Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria 99 (2012)

From the Clinical Virology1 and the Institute of Anatomy, Histology and Embryology2, Department for Pathobiology, the Biostatistics Group3, Department for Biomedical Sciences, and the Clinic for Diagnostic Imaging4, Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Austria Osteoarthritis in and the possible involvement of and feline

K. WEISSL1, V. BENETKA1, E. SCHACHNER2, A. TICHY3, M. LATIF1, E. MAYRHOFER4 and K. MÖSTL*1

received July 11, 2011 accepted for publication March 27, 2012

Keywords: osteoarthritis, retro- Schlüsselwörter: Osteoarthritis, , calicivirus, cats. Retroviren, Caliciviren, Katze. Summary Zusammenfassung

The occurrence of degenera- Untersuchungen zur Osteoar- untersucht. Die Blutproben wur- tive joint diseases (DJD) and the thritis bei Katzen und zur Beteili- den zusätzlich auf FIV-Antikörper possible involvement of Feline gung von felinem Calicivirus und getestet und im positiven Fall wur- Calicivirus (FCV), Feline Leukae- felinen Retroviren den die Gelenke auf FIV-spezi - mia (FeLV), Feline Immuno- sche Nukleinsäure untersucht. de ciency Virus (FIV) and Feline Einleitung Foamy Virus (FFV) infections In der Literatur gibt es diverse Ergebnisse were investigated in 54 eutha- Hinweise auf einen möglichen Zu- Die Röntgenbilder zeigten in nized cats chosen at random. sammenhang zwischen dem Vor- 88,9 % der Aufnahmen Zeichen Blood samples and synovial tis- handensein von Gelenksverände- von degenerativen Gelenksverän- sues were collected and bilateral rungen und Infektionen mit derungen. Am häu gsten waren radiographic images taken of the Felinem Calicivirus (FCV), Felinem die Ellbogengelenke von diesen shoulder, elbow, hip, sti e and Leukämievirus (FeLV), Felinem Im- Veränderungen betroffen (40 links hock. Samples were analysed for munde zienzvirus (FIV) und Feli- und 43 rechts), gefolgt von den viral nucleic acids using real-time nem Foamy Virus (FFV). Ziel der Hüftgelenken (39 beidseits) und PCR and blood samples were vorliegenden Studie war es, eine den Kniegelenken (39 links und 38 also tested for FIV antibodies. Pilotstudie zu dieser Fragestel- rechts). Bezüglich des Schwere- Radiographic images were evalu- lung unter Einsatz der modernen, grades der Veränderungen waren ated with 88.9 % of the cats hochsensitiven molekularvirologi- die Hüftgelenke (sechs links und showing signs of DJD. The most schen Verfahren durchzuführen. zehn rechts) am stärksten betrof- frequently affected joints were fen. Es konnte ein statistisch sig- the elbow and the hip joints with Material und Methode ni kanter Zusammenhang zwi- the hip showing the most severe Vierundfünfzig willkürlich aus- schen steigendem Alter der changes. There was a signi cant gewählte, aus unterschiedlichen Katzen und Anzeichen von Ge- correlation between signs of DJD Gründen euthanasierte Katzen lenksveränderungen nachgewie- and increasing age of the cats wurden auf die Präsenz von Ge- sen werden (r=0,475; p=0,004), (r=0.475; p=0.004). lenksveränderungen untersucht. nicht aber zwischen dem Alter der Nucleic acids of all viruses ex- Unmittelbar post mortem wurden Katzen und dem Schweregrad der cept FCV could be detected in bilaterale Röntgenaufnahmen von aufgetretenen Gelenksverände- the blood as well as in samples of Schulter, Ellbogen, Hüfte, Knie rungen (p=0,646). the joint capsules, whereas FCV und Tarsus angefertigt. Blutpro- Nukleinsäuren aller erwähnten Vi- was only found in synovial tissue ben und Bioptate der Synovial- ren (mit Ausnahme von FCV) konn- samples from one . A real- membran wurden gewonnen und ten sowohl im Blut als auch in den time PCR assay was established nach deren Weiterverarbeitung gesammelten Synovialmembranbi- to detect FFV-speci c nucleic mittels real-time PCR auf das Vor- optaten nachgewiesen werden. acids. FFV had the highest preva- handensein von viraler Nuklein- FCV wurde ausschließlich in den lence among all viruses (eight säuren von FCV, FeLV, und FFV Synovialmembranproben einer

