Chemical restrain of Baboons for samples and data collection for research purposes in AmboseliNational Park.
In Amboseli National Park, there is a well established baboon research center that was set up in collaboration between Institute of Primate Research (IPR), Dukes University, US and Kenya Wildlife Service (KWS). The research scientists and students in this center have been conducting research activities in specific baboon populations within or at the periphery of the park. Studies that are done with these animals include behavioural changes, movement patterns, survival instincts, genetic studies, feeds and feeding patterns. Some of these studies require that an animal be immobilized by darting for collection of the required samples and to take body measurements. The immobilization and anaesthetic monitoring process has to be done by a qualified wildlife veterinarian, during this time 4 baboons were captured and sampled for the study of genetic variation among different baboon families.
The animals were identified by assistant research scientists early in the morning and after identification darting was done using Pneu-darts propelled by a ” Blow pipe” dart gun. The drug used for immobilization was Telazol which is a combination of Tiletamine and Zolazepam (100mg/ml), the dosage used for adult baboon was 100mgs. The baboons were semi-habituated and darting was done from a very close range of less than 5 meters away. Four baboons were captured during the two days operation, all of them adults, 3 females and one male.
After chemical capture, the animal was placed on a soft blanket under a shade from where samples collection and morphometric measures were taken. Then the animal was placed into a cage which is covered by a green canvass from where the animal stays until it recovers fully from anaesthesia and released to join others.
Table 1: Summary of animals captured and vital parameters.
Immobilization drug & dosage
Type of samples collected from the baboons:
· Blood sample collected from saphenous vein and put in EDTA vacutainer tubes (purple-top).
· Blood sample collected from saphenous vein and put in Serum separator tubes (SST/red and black top).
· Blood sample collected from saphenous vein and placed in PaxGene tubes.
· Blood smears on microscope slides made from superficial ear-vein then fixed using methanol.
· Tissue samples collected from axillar region using biopsy punch preserved in a buffer for RNA analysis.
· Vaginal swabs collected from female baboons and stored in Stuarts transport media for bacteriological analysis.
· Ectoparasites including ticks collected and placed in 70% ethanol.
· Hair samples (Shavings) from shoulder region.
· Imprint of molar teeth casps was made using Polysiloxane agent dissolved in a Universal activator substance. The imprints would then help to know the preferred food types for these baboons by observing the wearing pattern of the teeth.
Blood samples processing:
The blood samples were basically collected for the genomic DNA and RNA analysis and they were processed according to Sarah Tishkoffs protocols.
Processing blood in EDTA Vacutainer tubes (purple-top)
These tubes contain blood for genomic DNA analysis that are processed in 4 ways:
1) Sarah Tishkoff’s protocol, saved as the white blood cell (WBC) fraction only in white cell lysis buffer (WCLB);
2) Tishkoff protocol, saved as the WBC fraction in White Blood Cell Lysis Buffer (WCLB), then as a 1:3 ratio in RNALater.(RNA Later is the medium for storing WBC fraction for genomic RNA analysis later).
3) Whole blood stored in 1:1 in blood storage buffer (BSB).
4) Whole blood stored 1:3 in RNALater.
Tishkoff protocol (produces above sample types 1 and 2):
This protocol uses 1 of the 2 purple-top (EDTA) samples collected from each darted individual (approx 3-4 mL of blood), and produces 2 mL of WBC in WCLB and 2 mL of WBC in WCLB at a 1:3 ratio in RNALater.
Preparations before starting:
1) Make a shallow 10% bleach bath for blood waste disposal (10 mL Jik bleach plus 90 mL water). Also check to make sure that a supply of 10% bleach is available on the bench top (stored in a 50 mL Falcon tube): this is for cleaning off the pipette barrel after pipetting from biological samples.
2) Set up the Mobile Spin centrifuge on a level surface.
3) Make sure there are at least 3-50 mL Falcon tubes filled with 1x red cell lysis buffer (RCLB). If not, resuspend the 50x RCLB solution by immersing the bottle in a hot water bath for several minutes. Make new aliquots of 1x RCLB in 50 mL Falcon tubes (1 mL 50x RCLB solution to 49 mL filtered, boiled water).
4) Label 2-15 mL Falcon tubes (Falcon tube #1 and Falcon tube #2) with the name of the individual from whom the sample was taken. Also label 4 Sarstedt tubes (ST) with the name of the individual, the date of the blood draw and the time of the blood draw; label both directly on the tubes and using tape. Of these 4 STs, 2 should also be labeled “WBC in WCLB” and the remaining 2 should be labeled “RNAL 3:1 WBC in WCLB.” Alternatively, label 2-2 mL orange Corning tubes with the same information, one labeled “WBC in WCLB” and the other “RNAL 3:1 WBC in WCLB.”
1) Open one purple-top (EDTA) blood tube and the two labeled 15 mL Falcon tubes. Pour half of the original blood sample into each Falcon tube (about 1.5-2 mL blood per Falcon tube).
2) Add 1x RCLB to each Falcon tube up to 12 mL volume—make sure the tubes are balanced.
