Antibody responses to inactivated (adjuvanted) and attenuated (non-adjuvanted) feline panleukopaenia virus vaccination,including in different injection sites

Abstract

Objectives

Vaccines have traditionally been administered to cats into the interscapular subcutaneous region (‘scruff’) for ease of restraining the cat while the vaccine is injected. Concern about feline injection-site sarcomas (FISSs) has prompted recommendations to use distal sites such as the hindlimb or tail, but evidence of adequate immune response at these sites is limited. This field study evaluated the antibody response to feline panleukopaenia virus (FPV) in kittens vaccinated with one of two commercial vaccines, injected at different sites.

Methods

An inactivated (killed), adjuvanted pentavalent vaccine (Fel-O-Vax 5; Boehringer Ingelheim Animal Health Australia) was injected subcutaneously into the scruff, left distal hindlimb or proximal tail. A modified-live virus (attenuated [MLV]), non-adjuvanted trivalent vaccine (Feligen RCP; Virbac Animal Health Australia) was given into the scruff only. For the latter, an inactivated, adjuvanted monovalent feline leukaemia virus vaccine was administered concurrently into the contralateral scruff, left distal hindlimb or proximal tail. Only one FPV-containing vaccine was administered to each cat/kitten: either the inactivated vaccine given in one of the three different anatomical regions or the MLV vaccine given into the scruff only. Cats were re-vaccinated in the same site and using the same vaccine type. Kittens were randomly allocated into study groups according to order of recruitment. Kittens were sampled at four time points over 12 months (T0, T1, T2 and T12). Protective FPV antibody titres were determined by haemagglutination inhibition (HI; negative <1:32, positive ⩾1:32).

Results

Samples from 100 kittens (T0, T1, T2) and 77 returning adult cats (T12) were available for FPV antibody testing (median age of kittens at T0 was 13 weeks, range 7–16). Eight kittens (8%) were found to be HI-positive at T0. Protective titres did not differ significantly among the three injection sites for the inactivated vaccine. Linear modelling demonstrated that antibody titres were higher in kittens given the MLV vaccine compared with kittens given the inactivated vaccine at T1 (P = 0.006) and T12 (P = 0.001). However, the proportion of kittens protected in each group (MLV vaccine vs inactivated vaccine) was the same. For most kittens (91%), a single vaccination was sufficient to produce protective antibody levels, irrespective of whether they were given the MLV (scruff) or inactivated vaccine (at any site). Age and sex had no effect on antibody titres after vaccination. Two kittens (2%) failed to achieve protective titres after two doses of vaccine and required re-vaccination between 16 and 18 weeks. Inexplicably, one (1%) adult cat lacked a protective antibody titre response at T12 despite earlier seroconversion.

Conclusions and relevance

Both inactivated and MLV vaccines induce protective titres against FPV. Although most kittens aged above 7 weeks are protected 1 month after a single vaccination, some require re-vaccination to develop protection. Some kittens require a final vaccination at 16 weeks or older and a booster 6–12 months later to develop robust protective titres. Comparable FPV antibody responses are elicited when inactivated vaccines are administered into the scruff, left distal hindlimb or proximal tail. Notably in this study, the MLV vaccine tended to produce higher antibody titres. These results support evidence-based recommendations on vaccination site selection to mitigate FISS risk in jurisdictions where this condition is prevalent.

Keywords

Antibodies fibrosarcoma feline panleukopaenia virus field study humoral immune response injection-site sarcoma vaccination veterinary science

Introduction

The most common injection site in cats, historically, has been the dorsal interscapular space (the ‘scruff’), involving subcutaneous and perhaps partly intramuscular injection. Its popularity is likely because of the ease of administration for veterinarians, who can simultaneously restrain the cat, raise a tent of skin and inject a vaccine (or antibiotic, anaesthetic or microchip). Despite an abundance of evidence from human medicine that intramuscular injection induces higher antibody titres, feline vaccine licensing studies usually involve the administration of vaccines subcutaneously, typically into the scruff.^1
–4

