PROTOCOLS FOR DRAWING BLOOD FROM
THE BRACHIAL PLEXUS OF DESERT TORTOISES:

Instructions for Ordering Equipment and Culture Media;
Summary of How to Draw Blood and Conduct Nasal Lavages; and
Information on How to Contract for Laboratory Analysis
and Ship Laboratory Samples


Prepared by

Kristin H. Berry and Tracy Goodlett
for Desert Tortoise Workshop on Health Evaluations, October 21-22, 2000
and modified March 2003 for work on Federal Fish and Wildlife Service Permit
No. TE-006556-11

Earlier versions were prepared for the

Marine Air Ground Task Force Training Command
Natural Resources and Environmental Affairs Division
Twentynine Palms, CA 92278

Revised June 2003

OVERVIEW OF THE PROTOCOL

Blood can be drawn from desert tortoises from several sources, such as the jugular vein, heart (cardiocentesis), or sinus associated with the eye. For research on hematological and plasma biochemical values of tortoises, the jugular vein is the appropriate source to obtain pure blood, without lymph (see Christopher et al., 1999, Christopher et al., submitted). For enzyme-linked immuno assay (ELISA) tests, blood can be drawn from the brachial plexus. The ELISA tests are for assessing exposure to herpes virus (Origgi et al., 2001) and Mycoplasma agassizii (see Jacobson et al., 1991; Brown et al., 1994, 1995; Schumacher et al., 1993), the bacterial pathogen that causes upper respiratory tract disease. ELISA tests do not require pure blood samples. Our protocol is designed to obtain either blood and/or lymph from the brachial plexus in the forelegs of large immature, subadult, and adult desert tortoises, that is tortoises >140 mm in carapace length at the midline.

The Protocol shown below starts with acquisition of equipment and ends with delivery of plasma and nasal lavage samples to the laboratory. It is designed to be used in conjunction with modified and expanded health profile data forms (see Attachment 1 and Berry and Christopher, 2001). The Protocol is also associated with Kristin Berry's research permit with the U. S. Fish and Wildlife Service, TE-006556-11.


List of Equipment to Acquire/Carry in Field Pack and Vehicle

Equipment for Day Pack (Lunch Carrying Case with Slots for Ice):

• Frozen ice packs (several)
• Microtainer Brand Tube with Lithium Heparin
• Nunc Cryotube vials
• 1 cc syringes with needles (22 gauge [larger] and 25 gauge [smaller])
• 3 cc syringes for nasal flush
• Betadine mix
• Rubbing alcohol
• Q-tips
• Disposable latex gloves
• Gauze
• Some frozen tryptose broth
• Rubber bands
• Small zip-lock bags
• Nalgene Lab Markers or similar for writing on glass/plastic tubes
• Lap pad or sheet for handling tortoise
• Clip board with pen with permanent black ink
• Data sheets
• Standard equipment for processing tortoises in the field, including 35 mm camera with close-up lens

• Ice Chest with as many ice packs that will fit
• More frozen tryptose broth (keep frozen in ice chest) for nasal lavage, culturing Mycoplasma
• Microtainer Brand Tube with lithium heparin
• Extra Nunc cryotube vials
• Extra syringes/needles of both sizes
• Extra syringes for nasal flush
• Bottle of Betadine
• Bottle of rubbing alcohol
• Box of Q-tips
• Box of gauze pads
• Disposable latex gloves
• Rubber bands
• Small zip-lock bags
• Nalgene Lab Markers or similar for writing on glass/plastic tubes
• Lap pad or sheet for handling tortoise
• Supply of data sheets
• Disinfectant (dilute solution of bleach, specifically sodium hypochlorite solution at 0.175%, made fresh each week ) for disinfecting equipment

1. Fisher Scientific Company: (1-800-766-7000)
   Be certain to check all catalog numbers; they may change. You will need to set up an account.
   
