Unusual stability of dyads during photochemical hydrogen production.

Dyads for photochemical water splitting often suffer from instability during irradiation with visible light. However, the use of bis(bidentate) phosphines forming a five-membered ring enhances their stability. The coordination of these phosphor based chelates to soft metals like Pd(ii) prolongs the photocatalytic activity to 1000 hours. To avoid contribution to hydrogen production by colloidal metal, a small amount of Hg is added to the reaction mixture. In the course of our investigations, it turned out that colloidal palladium was not able to produce hydrogen under our irradiation conditions. As soon as metallic palladium emerged in our reaction vessels, no further hydrogen production was detected. This is confirmed by the observation that the hydrogen production depends on the kind of ancillary ligands present in the dyads. The first dyads of the type [MI(bpy)2(dppcb)MII(bpy)](4+) are presented (MI = Os, MII = Pd (1); MI = Ru, MII = Pd (2); MI = Os, MII = Pt (3); MI = Ru, MII = Pt (4)). In [Os(bpy)2(dppcb)Pd(dppm)](PF6)4 (5) the ancillary ligand is varied. Furthermore, it is also possible to produce hydrogen in an intermolecular way. Using different bidentate diphosphines instead of a bis(bidentate) tetraphosphine leads to this intermolecular approach, where the chromophore and the water reduction catalyst (WRC) belong now to two molecules. In this case the TON is sensitive to the type of diphosphine, which is only possible if intact molecules act as catalysts and no free palladium(0) is formed.

Tumor-specific diagnostic marker for transmissible facial tumors of Tasmanian devils: immunohistochemistry studies.

Devil facial tumor disease (DFTD) is a transmissible neoplasm that is threatening the survival of the Tasmanian devil. Genetic analyses have indicated that the disease is a peripheral nerve sheath neoplasm of Schwann cell origin. DFTD cells express genes characteristic of myelinating Schwann cells, and periaxin, a Schwann cell protein, has been proposed as a marker for the disease. Diagnosis of DFTD is currently based on histopathology, cytogenetics, and clinical appearance of the disease in affected animals. As devils are susceptible to a variety of neoplastic processes, a specific diagnostic test is required to differentiate DFTD from cancers of similar morphological appearance. This study presents a thorough examination of the expression of a set of Schwann cell and other neural crest markers in DFTD tumors and normal devil tissues. Samples from 20 primary DFTD tumors and 10 DFTD metastases were evaluated by immunohistochemistry for the expression of periaxin, S100 protein, peripheral myelin protein 22, nerve growth factor receptor, nestin, neuron specific enolase, chromogranin A, and myelin basic protein. Of these, periaxin was confirmed as the most sensitive and specific marker, labeling the majority of DFTD cells in 100% of primary DFTD tumors and DFTD metastases. In normal tissues, periaxin showed specificity for Schwann cells in peripheral nerve bundles. This marker was then evaluated in cultured devil Schwann cells, DFTD cell lines, and xenografted DFTD tumors. Periaxin expression was maintained in all these models, validating its utility as a diagnostic marker for the disease.

A murine xenograft model for a transmissible cancer in Tasmanian devils.

The number of Tasmanian devils in the wild is rapidly declining owing to a transmissible cancer, devil facial tumor disease (DFTD). Although progress has been made to understand the spread of this disease, crucial research on the pathogenesis of DFTD has been limited because of the threatened status of the host species. Here, the authors describe the development of a NOD/SCID (nonobese diabetic / severe combined immunodeficiency) mouse model that reproduces DFTD and provides a much-needed model to undertake studies into this intriguing transmissible cancer. Histologically, the DFTD produced in NOD/SCID mice (xenografted DFTD) was indistinguishable from the DFTD identified in Tasmanian devils. At the protein level, all xenografted DFTD tumors expressed periaxin, a marker that confirmed the diagnosis of DFTD. The karyotype of DFTD in NOD/SCID mice reproduced similar chromosomal alterations as seen in diseased devils. Furthermore, each NOD/SCID mouse inoculated with cultured DFTD tumor cells developed tumors, whereas DFTD did not develop in any of the inoculated immune-competent BALB/c mice.

Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis is a fungus belonging to the Phylum Chytridiomycota, Class Chytridiomycetes, Order Chytridiales, and is the highly infectious aetiological agent responsible for a potentially fatal disease, chytridiomycosis, which is currently decimating many of the world's amphibian populations. The fungus infects 2 amphibian orders (Anura and Caudata), 14 families and at least 200 species and is responsible for at least 1 species extinction. Whilst the origin of the agent and routes of transmission are being debated, it has been recognised that successful management of the disease will require effective sampling regimes and detection assays. We have developed a range of unique sampling protocols together with diagnostic assays for the detection of B. dendrobatidis in both living and deceased tadpoles and adults. Here, we formally present our data and discuss them in respect to assay sensitivity, specificity, repeatability and reproducibility. We suggest that compliance with the recommended protocols will avoid the generation of spurious results, thereby providing the international scientific and regulatory community with a set of validated procedures which will assist in the successful management of chytridiomycosis in the future.

