Dogs with canine rheumatoid arthritis had signifi- cantly elevated levels of antibodies to canine distemper virus. This increase was particularly seen in.
Researchin VeterinaryScience1991, 50, 64-68
Canine distemper viral antigens and antibodies in dogs with rheumatoid arthritis S. C. BELL, S. D. CARTER, D. BENNETT, Veterinary Immunology, Departments o f Veterinary Pathology and Veterinary Clinical Science, University o f Liverpool, PO Box 147, Liverpool L69 3 B X
Dogs with canine rheumatoid arthritis had significantly elevated levels of antibodies to canine distemper virus. This increase was particularly seen in the synovial fluids, compared with paired sera, and was not found in dogs with infective arthropathies, osteoarthritis or in osteoarthritis secondary to rupture of the cranial cruciate ligament. Analysis of the immune complexes precipitated from synovial fluids showed immunoglobulins in all types of arthropathy. Western blotting analyses showed reactivity with antidistemper antisera in immune complexes from dogs with rheumatoid arthritis, but not in immune complexes from dogs with other joint diseases. These results suggest that there are increased immune responses to distemper in canine arthritis and that these may be due to the presence of this paramyxovirus in affected joints. The implications for the role of a possible infectious agent in rheumatoid arthritis in the dog are considerable.
al 1989) and collagen autoantibodies detected (Bari et al 1989). In both man and dog, the aetiology is unknown, although many agents, primarily infective, have been implicated. As the human disease often follows viral infection or vaccination (Phillips 1986), it was decided to investigate the possible involvement of viruses in canine joint diseases. A number of viruses have been implicated in human rheumatoid disease, including Epstein Barr virus (Catalano et al 1979), parvovirus (Cohen et al 1986), rubella (Tingle et al 1986) and a paramyxovirus, measles (Shirodaria et al 1979). Dogs undergo substantial exposure to another paramyxovirus, canine distemper virus (¢DV) which, like measles, is also a morbillivirus. Most dogs are exposed to this virus by either becoming infected or vaccinated and hence it is a suitable ubiquitous agent which could, in susceptible hosts, trigger mechanisms leading to disease. Consequently, a study was made to determine any involvement of c o y in canine rheumatoid arthritis.
ESTABLISHED criteria have allowed joint diseases in dogs to be classified into two main categories: degenerative joint disease (osteoarthritis) and the inflammatory arthropathies, the latter being subdivided further to include bacterial infective arthritis (Bennett and Taylor 1987) and immune-based arthritis (erosive and non-erosive forms) (Bennett 1987a,b,c). There are many similarities between human rheumatoid arthritis and its canine counterpart, including the distribution of age of onset, the symmetrical polyarthritis presentation of lameness, stiffness after rest and the involvement of mainly peripheral joints, which show swelling and pain. The course of canine rheumatoid arthritis is progressive and may lead to severe joint destruction and deformities. Auto-immune mechanisms have been shown in canine rheumatoid arthritis; rheumatoid factors and immune complexes were found (Carter et
Materials and methods
Dogs All the dogs with joint disorders were second opinion referral cases to the Small Animal Hospital, Liverpool University. The various joint diseases were categorised according to established criteria (Bennett 1987a,c,d, Bennett and Kelly 1987). The main divisions were (i) inflammatory arthropathies which included erosive and non-erosive forms (these are the canine rheumatoid arthritis cases), (ii) osteoarthritis, (iii) osteoarthritis secondary to cranial cruciate ligament rupture and (iv) infective arthropathies. In addition, a 'normal' population of dogs was sampled. This consisted of dogs which were presented to the hospital for reasons other than locomotory problems and which showed no evidence of articular disorders. Also, beagles from a breeding colony were taken at
Reprint requeststo Dr S. D. Carter 64
Distemper in canine arthritis necropsy as a source of 'normal' synovial fluids and sera.
65
Statistics Data were analysed by Student's t test and withindisease categories by the paired t test.
