SUMMARY
In 2007, a research team consisting of personnel from The Humane Society of the United States (HSUS) and the Tufts-Cummings School of Veterinary Medicine (TCSVM), with logistic and field support from the Fripp Island Property Owners Association, carried out the third year of a field test of the effects of long-acting contraceptive vaccines on reproduction and population growth of white-tailed deer on Fripp Island, SC. The objectives of this study are to examine the relative effectiveness, longevity, safety, and practicality of several different preparations of long-acting porcine zona pellucida (PZP) contraceptive vaccines, and to examine the effects of a large scale contraceptive program on deer population dynamics on Fripp Island.
Population surveys conducted in February 2007 suggested a modest decline in population density on Fripp Island since February 2005. Population densities on the Hunting Island control site were stable to increasing. Deer density on Fripp Island was approximately 162 deer/mi2 (vs. 186 deer/mi2 in 2005), with a fawn:doe ratio of 1.09 and a sex ratio of 1M:1.94F. Much lower deer densities continued to be found in Hunting Island State Park (68.6 deer/mi2 vs, 58 deer/mi2 in 2005), with a slightly more female-biased sex ratio (1M:2.26F). Sex ratio appears to be more-or- less stable on Fripp Island, but the female bias is diminishing somewhat on Hunting Island.
Twenty-eight (28) female deer were captured for the first time and ear-tagged in late winter and early spring 2007. Of these, 27 were hand-injected with an emulsion of PZP in modified Freund's Complete Adjuvant (mFCA) plus heat extruded timed-release pellets, and one received a sham injection of saline emulsified in mFCA. Pregnancy-testing using pregnancy-specific protein B indicated that 75% of the females were pregnant at time of initial capture. In addition, 30 females that had fawns in 2006 were given remote treatments of PZP in mFCA or Freund's Incomplete Adjuvant in August 2006 to further evaluate the effectiveness and longevity of boosters.
Fifty-seven percent (53/93) of the female deer captured and eartagged in late winter 2005, 90% (75/83) females captured and eartagged in late winter 2006, and 89% (25/28) females captured and eartagged in late winter 2007 were known to be alive and onsite in December 2007. Analysis suggests that high first-year mortality experienced by deer captured in 2005 was associated with Adjuvac treatments; no surplus mortality was seen in deer treated with Adjuvac in 2006.
Of 48 females recaptured in 2007, 12 were pregnant (25%), In contrast to the 2005-06 results, only 1 6% (3 of 19) females treated with SpayVac® in 2006 were pregnant, all of which had been treated with the non-aqueous form. Booster treatments delivered remotely in autumn 2006 reduced 2007 pregnancy rates (to 20%) among females treated with SpayVac in 2005 that had fawned in 2006. Cumulative results from 2006 and 2007 indicate that PZP primers and pellets are highly effective in the initial year, with only 12% (2/17) of pellet-treated females becoming pregnant.
During 2008, we will continue to collect follow-up efficacy and safety data on deer treated with various one-shot preparations, and to further examine the effectiveness and longevity of remotely-delivered boosters. However, we will somewhat shift focus to study the effects of our best available vaccine (PZP/mFCA with heat-extruded pellets) on population dynamics. We wilt also be exploring alternative techniques of population assessment and non-invasive pregnancy diagnosis.
INTRODUCTION
Conflicts with white-tailed deer have become commonplace in residential areas throughout the eastern and Midwestern United States. These conflicts include damage to ornamental plantings, deer-vehicle collisions, undesirable ecological impacts on natural areas, and an association with tick-borne zoonotic diseases including Lyme disease and ehrlichiosis, Although a variety of techniques exist for mitigating these conflicts, the public often views deer population control as an essential component of a comprehensive deer conflict resolution program. However, traditional deer population control methods such as public hunting may be unsafe, inappropriate, or publicly unacceptable in residential areas experiencing deer conflicts, and alternative methods are being explored.
Immunocontraception has proven to be a promising approach to deer population control in cities, towns, suburbs, and heavily used parks. Previous studies have shown that the porcine zona pellucida (PZP) vaccine dramatically reduces pregnancy rates in treated female white-tailed deer, and can stabilize and reduce deer populations over limited areas (Turner et al. 1992; Naugle et al. 2002; Rutberg et al, 2004). However, previous studies of PZP or) free-ranging white-tailed deer have employed and tested vaccines that require two initial shots and annual boosters to achieve full effectiveness. The need for repeat treatments significantly increases stress on treated animals, technical challenges of applying the vaccine, and cost of delivery, and limits the scope of potential management applications, Consequently, long-acting effectiveness with a single shot is extremely desirable for management purposes.
