Ocular disease severity varies by pathogen in cats.
Keywords: feline, PCR, feline herpesvirus, feline calicivirus, Chlamydophila felis, feline coronavirus, Mycoplasma felis, ocular
Feline herpesvirus-1 (FHV-1) has previously been implicated in causing feline ocular disease. The objective of this study was to identify pathogens using PCR and viral isolation in shelter cats with ocular and upper respiratory disease, and to identify factors associated with increased severity of clinical disease.
Oropharyngeal swabs from 90 cats in 11 animal shelters across the USA were obtained and tested by real-time PCR using a feline respiratory panel after clinical scoring for ocular and respiratory disease. A multivariable linear mixed model assessed the relationship between clinical scores and age, sex and pathogen. Viral isolation was performed on all samples, then 42/90 underwent DNA extraction and gel electrophoresis.
Five pathogens were detected by real-time PCR: feline calicivirus (FCV) (63%), FHV-1 (54%), Mycoplasma felis (50%), feline coronavirus (FCoV) (17%) and Chlamydophila felis (3%). A positive FHV-1 PCR result was associated with slightly higher respiratory clinical scores (95% CI= (0.050-1.351), p <0.05) and lower ocular clinical scores (95%CI= (-2.464,-0.597), p <0.01). Male cats (n=51) had higher ocular clinical scores than females (95%CI= (0.342-1.789), p < 0.01). Viral isolation detected virus in 84/90 samples within 7 days. Virus isolates from 18/21 samples with both FHV-1 and FCV detected by PCR showed no banding consistent with FHV-1 on electrophoresis after DNA extraction. Of these 18 samples, 15 were confirmed to contain FCV by PCR performed on first viral passage supernatant.
Conclusions and Relevance
Our finding that FHV-1 was associated with higher respiratory scores, but lower ocular scores, contradicts current understanding and warrants further research. The results of the viral isolation demonstrate that FCV may grow preferentially over FHV-1 in vitro. Overall, the results indicate that FCV may play a more significant role in infectious ocular disease in cats housed in animal shelters than previously realized.
Upper respiratory disease, both with and without ocular involvement, is an extremely common problem in shelter cats and the companion animal population (1). Common pathogens suggested to be responsible in the past include feline herpesvirus-1 (FHV-1), feline calicivirus (FCV), Chlamydophila felis, feline coronavirus (FCoV) and Mycoplasma felis. Specifically, FHV-1 has been associated with respiratory disease (2-4), ocular disease (2, 4) and skin disease (5). FCV has been associated with gingivostomatitis (6-12), respiratory disease (2, 3, 11) and ocular disease (2, 11). C.felis has been associated with respiratory disease (2) and ocular disease (2, 13). FCoV has been reported to cause mild respiratory signs, diarrhea and can mutate to cause feline infectious peritonitis (14). M. felis has been identified as a cause of pneumonia in cats (15), upper respiratory disease (16) and has been associated with ocular disease (13, 17-20). Current recommendations are that cats in a shelter environment should be vaccinated against both FHV-1 and FCV (21-23).
Previous studies have looked at the prevalence of these pathogens through the use of PCR in Spain (2), Switzerland (8), UK (24, 25), Germany (11), Canada (26, 27), Europe (3), Brazil (28), Korea (29), Japan (30), Australia (31) and the USA (32-34). However, the previous published studies that have taken place in the USA have been restricted geographically to California (32), New England (33) and Colorado (34).
Both ocular and oropharyngeal swabs from cats have been used to perform PCR for upper respiratory disease pathogens. The use of oropharyngeal swabs may increase the detection rate of pathogens (35).
The primary objectives of this study were to perform a survey of the pathogens causing upper respiratory and ocular disease in shelter cats across the USA, to identify associated pathogens using PCR and viral isolation, and to identify factors associated with increased severity of clinical disease in affected animals.
Materials and methods
Sample Acquisition and PCR
All procedures were performed in accordance with an approved University of Wisconsin-Madison Institutional Animal Care and Use Committee protocol. Cats in shelters with signs of respiratory disease with or without ocular involvement were identified by shelter veterinarians in eleven geographically distinct areas of the USA (Table 1, Figure 1). The veterinarians were instructed to include any animals showing clinical signs of respiratory disease of any severity with or without ocular involvement. The number of samples submitted from each location was limited by the number of animals showing clinical signs at the time of sampling. Samples were submitted between the months of August and December. Veterinarians at each shelter used a clinical scoring system (Table 2) to create a clinical score for each cat. The clinical signs were designated as either ocular, respiratory or generalized (Table 2). Two oropharyngeal swabs were taken from each cat by brushing the oropharyngeal area firmly for around 30 seconds. The swabs were then placed into a transport medium (Universal Viral Transport, Becton, Dickinson and Company), labelled and double bagged to prevent cross-contamination. Gloves were changed between animals. The swabs were shipped overnight to the Wisconsin Veterinary Diagnostic Laboratory for feline respiratory panel testing (FHV-1, FCV, C.felis, FCoV and M. felis) by real-time PCR and to the UW-Madison Brandt laboratory for viral isolation. The PCR test was validated as per accreditation protocols set by the American Association of Veterinary Laboratory Diagnosticians.
