The antemortem and postmortem sampling of tissues is necessary to fully understand the causes of lesions, disease, and mortality in living and dead marine turtles. In the living animal, sampling of single or multiple tissues is referred to as a biopsy. Biopsies are collected for biological and pathological studies. While biopsies have the potential to provide much information on the cause of illness in an animal, there are certain limitations. Ultimately when a population is affected or an individual is found dead, postmortem examinations are the best way to try and establish causes of mortality in marine turtles.
Whereas the postmortem examination of a human is referred to as an autopsy, in veterinary medicine, the postmortem examination of an animal is referred to as a necropsy. Although all dead turtles should be necropsied, the degree of decomposition will limit the amount of diagnostic information that can be gained. When investigating a population morbidity/mortality event, it is often more informative to euthanatize and necropsy a moribund turtle rather than one that died spontaneously. One is more likely to find active primary pathologic processes in the former case while chronic inflammatory responses and secondary infections may obscure these findings in the latter. Gross and histopathological evidence of an infectious process provides direction for further diagnostic workup.
Determining specific causes of death of a sea turtle are not possible in all cases. Even the best worked-up case may turn out to be a diagnostic conundrum. Many pesticides and pollutants may not result in light microscopic changes in tissues and trying to establish a causal relationship is difficult, especially since lethal doses for these compounds have not been determined. Still, necropsies provide invaluable information that cannot be derived through any other means. Thus it is surprising that so little effort (and funding) has been directed toward this area of investigation. While a tremendous amount of standing data is available for marine turtles in the United States, relatively few studies have been done to document the causes of mortality in these animals. Here we shall review necropsy techniques, discussing why they are important and when they should be done.
Whenever samples are collected, all information that relates to the sample needs to be thoroughly described and entered into either a field notebook or specific forms that have been developed to record this information. Information needs to be as detailed as possible. While you can always eliminate excess information, you will never be able to go back and retrieve information that is forgotten or has been missed.
Field Observations (Signalment, History, Clinical signs)
All detective work involves thorough description of the scene and preservation of the physical evidence. Careful and complete description of the health problem by the field biologist is the first and most critical step in arriving at a diagnosis. The species, age, size, and sex of animals affected (signalment), the onset, duration, and course of the problem (history), the observed clinical signs, and lesions need to be recorded and will define the problem and guide the selection of diagnostic approaches. For example, mass stranding of turtles in apparently good body condition following a sharp drop in water temperature might suggest a peracute infection or a hypothermic stunning event whereas a similar stranding event in the summer might result from a peracute infectious disease agent or a toxin. Although clinical signs such as weight loss or lethargy may be non-specific, any conclusions about etiology or pathogenesis based on results of diagnostic tests would have to be consistent with these observations.
Biopsies are routinely collected to better understand the nature of a lesion and to determine the most appropriate therapy. In biological science, biopsies may be collected from various tissues to ultimately provide information relative to the life history of the population being studied.
The total amount of blood that can be safely withdrawn from a reptile depends upon the reptile’s size and health status. The total blood volume of reptiles varies between species but as a generalization is approximately 5 to 8% of total body weight (Lillywhite and Smits, 1984; Smits and Kozubowski, 1985). Blood is a fluid tissue and represents the single most common tissue collected by biologists in the field. In juvenile and adult see turtles, blood is generally collected from the cervical sinus (Owens and Ruiz, 1980); in neonates, blood is often collected from the heart (cardiocentesis), with the needle passed through the overlying plastron. At either site, the integument should be cleansed with 70% ethanol prior to sampling. When cardiocentesis is performed, a surgical glue (cyanoacrylate; Nexaband, Veterinary Products Laboratory, Phoenix, AZ 85013), should be used to cover the hole remaining in the plastron after the needle is withdrawn. Otherwise, pathogens in water can migrate through the hole into the heart, resulting in infection (pericarditis).
Complete blood counts (CBCs) should be routinely performed on ill sea turtles. We recommend using microtainer tubes containing lithium heparin (Fisher Scientific Co., Orlando, Florida, USA). Immediately following collection of the sample, 0.6-ml of blood is added to a tube and the tube is inverted several times to prevent clotting. Other anticoagulants that can be used for CBCs include sodium heparin and ammonium heparin. Since potassium ethylenediaminetetraacetic acid (EDTA) results in haemolysis of chelonian red blood cells, this anticoagulant is not recommended (Jacobson, 1987).
In performance of CBCs, analyses include: 1) red blood cell counts; 2) white blood cell counts; 3) differential white blood cell counts; 4) packed cell volumes (PCV); and 5) hemoglobin concentrations. Red blood cell counts are determined using an automated coulter counter. White blood cell counts can be determined either manually utilizing a haemocytometer (Schermer, 1967) or as an estimated count from a blood film, similar to that used in birds (Campbell and Coles, 1986). Packed cell volumes are determined following centrifugation of a sample in a microhaematocrit tube. Haemoglobin values are determined utilizing a haemoglobinometer. Although total protein values are often determined utilizing a refractometer, the author’s experience suggests that accuracy of this method with reptile blood is questionable. Proper methods for determining total protein of reptile blood will be discussed under biochemical evaluations of blood. Normal haematological values can be found elsewhere (Jacobson, 1987).
Biochemical evaluations of blood generally involve analysis of plasma or serum samples for the following inorganic and organic constituents: sodium, potassium, chloride, calcium, phosphorus, glucose, urea, uric acid, creatinine, cholesterol, aspartate aminotransferase activity (AST; formerly GOT), alanine aminotransferase activity (ALT; formerly GPT), alkaline phosphatase activity (ALP), and total protein. Reference values for plasma analytes of green and loggerhead sea turtles can be found at: http://accstr.ufl.edu/blood_chem.htm. The author prefers to use plasma rather than serum since a greater volume of plasma can be collected per unit volume of blood compared with serum. Also, it is more common for clots to occur in serum than plasma. Serum removed from the blood sample following centrifugation may suddenly clot into a gelatinous mass. Although the causes of this phenomenon are unknown, clotting is more common in glass tubes and possibly the electric charge on the glass is an initiating factor.