123 Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria 99 (2012)

cats), whereas FIV and FeLV were Katze (in fünf Gelenken) nachge- statistisch nicht veri ziert werden only found in two cats and one wiesen. Für den Nachweis viraler (p=0,152). cat, respectively. Viral nucleic Nukleinsäuren von FFV wurde acids were found more frequently eine real-time PCR etabliert. Die Schlussfolgerung in the shoulder than in any other höchste Prävalenz zeigte FFV Die in dieser Studie eingesetz- joint. There was no signi cant dif- (acht Katzen, fünf Blutproben ten PCR-Methoden - einschließ- ference in sum of signs of DJD (PBMCs) positiv, 35 Synovial- lich der für FFV-spezi sche Nuk- (p=0.255) between virus-positive membranbioptate positiv), wo- leinsäure etablierten - erwiesen and virus-negative cats. hingegen FIV in lediglich zwei sich als vielversprechend für wei- Katzen (ein positives PBMC, drei tere Untersuchungen zu einer positive Synovialmembranbiop- möglichen viralen Ätiologie von tate), und FeLV in nur einer Katze osteoarthrotischen Veränderun- (ein positives PBMC, sieben po- gen bei der Katze. In dieser Pilot- sitive Synovialmembranbioptate) studie konnten sowohl FCV als nachgewiesen werden konnte. auch FeLV, FIV und FFV moleku- Am häu gsten gelang der Nukle- larvirologisch in Katzengelenken Abbreviations: Ct-values = threshold insäurenachweis in den Schulter- nachgewiesen werden, am häu- cycle values; DJD = degenerative gelenken. Unterschiede im Alter gsten FFV. Weitere Untersu- joint disease; EDTA = Ethylene-di- zwischen Nukleinsäure-positiven chungen sind erforderlich, um die amine-tetra-acetic acid; FCV = Feline (M=10,6; SD=5,0) und Nuklein- Beteiligung der angeführten Erre- Calicivirus; FeLV = Feline Leukaemia säure-negativen (M=10,2; SD=5,2) ger bei Osteoarthritis der Katze, Virus; FFV = Feline Foamy Virus; FIV = Feline Immunode ciency Virus; M = Tieren konnten nicht nachgewie- welche bei bemerkenswert vielen mean value; n.p.ch. = no pathological sen werden (p=0,848), ebenso Tieren nachgewiesen werden changes; OA = osteoarthritis; PBMC nicht hinsichtlich der Sum- konnte, weiter abzuklären. = peripheral blood mononuclear cell; me der Gelenksveränderungen PCR = polymerase chain reaction; (p=0,255). Auch das Vorliegen ei- RT-PCR = reverse transcription-poly- merase chain reaction; SD = standard ner Geschlechtsprädisposition deviation bei FFV positiven Tieren konnte

Introduction Degenerative joint disease (DJD), a general term to rst symptoms. GODFREY (2005) found clinical sym- describe degenerative alterations in any type of arti- ptoms in only one third of cats with radiologically culation (i.e. synovial, cartilaginous or brous), is the diagnosed OA, although there was a high prevalence most common form of arthritis. Terms such as of OA (22 %) in a group of osteoarthritic cats chosen osteoarthritis (OA) and osteoarthrosis (because of the at random. Radiographic evidence of DJD was found inconsistency of in ammation) are synonyms for this in 90 % of cats older than 12 years (HARDIE et al., condition (SUMEDHA, 2005). 2002) and in 61 % of cats older than six years Osteoarthritis (OA) is the term used to describe (SLINGERLAND et al., 2011). diseases of the whole joint due to articular cartilage Radiological signs of osteoarthritis are either an in- damage, which may be caused by mechanical over- crease in subchondral or periarticular bone density stress (due to high body weight or severe exercise), involving the capsule, ligaments and tendons (prolife- infections, injuries, neoplasms or systemic diseases rative form) or joint surface damage, reduced joint (HARDIE, 1997). A symmetrical polyarthritis of cats space, instability and bone destruction (deforming that resembles human rheumatoid arthritis has been type) (PEDERSEN et al., 1980). Previous studies found brie y described in European literature (BLÄHSER, more evidence of radiologic abnormalities in elbow, 1962; JOSHUA, 1965; WILKINSON, 1966). The disor- hip and sti e than in tarsus, carpus and shoulder der was subsequently shown to be a speci c disease (GODFREY, 2005; CLARKE and BENNETT, 2006; of cats and was named chronic progressive polyarth- ROE, 2006; LASCELLES, 2008). ritis (PEDERSEN et al., 1975). Two viral agents have been associated with joint The importance of feline OA is probably highly un- diseases in cats, namely the Feline Calicivirus (FCV) derestimated as cats show only mild clinical symp- and the Feline Foamy Virus (FFV). The importance of toms that are not easily recognized by owners or by the other feline retroviruses, Feline Leukaemia Virus veterinary practitioners. Changes in behaviour and (FeLV) and Feline Immunode ciency Virus (FIV), that level of activity such as reduced height jump, have been detected as coinfections in cats with OA is unwillingness to jump, a stiff gait and decreased currently unknown. Previous reports have suggested grooming (HARDIE, 1997; CLARKE and BENNETT, an association of FFV with feline polyarthropathy 2006; ROE, 2006; SLINGERLAND et al., 2011) are the (PEDERSEN et al., 1980) and a cofactor effect with