3) Spin the two Falcon tubes in the Mobile Spin centrifuge for 10 minutes. A visible reddish-white pellet should be visible on the bottom of the tubes after the spin is completed.
4) Remove the Falcon tubes from the centrifuge, and pour off the red supernatant, being careful to preserve the pellet, into the prepared 10% bleach bath. If necessary, wash the pellet in a little 1x RCLB in order to decant more of the red supernatant.
5) Repeat steps 2 through 4 for one to three additional spins. Before centrifuging, resuspend the pellet in the added 1x RCLB by inverting the tube repeatedly.
NOTES: Use your judgment on whether to try and purify the pellet again with an additional spin: in each spin, we lose a small fraction of white blood cells, so if it looks like the pellet is getting much smaller, it’s better to stop spinning and go on to the next step. At this point, the supernatant should be clear and the pellet should be mostly white. Stop agitating as soon as possible after the pellet resuspends in order to avoid lysing white blood cells.
6) Add 1.8 mL WCLB to Falcon tube #1 and 500 mL WCLB to Falcon tube #2. Resuspend both pellets by pipetting up and down.
IMPORTANT: after pipetting from any biological sample, clean the pipette barrel with a KimWipe and 10% bleach to avoid DNA cross-contamination.
7) From Falcon tube #1, pipette 1 mL of the homogenized solution into each of the 2 STs labeled “WBC in WCLB” (or all 2 mL into a single 2 mL orange Corning tube). Close the STs and discard Falcon tube #1.
8) From Falcon tube #2, pipette 250 mL of the homogenized solution into each of the 2 STs labeled “RNAL 3:1 WBC in WCLB” (or 500 mL into a single 2 mL orange Corning tube). Discard Falcon tube #2.
9) Add 750 mL RNALater to each of the 2 STs labeled “RNAL 3:1 WBC in WCLB,” which should also contain 250 mL of WBC in WCLB (or 1.5 mL RNALater to 500 mL WCB in WCLB if using a single orange Corning tube). Close the tubes.
10) Discard the 10% bleach bath containing the red cell fraction, serum, and RCLB in the fire pit, and wash out the bath with water.
Whole blood protocols (produces sample types 3 and 4)
This protocol uses 1 of the 2 purple-top (EDTA) tubes collected from darted individuals, and produces one EDTA tube containing whole blood 1:1 in blood storage buffer (BSB) and one EDTA tube containing whole blood 1:3 in RNALater.
1) Label the empty purple-top tube set aside from the Tishkoff processing protocol “RNAL 3:1 whole blood,” and the untouched purple-top tube “Whole blood 1:1 BSB.”
2) Open both tubes, and pour half of the blood in the filled tube into the empty tube (resulting in two purple-top tubes with about 1.5-2 mL whole blood in each).
3) Add approximately 4.5 mL RNALater to the tube labeled “RNAL 3:1 whole blood” and close the tube.
4) Add 1.5-2 mL BSB to the tube labeled “Whole blood 1:1 BSB” and close the tube.
5) Transfer the whole blood/BSB preparation into one 5 mL orange Corning tube and the whole blood/RNALater preparation into two 5 mL orange Corning tubes. Label the tubes directly on the tubes and also with green tape (individual, time of sampling, date of sampling, and method of preservation—either “RNAL 3:1 whole blood” or “whole blood 1:1 BSB.”
Serum Separator Tubes (SST/red and black top)
These tubes separate the serum fraction of whole blood from the cellular fraction after centrifugation in the field. The serum is then processed afterwards for analysis of viral DNA, but also save the clotted cellular fraction in the original tube, in case we need to try to extract baboon genomic DNA.
1) Pipette 250 mL of serum from the SST into each of 2 STs (or 500 mL serum if using 2 mL orange Corning tubes). If more serum is in the SST, pipette the rest of it up to 3 mL into a 15-mL Falcon tube. Do not discard the SST (place in the charcoal fridge with the other samples).
IMPORTANT: clean off the pipette barrel with a KimWipe and 10% bleach solution after pipetting biological samples to avoid DNA cross-contamination.
2) Add 750 mL of RNALater to each Serum tube (ST), (or 1.5 mL of RNALater to 2 mL orange Corning tubes), and 3x RNALater relative to the volume of serum in the Falcon tube to the Falcon tube.
3) Label the resulting set of STs and/or FTs with the name of the individual from whom the serum was drawn, the date of the blood drawn, and the time of the blood draw. Also write “RNAL 3:1 serum” on each tube. Label both directly on the tube and using tape. Do the same for the SST tube.
These tubes are not processed in the field. They are stored in a fridge along with the skin biopsy, vaginal swab samples, and the tubes resulting from the above processing steps. Label all tubes both directly on the tube and using tape.
Release of the animals:
After about 4 hours of recovery from the crate the baboon was driven to a nearby place where it could see the rest of its group members then released in a hidden place and joins others.
It is recommended that everyone who handles baboons during this exercise must be in a full protective gear. This includes putting on overall, gum boots, face masks, cap and gloves. These clothings should be washed and be disinfected immediately after the work. Baboons are carriers of some very important zoonotic diseases such as tuberculosis and haemorrhagic fevers that can easily be transmitted to humans if they are handled without proper protection.
