Abstract
In this pilot study, 12 adult, gang-housed cats that were known to be previously exposed (n=12) to feline herpesvirus-1 (FHV-1) and/or vaccinated against (n=2) feline calicivirus (FCV) and FHV-1 were randomly assigned to one of two groups of six cats each. Nasal and pharyngeal samples were collected from each cat on days −7, −3, and 0 prior to vaccination and on days 3, 7, 10, 14, 17, 21, and 28 after vaccination with an FHV-1, FCV, and panleukopenia (FVRCP) vaccine developed for intranasal (six cats) or parenteral (six cats) use. FHV-1 DNA was amplified from 1/12 cats (1/69 samples; 1.4%) prior to vaccination and 2/12 cats after vaccination (2/154 samples; 1.3%). FCV RNA was amplified from 2/12 cats (2/69 samples; 2.9%) prior to vaccination and 7/12 cats (12/154 samples; 7.8%) after vaccination. Positive molecular diagnostic assay results for FHV-1 and FCV were uncommon prior to or after vaccination in these cats.
Upper respiratory tract disease (URTD) continues to be a major problem in cats around the world. Feline herpesvirus-1 (FHV-1) and feline calicivirus (FCV) infections are commonly detected in cats with and without URTD. 1 –3 Treatment of individual cats with clinical disease thought to be associated with FHV-1 or FCV infections is difficult and many of the therapies like lysine (FHV-1), famciclovir (FHV-1), cidofovir (FHV-1), and interferons (FHV-1 and FCV) can be expensive, 4 have side-effects, or have minimal evidence for efficacy. 5,6 Thus, making an accurate diagnosis of FCV or FHV-1 infection is very important in cats exhibiting signs of URTD.
Molecular diagnostic assays are now being offered by some Veterinary Diagnostic Laboratories and include polymerase chain reaction (PCR) assays for the amplification of FHV-1 DNA and reverse transcriptase PCR (RT-PCR) assays for the amplification of FCV RNA. Positive test results in these assays can be used to prove current infection. However, intranasal or parenteral FHV-1, FCV, and panleukopenia (FVRCP) vaccines are thought to colonise vaccinated cats and current molecular assays do not differentiate vaccine and natural strains of FHV-1. 7 For example, in one study, FHV-1 DNA was amplified from samples collected from all 12 cats administered an intranasal FHV-1-containing vaccine. 8 Information concerning the effect of modified live FHV-1 vaccines intended for parenteral administration, or any type of FCV vaccines, on molecular diagnostic assay results from healthy adult cats is lacking. If these assays results are commonly positive as a result of modified live FVRCP vaccine administration, FHV-1- and FCV-associated clinical disease may be diagnosed erroneously.
In a similar study of adult previously vaccinated dogs, RNA of parainfluenza and adenovirus 2 were amplified from <3.0% of the 216 nasal or pharyngeal swabs collected after administration of modified live vaccines for intranasal or parenteral use. 9 Thus, in this study we hypothesize that healthy adult cats that were previously exposed to FHV-1 and FCV will have low percentage positive FHV-1 and FCV molecular assay results after administration of modified live vaccines. The objectives of this study were to determine the frequency of positive FHV-1 and FCV molecular diagnostic assay results before and after the administration of two different modified live FVRCP vaccines, and to determine whether positive assay results are obtained most frequently from cells obtained from the pharynx or nasal cavity of adult cats previously exposed to both viruses.
Materials and methods
Experimental design
This prospective study used 12, mixed sex, young-adult cats housed in a research facility. The cats had been originally obtained from a specific pathogen-free facility and subsequently gang-housed together and with other cats for up to 3 years prior to this study. One cat had been administered one dose of a modified live FVRCP vaccine (24 months prior) and one cat had been administered two doses of an inactivated FVRCP vaccine (25 and 26 months prior). The remaining 10 cats were never vaccinated but all had been experimentally-infected with FHV-1 14 months prior to this study using the USDA challenge strain of FHV-1. 10 All cats were assessed as healthy based on the current physical examination and there were no current signs of URTD in any cat.