Feline injection-site sarcomas (FISSs) were first reported in the USA in 1991, with lesions typically located in the interscapular region.⁵ Thereafter, incidence rates of one case per 1000–10,000 cats vaccinated (USA) and one case per 5000–12,500 vaccination visits (UK) have been reported.^6
–9 The incidence of FISS has not yet been described in Australia, although rare sporadic cases of post-vaccinal FISSs certainly occur.¹⁰ Some studies have noted an increased risk of FISS formation with adjuvanted vaccines containing aluminium compared with non-adjuvanted vaccines, in particular rabies and feline leukaemia virus (FeLV) vaccines, while others have not identified an increased risk.^5,6,11
–16 The current American Animal Hospital Association and the former American Association of Feline Practitioners (now the Feline Veterinary Medical Association) guidelines for feline vaccination recommend subcutaneous injection of vaccines into the left distal hindlimb (FeLV) or distal tail (rabies) to allow for wide excision by amputation should post-vaccinal complications occur. This is despite little work being done to demonstrate comparable immunological responses to vaccination at these alternative sites.¹⁷ In one pilot study, a modified-live virus (MLV) core vaccine containing feline panleukopaenia virus (FPV) and an inactivated rabies vaccine were administered either in the lateral hindlimbs subcutaneously below the stifles or intramuscularly in the distal third of the tail. No differences in antibody levels 1–2 months after vaccination were observed, and both sites of vaccination were well tolerated by cats, with minimal restraint required.¹ However, further evidence of comparable post-vaccinal antibody responses between different sites of vaccine administration is needed to assist the development of evidence-based consensus vaccination guidelines.

The site of vaccination has been shown to affect the post-vaccinal immune responses in some mammals. In rats and dogs, vaccination with an MLV vaccine produced the highest antibody response when administered subcutaneously into the Houhai acupoint (the centre part of the depression on the midline between the anus and the ventral base of the tail), compared with administration under the jaw, the popliteal fossa or the dorsal back.¹⁸ In human medicine, the standard recommendation is that vaccines should be administered intramuscularly, typically into the deltoid or the anterolateral aspect of the thigh to optimise the immunogenicity of the vaccine and to minimise adverse reactions at the injection site.^3,4,19 For example, administration of a hepatitis B vaccine into the upper arm produced an antibody titre up to 17 times greater than when administered into a person’s gluteal region.²⁰ In cats, we previously noted a significant difference in serological response to vaccination against FeLV, which might have been due to site of vaccine administration (scruff vs right flank fold).²¹ Theoretically, it is possible there is variation in blood supply and/or lymphatic drainage across different tissue types, which could affect the accessibility of immune cells, such as macrophages and dendritic cells, to the tissues where the vaccine is administered; these cells are essential for antigen recognition, processing and presentation to lymphocytes.^22
–24 Therefore, the hypothesis that there are differences in immune responses after vaccination at different anatomical locations is reasonable and warrants further investigation in cats.

The aim of this study was to determine the anti-FPV serological responses of kittens against a primary vaccination series with an inactivated (killed), adjuvanted vaccine (Fel-O-Vax 5; Boehringer Ingelheim Animal Health Australia) administered in different locations (the scruff, left distal hindlimb or proximal tail). A secondary aim of the study was to compare FPV antibody responses after administration of two different commercially available FPV vaccine formulations, the same inactivated, adjuvanted vaccine (Fel-O-Vax 5) and an MLV (attenuated), non-adjuvanted vaccine (Feligen RCP; Virbac Animal Health Australia).

Materials and methods

Study population

A total of 100 healthy kittens, aged 7 weeks or older, with no known history of exposure to FPV, were recruited via a rescue organisation in Sydney, NSW between January and August 2020 while awaiting rehoming. The kittens were sampled monthly at three time points (T0, T1, T2). Of these animals, 77 returned as adult cats on day 365 for re-vaccination and a fourth sampling (T12). Approval was obtained from the University of Sydney Animal Ethics Committee (approval number 2019/1165). When kittens were rehomed, owners consented to ongoing participation in the study, while also being given the option to opt out of the study. Owners who agreed to participate were asked to confine their kittens indoors for the duration of the study and to return for re-vaccination and sampling as per the study schedule (Figure 1).

Figure 1

Study schedule for feline panleukopaenia virus (FPV) vaccination and blood sampling. Fel-O-Vax 5 is an inactivated (adjuvanted) vaccine containing FPV, while Feligen RCP is a modified-live virus (non-adjuvanted) vaccine containing FPV. Fel-O-Vax 5 was administered into the scruff, left hindlimb or tail. Kittens administered Feligen RCP were concurrently administered a monovalent feline leukaemia virus (FeLV) vaccine (Fel-O-Vax Lv-K or Leucogen FeLV) into the scruff, left hindlimb or tail. See Table 1 for study group information and the ‘Study groups and vaccines administered’ section for further vaccine formulation and manufacturer details. T0 = day 0; T1 = day 28; T2 = day 56; T12 = day 365

Study groups and vaccines administered

Kittens were allocated into four different groups (Table 1). Kittens were enrolled into groups consecutively, in order of presentation. This group allocation enabled examination of the post-vaccinal FPV antibody response according to site of FPV vaccination (tail vs left hindlimb vs scruff; inactivated vaccine only; groups 2 vs 3 vs 4) and FPV vaccine formulation independent of site of vaccination (MLV vs inactivated; group 1 vs groups 2–4). The current study was performed in conjunction with a study investigating FeLV antibody response according to the site of FeLV vaccination (tail vs left hindlimb vs scruff) and FeLV vaccination formulation, as described.²⁵

