Abstract
The prevalence of various viral infections was examined in primary accession cases of feline lower urinary tract disease (FLUTD) and healthy control cats in Norway. Urine samples from 102 cats with clinical signs of FLUTD and 73 healthy control cats were tested for the presence of feline calicivirus (FCV), feline coronavirus (FCoV) and feline herpesvirus-1 (FHV-1) by polymerase chain reaction. All urinary samples were negative for FCV and FCoV. One (1%) of the FLUTD cats was found to be positive for FHV-1. The results did not indicate an association between the viral infections examined and signs of FLUTD in the study sample.
Introduction
Feline lower urinary tract diseases (FLUTDs) are characterised by clinical signs such as periuria, haematuria, dysuria, pollakuria and/or urinary obstruction. Although cats with signs of FLUTD are commonly presented in small animal practice, the pathogenesis of some manifestations of FLUTD remain incompletely understood.1–5 The clinical signs seen in cats with FLUTD may result from bacterial, fungal or parasitic infections, uroliths, urethral plugs, neoplasia, malformations, and traumatic, neurological and iatrogenic causes. There are, however, cats with FLUTD where no definite cause has been identified.1–4 These cats are diagnosed as having feline idiopathic cystitis (FIC). 5
Previously, viruses have been proposed as possible causative agents in cats with FIC.6–8 The hypothesis has been supported by the isolation of bovine herpesvirus-4 (BoHV-4), feline foamy virus/feline syncytium-forming virus (FFV) and feline calicivirus (FCV) from urine and tissue samples from cats with FLUTD.9–13
FCV was isolated from a Manx cat with spontaneous urethral obstruction. The virus induced obstructive uropathology in male cats after inoculation in the urinary bladder.14,15 In subsequent studies involving a larger number of cats with naturally-occurring FLUTD, FCV was not isolated by standard cell-culture inoculation.5,8 However, recent studies, where polymerase chain reaction (PCR) was used, have indicated a potential role for FCV as a causative agent in the pathogenesis of some cases of FIC. 5,16
The role of herpesviruses in cats with FLUTD has been questioned, as serum antibodies are found in healthy cats, as well as those with signs of FLUTD, and may also be induced by vaccination.6,7,17–19 Early studies indicated a possible connection between a gamma-herpesvirus and clinical signs of FLUTD. A cell-associated herpesvirus was isolated in studies of feline urolithiasis and later shown to induce clinical signs of FLUTD in specific pathogen-free male cats.6,8 Later studies showed that parts of the genome of the isolated virus were almost identical to BoHV-4, and both cats with naturally-occurring FLUTD and control cats have been screened serologically.1,5 However, positive BoHV-4 serology has not been shown to be associated with clinical signs of FLUTD or abnormal laboratory findings.5,8 In later reviews, authors conclude that an inability to isolate gamma-herpesviruses from additional cats with naturally-occurring FLUTD and failure to induce signs of FLUTD in healthy cats precluded assigning BoHV-4 a primary role in FIC. 5
Both feline herpesvirus-1 (FHV-1) and feline coronavirus (FCoV) are commonly occurring and may induce systemic infection and diseases. Although potential roles as causative agents in FLUTD have not been suggested previously, FHV-1 and FCoV were included in the study in addition to FCV because of their importance as feline pathogens.
The aim of this study was to determine the prevalence of FCV, FCoV and FHV-1 in the urine of Norwegian cats with signs of FLUTD and in a control group of healthy cats.
Materials and methods
Samples
The study population consisted of client-owned cats recruited from the Department of Companion Animal Clinical Sciences at the Norwegian School of Veterinary Science (NVH) from 2006 to 2009. Urine and serum samples were collected from 102 primary accession cases with signs of FLUTD and from a control group of 73 healthy cats. In addition to the owners’ informed consent, the inclusion criteria were clinical signs of FLUTD (periuria, haematuria, dysuria, pollakiuria and/or urinary obstruction) and a diagnosis consistent with FLUTD. A diagnosis of urolithiasis was given when uroliths were present in the urinary tract. Urethral obstruction by other material than uroliths resulted in a diagnosis of urinary plugs. Only cats that did not have concurrent urolithiasis or urinary plugs were given a diagnosis of bacterial cystitis when significant bacteriuria was detected by culturing [≥103 colony forming units (cfu)/ml and ≤2 types of bacteria]. Clinical signs of FLUTD with unknown cause after diagnostic investigation resulted in a diagnosis of FIC.
Exclusion criteria were treatment that could interfere with the diagnostics (medication-altering blood pressure, urine production and/or composition, hormones or medication with antimicrobial effect) and concurrent diseases likely to be of influence on the urinary findings, such as chronic kidney disease, diabetes mellitus or hyperthyroidism. No age, gender or breed restrictions were made.
