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Theory and Practice of Immunocontraception in Wild Author(s): Lisa I. Muller, Robert J. Warren, Donald L. Evans Source: Wildlife Society Bulletin, Vol. 25, No. 2, Deer Overabundance (Summer, 1997), pp. 504- 514 Published by: Allen Press Stable URL: http://www.jstor.org/stable/3783483 Accessed: 05/05/2010 12:26

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http://www.jstor.org 504 IMMUNOCONTRACEPTIONIN WILDMAMMALS

Theory and practice of immunocontraceptionin wild mammals

Lisa I. Muller, Robert J. Warren, and Donald L. Evans

Abstract Immunocontraceptionhas been proposed as a technique for managing wildlife popula- tions in urbanand suburbansettings where traditional,lethal control methods may not be publicly acceptable. Immunocontraceptionuses an animal's own to disrupt reproductivefunction. Proteinsof eggs, , fertilized eggs, and reproductive hormones have variously been proposed for use in developing a for fertilitycon- trol. The most widely tested immunocontraceptivevaccine for wild species is based on developing to the (ZP), which surroundsthe mammalianegg . This vaccine has successfully caused infertilityin some individual animals, but re- quires multiple treatments. Enhancementof immune response and efficiency of vaccine delivery will be necessary before this type of management strategy can be applied to wildlife control at the population level. Contraceptivetreatment may alter the health and behavior of wildlife populations and thereforemust be monitoredclosely.

Keywords contraceptives, ELISA,enzyme-linked immunoabsorbentassay, Equuscaballus, immuno- contraceptives, immunosterilization,Odocoileus virginianus,population management

Increasing attention is being focused on fertility American Association of Wildlife Veterinarians (re- control as a possible technique for controlling viewed by Nettles 1997). wildlife populations in urban and suburban areas. In 1991, Turner and Kirkpatrickreviewed the most In many of these areas, wildlife populations are be- recent developments in wildlife immunocontracep- coming overabundant. However, in such settings, tion. Since that time, a number of advances have traditional hunting or lethal control programs may taken place in this area of research. It is timely now not be publicly acceptable. A demand has arisen for to present an updated review. Our objectives are to contraceptive management techniques (e.g., "Bat- provide a brief review of the immunological theory tling animal -deer herds at heart of and biology underlying immunocontraception. Many park conflict" USA TODAY,25 Mar 1993), but fertil- wildlife managers may not be interested in acquiring ity control is not a panacea for population manage- a thorough knowledge of immunology; however, ment. First, effective contraception must be there are many important details of reproductive im- achieved, and delivery of the contraceptive must be munology that are directly related to the practical practical and cost effective. There also are many an- treatment of wildlife species with immunocontracep- imal health questions that must be resolved before tives. These details often are not of concern to re- the use of contraceptives for population control is productive specialists that work with either domestic considered (Nettles 1997). These concerns were species or human subjects. Therefore, we will re- also voiced in a proposed resolution on the use of view the relevant concepts in both immunology and fertility control in free-ranging wildlife by the wildlife management when considering immunology- Wildlife Disease Association (1994) and by the based fertility control for wildlife management.

Addressfor Lisa1. Muller: Department of Agricultureand NaturalResources, Delaware State University, Dover, DE 19901-2277, USA. Addressfor RobertJ. Warren:Daniel B. WarnellSchool of ForestResources, University of Georgia,Athens, GA 30602, USA. Address for Donald L. Evans:Department of MedicalMicrobiology, College of VeterinaryMedicine, University of Georgia,Athens, GA 30602, USA.

WildlifeSociety Bulletin 1997, 25(2):504-514 Peer refereed Immunocontraceptionin wild mammals * Mulleret al. 505

