兽研摘要
摘要
家猫种群过剩对公共卫生、生物多样性和动物福利构成风险。手术绝育虽被广泛采用，但存在诸多局限性。Gonacon是一种抗GnRH疫苗，提供了非手术替代方案；然而，对于生活在自然栖息地中的有主母猫以及同时接种狂犬病疫苗的数据尚缺乏。我们评估了宠物母猫同时接种Gonacon-狂犬病疫苗的1年免疫原性、安全性和有效性，并比较了室内猫与室外猫之间的反应。在一项前瞻性现场研究中，36只未绝育母猫（≥6月龄）接受了Gonacon-狂犬病疫苗接种（Gonacon-Rabies，n=30）或卵巢子宫切除术加狂犬病疫苗接种（Ovx-Rabies，n=6）。母猫仍由主人照料，并在0、3、6和12个月时进行监测。25只母猫完成了随访（22只Gonacon-Rabies，3只Ovx-Rabies）。注射部位结节仅在Gonacon疫苗接种后观察到（13.6 vs 0%；P=0.0564）。在3、6和12个月时，Gonacon-Rabies母猫的抗GnRH血清阳性率分别为68.2%、31.8%和27.3%。在应答者中（n=15），53.3%仅在3个月时呈阳性，6.7%持续阳性至6个月，40%在整个研究期间保持阳性。所有Gonacon疫苗接种无应答者均为室外母猫（室外：7/13；53.8%；室内：0/9；P=0.0167）。室外母猫的抗GnRH免疫持续时间较短（2.1±0.9 vs 8.3±1.5个月；P=0.0005）。生殖事件（发情或妊娠）在室外Gonacon-Rabies母猫中发生率为76.9%，室内为11.1%（P=0.0075）。AMH浓度与抗GnRH水平呈负相关（r=-0.53；P<0.0001）。狂犬病抗体保持保护水平（≥0.5 IU/mL），但室外母猫低于室内母猫（P=0.0490）。主人对室内Gonacon-Rabies母猫及无生殖事件母猫的满意度更高。同时接种Gonacon-狂犬病疫苗在1年内安全且部分有效，室内母猫的应答性更好。
通俗摘要
家猫繁殖力极强，导致种群过剩，对动物福利、野生动物生物多样性和公共卫生产生负面影响。尽管手术绝育被广泛用于防止猫繁殖，但其在实践、经济和伦理方面存在重要局限性。我们研究了在正常家庭条件下生活的雌性宠物猫中，与狂犬病疫苗同时接种的抗GnRH避孕疫苗（Gonacon）的安全性、免疫反应和生殖效果。联合疫苗接种总体安全，大多数母猫维持了针对狂犬病的保护性免疫。然而，避孕效果在动物间差异很大。室内母猫比室外母猫产生了更强、更持久的免疫反应，发情迹象和妊娠更少。相比之下，许多室外母猫的免疫反应较弱且持续时间较短。这些发现表明，联合疫苗接种可能为部分宠物猫的繁殖控制提供一种实用的非手术方法，但生活条件可强烈影响其有效性。
关键词：免疫避孕，抗GnRH疫苗，gonacon，母猫，家猫，狂犬病，猫种群管理
引言
家猫种群过剩在城市和农村地区均构成重大全球性挑战（Gunther et al. 2015, 2022, Ma et al. 2023）。全球家猫种群估计达数亿只，其中许多作为宠物饲养，且家庭饲养多只猫的情况很常见（Kennedy et al. 2020）。有主猫或宠物猫通常属于三种栖息类别之一：室内猫，仅局限于家庭内；室内/室外猫，在室内生活但可接触室外环境；室外猫，主要在室外生活但仍接受主人不同程度的照料，如食物、住所和医疗（Ma et al. 2023）。有主室外猫的失控繁殖，加上流浪猫和弃养猫的增多，导致全球多个地区出现了自由放养猫种群（Gunther et al. 2020, Kennedy et al. 2020, Ma et al. 2023）。
自由放养猫的过度繁殖与重大公共卫生风险相关，原因是人畜共患病向人类传播，如狂犬病、弓形虫病、猫抓病、钩端螺旋体病、Q热和弓蛔虫病（Gerhold & Jessup 2013, Roebling et al. 2014）。猫还可能介导疾病向家畜和野生动物传播（Wareth et al. 2017, Trouwborst et al. 2020）。此外，它们可能攻击人类、卷入交通事故，并造成滋扰，如猫尸体存在、噪音和污物，以及侵入私人或商业场所（Gunther et al. 2015, Crawford et al. 2019）。而且，自由放养猫和野猫是非本土捕食者，常捕杀鸟类、爬行动物、两栖动物、小型哺乳动物和鱼类，其中许多猎物物种濒临灭绝（Medina et al. 2011, Loss et al. 2022）。由于这些显著的生态破坏和野生动物减少，家猫已被列入世界100种最严重非本土入侵物种（Lowe et al. 2000, Loss et al. 2022）。此外，自由放养猫的过度繁殖与猫群中营养不良、疾病易感性和损伤的高患病率相关，对其福利和健康造成不利影响（Gunther et al. 2015, 2018, Ma et al. 2023）。
狂犬病是一种致死性人畜共患病毒病，影响人类和动物，在许多国家呈地方性流行（Boutelle et al. 2025）。根据世界卫生组织2020年至2024年发布的报告，狂犬病每年在全球造成估计59,000–60,000例人类死亡，绝大多数由犬介导传播所致。然而，在许多国家，包括美国，猫是最常报告患狂犬病的伴侣动物（Gerhold & Jessup 2013, Ma et al. 2024, Boutelle et al. 2025）。因此，在过去几十年中，已努力控制自由放养猫的过度繁殖并预防狂犬病（Roebling et al. 2014）。然而，不同栖息环境对家猫狂犬病疫苗免疫原性和反应性的影响仍不清楚。
目前，手术绝育是预防 owned cats 生育和管理自由放养猫种群的主要方法，通常通过诱捕-绝育-接种（狂犬病）疫苗-放归（TNVR）项目实施（Roebling et al. 2014, Murray et al. 2015, Gunther et al. 2022）。然而，手术方法有明显局限性。需要麻醉材料、专用设备、熟练兽医的专业知识，以及对自由放养猫而言，诱捕和运输至适当设施的额外费用，共同造成其巨大的财务和实践负担（Lohr et al. 2013, Benka et al. 2022, Gunther et al. 2022）。此外，手术操作给相关动物造成相当大的应激、疼痛和不适（Pereira et al. 2018, Crawford et al. 2019）。操作的侵入性，连同随后的恢复期和兽医诊所的不熟悉环境，导致猫经历痛苦，这不仅从伦理角度令人担忧，也因其不良健康影响而令人担忧（Adin 2011, Pereira et al. 2018, Klintip et al. 2022）。此外，猫的手术绝育操作可导致术中和术后后遗症，如麻醉并发症、出血、手术部位感染、伤口裂开、缝线不良反应、血清肿或血肿形成以及泌尿道问题（Adin 2011, Pereira et al. 2018）。因此，尽管其优势是为个体动物提供永久解决方案，但与成本、应激、健康影响和后勤挑战相关的内在限制阻碍了手术方法的可扩展性和有效性。实际上，手术方法未能减轻自由放养猫种群的大规模繁殖（Root Kustritz 2012, Roebling et al. 2014, Benka & Levy 2015, Crawford et al. 2019）。此外，许多猫主人因这些担忧或伦理和宗教信念而不愿让其宠物接受手术干预（Root Kustritz 2012, Murray et al. 2015）。然而，尽管进行了广泛研究，目前尚无用于 owned cats 和自由放养猫大规模非手术绝育的商业产品（Kutzler & Wood 2006, Benka & Levy 2015）。
Gonacon是由美国农业部动植物卫生检验局野生动物服务处国家野生动物研究中心开发的一种抗GnRH疫苗，旨在通过产生GnRH中和抗体来抑制生殖（Miller等，2008）。既往研究其对于猫的影响大多涉及非自然环境中的动物（Levy等，2011；Vansandt等，2017；Fischer等，2018），如实验室或研究群体的猫，这些猫与暴露于各种环境应激源和健康挑战的野猫和户外猫存在显著差异（Slater，2007；Gunther等，2015；Crawford等，2019）。值得注意的是，关于Gonacon与狂犬病疫苗同时接种于同一只猫时的免疫原性信息有限，这妨碍了依赖协调避孕和狂犬病控制策略的项目的循证决策。此外，关于Gonacon疫苗接种（单独或与狂犬病疫苗联合接种）在生活在自然环境（室内或室外）中的主人拥有的宠物猫中的数据非常有限。然而，我们最近报道了在主人拥有的公猫中联合抗GnRH和狂犬病疫苗接种的1年安全性、免疫原性和生殖效果（Novak等，2026）。然而，关于在自然条件下生活的主人拥有的母猫（queens）中联合Gonacon-狂犬病疫苗接种的安全性、免疫原性和有效性的信息仍然匮乏。为填补这一知识空白，我们还在主人拥有的母宠物猫中开展了一项平行的为期1年的研究，本文即对此进行报道。
本研究的主要目的是评估在自然环境中生活的主人拥有的母猫中同时接种Gonacon和狂犬病疫苗为期1年的安全性、免疫原性和有效性，并比较室内猫和室外猫之间的反应。我们的主要假设是：i）Gonacon-狂犬病疫苗接种将是安全的，不会导致不良反应；ii）针对GnRH和狂犬病的抗体反应将在整个研究期间维持，且室内和室外母猫之间无差异；iii）狂犬病抗体反应不会因同时接种而受到负面影响，并将在整个研究期间保持保护性水平（≥0.5 IU/mL）；iv）抗GnRH血清阳性将与生殖活动抑制相关。
材料与方法
动物与研究设计
本研究按照希伯来大学机构动物护理与使用委员会（IACUC）伦理批准（MD-15-1411-2）进行。以色列中部地区的猫主人通过向该地区兽医诊所发送电子邮件、向Koret兽医学院教职员工和学生发送邮件以及通过在线广告（如与动物相关的Facebook群组和网站）受邀将他们的母猫纳入本研究。联系研究团队的母猫主人获得了相关信息，动物由其主人自愿纳入，无报酬。所有主人在其母猫纳入研究之前均获得并签署了知情同意书。母猫在可参与时被纳入并接受 assigned 治疗；因此，个体母猫在全年不同时间进入研究。一名持证兽医执行了研究中的所有动物操作（S N）。纳入标准为6月龄或以上、兽医体格检查未发现异常的健康母猫。纳入研究的母猫在入组时根据主人报告的疫苗接种史和接种前未检测到狂犬病抗体浓度被认为是狂犬病疫苗未免疫状态。
最初共有36只母猫被招募进入这项现场研究，并分配到两组之一。在研究开始时，Gonacon-狂犬病组的母猫（n = 30）同时接种抗GnRH（Gonacon）和狂犬病疫苗，而Ovx-狂犬病组的母猫（n = 6）接受卵巢子宫切除术并接种狂犬病疫苗。在为期1年的研究期间，两组的母猫均留在其自然栖息地，由其主人护理和拥有。
治疗、采样和监测