Two technologies for single-shot effectiveness have emerged in recent years. One, named SpayVac® (ImmunoVaccine Technologies, Halifax), uses a liposome preparation of PZP (with adjuvant) to boost antibody effectiveness and longevity, and has shown long-term effectiveness in a variety of species (Brown et al. 1997; Fraker et al. 2002). The other, which uses timed-release pellets to simulate booster injections of PZP, has produced single-treatment, multi-year contraceptive effectiveness in wild horses (Turner et al. 2002, in press.).
In February 2005, we began a study on the effectiveness of one-shot PZP preparations on Fripp Island, South Carolina. The primary objectives of this study were (1) to evaluate the relative effectiveness, safety, and case-of-use of four different one-shot vaccine preparations, including two forms of SpayVac® and two forms of timed-release pellets; and (2) to examine the effects of PZP contraception on deer population density growth and composition in comparison to an untreated population at nearby Hunting Island State Park.
The study is a collaboration between The Humane Society of the United States (HSUS), TerraMar Environmental, Tufts-Cummings School of Veterinary Medicine (TCSVM), Wildlife Biometrics, and the Fripp Island Property Owners Association (FIPOA). It is being carried out under INAD 8840 from the FDA Center for Veterinary Medicine, belonging to the HSUS, and Protocol #G684-05 approved by the TCSVM Institutional Animal Care and Use Committee. Chemical immobilizing (CI) drugs used for capture are provided by and accounted for by Mark Guilloud, D.V.M. at the Animal Hospital of Lady's Island (Drug Enforcement Administration (DEA) number BG 3699026). Drug storage on site is provided by and overseen by the Fripp Island Police.
FIPOA and the Fripp Island Resort provide technical assistance, transportation and amenities. Housing is graciously provided by residents and property owners of Fripp Island. Funding for the immunocontraception project is provided by the generous residents of Fripp Island and by The HSUS. Numerous residents also volunteered to help with fieldwork.
METHODS
Population studies. During late winter 2005, 2006, and 2007, Mr. Frank Verret and his associates at Holterra Wildlife Management and Wildlife Biometrics conducted surveys of deer population density and group composition at Fripp Island and Hunting Island State Park. Counts were conducted rising distance sampling methods described by Burnham et al. (1980) and developed for deer by Gogan et al. (1986), Underwood et al. (1998), and Focardi et al. (2002).
Transects for sampling were selected randomly on Fripp Island and Hunting Island State Park. Salt marshes were not sampled in either location. In 2007, sampling on Fripp Island included 1485 acres (49%) of its 3000 acre total, and in Hunting Island State Park 2000 acres (40%) of the 5000 acres available, Surveys were performed during nighttime (2000-0530 hr) hours. Observers rode in the cab of a pickup truck and located deer with two million-candlepower hand-held spotlights. Observers determined distance to the center of deer clusters with a laser rangefinder and determined angles to the center of each cluster with a truck-mounted compass. Clusters were separated using nearest-neighbor criteria (LaGory 1986), location of deer, and their behavior in yards. In general, deer traveling together on the same or adjacent residential properties were considered one cluster.
All field data were entered into the computer program DISTANCE 5.1 (Laake et al. 1992) and density was derived from the model with the best Akiaiki Information Criteria and detection function with the lowest coefficient of variation.
We also attempted to determine buck:doe ratios and fawn:doe ratios.
Deer capture. Deer on Fripp Island were captured during January-March, 2007, using a combination of Xylazine HCl (at approximately 2.2 mg/kg) and Telazol HCI (at approximately 4.4 mg/kg). The drug combination was loaded into self-injecting 1 cc Pneu-Dart, Inc. transmitter darts with 1" needles and double wire barbs. Darts were delivered intramuscularly in the hip from a Dan-Inject CO2 powered Blo-jector at ranges of 3-12 meters. Dart transmitters had a tracking range of approximately 1 kilometer and were tracked with a Telonics TR-4 receiver and Yagi antenna.
