different groups, each of whichwith slightly different dissection techniques andanatomical features.

Before starting a post-mortem examination inreptiles, it is important to know that reptiles mayhave prolonged contractions of the skeletal andcardiac muscles, even a few hours after death. Thiscan sometimes be disturbing for the veterinarianperforming the necropsy and any observers. It is alsoimportant to ensure that the animal is truly deadbefore starting a necropsy. In some cases, especiallylarge reptiles, it may be difficult to assess death if rigormortis has not yet occurred or if it has completelyfinished. Some pathologists and veterinarians may liketo ensure that the animal is dead by exsanguinatingthe animal prior to starting the dissection.

LIZARDS AND CROCODILIANS

Lizards and crocodilians have a similar body shapeand can be examined in dorsal recumbency.However, some lizards, such as chameleons, whichare laterally compressed, are better examined inlateral recumbency by removing the skin, underlyingmuscles and rib cage of one entire side of the animal,in order to expose the coelomic cavity (Fig 3).Extreme caution should be taken when examiningvenomous lizards (Heloderma spp., refer to venomoussnakes in subsequent article). Once the animal isplaced in dorsal recumbency two different incisions

Fig. 1: Most tools necessary to perform a necropsyare easily retrievable in every veterinary clinic. Theplastic cassettes shown in the picture are useful whensampling small organs such as adrenal glands, thyroidsand parathyroids.

Fig. 2a: Four digits of the right front limb are missingmost likely due to low environmental humidity andmoulting difficulties.

Fig. 2b: Ball python with severe necro-suppurativestomatitis. (Photograph courtesy of Dr. Paolo SelleriCVS Rome.)

Page 3: Necropsy techniques in reptiles Part 1: General introduction and Lizards and Crocodilians

SMALL ANIMAL � EXOTICS ���UK Vet - Vol 15 No 5 June 2010 3

can be made to approach the coelomic cavity: eithera single incision is made on the ventral midline fromthe intermandibular region to the cloacal opening,or two ventrolateral incisions are made from the oralcommissures to the cloacal region. These incisionsshould follow the lateral margins of the ventralscales, removing the skin and underlying skeletalmuscles in one block (Figs. 4a and Fig. 4b).

Access to the cranial coelom is gained by cutting the ribsand intercostal muscles at the costochondral junctionfrom the last ribs to the thoracic inlet, bilaterally (Fig. 5).Another incision should be made over the long axisof the medial aspect of the thigh to collect the sciaticnerve, which runs immediately parallel and caudal to thefemur, and a sample of bone with bone marrow fromthe proximal femur. Skin and skeletal muscle collectionsmay be also taken at this stage.

The next step is to approach the oral/pharyngealregion in order to examine the oral cavity and freethe tongue, glottis and pharynx for examination andsampling. Two incisions should be made medially tothe mandibular rami through the skin (if still inplace) and intermandibular muscles extending intothe oral cavity. The resected tissues should bereflected caudally or laterally to visualise/examinethe tongue, oral cavity, pharynx, and larynx.Particular attention should be paid to theexamination of the gular valve in crocodiles, becauseit is a common site of diseases in these animals.Crocodiles have a pronounced caudal tracheal bendand have extrapulmonary bronchi immediatelycranial to the heart. The thyroids, parathyroids andthymus are also located between the intermandibularregion and the cranial aspect of the heart. Thethyroid gland in lizards and crocodilians may besingle, bilobated or paired. The location andanatomical appearance may vary among species. Ingreen iguanas, thyroid glands are paired andconnected by a thin band of thyroid tissue. Thepaired thyroids are located on the ventrolateral aspectof the caudal trachea cranial to the heart. Lizards mayhave either one or two pairs of parathyroid glandsdepending on the species. In green iguanas (Iguanaiguana) there are two pairs of parathyroids: one pair(cranial) is located between the medial aspect of themandibular rami and the trachea (often difficult tolocate); the second pair (caudal) is positioned at thebranch of the internal and external carotid arteries(Fig. 6). Crocodilians may have one or two pairs ofparathyroid glands positioned close to the commoncarotid artery. The thymus, in lizards, is usually apaired organ and is situated cranial to the heart inclose relationship with the common carotid artery,jugular vein and vagus nerve. In crocodilians thethymus is an elongated organ located cranial to theheart and projecting toward the cervical region inclose association with the common carotid artery,jugular vein and vagus nerve. The identification andlocalization of these organs may be difficult and, insome cases, it is advised to simply sample all thetissues immediately cranial to the heart, en bloc, andmake multiple serial sections after fixation in orderto locate these organs.