The cats were randomly selected to be vaccinated on day 0 with either a modified live FVRCP vaccine approved for either intranasal administration (UltraNasal FVRCP; Heska, Loveland, CO) or parenteral administration (Purevax Feline 3RCP; Merial, Athens, GA). Cats were group housed in separate rooms within vaccine type. The cats were fed ad libitum and perches and toys were available in each room for enrichment and stress relief. The cats were evaluated daily for clinical signs of URTD by both the investigators and the staff of the animal care facility. The project was approved by the Colorado State University Institutional Animal Care and Use Committee.
Sample collection
To serve as pre-vaccination control samples and to determine whether stress of sample collection would activate subclinical FHV-1 or FCV infection, samples were collected on days −7, −3, and 0 prior to vaccination. Samples were then collected on days 3, 7, 10, 14, 17, 21 and 28 from all cats after vaccination. All samples were collected without sedation. Nasal swabs were obtained from each cat by gently rolling a sterile transurethral culture swab (Ca alginate swabs; Ultrafine Al, product 14-959-78, Fischer Scientific) in the anterior aspect of the right naris. Oropharyngeal swabs were obtained using sterile, cotton-tipped applicators gently rotated in the oropharynx of each cat. Swabs were placed in sterile tubes for immediate transport to the laboratory and placed at −80°C until assayed. Upon removal from storage, the swabs were thawed for 30 min. One millilitre of phosphate buffered saline (PBS) was added to each swab and incubated at room temperature for 2–3 h. The swab was vortexed briefly and removed from the tube, with care taken to remove as much liquid from it as possible. The PBS was placed in a microcentrifuge tube and centrifuged for 5 min at 5000g. The resultant pellet was reconstituted in 150 μl PBS and processed through the QIAtractor robot using the DX Reagent Pack according to the manufacturer's instructions, and assayed immediately following extraction (Qiagen, Valencia, CA). A similar protocol was used to extract DNA/RNA from an aliquot of both vaccines. On day −7, 2 ml of blood was collected; serum was separated from each sample and stored at −80°C until assayed.
Assays
Previously reported conventional PCR assays for the amplification of FHV-1 DNA, glyceraldehyde phosphate dehydrogenase (GAPDH) (control DNA), and conventional RT-PCR assay for FCV RNA were performed on the DNA/RNA. 2 DNA/RNA extraction and the molecular diagnostic assays were performed in separate areas to avoid contamination and both positive and negative controls were included on each assay as previously reported. 2
Serum antibodies against FHV-1 and FCV were measured using previously reported enzyme-linked immunosorbent assays (ELISAs). 10 Results were reported as positive or negative or as mean absorbance values in the data analysis. Negative, positive, enzyme, and substrate controls were included on each ELISA. The positive cutoff chosen was that previously used to successfully predict resistance against challenge with virulent strains of the viruses. 10
Statistical analysis
Samples that were below detectable limits for GAPDH were excluded from the analysis. Data were entered into Microsoft Excel spreadsheet and percentage positive test results were calculated. Percentage positive results before and after vaccination were compared between vaccine types by using Fisher's exact test. Group mean FHV-1 and FCV ELISA absorbance values were compared by Student's t-test. Significance was defined as P<0.05.
Results
FHV-1 DNA and FCV RNA were amplified from both the vaccines. Over the course of the study, 119 nasal samples and 118 pharyngeal samples were collected. Generally, sample collection was well tolerated but one nasal sample (parenteral vaccine cat 5 on day −7) and two pharyngeal samples (parenteral vaccine cat 5 on day −7 and parenteral vaccine cat 2 on day −7) could not be collected because of resistance to the procedure (Table 1). GAPDH was amplified from all pharyngeal samples and 105/119 (88.2%) of the nasal samples (Table 1).
Distribution of conventional FHV-1 PCR assay and FCV RT-PCR assay results from pharyngeal and nasal samples collected from cats vaccinated with two different modified live FVRCP products via two different routes.