Deer that failed to become fully sedated or were difficult to restrain were given supplemental injections of Ketamine HCl (at approximately 5 mg/kg). When a subsequent darting was necessary we used either 1 or 2 cc standard Pneu-Dart darts with 1" needles and single wire barbs. These darts were fired from either a Dan-Inject CO2 Blo-jector or a Pneu-Dart Blo-jector.
To reverse the effects of Xylazine, Tolazoline was given intravenously and/or intramuscularly at approximately 4 mg/kg when the effects of the Telazol and/or Ketamine diminished (blinking, swallowing, tail twitching, etc.).
Deer were either captured as they were encountered along community streets while driving a golf cart or at 4 bait stations set up on empty lots with minimal exposure and risks to residents or visitors. Each bait station consisted of a single 30 gallon (114 liter) barrel feeder suspended from a tree branch. Each feeder had an automatic timer and electronic dispensing unit and was programmed to dispense approximately 3-5 pounds (1.4-2.3 kg) of whole corn one or two times daily depending on local deer activity.
Upon capture, each deer's eyes were treated with an ophthalmic ointment and the head and eyes covered with a cloth hood. Heads were elevated and vital signs (heart rate, respiration rate, body temperature, and coloration of mucosa) were checked at 10 minute intervals, When possible, heart rate and respiration were monitored continuously using a handheld pulse-oxymeter with the sensor attached to the tongue. Deer showing any signs of respiratory distress (SpO2 <90%) were given approximately 0.3-0.5 mg/kg of Dopram V at 20 mg/ml intravenously. Deer in severe respiratory distress (SpO2 <80%) were given Dopram V and/or the antagonist Tolazoline.
Once a deer was stabilized darts were removed and wounds were treated with a triple antibiotic topical ointment (Neosporin). Deer were then examined for injuries related and/or unrelated to the capture process. Deer were then ear-tagged with a large, red, plastic, individually numbered cattle tag in the right ear. Tags were labeled on the back with "EXPERIMENTAL ANIMAL, DO NOT CONSUME" and a telephone number where information could be obtained in the event an animal was killed or found. A small round, yellow hog tag with a corresponding number and label also was placed in the left ear.
Measurements were then taken on girth, hind-foot length, and body length. Each deer was weighed, and age (adult, yearling, fawn) was determined when possible by examining tooth wear. Deer were then assessed for any prior injuries and mammary glands were visually examined for signs of lactation. After examination each deer was injected subcutaneously with 2-3 ml Liquamycin (LA-200) antibiotic.
Pregnancy testing. At the time of capture, 10 ml of blood was collected from each deer to later determine pregnancy and PZP antibody titer level. Blood was cooled for a minimum of 30 minutes and centrifuged. After centrifugation the serum was poured off, labeled, and immediately frozen. Samples were sent to Biotracking (Moscow, Idaho) for pregnancy testing using ELISA tests for the presence of Pregnancy-Specific Protein B (PSPB; see http://www.biotracking.com for more information).
PZP treatments. After all standard procedures were completed, deer were hand injected in the hip with one of two treatments:
- Modified Freund's Complete Adjuvant (mFCA) sham-treated controls,
consisting of 0.5 ml of mFCA and 0.5 ml sterile saline.
- 100 µg PZP in 0.5 ml PBS emulsified with 0.5ml mFCA plus 550 µg PZP and 500 µg QA-21 prepared in three "heat-extruded" pulsed-release lactide glycolide pellets.
In preparation (2), the three pellets were loaded into 14 gauge needles and injected in the hip along with an emulsification of PZP in Adjuvac® using a trochar syringe supplied by Dr. John Turner of the Medical University of Ohio, Toledo.
PZP for primer and pellet preparations was produced at the Science and Conservation Center, Billings Montana. The PZP/QA-21 heat extruded pellets were engineered to release at 1 months, 3 months, and 12 months and were prepared in the laboratory of Dr. Douglas Flanagan, College of Pharmacy, University of Iowa.
In addition, 30 females that were observed to have fawns or were determined to have been pregnant by blood samples in 2007 were given remote treatments during August 2007 of 100 µg PZP in 0.5 ml PBS emulsified in 0.5 ml mFCA. Treatments were delivered in 1 cc Pneu-Dart darts with 1" barbless needles fired from a Dan-Inject Blo-jector into the hip. Each dart was then recovered and examined to confirm complete discharge.
Radiotelemetry. Radiocollared deer have been monitored since May 2005, with their locations mapped using a hand-held Magellan SportTrak Pro GPS unit.
