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Evaluation of a Gonadotropin Releasing Hormone Vaccine for the Humane Control of Female Feral Cats


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EVALUATION OF A GONADOTROPI N RELEASING HORMONE VACCINE FOR THE HUMANE CONTROL OF FEMALE FERAL CATS By JOHN FRIARY A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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ACKNOWLEDGMENTS I would like to thank Dr. Julie Levy for the opportunity to study under her and for her willingness to give second, even third, chances. She knew when I needed to be pushed even when I did not know it. I have very much appreciated and been inspired by Dr. Cynda Crawfords refusal to allow her indefatigable work habits to dull her sense of humor. I am grateful for Dr. Lowell Millers great patience while answering my seemingly endless questions regarding the vaccine, and for welcoming me to his laboratory in Colorado. I have benefited greatly from Dr. Karine Onclins ability to explain endocrinology to me and I thank her. Without the support and motivation of Dr. Paul Chadik and Jim Crocker, I would not have returned to school and I can not thank them enough. I would also like to thank Sylvia Tucker, Mike Reese, Kathy Kirkland, Audria West, and Rob Schleich for their technical assistance. The girls, of course, remind me every day why I have chosen this path. This study was made possible by funding received from the Morris Animal Foundation. ii

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS.........................................................................................ii LIST OF TABLES.....................................................................................................v LIST OF FIGURES...................................................................................................vi ABSTRACT...............................................................................................................vii IMPACT OF FERAL CATS.....................................................................................1 Human Health................................................................................................2 Animal Health................................................................................................4 Predation on Wildlife.....................................................................................5 METHODS TO MANAGE FERAL CATS..............................................................8 Lethal Methods..............................................................................................8 Non-Lethal Methods......................................................................................10 Adoption............................................................................................10 Relocation Programs..........................................................................10 Sanctuaries.........................................................................................10 TNR Programs...................................................................................11 Non-surgical Contraception...............................................................13 Feline reproductive endocrinology........................................14 Steroids and GnRH agonists..................................................17 Cell destruction......................................................................18 IMMUNOCONTRACEPTION.................................................................................19 Immunocontraception Targets.......................................................................19 Riboflavin Carrier Protein..................................................................19 Sperm Proteins...................................................................................20 Zona Pellucida...................................................................................20 Hypothalamic-Pituitary-Gonadal Axis..............................................20 Vaccine Delivery...........................................................................................21 GNRH VACCINE.....................................................................................................23 iii

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4 MATERIALS AND METHODS...............................................................................25 Cats................................................................................................................25 Vaccine Construction.....................................................................................25 Treatment.......................................................................................................26 Blood Collection............................................................................................27 Detection of GnRH Antibodies......................................................................27 Determination of Serum Estradiol-17 and Progesterone Concentrations.........................................................................................29 Breeding Trial................................................................................................29 Estrus Induction.................................................................................29 Assignment of Cats to Study Groups.................................................30 Fertility and Fecundity.......................................................................30 Body Phenotype.............................................................................................30 Statistical Analysis.........................................................................................31 RESULTS..................................................................................................................34 Reactions to Treatment..................................................................................34 Breeding Trial................................................................................................34 Detection of Estrus.............................................................................34 Fertility and Fecundity.......................................................................35 GnRH Antibody Titers...................................................................................35 Hormone Concentrations...............................................................................36 Progesterone.......................................................................................36 Estradiol-17......................................................................................37 Body Condition..............................................................................................37 Body Weight......................................................................................37 Falciform Fat Pad...............................................................................38 DISCUSSION...........................................................................................................43 REFERENCE LIST...................................................................................................48 BIOGRAPHICAL SKETCH.....................................................................................55 iv

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5 LIST OF TABLES Table Page 1. Body weight and percent weight gain...................................................................42 2. Falciform fat pad depth and area..........................................................................42 v

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6 LIST OF FIGURES Figure Page 1 Timeline................................................................................................................32 2 Lateral abdominal radiograph...............................................................................33 3 Breeding trial........................................................................................................39 4 GnRH antibody titers for female cats...................................................................40 5 GnRH antibody titers for cats with high titers .....................................................41 6 GnRH antibody titers for cats with variable titers................................................41 7 GnRH antibody titers of nonresponders...............................................................42 vi

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EVALUATION OF A GONADOTROPIN RELEASING HORMONE VACCINE FOR THE HUMANE CONTROL OF FEMALE FERAL CATS By John Friary August 2006 Chair: Julie Levy Major Department: Veterinary Medicine The unwanted cat population in the United States numbers in the tens of millions and current control measures have only had limited success in reducing it. Immunocontraception has the potential to humanely reduce this population. The purpose of this study was to investigate the effectiveness of a GnRH-based vaccine for immunocontraception of female cats. It was expected that the treated cats would produce antibodies against GnRH and there would be a positive correlation between high titer and contraception. Adult female cats were divided into a sham group (n = 5) and a treatment group (n = 15) that was immunized once with 200 g of synthetic GnRH coupled to keyhole limpet hemocyanin and combined with a mycobacterial adjuvant. GnRH antibody titer and serum concentrations of progesterone and estradiol-17were determined monthly. For the duration of the study the daily photoperiod was manipulated in an attempt to induce estrus. A male breeding cat was housed with the females during the long-day periods, and continuous videography was used to monitor vii

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for signs of estrus and breeding. GnRH antibodies were detected in all treated cats by 150 days after immunization, but when the titer in four cats fell below 16,000, they became pregnant and were classified as nonresponders. The titers of the remaining 11 cats (responders) never decreased below 16,000. These cats displayed no signs of behavioral estrus and did not become pregnant by the end of the study 24 months after immunization. All five sham cats became pregnant within one month of the introduction of the male cat. From 60 days after immunization until the end of the study, progesterone concentrations in all responders remained at basal levels, and increased two months before parturition in all cats that became pregnant. The responder cats gained more weight than the nonresponders during the 14 months after immunization (P = 0.004), which is the same response observed in surgically sterilized cats. A single dose of GnRH vaccine resulted in contraception in 73% of the cats for at least 24 months. viii

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IMPACT OF FERAL CATS The domesticated cat is the most numerous companion animal in the United States with 37.7 million households owning 90.5 million cats [1]. It is estimated that there are also an equal number of unowned, unwanted cats [2]. Considerable debate exists on what impact these cats have on human health through zoonotic diseases, animal health by acting as reservoirs for diseases that affect pet cats and other species, and wildlife through predation and competition. In addition, there is not agreement on the quality of life of the cats. The debate extends to the actions that should be taken to ameliorate the impact of these unowned cats. Accurately assessing the impact and designing solutions is made more difficult because cats are assigned to subpopulations according to different criteria. Cats are often described according to their ownership status (owned or unowned), lifestyle (indoor, indoor/outdoor, outdoor), or degree of socialization [3] (tame, feral). In addition, during its lifetime an individual cat may move from one subpopulation to another. For instance, a cat living indoors may be abandoned, forced to live outside, and over time become untrusting of humans; this tame, indoor, owned cat has become an unowned, feral cat. Many reports on the impacts of cats fail to clearly define which subpopulation of cats is being studied. For the purposes of this thesis, a feral cat will be defined as any unowned, free-roaming cat, regardless of its socialization status. 1

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2 Concerns have been expressed that feral cats serve as a reservoir for diseases that may be transmitted to humans, pet cats and wildlife [4,5]. In general, feral cats do not appear to pose a greater risk to humans or other cats than pet cats [3,6]. Cats can transmit disease directly through a bite or other physical contact; by shedding the pathogenic organism into the environment; or by concentrating pathogens that can be transferred to other hosts via vectors such as fleas, ticks and mosquitoes. The Center for Disease Control and Prevention considers eradicating wildlife to reduce disease reservoirs ineffective, but does support trap-vaccinate-release programs for wildlife [7]. Human Health The American Association of Feline Practitioners lists 40 potential feline zoonotic agents, but transmission of the disease from cat to human has not been documented or occurs only rarely with most of the organisms [8]. Rabies is often cited as a risk from feral cats, but none of the 57 human cases of rabies in the United States from 1980 to 2004 was attributable to cats [9,10]. The risk of rabies transmission from cat to human is very low. Bartonella henselae and Bartonella clarridgeiae can both be passed from cats to humans by biting or scratching. Infection by these bacteria is the most common direct zoonosis associated with cats, and it results in 25,000 cases of cat scratch disease in the United States each year [8]. Cat scratch disease is generally a mild illness, but in rare cases it can lead to serious disease. In Randolph County, North Carolina, 93 of 100 (93%) feral cats and 48 of 76 (63%) pet cats were seropositive for antibodies against B henselae [11]. In northern Florida 186 of 553 (33.6%) feral cats were seropositive for

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3 antibodies against B henselae, which is within the range of values found in several studies of pet cats [6]. Roundworms, hookworms and tapeworms can be shed by cats in their feces and can potentially cause infection in humans. However, the larvae of roundworms and hookworms must mature in the environment for at least 3 days and 3 weeks, respectively, before they can infect a new host [8]. Fleas containing tapeworm must be consumed for infection to occur [8]. The infection rate of roundworms in feral (21%) and pet (18%) cats in Randolph County, North Carolina, was not statistically different [11]. However, a study of 80 feral and 70 pet cats in California found infection rates of roundworms and tapeworms significantly higher in feral cats than pet cats. The roundworm infection rates were 54% and 4% for feral and pet cats, respectively, and tapeworm infection rates were 26% and 4% for feral and pet cats, respectively [3]. The protozoa Toxoplasma gondii, Cryptosporidium parvum, and some species of Giardia are shed in the feces of cats and can infect humans through the fecal-oral route. Infection with Cryptosporidium spp. and Giardia spp. in humans is common, but they are rarely directly linked to cats [8]. In Randolph County, North Carolina, the feral and pet cats did not have significantly different prevalences of infection with Cryptosporidium (7% and 6%) or Giardia (6% and 5%) [11]. Domestic cats and other felids are the only definitive hosts to shed T. gondii oocytes in their feces, thus contaminating the environment with infectious organisms. The seroprevalence of T. gondii in feral cats (63%) in Randolph County, North Carolina, was higher than for owned cats (34%) [11]. Feral cats from Northern Florida were found to have a seroprevalence of 12.1%, lower than seroprevalence rates found for pet cats in

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4 the United States (30%) [6]. Consumption of tissue containing T. gondii cysts allows transmission between intermediate hosts, such as pigs and humans. The relative importance of the two modes of infection in humans, ingestion of oocytes shed by cats and consumption of infected tissue, is unknown. The natural oropharyngeal flora of healthy cats includes Capnocytophaga and Pasteurella, which are frequently found in cat bite infections. Untreated infections with these organisms can cause death [12]. However, most feral cat bites are provoked [3]. Advising the public to avoid direct contact with feral cats and implementation of trap-vaccinate-return programs should reduce the zoonotic disease risk from feral cats. Animal Health The feline zoonotic diseases are transmissible between cats, but cats can also transmit diseases that do not affect humans, including feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and feline coronavirus (FCoV). Some feel that kitten mortality rates, which may be as high as 75%[13], and the general health of feral cats are compelling reasons to euthanize these cats[5]. They believe that feral cats suffer higher rates of injury and disease than owned cats[5]. However, feral cats brought to trap-neuter-return (TNR) clinics in northern Florida were lean, but not emaciated [14], had death rates due to complications of surgery comparable to pet cats, and had a euthanasia rate for humane reasons of only 0.4%[14,15]. In addition, several studies have found that disease prevalence rates are very similar between feral and owned cats[6,11,16,17]. In 2004, 5,259 feral cats and 12,779 pet cats were tested for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) in 145 animal shelters and 345 veterinary clinics in the United States, Puerto

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5 Rico and Canada. The infection rates for the feral cats were 2.0% (FIV) and 1.6% (FeLV) compared to the infection rates for the pet cats that were 2.7% (FIV) and 2.6% (FeLV) [16]. Between 1995 and 2000, a total of 1,876 feral cats from two trap-neuter-return programs in Gainesville, Florida and Raleigh, North Carolina, had infection rates for FIV and FeLV of 3.5% and 4.3%, respectively[17]. In addition, 553 feral cats in northern Florida had similar or lower prevalence rates of Mycoplasma haemofelis, Mycoplasma Haemominutum and Bartonella henselae compared to previous reports in pet cats [6]. However, it is possible that the overall health of feral cats brought in to TNR clinics is different than the health of feral cats in general. Predation on Wildlife It has been well-documented that cats can have adverse impacts on wildlife, particularly on islands. Prey species on island ecosystems are especially vulnerable to the introduction of non-native mammalian predators such as the cat [18]. Cats have been introduced to at least 65 island groups and are a major threat to many island bird species [18]. In 1949, cats were introduced to Marion Island, a 112-square mile sub-Antarctic island in the Indian Ocean. By 1975, it was estimated that they were killing 450,000 burrowing petrels annually and probably had driven the common diving petrel to local extinction [19]. On an island in New Zealand, the last remaining Stephens Island Wren (Xenicus [Traversia] lyalli) was killed by a cat [20]. There is evidence that cats contributed to the total extinction of the Little Barrier snipe (Coenocorypha aucklandica barrierensis) and the local extinction of the North Island Saddleback (Philesturnus carunculatus rufusater) on Little Barrier Island, New Zealand [21]. However, introduced rats also had a detrimental effect on the bird

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6 populations, and the relative contributions from the cat and the rat could not be determined [21]. In addition, seven years after eradication of cats was completed on the island, the bird numbers were similar to their numbers before the cat eradication program began [21]. It is believed that in some instances cats may actually protect island species by keeping the rodent population in check [19,22]. Determining the impact of cat predation in mainland settings is even more difficult than on islands because in most instances there are additional predators present. Outside of Canberra, Australia, domestic cats were found to be opportunistic predators, catching prey in proportion to the prey density. The conclusion reached was that cats had little effect on the local ecosystem [23]. Wildlife managers in Australia have been working to remove rabbits, another exotic species. However, in many ecosystems rabbits are the primary food for cats, and there is a fear that a sudden removal of rabbits will cause the opportunistic cats to switch to native prey. Individual ecosystems must be evaluated to determine the impact that cat predation has on wildlife and if removal is appropriate or necessary. Possible transmission of T. gondii from cats to California sea otters (Enhydra lutris nereis) is of current concern. Between 1997 and 2001, the seroprevalence of T. gondii was 42% for live otters and 62% for dead otters [24]. The source of the T. gondii is believed to be cat feces from sewage and surface runoff from coastal communities. However, whether the feces came from litter boxes of indoor cats, from owned cats allowed outdoors, or feral cats has not been investigated. There is also concern that native marsupials in Australia, which has non-native cats, may be more susceptible to T. gondii than ecosystems with native cats[25].

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7 Although the true impact of feral cats on the health and welfare of the public, animals, and the environment is difficult to quantify, it is appropriate public policy to develop effective cat control programs. The best approach to cat control is another area of intense controversy.

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METHODS TO MANAGE FERAL CATS The most common action taken regarding feral cats has been to do nothing [26]. Historically, lethal methods of control have been used in an attempt to eradicate particular groups of cats. In the United Sates, euthanasia is commonly performed at animal control shelters. Worldwide, however, trapping, poisoning, and hunting have been the most common methods for eradication of feral cats. The use of biological vectors has been utilized in at least three island eradications [20,27]. In some areas of the world, including the United States, there has been strong public pressure to devise more humane ways of dealing with feral cats, including trap and relocate, and TNR. Lethal Methods Since 1934, cats have been eradicated from 48 islands around the world. All but five of these islands had cat populations of less than 100, and only 10 of the islands are greater than 10 square kilometers [28]. The largest eradication effort occurred on Marion Island using trapping, poisoning, hunting with guns and dogs, and biological control, and took 19 years to complete [20]. It took three years to eradicate 151 cats from Little Barrier Island using traps, poison, hunting with guns and dogs, and biological control [21]. The poison used in most eradication campaigns was sodium monofluoracetate (1080), which has been used since the 1950s in New Zealand for pest control [29]. Sodium monofluoracetate kills by disrupting the Krebs cycle, thus inhibiting energy 8

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9 production by cells. Long-term exposure to sub-lethal doses can be harmful to people handling the poison. Non-target animals are also at risk; dogs are especially susceptible to 1080 [29]. For these reasons, the use of 1080 in the United States is restricted to the protection of livestock from coyotes [30]. Secondary poisoning by ingestion of rats poisoned with the anticoagulant, brodifacoum, contributed to cat eradication on four islands [28]. Feline panleukopenia virus (FPV) was the biological control agent used on Jarvis, Marion, and Little Barrier Islands [28]. FPV virus is spread through feces, urine, saliva, and vomit. Felids are the primary hosts of FPV, but raccoons and a few other mammals can also contract the disease. On Marion Island, 96 cats were trapped and inoculated with 1000 TCLD 50 of FPV and released by helicopter to 93 different locations on the island. The total cat population declined at a rate of 26% per year for five years, but stabilized as the population developed immunity [20]. On Little Barrier Island, the use of FPV was abandoned because the virulence among that population of cats was determined to be too low to be effective [21]. The use of biological controls has many inherent risks, including accidental release as happened with the rabbit calicivirus in Australia in 1995 [31], and non-target susceptibility. As was demonstrated on Marion and Little Barrier Islands, the target species may become resistant over time. Eradication or reduction of cats on the mainland is more difficult than on islands for several reasons. Continuous influx of cats into the area is likely, the risk of non-target species death increases with poisoning and hunting with dogs, hunting with guns is heavily restricted for human safety reasons, and biological control mechanisms would be nearly impossible to contain.