Plasma samples are collected from blood placed into 2 microtainer lithium heparin tubes. The tubes should be centrifuged immediately following collection and the plasma removed and submitted for evaluation. Since plasma potassium values will increase over the time period plasma is in contact with blood, it should be removed and frozen immediately following centrifugation. In performance of field work on blood biochemical values of free-ranging reptiles, plasma can be placed in cryotubes and frozen in liquid nitrogen. If the samples need to be sent to a laboratory, the samples should be transported frozen on dry ice.
To date, no information appears to have been published on the accuracy of different methodologies used in determining various plasma biochemical analytes in reptiles. A variety of automated machines have been developed for use in determination of plasma/serum biochemical profiles of humans. The veterinary profession also uses these machines for determining blood biochemical values of both domestic and non-domestic animals. Although it is quite simple to submit a sample and have it analyzed on an automated machine, it is difficult to determine the accuracy of these values. In studies the author has conducted on replicate plasma samples submitted from the same sample of blood and analyzed on different machines, flame photometry appear to be more accurate for determination of sodium and potassium values than machines utilizing ion-exchange electrodes.
With automated machines, procedures for determining total protein values are often based on the Biuret method and the author has found results by this method significantly different from values for the same samples determined by refractometry. The Biuret method appears to be more accurate and should be the preferred method. Albumen values are generally determined utilizing various dyes such as bromcresol green (BCG) and globulin values by subtracting albumen from total protein. When doing studies on blood proteins of reptile plasma, the author has found significant differences when comparing albumen and globulin values determined by electrophoresis with those determined utilizing BCG. Based on the author’s experience, it is likely that albumen values determined by dye techniques are inaccurate and methods utilizing these chemicals should be avoided. Serum/plasma protein electrophoresis is more costly to perform but is more accurate.
The most important points to remember when submitting plasma/serum samples for biochemical evaluations are try and utilize the same blood collection technique at all times.
- Handle the blood in a consistent fashion. Use the same anticoagulant and try and add the same volume of blood to the collection tube.
- Centrifuge the blood immediately following collection and remove the plasma immediately following centrifugation. The warmer the ambient temperature, the quicker potassium will move out of red blood cells into surrounding fluid, resulting in falsely elevated values.
- Freeze the sample following collection, preferably on dry ice, in liquid nitrogen, or in an ultracold freezer at -70°C.
- The sample should be transported frozen to the laboratory, preferably on dry ice.
- Try and use the same clinical pathology laboratory utilizing the same machine. Make sure the samples are handled similarly prior to analysis. For instance, small plasma volumes that have to be diluted in order to reach a minimum volume necessary for the machine will have a dilution error superimposed upon other technique errors.
The most common solid tissue biopsied is the skin. In most situations, a local anesthetic agent such as 2% lidocaine hydrochloride (Lidocaine, Phoenix Pharmaceuticals, St. Joseph, MO 64506) can be used around the site. The biopsy site and surrounding tissue should be cleansed with 3 alternating applications of 70% ethanol and a surigcal iodine soap (Betadine Surgical Scrub, The Purdue Frederick Co, Norwalk, CT 06856) before the sample is obtained. Sterile surgical gloves should be used. The sample can be obtained using a scalpel blade (#10 or #15) or a biopsy punch (Disposable Biopsy Punch, Premier Medical, Norristown, PA 19404). Following removal of the sample, the defect can either be sutured or left to heal by granulation. Depending upon the type of lesion being biopsied, single or multiple samples are collected. Subsequent preservation of the sample will depend upon the various diagnostic tests to be used. For histologic evaluation, a portion of each sample should be fixed in neutral buffered 10% formalin (NBF), with a tissue to fixative volume ratio of 1:10. Since formalin can only penetrate 6 mm in 24 hr, the tissue should be thin enough to allow adequate fixation. If tissues are to be stored beyond 48 hr in a fixative, they should be transferred from NBF to 70% ethanol at this time. If samples are to be submitted for microbial isolation attempts, they should be cleansed with sterile saline to remove the overlying alcohol and betadine scrub prior to being placed in an appropriate transport media or sterile container for shipment to a diagnostic laboratory. Since freezing results in crystallization artifact, tissues for histologic examination should never be allowed to freeze. For specifics on shipment of samples, the individual collecting the samples should contact a diagnostic laboratory in advance to receive specific information on transport of samples.
Biopsies also can be obtained from visceral structures. While potentially achievable in the field, in most situations this will be performed in a veterinary hospital, under general anesthesia. A gas anesthetic such as isoflurane (Aerrane, Ohmeda PPD inc, Liberty Corner, NJ 07938) is most commonly used. Biopsies can be obtained from the gastrointestinal tract using a flexible fiberopticscope and biopsy device. Biopsies from visceral structures such as the kidney or liver can be obtained either through a laporatomy incision or using a ultrasound guided technique and various automated biopsy devices. Again, consult a veterinary hospital for the various options available.
In order to determine cause(s) of death, a thorough postmortem evaluation should be performed. The quality of the necropsy will depend upon the background and training of the person doing the examination. Ideally, the person should have good experience and knowledge of sea turtle anatomy. Whether the necropsy is conducted in the field or in a veterinary diagnostic facility will determine the depth of the examination. Be prepared to collect the following samples: 1) tissues for histopathology; 2) tissues for electron microscopy; 3) samples for microbiology; 4) tissues for toxicology; 5) stomach content samples; and 6) parasites.
