Introduction

FPV, FCV, and FHV-1 are among the most significant pathogens affecting domestic cats worldwide. FPV, a member of Carnivore protoparvovirus 1, causes feline panleukopenia, characterized by severe leukopenia, enteritis, and high mortality rates (> 90%), especially in peracute cases1. This non-envoloped DNA virus is remarkably stable in the environment, contributing to persistent outbreaks if disinfection and vaccination are inadequate. FCV, an RNA virus of the Caliciviridae family, contributes to upper respiratory tract disease and oral ulceration. It is also highly contagious and environmentally stable, frequently detected in high-density settings such as animal shelters due to its capacity to persist on surfaces and its genetic diversity2. FHV-1, an alphaherpesvirus, causes feline viral rhinotracheitis, manifesting as conjunctivitis, nasal and ocular discharge, sneezing, and sometimes corneal ulcers. Nearly all cats that recover from primary FHV-1 infection become lifelong latent carriers with the virus sequestered in neurons; these latent infections can reactivate under stress, leading to recurrent shedding and clinical signs2. Together, FPV, FCV, and FHV-1 represent a serious health burden in cats globally, emphasizing the critical importance of effective vaccination programs in controlling these infections.

Vaccination remains the cornerstone of prevention for these viruses. In particular, feline trivalent vaccines combining antigens for FPV, FCV, and FHV-1 are considered core vaccines internationally3. The trivalent vaccine used in this study includes the inactivated virus to optimally stimulate protective immunity. Inactivated virus components are non-replicating and rely on adjuvants to produce immunity. Upon vaccination, cats develop active immunity primarily through production of virus-neutralizing antibodies that prevent viral attachment and replication in host tissues. For FPV, high titers of neutralizing IgG correlate strongly with protection from panleukopenia; in seronegative cats without maternal antibodies, a single MLV FPV vaccination can induce sustained antibody levels that confer long-term immunity1. In the case of FCV, vaccination does not always produce sterilizing immunity due to high genetic variability and immune escape, but it significantly reduces the severity of disease and virus shedding4,5. In addition to humoral immunity, cell-mediated immune responses are also induced – these are particularly important for intracellular pathogens such as FHV-1, where cytotoxic T cells and Th1 responses help control virus replication and prevent reactivation6. Thus, by inducing a combination of neutralizing antibodies and cellular immunity, the trivalent vaccine provides broad protection, and its repeated administration can further elevate and sustain immunity in the feline population.

While experimental studies have demonstrated robust protection conferred by vaccination, large-scale field data evaluating population-level immunity under real-world conditions remain scarce, particularly regarding factors such as seasonal influences, maternal antibody interference, individual immune variability, and environmental challenges. To address this gap, we conducted a comprehensive serological assessment involving 4,736 serum samples collected from domestic cats vaccinated with the trivalent vaccine Meowonder™ across diverse regions in China between 2024 and 2025. The primary objective of this study was to evaluate protective antibody titers elicited by Meowonder™ vaccine against FPV, FCV, and FHV-1. This research provides crucial insights into Meowonder™ vaccine’s protective efficacy and its capacity to enhance immunity through repeated administration, thereby offering valuable guidance for optimizing vaccination schedules and advancing feline vaccination programs in China.

Results

Cat population

A total of 4,736 feline serum samples were collected from veterinary clinics across 24 provinces in China between July 2024 and June 2025 for post-vaccination monitoring. The provinces contributing the most samples were Henan (n = 1,057), Guangdong (n = 1,033) and Jiangsu (n = 351) (Fig. 1A). All cats had received the Meowonder™ vaccine containing inactivated FPV strain 708, FCV strain 60 and FHV-1 strain 64. Of the 4,736 cats sampled, 2,145 (45.3%) were male, 1,978 (41.8%) were female, and 613 (12.9%) had unspecified sex (Fig. 1B). Age was categorized based on the AAHA/AAFP feline life-stage guidelines. Among the 4,736 cats included in this study, 3,169 (66.9%) were classified as kittens (≤ 1 year), 1,299 (27.4%) as young adults (1–<7 years), 193 (4.1%) as mature adults (7–<10 years), and 75 (1.6%) as seniors (≥ 10 years). The kitten period is widely recognized as a critical immunological window during which vaccines are most effective in eliciting robust and protective antibody responses. A more specific age distribution of the kitten cohort was shown in Fig. 1C. This detailed distribution highlights that a substantial proportion of kittens were sampled at 3–6 months of age. The breed was documented for all individuals, with the majority being domestic shorthair cats and the remainder comprising various pure breeds, including British Shorthair, Persian, Ragdoll, and the like. Vaccination records indicated that 107 cats received a single dose of the trivalent vaccine, 3,433 completed a two-dose regimen, 424 received a full three-dose primary series, and 409 received a booster vaccination.

