Overpopulation of free-roaming cats is a global problem in both urban and rural societies (1). It negatively impacts the cats' welfare and health (starvation, diseases, run over by cars, etc.), as well as causing public health risks due to the transmission of zoonotic diseases to humans (e.g., toxoplasmosis, rabies, cat scratch disease, leptospirosis, Q fever, toxocariasis, flea-borne typhus, etc.); some of these are reported to cause important health issues including abortion, blindness, pruritic skin rashes and other various symptoms, as well as mortality (2–6). Free-roaming abandoned and feral cats are non-native predators; they cause substantial wildlife destruction and ecosystem disruption, including the deaths of millions of birds, small mammals, reptiles, amphibians, and fish (7). Furthermore, disease transmission from cats to livestock and wild animals, attacks on humans, traffic accidents, and nuisance behavior, are just part of many concerns involved with this matter (6, 8, 9). Currently, surgical sterilization (ovariohysterectomy; orchiectomy) is the most common practice to control free-roaming cat reproduction, mainly via “Trap-Neutering-Return” (TNR) programs. It provides a permanent solution, should be done only once, and may positively affect the health and welfare of individual feral cats (10). However, the surgical approach is expensive, and requires surgical and anesthetic materials, equipment, as well as skilled veterinarians. Furthermore, it may cause significant stress to the animals (9, 11, 12). Therefore, the surgical approach practically failed to provide a solution for the millions of free-roaming cats that are confined and euthanized annually (9, 13–15). An efficient non-surgical sterilization/contraception method could have a significant impact on the control of cat overpopulation. However, to the best of our knowledge, despite significant efforts over the several last decades, there are currently no efficient commercial products that can be used for mass non-surgical sterilization/contraception of free-roaming cat populations (15, 16).

Rabies is a deadly, vaccine-preventable, zoonotic, viral disease, which may infect people and animals (17). According to the World Health Organization report (updated in April 2020), most human cases worldwide are due to disease transmission from domestic dogs. However, mandatory dog vaccination programs have halted the natural spread of rabies among domestic dogs in many countries. According to the information from the USA Centers for Disease Control and Prevention (CDC; updated on April 2020), as well as from other reports (4, 18, 19), in the USA, cats have become the companion animal species most commonly reported as rabid. Accordingly, in many endemic countries, including Israel and the USA, combined animal reproduction control and rabies vaccination programs are aspired.

Reproduction in the domestic queens is affected by photoperiod, as this species is considered as seasonal, polyestrous, long day breeder, with multiple ovulations induced following vaginal stimulation during mating (20, 21). Gonadotropin-Releasing Hormone (GnRH), which is released from the hypothalamus, is the master hormone of reproduction; it controls steroidogenesis and gametogenesis by stimulating the release of the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), triggering a cascade of endocrine effects that lead to sperm production in males and follicular development and ovulation in females. Gonacon is an anti-GnRH vaccine, developed by the National Wildlife Research Center, USDA APHIS Wildlife Services, USA. This immunocontraceptive vaccine was designed to trigger the production of antibodies that neutralize GnRH. It has been tested in many species, including white-tail deer (22), elk (23), horses (24, 25), cattle (26), ferrets (27), domestic, and wild pigs (28), as well as to a limited extent in dogs (29) and cats (30–33). However, none of the four previous studies conducted on cats included measurements of rabies antibodies in Gonacon-vaccinated cats, even in the studies in which cats were vaccinated with both vaccines (32, 33). Furthermore, in previous Gonacon studies conducted on cats, animals were not feral cats; they were either specific-pathogen-free (SPF) laboratory cats (30, 31), cats from research colony (32), or well-maintained friendly cats obtained from animal control agencies or from private individuals who posted cats for rehoming (33). All cats were kept in excellent health and body conditions, and were accustomed to human interactions. However, many previous reports documented that feral cats often suffer from high rates of morbidity and pre-mature mortality due to infectious (e.g., FIV, FeLV, parasites) and non-infectious diseases, malnutrition, poor body condition, traumas, etc. (9, 10, 34–36). Furthermore, most of these cats are typically too fearful and too wild to be handled awake, as they are not accustomed to close interactions with people. Therefore, feral cats, which are the main target population for non-surgical contraception/sterilization, often suffer from acute and chronic health problems and are under chronic stress due to their lifestyle and environment (5, 9, 10, 34–36). Potentially, these suboptimal conditions might weaken the immune response and antibody production following vaccination, such as to Gonacon and rabies vaccines, as has been documented for other vaccines and species (37–42). As recently reviewed by Zimmermann and Curtis (41), several factor might affect the immune respond and antibody production of individuals following vaccination; these include age, sex, genetics, comorbidities, infections, parasites, microbiota, pre-existing immunity, acute and choric stress, body condition, nutritional status, micronutrients (vitamins A, D, and E and zinc), as well as environmental factors, and vaccine factors (vaccine type, product, strain, and batch; vaccination route; combinations of vaccines, cet.). For example, some studies indicated poorer humoral response to influenza vaccine in humans with chronic diseases (37); Chronic inflammatory processes like rheumatoid arthritis alter the immune system and antibody formation (43); furthermore, depression and dementia as well as psychological stress are associated with poor antibody response and a higher range of inflammation markers (44). Thus, vaccines should optimally be tested in the typical target population and conditions.