On day −7, in the subcutaneous vaccine group, a nasal swab could not be collected from one cat and pharyngeal swabs could not be collected from that cat and one additional cat. All other missing samples did not have adequate DNA based on the GAPDH PCR assay results.
Antibodies against FHV-1 were detected in sera collected day −7 from all 12 cats. The mean ELISA absorbance values between groups were not statistically different (P=0.8271). FHV-1 DNA was amplified from one sample from one cat (pharyngeal sample; parenteral vaccine group) prior to vaccination; all other cats were negative on all pre-vaccination samples (Table 1). After vaccination, FHV-1 DNA was amplified from samples from two cats in the intranasal vaccine group (day 3 pharyngeal sample one cat; day 3 nasal sample one cat). There were no statistical differences between prevalence rates for FHV-1 DNA amplification rates before and after vaccination or between vaccine groups.
Antibodies against FCV were detected in sera collected day −7 from 6/12 cats; one in the subcutaneous group and five in the intranasal group. The mean ELISA absorbance values between groups were not statistically different (P=0.1822). Of the two cats that had been vaccinated prior to entering this study, FCV antibodies were only detected in the cat that was vaccinated twice with an inactivated product. FCV RNA was amplified from one sample from two cats prior to vaccination in the current study (Table 1); neither of these cats had been vaccinated before entering the study. One cat with a positive sample prior to vaccination (pharyngeal sample; day 0) was in the intranasal vaccine group and the other cat with a positive sample (pharyngeal sample; day 0) was in the parenteral vaccine group. After vaccination, FCV RNA was amplified from either nasal or pharyngeal swabs from 7/12 (58.3%) cats (Table 1). Of the seven cats with FCV RNA amplified from samples after vaccination, three were vaccinated with the parenteral vaccine and four were vaccinated with the intranasal vaccine. Five of these seven cats were seropositive for FCV antibodies on day −7. In the intranasal vaccine group, FCV RNA was amplified from one cat on days 14 and 17 (pharyngeal sample only), one cat on days 14 (pharyngeal and nasal sample) and 21 (pharyngeal sample only), one cat on days 7 and 14 (pharyngeal samples only), and one cat on days 21 (nasal sample only) and 28 (pharyngeal sample only). In the parenteral vaccine group, FCV RNA was amplified from one cat on day 10 (pharyngeal sample only), one cat on day 3 (pharyngeal sample only), and one cat on day 21 (nasal sample only). There were no statistical differences between prevalence rates for FCV RNA amplification rates before and after vaccination or between vaccine groups.
None of the samples collected from cats in either group over time had FCV RNA and FHV-1 DNA amplified concurrently.
Discussion
Molecular diagnostic assays for FHV-1 and FCV infections are becoming less expensive and more frequently used. Nasal and pharyngeal sample collection requires little training, does not require sedation, and is easily accomplished by veterinary or shelter personnel. Sample handling and shipment of samples for molecular diagnostic assays is also less cumbersome than some other diagnostic tests like virus isolation that require special transport media. In addition, molecular diagnostic assay results can return more quickly than those of virus isolation. Molecular assays also can be very sensitive and specific, although results do not prove the amplified DNA or RNA was from a live organism. However, it is generally believed that dead organisms are usually cleared by specific and non-specific immune defense mechanisms quickly, and so positive molecular assay results are likely to correlate to current infection. This is likely to be true for both FCV and FHV-1 as both organisms chronically colonise cats.