Fig. 1
figure 1

Geographic, demographic, and age distribution of vaccinated cats included in the study. (A) Geographic distribution of sampled cats across 24 regions in China. Shading intensity reflects the number of samples collected from each region, with darker red indicating higher sample density. Henan (1057) and Guangdong (1033) contributed the largest number of samples for each virus, followed by Jiangsu (351), Guangxi (297), Beijing (209), Hunan (187), Zhejiang (173), Sichuan (162), Shandong (154), Anhui (151), Hubei (124), Hebei (119), and Chongqing (70). (B) Gender distribution of vaccinated cats, categorized as male (2145), female (1978), or unknown (613). (C) Age distribution of cats ≤ 1 year old, categorized by month and cats ≥ 1 year old. The number of cats sampled at each age was as follows: 1 M (1 cat, 0.03%), 2 M (4, 0.13%), 3 M (309, 9.8%), 4 M (891, 28.1%), 5 M (699, 22.0%), 6 M (521, 16.4%), 7 M (282, 8.9%), 8 M (190, 6.0%), 9 M (136, 4.3%), 10 M (90, 2.8%), 11 M (46, 1.5%) and ≥ 1Y (1567, 33.1%).

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Serum FPV, FCV, FHV-1 antibody titer after clinical vaccination

Following trivalent vaccination, antibody responses varied across the three core pathogens. For FPV, statistical analysis showed that the neutralizing antibody titer in the three-dose group was higher than those in the one-dose and two-dose groups (p < 0.01) (Fig. 2A). However, the antibody positivity rates across all three FPV immunization groups remained high and comparable, at 95.0%, 96.0%, and 99.0%, respectively. For FCV, no statistically significant difference was observed between the two-dose and three-dose groups in terms of neutralizing antibody titers (Fig. 2B), with similar positivity rates of 90.0% and 89.0%, respectively. In the case of FHV-1, three-dose vaccination produced significantly higher antibody titers compared to the two-dose group (p < 0.01) (Fig. 2C), yet the positivity rates were close, at 87.0% and 88.0%. Booster vaccinations administered to cats with completed immunization history helped maintain high FPV seropositivity (98.0%), while FCV and FHV-1 booster responses exhibited slightly lower positivity rates of 74.0% and 85.0%, respectively.

Fig. 2
figure 2

Serum antibody titers against FPV (A), FCV (B), and FHV-1 (C) following sequential doses of triple vaccine in cats. Box plots display neutralizing antibody titers for each virus, with the y-axis presented on a base-10 logarithmic scale. (A) Mean FPV antibody titers following the first, second, and third vaccine doses, as well as the booster dose, were 11,202, 12,236, 13,715, and 12,529, respectively. (B) Mean FCV antibody titers after the first, second, third, and booster doses were 315.3, 275.6, 364.4, and 318.0. (C) Mean FHV-1 antibody titers after the first, second, third, and booster doses were 125.1, 111.4, 140.2, and 152.7, respectively. *p < 0.05, **p < 0.01, and ***p < 0.001.

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Seasonal trends in serum antibody titers following clinical vaccination

According to the seasonal categorization of months in China—spring (March–May), summer (June–August), autumn (September–November), and winter (December–February)—antibody titers were analyzed to assess seasonal variations in humoral immune responses. For FPV, antibody titers in spring were significantly higher than those observed in summer, autumn, and winter (Fig. 3A). For FCV, titers peaked in autumn, showing significantly higher levels compared with spring, summer, and winter (Fig. 3B). In the case of FHV-1, titers in spring, autumn, and winter were all significantly higher than those in summer (Fig. 3C). These results indicate clear seasonal fluctuations in antibody responses.