Ideally the necropsy should be performed as soon after death as possible. If the necropsy is delayed, the carcass should be either placed in a refrigerated room or placed on crushed ice. Avoid freezing the carcass since this will cause artifactual changes in tissues. To be most informative, necropsies should be done within 24 hr of death.
Marine turtle necropsy procedures have been described (Campbell, 1996) and a “Sea Turtle Necropsy Manual” (Wolke and George, 1981) was published in an attempt to try and standardize technique. Unfortunately this manual is outdated and this webpage is an attempt to update the methodology of performing sea turtle necropsies. A Necropsy Report Form was developed in the mid-1990s (Stamper et al, 1996) and since that time has gone through multiple revisions by various pathologists and biologists. The latest version is available here with the expectation that it will go through constant change as recommendations and suggestions are received. Images of major organs of sea turtles are linked to this form and new ones will be added as they become available. Understanding sea turtle anatomy is essential when performing a necropsy and an “Anatomy of Sea Turtles” by Jeanette Wyneken is available online.
Equipment needed for a necropsy are listed at the end of this page. It is important to wear appropriate clothing that can be washed following completion of the necropsy. This includes proper rubber boots or protective covering of shoes. Rubber gloves are essential and a face mask is recommended. When performing a necropsy, as much pertinent information should be collected and recorded including species of turtle, weight, carapace and plastron length and width, and sex. Weather conditions should be noted. The times at start and finish of the necropsy should be noted. For captive animals, a summary of the clinical course of the turtle should be recorded. For wild turtles found dead in the field, the stranding data sheet should be attached to the necropsy report. Photographs should be taken of the entire carcass, both dorsally and ventrally. Photographs should also be taken of any lesions recognized.
Necropsies start on the outside and move internally in a methodical manner. The exterior of the turtle should be thoroughly examined, describing all gross abnormalities. Drawings of marine turtles, both dorsally and ventrally, should be used to indicate location of lesions. Wounds to the shell and soft tissues are noted. Any other changes such as swellings to join spaces of long bones and cutaneous or subcutaneous masses are recorded. Samples of all significant lesions should be collected for histopathology. Samples are placed in neutral buffered 10% formalin (NBF). NBF will only penetrate 6 mm in 24 hr, so make sure tissues are thin enough to allow adequate fixation. The NBF to tissue volume ratio should be 10:1. If hard tissue such as long bone is collected, it should be fixed in a container separate from the soft tissues to allow adequate penetration and fixation.
The overall appearance of the turtle will dictate whether to continue with a full necropsy. If the turtle is in an advanced state of postmortem change, such as being bloated with gas, skin discolored, or scutes falling from the shell, collection of tissues for histopathologic evaluation will be unrewarding.
The necropsy progresses with the turtle in dorsal recumbency (plastron up). The plastron is removed intact by separating it from the carapace along the marginal bridge, on both sides, and from the skin at areas of attachment. When this is done, the plastron is removed from the carcass. The gular area of the lower jaw is incised just medial to, and along the edges of the mandible. The incision is extended into the oropharynx, and when done, the tongue, glottis, and proximal trachea are lifted and exteriorized. This allows visualization of the oral cavity, with sampling of tissues as needed. Portions of tongue and glottis are collected for histology. As tissues are sampled for histology, the transition area between healthy and abnormal tissue should be collected. This is often an important area to look for pathogens. The trachea and esophagus are severed just cranial to the base of the forelimbs and removed from the carcass as a unit. Next, the forelimbs and hindlimbs and their associated girdles are removed. When this is done, the entire coelomic cavity can be visualized.
Before any further samples are collected, this is a good time to scan the coelomic cavity for any obvious lesions. All lesions noted should be described in terms of size, color, and consistency. If excess or discolored fluid is seen in the coelomic cavity, a sample should be obtained for culture. A small amount of fluid can be placed on a microscopic slide and a smear made for future cytological examination. Visually scanning the coelomic cavity for changes and collecting samples at this stage of the necropsy is important in order to ensure that minimally contaminated samples are collected for microbiology. As the necropsy progresses contamination of tissues is inevitable. Samples of lesions may be swabbed with appropriate culturettes or portions collected aseptically (using either sterile or flamed instruments), placed in a sterile container, and transported to a diagnostic laboratory for culture. The manner in which the sample is transported will depend upon the cultures attempted. For the most part, samples should be transported either on crushed or dry ice. If the animal is recently dead (within 1 hr), heart blood can be collected for culture of aerobic organisms. Again, consult a veterinarian or diagnostic laboratory for selection of appropriate transport media.
A complete necropsy should include collection and archiving of fixed (neutral buffered 10% formalin) and frozen tissue (at least -70 C) samples from all tissues, so that materials are available for retrospective studies and research, e.g. toxin analysis, nutrient analysis, virus isolation, transmission studies, immunodiagnostic and molecular diagnostic tests. Normal tissue specimens should be saved in addition to obvious lesions. Specimens from lesions should be representative of the entire lesion and large enough to include adjacent normal tissue. This not only facilitates comparison of pathologic tissue with normal but often the active process and the primary etiologic agent are found at the edges of a lesion.
Examination of touch impressions and wet mounts of various lesions is extremely helpful in diagnosing disease problems in reptiles. Because of the ease and rapidity of processing these samples, much information can be gathered in a short time. For external lesions, samples can be collected with relative ease. Depending upon the size and nature of the patient, manual restraint alone may be all that is needed. In larger more fractious reptiles, or in those patients with internal masses, sedation or anesthesia will be required.
