USFWS Eastern Indigo Snake Handling Protocol

EASTERN INDIGO SNAKE

(Drymarchon couperi)

CAPTURING, HANDLING, BLOOD AND TISSUE SAMPLING,

MARKING, PIT TAG IMPLANTATION, AND SURGICAL

PROTOCOL

U.S. FISH AND WILDLIFE SERVICE

Dated: August 17, 2016

The U.S. Fish and Wildlife Service (Service) authorizes capture, handling, blood and

tissue sampling, marking (scale clipping), PIT (passive integrated transponder) tag

insertion, and radio telemetry (including surgical implantation of transmitters) of eastern

indigo snakes, pursuant to section 10(a)(1)(A) of the Endangered Species Act (Act),

through issuance of permits to take eastern indigo snakes in response to requests from

individuals for such permits. Permits will only be issued for activities conducted by

qualified persons, as described in permit applications, which benefit the recovery of the

eastern indigo snake.

The Service has developed this protocol which identifies specific capturing, handling,

blood and tissue sampling, marking, PIT tag insertion, and surgical transmitter

implantation techniques to be followed when conducting these activities. This protocol

will be attached to all future permits authorizing these activities. The Service will up-

date the protocol as new information is obtained from on-going studies.

CAPTURING

Capturing eastern indigo snakes, even in areas where they are known to occur can be

difficult. Smith and Dyer (2003) conducted a study to determine whether road running

(road cruising), using drift fencing with traps, or using a camera to scope gopher tortoise

burrows would be valid techniques for detecting indigo snakes, and thus offer an

opportunity either directly or indirectly for their capture. The results were disappointing.

In fact, incidental sightings, outside the scope of the research project, resulted in the most

encounters with eastern indigo snakes. Researchers familiar with indigo snakes find that

this is frequently still the case. The Service is continuing to pursue a more accurate

survey method for this species, including the use of detector dogs. With the issuance of a

permit, eastern indigo snakes may be captured by hand after being observed during road

cruising, or other direct observation during general field surveys, and/or by trapping

using stationary cover objects or large box traps (see Rudolph et al. 1999 for a

description of box traps).

Gopher tortoise burrows

If an eastern indigo snake is present or suspected of being present in a gopher tortoise

burrow, a large single-opening funnel trap placed at the mouth of the burrow may be used

to capture snakes as they exit the burrow. Shade the funnel traps and check them 3 to 4

times per day during daylight hours.

HANDLING

Captured eastern indigo snakes may be measured, weighed, sexed, photographed, scale

clipped and/or implanted with a PIT tag; sampled for genetic studies or diagnostic

purposes via blood sampling, skin biopsies, or body (cloacal, oral, and/or skin) swabbing;

and/or surgically implanted with a radio telemetry device (see specific protocols for these

techniques below). Disease may be transmitted within and among eastern indigo snake

populations during handling if appropriate precautions are not taken. Avoid handling

multiple snakes at one time if possible. Use extra precautions when handling ill snakes,

especially if snake fungal disease (SFD) (Ophidiomyces ophiodiicola) is a possibility.

A simple field kit can be made using leak proof containers to hold gauze soaked with a

solution of povidone-iodine, such as Betadine

, to be used for scrubbing, and alcohol for

cleaning areas where scales are clipped and/or sites where PIT tags are inserted.

Appropriate sized examination gloves can be placed in zip top bags.

When handling eastern indigo snakes, the following protocols must be used when

handling snakes for mark-recapture, telemetry, and other purposes:

Disinfection prior to handling

• Examination gloves should be worn whenever possible when performing invasive

procedures. If not possible, wash hands with soap and water if a vehicle is

available near the processing site, or with disinfectant hand wash. Repeat after

handling each snake or wear separate disposable latex gloves for each snake.

• All equipment used for holding snakes should be washed prior to use (e.g. cloth

bags or pillowcases) or disinfected between snakes.

• Clean handling equipment used in the field with isopropyl alcohol or other

appropriate disinfectant (i.e., dilute bleach, chlorhexidine, or other environmental

disinfectant) between each snake. Contact time with the disinfectant is important;

a minimum of 10 minutes of contact time should be used.

• Care should be taken to ensure that contaminated items, such as used examination

gloves, swabs, gauze, disinfectant wipes, etc., do not contact cleaned or sterile

equipment or snakes. Contaminated materials should be isolated and disposed of

in sealed bags.