Fig. 3
figure 3

Seasonal distribution of serum antibody titers against FPV, FCV, and FHV-1 following triple vaccination. Seasonal distributions of serum antibody titers for FPV, FCV, and FHV-1 from July 2024 to June 2025 are illustrated using box plots to visualize dynamic changes in humoral immune responses over time. The y-axis represents the neutralizing antibody titer on a logarithmic scale with base 10. (A) Mean FPV antibody titers in spring, summer, autumn, and winter were 13422.4, 12093.5, 11748.6, and 12038.6, respectively. (B) Mean FCV antibody titers were 231.9, 225.8, 414.5, and 260.6, respectively. (C) Mean FHV-1 antibody were 108.8, 81.1, 131.6, and 140.1, respectively. *p < 0.05, **p < 0.01, and ***p < 0.001, ****p < 0.0001.

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Age-associated susceptibility following triple vaccination

Based on 4736 serum samples from cats receiving two or three doses of the trivalent vaccine, humoral immune responses to FPV, FCV, and FHV-1 were examined across different age group (Fig. 4). In both the 2-dose and 3-dose groups, neutralizing antibody titers against FPV, FCV, and FHV-1 were generally comparable across most age groups, with no statistically significant differences, except in 7-month-old cats, where FCV titers were significantly higher in the 3-dose group than in the 2-dose group (Fig. 4E). Notably, kittens aged ≤ 3 months (Fig. 4A) exhibited lower FPV neutralizing antibody titers compared to older age groups, regardless of receiving two or three doses. A marked increase in FPV antibody titers was observed between 3 and 7 months of age, followed by a plateau in older cats. In the 2-dose group, mean FPV titers rose from 7,866.8 at 3 months to 14,288.3 at 7 months (Fig. 4E), while in the 3-dose group, titers increased from 4,781.7 at 3 months to 14,705.3 by 6 months (Fig. 4D). Beyond 7 months, titers stabilized in both groups, consistently exceeding 14,000. Supporting this, 460 serum samples out of 4736 exhibited relatively low FPV titers (102.3 ≤ X ≤ 1023.3). Among these, 421 cats had received two doses, with 69% being 3- or 4-month-old kittens, and 26 cats had received three doses, of which 62% were also within the 3–4 month age range (Table 1).

Fig. 4
figure 4

Age-stratified neutralizing antibody titers against FPV, FCV, and FHV-1 following trivalent vaccination in cats aged ≤ 3 to ≥ 12 months. The y-axis represents the neutralizing antibody titer on a logarithmic scale with base 10. Box plots illustrate serum antibody titers by age group, from ≤ 3 months to ≥ 12 months, showing humoral immune responses to FPV, FCV, and FHV-1 in cats receiving either two or three doses of the trivalent vaccine. * indicates p < 0.05, ns indicate p > 0.05.

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Table 1 Distribution of cats’ vaccination ages showing lower effective serum FPV neutralizing antibody titers after Meowonder™ vaccination (102.3 ≤ X ≤ 1023.3).
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Wild-type FPV infection before and after vaccination

Among 435 vaccinated cats which were clinically healthy showed in Fig. 5, serum neutralizing antibody titers against FPV exceeded 1:8000, indicating a strong humoral immune response. In contrast, the corresponding titers against FHV-1 and FCV in the same individuals remained below 1:50, although potentially providing partial protection, indicate that a larger proportion of cats may fall short of the commonly accepted threshold for protective immunity. Further follow-up revealed that these cats had a history of contact with FPV-infected individuals both before and after vaccination based on the owner’s recollections and feedback, suggesting the possibility of antigenic stimulation through environmental exposure.

Fig. 5
figure 5

Irregular serum antibody titers against FPV, FCV, and FHV-1. Violin plots display serum antibody titers measured in 435 cats for each virus. Mean titers were highest for FPV (17,445), followed by FHV-1 (25.42) and FCV (23.98).