Serological responses against the pathogenic dimorphic fungus Mucor amphibiorum in populations of platypus (Ornithorhynchus anatinus) with and without ulcerative mycotic dermatitis.

Mucor amphibiorum, a dimorphic fungus, causes ulcerative dermatitis and systemic infections in the platypus Ornithorhynchus anatinus in some river systems in Tasmania but apparently not in other regions of Australia. As yet there are no suitable tests for population surveys, nor for detection of internal lesions in live animals. Consequently, immunoglobulins were purified from the serum of platypuses and anti-immunoglobulin antisera were prepared in rabbits in order to develop an enzyme-linked immunosorbent assay (ELISA) for anti-M. amphibiorum antibodies. Antigens from plate-grown cultures resulted in greater signal-to-noise ratios in indirect ELISA than those from broth-grown cultures. Platypuses with clinical ulcerative dermatitis had elevated anti-Mucor antibody levels compared to apparently unaffected individuals. Seroconversion was observed in one animal coincident with the development of cutaneous ulcers. The results suggested that platypuses in affected rivers were exposed to M. amphibiorum at a higher frequency than the occurrence of clinical disease. Some platypuses from New South Wales had elevated antibody levels but these increased significantly with age suggesting exposure to cross-reactive antigens, although exposure to M. amphibiorum cannot be excluded. Further studies are warranted to determine factors that result in progression from infection to disease, the occurrence of the fungus in areas where disease has not been observed and the specificity of antigen used in ELISA.

Surveillance and monitoring of wildlife diseases.

It is now recognised that those countries which conduct disease surveillance of their wild animal populations are more likely to detect the presence of infectious and zoonotic diseases and to swiftly adopt counter measures. The surveillance and monitoring of disease outbreaks in wildlife populations are particularly relevant in these days of rapid human and animal translocation, when the contact between wild and domestic animals is close and the threat of a bioterrorist attack is very real. The authors describe the problems inherent in wildlife disease surveillance and stress the importance of the establishment of national strategies for disease detection. The various sampling methods employed for monitoring outbreaks of disease and mortality in wildlife populations are discussed and their strengths and weaknesses described. A major advantage of an efficient disease monitoring programme for wildlife is the early detection of new and 'emerging' diseases, some of which may have serious zoonotic and economic implications. The authors conclude that wildlife disease monitoring programmes that are integrated within national animal health surveillance infrastructures should have the capacity to respond promptly to the detection of unusual wildlife mortality and to institute epizootiological research into new and emerging wildlife diseases.

HPLC with simultaneous UV and reductive electrochemical detection at the hanging mercury drop electrode: a highly sensitive and selective tool for the determination of benzodiazepines in forensic samples.

A versatile, sensitive, and selective high-performance liquid chromatographic (HPLC) procedure for the determination of common benzodiazepines and some of their most frequently occurring metabolites in forensic samples was developed and optimized with respect to effective and rapid sample preparation and high selectivity of the analytical assay. The optimized method includes liquid-liquid extraction of the drugs with chloroform followed by isocratic reversed-phase chromatography on a LiChrospher-100 RP-8ec column (150 x 4.6-mm i.d.) with a mobile phase consisting of 0.03 mol/L acetate buffer (pH 4.6)/acetonitrile (55:45, v/v). The use of dual-mode detection made up of UV-detection (250 nm) in series with reductive electrochemical detection (-1.4 V vs. Ag/AgCl) at the hanging mercury drop electrode permits the detection and quantitation of benzodiazepines even in degraded samples with higher selectivities than usually reached with conventional HPLC techniques. Depending on the actual benzodiazepine species, detection limits are in the range of 2.0 to 14.1 ng/mL. Mean recovery values of the drugs from blood range from 82 to 92%; within-day and day-to-day repeatabilities typically lie between 3 and 9%. Several case work examples demonstrate the high selectivity and remarkably low matrix sensitivity of the described assay.

Selective and sensitive assay for the determination of benzodiazepines by high-performance liquid chromatography with simultaneous ultraviolet and reductive electrochemical detection at the hanging mercury drop electrode.

