Equine immunocontraception using porcine zona pellucida: a new method for remote delivery and characterization of the immune response.

Summary The purpose of this study was to investigate a new method of remote vaccine delivery and characterize the antibodies produced by the horse following vaccination with porcine zona pellucida (pZP). Three mares were initially immunized with 400 μg and boosted with 200 μg of pZP combined with a synthetic trehalose dicorynomycolate glycolipid adjuvant and packaged in a biodegradable bullet. Four control mares were vaccinated in the same manner, but with placebos. Contraception efficacy was determined by pregnancy rates. Serum antibody levels were measured by enzyme linked immunosorbent assay (ELISA) and expressed as an absorbance at a dilution of 1:l,000 of the mares' serum. Immunoblots using pZP and equine zona pellucida (eZP) were used to determine specificity and cross-reactivity of the antibodies. The delivery system investigated proved to be easy to administer, effective, and did not result in abscess formation or produce any clinical problems. Treatment with pZP resulted in a decreased conception rate during the first year (0/3) and second year (0/3) when compared to control mares for each year (3/4 and 4/4, respectively). ELISA revealed that the treated mares had absorbance levels that varied from 0.20 to 2.28 and lasted for at least 2 breeding seasons. The sera from these mares reacted with all three pZP glycoproteins and varied in their specific response to eZP glycoproteins. This variation was demonstrated by recognition of the 90-106 and 45-55 kDa eZP bands by serum of one mare while the 75-90 and 40-50 kDa glycoproteins bands were recognized with another mare's serum. In summary, immunocontraception for two breeding seasons was achieved using a new remote delivery system for vaccination of mares with pZP and resulting serum antibodies cross-reacted with specific eZP glycoproteins. In 1971, feral horse populations came under government protection with the passage of the Wild Free-Roaming Horse and Burro Act which directs the Secretaries of Agriculture and Interior to protect, manage, and control these populations on public land. In the early 1970's the population was estimated to be around 17,500 horses and burros and increased to an estimated 42,756 horses and 7,665 burros by 1985.1 In 1985, over 18,000 excess animals were removed and plans were made for subsequent yearly removal of additional animals. The Wild and Free-Roaming Horse and Burro Act of 1971 was an act meant to protect dwindling populations of feral horses. It did not provide a means for managing the populations once their numbers had been reestablished and does not allow for any type of management that involves death of the animals. Since 1985 excess animals have been removed and put up for public sale through the “Adopt-A-Horse” program. However, many of the animals are not desirable for domestic use and remain in holding pens until natural death. Reduced budget trends reflected an increased concern in the United States Congress about management levels and the rapidly escalating cost of maintaining unadopted excess animals. In 1985, over $5 million was spent to maintain up to 10,000 animals at a time.1 Traditional methods of animal population control including hunting, trapping, relocating, and poisoning are often less than desirable.2 Controlled hunting cannot be used in urban areas or National Parks. Trapping, particularly with leg-holding devices, is now illegal in many states, provinces, and countries. Live-trapping and relocation of animals is expensive and works only where sufficient suitable habitat for relocation exists. Poisoning is unacceptable because it is notoriously nonspecific. Recently, a great deal of emphasis has been placed on immunological methods of fertility control to regulate population growth for both humans and wildlife. Long-term contraception via stimulation of the immune system has been investigated in feral mares.3,4,5,6 These investigations used heat-solubilized porcine zona pellueida (pZP) as the contraceptive immunogen. The most detailed investigation was reported by Liu et al.3 using a series of four injections containing either 2000 or 5000 pZP each, along with either aluminum hydroxide gel or Freund's as the adjuvant. Contraception occurred in 86% (12/14) of the fertile mares studied; however, one of the mares developed an abscess at the injection site. Although mare anti-pZP sera cross-reacted with intact equine oocytes, the highest reactive serum dilution for equine oocytes was 1:20 while it was 1:800 for porcine oocytes. Serum titers, reported as relative absorbance levels, at 8 months post-injection remained high (>0.8) for seven of the feral mares while two had near zero levels ( Subsequently, Kirkpatrick et al.4 investigated the applicability of this type of immunocontraceptive vaccine to feral horse populations. This vaccine also contained 5,000 heat-solubilized pZP (64.3 μg protein) emulsified with an equal volume of Freund's complete adjuvant. A final volume of 1 cc was loaded into a 3.0 cc self-injecting, plastic dart tipped with a 3.81 cm barbless needle. Three of the 26 horses treated developed abscesses at the injection site and difficulty was encountered in relocating mares initially injected. The result was that 18 mares received three injections while 8 received only two. Vaccination had no deleterious effect on current pregnancies since 57.6% (14/26) of the mares delivered live foals approximately 1 to 3 months after the final injection. The foaling rate for the year following vaccination decreased significantly for treated mares (3.8%) when compared to the previous two pretreatment years (53.8%), as well as placebo treated (50.0%) and untreated sexually-mature mares in the same area during the posttreatment year (45.4%). A booster injection given to 14 mares one year after the initial vaccinations again significantly decreased foaling rates to 7% (1/14) for boosted mares as compared to 50% (3/6) for placebo treated and 44% (7/16) for untreated mares.5 During the third year of study Kirkpatrick et al.6 administered a second annual booster to 10 mares. In this study behavioral estrus and ovarian function were monitored by observation and urinary hormone analysis, respectively, for 7/10 treated and 4/20 control mares. Results indicated that none of the ten mares boosted for a third consecutive year became pregnant as opposed to 55% (11/20) of control mares. They also could find no evidence of ovulation during the ovulatory season and urinary estrogen concentrations in 4 of 7 treated mares indicated acyclicity. The authors suspected possible altered ovarian function as the cause. This would be consistent with data from similar vaccines used in other species.7,8,9 Thus far, studies using immunocontraceptive vaccines in the horse have involved either animal capture or remote delivery via dart.3,4,5,6 Animal capture was not an applicable method for delivery of a vaccine to large numbers of wild animals due to the high cost, labor, and risk of injury to both horses and handlers. Although the dart offered the advantage of remote delivery, it has been shown to be difficult to deliver. In addition, dart recovery was less than optimal,10 which led to the potential for environmental contamination. The current investigation was undertaken to test a new remote delivery system for mare immunocontraception that would not lead to environmental contamination, and that would decrease the incidence of abscess formation at the injection site. In addition, this investigation sought to more precisely characterize the antibody response produced following vaccination.