   

   Latex gloves, large: powder-free.		Catalog No: 11-394-5C
   Microtainer with lithium heparin 50/pk		Catalog No: 0266875
   Cryotube 1.8 ml, 500/pack		Catalog No: 12565170N
   Syringe/needle TB, 1cc, 25GX5/8", 100/pk		Catalog No: 1482688
   Luer-LOK Syringe, 3cc, 100/pk		Catalog No: 1482340
   Syringe TB 21GX1, 1cc, 100/pk		Catalog No: 1482910D
   Isopropyl alcohol 70%, 500 ml		Catalog No: LC157601
   Betadine solution 16 oz		Catalog No: 19027133
   Gauze pads sterile 3 x 3, 100/pk		Catalog No: 19027227
   Nalgene Lab Markers		Catalog No: 13-382-50, 6310-0010



2. Frozen sterile tryptose /SP4 broth for culturing Mycoplasma: Order directly from Dr. Mary Brown, Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611-0633, (352) 392-4700, ext. 6188, (Barbara Crenshaw is at 352-392-4700, ext. 3968). Email address for Dr. Brown: mbbrown@nersp.nerdc.ufl.edu. Dr. Brown will FEDEX the tryptose broth frozen; you will need to be present to place it in the freezer when it arrives. Dr. Brown has provided the frozen broth free of charge.



3. Lap pads for handling tortoises: 28" x 36 A underpads. Available from a medical supply house or discount center

1. For the Fish and Wildlife Service research permit No. TE-006556-11 held by Kristin H. Berry, two field workers are required to work together on drawing blood and conducting nasal lavages. One field worker must be specifically on the portion of the permit described for drawing blood. No field work on this topic can occur without Dr. Berry issuing a special letter authorizing this activity specifically and with the names of the individuals listed on the cover letter.



2. Prepare Centrifuge for Use. The centrifuge, if one of the more basic and sturdy models, can be run by using an inverter attached to the cigarette lighter of a vehicle. A good place to put the centrifuge is on the floor of the car in front of the front seat (in the shade). Be sure to test the centrifuge system with the vehicle prior to leaving for the field, because the centrifuge/inverter system may not work on some of the older, smaller vehicles.
   
   Balance the centrifuge on the opposite side, e.g., put water in Nunc tubes.



3. Prepare Day Pack For Use. Drawing on the field equipment and ice packs in the car, prepare all necessary equipment for drawing blood and collecting nasal lavages away from the vehicle. Keep needles, gauze, and other equipment sterile. Have lap sheet or pad available to keep tortoise from clothes and limbs. Use a fresh pair of gloves for each tortoise. Prepare clipboard with pen (with permanent black ink) and data sheets for Live Desert Tortoises and Health Profiles (expanded format).
   
   Dilute Betadine to 10 parts water and 1 part Betadine in a container for use in the day pack. Take care to ensure that the Betadine does not get on clothes and pack, because it stains.
   
   If ice packs thaw, return to the vehicle to get fresh ice (and centrifuge the blood).



4. Reducing Stress to the Desert Tortoise. Go to the desert tortoise in the field, carrying field equipment in the day pack. Do not plan to carry the tortoise to the vehicle, carry it in a pack, or drive it in the car to a laboratory or a work site. The system described in this protocol will function best when the field workers go to the cover site or otherwise locate the Tortoise in the field, whether under a shrub or out in the open. In our experience, the tortoise will void less if not moved from his field site, even 50 meters.
   
   Once the tortoise is observed, prepare a shady place for the tortoise and the field workers away from the cover site so that the cover site is not disturbed. This can be done by placing a small, light-weight blanket or canvas or shade cloth or other similar light-weight material over a large shrub or group of shrubs, thereby creating a block of shade. Soil temperatures, if warm or hot, can be reduced by clearing away the top few inches. Do not disturb the area around the cover site, the burrow mound, or initial resting place of the tortoise. Instead, prepare the shady site a few meters away. Ensure that area where tortoise will be handled is clean. Lay out equipment in the order that it will be used. Determine who will be holding the tortoise and who will be taking notes and drawing blood.



