• View in gallery
    Figure 1—

    Mean overall clinical scores for shelter cats with URTD that received 1 drop of HuIFN solution (1 × 106 U/mL [group 1; n = 12; squares]), FeIFN solution (1 × 106 U/mL [group 2; 12; circles]), or saline (0.9% NaCl) solution (group 3; 12; triangles) in each eye twice daily for 14 days (beginning day 1). Clinical signs of ocular and systemic disease were subjectively scored for each cat on days 0, 3, 7, 10, and 14; higher scores were indicative of greater severity (maximum possible score for ocular or systemic signs was 11 in each category [33 cumulatively]). At each time point, scores for each eye and signs of systemic disease in each cat were combined to calculate a mean overall clinical score for each group. In all groups, the overall clinical score at day 14 was significantly decreased from day 0 findings; however, there was no difference among groups at any time point.

  • View in gallery
    Figure 2—

    Results of VI for FHV-1 in conjunctival (conj) and oropharyngeal (oro) swab samples collected from shelter cats with URTD that received 1 drop of HuIFN solution (1 × 106 U/mL [group 1; n = 12]), FeIFN solution (1 × 106 U/mL [group 2; 12]), or saline solution (group 3; 12) in each eye twice daily for 14 days. For each cat, 1 swab from the oropharyngeal region and 1 swab from each conjunctival fornix was obtained before (day 0 [black bars]) and after 14 days of treatment (day 14 [gray bars]; treatments were initiated on day 1); at each time point, the samples from the 2 eyes were pooled together. Data are reported as the number of cats for which virus was detected. *For a given sample collection site, value at day 14 is significantly (P < 0.05) less than the value at day 0.

  • View in gallery
    Figure 3—

    Results of VI for FCV in conjunctival (conj) and oropharyngeal (oro) swab samples collected from shelter cats with URTD before (day 0) and after (day 14) twice-daily topical administration of HuIFN solution (group 1; n = 12), FeIFN solution (group 2; 12), or saline solution (group 3; 12) in each eye for 14 days. †Within a group at day 0, value derived from oropharyngeal swab samples was significantly (P < 0.05) higher than the value derived from conjunctival swab samples. ‡Within a group at day 14, value derived from oropharyngeal swab samples was significantly (P < 0.05) higher than the value derived from conjunctival swab samples. See Figure 2 for remainder of key.

  • View in gallery
    Figure 4—

    Results of RT-qPCR assay for FHV-1 in conjunctival (conj) and oropharyngeal (oro) swab samples collected from shelter cats with URTD before (day 0) and after (day 14) twice-daily topical administration of HuIFN solution (group 1; n = 12), FeIFN solution (group 2; 12), or saline solution (group 3; 12) in each eye for 14 days. See Figure 2 for key.

  • View in gallery
    Figure 5—

    Results of RT-qPCR assay for FCV in conjunctival (conj) and oropharyngeal (oro) swab samples collected from shelter cats with URTD before (day 0) and after (day 14) twice-daily topical administration of HuIFN solution (group 1; n = 12), FeIFN solution (group 2; 12), or saline solution (group 3; 12) in each eye for 14 days. See Figures 2 and 3 for key.

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Effects of topical ocular administration of high doses of human recombinant interferon alpha-2b and feline recombinant interferon omega on naturally occurring viral keratoconjunctivitis in cats

Jessica M. SlackDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

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Jean StilesDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

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Christian M. LeuteneggerMolecular Diagnostics Laboratory, IDEXX, 2825 Kovr Dr. West Sacramento, CA 95605.

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George E. MooreDepartment of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

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Roman M. PogranichniyDepartment of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

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Abstract

Objective—To determine whether 14-day topical ocular administration of high doses of feline recombinant interferon omega (FelFN) or human recombinant interferon alpha-2b (HulFN) solution improves clinical disease and decreases virus shedding in cats with naturally acquired viral keratoconjunctivitis.

Animals—36 cats with upper respiratory tract disease and ocular involvement.

Procedures—Cats received 1 drop of FelFN solution (1 × 106 U/mL), HulFN solution (1 × 106 U/mL), or saline (0.9% NaCl) solution (12 cats/group) in each eye twice daily for 14 days (beginning day 1). Oropharyngeal and conjunctival swab samples were collected from each cat before (day 0) and on day 14 of treatment for virus isolation (VI) and real-time quantitative PCR (RT-qPCR) testing to detect feline herpesvirus-1 and feline calicivirus. Subjective clinical scores were recorded on days 0, 3, 7, 10, and 14.

Results—The number of cats for which feline herpesvirus-1 was detected via VI or RT-qPCR assay was generally (albeit not always significantly) lower on day 14, compared with day 0 findings; however, findings on days 0 or 14 did not differ among groups. The number of cats for which feline calicivirus was detected via VI or RT-qPCR assay did not differ significantly between days 0 and 14 for any group. Clinical scores significantly decreased over the 14-day period but did not differ among groups.

Conclusions and Clinical Relevance—In cats with naturally occurring viral keratoconjunctivitis, bilateral ocular administration of high doses of FelFN or HulFN twice daily for 14 days did not improve clinical disease or virus shedding, compared with treatment with saline solution.