Wildlife biologists in the future may become more in- tration through the ZP and then fusion with the egg volved in the use of contraceptives for population plasma membrane (reviewed in Yanagimachi 1994). management than they have in the past (Garrott Warren should 1995, 1995). Biologists understand A brief review of the immunocontraceptive techniques and the advan- immunology tages or disadvantages of contraceptive fertility con- relative to immunocontraception trol in order to make informed recommendations for wildlife population management. B-cell versus T-cell immune responses We review the concepts in reproductive biology When a foreign (i.e., an ) enters the and immunology necessary to understand how po- body of an animal, the undergoes a tential could cause infertility. Then, we re- complex series of events to protect the animal. The view selected reproductive that have been immune response consists of the humoral, or anti- tested as immunocontraceptives in wildlife. This re- body response, and the cell-mediated response. view is not designed to be exhaustive, but rather, to These responses are carried out by . facilitate understanding of how immunocontracep- The 2 types of lymphocytes responsible for the divi- tives have been applied to wildlife species. Many sion of labor are B cells and T cells. The B cells pro- other proteins are being isolated and tested for use as duce antibodies responsible for the humoral re- immunocontraceptive vaccines, all of which rely on sponse. The T cells are either involved in target-cell an animal's own immune system to disrupt reproduc- death (cytotoxic T cells) or provide help for both hu- tive function. Immunological concepts also are im- moral and cell-mediated immune responses (T helper portant for understanding vaccine delivery tech- cells). Activated T helper cells release factors that af- niques. Finally, we consider selected topics in popu- fect growth and differentiation. A subset lation health and behavior in regard to contraceptive of T helper cells is involved in delayed-type hyper- management techniques. sensitivity reaction, which includes granulomatous inflammation. A brief review of reproductive Factors affecting the immune response biology relative to from reproductive proteins immunocontraception When the body encounters foreign pathogenic proteins (), generally there is a response Many elements combine to bring about successful from the immune system. Most reproductive protein reproduction in mammals, including precise coordi- targets for immunocontraception are proteins nor- nation of hormones, production, fusion of mally encountered by the immune system; therefore, oocyte (egg) with sperm, and maintenance of preg- the immune response against these "self-antigens" nancy. The hypothalamic-pituitary-gonadal axis may be weak (Alexander and Bialy 1994). The ZP is a controls sperm and egg production. Successful fertil- normal protein component of the , and there- ization occurs when the sperm is deposited, travels fore is a "self-antigen." up the female reproductive tract, and finally fuses Sperm may introduce foreign proteins in the fe- with the egg (reviewed in Yanagimachi 1994). male. However, the system is adapted to allow The mammalian egg is surrounded by 3 layers: the sperm entry to the female reproductive tract for fer- vitelline membrane, the ZP (a matrix of protein with tilization without generating an immune system at- attached carbohydrate groups), and a cumulus cell tack. layer (mass of loosely packed cells). Ejaculated response in the female genital tract may sperm, coated with seminal proteins undergo many be under hormonal influence (Wira and Sandoe 1977, changes before passing through the layers surround- Murdoch et al. 1982, Wira and Sandoe 1987). A drop ing the egg. Sperm membrane structure and protein in immune response in the cervical mucus at the time composition change (i.e., capacitation) as they travel of ovulation probably improves conditions for sperm through the female's reproductive tract. Seminal pro- penetration. Additionally, Parrand Parr(1990) found teins are removed as the sperm undergo capacitation. reduced antigen uptake in the vagina at the time of Capacitation prepares the sperm for the acrosome re- estrus. Reduced immune response in the reproduc- action and allows penetration of the cumulus cells (in tive tract at ovulation may be a mechanism for ensur- those species where present). Sperm-egg recogni- ing successful fertilization. tion occurs at the ZP and is followed by the acrosome Similarly sperm may have evolved mechanisms to reaction. This reaction is necessary for sperm pene- reduce their immunogenicity in the female reproduc- 506 Wildlife Society Bulletin 1997, 25(2):504-514 tive tract. Seminal plasma provides most of the vol- with remote delivery of ZP vaccines, researchers ume of a normal ejaculate and is a complex mixture used Freund's Complete Adjuvant (FCA). This adju- of secretions from the male reproductive tract. Sem- vant is very potent, but often causes severe