5. Drawing blood. Squeeze the limb to improve circulation; consider using a rubber band as a tourniquet posterior to the tendon. Swab the posterior side of one forelimb, either right or left, with diluted Betadine. Rinse with rubbing alcohol. Palpate the area, feeling for the brachial vein. After locating the vein, insert the 25 gauge needle at 45 degree angle into the brachial vein, anterior to the tendon and below the muscle. Slowly draw out at least 0.255 and not more than 0.5 cc blood. Be careful not to push the plunger into the syringe: air bubbles must be avoided. If the needle stick is dry, you may attempt to draw blood with a maximum of 3 needle sticks in each foreleg. If six attempts are unsuccessful, do not continue. Once the needle is inserted into the foreleg, do not move it about to search for the vessel. Lymph may be withdrawn or a mixture of blood and lymph. Lymph may be as effective in the ELISA test as blood (the scientific demonstration of this has not bee completed, so a mixture of blood and lymph may be satisfactory. The area where the typical needle stick is done is associated with a brachial plexus where both blood and lymph come together.
   
   If blood and/or lymph are successfully drawn, apply a gauze pad to the puncture site and apply pressure to prevent bleeding. Hold the gauze tightly pressed to the leg for a few minutes. Inject the blood into a Microtainer Brand Tube and gently mix about 10-15 times. Label as plasma (see item 7 below) and place on ice. Do not freeze because the blood must be centrifuged prior to freezing.



6. Nasal lavages. Thaw broth by holding in hands for a few minutes. Swab nares with rubbing alcohol on a Q-tip. Remove the needle from the 22 gauge syringe and place it on the 3 cc syringe. With the syringe, draw about 1 cc of the broth from its container. Remove the needle and place the open end of the 3 cc syringe against the nare. Inject with the 3 cc syringe about 1/2 of the broth into one nare, then draw it back into the syringe by withdrawing the plunger slowly. Inject the remaining broth into the other nare using the same procedure. Inject the material in the syringe into a Nunc tube taking care not to touch the Nunc tube inside or the top. Label as nasal (see item 7 below) and place on ice.
   
   It is important that the nares and beak be clean for this procedure and that the syringes and needles remain uncontaminated. The nasal fluid is used to culture organisms in the upper respiratory tract, specifically Mycoplasma. Mycoplasmas require weeks to growth and cultures can be rapidly overgrown by other bacterial organisms, thus obscuring or inhibiting growth of the species of interest to us.



7. Required procedure for labeling vials: use a Nalgene Lab Marker Pen (other markers will not retain the ink on the glass and the specimen will become valueless). Place the following information on the vials:
   
   G. agassizii
   Tortoise number and sex
   Date
   Observer
   Study site name
   Plasma or nasal sample



8. Fill out the Health Profile and Live Tortoise Data Sheets. Once the tortoise is located in the field, the data sheets should be filled out. The data sheets are an integral and essential part of the health assessment. The eyes and nares should be evaluated prior to drawing blood and taking the nasal lavage, preferably when the tortoise has its head and beak naturally exposed.



9. Photograph the tortoise. Close-up views of the carapace, plastron, beak, and eyes (both sides of the face) should be taken with a 35-mm camera equipped with close-up lenses. Each slide must be labeled with the tortoise identification number, date, study site name, observer, and location details (UTMs or latitude/longitude in decimal degrees, county, state).



10. Return the tortoise to the cover site or shady site adjacent to where collected.



11. Disinfect all equipment/materials/your hands or arms that have come in contact with the tortoise, the tortoise cover site (probes), or similar using a dilute solution of bleach. Do not use alcohol or other substances. The bleach solution must be made fresh each week and kept out of the sun.