Therefore, the objective of the current study was to examine the short-term safety and efficacy of the anti-GnRH vaccine (Gonacon), given either as a single dose, or two doses 3 weeks apart, in combination with rabies vaccine, in mature feral female cats. Our main hypotheses were that: (1) in the short term (14 weeks), the combined vaccination with Gonacon and rabies will not be harmful and will not have adverse side effects; (2) antibody titers against GnRH and rabies will increase following the combined vaccination; (3) as compared to a single dose Gonacon vaccination, two doses of Gonacon given 3 weeks apart (booster) will induce higher anti-GnRH titer, but anti-rabies antibodies titer will not differ; (4) development of anti-GnRH antibodies will be associated with suppression of the ovarian function; and (5) antibody titers against rabies will not be negatively influenced by the simultaneous vaccination approach and will remain protective during the study period.

The rationale of this study was to examine the short-term safety and efficacy of combined vaccination with anti-GnRH (Gonacon) and rabies vaccines, specifically in female feral cats, which often suffer from disrupted health conditions and experience high-stress levels, which potentially may alter vaccination efficacy. Our study indicated that in the short term, the combined vaccination with Gonacon and rabies is safe and effective in female feral cats, with no apparent benefits for two Gonacon vaccinations as compared to a single dose. Over the study period, no local or systemic adverse health concerns were detected, in accordance with our hypothesis. There were no differences in serum rabies antibody titers among groups, and queens kept a protective titer throughout the study. Anti-GnRH antibodies were detected in all Gonacon-vaccinated queens, excluding one queen (GonaconX2-Rabies group). Furthermore, evaluation of serum AMH concentrations, vaginal cytology, and ovarian histology suggested that reproductive cyclicity was indeed suppressed in Gonacon-vaccinated queens, as hypothesized.

Different from previous studies in which researchers examined the effects of Gonacon in experimental cats or pets cats from colonies, in the current study we utilized feral cats. In previous Gonacon studies (30–33), animals were domestic short hair cats kept in excellent health and body conditions, and were accustomed to human interactions. In the studies of Levy et al. (30, 31), specific-pathogen-free (SPF) males and females, respectively, were acquired from a commercial vendor and housed in climate-controlled indoor spaces with controlled light cycles (30, 31). Vansandt et al. (32) used ovariohysterectomized cats from a research colony at the Cincinnati Zoo and Botanical Garden's Center for Conservation and Research of Endangered Wildlife. In the study conducted by Fischer et al. (33), well-maintained friendly cats were obtained from animal control agencies, or from private individuals who posted cats for rehoming on a classified advertisement website; all cats were in excellent body condition and health. However, it is well-documented that feral cats often suffer from high rates of pre-mature mortality and morbidity due to infectious and non-infectious diseases, internal and external parasites, starvation and poor body condition, weather extremes, trauma, etc. (9, 10, 34–36). Furthermore, most of these cats are not accustomed to contact with people and are typically too fearful and too wild to be handled. Therefore, feral cats often suffer from health issues and are under chronic stress conditions, which might weaken the immune response to vaccination (41), such as to the combined vaccination with Gonacon and rabies vaccines. Therefore, although some adjustments were needed in order to perform a study with feral cats as compared to well-maintained friendly cats (in regards to capturing, management, handling, cet.), the study was designed to examine the short term safety and efficacy of our combined vaccination approach specifically in feral queens, as they are the main target population for non-surgical contraception/sterilization.

