Abstract
The objective of this study was to determine whether intranasal administration of a commercially available FVRCP vaccine to kittens lessened clinical signs and feline herpesvirus 1 (FHV-1) viral shedding when compared to unvaccinated control kittens after FHV-1 challenge. Three groups of 10 unvaccinated kittens were administered one dose of vaccine 6 days (group 1), 4 days (group 2), or 2 days (group 3) before challenge, respectively. One group was maintained as unvaccinated controls (group 4). FHV-1 challenge was then induced and the kittens were observed for 14 days. When the grouped vaccinated kitten results (groups 1–3) were compared to group 4 results, clinical scores following challenge were significantly lower (P<0.05) and significantly lower body temperatures (P<0.05) were detected on days 0, 5 and 9 post-challenge. When evaluated by individual group, group 1 and group 2 kittens had significantly lower clinical scores (P<0.05) than group 4 kittens post-challenge. In addition, FHV-1 shedding was lower in group 1 kittens when compared to group 4 kittens on day 6 after challenge (P<0.05). Administration of this vaccine within several days prior to exposure lessened clinical signs of disease and FHV-1 shedding compared to unvaccinated kittens.
Feline herpesvirus 1 (FHV-1) infection is common in kittens, is extremely contagious between kittens, and frequently results in severe clinical disease (Harbour et al 1991, Nasisse et al 1993, Binns et al 2000, Stiles 2003, Veir et al 2004). Kittens are at most risk for development of severe clinical disease: fever, sneezing, nasal discharge, conjunctivitis, keratitis, cough, dyspnea, and death can occur. Kittens housed with other kittens are very likely to become infected and clinical disease may be exacerbated by crowding and stress. For example, in a recent prevalence study at a humane society in north central Colorado, FHV-1 DNA was amplified from throat swabs or nasal discharges collected from 52 of the 61 kittens (85.2%) tested (Veir et al 2004). In a separate study, FHV-1 shedding rates of kittens on admission to a shelter and 1 week later were 4% and 52%, respectively, showing that spread of this virus in crowded environments is rapid (Pedersen et al 2004). Thus, it is important to induce immunity in kittens by vaccination quickly, particularly in populations at high risk of exposure.
Multiple FHV-1 containing vaccines are commercially available throughout the world. The vaccines generally contain FHV-1, feline calicivirus (FCV), and feline panleukopenia virus (FPV) and are collectively called feline rhinotracheitis, calicivirus, and panleukopenia (FVRCP) vaccines. Several modified live FVRCP vaccines for parenteral administration, a killed FVRCP vaccine for parenteral administration, and a modified live FVRCP vaccine for intranasal administration are available in the United States. When efficacy is assessed on challenge with virulent strains, FPV infection is prevented and FCV and FHV-1 clinical disease is attenuated (Scott and Geissinger 1999, Lappin et al 2002). Of the three vaccine components, the protection induced against FHV-1 is generally the least effective (Scott and Geissinger 1999, Lappin et al 2002). Because of risks associated with parenteral injections in kittens, alternate routes of vaccine administration to kittens are being studied further (Burton and Mason 1997, Lappin et al 2005a). Over the years, several modified live FHV-1 strains have been administered to kittens intranasally with subsequent challenge with virulent FHV-1 (Davis and Beckenhauer 1976, Slater and York 1976, Folkers and Hoogenboom 1978, Orr and Gaskell 1980, Cocker et al 1983, 1986, Weigler et al 1997, Edinboro et al 1999). In most of the studies, the immunogen was relatively safe and an early protective effect has been reported (Folkers and Hoogenboom 1978, Cocker et al 1983, 1986). A new formulation of a modified live FVRCP for intranasal administration (Feline UltraNasal; FVRCP Vaccine, Heska Corporation, Loveland, CO) has just received a license in the United States. The objective of this study was to determine whether intranasal administration of one dose of this vaccine to kittens 2, 4, and 6 days before virulent FHV-1 challenge would lessen clinical signs and FHV-1 shedding when compared to unvaccinated control kittens.