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FIV in naturally infected cats (ZENGER et al., 1993; (1980) isolated FFV from blood, synovial  uid or syno- WINKLER et al., 1999; GREENE, 2006). vial tissue in 83 % of cats with chronic progressive Feline Calicivirus (FCV, family Caliciviridae, genus polyarthritis; 80 % of these cats were also infected Vesivirus) is widespread especially in catteries and with FeLV. This may be a coincidence but may also households with more than one cat due to asympto- point towards a trigger function of one virus to the matic carriers that shed the virus continuously from other. the oropharynx (POVEY and JOHNSON, 1970; Similarly to FFV, FIV is also transmitted largely WALTON, 1971) over months and even years (POVEY through bite wounds, so is more prevalent in free et al., 1973). At any given time, about a fth of symp- ranging or sheltered cats than in cats kept in private tomatic or asymptomatic cats from different environ- homes. The part played by FIV in feline OA is unclear ments are virus shedders (HARBOUR et al., 1991; but, like FeLV, FIV may be a cofactor, as stated by TENORIO et al., 1991; MOCHIZUKI et al., 2000; BECKER et al. (1994). CAI et al., 2001). FCV is involved in causing upper Feline OA is suspected to be related to infections respiratory tract disease of the cat and is associated with FCV, FeLV, FIV and FFV. However, there is still no in particular with acute and chronic gingivitis/stomati- proof of a direct link between the occurrence of OA tis and oral ulceration (REUBEL et al., 1992; ZETNER and the expected viral infection. We now report a pilot et al., 2004, 2006). Furthermore, FCV has been linked study in a population of cats on the occurrence of to the ‘limping syndrome‘, a certain kind of lameness feline OA (based on radiological examination) and a observed following acute infection and viraemia. The possible association with the viruses mentioned disease is most likely immune complex related or above using highly sensitive molecular methods that connected with acute phase proteins, such as detect nucleic acids in blood samples and articular α1-acid-glycoprotein (BENNETT et al., 1989; tissues. HARTMANN and HEIN, 2008a,b), but the exact pa- thogenesis has not been clari ed. Lameness and Material and methods subsequent development of polyarthritis have been associated with both eld virus infection and FCV Animals vaccination (PEDERSEN et al., 1983). After FCV infec- tion/vaccination, acute lesions are evident, consisting A total of 54 cats were available for this study. The of deposition of brin on the synovial membrane sur- animals had been patients of the Clinic for Small face. Animals of the University of Veterinary Medicine Three feline retroviruses are known: FFV (subfamily Vienna, Austria, or a private practice (‘Tierklinik , genus Spumavirus) and the two Breitensee’) also in Vienna. They had been eutha- members of the subfamily , FeLV nized in the stage of terminal illness or had suffered (genus Gammaretrovirus) and FIV (genus ). acute trauma. No information about lameness was FFV is widespread in feline populations studied: available. 29 % in domestic cats in Vietnam (MIYAZAWA et al., The investigated group consisted of 44 European 1998), 30 % in domestic cats in the USA (PEDERSEN short hair, four European long hair, two British short et al., 1980) and 57 % in domestic cats in South Aus- hair and four Persian cats. The gender distribution tralia, with up to 73 % prevalence in cats aged nine was 21 female (eleven neutered) and 33 male (24 neu- years or more (WINKLER et al., 1999). The pathways tered) cats. All animals originated from Vienna or its of FFV transmission paths have not been conclusively surrounding areas. Age of the cats ranged from six identi ed but vertical transmission in utero as well as months to 22 years, with an average of 10.33 years, horizontal transmission through bite wounds are the and was unknown in two patients. Gender and the most probable ways of infection (GREENE, 2006; different age groups are shown in table 1. GERMAN et al., 2008). The importance of FFV is un- clear: the infection has been described as asympto- Radiographic images matic but FFV has been isolated from cats with poly- arthritis. Male cats between one and ve years of age After euthanasia, radiographic images of ve joints seem to be predisposed to this infection (PEDERSEN (shoulder, elbow, hip, sti e, hock joint) were taken et al., 1975, 1980; MOISE and CRISSMAN, 1982). bilaterally in two directions (transversal, sagittal). The PEDERSEN et al. (1980) described the onset of di- images of the hip and hock joints for cat no. 26 were sease as tendosynovitis and synovitis with subse- not available. The joints were positioned as described quent changes in the articular cartilage and periostal by WAIBL et al. (2004) and the images were evalua- bone. After three months, granulation tissue origina- ted for abnormalities. Based on the presence and ting from the in amed synovium of the joint capsule severity of radiographic changes consistent with erodes the subchondral bone at the joint margins and DJD, the abnormalities were characterized as mild, extends over the borders of the articular surfaces as moderate and severe, similarly to the grading system pannus (PEDERSEN et al., 1980). PEDERSEN et al. of MORGAN (1999). Signs of degenerative joint