Table 1

Study schedule for recruited kittens (n = 100)*

Group	FPV vaccine	FPV vaccination site	FeLV vaccine	FeLV vaccination site²⁵
1	Feligen RCP (n = 69)	Scruff	Fel-O-Vax Lv-K Leucogen FeLV	Scruff, left hindlimb or tail Scruff, left hindlimb or tail
2	Fel-O-Vax 5 (n = 12)	Tail	Fel-O-Vax 5	Tail
3	Fel-O-Vax 5 (n = 10)	Left hindlimb	Fel-O-Vax 5	Left hindlimb
4	Fel-O-Vax 5 (n = 9)	Scruff	Fel-O-Vax 5	Scruff

Fel-O-Vax 5 is an inactivated (adjuvanted) vaccine containing feline panleukopaenia virus (FPV) while Feligen RCP is a modified-live virus (non-adjuvanted) vaccine containing FPV. See the ‘Study groups and vaccines administered’ section for vaccine formulation and manufacturer details. Kittens in group 1 had two vaccines administered concurrently at each time point, while kittens in groups 2–4 only received a single vaccine per time point. This meant some kittens in group 1 had two vaccines injected into the scruff, at slightly different locations

FeLV = feline leukaemia virus

Fel-O Vax 5 is an inactivated (killed), adjuvanted pentavalent vaccine that comprises FPV, feline herpesvirus-1 (FHV-1), feline calicivirus (FCV), Chlamydia felis and FeLV antigens. Feligen RCP is a core MLV (attenuated) non-adjuvanted vaccine containing FPV, FHV-1 and FCV. Fel-O-Vax Lv-K and Leucogen FeLV are monovalent, adjuvanted FeLV vaccines. Fel-O Vax 5 and Fel-O-Vax Lv-K are sold by Boehringer Ingelheim Animal Health, Australia, and manufactured in Fort Dodge, IA, USA. Fel-O Vax 5 is also sold as Fel-O-Vax Lv-K IV in North America (Elanco Animal Health). Leucogen FeLV and Feligen RCP are manufactured and distributed by Virbac Animal Health Australia.

It was intended that vaccines administered into the left hindlimb were injected distal to the stifle. This may not have always been achieved, and some vaccinations may have been administered at the level of the stifle. All tail vaccinations were administered into the proximal third of the tail.

Vaccination schedule

Animals received the vaccination schedule shown in Figure 1. Kittens were vaccinated three times (days 0, 28 and 56), with blood collected at each time point. An exception was kittens that were aged 20 weeks or older at the second time point (day 28). These animals only had two doses of vaccine administered (days 0 and 28), with the third time point (day 56) involving only blood collection.

Sample collection and processing

Sampling was performed in kittens on day 0 (T0), day 28 (T1) and day 56 (T2), and 1 year later in adult cats on day 365 (T12) (Figure 1). Approximately 1 ml of blood was collected from the jugular vein. Blood was aliquoted into two tubes, 0.5 ml into a lithium heparin tube and 0.5 ml into an EDTA tube. Lithium heparinised blood samples were centrifuged at 15,800 g for 3 mins, plasma was transferred to plain cryovials and all samples were stored at −80°C. At the end of the study, frozen plasma samples were transported on dry ice to Veterinary Diagnostic Services (VDS), University of Glasgow for batch serology testing.

Feline immunodeficiency virus and FeLV testing

Fresh EDTA-anticoagulated whole blood was used for testing with Anigen Rapid FIV/FeLV point-of-care (POC) test kits (BioNote) to detect FeLV capsid antigen (p27) and/or antibodies to feline immunodeficiency virus (FIV) envelope glycoprotein (gp40). Stored EDTA samples were used to perform semi-quantitative FeLV proviral real-time PCR (qPCR) testing at the University of Sydney.^26,27 FeLV-positive cats identified at any time point were excluded, while FIV-positive animals were retained in the study.

FPV serology testing for evaluation of post-vaccination antibody response

Haemagglutination inhibition (HI) testing was performed by VDS on thawed plasma samples, as described previously.²⁸ HI is the standard serological test used to determine the presence of adequate antibody-mediated immunity against FPV, with the presence of anti-FPV antibodies correlating strongly with protection from FPV challenge.^29
–32 In the current study, HI-negative samples were defined by a reciprocal HI titre of <32, due to observations of occasional non-specific agglutination up to a reciprocal titre of 16.²⁸ HI-positive samples were defined by having a reciprocal HI titre ⩾32.^{28,29,33
–35} Samples positive at a screening dilution (1:4) were serially diluted (two-fold) until they tested negative, with the maximum dilution of plasma where HI was observed reported as the titre. Reciprocal titres are reported herein (ie, 1:4 = 4, 1:8 = 8, etc).