Materials and methods
Animals
Mixed sex, specific-pathogen free, 13- to 17-week-old kittens (n=40) were purchased from commercial breeders (Harlan Sprague–Dawley, Madison, WI; Cedar River Laboratories, Mason City, IA). Before vaccination or challenge, the kittens were shown to be negative for serum antibodies against FHV-1 by serum neutralization (Diamond Animal Health, Des Moines, IA), negative for virus isolation on throat swabs (Diamond Animal Health), and negative for FHV-1 DNA on throat swabs as determined by polymerase chain reaction (PCR) assay (Veir et al 2004). The kittens were randomly divided into four groups of 10 kittens, housed by group for the duration of the study, and handled as separate groups to avoid cross-contamination between groups. The kittens were fed and cared for according to normal husbandry practices for the research site (Diamond Animal Health). The investigators were responsible for compliance with all applicable federal, state, and local laws, ordinances, rules, and regulations pertaining to animal welfare.
Experimental design
Clinical evaluations were performed on all kittens daily for 9 days before vaccination, through the vaccination period, and for 14 days after challenge using the system approved by the Center for Veterinary Biologicals in the immunogenicity study for the original licensure of the vaccine (Table 1). Body temperatures of all kittens were recorded for 4 days prior to vaccination and then daily from the start of the vaccination period until the end of the study. On the day of vaccination, the lyophilized portion of the vaccine (FHV and FCV portion) was reconstituted using the liquid portion of the vaccine (FPV) and appropriate kittens were administered approximately equal portions of the vaccine into each nostril, following label instructions. Groups 1, 2, and 3 were vaccinated 6 days, 4 days, or 2 days before challenge, respectively; group 4 was not vaccinated. On the day of challenge (day 0), all kittens were sedated and the Feline Rhinotracheitis Challenge Virus strain SGE (Lot number 96–13; Center for Veterinary Biologics-Policy, Evaluation, and Licensing, Ames, IA) diluted 1:200 in DME High-Glucose (FHV-1 virus titer of 105.3 TCID50/ml) was administered by atomizer (152 Atomizer, The DeVilbiss Company, Somerset, PA) with 0.25 ml given into each of the nostrils and 0.5 ml into the oropharyngeal region.
Table 1.
Clinical scoring system used for FHV-1 challenge
| Clinical signs | Days abnormality present * | Score |
|---|---|---|
| Conjunctivitis with serous discharge | 1–3 days | 1 |
| ≥4 days | 2 | |
| Conjunctivitis with mucopurulent discharge | 1–2 days | 2 |
| 3–5 days | 4 | |
| ≥6 days | 6 | |
| Serous nasal discharge | 1–3 days | 1 |
| ≥4 days | 2 | |
| Mucopurulent nasal discharge | 1–2 days | 2 |
| 3–5 days | 4 | |
| ≥6 days | 6 | |
| Sneezing | Scored daily | 1 |
| Dyspnea with audible rales | Scored daily | 2 |
| Coughing | Scored daily | 2 |
| Open mouth breathing | Scored daily | 3 |
| Anorexia | Scored daily | 1 |
| Dehydration | 1–2 days | 3 |
| ≥3 days | 4 | |
| Hypothermia <99°F | Scored daily | 2 |
| Oral ulcers (lingual or oral mucosa) | ||
| 1 ulcer <4 mm | 1–5 days | 2 |
| 6–9 days | 3 | |
| ≥10 days | 4 | |
| Multiple ulcers <4 mm | 1–4 days | 3 |
| 5–8 days | 5 | |
| ≥9 days | 7 | |
| 1 or more ulcers ≥4 mm | 1–4 days | 5 |
| 5–8 days | 7 | |
| ≥9 days | 9 | |
| Salivating | Scored daily | 1 |
| Non-bleeding ulcer on the lips or nares | Scored once if present | 4 |
| Bleeding ulcer on the lips or nares | Scored once if present | 6 |
| Death | Scored once | 15 |
Those clinical abnormalities with a range of days are only assigned the designated score once.