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Suboptimal immune response

Among the 4736 feline serum samples analyzed, 98 (2.07%) exhibited notably low neutralizing antibody titers (Fig. 6). The detailed background information of the 98 cats—including province of origin, breed, sex, clinical manifestation, age at first Meowonder™ vaccination, immunization status, and time of blood collection—is recorded in supplementary data. The average FPV titer in this group was 297.5 with a seropositivity rate of 62%; for FCV, the mean titer was 21.2 with 61% positivity; and for FHV-1, the mean titer was 17.7 with a positivity rate of 39%. These findings suggest that approximately 2% of the population may exhibit suboptimal immune responses or reduced sensitivity to vaccine antigens, resulting in inadequate post-vaccination antibody levels.

Fig. 6
figure 6

Comparison of sub-threshold serum antibody titers against FPV, FCV, and FHV-1. Violin plots display serum antibody titers measured in 98 cats whose antibody responses did not reach the defined threshold for protective immunity. Mean titers were highest for FPV (297.5), followed by FCV (21.2), FHV-1 (17.7).

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Discussion

According to international guidelines such as those from the WSAVA, vaccination is considered the primary strategy for eliminating the threat posed by FPV, FCV, and FHV-1 globally3. However, data describing antibody responses under real-world conditions and across large, diverse cat populations in China remain limited. This study provided a comprehensive evaluation of humoral immune responses to a trivalent feline vaccine, addressing protective antibody titers, seasonal influences on vaccine effectiveness, and variability in immune responses due to maternal antibody interference and genetic factors.

Based on the background information of the sampled cats, only 14 individuals-initiated vaccination at or before two months of age (≤ 8 weeks), while the remaining cats received their first dose at three months of age (≥ 12 weeks) or later. Consequently, the majority of cats received their second vaccination at 16 weeks of age or older, aligning with the vaccination schedule recommended by the WSAVA3. According to the collection of data from 4736 cats, two doses of the multivalent vaccine were sufficient to induce strong protective antibody titers against FPV, FCV, and FHV-1 in the majority of cats. Although the antibody titers following two doses were slightly lower than those observed after three doses, they still exceeded the seropositivity threshold7, indicating adequate immunogenicity. However, the positivity rates following the booster dose for FHV-1 and FCV were lower than those observed after the two- or three-dose regimen. This unexpected observation may be explained by inaccuracies in the timing of booster administration and the small sample size. Some pet owners may forget to bring their cats for booster vaccinations, causing the interval between the booster and the initial vaccination to exceed one year. In such cases, the antibody response after the booster may be lower than if it were given within a year. Moreover, the limited number of cats that received the booster could introduce bias into the analysis, leading to the unexpected finding that the FCV and FHV-1 antibody positivity rates after boosting appear lower. Additionally, the two-dose regimen demonstrates comparable immune efficiency and presents several practical advantages. Firstly, the positivity rate after the second dose approached a plateau, suggesting the successful induction of immunological memory. This plateau likely results from the activation and proliferation of antigen-specific B cells, which differentiate into memory B cells and long-lived plasma cells capable of continuously secreting protective antibodies8. Secondly, It reduces the likelihood of adverse reactions and minimizes the number of veterinary visits, thereby reducing stress in pets induced by clinical environments and lowering the risk of hospital-acquired infections. Additionally, given the existing pressures of work and daily life, the two-dose protocol can save time and lessen the burden on pet owners. Previous studies on FPV, FCV, and FHV-1 vaccines have also suggested that less frequent vaccination can provide durable protection3,5, with a two-dose regimen proving effective9. Together, these findings suggest that, under appropriate conditions —such as in cats older than 3 months—a two-dose primary vaccination can effectively induce robust and sustained immunity, offering a more practical alternative to the three-dose regimen.