研究团队在整个研究期间对母猫进行了四次检查和采样：在研究开始时（时间0，即进行初始治疗时；即疫苗接种和卵巢子宫切除术），以及疫苗接种后3、6和12个月。母猫通过联合使用右美托咪定（0.02–0.04 mg/kg；Orion Pharma，芬兰）、布托啡诺（0.2–0.4 mg/kg；肌肉注射；Richter Pharma AG，奥地利）和咪达唑仑（0.1–0.25 mg/kg肌肉注射；Rafa Laboratories，以色列）进行镇静，以进行兽医体格检查、疫苗接种和血液样本采集。操作结束时通过皮下注射阿替美唑（0.05–0.10 mg/kg；Orion Pharma，芬兰）拮抗右美托咪定的镇静作用以促进恢复。母猫仅在完全从镇静中恢复且精神活泼、警觉、反应灵敏后才被释放回其家中由其主人护理。
在研究开始时，每只母猫通过左大腿皮下注射一种获批的狂犬病疫苗（Rabisin；Merial，法国）进行狂犬病免疫接种。同时，使用USDA-APHIS NWRC（美国科罗拉多州柯林斯堡）提供的疫苗材料肌肉注射Gonacon免疫制剂。如先前所述（Novak et al. 2021, 2026），将500 μL剂量注射到右侧股四头肌。注射前剪去每个注射部位的毛发，并用70%异丙醇对皮肤进行消毒准备。
每次检查时，由认证兽医进行完整的体格检查，包括采用5分制评估体况（Baldwin et al. 2010）以及检查注射部位。通过颈静脉穿刺将血液采集到不含抗凝剂的普通管和含钾-EDTA的试管中。普通管中的血液待凝固后离心，血清样本储存于−80°C直至研究完成时进行分析；这些分析包括抗狂犬病和抗GnRH抗体水平、FeLV和FIV血清学检测以及血清抗苗勒管激素（AMH）浓度分析。含钾-EDTA试管中的血液样本用于测量红细胞压积（PCV），使用粘土密封的血细胞比容管离心约3分钟；血浆总固体（TS）通过将血浆直接置于折光仪（Clinical Refractometer RHC-200 ATC，Cmall，Medent，以色列）上测量。
每3个月，在定期检查期间或通过电话随访，要求主人填写一份关于其母猫饲养条件（即室内或室外）、总体健康状况和生殖行为的问卷。此外，在整个研究期间，指导主人实时向研究团队报告任何行为或健康异常、发情迹象或生殖相关事件，包括交配、妊娠或分娩。
FIV/FeLV血清学检测
为进一步评估总体健康状况，对研究 midpoint（疫苗接种后6个月）采集的血清进行猫免疫缺陷病毒（FIV）抗体和猫白血病病毒（FeLV）p27抗原检测。使用商业FIV/FeLV检测试剂盒（SNAP Combo Plus，IDEXX，美国）按照制造商说明进行分析。母猫的FeLV疫苗接种史未知；然而，此类疫苗接种预计不会干扰使用SNAP Combo Plus检测法进行FeLV抗原检测（Little et al. 2020）。
抗GnRH抗体的检测
在0、3、6和12个月采集的血清样本冷冻保存，并在研究完成时运送至USDA-APHIS国家野生动物研究中心（美国科罗拉多州柯林斯堡），按照先前发表的方法（Levy et al. 2011, Novak et al. 2021, 2026）通过酶联免疫吸附试验（ELISA）分析抗GnRH抗体。血清样本用磷酸盐缓冲液（PBS）按1:8,000稀释。所有样本均做双份分析，抗体反应通过光密度（OD）测量进行定量。每个检测板上均包含双份阳性对照样本以监测检测性能。抗GnRH血清阳性的阈值使用所有母猫的疫苗接种前样本确定，定义为平均OD值加三个标准差。OD值超过该阈值的样本被归类为血清阳性，而低于阈值的样本被视为血清阴性。为尽量减少分析变异，所有样本均由同一操作者分析，该操作者对样本身份、治疗组和饲养状态不知情。此外，同一只母猫的所有样本在同一检测批次内分析。批内和批间变异系数分别为12.3%和9.5%。
抗狂犬病抗体的检测
在0、3、6和12个月采集的血清样本使用快速荧光灶抑制试验（RFFIT）分析狂犬病病毒中和抗体，该试验由Kimron兽医研究所（以色列农业部兽医服务与动物卫生）的WOAH认证狂犬病参考实验室按照先前所述方法（Yakobson et al. 2015, Novak et al. 2021, 2026）进行。RFFIT被认为是测量狂犬病病毒中和抗体的参考方法，并已证明具有高诊断性能（即100%灵敏度和89%特异性）；因此被世界卫生组织视为金标准（Bahloul et al. 2005, Yakobson et al. 2015）。简言之，系列血清稀释液在阳性和阴性对照存在下与狂犬病病毒孵育，随后加入小鼠神经母细胞瘤细胞（CDC，美国佐治亚州亚特兰大）。孵育后，细胞固定，用异硫氰酸荧光素（FITC）偶联的抗狂犬病抗体（Fujirebio，美国）染色，并通过荧光显微镜（Olympus BX40；激发波长470 nm；×200放大倍数）检查。计算狂犬病病毒中和抗体浓度并以国际单位每毫升（IU/mL）表示。抗体浓度≥0.5 IU/mL被认为表明具有足够的血清学保护（Yakobson et al. 2015）。
血清抗苗勒管激素分析
使用商业ELISA试剂盒（AMH Gen II；Beckman Coulter Inc.，美国），按照制造商说明及既往已验证适用于猫样本的发表方法（Axner & Strom Holst 2015, Novak et al. 2021），对所有采集样本中的血清AMH浓度进行定量。简言之，将300 μL AMH Gen II检测缓冲液与60 μL校准品、质控品或血清样本混合，取120 μL该混合物双份加入检测板。孵育并洗涤后，加入100 μL生物素标记的抗AMH抗体。再次孵育并洗涤后，加入100 μL链霉亲和素-酶结合物。第三次孵育和洗涤循环后，加入100 μL TMB底物溶液，加入100 μL提供的终止液终止反应。使用SpectraMax Paradigm多模式微孔板读数仪（SpectraMax Paradigm Multi-Mode Detection Platform，Molecular Devices，奥地利）在450 nm处测定吸光度，以620 nm作为参考校正。该检测的批间和批内变异系数分别为8.2%和2.4%。
主人满意度
在1年随访期结束时，要求Gonacon–Rabies组母猫的主人完成一份简要问卷，评估其对Gonacon–狂犬病疫苗接种策略的总体满意度以及向其他未绝育母猫主人推荐该方案的可能性。问卷包括两个项目：i）主人是否对Gonacon–狂犬病疫苗接种抑制其母猫生殖相关体征的有效性感到满意；ii）他们是否会向其他猫主人推荐这种疫苗接种方式。在整个研究过程中，主人对其猫获得的激素和免疫学结果保持盲态。
统计分析
使用Statistix 8软件（Analytical Software，美国）进行统计分析；使用Prism 5.01（GraphPad Software，美国）绘制图表。数据在Gonacon–Rabies组与Ovx–Rabies组之间以及室内与室外猫栖息组之间进行比较。连续数据采用重复测量方差分析，评估组别（受试者间因素）、时间（受试者内因素）及组别×时间交互作用的影响，随后进行Tukey HSD全配对比较检验。接种或采样的月份和季节对统计结果无影响，因此被排除在最终分析模型之外，这与既往报道一致，即包括以色列在内的温暖温带国家的家猫可在一年中大部分时间繁殖（Dards 1983, Gunther et al. 2015）。在适用情况下，使用Wilcoxon秩和检验比较两组。血清AMH浓度与抗GnRH抗体水平（OD）之间的相关性采用Pearson相关检验评估。接种后不同时间点抗GnRH抗体水平阴性 vs 阳性母猫的血清AMH浓度采用一般AOV/AOCV分析。比例比较使用Fisher精确检验或卡方检验。P < 0.05认为差异显著。除非另有说明，结果以平均值 ± 标准误表示。
结果
最初纳入的36只母猫中，25只完成了1年随访，包括Gonacon–Rabies组22只（室内9只，室外13只）和Ovx–Rabies组3只。治疗启动时间分布于全年。在完成随访的22只Gonacon–Rabies母猫中，3只（13.6%）在秋季接受治疗，8只（36.4%）在冬季，3只（13.6%）在春季，8只（36.4%）在夏季。3只Ovx–Rabies母猫中，两只在夏季接受治疗，一只在冬季。11只母猫失访，对应30.6%的脱落率。失访原因包括：母猫在初次检查后难以捕获进行重复检测（n = 5）、被车撞死（n = 2）、失踪（n = 1）或更换主人（n = 1）。此外，两只母猫的主人在初次接种后停止合作。有趣的是，这11只失访猫中有10只为室外猫，表明与室内猫相比，室外猫失访风险明显更高（室内：1/10，10% vs 室外：10/26，38.5%；比值比 = 5.6；P = 0.0968）。
体格检查、PCV/TS及FeLV/FIV分析
研究期间，没有母猫在狂犬病疫苗注射部位出现肿胀、炎症体征或疼痛。然而，三只母猫在Gonacon疫苗注射部位出现局部肿块，分别在接种后3、6和12个月的计划检查中首次发现。3个月时检测到的肿块相对柔软，大小约3 × 2 cm；随后变硬，至12个月时缩小至约2 × 1 cm。6个月时首次检测到的肿块质硬、椭圆形，大小约3.7 × 2 cm，随访期间逐渐缩小。12个月时首次检测到的肿块质硬，大小约2.5 × 2 cm。相应统计分析表明，与狂犬病疫苗接种相比，Gonacon接种后注射部位反应风险有升高趋势（Gonacon：3/22，13.6% vs 狂犬病：0/25，0%；P = 0.0564）。
所有母猫体格状况良好。Gonacon–Rabies组与Ovx–Rabies组的平均体况评分（BCS）无差异，但总体而言，其在研究期间呈改善趋势（补充图1A（见文末补充材料部分），时间因素，P = 0.0633）；个体猫的BCS保持不变或在5分制BCS量表（Baldwin等，2010）上改善最多1分。检查时，生命体征（TPR：体温、脉搏和呼吸频率）保持在正常范围内，组间及随时间均无显著差异（补充图1B、C、D）。PCV和TS值均在正常范围内，Gonacon–Rabies组与Ovx–Rabies组之间及随时间均无显著差异（补充图1E和F）。在Gonacon–Rabies组中，室内与室外母猫的生命体征（TPR）和TS无差异；然而，室外猫的PCV总体较低（P = 0.0488）。研究中的所有母猫FeLV/FIV均为阴性。
GnRH抗体
如预期，接种疫苗后，Gonacon–Rabies组的抗GnRH抗体光密度（OD）读数高于Ovx–Rabies组（图1A，P = 0.0235）。总体而言，68.2%（15/22）的Gonacon疫苗接种母猫检测到抗GnRH血清阳性，表明无应答率为31.8%。在接种疫苗后0、3、6和12个月，Gonacon疫苗接种母猫中检测到抗GnRH抗体阳性的比例分别为0%、68.2%、31.8%和27.3%，表明接种疫苗后抗GnRH抗体显著升高，随后随时间下降（图1A和B）。在对Gonacon呈阳性应答的母猫（n = 15）中，40%（6/15）在所有检查时（3、6和12个月）均保持阳性；6.7%（1/15）在3和6个月时阳性但12个月时不是；53.3%（8/15）仅在3个月检查时阳性。
[图：Gonacon–Rabies组和Ovx–Rabies组以及室内与室外Gonacon疫苗接种母猫的血清抗GnRH抗体水平]
