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 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.

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.

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.

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.

Evidence for the classification of a crayfish pathogen as a member of the genus Coxiella.

AIMS: The study aimed to provide characterization of a potential new species of Coxiella, identified following a series of outbreaks of disease in Australian native freshwater crayfish. METHODS AND RESULTS: PCR primers designed for amplification of Coxiella burnetii genes including 16S rDNA, com1 and sodB were used to amplify homologues in the Coxiella-like crayfish pathogen. Products were then cloned and sequenced. The organism demonstrated a high degree of sequence homology in the highly conserved 16S rDNA (96%) and sodB (99%) genes, as well as the Coxiella sp. specific com1 (100%) gene. Regions flanking the sodB coding sequence demonstrated homology to C. burnetii antioxidant AhpC/Tsa family protein and dihydrodipicolinate reductase gene. CONCLUSIONS: The degree of homology between the genes selected and flanking regions suggested the two organisms were sufficiently closely related to belong to the same genus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided evidence for a potential new species in the currently monospecific genus Coxiella, with the only described member being C. burnetii, a category B biological warfare agent.

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.

The effect of transportation on the immune status of Bos indicus steers.

This study investigated the effect of 72 h of road transport on the immune status of Bos indicus steers (n = 10; age = 15 to 18 mo). Total and differential leukocyte numbers and lymphocyte function were determined at 2 d before transport (-48 h), immediately after 72 h of transport (72 h), and 6 d after transport (216 h). Phytohemagglutinin (PHA)-stimulated lymphocyte proliferation, interferon-gamma production, and tetanus-toxoid specific antibody levels were determined. Total leukocyte and eosinophil numbers showed a transient decrease at 72 h (immediately after transport; P < 0.05) and returned to baseline values by 6 d after transport. Lymphocyte numbers and antibody titers were unaffected by transportation. The PHA-stimulated lymphocyte proliferation decreased (P < 0.05) at 72 h and returned to baseline levels 6 d after transport. This study demonstrated that transportation of mature Bos indicus steers caused transient decreases in leukocyte numbers and lymphocyte function, although all measures recovered by 6 d after transport. Therefore, Bos indicus cattle may be vulnerable to infection during this period.

Adaptive immunity in melioidosis: a possible role for T cells in determining outcome of infection with Burkholderia pseudomallei.

Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia pseudomallei. Individuals with subclinical melioidosis have no apparent clinical signs or symptoms, and are identified only by positive serology. The present study is the first to investigate cell-mediated immune (CMI) responses following in vitro stimulation with B. pseudomallei antigens in peripheral blood mononuclear cells (PBMC), collected under field conditions in Papua New Guinea (PNG) from individuals with exposure to B. pseudomallei (n = 13). While five had a clinical history of melioidosis (C(+)), the remaining individuals (n = 8) were seropositive, yet healthy with no clinical history of melioidosis (S(+)/C(-)). Proliferation and IFN-gamma production were significantly greater in lymphocyte cultures from S(+)/C(-) individuals compared to C(+) individuals (P < 0.001 and P < 0.05, respectively). These findings demonstrate that compared to C(+) patients, individuals with subclinical melioidosis have a stronger CMI response to B. pseudomallei antigens in vitro. Such a response may be essential for protection against disease progression.

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.

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.

Induction of multiple chemokine and colony-stimulating factor genes in experimental Burkholderia pseudomallei infection.