Treatment of adult female elephant in Kitende area of AmboseliNational Park.
This case was reported by the senior Warden of Amboseli National Park that an adult female elephant had been sighted in Kitende area just about 15 Kilometers away from the park boundary towards Mount Kilimanjaro side. The animal was said to be limping with the right front leg greatly swollen and it had been left by the rest of the family members because it was unable to keep pace with them, it was the matriarch in that family according to the information we got from the elephant research team in Amboseli.
It took us along time to find the animal as it had moved from where it was and the veterinary team, elephant research team, community wildlife scouts and the senior warden all joined up to find out where it was. Later on it was found in a bushy and stony area. It was then darted on foot and got immobilized after about 5 minutes.
Treatment of a speared elephant in Amboseli National park.
The wound was quite deep and went through the right front limb carpal joint and there were chances of developing arthritis. It was properly debrided using 10% hydrogen peroxide and water then a tincture of iodine applied on it. Other treatments included intramuscular antibiotic administration and dexamethasone for antiinflammation and to relieve pain and swelling.
Blood samples were collected for laboratory analysis, these included blood smears, whole blood and serum. The elephant was then revived from anaesthesia but would not move fast because of pain. We left the community scouts being assisted by the elephant monitoring team to keep monitoring its response and report any further complications with the animal.
Treatment of an adult male elephant in Kimana ranch.
This was and adult male elephant in Kimana ranch we got the information about it from the elephant research group just after treating the first elephant. It had two wounds, one on the right front limb on the lateral side distal to the carpal joint, this was a superficial wound with pus exudation. The other wound was on the right hind leg on the lateral side but proximal to the tarsal joint, it was fairly deep and narrow suspected to have been caused by a sharp and pointed object.
This animal was found in an open area of the ranch and was darted from barely 10 meters away using 17 mgs of etorphine hydrochloride combined with 1500 i.u of hyaluronidase. It took about 6 minutes to become recumbent. The wounds were then cleaned debrided and treated with a tincture of iodine solution. Blood samples were collected for laboratory analysis, these included blood smears, whole blood and serum.
Treatment and release of a wounded elephant in Kimana ranch.
Other treatments included intramuscular antibiotic administration and dexamethasone for antinflammation effect and pain relief. It was also supposed to be monitored and its response be reported to the veterinarian for action.
Desnaring of two Waterbucks in Mount Elgon National Park
One of these waterbucks was an adult male aged about 6 years that had a 3 meter long wire tied to its left hind limb. The animal had difficulties in walking because the wire kept grasping trees and other vegetation as the animal walked causing much irritation and it was restless and not feeding most of the time. We found it just at the edge of the forest next to one of the campsites in mount Elgon and it was immobilized by darting using 6mgs of etorphine hydrochloride combined with 20 mgs of xylazine hydrochloride and 1000 iu of hyaluronidase. It took off into the thicket after darting but we tracked and found it recumbent inside the forest. The wire was still loose and it was cut off using a wire-cutter. The animal was then revived from anaesthesia using 24mgs of diprenorphine hydrochloride combined with 5mgs of atipamezole hydrochloride administered through the ear vein.
Second case of Waterbuck in Mount Elgon National Park.
The second waterbuck was a two year old male that was found lying just next to the park offices, this was immobilized by darting using 4mgs of etorphine hydrochloride combined with 20 mgs of xylazine hydrochloride and 1000 iu of hyaluronidase. It took about 5 minutes to become recumbent. It had a tight copper wire that was cutting through the soft tissues around the distal metatarsal region. This wire was pulled out and cut using a wire cutter, then the wound debrided and treated using a tincture of iodine, antibiotic spray and intramuscular antibiotics administered. It was then revived from anaesthesia and released back to the wild.
In these two cases of waterbucks, the prognosis was good and they had very good chances of recovery since the irritant had been removed.
Treatment of a wounded Masai giraffe in Masai Mara National Reserve.
The giraffe was found next to Keekorock lodge and the veterinary team was informed that the animal had a large wound just above the corium on the planter side of the right hind limb. It was so easy to find because it could not move over a long distance due to pain experienced from the limb. It was an adult male giraffe, we found it browsing near a small stream next to the lodge.
The giraffe was immobilized by darting from a vehicle using 13mgs of etorphine hydrochloride combined with 30mgs of xylazine hydrochloride and it took about 6 minutes for the drug to take effect then finally it was roped down for treatment. The wound was quite large with a lot of pus exudation, it was cleaned and debrided by 10% hydrogen peroxide and a tincture of iodine. It was also treated by antibiotic drugs and anti-inflammatory drugs. The animal was then revived from anaesthesia and assisted to rise up, prognosis was good because the wound was quite superficial and had no foreign material.
During the month of June, 2007, the number of cases of snaring and animal injuries remained high especially in Naivasha area, few of these cases were reported to the veterinary team but most of the cases were met by the team while patrolling in the field. The team was also involved in a number of research activities including baboon research in Amboseli. Disease surveillance in Lake Nakuru National park was another major activity which the team was involved in.