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10 Euthanasia at animal shelters is the leading cause of death of cats in the United States, with an estimated 3 million cats per year [26,32]. Euthanasia has not been shown to effectively reduce feral cat populations. In 1996 in Ohio 72.2% of cats admitted to shelters were euthanized and in 2004, the euthanasia rate was 68.8% [33]. It is hypothesized that removing cats from a habitat simply allows other cats to fill the vacated niche [26,34]. Non-Lethal Methods More humane solutions include trapping cats, sterilizing them, and adopting them into homes, relocating them to a more acceptable outdoor location, placing them in sanctuaries, or returning them to the location of their capture. Adoption Seventy-five percent of the cats euthanized in animal shelters in the United States are classified as adoptable [3], but there are not sufficient homes to accept them. Increasing the pool of cats waiting to be adopted with less socialized cats is clearly not a viable solution to the overall problem. Relocation Programs Relocation of cats to a non-enclosed site is difficult because it is time-consuming to acclimate the cats to their new environment and the cats often have low survival rates at the new sites [26]. Sanctuaries Another alternative is to remove cats to permanent sanctuaries where they can live out their lives in confinement. Although sanctuaries can be a small-scale solution the overall number of feral cats is too large to be accommodated in sanctuaries. Three

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11 sanctuaries that have reached capacity and only accept cats on a very limited basis are Best Friends Animal Sanctuary in Utah, the Chico Cat Coalition in California, and a program instituted by the National Humane Education Society. In addition to their sanctuaries, Best Friends Animal Sanctuary and National Humane Education Society operate TNR programs [3]. TNR Programs The goal of TNR programs is to sterilize feral cats and return them to the location where they were trapped. TNR programs are generally run as grass-roots operations that depend on donations and volunteers. However, some municipalities, such as Orange County, Florida, use TNR programs as a cost-effective alternative to trapping and euthanizing cats and have incorporated TNR into their animal control efforts. The average cost of sterilizing each cat was $56 compared to the estimated $139 per cat for impounding, sheltering and processing [26]. Other agencies that have incorporated TNR into their animal control programs include Tomkins County Society for the Prevention of Cruelty to Animals, New York; Maricopa County Animal Care and Control, AZ; New York City Center for Animal Care and Control; San Francisco Society for the Prevention of Cruelty to Animals; and the American Society for the Prevention of Cruelty to Animals. At a minimum, TNR programs sterilize the cats and return them to their colonies. Some programs provide food, shelter and veterinary care, vaccinate against rabies and other diseases, and test for diseases such as FIV and FeLV. Removal of the tip of an ear is recognized internationally as a sign that the cat has been sterilized. For control to be effective it is essential that colonies are monitored for new arrivals and for

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12 the birth of kittens. A TNR program was started and stopped on a Florida university campus; once the program stopped, the feral cat population began increasing [2]. One long-term goal of many TNR programs is to reduce the colony size through adoption and attrition. Complete elimination of large colonies is uncommon, but there are many examples of colonies being greatly reduced in size. In 1991, a TNR program was instituted on the University of Central Florida campus and by 2002, the original 11 colonies containing a total of 155 cats had decreased to eight colonies comprised of 23 cats [2]. In addition to a decrease of 132 cats, other benefits of the program included prevention of the birth of an estimated 300 to 700 kittens, and medical care or euthanasia for sick and injured cats in the colonies. In a three-year period ending in 2002, 1,116 California Veterinary Medical Association veterinarians neutered 170,334 feral cats through the Feral Cat Altering Program [35]. Maddies Fund awarded nearly $9.5 million to the California Veterinary Medical Association for the program [35]. ATNR program on a Texas university campus resulted in a 30% decrease in the feral cat population in two years [26]. Some wildlife advocates believe TNR is inappropriate because the cats are not removed from the wild, some critics believe euthanasia is more humane than returning the cats to their colonies, and others believe TNR is ineffective. Indeed, critics of TNR often cite two county parks in south Florida, A.D. Barnes Park and Crandon Marina, as examples of TNR failure. The high visibility of the TNR programs in these parks encouraged continual cat abandonment at the sites, resulting in a net increase in the colony size despite a decrease in the original cat populations [36].

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13 Drawbacks recognized by proponents of TNR include expenses for traps, surgical equipment, medical supplies, and veterinary fees; intensive labor for trapping, transportation, surgery, recovery and return to the colony; and requirement for the technical expertise of veterinarians. Mathematical models have been used to estimate the percent of feral cats that would need to be neutered in order to cause an overall decline in a population. One population model predicted that the annual percent sterilization necessary for stabilization of population growth would be 14% of the 241,000 cats in San Diego County, CA and 19% for the 36,000 cats in Alachua County, FL [34]. Another study [37] predicted that there would have to be a 75% neutering rate annually in order to have a population decline. This study predicted that a trap and euthanize program would only have to trap 50% of the population to have a population decline. A limitation to the mathematical models is the difficulty of determining parameters that determine feral cat reproductive capacity, such as the carrying capacity of a particular habitat [34]. While surgical TNR can be effective for reducing targeted cat populations, it is extremely resource-intensive and difficult to implement on a regional or national scale. Non-Surgical Contraception Non-surgical methods of contraception or sterilization have the potential for more efficient implementation, less risk to cats, lower cost, and lower technical requirement when compared to surgery. Surgical sterilization achieves sterility by removal of the gonads, thus preventing the production of gametes. Successful non-surgical contraception or sterilization methods disrupt some aspect of the hypothalamic-pituitary-gonadal axis or interfere with fertilization of the egg or maintenance of the pregnancy.

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14 Feline reproductive endocrinology Cats will only breed when they are in estrus, which is characterized by a rapid increase in estradiol-17 concentrations. The estrogen is released by the follicles, which are stimulated to grow by the gonadotrope, follicle-stimulating hormone (FSH). FSH is synthesized and released at a basal level, but the rate of synthesis increases greatly with the release of gonadotropin releasing hormone (GnRH). Female cats are photoperiod sensitive and they begin estrous cycling with an increasing daylight-length, as occurs in January and February in the northern hemisphere. Cats will stop estrus with a decreasing daylight-length as occurs in September or October. Cats in the laboratory can be induced to enter estrus by increasing the daylight-length to 14 hours or greater. It is possible that the pineal gland responds to increasing daylight-length by decreasing the production of melatonin, which inhibits the release of FSH [38]. Conversely, decreasing the amount of light appears to increase the production of FSH and the cats will enter anestrous, meaning that estrous cycling stops. During estrus, female cats are receptive to male cats and sufficient breeding triggers a cascade that leads to ovulation. As an induced ovulating species, cats release GnRH within minutes of genital somatosensory stimuli, as occurs during breeding [39]. GnRH causes the release of a second gonadotrope, luteinizing hormone (LH), within 5 minutes of the GnRH release [40]. LH concentration increase does not necessarily lead to ovulation, however. With a single copulation only about 50% of cats will ovulate, but repeated copulations probably trigger successive GnRH releases which leads to cumulative increments in LH levels [39].

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15 GnRH is synthesized in hypothalamic neurons whose axons extend into the pituitary stalk, which is not within the blood-brain barrier. Upon appropriate stimulation, GnRH is released by exocytosis into the capillary plexus that emanates from the superior hypophyseal artery. GnRH is carried down the pituitary stalk in portal veins which give rise to a second capillary plexus that supplies the endocrine cells of the anterior pituitary. GnRH binds to gonadotrophs which synthesize and release the two gonadotropins, LH FSH. However, the duration and the amplitude of the gonadotropin release depends on the pulsatile and periodic release of GnRH. For a proper functioning hypothalamic-pituitary-gonadal axis (HPGA), GnRH must be released with the proper pulsatility and periodicity; the characteristics of the GnRH release differ in male and female cats, with the age of the cat, and the stage of estrus with the female cat. LH binds to Leydig cells in the male cat and thecal cells in the female cat and stimulates the synthesis and secretion of androgens. FSH binds to Sertoli cells in the male cat and granulosa cells in the female cat and stimulates estrogen synthesis. FSH also stimulates synthesis in the Sertoli and granulosa cells of various protein products, including activin and inhibin, two proteins involved in feedback mechanisms within the HPGA. In addition, FSH increases the number of LH receptors on the granulosa cells, which amplifies the sensitivity of granulosa cells to LH. If the female cat does not breed during estrus, or if there is insufficient breeding to induce ovulation, then the estrogen levels decrease after three to sixteen days [41] and the cat enters interestrous, or a phase between successive estrous periods. The cat will continue to cycle between estrus and anestrous until ovulation occurs or the breeding season comes to an end with decreasing daylight-length.

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16 If the cat ovulates and the egg or eggs are successfully fertilized then the cat begins a gestation period of approximately 65 days. If the cat ovulates, but the egg or eggs are not fertilized, then the cat enters pseudopregnancy which lasts approximately 45 days. In both pregnant and pseudopregnant cats, the follicles that have matured have released their eggs from the ovary. The portion of the follicles that remain behind are termed corpus lutea and within 48 hours of ovulation they begin secreting progesterone. Prior to the release of progesterone by the corpus lutea, the basal level of progesterone is less than 2 ng/ml. Within 14 to 18 days after ovulation the progesterone concentrations are greater than 20 ng/ml. The lifespan of the corpus lutea is approximately 35 days, so the progesterone concentration in a pseudopregant cat returns to baseline after 35 or 40 days. However, a pregnant cat maintains progesterone concentrations above baseline because the placenta secretes the hormone after 30 days of gestation [41]. GnRH, the gonadotropes and the sex hormones form a complex feedback web. GnRH induces the release of FSH and LH, which in turn induce the release of testosterone and estrogen. However, testosterone and estrogen provide negative feedback and inhibit the release of LH and GnRH. In addition, the gonads produce three other hormones that act on the gonadotrophs. Activin stimulates the release of FSH, inhibin and follistatin inhibit release of FSH [41]. Additional hormones that are produced outside the gonads and affect fertility include leptin, prolactin, growth hormone and insulin-like growth factor 1 (IGF-1). Leptin is required for fertility [42], while high concentrations of prolactin inhibit the

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17 secretion of FSH and LH in both sexes. Growth hormone stimulates synthesis of insulin-like growth factor 1 which stimulates sex hormone synthesis [42]. The reproductive system in cats provides a number of mechanisms and potential targets to inhibit fertility, including the use of steroid and peptide hormones to disrupt the necessary hormone balance, destruction of reproduction-related cells, and immunocontraception to bind a hormone or hormone receptor in the HPGA. Steroid hormones and GnRH agonists Steroids related to estrogen and progesterone have been used since the 1960s for contraception of some species. Diethylstilbestrol (DES) is a synthetic estrogen that can reduce fertility in animals, but it must be administered with precise timing, lasts a limited amount of time, accumulates in body tissues, and has potential adverse health effects in treated animals [43]. These characteristics preclude DES and other estrogen-related compounds from intense use in feral cat population control. Progestins, which are synthetic progesterones, induce contraception, but the mechanism of contraception is not well understood. The mechanism most likely involves one or more of the following: negative feedback on GnRH release, disruption of oocyte transport and fertilization, and altered receptivity of the endometrium [44]. However, limited duration of contraception and potential harm to target animals also preclude progestins from use in feral cats. Administration of GnRH agonists disrupt the pulsatile release of GnRH necessary for secretion of pituitary follicle-stimulating (FSH) and lutenizing hormones (LH) [45]. This method disrupts gonadal hormone production to produce contraception, but the short duration of effectiveness makes this method unsuitable for feral cats.

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18 Cell destruction Targeted destruction of specific cells or tissues can be used to disrupt the reproductive process. A GnRH analogue conjugated to a cytotoxin, pokeweed antiviral protein, resulted in lower serum testosterone concentrations in dogs [46]. It is believed that the GnRH analogue portion of the conjugate attaches to GnRH receptors on the gonadotroph cells in the pituitary gland. The molecule is taken into the cells by endocytosis and the cytotoxic portion of the conjugate destroys the cells ability to synthesize proteins, which leads to cell death. It was hoped that the effect would be permanent, but a portion of the treated dogs demonstrated increasing FSH and LH serum concentrations by week 36. Ovarian follicles in rats have been destroyed by administration of 4-vinylcyclohexene diepoxide (VCD), a metabolite of an industrial chemical [47]. Female mammals are born with their entire lifetime supply of oocytes contained within primordial ovarian follicles. VCD targets and destroys the primordial follicles and the oocytes they contain leaving the animal devoid of oocyte stores and unable to ovulate successfully. This method provides permanent sterility and cessation of estrus cycling. However, VCD has only proven safe and effective in rodents and more research needs to be done in other species. In addition, the current treatment requires daily doses for 15 days, which is not practical for feral cats.

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IMMUNOCONTRACEPTION The goal of immunocontraception is to induce an immune response against critical elements of the reproductive process that are not used for other functions. There are several targets that meet this criterion and have been investigated for their immunocontraceptive potential, including riboflavin carrier protein (RCP), sperm proteins, zona pellucida (ZP), lutenizing hormone receptor (LH-R), and GnRH. An acceptable immunocontraceptive agent for feral cats would be effective in both sexes and only a single treatment should be used because retrapping of free-roaming cats for repeated treatments would be impractical. In addition, the vaccine should block the production of sex hormones so as to eliminate nuisance behaviors such as calling, spraying, wandering, and fighting. In addition to these minimum standards, an ideal agent would be permanent, have a quick onset of contraceptive effect, be effective in all ages, be safe to the cat and the environment, be inexpensive, and be easy to administer. Immunocontraception Targets Riboflavin Carrier Protein RCP is the prime mediator of riboflavin supply to the developing zygote in mammals and is necessary for maintenance of pregnancy. Three monthly treatments with an RCP vaccine followed by repeated treatments every four months elicited high antibody titers and interfered with pregnancy in Bonnet monkeys, but it was not determined if conception or implantation was affected [45]. The need for multiple 19

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20 treatments and failure to block sex hormone production make this approach impractical for feral cats. Sperm Proteins Provoking an immune response against sperm proteins has the potential to be effective in both sexes because antibodies to the proteins could interfere with sperm development and viability in the male or after insemination in the female [43]. Targeting sperm would not interfere with sex hormone production, therefore, immunocontraceptives against sperm proteins are not the best candidate for feral cats. Zona Pellucida Zona pellucida (ZP) is a glycoprotein layer surrounding the mammalian egg and is involved in sperm-egg interaction [43]. The use of ZP antigens for immunocontraception has been successful in preventing pregnancy in many species, but not in cats [49,50]. When ZP from pigs, cows, ferrets, dogs and mink was used, the cats developed high antibody titers to the xenogenic proteins, but these antibodies failed to cross-react with the cats native ZP. Immunization with antigens from feline zona pellucida evoked a poor immune response in the cats, possibly because feline ZP was recognized as a self protein. Hypothalamic-Pituitary-Gonadal Axis Seven adult female cats immunized against LH-R and given four booster treatments had absence of behavioral estrus for 500 days, at which point the cats showed signs of recovering normal ovarian function [51]. While immunization against LH-R has the potential to block the production of sex hormones, multiple treatments were used and the goal of a single treatment was not reached. However, it is possible that a variation on

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21 this method that extended the duration of immunity could lead to an effective single-treatment vaccine. In a previous study, six male cats immunized three to four times with GnRH conjugated to tetanus toxoid produced GnRH antibody titers, but there was no resulting decrease in testosterone concentrations below the contraceptive level [52]. In another study 10 female and four male cats were immunized against GnRH at eight weeks, boosted four weeks and 100 weeks later, and housed with a proven male. GnRH antibodies were detected in all 10 female cats. They had basal progesterone concentrations, did not display estrous behavior and failed to become pregnant for the entire observation period of 20 months. Three of the four males had castrate levels of testosterone for the entire duration of the study. A rise in testosterone in the 4 th male was associated with a decline in GnRH antibody titer [53]. Vaccine Delivery Vaccines can be delivered orally, by a biological vector, or by injection. Delivery of the vaccine through bait would be easier than the other methods, but dose is much more difficult to control, and consumption of the vaccine by non-target species would be a risk. The advantages of a biological vector for immunocontraception include the low expense for vectors that are self-disseminating; possibly low environmental impact compared to poisons; and it is considered humane [54]. Disadvantages include the risk to non-target species and the inherent risk associated with releasing a potential pathogen into the environment. As designed, the vector is not pathogenic, but there is always a chance for reversion to virulence or unexpected effects on non-target species. There is

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22 great concern that other species would be transfected by the vector, so this approach could not be used where other potentially vulnerable species are present. This limitation applies to almost any mainland area. Biological vectors may play a role in immunocontraception of cats on islands where the risk of the vector reaching the mainland is low. Injection requires the targeting of individual animals, which increases the effort required for control programs. Injectable vaccines are often delivered with a dart rifle for large animals [55], but it requires a skilled marksman and may not be practical or safe for cats due to their small size. TNR programs typically use live traps to capture cats for transport to veterinary clinics the cats to veterinarians for neutering. The same method of capture would allow delivery of the vaccine by intramuscular or subcutaneous injection in the field without need for transporting the cats to a central facility for surgical sterilization. An implant, which would allow timed-release of a vaccine, could also be delivered in this way.

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GNRH VACCINE Blocking GnRH from binding to the gonadotrophs disrupts the hormone cascade, leading to cessation of both fertility and sexual behavior. GnRH has the potential to satisfy many of the requirements of an ideal antigen for an immunocontraceptive vaccine. Since GnRH is a decapeptide, it is a hapten which is a very weak immunogen. The National Wildlife Research Center (NWRC) developed a GnRH vaccine that uses keyhole limpet hemocyanin (KLH) as a protein carrier and AdjuVac as an adjuvant. The GnRH peptides are attached to the KLH in such a way as to mimic molecular patterns found on many microorganisms; these patterns activate the innate immune system [56]. AdjuVac was developed as an alternative to Freunds complete adjuvant (FCA). FCA is a very effective adjuvant, but it can result in harm to the immunized animal including necrosis and development of granulomas. AdjuVac contains inactivated Mycobacterium avium in oil. In a previous pilot study in male cats, GnRH/KLH-AdjuVac resulted in testosterone concentrations below the contraceptive level following a single treatment in six of nine male cats for the duration of the 6-month observation period [57]. A single injection also prevented pregnancy in female feral pigs for 36 weeks [58] and was effective in female deer [55]. The hypothesis was that a single-dose GnRH vaccine, (GnRH/KLH-AdjuVac), would produce long-term immunocontraception in female cats. The specific objectives 23

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24 were to determine the proportion of cats that respond, the duration of the response, and the safety of the vaccine over a 24 month period.