Observations of study animals. During August 2007, as many tagged deer as possible were relocated and observed to gather fawning data. Deer were observed for associations with fawns and signs of extended udders to attempt to corroborate the winter pregnancy test results.
RESULTS
Population studies. Fifteen (15) sampling sessions were conducted during 24 February - 4 March 2007. This number achieved the 60 or more deer clusters at both Fripp Island and Hunting Island State Park needed to generate a robust estimate of population abundance (Buckland et al. 1993). Investigators randomly sampled 24.9 miles of roads on Fripp Island and systematically sampled 48.5 miles of road within Hunting Island State Park. One new transect line was added at Hunting Island, consisting of a segment of U.S. 21 from the north entrance of Hunting Island to the bridge at the entrance of Fripp Island.
A total of 317 fawn, yearling, and adult deer in 112 clusters was detected on Fripp Island, and 123 fawn, yearling, and adult deer in 62 clusters were detected within Hunting Island State Park, Deer densities for Fripp Island and Hunting Island State Park were 161.5 deer/mi2 (90% C.I.: 133.8-207.2 deer/mi2 ) and 68.6 deer/mi2 (90% C.I.: 48.7-90.4 deer/mi2), respectively. The estimated sex ratio was 1 M:1.94F at Fripp Island and 1M:2.26F at Hunting Island State Park (standard errors of estimates of sex ratios <15%), with 1.09 fawns per doe observed at Fripp and 1 .62 fawns per doe observed at Hunting Island.
Since 2005, estimated population density has declined at about 7% per year on Fripp Island, while estimated population density has increased at about 9% per year an Hunting Island (Fig. 1).
Figure 1. Population density trends at Fripp Island and Hunting Island State Park, 2005-2007
Deer captures and treatments. During 27 days in late January-March 2007, 28 new deer and 55 previously tagged deer were captured on Fripp Island using chemical immobilization. A total of 446 person-hours was spent capturing and handling the deer at an average of 16.5 person hours/day, 3.1 deer captured/day, and 5.4 person-hours/deer captured.
Three (3) of the 83 deer captured had to be darted multiple times to insure capture. An additional 15 deer were darted and not recovered before their transmitter batteries died (3-5 days post-injection). Three darts missed deer and both were recovered. Deer not captured carried darts ranging from <1 days to 8 days. As a result, 10 telemetry darts and transmitters were not recovered. No deer sustained serious injuries as a result of darting or captures, but two females were found dead 24-48 hours post-release. Necropsies revealed that both deer were old (>8 years old) and that their rumens and fur contained various parasites. Both deer were buried on site.
All 28 new deer captured were females. Of these, 27 females were treated with PZP emulsified in mFCA plus 3 heat-extruded pellets. One female was given an mFCA placebo to replace a control that had died or disappeared.
The 55 recaptures were each bled to determine pregnancy. One previously tagged female that lost both tags received a radio-collar to replace a collared deer that had died in 2007. Two previously tagged deer were captured and given new tags.
Deer survival. An inventory of tagged females in December 2007 found that some animals who were not sighted in September 2006 surveys were in fact still alive, and that some earlier mortality estimates were slightly overstated. Approximately 72% of animals tagged in 2005 or 2006 were seen alive in December 2007, and 89% of animals tagged in winter-spring 2007 were seen alive in December 2007 (Table 1). During the time between tagging in 2005 and September 2007, 12 radio-collared deer had died or disappeared.
Table 1. Survival of tagged females through December 2007
| Year Tagged | Known Dead | Missing | Alive | Total |
| 2005 | 40 (43%) | 1 (1%) | 53 (57%) | 94 |
| 2006 | 8 (10%) | 0 (0%) | 75 (90.4%) | 83 |
| 2007 | 2 (7%) | 1 (4%) | 25 (89%) | 28 |
| Total | 50 (24%) | 2 (1%) | 153 (75%) | 205 |
Survival to December 2007 differed by year and adjuvant type. Deer treated with Adjuvac® in 2005 showed significantly lower survival to September 2006 than deer treated with mFCA (Fisher's Exact test, 2-tailed P = 0.044; Table 2). This result was consistent regardless of whether the Adjuvac was administered with Spay-Vac, pellets, or sham controls. However, survival to December 2007 of deer treated with Adjuvac in 2006 was quite high (70/78, or 90%), matching 2005-2006 survival rates of deer treated with mFCA.