In the study described here, the cats generally tolerated the collection of samples from either site. However, some cats became increasingly refractory to sample collection over time. This may explain the small percentage of nasal samples that were negative for GAPDH. When adequate samples were obtained from both sites, FCV RNA was amplified more frequently from pharyngeal swabs. In a previous study of clinically ill cats, both sampling sites were found to be adequate for FHV-1 PCR and bacterial culture, but none of the cats were positive for FCV RNA in that study. 2 Overall, the results of the study described here suggest that pharyngeal samples are superior to nasal swabs for sampling cats for FCV molecular diagnostic assays. However, that may not be true for cats with clinical signs of URTD. Levels of agreement between the two sample sites were not calculated in this study because of the low overall percentage of positive test results. Another explanation for detection of FCV RNA in a greater number of pharyngeal samples is the propensity for this virus to colonise the oral cavity. 1
The cats used in this study were selected based solely on availability and current clinical normalcy. As the cats had all been infected with FHV-1 previously, 1/12 cats had been directly vaccinated with a modified live FCV containing product, and all 12 cats had been gang-housed with other cats known to be vaccinated with modified live FCV vaccines, all were presumed to have been exposed to both viruses. Antibody levels considered predictive of resistance on challenge were detected for FHV-1 in all 12 cats and for FCV for six cats. There were no differences in mean ELISA absorbance values for FHV-1 or FCV antibodies between groups. It would have been optimal experimentally to have all 12 cats managed identically prior to entering this study and so the results should be interpreted carefully. However, the variable history in regards to FHV-1 and FCV exposure may be representative of healthy previously exposed or vaccinated adult cats in the field.
While all cats in this study had previously been infected with FHV-1, only one sample from one cat was positive for FHV-1 DNA in the pre-vaccination control samples and respiratory signs were not noted before vaccination. FHV-1 DNA was amplified from samples from 2/12 cats for 1 day after vaccination (day 3; both in the intranasal vaccine group). From this data, it cannot be determined whether these two positive samples were from spontaneous shedding of FHV-1 from the previous infection or from shedding of the vaccine strains of FHV-1. Recrudescence of FHV-1 associated illness can occur as soon as 2 days after administration of methylprednisolone acetate. 6 However, reactivation of FHV-1 infection is variable. For example, attempted induction of stress via housing changes did not reactivate respiratory signs of FHV-1 infection over time. 11
As only 1.3% of the total post-vaccination sample set was positive for FHV-1 DNA, we believe that the hypothesis that administration of these two vaccines to previously infected, healthy cats would result in a low percentage of positive FHV-1 PCR assay results has been supported. However, this conclusion should not be over-interpreted as there are multiple FHV-1 strains in the field, only two vaccines were studied, and only one FHV-1 PCR assay was assessed. The results described here differ from a previous study where all 12 kittens shed FHV-1 virus as determined by PCR after administration of a different formulation of an intranasal vaccine. 8 However, the previous study used 18-week-old specific pathogen-free kittens rather than previously exposed or vaccinated adult cats. Additionally, the vaccine product was administered topically on both the ocular and nasal surface.
There were no differences in FCV RT-PCR assay results between groups of cats before or after vaccination. Overall, the number of FCV positive samples after vaccination was 7.8%, positive results were detected in 7/12 cats, three of the positive cats were in the subcutaneous vaccine group, four of the positive cats were in the intranasal vaccine group, and no individual cat was positive for FCV RNA on more than two sample dates. Thus, we believe that the hypothesis that administration of these two vaccines to healthy cats that are likely to have been previously exposed to the virus would result in a low percentage of positive FCV RT-PCR assay results, has been supported. While the numbers of FCV RNA positive cats and samples that were positive for FCV RNA increased numerically after vaccination when compared to the pre-vaccination control samples, it cannot be determined whether the post-vaccination positive samples were from spontaneous shedding of FCV from a previous infection or from shedding of the vaccine strains of FCV. However, if the positive samples detected after vaccination were related to the vaccines, the fact that a numerically greater percentage positive rate was detected after vaccination in the intranasal group (11.3%) when compared to the subcutaneous group (4.1%) may relate to the local delivery of the vaccine virus.
Commercially available molecular diagnostic assays for FHV-1 and FCV may be applied to cats that develop clinical signs of URTD shortly after vaccination. As the PCR assays used here, or in commercial laboratories, cannot differentiate vaccine strains and naturally occurring strains, future studies could also evaluate whether there are molecular markers that could easily and inexpensively be used to stringently differentiate strains of these viruses. Further studies of kittens after primary vaccination with modified live FHV-1 or FCV containing products should be performed.