Genetic analysis of porcine circovirus type 2 (PCV2) in Queensland, Australia.

OBJECTIVE: To determine the current porcine circovirus type 2 (PCV2) genotypes circulating in pigs in Queensland (QLD). METHODS: The PCV2 infection status of pigs was determined by real-time PCR testing of 210 lymph nodes and 30 serum samples derived from 45 QLD farms. PCV2-positive samples from 22 pigs from 15 farms were subjected to conventional polymerase chain reaction (PCR) and sequencing of the full PCV2 genome. Phylogenetic analysis of 17 of these sequences in relation to published PCV2 sequences was then performed, and the genotypes were compared. RESULTS: PCV2 DNA was detected in 95 lymph nodes and 15 serum samples. Phylogenetic analysis of 17 PCV2 sequences demonstrated that seven belonged to genotype PCV2b, two to PCV2d, one to PCV2f and seven to an "intermediate group" that clustered with PCV2d on the full genome analysis. CONCLUSION: This work confirms earlier studies reporting the presence of PCV2b in Australia. It is the first study to report that PCV2d and PCV2f are also present in this country. PCV2d is currently a fast-spreading genotype globally, with reported high virulence. The potential implications of these findings with respect to pathogenicity and vaccine efficacy require further investigation.

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.

Genetic analysis of canine parvovirus from dogs in Australia.

OBJECTIVE: To determine the genetic variants of canine parvovirus-2 (CPV) present in domestic dogs in Australia and to investigate 26 cases of apparent vaccine failure. DESIGN: Thirty-three samples of faeces or intestinal tissues and 16 cell culture virus isolates collected over a period from 1980 to 2005 from five Australian states were analysed. Procedure DNA was extracted from the samples and a 1975 bp fragment of the VP1/2 gene of CPV was amplified by polymerase chain reaction (PCR) and sequenced. Sequences were compared to published strains of CPV-2, CPV-2a, CPV-2b and CPV-2c. RESULTS: Forty-one of 43 PCR-positive samples contained CPV-2a viruses. One sample collected in 2002 from a pup in northern NSW contained a CPV-2b virus. One sample that had been included in the study as a CPV-antigen negative control sample contained a CPV-2 virus. CONCLUSION: CPV-2a remains the predominant genetic variant of CPV in dogs in Australia and has not been replaced by CPV-2b or CPV-2c as in many other countries. The vaccine failures investigated in the study were likely caused not by genetic variation of field viruses but by maternal antibody interference in the response of pups to vaccination.

Co-infection with different subtypes of feline immunodeficiency virus can complicate subtype assignment by phylogenetic analysis.

Phylogenetic analyses of the V3-V5 region of the env gene are used to determine Feline immunodeficiency virus (FIV) subtypes but can be complicated by co-infection with different subtypes or the presence of recombinant subtypes. FIV in blood samples from 30 domestic cats in New Zealand was subtyped by sequencing three overlapping fragments of the V3-V5 region of the env gene and 467 bp of the gag gene. Phylogenetic analyses revealed that the isolates clustered with subtype A and C viruses. Seven samples showed discrepancies in subtype assignment from analyses of their env gene sequences. Nucleotide differences of 19.6% and 20.9% in overlapping regions in two cats suggest co-infection with subtypes A and C.

Phylogenetic analysis to define feline immunodeficiency virus subtypes in 31 domestic cats in South Africa.

Feline immunodeficiency virus (FIV), a lentivirus, is an important pathogen of domestic cats around the world and has many similarities to human immunodeficiency virus (HIV). A characteristic of these lentiviruses is their extensive genetic diversity, which has been an obstacle in the development of successful vaccines. Of the FIV genes, the envelope gene is the most variable and sequence differences in a portion of this gene have been used to define 5 FIV subtypes (A, B, C, D and E). In this study, the proviral DNA sequence of the V3-V5 region of the envelope gene was determined in blood samples from 31 FIV positive cats from 4 different regions of South Africa. Phylogenetic analysis demonstrated the presence of both subtypes A and C, with subtype A predominating. These findings contribute to the understanding of the genetic diversity of FIV.