reactions inal plasma contains factors that suppress immune including granuloma formation and ulceration of function (reviewed in James and Skibinski 1995). overlying skin (Anderson and Alexander 1983, Stills Also, sperm do not express major histocampatibility and Bailey 1991). Many synthetic adjuvants are being complex (MHC) class I molecules (Guillaudeux et al. developed to reduce these side-effects, while pro- 1996) which may be a factor in reduced female im- moting an immunological response (Sager 1992). mune response to sperm. Modifications of the antigen can be used to over- Quantifying antibody production after come the normally weak immune response expected immunocontraceptive treatment from reproductive proteins (Skinner et al. 1994). A quantitative measure of the humoral immune re- When ZP from a different species is injected into the sponse (antibody production) is often determined female reproductive tract, it may elicit a stronger im- with an enzyme-linked immunosorbent assay mune response than ZP from the same species. Most (ELISA).This assay measures antibody titers. Several of the initial work with ZP vaccines has been done studies evaluating potential wildlife contraceptives with protein isolated from porcine . Porcine ZP have documented the relationship between systemic (PZP) has been successful in stimulating the immune antibody titer and infertility (Liu et al. 1989, Willis et response in other species (such as and deer), by al. 1994). Although there was apparent individual means of or antigenic determinants, sites on variation in immune response, mares with higher an- the antigen that interact with antibodies or T-cell re- tibody titers did not conceive (Liu et al. 1989, Willis ceptors. However, an immune response to PZP also et al. 1994). As antibody titers of mares treated with produces antibodies that are directed against common PZP declined, fertility resumed (Liu et al. 1989). epitopes shared among different species (Skinneret al. 1994). Many of these common epitopes are involved Preferentially targeting the cell- in fertilization or and follicular development mediated immune response with (reviewed in Skinner et al. 1994). Anti-PZPantibodies immunocontraceptives have been shown to react with the ZP isolated from Cell-mediated responses may be stimulated by white-tailed deer (Odocoileus virginianus; Milleret al. some epitopes of gamete proteins. Gamete cell de- 1992a, Skinner et al. 1994) and horses (Equus cabal- struction may be a target for irreversible contracep- lus; Milleret al. 1992b). tion or sterilization. This may be especially appropri- Immune response of antigens depends on other ate in situations where it is infeasible to repeatedly factors that may be manipulated. Amino acid com- treat free-rangingwildlife with a contraceptive agent. position, protein structure, and carbohydrate side Matschke (1980) argued that efficient and practical chains of target proteins may affect these proteins' management of deer populations in the absence of ability to stimulate the immune system (Dunbar et al. regulated hunting required a contraceptive capable 1994). Molecular biology techniques have been used of lasting the reproductive life of an animal. The pre- to express the complementary DNA (cDNA) of ferred mode of action in that case may be cell-medi- mouse, hamster, and rabbit ZP proteins in various ated tissue destruction causing sterility. cell lines (Dunbar et al. 1994). In this process DNA is Yewdell and Bennink (1993) reviewed potential inserted into a foreign cell, which then manufactures antigen processing as a means of specifically target- the new protein. The protein can be more efficiently ing humoral or cell-mediated responses. This differ- produced in large quantities by this process as com- ential direction of response has been demonstrated pared to lengthy purification procedures for ZP isola- with various ZP epitopes (Millaret al. 1989, Tung et tion from whole ovaries. Although, the protein ex- al. 1994). Epifano and Dean (1994) reported 1 out of pressed by the foreign cells duplicate the composi- 5 different ZP tested caused oophoritis (a tion exactly, it may not include modifications found destructive inflammatory disease of the ovary) in an in the naturally produced proteins (Dunbar et al. inbred strain of mice. This was 16 amino 1994). These modifications include glycosylation of acids long and caused a cell-mediated response in the ZP (addition of carbohydrate groups to the protein), mice. Rhim et al. (1992) found a 15-amino-acid ZP which may be important for optimum immune re- peptide caused oophoritis in some but not all strains sponse (Dunbar et al. 1994, Prasad et al. 1995). of mice. In their study, oophoritis probably was me- An adjuvant is a substance that enhances the spe- diated via T cells. Upadhyay et al. (1989) found that cific immune reaction to an antigen. In early work the adjuvant used with the ZP antigen could have an Immunocontraceptionin wild mammals * Mulleret al. 507 effect on how infertility was associated