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disease were de ned as an increase of subchondral Extraction of viral nucleic acids or periarticular bone density affecting the capsule, ligaments and tendons, or as joint surface damage, 140 µl of the synovial membrane lysate or the leu- reduced joint space, instability and bone destruction cocyte pellet served as template for nucleic acid (PEDERSEN et al., 1980; HARDIE et al., 2002). The extraction using a commercially available kit ndings were graded by at least two radiologists (QIAamp® Viral RNA Kit) as instructed by the manu- supervised by an expert. In addition, a weighted sum facturer. Negative controls consisting of the com- index was calculated using the sum of degenerative ponents of the kit were run together with the samp- joints weighted by the assigned grade. The term ‘sum les throughout sample preparation and extraction of signs‘ refers to this index. procedures. Extracts (60 µl) were stored at -20 °C until PCR analysis. Blood samples Real-time PCR assays for FCV, FeLV, FIV and FFV Blood samples (supplemented with EDTA) were collected in the course of (immediately after) The PCR mixture was prepared using the compo- euthanasia and stored at 4 °C until further analysis nents of a commercially available kit (SuperScript within 24 hours. TM III Platinum One-Step qRT-PCR System) in a re- action volume of 25 µl (2.5 µl template and 22.5 µl Isolation of peripheral blood mononuclear cells PCR mixture) using the cycling conditions instructed (PBMC) by the manufacturer and an annealing temperature of 60 °C. Blood samples were centrifuged at 3,400 g at 4 °C The real-time PCR assay for FeLV was carried out for 15 min. The buffy coat was incubated in erythro- using primers and probe described by TANDON et al. cyte lysis buffer (Buffer EL) on ice for 10 min and (2005), for FIV using the ones by PINCHES et al. (2007) centrifuged at 470 g at 4 °C for 10 min. The superna- and for FCV following instructions of LESCHNIK et al. tant was discarded and the washing step repeated (2007). until the pellet looked white to the naked eye. Finally, For the real-time PCR assay for FFV, primers and the pellet was incubated in 180 µl Buffer ATL and probe were designed using the program Primer Ex- 20 µl Proteinase K at 56 °C until complete lysis. press®, v2.0 and the GenBank reference sequence (www.ncbi.nlm.nih.gov) with the Acc.No. AJ564745. Detection of FIV specifi c antibodies The sequences are the following: forward primer (pos. 2641): 5‘-CGCAACCTCCTCGTGGAA-3‘, reverse pri- To detect FIV-speci c antibodies in plasma mer (pos. 2744): 5‘-TCGTCGGTACGGGTTTGGT-3‘, samples, a commercially available test (Witness FIV) probe: FAM-5‘-AATCCGCAGCAGCCTCAGCGC-3‘- was used according to the manufacturer’s TAMRA. PCR mixture and cycler scheme were used instructions. All blood samples were tested for FIV as described above. antibodies. Extracts from PBMCs were always analysed as in- dividual samples, whereas joint samples were pooled Synovial membrane samples (up to four joints from each cat) for FCV, FeLV and FFV PCR assays. If one of the pooled samples tested po- Surgical approaches to the joints (shoulder, elbow, sitive, all eleven extracts per cat were retested sepa- hip, sti e, hock joint; bilaterally) were carried out fol- rately. lowing the instructions of FOSSUM (2007) and PIER- The samples of synovial membranes of cats tested MATTEI and BRUMM (1996) immediately after taking positive for FIV antibodies were further tested for FIV the radiographic images. To minimize bleeding and speci c nucleic acids by PCR assay. the resulting contamination of the samples with blood, the access to the synovial membrane was per- Specifi city of the newly designed FFV specifi c formed to cause as little damage to the local blood PCR assay vessels as possible. Of each synovial membrane, a sample of approximately 4 mm3 was collected using Ampli cation products were extracted from the sterile scalpel, scissors and forceps and stored in Ep- PCR product after real-time PCR using a commer- pendorf tubes at -80 °C until further analysis. Samp- cially available kit (QIAquick PCR puri cation kit). les were homogenized using Proteinase K digestion The extracts were submitted to sequencing PCR as described above. Additionally, synovial membrane (BigDye Terminator Cycle sequencing kit) and the samples of the same size were stored in Eppendorf forward and reverse sequences were determined tubes containing formalin for histopathological exa- using the ABI Prism 310 Genetic Analyser. The alig- minations. The results of the histopathological exami- ned sequences were submitted to a blast search in nations were described by SCHACHNER (2010). the GenBank.