Fig. 3: The white dotted line indicates the portion ofskin that should be removed in order to expose theentire coelomic cavity in a chameleon post-mortemexamination (Photograph courtesy of Dr. Paolo SelleriCVS Rome).

Fig. 5: The entire coelomic cavity is open and all themajor organs are exposed for examination: 1. Heart;2. Liver; 3. Stomach; 4. Ovary with ova; 5. Fat bodies;6. Small intestine; 7. Large intestine; 8. Kidneys.

Fig. 4b: The skin and underlying skeletal muscles havebeen removed in one block (asterisk).

Fig. 4a: Single incision on the ventral midline (white).Two ventrolateral incisions (red).

Page 4: Necropsy techniques in reptiles Part 1: General introduction and Lizards and Crocodilians

The next organ system that should be examined isthe cardiovascular system. Lizards have a threechambered heart with two atria, one ventricle andtwo aortas (left and right) (Fig. 7). The heart shouldbe removed by cutting the major blood vessels andexamined by bisecting the entire heart starting at theapex of the ventricle and continuing cranially intoboth atria and the major blood vessels. Once thethree chambers have been opened, the endocardialsurfaces, valves, and major blood vessels can beexamined. Crocodilians are the only reptiles thathave an interventricular septum with two ventricles,two atria and two aortas (left and right).Crocodilians, lizards and chelonians have a fibrousgubernaculum cordis between the apex of theventricle and the pericardium, which should not bemistaken for a pathologic adhesion (Fig. 8).

Two, large, dark-yellow, elongated structures (fatbodies) are located within the caudo-ventral portionof the coelomic cavity (Fig. 5). The fat bodies arepaired organs in lizards and a single organ incrocodilians and can be used as indicators of thenutritional status.

The remaining organs can be removed, en bloc, bypulling gently on the tongue caudally, cutting thedorsal connective tissue attachments as needed. Inlarger animals, the liver (Fig. 9), gastrointestinal tract

and remaining caudal coelomic organs can beremoved separately from the lungs, trachea, andtongue by cutting the oesophagus at the gastriccardia. The colon is transected as close as possible tothe cloaca or can be removed with it. Once out, theliver can be examined. It should have ahomogeneous dark-brown to red coloration (Fig.10). Often this colour will change if there are anypathologic processes present (Fig. 9). The gall bladderin lizards is usually closely associated with the liver.The spleen and pancreas are closely associated witheach other and the spleen is usually an ovoid shape(Fig. 11). The intestine is a simple tubular structureand sometime it is difficult, at least macroscopically,to discern between the different segments. The wallof the small intestine is usually thicker than the largeintestine. The entire gastrointestinal tract should beopened longitudinally for an accurate macroscopicexamination of the mucosal surface. Samples of allthe above mentioned organs can be collected aftertheir examination. The remaining caudal coelomicorgans can be examined and sampled at this stage.

The kidneys are located within the pelvic canal andcannot be examined without removing the pelvicfloor. If the kidneys are easily visible without

SMALL ANIMAL � EXOTICS ��� UK Vet - Vol 15 No 5 June 20104

Fig. 6: Parathyroid gland positioned at the branch ofthe internal and external carotid arteries: Parathyroidgland (red arrow), carotid artery (blue arrow),trachea (1) and heart (2).

Fig. 7: The two aortas are visible adhered to thedorsal aspect of the coelomic cavity adjacent to thespinal column (arrows). The heart (1) and left lung(2) are also visible.

Fig. 8: Crocodilians, lizards and chelonians have afibrous gubernaculum cordis between the apex ofthe ventricle and the pericardium (red arrow),atrium (1) and ventricle (2).

Fig. 9: The gall bladder in lizards is usually closelyassociated with the liver. The colour of the liver isabnormal due to severe hepatic lipidosis.

Page 5: Necropsy techniques in reptiles Part 1: General introduction and Lizards and Crocodilians

SMALL ANIMAL � EXOTICS ���UK Vet - Vol 15 No 5 June 2010 5

removing the pelvis, there is likely to be significantrenal pathology present (Fig. 12). The adrenal glandsare elongated paired, light yellow organs, usuallytightly associated with the gonads and are locatedwithin the connective tissue immediately next to thetesticles and ovaries (Fig. 12). The urinary bladder canbe also examined and sampled at this stage (Fig. 13).

The examination of the respiratory tract first involvesopening of the pharynx, trachea and major airways(Fig. 14). If the animal is large enough, the smallerairways should be opened as far as possible. Theoesophagus should be opened along its entire lengthand the tongue, larynx and oesophagus should beexamined. Samples of the respiratory tract, oesophagusand tongue can be taken after their examination.