In this study, antibody titers displayed seasonal fluctuations, with FPV peaking in spring, FCV in autumn, and FHV-1 in autumn and winter (Fig. 3). These patterns likely reflect increased vaccination efforts during times aligned with periods of higher disease prevalence. The rise in antibody titers during the spring and autumn likely reflects renewed vaccination efforts and increased environmental exposure to viruses. Additionally, antibody levels remained relatively stable throughout the winter, which may be due to decreased outdoor activities. This phenomenon aligns with previous veterinary epidemiological findings, which indicate that stray cats exhibit significantly higher FCV antibody titers than owned cats, suggesting that increased outdoor activity enhances antibody responses10. The observed decline in antibody levels for FPV during autumn and for FHV-1 and FCV during summer coincides with the peak of kitten season11, likely reflecting an increased the number of newborn kittens carrying MDAs. The maternal immunoglobulin can impede the kitten’s ability to develop its own immune response when vaccinated at a young age1. The age-stratified antibody responses further corroborate the inhibitory role of MDAs, as lower FPV titers in kittens ≤ 3 months of age gradually increase after 4 months, reflecting the natural decline of MDA levels and improved vaccine responsiveness. These findings align with WSAVA recommendations, which advocate for the completion of core vaccination by 16 weeks of age, followed by a booster at 6 or 12 months depending on MDA interference risk. Previous studies have shown that MDAs capable of interfering with vaccine efficacy can still be detected in the serum of 5-month-old (20-week-old) kittens, or even older1. Maternal antibodies against FCV and FHV-1 are typically very low or absent in kittens by six weeks of age. The duration of these MDAs varies between viruses, with FHV-1 MDA persisting for approximately 2 to 10 weeks12,13,14. At six weeks of age, the average FHV-1 MDA titer is around 1:8 12. Given the very low antibody levels observed at this age, no further analysis was performed for kittens younger than six weeks. These results emphasize the importance of timing the final vaccine dose at ≥ 16 weeks, in line with WSAVA recommendations, to ensure strong and sustained immunity.

Mechanistically, there are several ways MDAs inhibit active immunity. Firstly, maternal antibodies cover the epitopes of the pathogen or vaccine, preventing the kitten’s B cells from recognizing those sites15. Secondly, immune complexes formed by maternal antibodies and antigens can activate inhibitory Fcγ receptors on B cells, preventing B-cell activation and antibody production15. Additionally, MDAs can also weaken the adaptive immune response by interfering with T cells activation16. When vaccine antigens are neutralized or masked by pre-existing maternal antibodies, they are less efficiently captured and processed by antigen-presenting cells. As a result, MHC molecules present fewer peptide epitopes, interfering the activation of T cells. Subsequently, CD4⁺ T cells may not help B cells sufficiently, and CD8⁺ T cells may not eliminate infected cells, leading to a weak or insufficient antibody response. Thus, young kitten’s active immunity and vaccination are readily interfered by MDAs. Nonetheless, overall vaccination has dramatically reduced the incidence and severity of these diseases, and maintaining high vaccine coverage is critical for population health3. Maternal antibody levels decline over time, but the rate of decay and the initial levels can vary widely between individuals17. To follow the optimal vaccination regimen, two doses are sufficient to establish stable immunity, provided that primo vaccination begins at or after 12 weeks of age. If kittens are vaccinated before 12 weeks, our data suggest that three doses are necessary to overcome MDA. Therefore, MDAs temporarily affect overall positivity rate, a concept supported by previous work examining maternal antibody interference18,19.