126 Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria 99 (2012)

Confi rmation of FFV positive samples by PCR periarticular margins. When the joints were checked for severity of DJD, the left and right shoulders as Samples positive for FFV by real-time PCR were well as the left and right hocks, were most frequent- retested using the primers FUV2610s and FUV3107a ly free of pathological changes (36, 32, 35 and 29 published by WINKLER et al. (1998) amplifying 498 bp joints, respectively). Mild signs of DJD were most of the gag/pol domain of the viral genome. The two frequently found in the left and right elbow and the samples having the lowest Ct-values re ecting the left and right hip joints (35, 35, 26 and 23 affected highest quantity of viral nucleic acid in the sample of joints, respectively). The left and right hip and the each cat were chosen for retesting. left and right sti e showed moderate signs of DJD (signs of grade 2) in 7, 6, 11 and 13 cases, respec- Statistics tively. The most severely affected joints were the left and right hip joints (six and eight joints, respectively). All statistical analyses were performed using The distribution of the radiographic changes in the SPSS v17 for Windows. Differences between virus- joints is shown in table 2 and gure 1. In general, positive and -negative cats, in their mean age and the mean sum of osteoarthrotical changes in their joints were analysed using t-tests for independent samples. The assumption of normal distribution was tested using Kolmogorov-Smirnov-Test. Differences bet- ween virus-positive and -negative cats in frequency distribution of blood negative/positive and sex were analysed using the Chi-test. Correlations between age and severity of degenerated joints were investi- gated using the nonparametric Spearman’s correlati- on. Probability values of p<0.05 were considered signi cant for all statistical analyses.

Source of supply Fig. 1: Severity of radiographic changes in the investigated joints ABI Prism 310 Genetic Analyser (Applied Biosystems, of 54 cats CA, USA); BigDye Terminator Cycle sequen- cing kit (Applied Biosystems, CA, USA); Buffer ATL most pathological changes were found bilaterally in (Qiagen, Hilden, Germany); Buffer EL (Qiagen, Hilden, the elbows (40 left and 43 right), the hip joints (39 Germany); Primer Express®, v2.0 (Applied Biosys- bilateral) and the sti es (39 left and 38 right). tems, CA, USA); Proteinase K (Qiagen, Hilden, Germany); QIAamp Viral RNA kit (Qiagen, Valencia, Detection of FIV antibodies in blood samples CA, USA); QIAquick®PCR puri cation Kit (Qiagen, Hilden, Germany); SuperScript III Platinum One-Step One blood sample tested positive for FIV (no. 57). Quantitative RT-PCR System (Invitrogen Corporation, Four samples (no. 5, 20, 33, 34) exhibited only a very Carlsbad, CA, USA); ViraCHEK/FeLV (Synbiotics faint change in colour in the diagnostic eld indicating Corp., MO, USA); Witness FIV (Synbiotics Corp., MO, a positive result, or a change in colour after the time USA) period in which the result has to be recorded accor- ding to the manufacturer had elapsed. Therefore Results these results were interpreted as equivocal. The other 49 blood samples tested negative. Radiographic images Real-time PCR assays for viral nucleic acids in Signs of DJD were evident in 48 (88.9 %) of the synovial membranes and PBMCs cats investigated. Only six cats showed no signs of DJD (No. 24, 25, 40, 51, 55, 56). One of these was Results of real-time PCR assays are shown in younger than one year (six months, female), three table 3. FCV speci c nucleic acids were detected were one year old (two female, one male), one was bilaterally in the sti e and shoulder, and in the right two years of age (male) and the oldest was seven hip of one cat (no. 3). (male). In one cat (no. 18), FeLV-specific nucleic acids The radiographic ndings demonstrated several were detected in the PBMCs and in all joints exa- kinds of morphological changes, such as diffuse mined except the left stifle and the left and right soft tissue swelling, subchondral bone erosion, nar- hip joints. Additionally, cat 18 was positive for rowing of the joint spaces, small enthesiophytes or FeLV antigen in plasma (ViraCHEK/FeLV, data not small osteophytes and slight roughening of the shown).

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FIV PCR gave a positive result in the PBMCs as well there was no signi cant difference in age between as in two joints (right hock and shoulder) of cat 57. In viral-positive (M=10.6; SD=5.0) and -negative (M=10.2; cat 5, FIV PCR gave a positive result in one joint. All SD=5.2) cats (p=0.848). The mean sum of signs of other samples of those cats, which had not clearly DJD between viral-positive (in blood and joints) tested negative for FIV antibodies (20, 33, 34), were (M=9.7; SD=6; Median=7.5; 1st quartile=6.25; 3rd quar- negative for FIV speci c nucleic acids. tile=9.5) and -negative cats (M=7.7; SD=4.9; Medi- FFV was detected in eight out of 54 cats (no. 10, 26, an=7; 1st quartile=4; 3rd quartile=12.5) did not differ 28, 32, 45, 49, 50 and 57). In one of them, it was found signi cantly (p=0.255; Fig. 3). Furthermore there were only in the PBMCs (no. 57), in four cats (no. 10, 26, 32, no differences between males and females among 45) it was detected in the PBMCs, as well as in three, viral positives (p=0.491) or the sum of signs (males: ve, one and all joints, respectively. In cats 28, 49 and M=8.18; SD=5.6; females: M=8.33; SD=4.6; 50, viral nucleic acids were evident in several joints. p=0.919) of DJD. The mean age of the cats that tested positive for FFV There was no signi cant age difference between was 13.4 years and the group consisted of six neute- FFV-infected (M=13.3; SD=2.7) and non-infected red males (no. 28, 32, 45, 49, 50, 57) and two females (M=9.9; SD=5.4) cats (p=0.114) and no difference in (no. 10, 26). gender (p=0.152).