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MATERIALS AND METHODS Cats Twenty-four 8to 14-month-old specific-pathogen-free female domestic shorthair cats were acquired from a commercial vendor (Liberty Research, Waverly, NY, USA). All 24 cats were group-housed in the Animal Care Services facilities at the University of Florida College of Veterinary Medicine, which are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Cat housing consisted of one large room with raised resting benches and was climate controlled to maintain ambient temperatures between 21 and 23C with controlled lighting. Food and water were provided ad libitum. The experimental design was approved by the UF Institutional Animal Care and Use Committee. All cats and their offspring underwent surgical sterilization and were adopted to private homes at the conclusion of the study. Vaccine Construction A GnRH vaccine was constructed using a synthetic GnRH peptide with the sequence [ pEHWSYGLRPG GGC-SH] produced by the Fmoc/tBU protection method (Global Peptide Services, Fort Collins, CO, USA). Immunogenicity was enhanced by coupling GnRH peptides to a protein carrier, keyhole limpet hemocyanin (KLH; Pierce Endogen, Rockford, IL, USA), in a 1:3 GnRH:KLH mass ratio. The underlined amino acids represent the native GnRH molecule and pE signifies pyro-glutamate. Two glycines were added at the C terminus as a spacer and a cysteine was added to ensure consistent 25

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26 alignment of the peptide to the maleimide-activated KLH. The aqueous-based GnRH-KLH conjugate (200 g) was combined in a 1:1 ratio by volume with a novel adjuvant, AdjuVac AdjuVac was produced by diluting a USDA-licensed Johnes disease vaccine containing inactivated Mycobacterium avium in mineral oil (Mycopar ; Fort Dodge Animal Health, Fort Dodge, IA, USA) Treatment Upon arrival, the 24 cats were housed for 30 days in a photoperiod regime (8 hours light:16 hours dark) that is inhibitory of estrus. Fifty days prior to treatment (Day -50), the photoperiod regime was reversed to 16 hours light:8 hours dark, which stimulates estrous cycling within 15 days in 85% of cats [59]. Serum hormones (progesterone and estradiol-17) were measured on Day -60, then every other day from Day -50 to Day -30. The magnitude, duration and rate of change of estradiol-17 concentrations in each cat from Day -50 to Day -30 were used to confirm normal estrous cycling in cats selected for this study. Confirmation of normal hormonal responses to the lighting change, including evidence of estrous cycling and docile temperament, were used to select 20 of the 24 cats to continue in the study. The 20 cats were randomized into a sham group (n = 5) and a treatment group (n = 15) based on maximum estradiol-17 concentrations. The sham group received placebo vaccines containing all components except GnRH-KLH. The 15 treated cats received vaccines containing 200 g GnRH-KLH. Brief anesthesia was induced by administration of isoflurane (IsoFlo ; Abbott Laboratories, North Chicago, IL, USA) by face mask. The hair of the right cranial thigh was clipped, and the injection site was cleaned with 70% isopropyl alcohol. The vaccine (0.5 mL) was injected into the

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27 quadriceps muscle group. The right pinna was tattooed with a treatment code, specifically, C1 through C5 for the sham cats and T1 through T15 for the treatment cats. Potential adverse reactions to treatment were evaluated by daily physical examination, including inspection of the injection site and measurement of body temperature for one week following treatment. Blood Collection Blood (4 mL) was collected by jugular venipuncture monthly into serum separator tubes for determination of GnRH antibody titer and concentration of estradiol-17 and progesterone. In addition, blood for estradiol-17 and progesterone concentration determination was collected every other day for 20 days following each of four estrus-inducing photoperiod changes. Serum was separated by centrifugation and stored at 20 C until analysis. Detection of GnRH Antibodies Serum was tested for GnRH antibodies using an enzyme-linked immunoabsorbant assay (ELISA). Bovine serum albumen (BSA) was coupled to GnRH in a 1:1 GnRH:BSA mass ratio and 200 ng of GnRH-BSA buffered with 50 l bicarbonate was used to coat the wells in each 96-well microtiter plate and incubated overnight at 4 o C. GnRH-BSA was used so that only anti-GnRH antibodies would be detected, not antibodies against the KLH component of the vaccine. The wells were washed twice with 200 l of phosphate buffered saline (PBS) and 0.05% Tween 20 (Sigma Chemical Co., St. Louis, MO, USA), blocked with salmon serum (SeaBlock, East Coast Bio, Inc., North Berwick, ME, USA) overnight at 4 o C. The wells were aspirated and 100 l of cat serum diluted 1:1,000 in PBS was added in the top row of the plate, with each column

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28 containing a sample from a different cat. Two negative controls were run on each plate; one negative control was buffer without cat serum and the other was pre-vaccination cat serum. The last column was used as a positive control with serum from a cat with a known high antibody titer. All remaining wells were filled with 50 l PBS and serial dilutions were made from 1:1,000 to 1:128,000 by taking 50 l from each well in row 1 and mixing it into the corresponding well in row two. This process was repeated for the remaining six rows. The plates were then incubated for two hours at room temperature on a shaker. The wells were washed twice with 200 l PBS/0.05% Tween 20. Antibody to GnRH was detected by adding 50 l goat anti-cat IgG (Sigma Chemical Co.) diluted 1:10,000 in PBS/1% Sea Block to each well. The plate was incubated at room temperature on a shaker for 2 hours. The wells were washed twice with 200 l PBS/0.05% Tween 20. Fifty microliters of rabbit anti-goat coupled to horseradish peroxidase conjugate (Sigma Chemical Co.) diluted 1:3,000 in PBS/1% Sea Block was added to each well and incubated for two hours at room temperature on a shaker. The wells were washed three times with 200 l PBS/0.05% Tween 20. Fifty microliters of Tetramethylbenzidine/phosphate-citrate buffer (Sigma Chemical Co.) was added to each well and the plate was incubated for three to five minutes or until a blue color change developed. Fifty microliters of 2M sulfuric acid was added to each well to stop the reaction and the plates were read on a plate reader (Dynatech, Horsham, PA) at 450 nm. The endpoint dilution was considered positive based on the positive control with the titer equal to the reciprocal of the endpoint dilution.

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29 Determination of Serum Estradiol-17 and Progesterone Concentrations In cats, behavioral estrus is preceded by a surge in estradiol-17 released by developing follicles. After ovulation progesterone concentrations rise significantly within four days [60]. Serum was tested monthly for estradiol-17 and progesterone concentration. In addition, blood was collected every other day for 20 days following each of four estrus-inducing photoperiod changes. Serum samples were analyzed for total estradiol-17 and progesterone by radio-immunoassay (Coat-A-Count ; Diagnostic Products Corporation, Los Angeles, CA, USA) according to the manufacturers instructions. The manufacturer reports an estradiol-17 sensitivity of 8 pg/mL with within-run coefficient of variation (CV) of 4% and between-run CV of 4%, depending on the estradiol-17 concentration. The manufacturer reports a progesterone sensitivity of 0.02 ng/mL with within-run CV of 2% and between-run CV of 4%, depending on the progesterone concentration. Breeding Trial Estrus Induction The effects of treatment on normal hormone responses and fertility were evaluated beginning four months after treatment and continuing for the duration of the 26-month study. The photoperiod length was alternated between the estrus-inhibiting regime (16 hours dark:8 hours light) and the estrus-inducing regime (8 hours dark:16 hours light). From Day 0 to Day 120, Day 330 to Day 360 and Day 510 to Day 540, the cats were exposed to the estrus-inhibiting regime (26% of post-treatment time). For the remainder of the study the cats were exposed to estrus-inducing light regime (74% of post-treatment time) (Figure 1). The switch from the short-day photoperiod to the long-day photoperiod

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30 was intended to induce estrus in any of the cats that were capable of an estrous response. A breeding male was housed continuously with the females during all of the long-day photoperiods after Day 120. Three breeding males were alternated to assure that inter-cat incompatibility was not an issue. Time-lapse videography was used to monitor breeding activity during periods of long-day photoperiod. The tapes were reviewed daily and the number of attempted and successful breedings was recorded. Distinctive shaving of the cats fur was used to allow differentiation of similar-looking cats. Assignment of Cats to Study Group During the pre-treatment long-day photoperiod (Day -50 to Day -30), 19 of 24 cats (79%) had a pattern of estradiolconcentrations consistent with estrus based on the peak concentrations and the rate of change of the concentrations. These 19 cats and one cat that did not display estrus were divided among the sham and treatment groups in such a way that the two groups did not have significantly different maximum estradiol-17 concentrations (P = 0.4). Fertility and Fecundity Fertility was defined by the proportion of cats becoming pregnant. Fecundity was defined as the number of live births. Pregnancy was scored as a treatment failure, and the cats were removed from the study after parturition. The treated cats were divided into responders, which failed to become pregnant during the 19 months following treatment, and nonresponders, which became pregnant during the study. Body Phenotype It has been well-documented that cats gain weight after surgical neutering, but it is unknown if immunocontraception has the same effect. To evaluate for this effect, body

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31 weight was recorded for each cat on Day 0 and Day 420, and percent weight gain was calculated. Radiographic measurement of the falciform fat pad was performed 14 months (Day 420) after treatment for further evaluation of phenotypic changes. For fat pad measurement, the cats were anesthetized with medetomidine 40 mcg/kg IM and radiographed in right lateral recumbancy. Using computed radiography (Kodak, Rochester, New York, USA), the depth (mm) of the falciform fat pad was measured by dropping a perpendicular line from the center of the body of the 12 th thoracic vertebra to the ventral body wall and measuring the distance between the caudoventral angle of the liver and the ventral body wall. The area (mm 2 ) of the fat pad was defined as the area outlined by the line used for the depth measurement, the ventral border of the liver, the diaphragm, and the ventral body wall (Figure 2). Statistical Analysis Descriptive statistics (mean, error, range) were calculated for each group for hormone concentrations, GnRH antibody titers, fecundity and body weight. The fertility of the sham and treatment groups was compared by the two-sided Wilcoxon rank sum test. Fecundity of the sham and nonresponder groups was compared by one-way analysis of variance. Differences in GnRH antibody titer and hormone concentrations between the groups were tested by Kruskal-Wallis one-way analysis of variance on ranks (Kruskal-Wallis ANOVA). Differences between the three groups (responder, nonresponder and sham) with regard to body weight, percent change of body weight, falciform fat pad depth, and falciform fat pad area were analyzed using Kruskal-Wallis ANOVA. The Holm-Sidak method for pairwise comparison was used for all of the Kruskal-Wallis ANOVA tests that indicated a difference between at least two of the groups. Differences

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32 were considered significant when P < 0.05. All tests were performed using SigmaStat statistics software, version 3.0.1 (SPSS Inc., Chicago, IL, USA). -6060180300420540660Days post-treatment Long-day lighting Short-day lighting Breeding period Blood collection Immunization Radiography Body weight Figure 1. Timeline for 26-month study showing alternating periods of long-day (16 hr light:8 hr dark) and short-day (8 hr light:16 hr dark); periods during which the breeding male had access to the females (Breeding trial); blood collection times; the treatment (Day 0); falciform fat pad radiograph; and body weight times.

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33 Figure 2. Lateral abdominal radiograph of a cat with falciform fat pad measurements marked. The vertical line extends from the center of the body of T12 to the ventral abdominal wall with the solid portion of the line measuring the fat pad depth. The three solid lines demarcate the area for the falciform fat pad area measurement.

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RESULTS Reactions to Treatment Body temperature in all of the cats remained normal after treatment, and there was no inflammation or tenderness at the injection sites. One treatment cat, (T4), died suddenly 45 days after treatment; necropsy revealed no gross or histological abnormalities to explain the death. This cat was replaced with a two-year old cat of proven fertility from the research colony which was tattooed with the code T16 and vaccinated with a vaccine containing GnRH. This replacement cat was exposed to the same lighting regimen and blood collection schedule as the other 19 cats. Twenty-four months after immunization a 3 cm x 4 cm mass was discovered by palpation at the injection site. A biopsy was performed and the mass was found to be neither cancerous or infected. It was concluded that the mass was a granuloma that had formed in response to the adjuvant. One cat had multiple 1 cm masses near the injection site and three other cats had a single 1 cm mass near the injection site. Breeding Trial Detection of Estrus The breeding male cat was introduced on Day 120 at the beginning of the second long-day regime. All sham cats displayed behavioral signs of estrus and were receptive to breeding attempts by the male. Time-lapse videography revealed that the male bred each of the sham cats at least 15 times between Day 122 and Day 155. One cat was observed breeding in two intervals during gestation (days 7-11, 25-26 of gestation). Four treated 34

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35 cats displayed estrous behavior and began breeding on Day 128 (T9), Day 418 (T8), Day 469 (T15), and Day 504 (T5). These four nonresponder cats were the treated cats with the lowest GnRH antibody titers. Fertility and Fecundity Based on an average gestation period of 65 days, the date of parturition was used to estimate the date of successful breeding. All five sham cats became pregnant between five and 26 days after the male cat was introduced (Day 125 to Day 146) and four of them within six days of each cats first observed breeding. Nonresponder cat T9 was bred in three different estrous intervals from Day 129 to Day 162 before becoming pregnant. In contrast, nonresponder cats T8, T15, and T5 became pregnant in their first breeding cycles (Day 418, Day 469, and Day 504, respectively) (Figure 3). The mean number of live kittens per litter was 3.8 (range 3 to 5) in the sham group, which was significantly higher (P = 0.03) than the mean of 2.3 (range 1 to 3) live kittens in the nonresponder group. Based on maintenance of contraception in 11 of 15 treated cats over a 24-month period, the vaccine had a 73% success rate. GnRH Antibody Titer The mean GnRH antibody titer for the responder group was significantly higher than the titer of the nonresponder group by Day 150 and remained significantly higher for most of the remainder of the study (Figure 4). All of the sham cats had negative titers throughout the study. Three patterns of antibody response were observed among the treated cats: high titers, variable titers, and low titers. Five of the 11 responder cats (T1, T7, T10, T14, and T16) had GnRH antibody titers of 128,000 by Day 30 and maintained a titer of 64,000 or

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36 above for the remaining 20 months of the study (Figure 5). The antibody titers of five other responders (T2, T6, T11, T12, and T13) peaked, then decreased and subsequently increased again. None of these variable titers decreased below 16,000, and after a trough level of three to six months, all the titers increased again to at least 64,000 by Day 540 (Figure 6). All four nonresponders became pregnant after their GnRH antibody titer decreased to 8,000 or below (Figure 7). For the last seven months of the study, 10 of 11 responders had a GnRH titer of 64,000 or above. The responder cat with the lowest GnRH antibody titer, T3, remained at a titer of 16,000 for the last 13 months of the study. Two of the nonresponder cats had an initial high titer that was not sustained and two had titers at 32,000 or below throughout the study. These four cats became pregnant when titers decreased below 16,000, which suggests that a titer of 16,000 is contraceptive in female cats. Poor antibody responses in the nonresponder group delayed, but did not prevent pregnancy. Hormone Concentrations Progesterone In cats, ovulation accompanied by fertilization results in pregnancy, whereas ovulation without fertilization results in pseudopregnancy. In both cases, serum progesterone increases from a baseline of < 0.5 ng/mL to a peak above 20 ng/mL between 16 and 26 days after ovulation [41]. In pregnancy, progesterone concentrations are higher than in pseudopregnancy and remain above baseline for the entire gestation period. In pseudopregnancy, progesterone concentrations return to baseline by 50 days. The nine cats (five sham, four nonresponder) that became pregnant had substantial increases in progesterone concentrations for two months prior to parturition. This finding

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37 was expected given the average gestation period of 65 days. Prior to the introduction of the male (Day 120), two of the sham cats, two of the responders, and two of the nonresponders had serum progesterone concentrations higher than 2.0 ng/mL, indicating ovulation. T9, the first nonresponder to become pregnant, had progesterone concentrations indicative of pseudopregnancy on Day -30 and Day 60. A second nonresponder, T5, had elevated progesterone concentration on Day 30. T1 and T2, both responders, had progesterone concentrations of 7 and 20 ng/mL respectively, on Day 30. It is likely that these cats experienced nonfertile ovulation and pseudopregnancy prior to the development of contraceptive titers of GnRH antibody. After Day 30, none of the responder cats had evidence of ovulation. Estradiol-17 It was expected that the sham groups estradiol-17 concentrations would be consistent with estrous cycling until they became pregnant. Four of the five control cats became pregnant during the first sustained estradiol-17 elevation (the first estrous cycle) following the introduction of the breeding male and one cat, C2, became pregnant during the second estrous cycle. Body Condition Body Weight The mean weights of the sham, nonresponder, and responder cats on Day 0 were not significantly different from each other (P = 0.5). Similar to cats undergoing surgical ovariohysterectomy, responding cats had a higher percent gain in body weight than the nonresponder cats (P = 0.004) and sham cats (P = 0.02) (Table 1). The percent weight gain was not significantly different between the sham and nonresponder groups (P =

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38 0.08). The mean weight on Day 420 of the responder group was significantly greater than the nonresponder group (P = 0.002), but not significantly greater than the sham group (P = 0.1) (Table 1). The mean weights of the sham and nonresponder groups on Day 420 were not significantly different (P = 0.1). Falciform Fat Pad At 14 months post-treatment, the mean falciform fat pad depth for the responder group was greater than the mean for the nonresponder group (P = 0.046) and sham group (P = 0.03) (Table 2). The mean falciform fat pad areas of the three groups were not significantly different from each other (P = 0.1) (Table 2).

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39 0%25%50%75%100%120270420570720Days post-treatmentProportion of cats pregnant Sham GnRH Figure 3. Breeding trial. Change to a long day-length cycle (Day 120) resulted in the rapid induction of behavioral estrus and breeding in all five sham cats, whereas only one of 15 immunized cats was observed to be breeding. All six cats that bred shortly after Day 120 became pregnant. During the same period three additional immunized cats displayed behavioral estrus and became pregnant.

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40 0408012016020030130230330430530630Days post-treatmentGnRH antibody titer (x1000) Sham (n=5) Non-responder (n=4) Responder (n=11) Figure 4. GnRH antibody titer for female cats (mean + SE). The sham group had baseline titer for the duration of the study. For nine of the 18 post-treatment time points the responder cats (cats that did not become pregnant during the study) had significantly higher GnRH antibody titers than the nonresponder cats. (*P<0.05.) None of the responder cats had antibody titers that decreased below 16,000 and all four nonresponder cats (cats that became pregnant during the study) decreased below 16,000 and became pregnant within 100 days. The contraceptive titer for these cats ( ) was 16,000.

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41 05010015020025030030130230330430530630Days post-treatmentGnRH antibody titer (x1000) T1 T7 T10 T14 T16 Figure 5. GnRH antibody titers for cats with high titers from Day 30 through Day 630. These five responders (cats that did not become pregnant during the study) (T1, T7, T10, T14, T16) had GnRH antibody titers of 128,000 by Day 30 and none decreased below a titer of 64,000 for the entire 24-month treatment period, which was above the contraceptive titer of 16,000 ( ). 05010015020025030030130230330430530630Days post-treatmentGnRH antibody titer (x1000) T2 T6 T11 T12 T13 Figure 6. GnRH antibody titers for cats with variable titers. These five responders (cats that did not become pregnant during the study) (T2, T6, T11, T12, T13) had titers that peaked, decreased, and then increased again. After the second increase all five cats maintained this new titer through Day 630. The titers for these cats did not decrease below the contraceptive titer ( ) of 16,000.