有趣的是，所有Gonacon无应答母猫均为室外猫（无应答率——室内：0%，0/9；室外：53.8%，7/13；P = 0.0167）。此外，室外母猫的GnRH抗体水平（OD）显著低于室内母猫（图1C），Gonacon疫苗接种后不同时间点抗GnRH阳性应答的母猫比例亦然（图1D）。因此，尽管母猫间存在变异性，室内母猫的抗GnRH血清阳性平均持续时间长于室外母猫（室内：8.3 ± 1.5个月 vs 室外：2.1 ± 0.9个月；P = 0.0005）。总体而言，如狂犬病疫苗所示，Gonacon疫苗在室外母猫中的免疫原性低于室内母猫。
狂犬病抗体
所有母猫在接种疫苗后均产生抗狂犬病抗体，抗体水平在整个研究期间维持在0.5 IU/mL以上，该水平被认为具有保护性（Mansfield等，2004；Shiraishi等，2014）。Gonacon–Rabies组与Ovx–Rabies组之间的抗狂犬病抗体无显著差异（图2A，P = 0.4428）。然而有趣的是，室外母猫的狂犬病抗体浓度低于室内母猫（图2B，P = 0.0490）。
[图：接受Gonacon–Rabies疫苗接种或Ovx–Rabies治疗的母猫血清狂犬病毒中和抗体浓度]
抗苗勒管激素血清浓度
在Ovx–Rabies组中，血清AMH浓度从卵巢子宫切除术前的7.1 ± 1.2 ng/mL显著降至3、6和12个月后的不可检测值（图3A）。在Gonacon–Rabies组中，平均AMH浓度在接种疫苗后3个月较接种前显著降低，但在接种后6和12个月无差异（图3A）。接种Gonacon的室外与室内母猫之间AMH浓度无差异（图3B），但室内母猫在接种后3个月时AMH浓度有低于室外母猫的趋势（P = 0.0727）。有趣的是，在二分类分析中，抗GnRH应答阳性的血清样本AMH浓度低于应答阴性的样本（P < 0.0001；图3D）。此外，发现抗GnRH抗体（OD）与AMH之间呈负相关，Pearson相关系数为−0.5324（P < 0.0001；图3C）。随着抗GnRH抗体水平升高，尤其是高于临界值时，AMH水平下降；然而，当抗GnRH抗体下降时，AMH浓度升高。此外，对接种疫苗后不同时间点影响AMH浓度的因素分析显示，采样时抗GnRH抗体应答状态（阳性或阴性）显著影响AMH浓度（图3E）。
[图：Gonacon–Rabies组和Ovx–Rabies组以及室内和室外Gonacon疫苗接种母猫的血清AMH浓度]
生殖事件
如前所述，研究人员要求主人报告发情迹象以及交配、妊娠或分娩等繁殖事件。正如预期，Ovx–Rabies组的母猫没有出现任何繁殖事件（图4A）。然而，在Gonacon–Rabies组中，50%（11/22）的母猫未报告繁殖事件，且在整个1年监测期内似乎一直处于生殖抑制状态。在其余11只母猫中，10只被报告为妊娠或已分娩幼猫，1只出现发情迹象。接种疫苗后前6个月内未报告任何繁殖事件；所有首次报告的繁殖事件均发生在Gonacon疫苗接种后6至12个月之间（图4）。有趣的是，绝大多数（10/11）发生在室外母猫中（图4B）。总体而言，接种Gonacon的室外母猫中有76.9%（10/13）在研究期间被报告为妊娠或已分娩幼猫，而在室内母猫中，仅1只（1/9，11.1%）出现发情迹象（Fisher精确检验，P = 0.0075）。如上所述，繁殖事件与无应答者、抗GnRH抗体水平降低以及血清AMH浓度重新升高相关。
[图：Gonacon–Rabies组和Ovx–Rabies组以及室内和室外接种Gonacon疫苗的母猫中报告的繁殖事件]
主人满意度
研究结束时，59%（13/22）的主人表示对Gonacon–rabies疫苗接种方案感到满意，并表示会向其他母猫主人推荐。平均而言，表示满意的主人其母猫抗GnRH抗体阳性持续时间长于不满意的主人（6.7 ± 1.4个月 vs 1.7 ± 0.7个月；P = 0.0161）。室内母猫主人的满意度高于室外母猫主人（分别为88.9%，8/9 vs 38.5%，5/13；P = 0.0306）。此外，在1年研究期间未发生繁殖事件的猫的主人满意度（100%，11/11）显著高于母猫出现发情迹象或妊娠的主人（18.2%，2/11；P = 0.0001）。
讨论
本研究旨在调查Gonacon–rabies疫苗对生活在自然栖息地、由主人照管的宠物母猫的安全性、免疫原性和有效性。此外，尽管纳入了一个小型Ovx–Rabies组作为参照，但本研究重点关注Gonacon–Rabies组中室内和室外母猫在疫苗接种反应和繁殖结局方面的差异。正如预期，Ovx–Rabies组的母猫被永久绝育，并产生了具有保护水平的抗狂犬病抗体。在Gonacon–Rabies组中，狂犬病疫苗诱导了强劲的免疫反应；然而，其在室外母猫中的免疫原性较差，导致狂犬病抗体水平显著低于室内母猫。我们发现，Gonacon–Rabies疫苗接种在一些宠物母猫中产生了阳性抗GnRH抗体反应，这与明显的避孕效果相关。然而，室外母猫对Gonacon疫苗的免疫原性低于室内母猫，且数只雌猫在接种Gonacon后出现了繁殖事件。主人对Gonacon–Rabies疫苗接种方案的满意度因饲养条件和繁殖结局而异，凸显了制定针对性种群控制策略的必要性。总体而言，本研究强调了在狂犬病疫苗效力和用于种群管理的免疫避孕工作中考虑环境因素和母猫栖息地的重要性。
在本研究中，狂犬病疫苗在所有接种母猫中引发了强劲的免疫反应，保护性抗体水平在整个研究期间得以维持。这一发现与先前证明猫狂犬病疫苗有效性的研究一致（Fogelman等，1993；Mansfield等，2004；Roebling等，2014；Shiraishi等，2014）。然而，近期报道记录了既往接种疫苗的犬和猫发生狂犬病病例，突出表明疫苗接种并不总能保证完全保护，并强调了在高风险人群中监测免疫力的重要性（Nathan等，2025）。在本研究中，室外母猫的狂犬病抗体浓度显著低于室内母猫，这引发了对自由 roaming猫群中疫苗诱导免疫一致性和持久性的担忧。与这一发现一致，我们近期报道，接种Gonacon和狂犬病疫苗的室外公猫的狂犬病抗体水平显著低于室内公猫（Novak等，2026）。其他一些先前研究也发现了在室外猫中实现和维持长期充足狂犬病免疫力的挑战，提示疫苗递送、野生动物储存宿主暴露和环境应激等因素可能在狂犬病疫苗免疫原性中发挥作用（Mansfield等，2004；Roebling等，2014；Shiraishi等，2014）。
既往使用高剂量GnRH拮抗剂和激动剂的研究已显示母猫生殖活动可被暂时抑制，表明调节下丘脑-垂体-性腺轴是一种可行的避孕途径（Goericke-Pesch等，2014；Velkovska等，2025）。家猫及其他动物物种的免疫避孕策略基于针对关键生殖抗原的主动免疫以及抗体刺激，抗体进而中和相关因子并改变生育力（Kutzler & Wood，2006；Levy等，2011）。Gonacon特异性靶向促性腺激素释放激素（GnRH），即生殖的主调控因子。该疫苗含有与大型载体蛋白连接的GnRH肽，并以含分枝杆菌细胞壁成分的油基佐剂配制，旨在增强并延长抗体产生（Benka & Levy，2015；Novak等，2026）。通过诱导抗GnRH抗体，Gonacon预期可破坏GnRH信号传导，从而减少促性腺激素分泌，进而下游抑制卵泡发生和生殖活动。在本研究中，通过测量抗GnRH抗体水平评估了Gonacon疫苗的免疫原性，抗体水平在接种母猫间总体存在差异，且随时间变化。虽然多数猫表现出初始阳性抗体水平，但一部分个体未能产生足够的免疫应答（无应答率约为32%），或其应答相对短暂并随时间下降，尤其是在户外猫中。其他研究者此前在管理良好的实验室环境中饲养的猫中研究Gonacon免疫原性（Levy等，2004，2011；Vansandt等，2017；Fischer等，2018），以及我们在被捕获并圈养监测14周的野化母猫中（Novak等，2021）和在对宠物公猫接种Gonacon-狂犬病疫苗后监测1年的研究中（Novak等，2026），均报道了疫苗应答的个体差异。同样，Johnson等（2020）在公猫中测试了一种新型噬菌体基GnRH疫苗，观察到免疫应答和生殖抑制存在显著的个体差异，凸显了在不同GnRH基疫苗平台上实现均匀避孕效果的难度。然而，据我们所知，本研究是首次调查Gonacon疫苗与狂犬病疫苗联合在居住在自然栖息地、由主人照护的宠物母猫中的免疫原性和有效性，从而反映了需要生育控制和狂犬病控制的部分目标人群的真实生活场景。观察到的室内猫与户外猫之间免疫原性的差异提示环境因素可能影响个体疫苗效力，在管理规划中应予以认真考虑。与户外生活相关的应激源，如病原体暴露、环境毒素和社会应激，可能损害免疫功能并削弱疫苗应答（Luzardo等，2023；Ma等，2023）。这与2020年AAHA/AAFP猫疫苗接种指南一致，该指南强调生活方式因素，包括户外活动、环境应激源和整体健康状况，可能显著影响猫的疫苗诱导免疫力和对传染病的易感性（Stone等，2020）。
Gonacon与狂犬病疫苗同时接种总体看来是安全的，未观察到不良反应， except for对Gonacon疫苗的注射部位反应见于三只母猫（狂犬病疫苗无此反应）；一只室内猫和两只户外猫。这一发现与既往研究报告部分猫在Gonacon给药后出现轻度局部反应一致（Levy等，2004，2011；Vansandt等，2017；Fischer等，2018；Novak等，2026）。有趣的是，这三只出现注射部位反应的母猫抗GnRH抗体水平持续较高，至少维持12个月。这三只母猫的血清AMH浓度也持续较低，研究期间未报告生殖事件。综合这些观察结果，提示注射部位反应与Gonacon免疫原性和避孕效力增强之间可能存在关联。然而，这些注射部位反应值得进一步研究，以阐明潜在促成因素并评估其临床意义、管理及对疫苗使用的影响。
该研究证明，只要疫苗接种后的母猫体内产生更高水平的抗GnRH抗体，Gonacon疫苗就能够抑制其生殖活动，而高抗GnRH水平与成功接种疫苗的猫只出现低发情率和低妊娠率相关。这一发现与既往研究一致（Levy et al. 2004, 2011, Vansandt et al. 2017, Fischer et al. 2018, Novak et al. 2021）。此外，抗GnRH抗体阳性的母猫血清AMH浓度降低，这与我们此前发表的研究结果一致（Novak et al. 2021）。我们的研究结果进一步支持将AMH降低作为生殖抑制和疫苗避孕效果的标志物。随着抗GnRH抗体水平逐渐下降（在户外母猫中表现得更为明显），AMH浓度升高，生殖事件也变得更加常见。这一模式与使用deslorelin植入剂治疗的母猫的研究结果一致，即在取出植入剂后随着卵巢活动恢复，AMH浓度升高（Goericke-Pesch et al. 2014, Ferre-Dolcet et al. 2022）。在使用Improvac（Zoetis）治疗的猫中也报告了类似发现，该产品是一种获批用于预防猪公猪膻味的GnRH疫苗，随着避孕效果减弱，发情和妊娠恢复，但加强免疫剂量可延长母猫的抑制效果（Morgan et al. 2019, Florencia et al. 2025）。抗GnRH抗体水平与血清AMH浓度之间的关联为深入了解Gonacon避孕作用的机制以及猫卵巢功能潜在生物标志物提供了宝贵信息，尤其是因为已有报告显示家猫的AMH不受季节影响（Place et al. 2011, Axner & Strom Holst 2015, Claassen et al. 2023），并且在完整母猫与卵巢子宫切除术母猫之间表现出明显差异，即使在未排卵的完整母猫中也有轻微的周期性变化（Johnson et al. 2023）。此外，我们的结果凸显了Gonacon疫苗的潜力，但也强调需要进一步探索可能更有效的方案，例如提高疫苗接种剂量、实施Gonacon加强免疫接种（如已在其他物种中所示）、开发免疫原性更强的疫苗配方，或采用补充性方法，以提供更一致、有效且持久的策略来应对自由放养猫群体的生殖控制问题（Baker et al. 2018, Pinkham et al. 2022）。此外，未来研究还应确定卵巢功能的替代生物标志物，以改善对免疫避孕个体避孕效果的监测。