Statistical analysis

Statistical analyses were performed using the statistical software Genstat 22nd Edition (VSN International) and R Version 4.3 (The R Foundation). A P value <0.05 was considered significant.

Anti-FPV antibody titres were compared based on HI dilution at which a positive result was obtained using a log₂ transformation. Two-tailed Fisher’s exact tests were applied to the results to assess statistical significance in a binomial test for proportions based on seronegativity vs seropositivity. Linear modelling was used to assess the effect of age, sex, time point (T0 vs T1 vs T2 vs T12), vaccine type (MLV [group 1] vs inactivated [groups 2–4]) and site of administration (scruff vs left hindlimb vs tail; inactivated vaccine only) on anti-FPV antibody titres before and after vaccination. The effect of an interaction between vaccine type and time point was also investigated and post-hoc Tukey’s tests were utilised to obtain pairwise differences. Some cats did not have sufficient samples available for all testing at every time point.

Results

Study population: kittens (n = 100)

A total of 100 kittens were available for FPV antibody testing, comprising 56 female and 44 male kittens (Table 2). Overall, the median age at recruitment was 13 weeks (range 7–16, interquartile range 9–14).

Table 2

Recruitment numbers and signalment details for feline panleukopaenia virus antibody testing in kittens (n = 100)

Vaccination site	Feligen RCP (MLV) (n = 69) (n = 53)	Fel-O-Vax 5 (inactivated) (n = 31) (n = 24)
Tail	–	12 (10) – group 2, median age 12 weeks; 8 F, 4 M
Left hindlimb	–	10 (9) – group 3, median age 13.5 weeks; 6 F, 4 M
Scruff	69 (53) – group 1, median age 13 weeks; 38 F, 31 M	9 (5) – group 4, median age 13 weeks; 4 F, 5 M

Adult cats that returned for re-vaccination and a fourth sample collection on day 365 (T12) are shown second in brackets (n = 77). See the ‘Study groups and vaccines administered’ section for vaccine formulation and manufacturer details

F = female; M = male; MLV = modified-live virus

Three FIV-positive kittens were included in the analysis, each of which tested FIV antibody-positive at all three kitten time points. All kittens were FeLV-negative (via antigen and proviral DNA qPCR testing) for the duration of the study.

Study population: adult cats (n = 77)

Of the 100 kittens, 77 returned as adult cats on day 365 (T12) for re-vaccination and a fourth sampling (44 female and 33 male cats) (Table 2). No FIV-positive cats returned for sampling at T12. All cats were FeLV-negative (via antigen and proviral DNA qPCR testing) on day 365.

Exclusion of kittens found to be HI-positive before vaccination

Of the 100 kittens, eight (8%) from three different litters tested FPV HI-positive on day 0 and were excluded from analysis (four females and four males; five kittens in group 1, two kittens in group 2 and one kitten in group 3; median age 12 weeks, range 8–12). Of the eight kittens, five were persistently HI-positive, with antibody titres in the range of 256–512 at T0, rising to 2048–4096 at T2, likely reflecting the presence of antibodies from a low level of prior natural FPV exposure presumably boosted by vaccination (kittens 28, 29, 30, 46 and 47 from two separate litters). The other three kittens had antibody titres that declined from 32–64 at T0 to 16 at T1, likely reflecting the presence of low levels of maternally derived antibodies (MDA) blocking response to vaccination (kittens 58, 59 and 60, all from one litter). One of these kittens (kitten 59) remained HI-negative at T2. All eight kittens had protective titres of antibodies at T12 (Table 3).

Table 3

Feline panleukopaenia virus (FPV) antibody results for eight kittens that were haemagglutination inhibition (HI)-positive at T0 (day 0, before vaccination)

Study	Group	Age	Sex (weeks)	HI reciprocal titre
Study	Group	Age	Sex (weeks)	T0	T1	T2	T12
Kitten 28*	2	12	M	512	2048	4096	4096
Kitten 29*	1	12	M	256	1024	2048	512
Kitten 30*	1	12	M	512	512	2048	2048
Kitten 46^†	2	12	M	256	1024	2048	1024
Kitten 47^†	1	12	F	256	1024	2048	4096
Kitten 58^‡	3	8	F	32	16	N/A	128
Kitten 59^‡	1	8	F	32	16	8	1024
Kitten 60^‡	1	8	F	64	16	1024	4096

Anti-FPV antibody titres were measured using serial dilutions, with reciprocal titres presented. T0 = day 0, T1 = day 28, T2 = day 56, T12 = day 365. Age at recruitment and first vaccination is shown

*,†,‡

Kittens from the same litter

F = female; M = male; N/A = not available

FPV post-vaccination antibody production

Effect of age

Overall, there was no significant effect of age on the FPV antibody result (P = 0.57 in kittens, P = 0.64 in adult cats). No effect of age at first vaccination (<8 vs 8–12 vs 12–16 weeks) on subsequent FPV antibody titres was observed (P = 0.35).