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42 030609012015030130230330430530630Days post-treatmentGnRH antibody titer (x1000) T5 T8 T9 T15 Figure 7. GnRH antibody titers of nonresponders (treated cats that became pregnant during the study). Cats became pregnant on Day 569 (T5), Day 483 (T8), Day 229 (T9), and Day 534 (T15). The titer of these cats decreased below the contraceptive titer ( ) of 16,000. Table 1. Body weight and percent weight gain in cats over a 14-month period following GnRH immunocontraception. The mean weights of the three groups were not significantly different from each other on Day 0. The percent weight gain of the responder group was significantly greater than the nonresponder group and sham group. Group Weight (kg), mean (range) % Weight gain, mean (range) Day 0 (Treatment Day) Day 420 Day 0 to Day 420 Sham 3.54 (3.30 4.18) 4.05 (3.48 4.66) 15 ( 1 41) Nonresponder 3.21 (2.77 3.50) 3.31 (2.62 4.00) 3.5 (-14 31) Responder 3.34 (2.64 4.20) 4.63 (3.70 5.98) 40 (12 73) Table 2. Falciform fat pad depth and area in cats 14 months after GnRH immunization. Fat pad depth of the responder group was significantly greater than the nonresponder group and sham group. The falciform fat pad areas were not significantly different between the groups. Group Fat Pad Depth (mm) mean (range) Fat Pad Area (mm 2 ) mean (range) Sham 18.6 (13.2 23.8) 728 (367 1210) Nonresponder 18.6 (15.2 22.4) 839 (543 1270) Responder 27.8 (17.2 42.9) 1194 (489 2020)

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DISCUSSION In this study, 15 sexually intact female cats were immunized once against GnRH. Twenty-four months after immunization it was discovered that five of the treated cats had granulomas near their injection site. There was no lameness evident and the masses did not appear to cause the cats any discomfort. It is possible that a lower dose of vaccine would still be effective, but not cause this type of reaction at the injection site. No systemic or local adverse reactions were noted during the 24-month observation period. For the 24 months following treatment, 73% of the treated cats did not become pregnant or display signs of behavioral estrus. The four nonresponders had GnRH antibody titers at or below 8,000 when they became pregnant, while none of responders had a titer below 16,000 during the study. This suggests that the contraceptive titer in female cats is 16,000. As expected, the responder cats reacted to the immunocontraceptive treatment in ways similar to cats that have undergone surgical sterilization. Behavioral estrus was suppressed and they gained more weight than the sham and nonresponder cats. However, surgically sterilized female cats have estradiol-17 plasma concentrations consistent with cats in anestrous, whereas the responder cats intermittently demonstrated estradiol-17 concentrations well above expected anestrous values and possibly indicative of estrus cycling. However, the females were never receptive nor attractive to the male, so if the cats were cycling, they were not cycling normally. 43

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44 Many studies have demonstrated weight gain following surgical sterilization [61-63] and the percent weight gain in these studies (28% to 39%) was consistent with the 40% weight gain found in the responder cats in the present study. The number of live kittens per litter born to the nonresponder cats was significantly less than the number for sham cats. Immunization against GnRH may not prevent pregnancy in all cats, but it might interfere with some aspect of pregnancy that results in lower fecundity. One possibility is that decreased LH, FSH, and estradiol-17 led to fewer follicles developing and fewer ovulated follicles. However, the sample size was very low and the difference found may have been due to normal variation. In a study of nine male cats immunized with the same vaccine used in the current study (50 to 200 g), six cats had a GnRH antibody titer above 64,000, undetectable testosterone concentrations and testicular atrophy. The three cats with an antibody titer below 64,000 had measurable testosterone concentrations and normal semen quantity [57]. In a study that targeted a hypothalamic-pituitary-gonadal axis component downstream from GnRH, seven adult female cats were immunized with lutenizing hormone receptor (LH-R) encapsulated in an implant and given four booster injections. LH-R antibodies were detected, progesterone concentrations remained near basal levels, and no behavioral estrus was observed for approximately 500 days, at which point antibody titers began to decrease and the cats showed signs of recovering normal physiologic ovarian function. Estradiolconcentrations were not significantly different between the sham cats and treated cats, even though estrus was observed in the sham cats, but not in the treated cats

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45 [51]. Determining hormonal estrous cycling based on estradiol-17 concentrations requires estrogen be measured very frequently over the time frame of interest. In the current study, there were four clusters of 11 every other day blood collection over a 26-month span. The four clusters of blood collections, each 20 days long, were likely to reveal an estrous cycle if one occurred during one of those intervals. However, it is possible that an estrous cycle occurred, but was missed, during the twoto seven-month intervals between these clustered blood collections. In addition, the basal levels and estrus profile of estradiol-17 concentrations are highly variable among cats and other components such as plasma protein content and steroid hormone binding protein may interfere with the measurement [64]. Increasing the frequency of estrogen measurement would aid in more precise determination of estrous cycling regardless of basal and maximum concentrations. However, continuous collection of blood samples would create welfare and health issues for cats and monitoring of fecal estrogen would necessitate individual housing. To increase the accuracy of the estradiol-17 assay, a portion of each sample can be used to prepare an estradiol-17-free aliquot. The steroid is removed by passing the aliquot through a charcoal column. The estradiolconcentration in each sample is determined by subtracting the value in the aliquot (sample blank) from the estradiolconcentration value in the unfiltered portion [64] Immunization against GnRH in other species has been used as an alternative to gonadectomy to prevent undesirable behavior or characteristics caused by sex hormones for more than 25 years [65]. It has been primarily used in juvenile male food animals and has resulted in decreased aggression, testosterone concentration, and testes size in bulls

PAGE 54

46 [66-70], boars [58,71-73] and rams [74,75]. In female pigs [58,76], sheep [77] and horses [78] estrus was suppressed; concentrations of lutenizing hormone, progesterone, and inhibin A were decreased; and ovarian and uterine weights were reduced. Most of these animals received multiple treatments, and the observation periods were short since most of the animals were sent to slaughter. GnRH immunization has been proposed as a humane solution to overpopulation of wildlife species and has been tested in deer, bison, rats, swine, rabbits, squirrels, coyotes and horses [79]. In white-tailed deer, three or four treatments of GnRH vaccine resulted in a fawning rate reduction up to 88% [55]. GnRH vaccination was 100% effective with three treatments in male and female Norway rats for up to 17 months [55]. A single treatment in male and female feral swine resulted in reduced testicular and ovarian sizes, reduced concentrations of testosterone and progesterone, and a 90% reduction in pregnancy for 36 weeks [58]. A single treatment in bison led to a decrease in progesterone concentrations and prevented pregnancies for one year [80]. There is a protein very similar to GnRH, named GnRH-II, whose function has yet to be elucidated. However, there is concern that a vaccine against GnRH could also bind to GnRH-II and disrupt a non-reproduction function [81]. The safety of the GnRH vaccine has been widely investigated, though, and no significant safety concerns have been found. A study in male rats and rabbits found no differences in hematological or biochemical findings between GnRH-immunized animals and surgically sterilized animals [82]. At necropsy the only abnormalities were detected in the reproductive organs. Studies in many species including horses [78], deer [83], and male cats [57], found no health concerns associated with GnRH immunocontraception.

PAGE 55

47 Contraceptive GnRH antibody titer and effective dose may be different for male and female animals. The GnRH antibody titer needed to induce contraception in the current study in female cats (16,000) was lower than the antibody titer needed for male cats (64,000) treated with the same vaccine [57]. A similar sex difference has been noted in horses with a contraceptive titer in males of 1,000 and in females of 300 [78]. The effective dose of GnRH vaccine was found to be lower in male than female swine [58]. Sex differences may be related to the cyclic nature of hormone secretion in females that is largely absent in males [58]. Since males continuously produce GnRH, it is possible that the store of anti-GnRH antibodies is depleted more quickly at the sites of interaction or that GnRH must be suppressed to a greater degree to block reproductive functions in males than in females. GnRH has been shown previously to be an effective immunocontraceptive target in many species, including cats, but in reports the contraceptive activity was not effective in all of the animals, required multiple treatments, and faded over time. In contrast, a practical contraceptive for feral cats must be effective in a large fraction of cats for a substantial duration following a single treatment. In this report, a single treatment of GnRH conjugated with KLH and adjuvanted with M. avium and oil achieved long-term contraception in female cats, meeting the minimum requirements for contraception in feral cats. Targeting of GnRH had the additional benefit of curbing nuisance behavior associated with estrous cycling. Additional studies are needed to investigate the full duration of immunity, rate of efficacy, and safety in both sexes and all ages of cats.

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REFERENCE LIST [1] APPMAs 2005/2006 National Pet Owners Survey. American Pet Products Manufacturing Association. Available at http://www.appma.org/press_industrytrends.asp. Accessed May 1, 2006. [2] Levy JK, Gale DW, Gale LA. Evaluation of the effect of a long-term trap-neuter-return and adoption program on a free-roaming cat population. J Am Vet Med Assoc 2003;222:42-46. [3] Levy JK, Crawford PC. Humane strategies for controlling feral cat populations. J Am Vet Med Assoc 2004;225:1354. [4] Barrows, PL. Professional, ethical, and legal dilemmas of trap-neuter-release. J Am Vet Med Assoc 2004;225:1365-1369. [5] Jessup DA. The welfare of feral cats and wildlife. J Am Vet Med Assoc 2004;225:1377. [6] Luria BJ, Levy JK, Lappin MR, Breitschwerdt EB, Legendre AM, Hernandez JA, et al. Prevalence of infectious diseases in feral cats in northern Florida. J of Feline Med and Surgery 2004;6:287-296. [7] Hanlon CA, Childs JE, Nettles VF. Recommendations of the working group on rabies. Article III: Rabies in wildlife. J Am Vet Med Assoc 1999;215:1612-1618. [8] Brown RR, Elston TH, Evans L, Glaser C, Gulledge ML, Lappin MR, Marcus LC. American association of feline practitioners 2003 report on feline zoonoses. J Feline Med Surg 2005; 7:243-274. [9] Noah DL, Drenzek CL, Smith JS, Krebs JW, Orciari L, Shaddock J, Sanderlin D, Whitfield S, Fekadu M, Olson JG, Rupprecht CE, Childs JE. Epidemiology of human rabies in the United States, 1980 to 1996. Ann Intern Med. 1998;128:922-930. [10] CDC. Human RabiesFlorida, 2004. MMWR Morb Mortal Wkly Rep 2005; 54:767-769. 48

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49 [11] Nutter FB, Dubey JP, Levine J F, Breitschwerdt EB, Ford RB, Stoskopf MK. Seroprevalences of antibodies against Bartonella henselae and Toxoplasma gondii and fecal shedding of Cryptosporidium spp, Giardia spp, and Toxocara cati in feral and pet domestic cats. J Am Vet Med Assoc 2004;225:1394-1398. [12] Greene CE, Levy JK. Chapter 99: Immunocompromised People and Shared Human and Animal Infections: Zoonoses, Sapronoses, and Anthroponoses. In: Infectious Diseases of the Dog and Cat, Greene CE (Ed.), St. Louis, Missouri: Elsevier B.V., 2006, pp. 1051-1068. [13] Nutter FB, Levine JF, Stoskopf MK. Reproductive capacity of free-roaming domestic cats and kitten survival rate. J Am Vet Med Assoc 2004; 225:1399-1402. [14] Scott KC, Levy JK, Crawford PC. Characteristics of free-roaming cats evaluated in a trap-neuter-return program. J Am Vet Med Assoc 2002;221:1136-1138. [15] Levy JK, Scott HM, Lachtara JL, Crawford PC. Seroprevalence of feline leukemia virus and feline immunodeficiency virus infection among cats in North America and risk factors for seropositivity. J Am Vet Med Assoc 2006;228:371-376. [16] Lee IT, Levy JK, Gorman SP, Crawford PC, Slater MR. Prevalence of feline leukemia virus infection and serum antibodies against feline immunodeficiency virus in unowned free-roaming cats. J Am Vet Med Assoc 2002;220:620. [17] Wallace JL, Levy JK. Population characteristics of feral cats admitted to seven trap-neuter-return programs in the United States. J of Feline Med and Surgery (2006). [18] Courchamp F, Langlais M, Sugihara G. Cats protecting birds: modeling the mesopredator release effect. J of Animal Ecology 1999;68:282-292. [19] Birdlife International 2004: Traversia lyalli. In IUCN 2004. 2004 IUCN Red List of Threatened Species. http://www.redlist.org. Accessed May 1, 2006. [20] Bester MN, Bloomer JP, van Aarde RJ, Erasmus BH, van Rensburg PJJ, Skinner JD, Howell PG, Naude TW. A review of the successful eradication of feral cats from sub-Antarctic Marion Island, southern Indian Ocean. South African J of Wildlife Research 2002;32: 65-73. [21] Veitch CR. The eradication of feral cats (Felis catus) from Little Barrier Island, New Zealand. New Zealand J of Zoology 2001;28:1-12.

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50 [22] Fan M, Yang K, Feng Z. Cats protecting birds revisited. Bulletin of Mathematical Biology 2005;67:1081. [23] Barratt DG. Predation by House cats, Felis catus (L.), in Canberra, Australia. I. Prey composition preference. Wildlife Research 1997;24:263-277. [24] Miller MA, Gardner IA, Kreuder C, Paradies DM, Worcester KR, Jessup DA, Dodd E, Harris MD, Amesf JA, Packham AE, Conrad PA. Coastal freshwater runoff is a risk factor for Toxoplasma gondii infection of southern sea otters (Enhydra lutris nereis). Int J Parasitol 2002;32:997-1006. [25] Tenter AM, Heckenroth AR, Weiss LW. Toxoplasma gondii: from animals to humans. Int J Parasitol 2000;30:1217. [26] Hughes KL, Slater MR, Haller L. The effects of implementing a feral cat spay/neuter program in a Florida county animal control service. J Appl Anim Welf Sci. 2002;5:285-298. [27] Girardet SAB, Veitch CR, Craig JL. Bird and rat numbers on Little Barrier Island, New Zealand, over the period of cat eradication 1976-1980. New Zealand J of Zoology 2001;28:13-29. [28] Nogales M, Martin A, Tershy B, Donlan C, Veitch D, Puerta N, Wood B, Alonso J. A review of feral cat eradication on islands. Conservation Biology 2004;18:310-319. [29] Eason, C. Sodium monofluoroacetate (1080) risk assessment and risk communication. Toxicology 2002;181-182:523-530. [30] Eisler, R. Sodium monofluoroacetate (1080) hazards to fish, wildlife, and invertebrates: synoptic review. U.S. Department of the Interior National Biological Service Biological Report 1995:27, 47. [31] Nagesha, HS, Wang LF, Hyatt AD. Virus-like particles of calicivirus as epitope carriers. Archives of Virology 1999;144:2429-2439. [32] Wenstrup J, Dowidchuk A. Pet overpopulation: data and measurement issues in shelters. J Appl Anim Welf Sci 1999;2:303-319. [33] Lord LK, Wittum TE, Ferketich AK, Funk JA, Rajala-Schultz P, Kauffman RM. Demographic trends for animal care and control agencies in Ohio from 1996 to 2004. J Am Vet Med Assoc 2006;229:48-54. [34] Foley P, Foley JE, Levy JK, Paik T. Analysis of the impact of trap-neuter-return programs on populations of feral cats. J Am Vet Med Assoc 2005;227:1775-1781.

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51 [35] Berkeley EP. TNR Past Present and Future, a History of the Trap-Neuter-Return Movement. Washington, DC: Alley Cat Allies, 2004, p 30. [36] Castillo D, Clarke AL. Trap/neuter/release methods ineffective in controlling domestic cat colonies on public lands. Nat Areas J 2003;23:247-253. [37] Andersen M, Martin BJ, Roemer GW. Use of matrix population models to estimate the efficacy of euthanasia versus trap-neuter-return for management of free-roaming cats. J Am Vet Med Assoc 2004;225:1871-1876. [38] Leyva H, Madley T, Stabenfeldt GH. Effect of light manipulation on ovarian activity and melatonin and prolactin secretion in the domestic cat. J Reprod Fertil Suppl 1989;39:125-33. [39] Bakker J, Baum MJ. Neuroendocrine regulation of GnRH release in induced ovulators. Front Neuroendocrinol 2000;21:220-62. [40] Johnson LM, Gay VL. Luteinizing hormone in the cat, mating-induced secretion. Endocrinology 1981;109:247-252. [41] Feldman EC, Nelson, RW, eds. Canine and Feline Endocrinology and Reproduction. 3rd ed. St. Louis, Missouri: WB Saunders Co, 2004;10221024. [42] Martin LJ, Siliart B, Dumon HJ, Nguyen P. Spontaneous hormonal variations in male cats following gonadectomy. J Feline Med Surg 2006. [43] Fagerstone, KA, Coffey MA, Curtis PD, Dolbeer RA, Killian GJ, Miller LA, Wilmot LM. Wildlife fertility control. Wildl Soc Tech Rev 2002;02-2:29. [44] Munson L. Contraception in felids. Theriogenology 2006;66:126-134. [45] Munson, L. Bauman JE, Asa C, Jochle M, Trigg TE. Efficacy of the GnRH analogue deslorelin for suppression of oestrus cycle in cats. J Reprod Fertil Suppl 2001;57:269-273. [46] Sabeur K, Ball BA, Nett TM, Ball HH, Liu IK. Effect of GnRH conjugated to pokeweed antiviral protein on reproductive function in adult male dogs. Reproduction 2003;125:801-806. [47] Hoyer PB, Devine PJ, Hu X, Thompson KE, Sipes IG. Ovarian toxicity of 4-vinylcyclohexene diepoxide: a mechanistic model. Toxicol Pathol 2001;29:91-99. [48] Adiga PR, Subramanian S, Rao J, Kumar M. Prospects of riboflavin carrier protein (RCP) as an antifertility vaccine in male and female mammals. Hum Reprod Update 1997;3:325-334.

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52 [49] Levy JK, Mansour M, Crawford PC, Pohajdak B, Brown RG. Survey of zona pellucida antigens for immunocontraception of cats. Theriogenology 2005;63:1334-1341. [50] Gorman SP, Levy JK, Hampton AL, Collante WR, Harris AL, Brown RG. Evaluation of a porcine zona pellucida vaccine for the immunocontraception of domestic kittens (Felis catus). Theriogenology. 2002;58:135-149. [51] Saxena BB, Clavio A, Singh M, Rathnam P, Bukharovich EY, Reimers Jr TJ, Saxena A, Perkins S. Effect of immunization with bovine luteinizing hormone receptor on ovarian function in cats. Am J Vet Res 2003;64:292. [52] Ladd A, Tsong YY, Walfield AM, Thau R. Development of an antifertility vaccine for pets based on active immunization against luteinizing hormone-releasing hormone. Biol Reprod 1994;51:1076. [53] Robbins SC, Jelinski MD, Stotish RL. Assessment of the immunological and biological efficacy of two different doses of a recombinant GnRH vaccine in domestic male and female cats (Felis catus). J Reprod Immunol 2004;64:107-119. [54] Courchamp, Franck, S. Cornell. Virus-vectored immunocontraception to control feral cats on islands: a mathematical model. J of Applied Ecology 2000;37:903-913. [55] Curtis PD, Pooler RL, Richmond ME, Miller LA, Mattfeld GF, Quimby FW. Comparative effects of GnRH and porcine zona pellucida (PZP) immunocontraceptive vaccines for controlling reproduction in white-tailed deer (Odocoileus virginianus). Reprod Suppl. 2002;60:131-141. [56] Abbas, Abul, A. Lichtman, J. Pober. Cellular and Molecular Immunology, Fourth Edition. St. Louis, Missouri: W.B. Saunders Company 2000. [57] Levy, JK, LA Miller, PC Crawford, JW Ritchey, MK Ross, KA Fagerstone. GnRH immunocontraception of male cats. Theriogenology 2004;62:1116-1130. [58] Killian, G, Miller L, Rhyan J, Dees T, Perry D, Doten H. Evaluation of GnRH contraceptive vaccine in captive feral swine in Florida. Am J Reprod Immunol 2006;55:378-84. [59] Michel, Chantal. Induction of oestrus in cats by photoperiod manipulation and social stimuli. Laboratory Animals 1993;27:278-280. [60] Wildt, DE, Chan SYW, Seager SWJ, Chakraborty PK. Ovarian activity, circulating hormones, and sexual behavior in the cat. I. Relationships during the coitus-induced luteal phase and the estrous period without mating. Biology of Reproduction 1981;25:15.