Abstract

Objective—To determine whether 14-day topical ocular administration of high doses of feline recombinant interferon omega (FelFN) or human recombinant interferon alpha-2b (HulFN) solution improves clinical disease and decreases virus shedding in cats with naturally acquired viral keratoconjunctivitis.

Animals—36 cats with upper respiratory tract disease and ocular involvement.

Procedures—Cats received 1 drop of FelFN solution (1 × 106 U/mL), HulFN solution (1 × 106 U/mL), or saline (0.9% NaCl) solution (12 cats/group) in each eye twice daily for 14 days (beginning day 1). Oropharyngeal and conjunctival swab samples were collected from each cat before (day 0) and on day 14 of treatment for virus isolation (VI) and real-time quantitative PCR (RT-qPCR) testing to detect feline herpesvirus-1 and feline calicivirus. Subjective clinical scores were recorded on days 0, 3, 7, 10, and 14.

Results—The number of cats for which feline herpesvirus-1 was detected via VI or RT-qPCR assay was generally (albeit not always significantly) lower on day 14, compared with day 0 findings; however, findings on days 0 or 14 did not differ among groups. The number of cats for which feline calicivirus was detected via VI or RT-qPCR assay did not differ significantly between days 0 and 14 for any group. Clinical scores significantly decreased over the 14-day period but did not differ among groups.

Conclusions and Clinical Relevance—In cats with naturally occurring viral keratoconjunctivitis, bilateral ocular administration of high doses of FelFN or HulFN twice daily for 14 days did not improve clinical disease or virus shedding, compared with treatment with saline solution.

Feline herpesvirus-1 and FCV are common causes of surface ocular disease in cats, resulting in conjunctivitis, ulcerative and nonulcerative keratitis, corneal scarring, and in some cases, blindness.1 In addition, these viruses cause URTD, which is associated with considerable morbidity and occasionally death in affected individuals. Feline herpesvirus-1 is ubiquitous in the domestic cat population; > 80% of cats are subclinical carriers, with 45% to 50% of those animals affected by episodes of recrudescent disease at some time in life.2 Specific treatment options for FHV-1 infection include oral administration of famciclovir3 or lysine4 and topical administration of agents such as cidofovir,5 idoxuridine, trifluridine, and vidarabine.6 The topical antiviral drugs are nucleoside analogues; they are virostatic and, with the exception of cidofovir, require frequent application.7 Because these drugs target DNA synthesis, they are ineffective against FCV, which is an RNA virus.

Interferons are a group of multifunctional secreted proteins, or cytokines, produced by the body in response to stressful stimuli including the detection of certain molecules such as viral nucleic acids, lipopolysaccharide, interleukin-1, or tumor necrosis factor.8 Interferons do not act directly on viruses during infection but instead induce an antiviral state in host cells. This mechanism of establishing an antiviral state, rather than exerting an effect directly on viruses as do the traditional nucleoside analogue drugs, has led to investigations into IFNs as potential therapeutic agents for treating a myriad of viral diseases. In the presence of IFNs, host cells are stimulated to synthesize enzymes that interfere with cellular and viral processes, limiting replication and spread of viruses.8 Interferons are categorized into 2 main types: type I (viral) and type II (immune) IFNs. Type I IFNs are produced by various cell types, such as leukocytes and fibroblasts, in direct response to virus infection, whereas type II IFNs are produced in response to recognition of infected cells by T lymphocytes and natural killer cells of the host's immune system.8 The type I group, which includes HuIFN and FeIFN, is the most widely studied IFN subset in terms of clinical applications.

Controlled studies to evaluate the in vivo use of topically applied IFNs in cats have been limited. Moreover, to our knowledge, there are no studies to date evaluating the clinical efficacy of IFNs against FCV infection. The purpose of the study reported here was to determine whether IFNs at higher doses than previously evaluated would lead to faster resolution of clinical disease and decreased viral shedding in cats with naturally acquired viral keratoconjunctivitis.

Materials and Methods

Animals—Cats with evidence of URTD and ocular involvement at The Humane Society of Indianapolis were evaluated for possible inclusion in the study. All cats were screened for FeLV infection (some cats were also tested for FIV infection) and were vaccinated against feline viral rhinotracheitis, calicivirus, and panleukopenia with a combination vaccinea as part of standard operating protocol at shelter intake. Cats were designated as affected with URTD by shelter staff on the basis of subjective assessment and observations of signs including sneezing and oculonasal discharge. Cats deemed severely ill with systemic signs such as anorexia and lethargy upon initial evaluation were excluded from enrollment in the study. On the day of enrollment in the study (day 0), each cat underwent a brief physical examination and complete ophthalmic examination, including fluorescein staining of the cornea,b slit-lamp biomicroscopy,c and indirect binocular ophthalmoscopy.d The protocol for use of animals in the study was approved by the Purdue University Animal Care and Use Committee and conformed to the Association of Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

Treatment and control groups—At the time of study enrollment (day 0), each cat was assigned to 1 of 3 groups; cats in group 1 were to receive treatment with HuIFN solutione (concentration, 1 × 106 U/mL; 1 drop/eye twice daily for 14 days), cats in group 2 were to receive treatment with FeIFN solutionf (concentration, 1 × 106 U/mL; 1 drop/eye twice daily for 14 days), and cats in group 3 were to receive treatment with saline (0.9% NaCl) solution (1 drop/eye twice daily for 14 days). Cats were assigned to a group in alternating order until 36 cats were enrolled, with 12 cats in each of the 2 treatment groups and 12 cats in the control group. The primary investigator (JMS) was masked to the identity of drug for each treatment group for the duration of the study.

