Serological evidence of Coxiella burnetii exposure in native marsupials and introduced animals in Queensland, Australia.

The state of Queensland has the highest incidence of Q fever in Australia. In recent years, there has been an increase in human cases where no contacts with the typical reservoir animals or occupations were reported. The aim of this study was to determine the seroprevalence of Coxiella burnetii in Australian native animals and introduced animals in northern and southeastern Queensland. Australian native marsupials sampled included the brushtail possum (Trichosurus vulpecula) and common northern bandicoot (Isoodon macrourus). Introduced species sampled included dingoes (Canis lupus dingo), cats (Felis catus), foxes (Vulpes vulpes) and pigs (Sus scrofa). Serum samples were tested by ELISA for both phase II and phase I antigens of the organism using an Australian isolate. The serological evidence of C. burnetii infection demonstrated in these species has public health implications due to their increasing movement into residential areas in regional Queensland. This study is the first known investigation of C. burnetii seroprevalence in these species in northern Queensland.

Immunohistochemical analysis of structural changes in collagen for the assessment of osteoarthritis.

Collagen fibrillation within articular cartilage (AC) plays a key role in joint osteoarthritis (OA) progression and, therefore, studying collagen synthesis changes could be an indicator for use in the assessment of OA. Various staining techniques have been developed and used to determine the collagen network transformation under microscopy. However, because collagen and proteoglycan coexist and have the same index of refraction, conventional methods for specific visualization of collagen tissue is difficult. This study aimed to develop an advanced staining technique to distinguish collagen from proteoglycan and to determine its evolution in relation to OA progression using optical and laser scanning confocal microscopy (LSCM). A number of AC samples were obtained from sheep joints, including both healthy and abnormal joints with OA grades 1 to 3. The samples were stained using two different trichrome methods and immunohistochemistry (IHC) to stain both colourimetrically and with fluorescence. Using optical microscopy and LSCM, the present authors demonstrated that the IHC technique stains collagens only, allowing the collagen network to be separated and directly investigated. Fluorescently-stained IHC samples were also subjected to LSCM to obtain three-dimensional images of the collagen fibres. Changes in the collagen fibres were then correlated with the grade of OA in tissue. This study is the first to successfully utilize the IHC staining technique in conjunction with laser scanning confocal microscopy. This is a valuable tool for assessing changes to articular cartilage in OA.

Q fever cases at a North Queensland centre during 1994-2006.

This study reviewed the epidemiological features, management and outcomes of patients with Q fever treated at a tertiary facility in North Queensland during the period from July 1994 to January 2006. Twenty-seven patients were identified. Our findings were consistent with the observations about Q fever that have been made in other regions of Australia. A diagnosis of Q fever should be considered in patients with a non-specific febrile illness.

Burkholderia pseudomallei virulence: definition, stability and association with clonality.

Clinical presentations of melioidosis, caused by Burkholderia pseudomallei are protean, but the mechanisms underlying development of the different forms of disease remain poorly understood. In murine melioidosis, the level of virulence of B. pseudomallei is important in disease pathogenesis and progression. In this study, we used B. pseudomallei-susceptible BALB/c mice to determine the virulence of a library of clinical and environmental B. pseudomallei isolates from Australia and Papua New Guinea. Among 42 non-arabinose-assimilating (ara(-)) isolates, LD(50) ranged from 10 to > 10(6) CFU. There were numerous correlations between virulence and disease presentation in patients; however, this was not a consistent observation. Virulence did not correlate with isolate origin (i.e. clinical vs environmental), since numerous ara(-) environmental isolates were highly virulent. The least virulent isolate was a soil isolate from Papua New Guinea, which was arabinose assimilating (ara(+)). Stability of B. pseudomallei virulence was investigated by in vivo passage of isolates through mice and repetitive in vitro subculture. Virulence increased following in vivo exposure in only one of eight isolates tested. In vitro subculture on ferric citrate-containing medium caused attenuation of virulence, and this correlated with changes in colony morphology. Pulsed-field gel electrophoresis and randomly amplified polymorphic DNA typing demonstrated that selected epidemiologically related isolates that had variable clinical outcomes and different in vivo virulence were clonal strains. No molecular changes were observed in isolates after in vivo or in vitro exposure despite changes in virulence. These results indicate that virulence of selected B. pseudomallei isolates is variable, being dependent on factors such as iron bioavailability. They also support the importance of other variables such as inoculum size and host risk factors in determining the clinical severity of melioidosis.

Reconstruction of the block matching profiles.