Table 2. Survival to September 2006 of females treated with vaccines emulsified in Adjuvac® or mFCA.
| Dead or Missing | Alive | Total | |
| Adjuvac | 36 (44%)* | 45 (56%) | 81 |
| mFCA | 1 (10%) | 9 (90%) | 10 |
| Total | 37 (41%) | 54 (59%) | 91 |
*One Adjuvac deer missing >1 year; the rest known dead
Deer reproduction and contraceptive efficacy. Pregnancy diagnoses using PSPB indicated that 12 of 48 females (25%) recaptured in 2007 were pregnant, By contrast, 15 of 20 adult females (75%) and 3 of 7 yearling females (43%) were pregnant at the time of initial capture. Overall, from 2005-2007, 159 of 195 females (82%) were pregnant at the time of initial capture, presumably reflecting the baseline pregnancy rate among untreated female deer at Fripp.
Several important results are emerging from the 2006-07 work.
- The 2006 batch of SpayVac® (SV) was highly effective in preventing
pregnancies. Whereas 73% of females treated with SV in 2005 were
pregnant in 2006, only 3 of 19 (16%) of females treated with SV in 2006
were diagnosed as pregnant via blood tests in 2007. Pregnancy diagnoses
were largely corroborated by observations; 5 of 21 SV-treated deer (24%)
were observed with fawns or udders. All 3 deer diagnosed as pregnant by
blood test and all 5 deer seen with fawns and udders had been treated
with the non-aqueous form (N = 10 and N = 12, respectively), This
suggests that the non-aqueous SV may be somewhat less effective than the
aqueous form.
- Remotely-delivered booster injections of PZP/MFA in autumn 2006 to
deer treated with the problematic 2005 SV were highly effective in
preventing pregnancies. Only 2 of 10 females receiving boosters in
autumn 2006 were pregnant in 2007 (again, both were initially treated
with the non-aqueous SV). In contrast, 6 of 8 females treated with SV in
2005 and not boosted were pregnant in 2007 (Fisher's exact test,
1-tailed P = 0.03).
- Both cold-evaporated and heat-extruded pellets were highly effective in preventing pregnancies for at least one year, and preliminary results suggest that they may be at least somewhat effective for a second year (Table 3).
Table 3. Pregnancy rates of deer treated with a priming dose of PZP/mFCA or PZP/Adjuvac® plus cold-evaporated or heat extruded pellets in 2005 and 2006
| Initial Capture | Year One | Year Two | |
| PZP/adj + cold- evaporated pellets | 83% (10/12) | 25% (2/8) | 0/1 |
| PZP/adj + heat- extruded pellets | 81% (25/31) | 0% (0/9) | 1/3 |
| Total | 81% (35/43) | 12% (2/17) | 25% (1/4) |
Telemetry. In 2007 one tracker spent 7 days and approximately 23 hours or 3.3 hours/day tracking movements of radio-collared deer on Fripp Island. A detailed report on these activities is still in progress.
Remote PZP treatments. Over a 4 day period in August two persons remotely treated 30 deer with 100 µg PZP in PBS emulsified in mFCA and 3 deer with 100 µg PZP in PBS emulsified in FIA. A total of 56 person-hours was spent darting at an average of 8.3 deer/day mid 1.7 person-hours/treatment.
Of the 30 females remotely treated with PZP/mFCA, 17 females had been hand-injected in the winter of 2005 with Spay-Vac® (10 non-aqueous and 7 aqueous), 11 females had been hand-injected in the winter of 2006 with Spay-Vac® (non-aqueous), and 2 females had been hand-injected with cold-evaporated pellets and Adjuvac® in 2005. Of the 3 females remotely treated with PZP/FIA, 1 female had been hand-injected with cold-evaporated pellets and mFCA in 2005, 1 female had been injected with heat extruded pellets and mFCA in 2005, and 1 female had been treated with Spay-Vac aqueous in 2005, and given a remote treatment of mFCA and 100 µg PZP in 2006.
Miscellaneous. An additional 50-60 person-hours were spent in 2007 preparing drugs and darts to capture deer, collecting and labeling blood serum samples, recording data, and on other aspects of fieldwork.
Costs. The cost for vaccine, supplies, equipment, travel, and other related expenses in 2007 was approximately $43,875.00 (Table 4). Not included in that cost is the time of salaried HSUS, Tufts, and FIPOA employees, which was donated to the project.