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53 [61] Kanchuk ML, Backus RC, Calvert CC, Morris JG, Rogers QR. Weight gain in gonadectomized normal and lipoprotein lipasedeficient male domestic cats results from increased food intake and not decreased energy expenditure. J Nutri 2003;133:1866-1874. [62] Fettman MJ, Stanton CA, Banks LL, Hamar DW, Johnson DE, Hegstad RL, Johnston S. Effects of neutering on body weight, metabolic rate and glucose tolerance of domestic cats. Res Vet Sci 1997;62:131-136. [63] Harper EJ, Stack DM, Watson TD, Moxham G. Effects of feeding regimens on bodyweight, composition and condition score in cats following ovariohysterectomy. J Small Animal Prac 2001;42:433-438 [64] Onclin K, Murphy B, Verstegen JP. Comparisons of estradiol, LH and FSH patterns in pregnant and nonpregnant beagle bitches. Theriogenology 2002;57:1957-1972. [65] Robertson IS, Fraser HM, Innes GM, Jones AS. Effect of immunological castration on sexual and production characteristics in male cattle. Vet Rec 1982;111:529-531. [66] Huxsoll CC, Price EO, Adams TE. Testis function, carcass traits, and aggressive behavior of beef bulls actively immunized against gonadotropin-releasing hormone. J Anim Sci 1998;1760. [67] Finnerty M, Enright WJ, Roche JF. Testosterone, LH and FSH episodic secretory patterns in GnRH-immunized bulls. J Reprod Fertil 1998;114:8594. [68] Cook RB, Popp JD, Kastelic JP, Robbins S, Harland R. The effects of active immunization against GnRH on testicular development, feedlot performance, and carcass characteristics of beef bull. J Anim Sci 2000;78:2778. [69] Adams TE, Daley CA, Adams BM, Sakurai H. Testes function and feedlot performance of bulls actively immunized against gonadotropin-releasing hormone: effect of age at immunization. J Anim Sci 1996;74:950. [70] Jago JG, Cox NR, Bass JJ, Matthews LR. The effect of prepubertal immunization against gonadotropin releasing hormone on the development of sexual and social behavior of bulls. J Anim Sci 1997;75:2609. [71] Zeng XY, Turkstra JA, Meloen RH, Liu XY, Chen FQ, Schaaper WMM, Oonk HB, Guo da Z, van de Wiel DF. Active immunization against gonadotrophin-releasing hormone in Chinese male pigs: effects of dose on antibody titer. Theriogenology 2002;70:223. [72] Meloen RH, Turkstra JA, Lankhof H, Puijk WC, Schaaper WM, Dijkstra G, Wensing CJ, Oonk RB. Efficient immunocastration of male piglets by

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54 immunoneutralization of GnRH using a new GnRH-like peptide. Vaccine 1994;12:741. [73] Bonneau M, Dufour R, Chouvet C, Roulet C, Meadus W, Squires EJ. The effects of immunization against luteinizing hormone-releasing hormone on performance, sexual development, and levels of boar taint-related compounds in intact male pigs. J Anim Sci 1994;72:14. [74] Kiyma Z, Adams TE, Hess BW, Riley ML, Murdoch WJ, Moss GE. Gonadal function, sexual behavior, feedlot performance, and carcass traits of ram lambs actively immunized against GnRH. J Anim Sci 2000;78:2237. [75] Brown BW, Mattner PE, Carroll PA, Holland EJ, Paull DR, Hoskinson RM, Rigby RD. Immunization of sheep against GnRH early in life: effects on reproductive function and hormones in rams. J Reprod Fertil 1994;101:15. [76] Zeng XY, Turkstra JA, Tsigos A, Meloen RH, Liu XY, Chen FQ. Effects of active immunization against GnRH on serum LH, inhibin A, sexual development and growth rate in Chinese female pigs. Theriogenology 2002;58:1315. [77] Brown BW, Mattner PE, Carroll PA, Hoskinson RM, Rigby RD. Immunization of sheep against GnRH early in life: effects on reproductive function and hormones in ewes. J Reprod Fertil 1995;103:131. [78] Tshewang U, Dowsett KF, Knott LM, Trigg TE. Preliminary study of ovarian activity in fillies treated with a GnRH vaccine. Aust Vet J 1997;75:663-667. [79] Fagerstone KA, Miller LA. GnRH immunocontraception: a possible control for populations of feral cats. In: Proc Int Symp Nonsurgical Methods for Pet Population Control, 2002; Pine Mountain, GA, p. 48. [80] Miller LA, Rhyan JC, Drew M. Contraception of bison by GnRH vaccine: a possible means of decreasing transmission of brucellosis in bison. J Wildl Dis 2004;40:725-730. [81] Ferro VA, Khan MAH, Latimer VS, Brown D, Urbanski HF, Stimson WH. Immunoneutralisation of GnRH-I, without cross-reactivity to GnRH-II, in the development of a highly specific anti-fertility vaccine for clinical and veterinary use. J Repro Immun 2001;51:109-129. [82] Kumar N, Savage T, DeJesus W, Tsong YY, Didolkar A, Sundaram K. Chronic toxicity and reversibility of antifertility effect of immunization against gonadotropin-releasing hormone in male rats and rabbits. Toxicol Sci 2000;53:92-99. [83] Miller LA, Johns BE, Killian GJ. Immunocontraception of white-tailed deer with GnRH vaccine. Am J Reprod Immunol 2000;44:2664.

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BIOGRAPHICAL SKETCH John Friary was born July 24, 1970, in Laconia, New Hampshire. He attended secondary school in St. Petersburg, Florida, studied for one year at the Georgia Institute of Technology in Atlanta and then moved to Gainesville, Florida. John graduated from the University of Florida with a B.S. in environmental engineering in 2004. John became interested in research to benefit feral cats after volunteering at Operation Catnip, a trap-neuter-return clinic in Gainesville, Florida. He entered a graduate program at the College of Veterinary Medical at the University of Florida to investigate the potential for immunocontraception to reduce the population of feral cats under the advisement of Dr. Julie Levy. He received his M.S. in 2006. John will continue research related to the humane control of feral cats. 55


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Title: Evaluation of a Gonadotropin Releasing Hormone Vaccine for the Humane Control of Female Feral Cats
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
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EVALUATION OF A GONADOTROPIN RELEASING HORMONE VACCINE
FOR THE HUMANE CONTROL OF FEMALE FERAL CATS
















By

JOHN FRIARY


A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF
FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006















ACKNOWLEDGMENTS

I would like to thank Dr. Julie Levy for the opportunity to study under her and for

her willingness to give second, even third, chances. She knew when I needed to be

pushed even when I did not know it. I have very much appreciated and been inspired by

Dr. Cynda Crawford's refusal to allow her indefatigable work habits to dull her sense of

humor. I am grateful for Dr. Lowell Miller's great patience while answering my

seemingly endless questions regarding the vaccine, and for welcoming me to his

laboratory in Colorado. I have benefited greatly from Dr. Karine Onclin's ability to

explain endocrinology to me and I thank her. Without the support and motivation of Dr.

Paul Chadik and Jim Crocker, I would not have returned to school and I can not thank

them enough. I would also like to thank Sylvia Tucker, Mike Reese, Kathy Kirkland,

Audria West, and Rob Schleich for their technical assistance. The girls, of course,

remind me every day why I have chosen this path.

This study was made possible by funding received from the Morris Animal

Foundation.















TABLE OF CONTENTS
Page

A C K N O W L E D G M EN T S .............................................................. .....................ii

LIST OF TABLES ............................... ................. ....... .. ..............v

LIST OF FIGURES ............ .................................. .... ...... ................. vi

A B ST R A C T ............................................................................................................... vii

IM PA CT OF FERAL CA TS .................................. ..........................................

Human Health ........................................................................................................ 2
A nim al H health ..........................................................................................4
Predation on W wildlife ................................. ...................... ............... 5

METHODS TO MANAGE FERAL CATS ............. .............. ............... 8

L ethal M ethods ....................................................... 8
Non-Lethal M ethods ......... .... .......... ......... ...... ............... 10
A adoption ................................................ ............... 10
Relocation Programs .......... ...................... .. ............... 10
Sanctuaries ........................ .... ........ ............................... 10
TNR Programs .............................. ....... ... 11
N on-surgical Contraception...................................... .......... ......... 13
Feline reproductive endocrinology .......................................14
Steroids and G nRH agonists ............. ................................... 17
Cell destruction ............... .............................................18

IM M UNOCONTRACEPTION ....................................................... .... ........... 19

Im m unocontraception Targets ................................................... ........ 19
Riboflavin Carrier Protein............... ......... ............. .. .............. 19
Sperm Proteins ..... .. ........ .... ........................ ... .... ... ..... .... 20
Zona Pellucida .......................... .... ... .... ........... ........ ......20
Hypothalamic-Pituitary-Gonadal Axis ...........................................20
V accine D delivery ..................... .. ......................... .... .. ........... 2 1

GNRH VACCINE .................................... ..... .......... .............. .. 23















M ATERIALS AND M ETHODS......................................... .......................... 25

C ats ....................................................... 2 5
Vaccine Construction................................................ 25
Treatment .............. ......... ............. ......... 26
B lood C collection ............................................ .............. ....27
Detection of GnRH Antibodies.................. ........ ..............27
Determination of Serum Estradiol-173 and Progesterone
Concentrations ...................... ...... ........ ................ 29
B reeding T rial ............................................................2 9
E strus Induction ............................................. ........ .......... 29
Assignment of Cats to Study Groups ..................................30
F ertility and F ecundity .................................................... ............... 30
B ody Phenotype ...................................... .... ............. .. .............. .. ... 30
S statistical A n aly sis............ .... .......................................... ........ .............. 3 1

R E S U L T S ................................................................3 4

Reactions to Treatm ent ........................................................................... 34
B reedin g T rial ............................................................34
D election of E strus..................................... ............................ 34
F ertility and F ecundity .................................................... ............... 35
G nR H A ntibody Titers........................................................ ............... 35
H orm one C concentrations ........................................ .......................... 36
Progesterone ......... .. .. ......... .....................36
Estradiol-17.. ...... ....................................37
B o dy C o n d itio n ......... ....... .................................................... .. .... .... .. ....3 7
B ody W eight .................................... .... ...... .... .............. ..37
F alciform F at P ad ............................. .. ........ ...... .. .. .......... 38

DISCUSSION ......................................... ................ 43

R E F E R E N C E L IST ......................................................................... ....................48

BIOGRAPHICAL SKETCH .............................................................................55















LIST OF TABLES


Table
Page

1. Body weight and percent w eight gain........................................ ............... 42

2. Falciform fat pad depth and area ............................................... ............... 42
















LIST OF FIGURES


Figure Page

1 Timeline ......... ................. ................... ...... 32

2 Lateral abdominal radiograph.......................... .......................... ....... ........ 33

3 B reedin g trial ...............................................................3 9

4 GnRH antibody titers for female cats ....................................... ............... 40

5 GnRH antibody titers for cats with high titers ............................................. 41

6 GnRH antibody titers for cats with variable titers .............................................41

7 GnRH antibody titers of nonresponders. ............................................................42















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EVALUATION OF A GONADOTROPIN RELEASING HORMONE VACCINE
FOR THE HUMANE CONTROL OF FEMALE FERAL CATS

By

John Friary

August 2006

Chair: Julie Levy
Major Department: Veterinary Medicine

The unwanted cat population in the United States numbers in the tens of millions

and current control measures have only had limited success in reducing it.

Immunocontraception has the potential to humanely reduce this population. The purpose

of this study was to investigate the effectiveness of a GnRH-based vaccine for

immunocontraception of female cats. It was expected that the treated cats would produce

antibodies against GnRH and there would be a positive correlation between high titer and

contraception. Adult female cats were divided into a sham group (n = 5) and a treatment

group (n = 15) that was immunized once with 200 |tg of synthetic GnRH coupled to

keyhole limpet hemocyanin and combined with a mycobacterial adjuvant. GnRH

antibody titer and serum concentrations of progesterone and estradiol-173 were

determined monthly. For the duration of the study the daily photoperiod was

manipulated in an attempt to induce estrus. A male breeding cat was housed with the

females during the long-day periods, and continuous videography was used to monitor









for signs of estrus and breeding. GnRH antibodies were detected in all treated cats by

150 days after immunization, but when the titer in four cats fell below 16,000, they

became pregnant and were classified as nonresponders. The titers of the remaining 11

cats responderss) never decreased below 16,000. These cats displayed no signs of

behavioral estrus and did not become pregnant by the end of the study 24 months after

immunization. All five sham cats became pregnant within one month of the introduction

of the male cat. From 60 days after immunization until the end of the study, progesterone

concentrations in all responders remained at basal levels, and increased two months

before parturition in all cats that became pregnant. The responder cats gained more

weight than the nonresponders during the 14 months after immunization (P = 0.004),

which is the same response observed in surgically sterilized cats. A single dose of GnRH

vaccine resulted in contraception in 73% of the cats for at least 24 months.















IMPACT OF FERAL CATS

The domesticated cat is the most numerous companion animal in the United

States with 37.7 million households owning 90.5 million cats [1]. It is estimated that

there are also an equal number of unowned, unwanted cats [2]. Considerable debate

exists on what impact these cats have on human health through zoonotic diseases, animal

health by acting as reservoirs for diseases that affect pet cats and other species, and

wildlife through predation and competition. In addition, there is not agreement on the

quality of life of the cats. The debate extends to the actions that should be taken to

ameliorate the impact of these unowned cats.

Accurately assessing the impact and designing solutions is made more difficult

because cats are assigned to subpopulations according to different criteria. Cats are often

described according to their ownership status (owned or unowned), lifestyle (indoor,

indoor/outdoor, outdoor), or degree of socialization [3] (tame, feral). In addition, during

its lifetime an individual cat may move from one subpopulation to another. For instance,

a cat living indoors may be abandoned, forced to live outside, and over time become

untrusting of humans; this tame, indoor, owned cat has become an unowned, feral cat.

Many reports on the impacts of cats fail to clearly define which subpopulation of cats is

being studied. For the purposes of this thesis, a feral cat will be defined as any unowned,

free-roaming cat, regardless of its socialization status.









Concerns have been expressed that feral cats serve as a reservoir for diseases that

may be transmitted to humans, pet cats and wildlife [4,5]. In general, feral cats do not

appear to pose a greater risk to humans or other cats than pet cats [3,6]. Cats can transmit

disease directly through a bite or other physical contact; by shedding the pathogenic

organism into the environment; or by concentrating pathogens that can be transferred to

other hosts via vectors such as fleas, ticks and mosquitoes. The Center for Disease

Control and Prevention considers eradicating wildlife to reduce disease reservoirs

ineffective, but does support trap-vaccinate-release programs for wildlife [7].

Human Health

The American Association of Feline Practitioners lists 40 potential feline zoonotic

agents, but transmission of the disease from cat to human has not been documented or

occurs only rarely with most of the organisms [8]. Rabies is often cited as a risk from

feral cats, but none of the 57 human cases of rabies in the United States from 1980 to

2004 was attributable to cats [9,10]. The risk of rabies transmission from cat to human is

very low.

Bartonella henselae andBartonella clarridgeiae can both be passed from cats to

humans by biting or scratching. Infection by these bacteria is the most common direct

zoonosis associated with cats, and it results in 25,000 cases of cat scratch disease in the

United States each year [8]. Cat scratch disease is generally a mild illness, but in rare

cases it can lead to serious disease. In Randolph County, North Carolina, 93 of 100

(93%) feral cats and 48 of 76 (63%) pet cats were seropositive for antibodies against B

henselae [11]. In northern Florida 186 of 553 (33.6%) feral cats were seropositive for









antibodies against B henselae, which is within the range of values found in several

studies of pet cats [6].

Roundworms, hookworms and tapeworms can be shed by cats in their feces and

can potentially cause infection in humans. However, the larvae of roundworms and

hookworms must mature in the environment for at least 3 days and 3 weeks, respectively,

before they can infect a new host [8]. Fleas containing tapeworm must be consumed for

infection to occur [8]. The infection rate of roundworms in feral (21%) and pet (18%)

cats in Randolph County, North Carolina, was not statistically different [11]. However, a

study of 80 feral and 70 pet cats in California found infection rates of roundworms and

tapeworms significantly higher in feral cats than pet cats. The roundworm infection rates

were 54% and 4% for feral and pet cats, respectively, and tapeworm infection rates were

26% and 4% for feral and pet cats, respectively [3].

The protozoa Toxoplasma gondii, Cryptosporidium parvum, and some species of

Giardia are shed in the feces of cats and can infect humans through the fecal-oral route.

Infection with Cryptosporidium spp. and Giardia spp. in humans is common, but they are

rarely directly linked to cats [8]. In Randolph County, North Carolina, the feral and pet

cats did not have significantly different prevalences of infection with Cryptosporidium

(7% and 6%) or Giardia (6% and 5%) [11].

Domestic cats and other felids are the only definitive hosts to shed T gondii

oocytes in their feces, thus contaminating the environment with infectious organisms.

The seroprevalence of T gondii in feral cats (63%) in Randolph County, North Carolina,

was higher than for owned cats (34%) [11]. Feral cats from Northern Florida were found

to have a seroprevalence of 12.1%, lower than seroprevalence rates found for pet cats in









the United States (30%) [6]. Consumption of tissue containing T. gondii cysts allows

transmission between intermediate hosts, such as pigs and humans. The relative

importance of the two modes of infection in humans, ingestion of oocytes shed by cats

and consumption of infected tissue, is unknown.

The natural oropharyngeal flora of healthy cats includes Capnocytophaga and

Pasteurella, which are frequently found in cat bite infections. Untreated infections with

these organisms can cause death [12]. However, most feral cat bites are provoked [3].