FHV-1 quantitative PCR
Throat swabs were collected from the group 1 and group 4 kittens 6 days before challenge, 3 days before challenge, day 0, and post-challenge days 4, 6, 8, and 12. Swabs were placed in sterile saline, incubated for 2 h at room temperature, and frozen at −70°C until transported on dry ice for assay. FHV-1 and feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA were amplified from each sample by use of a previously described fluorogenic PCR assay (Veir et al 2004). Results for the fluorogenic assay for FHV-1 DNA were compared to cell count derived from a standard curve for GAPDH/cell to ensure an adequate cell count was present on the swab for analysis.
Statistical methods
For clinical response variables, the 14 day cumulative clinical score after challenge from each group of vaccinated kittens was compared to the control kittens using the Mann–Whitney modification of Wilcoxon's two sample rank sum test. Rectal temperatures were analyzed using a mixed model appropriate for a repeated measures experiment (the MIXED procedure in SAS, SAS Institute, Version 8, Cary, NC). The model included the fixed effects of treatment group, time, and the group by time interaction. Effects were deemed statistically significant if P<0.05. If the group by time interaction was significant, within time group effects were evaluated using Fisher's least significant difference test. Otherwise, the main effect of group was evaluated directly. Eight of 10 control kittens needed to show clinical signs of disease in order for the study to be valid.
Fluorogenic FHV-1 PCR results from group 1 and group 4 kittens were log transformed (natural log [copy number+1]) before statistical analysis. Results were analyzed as described above for temperature. Geometric means are presented.
Results
Clinical observations
Ten of the 40 kittens had rectal temperatures >103°F (>39.44°C) before vaccination, including 2/10 kittens in group 1, 3/10 kittens in group 2, 2/10 kittens in group 3, and 3/10 in group 4 kittens. No abnormal clinical signs were observed in any of the kittens after vaccination. In kittens in groups 1–3, the temperatures after vaccination and before challenge ranged from 100.1°F (38.39°C) to 102.9°F (39.39°C). After challenge, the temperatures ranged from 98.0°F (36.67°C) to 104.4°F (40.22°C), with 42 of the 420 temperatures (10%) measured being >103°F (>39.44°C). In group 4 kittens, the temperatures before challenge ranged from 99.6°F (37.56°C) to 104.2°F (40.11°C), with five of the 10 temperatures (50%) measured being >103°F (>39.44°C). After challenge, the temperatures ranged from 99.0°F (37.22°C) to 105.5°F (40.83°C), with 24 of the 140 temperatures (17%) measured being >103°F (>38.44°C). A significant time by treatment interaction was detected (P<0.05); thus differences between groups within a day were evaluated. On days 0, 5 and 9 post-challenge, treatment effects were detected (P<0.05). Within day, temperatures of group 1 kittens were lower than group 4 kittens on day 9 post-challenge (P<0.05), and temperatures of group 2 and 3 kittens were lower than group 4 kittens on days 0, 5 and 9 post-challenge (P<0.05).
The most common signs observed after challenge (ie, signs seen in at least 5% of the observations) were sneezing, serous and mucopurulent conjunctival discharge, serous and mucopurulent nasal discharge, and ulcers on the nares or lips. Cumulative clinical scores from days 0 to 14 after challenge were compared in a pairwise fashion, comparing group 4 results to each of the vaccinated groups (groups 1–3) in a one-sided test (Fig 1). Group 1 and group 2 kittens had significantly lower clinical scores (P<0.05) than group 4 kittens. In addition, results from all vaccinated kittens (groups 1–3) were pooled and compared to group 4. Vaccinated kittens had significantly lower clinical scores (P<0.05) than control kittens (Fig 2).