Specifi city of the designed real-time PCR assay Discussion for FFV The occurrence of signs of DJD in radiographic ima- The sequence of the ampli cation product obtained ges and viral infection with FCV, FFV, FeLV and FIV in from the left hock of cat 45 (110 bp) after real-time ten joints and blood samples of each of 54 cats was PCR for FFV was submitted to a blast search in the investigated. The cats were picked at random and had GenBank and showed identities of 97–98 % with se- been put down for various reasons: some were in the veral sequences available (AJ223851, AB052796, terminal stages of illness and others had suffered an Y08851, X98741, AJ564745, U85043, AB052798, acute trauma, so the sample study did not solely com- AB052797). prise old cats. The mean age of the 54 cats was 10.33 years. No information about lameness or behavioural Confi rmation of FFV positive samples by PCR changes was available. To identify signs of DJD we evaluated radiographic images of ten synovial joints of In ve (no. 10, 26, 28, 45, 49) of the cats that tested each patient (with the exception of one cat). To avoid positive for FFV by real-time PCR the result could be intra- and inter-observer variation, the images were al- con rmed in one or both samples analysed by PCR ways evaluated by the same persons supervised by an amplifying 498 bp of the viral genome (WINKLER et experienced radiologist. Only 11.1 % of the patients in al., 1998). this study were free of radiographic signs for DJD. This is similar to the ndings of HARDIE et al. (2002) where Comparison of the results of radiological and only 10 % of the investigated cats (n=100) were free of virological investigations of the joints any signs of DJD, but a lower percentage than described by SLINGERLAND et al. (2011), who identi- Refer to table 3 for results of real-time PCR assays as ed 39 % of cats without radiological changes. The well as severity of radiographic changes in the joints. most commonly affected joint was the elbow, which is All cats that tested positive for one or more of the similar to the results of HARDIE et al. (2002) and four viral agents showed pathological changes in at SLINGERLAND et al. (2011). The most severe changes least one joint on the radiographic images. Both cats were evident in the hip joints, whereas SLINGERLAND (no. 5, 57) positive for FIV showed mild to moderate et al. (2011) observed the highest frequency of severe signs of DJD in the positive joints (except right hock changes in the elbows. In contrast, HARDIE et al. of cat no. 57). Five out of seven FeLV-positive joints of (2002) reported that the hip joints examined did not cat 18 showed signs of DJD. 16.7 % of the cats with exhibit DJD changes of grade 2 or 3. radiographically identi ed osteoarthrotic lesions were The histopathological examination of the samples of FFV-positive. 23 out of 35 FFV-positive joints showed synovial membranes used in this study showed that the different stages of severity of DJD. tarsal joint was most commonly affected, followed by the elbow, sti e, shoulder and hip (SCHACHNER, 2010). Statistics This nding is not in accordance with the results of the radiological investigations, which showed the hip as the The correlation between age and sum of signs of most affected joint. Nevertheless, the ranking of the re- DJD was signi cant (r=0.475; p=0.004; Fig. 2). There mainder of the joints was similar. was no signi cant correlation between age and Although even the younger patients showed a wide severity of degenerated joints (p=0.646). Additionally, range of different stages of DJD, there was a