Preparation of IFNs—Reconstitution of IFNs to form ophthalmic solutions was performed under a sterile hood via aseptic technique to achieve final concentrations of 1 × 106 U/mL for each compound. The FeIFN productf is currently unavailable in the United States; permission was obtained from the FDA for its use in the study. Saline solution alone was used as the placebo control. The solutions were clear and colorless with no grossly distinguishing features. Each solution was placed in sterile dropper bottlesg; bottles were color-coded.

Treatment procedures—Depending on group allocation, each cat was treated with 1 drop of the designated solution in each eye twice daily (at 8 am and 5 pm) for 14 days. Treatment was initiated on day 1. All solutions (1 bottle of solution was used/group) were refrigerated (4°C) at the facility where cats were housed during the treatment period. Cats were not treated with other topical medications or any antiviral medications during the 14-day study period. Some cats received concurrent oral or transdermal administration of antimicrobials in accordance with established shelter protocol for treatment of cats with URTD.

Clinical assessment—On day 0, each cat underwent a complete ophthalmic examination (including slit-lamp biomicroscopy and fluorescein staining of each eye) and during physical examination, clinical signs of ocular and systemic disease were subjectively scored (Appendix) by a veterinarian (JMS). Subsequently, slit-lamp biomicroscopy and fluorescein staining of each eye were performed and clinical signs of ocular and systemic disease were subjectively scored for each cat by the same veterinarian (JMS) on days 3, 7, 10, and 14. At each time point, each cat was assigned a systemic sign score and each eye of each cat was assigned an ocular sign score. These scores (those for each eye and the systemic score) were combined to equal a single cumulative disease score for each time point.

During the treatment period, cats were monitored for signs of adverse reaction in the form of topical ocular irritation. Topical ocular irritation was defined as the development of signs of increased (compared with pretreatment findings) conjunctival hyperemia, epiphora, or blepharospasm within 5 to 10 minutes after solution application.

Collection of swab samples—Swab samples for VI (detection of FHV-1 and FCV) and RT-qPCR assays (detection of FHV-1, FCV, Chlamydophila felis, Mycoplasma felis, and Bordetella bronchiseptica) were obtained from each cat on days 0 and 14. For each cat, 1 swab from each conjunctival fornix was obtained and the samples from the 2 eyes were pooled together (1 tube containing 2 swabs); swab samples were taken from the conjunctival sac prior to instillation of topical anesthetic agent or fluorescein dye. A single swab sample was also obtained from the oropharynx of each cat. Viral medium transport swabsh were used for VI sample collection. Cotton-tipped applicators with plastic handles and sterile blood collection tubes (no additives) were used for RT-qPCR sample collection. Samples for VI were stored in a −80°C freezer until processing. Samples for RT-qPCR testing were stored on ice until transported to the laboratory.

VI and FA staining—Swab samples immersed in viral transport medium were thawed, and the liquid components were gently agitated to ensure even distribution of contents of the swabs into the liquid. The samples were then passed through a syringe filteri and stored in microtubes; portions of each sample not immediately processed for VI were stored in a −80°C freezer.

Crandell-Rees feline kidney cells were grown in 48- and 96-well plates until approximately 90% confluent. Samples (100-μL aliquots) were added to the 48-well plates. All samples were run in duplicate. The plates were incubated in 5% CO2 at 37°C for 5 days and examined daily for cytopathic effect. For positive controls, FHV-1 and FCV were added to the wells; for negative controls, wells contained CRFK cells in medium only. The 48-well plates were then frozen and thawed, and then 50 μL of medium from each well was transferred to the 96-well plates containing CRFK cells and allowed to incubate for 24 to 48 hours before undergoing FA staining.

For FA staining, the 96-well plates were fixed with 80% acetone for 10 minutes and then air-dried at room temperature (approx 21°C). On each plate, designated well rows were stained with a direct polyclonal FHV-1–specific FAj or an indirect FCV fluorescent monoclonal antibodyk and then examined under an inverted microscope to observe for fluorescence.

RT-qPCR assays—Real-time quantitative PCR assays targeting 5 infectious agents that contribute to UTRD in cats were performed on swab samples.l These infectious agents included B bronchiseptica, C felis, FHV-1,9 FCV, and M felis. All assays were designed and validated according to industry standards.m Target genes for each application were as follows: B bronchiseptica, hemagglutinin fusion protein gene (GenBank accession No. AF140678); C felis, outer membrane protein A (GenBank accession No. AP006861); FHV-1, glycoprotein B; FCV, open reading frame 1 (GenBank accession No. AF109465); and M felis, small subunit rRNA (18S rRNA)–internal transcribed region 1 (GenBank accession No. AF443608).

