Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide, associated with declines in amphibian populations. Diagnosis of chytridiomycosis to date has largely relied upon histological and immunohistochemical examination of toe clips. This technique is invasive and insensitive particularly at early stages of infection when treatment may be possible. We have developed a real-time PCR Taqman assay that can accurately detect and quantify one zoospore in a diagnostic sample. This assay will assist the early detection of B. dendrobatidis in both captive and wild populations, with a high degree of sensitivity and specificity, thus facilitating treatment and protection of endangered populations, monitoring of pristine environments and preventing further global spread via amphibian trade. KEY WORDS: Chytrids · Batrachochytrium dendrobatidis · Amphibian declines · Real-time PCR Taqman assay · Chytridiomycosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 60: [141][142][143][144][145][146][147][148] 2004 examination via haematoxylin and eosin staining of toe clips or skin scrapings (Daszak et al. 1999, see also www.jcu.edu.au/school/phtm/phtm/frogs/histo/ chhisto.htm). Immunoperoxidase staining with polyclonal antibodies to B. dendrobatidis increases the specificity and sensitivity of detection (Berger et al. 2002). More recently, a staining protocol has been described which enhances diagnosis by the colocalisation of B. dendrobatidis and keratin in the skin of amphibians (V. Olsen, D. Boyle, D. Mendez, A. D. Hyatt unpubl.). However, the drawbacks of histological testing include the degree of expertise needed for identification, the invasiveness of sampling technique -typically performed by toe-clipping of live animals -the length of time required, the low sensitivity of the test, and the variability of infection levels amongst toes. In the field, examination of oral disc abnormalities in tadpoles with a 10 × hand lens has been recommended as a preliminary indication of chytridiomycosis (Fellers et al. 2001), although these abnormalities can also be caused by DDT intoxication. The fungus can be identified by isolation and culture , but this requires a high degree of expertise and time.Early detection of Batrachochytrium dendrobatidis is vital to the control of spread of the disease via global amphibian trade. B. dendrobatidis has been identified in animals imported for zoo collections , in international pet trade (Mutschmann et al. 2000), in the food trade (Mazzoni et al. 2003) and in laboratory animal trade (Parker et al. 2002). Early detection of infection would allow possible curative treatment to be undertaken, at least in captive populations where formalin/malachite green solution has been shown to be effective on adult Xenopus tropicalis (Parker et al. 2002). Itraconazole baths (0.01%) were found to be effective in treating the terrestrial species Dendrobates tinctorius (Nichols et al. 2001), although whether this treatment is effective and safe...

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.

Chytridiomycosis is a major cause of mortality in free-living and captive amphibians in Australia and mortality rate increases at lower temperatures.

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide. It has been associated with catastrophic declines of frog populations including those in pristine habitats in Queensland, Australia. To facilitate genetic and disease studies of this fungus and related species, it is essential to have a reliable long-term storage method to maintain genetic integrity of isolates. We have adapted well-established techniques used for the long-term storage of tissue-culture cell lines to the preservation of B. dendrobatidis and other chytridiomycetes. This simple method has allowed us to recover these fungi from storage at -80°C and in liquid nitrogen over an extended period. With this technique it is now possible to preserve saprobic and parasitic isolates from a variety of environmental and disease situations for comparative genetic and biological studies. KEY WORDS: Chytrids · Batrachochytrium dendrobatidis · Storage · Cryopreservation · Amphibian declines Resale or republication not permitted without written consent of the publisherDis Aquat Org 56: [59][60][61][62][63][64] 2003 to the origin and spread/transmission of disease and specific cause of death must be addressed. To address these questions it is essential that the many isolates be effectively frozen and archived until required. The development of such a procedure would be invaluable for future research as questions relating to attenuation (via passage), contamination and alterations in the genome (e.g. site mutations) can be minimized. B. dendrobatidis can be isolated from infected frogs using TGhL agar and then maintained on agar or in TGhL broth at 23°C (Longcore et al. 1999). Cultures also grow well at 15°C and survive many months at 4°C. These procedures are, however, labour intensive since cultures require regular examination for contamination, growth failure and passage every 14 to 21 d on nutrient agar plates at room temperature or every 4 to 5 mo when grown in liquid medium and stored at 4 to 6°C.Previous methods for long-term storage of Chytridiomycetes at low temperatures have produced survival rates of only 16% (Smith 1982) while Hohl & Iselin (1986) had limited success with storage in liquid nitrogen. We have developed a simple method of freezing, based on established methods for freezing tissue-culture cell lines that have had 100% success in retrieval of Batrachochytrium dendrobatidis from storage over 12 mo. The method was also successfully applied to other fungi from the phylum Chytridiomycota. MATERIALS AND METHODSCulture and harvest of Batrachochytrium dendrobatidis. TGhL (13 ml) broth (16 g tryptone, 4 g gelatin hydrolysate, 2 g lactose, 1000 ml distilled water) in a 25 cm 2 disposable tissue-culture flask was inoculated with 2 ml of an actively growing 7 d old broth culture of B. dendrobatidis (Isolate A98 1810/3) and incubated at 17 to 23°C for 3 to 21 d (Longcore et al. 1999). All cultures contained active released zoospores and sporangia (empty or containing zoospores), both in the media and attached to the plasti...