Effect of sex

Overall, no significant effect of sex on the FPV antibody result was observed (P = 0.58 in kittens, P = 0.91 in adult cats).

Effect of time point (number of vaccinations), vaccine type (MLV vs inactivated) and site of vaccination (inactivated vaccine only)

Table 4 summarises the protective titres (HI ⩾32) afforded by vaccination at each time point by group. Figure 2 presents antibody titres by group and time point. Overall, time point (number of vaccinations) was observed to be a significant effect, with FPV antibody titres higher (on average) at each successive sampling (ie, T12 > T2 > T1 > T0; P <0.001). When individual group (ie, groups 1–4) and time were considered together, there were no significant differences between FPV antibody titres produced. This included there being no difference between kittens vaccinated with the inactivated vaccine in the three different anatomical locations (P = 0.17).

Table 4

Level of protection against feline panleukopaenia virus (FPV) produced by vaccination with either the modified-live virus (MLV) vaccine administered into the scruff (Feligen RCP; group 1) or inactivated vaccine administered into three different anatomical sites (Fel-O-Vax 5; groups 2–4), by time point

Group	FPV vaccination site (and vaccine type)	Proportion of HI-positive (reciprocal titres ⩾32) kittens
Group	FPV vaccination site (and vaccine type)	T0	T1	T2	T12
1	Scruff (MLV)	0/64 (0)	55/62 (89)	57/58 (98)	49/49 (100)
2	Tail (inactivated)	0/10 (0)	10/10 (100)	10/10 (100)	6/7 (86)
3	Left hindlimb (inactivated)	0/9 (0)	7/7 (100)	9/9 (100)	9/9 (100)
4	Scruff (inactivated)	0/9 (0)	8/9 (89)	7/7 (100)	5/5 (100)

Data are n (%). Anti-FPV antibody titres were measured using serial dilutions and haemagglutination inhibition (HI), with reciprocal titres presented. No differences between the MLV vaccine and inactivated vaccine (ie, group 1 vs groups 2–4), or between kittens vaccinated in the three different locations (group 2 vs group 3 vs group 4), were observed. Eight kittens that were HI-positive (⩾32) at T0 are not shown. Some cats did not have sufficient samples available for HI testing at every time point

T0 = day 0; T1 = day 28; T2 = day 56; T12 = day 365

Figure 2

Results by study group and time point. There were no significant differences between feline panleukopaenia virus (FPV) antibody titres in groups 1–4 when vaccine type and time point were considered together (P = 0.17). Data points are plotted as circles, centre lines show the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. The red line shows the study cut-off for seropositivity (ie, assumed protection; haemagglutination inhibition [HI] ⩾32). Eight kittens that were HI-positive (⩾32) at T0 are not shown. inactivated vaccine = pentavalent vaccine (Fel-O-Vax 5); MLV = modified-live virus trivalent vaccine (Feligen RCP); T0 = day 0; T1 = day 28; T2 = day 56; T12 = day 365. Figure made with R Version 4.3 (The R Foundation)

At T1 (1 month after a single FPV vaccination), based on the assigned HI cut-off, 91% of kittens had protective titres of antibodies against FPV (n = 80/88; four samples missing). This comprised 55/62 (89%) cats vaccinated with the MLV vaccine and 25/26 (96%) administered the inactivated vaccine (no difference in protection between vaccine type [MLV vs inactivated] or site of vaccination [inactivated vaccine only]; P = 0.43 and P = 0.62, respectively; two-tailed Fisher’s exact tests). The eight HI-negative kittens (five males and three females) comprised seven kittens from group 1 (MLV vaccine into the scruff) and one kitten from group 4 (inactivated vaccine into the scruff), with a median age at first vaccination of 14 weeks (range 9–15).

At T2 (after two FPV vaccinations), based on the assigned HI cut-off, 99% of kittens had protective titres of antibodies against FPV (n = 83/84; eight samples missing). This comprised 57/58 (98%) cats vaccinated with the MLV vaccine and 26/26 (100%) administered the inactivated vaccine (no difference in protection between vaccine type or site of vaccination, P = 1.0 and P = 1.0, respectively; two-tailed Fisher’s exact tests). Overall, the number of protected kittens increased significantly from T1 to T2 (P = 0.035; Fisher’s exact test).