Oropharyngeal and conjunctival swabs were processed separately with protocols adapted as reported.10,11 Briefly, swabs were submerged in lysis solution and incubated for 10 minutes. Lysates were extracted with syringe filtersi in a tractor platform.n Nucleic acids were eluted into 150 μL of PCR-grade nuclease-free water,o and 5 μL of that mixture underwent RT-qPCR amplification. For the FCV-specific RT-qPCR assay and the preanalytic RNA control, 20 μL of total nucleic acid was reverse transcribed into cDNA with random hexamer primers and reverse transcriptasep in a final volume of 40 μL. Five microliters of a diluted cDNA solution (final volume after the real-time step, 100 μL) was used for the FCV-specific RT-qPCR assay.

Analysis was performed on a multiwall-plate-based RT-qPCR platform,q and raw data were analyzed via a second-derivative maximum method with high sensitivity settingsr to generate crossing points. Each RT-qPCR assay was performed with 7 quality controls including PCR positive controls (to assess the functionality of the PCR protocol), PCR negative controls (to confirm the absence of contamination in the reagents), negative extraction controls (to confirm the absence of cross-contamination during the extraction process), a DNA preanalytic quality control targeting the host small subunit rRNA (18S rRNA) gene complex (to assess the quality and integrity of the DNA as a measure of sample quality), an RNA preanalytic quality control targeting the host small subunit rRNA gene complex (to assess the quality and integrity of the RNA as a measure of sample quality and to assess the real-time protocol), an internal positive control spiked into the lysis solution (to assess the functionality of the PCR protocol and to confirm the absence of PCR inhibitory substances as a carryover from the sample matrix), and an environmental contamination-monitoring control (to confirm the absence of contamination in the laboratory).

Analytic and clinical validation of assays—Realtime PCR tests underwent analytic and clinical validation. For the analytic validation, each assay had to pass 6 validation criteria, including amplification efficiency, linearity, intrarun reproducibility, interrun reproducibility, 2-square value, and signal-to-noise ratio of the fluorescent signal. Clinical samples were selected on the basis of a reference method for each test, and a correlation assessment was performed. Positive signals from clinical samples were confirmed via resequencing. Diagnostic sensitivity and specificity on the basis of comparison to reference testing methods were > 90% for each RT-qPCR test.

Statistical analysis—For each group, a mean clinical score was calculated at days 0, 3, 7, 10, and 14. The VI and RT-qPCR findings were reported as the number of cats with positive results. Data were compared within and between groups at the various time points. Categorical data (VI results) were compared via a χ2 test, and nonparametric numeric data (clinical scores and RT-qPCR test results) were compared via a Wilcoxon rank sum test. Correlation between nonparametrically distributed numeric variables was assessed by calculation of the Spearman rank correlation coefficient (ρ). Any relationship between clinical score at day 0 and virus detection (positive or negative) was also compared. Significance was set at a value of P ≤ 0.05.

Results

Animals—Cats meeting inclusion criteria were openly enrolled until the appropriate number (n = 36) for statistical power was reached. The estimated ages of the 36 cats, as provided by shelter personnel and intake records, ranged from 0.16 to 13 years (median, 1.29 years) and did not differ significantly (P = 0.381) among the 3 groups. When evaluating sex and neuter status among the 36 cats, 18 (50%) were castrated males, 13 (36.11%) were spayed females, 3 (8.33%) were sexually intact females, and 2 (5.56%) were sexually intact males; sex did not differ significantly (P = 0.587) among groups. Breeds represented included domestic shorthair (22 [61.11%]), domestic medium hair (6 [16.67%]), domestic longhair (3 [8.33%]), Maine Coon mix (2 [5.56%]), Persian mix (2 [5.56%]), and Siamese (1 [2.78%]). Breed differed significantly (P = 0.043) among groups because of the impact of the few breeds represented in small numbers across the 3 groups. There were few cats of breeds other than domestic shorthair, domestic medium hair, or domestic longhair enrolled in the study, and those cats were distributed randomly into each treatment group; as a result, there were more purebred or mixed purebred cats in group 1 (1 Maine Coon mix and 2 Persian mix cats), compared with group 2 (1 Maine Coon mix cat) or group 3 (1 Siamese cat).

At shelter intake, all 36 cats were confirmed to be negative for FeLV infection; for those cats tested for FIV infection, results were also negative. Each cat was vaccinated against feline viral rhinotracheitis, calicivirus, and panleukopenia (median interval from vaccination to enrollment in the study, 10.5 days [range, 1 to 37 days]).

One cat in group 3 was euthanatized after assessment by the shelter veterinarian on day 4 of the study because of marked decline in systemic health. One cat in group 2 was adopted on day 10 of the study. The data points for these 2 individuals up to the time of removal from the study were included in the statistical analyses. Two cats in group 2 underwent surgical procedures during the course of the study, one for ovariohysterectomy and the other for orbital exploration of an enucleation site infection; these cats remained in the study for its duration.

Clinical scores—Mean overall clinical scores were significantly (P < 0.001) decreased at day 14, compared with day 0 findings; however, at each time point, clinical scores did not differ (P = 0.901) among groups (Figure 1). There was no significant correlation between FHV-1 RT-qPCR value and clinical score (ρ = 0.127; P = 0.466) or FCV RT-qPCR value and clinical score (ρ = 0.126; P = 0.473) on day 0. With regard to FHV-1, the overall clinical score for VI-positive cats was significantly greater than that for VI-negative cats at day 0 in group 3 (rank sum P = 0.014), but not in group 1 (P = 0.673) or group 2 (P = 0.906). With regard to FCV, there was no significant difference in overall clinical scores between VI-positive and VI-negative cats at day 0 (rank sum P = 0.208) in any of the 3 groups. No adverse reactions to any solution were observed in any cat during the course of the study.