Advising the public to avoid direct contact with feral cats and implementation of trap-

vaccinate-return programs should reduce the zoonotic disease risk from feral cats.

Animal Health

The feline zoonotic diseases are transmissible between cats, but cats can also

transmit diseases that do not affect humans, including feline leukemia virus (FeLV),

feline immunodeficiency virus (FIV), and feline coronavirus (FCoV). Some feel that

kitten mortality rates, which may be as high as 75%[13], and the general health of feral

cats are compelling reasons to euthanize these cats[5]. They believe that feral cats suffer

higher rates of injury and disease than owned cats[5]. However, feral cats brought to

trap-neuter-return (TNR) clinics in northern Florida were lean, but not emaciated [14],

had death rates due to complications of surgery comparable to pet cats, and had a

euthanasia rate for humane reasons of only 0.4%[14,15].

In addition, several studies have found that disease prevalence rates are very

similar between feral and owned cats[6,11,16,17]. In 2004, 5,259 feral cats and 12,779

pet cats were tested for feline immunodeficiency virus (FIV) and feline leukemia virus

(FeLV) in 145 animal shelters and 345 veterinary clinics in the United States, Puerto









Rico and Canada. The infection rates for the feral cats were 2.0% (FIV) and 1.6% (FeLV)

compared to the infection rates for the pet cats that were 2.7% (FIV) and 2.6% (FeLV)

[16]. Between 1995 and 2000, a total of 1,876 feral cats from two trap-neuter-return

programs in Gainesville, Florida and Raleigh, North Carolina, had infection rates for FIV

and FeLV of 3.5% and 4.3%, respectively[17]. In addition, 553 feral cats in northern

Florida had similar or lower prevalence rates of Mycoplasma haemofelis, Mycoplasma

Haemominutum and Bartonella henselae compared to previous reports in pet cats [6].

However, it is possible that the overall health of feral cats brought in to TNR

clinics is different than the health of feral cats in general.

Predation on Wildlife

It has been well-documented that cats can have adverse impacts on wildlife,

particularly on islands. Prey species on island ecosystems are especially vulnerable to the

introduction of non-native mammalian predators such as the cat [18]. Cats have been

introduced to at least 65 island groups and are a major threat to many island bird species

[18]. In 1949, cats were introduced to Marion Island, a 112-square mile sub-Antarctic

island in the Indian Ocean. By 1975, it was estimated that they were killing 450,000

burrowing petrels annually and probably had driven the common diving petrel to local

extinction [19]. On an island in New Zealand, the last remaining Stephens Island Wren

(Xenicus [Traversia] lyalli) was killed by a cat [20].

There is evidence that cats contributed to the total extinction of the Little Barrier

snipe (Coenocorypha aucklandica barrierensis) and the local extinction of the North

Island Saddleback (Philesturnus carunculatus rufusater) on Little Barrier Island, New

Zealand [21]. However, introduced rats also had a detrimental effect on the bird









populations, and the relative contributions from the cat and the rat could not be

determined [21]. In addition, seven years after eradication of cats was completed on the

island, the bird numbers were similar to their numbers before the cat eradication program

began [21]. It is believed that in some instances cats may actually protect island species

by keeping the rodent population in check [19,22].

Determining the impact of cat predation in mainland settings is even more

difficult than on islands because in most instances there are additional predators present.

Outside of Canberra, Australia, domestic cats were found to be opportunistic predators,

catching prey in proportion to the prey density. The conclusion reached was that cats had

little effect on the local ecosystem [23]. Wildlife managers in Australia have been

working to remove rabbits, another exotic species. However, in many ecosystems rabbits

are the primary food for cats, and there is a fear that a sudden removal of rabbits will

cause the opportunistic cats to switch to native prey. Individual ecosystems must be

evaluated to determine the impact that cat predation has on wildlife and if removal is

appropriate or necessary.

Possible transmission of T. gondii from cats to California sea otters (Enhydra

lutris nereis) is of current concern. Between 1997 and 2001, the seroprevalence of T

gondii was 42% for live otters and 62% for dead otters [24]. The source of the T gondii

is believed to be cat feces from sewage and surface runoff from coastal communities.

However, whether the feces came from litter boxes of indoor cats, from owned cats

allowed outdoors, or feral cats has not been investigated. There is also concern that

native marsupials in Australia, which has non-native cats, may be more susceptible to T.

gondii than ecosystems with native cats[25].






7


Although the true impact of feral cats on the health and welfare of the public,

animals, and the environment is difficult to quantify, it is appropriate public policy to

develop effective cat control programs. The best approach to cat control is another area

of intense controversy.















METHODS TO MANAGE FERAL CATS

The most common action taken regarding feral cats has been to do nothing [26].

Historically, lethal methods of control have been used in an attempt to eradicate

particular groups of cats. In the United Sates, euthanasia is commonly performed at

animal control shelters. Worldwide, however, trapping, poisoning, and hunting have

been the most common methods for eradication of feral cats. The use of biological

vectors has been utilized in at least three island eradications [20,27]. In some areas of the

world, including the United States, there has been strong public pressure to devise more

humane ways of dealing with feral cats, including trap and relocate, and TNR.

Lethal Methods

Since 1934, cats have been eradicated from 48 islands around the world. All but

five of these islands had cat populations of less than 100, and only 10 of the islands are

greater than 10 square kilometers [28]. The largest eradication effort occurred on Marion

Island using trapping, poisoning, hunting with guns and dogs, and biological control, and

took 19 years to complete [20]. It took three years to eradicate 151 cats from Little

Barrier Island using traps, poison, hunting with guns and dogs, and biological control

[21].

The poison used in most eradication campaigns was sodium monofluoracetate

(1080), which has been used since the 1950s in New Zealand for pest control [29].

Sodium monofluoracetate kills by disrupting the Krebs cycle, thus inhibiting energy









production by cells. Long-term exposure to sub-lethal doses can be harmful to people

handling the poison. Non-target animals are also at risk; dogs are especially susceptible to

1080 [29]. For these reasons, the use of 1080 in the United States is restricted to the

protection of livestock from coyotes [30]. Secondary poisoning by ingestion of rats

poisoned with the anticoagulant, brodifacoum, contributed to cat eradication on four

islands [28].

Feline panleukopenia virus (FPV) was the biological control agent used on Jarvis,

Marion, and Little Barrier Islands [28]. FPV virus is spread through feces, urine, saliva,

and vomit. Felids are the primary hosts of FPV, but raccoons and a few other mammals

can also contract the disease. On Marion Island, 96 cats were trapped and inoculated

with 1000 TCLD5s of FPV and released by helicopter to 93 different locations on the

island. The total cat population declined at a rate of 26% per year for five years, but

stabilized as the population developed immunity [20]. On Little Barrier Island, the use of

FPV was abandoned because the virulence among that population of cats was determined

to be too low to be effective [21]. The use of biological controls has many inherent risks,

including accidental release as happened with the rabbit calicivirus in Australia in 1995

[31], and non-target susceptibility. As was demonstrated on Marion and Little Barrier

Islands, the target species may become resistant over time.

Eradication or reduction of cats on the mainland is more difficult than on islands

for several reasons. Continuous influx of cats into the area is likely, the risk of non-target

species death increases with poisoning and hunting with dogs, hunting with guns is

heavily restricted for human safety reasons, and biological control mechanisms would be

nearly impossible to contain.









Euthanasia at animal shelters is the leading cause of death of cats in the United

States, with an estimated 3 million cats per year [26,32]. Euthanasia has not been shown

to effectively reduce feral cat populations. In 1996 in Ohio 72.2% of cats admitted to

shelters were euthanized and in 2004, the euthanasia rate was 68.8% [33]. It is

hypothesized that removing cats from a habitat simply allows other cats to fill the vacated

niche [26,34].

Non-Lethal Methods

More humane solutions include trapping cats, sterilizing them, and adopting them

into homes, relocating them to a more acceptable outdoor location, placing them in

sanctuaries, or returning them to the location of their capture.

Adoption

Seventy-five percent of the cats euthanized in animal shelters in the United States

are classified as adoptable [3], but there are not sufficient homes to accept them.

Increasing the pool of cats waiting to be adopted with less socialized cats is clearly not a

viable solution to the overall problem.

Relocation Programs

Relocation of cats to a non-enclosed site is difficult because it is time-consuming

to acclimate the cats to their new environment and the cats often have low survival rates

at the new sites [26].

Sanctuaries

Another alternative is to remove cats to permanent sanctuaries where they can live

out their lives in confinement. Although sanctuaries can be a small-scale solution, the

overall number of feral cats is too large to be accommodated in sanctuaries. Three









sanctuaries that have reached capacity and only accept cats on a very limited basis are

Best Friends Animal Sanctuary in Utah, the Chico Cat Coalition in California, and a

program instituted by the National Humane Education Society. In addition to their

sanctuaries, Best Friends Animal Sanctuary and National Humane Education Society

operate TNR programs [3].

TNR Programs

The goal of TNR programs is to sterilize feral cats and return them to the location

where they were trapped. TNR programs are generally run as grass-roots operations that

depend on donations and volunteers. However, some municipalities, such as Orange

County, Florida, use TNR programs as a cost-effective alternative to trapping and

euthanizing cats and have incorporated TNR into their animal control efforts. The

average cost of sterilizing each cat was $56 compared to the estimated $139 per cat for

impounding, sheltering and processing [26]. Other agencies that have incorporated TNR

into their animal control programs include Tomkins County Society for the Prevention of

Cruelty to Animals, New York; Maricopa County Animal Care and Control, AZ; New

York City Center for Animal Care and Control; San Francisco Society for the Prevention

of Cruelty to Animals; and the American Society for the Prevention of Cruelty to

Animals. At a minimum, TNR programs sterilize the cats and return them to their

colonies. Some programs provide food, shelter and veterinary care, vaccinate against

rabies and other diseases, and test for diseases such as FIV and FeLV. Removal of the tip

of an ear is recognized internationally as a sign that the cat has been sterilized. For

control to be effective it is essential that colonies are monitored for new arrivals and for









the birth of kittens. A TNR program was started and stopped on a Florida university

campus; once the program stopped, the feral cat population began increasing [2].

One long-term goal of many TNR programs is to reduce the colony size through

adoption and attrition. Complete elimination of large colonies is uncommon, but there

are many examples of colonies being greatly reduced in size. In 1991, a TNR program

was instituted on the University of Central Florida campus and by 2002, the original 11

colonies containing a total of 155 cats had decreased to eight colonies comprised of 23

cats [2]. In addition to a decrease of 132 cats, other benefits of the program included

prevention of the birth of an estimated 300 to 700 kittens, and medical care or euthanasia

for sick and injured cats in the colonies. In a three-year period ending in 2002, 1,116

California Veterinary Medical Association veterinarians neutered 170,334 feral cats

through the Feral Cat Altering Program [35]. Maddie's Fund awarded nearly $9.5

million to the California Veterinary Medical Association for the program [35]. ATNR

program on a Texas university campus resulted in a 30% decrease in the feral cat

population in two years [26].

Some wildlife advocates believe TNR is inappropriate because the cats are not

removed from the wild, some critics believe euthanasia is more humane than returning

the cats to their colonies, and others believe TNR is ineffective. Indeed, critics of TNR

often cite two county parks in south Florida, A.D. Barnes Park and Crandon Marina, as

examples of TNR failure. The high visibility of the TNR programs in these parks

encouraged continual cat abandonment at the sites, resulting in a net increase in the

colony size despite a decrease in the original cat populations [36].









Drawbacks recognized by proponents of TNR include expenses for traps, surgical

equipment, medical supplies, and veterinary fees; intensive labor for trapping,

transportation, surgery, recovery and return to the colony; and requirement for the

technical expertise of veterinarians.

Mathematical models have been used to estimate the percent of feral cats that

would need to be neutered in order to cause an overall decline in a population. One

population model predicted that the annual percent sterilization necessary for stabilization

of population growth would be 14% of the 241,000 cats in San Diego County, CA and

19% for the 36,000 cats in Alachua County, FL [34]. Another study [37] predicted that

there would have to be a 75% neutering rate annually in order to have a population

decline. This study predicted that a trap and euthanize program would only have to trap

50% of the population to have a population decline. A limitation to the mathematical

models is the difficulty of determining parameters that determine feral cat reproductive

capacity, such as the carrying capacity of a particular habitat [34].

While surgical TNR can be effective for reducing targeted cat populations, it is

extremely resource-intensive and difficult to implement on a regional or national scale.

Non-Surgical Contraception

Non-surgical methods of contraception or sterilization have the potential for more

efficient implementation, less risk to cats, lower cost, and lower technical requirement

when compared to surgery. Surgical sterilization achieves sterility by removal of the

gonads, thus preventing the production of gametes. Successful non-surgical

contraception or sterilization methods disrupt some aspect of the hypothalamic-pituitary-

gonadal axis or interfere with fertilization of the egg or maintenance of the pregnancy.









Feline reproductive endocrinology

Cats will only breed when they are in estrus, which is characterized by a rapid

increase in estradiol-173 concentrations. The estrogen is released by the follicles, which

are stimulated to grow by the gonadotrope, follicle-stimulating hormone (FSH). FSH is

synthesized and released at a basal level, but the rate of synthesis increases greatly with

the release of gonadotropin releasing hormone (GnRH). Female cats are photoperiod

sensitive and they begin estrous cycling with an increasing daylight-length, as occurs in

January and February in the northern hemisphere. Cats will stop estrus with a decreasing

daylight-length as occurs in September or October. Cats in the laboratory can be induced

to enter estrus by increasing the daylight-length to 14 hours or greater. It is possible that

the pineal gland responds to increasing daylight-length by decreasing the production of

melatonin, which inhibits the release of FSH [38]. Conversely, decreasing the amount of

light appears to increase the production of FSH and the cats will enter anestrous, meaning

that estrous cycling stops.

During estrus, female cats are receptive to male cats and sufficient breeding

triggers a cascade that leads to ovulation. As an induced ovulating species, cats release

GnRH within minutes of genital somatosensory stimuli, as occurs during breeding [39].

GnRH causes the release of a second gonadotrope, luteinizing hormone (LH), within 5

minutes of the GnRH release [40]. LH concentration increase does not necessarily lead

to ovulation, however. With a single copulation only about 50% of cats will ovulate, but

repeated copulations probably trigger successive GnRH releases which leads to

cumulative increments in LH levels [39].









GnRH is synthesized in hypothalamic neurons whose axons extend into the

pituitary stalk, which is not within the blood-brain barrier. Upon appropriate stimulation,

GnRH is released by exocytosis into the capillary plexus that emanates from the superior

hypophyseal artery. GnRH is carried down the pituitary stalk in portal veins which give

rise to a second capillary plexus that supplies the endocrine cells of the anterior pituitary.

GnRH binds to gonadotrophs which synthesize and release the two gonadotropins, LH

FSH. However, the duration and the amplitude of the gonadotropin release depends on

the pulsatile and periodic release of GnRH. For a proper functioning hypothalamic-

pituitary-gonadal axis (HPGA), GnRH must be released with the proper pulsatility and

periodicity; the characteristics of the GnRH release differ in male and female cats, with

the age of the cat, and the stage of estrus with the female cat.

LH binds to Leydig cells in the male cat and thecal cells in the female cat and

stimulates the synthesis and secretion of androgens. FSH binds to Sertoli cells in the

male cat and granulosa cells in the female cat and stimulates estrogen synthesis. FSH

also stimulates synthesis in the Sertoli and granulosa cells of various protein products,

including activin and inhibin, two proteins involved in feedback mechanisms within the

HPGA. In addition, FSH increases the number of LH receptors on the granulosa cells,

which amplifies the sensitivity of granulosa cells to LH.

If the female cat does not breed during estrus, or if there is insufficient breeding to

induce ovulation, then the estrogen levels decrease after three to sixteen days [41] and the

cat enters interestrous, or a phase between successive estrous periods. The cat will

continue to cycle between estrus and anestrous until ovulation occurs or the breeding

season comes to an end with decreasing daylight-length.









If the cat ovulates and the egg or eggs are successfully fertilized then the cat

begins a gestation period of approximately 65 days. If the cat ovulates, but the egg or

eggs are not fertilized, then the cat enters pseudopregnancy which lasts approximately 45

days. In both pregnant and pseudopregnant cats, the follicles that have matured have

released their eggs from the ovary. The portion of the follicles that remain behind are

termed corpus lutea and within 48 hours of ovulation they begin secreting progesterone.

Prior to the release of progesterone by the corpus lutea, the basal level of

progesterone is less than 2 ng/ml. Within 14 to 18 days after ovulation the progesterone

concentrations are greater than 20 ng/ml. The lifespan of the corpus lutea is

approximately 35 days, so the progesterone concentration in a pseudopregant cat returns

to baseline after 35 or 40 days. However, a pregnant cat maintains progesterone

concentrations above baseline because the placenta secretes the hormone after 30 days of

gestation [41].

GnRH, the gonadotropes and the sex hormones form a complex feedback web.

GnRH induces the release of FSH and LH, which in turn induce the release of

testosterone and estrogen. However, testosterone and estrogen provide negative feedback

and inhibit the release of LH and GnRH. In addition, the gonads produce three other

hormones that act on the gonadotrophs. Activin stimulates the release ofFSH, inhibin

and follistatin inhibit release of FSH [41].

Additional hormones that are produced outside the gonads and affect fertility

include leptin, prolactin, growth hormone and insulin-like growth factor 1 (IGF-1).

Leptin is required for fertility [42], while high concentrations of prolactin inhibit the









secretion of FSH and LH in both sexes. Growth hormone stimulates synthesis of insulin-

like growth factor 1 which stimulates sex hormone synthesis [42].

The reproductive system in cats provides a number of mechanisms and potential

targets to inhibit fertility, including the use of steroid and peptide hormones to disrupt the

necessary hormone balance, destruction of reproduction-related cells, and

immunocontraception to bind a hormone or hormone receptor in the HPGA.

Steroid hormones and GnRH agonists

Steroids related to estrogen and progesterone have been used since the 1960's for

contraception of some species. Diethylstilbestrol (DES) is a synthetic estrogen that can

reduce fertility in animals, but it must be administered with precise timing, lasts a limited

amount of time, accumulates in body tissues, and has potential adverse health effects in

treated animals [43]. These characteristics preclude DES and other estrogen-related

compounds from intense use in feral cat population control. Progestins, which are

synthetic progesterones, induce contraception, but the mechanism of contraception is not

well understood. The mechanism most likely involves one or more of the following:

negative feedback on GnRH release, disruption of oocyte transport and fertilization, and

altered receptivity of the endometrium [44]. However, limited duration of contraception

and potential harm to target animals also preclude progestins from use in feral cats.