At T12 (1 year after the final kitten vaccination), based on the assigned HI cut-off, 99% of cats showed protective titres of antibodies against FPV (n = 69/70). This comprised 49/49 (100%) cats vaccinated with the MLV vaccine and 20/21 (95%) administered the inactivated vaccine (no difference in protection between vaccine type or site of vaccination, P = 0.30 and P = 0.60, respectively; two-tailed Fisher’s exact tests). Results from the one HI-negative kitten and one HI-negative adult cat at T2 and T12, respectively, are shown in Table 5.

Table 5

Feline panleukopaenia virus (FPV) antibody results for one kitten that was haemagglutination inhibition (HI)-negative at T2 (kitten 71) and one cat that was HI-negative at T12 (kitten 37)

Study	Group	Age at recruitment (weeks)	Sex	HI reciprocal titre
Study	Group	Age at recruitment (weeks)	Sex	T0	T1	T2	T12
Kitten 37	2	10	F	4	64	512	16
Kitten 71	1	14	M	16	8	8	1024

Anti-FPV antibody titres were measured using serial dilutions, with reciprocal titres presented. A result <32 was deemed HI-negative. Age at recruitment and first vaccination is shown

F = female; M = male; T0 = day 0; T1 = day 28; T2 = day 56; T12 = day 365

Although the level of protection (ie, proportion of HI-positive kittens after vaccination) was not significantly different between the MLV vaccine (group 1) and inactivated vaccine (groups 2–4) at any time point, when vaccine type and time point were considered together, there were significant differences in the magnitude of the antibody response produced. Vaccination of kittens with the MLV vaccine into the scruff (group 1) produced higher FPV antibody titres than vaccination of kittens with the inactivated vaccine into the three different sites combined (groups 2–4) at T1 (23% higher antibody titres; P = 0.006) and T12 (20% higher antibody titres; P = 0.001), but not T2 (P = 0.90) (Figure 3).

Figure 3

Results when vaccine type (MLV vs inactivated) and time point were considered together. Vaccination of kittens with the MLV vaccine into the scruff (group 1) produced higher feline panleukopaenia virus (FPV) antibody titres than vaccination with the inactivated vaccine into one of the three different anatomical locations (scruff, left hindlimb or tail; groups 2–4) at T1 (P = 0.006) and T12 (P = 0.001). Centre lines show the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. The red line shows the study cut-off for seropositivity (ie, assumed protection; haemagglutination inhibition (HI) ⩾32). Eight kittens that were HI-positive (⩾32) at T0 are not shown. inactivated vaccine = pentavalent vaccine (Fel-O-Vax 5); MLV = modified-live virus trivalent vaccine (Feligen RCP); T0 = day 0; T1 = day 28; T2 = day 56; T12 = day 365. Figure made with R Version 4.3 (The R Foundation)

Discussion

This study demonstrated that administration of an inactivated, adjuvanted vaccine containing FPV (Fel-O-Vax 5), injected subcutaneously into three alternate locations (scruff, left hindlimb or tail), did not elicit significantly different anti-FPV antibody responses. Based on the results of this study, the hypothesis that different anatomical sites of vaccine administration in the feline patient induce significant differences in the post-vaccinal immune response is rejected. This conclusion supports results from a similar study involving the same cohort of kittens, where no difference in antibody response was observed with differing sites of FeLV vaccination.²⁵ Consequently, these results provide an evidence base for recommendations from major vaccine guideline bodies in Europe and North America advising vaccination elsewhere than the scruff when possible, including subcutaneously into one of the hindlimbs or the tail.^1,17,36,37

Regarding the secondary aim of the current study, we observed at T1 (1 month after a single FPV vaccination) and T12 (1 year after the initial vaccination) that animals administered an MLV, non-adjuvanted vaccine subcutaneously into the scruff (Feligen RCP) developed significantly higher post-vaccinal reciprocal antibody titres against FPV compared with animals receiving the inactivated vaccine (Fel-O-Vax 5). These differences, however, did not translate into any difference in presumptive protection from FPV challenge, based on the pre-assigned antibody cut-off (HI ⩾32). The effect of concomitant FeLV vaccination on FPV antibody response (cats administered the MLV containing FPV also received a separate dose of a monovalent, adjuvanted FeLV vaccine) was considered unlikely to have any effect on study findings, given that MLV are highly immunogenic and comparable results have been reported previously.^34,36

