Prevalence of canine enteric coronavirus in a cross-sectional survey of dogs presenting at veterinary practices.

In order to determine the prevalence of canine enteric coronavirus (CECoV) in the general dog population, faecal samples were obtained in a cross-sectional study of 249 dogs presenting for any reason at veterinary practices randomly selected from across the UK. Demographic and clinical data was obtained for each of the samples, including signalment, number of dogs in the household, reason for visiting the practice, and any recent history of diarrhoea. The samples were tested by RT-PCR for the presence of both type I and type II CECoV. Seven samples were positive (three from dogs in the same household), a prevalence of 2.8% (95% confidence intervals 1.1-5.7). Phylogenetic analysis of partial M gene sequences revealed that all seven positive samples grouped with type I CECoV, the first report of this virus in the UK. None of the positive dogs presented for gastrointestinal disease. Interestingly five of the positive dogs from three separate households were aged over 6 years, suggesting that older dogs may play an important role in the persistence of CECoV in such populations.

Prevalence of Campylobacter spp. in a cross-sectional study of dogs attending veterinary practices in the UK and risk indicators associated with shedding.

Campylobacteriosis is a major cause of gastroenteritis in humans and some studies have suggested that dog ownership is a risk factor for the condition. To determine the prevalence, species distribution, and risk indicators for Campylobacter spp. infecting dogs attending veterinary practices in UK, faecal samples were collected in a cross-sectional study from 249 dogs with and without clinical signs. The prevalence of Campylobacter spp. was 38% (95% CI 32, 44), with Campylobacter upsaliensis accounting for 94 (98%) of the isolates and Campylobacter jejuni for the remainder. Multivariable analysis indicated that younger dogs were more likely to carry C. upsaliensis and the high prevalence of this pathogen supports the hypothesis that dogs, particularly younger animals, may be an important source of C. upsaliensis infection for humans. However the prevalence of C. jejuni, the most common Campylobacter spp. associated with disease in humans, was low (1.2%, 95% CI 0.3, 3).

Brain copper elevation and neurological changes in north ronaldsay sheep: a model for neurodegenerative disease?

This study in North Ronaldsay (NR) sheep showed that copper was elevated in both the liver and brain of older animals and that the elevation in these two sites was highly correlated. The accumulation of copper in the liver culminated in chronic active hepatitis. Evidence of tissue damage in the brain was equivocal, but the astrocytes showed strong immunoreactivity for metallothionein. The study suggested that the blood-brain barrier of NR sheep possesses unusual features in respect of the import of copper into the brain, and that NR sheep may provide a useful animal model for the investigation of brain copper homeostasis.

The use of reference strand-mediated conformational analysis for the study of cheetah (Acinonyx jubatus) feline leucocyte antigen class II DRB polymorphisms.

There is now considerable evidence to suggest the cheetah (Acinonyx jubatus) has limited genetic diversity. However, the extent of this and its significance to the fitness of the cheetah population, both in the wild and captivity, is the subject of some debate. This reflects the difficulty associated with establishing a direct link between low variability at biologically significant loci and deleterious aspects of phenotype in this, and other, species. Attempts to study one such region, the feline leucocyte antigen (FLA), are hampered by a general reliance on cloning and sequencing which is expensive, labour-intensive, subject to PCR artefact and always likely to underestimate true variability. In this study we have applied reference strand-mediated conformational analysis (RSCA) to determine the FLA-DRB phenotypes of 25 cheetahs. This technique was rapid, repeatable and less prone to polymerase chain reaction (PCR)-induced sequence artefacts associated with cloning. Individual cheetahs were shown to have up to three FLA-DRB genes. A total of five alleles were identified (DRB*ha14-17 and DRB*gd01) distributed among four genotypes. Fifteen cheetahs were DRB*ha14/ha15/ha16/ha17, three were DRB*ha15/ha16/ha17, six were DRB*ha14/ha16/ha17 and one was DRB*ha14/ha15/ha16/ha17/gd01. Sequence analysis of DRB*gd01 suggested it was a recombinant of DRB*ha16 and DRB*ha17. Generation of new alleles is difficult to document, and the clear demonstration of such an event is unusual. This study confirms further the limited genetic variability of the cheetah at a biologically significant region. RSCA will facilitate large-scale studies that will be needed to correlate genetic diversity at such loci with population fitness in the cheetah and other species.