Administration of GnRH agonists disrupt the pulsatile release of GnRH necessary for

secretion of pituitary follicle-stimulating (FSH) and lutenizing hormones (LH) [45]. This

method disrupts gonadal hormone production to produce contraception, but the short

duration of effectiveness makes this method unsuitable for feral cats.









Cell destruction

Targeted destruction of specific cells or tissues can be used to disrupt the

reproductive process. A GnRH analogue conjugated to a cytotoxin, pokeweed antiviral

protein, resulted in lower serum testosterone concentrations in dogs [46]. It is believed

that the GnRH analogue portion of the conjugate attaches to GnRH receptors on the

gonadotroph cells in the pituitary gland. The molecule is taken into the cells by

endocytosis and the cytotoxic portion of the conjugate destroys the cell's ability to

synthesize proteins, which leads to cell death. It was hoped that the effect would be

permanent, but a portion of the treated dogs demonstrated increasing FSH and LH serum

concentrations by week 36. Ovarian follicles in rats have been destroyed by

administration of 4-vinylcyclohexene diepoxide (VCD), a metabolite of an industrial

chemical [47]. Female mammals are born with their entire lifetime supply of oocytes

contained within primordial ovarian follicles. VCD targets and destroys the primordial

follicles and the oocytes they contain leaving the animal devoid of oocyte stores and

unable to ovulate successfully. This method provides permanent sterility and cessation of

estrus cycling. However, VCD has only proven safe and effective in rodents and more

research needs to be done in other species. In addition, the current treatment requires

daily doses for 15 days, which is not practical for feral cats.

















IMMUNOCONTRACEPTION

The goal of immunocontraception is to induce an immune response against

critical elements of the reproductive process that are not used for other functions. There

are several targets that meet this criterion and have been investigated for their

immunocontraceptive potential, including riboflavin carrier protein (RCP), sperm

proteins, zona pellucida (ZP), lutenizing hormone receptor (LH-R), and GnRH.

An acceptable immunocontraceptive agent for feral cats would be effective in

both sexes and only a single treatment should be used because retrapping of free-roaming

cats for repeated treatments would be impractical. In addition, the vaccine should block

the production of sex hormones so as to eliminate nuisance behaviors such as calling,

spraying, wandering, and fighting. In addition to these minimum standards, an ideal

agent would be permanent, have a quick onset of contraceptive effect, be effective in all

ages, be safe to the cat and the environment, be inexpensive, and be easy to administer.

Immunocontraception Targets

Riboflavin Carrier Protein

RCP is the prime mediator of riboflavin supply to the developing zygote in

mammals and is necessary for maintenance of pregnancy. Three monthly treatments with

an RCP vaccine followed by repeated treatments every four months elicited high

antibody titers and interfered with pregnancy in Bonnet monkeys, but it was not

determined if conception or implantation was affected [45]. The need for multiple









treatments and failure to block sex hormone production make this approach impractical

for feral cats.

Sperm Proteins

Provoking an immune response against sperm proteins has the potential to be

effective in both sexes because antibodies to the proteins could interfere with sperm

development and viability in the male or after insemination in the female [43]. Targeting

sperm would not interfere with sex hormone production, therefore,

immunocontraceptives against sperm proteins are not the best candidate for feral cats.

Zona Pellucida

Zona pellucida (ZP) is a glycoprotein layer surrounding the mammalian egg and

is involved in sperm-egg interaction [43]. The use of ZP antigens for

immunocontraception has been successful in preventing pregnancy in many species, but

not in cats [49,50]. When ZP from pigs, cows, ferrets, dogs and mink was used, the cats

developed high antibody titers to the xenogenic proteins, but these antibodies failed to

cross-react with the cats' native ZP. Immunization with antigens from feline zona

pellucida evoked a poor immune response in the cats, possibly because feline ZP was

recognized as a self protein.

Hypothalamic-Pituitary-Gonadal Axis

Seven adult female cats immunized against LH-R and given four booster

treatments had absence of behavioral estrus for 500 days, at which point the cats showed

signs of recovering normal ovarian function [51]. While immunization against LH-R has

the potential to block the production of sex hormones, multiple treatments were used and

the goal of a single treatment was not reached. However, it is possible that a variation on









this method that extended the duration of immunity could lead to an effective single-

treatment vaccine.

In a previous study, six male cats immunized three to four times with GnRH

conjugated to tetanus toxoid produced GnRH antibody titers, but there was no resulting

decrease in testosterone concentrations below the contraceptive level [52]. In another

study 10 female and four male cats were immunized against GnRH at eight weeks,

boosted four weeks and 100 weeks later, and housed with a proven male. GnRH

antibodies were detected in all 10 female cats. They had basal progesterone

concentrations, did not display estrous behavior and failed to become pregnant for the

entire observation period of 20 months. Three of the four males had castrate levels of

testosterone for the entire duration of the study. A rise in testosterone in the 4th male was

associated with a decline in GnRH antibody titer [53].

Vaccine Delivery

Vaccines can be delivered orally, by a biological vector, or by injection. Delivery

of the vaccine through bait would be easier than the other methods, but dose is much

more difficult to control, and consumption of the vaccine by non-target species would be

a risk.

The advantages of a biological vector for immunocontraception include the low

expense for vectors that are self-disseminating; possibly low environmental impact

compared to poisons; and it is considered humane [54]. Disadvantages include the risk to

non-target species and the inherent risk associated with releasing a potential pathogen

into the environment. As designed, the vector is not pathogenic, but there is always a

chance for reversion to virulence or unexpected effects on non-target species. There is









great concern that other species would be transfected by the vector, so this approach

could not be used where other potentially vulnerable species are present. This limitation

applies to almost any mainland area. Biological vectors may play a role in

immunocontraception of cats on islands where the risk of the vector reaching the

mainland is low.

Injection requires the targeting of individual animals, which increases the effort

required for control programs. Injectable vaccines are often delivered with a dart rifle for

large animals [55], but it requires a skilled marksman and may not be practical or safe for

cats due to their small size.

TNR programs typically use live traps to capture cats for transport to veterinary

clinics the cats to veterinarians for neutering. The same method of capture would allow

delivery of the vaccine by intramuscular or subcutaneous injection in the field without

need for transporting the cats to a central facility for surgical sterilization. An implant,

which would allow timed-release of a vaccine, could also be delivered in this way.















GNRH VACCINE

Blocking GnRH from binding to the gonadotrophs disrupts the hormone cascade,

leading to cessation of both fertility and sexual behavior. GnRH has the potential to

satisfy many of the requirements of an ideal antigen for an immunocontraceptive vaccine.

Since GnRH is a decapeptide, it is a hapten which is a very weak immunogen.

The National Wildlife Research Center (NWRC) developed a GnRH vaccine that uses

keyhole limpet hemocyanin (KLH) as a protein carrier and AdjuVacTM as an adjuvant.

The GnRH peptides are attached to the KLH in such a way as to mimic molecular

patterns found on many microorganisms; these patterns activate the innate immune

system [56]. AdjuVacTM was developed as an alternative to Freund's complete adjuvant

(FCA). FCA is a very effective adjuvant, but it can result in harm to the immunized

animal including necrosis and development of granulomas. AdjuVacTM contains

inactivated Mycobacterium avium in oil.

In a previous pilot study in male cats, GnRH/KLH-AdjuVacTM resulted in

testosterone concentrations below the contraceptive level following a single treatment in

six of nine male cats for the duration of the 6-month observation period [57]. A single

injection also prevented pregnancy in female feral pigs for 36 weeks [58] and was

effective in female deer [55].

The hypothesis was that a single-dose GnRH vaccine, (GnRH/KLH-AdjuVacTM),

would produce long-term immunocontraception in female cats. The specific objectives






24


were to determine the proportion of cats that respond, the duration of the response, and

the safety of the vaccine over a 24 month period.















MATERIALS AND METHODS

Cats

Twenty-four 8- to 14-month-old specific-pathogen-free female domestic shorthair

cats were acquired from a commercial vendor (Liberty Research, Waverly, NY, USA).

All 24 cats were group-housed in the Animal Care Services facilities at the University of

Florida College of Veterinary Medicine, which are accredited by the Association for

Assessment and Accreditation of Laboratory Animal Care. Cat housing consisted of one

large room with raised resting benches and was climate controlled to maintain ambient

temperatures between 21 and 23 C with controlled lighting. Food and water were

provided ad libitum. The experimental design was approved by the UF Institutional

Animal Care and Use Committee. All cats and their offspring underwent surgical

sterilization and were adopted to private homes at the conclusion of the study.

Vaccine Construction

A GnRH vaccine was constructed using a synthetic GnRH peptide with the sequence

[pEHWSYGLRPGGGC-SH] produced by the Fmoc/tBU protection method (Global

Peptide Services, Fort Collins, CO, USA). Immunogenicity was enhanced by coupling

GnRH peptides to a protein carrier, keyhole limpet hemocyanin (KLH; Pierce Endogen,

Rockford, IL, USA), in a 1:3 GnRH:KLH mass ratio. The underlined amino acids

represent the native GnRH molecule and "pE" signifies pyro-glutamate. Two glycines

were added at the C terminus as a spacer and a cysteine was added to ensure consistent









alignment of the peptide to the maleimide-activated KLH. The aqueous-based GnRH-

KLH conjugate (200 [tg) was combined in a 1:1 ratio by volume with a novel adjuvant,

AdjuVac. AdjuVac was produced by diluting a USDA-licensed Johne's disease

vaccine containing inactivated Mycobacterium avium in mineral oil (Mycopar; Fort

Dodge Animal Health, Fort Dodge, IA, USA)

Treatment

Upon arrival, the 24 cats were housed for 30 days in a photoperiod regime (8 hours

light: 16 hours dark) that is inhibitory of estrus. Fifty days prior to treatment (Day -50),

the photoperiod regime was reversed to 16 hours light:8 hours dark, which stimulates

estrous cycling within 15 days in 85% of cats [59]. Serum hormones (progesterone and

estradiol-173) were measured on Day -60, then every other day from Day -50 to Day -30.

The magnitude, duration and rate of change of estradiol-173 concentrations in each cat

from Day -50 to Day -30 were used to confirm normal estrous cycling in cats selected for

this study.

Confirmation of normal hormonal responses to the lighting change, including

evidence of estrous cycling and docile temperament, were used to select 20 of the 24 cats

to continue in the study. The 20 cats were randomized into a sham group (n = 5) and a

treatment group (n = 15) based on maximum estradiol-173 concentrations. The sham

group received placebo vaccines containing all components except GnRH-KLH. The 15

treated cats received vaccines containing 200 [g GnRH-KLH. Brief anesthesia was

induced by administration of isoflurane (IsoFlo; Abbott Laboratories, North Chicago,

IL, USA) by face mask. The hair of the right cranial thigh was clipped, and the injection

site was cleaned with 70% isopropyl alcohol. The vaccine (0.5 mL) was injected into the









quadriceps muscle group. The right pinna was tattooed with a treatment code,

specifically, Cl through C5 for the sham cats and T through T15 for the treatment cats.

Potential adverse reactions to treatment were evaluated by daily physical

examination, including inspection of the injection site and measurement of body

temperature for one week following treatment.

Blood Collection

Blood (4 mL) was collected by jugular venipuncture monthly into serum separator

tubes for determination of GnRH antibody titer and concentration of estradiol-173 and

progesterone. In addition, blood for estradiol-173 and progesterone concentration

determination was collected every other day for 20 days following each of four estrus-

inducing photoperiod changes. Serum was separated by centrifugation and stored at

-20 C until analysis.

Detection of GnRH Antibodies

Serum was tested for GnRH antibodies using an enzyme-linked immunoabsorbant

assay (ELISA). Bovine serum albumen (BSA) was coupled to GnRH in a 1:1

GnRH:BSA mass ratio and 200 ng of GnRH-BSA buffered with 50 [tl bicarbonate was

used to coat the wells in each 96-well microtiter plate and incubated overnight at 40 C.

GnRH-BSA was used so that only anti-GnRH antibodies would be detected, not

antibodies against the KLH component of the vaccine. The wells were washed twice

with 200 [tl of phosphate buffered saline (PBS) and 0.05% Tween 20 (Sigma Chemical

Co., St. Louis, MO, USA), blocked with salmon serum (SeaBlock, East Coast Bio, Inc.,

North Berwick, ME, USA) overnight at 40 C. The wells were aspirated and 100 [tl of cat

serum diluted 1:1,000 in PBS was added in the top row of the plate, with each column









containing a sample from a different cat. Two negative controls were run on each plate;

one negative control was buffer without cat serum and the other was pre-vaccination cat

serum. The last column was used as a positive control with serum from a cat with a

known high antibody titer.

All remaining wells were filled with 50 [tl PBS and serial dilutions were made from

1:1,000 to 1:128,000 by taking 50 [tl from each well in row 1 and mixing it into the

corresponding well in row two. This process was repeated for the remaining six rows.

The plates were then incubated for two hours at room temperature on a shaker. The wells

were washed twice with 200 [tl PBS/0.05% Tween 20.

Antibody to GnRH was detected by adding 50 [tl goat anti-cat IgG (Sigma Chemical

Co.) diluted 1:10,000 in PBS/1% Sea Block to each well. The plate was incubated at

room temperature on a shaker for 2 hours. The wells were washed twice with 200 [tl

PBS/0.05% Tween 20. Fifty microliters of rabbit anti-goat coupled to horseradish

peroxidase conjugate (Sigma Chemical Co.) diluted 1:3,000 in PBS/1% Sea Block was

added to each well and incubated for two hours at room temperature on a shaker. The

wells were washed three times with 200 [tl PBS/0.05% Tween 20. Fifty microliters of

Tetramethylbenzidine/phosphate-citrate buffer (Sigma Chemical Co.) was added to each

well and the plate was incubated for three to five minutes or until a blue color change

developed. Fifty microliters of 2M sulfuric acid was added to each well to stop the

reaction and the plates were read on a plate reader (Dynatech, Horsham, PA) at 450 nm.

The endpoint dilution was considered positive based on the positive control with the titer

equal to the reciprocal of the endpoint dilution.









Determination of Serum Estradiol-17p and Progesterone Concentrations

In cats, behavioral estrus is preceded by a surge in estradiol-173 released by

developing follicles. After ovulation progesterone concentrations rise significantly within

four days [60]. Serum was tested monthly for estradiol-173 and progesterone

concentration. In addition, blood was collected every other day for 20 days following

each of four estrus-inducing photoperiod changes. Serum samples were analyzed for total

estradiol-17P and progesterone by radio-immunoassay (Coat-A-Count; Diagnostic

Products Corporation, Los Angeles, CA, USA) according to the manufacturer's

instructions. The manufacturer reports an estradiol-173 sensitivity of 8 pg/mL with

within-run coefficient of variation (CV) of 4-7% and between-run CV of 4-8%,

depending on the estradiol-17P concentration. The manufacturer reports a progesterone

sensitivity of 0.02 ng/mL with within-run CV of 2-9% and between-run CV of 4-10%,

depending on the progesterone concentration.

Breeding Trial

Estrus Induction

The effects of treatment on normal hormone responses and fertility were evaluated

beginning four months after treatment and continuing for the duration of the 26-month

study. The photoperiod length was alternated between the estrus-inhibiting regime (16

hours dark:8 hours light) and the estrus-inducing regime (8 hours dark: 16 hours light).

From Day 0 to Day 120, Day 330 to Day 360 and Day 510 to Day 540, the cats were

exposed to the estrus-inhibiting regime (26% of post-treatment time). For the remainder

of the study the cats were exposed to estrus-inducing light regime (74% of post-treatment

time) (Figure 1). The switch from the short-day photoperiod to the long-day photoperiod









was intended to induce estrus in any of the cats that were capable of an estrous response.

A breeding male was housed continuously with the females during all of the long-day

photoperiods after Day 120. Three breeding males were alternated to assure that inter-cat

incompatibility was not an issue. Time-lapse videography was used to monitor breeding

activity during periods of long-day photoperiod. The tapes were reviewed daily and the

number of attempted and successful breeding was recorded. Distinctive shaving of the

cats' fur was used to allow differentiation of similar-looking cats.

Assignment of Cats to Study Group

During the pre-treatment long-day photoperiod (Day -50 to Day -30), 19 of 24 cats

(79%) had a pattern of estradiol-173 concentrations consistent with estrus based on the

peak concentrations and the rate of change of the concentrations. These 19 cats and one

cat that did not display estrus were divided among the sham and treatment groups in such

a way that the two groups did not have significantly different maximum estradiol-17 3

concentrations (P = 0.4).

Fertility and Fecundity

Fertility was defined by the proportion of cats becoming pregnant. Fecundity was

defined as the number of live births. Pregnancy was scored as a treatment failure, and the

cats were removed from the study after parturition. The treated cats were divided into

responders, which failed to become pregnant during the 19 months following treatment,

and nonresponders, which became pregnant during the study.

Body Phenotype

It has been well-documented that cats gain weight after surgical neutering, but it is

unknown if immunocontraception has the same effect. To evaluate for this effect, body









weight was recorded for each cat on Day 0 and Day 420, and percent weight gain was

calculated. Radiographic measurement of the falciform fat pad was performed 14 months

(Day 420) after treatment for further evaluation of phenotypic changes. For fat pad

measurement, the cats were anesthetized with medetomidine 40 mcg/kg IM and

radiographed in right lateral recumbancy. Using computed radiography (Kodak,

Rochester, New York, USA), the depth (mm) of the falciform fat pad was measured by

dropping a perpendicular line from the center of the body of the 12th thoracic vertebra to

the ventral body wall and measuring the distance between the caudoventral angle of the

liver and the ventral body wall. The area (mm2) of the fat pad was defined as the area

outlined by the line used for the depth measurement, the ventral border of the liver, the

diaphragm, and the ventral body wall (Figure 2).

Statistical Analysis

Descriptive statistics (mean, error, range) were calculated for each group for hormone

concentrations, GnRH antibody titers, fecundity and body weight. The fertility of the

sham and treatment groups was compared by the two-sided Wilcoxon rank sum test.

Fecundity of the sham and nonresponder groups was compared by one-way analysis of

variance. Differences in GnRH antibody titer and hormone concentrations between the

groups were tested by Kruskal-Wallis one-way analysis of variance on ranks (Kruskal-

Wallis ANOVA). Differences between the three groups (responder, nonresponder and

sham) with regard to body weight, percent change of body weight, falciform fat pad

depth, and falciform fat pad area were analyzed using Kruskal-Wallis ANOVA. The

Holm-Sidak method for pairwise comparison was used for all of the Kruskal-Wallis

ANOVA tests that indicated a difference between at least two of the groups. Differences










were considered significant when P < 0.05. All tests were performed using SigmaStat

statistics software, version 3.0.1 (SPSS Inc., Chicago, IL, USA).