Protection against FPV was provided by both vaccine formulations after only a single vaccine dose had been administered to kittens with a median age of 13 weeks. At T1 (1 month after the first vaccine), 80/88 (91%) kittens developed protective titres of antibodies against FPV, with no difference in proportionate seropositivity between vaccine types (MLV vs inactivated) found. This finding is consistent with other studies that have shown that a single dose of either an MLV or inactivated vaccine induces seroconversion against FPV in most kittens aged 9 weeks and 10 months.^38,39 Notably, with inactivated vaccines, time to seroconversion has been shown to be marginally longer (up to 1 week slower) when compared with MLV vaccines.⁴⁰ A report of feral cats receiving a single dose of vaccine (either inactivated or MLV) immediately on completion of neutering surgery observed no differences in the proportion of cats protected against FPV, with both preparations producing protection in most cases.³⁴ In contrast, one study found a significantly higher response rate 14 days after vaccination with a single MLV vaccine compared with a single inactivated vaccine (85% vs 31% protective titres), based on an FPV reciprocal antibody titre cut-off of 40 or higher.³³ Regardless, despite 91% of kittens developing protective titres with one dose of vaccine in the current study, the kittens at T1 without protective antibody titres are a reminder of the importance of recommending the second core vaccination for cats, irrespective of the type of vaccine used.³⁶

Overall, FPV antibody titres increased after administration of a second dose of vaccine, consistent with the anamnestic effects of repeat vaccination, although the increase in reciprocal titre after the second vaccination was not as marked as the increase in titre after the first vaccination. At T2 (1 month after the second vaccine), 83/84 (99%) kittens had protective titres of antibodies against FPV, again with no difference in proportionate protection based on vaccine type (MLV vs inactivated). In comparison, a field study in Germany using three different vaccines given at 8, 12 and 16 weeks reported that only 45% of kittens had protective antibody titres against FPV after two vaccinations, and 37% of kittens failed to seroconvert after three vaccines (as defined by a minimum four-fold increase in reciprocal antibody titres in consecutive sera).⁴¹ We observed two kittens in the current study that tested HI-negative at T2 and were therefore possibly not protected. One female kitten (kitten 59) was HI-positive on day 0 (antibody titre 32) and aged 8 weeks when first vaccinated (excluded). This animal had declining reciprocal antibody titres over the three kitten time points (T0: 32, T1: 16, T2: 8) (Table 3). The other was a male kitten (kitten 71) that was first vaccinated at the age of 14 weeks (MLV into the scruff) and had a low antibody titre of 16 on day 0 (included). This kitten also had declining titres over the three kitten time points (T0: 16, T1: 8, T2: 8) (Table 5). Both kittens were non-responsive to the first two vaccinations; it is likely that low levels of MDA interfered with the FPV vaccination.^40,42 These kittens were in group 1 (MLV vaccine administered into the scruff) and were vaccinated a third time at T2; at T12 both kittens showed protective titres of 1024. A further two kittens (kittens 58 and 60, excluded) presumably also had high FPV MDA, which delayed their response to vaccination; again, both developed protective titres by T12 (Table 3).⁴⁰ This group of kittens demonstrate the importance of a third FPV vaccine for some animals, when the kitten is older than 18 weeks, in accordance with similar results from other studies and recommendations from major vaccine consensus guideline bodies.^17,36

At T12 (1 year after the initial vaccination), 99% of adult cats showed protective antibody titres against FPV, with no difference observed in the proportionate protection between vaccine types (MLV vs inactivated). A single cat was FPV HI-negative at T12 (reciprocal titre 16); this animal (kitten 37) had received three doses of inactivated vaccine into the tail and was HI-positive at T2 after only two doses of vaccine (reciprocal titre 512) (Table 5). This kitten successfully mounted a protective immune response by the end of her kitten vaccine course, but inexplicably this cat’s antibody titre dramatically decreased between T2 and T12. Despite the decrease in antibody titre, this cat may still have been afforded protection against FPV challenge at T12. In an experimental challenge trial, 6/25 vaccinated cats had negative antibody titres after vaccination measured by virus neutralisation (reciprocal titres <10); however, there was no evidence of panleukopaenia after challenge with FPV.⁴³ The HI cut-off used in the current study to define seropositivity may be overly cautious, with other studies reporting reciprocal antibody titres of 20 to indicate a successful post-vaccination response and predict protection from FPV-related disease.^43,44 A cut-off one dilution lower (ie, reciprocal titre ⩾16) would have classified all adult cats at T12 as having protective antibody titres against FPV. The World Small Animal Veterinary Association vaccination guidelines recommend re-vaccination of cats aged approximately 6 months, rather than the traditional 16 months, narrowing the window of susceptibility for kittens that fail to mount a protective immune response after the first course of vaccination.³⁶ Our findings support this recommendation.