Accordingly, in our study, the feral queens were captured by traps, and were maintained under ambient summer temperature and light. The study was conducted during the Israeli summer to avoid suppressing effects of the photoperiod or climate conditions on the reproductive system (8, 21, 53, 54). Furthermore, queens were vaccinated 24–72 h after they were captured, while their health condition was still typical to that of feral cats. Indeed, our physical examination and blood tests performed at study initiation, just prior to vaccination, indicated that many of the queens were in suboptimal body condition, and some had signs of anemia, inflammation, or muscle injuries. Furthermore, many cats had a high eosinophilic count in their blood, suggesting high infestation of gastrointestinal or other parasites. Despite the suboptimal health condition of the feral cats in the current study, the combined vaccination with Gonacon and rabies was safe and effective in the short term. None of the animals showed any local or systemic adverse reactions. In the study of Levy et al. (31), late-onset (2 years post-treatment) granulomatous injection-site masses developed in 5/15 (33%) of the Gonacon-vaccinated female cats. However, in another study conducted by the same group, no injection site masses were detected in male cats at a period of 6 months post-injection (30). In the study conducted by Vansandt et al. (32), four cats developed a sterile, painless, self-limiting mass at the site of Gonacon injection, after only few weeks. Fisher et al. (33) reported injection-site reactions ranging from swelling to transient granulomatous masses in 45% (n = 9/20) of vaccinated cats, beginning at least 1 month after treatment. Furthermore, in another study conducted in our laboratory (Novak and Raz, unpublished data), we also documented injection-site masses 3 to 12 months post-Gonacon vaccination in 7/44 (16%) owner-owned cats. Therefore, it is possible that in the current study, masses were not detected at all as the monitoring period of 14 weeks was relatively short; furthermore, we cannot rule out the possibility of local reactions later on, after the queens were released back to their environment or adopted. Regarding the systemic health conditions of the queens, although it was initially suboptimal for many of the animals, as typical for feral cats in Israel and other places, it improved over the study period. This improvement in the cats' health is probably due to the housing conditions during the study which allow the queens to recover; i.e., good availability of food, water, and shelter; reduced risks of infectious diseases and injuries; as well as reduced stress due to the acclimation of the cats to their environment.

Despite the initial suboptimal health condition of the feral cats in the current study, the combined vaccination with Gonacon and rabies was effective in the short term. Following vaccination with rabies, all queens from all the groups produced antibodies against rabies, which remained above the level considered protective. Bender et al. (29) vaccinated dogs with both Gonacon and a commercial rabies vaccine (Defensor 3, Pfizer, Inc., New York, NY, USA), and reported no adverse effects of simultaneous vaccination on rabies virus neutralizing antibody production. None of the previous studies measured rabies antibodies in Gonacon-vaccinated cats, even if cats were vaccinated with both vaccines (32, 33). Nevertheless, our results support our hypothesis that antibody titers against rabies will not be negatively influenced by the simultaneous vaccination approach and will remain protective during the study period.

Following vaccination with Gonacon, anti-GnRH antibodies titer increased in all vaccinated queens within 2–4 weeks, except for one non-responder queen from the GonaconX2-Rabies group. In all responder queens, anti-GnRH titer remained high during the study period, except for one queen from the GonaconX1-Rabies group, in which it reduced in the last two examinations. This queen was identified as FIV positive, which could have affected the immunogenicity of the Gonacon vaccine; however, the production of anti-rabies antibodies did not appear to be negatively affected in this queen. Nevertheless, the possibility of immunogenicity alteration of any immunocontraception vaccine due to FIV infection is interesting and warrants further investigation, as FIV might be quite prevalent among free-roaming cat populations (35, 55–57). There was no significant difference in GnRH antibodies between the GonaconX1-Rabies and GonaconX2-Rabies groups. However, as our study focused on the short-term effects, we cannot rule out the possibility that repeated Gonacon vaccinations would extend the effective period, and potentially provide extended contraception. Furthermore, as our assay was limited to series dilutions from 1:8,000 to 1:64,000, it is possible that the double Gonacon vaccination could have shown positive results in higher dilutions than 1:64,000. Previous studies showed that some cats did not respond to the Gonacon vaccine (i.e., no anti-GnRH antibodies), while others responded to the vaccine with anti-GnRH antibodies detected for various periods after injection. The only other study which compared a single to double Gonacon vaccination could not detect differences in the anti-GnRH antibody at 1:1,024 dilution at 4-month post-vaccination of ovariectomized queens (3 cats in each group) (32). Nevertheless, our hypothesis that two doses of Gonacon would induce higher anti-GnRH titer, as compared to a single vaccination, require further long-term study.