Table 4. Summary of expenditures on Fripp Island deer research. (Figures are estimates.)
| Description |
Cost |
| Equipment and Supplies | $ 8,000.00 |
| PZP vaccine, including pellets | 9,400.00 |
| Blood Analysis | 2,075.00 |
| Travel - Airfares | 2,500.00 |
| Travel - Food, mileage, car rental | 2,500.00 |
| Population Survey - Wildlife Biometrics | 7,900.00 |
| Consultant labor - HSUS | 11,500.00 |
| Total | $43,875.00 |
DISCUSSION
Overall, the results on PZP vaccine efficacy have been very encouraging. Deer treated with (2006) SpayVac® or a primer dose of PZP plus pellets showed very high efficacy in the initial year. We do not yet have second year data for the 2006 SV, but a small sample of PZP pellet treated deer did show indications of reduced pregnancy rates in year 2. As of December 2007, 50 deer treated with SV in 2006 and 20 deer treated with heat-extruded pellets in 2006 were alive and present on the study site; these deer should provide significant opportunities to evaluate the second-year effectiveness of the test vaccines in 2008.
It was also encouraging that deer treated with 2005 SV and fawned in 2006 showed markedly reduced pregnancy rates when treated with a single booster of PZP and mFCA in autumn 2006. This suggests that the 2005 SV did provide enough antigen recognition to allow boosting to elevate titers to contraceptive levels.
Survival data suggest that the 2005 batch of Adjuvac was associated with significantly increased mortality among treated deer. Survival was high among deer treated with Adjuvac in 2006, however. All 2007 treatments used either mFCA or (for 3 remotely boosted deer) FIA, neither of which has been associated with increased mortality in any context known to us.
After two years of PZP treatments, we had no expectation of seeing clear effects on deer population parameters on Fripp Island, especially since the 2005 SV treatments were ineffectual. However, fawn-doe ratios were conspicuously lower on Fripp Island than on the Hunting Island State Park control site in February 2007. Although small size differences between fawns and yearlings at the time of sampling again hindered the effort to evaluate fawn-doe ratios, it is unlikely that this imprecision can account fully for the differences between sites. Antlers on yearling bucks were difficult to see and it made evaluation of herd composition somewhat difficult.
The deer population on Fripp Island is either stable or trending modestly downward. As of December 2007, there were approximately 138 female deer living on Fripp Island that have been treated with some preparation of PZP. (There were also approximately 14 ear-tagged untreated controls.) The February 2007 population estimates of 375 deer and a sex ratio of 1M:1.94F implies that there were about 250 females on Fripp at that time. However, unsystematic surveys conducted in April-August 2007 suggest that >85% of regularly sighted females were tagged, suggesting a female population size closer to 180 (this is probably biased low, since females that are rarely sighted may be less likely to be tagged). Taken together, however, these observations suggest that between 55-77% of female deer currently living on Fripp Island have been treated with contraceptives over the last three years. We may thus see a more dramatic population effect evident in the upcoming 2008 survey.
RESEARCH PLAN FOR 2008
Goals. After three years of research using different adjuvants and one-shot vaccines, we are planning to shift focus somewhat. We will continue to collect follow-up efficacy and safety data on deer treated with SpayVac and the various PZP pellet preparations; existing sample sizes should be sufficient for the different preparations to allow us to discern longevity differences of interest to us. In addition, we plan to further examine the effectiveness and longevity of single boosters delivered remotely in the fall, using mFCA as an adjuvant, or FIA for deer previously treated with mFCA.
However, concerns over the safety, consistency, and availability of Adjuvac and SpayVac are causing us to focus on the pellet technology delivered with PZP/mFCA primers. Although the cold-evaporated pellets have proven somewhat effective in this study, in vitro studies of antigen release rates and unsuccessful trials on captive horses both suggest limited effectiveness for this method of pellet preparation, Thus, we will focus future studies on pellets prepared by the heat-extrusion method, which have shown high effectiveness in this study and in captive and free-ranging wild horses (Turner et al. 2007).
We will also place greater emphasis on population studies, as we step up the use of reliable vaccines of known effectiveness.
Monitoring of Population Density. Distance sampling in Hunting Island State Park and on Fripp Island will be repeated in February 2008, following the methodology used in previous years.