Fig 1.
Mean ranks of clinical scores between groups of kittens vaccinated 6 days (group 1), 4 days (group 2), or 2 days (group 3) before challenge and control kittens (group 4). Data are from the cumulative score by group collected days 0–14 after challenge with FHV-1. *=statistically different than group 4.
Fig 2.
Mean ranks of combined clinical scores between groups of vaccinated kittens (groups 1–3) and control kittens (group 4). Data are from the cumulative score by group collected days 0–14 after challenge with FHV-1. *=statistically different from group 4 at P<0.05 by Wilcoxon's rank sum test.
FHV-1 quantitative PCR results
GAPDH was detected in all samples and so it was assumed each contained feline cells and were considered adequate for analysis. The group by time interaction was significant in the statistical model (P=0.01). FHV-1 was first detected in throat swabs collected from group 1 kittens on day 0 and from group 4 kittens on day 4 (Table 2). On day 6 post-challenge, group 1 kittens had significantly less FHV-1 DNA on throat swabs than did group 4 kittens. The presence of FHV-1 on throat swabs on day 0 from group 1 kittens was attributed to presence of the modified live vaccine virus administered 6 days previously.
Table 2.
Change in estimated FHV-1 copy number over time
| Study day | Group 1 kittens | Group 4 kittens | P-value |
|---|---|---|---|
| −6 | 0 | 0 | 1.0000 |
| −3 | 0 | 0 | 1.0000 |
| 0 | 1.763 | 0 | 0.0473* |
| 4 | 46.224 | 18.207 | 0.1278 |
| 6 | 2.408 | 17.031 | 0.0013* |
| 8 | 4.054 | 7.729 | 0.2834 |
| 12 | 0.684 | 0.671 | 0.2979 |
Group 4 kittens were unvaccinated controls and group 1 kittens had been vaccinated 6 days before challenge of both groups on study day 0. FHV-1 copy numbers were estimated by comparison of FHV-1 DNA results to a cell count derived from a standard curve for GAPDH/cell. The data consist of geometric means and within time point comparisons. The group by time interaction was significant in the statistical model (P=0.0100); points marked by * are statistically different between the two groups.
Discussion
The vaccine used here is a reformulation of a previously licensed vaccine (Heska Trivalent Intranasal/Intraocular Vaccine, Heska Corporation, Fort Collins, CO). Because FHV-1 infection is so common (Veir et al 2004) and spreads so rapidly (Pedersen et al 2004), we believed it important to determine whether this new product could induce early responses to vaccination. To improve ease of administration, the new formulation does not require topical ocular administration and is of a smaller volume than the previous formulation.
Clinical abnormalities that could be attributed to administration of this formulation were not detected in these experimentally inoculated, laboratory-reared kittens. In a field safety study performed in 501 client-owned kittens (required for USDA licensure of the vaccine), transient sneezing was seen after 30% of vaccinations, of which most (52%) sneezed only 1 (20%), 2 (20%) or 3 (12%) days (Data on file; Heska Corporation). Other respiratory signs were seen in 3.4%, gastrointestinal signs in 4.8%, nasal or oral ulcers in 1.2% and other signs in 0.8% of vaccinations. While clinical signs that could be attributed to administration of the vaccine were not detected between vaccine administration and challenge of cats in this study, it is possible that some of the clinical abnormalities noted after challenge were from the vaccine. Other modified live FHV-1 strains studied for intranasal administration have also induced clinical signs of disease in laboratory-reared kittens (Cocker et al 1983, 1986, Davis and Beckenhauer 1976, Slater and York 1976, Folkers and Hoogenboom 1978, Orr and Gaskell 1980, Weigler et al 1997). The clinical signs are attributed to replication of the modified live viruses locally and so when this vaccine is used, potential side effects should be discussed with the owners.