An isocratic chromatographic method for the simultaneous determination of 10 benzodiazepines is presented. The selectivity of the assay was optimized by variation of stationary phase, temperature, as well as ionic strength, composition and pH of the mobile phase and the dependence of the detector response on the applied potential was investigated. The best results with respect to resolution at moderate retention times were obtained with a mixture of 0.02 mol/l phosphate buffer (pH 6) and acetonitrile in a volume ratio of 55:45 (v/v) on a LiChrospher-100 RP-8ec column (150x4.6 mm I.D.). Considerable improvement of selectivity was achieved if the column temperature was kept constant at 12 degrees C. Two detection modes were applied, UV detection at 250 nm, inserted upstream to the electrochemical detector, and reductive electrochemical detection at the hanging mercury drop electrode at -1.4 V (vs. Ag/AgCl), which proved to be especially sensitive in case of nitrophenyl-containing benzodiazepine species. The elaborated assay showed to be linear up to at least 2 mg/l for each compound. Detection limits generally were in the range of 6.5-123 ng/ml (130 pg-2.46 ng on-column, using a 20-microl loop) with relative standard deviations between 1.1 and 8.6% depending on the actual benzodiazepine investigated.

Gross, histological and immunohistochemical features of mucormycosis in the platypus.

Nine male and five female adult free-living platypuses, obtained in a prospective capture-release study from northern Tasmania, exhibited gross features of cutaneous mycosis caused by Mucor amphibiorum. The lesions were present on the hind limbs (six cases), front limbs (four), tail (five), dorsal trunk (three) and ventral trunk (one). They varied in size, and ranged from raised red nodules or plaques, which sometimes exuded purulent material, to ulcerated lesions with central cavitation, red exuding centres and raised epidermal margins. Older lesions were covered either partly or fully by thickened and irregular epidermis. Histological examination of skin biopsies revealed discrete, poorly encapsulated granulomas, or more commonly a diffuse granulomatous or pyogranulomatous inflammation. Inflammatory cells consisted of neutrophils or eosinophils, sparse plasma cells and lymphocytes, many macrophages and occasional multinucleated giant cells. Fibrovascular tissue was diffusely and irregularly scattered in the granulomatous regions. Sphaerules characteristic of M. amphibiorum infection were observed in all lesions. The cutaneous distribution of the lesions and the natural history of the platypus indicated that entry of M. amphibiorum may have been via superficial skin wounds. T cells were the predominant infiltrating lymphoid cells in the diffuse lesions, indicating the importance of the cell-mediated response to infection.

Pathology of experimental toxoplasmosis in eastern barred bandicoots in Tasmania.

Wild-caught eastern barred bandicoots (Perameles gunnii) initially seronegative to Toxoplasma gondii, were inoculated orally with approximately 100 T. gondii oocysts. The bandicoots were maintained in indoor pens under laboratory conditions and observed daily. Serial blood samples were tested for agglutinating antibodies to T. gondii. Inoculated bandicoots died 15 and 17 days post infection. A rise in Direct Agglutination Test (DAT) titres was detected at the time of death (1:256, 1:64 respectively). Clinical observations, serological changes, gross findings at necropsy, and histopathological changes were consistent with acute toxoplasmosis. The findings indicate that eastern barred bandicoots are likely to die from primary T. gondii infection, often even before detectable antibodies are produced, reinforcing the significance of toxoplasmosis as a potential contributor to the reduction in numbers of wild populations of eastern barred bandicoots.

Earthworms as paratenic hosts of toxoplasmosis in eastern barred bandicoots in Tasmania.

An experimental feeding study was designed to assess the role of earthworms in the transmission of Toxoplasma gondii infection to eastern barred bandicoots (Perameles gunnii). Six animals with no agglutinating antibodies to T. gondii were fed artificially cultured earthworms that had been maintained in autoclaved nutrient-enriched soil. Two animals were given earthworms that had been maintained in soil contaminated with T. gondii oocysts (P89/VEG strain); two animals were fed on earthworms, which initially had been exposed to soil containing T. gondii oocysts then transferred through three changes of sterile soil; two control bandicoots were fed earthworms maintained in sterile soil. Both bandicoots fed earthworms maintained in T. gondii contaminated soil died 11 and 14 days after feeding. The necropsy findings were consistent with acute toxoplasmosis. Bandicoots fed earthworms exposed to oocysts but then transferred through changes of sterilized soil remained healthy as did control animals. All surviving animals remained seronegative over the 6 wk observation period after feeding. These findings confirm that earthworms, a major component of the natural diet of P. gunnii, can transmit T. gondii infection. It appears that oocysts present in the alimentary tracts of the worms, rather than infective stages of T. gondii in worm somatic tissues, are responsible for these infections.