Because a high proportion of females on Fripp Island is marked, we will also attempt to develop a mark-resight population estimation protocol to corroborate distance sampling estimates of female population size. To provide an accurate and precise estimate of the number of marked females an the island, we will begin by inventorying all marked females on the island over a one-week period. We will then establish a series of road transects and carry out 3-5 surveys at dusk or dawn over a week, counting the number of marked and unmarked females encountered (following Focardi et al. 2002). Female population estimates will be obtained using a modified Lincoln-Petersen model, or with appropriate models incorporated into the MARK software (White et at. 2007). Multiple surveys allow the calculation of standard errors of the mean.
Vaccination Procedures. Observations of female deer on Fripp Island in April-August 2007 suggest there may only be 30-40 untagged adult and yearling females remaining. Vaccinations conducted in 2008 will consist of PZP/mFCA primers plus PZP in heat-extruded pellets, which in 2006-07 yielded the lowest pregnancy rates and highest antibody titers, but for which the sample remains small compared to SpayVac-treated deer. We will also recruit new controls to replace those that died in 2006 and early 2007.
Beginning in late January or early February, we will capture up to 40 adult female deer using darting and handling procedures described above. These will be assigned to one of two treatment groups:
- Untreated controls or
- 100 µg PZP in 0.5 ml PBS emulsified with 0.5 ml mFCA, plus 550 µg PZP and 500 µg QA-21 prepared in three heat-extruded lactide-glycolide pellets.
In addition, during late summer/early fall 2008, treated deer that became pregnant after treatment may receive remote inoculations of PZP and mFCA or FIA. FIA will be administered to deer that had previously been treated with mFCA. Also, additional deer will be collared to replace deer with collars that have stopped working or deer that have died or disappeared.
Pregnancy and fawning assessments. Using capture methods described above, we will recapture as many as possible of the surviving females (up to 60) that were treated and tagged in the winters of 2005-2007. Blood samples will be taken, and sent to BioTracking (Moscow, Idaho) for pregnancy testing using Pregnancy-Specific Protein B. Blood samples will also be taken from females captured for the first time in 2008 and submitted for pregnancy diagnosis.
Because capturing deer for blood sampling is time-consuming, invasive, and risky, we will also explore the use of fecal progesterone assays for pregnancy diagnosis. Fecal steroids such as estrone conjugates (E1C) and progesterone metabolites (pregnanediol-3-diglucuronide, PdG) have been used to diagnose pregnancy reliably in Tule elk, sika deer, and other species, but results for white-tailed deer have been mixed (Stoops et al. 1999; Hamasaki et al. 2001; Kapke et al. 1999; Walter et al. 2002), Consequently, it will be important to validate pregnancy diagnoses from fecal steroids with P-SPB results and ground observations. Because timing of collection may be an important variable in pregnancy diagnosis (J. F. Kirkpatrick, pers. comm.), we will collect serial fecal samples over a period from late February to mid-April to determine what dates yield the most accurate diagnoses. Fecal samples will be obtained from all deer captured in February-March 2008. We will then attempt to collect follow up samples in April (without capture) from up to 15 deer first captured in 2008 and 15 PZP-treated deer recaptured in 2008. This sampling scheme should provide a rough balance of pregnant and non-pregnant animals, allow serial comparisons of the same animals, and allow comparisons of pregnancy diagnoses with P-SPB results. Fecal samples will be frozen within 12 h of collection and sent to the Science and Conservation Center in Billings, MT, for assay for PdG via ELISA.
Between May and September, 2008, all tagged deer will be relocated and observed at least monthly to gather fawning data. Deer will again be observed for signs of lactation (extended udder), the repeated presence or absence of associated fawns, and nursing behavior by fawns.
Radiotelemetry. Because of the valuable data on causes of death from radio-collared animals, and an interest in observing potential behavioral effects of treatments, radiotelemetry will continue throughout 2008. Problems with radio collars will continue to be monitored and corrected when necessary.
Necropsies and Tissue collection. Deer carcasses located in adequate condition will be grossly necropsied opportunistically. Because of informal reports from other investigators of internal abscesses at injections sites involving Adjuvac, special attention will be paid to tissues near injection sites of all deer previously treated with Adjuvac or mFCA. Reproductive tracts will also be removed opportunistically from study subjects that are killed by cars or suffer other mortality incidental to the study. These tissues will be preservation for histological examination. All personnel will wear disposable rubber gloves and take other appropriate precautions while conducting necropsies and collecting and handling tissues from dead deer.
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