Block matching is a valuable tool for selecting donors for bone marrow transplantation. Identical, electrophoretic profiles of unrelated bone marrow donor-recipient pairs have been shown to be associated with long-term survival and a reduction of graft versus host disease (GVHD). This study was undertaken to determine the sequences of the PCR products which are generated. PCR products obtained with beta-block primers following the amplification of DNA extracted from cell lines homozygous for 7.1 and 8.1 ancestral haplotypes were cloned and sequenced. The PCR products were characterised and the beta block profiles reconstructed. The data indicate that the profiles consist of homoduplexes and heteroduplexes which are formed by the products of probably 3 different sequence locations.

MHC haplotype analysis by artificial neural networks.

Conventional matching is based on numbers of alleles shared between donor and recipient. This approach, however, ignores the degree of relationship between alleles and haplotypes, and therefore the actual degree of difference. To address this problem, we have compared family members using a block matching technique which reflects differences in genomic sequences. All parents and siblings had been genotyped using conventional MHC typing so that haplotypes could be assigned and relatives could be classified as sharing 0, 1 or 2 haplotypes. We trained an Artificial Neural Network (ANN) with subjects from 6 families (85 comparisons) to distinguish between relatives. Using the outputs of the ANN, we developed a score, the Histocompatibility Index (HI), as a measure of the degree of difference. Subjects from a further 3 families (106 profile comparisons) were tested. The HI score for each comparison was plotted. We show that the HI score is trimodal allowing the definition of three populations corresponding to approximately 0, 1 or 2 haplotype sharing. The means and standard deviations of the three populations were found. As expected, comparisons between family members sharing 2 haplotypes resulted in high HI scores with one exception. More interestingly, this approach distinguishes between the 1 and 0 haplotype groups, with some informative exceptions. This distinction was considered too difficult to attempt visually. The approach provides promise in the quantification of degrees of histocompatibility.

The significance of HLA matching in cardiac transplantation.

It is argued that HLA matching is not worthwhile in heart transplantation. However, transplanting HLA compatible hearts enhances graft survival and should significantly reduce infection and malignancies related to aggressive immunosuppression. It is our view that the problem is technical and we offer a potential solution.

Exposure to Burkholderia pseudomallei induces cell-mediated immunity in healthy individuals.

Melioidosis is an emerging tropical infection caused by the intracellular bacterium Burkholderia pseudomallei, and is associated with high mortality rates. Previous studies investigating the prevalence of melioidosis have based conclusions on serological evidence. However, cell-mediated immunity is more relevant for protection against an intracellular pathogen such as B. pseudomallei. This is the first demonstration that exposure to B. pseudomallei may lead to the formation of specific antibodies and the development of cell-mediated immunity in a healthy individual.

Macrophage-lymphocyte interactions mediate anti-Burkholderia pseudomallei activity.

The mechanisms involved in the pathogenesis of melioidosis, caused by the intracellular bacterium Burkholderia pseudomallei, are unclear. C57BL/6 mice are resistant to infection, while BALB/c mice are highly susceptible. Previous studies have demonstrated that peritoneal exudate cell preparations enriched for macrophages are capable of effectively eliminating intracellular pathogens. In this study we present evidence showing that interaction of macrophages with lymphocytes is necessary for efficient anti-B. pseudomallei activity.

Proinflammatory cytokine mRNA responses in experimental Burkholderia pseudomallei infection in mice.

Melioidosis is a potentially fatal disease of both human and animals caused by the bacterium Burkholderia pseudomallei. Disease is endemic in tropical and subtropical regions of Southeast Asia and Northern Australia. The pathogenesis of melioidosis is poorly understood. In particular, the host responses that occur following infection, and the specific host-pathogen interactions that result in the development of either acute or chronic infection are unclear. Using an established murine model, we investigated early proinflammatory cytokine responses believed to be critical in the development of acute and chronic B. pseudomallei infection. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to assess levels of mRNA for tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta) and interleukin 6 (IL-6) in the liver of mice following infection. We demonstrate that the level of mRNA for these cytokines increase moderately in chronic infection in C57BL/6 mice. However, in acute infection in BALB/c mice, mRNA responses for these cytokines were shown to be comparatively greater. These results demonstrate that early proinflammatory cytokine responses are important in the immunopathogenesis of melioidosis.

Assessment of immunogenicity and viability of homologous human cardiac valves in vitro.