In addition to maternal antibody interference and seasonal factors, this study also investigates the potential influence of environmental and genetic variables on vaccine-induced antibody titers. Building on the role of outdoor exposure discussed previously, our findings revealed that 435 vaccinated cats exhibited unusually high FPV antibody titers, exceeding those observed in laboratory-negative cats after vaccination7. Because all cats were clinically healthy and exhibited no characteristic signs of FPV infection, no pre-vaccination health assessment was performed; therefore, it was not possible to determine whether FPV exposure occurred prior to or following vaccination. However, the magnitude of the responses in these animals suggests possible exposure to FPV either prior to or following immunization. The information provided by the pet owners also supports our hypothesis. Studies have reported that the fecal FPV positivity rate reached 33% among 50 clinically healthy cats housed in UK shelters20. Additionally, FPV is highly stable in the suitable external environment and can persist for at least one years21. Even indoor-only cats may be indirectly exposed through their owners’ contaminated hands, shoes, or clothing22. In addition, several cats displayed disproportionately high FPV titers but low FCV and FHV-1 titers, which may indicate immune suppression associated with FPV-induced leukocyte reduction23,24, potentially impairing responses to the other vaccine components. As demonstrated in Dall’ Ara et al. (2023), post-vaccination field exposure can substantially elevate antibody titers beyond levels typically induced by vaccination alone10. However, FPV infection is known to induce leukopenia, as demonstrated in Carman’s study23. Sustained or intense FPV exposure may impair immune function. FPV targets rapidly dividing cells, including bone marrow cells, can induce panleukopenia, resulting in a marked reduction in circulating leukocytes and significant immunosuppression25. Such persistent antigenic stimulation, coupled with FPV-induced leukopenia, imposes a considerable immunological burden, potentially compromising the host’s ability to mount effective immune responses to concurrent or subsequent antigens. Collectively, these findings support the hypothesis that FPV exposure before or after vaccination contributed to the unusually elevated FPV antibody titers observed in this subgroup.

For cats with suboptimal immune systems, congenital immunodeficiencies may result from potential genetic variation in FLA. The major histocompatibility complex (MHC), referred to in cats as the FLA, is highly polymorphic and plays a central role in regulating immune responses26. Similar to other species, feline MHC class I and II molecules are responsible for presenting viral peptide epitopes to T cells, which is a key step in initiating protective immunity. Class I molecules present intracellular peptides to CD8⁺ cytotoxic T cells to trigger cell-mediated responses, while class II molecules present extracellular peptides to CD4⁺ helper T cells to support B cell activation and antibody production27. The strength of a cat’s vaccine response depends heavily on whether its specific FLA alleles can effectively bind and present these viral peptides to T cells. These findings underscore the importance of considering both environmental antigenic pressure and host genetic variability when evaluating heterogeneity in vaccine responsiveness within feline populations.

While neutralizing antibodies are a critical component of protective immunity, they do not fully explain vaccine efficacy for all pathogens in the trivalent formulation. FHV-1 establishes latent infections in the trigeminal ganglia, cell-mediated immunity, particularly the role of CD8 + T cells and IFN-γ production, is essential for controlling virus replication and preventing recurrent shedding6. Therefore, cats with low or undetectable neutralizing antibody titers may still possess effective cellular immunity against FHV-1. In contrast, neutralizing antibodies are an important marker of immune protection against FCV, but their effectiveness is limited by the virus’s genetic diversity. FCV has a broad range of strains with significant antigenic variation, meaning that neutralizing antibodies induced by a vaccine may not offer protection against all strains. As such, the antigenic diversity of FCV underscores the challenges in developing a universally effective vaccine and highlights the importance of considering strain-specific immunity in vaccine design3,28. These considerations highlight that both the quality of the immune response and the genetic variability of viral antigens must be included for when evaluating vaccine performance.

In conclusion, this large-scale serological assessment underscores the efficacy of the trivalent feline vaccine in producing robust and sustained humoral immunity against FPV, FCV, and FHV-1. For cats aged 3 months or older, two primary doses effectively established protective immunity for the majority of the feline population, with minimal additional benefit observed from a third dose. Seasonal variations significantly influenced antibody titers, suggesting optimal vaccination timing is crucial for maximizing immunity. MDAs notably impacted immune responses in younger kittens, emphasizing the need for age-specific vaccination strategies to overcome this limitation. Furthermore, environmental antigen exposure and genetic variability were identified as critical factors influencing vaccine responsiveness, highlighting the importance of considering individual and environmental contexts in feline immunization programs. Collectively, these results provide valuable guidance for optimizing feline vaccination protocols, thereby enhancing feline population health and disease prevention strategies.

Materials and methods

Virus and cell line

Feline kidney cells (F81 cells, preserved in our laboratory and were originally donated by the Center for Excellence in Molecular Cell Science) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, 61870036), supplemented with 8% fetal bovine serum (Cegrogen Biotech, A0500), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO2. FPV strain 708, FHV-1 strain 64, and FCV strain 60 utilized in this study were isolated from anal swab, eyelid and nasal swab suspensions of virus-infected cats in Henan Province in 2016. Virus isolation procedures followed previously established protocols described in prior studies7,29,30.