Collection and Preparation
The collection method used will depend upon the nature and consistency of the lesion being sampled. Prior to collection of the sample, the distribution, color, morphological description, size, and odour (if present) of the lesion should be noted. For cutaneous lesions, scrapings can be collected from keratinized structures such as shell lesions of chelonians or hyperkeratotic surfaces. A sample can be suspended in a small quantity of saline and a coverslip placed over the sample. Next, by applying gentle pressure to the coverslip using a fingertip (in a twisting motion), a “squash-preparation” can be made. This will help in detecting pathogens contained within keratinized material. Potassium hydroxide can be used for digesting keratinized material and improving examination for pathogens.
Lesions containing fluid or purulent material can easily be sampled with a needle. If the lesion is cutaneous, it should be cleaned with a small amount of 70% ethanol and allowed to dry prior to sampling. As discussed earlier, fine-needle aspiration biopsy specimens also can be collected from firm masses.
Aspirates should be examined first as a wet mount. This will give the clinician an idea of what additional preparations should be made. Some organisms such as protozoa and nematode larvae are better appreciated in a wet mount than in a stained preparation. Aspirates having good cellularity can be used to prepare direct smears using the conventional wedge method or the method used for preparing blood films on coverslips. Aspirates containing tenacious material with thick cellular fragments should be prepared using a squash technique. After the sample is placed on a microscopic slide, another slide is used to flatten the material and both slides are quickly pulled apart.
Lung washes should be routinely collected from live turtles with respiratory disease. While samples can be collected from some turtles using manual restraint, other patients will have to be sedated or anesthetised. The jaws of the turtle are held apart and a sterile catheter guided through the glottis into the lung field. The clinician needs to know the location of the lung(s) before collecting a lung wash. With a syringe attached to the catheter, sterile saline (2 ml for a 1 kg turtle snake) can be introduced into the lung field and aspirated several times. Material collected can be used for both cytological evaluation and microbiological culture.
If the turtle is large enough, samples can be collected via bronchoscopy. Bronchoscopy has an added advantage of permitting the lower respiratory tract to be examined directly. The author prefers the patient to be anesthetised when performing bronchoscopy with the flexible fiberoptic bronchoscope passed through the endotracheal tube. A t-tube connected to the endotracheal tube can be used to allow the technique to be performed while the patient remains connected to the gas anaesthesia machine. Utilizing this technique, the tracheobronchial system can be methodically examined and sampling procedures such as lavage, culture, brushing, and transbronchial biopsy can be performed on specific areas of the respiratory tract. For collecting samples for culture, the plugged telescoping catheter brush system is the method of choice.
Aspirates and lung washes of low cellularity can be concentrated utilizing a number of techniques. Using the conventional wedge method, cells can be marginated by lifting the spreader slide from the smear slide just prior to reaching the end of the smear. Cells also can be concentrated by simple centrifugation in a plastic capped tube followed by examination of the pelleted sediment. If samples are sent to a clinical pathology laboratory, cells can be concentrated on a microscopic slide using a cytocentrifuge.
The collection of biopsy specimens has already been discussed and in addition to histopathology, biopsy specimens can be evaluated cytologically. The cut surface of a specimen can either be scraped with a sterile scalpel blade or touched several times by a microscopic slide to make impression smears. If the surface of the specimen contains a moderate amount of blood or clots of blood, this can be removed by gently cleaning the surface with sterile saline via a syringe and needle or gently rolling a saline moistened cotton swab across the surface. A swab also can be used for collecting cellular samples for examination on a microscopic slide.
Depending upon the suspected disease or presence of pathogenic organisms, a variety of staining techniques can be used in evaluating smears. The method of fixation of the smear to the slide will depend upon the staining procedure used.
The most common method used for initial evaluation of smears is the Wright’s-Giemsa stain. Prior to staining, the smear is fixed in absolute methanol for approximately 10 seconds. Quick staining techniques are commercially available and allow staining of smears in a few seconds. Other stains that are commonly used in evaluating smears of lesions from reptiles include Gram’s stain for bacteria, acid-fast stain for mycobacteria, and new methylene blue for fungi.
Handling Tissues for Light Microscopy
Representative sections of all major organs are collected including the following: tongue, skeletal muscle, glottis, trachea, lungs, thymus, thyroid, adrenal gland, pancreas, heart, liver, gall bladder, esophagus, stomach, small intestine, large intestine, bladder, reproductive organs and tract, and brain. Tissue for light microscopy should be fixed in 10% neutral buffered formalin (NBF). Tissue to volume of fixative ratio should be about 1:10. Formalin can only penetrate 6 mm of tissue in 24 hrs so section should be thin enough to allow adequate penetration. It is recommended that in 24 to 48 hr, tissue to be moved to 70% ethanol where it can be stored for long periods of time.
Handling Tissues for Transmission Electron Microscopy
For electron microscopy, small portions (1 mm3) of relevant tissue should be fixed in either Trumps solution (McDowell and Trump, 1976) or 2.5% glutaraldehye. After 24 hr of fixation, the specimen should be transferred to an appropriate buffer such as phosphate buffer. If an interesting change suggestive of a viral infection is found by light microscopic examination of NBF-fixed tissue, a small portion of tissue can be processed for electron microscopy. It is even possible to use paraffin embedded tissue in identifying the presence of viruses. Most viruses are preserved fairly well in paraffin.