Transport and Temporary Captivity

• Use a separate clean cloth bag or pillowcase for each snake. Do not reuse until

washed with soap, bleach, and hot water.

• Place each individual snake in separate, well-ventilated, plastic containers for

transport. Their containers should have some substrate or a towel to make the

snake more comfortable and to soak up urine and feces.

• Avoid overheating or cool temperatures during transport. The preferred range of

transport temperature is 60 to72 degrees Fahrenheit (º F) (16 to 22 degrees

Centigrade (ºC)).

• Containers housing snakes must be secured during transport to prevent sliding,

bumping into other surfaces, and/or tipping over.

• Avoid housing snakes in captivity unless necessary for surgery, to treat injuries,

or for placement in the captive breeding colony as part of a Service-approved

action. Currently, only the Central Florida Zoo’s Orianne Center for Indigo

Conservation (OCIC) has approval under this protocol to retain individuals

permanently in captivity for the purpose of breeding and reintroductions. Snakes

captured for the breeding colony must be transported within 3 days of their

capture to OCIC. Each captured snake being temporarily held for the captive

colony must have its own well-ventilated enclosure and kept at the indigo snake’s

preferred temperature zone of 70º to 85º F (21º to 29 º C). The enclosure should

be easily disinfected (dilute bleach, a quaternary ammonia compound such as

Roccal

, or other broad spectrum disinfectant). Wood enclosures should be

avoided, because they cannot be disinfected. More specifics on housing are

provided below under the SURGICAL TRANSMITTER INSERTION section.

TISSUE AND BLOOD SAMPLING

Shed skins

Shed skins may be used for genetic analysis. They should be stored individually (after

drying to prevent mold buildup) in paper envelopes, brown paper bags, or zip top bags

(The Orianne Society undated). Shed skins should be transferred to a freezer as soon as

possible (The Orianne Society undated). Data recorded for each sample should include: a

sample identification number, date collected, state and county where sample collected,

specific location (latitude/longitude, UTM coordinates), and a physical description of the

site where the sample was collected (The Orianne Society undated).

Scale clipping

Examination gloves should be worn when performing invasive procedures such as scale

clipping. If examination gloves cannot be worn, then hands should be free of dirt and

thoroughly cleaned prior to performing any of these procedures. Ventral scale clipping is

one of the most commonly used marking techniques in snake studies. Brown and Parker

(1976) described the technique for ventral scale clipping and provided a scale-clipping

system which can be used for eastern indigo snakes. Scale clips should be taken from the

ventral scales 5 to 25 scales anterior to the vent. The area to be clipped should be cleaned

at least once with a

povidone-iodine scrub and then with a 70 percent isopropyl alcohol

soaked gauze. Examination gloves should be used by the person doing the procedure.

Scales are clipped to remove a rectangular section of scute approximately 0.125 to 0.16

inch (in) (3 to 4 millimeters (mm)) deep and 0.16 to 0.31 in (4 to 8 mm) wide. Use sharp-

pointed dissecting or micro-surgical scissors that have been heat-sterilized (autoclave) or

soaked in isopropyl alcohol or chlorhexidine prior to the procedure. If multiple snakes are to

be clipped, sterilize for at least 10 minutes between snakes

. Care must be taken to remove

all layers of the skin to expose the underlying ventral musculature in a section large

enough to cover half of a given ventral scute (Brown and Parker 1976). The incision site

should not bleed afterwards, but if bleeding occurs use a sterile compress (cotton ball,

gauze) to staunch the bleeding. A liquid bandage may be applied to the site if available.

Scale clips may be taken and used for genetic analysis. If used for this purpose, scale

clips should be immediately placed in individually labelled 1.5 ml (medical standard)

tubes (with screw tops and O-rings) filled with 95 to 100 percent ethanol containing no

additives (The Orianne Society undated). Scale clipping should be done by two

individuals whenever possible; one person to hold the snake and the second to do the

clipping.

Blood sampling

Small blood samples may be taken from the caudal ventral tail vein for genetic analysis

or diagnostic testing. Depending on the diagnostic testing needs, a 25-gauge needle on a

heparinized 3-ml (medical standard) syringe can be utilized. Alternatively, if only

samples for genetics or infectious disease testing using PCR (polymerase chain reaction

analysis) are needed, insulin syringes work well for small samples and are widely

available at pharmacies. The caudal ventral tail vein (coccygeal vein) is located on the

ventral midline of the tail and can generally be easily accessed. Male snakes should be

sampled distal to the hemipenal sacs. The sampling site should be cleaned thoroughly

with 70 percent isopropyl alcohol. Only veterinarians, or trained field technicians, with

experience taking blood from snakes will be permitted for this procedure.