Sera and synovial fluids Serum was obtained from clotted blood and stored at - 2 0 ° C until required. Synovial fluids were aspirated aseptically from the joint, centrifuged to remove cells and debris and stored at - 2 0 ° C .
Measurement of anti-cDV antibodies Anti-cDv antibodies were detected in sera and synovial fluids by an ELISA technique. Virus was obtained from a CDV vaccine (Intervet) by salination to 330 mM sodium chloride, precipitation with polyethylene glycol 6000 (7 per cent) at 4°C and centrifugation at 3000 g. Viral proteins Were obtained by disruption with freezing and thawing in 15 mM sodium chloride and stripping of the surface proteins by a non-ionic detergent (Nonidet P40) treatment at 37°C for three hours. The unseparated viral constituents were dialysed against phosphate buffered saline (PBS) and stored at - 2 0 ° C . Aliquots were bound to a 96-well ELISAplate (Flow Laboratories) at 10/zg ml -~ in PBS and then serum or synovial fluid (10 -2) added before incubation at 37°C for one hour. Unbound molecules were washed off with PBS/ Tween (0-05 per cent). Alkaline phosphataseconjugated rabbit anti-dog Ig (10 -3, Sigma) was added (37°C for one hour), followed by the substrate, p-nitrophenyl phosphate. Serum from an unvaccinated, 10-week-old puppy provided a negative control and a serum from a high CDV-titre dog were both included with each plate as internal controls. It was necessary to establish whether the ELISA was effective and correlated with the more frequently used assay for immune responses to CDV, the virus neutralising test. Thus, some sera were tested in parallel in both systems.
Results
Anti-cDv antibodies Antibodies to CDV were detected in the sera and synovial fluids (Table 1) of 'normal' dogs and dogs presenting with different joint disorders. However, it was only in the synovial fluids of dogs with rheumatoid arthritis that there was a significant increase in the percentage of specific CDv-antibodies (88 per cent) compared to the control animals. That these antibodies were produced within (or specifically localised to) the joint and did not arise by diffusion from the serum can be demonstrated by the ratios of the specific anti-CDV antibodies in the serum and synovial fluid from the same dog. In a control experiment, the CDv-free tissue culture supernatant, from the cell line used to generate the vaccine, was treated as for the vaccine and used as antigen in the ELISA, no dog sera reacted, even at low dilution (data not shown). ELISA plates coated with measles virus components were also used to detect any possible crossreactivity with other virus responses. Only one dog ('normal') out of 32 reacted and then at a very low level (data not shown). The ELISAdata were shown to correlate well with the serum neutralisation titres, suggesting that they primarily identified the same antigen (Fig 1).
Immune complex analysis Coomassie blue staining of immune complexes separated by PAGE showed many constituents (Fig 2). In all cases, the major proteins were immunoglobulins, mainly IgG, as shown by the bands at 52 kD (heavy chain) and 26 kD (light chain). Increasing the protein loading resulted in a high number of minor
Distemper antigens in immune complexes Immune complexes were precipitated from synovial fluids with 2- 5 per cent polyethylene glycol (Carter et al 1984, 1989). The precipitated proteins were separated, under reducing conditions, by polyacrylamide gel electrophoresis (PAGE)in vertical gels of 5 to 15 per cent acrylamide (Laemmli 1970). Duplicate gels were either stained with Coomassie blue or electroblotted on to cellulose acetate paper. The transferred proteins were probed with rabbit antisera to CDV and parvovirus and developed with alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma) and the substrate (o-dianisidine and /3naphthyl acid phosphate).
TABLE 1: Anti-CDV antibodies in dog sera and synovial fluids (ELISA: data = absorbance at OD405nm) Control
Infectious arthritis
Cruciate
0.934 0.246 11
O'826 0"O67 3
0-631 O. 272 20
0"970 O-379 10
Synovial fluids Mean 0.389 SD 0" 127 n 27
0.458 0-264 4
0.482 0.266 25
0.731" 0"359 50
Sera Mean SD n
* P