Figure 1—
Figure 1—

Mean overall clinical scores for shelter cats with URTD that received 1 drop of HuIFN solution (1 × 106 U/mL [group 1; n = 12; squares]), FeIFN solution (1 × 106 U/mL [group 2; 12; circles]), or saline (0.9% NaCl) solution (group 3; 12; triangles) in each eye twice daily for 14 days (beginning day 1). Clinical signs of ocular and systemic disease were subjectively scored for each cat on days 0, 3, 7, 10, and 14; higher scores were indicative of greater severity (maximum possible score for ocular or systemic signs was 11 in each category [33 cumulatively]). At each time point, scores for each eye and signs of systemic disease in each cat were combined to calculate a mean overall clinical score for each group. In all groups, the overall clinical score at day 14 was significantly decreased from day 0 findings; however, there was no difference among groups at any time point.

Citation: American Journal of Veterinary Research 74, 2; 10.2460/ajvr.74.2.281

VI—During the study, 140 swab samples were collected for VI; samples were not collected at day 14 from 1 cat in group 3 and 1 cat in group 2. Among the 140 collected samples, FHV-1 was isolated from 36 (25.7%) and FCV was isolated from 15 (10.7%). The number of cats for which FHV-1 was detected via VI was significantly lower at day 14, compared with day 0 findings for group 3 (P = 0.011) but not for group 1 (P = 0.086) or group 2 (P = 0.460); results of VI of FHV-1 did not differ by collection site in any group at day 0 or day 14 (Figure 2). The number of cats for which FCV was detected via VI did not significantly (all P > 0.410) differ between day 0 and 14 in any group (Figure 3). At days 0 and 14, the frequency of detection of FCV via VI in all 3 groups of cats was significantly (P = 0.006) greater for the samples collected from the oropharynx, compared with the samples collected from the eyes.

Figure 2—
Figure 2—

Results of VI for FHV-1 in conjunctival (conj) and oropharyngeal (oro) swab samples collected from shelter cats with URTD that received 1 drop of HuIFN solution (1 × 106 U/mL [group 1; n = 12]), FeIFN solution (1 × 106 U/mL [group 2; 12]), or saline solution (group 3; 12) in each eye twice daily for 14 days. For each cat, 1 swab from the oropharyngeal region and 1 swab from each conjunctival fornix was obtained before (day 0 [black bars]) and after 14 days of treatment (day 14 [gray bars]; treatments were initiated on day 1); at each time point, the samples from the 2 eyes were pooled together. Data are reported as the number of cats for which virus was detected. *For a given sample collection site, value at day 14 is significantly (P < 0.05) less than the value at day 0.

Citation: American Journal of Veterinary Research 74, 2; 10.2460/ajvr.74.2.281

Figure 3—
Figure 3—

Results of VI for FCV in conjunctival (conj) and oropharyngeal (oro) swab samples collected from shelter cats with URTD before (day 0) and after (day 14) twice-daily topical administration of HuIFN solution (group 1; n = 12), FeIFN solution (group 2; 12), or saline solution (group 3; 12) in each eye for 14 days. †Within a group at day 0, value derived from oropharyngeal swab samples was significantly (P < 0.05) higher than the value derived from conjunctival swab samples. ‡Within a group at day 14, value derived from oropharyngeal swab samples was significantly (P < 0.05) higher than the value derived from conjunctival swab samples. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 74, 2; 10.2460/ajvr.74.2.281

RT-qPCR assays for FHV-1 and FCV—During the study, 140 swab samples were collected for RT-qPCR assays; samples were not collected at day 14 from 1 cat in group 3 and 1 cat in group 2. Among the 140 collected samples, 74 (52.9%) were positive for FHV-1 and 14 (10%) were positive for FCV. In all 3 groups, the number of cats for which FHV-1 was detected via RT-qPCR assay was significantly (P < 0.001) lower at day 14, compared with day 0 (Figure 4). Results of RT-qPCR assay for FHV-1 did not differ (P = 0.097) by collection site in any group at day 0 or day 14. The number of cats for which FCV was detected via RT-qPCR assay did not differ significantly (P = 0.492) between days 0 and 14 in any group (Figure 5). At days 0 and 14, the frequency of detection of FCV via RT-qPCR assay in all 3 groups of cats was significantly (P = 0.006) greater for the samples collected from the oropharynx, compared with the samples collected from the eyes.

Figure 4—
Figure 4—

Results of RT-qPCR assay for FHV-1 in conjunctival (conj) and oropharyngeal (oro) swab samples collected from shelter cats with URTD before (day 0) and after (day 14) twice-daily topical administration of HuIFN solution (group 1; n = 12), FeIFN solution (group 2; 12), or saline solution (group 3; 12) in each eye for 14 days. See Figure 2 for key.