The last organ system to be examined is the centralnervous system. The head should be detached fromthe neck by disarticulating the atlanto-occipital jointand transecting the cervical spinal cord. After thehead has been removed the skin over the skull can bereflected and the bone can be cut by using a smallrotary tool (Dremel®) or a cast cutting saw(Stryker®), depending on the size of the animal. Twolateral cuts can be made on the skull extending fromthe foramen magnum to the mid orbit on either side(Fig. 15). A single transverse cut connects the twolateral cuts, rostrally (Fig. 15). Once the bone has

Fig. 10: Normal colour of a liver in an iguana. Fig. 14: Both lungs (asterisks), aortas (white arrows)and ovaries (1) are seen in this picture.

Fig. 15: Two lateral cuts can be made on the skullextending from the foramen magnum to the mid orbiton either side (white dotted lines). A single transversecut connects the two lateral cuts, rostrally (reddotted line). Once the bone and overlying skeletalmuscle and skin have been removed the brain (1) canbe collected.

Fig. 11: The spleen (1) and pancreas (2) are closelyassociated with each other and the spleen is usually an ovoid shape. The stomach (3) and small intestine(4) are also visible in this picture.

Fig. 12: Caudal coelomic cavity of a green iguana. Theadrenal glands are visible (arrows), as well as the kidneys(1) and one ovary (2). The cranial pole of both kidneysextends over the cranial edge of the pelvic canal (arrow).(Image courtesy of Dr. Paolo Selleri CVS Rome.)

Fig. 13: The urinary bladder is visible on the caudalportion of the coelomic cavity (1), as well as bothoviducts (2). (Image courtesy of Dr. Paolo Selleri CVSRome.)

Page 6: Necropsy techniques in reptiles Part 1: General introduction and Lizards and Crocodilians

been removed the brain can be collected intact bytransecting the cranial nerve attachments (Fig. 16)and placed in formalin. For very small lizards, theentire head including the brain, with the top of theskull removed, can be placed in formalin. The spinalcord can be also examined and collected, if necessary.A rotary tool or cast cutting saw can be used for thisalso. The spinal cord can be accessed by performinga dorsal laminectomy.

Both eyes are collected before disposing of the head.

LIST OF TISSUES TO BE SAMPLED

● Eyes (both)● Skin (ventral abdomen)● Skeletal muscle (hind limb)● Sciatic nerve (hind limb)● Bone marrow with a section of bone from thefemur

● Thyroids, parathyroids and thymus ● Tongue● Pharyngeal and laryngeal regions● Oesophagus and trachea together● Lung (4-5 sections from different segments)● Right and left atria and ventricle (whole heartfor small reptiles)

● Aorta ● Adrenal glands, which should be left associatedwith the gonads to make their differentiationeasier when fixed

● Stomach● Duodenum with pancreas● Small intestine● Large intestine and cloaca● Liver (4-5 sections from different segments)● Gall bladder (gall bladder, pancreas and spleenshould be left together for better identificationafter fixation)

● Spleen● Kidney (one section of each)● Bladder (if present)● Ovaries or testicles ● Brain● Spinal cord (if necessary).

All lesions should be sampled and labelled; inaddition all small specimens should be placed insmaller containers and labelled for easieridentification after fixation.

REFERENCES

JACOBSON, E. R., Infectious Diseases and Pathology of Reptiles, CRC

Press, 2007.

MADER, D. R., Reptile Medicine and Surgery, Elsevier Inc., 2006.

NILLSON, S., The crocodilian heart and central hemodynamics,

Cardioscience, 1994 Sep;5 (3):163-6.

RIVERA, S., LOCK B., The reptilian thyroid and parathyroid glands,

Veterinary Clinics of North America: Exotic Animal Practice 2008

Jan;11(1):163-75, viii.

PUTTERILL, J. F., SOLEY, J. T,. Morphology of the gular valve of the Nile

crocodile, Crocodylus niloticus (Laurenti, 1768).J Morphol. 2006

Aug;267(8):924-39.

SMALL ANIMAL � EXOTICS ��� UK Vet - Vol 15 No 5 June 20106

Fig. 16: The brain can be collected intact bytransecting the cranial nerve attachments.

C O N T I N U I N G P RO F E S S I O N A LD E V E L O P M E N T S P O N S O R E D B YB AY E R A N I M A L H E A LT H

1. To which organ is the pancreas usually associated

with in lizards:

a. Spleen

b. Gall bladder

c. Liver

2. How many pairs of parathyroids are generally

present in lizards:

a. 1

b. 2

c. 3

3. Fat bodies in reptiles are used as indicators of

which of the following:

a. Nutritional status

b. Age

These multiple choice questions are based on the abovetext. Answers appear on page 99.