Several findings of this study suggest that administration of this intranasal vaccine has potential clinical benefits when administered between 2 and 6 days before challenge, lessening fever and clinical scores at some time points after challenge. The most notable being the significantly lower mean rank clinical scores after challenge in the kittens vaccinated 6 days (group 1) or 4 days (group 2) before challenge when compared to control kittens (group 4). In addition, in kittens vaccinated 6 days before challenge (group 1), less FHV-1 DNA was shed on day 6 after challenge when compared to control kittens (group 4). Our findings were similar to those of others that have shown early protection against clinical signs of FHV-1 infection as well as decreased FHV-1 shedding in kittens vaccinated with modified live FHV-1 intranasally and then challenged with virulent FHV-1 (Slater and York 1976, Folkers and Hoogenboom 1978, Cocker et al 1983, 1986). However, while intranasal administration of modified-live FHV-1 containing vaccines may lessen FHV-1 shedding and clinical signs of disease in vaccinates, it has not been shown to protect against the development of latent FHV-1 infections (Cocker et al 1986, Weigler et al 1997).
How intranasal FHV-1 containing vaccines mediate early protection has not been completed elucidated. In one study, intranasal administration of the modified live strain of FHV-1 protected against FHV-1 but not FCV challenge, suggesting the FHV-1 protection was specific (Cocker et al 1986). However, when FHV-1-specific immune responses were studied during the period of rapid protection, FHV-1 specific humoral responses could not be detected in serum and FHV-1 antigen-specific induction of lymphoblast transformation using peripheral blood lymphocytes was variable (Cocker et al 1986). FHV-1 specific IgM and IgA could be detected by enzyme-linked immunosorbent assay (ELISA) in nasal washings by day 6 after vaccination suggesting the specific mediation of protection was local (Cocker et al 1986). Immune responses of the kittens were not measured in the study described here; further experiments will be required to determine mediation of early protection and to determine whether the presence of modified-live FCV and FPV in the vaccine accentuates FHV-1-specific or non-specific immune responses. In another study, we compared the immune responses of kittens to four of the market-leading parenterally administered FVRCP vaccines and the modified-live intranasal FVRCP vaccine studied here. When compared to the parenteral vaccines, it was shown that the intranasal product induced equivalent or superior FHV-1, FCV, and FPV humoral immune responses, equivalent or superior FHV-1 specific lymphoblast transformation, and equivalent or superior lymphoblast transformation in response to the non-specific mitogen, concanavalin A (Lappin et al 2005b).
Administration of FVRCP vaccines parenterally or intranasally has generally been considered to be relatively safe and their use widely recommended (Richards et al 2001). However, injection site sarcomas have developed at the site of modified live or killed FVRCP inoculations (Burton and Mason 1997). In addition, most FVRCP vaccine viruses manufactured in the United States are grown on the Crandall Reese feline kidney (CRFK) cell line. This cell line was originally developed from cells derived from feline kidneys and small amounts of the cell line contaminate the FVRCP vaccines grown upon it (Lappin et al 2004, 2005a). When compared to kittens vaccinated with the intranasal FVRCP product used here, kittens administered FVRCP vaccines parenterally developed greater antibody responses against CRFK cell extracts (Lappin et al 2004). We believe this finding relates to the fact that when most inactivated antigens contact mucous membranes they are excluded and fail to induce systemic immune responses but if the antigen is given by injection, it is taken up by antigen-presenting cells for clearance, and ultimately can result in antigen specific antibody production. In one study of parenterally vaccinated and CRFK hypersensitized kittens, the antibodies induced also recognized feline renal cell extracts suggesting that the CRFK cell line maintains antigenic relatedness to feline cells (Lappin et al 2004). However, it is important to note that to date their significance is unknown as the presence of these antibodies have not been linked to disease.
Acknowledgement
This study was funded by a grant from Heska Corporation.
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