Long-day lighting
Short-day lighting
-Breeding period
* Blood collection
A Immunization
Radiography
Body weight


-60 60 180 300 420 540 660
Days post-treatment


Figure 1. Timeline for 26-month study showing alternating periods of long-day (16 hr
light:8 hr dark) and short-day (8 hr light: 16 hr dark); periods during which the
breeding male had access to the females (Breeding trial); blood collection times;
the treatment (Day 0); falciform fat pad radiograph; and body weight times.































figuree 2. Lateral abdominal radiograph ot a cat with talcitorm tat pad measurements
marked. The vertical line extends from the center of the body of T12 to the
ventral abdominal wall with the solid portion of the line measuring the fat pad
depth. The three solid lines demarcate the area for the falciform fat pad area
measurement.















RESULTS

Reactions to Treatment

Body temperature in all of the cats remained normal after treatment, and there was

no inflammation or tenderness at the injection sites. One treatment cat, (T4), died

suddenly 45 days after treatment; necropsy revealed no gross or histological

abnormalities to explain the death. This cat was replaced with a two-year old cat of

proven fertility from the research colony which was tattooed with the code "T16" and

vaccinated with a vaccine containing GnRH. This replacement cat was exposed to the

same lighting regimen and blood collection schedule as the other 19 cats.

Twenty-four months after immunization a 3 cm x 4 cm mass was discovered by

palpation at the injection site. A biopsy was performed and the mass was found to be

neither cancerous or infected. It was concluded that the mass was a granuloma that had

formed in response to the adjuvant. One cat had multiple 1 cm masses near the injection

site and three other cats had a single 1 cm mass near the injection site.

Breeding Trial

Detection of Estrus

The breeding male cat was introduced on Day 120 at the beginning of the second

long-day regime. All sham cats displayed behavioral signs of estrus and were receptive to

breeding attempts by the male. Time-lapse videography revealed that the male bred each

of the sham cats at least 15 times between Day 122 and Day 155. One cat was observed

breeding in two intervals during gestation (days 7-11, 25-26 of gestation). Four treated









cats displayed estrous behavior and began breeding on Day 128 (T9), Day 418 (T8), Day

469 (T15), and Day 504 (T5). These four nonresponder cats were the treated cats with

the lowest GnRH antibody titers.

Fertility and Fecundity

Based on an average gestation period of 65 days, the date of parturition was used to

estimate the date of successful breeding. All five sham cats became pregnant between

five and 26 days after the male cat was introduced (Day 125 to Day 146) and four of

them within six days of each cat's first observed breeding. Nonresponder cat T9 was bred

in three different estrous intervals from Day 129 to Day 162 before becoming pregnant.

In contrast, nonresponder cats T8, T15, and T5 became pregnant in their first breeding

cycles (Day 418, Day 469, and Day 504, respectively) (Figure 3). The mean number of

live kittens per litter was 3.8 (range 3 to 5) in the sham group, which was significantly

higher (P = 0.03) than the mean of 2.3 (range 1 to 3) live kittens in the nonresponder

group. Based on maintenance of contraception in 11 of 15 treated cats over a 24-month

period, the vaccine had a 73% success rate.


GnRH Antibody Titer

The mean GnRH antibody titer for the responder group was significantly higher than

the titer of the nonresponder group by Day 150 and remained significantly higher for

most of the remainder of the study (Figure 4). All of the sham cats had negative titers

throughout the study.

Three patterns of antibody response were observed among the treated cats: high titers,

variable titers, and low titers. Five of the 11 responder cats (T1, T7, T10, T14, and T16)

had GnRH antibody titers of 128,000 by Day 30 and maintained a titer of 64,000 or









above for the remaining 20 months of the study (Figure 5). The antibody titers of five

other responders (T2, T6, T11, T12, and T13) peaked, then decreased and subsequently

increased again. None of these variable titers decreased below 16,000, and after a trough

level of three to six months, all the titers increased again to at least 64,000 by Day 540

(Figure 6). All four nonresponders became pregnant after their GnRH antibody titer

decreased to 8,000 or below (Figure 7).

For the last seven months of the study, 10 of 11 responders had a GnRH titer of

64,000 or above. The responder cat with the lowest GnRH antibody titer, T3, remained at

a titer of 16,000 for the last 13 months of the study. Two of the nonresponder cats had an

initial high titer that was not sustained and two had titers at 32,000 or below throughout

the study. These four cats became pregnant when titers decreased below 16,000, which

suggests that a titer of 16,000 is contraceptive in female cats. Poor antibody responses in

the nonresponder group delayed, but did not prevent pregnancy.

Hormone Concentrations

Progesterone

In cats, ovulation accompanied by fertilization results in pregnancy, whereas

ovulation without fertilization results in pseudopregnancy. In both cases, serum

progesterone increases from a baseline of < 0.5 ng/mL to a peak above 20 ng/mL

between 16 and 26 days after ovulation [41]. In pregnancy, progesterone concentrations

are higher than in pseudopregnancy and remain above baseline for the entire gestation

period. In pseudopregnancy, progesterone concentrations return to baseline by 50 days.

The nine cats (five sham, four nonresponder) that became pregnant had substantial

increases in progesterone concentrations for two months prior to parturition. This finding









was expected given the average gestation period of 65 days. Prior to the introduction of

the male (Day 120), two of the sham cats, two of the responders, and two of the

nonresponders had serum progesterone concentrations higher than 2.0 ng/mL, indicating

ovulation. T9, the first nonresponder to become pregnant, had progesterone

concentrations indicative of pseudopregnancy on Day -30 and Day 60. A second

nonresponder, T5, had elevated progesterone concentration on Day 30. T1 and T2, both

responders, had progesterone concentrations of 7 and 20 ng/mL respectively, on Day 30.

It is likely that these cats experienced nonfertile ovulation and pseudopregnancy prior to

the development of contraceptive titers of GnRH antibody. After Day 30, none of the

responder cats had evidence of ovulation.


Estradiol-17p

It was expected that the sham group's estradiol-173 concentrations would be

consistent with estrous cycling until they became pregnant. Four of the five control cats

became pregnant during the first sustained estradiol-173 elevation (the first estrous cycle)

following the introduction of the breeding male and one cat, C2, became pregnant during

the second estrous cycle.

Body Condition

Body Weight

The mean weights of the sham, nonresponder, and responder cats on Day 0 were not

significantly different from each other (P = 0.5). Similar to cats undergoing surgical

ovariohysterectomy, responding cats had a higher percent gain in body weight than the

nonresponder cats (P = 0.004) and sham cats (P = 0.02) (Table 1). The percent weight

gain was not significantly different between the sham and nonresponder groups (P =









0.08). The mean weight on Day 420 of the responder group was significantly greater than

the nonresponder group (P = 0.002), but not significantly greater than the sham group (P

= 0.1) (Table 1). The mean weights of the sham and nonresponder groups on Day 420

were not significantly different (P = 0.1).

Falciform Fat Pad

At 14 months post-treatment, the mean falciform fat pad depth for the responder

group was greater than the mean for the nonresponder group (P = 0.046) and sham group

(P = 0.03) (Table 2). The mean falciform fat pad areas of the three groups were not

significantly different from each other (P = 0.1) (Table 2).









-Sham


100%


75%


50%


25%


0%


120


270 420


570


720


Days post-treatment

Figure 3. Breeding trial. Change to a long day-length cycle (Day 120) resulted in the
rapid induction of behavioral estrus and breeding in all five sham cats, whereas
only one of 15 immunized cats was observed to be breeding. All six cats that
bred shortly after Day 120 became pregnant. During the same period three
additional immunized cats displayed behavioral estrus and became pregnant.


-GnRH







40





200


1 160


@ 120 T I X_
S- T I --Sham (n=5)
-o -Non-responder (n=4)
0 1 Responder (n=11)
= 80

C



0
30 130 230 330 430 530 630
Days post-treatment


Figure 4. GnRH antibody titer for female cats (mean + SE). The sham group had
baseline titer for the duration of the study. For nine of the 18 post-treatment
time points the responder cats (cats that did not become pregnant during the
study) had significantly higher GnRH antibody titers than the nonresponder
cats. (*P<0.05.) None of the responder cats had antibody titers that decreased
below 16,000 and all four nonresponder cats (cats that became pregnant during
the study) decreased below 16,000 and became pregnant within 100 days. The
contraceptive titer for these cats ( ) was 16,000.







41



300


250
o

200
3--I-T1
T7
S150 -AT10
0 1- -- -- 0--T14
C T16
S100

50


0
30 130 230 330 430 530 630
Days post-treatment

Figure 5. GnRH antibody titers for cats with high titers from Day 30 through Day 630.
These five responders (cats that did not become pregnant during the study) (T1,
T7, T10, T14, T16) had GnRH antibody titers of 128,000 by Day 30 and none
decreased below a titer of 64,000 for the entire 24-month treatment period,
which was above the contraceptive titer of 16,000 ( ).


300


250
o

200
a -4--T2

150 --T11
-0-T12
--T13
M 100


50
50
0 _-_ ., ,/__,_

30 130 230 330 430 530 630
Days post-treatment

Figure 6. GnRH antibody titers for cats with variable titers. These five responders (cats
that did not become pregnant during the study) (T2, T6, T11, T12, T13) had
titers that peaked, decreased, and then increased again. After the second
increase all five cats maintained this new titer through Day 630. The titers for
these cats did not decrease below the contraceptive titer ( ) of 16,000.










150


6" 120

o



60
I
30


-0--T5
-W-T8
--A-T9
---T15


130 230 330 430 530 630
Days post-treatment


Figure 7. GnRH antibody titers of nonresponders (treated cats that became pregnant
during the study). Cats became pregnant on Day 569 (T5), Day 483 (T8), Day
229 (T9), and Day 534 (T15). The titer of these cats decreased below the
contraceptive titer ( ) of 16,000.

Table 1. Body weight and percent weight gain in cats over a 14-month period following
GnRH immunocontraception. The mean weights of the three groups were not
significantly different from each other on Day 0. The percent weight gain of the
responder group was significantly greater than the nonresponder group and
sham group.
p Weight (kg), % Weight gain,
mean (range) mean (range)
Day 0 (Treatment Day) Day 420 Day 0 to Day 420
Sham 3.54 (3.30 4.18) 4.05 (3.48 -4.66) 15(1 -41)
Nonresponder 3.21 (2.77 3.50) 3.31 (2.62 4.00) 3.5 (-14 31)
Responder 3.34 (2.64 4.20) 4.63 (3.70 5.98) 40 (12-73)


Table 2. Falciform fat pad depth and area in cats 14 months after GnRH immunization.
Fat pad depth of the responder group was significantly greater than the
nonresponder group and sham group. The falciform fat pad areas were not
significantly different between the groups.

Fat Pad Depth (mm) Fat Pad Area (mm2)
Group mean (range) mean (range)
Sham 18.6 (13.2 23.8) 728 (367 1210)
Nonresponder 18.6 (15.2 22.4) 839 (543 1270)
Responder 27.8 (17.2 42.9) 1194 (489 2020)















DISCUSSION

In this study, 15 sexually intact female cats were immunized once against GnRH.

Twenty-four months after immunization it was discovered that five of the treated cats had

granulomas near their injection site. There was no lameness evident and the masses did

not appear to cause the cats any discomfort. It is possible that a lower dose of vaccine

would still be effective, but not cause this type of reaction at the injection site.

No systemic or local adverse reactions were noted during the 24-month observation

period. For the 24 months following treatment, 73% of the treated cats did not become

pregnant or display signs of behavioral estrus. The four nonresponders had GnRH

antibody titers at or below 8,000 when they became pregnant, while none of responders

had a titer below 16,000 during the study. This suggests that the contraceptive titer in

female cats is 16,000.

As expected, the responder cats reacted to the immunocontraceptive treatment in

ways similar to cats that have undergone surgical sterilization. Behavioral estrus was

suppressed and they gained more weight than the sham and nonresponder cats. However,

surgically sterilized female cats have estradiol-17 3 plasma concentrations consistent with

cats in anestrous, whereas the responder cats intermittently demonstrated estradiol-173

concentrations well above expected anestrous values and possibly indicative of estrus

cycling. However, the females were never receptive nor attractive to the male, so if the

cats were cycling, they were not cycling normally.











Many studies have demonstrated weight gain following surgical sterilization [61-63]

and the percent weight gain in these studies (28% to 39%) was consistent with the 40%

weight gain found in the responder cats in the present study.

The number of live kittens per litter born to the nonresponder cats was significantly less

than the number for sham cats. Immunization against GnRH may not prevent pregnancy

in all cats, but it might interfere with some aspect of pregnancy that results in lower

fecundity. One possibility is that decreased LH, FSH, and estradiol-173 led to fewer

follicles developing and fewer ovulated follicles. However, the sample size was very low

and the difference found may have been due to normal variation.

In a study of nine male cats immunized with the same vaccine used in the current

study (50 to 200 [tg), six cats had a GnRH antibody titer above 64,000, undetectable

testosterone concentrations and testicular atrophy. The three cats with an antibody titer

below 64,000 had measurable testosterone concentrations and normal semen quantity

[57].

In a study that targeted a hypothalamic-pituitary-gonadal axis component downstream

from GnRH, seven adult female cats were immunized with lutenizing hormone receptor

(LH-R) encapsulated in an implant and given four booster injections. LH-R antibodies

were detected, progesterone concentrations remained near basal levels, and no behavioral

estrus was observed for approximately 500 days, at which point antibody titers began to

decrease and the cats showed signs of recovering normal physiologic ovarian function.

Estradiol-17P concentrations were not significantly different between the sham cats and

treated cats, even though estrus was observed in the sham cats, but not in the treated cats









[51]. Determining hormonal estrous cycling based on estradiol-173 concentrations

requires estrogen be measured very frequently over the time frame of interest. In the

current study, there were four clusters of 11 every other day blood collection over a 26-

month span. The four clusters of blood collections, each 20 days long, were likely to

reveal an estrous cycle if one occurred during one of those intervals. However, it is

possible that an estrous cycle occurred, but was missed, during the two- to seven-month

intervals between these clustered blood collections. In addition, the basal levels and

estrus profile of estradiol-173 concentrations are highly variable among cats and other

components such as plasma protein content and steroid hormone binding protein may

interfere with the measurement [64].

Increasing the frequency of estrogen measurement would aid in more precise

determination of estrous cycling regardless of basal and maximum concentrations.

However, continuous collection of blood samples would create welfare and health issues

for cats and monitoring of fecal estrogen would necessitate individual housing. To

increase the accuracy of the estradiol-173 assay, a portion of each sample can be used to

prepare an estradiol-173-free aliquot. The steroid is removed by passing the aliquot

through a charcoal column. The estradiol-173 concentration in each sample is

determined by subtracting the value in the aliquot (sample blank) from the estradiol-173

concentration value in the unfiltered portion [64]

Immunization against GnRH in other species has been used as an alternative to

gonadectomy to prevent undesirable behavior or characteristics caused by sex hormones

for more than 25 years [65]. It has been primarily used in juvenile male food animals and

has resulted in decreased aggression, testosterone concentration, and testes size in bulls









[66-70], boars [58,71-73] and rams [74,75]. In female pigs [58,76], sheep [77] and horses

[78] estrus was suppressed; concentrations of lutenizing hormone, progesterone, and

inhibin A were decreased; and ovarian and uterine weights were reduced. Most of these

animals received multiple treatments, and the observation periods were short since most

of the animals were sent to slaughter.

GnRH immunization has been proposed as a humane solution to overpopulation of

wildlife species and has been tested in deer, bison, rats, swine, rabbits, squirrels, coyotes

and horses [79]. In white-tailed deer, three or four treatments of GnRH vaccine resulted

in a fawning rate reduction up to 88% [55]. GnRH vaccination was 100% effective with

three treatments in male and female Norway rats for up to 17 months [55]. A single

treatment in male and female feral swine resulted in reduced testicular and ovarian sizes,

reduced concentrations of testosterone and progesterone, and a 90% reduction in

pregnancy for 36 weeks [58]. A single treatment in bison led to a decrease in

progesterone concentrations and prevented pregnancies for one year [80].

There is a protein very similar to GnRH, named GnRH-II, whose function has yet to

be elucidated. However, there is concern that a vaccine against GnRH could also bind to

GnRH-II and disrupt a non-reproduction function [81]. The safety of the GnRH vaccine

has been widely investigated, though, and no significant safety concerns have been

found. A study in male rats and rabbits found no differences in hematological or

biochemical findings between GnRH-immunized animals and surgically sterilized

animals [82]. At necropsy the only abnormalities were detected in the reproductive

organs. Studies in many species including horses [78], deer [83], and male cats [57],

found no health concerns associated with GnRH immunocontraception.









Contraceptive GnRH antibody titer and effective dose may be different for male and

female animals. The GnRH antibody titer needed to induce contraception in the current

study in female cats (16,000) was lower than the antibody titer needed for male cats

(64,000) treated with the same vaccine [57]. A similar sex difference has been noted in

horses with a contraceptive titer in males of 1,000 and in females of 300 [78]. The

effective dose of GnRH vaccine was found to be lower in male than female swine [58].

Sex differences may be related to the cyclic nature of hormone secretion in females that

is largely absent in males [58]. Since males continuously produce GnRH, it is possible

that the store of anti-GnRH antibodies is depleted more quickly at the sites of interaction

or that GnRH must be suppressed to a greater degree to block reproductive functions in

males than in females.

GnRH has been shown previously to be an effective immunocontraceptive target in

many species, including cats, but in reports the contraceptive activity was not effective in

all of the animals, required multiple treatments, and faded over time. In contrast, a

practical contraceptive for feral cats must be effective in a large fraction of cats for a

substantial duration following a single treatment.

In this report, a single treatment of GnRH conjugated with KLH and adjuvanted with

M. avium and oil achieved long-term contraception in female cats, meeting the minimum

requirements for contraception in feral cats. Targeting of GnRH had the additional

benefit of curbing nuisance behavior associated with estrous cycling. Additional studies

are needed to investigate the full duration of immunity, rate of efficacy, and safety in

both sexes and all ages of cats.















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BIOGRAPHICAL SKETCH

John Friary was born July 24, 1970, in Laconia, New Hampshire. He attended

secondary school in St. Petersburg, Florida, studied for one year at the Georgia Institute

of Technology in Atlanta and then moved to Gainesville, Florida. John graduated from

the University of Florida with a B.S. in environmental engineering in 2004. John became

interested in research to benefit feral cats after volunteering at Operation Catnip, a trap-

neuter-return clinic in Gainesville, Florida.

He entered a graduate program at the College of Veterinary Medical at the

University of Florida to investigate the potential for immunocontraception to reduce the

population of feral cats under the advisement of Dr. Julie Levy. He received his M.S. in

2006.

John will continue research related to the humane control of feral cats.