与我们最初的假设相反，室内母猫和户外母猫对抗GnRH疫苗和狂犬病疫苗的反应性存在显著差异。我们的研究揭示了在免疫原性、生殖结局和主人满意度方面的显著差异。户外母猫对Gonacon疫苗和狂犬病疫苗的免疫反应均较低，经历的生殖事件更多，其主人的满意度也低于室内母猫的主人。有趣的是，在我们对Gonacon-狂犬病疫苗接种后监测1年的宠物公猫进行的平行研究中，我们观察到了类似的室内-户外差异，提示存在更广泛的、与栖息环境相关的效应（Novak et al. 2026）。与室内猫相比，自由放养的户外猫可能从主人处获得较少的照料和关注，并可能承受更大的社会应激（de Assis & Mills 2020, Ma et al. 2023）。它们通常面临许多挑战，包括暴露于恶劣天气和噪音，以及来自其他动物、人和车辆造成伤害的风险（Slater 2007, Gerhold & Jessup 2013, Gunther et al. 2015, 2018, Crawford et al. 2019）。此外，它们易感染传染病，健康状况受损的风险更高，导致更高的发病率和死亡率（Slater 2007, Gunther et al. 2018, Crawford et al. 2019）。事实上，我们的研究发现户外母猫的PCV较低，可能提示其中部分猫存在贫血。此外，户外母猫的失访率更高，凸显了与其栖息环境相关的风险。户外母猫的次优状况可能促成接种Gonacon疫苗和狂犬病疫苗后免疫反应和抗体产生降低，正如其他疫苗和物种中所提示的那样（Zimmermann & Curtis 2019, Tizard 2021）。我们的发现凸显了管理野猫和自由放养猫群体及相关公共卫生风险方面的关键挑战，尤其是考虑到大多数自由放养猫一生中仅接种一次狂犬病疫苗，通常是在TNVR期间。旨在改善户外猫健康和福利的策略，如栖息地丰富化、合理喂养和疫苗接种活动，可能有助于减轻户外生活的负面影响并促进自由放养猫群体的种群控制（Levy et al. 2003, de Assis & Mills 2020, Lawson et al. 2020, Ma et al. 2023）。
主人对Gonacon-狂犬病疫苗接种的满意度因饲养条件和繁殖结局而异。室内母猫的主人报告的满意度显著高于户外猫主人，这可能是由于室内环境中繁殖事件发生率较低以及主人对Gonacon疫苗效力的感知。同样，未经历繁殖事件猫的主人更可能报告对Gonacon-狂犬病免疫接种更高的满意度，凸显了实现预期避孕结局的重要性。值得注意的是，主人满意度不受母猫对狂犬病疫苗免疫应答的影响，因为主人对这些实验室检查结果并不知情。这揭示了一个值得关注的问题：狂犬病免疫应答不足若发生，可能在临床上不被察觉，从而在实践中被忽视。未来研究还应考察主人对自由 roaming 猫狂犬病疫苗应答不足后果的理解程度，否则这些缺陷可能被忽视。此外，更广泛地需要加强与主人以及兽医、动物福利组织、市政当局、政策制定者和其他利益相关方的沟通，以确保期望和管理实践与免疫避孕和狂犬病控制策略的现实结局和限制条件相一致（de Assis & Mills 2020, Luzardo et al. 2023）。
总体而言，本研究为生活在自然栖息环境中的有主母猫联合接种Gonacon-狂犬病疫苗的安全性、效力和主人满意度提供了新见解。我们的研究结果表明，室内和户外母猫在狂犬病和Gonacon疫苗接种后的免疫原性和繁殖结局方面存在显著差异，凸显了在实施繁殖和狂犬病控制策略时环境和饲养条件的重要性。未来研究应旨在阐明驱动疫苗应答个体差异的机制，并开发提高避孕效力的靶向方法，尤其是在户外猫中。总体而言，这项工作增进了对猫繁殖健康管理的理解，并支持基于证据的策略，以减少猫过度繁殖，同时保障公共卫生和动物福利。
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资助
本工作由以色列农业部首席科学家（资助号12-11-0009）和希伯来大学资助。该研究还部分得到美国农业部动植物卫生检验局的支持。
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SN参与了调查、数据管理、可视化以及初稿撰写。BY参与了概念构思、资源提供、验证以及写作（审阅和编辑）。RK参与了概念构思、方法学以及写作（审阅和编辑）。LJ参与了调查、形式分析和写作（审阅和编辑）。DCE参与了概念构思、资源提供、验证以及写作（审阅和编辑）。TR参与了监督、概念构思、方法学、形式分析、资源提供、资金获取、可视化、初稿撰写以及手稿的审阅和编辑。
致谢
我们感谢Nathalie Weizmann女士、Ron Sicsic博士、Liat Morgan博士、Idit Gunther博士、Hagai Akiva博士和Shaked Druker博士在宣传本研究招募猫、提供技术援助以及参与小组会议中有价值讨论方面给予的帮助。
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Abstract
Abstract
Overpopulation of domestic cats poses risks to public health, biodiversity, and animal welfare. Surgical sterilization is widely used but has substantial limitations. Gonacon, an anti-GnRH vaccine, provides a nonsurgical alternative; however, data are lacking for owned queens living in natural habitats and for simultaneous vaccination against rabies. We evaluated the 1-year immunogenicity, safety, and efficacy of simultaneous Gonacon–rabies vaccination in pet queens and compared responses between indoor and outdoor cats. In a prospective field study, 36 intact queens (≥6 months) received Gonacon–rabies vaccination (Gonacon–Rabies, n = 30) or ovariohysterectomy with rabies vaccination (Ovx–Rabies, n = 6). Queens remained under their owners’ care and were monitored at 0, 3, 6, and 12 months. Twenty-five queens completed the follow-up (22 Gonacon–Rabies, 3 Ovx–Rabies). Injection-site nodules were observed only following Gonacon vaccination (13.6 vs 0%; P = 0.0564). Anti-GnRH seropositivity was detected in 68.2, 31.8, and 27.3% of Gonacon–Rabies queens at 3, 6, and 12 months, respectively. Among responders (n = 15), 53.3% were positive only at 3 months, 6.7% remained positive up to 6 months, and 40% remained positive throughout the study. All Gonacon-vaccinated nonresponders were outdoor queens (outdoor: 7/13; 53.8%; indoor: 0/9; P = 0.0167). The duration of anti-GnRH immunity was shorter in outdoor queens (2.1 ± 0.9 vs 8.3 ± 1.5 months; P = 0.0005). Reproductive events (estrus or pregnancy) occurred in 76.9% of outdoor vs 11.1% of indoor Gonacon–Rabies queens (P = 0.0075). AMH concentrations were inversely correlated with anti-GnRH levels (r = −0.53; P < 0.0001). Rabies antibodies remained protective (≥0.5 IU/mL) but were lower in outdoor vs indoor queens (P = 0.0490). Owner satisfaction was higher for Gonacon–Rabies indoor queens and those without reproductive events. Simultaneous Gonacon–rabies vaccination was safe and partially effective over 1 year, with better responsiveness in indoor queens.