Resolution of complex feline leukocyte antigen DRB loci by reference strand-mediated conformational analysis (RSCA).

The DRB genes of the domestic cat are highly polymorphic. Studies based on clonal sequence analysis have suggested the existence of two distinct loci within individual animals and good evidence for 24 distinct FLA-DRB alleles. This variability, the complexity of clonal sequence analysis and its susceptibility to PCR-induced artefacts has represented a bottleneck to further progress. In this study we have applied reference strand-mediated conformational analysis (RSCA) to FLA-DRB. This protocol has been shown to be highly reproducible. Using five reference strands including two derived from non-domestic felines, we could distinguish 23 FLA-DRB alleles. We used RSCA to explore genetic polymorphism of FLA-DRB in 71 cats including 31 for which clonal sequence analysis was also available. On average, RSCA identified 0.9 more alleles within cats than clonal sequence analysis. Reference strand-mediated conformational analysis was also able to identify animals containing new alleles that could be targeted for sequence analysis. Analysis of allele patterns showed clear evidence for different allele distributions between breeds of cats, and suggested the Burmese breed may have highly restricted FLA-DRB polymorphism. Results from two families provided clear evidence for variation in the number of DRB genes on different haplotypes, with some haplotypes carrying two genes and some containing three. This study highlights the utility of RSCA for the resolution of complex amplicons containing up to six distinct alleles. A simple, rapid method for characterizing FLA-DRB makes possible studies on vaccine response and susceptibility/resistance to viral infections, which are a significant clinical problem in cats.

Molecular analysis of isolates of feline calicivirus from a population of cats in a rescue shelter.

Two visits, six weeks apart, were made to a cat rescue shelter and single oropharyngeal swabs were taken from all the compliant cats. Feline calicivirus was isolated from 14 of 45 swabs (31 per cent) taken on the first visit and 12 of 46 swabs (26 per cent) taken on the second visit. Nucleotide sequences were obtained for nine isolates from the first visit, six isolates from the second visit, and for the vaccine virus used in the cattery. Distance analysis showed that the majority of the isolates could be assigned to one of two groups. All the isolates obtained from cats sharing the same pen or isolates obtained from the same cat on successive visits, were less than 5 per cent distant, whereas most of the isolates from cats in different pens were more than 20 per cent distant. Phylogenetic analysis showed that at least seven distinct field isolates were present in the cattery. The only good evidence for virus transmission within the cattery was a case in which two viruses isolated from cats in different pens had sequences that were less than 5 per cent distant.

Endemic infection of a cat colony with a feline calicivirus closely related to an isolate used in live attenuated vaccines.

We have typed three feline calicivirus (FCV) isolates obtained over a 5-month-period from an endemically infected cat colony. Sequence analysis from variable region E of the capsid gene from these isolates strongly suggests they are minor variants of a single FCV strain, and that this strain is closely related to the one used in many live-attenuated FCV vaccines. Such a vaccine was last used approximately 2 months before the first of the isolates in this study was obtained. Sequence differences between the 'colony isolate' and the vaccine virus suggest that the colony virus has evolved from the vaccine virus and was persisting in the colony. The extent to which vaccine virus may contribute to the continued high prevalence of FCV needs to be determined.

Comparison of serological and sequence-based methods for typing feline calcivirus isolates from vaccine failures.

Feline calicivirus (FCV) can be typed by exploiting antigenic differences between isolates or, more recently, by the sequence analysis of a hypervariable region of the virus's capsid gene. These two methods were used to characterise FCV isolates from 20 vaccine failures which occurred after the use of a commercial, live-attenuated vaccine. Using virus neutralisation, the isolates showed a spectrum of relatedness to the vaccine; depending on the criterion adopted for identity, 10 to 40 per cent of them appeared to be similar to the vaccine virus. Using sequence analysis, the isolates fell into one of two categories; 20 per cent had a similar sequence to the vaccine (0-67 to 2-67 per cent distant), and the remainder had a dissimilar sequence (21-3 to 36-0 per cent distant). Sequence analysis identified one cat that appeared to be infected with two distinct FCVs. The serological and sequence-based typing methods gave the same result in 80 to 95 per cent of individual cases, depending on the criterion adopted for serological identity. It is suggested that molecular typing is a more definitive method for characterising the relatedness of FCV isolates.

Investigation of vaccine reactions and breakdowns after feline calicivirus vaccination.