Haematological, serum biochemical and serological features of platypuses with and without mycotic granulomatous dermatitis.

OBJECTIVE: To determine whether there are haematological, serum biochemical and serological differences between platypuses (Ornithorhynchus anatinus) with and without granulomatous dermatitis due to Mucor amphibiorum infection. An additional objective was to establish reference haematological and serum biochemical ranges for the species in Tasmania. DESIGN: A clinicopathological and serological study. ANIMALS: A total of 37 free-living adult platypuses captured from streams and dams in Northern Tasmania were used in the clinicopathological study. Twenty-seven were clinically normal and 10 had mycotic granulomatous dermatitis. A total of 22 platypuses (20 adult and 2 juvenile) were used for the serosurvey. Eighteen were captured from streams in Northern Tasmania, and four were submitted for necropsy. RESULTS: Platypuses with mycotic ulcerative dermatitis had significantly smaller packed red cell volumes, haemoglobin concentrations, lymphocyte counts, serum cholesterol and calcium concentrations, and higher serum globulin and potassium concentrations than clinically normal animals. The lymphopenia and hyperkalaemia were thought to be clinically significant. Numbers of Trypanosoma binneyi in blood smears were similar between the two groups. Diseased platypuses had higher concentrations of serum antibody against Mucor amphibiorum as determined by ELISA compared to clinically normal platypuses. CONCLUSION: Platypuses affected by mycotic granulomatous dermatitis showed haematological and serum biochemical changes when compared to clinically normal animals from the same Tasmanian sites. A serological survey may be a useful method for detecting the prevalence of exposure to Mucor amphibiorum and humoral immunity in platypus populations both in Tasmania and the mainland of Australia.

Determination of Sn(II) in technetium cold kits by voltammetry at the hanging mercury drop electrode (HMDE) and relevant radiopharmaceutical applications.

A novel approach for the determination of the stannous content in cold kits for labelling with 99mTc is described. The method is based on differential pulse polarography on the hanging mercury drop electrode in a methanol/water/perchloric acid mixture and is easy to perform. Examples for the determination of tin(II) in fractionated technetium cold kits are shown. The stability of tin(II) in solution was mainly dependent on the storage temperature and the kit composition. The low stability of stannous ions under certain conditions was shown to be the main reason for low radiochemical purity. Limits and dangers of fractionating kits are discussed and related to content and instability of tin(II).

Comparison of the inflammatory responses of mice infected with American and Australian Trichinella pseudospiralis or Trichinella spiralis.

This study was designed to determine if the Tasmanian devil isolate of Trichinella pseudospiralis suppressed inflammation as does the original isolate. While adult worm numbers were similar in all groups, lower enteritis occurred in devil isolate-infected mice compared with mice infected with the original isolate of T. pseudospiralis or with Trichinella spiralis. Diaphragm muscle inflammation was greater in T. spiralis-infected than in mice infected with either isolate of T. pseudospiralis or concurrently infected with T. spiralis and either of the isolates of T. pseudospiralis. Granuloma inflammation was lower in mice infected with either isolate of T. pseudospiralis compared with uninfected or T. spiralis-infected mice. The devil isolate down-regulated inflammation more profoundly during the intestinal phase than the muscle phase compared to the original isolate, differences which may be related to the biology of their natural hosts.

Direct determination of oxygen by HPLC. 1. Basic principles of a sensitive and selective oxygen sensor.

The basic principles of a novel, versatile, sensitive, and selective oxygen-sensing assay are presented in this paper. For the first time, liquid chromatography with electrochemical detection (at the hmde) has been used for the determination of oxygen. All factors concerning optimization of the chromatographic separation conditions and electrochemical detection with respect to direct determination of oxygen even in complex biological samples are discussed. Due to the combination of a chromatographic technique with amperometric detection, a high selectivity can be achieved. A direct and linear relationship between the oxygen concentration in the sample and the reduction current was verified in a large concentration range from saturation down to trace level oxygen concentrations. The novel oxygen-sensing assay provides a much higher sensitivity compared to conventional oxygen sensors. In principle, O(2) concentrations down to 4.5 × 10(-)(9) mol L(-)(1) O(2) (corresponding to a signal-to-noise ratio of 3) can be detected. Precision was determined by repeated measurements (n = 6) of air-saturated solutions (2.5 × 10(-)(4) mol L(-)(1) O(2), 20 °C, 920 mbar) which yielded relative standard deviations of lower than 0.2%.

Direct determination of oxygen by HPLC. 2. Chamber and sample application system for determination of o(2) at trace levels.