Lay summary
Domestic cats are highly prolific, contributing to overpopulation that negatively affects animal welfare, wildlife biodiversity, and public health. Although surgical sterilization is widely used to prevent reproduction in cats, it has important practical, economic, and ethical limitations. We studied the safety, immune response, and reproductive effects of a contraceptive anti-GnRH vaccine (Gonacon) administered concurrently with a rabies vaccine in female pet cats living under normal home conditions. The combined vaccination was generally safe, and most queens maintained protective immunity against rabies. However, the contraceptive effects varied considerably among animals. Indoor queens developed stronger, longer-lasting immune responses and experienced fewer signs of estrus and fewer pregnancies than outdoor queens. In contrast, many outdoor queens showed weaker and shorter-lasting immune responses. These findings suggest that combined vaccination may provide a practical nonsurgical approach to controlling reproduction in some pet cats, but living conditions can strongly influence its effectiveness.
Keywords: immunocontraception, anti-GnRH vaccine, gonacon, queens, felis catus, rabies, feline population management
Introduction
Overpopulation of domestic cats poses a significant global challenge in both urban and rural regions (Gunther et al. 2015, 2022, Ma et al. 2023). The global domestic cat population is estimated at several hundred million individuals, many of which are owned as pets, and household ownership of multiple cats is common (Kennedy et al. 2020). Owned or pet cats typically fall into one of three habitat categories: indoor cats, confined solely within the household; indoor/outdoor cats, living indoors with access to outdoor environments; and outdoor cats, living predominantly outdoors but still receiving varying degrees of care from their owners, such as food, shelter, and medical treatment (Ma et al. 2023). The uncontrolled breeding of owned outdoor cats, coupled with the proliferation of stray and abandoned cats, has led to the emergence of free-roaming cat populations across numerous regions worldwide (Gunther et al. 2020, Kennedy et al. 2020, Ma et al. 2023).
Overpopulation of free-roaming cats is associated with substantial public health risks due to the transmission of zoonotic diseases to humans, such as rabies, toxoplasmosis, cat scratch disease, leptospirosis, Q fever, and toxocariasis (Gerhold & Jessup 2013, Roebling et al. 2014). Cats may also mediate the transmission of diseases to livestock and wild animals (Wareth et al. 2017, Trouwborst et al. 2020). In addition, they may attack humans, be involved in traffic accidents, and cause nuisances, such as the presence of cat carcasses, noise and dirt, and invasion into private or business grounds (Gunther et al. 2015, Crawford et al. 2019). Moreover, free-roaming and feral cats are non-native predators that commonly kill birds, reptiles, amphibians, small mammals, and fish, with many of these prey species being on the verge of extinction (Medina et al. 2011, Loss et al. 2022). Due to these significant ecological disruptions and wildlife depletion, the domestic cat has been included among the world’s top 100 worst non-native invasive species (Lowe et al. 2000, Loss et al. 2022). In addition, overpopulation of free-roaming cats is associated with high prevalences of malnutrition, disease susceptibility, and injuries among cat populations, leading to adverse effects on their welfare and health (Gunther et al. 2015, 2018, Ma et al. 2023).
Rabies, a lethal zoonotic viral disease affecting both humans and animals, is endemic in many countries (Boutelle et al. 2025). According to World Health Organization reports published between 2020 and 2024, rabies causes an estimated 59,000–60,000 human deaths annually worldwide, with the vast majority resulting from dog-mediated transmission. Nevertheless, in many countries, including the United States, cats are the most frequently reported rabid companion animal (Gerhold & Jessup 2013, Ma et al. 2024, Boutelle et al. 2025). Consequently, in the last few decades, efforts have been made to control the overpopulation of free-roaming cats and prevent rabies (Roebling et al. 2014). However, the influence of different habitats on rabies vaccine immunogenicity and responsiveness in domestic cats remains unclear.
Currently, surgical sterilization is the primary method for preventing fertility in owned cats and for managing free-roaming cat populations, typically through trap–neuter–vaccinate (for rabies)-release (TNVR) programs (Roebling et al. 2014, Murray et al. 2015, Gunther et al. 2022). Nevertheless, the surgical approach has clear limitations. The need for anesthetic materials, specialized equipment, the expertise of skilled veterinarians, and, in the case of free-roaming cats, the additional costs of trapping and transporting them to a proper facility collectively contribute to its substantial financial and practical burdens (Lohr et al. 2013, Benka et al. 2022, Gunther et al. 2022). Moreover, surgical procedures cause considerable stress, pain, and discomfort to the animals involved (Pereira et al. 2018, Crawford et al. 2019). The invasiveness of the procedures, along with the subsequent recovery period and the unfamiliar surroundings of veterinary clinics, contribute to the distress experienced by cats, which is a concern not only from an ethical standpoint but also due to adverse health effects (Adin 2011, Pereira et al. 2018, Klintip et al. 2022). Furthermore, surgical sterilization procedures in cats can lead to intraoperative and postoperative sequelae, such as anesthetic complications, bleeding, surgical-site infections, wound dehiscence, adverse reactions to sutures, seroma or hematoma formation, and urinary tract issues (Adin 2011, Pereira et al. 2018). Hence, despite its advantage of providing a permanent resolution for the individual animal, the intrinsic constraints related to cost, stress, health implications, and logistical challenges have hindered the scalability and efficacy of the surgical approach. Practically, the surgical approach has failed to mitigate the extensive reproduction of free-roaming cat populations (Root Kustritz 2012, Roebling et al. 2014, Benka & Levy 2015, Crawford et al. 2019). In addition, many cat owners are reluctant to subject their pets to surgical interventions due to these concerns or ethical and religious convictions (Root Kustritz 2012, Murray et al. 2015). Nevertheless, commercial products for mass non-surgical sterilization of owned and free-roaming cats are currently unavailable despite extensive research efforts (Kutzler & Wood 2006, Benka & Levy 2015).
Gonacon is an anti-GnRH vaccine developed by the National Wildlife Research Center, USDA APHIS Wildlife Services, USA, that aims to suppress reproduction by generating GnRH-neutralizing antibodies (Miller et al. 2008). Previous studies examining its effects on cats have mostly involved animals in non-natural settings (Levy et al. 2011, Vansandt et al. 2017, Fischer et al. 2018), such as laboratory or research colony cats, that differ significantly from feral and outdoor cats exposed to a variety of environmental stressors and health challenges (Slater 2007, Gunther et al. 2015, Crawford et al. 2019). Notably, there is limited information regarding the immunogenicity of Gonacon and rabies vaccines when administered concurrently to the same cat, which hampers evidence-based decision-making for programs that rely on coordinated contraceptive and rabies control strategies. Furthermore, data on Gonacon vaccination, either alone or in combination with rabies vaccination, in owner-owned pet cats living in their natural environments (indoors or outdoors) have been very limited. Recently, however, we reported the 1-year safety, immunogenicity, and reproductive effects of combined anti-GnRH and rabies vaccination in owner-owned male cats (Novak et al. 2026). Information regarding the safety, immunogenicity, and efficacy of combined Gonacon–rabies vaccination in owner-owned female cats (queens) living under natural conditions, however, remains scarce. To address this knowledge gap, we also conducted a parallel 1-year study in owner-owned female pet cats, which we report herein.
The primary objective of the current study was to evaluate the safety, immunogenicity, and efficacy of simultaneous Gonacon and rabies vaccination over a 1-year period in owner-owned queens living in their natural environments and to compare responses between indoor and outdoor cats. Our main hypotheses were that: i) Gonacon–rabies vaccination would be safe and would not result in adverse effects; ii) antibody responses against GnRH and rabies would be maintained throughout the study period and would not differ between indoor and outdoor queens; iii) rabies antibody responses would not be negatively affected by simultaneous vaccination and would remain protective (≥0.5 IU/mL) throughout the study period; and iv) anti-GnRH seropositivity would be associated with suppression of reproductive activity.
Materials and methods
Animals and study design
The study was conducted in accordance with the Hebrew University Institutional Animal Care and Use Committee (IACUC) ethical approval (MD-15-1411-2). Cat owners in the central area of Israel were invited to enroll their queens in this study via email to veterinary clinics in the region, to Koret School of Veterinary Medicine staff and students, and through online advertisements (e.g. animal-related Facebook groups and websites). Owners of queens who contacted the research team received the relevant information, and the animals were enrolled voluntarily by their owners, without compensation. Informed consent was obtained and signed by all owners prior to their queen’s enrollment in the study. Queens were enrolled and received their assigned treatment when they became available for participation; consequently, individual queens entered the study at different times throughout the year. A licensed veterinarian performed all animal procedures in the study (S N). The inclusion criteria were intact queens aged 6 months or older, with no abnormalities detected during veterinary physical examination. Queens included in the study were considered rabies vaccine naïve at enrollment based on owner-reported vaccination history and the absence of detectable rabies antibody concentrations prior to vaccination.
A total of 36 queens were initially recruited into this field-based study and allocated to one of two groups. At study initiation, queens in the Gonacon–Rabies group (n = 30) were vaccinated with both anti-GnRH (Gonacon) and rabies vaccines, whereas in the Ovx–Rabies group, queens (n = 6) underwent surgical ovariohysterectomy and were vaccinated against rabies. Throughout the 1-year study, queens in both groups remained in their natural habitat, under the care and possession of their owners.