Handling Tissues for Negative Staining Electron Microscopy
Negative staining (NEM) in conjunction with transmission electron microscopy (TEM) is a useful and rapid method of examining clinical specimens. The principle of NEM is that there is no reaction between the stain and the specimen. The most commonly used negative stains are uranyl acetate (0.5-1.0%) and potassium phosphotungstate (PTA) (0.5-3.0%). In cases where a viral agent is suspected, this technique may be used in a variety of specimens. Depending on the nature of the tissue, different ways of processing the sample are required to detect viral particles. Fluid from vesicles can be obtained with a sterile pipette and may be placed directly on a Formvar-coated 200-mesh copper grid, while large amounts of fluid (serum, urine, liquor) require centrifugation for clarification. In these cases the supernatant after low speed centrifugation (1,500 g) or the diluted pellet after high speed centrifugation (15,000 g) is placed on the grid for staining. Faecal material requires suspension and concentration and will be placed in distilled water or phosphate buffered saline (PBS). A very useful method is to mix faecal material with PBS to give a 20% suspension in 5 ml. After centrifugation a drop of the supernatant is placed on a grid and negatively stained. Alternatively one drop of a 1:1 mixture of supernatant and stain is placed directly on a grid for examination. If needed, bacitracin as a wetting agent may be added. If the grids will be stored or cannot be examined immediately, the stain of choice is uranyl acetate because it will not have adverse effects.
Tissues such as liver, kidney, spleen, etc., require grinding to obtain a homogenate that will be processed in the same way as the above samples. In general the method used for processing specimens depends on the concentration of viruses suspected in the sample.
For heavy metal analysis, samples of kidney, liver, and skeletal muscle can be collected, placed in separate plastic bags, and frozen on dry ice or in an ultrafreezer until submitted. For pesticide analysis, fat, liver, kidney, and skeletal muscle should be wrapped in aluminum foil, placed in a plastic bag, and similarly frozen. Approximately 200 grams of tissue should be the minimal amount collected.
A thorough diagnostic workup of suspected viral, bacterial, fungal, and parasitic diseases would include attempts to isolate and identify the pathogen. Specimens must be collected and transported in a way that preserves pathogen viability, minimizes contamination by commensal and incidental microorganisms, with minimal changes in the floral composition caused by overgrowth of the specimen by faster growing species. Blood, tissue fluids, exudates, or tissue biopsies to be submitted for microbial culture must be collected under aseptic conditions using sterile instruments and technique so that the specimen is representative of the microbes found in the lesion rather than contaminants. These samples can yield spurious and confounding results and are not worth collecting if they cannot be handled properly and in a timely manner to an experienced clinical microbiological laboratory.
Contact should be made with receiving laboratory well in advance so that they can advise the field worker about the laboratory’s capabilities, submission deadlines, proper collection materials, and transport media. It is important to realize that, although many species of bacteria and fungi can be cultured using standard media and procedures, many other microorganisms such as Mycoplasma spp. and Mycobacterium spp. require specialized culture media and conditions and other organisms such as Chlamydophila spp. and viruses require a permissive cell culture system for isolation. One must find a diagnostic laboratory that has access to specialized media and cell lines and be prepared to carry out the special culture procedures required by these agents. Failure to isolate a certain microorganism does not rule it out as a potential cause of the disease under study. Appropriate culture systems for some potential pathogenic bacteria, fungi, and viruses have not been developed yet.
Concepts of Infectious Disease
To understand infectious disease in populations one must understand the distinction between being infected with a disease causing agent and having disease (overt illness) caused by that agent. As a rule, infection will be relatively common in a population but clinical disease rare. For any disease agent in a population of turtles there will be individuals that have never been infected, individuals that are infected but are not sick, those that are both infected and sick, and individuals that were infected but are now immune. The interactions of factors that influence whether infection is expressed as clinical disease in a population can be very complex.
Different diagnostic tests can be used to detect or monitor prior or current infection or clinical illness. The results of any single diagnostic test must be interpreted in the context of the entire picture, including the history and pattern of disease in the population, clinical signs, results of other tests, and the gross and histopathological lesions. Detection or isolation of an infectious agent or detection of antibodies to that agent provide only partial information in an investigation of a morbidity / mortality problem. In some instances, findings may be completely incidental to the real cause of the disease
Serodiagnostic Tests (Serology)
Serodiagnostic tests are tests performed on serum or plasma to detect either the presence of antibodies to a particular disease causing agent or the presence of circulating antigens from the disease-causing agent itself. The former type of test is used to determine whether individuals in a population have ever been exposed to a particular disease causing agent, by the fact that they have mounted a humoral immune (antibody) response against it. The latter type of test is used to determine whether the individual has an ongoing exposure (e.g. active infection), by the fact that they presently have foreign substances (antigens) derived from the causative agent circulating in their blood. The high sensitivity and specificity of these types of tests make them extremely valuable in population health monitoring (disease surveillance), where most infections are subclinical, and in testing specific hypotheses (differential diagnosis) about the causes of specific disease outbreaks (epizootics).
The fact that antibodies and some antigens are stable in frozen plasma for many years, makes it possible to perform retrospective epizootiological studies that can yield valuable information on the long-term health history of turtle populations. This will help pinpoint the time, perhaps long before clinical disease became recognized, when a new infectious agent entered a population.
Molecular Diagnostic Tests
The science of detection and characterization of pathogenic organisms has made tremendous advances with the development of nucleic acid hybridization (Southern and Northern Blotting, in situ hybridization) and amplification techniques [polymerase chain reaction (PCR)] and the ever-increasing availability of specific nucleic acid probes and primers. While molecular diagnostic tests exist for many bacteria and fungi shared between turtles and other vertebrates, those pathogenic organisms unique to marine turtles await development. Nevertheless, turtle biologists should anticipate the eventual availability of these tests and collect the appropriate specimens. Fortunately, either formalin fixed or deep frozen tissues (< -70 C) can be used for many applications. For research requiring non-degraded DNA and RNA, immediate freezing and storage of fresh tissue samples in liquid nitrogen would be preferable.