Polyclonal antibodies were produced for diagnosing chytridiomycosis in amphibians. Two sheep and 4 rabbits were inoculated with homogenized whole culture of Batrachochytrium dendrobatidis in Freund's complete adjuvant or triple adjuvant. Antisera from all animals reacted strongly with all stages of B. dendrobatidis and stained the walls, cytoplasm, rhizoids and zoospores in an indirect immunoperoxidase test. Significant cross-reactivity occurred only with some fungi in the Chytridiomycota, and there are no members of this phylum besides B. dendrobatidis that infect frogs. The immunoperoxidase stain is a useful screening test when combined with recognition of the morphology and infection site of B. dendrobatidis. KEY WORDS: Batrachochytrium dendrobatidis · Chytridiomycosis · Fungus · Amphibians · Immunoperoxidase · Polyclonal antibodies · Diagnosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 48: [213][214][215][216][217][218][219][220] 2002 diagnosis is insensitive when dealing with light infections in healthy animals or autolysed samples, or when tests are performed by inexperienced workers.Diagnostic methods with improved sensitivity and ease of testing are needed. The production of polyclonal antibodies and the introduction of an immunoperoxidase (IPX) stain are the first steps in the development of more sophisticated tests for the detection of antigen. MATERIALS AND METHODS Antigen for immunization.A culture of Batrachochytrium dendrobatidis (isolate A98 1810/3) was obtained from a sick, wild adult Australian lacelid (Nyctimystes dayi). The culture was maintained on tryptone, gelatin hydrolysate agar ) for 5 mo before use. Sporangia were harvested by lightly scraping a 10 d old culture. Thirty milligrams of culture was mixed with 1 ml distilled water and left for 24 h at room temperature, then manually homogenized in a sterile petri dish and frozen at -80°C. After defrosting, the mixture was diluted with phosphate buffered saline (calciumand magnesium-free) (PBSA) to a final concentration of 3.25 mg ml -1 . The protein concentration was determined using a Pyr Unicam PU8800 UV/VIS spectrophotometer at 280 nm, by interpolation of its absorbance from a standard curve calculated from known concentrations of bovine serum albumin (0.25, 0.5, 1.0 and 5.0 mg ml -1 ). Freund's complete adjuvant and triple adjuvant (Quil A, DEAE-dextran, Montanide 888 oil) (Prowse 2000) were prepared using a Sorvall Omnimixer for emulsification. The final protein concentration of antigen in both preparations was 0.5 mg ml -1 antigen.Immunization. Two rabbits (666 and 667) and 1 sheep (322) were inoculated with triple adjuvant intradermally with boosters at 7 and 11 wk post inoculation (pi). Two rabbits (668 and 669) and 1 sheep (386) were inoculated with Freund's complete adjuvant subcutaneously and boosted with Freund's incomplete adjuvant at 7 and 11 wk pi. At each inoculation, the rabbits received 0.5 mg fungus in 1 ml adjuvant and the sheep received 1 mg fungus in 2 ml...

Archey's frog Leiopelma archeyi is a critically endangered New Zealand endemic species. The discovery of the emerging infectious disease, chytridiomycosis, in wild populations of this frog raised concern that this disease may drive the species to extinction. Twelve wild-caught Archey's frogs naturally infected with the amphibian chytrid fungus Batrachochytrium dendrobatidis were monitored in captivity by observing clinical signs, measuring weight gain, and performing repeated PCR tests. Eight frogs were treated with topical chloramphenicol, without PCR results being available, for B. dendrobatidis at the day of entry of the frog into the trial. Eleven of the 12 frogs (92%) cleared their infection within 3 mo of capture, even though they were held at 15°C and in high humidity, conditions that are ideal for the survival and propagation of B. dendrobatidis. B. dendrobatidis in the remaining frog tested positive for the fungus was eliminated after treatment with topical chloramphenicol. None of the 8 frogs exposed to chloramphenicol showed any acute adverse reactions. Archey's frog appears to have a low level of susceptibility to the clinical effects of chytridiomycosis. Individual frogs can eliminate B. dendrobatidis and Archey's frog can apparently be treated with topical chloramphenicol with no acute adverse reactions. However, the small number of specimens treated here requires that more extensive testing be done to confirm the safety of chloramphenicol. The significance of the amphibian chytrid fungus for wild populations of Archey's frog needs to be determined by a longitudinal study in an infected wild population to correlate the presence of B. dendrobatidis in individual frogs. Such a study should occur over a period of at least 3 yr with clinical assessment and monitoring of survival, growth and body condition parameters.

Over 850,000 people living in the United Kingdom have been diagnosed with dementia, yet knowledge about this condition amongst the general population remains relatively poor. Many studies have evaluated the level of public knowledge and understanding about dementia from a research and professional service perspective, however none have considered this condition from the perspective of the wider public. In this preliminary overview, we analyse and describe high level narratives collected from 143 respondents to a dementia Directive commissioned to the Mass Observation Project. These narratives present a perspective on the public knowledge and understanding about dementia not previously considered, where respondents have written openly about their own experiences, and reflected on their perception of the wider public's knowledge and understanding about dementia. This unique perspective importantly enhances our knowledge about the public's understanding and awareness of dementia, and informs the main areas of public concern found in the analysis: care responsibilities, impact on relationships, and fears about developing dementia.

Floods become disasters when people and property are placed in harm's way. Yet stakeholders, those at risk of flooding, often take no action to reduce their vulnerability. We demonstrated an approach for improving flood risk communication using the process of realistic interactive visualisation. Our goal was to communicate information about flood risk at the community level and increase stakeholders’ intent to take actions to reduce their risk. Realistic visualisation promotes action based on emotional connection to images. Interactive visualisation is the direct construction of the model by stakeholders. As a reference, we also tested a nationally‐recognised model designed for the U.S. Federal Emergency Management Agency (FEMA). Both methods resulted in significant learning about community‐specific flood risk and risk reduction options. To maximise the intent by stakeholders to take actions to reduce risk, the realistic interactive visualisation method used in high‐quality meeting facilities performed best.