Treatments, sampling, and monitoring
Queens were examined and sampled by the research team four times throughout the study: at study initiation (time 0, when the initial treatments were performed; i.e. vaccinations and ovariohysterectomy), and at 3, 6, and 12 months after vaccination. Queens were sedated for veterinary physical examination, vaccinations, and blood sample collections by combining medetomidine (0.02–0.04 mg/kg; Orion Pharma, Finland), butorphanol (0.2–0.4 mg/kg; intramuscular injection; Richter Pharma AG, Austria), and midazolam (0.1–0.25 mg/kg intramuscular injection; Rafa Laboratories, Israel). Medetomidine’s sedative effects were counteracted at the end of the procedure by administering atipamezole subcutaneously (0.05–0.10 mg/kg; Orion Pharma, Finland) to facilitate recovery. The queens were released back to their homes and the care of their owners only after they had fully recovered from the sedation and were bright, alert, and responsive.
At study initiation, each queen was vaccinated against rabies by subcutaneous injection of a licensed rabies vaccine (Rabisin; Merial, France) in the left thigh. Simultaneously, Gonacon immunization was delivered intramuscularly using vaccine material provided by USDA-APHIS NWRC (Fort Collins, CO, USA). A 500 μL dose was injected into the right quadriceps, as previously described (Novak et al. 2021, 2026). The fur over each injection site was clipped, and the skin was prepared with 70% isopropyl alcohol before administration.
At each examination, a complete physical examination was performed by a certified veterinarian, including evaluation of body condition on a 5-point scale (Baldwin et al. 2010) and examinations of the injection sites. Blood samples were collected by jugular venipuncture into plain tubes without anticoagulant and into potassium-EDTA tubes. Blood in plain tubes was allowed to clot, centrifuged, and the serum samples were stored at −80°C until analysis at study completion; these analyses included anti-rabies and anti-GnRH antibody levels, serology for FeLV and FIV, and analysis of serum anti-Müllerian hormone (AMH) concentration. Blood samples in potassium-EDTA tubes were used to measure packed cell volume (PCV), using a hematocrit tube sealed with clay that was centrifuged for approximately 3 min; plasma total solids (TS) were measured by placing the plasma directly onto a refractometer (Clinical Refractometer RHC-200 ATC, Cmall, Medent, Israel).
Every 3 months, either during scheduled examinations or by telephone follow-up, owners were asked to complete a questionnaire regarding their queen’s housing conditions (i.e. indoor or outdoor), general health status, and reproductive behavior. In addition, throughout the study period, owners were instructed to report to the research team in real time any behavioral or health abnormalities, signs of estrus, or reproductive-related events, including mating, pregnancy, or parturition.
FIV/FeLV serology
To further assess overall health status, serum collected at the midpoint of the study (6 months post-vaccination) was tested for feline immunodeficiency virus (FIV) antibodies and feline leukemia virus (FeLV) p27 antigen. Analyses were performed using a commercial FIV/FeLV test kit (SNAP Combo Plus, IDEXX, USA) according to the manufacturer’s instructions. The queens’ FeLV vaccination histories were unknown; however, such vaccination is not expected to interfere with FeLV antigen detection using the SNAP Combo Plus assay (Little et al. 2020).
Detection of anti-GnRH antibodies
Serum samples collected at 0, 3, 6, and 12 months were stored frozen and shipped on study completion to the USDA-APHIS National Wildlife Research Center (Fort Collins, CO, USA) for analysis of anti-GnRH antibodies by enzyme-linked immunosorbent assay (ELISA), following previously published procedures (Levy et al. 2011, Novak et al. 2021, 2026). Serum samples were diluted 1:8,000 in phosphate-buffered saline (PBS). All samples were analyzed in duplicate, and antibody responses were quantified using optical density (OD) measurements. A positive control sample was included in duplicate on each assay plate to monitor assay performance. The threshold for anti-GnRH seropositivity was established using the pre-vaccination samples from all queens and was defined as the mean OD value plus three standard deviations. Samples with OD values exceeding this threshold were classified as seropositive, whereas those below the threshold were considered seronegative. To minimize analytical variation, all samples were analyzed by the same operator, who was blinded to sample identity, treatment groups, and housing status. In addition, all samples from an individual queen were analyzed within the same assay run. The intra-assay and interassay coefficients of variation were 12.3 and 9.5%, respectively.
Detection of anti-rabies antibodies
Serum samples collected at 0, 3, 6, and 12 months were analyzed for rabies virus-neutralizing antibodies using the rapid fluorescent focus inhibition test (RFFIT), performed by the WOAH-accredited Rabies Reference Laboratory at the Kimron Veterinary Institute (Veterinary Services and Animal Health, Ministry of Agriculture, Israel), as previously described (Yakobson et al. 2015, Novak et al. 2021, 2026). RFFIT is considered the reference method for the measurement of rabies virus-neutralizing antibodies and has demonstrated high diagnostic performance (i.e. 100% sensitivity and 89% specificity); it is therefore considered the gold standard by the World Health Organization (Bahloul et al. 2005, Yakobson et al. 2015). Briefly, serial serum dilutions were incubated with rabies virus in the presence of positive and negative controls, followed by the addition of mouse neuroblastoma cells (CDC, Atlanta, GA, USA). After incubation, the cells were fixed, stained with a fluorescein isothiocyanate (FITC)-conjugated anti-rabies antibody (Fujirebio, USA), and examined by fluorescence microscopy (Olympus BX40; excitation, 470 nm; ×200 magnification). Rabies virus-neutralizing antibody concentrations were calculated and expressed as international units per milliliter (IU/mL). Antibody concentrations ≥0.5 IU/mL were considered indicative of adequate serological protection (Yakobson et al. 2015).
Serum anti-Müllerian hormone analysis
Serum AMH concentrations were quantified in all collected samples using a commercial ELISA kit (AMH Gen II; Beckman Coulter Inc., USA), according to the manufacturer’s protocol and previously published methods validated for feline samples (Axner & Strom Holst 2015, Novak et al. 2021). Briefly, 300 μL of AMH Gen II assay buffer was combined with 60 μL of calibrator, control, or serum sample, and 120 μL of this mixture was dispensed in duplicate into the assay plate. After an incubation and subsequent washing, 100 μL biotin-labeled anti-AMH antibody was added. Plates were incubated and washed again before adding 100 μL streptavidin–enzyme conjugate. Following a third incubation and wash cycle, 100 μL TMB substrate solution was introduced, and the reaction was terminated by adding 100 μL supplied stop solution. Absorbance was measured at 450 nm with reference correction at 620 nm using a SpectraMax Paradigm multi-mode microplate reader (SpectraMax Paradigm Multi-Mode Detection Platform, Molecular Devices, Austria). The assay demonstrated inter- and intra-assay coefficients of variation of 8.2 and 2.4%, respectively.
Owner satisfaction
At the completion of the 1-year follow-up period, owners of queens in the Gonacon–Rabies group were asked to complete a brief questionnaire evaluating their overall satisfaction with the Gonacon–rabies vaccination strategy and their likelihood of recommending it to other owners of intact female cats. The questionnaire included two items: i) whether the owner was satisfied with the effectiveness of Gonacon–rabies vaccination in suppressing reproductive-related signs in their queen; ii) whether they would recommend this vaccination approach to other cat owners. Throughout the study, owners were blinded to the hormonal and immunological findings obtained from their cats.
Statistical analysis
Statistical analyses were performed using Statistix 8 software (Analytical Software, USA); plots were produced using Prism 5.01 (GraphPad Software, USA). Data were compared between the Gonacon–Rabies and Ovx–Rabies groups, and between the indoor and outdoor cat habitat groups. Continuous data were analyzed by Repeated Measures ANOVA, to evaluate the effects of Group (between-subject factor), Time (within-subject factor), and Group-by-Time interactions, followed by Tukey HSD All-Pairwise Comparisons Test. The month and season of vaccination or sampling had no impact on the statistical outcomes and were therefore excluded from the final analytic model, consistent with previous reports indicating that domestic cats in warm and temperate countries, including Israel, can breed throughout most of the year (Dards 1983, Gunther et al. 2015). When applicable, two groups were compared using the Wilcoxon Rank Sum Test. The correlation between serum AMH concentrations and anti-GnRH antibody levels (OD) was evaluated by the Pearson Correlation Test. Serum AMH concentrations in queens with negative vs positive anti-GnRH antibody levels at different time points post-vaccination were analyzed using a General AOV/AOCV. Fisher’s Exact Test or the Chi-square Test was used to compare proportions. Differences were considered significant at P < 0.05. Unless otherwise noted, results are presented as mean ± SEM.
Results
Of the 36 queens initially enrolled, 25 completed the 1-year follow-up, including 22 from the Gonacon–Rabies group (9 indoor, 13 outdoor) and 3 from the Ovx–Rabies group. Treatment initiation was distributed throughout the year. Among the 22 Gonacon–Rabies queens that completed follow-up, 3 (13.6%) were treated in fall, 8 (36.4%) in winter, 3 (13.6%) in spring, and 8 (36.4%) in summer. Of the three Ovx–Rabies queens, two were treated in summer and one in winter. The loss to follow-up of 11 queens corresponds to a 30.6% dropout rate. Reasons for loss to follow-up included queens that were difficult to capture for repeated testing beyond the initial examination (n = 5), were hit by a car and died (n = 2), disappeared (n = 1), or changed ownership (n = 1). In addition, the owners of two queens discontinued cooperation following the initial vaccination. Interestingly, 10 of these 11 follow-up losses were outdoor queens, indicating a clear tendency for a higher risk for follow-up loss among outdoor cats compared to indoor cats (indoor: 1/10, 10% vs outdoor: 10/26, 38.5%; odds ratio = 5.6; P = 0.0968).
Physical examinations, PCV/TS, and FeLV/FIV analyses
During the study, none of the queens exhibited swelling, signs of inflammation, or pain at the rabies vaccine injection sites. However, three queens developed localized masses at the Gonacon vaccine injection site, first identified during scheduled examinations at 3, 6, and 12 months after vaccination, respectively. The mass detected at 3 months was relatively soft and measured approximately 3 × 2 cm; it subsequently became firm and decreased in size to approximately 2 × 1 cm by 12 months. The mass first detected at 6 months was firm and oval, measuring approximately 3.7 × 2 cm, and gradually decreased in size during follow-up. The mass first detected at 12 months was firm and measured approximately 2.5 × 2 cm. The corresponding statistical analysis indicated a tendency toward a higher risk of injection–site reaction following Gonacon vaccination compared with rabies vaccination (Gonacon: 3/22, 13.6%, vs Rabies: 0/25, 0%; P = 0.0564).