Degenerative changes in a proportion of implanted homologous cardiac valves are considered to be due to the immunologic tissue reactions initiated by the donor valves. Sterilization and storage protocols can be used to modulate the immunogenicity of donor valves prior to implantation. Therefore, it is essential to assess the effect of treatment protocols on the immunogenicity and viability of donor valve tissue. The optimal conditions for two novel in vitro tests to assess the immunogenicity and viability of human cardiac valve tissue, the valve cusp cell/responder lymphocyte reaction to assess immunogenicity and the tetrazolium based colorimetric assay to assess viability of valve tissue, are described. The in vitro tests that have been developed in this study will be helpful in assessing the effect of various treatment protocols on the immunogenicity and viability of homologous human cardiac valves.

In vitro immunogenicity of allogeneic cardiac valves.

The immunogenicity of allogeneic cardiac valves (ACV) has not been previously demonstrated in vitro, though valve failure due to tissue degeneration has been attributed to adverse immunological reactions. A novel in vitro assay has been developed in a Brown Norway (BN; RT1n)-Lewis (RT1; donor-recipient) rat model system that demonstrates the immunogenicity of ACVs. A single cell suspension of viable cardiac valve conduit (CVC) cells was obtained by collagenase treatment of BN rat aortic valve conduits. Brown Norway rat CVC cells (5 x 10(4)) and Lewis responder lymphocytes (10(5)) were co-cultured in 96 well plates in RPMI 1640 plus 2.5% (v/v) non heat-inactivated Lewis rat serum and supplements with appropriate controls. Stimulation of responder lymphocytes by CVC cells was measured by 3H-thymidine incorporation into DNA. The counts obtained between 96-192 h of co-culture in the CVC cell/responder lymphocyte reaction were significantly higher (P < 0.05) than those of responder cell controls as assessed by analysis of variance. These results indicate the presence of potent immunostimulatory cells in viable ACVs and the possibility of using a sensitive and reproducible in vitro assay to evaluate ACV immunogenicity.

Invasive group A streptococcal disease in North Queensland (1996 - 2001).

BACKGROUND & OBJECTIVES: The incidence of group A streptococcal (GAS) invasive infections have been increasing worldwide. The aim of this study was to characterize clinical and microbiological features of isolates obtained from invasive GAS infections in North Queensland, Australia between 1996 and 2001. METHODS: Clinical and demographic data were collected prospectively. Isolates were biotyped, emm sequenced, M typed and tested for antibiotic sensitivity using E-test. Detection of the presence of the streptococcal pyrogenic exotoxin (spe) and fibronectin binding protein (prtF1) genes was also carried out. RESULTS: There were 109 isolates from blood and sterile sites. All isolates were sensitive to penicillin. Tetracycline and erythromycin resistance was seen in 11 and 2.7 per cent of isolates respectively. The isolates were evenly distributed by age and sex. The overall mortality was 7 per cent and there were 18 cases of streptococcal toxic shock syndrome (STSS) in which the mortality was 22 per cent. Indigenous patients had a crude incidence rate of 82.5 per 100,000 per year compared with 10.3 per 100,000 per year in the non-indigenous patients. There was no predominance of emm / M type or association of spe type with STSS. There was also no relationship between the presence of the prtF1 gene and invasive disease. INTERPRETATION & CONCLUSION: Invasive group A streptococci from North Queensland are similar to those from the Northern Territory of Australia in that no single strain is predominant. The indigenous population is overrepresented. Invasiveness and the development of streptococcal toxic shock is not related to the presence of the prtF1 gene or spe a or c.

Donor valves as substitutes for heart valve replacement.

The main problems of heart valve replacement in Sri Lanka are the cost of prosthetic valves and anticoagulant related complications. The use of human donor heart valves (homografts; allogeneic heart valves [AHV]) will alleviate these shortcomings. Recipients of AHV do not require anticoagulant therapy. Moreover, cryopreservation of AHV offers the opportunity for the storage of valves for an extended length of time with the preservation of valve integrity which is essential for their function after implantation. A donor valve bank can potentially provide diameter matched valves for recipients. Current research suggests that the adverse immunological reactions initiated by AHV cause tissue degeneration in a proportion of these implants. However, the grafts may be improved before implantation during the disinfection and storage of the valves. In this essay an overview on the advantages of using AHV, current concepts of valve banking, recent advances in the understanding of AHV immunogenicity, emerging techniques for immunomodulation of AHV and the possibility of setting up a donor heart valve bank in Sri Lanka are discussed.

Dietary composition of carbohydrates contributes to the development of experimental type 2 diabetes.