The control cats were recruited among patients brought to the clinic for healthcare reasons which required sedation or anaesthesia, and would not interfere with the urine analyses (castration, spaying, dental problems or minor surgery). With the exception of clinical signs and a diagnosis consistent with FLUTD, the cats in the healthy control group met the same inclusion and exclusion criteria. Permission to collect the data was granted from the Committee of Research and Ethics at NVH.
All the cats were given a physical examination, and blood samples for haematology and biochemistry were collected. The urine samples were obtained in the acute phase, when the cat was admitted to the clinic, and sent directly to the microbiology department for bacteriological and virological examinations. Thus, the study included all cats that received a final diagnosis consistent with FLUTD, not only those later to be diagnosed with FIC. The samples for virological testing were frozen at -70°C without delay and stored for a maximum of 4 weeks before they were tested for the presence of FCV and FCoV by reverse transcriptase (RT)-PCR and for FHV by PCR. In addition, standard urinalysis was performed, including commercial dipstick analysis (Krulab; Kruuse), measurement of urine specific gravity by refractometer (URC-Ne; Atago) and microscopic examination of urine sediment (native samples and samples stained with Sternheimer-Malbins). The majority of FLUTD cats received an ultrasound examination (84%) or a radiological examination (7%).
FCV and FCoV
Viral RNA was isolated from 140 µl urine using the QiAmp Viral RNA mini kit (Qiagen) following the manufacturer’s instructions. This extraction procedure is designed for the isolation of viral RNA from cell-free body fluids. Owing to the addition of carrier RNA during the procedure, the concentration of extracted RNA was not measured. One-step RT-PCR was carried out using Ready-To-Go RT-PCR Beads (Amersham Pharmacia) in 50 μl reaction volume with random hexamers [pd(N)6; Amersham] as primers in the cDNA synthesis. To avoid possible interference by multiplexing, FCV- or FCoV-specific primers were run in separate PCR reactions. Primers are listed in Table 1. For each sample RNA was added and incubated at 25°C for 10 mins and then at 48°C for 30 mins for the cDNA synthesis. Cycling conditions were 94°C for 5 mins followed by 40 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 1 min with a final extension at 72°C for 15 mins. Amplified products were resolved by electrophoresis on a 3% NuSieve 3:1 agarose (FMC Bioproducts) and visualised under ultraviolet light after ethidium bromide staining. The additional primer pair FCoV MF151/MR257 was used in nested PCR for a total of 19 samples. Positive controls were run for each gel and consisted of cell culture supernatants for FCV (strain F9) and peritoneal fluid from a Norwegian cat with feline infectious peritonitis for FCoV. The amplicons from positive controls were verified by sequencing (GATC Biotech) and, subsequently, analysis in BLASTN. 20
Primers used in polymerase chain reactions
In order to test for the eventual inhibitory effect of urine, 0.1, 1.0, 10 or 50 µl cell supernatant containing FCV were added to urine (final volume 140 µl) prior to RNA extraction.
FHV-1
In order to test for the presence of FHV-1, the cells in urine samples were targeted. After material for FCV and FCoV had been collected, the urine samples were centrifuged at 700 × g for 10 mins, the pellet was washed in phosphate buffered saline (PBS; 0.14 M NaCl, 2.7 mM KCl, 0.88 mM KH2PO4, 7.6 mM NaHPO4, pH 7.4) and then dissolved in 100 µl PBS. A DNA extraction procedure suitable for washed cells from urine was employed (QiAmp DNA minikit; Qiagen), according to the manufacturer’s instructions. A total of 5 µl DNA solution was used in a PCR reaction volume of 50 µl. Primers were chosen to target a conserved part of the FHV-1 genome (thymidine kinase). To test for the eventual inhibitory effect on the PCR, FHV-1 containing cell culture supernatant (5 µl) was added to a solution of dissolved pellet. The cycling conditions were 94°C for 5 mins followed by 40 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min and a final extension at 72°C for 15 mins. The positive control was cell culture supernatants for FHV (in-house isolate), and amplicons were verified by sequencing (GATC-Biotech AG). PCR products were resolved as described for the RNA viruses.
Results
Cats
Distributions of age, weight, gender, breed and method of urine collection for the 102 FLUTD cats and the 73 control cats included in the study are given in Table 2. The final diagnosis is also included for the 102 FLUTD cats. Forty-nine percent of the FLUTD cats were predominantly indoor cats and 43% had outdoor access, while this information was missing for the other 8%.
Feline lower urinary tract disease (FLUTD) cats and controls: age, weight, gender and breed
Urine samples
The primers recognised the positive controls for FCV, FCoV and FHV-1, and the specificities of the amplicons were verified by nucleotide sequencing. No inhibitory effects were observed in RT-PCR when cell culture supernatant was added to urine; even addition of as little as 0.1 µl FCV-containing cell culture supernatant to 140 µl urine was positive. Similarly, no inhibitory effect was observed in PCR when FHV-containing cell culture supernatant was added to the solution of dissolved pellet before DNA extraction.