All queens were in good physical condition. Mean body condition score (BCS) did not differ between the Gonacon–Rabies and Ovx–Rabies groups, but overall, it tended to improve over the study period (Supplementary Fig. 1A (see the section on Supplementary materials given at the end of the article), Time factor, P = 0.0633); BCS of individual cats stayed the same or improved by up to one point in a 5-point BCS scale (Baldwin et al. 2010). Upon examination, vital signs (TPR: temperature, pulse, and respiratory rate) remained within normal limits, with no significant differences between the groups or over time (Supplementary Fig. 1B, C, D). The values of PCV and TS were within normal limits, with no significant differences between the Gonacon–Rabies and Ovx–Rabies groups or over time (Supplementary Fig. 1E and F). In the Gonacon–Rabies group, vital signs (TPR) and TS did not differ between indoor and outdoor queens; however, PCV was lower overall in outdoor cats (P = 0.0488). All queens in the study were negative for FeLV/FIV.
GnRH antibodies
As expected, following vaccination, the optical density (OD) readings of anti-GnRH antibodies were higher in the Gonacon–Rabies group as compared to the Ovx–Rabies group (Fig. 1A, P = 0.0235). Overall, anti-GnRH seropositivity was detected in 68.2% (15/22) of Gonacon-vaccinated queens, indicating a non-response rate of 31.8%. Positive anti-GnRH antibody levels were detected in 0, 68.2, 31.8, and 27.3% of the Gonacon-vaccinated queens at 0, 3, 6, and 12 months after vaccination, respectively, indicating a significant increase in anti-GnRH antibodies after vaccination, followed by a reduction over time (Fig. 1A and B). Among the queens that responded positively to Gonacon (n = 15), 40% (6/15) remained positive at all examinations (3, 6, and 12 months); 6.7% (1/15) were positive at 3 and 6 months but not at 12 months; and 53.3% (8/15) were positive only at the 3-month examination.
[图：Serum anti-GnRH antibody levels in the Gonacon–Rabies and Ovx–Rabies groups and in indoor vs outdoor Gonacon-vaccinated ]
Interestingly, all Gonacon non-responder queens were outdoor cats (non-responder rate – indoor: 0%, 0/9; outdoor: 53.8%, 7/13; P = 0.0167). Moreover, GnRH antibody levels (OD) were significantly lower in outdoor vs indoor queens (Fig. 1C), as well as the proportions of queens with positive anti-GnRH response at the different time points after Gonacon vaccination (Fig. 1D). Accordingly, despite the variability among queens, the mean duration of anti-GnRH seropositivity was longer in indoor as compared to outdoor queens (indoor: 8.3 ± 1.5 months vs outdoor: 2.1 ± 0.9 months; P = 0.0005). Overall, as shown for the rabies vaccine, the immunogenicity of the Gonacon vaccine was lower in outdoor queens compared to indoor queens.
Rabies antibodies
All queens produced antibodies against rabies following vaccination, with antibody levels maintained above 0.5 IU/mL throughout the study, a level that is considered protective (Mansfield et al. 2004, Shiraishi et al. 2014). There were no significant differences in the anti-rabies antibodies between the Gonacon–Rabies and Ovx–Rabies groups (Fig. 2A, P = 0.4428). Interestingly, however, rabies antibody concentrations were lower in outdoor queens than in indoor queens (Fig. 2B, P = 0.0490).
[图：Serum rabies virus-neutralizing antibody concentrations in queens receiving Gonacon–Rabies vaccination or Ovx–Rabies tre]
Anti-Müllerian hormone serum concentrations
In the Ovx–Rabies group, serum AMH concentrations reduced significantly from 7.1 ± 1.2 ng/mL before ovariohysterectomy to nondetectable values at 3, 6, and 12 months later (Fig. 3A). In the Gonacon–Rabies group, mean AMH concentrations reduced significantly at 3 months after vaccination as compared to before vaccination, but did not differ at 6 and 12 months after vaccination (Fig. 3A). AMH concentrations did not differ between outdoor vs indoor queens vaccinated with Gonacon (Fig. 3B), but there was a tendency for lower AMH concentration at 3 months after vaccination in indoor vs outdoor queens (P = 0.0727). Interestingly, in a dichotomous analysis, serum samples with positive anti-GnRH response had lower AMH concentrations than samples with negative response (P < 0.0001; Fig. 3D). Furthermore, the relationship between anti-GnRH antibodies (OD) and AMH was found to be negatively correlated, with a Pearson correlation coefficient of −0.5324 (P < 0.0001; Fig. 3C). As the levels of anti-GnRH antibodies increased, particularly above the cutoff, the level of AMH decreased; however, when the anti-GnRH antibodies decreased, the AMH concentration increased. Moreover, an analysis of factors affecting AMH concentrations at different time points after vaccination revealed that the status of the anti-GnRH antibody response (positive or negative) at the time of sampling significantly influenced AMH concentrations (Fig. 3E).
[图：Serum AMH concentrations in the Gonacon–Rabies and Ovx–Rabies groups, in indoor and outdoor Gonacon-vaccinated queens, a]
Reproductive events
As noted, owners were asked to report signs of estrus and reproductive events such as mating, pregnancy, or parturition. As expected, queens in the Ovx–Rabies group had no reproductive events (Fig. 4A). However, in the Gonacon–Rabies group, 50% (11/22) of the queens had no reported reproductive events and appeared to remain reproductively suppressed throughout the 1-year monitoring period. Of the remaining 11 queens, 10 were reported as pregnant or having delivered kittens, and one showed signs of estrus. No reproductive events were reported during the first 6 months after vaccination; all first reported reproductive events occurred between 6 and 12 months after Gonacon vaccination (Fig. 4). Interestingly, the vast majority (10/11) occurred in outdoor queens (Fig. 4B). Overall, 76.9% (10/13) of the outdoor queens vaccinated with Gonacon were reported as pregnant or having delivered kittens during the study, while among the indoor queens, only one (1/9, 11.1%) showed signs of estrus (Fisher Exact Test, P = 0.0075). Reproductive events were associated with non-responders, reduced anti-GnRH antibody levels, and re-elevation of serum AMH concentrations, as reported above.
[图：Reported reproductive events in the Gonacon–Rabies and Ovx–Rabies groups and in indoor and outdoor Gonacon-vaccinated qu]
Owner satisfaction
At the conclusion of the study, 59% (13/22) of owners expressed satisfaction with the Gonacon–rabies vaccination approach and indicated they would recommend it to other queen owners. On average, queens of owners who expressed positive satisfaction had a longer duration of positive anti-GnRH antibody levels than queens of owners who were not satisfied with it (6.7 ± 1.4 months vs 1.7 ± 0.7 months; P = 0.0161). Higher satisfaction levels were reported among owners of indoor queens than among those with outdoor queens (88.9%, 8/9 vs 38.5%, 5/13, respectively; P = 0.0306). Furthermore, owner satisfaction was significantly higher among owners whose cats did not experience reproductive events during the 1-year study period (100%, 11/11) compared with owners whose queens exhibited signs of estrus or became pregnant (18.2%, 2/11; P = 0.0001).
Discussion
Our study aimed to investigate the safety, immunogenicity, and efficacy of Gonacon–rabies vaccination in owner-owned pet queens residing in their natural habitats under their owners’ care. In addition, although a small Ovx–Rabies group was included as a reference, our study focused on differences in vaccination responses and reproductive outcomes between indoor and outdoor queens in the Gonacon–Rabies group. As expected, queens in the Ovx–Rabies group were permanently sterilized and developed protective levels of anti-rabies antibodies. In the Gonacon–Rabies group, the rabies vaccine induced robust immune responses; however, its immunogenicity was inferior in outdoor queens, resulting in significantly lower rabies antibody levels than in indoor queens. We found that Gonacon–Rabies vaccination generated positive anti-GnRH antibody responses in some owner-owned pet queens, which was associated with apparent contraceptive efficacy. However, outdoor queens showed lower immunogenicity to the Gonacon vaccine than indoor queens, and several females experienced reproductive events post-Gonacon vaccination. Owner satisfaction with the Gonacon–Rabies vaccination approach varied by housing conditions and reproductive outcomes, underscoring the need for tailored population control strategies. Overall, our study emphasizes the importance of considering environmental factors and queens’ habitats in rabies vaccine efficacy and immunocontraception efforts for population management.
In our study, the rabies vaccine elicited a robust immune response in all vaccinated queens, with protective antibody levels maintained throughout the study period. This finding is consistent with previous research demonstrating the efficacy of rabies vaccination in cats (Fogelman et al. 1993, Mansfield et al. 2004, Roebling et al. 2014, Shiraishi et al. 2014). However, recent reports have documented rabies cases in previously vaccinated dogs and cats, highlighting that vaccination does not always guarantee full protection and emphasizing the importance of monitoring immunity in at-risk populations (Nathan et al. 2025). In the current study, outdoor queens exhibited significantly lower rabies antibody concentrations than indoor queens, raising concerns regarding the consistency and durability of vaccine-induced immunity in free-roaming cat populations. Consistent with this finding, we recently reported that outdoor toms vaccinated with Gonacon and rabies exhibited significantly lower rabies antibody levels than indoor toms (Novak et al. 2026). Some other previous studies have also identified challenges in achieving and maintaining long-term, sufficient rabies immunity in outdoor cats, suggesting that factors such as vaccine delivery, exposure to wildlife reservoirs, and environmental stressors may play a role in rabies vaccine immunogenicity (Mansfield et al. 2004, Roebling et al. 2014, Shiraishi et al. 2014).