Skin biopsies

Snake fungal disease (SFD) (Ophidiomyces ophiodiicola) is becoming more common in

various species of snakes including eastern indigo snakes (Lorch et al. 2015, T. Norton et

al. 2015 unpublished data). If skin lesions are present, a diagnosis can be made by taking

a full thickness skin biopsy for histopathology and PCR. In most situations, some form of

local or injectable anesthesia will be needed to perform the procedure. To get accurate

results, the biopsy must be full skin thickness. Observing changes in the dermis is

important for distinguishing SFD from other infections. Also, collecting adjacent healthy

skin with the biopsy is helpful for observing necrosis patterns that are somewhat

characteristic of SFD. Finally, detection of O. ophiodiicola alone should not be

considered a positive diagnosis without supportive histopathology. Samples should be

fixed in buffered 10 percent formalin for histopathology and frozen in an ultralow freezer

or shipped immediately to the participating laboratory on ice packs for disease

identification through use of PCR. Samples must be collected by a veterinarian or well-

trained biologist. If skin lesions are on the head, an experienced veterinarian will need to

do the biopsies. Sterile technique should always be utilized and post-operative pain

management should be considered when appropriate.

Other sampling

Swabbing snakes (oral cavity, cloaca, skin), in the lab or field, may be conducted for

diagnostic purposes.

PIT TAG INSERTION

PIT tags are electronic microchips encased in biocompatible glass that vary in size

between approximately 0.4 and 0.55 in (10 and 14 mm) long and 0.08 in (2 mm) wide

(Gibbons and Andrews 2004). In small snakes weighing less than 7 ounces (oz) (200

grams (g)) and with a total length less than approximately 3.3 feet (100 centimeters), the

smallest PIT tags should be used. Once implanted, PIT tags can be used to verify if

individuals have been previously captured. PIT tags may be inserted subcutaneously

(SQ) by appropriately trained individuals, or implanted into the body cavity by a

qualified veterinarian during radio transmitter placement surgery. Test the tag to be used

with a PIT tag reader before it is injected to make sure it is reading properly.

Subcutaneous implantation

Examination gloves should be worn when performing invasive procedures such as PIT

tagging. If examination gloves cannot be worn, then hands should be free of dirt and

thoroughly cleaned with a disinfectant soap or solution prior to handling each snake.

Clean the area selected for tag insertion with a povidone-iodine scrub and 70 percent

isopropyl alcohol. Optimally, at least two scrubs of each should be done prior to

injecting the PIT tag under the skin with the insertion device and needle provided with it.

Only packaged and sterilized needles and PIT tags should be utilized. If reusing needles

and placing the PIT tags in the needle manually, they should be gas-sterilized rather than

sterilized in liquid disinfectant such as alcohol or chlorhexidine. The PIT tag should be

inserted SQ in the area that was previously cleaned, approximately 15 to 30 scale rows

anterior to the vent (cloaca), along the side of the body (approximately two-thirds of the

snout-vent length from the head). Insertion location is very important in order to help

prevent injury to internal organs. The PIT tag should be placed along the upper side of

the snake, approximately two thirds of the way up between the edge of the ventral scales

and the crest of the vertebrae. To inject the tag, grasp a fold of skin between your

fingers; insert the needle into this fold and depress the plunger completely (The Orianne

Society undated). Keep the needle parallel to the snake’s body; do not force the needle

into the muscle tissue or between ribs. Post-injection, sterilize the site using rubbing

alcohol and use tissue glue to seal and protect the injection site. Scan the tag to ensure it

is reading correctly.

Body cavity implantation

Using sterile technique, a qualified veterinarian may manually place a PIT tag into the

body cavity of an anesthetized indigo snake receiving a radio transmitter (see

SURGICAL TRANSMITTER INSERTION, below). Female snakes will be palpated

to determine if eggs are present and, if present, care taken to avoid impacting them during

tag implantation.

SURGICAL TRANSMITTER INSERTION

Eastern indigo snakes are particularly amenable to animal studies using implanted radio

transmitters due to their large size, long-range movements, and large home range sizes.