All oxygen measurement systems so far available are characterized by a lack of suitable precision and/or required limit of detection, which would be essential for a great variety of applications. In this paper, a novel oxygen chamber together with a completely new concept of sample application ("two-chamber-siphon technique") is presented which can be used in combination with the previously reported chromatographic oxygen sensor (part 1). This new oxygen-sensing assay exhibits several advantages in comparison to conventional oxygen measurement systems: e.g., the uncontrollable influence of the surrounding atmosphere as well as oxygen consumption and storage processes are excluded. For the first time, measurements of molecular oxygen below 1 × 10(-)(7) mol L(-)(1) can be performed. Reliable quantification of oxygen in liquids and also in gaseous and solid samples can be achieved with utmost sensitivity (LOD 4.9 × 10(-)(9) mol L(-)(1) O(2) = 98 fmol of oxygen on column) and precision (RSD = 0.7%, n = 8).

Detection of agglutinating antibodies to Toxoplasma gondii in sera from free-ranging eastern barred bandicoots (Perameles gunnii).

Sera from 150 eastern barred bandicoots (Perameles gunnii) were collected from two study sites in southern Tasmania between 1992 and 1995. Samples were tested for antibodies to the protozoan parasite, Toxoplasma gondii, using formalin-treated tachyzoites as the antigen in direct (DAT) and modified agglutination tests (MAT). Cut-off titers were set based on confirmed cases of toxoplasmosis in this species. A total of 133 animals (89%) were classified as negative, seven (4.6%) had suspicious reactions, and 10 (6.7%) were diagnosed as positive. Five of the 10 positive animals were not retrapped after initial seroconversion; another three animals recorded high MAT titers on two consecutive bleedings, three months apart. Of the remaining two sero-positive bandicoots, one was found dead in a trap and generalized toxoplasmosis was diagnosed at necropsy, while the other animal had central nervous system disabilities consistent with toxoplasmosis but was accidently released and never recaptured. Based on these findings we propose that eastern barred bandicoots are likely to be highly susceptible to primary T. gondii infection.

Protozoan parasites and pathological findings in Hydromys chrysogaster (Muridae: Hydromyinae) from Queensland.

Tissues from 23 Australian water rats (Hydromys chrysogaster) collected from five localities in central and northern Queensland, Australia, between February 1992 and May 1993, were examined for protozoan parasites and additional pathological changes. We found Klossiella hydromyos in the kidneys, Toxoplasma gondii in the brain and skeletal muscles and Sarcocystis sp. in the somatic musculature. Other pathological findings, including interstitial nephritis, interstitial pneumonia and a tongue abscess, as well as helminth-induced lesions in the lungs, mesenteries, stomach wall and cecal wall were also noted.

Determination of trace levels of niguldipine in urine and blood by adsorptive stripping voltammetry at the hanging mercury drop electrode.

A relatively simple electroanalytical procedure for the determination of niguldipine in biological samples is described. The technique involves adsorptive accumulation of the drug at the hanging mercury drop electrode (HMDE) followed by a differential-pulse polarographic determination of the preconcentrated species. The adsorptive stripping response is evaluated with respect to various experimental conditions, such as solvent composition and pH of the supporting electrolyte, accumulation potential and accumulation time. After a simple sample preparation, the method can be used for the determination of niguldipine in blood and urine. Interfering substances are simply removed by precipitation, adding a small amount of 5% ZnSO4 solution and ethanol to the urine or blood sample and centrifuging the mixture. A limit of detection of 6.7 ng per ml of urine and 41 ng per ml of blood is found with a mean recovery of 96% in urine and 71% in blood. The mean relative errors are 8.4% and 2.2%, respectively.

Adsorptive stripping voltammetry of nicardipine at a HMDE; determination of trace levels nicardipine in blood and urine.

The redox behaviour of nicardipine, a 1,4-dihydropyridine calcium antagonist, has been studied in different media on mercury, glassy carbon, gold and platinum electrodes using various voltammetric techniques. A highly sensitive adsorptive stripping voltammetric method for the determination of nicardipine based on adsorption of the drug onto mercury, followed by differential pulse voltammetric determination of the surface species, is described. All factors (pH, supporting electrolyte, accumulation potential and time, etc.) influencing adsorption as well as voltammetric response are discussed. The application of adsorptive stripping voltammetry at the hanging mercury drop electrode (HMDE) to the determination of trace levels of nicardipine in human urine and blood is illustrated, without an extraction procedure being necessary prior to the voltammetric measurement. A limit of detection of 4.8 ng per ml urine and 34 ng per ml blood is found with a mean recovery of nicardipine in urine and blood of 97%. The mean relative error does not exceed 6.5%.