There are several relevant approaches to evaluate the function of the reproductive system following the administration of an immunocontraceptive agent, but it is clear that breeding provides the most definitive results. However, breeding trials, particularly in overpopulated species like cats, are practically challenging, require time and resources, as well as carry some ethical dilemmas, as to the destiny of the newborn offspring (or performing ovariohysterectomy of pregnant animals). Therefore, in the current study, we were looking for other alternatives. Accordingly, we evaluated the effects of Gonacon vaccination on the reproductive system by a combination of serum AMH concentrations measurements, vaginal cytology, and ovarian histology; which all suggested that the reproductive cyclicity was indeed suppressed in Gonacon-vaccinated queens. In females, AMH is a hormone produced in the ovaries by small and developing follicles, and has a pivotal role in the regulation of ovarian follicle reserve and folliculogenesis. Studies in other species have shown that there is a correlation between the number of developing follicles in the ovary and serum AMH concentration (58). It has also been found that when a queen is spayed, AMH serum concentration drops abruptly (50, 59). Accordingly, we initially hypothesized that AMH serum concentrations would be reduced following ovariohysterectomy, as well as following vaccination with Gonacon, due to the vaccine potential suppression effect on gonadotropins and folliculogenesis. Indeed, in the current study, AMH serum concentration was extremely low following ovariohysterectomies, but it remained high in intact queens, in agreement with previous studies in cats and other species (50, 59, 60). Interestingly, in queens vaccinated with Gonacon, the reduction in AMH concentration was gradual. Eventually, at study completion, AMH serum concentrations were lower in all Gonacon-vaccinated queens, as compared to study initiation, except in the non-responder queen from the GonaconX2-Rabies group, in which AMH concentrations remained stable throughout the study. Furthermore, statistical analysis of all serum samples obtained from Gonacon-vaccinated queen revealed that AMH concentrations were significantly lower in serum samples with high anti-GnRH titers (titer 0, 6.7 ± 1.1 ng/mL, vs. titer 1:64, 3.9 ± 0.7 ng/mL; p = 0.0429). Overall, these findings suggest, for the first time to the best of our knowledge, that measuring AMH serum concentration is a valuable method for evaluating the efficacy of immunocontraception.

Evaluation of vaginal cytology smears and histology of the ovaries at study completion indicated that none of the Gonacon-vaccinated queens was in estrus, except the non-responder queen. However, the assessment of these results, particularly those of the vaginal cytology, could have been clearer if other analyses could have done, such as measurements of other sexual hormones (e.g., estrogen, LH, and FSH), and ultrasonography of the reproductive tract. Nevertheless, the typical histological finding in the ovaries of Gonacon-vaccinated queens was the presence of a high number of degenerated oocytes, and lack of antral follicles, alongside a low percentage of superficial cells in vaginal smears. In contrast, in intact animals (samples of the Ovx-Rabies group just before spaying; and samples of Intact-Rabies at study completion), ovaries had normal histology, with various follicles (primary, secondary, and antral follicle), and very few degenerative oocytes. Interestingly, the ovaries of the non-responder queen from the GonaconX2-Rabies group were histologically similar to those of the controls, and the vaginal cytology smear had a high number of superficial cells (~90%) indicated that this female was in estrus. In our histologic evaluation, we were not able to detect apparent differences in the primordial follicle reserve in the ovaries, nor differences in the endometrium. Future studies should consider exploring the possible short and long-term effects of immunocontraceptive agents on the primordial follicle reserve, as the reduction or elimination of this pool would be beneficial for long-term contraception/sterilization.