Collecting Tissues for Diagnosing Pathogens
The following sections briefly describe the types of samples that would be needed for the major infectious disease agents.
Preliminary diagnosis of viral disease usually comes from histopathological examination of fixed tissues obtained by biopsy or at necropsy. Coupled with history and clinical signs, the occurrence of characteristic cytopathology such as cell degeneration (swelling and lysis), syncytia formation (fusion of adjacent cells), and intranuclear or intracytoplasmic inclusion bodies, provides the first clue that a viral agent may be involved. Electron microscopic examination of these fixed tissues may confirm the presence of virus-like particles within the lesion and provide a preliminary identification of the agent. Complete diagnosis is achieved by virus isolation from fresh or frozen (< -70 C) samples in an appropriate tissue culture system, followed by immunological and molecular characterization of the isolate. In cases where an appropriate cell culture system has not been developed for the agent, further identification may be achieved by agent specific immunohistochemical techniques using agent specific antibodies or by molecular biochemical techniques using agent specific nucleic acid probes (Southern blotting; PCR). Although some viruses may remain intact and infectious for very long times at ambient temperature, the most environmentally sensitive viruses usually retain some infectivity if they are rapidly frozen stored at -70 C. Fresh tissue samples placed in virus transport media (serum-free cell culture media containing antibiotics and antifungals) can be shipped on ice to a laboratory set up with a variety of cell lines (including sea turtle cell lines) for virus isolation.
Gross and histologic examination of lesions usually provides first evidence of bacterial or fungal disease. In addition to routine hematoxylin and eosin, special tissue stains, such as tissue Gram stains (Brown and Brenn), silver impregnation stains (Warthin-Starry; Gomori Methamine Silver), and acid fast stains (Ziehl-Neelsen), can help narrow the range of possible agents. Smear preparations of lesion exudates or impression smears of affected tissues can be made, stained, and examined in the field. Submission of specimens for bacterial and fungal culture should follow the guidelines discussed above (clinical microbiology). Immunodiagnostic and molecular diagnostic techniques can also be applied to fixed or frozen tissues or to culture isolates.
The protozoal diseases that have been described in marine turtles so far are primarily pathogens of the gastrointestinal tract. While fecal analysis (direct smears, floatation) can be an aid in diagnosis, many gastrointestinal protozoans may be commensals and finding the organisms within characteristic histological lesions is the best way to identify pathogenic species. Protozoal infections of other organs will also require histological diagnosis.
Most organs in sea turtles have the potential to contain parasitic worms (helminths). To facilitate finding as many of the helminths as possible, one must use a systematic approach and work with the pathologist doing the necropsy. Pathologists will facilitate having all organs examined for lesions possibly related to the parasites present and having tissues collected for fixation for either light or electron microscopy, freezing for pesticide work or virus isolation. Tubular organs will be opened in their entirety and solid organs will be slices and compressed to expel any worms present. All liquids and particulate material will be collected from each organ individually and then this material will be washed in a container of water before being placed into a #50 standard sieve and washed with a spray from a pistol grip nozzle on a normal garden hose. The washed contents will be saved and viewed in a light box for visualization for recovery of helminths. Begin with a set of labeled plastic containers including esophagus, stomach upper intestine, middle intestine, lower intestine, liver, urinary bladder, heart, major vessels, pancreas, kidney, lung, spleen, mesentery, feces, and brain. Small organs and feces could be placed into screw top urine cups. Larger organs could be place into 1000 mL plastic containers and medium sized organs could be placed into 500 mL containers.
Organ by organ recovery of helminths
This is best done in sections. The esophagus can be separated from the glottis and the stomach and placed into a container. The stomach is also separated and placed into its own container. The intestines may be separated from the mesentery and laid out on the necropsy table. If you fold it back on itself twice, you can subdivide the gut into three equal lengths. Keep tract of which is the upper end versus the lower end as the pathologist will want sections for fixation. If lesions are observed after the gastrointestinal (GI) tract is opened, the pathologist will want to know where in the tract the lesions were located. It is best to let the pathologist examine the opened gut mucosa of the various sections and select areas to collect for histology. Furthermore, there are helminth communities in these sections that can vary markedly. The segment of the GU tract must be opened over a container or large tray to catch the contents. Once the pathologist has examined the mucosa, place the opened gut between two fingers and pull the gut through your tightly clasped fingers over the container so the stripped mucosa and worms will fall into the catch container. The gut contents may be placed into the sieve and washed. Once the water comes through clear, concentrate the remaining ingesta and back flush it into the labeled container. Repeat for each section of the gut. Do not forget to collect a fecal sample from as distal as possible. Rinse the mucosa again and examine for helminths still adhering. You can strip it twice if you feel the need to do so. Examine the serosa for small white bodies in the gut wall as these might be embedded in the wall. These will be tapeworm or nematode larvae and the tissue around it should be removed and saved fresh to dissect out these parasites. These will need to be dissected open in the lab under a stereo-microscope, or sectioned to see what they are. Occasionally, you will find blackened, raised ridges running longitudinally of the gut for short sections of the gut. These are fluke eggs that have not cleared the blood vessels in which they are produced. Histological sections will demonstrate the eggs and occasionally the flukes producing the eggs.
Open the urinary bladder over the container and rinse the mucosa into the cup and strip the mucosa lining after you have looked for lesions and collected a piece for histology. Place the kidney on a cutting board. Let the pathologist look for lesions and harvest tissue for later use. The remainder of the kidney should be sliced into slabs about 1 cm thick and then compress the kidney. Continue until the kidney has been fully sliced and then wash the entire kidney into a container, add water and swish the water around the pieces. Remove the pieces and pass the debris and blood through the sieve and wash until clear and process as for gut.