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.

Equine laminitis: increased transcription of matrix metalloproteinase-2 (MMP-2) occurs during the developmental phase.

REASONS FOR PERFORMING STUDY: The dysadhesion and destruction of lamellar basement membrane of laminitis may be due to increased lamellar metalloproteinase activity. Characterising lamellar metalloproteinase-2 (MMP-2) and locating it in lamellar tissues may help determine if laminitis pathology is associated with increased MMP-2 transcription. OBJECTIVES: To clone and sequence the cDNA encoding lamellar MMP-2, develop antibody and in situ hybridisation probes to locate lamellar MMP-2 and quantitate MMP-2 transcription in normal and laminitis tissue. METHODS: Total RNA was isolated, fragmented by RT-PCR, cloned into vector and sequenced. Rabbit anti-equine MMP-2 and labelled MMP-2 riboprobe were developed to analyse and quantitate MMP-2 expression. RESULTS: Western immunoblotting with anti-MMP-2 detected 72 kDa MMP-2 in hoof tissue homogenates and cross-reacted with human MMP-2. Immunohistochemistry and in situ hybridisation detected MMP-2 in the cytoplasm of basal and parabasal cells in close proximity to the lamellar basement membrane. Northern analysis and quantitative real-time PCR showed MMP-2 expression significantly (P < 0.01) elevated in laminitis affected tissues. CONCLUSION: The lamellar pathology of laminitis is associated with increased transcription of MMP-2. POTENTIAL RELEVANCE: Real-time PCR analysis of lamellar MMP-2 accurately monitors laminitis development at the molecular level and can be used diagnostically and for testing preventive strategies. Controlling increased MMP-2 transcription may ameliorate or prevent laminitis in high risk clinical situations. Our findings represent a warning to clinicians that the basement membrane lesion of laminitis is insidious and well under way before clinical signs are apparent.

Productive infection of the bone marrow cells in feline immunodeficiency virus infected cats.

Bone marrow stromal cells (BMSC) from cats experimentally infected with feline immunodeficiency virus (FIV) were shown to contain FIV provirus using polymerase chain reaction and viral products were detected in culture supernatant using reverse transcriptase and enzyme linked immunosorbent assay techniques. Peripheral blood mononuclear cells from FIV-free cats co-cultured with infected bone marrow cells became productively infected with FIV. Such evidence supports the hypothesis that BMSC are a reservoir for FIV. Furthermore, BMSC produced virions capable of infecting susceptible cells and may represent an important source of infectious virus to cells of the macrophage lineage and/or hemopoietic progenitor cells, both of which ultimately become widely disseminated throughout the body.

In vitro evidence for a bacterial pathogenesis of equine laminitis.

Utilizing an in vitro laminitis explant model, we have investigated how bacterial broth cultures and purified bacterial proteases activate matrix metalloproteinases (MMPs) and alter structural integrity of cultured equine lamellar hoof explants. Four Gram-positive Streptococcus spp. and three Gram-negative bacteria all induced a dose-dependent activation of MMP-2 and MMP-9 and caused lamellar explants to separate. MMP activation was deemed to have occurred if a specific MMP inhibitor, batimastat, blocked MMP activity and prevented lamellar separation. Thermolysin and streptococcal pyrogenic exotoxin B (SpeB) both separated explants dose-dependently but only thermolysin was inhibitable by batimastat or induced MMP activation equivalent to that seen with bacterial broths. Additionally, thermolysin and broth MMP activation appeared to be cell dependent as MMP activation did not occur in isolation. These results suggest the rapid increase in streptococcal species in the caecum and colon observed in parallel with carbohydrate induced equine laminitis may directly cause laminitis via production of exotoxin(s) capable of activating resident MMPs within the lamellar structure. Once activated, these MMPs can degrade key components of the basement membrane (BM) hemidesmosome complex, ultimately separating the BM from the epidermal basal cells resulting in the characteristic laminitis histopathology of hoof lamellae. While many different causative agents have been evaluated in the past, the results of this study provide a unifying aetiological mechanism for the development of carbohydrate induced equine laminitis.