with ovarian jected female red foxes (Vulpes vulpes) with whole morphological changes. sperm directly into the PP. These females produced Ovarian dysfunction was demonstrated in dogs anti-sperm antibodies in their sera and vaginal secre- treated with a ZP vaccine (Mahi-Brown et al. 1988). tions. The anti-sperm antibodies were apparently re- However, in this study, ovarian histopathology (de- sponsible for embryonic failure. fective follicular development and cyst formation) In other studies, IM injection of sperm plasma apparently occurred, but a cell-mediated response membranes did not affect fertility (Willis 1993, White was not detected. Selection of ZP epitopes that 1995). In these studies the animals developed high cause tissue destruction or dysfunction may provide titers of anti-sperm antibodies in their sera (antibody a better option for wildlife fertility control than a hu- titers in the female reproductive tracts were not mea- moral response (antibody production) that only tem- sured). It is possible that the lack of contraception porarily interferes with ovarian function. was due to an immune response to sperm proteins not necessary for fertilization. Alternatively, infertil- Peripheral systemic versus mucosal ity may have resulted from an inadequate mucosal im- immune system and the reproductive mune response in the genital tract. Alexander et al. tract (1992) found uterine wash IgA and IgG concentra- The immune system can be divided into the pe- tions resulting from an injected protein did not mir- ripheral, systemic and separate, mucosal immune ror levels. It is likely the female reproductive system. The lymphoid tissues of the systemic im- tract is protected by both mucosal and systemic im- mune system are responsible for antibody types munity (McGhee et al. 1994). However, a stronger found in the blood circulation. When an antigen antibody response in the reproductive tract may be challenges the immune system either through intra- necessary to neutralize the large number of sperm muscular (IM), intraperitoneal (IP), or intravenous (Alexander et al. 1992). (IV) entry, then the systemic immune system be- Generally only 1 to a few eggs are ovulated per cy- comes involved in neutralization of the antigen. cle; therefore, only a small number of antibodies may There is an initial lag in response, followed by pro- be needed to interfere with ZP function compared to duction of antibodies by activated B cells. If the sys- the larger antibody concentrations necessary to neu- tem is challenged later with the same antigen, then tralize great numbers of sperm (Dunbar et al. 1994). there is a quicker response. The booster response The ZP is exposed to antibodies in the follicular fluid generally has greater magnitude and affinity for the and therefore only a minimal mucosal immune re- antigen than the initial response. The antibodies sponse in the genital tract may be necessary for infer- usually are of the immunoglobulin G (IgG) isotype. tility. Studies that demonstrated infertility using PZP There are several isotypes or classes of immunoglob- vaccine in horses reported systemic anti-PZPantibod- ulins; each has different molecular weights and bio- ies (Liu et al. 1989, Willis et al. 1994). These investi- logical properties. gators did not know what the antibody titers were in The mucosal immune system functions separately the genital tract. However, there must have been suf- as a distinct component of the animal's immune sys- ficient antibody titers around the ovulated eggs to al- tem. The mucosal immune system communicates ter ZP function and cause infertility. throughout the body and includes tissues with secre- tions in the gut; respiratory tract; mammary, salivary A review of and lacrimal glands; as well as the reproductive tract potential (Kutteh et al. 1988, McGhee et al. 1994). Antibodies immunocontraceptive found in external secretions from these mucosal im- protein targets mune sites are usually of the IgA isotype (Kutteh et al. 1988, Kagnoff 1993, McGhee et al. 1994). Kutteh et Ovarian protein targets al. (1988) demonstrated that IgA was produced by tis- The immunocontraceptive that has been most sues of the female reproductive tract. Therefore, the widely tested in wild species is based on developing mucosal immune system can profoundly affect the antibodies to the ZP. Antibodies to ZP may reduce function of immunologically based fertility control. fertility by inhibiting sperm binding with, or penetra- In the mucosal immune system there are discrete, tion of, the egg (reviewed in Skinner et al. 1990). Ad- inductive sites where antigen uptake and processing ditionally, ovarian function may be disrupted by ZP occur (Kagnoff 1993, McGhee et al. 1994). These in- antibodies (Skinner et al. 1984; Fig. 1). ductive sites include gut-associated lymphoreticular Porcine ZP antigen has been used successfully as tissues of the Peyer's patches (PP). Bradley (1994) in- an immunocontraceptive in penned white-tailed deer 508 Wildlife Society Bulletin 1997, 25(2):504-514