Melioidosis is a disease of the tropics caused by the facultative intracellular bacterium Burkholderia pseudomallei. In human infection, increased levels of IFN-gamma in addition to the chemokines interferon-gamma-inducible protein 10 (IP-10) and monocyte interferon-gamma-inducible protein (Mig) have been demonstrated. However, the role of these and other chemokines in the pathogenesis of melioidosis remains unknown. Using BALB/c and C57BL/6 mice as models of the acute and chronic forms of human melioidosis, the induction of mRNA was assessed for various chemokines and CSF (G-CSF, M-CSF, GM-CSF, IP-10, Mig, RANTES, MCP-1, KC and MIP-2) in spleen and liver following B. pseudomallei infection. Patterns of chemokine and CSF induction were similar in liver and spleen; however, responses were typically greater in spleen, which reflected higher tissue bacterial loads. In BALB/c mice, high-level expression of mRNA for all chemokines and CSF investigated was demonstrated at day 3 postinfection, correlating with peak bacterial load and extensive infiltration of leucocytes. In contrast, increased mRNA expression and bacterial numbers in C57BL/6 mice were greatest between 4 and 14 days following infection. This paralleled increases in the size and number of abscesses in liver and spleen of C57BL/6 mice at days 3 and 14 postinfection. Earlier induction of cytokine-induced neutrophil chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) mRNA was demonstrated in spleen, while MIP-2, MCP-1, IP-10 and Mig were demonstrated in liver of BALB/c mice when compared to spleen and liver of C57BL/6. The magnitude of cellular responses observed in the tissue correlated with increased levels of the chemokines and CSF investigated, as well as bacterial load. Compared with C57BL/6 mice, greater infiltration of neutrophils was observed in liver and spleen of BALB/c mice at day 3. In contrast, early lesions in C57BL/6 mice predominantly comprised macrophages. These results suggest that the inability of BALB/c mice to contain the infection at sites of inflammation may underlie the susceptible phenotype of this mouse strain towards B. pseudomallei infection.

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.

Cytokine gene expression in innately susceptible BALB/c mice and relatively resistant C57BL/6 mice during infection with virulent Burkholderia pseudomallei.

Production of cytokines including gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) is an important early-stage host response following infection with intracellular pathogens. Development of immunity to these pathogens is determined to a large extent by the timing and relative level of expression of the cytokines. Numerous studies have shown that early cytokine responses involving interleukin-12 (IL-12) and IFN-gamma are important for resistance to intracellular pathogens, whereas responses involving IL-4 and IL-10 increase host susceptibility. These often-indistinct early cytokine responses influence the differentiation of naïve CD4(+) T helper cells, which later develop into what have commonly been termed Th1- and Th2-type cells. The characterization of CD4(+) T-helper-cell responses as Th1 or Th2 type is based largely on the cytokine profiles during the specific phase and has been used in recent years to account for the innate resistance and susceptibility of different inbred strains of mice to several intracellular pathogens. Studies investigating cytokine production in terms of CD4(+) T-helper-cell polarization in Burkholderia pseudomallei infection have not been undertaken. In this study, we used semiquantitative reverse transcription-PCR to assess induction of cytokine mRNA in liver and spleen of B. pseudomallei-susceptible BALB/c and relatively resistant C57BL/6 mice following infection with virulent B. pseudomallei. The levels of mRNA for IFN-gamma, TNF-alpha, IL-1beta, IL-6, IL-10, and IL-12 increased in both BALB/c and C57BL/6 mice 24 to 36 h after infection. A comparison of BALB/c and C57BL/6 responses revealed the relative levels of expression of mRNA for several of these cytokines, including IFN-gamma, were greater in BALB/c mice, suggesting a role for endotoxic shock and cytokine-mediated immunopathology in the development of acute melioidosis. Early induction of mRNA for the cytokines classically associated with development of Th1- and Th2-type responses was absent or minimal, and induction levels in both strains of mice were similar. During the specific phase, cytokine mRNA profiles occurred as a combination of Th1- and Th2-type patterns. Collectively, these results demonstrate that cytokine mRNA responses in BALB/c and C57BL/6 mice following infection with virulent B. pseudomallei do not develop as polarized Th1- or Th2-type profiles. Considering the role of TNF-alpha and IFN-gamma in the processes of endotoxic shock, these results also indicate that selected cytokines, while important for resistance to B. pseudomallei infection, are also potential contributors to immunopathology and the development of acute fulminating disease.

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.

Disseminated infection due to Nocardia transvalensis coincident with Cryptococcus neoformans variety gattii meningitis.

A case of meningitis due to Cryptococcus neoformans var. gattii coincident with disseminated Nocardia transvalensis infection is reported. Nocardia infection initially progressed despite high-dose antimicrobial therapy. Although a specific immunologic defect could not be defined, in vitro lymphocyte proliferation in response to stimulation with the Nocardia isolate was reduced. It is proposed that coinfection with Cryptococcus neoformans may have contributed to the observed impairment of lymphocyte function, leading to disseminated Nocardia disease and a suboptimal treatment response.