Shelter-housed cats show no evidence of faecal shedding of canine parvovirus DNA.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are deoxyriboncucleic acid (DNA) viruses in the taxon Carnivore protoparvovirus 1. Exposure of cats to either CPV or FPV results in productive infection and faecal shedding of virus. Asymptomatic shedding of CPVs by one-third of shelter-housed cats in a UK study suggests that cats may be an important reservoir for parvoviral disease in dogs. The aim of this cross-sectional study was to determine the prevalence of faecal shedding of CPVs in asymptomatic shelter-housed cats in Australia. Faecal samples (n=218) were collected from cats housed in three shelters receiving both cats and dogs, in Queensland and NSW. Molecular testing for Carnivore protoparvovirus 1 DNA was performed by polymerase chain reaction (PCR) amplification followed by DNA sequencing of the VP2 region to differentiate CPV from FPV. Carnivore protoparvovirus 1 DNA was detected in only four (1.8%, 95% confidence interval 0.49-4.53%) faecal samples from a single shelter. Sequencing identified all four positive samples as FPV. Faecal shedding of CPV by shelter-cats was not detected in this study. While the potential for cross-species transmission of CPV between cats and dogs is high, this study found no evidence of a role for cats in maintaining CPV in cat and dog populations through faecal shedding in the regions tested.

Advanced glycation endproducts in horses with insulin-induced laminitis.

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6h, 12h and 24h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.

The developmental and acute phases of insulin-induced laminitis involve minimal metalloproteinase activity.

Metalloproteinases have been implicated in the pathogenesis of equine laminitis and other inflammatory conditions, through their role in the degradation and remodelling of the extracellular matrix environment. Matrix metalloproteinases (MMPs) and their inhibitors are present in normal equine lamellae, with increased secretion and activation of some metalloproteinases reported in horses with laminitis associated with systemic inflammation. It is unknown whether these enzymes are involved in insulin-induced laminitis, which occurs without overt systemic inflammation. In this study, gene expression of MMP-2, MMP-9, MT1-MMP, ADAMTS-4 and TIMP-3 was determined in the lamellar tissue of normal control horses (n=4) and horses that developed laminitis after 48 h of induced hyperinsulinaemia (n=4), using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Protein concentrations of MMP-2 and MMP-9 were also examined using gelatin zymography in horses subject to prolonged hyperinsulinaemia for 6h (n=4), 12h (n=4), 24h (n=4) and 48 h (n=4), and in normal control horses (n=4). The only change in gene expression observed was an upregulation of MMP-9 (p<0.05) in horses that developed insulin-induced laminitis (48 h). Zymographical analysis showed an increase (p<0.05) in pro MMP-9 during the acute phase of laminitis (48 h), whereas pro MMP-2 was present in similar concentration in the tissue of all horses. Thus, MMP-2, MT1-MMP, TIMP-3 and ADAMTS-4 do not appear to play a significant role in the pathogenesis of insulin-induced laminitis. The increased expression of MMP-9 may be associated with the infiltration of inflammatory leukocytes, or may be a direct result of hyperinsulinaemia. The exact role of MMP-9 in basement membrane degradation in laminitis is uncertain as it appears to be present largely in the inactive form.

Equine laminitis: membrane type matrix metalloproteinase-1 (MMP-14) is involved in acute phase onset.

REASONS FOR PERFORMING STUDY: Enzymatic separation at the hoof lamellar dermal-epidermal interface may play a role in the development of laminitis and characterising and locating matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of MMPs or TIMPs) in lamellar tissues may further understanding of pathogenesis. OBJECTIVES: To clone and sequence the cDNA encoding lamellar MMP-14 and TIMP-2, and quantify their transcription in normal and laminitic tissue; and to develop antibody to locate MMP-14 in lamellar tissues. METHODS: Tissue samples were obtained from an oligofructose induced model of laminitis. Total RNA was isolated, amplified by RT-PCR, cloned into a vector and sequenced. Real-time PCR was used to quantify MMP-14 and TIMP-2 expression. Rabbit anti-equine MMP-14 antibody was developed to analyse MMP-14 proteins from hoof tissues. RESULTS: Immunohistochemistry detected MMP-14 in the cytoplasm of normal lamellar basal and parabasal cells in close proximity to the lamellar basement membrane. In laminitis affected tissue MMP-14 immunostaining was depleted in lamellar basal cells. Quantitative real-time PCR showed MMP-14 and TIMP-2 expression significantly (P<0.05) elevated and lowered respectively in laminitis affected tissues. CONCLUSION: MMP-14, located in the cytoplasm of normal lamellar basal cells, disappears during laminitis development. The pathology of laminitis is associated with increased and lowered transcription of MMP-14 and TIMP-2, respectively. POTENTIAL RELEVANCE: Enzymes have a role in laminitis pathology and inhibition of their activity may prevent laminitis.

Molecular subtyping of feline immunodeficiency virus from domestic cats in Australia.