Liver, lungs, and pancreas
The lliver, lungs, and pancreas are processed the same way as the kidney. Bile should be removed from the gall bladder and examined.
The heart should be separated from the major vessels and then all three chambers be opened and grossly examined for flukes. The endocardium should be washed thoroughly into the cup for the heart. The vessels such as aorta, carotids and any other major ones will be slit open, examined for flukes and then washed by placing in a container, adding water to half full and shaking the closed container to wash the flukes free of the vessels. These can be kept separate from the heart material. Remnants from both can be passed through the sieve to get rid of blood and clots. Clear the sieve as above.
The mesentery supporting the intestine is well vascularized and by using a 12.5 cm diameter glass clear light fixture, the mesentery can be draped over it in the Pyrex casserole and the blood can be forced to move through the vessels to assist in seeing adults. Alternatively, by stripping the mesentery as was done for the intestine, the spirorchiid flukes might be expressed from the vessels and then the mesentery could be washed in water to remove the flukes. Use the sieve for removal of blood and lymph.
Brain and spinal chord should be removed as intact as possible and viewed under a stereo-microscope to look for masses of eggs and adult flukes in the superficial blood vessels. The adults need to be dissected out of the vessels and preserved. The removal is very difficult. It may be easier to cut the vessels containing the individual worms and fix the worms in situ and then process the worms for identification in situ. Eggs are helpful in categorizing the spirorchiids so record egg size and shape.
The spleen should be teased apart and then pepsin digested. There are some spirorchiids that live in the vessels of the spleen. The digestion will release eggs that have been filtered out of the blood and give a better indication of spirorchiid presence than trying to find the adults.
Once all the containers contain their appropriate samples, return to the lab to recover the helminths. Have a series of 6 cm petri dishes ready to be labeled by organ be used for initial collection of helminthes. They will be transferred to containers where they will be fixed, stored, and labeled by organ in preparation for submission to a parasitologist. One can use a regular petri dish and view all washed materials under a stereo microscope and pick out the helminths or once you recognize helminths, you can use a rectangular Pyrex casserole and place it on a countertop radiograph light or a light box and then pick the helminths without the use of magnification. There are no helminths of sea turtles that cannot be visualized with the naked eye after they have been washed as outlined above. The primary helminths of sea turtles are digenetic flukes.
Flukes should be fixed in AFA (85 parts 85% ethanol, 10 parts commercial formalin and 5 parts stock glacial acetic acid) and stored in vials into which the turtle species, ID, date, and organ will be written with soft pencil (or indelible ink that is resistant to being dissolved in ethanol on slips of bond paper which can be inserted with the worms into the vial. Fat bodied flukes or flukes that normally are flexed after removal from the host need to be flattened with slight pressure between two slides and the fixative allowed to diffuse around the helminths. Keep in this position for about 5 minutes and then place flukes into the vial and fill with AFA. Most flukes recovered from marine turtles have relaxed by the time you recover them. Nematodes are sometimes numerous in loggerhead sea turtles. These can be placed into glycerine alcohol (90 parts of 70% ethanol and 10 parts glycerin). The labeling should be the same as for the flukes. Keep a written record of the numbers of helminths you have recovered from each organ and indicate which organs did not contain helminthes as sometimes organs contain parasites and other times they will not and you want the parasitologist to know that the organ was examined and no helminths were recovered. It is a good idea to contact the parasitologist you will send your specimens to in advance and see if they have any special recommendations. Also forewarn them when you have samples headed their way.
Feces should be examined by both sedimentation and flotation procedures. Flotation is done for stages that normally float in special media. In the feces of marine turtles, these include the nematode eggs and the coccidian oocysts. The trematode eggs do not usually float and are sedimented. It is best to minimally use one gram of feces for both procedures. About 15 ml of the flotation medium is placed into a urine cup and the feces is added and the feces is broken up to release the eggs. The contents are then pour through a layer of gauze into a 15 ml centrifuge tube and this is topped off with flotation medium until a slight positive meniscus is formed. A cover slip is placed over the top and the specimen allowed to stand for 10 minutes. Next, lift the cover slip straight up and place on a labeled slide. Read the entire cover slip under the 10x objective of a microscope.
The sedimentation procedure uses soapy water (1 squirt of inexpensive dish soap to 500 ml of tap water in a squeeze bottle) as the aquatic medium. The feces is mixed in the soapy water (<45 ml) in a urine cup and then passed through a layer of gauze into a 50 ml centrifuge tube. You allow the solution to stand for 5 minutes, decant or aspirate off supernatant, refill with soapy water and resuspend debris at bottom of tube, let stand another 5 minutes and then continue with this process until the solution remains clear. Examine the sediment under 100x magnification. Record size and characteristics of eggs and photograph if possible.
At the end of the necropsy, the carcass should be disposed of in a manner consistent with those approved by both federal and state permitting agencies. Sea turtles may harbor a number of bacterial species that are known human pathogens or are opportunistic pathogens in a wide range of vertebrate species. These include Mycobacterium spp, Salmonella, Vibrio, and Chlamydophila spp. While it is unlikely that field workers will use universal precautions in handling sea turtles turtles, they should realize that risks exist and have appropriate materials available for disinfecting wounds received while handling these animals. Workers should seek medical attention if wounds become infected or if they become systemically ill after working with turtles. Gloves should always be worn when performing necropsies.