ZP Protein is combined with an adjuvant (to enhance the immune response)

The systemic Immunocontraceptive \ svaccine 1 immune system^^ ^ responds to the The mucosal antigen after immune system intramuscular, responds to the intraperitoneal, and/or antigen or intravenous presented in injection of the antigen |

Antibodies are / \ -ijjiiiiiii produced by B cells. Antibody Anid Specific epitopes t^^~nmay> cause activation of cytotoxic T _ Antigen cells that kill binding site the target cell.

Antibody binds to specific antigenic determinants ame - L.

Antibodies bound to the ZP binding sites prevent spezm attachment and fertilization does not occur.

Fig. 1. Immunocontraception using an immune response to zona pellucida (ZP). The ZP is a matrix surrounding the mam- malian egg cell. The ZP is isolated, combined with an adjuvant, and injected into an animal. This animal produces an immune re- sponse to the ZP antigen in the systemic and mucosal immune system. The immune response to the ZP antigen can inhibit sperm-egg binding or disrupt ovarian function.

(Turner et al. 1992). In this study, deer were housed crospheres, and none of 3 treated with osmotic mini- in an 0.5-ha enclosure and treated with PZP vaccine pumps, produced fawns. However, some of the in- (64.3 tg protein in phosphate buffer) and FCA adju- fertile PZP-treated does continued breeding into Feb- vant to boost the immune response. The vaccine was ruary and March. Control does were not observed administered through the fence with a blow pipe. breeding after January. Extension of the normal The deer were vaccinated initially in October and breeding season with contraceptive treatment is an given booster inoculations 3 and 6 weeks later. None important consideration in this method of population of the PZP-treated does produced fawns; however, management. the necessity of 3 vaccinations in the first year with Use of PZP also has been evaluated in horses (Liu et this method makes it infeasible for treating wild deer al. 1989; Kirkpatrick et al. 1990, 1991,1992; Willis et al. populations. 1994). Liu et al. (1989) treated mares with 4 inocula- Alternative treatment regimes of PZP have been tions of PZP. This treatment was effective for contra- evaluated (White 1995, Turner et al. 1996). Turner et ception and lasted >7 months. Using darts for remote al. (1996) tested single-injections of PZP using im- delivery of PZP to horses, Kirkpatrick et al. (1990) planted osmotic mini-pumps or microspheres con- demonstrated effective fertility control with 2-3 inocu- taining PZP. Only 2 of 7 does treated with PZP mi- lations. After antibody titers in the horses were raised Immunocontraceptionin wild mammals * Mulleret al. 509 with the multiple vaccinations, Kirkpatricket al. (1991) Anti-sperm antibodies may be involved in postfer- demonstrated that titers could be boosted with an an- tilization loss of the (Menge and Naz 1988, nual inoculation. Kirkpatricket al. (1992) also found Bradley 1994). This may indicate that some sperm- that PZP treatment for 3 years caused altered ovarian surface antigens become incorporated into function in some mares. Furtherattempts to make PZP and are involved in normal embryonic development treatment more efficient for horses were evaluated by (Menge and Naz 1988). Anti-sperm antibodies may Willis et al. (1994). In this study, 2 treatments of a disrupt embryo development resulting in postfertil- higher dose of PZP (remotely delivered with a biobul- ization reproductive failure (Menge and Naz 1988). let) resulted in contraception for 2 breeding seasons in When determining potential targets for sperm- 2 mares. A third mare was given a third treatment after based immunocontraception in the female, the target which she became infertile. protein must be accessible to the anti-sperm antibod- ies (i.e., ejaculated vs. capacitated sperm proteins). Sperm protein targets Okabe et al. (1986) demonstrated an inconsistent re- Sperm proteins also have received attention as pos- sponse of an anti-sperm monoclonal antibody bind- sible protein targets for immunocontraception. ing to sperm. A monoclonal antibody is a product of Sperm proteins recently have been tested in white- a cloned hybridoma cell (derived from biotechnolog- tailed deer (White 1995). This study did not demon- ical fusion of an antibody-producing white blood cell strate effective contraception using whole-sperm with a malignant tumor cell) that is specific for only 1 plasma-membraneproteins. However, Bradley(1994) part of the antigenic protein. The anti-sperm anti- showed apparent embryonic failure in red fox females body appeared to react with this antigenic compo- vaccinated with whole sperm. Future research by nent that was only available on the surface of the Bradley (1994) will evaluate specific protein compo- sperm after capacitation. nents of fox sperm for potential immunocontracep- A successful sperm-based contraceptive vaccine tives. Several other different spermatozoa antigens must be directed against sperm antigens that target also have been considered for use as immunocontra- specific tissues, affect fertilization, and raise antibody ceptives (Anderson et al. 1987, Primakoffet al. 1988, titers in genital tracts (Naz and Menge 1990). In the Herr et al. 1990, Naz and Menge 1990, Isahakia and future, there may be more effective target proteins Bambra 1992, O'Hem et al. 1995). identified as researchers refine and address these spe- Anti-sperm vaccination may cause infertility in the cific concerns for a successful sperm-based immuno- male or female. In the male, anti-sperm antibodies contraceptive. may cause an autoimmune response (recognition of self proteins as foreign) to the sperm, thus resulting in Other reproductive proteins infertility (Mathur et al. 1988). However, sperm anti- Other potential protein targets for immunocontra- gens rarely cause an immune response in the male be- ception interfere with the reproductive endocrine cause the blood-testes barrier causes isolation of the system. Disruption of the hypothalamic-pituitary- testes from the immune system (reviewed in Ander- gonadal axis can occur when antibodies to gonado- son and Alexander 1983). Naz and Bhargava(1990) trophin-releasing hormone (GnRH;released from the found that antibodies to fertilization antigen-1 (FA-1) ) or follicle-stimulating hormone (FSH; were directed to the late stages of spermatogenesis in released from the pituitary) are produced (reviewed the male. The antibodies gained access to the sperm in Alexander and Bialy 1994). An anti-LHRH(type of in the and vas deferens. These anti-FA-1 GnRH) vaccine was used unsuccessfully in feral antibodies did not react with somatic tissues, such as horses (Goodloe 1991). Other studies have achieved muscle, heart, blood cell, liver, and kidney. infertility with this type of approach (Fraser 1983), In the female, anti-sperm antibodies may cause but there is a potential for altered health and behav- clumping of sperm (reviewed in Shulman 1986), or ior with immunocontraceptives that cause endocrine reduced penetration of sperm through the cervical dysfunction. mucus (Clarke 1988). Sperm proteins also may be Proteins involved in maintenance of pregnancy important for penetration of the cumulus-cell layer also have been considered for immunocontracep- around the egg (Lin et al. 1994); antibodies that in- tion. Antibodies to P-human chorionic gonadotro- terfere with these proteins may prevent sperm from phin (,/hCG) have been targeted (reviewed in reaching the egg. Anti-sperm antibodies also may Alexander and Bialy 1994). Additional proteins asso- prevent sperm attachment and penetration (Gocial et ciated with pregnancy may provide immunocontra- al. 1988) or alter sperm binding to the ZP (Bronson et ception. All of these methods would interfere with al. 1982, Coddington et al. 1992, Naz et al. 1992). pregnancy and cause in the treated animal. 510 Wildlife Society Bulletin 1997, 25(2):504-514