No positive RT-PCR results were found for FCV or FCoV in samples from cats with FLUTD or in controls. However, PCR amplicons with an approximate size of 160 base pairs (bp) (expected size should be 140 bp) were found in four samples from the FLUTD group tested for FCV. These amplicons were cloned and sequenced, but no sequence similarity to FCV was found and they were, therefore, considered to be non-specific amplifications. Samples that were tested for FCoV and that gave smearing in the gel in the approximate size range of the expected amplicon were run in a nested reaction using the FCoV MF151 and FCoV MR257 primers. A total of 19 samples were run in nested PCR, but none were positive. One sample from a cat with signs of FLUTD was found to be positive for FHV-1 by PCR.
Discussion
There was no indication of an association between the presence of FCV, FCoV or FHV-1 in urine and signs of FLUTD in this study. This may be owing to technical aspects, as urine is recognised as a difficult substrate for recovery and amplification of nucleic acids. 21 However, inhibition of the PCR was not found to be present as the positive controls could be amplified after addition of urine before RNA extraction and addition of virus supernatant to urinary cell pellet before DNA extraction. The silica gel-based columns for RNA extractions used in this study have been shown previously to be suitable for urinary samples. 21 The urine samples were received at the laboratory within a few hours of collection and frozen without delay; decay of virus or viral nucleic acid due to storage was considered unlikely.
A potential reason for false-negative results in this study is the possibility of virus levels being below the threshold of a single-round classical PCR. At the time this study was initiated real-time PCR was not available; this would have been the method of choice if the study was initiated at a later time point. A previous study of FCV from conjunctival and pharyngeal swabs showed that the use of nested PCR increased the sensitivity. 22 However, a number of samples in the present study were run in a FCoV-nested reaction but none was found to be positive. Although virus levels below the threshold of the test cannot be ruled out, the biological importance of such low virus levels may be questioned with regard to a cause-and-effect relationship, ie, between virus and signs of FLUTD. There is also the possibility of fluctuating loads of virus in the urine. However, if that was the case, a limited number of positive samples would be expected.
FCV is known as a widespread and highly contagious pathogen, and the prevalence of FCV antibodies reported varies extensively depending on local variation, as well as the extent of vaccination.5,23,24Although the prevalence of FCV antibodies may be high in some cat populations, the number of cats with FCV viruria was relatively low in previously published studies based on PCR and a modified virus isolation method.5,25 In these studies, viruria was detected in 6% of the FIC cats by PCR and in 5% of the cats by the modified isolation method.5,25 The vaccination status was unknown for a majority of the cats included in the present study. The influence of vaccination on the results is, therefore, uncertain and a large proportion of vaccinated cats in the study sample may have caused an underestimate of FCV among Norwegian cats with FLUTD.
Caliciviruses do show high genetic variability23,25–27 and, therefore, the FCV primers were degenerated in order to enable recognition of all FCV sequences registered in Genbank. However, the use of degenerated primers may not be optimal regarding the sensitivity in PCR. 28 Failure to pick up local strains of FCV may have represented another potential reason for false-negative results. The use of primers based upon a positive control in form of a FCV isolate from a Norwegian cat may have mirrored the field situation in a more appropriate way. However, virus strains used in vaccines are often based on the F9 strain, which was used as a positive control in this study. There are, to our knowledge, no indications of failure of the vaccination regime in Norway. The primers used in this study may, therefore, be expected to pick up the field variants of FCV.
In the present study, only one cat with signs of FLUTD was found to be positive for FHV-1 in the urine and statistical evaluation was thus precluded. The cat was a 5-year-old overweight intact male Norwegian Forest cat that was kept primarily indoors. Bacteria, uroliths or crystals were not found in its urine. The same cat also tested positive for FIV antibodies in serum. All control cats were found to be negative for FHV-1. Although the nature of FHV-1 must be taken into consideration, with its latent infection and intermittent viral shedding, these results do not support an important aetiological role for FHV-1 in the pathogenesis of FLUTD.
Conclusions
The results did not indicate an association between the presence of FCV, FCoV or FHV-1 in the urine and FLUTD in the study sample.
Footnotes
Acknowledgements
The authors would like to thank Berit Gamnes for technical assistance and the veterinarians and veterinary nurses at the Department of Companion Animal Clinical Sciences for their help and assistance in collecting the material.
Funding
This work was supported by two not-for-profit foundations: Astri and Birger Torsted’s Foundation and Veterinary Smidt’s Foundation.
Conflict of interest
The authors declare that there is no conflict of interest.