Citation: American Journal of Veterinary Research 74, 2; 10.2460/ajvr.74.2.281

Figure 5—
Figure 5—

Results of RT-qPCR assay for FCV in conjunctival (conj) and oropharyngeal (oro) swab samples collected from shelter cats with URTD before (day 0) and after (day 14) twice-daily topical administration of HuIFN solution (group 1; n = 12), FeIFN solution (group 2; 12), or saline solution (group 3; 12) in each eye for 14 days. See Figures 2 and 3 for key.

Citation: American Journal of Veterinary Research 74, 2; 10.2460/ajvr.74.2.281

Identification of virus by VI or RT-qPCR assay—Thirty-five of 36 cats had a positive result for at least 1 virus (FHV-1 or FCV) via 1 testing method (VI or RT-qPCR assay). The proportion of swab samples positive for FHV-1 via RT-qPCR assay was significantly (P < 0.001) greater than proportion of swab samples positive for FHV-1 via VI. The proportion of samples positive for FCV via VI and RT-qPCR assay did not differ significantly.

Concurrent viral infections—Via RT-qPCR assays, concurrent infection with FHV-1 and FCV was detected in 8 of 36 (22.2%) cats. Via VI, concurrent infection with FHV-1 and FCV was detected in 6 of 36 (16.6%) cats.

Other etiologic agent DNA detected via RT-qPCR assay—Among the 140 collected swab samples, 12 (8.6%) were positive for B bronchiseptica DNA and 12 (8.6%) were positive for C felis DNA. Mycoplasma felis DNA was detected in 55 of 140 (39.3%) swab samples.

Discussion

The results of the present study indicated that topical ocular application of high doses of HuIFN or FeIFN solution twice daily for 14 days did not shorten the duration of active disease or reduce viral shedding in cats with naturally occurring viral keratoconjunctivitis. Although the number of cats for which FHV-1 was detected via VI was significantly lower at day 14, compared with the pretreatment (day 0) findings in cats that received saline solution, a similar change was not detected in cats that received either IFN treatment. Additionally, FHV-1 detection via RT-qPCR assay was significantly lower at day 14, compared with day 0 findings, in all groups. These results likely reflected the self-limiting course of FHV-1–induced disease and natural diminution of viral replication and shedding over time. The number of cats for which FCV was detected via VI or RT-qPCR assay on days 0 and 14 did not differ in any group.

Since the discovery of IFNs in the 1950s,12 researchers in human medicine have endeavored to demonstrate the clinical activity of these cytokines. Interferons have historically been and are currently used to treat a variety of neoplastic and immune-mediated conditions in humans.13–15 Most of the studies that used IFNs to treat herpetic keratitis in humans were undertaken in the late 1970s and early 1980s. One such study16 revealed that topical ocular administration of high-dose (3 × 106 U/mL) type I IFN accelerated healing and decreased viral shedding in humans with keratitis caused by herpes simplex virus-1. That study16 was preceded and followed by reports17,18 from the same authors regarding the effect of human leukocytic IFN, compared with that of mechanical debridement, on epithelial corneal ulcers in humans. It was concluded that topically applied IFN had some value in hastening resolution of herpetic erosions, but could not replace conventional treatment with mechanical debridement. Other studies19,20 reported in the human medical literature have evaluated, in vitro and in a clinical setting, ocular treatment with IFN in combination with virostatic nucleoside analogues. Results of those studies were promising, and it was concluded that the evaluated compounds may work synergistically in speeding recovery from active viral ocular infection; however, the benefit of IFNs as sole therapeutic agents could not be definitively proven.

A single study21 evaluated the in vitro effects of feline and recombinant human leukocytic IFNs on FHV-1 and FCV as well as on vesicular stomatitis virus in feline lung cell cultures pretreated with the IFNs and subsequently challenged with the viruses. Results of that study21 indicated that pretreatment with the IFNs reduced viral yield by a minimum of 1.4 log 10 and 1.6 log 10 plaque-forming units for FHV-1 and FCV, respectively, compared with control cultures that were not pretreated with an IFN. To our knowledge, other studies evaluating the effects of IFNs on FCV are lacking, and studies22–24 evaluating the in vitro efficacy of high dose (1 × 106 U/mL) IFNs against FHV-1 are relatively limited. In 1 study,22 the efficacy of HuIFN and FeIFN on replication of FHV-1 in CRFK cells was assessed and results indicated that there was a significant antiviral effect only at higher concentrations of IFNs (1 × 105 U/mL to 5 × 105 U/mL). On the basis of results of another study23 to investigate the effects of HuIFN on FHV-1 replication and cytopathic changes, it was concluded that treatment significantly reduced the cytopathic effect and virus titers of FHV-1 at concentrations of 1 × 105 U/mL but did not inhibit the virus from infecting cells. In an investigation24 of the antiviral activity of acyclovir alone and in combination with low-dose human leukocytic IFN (10 or 100 U/mL) on FHV-1 in cultures of feline embryo cells, both treatments inhibited viral infection of cells; however, use of acyclovir and the IFN in combination had a synergistic effect.

To our knowledge, results of only 1 placebo-controlled study25 of the in vivo use of IFN in cats to treat ocular signs associated with FHV-1 infection have been reported. In that study, which evaluated the efficacy of pretreatment with FeIFN (10,000 U administered topically in the eyes and 20,000 U administered orally, twice daily, for 2 days prior to infection) on the course of disease in experimentally infected cats, no beneficial effect of FeIFN treatment was observed. However, cats were not treated during the active disease phase.