A review of delivery techniques land settings was cheaper and more efficient than for immunologically based darting wary horses on western rangelands. control fertility Oral delivery of immunocontraceptives Another method for Intramuscular vaccine proposed immunocontracep- injection by tive vaccine delivery is through oral dosing. Antigen remote delivery uptake and processing following oral immunocontra- Vaccine administration is also an important con- ceptive vaccine delivery can target discrete inductive sideration for immunocontraceptive management sites of the gut and generate a mucosal immune re- of wildlife. Many techniques have been developed sponse (Kagnoff 1993). This response is reflected in and already evaluated for administration of vac- the reproductive tract because there appears to be a cines in wild populations. Remote delivery is a common mucosal immune system (Kagnoff 1993, more efficient method for delivering contraceptive McGhee et al. 1994). vaccines than methods requiring trapping and im- Enzymatic degradation and conformation changes mobilization of animals. Porcine ZP antigen has of proteins in the gut present obstacles to effective been used successfully as a remotely delivered im- oral delivery of vaccines (Shalaby 1995). Combining munocontraceptive in free-ranging white-tailed the antigen with polymers in microspheres may pro- deer (Turner et al. 1992) and horses (Kirkpatrick et tect the protein from enzymatic digestion. Also, the al. 1990, 1991, 1992). In these applications, dart ri- vaccine could be administered using recombinant- fles, FCA, and multiple booster injections were DNA technology. or bacterial genes could be used, all of which are limiting when applied to free- altered to express specific immunocontraceptive ranging populations. Darts are difficult to deliver protein targets as live vectors (Tyndale-Biscoe 1994, and unretrieved darts may pose a threat as an envi- Srinivasan et al. 1995). Some viruses and bacteria ronmental hazard. have great affinity for PP (Shalaby 1995). These mi- A remote biobullet delivery system has been devel- crospheres and live vectors would escape degrada- oped (DeNicola et al. 1996). This system uses an air tion in the gut and possibly promote preferential mu- gun to project a 0.25-caliber biodegradable bullet cosal-immune responses (Hruby 1993, Bradley 1994). filled with vaccine. The bullets are accurate for <25 Microspheres may be safer for oral vaccine deliv- m, and lost bullets degrade quickly in the environ- ery than using a live vector because of the risks asso- ment. Biobullets have been used to remotely treat ciated with releasing living, replicating organisms free-ranging feral horses with an anti-LHRH vaccine that promote infertility (Tyndale-Biscoe 1994). Re- (Goodloe 1991), as well as captive horses (Willis et searchers are aware of the risks associated with these al. 1994) and white-tailed deer (White 1995) with live vectors and are targeting species-specific vectors. anti-PZP vaccine. The myxoma appears to only affect rabbits and The use of darts or biobullets to remotely inject a therefore is being examined for potential as a recom- vaccine IM is difficult and may require multiple binant carrier for immunocontraception (reviewed in boosters. Booster vaccines often are necessary to Tyndale-Biscoe 1994, Robinson et al. 1997). Perhaps maintain the immune response at a sufficient level to an ideal vector to maintain species specificity would prevent fertility. The use of microspheres containing be based on a sexually transmitted herpes-type vector the antigen and adjuvant are being evaluated as a re- (Barlow 1994). placement for multiple boosters (Turner et al. 1996). Research has shown that the strongest mucosal im- The microspheres contain inert polymers that allow mune response may be in response to an initial prim- for slow, controlled release of antigen (Shalaby ing of the systemic immune system through IM or IP 1995). Presumably these microspheres could be de- injection, followed by mucosal booster (Pierce and livered remotely by dart or biobullet to provide a "1- Gowans 1975, Alexander et al. 1992, Eldridge et al. shot" vaccine. In addition to controlling the rate of 1993). Pierce and Gowans (1975) used an initial IP antigen release, microspheres may function as an ad- injection with either an oral or intraintestinal booster juvant (Eldridge et al. 1993). to generate a high IgA response. If the booster was Some species may be more suited to IM remote de- delivered IP, then there was only a weak mucosal re- livery of vaccines than others. For example, forest sponse. dwelling white-tailed deer would be more difficult to Immunocontraceptives administered initially by re- dart than relatively tame horses in a field. Garrott et motely delivered IM injection (e.g., via biobullet) fol- al. (1992) found that certain situations affected the lowed by oral boosters (microencapsulated so that ease of darting. Darting semi-tame feral horses on is- the antigen is exposed to the mucosal immune sys- Immunocontraceptionin wild mammals * Mulleret al. 511 tem in the intestines) may provide an