Characterisation of lymphosarcomas in Australian cats using polymerase chain reaction and immunohistochemical examination.

OBJECTIVE: To examine tumour tissue of cats with lymphosarcoma for the presence of feline leukaemia virus and feline immunodeficiency virus and analyse the immunophenotype of the tumours. DESIGN: A retrospective study of feline lymphosarcoma cases. METHODS: Formalin-fixed, paraffin-embedded tumour tissue of 14 feline lymphosarcomas was examined for the presence of feline leukaemia virus and feline immunodeficiency virus by polymerase chain reaction and immunohistochemistry. Using polyclonal and monoclonal antibodies against T and B lymphocytes, the phenotypic expression of the tumours was characterised. RESULTS: No feline leukaemia virus antigen or proviral sequences were detected. Feline immunodeficiency virus proviral sequences were detected in two cases by polymerase chain reaction. Immunophenotyping of all 14 cases resulted in seven cases being classified as B-cell phenotype, four as T-cell phenotype, and the remaining three undetermined. CONCLUSIONS: In contrast to previous reports overseas, our results suggest that feline leukaemia virus infection appears to be an infrequent cause of lymphosarcoma in the cats that were necropsied. Feline immunodeficiency virus may have a role in lymphomagenesis. The potential role of feline immunodeficiency virus needs to be explored in more depth. Compared with most previous reports, B-cell tumours were more common than T-cell tumours in this series of cats.

T-cell-rich B-cell lymphoma in the cat.

The clinical and pathological features of eight cases of feline T-cell-rich B-cell lymphoma are described. The disease occurred in older cats (mean age 11.4 +/- 3.9 years), which on initial examination generally showed enlargement of a single submandibular or cervical lymph node. After excision, there was no recurrence of the lesions at 6 months in three cats. In one further case, however, the lesion had recurred 6 months later; it was again excised but recurred after an additional 6 months. Microscopically, there was effacement of normal lymph node architecture by a nodular (n = 4) or diffuse (n = 4) proliferation of small to blastic lymphocytes, accompanied by a characteristic population of bizarre giant, or multinucleate, cells. The mitotic rate was low and mitoses were restricted to the atypical population. Immunophenotyping revealed the smaller lymphocytes to be a mixture of CD3+ MHC Class II+ T lymphocytes and BLA36+CD79variable MHC Class IIvariable B lymphocytes. The atypical cells were of the B-cell lineage (BLA36+MHC Class IIvariable). Polymerase chain reaction analysis revealed no proviral DNA products of feline leukaemia virus or feline immunodeficiency virus in tissue from any tumour, confirming that these neoplasms were not associated with either virus. The clinical, histological and immunophenotypic findings in these cats were identical with those of "nodular lymphocyte predominance (lymphocytic and histiocytic/L&H) Hodgkin's disease" in man.

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.

Feline immunodeficiency virus replicates in salivary gland ductular epithelium during the initial phase of infection.

Feline immunodeficiency virus (FIV) antigen was detected by immunochemistry in salivary glands of cats experimentally inoculated with West Australian isolate T91. Six cats were inoculated subcutaneously with 1.0 ml of tissue culture supernatant fluid from a feline T-lymphoblastoid cell line (MYA-1) infected with T91. FIV antigens were detected in the interlobular ducts of the salivary gland of cats infected with FIV 2, 4 and 6 weeks previously. FIV antigen was not detected in the salivary glands of three FIV negative cats and one naturally infected cat. Further, FIV antigen was located only in interlobular duct epithelial cells. The distribution of FIV in the interlobular ducts confirms the important role of salivary glands as a major reservoir of FIV in the early phase of infection and strengthens suggestions that the salivary route is an important mode of transmission of FIV.

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.