In the present study, no adverse effects attributable to ocular HuIFN or FeIFN administration developed in any cat. Both HuIFN and FeIFN have been shown to be noncytotoxic when used in vitro on CRFK cells and feline corneal epithelial cells.22,23 Additionally, it has been reported that no adverse effects developed when FeIFN was applied topically in the eyes of cats that were naturally infected with FHV-1.s That study differed from the present study in that it lacked a control group; thus, conclusions could not be drawn regarding the effect of FeIFN, compared with the natural resolution of FHV-1–induced disease.

Some variation in the ability to detect FCV via VI or RT-qPCR assay depending on the sample collection site was evident in the present study; however, this was not true of FHV-1, for which detection by the 2 methods was not influenced by sample collection site. For both VI and RT-qPCR C, the number of cats for which FCV was detected was significantly greater in samples obtained from the oropharynx were evaluated, compared with findings in samples obtained from the conjunctival sac were evaluated. This influence of sample collection site was in agreement with results of other studies.26–28 Another finding of the present study that was supported by reported data was the greater frequency of FHV-1 detection via RT-qPCR assay than via VI.29–31 Polymerase chain reaction testing is reported to have significantly higher sensitivity and specificity for the detection of FHV-1 in samples than does VI or indirect FA staining.30 It is known that positive PCR assay results must be interpreted with caution because very small amounts of viral nucleic acid can be detected via PCR amplification.32 Also, PCR testing has detected FHV-1 DNA in healthy carrier cats.30,33 This must be considered when determining whether the presence of viral DNA is causing clinical disease. Results of quantitative RT-qPCR procedures, as performed in the present study, can be helpful in elucidating whether the presence of FHV-1 corresponds with clinical disease by identifying the amount of virus within a sample; as the amount of detected virus increases, so does the likelihood of active disease.32

Concurrent infection with FHV-1 and FCV in cats can occur. In a study34 in Belgium, analysis of oropharyngeal samples revealed that the prevalence of coinfection with FHV-1 and FCV in shelter cats was 10%. In the present study, the prevalence of coinfection in shelter cats was 16% and 22%, as detected via VI and RT-qPCR assay, respectively. In the veterinary medical literature, most reported studies30,35–44 to investigate the prevalence of pathogens causing conjunctivitis in cats used PCR techniques to analyze collected samples for the presence of specific nucleic acids. In cats, the prevalence of FHV-1 infection is 14% to 54%30,35–41 and the prevalence of FCV infection is 14% to 21%38,41; the prevalence of infection with C felis, B bronchiseptica, or Mycoplasma spp is 0% to 59%,35,39–41 approximately 5% to 11%,42,43 and 16% and 25%, resepctively.35,44 The overall prevalence of single agents implicated in conjunctivitis in cats and screened for in the present study was in accordance with those reported values, with the exception of M felis, which was detected in a higher percentage of cats (39%) in the present study.

The daily dosing frequency for IFN administration used in the present study was selected on the basis of a consistent standard of once or twice daily treatments reported for humans.16,18,19 It is possible that this dosing frequency was too low to result in observable effects in the treated cats. Future studies, if undertaken, could perhaps evaluate a higher dosing frequency or take steps to more quantitatively measure IFN activity in individual cats. In the present study, the decision to compare a human and a feline recombinant IFN was made on the basis of the known potential for species specificity in IFN activity. We sought to determine whether a feline IFN might be more efficacious in the treatment of cats with naturally acquired viral keratoconjunctivitis than a human IFN, possibly due to increased binding affinity for IFN cell receptors. On the basis of the results of the present study, no conclusions regarding any superior effect of a feline IFN in cats and its benefit over administration of a human IFN could be drawn.

As part of the present study, we also attempted to determine whether severity of the cats' clinical signs, and thus magnitude of clinical score, could be correlated to detection of the viruses in swab samples via VI and RT-qPCR assay. This association was investigated prior to initiation of treatment (ie, day 0) because theoretically, this was the time point at which cats would be assigned a maximal clinical score. No significant association between the magnitude of clinical score and the detection of FHV-1 or FCV via VI or RT-qPCR assay could be identified.