This technique has been extensively studied and is considered an established method for

the study of snake ecology and conservation (Dorcas and Willson 2009). It is described

in detail in Hyslop et al. (2009).

Radio-telemetry devices may be surgically placed in the body cavity of eastern indigo

snakes by a veterinarian** having knowledge of snake anatomy and significant

experience with snake anesthesia and surgery, and holding the appropriate permits

necessary to treat species listed by State regulations and the Act. All snakes must be in

good body condition prior to surgery. Snakes will be prepared for sterile surgery in a

standard accepted fashion. Recently there have been advances in snake analgesia (pain

management) and anesthesia. In the past, surgery on snakes involved using gas

anesthesia (isoflurane) alone. Isoflurane alone was typically administered while the

snake was in a clear plastic tube, and once the snake was relaxed, it was intubated with an

un-cuffed endotracheal tube and maintained on isoflurane. However, isoflurane does not

have any pain management properties so some form of pain management is needed. A

second surgical method provides pain management during surgery and post-operatively

and is preferred for eastern indigo snakes unless professional expertise allows for an

alternate method of pain management. This method, developed by Dr. Darryl Heard

from the University of Florida, has been used successfully on eastern indigo snakes by

Dr. Terry Norton from the Georgia Sea Turtle Center and is summarized below by Dr.

Norton. Metric units are not converted as these units are standard in medical practice.

Surgery using a pain management methodology:

Administer ketamine 5 mg/kg (body weight) + dexmedetomidine 50 micrograms

per kg + buprenorphine 0.03 mg/kg intramuscularly (IM). Place the snake back in

a secure container for 15 to 20 minutes. After this time period, the snake will be

sedated but not fully anesthetized. Then, place the snake in the plastic tube, start

isoflurane at 1 percent and work up to higher level until sedation/anesthesia level

allows intubation with an appropriate sized, un-cuffed endotracheal tube. Intubate

and maintain on isoflurane. Manually ventilate 1 to 2 breaths per minute and less

frequently during recovery. Turn isoflurane off prior to the end of the procedure

and keep as low as possible to maintain the snake under appropriate levels of

anesthesia throughout. Once the isoflurane has been discontinued, administer

oxygen for a few minutes, and then switch to a manual resuscitator such as an

Ambu

bag to allow CO

levels to rise which will stimulate the snake to breathe.

Reverse the dexmedetomidine with atipamezole at the same volume as the

dexmedetomidine given IM. Use ultrasound for monitoring heart rate; Total CO

monitoring may also be used. Use meloxicam at 0.2 mg/kg SQ at the start of the

procedure for additional pain management.

During surgery, maintain snakes at a temperature around 80º F (27º C). Avoid sudden

temperature changes. Using standard sterile techniques, make a 1.2 in to 2 in (3 to 5 cm)

lateral skin incision and reflect skin ventrally. Follow with a ventral midline incision

through the coelomic (main body) lining to allow access to the coelomic cavity. Place

the telemetry device intracoelomically (within the body cavity). Use transmitters of

appropriate weight and shape. For reptiles, it is generally recommended that transmitters

should not exceed 5 percent of body mass (Plummer and Ferner 2012). However, since

the eastern indigo is a protected species under State and Federal laws, the Service is

requiring that a more conservative approach be taken and that transmitters be no more

that 3 percent of body mass (British Columbia Ministry of Environment 1998). Since 0.2

oz (5 g) transmitters are now available, juvenile snakes of 7 oz (200 g) or greater may be

implanted as long as the required body mass ratio is met. Researchers should be mindful

that a transmitter, meeting the less than 3 percent of body mass standard, may be too

wide, especially in young, slim, snakes. Researchers must strive to minimize the ratio of

transmitter width to body width to the extent possible. Therefore, this might mean that in

some cases, elongate, flattened transmitter packages will be preferred over short,

cylindrical ones depending on their weight and width. Thread sterilized copper tubing, or

similar product, subcutaneously anteriorly, and then thread the transmitter antenna through it.

Make a small skin incision in the area of the anterior end of the tubing and remove the

tubing, leaving the antenna in place. This skin incision is typically very small so suturing

here is usually not needed.

Suture material (for example, size 3.0 to 4.0 polydioxanone

(PDS

) or nylon) for the main incision in the body cavity is used in a horizontal mattress

or simple interrupted suture pattern to close the skin incision. The coelomic lining is too

thin to suture. To support full recovery, snakes must be held post-surgery at least 2 to 3

days to monitor them for medical complications and to document their resumption of normal

behaviors; all else being equal, the less time held in captivity, the better.