effective and responded weakly to all antigenic challenges, then it easily administered method for some wildlife popula- probably would have an increased risk of mortality tions. Remotely treating wild animals with contra- from environmental pathogens and also would be se- ceptives is labor intensive. However, biobullet deliv- lected against. ery of booster injections would be even more time Other population-health considerations relate to al- and cost prohibitive compared to oral boosters. Oral tered reproductive behavior and population dynamics. administration of these boosters would probably be Health and fitness of a population may be affected if all more efficient. Furthermore, nontarget species females continue estrous cycling beyond the normal would not be affected by eating the oral bait because breeding season. This problem may have serious im- they would not have been exposed to the initial bio- plications for a seasonally breeding species such as bullet (i.e., IM) treatment. Administration of a single white-tailed deer in which does may continue estrous oral vaccination to nontarget species should be inef- cycling for <7 months (Knox et al. 1988). Rutting fective in generating a mucosal immune response bucks reduce food intake, resulting in significantbody (Pierce and Gowans 1975, Parr et al. 1988). weight losses (Warrenet al. 1981), during the breeding season. Bucks may continue breeding infertile females Use of biological markers to monitor beyond the normal breeding season, and thereby ex- immunocontraceptive treatment pend more of their body reserves. Therefore, extend- The efficiency of contraceptive treatments will ing the rutting period by treating a population with need to be monitored very closely. Biomarkers may contraceptives could increase over-wintermortality. be used to evaluate the affects of contraception on Increased over-winter mortality of bucks resulting population growth. Also biomarkers may be useful from immunocontraceptive treatment of does in a to evaluate exposure of nontarget animals to vaccine population should be closely monitored. However, a treatments. Savarie et al. (1992) reviewed biomark- study by McShea et al. (1997) indicated that buck be- ers used to monitor animal movements and to deter- havior may shift during the breeding season. In a mine bait acceptance by target and nontarget ani- population in which ZP-treated does continued cy- mals. Tetracycline, rhodamine B, and iophenoxic cling beyond the usual breeding season, the re- acid have been evaluated as biomarkers for potential searchers found that larger bucks ceased breeding as oral vaccine programs (Fletcher et al. 1990, Hable et the winter progressed. The ZP-treateddoes that con- al. 1992) tinued cycling until March were bred by smaller bucks. Long-term effects of contraception on deer social behavior are not known. A brief review of population In addition to potential effects of contraceptives on health and behavior concerns male breeding physiology, there may be important relative to immunocontraception changes in female reproductive behavior. Knox et al. (1988) reported that captive, unbred, female white- There may be broad ecological effects where tailed deer exhibited 2-7 recurrent estrous cycles. wildlife contraception is used as a management tool. The last estrus was detected on 7 April 1986. At the It has been proposed that only those animals with the same facility in 1990-1991, White et al. (1995) evalu- "best"immune response to an immunocontraceptive ated the potential effects of breeding stimulus on es- would become infertile (Mitchison 1995, Nettles trus duration. They monitored recurrent estrous cy- 1997). Those animals with poor immune responses cles of 9 does. Each estrous cycle was assigned ran- would be affected less. Thereby, immunocontracep- domly to: (1) no stimulus, (2) with a tion could artificially select for those individuals that vasectomized male, or (3) injection of gonadotrophin were immunodeficient and therefore more suscepti- releasing hormone (GnRH). The does underwent 3-9 ble to pathogenic organisms. This concern warrants estrous cycles with the last estrus detected on 6 June close attention; there is evidence that vaccine-in- 1992. Although there were many confounding fac- duced antibody responses may be controlled geneti- tors involved, the stimulus of breeding without con- cally (Newman et al. 1996). However, not all indi- ception may have lengthened the breeding season. viduals respond equally to all antigenic determinants. This potential extension of recurrent estrous cycles Rhim et al. (1992) found that only certain strains of from a repeated breeding stimulus (as would occur mice were susceptible to oophoritis from injection of with ZP immunocontraception) should be evaluated a 15-amino-acidZP peptide combined with adjuvant. carefully. Unpredictable health effects could result if The strains of mice that did not respond to the ZP a population treated with contraceptives became peptide were not immunocompromised. If an animal aseasonal in its reproductive chronology. 512 Wildlife Society Bulletin 1997, 25(2):504-514