Another concern is the possible accidental (iatrogenic) spread of infection among turtles by turtle biologists who fail to take precautions to prevent this from happening. Needles, tags and tag applicators, laparascopes, endoscopes, stomach tubes, etc. can efficiently transfer infectious agents. Inexpensive disposable materials such as scalpel blades and needles should not be used on more than one animal. Instruments that are used repeatedly must be sanitized or disinfected between animals. Adapting decontamination techniques to the field, although difficult, should be done for equipment that is to be reused. Sodium hypochlorite (10% bleach solution) is an excellent and inexpensive disinfectant but it is corrosive and is rapidly deactivated by organic debris. Washing in hot water with a strong detergent is also very useful. Glutaraldehyde solutions or formalin are powerful diinfectants but are very toxic. Chlorhexidine solutions and povidone iodine solutions are effective and less toxic. Alcohol is not an effective disinfectant unless the instrument is flamed or left for very long periods of time.
As marine turtle resources and marine ecosystems become more intensively managed, with individual turtles and populations being manipulated within and possibly moved between natural habitats and artificial enclosures, the potential impact of infectious diseases will become more and more apparent. Health monitoring will become an important part of overall management so that new potentially devastating diseases can be discovered before they threaten management efforts and so that diseases already having such effects can be monitored and controlled. Presently, much of the diagnostic work performed on marine turtles is performed in retrospect, at necropsy or in the face of a disease outbreak. It will be important to make population health monitoring more prospective by developing and using mass screening diagnostic tests for disease agents of concern.
Serodiagnostic tests are highly sensitive yet highly specific for a particular pathogen and are important components of prospective population health monitoring. Development of serodiagnostic tests for marine turtles health assessment are in early stages of development. Significant progress has occurred with the production of monoclonal antibodies specific for green turtle immunoglobulin classes (Herbst and Klein, 1995). With these reagents, antibody responses of green turtles, Chelonia mydas to any foreign antigens, including infectious agents and toxins, can be detected if reliable sources of target antigen can be found. Some of these monoclonal antobodies can also be used with other marine turtle species (Herbst and Klein, 1995). The limitations on applying these reagents widely in standardized tests has been the paucity of antigens. While the monoclonal antibodies provide half of the requirement for reliable, repeatable, standardized serodiagnostic tests, we do not yet have reliable sources for well characterized and standardized test antigens with which to monitor any disease. Although some preliminary immunodiagnostic tests have been produced (Herbst 1995) they require further development and refinement before they are available for wide application. Nevertheless, it cannot be emphasized enough that plasma specimens should be collected and archived in an ultrafreezer since they will provide a snapshot in time of the disease exposure of a turtle population being studied. All field biologists who are handling marine turtles for other purposes (e.g. tag and release) are urged to consider collecting plasma to archive for future testing. This recommendation points to the obvious need to establish a registry or plasma bank to curate these samples.
- Campbell TW. 1996. Sea Turtle Rehabilitation. Section VII. Appendix, in Reptile Medicine and Surgery, Mader DR (ed.), W.B. Saunders Co, Philadelphia, pp.427-436.
- Campbell TN, Coles EH. 1986. Avian clinical pathology, in Veterinary Clinical Pathology, Coles EH (ed), W.B. Saunders Co., Philadelphia, pp. 279-301.
- Herbst LH. 1995. The etiology and pathogenesis of green turtle fibropapillomatosis. PhD Diss. University of Florida, Gainesville, Fl.
- Herbst LH, Klein PA. 1995. Monoclonal antibodies for the measurement of class-specific antibody responses in the green turtle, Chelonia mydas. Vet Immunol Immunopathol 46:317–335.
- Jacobson ER. 1987. Reptiles, in Veterinary Clinics of North America: Small Animal Practice, J. Harkness (ed), W.B. Saunders, Philadelphia, pp. 1203-1225.
- Lillywhite HB, Smits AN. 1984. Lability of blood volume in snakes and its relationship to activity and hypertension. J Exp Biol 110:267-274.
- McDowell EM, Trump BF. 1976. Historical fixatives suitable for diagnostic light and electron microscopy. Archiv Pathol Lab Med 100:405-414.
- Owens DW, Ruiz GJ. 1980. New methods of obtaining blood and cerebrospinal fluid from marine turtles. Herpetologica 36:17-20.
- Rainey WE.1981. Guide to Sea Turtle Visceral Anatomy. NOAA Technical Memorandum NMFS SEFC-82, U.S. Department of Commerce, Panama City, Fl.
- Smits AW, Kozubowski MM. 1985. Partitioning of body fluids and cardiovascular responses to circulatory hypovolemia in the turtle Pseudemys scripta elegans. J Exp Biol 116:237-250
- Stamper MA, Cornish T, Lewbart G, et al. 1996. Cooperative efforts between veterinary diagnostic facilities and government agencies in assessing two sea turtle stranding episodes. International Sea Turtle Conference, Orlando, Fl.
- Wolke RE, George A. 1981. Sea Turtle Necropsy Manual. NOAA Technical Memorandum NMFS SEFC-24, U.S. Department of Commerce, Panama City, Fl.
Necropsy equipment list
- Coveralls or other appropriate clothing
- Rubber boots or shoe covers
- Rubber gloves
- String, labels, assorted bottles, water proof pen
- Forceps – several sizes
- Tissue cutting board
- Necropsy knives and sharpener
- Scalpel blades (#20 and #10) and handle
- Postmortem shears
- Alcohol lamp or butane burner
- Matches or lighter
- 70% alcohol
- Containers with neutral buffered formalin
- Fixative for electron microscopy such as Trumps solution (should be kept chilled)
- Sterile whirl-pack bags
- Microbial culturette swabs
- Microbial transport media
- Dry ice and ice chest or cooler
- Stryker saw
- Microscope slides
- Necropsy sheet and notebook
- Camera: digital or conventional SLR