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signi cant correlation between increasing age of the with FFV than in FFV-negative cats (CARRO, 1994). In cats and osteoarthrotical changes. This is in ac- the present study 16.7 % of the cats with radiographi- cordance with previous studies (HARDIE et al., 2002; cally identi ed osteoarthrotic lesions were FFV positi- GODFREY, 2005; CLARKE and BENNETT, 2006; ve. This is a much lower percentage than that descri- SLINGERLAND et al., 2011). bed by PEDERSEN et al. (1980), who detected FFV in The occurrence of DJD may be associated with in- 83 % of cats with chronic progressive polyarthritis. creased body mass, as it is known for humans, mice, The age of the FFV-positive cats ranged from 10 to 17 guinea pigs and companion animals (GERMAN, 2006; years, which is much older than the FFV-positive cats GRIFFIN and GUILAK, 2008; KEALY et al., 2000). As described by PEDERSEN et al. (1980) with an age no data on body mass was available for the cats pre- range from one and a half to ve years. In our study, sented in this study, this parameter could not be eva- FFV-positive cats seem generally to be older than luated. FFV-negative cats, although the difference was not In eleven out of 54 cats, nucleic acids of at least one statistically signi cant (p=0.114). Six of the FFV-posi- virus could be detected in at least one sample. Be- tive cats were neutered males and two were females. cause of the precautionary measures taken, it seems No signi cant correlation between the gender of cats unlikely that positive results were missed because of and the detection of FFV was found, possibly resul- pooling. However, it cannot be excluded completely ting from the small sample size. Nevertheless, these that very weakly positive samples were missed. In two aspects should be taken into account in future any case, the percentage of cats positive for at least studies. one of the viruses investigated is rather high (20.4 %). An interaction between FFV, FIV and FeLV has been Although blood contamination was avoided as far as hypothesized by PEDERSEN et al. (1980), ZENGER et possible while collecting the synovial membrane al. (1993) and BECKER et al. (1994). There is frequent samples, it cannot be completely excluded. However, co-infection with FFV and FIV, as both viruses are as no viral nucleic acids could be detected in the transmitted through bite wounds (GREENE, 2006). In blood cells of ve of the cats with positive results in our study a co-infection of FFV and FIV was detected the joints, the possibility of contamination seems un- in only one cat. The overall seroprevalence of FIV was likely, at least for these animals. only 1.9 %, which is lower than the (serological) pre- A number of studies on FCV have found a correlati- valence in a preselected population in Austria (4.6 % on between virus infection and the onset of lameness of 263 sera, sampled from 1.1.2008 to 31.3.2011, in cats (CRANDELL and MADIN, 1960; PEDERSEN et Clinical , University of Veterinary Medicine Vi- al., 1983; BENNETT et al., 1989; CHURCH, 1989; enna; personal communication). LEVY and MARSH, 1992; DAWSON et al., 1994). For FeLV, PEDERSEN et al. (1980) described the in- LEVY and MARSH (1992) documented one case of a cidence of polyarthritis to be four to 60 times higher in with severe signs of lameness associated with infected cats compared to FeLV-negative cats. Of our administering a vaccine containing modi ed-live patients, cat no. 18 was the only one tested positive Calicivirus. In the present study, FCV-speci c nucleic for FeLV and was obviously viraemic. Not only was acids were detected in only one cat (in ve joints). Vi- the FeLV speci c nucleic acid detected in the PBMC; ral antigen has previuosly been detected in synovial viral nucleic acids could also be detected in seven macrophages in ve cats (BENNETT et al., 1989). joints. No co-infection with FFV was detected, which Nevertheless, calicivirus itself as a primary cause for has been hypothesized to increase the pathogenic the development of arthritis needs further research. effect of FFV (PEDERSEN et al., 1980). The real-time PCR assay established in this study PEDERSEN et al. (1980) stated that in cats suffe- was able to detect FFV-speci c nucleic acids, as ring from OA the lower appendicular joints (carpus shown by sequencing of an ampli cation product and and hock joints) were primarily affected, which by verifying positive results with published PCR seems to be characteristic for a viral aetiology of os- assay. The positive real-time PCR result could be teoarthritis in the early stages (PEDERSEN et al., proven in ve of the eight positive cats, verifying the 1980; BENNETT et al., 1989; CARRO, 1994). In con- speci city of the real-time PCR assay in these samp- trast, the most affected joints in the present study les. The reason why three samples were not detected were the elbows and hips, which were affected bila- positive by conventional PCR may be related to the terally. However, the carpal joints were not conside- higher sensitivity of real-time PCR. Using real-time red in our investigation. PCR assay for FFV, speci c nucleic acid was The PCR assays used and the newly established detected in blood samples as well as in synovial tis- FFV real-time PCR assay are promising tools for sue of eight cats. Case studies have described the further investigations of the etiologic role of viruses onset of lameness in neutered male cats seropositive in the development of OA in cats. In the present pilot for FFV (BECKER et al., 1994; FELDMAN, 1994). The study we were able to detect all viruses investigated incidence of chronic progressive polyarthritis was de- in synovial membrane samples, with FFV having the scribed to be two to four times higher in cats infected highest prevalence. Further research will be needed

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to clarify the role of the retroviruses in osteoarthriti- Acknowledgements cal changes, which occur in cats with a remarkable We should like to thank Dr Britta Vidoni for giving a brie- frequency. ng in performing the surgical approaches to the joints.

Fig. 2: Correlation between age and sum of signs (weighted sum Fig. 3: Sum of signs (weighted sum index calculated using the index calculated using the sum of degenerative joints weighted by sum of degenerative joints weighted by the assigned grade) of de- the assigned grade) of degenerative joint disease (DJD), marked generative joint disease (DJD) for virus-positive (n=11) and virus- by virus-positive (n=11) and virus-negative cats (n=43) negative cats (n=43)

Tab. 1: Gender-dependent distribution of the age groups (age in years) and the percentage of each age group compared to cats examined male female age group male female percentage neutered neutered <1–3 2 1 2 1 11.1 4–6 0 3 1 2 11.1 7–9 2 3 1 1 13.0 10–15 3 12 4 4 42.6 16–18 1 4 1 1 13.0 >18 0 1 0 2 5.6 unknown 1 0 1 0 3.7 average age* 8.6 11.3 9.2 11

*The average age is the mean value over all age groups shown in the table.