12. Temperature limitations. The drawing of blood and nasal lavages requires careful attention to air and surface temperatures and other weather conditions, because the desert tortoise can easily become stressed and overheated on warm spring days. Desert tortoises generally are not active above ground when air temperatures at 1.0 – 1.5 meters exceed 33° C. Once air temperatures reach this point, then field workers must take great care not to stress the tortoise with overheating. If there is no cloud cover when air temperatures are at 33° C, ground temperatures can exceed 50° C, which is far too hot for the tortoise. As the permit holder, I hesitate to put a fixed figure on air and surface temperatures at which field workers must stop processing tortoises, because extenuating circumstances often occur, such as heavy cloud cover or rain (soil temperatures may remain very cool and tortoises may emerge), deep and permanent shade (soil temperatures will not rise above the air temperature), or the emergence of a cool tortoise from deep within a cover site, etc. Therefore experience, common sense, and good judgment are essential attributes for the fieldworker performing this type of work.



13. Centrifuging the blood. The blood and nasal lavages must be kept on ice at all times. If the ice melts, then the fieldworker must return to the vehicle and get fresh ice. The blood must be centrifuged as soon as possible, preferably within 2 to 3 hours. The longer the blood sits, the more likely it is to clot and have the sample spoiled. We have experienced clotting within 15 minutes. Before starting the centrifuge, ensure that the centrifuge is properly balanced. Spin the blood for 3 minutes. The centrifuged blood in the Microtainer Brand tube may have a layer of plasma on the top, a layer of clay in the middle, and a layer of blood at the bottom (depending on the type of Microtainer Brand tube). With a 22 gauge needle and syringe, draw out the plasma only, without getting clay or blood/red blood cells. Inject the plasma into a Nunc cryotube, labeling the tube as plasma. Freeze the plasma and centrifuged blood in the Microtainer Brand tube. Label the Nunc tube as described in item 7. Do not leave the tubes exposed where they can heat up or warm at any time.

1. Evaluation of the blood samples with the ELISA test and the nasal lavage using cultures and the polymerase chain reaction (PCR) tests. For those individuals not involved with Dr. Berry and her FWS Scientific Research permit, a contract will need to be established with Dr. Mary Brown at the University of Florida . The research scientists and University of Florida hold the ELISA under a patent or similar. In the past, they have provided outstanding service. We recommend requesting a price for all tests for each tortoise. (Department of the Interior agencies have paid about $50 for the set of Mycoplasma tests for each tortoise in the past, as well as additional fees for the herpes virus tests.) The University of Florida does not want to handle charges on a single tortoise basis, so it is most effective to set up a one- to two-year contract for about 50 sets of tests at a time, or more, depending on research plans. The USGS has a contract with the University of Florida for ELISA tests, PCR tests, and cultures. Dr. Berry manages this contract and will manage the blood and nasal samples sent to the University of Florida under her research permit.



2. Preparing the samples for shipping to Dr. Berry or to the University of Florida. For those individuals working on Dr. Berry’s permit and projects, shipping will be directly to Dr. Berry. For those individuals working under other permits, shipping may be directly to the University of Florida. In any event, samples should be stored in layers of zip-lock bags and kept frozen until shipping. They should be shipped via FEDEX on dry ice via overnight mail only after confirmation from Dr. Berry or her staff that they are ready to receive them and will look for them. You do not want to have the samples accidentally lost or thawed. For this reason, only plan to ship Monday through Thursday. Check the shipping address in advance with Dr. Berry and staff. For individuals working on other Scientific Research permits and who have separate contracts with the University of Florida and Dr. Brown, contact Dr. Brown and her staff prior to shipping because the shipping address differs from the mailing address. In any case, the vials should be carefully packed to avoid breakage (we have lost vials and samples to breakage) and must be very carefully labeled with the appropriate ink. We have lost valuable data because the ink washed off sample vials during shipment!
   
   Each shipment should be accompanied by a letter and list of the samples with details. The letter and packing list should be placed in a zip-lock bag with the samples. For samples that are sent to the University of Florida under research permits other than Dr. Berry’s, the advance announcement of arrival should also be sent via facsimile message to Dr. Brown or Ms. Crenshaw on the date of the FEDEX shipment. The following is an example of a table that would be included in a letter accompanying the samples:

References