Initial viral isolation and creation of viral stocks
Samples were immediately refrigerated on receipt prior to viral isolation. A 1 ml aliquot of each sample was added to individual 100 mm tissue culture plates with maximally confluent Crandell Rees feline kidney cells (CRFK) along with 1 ml of Dulbecco’s modified Eagle’s medium containing 2% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin sulfate and 250 µg/ml amphotericin B (DMEM) before being incubated at 37°C for 60 minutes. An additional 4 ml of DMEM was then added to each plate before being incubated at 37°C and checked daily for 7 days until 100% cytopathic effect (CPE) was observed. The cells and media were scraped and pipetted from the plate and placed in a conical tube for centrifugation at 600 X g for 10 minutes at 4°C in a Sorvall X1R Legend centrifuge. The supernatant was removed and the pellet was re-suspended in 750 µL of the reserved culture medium. The remainder of the culture supernatant was stored at 4°C. The re-suspended pellet was subjected to three freeze-thaw cycles and centrifuged at 600 X g for 10 minutes at 4°C (Sorvall X1R Legend centrifuge). The resultant supernatant was combined with the saved culture medium and stored in 200 µL aliquots at -80°C.
Viral DNA preparation
Viral DNA was prepared using a modification of a previously published protocol (36). Briefly, a thawed vial of virus stock was added to 12ml of DMEM in a 15 ml conical tube. Two ml of this mixture per plate was added to 6 confluent 100 mm tissue culture plates of CRFK cells and incubated at 37°C for 60 minutes. An additional 4 ml of DMEM was added to each plate before being incubated at 37°C and checked daily until 100% CPE was observed. The cells and media were scraped and added to a single 50 ml conical tube before being centrifuged at 600 X g for 10 minutes at 4°C (Sorvall Legend X1R centrifuge). The supernatant was then stored at 4°C. The pellets were re-suspended in 5 ml of the saved supernatant and subjected to three freeze-thaw cycles. All supernatants were combined and centrifuged at 600 X g for 10 minutes at 4°C. The resultant supernatant was then centrifuged at 600 X g for 5 minutes at 4°C. The supernatant was layered onto a 36% sucrose cushion in 0.1 M phosphate-buffered saline and centrifuged for 80 min at 24,000 X g at 4°C (Sorvall WX Ultra Series ultracentrifuge). The pellet was re-suspended in 1 ml of TE buffer per tube (10 mM Tris [pH 7.4], 1 mM EDTA) with 0.15 M sodium acetate and 50 μg/ml RNase A and then incubated for 15 min at 37°C. Proteinase K and SDS (50 μg/ml and 0.1%, final concentrations respectively) were added, and the solution was incubated for 15 min at 37°C. The viral DNA was purified by phenol-chloroform extraction and ethanol precipitation, incubated with 50 μg/ml RNase A for a further 15 minutes, re-suspended in deionized water, and stored at −20°C. DNA purity and concentration was analyzed using a Nanodrop Lite Spectrophotometer (Thermo Scientific).
Gel Electrophoresis and PCR Confirmation of Pathogens
Viral DNA samples obtained from second viral passage supernatants were digested with Bam H1 according to the manufacturer’s instructions (Promega) for 12 hours at 37°C and electrophoresed in 0.8% agarose. DNA from a plaque-purified field isolate that was verified as FHV-1 by immunofluorescence with FHV–1-specific antiserum (37) was included as a positive control.
Supernatant obtained after first passage of each virus which was positive for both FHV-1 and FCV on PCR but showed no banding consistent with FHV-1 on electrophoresis after DNA extraction was submitted to the Wisconsin Veterinary Diagnostic Laboratory for real-time PCR panel testing (FHV-1, FCV).
The following variables were analyzed statistically; total clinical score, respiratory clinical score, ocular clinical score, generalized signs clinical score, sex (male/female), age (juvenile, ≤ 5 months; young, 6-23 months; adult, ≥ 24 months), PCR status for FHV-1/FCV/FCoV/M. felis/C. felis (positive or negative) and geographic collection site (Eastern USA, Western USA, Southern USA, Midwestern USA). For the purposes of clustering the shelters by geographic location, designations as shown in Table 1 were used. Before fitting a multivariable model to the data, different univariate relationships in the data were explored. The effect of geographic collection site, sex, age, total number of pathogens per sample, PCR status for pathogens and prior anti-viral use on different clinical scores were explored graphically (38) and numerically when appropriate through the use of non-parametric testing procedures (Kruskal-Wallis or Mann-Whitney rank sum tests).
Following the initial exploration of the data it was decided that the relationship between both FHV-1 and FCV and ocular and respiratory clinical scores would be the primary focus of further investigation.
To estimate the relationship between FHV-1 and both ocular and respiratory clinical scores, two separate linear mixed models (LMM) were fit to the data (one for each clinical symptom score) using the restricted maximum likelihood (REML) criterion in the lme4 (V 1.1-12) package for R (V 3.3.2) (39, 40). Both models were adjusted for age, sex, total number of pathogens detected by PCR, FCV PCR status, FCoV PCR status and FHV-1 PCR status, while collection site was modeled as a random effect. For the respiratory model, the random effect variance was estimated to be zero by the model, hence the LMM estimates were identical to those of a fixed-effects multiple linear regression. A more complex model, which included a random effect for the region of the shelter (with an identical fixed-effects specification) was found to not fit the data significantly better than our proposed model (LRT
Figure 3 – Examples of results of DNA extraction and gel electrophoresis of viral isolates from 8 cats. The 4 lanes on the left of the image (NEWY02, OREG02, SANF05 and PHIL05, all FHV-1 PCR positive and FCV PCR positive) do not demonstrate banding typical of the presence of Feline Herpesvirus-1 DNA. The 4 lanes on the right of the image (PHIL10, KANS08, NEWY03 and WASH01, all FHV-1 PCR positive and FCV PCR negative) demonstrate banding typical of the presence of Feline Herpesvirus-1 DNA
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