Previous studies using high-dose GnRH antagonists and agonists have shown temporary suppression of reproductive activity in female cats, indicating that modulation of the hypothalamic–pituitary–gonadal axis is a viable contraceptive approach (Goericke-Pesch et al. 2014, Velkovska et al. 2025). Immunocontraception strategies in domestic cats and other animal species are based on active immunization against key reproductive antigens and on antibody stimulation, which, in turn, neutralize the relevant factor(s) and alter fertility (Kutzler & Wood 2006, Levy et al. 2011). Gonacon specifically targets gonadotropin-releasing hormone (GnRH), the master regulator of reproduction. The vaccine contains a GnRH peptide linked to a large carrier protein and formulated with an oil-based adjuvant containing mycobacterial cell wall components, aiming to enhance and prolong antibody production (Benka & Levy 2015, Novak et al. 2026). By inducing anti-GnRH antibodies, Gonacon is anticipated to disrupt GnRH signaling, thereby reducing gonadotropin secretion, leading to downstream suppression of folliculogenesis and reproductive activity. In our study, the immunogenicity of the Gonacon vaccine was evaluated by measuring anti-GnRH antibody levels, which varied overall among vaccinated queens and over time. While most cats exhibited initial positive antibody levels, a subset of individuals did not mount a sufficient immune response (a non-responder rate of approximately 32%), or their response was relatively short-lived and declined over time, particularly among outdoor cats. Individual variability in vaccine response has been reported in previous studies by others investigating the immunogenicity of Gonacon in cats housed in well-managed laboratory environments (Levy et al. 2004, 2011, Vansandt et al. 2017, Fischer et al. 2018), and by us in feral queens that were trapped and kept in captivity for a 14-week monitoring period (Novak et al. 2021), and in the study we performed in pet toms monitored for 1 year after Gonacon–rabies vaccination (Novak et al. 2026). Likewise, Johnson et al. (2020) tested a novel phage-based GnRH vaccine in male cats and observed substantial individual variability in immune responses and reproductive suppression, underscoring the difficulty of achieving uniform contraceptive efficacy across different GnRH-based vaccine platforms. However, to our knowledge, the current study represents the first investigation into the combined immunogenicity and efficacy of the Gonacon vaccine with the rabies vaccine in pet queens residing in their natural habitats under the care of their owners, thus reflecting the real-life scenario for a segment of the target population requiring fertility and rabies control. The observed differences in immunogenicity between indoor and outdoor cats suggest that environmental factors may influence individual vaccine efficacy and should be seriously considered in management programs. Stressors associated with outdoor living, such as exposure to pathogens, environmental toxins, and social stress, may impair immune function and compromise vaccine responses (Luzardo et al. 2023, Ma et al. 2023). This is consistent with the 2020 AAHA/AAFP Feline Vaccination Guidelines, which emphasize that lifestyle factors, including outdoor access, environmental stressors, and overall health status, may significantly influence vaccine-induced immunity and susceptibility to infectious diseases in cats (Stone et al. 2020).
The simultaneous Gonacon and rabies vaccines appeared overall safe, with no observed adverse reactions, except for injection–site reactions to the Gonacon vaccine in three queens (none to the rabies vaccine); one indoor and two outdoor queens. This finding is consistent with previous studies reporting mild local reactions following Gonacon administration in some cats (Levy et al. 2004, 2011, Vansandt et al. 2017, Fischer et al. 2018, Novak et al. 2026). Interestingly, the three queens with injection-site reactions had consistently high anti-GnRH antibody levels, which persisted for at least 12 months. These three queens also had consistently low serum AMH concentrations, with no reproductive events reported during the study. Together, these observations suggest a possible association between injection-site reactions and increased Gonacon immunogenicity and contraceptive efficacy. However, these injection-site reactions warrant further investigation to elucidate potential contributing factors and to evaluate their clinical significance, management, and implications for vaccine use.
The study demonstrates the ability of the Gonacon vaccine to suppress reproductive activity in vaccinated queens, provided that higher anti-GnRH levels are developed, which is associated with a low incidence of estrus and pregnancy among successfully vaccinated cats. This finding aligns with previous studies (Levy et al. 2004, 2011, Vansandt et al. 2017, Fischer et al. 2018, Novak et al. 2021). In addition, queens with positive anti-GnRH levels exhibited reduced serum AMH concentrations, consistent with our previous publication (Novak et al. 2021). Our findings further support the value of reduced AMH as a marker for reproductive suppression and the vaccine’s contraceptive effects. As anti-GnRH levels gradually reduced, as illustrated more clearly in outdoor queens, AMH concentrations increased, and reproductive events became more common. This pattern is consistent with findings from queens treated with deslorelin implants, in which AMH concentrations rose as ovarian activity resumed following implant removal (Goericke-Pesch et al. 2014, Ferre-Dolcet et al. 2022). A comparable finding was also reported in cats treated with Improvac (Zoetis), which is a GnRH vaccine licensed for boar taint prevention in pigs, where estrus and pregnancy returned as the contraceptive effect waned, but a booster dose prolonged suppression in queens (Morgan et al. 2019, Florencia et al. 2025). The association between anti-GnRH antibody levels and serum AMH concentrations provides valuable insights into the mechanisms underlying the contraceptive effects of Gonacon and the potential biomarkers of ovarian function in cats, particularly since AMH has been reported not to be affected by season in domestic cats (Place et al. 2011, Axner & Strom Holst 2015, Claassen et al. 2023) and shows clear differences between intact and ovariohysterectomized queens, with additional minor cyclic variation even in nonovulating intact queens (Johnson et al. 2023). Moreover, our results highlight the potential of the Gonacon vaccine, but also underscore the need to explore further potentially more effective regimens, such as higher vaccination doses, administration of Gonacon booster vaccinations (as has been shown in other species), more potent immunogenic vaccine formulas, or complementary approaches that would provide more consistent, effective, and longer-lasting strategies to address reproductive control in free-roaming cat populations (Baker et al. 2018, Pinkham et al. 2022). In addition, future research should also identify alternative biomarkers of ovarian function to improve monitoring of contraceptive efficacy in immunocontracepted individuals.
Contrary to our original hypothesis, indoor and outdoor queens differed substantially in their responsiveness to both anti-GnRH and rabies vaccines. Our study revealed notable differences in immunogenicity, reproductive outcomes, and owner satisfaction. Outdoor queens showed lower immune responses to both the Gonacon and rabies vaccines, experienced more reproductive events, and their owners expressed lower satisfaction than those of indoor queens. Interestingly, in our parallel study of pet toms monitored for 1 year after Gonacon–rabies vaccination, we observed similar indoor–outdoor differences, suggesting a broader, habitat-related effect (Novak et al. 2026). Free-roaming outdoor cats may receive less care and attention from their owners than indoor cats and may be exposed to greater social stress (de Assis & Mills 2020, Ma et al. 2023). They typically face many challenges, including exposure to harsh weather and noise, as well as risks of injury from other animals, people, and vehicles (Slater 2007, Gerhold & Jessup 2013, Gunther et al. 2015, 2018, Crawford et al. 2019). Moreover, they are susceptible to infectious diseases and at greater risk of compromised health, resulting in higher morbidity and mortality rates (Slater 2007, Gunther et al. 2018, Crawford et al. 2019). Indeed, our study found lower PCV in outdoor queens, possibly indicating anemia in some of them. Furthermore, the loss-to-follow-up rate was higher among outdoor queens, highlighting the risks associated with their habitat. Potentially, the suboptimal conditions of outdoor queens may contribute to reduced immune response and antibody production following vaccination with Gonacon and rabies vaccines, as has been suggested for other vaccines and species (Zimmermann & Curtis 2019, Tizard 2021). Our findings highlight key challenges in managing feral and free-roaming cat populations and the associated public health risks, especially given that most free-roaming cats receive only a single lifetime rabies vaccination, typically during TNVR. Strategies aimed at improving the health and welfare of outdoor cats, such as habitat enrichment, proper feeding, and vaccination campaigns, may help mitigate the negative impacts of outdoor living and promote population control in free-roaming cat populations (Levy et al. 2003, de Assis & Mills 2020, Lawson et al. 2020, Ma et al. 2023).
Owner satisfaction with Gonacon–rabies vaccination varied with housing conditions and reproductive outcomes. Indoor queens’ owners reported significantly higher satisfaction levels than outdoor cat owners, likely due to the lower incidence of reproductive events and the perceived efficacy of the Gonacon vaccine in indoor settings. Similarly, owners whose cats did not experience reproductive events were more likely to report greater satisfaction with Gonacon–rabies immunization, underscoring the importance of achieving the desired contraceptive outcome. Notably, owner satisfaction was not influenced by the queens’ immune response to the rabies vaccine, as owners were unaware of these laboratory findings. This highlights a concern, as inadequate rabies responsiveness, if it occurs, may go unnoticed clinically and therefore be overlooked in practice. Future work should also examine how well owners understand the implications of inadequate rabies vaccine responses in free-roaming cats, as these deficiencies may otherwise go unnoticed. Moreover, there is a broader need to strengthen communication not only with owners but also with veterinarians, animal welfare organizations, municipal authorities, policy makers, and other stakeholders, to ensure that expectations and management practices are aligned with the realistic outcomes and constraints of immunocontraceptive and rabies-control strategies (de Assis & Mills 2020, Luzardo et al. 2023).
Overall, our study provides new insights into the safety, efficacy, and owner satisfaction associated with combined Gonacon–rabies vaccination in owner-owned queens living in their natural habitats. Our findings demonstrate marked differences in immunogenicity and reproductive outcomes following rabies and Gonacon vaccination between indoor and outdoor queens, underscoring the importance of environmental and housing conditions when implementing reproductive and rabies control strategies. Future research should aim to clarify the mechanisms driving individual variability in vaccine responses and to develop targeted approaches that enhance contraceptive efficacy, particularly in outdoor cats. Overall, this work advances the understanding of feline reproductive health management and supports evidence-based strategies to reduce cat overpopulation while safeguarding public health and animal welfare.
Supplementary materials
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Declaration of interest
The authors declare that they have no known competing financial or personal interests that could have appeared to influence the work reported in this paper.
Funding
This work was supported by the Israel Chief Scientist, Ministry of Agriculture and Rural Development (grant number 12-11-0009) and the Hebrew University. The research was also supported in part by the U.S. Department of Agriculture, Animal and Plant Health Inspection Service.
Author contribution statement
SN contributed to investigation, data curation, visualization, and preparation of the initial draft. BY contributed to conceptualization, resources, validation, and writing (review and editing). RK contributed to conceptualization, methodology, and writing (review and editing). LJ contributed to investigation, formal analysis and writing (review and editing). DCE contributed to conceptualization, resources, validation, and writing (review and editing). TR contributed to supervision, conceptualization, methodology, formal analysis, resources, funding acquisition, visualization, writing of the original draft, and review and editing of the manuscript.
Acknowledgments
We are grateful to Ms. Nathalie Weizmann, Dr Ron Sicsic, Dr Liat Morgan, Dr Idit Gunther, Dr Hagai Akiva, and Dr Shaked Druker for their help in advertising the study for cat recruitment, providing technical assistance, and participating in valuable discussions during group meetings.
References
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Associated Data
Supplementary Materials
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