All indigo snakes in captivity for surgery or other health reasons should be kept in strict

isolation. They must be kept in a separate room from all other reptiles, including indigo

snakes from different sites or permanent captives. When holding snakes prior to and

after surgeries, large, well-ventilated terraria under temperature-controlled conditions

with supplemental heating, hide boxes, water bowls, and paper bedding must be supplied

for each individual snake. Snakes must be monitored at least daily and supplied with

clean water and paper bedding as needed. Protective clothing such as gloves and

coveralls should be worn when handling these captive snakes. Antibiotics are not

typically needed if sterile technique was used during surgery. However, ceftazidime at

20 mg/kg SQ or IM, given every 72 hours, is a good broad spectrum antibiotic to use if

deemed necessary.

Eastern indigo snakes should not be captured for transmitter implantation unless daytime

temperatures rise to at least 70º F (21º C). Prior to release, ensure that a minimum of 10

days of thermoregulatory temperatures (70 to 80º F (21 to 27º C)) are predicted in order

to ensure proper healing in the wild, post-surgery. If these conditions are not likely to be

met post-surgery, do not implant radio transmitters.

At the end of studies, all radio transmitters must be surgically removed from eastern

indigo snakes by a qualified veterinarian.

**WARNING: Caution to veterinarians that Ivermectin is NOT to be used as a

prophylactic. It has caused mortality in eastern indigo snakes.

REFERENCES

British Columbia Ministry of Environment. 1998. Wildlife radio-telemetry. Standards

for components of British Columbia’s Biodiversity. No. 5. Website:

https://www.for.gov.bc.ca/hts/risc/pubs/tebiodiv/wildliferadio/rtelml20-

07.htm#p1516_113774 viewed April 8, 2016.

Brown, W.S. and W.S. Parker. 1976. A ventral scale clipping system for permanently

marking snakes (Reptiles, Serpentes). Journal of Herpetology 10:247-249.

Dorcas, M.E. and J.D. Willson. 2009. Innovative methods for studies of snake

conservation. Pgs. 5-14 In: S.J. Mullin and R.A. Seigel, eds. Snakes. Ecology and

Conservation. Comstock Publishing Associates, Ithaca, New York.

Gibbons, J.W. and K.M. Andrews. 2004. PIT tagging: simple technology at its best.

BioScience 54:447-454.

Hyslop, N.L., J.M. Meyers, R.J. Cooper, and T.M. Norton. 2009. Survival of radio-

implanted Drymarchon couperi (eastern indigo snake) in relation to body size and

sex. Herpetologica 65:199-206.

Lorch, J.M., J. Lankton, K. Werner, E.A. Falendysz, K. McCurley, and D.S. Blehert.

2015. Experimental infection of snakes with Ophidiomyces ophiodiicola causes

pathological changes that typify snake fungal disease. Download from open access

journal published by American Society for Microbiology: mbio 6(6); e01534-15.

Plummer, M.V. and J.W. Ferner. 2012. Marking reptiles. Pgs. 143-150 In: R.W.

McDiarmid, M.S. Foster, C. Guyer, J.W. Gibbons, and N. Chernoff, eds. Reptile

Biodiversity: Standard Methods for Inventory and Monitoring. University of

California Press, Berkeley, California.

Rudolph, C.C., S.J. Burgdorf, R.N. Conner, and R.R. Schaefer. 1999. Preliminary

evaluation of the impact of roads and associated vehicular traffic on snake

populations in eastern Texas. Pgs. 129-136 In: G.L. Evink, P. Garrett, and D. Zeigler,

eds. Proceedings of the 3

International Conference on Wildlife Ecology and

Transportation. FL-ER-73-99. Florida Department of Transportation, Tallahassee,

Florida.

Smith, R.B. and K.J. Dyer. 2003. Preliminary testing and comparisons of herpetological

survey techniques for eastern indigo snakes (Drymarchon couperi). Unpublished

report submitted to U.S. Fish and Wildlife Service, Jackson Field Office, Jackson,

Mississippi. 15 pp. + figures

The Orianne Society. Undated. Protocol for PIT tagging eastern indigo snakes and

collecting and storing eastern indigo snake tissue for genetic analysis. 5 pp.