OBJECTIVE: To determine the prevalent subtypes of feline immunodeficiency virus (FIV) present in the domestic cat population of Australia. METHOD: Blood samples were collected from 41 FIV antibody positive cats from four cities across Australia. Following DNA extraction, polymerase chain reaction (PCR) was performed to amplify the variable V3-V5 region of the envelope (env) gene. Genotypes were assessed by direct sequencing of PCR products and comparison with previously reported FIV sequences. Phylogenetic analysis allowed classification of the Australian sequences into the appropriate subtype. RESULTS: Of the 41 FIV samples, 40 were found to cluster with previously reported subtype A isolates, whilst the remaining sample grouped within subtype B. CONCLUSIONS: Subtype A was found to be the predominant FIV subtype present in Australia, although subtype B was also found. These results broaden our knowledge of the genetic diversity of FIV and the associated implications for preventative, diagnostic and therapeutic approaches.

Transmission of human and feline immunodeficiency viruses via reused suture material.

Several documented cases of human immunodeficiency virus (HIV) infection have involved unconventional or unknown modes of transmission of the virus. Some such cases have occurred within a surgical setting. We investigated the potential for transmission of HIV on suture material that had been reused following passage through an HIV-infected patient. Initial experiments were conducted in vitro using HIV. To provide stronger evidence that HIV could be transmitted via this route, further experiments were undertaken in vivo using a feline immunodeficiency virus (FIV)/cat model. Both methods indicated the possibility of transmission of virus if suture materials were reused.

Quasispecies and naturally occurring superinfection in feline immunodeficiency virus infection.

Analysis of individual clones containing the V1 and V2 domains of the segment of the FIV env gene present in a naturally infected cat (T) was carried out. The polymerase chain reaction (PCR) was used to amplify proviral FIV DNA extracted from peripheral blood mononuclear cells (PBMCs) obtained in October 1994 from this cat. The PCR products were cloned and the DNA sequences determined for 11 clones. Sequences obtained were aligned with sequences corresponding to FIV isolates (T90, T91, T92) previously obtained from the same cat in 1990, 1991 and 1992. Phylogenetic analysis was performed which included consensus sequences of another Australian isolate, N91, as well as UK, US, Swiss and Japanese isolates of FIV. All clones varied from each other, and none of these clones was identical to the consensus sequences of the isolates obtained previously from the same cat (the T-series). However, most of these clones appeared to have originated from the ancestor of the most recent isolate (T92). In addition, 2 of the clones (7&11) are closely related to another Australian isolate N91, obtained from a different cat (N) in 1991. Because these two cats (T and N) were housed together for at least 3 years (1990-1993) it is suggested that the first cat (T) has become superinfected with an isolate from a second cat (N) under natural conditions. The identification of clones of differing sequences, which were not identical to each other nor to their ancestors, emphasises the rapid mutation of lentiviruses within the env region, and the difficulty of developing an effective FIV vaccine. More importantly, the possibility of natural superinfection with FIV in cats has implications for the development of a successful lentiviral vaccine.

Epstein-Barr virus-specific cytotoxic T cell response in cardiac transplant recipients.

PBLs from 10 normal seropositive donors, 15 precardiac transplant patients, and 17 postcardiac transplant patients have been assayed for their ability to mount a cytotoxic T cell response to both A- and B-type EBV. Compared with the results obtained with healthy seropositive donors, pre- and posttransplant patients had significantly weaker T cell responses against both A-type and B-type EBV. Analysis of individual patients showed a preferential T cell response to B-type EBV in 4/15 pre- and 6/17 posttransplant patients and a preferential T cell response to A-type EBV in 1/15 pretransplant and 2/17 posttransplant recipients. PBMCs were obtained from patients and analyzed for the presence of A- and B-type EBV using polymerase chain reaction. EBV types detected in the PBMCs of each individual were correlated with their EBV-specific CTL response. The results obtained indicated that the EBV-specific cytotoxic T cell response of these patients matched the EBV types with which they were infected.

The induction of in vivo superinfection and recombination using feline immunodeficiency virus as the model.

This study investigated the hypothesis that under certain conditions, superinfection of cats with feline immunodeficiency virus (FIV), may occur. One FIV isolate (T91) was used to inoculate three FIV and FeLV-free cats. Blood from an FIV-infected cat (N), which contained two variants and differed from T91 by at least 5% in nucleotide sequence in the env gene, was inoculated into a fourth cat. Both T91 and blood from N were inoculated simultaneously into a fifth cat. After 22 weeks, two of the three cats initially infected with T91 were challenged with blood from N. At 30 weeks following initial infection, peripheral blood mononuclear cells were obtained from all cats, DNA was extracted, and a segment of the env gene was PCR amplified, cloned and sequenced. Nucleotide sequence analysis of the cloned PCR product showed that virus strains used in initial infection were recovered from cats not challenged with a second variant. Challenge of cats with the blood of N following initial infection with T91 resulted in superinfection occurring in one cat and recombination occurring in the other. Furthermore, the use of blood as a source of challenge, in cats where superinfection and simultaneous infections were attempted, may have induced the appearance of variants which more closely resembled the most heterologous strain present in the infectious source.