Tab. 2: Distribution of the radiographic changes in the investigated joints of 54 cats (in total numbers and %) no changes mild moderate severe number of joint (n; %) (n; %) (n; %) (n; %) examined joints shoulder left 36; 66.7 14; 25.9 2; 3.7 2; 3.7 54 shoulder right 32; 59.3 18; 33.3 2; 3.7 2; 3.7 54 elbow left 14; 25.9 35; 64.8 3; 5.6 2; 3.7 54 elbow right 11; 20.4 35; 64.8 5; 9.3 3; 5.6 54 hip left* 14; 26.4 26; 49.1 7; 13.2 6; 11.3 53 hip right* 14; 26.4 23; 43.4 6; 11.3 10; 18.9 53 sti e left 15; 27.8 25; 46.3 11; 20.4 3; 5.6 54 sti e right 16; 29.6 22; 40.7 13; 24.1 3; 5.6 54 hock left* 35; 66.0 11; 20.8 5; 9.4 2; 9.4 53 hock right* 29; 54.7 20; 37.7 3; 5.7 1; 1.9 53

* radiographic images of the hips and hocks were not available for cat 26.

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Tab. 3: Results of FeLV antigen and FIV antibody detection and of real-time PCR assays as well as severity of radiographic changes in the joints plasma (Fast test) PBMC shoulder elbow hip sti e hock shoulder elbow hip sti e hock FeLV, FIV left left left left left right right right right right

Cat 3 /0 0 3 /0 0 /0 0 /3 /0 0 m, 0,75 y ◆ ◆ ◆ ◆ ◆ Cat 5 m, ?? ◼* 0 1 1 2 0 1 1 1 ◼/2 1

Cat 10 0 1 1 2 3 2 /1 /1 2 /2 f, 14 y ▲ ▲ ▲ ▲ Cat 18 f, ?? ● ● ●/0 ●/1 1 1 ●/0 ●/1 ●/1 1 ●/1 ●/1

Cat 20 1 0 1 3 1 1 1 1 3 1 fn, 5 y ◼*

Cat 26 /2 /1 ? 0 /? /1 1 ? 0 /? f, 15 y ▲ ▲ ▲ ▲ ▲ ▲

Cat 28 /0 /1 /1 /1 /0 /0 /1 /1 /1 /1 mn, 11 y ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲

Cat 32 0 1 1 1 0 /0 1 1 1 0 mn, 12 y ▲ ▲

Cat 33 0 0 1 2 0 0 1 1 2 1 mn, 5 y ◼*

Cat 34 1 1 2 0 1 1 1 2 0 1 mn, 13 y ◼*

Cat 45 /0 /0 /1 /2 /0 /0 /1 /1 /2 /0 mn, 16 y ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲

Cat 49 /3 3 2 3 3 3 /3 2 3 /3 mn, 17 y ▲ ▲ ▲

Cat 50 /1 /2 1 1 1 /0 2 3 1 1 mn,12 y ▲ ▲ ▲

Cat 57 1 1 0 1 0 1 1 1 mn, 10 y ◼ ◼▲ ◼/1 ◼/0 ◼ = FIV positive; ◼* = FIV equivocal result; ● = FeLV positive; ◆ = FCV positive; ▲ = FFV positive; severity of changes in radiographic images: 0 = no signs; 1 = mild; 2 = moderate; 3 = severe; ? = radiographic images of the hips and hocks were not available; m = male, f = female, fn = female neutered, mn = male neutered; y = years of age

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Epidemiology of Feline Foamy Virus and Feline Immuno- Katze. Prakt Tierarzt 85, 798–807. de ciency Virus infections in domestic and feral cats: a ZETNER, K., STOIAN, C., BENETKA, V., MÖSTL, K., seroepidemiological study. J Clin Microbiol 37, 2848–2851. GROISS, S., SAALMÜLLER, A. (2006): Klinische Ergeb- ZENGER, E., BROWN, W.C., SONG, W., WOLF, A.M., nisse einer neuen Therapiemöglichkeit der chronischen PEDERSEN, N.C., LONGNECKER, M., LI, J., COLLIS- Gingivostomatitis der Katze mittels eines Immunmodula- SON, E.W. (1993): Evaluation of cofactor effect of feline tors (Zylexis®). Prakt Tierarzt 87, 678–687. syncytium-forming virus on feline immunode ciency vi- rus infection. Am J Vet Res 54, 713–718. Corresponding author’s address: ZETNER, K., STOIAN, C., BENETKA, V., KLEIN, D., MÖSTL, Prof. Dr. Karin Möstl, K. (2004): Der Ein uss von rekombinantem, felinen Ome- Veterinärplatz 1, A-1210 Vienna. ga-Interferon auf die chronische Gingivostomatitis der e-mail: [email protected]

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