Evidence has emerged supporting a link between high glycaemic index (GI) diets and type 2 diabetes (T2D). The aim of this study was to determine if dietary GI influences the development of hyperglycaemia in C57BL/6 mice to more closely reflect T2D. Male C57BL/6 mice (n=30) were randomly divided into 3 dietary groups consisting of either standard rodent chow (4.8 % fat, 20 % protein), or a high fat (HF) diet (21-23 % fat, 19 % protein) with low GI (15.4 % starch; HF-LG) or high GI (50.5 % dextrose; HF-HG) ad libitum for 10 weeks. Body weight, blood glucose, glucose tolerance, and circulating cholesterol and triglyceride levels were measured for the duration of the study. We found that increasing the GI of a moderately HF diet induces severe hyperglycaemia and insulin resistance in C57BL/6 mice, reflective of criteria for diagnosis of T2D, whilst littermates consuming an equivalent low GI diet maintain glucose homeostasis. This study demonstrates the significant contribution of both dietary carbohydrate and fat composition in the aetiopathogenesis of T2D.

Detection of Coxiella burnetii DNA in wildlife and ticks in northern Queensland, Australia.

Wild animals and the tick species that feed on them form the natural transmission cycle and reservoir of Coxiella burnetii. The objective of this study was to determine whether C. burnetii was present in the blood of host animals and their ticks in northern Queensland, Australia. Three genomic targets were detected using real-time PCR assays-the Coxiella-specific outer membrane protein coding gene (Com1), the multicopy insertion element (IS1111), and the isocitrate dehydrogenase gene (Icd). Quantification of the single-copy targets identified a range of 1.48×10(1) to 4.10×10(3) C. burnetii genome equivalents per microliter in the ticks tested. The detection of Coxiella based on the presence of the genomic targets indicated the occurrence of C. burnetii in both the ticks and whole blood of a variety of native Australian marsupials and confirms these animals are capable of acting as reservoirs of Q fever in northern Queensland.

Veterinary infection control in Australia: is there control?

There is little information relating to infection control procedures in Australian veterinary practices. This review summarises the findings of international studies in the area of zoonoses and infection control, and discusses potential reasons for the apparent complacency about these issues in veterinary practice. It is the authors' opinion that legislative changes governing veterinary practice in Australia should be implemented. The curricula in veterinary schools should also emphasise infection control. These measures would significantly improve safety issues associated with the control of zoonoses in veterinary practice.

Serological evidence of Coxiella burnetii infection in dogs in a regional centre.

OBJECTIVE Investigate the seroprevalence of the causative agent of Q fever, Coxiella burnetii in domestic dogs in the Townsville region, North Queensland, Australia. METHOD Blood samples were collected from dogs attending veterinary clinics for routine procedures. RESULTS An overall seropositivity of 21.8% (95% confidence interval (CI) 21.6-22.1%) was observed. A retrospective study of samples collected in the same region during 1984-85 was also performed, with an overall seropositivity of 16.0% (95% CI 15.9-16.2). CONCLUSION Evidence of C. burnetii infection in domestic dogs may have public health implications for dog owners, as well as veterinarians because of occupational exposure. This study is the first known investigation of C. burnetii seroprevalence in dogs in Queensland.

Serological evidence of Coxiella burnetii infection in beef cattle in Queensland.

BACKGROUND: Queensland has the highest incidence of Q fever in Australia. The aim of this study was to undertake a cross-sectional seroprevalence survey of Coxiella burnetii, the causative agent of Q fever, in beef cattle in Queensland. METHODS: Serum samples were tested by ELISA for both phase II and phase I antigens of the organism using an Australian isolate. Blood samples were collected at an abattoir that processes beef cattle originating from northern and north-western Queensland, in addition to blood samples taken from beef cattle across Queensland as part of a second survey. RESULTS: Seropositivity was 16.8% (95% confidence interval 16.7-16.8%). CONCLUSION: Evidence of C. burnetii infection in beef cattle has public health implications for occupational exposure of primary producers and veterinarians and for the proximity of beef cattle properties to residential areas in regional Queensland. This study is the first known investigation of C. burnetii seroprevalence in beef cattle in Queensland and the first known use of an Australian C. burnetii isolate for screening using both phase II and phase I antigens.

An alternative technique for the induction of autoimmune valvulitis in a rat model of rheumatic heart disease.

We currently use a rat model in our investigations into human rheumatic heart disease (RHD). This model traditionally involves footpad immunization with antigen emulsified in complete Freund's adjuvant (CFA). Trials to find an alternative adjuvant to CFA which produced a Th1 type response in the rats resulting in carditis were unsuccessful. However, hock immunization was found to produce the desired valvular pathology without the adverse inflammatory side-effects associated with CFA. We therefore consider the hock an ideal site for immunization, particularly when using CFA.