Vaccine and study design

The Meowonder™ vaccine containing inactivated FPV strain 708, FCV strain 60 and FHV-1 strain 64 utilized in this study had approval by the Ministry of Agriculture and Rural Affairs of the People’s Republic of China. Antigen content was quantified using the relative potency method, and all three components exceeded a potency ratio of 1.0. The vaccine was produced by PULIKE Biological Engineering, Inc. in a GMP-certified facility.

A standard two-dose vaccination schedule was implemented in clinical veterinary hospital settings, with doses administered at 3- to 4-week intervals, followed by 1 booster annually. While most cats received two doses according to protocol, some received a third dose based on veterinarians’ clinical discretion, typically administered at 3–4 week intervals with the final dose given at 16 weeks of age or older. The feline serum samples analyzed in this study were collected over a 12-month period (from July 2024 to June 2025) as part of routine veterinary care and vaccination follow-up in multiple animal clinics across 24 provinces in China. May 2024 marked the beginning of the vaccination campaign in participating veterinary clinics, while sample collection began in July 2024. All samples were obtained with the informed consent of owners prior to analysis. For blood sampling timepoint, it is required that a serum sample be collected from each cat 3–4 weeks after receiving the final dose (either the 2nd or 3rd dose) to test neutralizing antibody titers.

Feline population

Most of cats, key demographic and clinical information was recorded, including: (1) the cat’s origin; (2) sex, categorized as male, female, or unspecified; (3) age; (4) breed was directly recorded for each individual; (5) vaccination history, specifying whether the individual received a primary series (1–3 doses) or booster immunization with the trivalent vaccine. All cats were primarily housed indoors but were occasionally allowed supervised outdoor access. Most cats resided in a single-cat household. Serum samples were collected, transported under cold chain conditions, and stored at − 20 °C until serological analysis to detect antibody titer. Animal experiments were approved by the Animal Experiment Ethics Committee of the National Veterinary Research Center (Approval No.: 202404001). All blood samples were collected by skilled veterinarians. All the methods and procedures of this study comply with the requirements of the ARRIVE Guidelines.

Antibody titer measurement

Serum antibody titers against FPV, FHV-1, and FCV were determined using standardized virus neutralization (VN) assays6. Serum samples were heat-inactivated at 56 °C for 30 min prior to testing.

For FPV, FHV-1 and FCV, neutralizing antibody titers were determined by a two-fold serial dilution from 1:2 to 1:256, as per the previous method. Equal volumes of dilution serum and 200 TCID₅₀ of FPV strain 708, FCV strain 60, FHV-1 strain 64 were mixed and incubated at 37 °C for 1 h, followed by the addition of 2 × 10⁴ F81 cells in 100 µL. Neutralizing antibody titers were determined using a constant-virus, serum-dilution assay, in which a fixed concentration of FPV, FCV, and FHV-1 was employed for all neutralization tests. After incubation at 37 °C with 5% CO₂ for 4–5 days, virus neutralization was assessed by CPE and titers were reported as the reciprocal of the highest serum dilution that inhibited infection of the F81 cells in 50% of the culture wells.

The antibody threshold values were based on Mouzin’s study31. Additionally, the standard for antibody protection threshold values were slightly adjusted upward in the present analysis.

Statistical analysis

Antibody titer values were log10-transformed prior to analysis to normalize the data distribution. Monthly variation (July 2024-June 2025) was tested with one-way ANOVA on log10 titers followed by Tukey’s multiple-comparisons test (α = 0.05). To further detect potential “assay-session” effect, we repeated the analyses in a sensitives framework that adjusted p-values using a Benjamin-Hochberg FDR of 0.10 across month wise contrasts and collapsed months into seasons (Spring: March–May, Summer: June–August, Autumn: September–November, Winter: December–February) to increase power and reduce session-to-session noise. All statistical analyses were conducted using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, USA). A significance threshold of p < 0.05 was applied.