The clinical and epidemiological features of feline calicivirus associated vaccine reactions and breakdowns were investigated. Twenty per cent of 123 vaccine reactions were associated with acute oral/respiratory disease alone, and 80 per cent were associated with lameness either alone or in association with other clinical signs. Feline calicivirus was isolated from oropharyngeal swabs from 69 per cent of the vaccine reaction cases. Twenty-four of 31 vaccine breakdowns were associated with acute oral/respiratory disease and only seven with lameness; the virus was isolated from 28 of the 31 breakdowns. Some of the viruses isolated from the vaccine reactions were compared with the appropriate vaccine virus in virus neutralisation tests; the majority appeared to be different from the vaccine virus and were presumably field viruses. However, some appeared more similar to the vaccine virus and the majority of these were associated with lameness after the first vaccination.

Biological changes in the annulus fibrosus in patients with low-back pain.

The anterior annulus from patients undergoing surgery for low-back pain was compared with the same region removed at autopsy. Collagen, proteoglycan, and water content were measured in different regions across the annulus. Water and proteoglycan contents increased from the outer to the inner annulus, whereas the collagen contents declined. Quantitative differences were found between agematched surgical and autopsy specimens. Discs from most patients showed lowered proteoglycan contents, and a few of these showed high water contents. Loss of collagen in isolated areas of the annulus was demonstrated in some patients. Collagen loss occurred most frequently in the lumbosacral disc, whereas changes in proteoglycan and water were more frequent higher in the lumbar region. No qualitative changes in collagen types were detected immunochemically. In many patients, the levels of proteoglycan which aggregated in vitro were extremely low. Changes in hexosamine molar ratio of disc proteoglycan and in the length of the chondroitin sulfate side chains were observed with aging in this group of patients.

Anti-collagen antibodies in sera from rheumatoid arthritis patients.

Anti-cartilage antibodies, demonstrable by immunofluorescence, were found in 3.3% of rheumatoid arthritis patients. In most of these patients antibodies to type II collagen were detected. In specificity studies on these anti-collagen antibodies, they appeared to be type specific, showing no reaction with collagen types I and III. Denatured type II collagen reacted much less well than native type II, but isolated peptides from different regions of the collagen molecule were differentiated by individual sera. Removal of the glycoside side chains from native type II collagen had no effect on its antigenicity. The findings suggest that these patients produce highly specific antibodies which react with the triple helix of type II collagen.

Immunochemical localization of collagen types and proteoglycan in pig intervertebral discs.

Antisera were produced which recognized specifically native type I and type II collagens and proteoglycan. These were used in immunofluorescence studies to investigate the distribution of collagens and proteoglycan in intervertebral discs from adult and newborn pigs. Cervical, thoracic and lumbar discs gave similar staining patterns. In the adult, the outer 1 mm of the annulus fibrosus resembled a perichondrium and was negative for type II collagen. The inner regions of the annulus contained proteoglycan and both types of collagen, but these molecules appeared to have separate distributions. The nucleus showed no staining for type I collagen. Newborn pig discs differed from those of the adult in that type II collagen was restricted to the central notochord and to a narrow zone surrounding it. The newborn annulus was negative for type II collagen but reacted strongly with antibodies to both type I collagen and proteoglycan. It is suggested that during development of the pig annulus fibrosus, cells producing type II collagen may migrate into this area from the central regions.

Anomalous reactions in the haemagglutination assay for anti-collagen antibodies: studies on patients with rheumatoid arthritis or chronic low back pain.

Sera from 23 patients with chronic low back pain, 20 rheumatoid patients and 16 normal controls were tested for antibodies to collagen types I, II and III, both native and denatured, by haemagglutination. Weak reactions against denatured collagen types I and II were found in 30-40% of the sera. Sera from individuals with rheumatoid arthritis or chronic low back pain behaved similarly, while only one normal serum showed any positive reaction. Reactions to denatured collagen type III and to native collagen of all 3 types were largely negative. Non-antibody serum components were thought to be responsible for these haemagglutination reactions since weakly positive reactions were abolished by cryoprecipitation and could not be confirmed by a solid-phase fluorimetric assay. Using the latter technique sera from 62 rheumatoid patients were screened for antibodies to type II collagen (native and denatured) and only one positive serum found. We conclude that haemagglutination is subject to false positive reactions and that the incidence of anticollagen antibodies in sera from patients with rheumatoid arthritis or chronic low back pain is low.