Out of the three previous publications regarding Gonacon vaccination in female cats, two studies examined the contraceptive effect. In the long-term study conducted by Levy et al. (31), 15 queens were vaccinated with the early generation Gonacon vaccine, and were exposed to intact males. Gonacon-vaccinated queens re-gained fertility at a later time, as compared to controls; of the 15 vaccinated queens, 93% were infertile for at least 1 year, 73% for 2 years, 53% for 3 years, and 40% for 4 years; 27% were still infertile at the conclusion of the 5-year study. In the study conducted by Vansandt et al. (32), ovariectomized queens were used, and therefore, the possible effects of Gonacon vaccination on the reproductive system could not be determined. Later on, Fischer et al. (33) reported poor contraceptive efficacy of the Gonacon vaccine in colony cats. In their study, queens were exposed to intact males 4 months after vaccination with the newer Gonacon version. All control queens (n = 10/10) and 60% (n = 12/20) of Gonacon-vaccinated queens became pregnant within 4 months of the introduction of males. Two additional vaccinated queens became pregnant (70%; n = 14/20) within 1 year of treatment. Overall, vaccinated queens had a significantly longer (P = 0.0120) median time to conception (212 days), and lower fetal counts. Nevertheless, the results of their study were disappointing. In the current study, which utilized the same Gonacon formulation as in the study of Fischer et al. (33) but of different batch, our approach indicated that ovarian cyclicity was suppressed in all queens who respond to the Gonacon vaccine, as detailed above. The differences among studies could be due to variability in the vaccine itself [overtime differences in formulas, as reviewed by Benka et al. (15); different batches]; differences between cats (laboratory vs. free-roaming vs. stray cats); environmental conditions; as well as individual variability.

There are several limitations to our study. It is possible that due to the small number of cats in each of the groups, as well as the short duration of the study, we were not able to detect uncommon adverse reactions, or other safety issues, that could have been detected in a large group of vaccinated cats or at a later stage. Also, the validity of the vaccines' immunogenicity and the assessment of the proportion of non-responder cats (i.e., no antibody formation), as well as the assessment of reproductive effects, based on a small number of vaccinated cats examined over a short period, is limited. In regards to the assessment of the cats' health before and after vaccination, future studies should consider performing additional lab tests, such as urinalysis and fecal examination for the detection of intestinal parasites, which were not performed in our study, and could have provided a better understanding of the cat health status at vaccination. In regards to reproductive effects, other methods, such as assessment of other sexual hormones (e.g., estrogen, LH, FSH), ultrasonography of the reproductive tract, and breeding, should be considered and combined. In addition, during our study period, the queens were housed in individual cages, in conditions that were not similar to that of feral cats in an urban environment. Our study emphasizes the need to explore the safety and efficacy of the Gonacon vaccine in several target populations, and we therefore believe that further long-term studies are warranted, including in populations of stray cats in their natural environment, as well as in pet indoor vs. outdoor cats.

In summary, to the best of our knowledge, this is the first study that examined the short-term safety and efficacy of combined vaccination approach, which included anti-GnRH vaccine (Gonacon), given either as a single dose, or as two doses 3 weeks apart, in parallel with rabies vaccine, specifically in female feral cats. Overall, our results indicate that in the short term, the combined vaccination with Gonacon and rabies is safe and effective in female feral cats, with no apparent benefits for two Gonacon vaccinations as compared to a single dose. Following the combined vaccination, anti-GnRH and rabies antibodies were detected, despite the suboptimal health condition of the queens. Furthermore, evaluation of serum AMH concentrations, vaginal cytology, and ovarian histology suggested that reproductive cyclicity was indeed suppressed in Gonacon-vaccinated queens for at least 3 months. However, future studies should examine the long-term effects of contraceptive agents in free-roaming cats in their natural environment.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by The Hebrew University's Institutional Animal Care and Use Committee.

SN: investigation, data analysis and interpretation, methodology, and writing—original draft. BY: conceptualization, investigation, data curation, writing—review, and editing. SS: investigation, data curation, writing—review, and editing. LM, ST, RN, and LJ: investigation, writing—review, and editing. RK: conceptualization, writing—review, and editing. DE: providing Gonacon vaccines, investigation, data analysis and interpretation, writing—review, and editing. TR: conceptualization, supervision, data curation, formal analysis, funding acquisition, project administration, investigation, methodology, validation, visualization, writing—review, and editing. All authors: contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.