Conclusions ANDERSON, D. J., P. M. JOHNSON, N. J. ALEXANDER,W. R. JONES, AND P. D. GRIFFIN.1987. Monoclonal antibodies to human trophoblast and sperm antigens: of two work- Immunological methods of or steril- Report WHO-sponsored contraception shops, June 30, 1986, Toronto, Canada. J. Reprod. Immunol. ization show promise for effective and efficient fertil- 10:231-257. ity control of individual animals. However, many fac- BARLOW, N. D. 1994. Predicting the effect of a novel vertebrate tors must be considered before implementing this biocontrol agent: a model for viral-vectored immunocontracep- tion of type of control on wild animal populations. Knowl- possums. J. Appl. Ecol. 31:454-462. BRADLEY,M. P. 1994. Experimental strategies for the edge of immune function may aid in an effective development of an immunocontraceptive vaccine for the red fox choice of and European antigen delivery method. Such knowl- (Vulpes vulpes). Reprod. Fertil. Dev. 6:307-317. edge should be combined with an understanding of BRONSON,R. A., G. W. COOPER,AND D. L. ROSENFIELD.1982. Sperm- the normal behavior and the reproductive biology of specific isoantibodies and autoantibodies inhibit the binding of an animal. With these tools, population-management human sperm to the human zona pellucida. Fertil. Sterility alternatives be evaluated where traditionalmeth- 38:724-729. may CLARKE,G. N. 1988. class and ods are not Immunoglobulin regional specificity possible. of antispermatozoal autoantibodies blocking cervical mucus Further research should concentrate on the applic- penetration by human spermatozoa. Am. J. Reprod. Immunol. ability of immunocontraceptive techniques to wild 16:135-138. animal populations. Successful immunocontracep- CODDINGTON, C. C., N. J. ALEXANDER, I). FuIG(HAM, M. MAHONY, D. tives are available and new ones are to be de- JOHNSON,AND G. D. HODGEN. 1992. Hemizona assay (HZA) likely demonstrates effects of characterized mouse antihuman soon. Success of wildlife at sperm veloped contraceptives antibodies on sperm zona binding. Andrologia 24:271-277. the individual-animallevel may not translate into ef- DENICOLA,A. J., D. J. KESLER,ANI) R. K. SWIHART.1996. Ballistics of fective fertility control at the population level (War- a biobullet delivery system. Wildl. Soc. Bull. 24:301-305. ren 1995). Delivery techniques must become more DUNBAR, B. S., S. AVERY, V. LEE, S. PRASAD,D. SCHWAHN, E. SCHWOEBEL, S. SKINNER,AND B. WILKINS. 1994. The mammalian zona practical and efficient to treat populations. Targeting pellu- cida: its biochemistry, molecular both the and mucosal immune immunochemistry, biology, systemic system ap- and developmental expression. Reprod. Fertil. Dev. 6:331- pears to be a promising method for efficient vaccine 347. delivery. Systemic priming with an immunocontra- ELDRIDGE,J. H., J. K. STAAS,D. CIIEN, P. A. MARX,T. R. TiCE,AND R. M. ceptive vaccine delivered IM followed by oral boost- GILLEY.1993. New advances in vaccine delivery systems. ers needs further evaluation. Semin. Hematology 30:16-25. EPIFANO,O., ANDJ. DEAN.1994. and structure of the zona Irreversible control or im- Biology immunological fertility pellucida: a target for immunocontraception. Reprod. Fertil. munosterility also deserves further attention. Im- Dev. 6:319-330. munosterility may be achieved by means of specific FLETCHER,W. O., T. E. CREEKMORE,M. S. SMITH, AND V. F. NETTLES. epitopes that stimulate cell-mediated or delayed-type 1990. A field trial to determine the feasibility of delivering oral vaccines to wild swine. Wildl. Dis. 26:502-510. hypersensitivity reactions in J. resulting gamete dys- FRASER,H. M. 1983. 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BAILEY.1991. The use of Freund's com- Auburn University and plete adjuvant. Lab. Anim. 20(4):25-30. her Ph.D. in wildlife man- TUNG, K. S. K., Y.-H. Lou, A.-M. Luo, AND J. ANG. 1994. Contra- agement from the Univer- ceptive vaccine assessment based on a murine ZP3 mini-au- sity of Georgia. Her re- toantigen. Reprod. Fertil. Dev. 6:349-355. search interests include wildlifephysiology, wild- TURNER, J. W., AND J. F. KIRKPATRICK. 1991. New developments in life damage management, feral horse contraception and their potential application to and of wildlife wildlife. Wildl. Soc. Bull. 19:350-359. ecology populations. Robert I. TURNER, W., F. KIRKPATRICK,AND I. K. M. LIu. 1996. Effective- J. J. (Bob) Warren is a profes- ness, reversibility and serum antibody titers associated with im- sor of wildlife ecology munocontraception in captive white-tailed deer. J. Wildl. Man- and management in the age. 60:45-51. Daniel B. Warnell School TURNER,J. W., I. K. M. Liu, ANDJ. F. KIRKPATRICK.1992. Remotely of Forest Resources and delivered immunocontraception in captive white-tailed deer. Directorof the National Park Service Technical Center at the of J. Wildl. Manage. 56:154-157. Support University Georgia. He was on the wildlife at Texas Tech University. C. H. 1994. Virus-vectored formerly faculty TYNDALE-BISCOE, immunocontracep- Bob obtainedhis B.S. in zoology fromOklahoma State University tion of feral mammals. Reprod. Fertil. Dev. 6:281-287. and his M.S.and Ph.D.in wildlifefrom Virginia Polytechnic Institute UPADHYAY, S. N., P. THILLAIKOOTHAN,A. BAMEZAI,S. JAYARAMAN,AND G. and State University. He has served on numerous committees for P. TAIWAR. 1989. Role of adjuvants in inhibitory influence of The WildlifeSociety (TWS), the SoutheasternSection of TWS(SE- immunization with porcine zona pellucida antigen (ZP-3) on TWS),and the Georgiaand Texaschapters of TWS. Bobwas presi- ovarian folliculogenesis in bonnet monkeys: a morphological dentof SE-TWSin 1992 and 1993, and he servedas associateeditor study. Biol. Reprod. 41:665-673. of the WildlifeSociety Bulletin and the Proceedingsof the Annual WARREN,R. J. 1995. Should wildlife biologists be involved in Conference of the Southeastern Association of Fish and Wildlife Bob has edited newslettersfor SE-TWSand the Texas wildlife contraception research and management? Wildl. Soc. Agencies. His research interests include Bull. 23:441-444. Chapter. physiology, reproduction, nutrition, genetics, and ecology of wildlife populations. Donald L. WARREN, R. J., R. L. KIRKPATRICK,A. OELSCHLAEGER,P. F. SCANLON, AND (Don)Evans is a professorin medicalmicrobiology at the Collegeof F. C. GWAZDAUSKAS.1981. Dietary and seasonal influences on VeterinaryMedicine, University of Georgia.Previously, he did post- nutritional indices of adult male white-tailed deer. J. Wildl. doctoraltraining in the Departmentof Virologyat the Universityof Manage. 45:926-936. TexasSystem Center in Houston,Texas. Don receivedhis WHITE,L. M. 1995. Experimental evaluation of fertility control B.S.and M.S. at the Universityof Missouri,and a Ph.D.from the Uni- methods and delivery techniques for managing white-tailed versityof Arkansas. His researchinterests include molecularim- deer populations. Ph.D. Diss., Univ. Georgia, Athens. 156pp. munologyand cell signaling. WHITE,L. M., D. A. HOSACK,R. J. WARREN,AND R. A. FAYRER-HOSKEN. 1995. Influence of mating on duration of estrus in captive white-tailed deer. J. Mammal. 74:1159-1163. Associate Editor: Barnes