The present study had some limitations, including the recent vaccination of study cats, the poorly understood stability of IFNs for ophthalmic use after reconstitution into solution, and the possible role of nonviral infectious agents in causing clinical disease in the evaluated shelter cats. The cats used in the study were vaccinated with a modified-live virus at the time of shelter intake. The median time from vaccination to enrollment in the study was 10.5 days (range, 1 to 37 days). This procedure was part of the shelter's standard operating protocol for intake of cats; given our intent to perform the study on a population of shelter cats, vaccination of the study cats was unavoidable. It is possible that some of the VI and RT-qPCR assay results identified vaccine-associated viruses rather than wild-type viruses; however, a recent study45 revealed that, at least in terms of molecular diagnostic assays, positive test results are uncommon in adult cats parenterally or intranasally vaccinated with modified live FHV-1 and FCV. The stability of IFNs after reconstitution into solution has been minimally investigated, to our knowledge. The influences of packaging material and buffers on the stability of a liquid HuIFN have been investigated.46 It was determined that there was considerable adsorption of HuIFN to type I borosilicate glass ampoules but that this adsorption was ameliorated by the addition of certain vehicles.46 Additionally, degradation of HuIFN was accelerated by contact of the cytokine with chlorobutyl stoppers; other materials, such as plastic, were not tested.46 In another study,47 an extemporaneously prepared HuIFN eyedrop formulation was evaluated for stability in various storage conditions over time. That HuIFN formulation was stable at 5°C for as long as 15 days, and at 28°C for as long as 7 days. We are not aware of published data regarding the stability of FeIFN formulations. Further studies are needed to evaluate the stability of various IFNs and investigate how stability impacts efficacy of those cytokines in clinical settings. Other nonviral infectious agents detected via RT-qPCR assays in samples collected from the cats in the present study could be confounding factors in light of their potential contribution to clinical disease. The role of other nonviral infectious agents was not evaluated in this study, because our specific goals were to evaluate IFNs in the treatment of FHV-1– and FCV-induced disease in cats. Thus, the importance of infection with nonviral infectious agents in the cats in present study is not known.

Small sample size was an additional limitation of the present study. Evaluation of larger numbers of cats in each study group would have increased statistical power and may have allowed detection of significant differences in clinical disease score and virus shedding data among treatment and control groups. However, results of the present study indicated that topical ocular application of the evaluated high-dose HuIFN and FeIFN solutions were not effective in shortening the duration of clinical disease or decreasing virus shedding in shelter cats with naturally occurring viral keratoconjunctivitis.

ABBREVIATIONS

CRFK

Crandell-Rees feline kidney

FA

Fluorescent antibody

FCV

Feline calicivirus

FelFN

Feline recombinant interferon omega

FHV

Feline herpesvirus

HulFN

Human recombinant interferon alpha-2b

IFN

Interferon

RT-qPCR

Real-time quantitative PCR

URTD

Upper respiratory tract disease

VI

Virus isolation

a.

Felocell 3, Pfizer, New York, NY.

b.

Flu-Glo fluorescein sodium ophthalmic strips USP 1.0 mg, Akorn, Lake Forest, Ill.

c.

Kowa SL-14 biomicroscope, Kowa, Nagoya, Japan.

d.

Keeler Vantage binocular indirect headset, Keeler Instruments Inc, Broomall, Pa.

e.

Intron A, Schering Corp, Kenilworth, NJ.

f.

Virbagen Omega, Virbac, Carros, France.

g.

Steri-Dropper, The Medi-Dose Group, Warwick Township, Pa.

h.

Universal Viral Transport Standard Kit, BD Diagnostics, Franklin Lakes, NJ.

i.

Syringe filters 0.2 μm, Whatman Inc, Piscataway, NJ.

j.

FHV-1 FA, Custom Monoclonals International, West Sacramento, Calif.

k.

FCV FA, Veterinary Medical Research and Development, Pullman, Wash.

l.

RealPCR TaqMan FURD panel test code 2512, IDEXX Laboratories, Sacramento, Calif.

m.

Applied Biosystems User Bulletin No. 3, Life Technologies, Foster City, Calif.

n.

Corbett X-Tractor platform, Qiagen, Valencia, Calif.

o.

Fisher PCR grade nuclease free water, Thermo Fisher Scientific, Waltham, Mass.

p.

SuperScript III, Invitrogen, Carlsbad, Calif.

q.

Roche LightCycler 480, Roche Applied Science, Indianapolis, Ind.

r.

Roche LightCycler 480 software, version 1.5.0 (SP3), Roche Applied Science, Indianapolis, Ind.

s.

Verneuil M. Topical application of interferon omega in the treatment of herpetic keratitis in the cat: preliminary study (abstr). Vet Ophthalmol 2004;7:427.

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Appendix

Clinical scoring system used to assess signs of ocular and systemic disease in shelter cats with URTD.

SignScore (per eye)
Ocular
   Conjunctival hyperemia0 = none; 1 = mild; 2 = moderate; 3 = severe
   Chemosis0 = none; 1 = mild; 2 = moderate; 3 = severe
   Ocular discharge0 = none; 1 = mild; 2 = moderate; 3 = severe
   Symblepharon0 = none; 1 = present
   Corneal ulcer0 = none; 1 = present
Systemic
   Nasal discharge0 = none; 1 = mild serous; 2 = moderate serous; 3 = purulent
   Dyspnea0 = none; 1 = mild; 2 = moderate; 3 = severe
   Cough0 = none; 1 = present
   Sneeze0 = none; 1 = present
   Demeanor0 = bright, alert, and responsive; 1 = quiet and lethargic; 2 = signs of depression but responsive; 3 = signs of severe depression and unresponsive

Contributor Notes

Dr. Slack's present address is Department of Clinical Sciences, Kansas State University Manhattan, KS 66506.

Supported in part by the Moustaki Graduate Research Fund, Purdue University College of Veterinary Medicine.

Presented in abstract form at the 42nd Annual Meeting of the American College of Veterinary Ophthalmologists, Hilton Head, SC, October 2011.

Address correspondence to Dr. Stiles (stilesj@purdue.edu).