Testing of cats at the POC allows the clinician to evaluate the requirement for re-vaccination, simultaneously identifying cats at risk and avoiding administration of unnecessary FPV vaccines. The findings of this study suggest that the majority of cats retain protective titres to adulthood, and therefore illustrate the value of pre-vaccination utilisation of commercial POC ELISA kits (validated against HI) to measure anti-FPV antibodies.^35,44,45 Notwithstanding this individualised approach, it is also important to note that ‘herd immunity’ (>70% of animals protected in a population) is a fundamental concept for veterinarians to espouse to reduce FPV outbreaks; vaccinating more unprotected cats, even if a cat receives only a single FPV vaccine (MLV or inactivated), will achieve far more than re-vaccination of a small proportion of protected pet cats within a population.^36,46

In this field study (ie, client-owned animals instead of specific pathogen-free kittens housed inside a laboratory), owners were advised to keep their kittens indoors after rehoming until the primary kitten vaccination series was completed. Despite this, natural exposure to FPV cannot be entirely ruled out. Five of the eight kittens excluded because they were FPV HI-positive on day 0 (kittens 28, 29, 30, 46 and 47) originated from two different litters, and based on their sustained high anti-FPV titres (persistently ⩾256) (Table 3) were likely naturally exposed to FPV before entering the shelter.³⁰ HI detects antibodies produced by both natural exposure and vaccination; therefore, it was impossible to identify and exclude individuals that had been exposed to field challenge during the study period. Despite eight kittens being HI-positive (⩾32) before vaccination at T0, 6/8 were HI-positive at T2 with increasing reciprocal titres between T0 and T2, and all eight kittens were HI-positive at T12. One previous study found a pre-vaccination reciprocal antibody titre of 40 and above was significantly associated with an inadequate response to vaccination compared with kittens with a pre-vaccination titre below 40.³⁵ It is possible that FPV antibody production in some animals during the study was the result of natural FPV exposure rather than vaccination or as well as vaccination. Although unlikely because of the age of recruits, it is also possible that some of the HI-positive results in the current study represented kittens that had been unknowingly vaccinated before entering the rehoming facility. This reflects a reality of clinical practice and shelters worldwide; therefore, it was encouraging to observe that all eight seropositive kittens at T0 retained protective titres against FPV throughout the study to T12.

Assuming all eight T0 seropositive kittens represented natural exposure in NSW, the 8% (n = 8/100) positivity rate is far lower than the 34% FPV exposure rate reported in kittens (aged <6 months) entering a Florida animal shelter.³⁰ This difference in FPV prevalence rates may be attributable to the reduced incidence of FPV in Australia, where there were no cases reported over a 30-year period. The virus only re-emerged in Australia in 2014, primarily in shelter-housed cats.⁴⁷

Conclusions

Vaccination of cats subcutaneously into the scruff, distal left hindlimb or proximal tail with an inactivated, adjuvanted FPV vaccine formulation induced a comparable and protective humoral immune response. Veterinarians can therefore have confidence that vaccinating using any of these three locations will produce a robust antibody response against FPV. This finding provides further evidence to support recommendations promoted by major feline vaccination guidelines in Europe and North America to vaccinate elsewhere from the scruff when possible, if there is a sufficiently high prevalence of FISSs in that region. Most kittens (91%) were protected against FPV after a single vaccination, irrespective of vaccine formulation (MLV vs inactivated). All cats except for one had protective antibody titres present 1 year after the initial vaccination. Veterinarians in Australia should continue to recommend vaccination of at-risk kittens and young cats against FPV, with this study suggesting ongoing risk of natural exposure within Australia. Vaccination into the scruff remains an acceptable choice for veterinarians where there is a reduced risk and incidence of FISSs.

Footnotes

Acknowledgements

We are grateful for the significant in-kind contribution of Anigen Rapid FIV/FeLV diagnostic kits by BioNote, without which this study would not have been possible. Thanks also to all the cats and owners who supported this study.

Accepted: 15 April 2026

Author note

All the data presented in this article are available on request.

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this research was supported financially by the Australian Companion Animal Health Foundation (grant numbers 008/2020 and 002/2023) and by the Cat Protection Society of NSW. Richard Malik was supported by the Valentine Carlton Bequest.

Ethical approval

The work described in this manuscript involved the use of non-experimental (owned or unowned) animals and procedures that differed from established internationally recognised high standards (‘best practice’) of veterinary clinical care for the individual patient. The study therefore had prior ethical approval from an established (or ad hoc) committee as stated in the manuscript.

Informed consent

Informed consent (verbal or written) was obtained from the owner or legal custodian of all animal(s) described in this work (experimental or non-experimental animals, including cadavers, tissues and samples) for all procedure(s) undertaken (prospective or retrospective studies). No animals or people are identifiable within this publication, and therefore additional informed consent for publication was not required.

ORCID iD

Mark E Westman

Evelyn Hall

Jacqueline M Norris

William Weir

Dawn Dunbar

Yasmin Parr

Caitlin Martin

Maira Nascimento Meggiolaro

Mizuho Nakamura

Richard Malik

Margaret J Hosie

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