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.

Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay.

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.

Cryo-archiving of Batrachochytrium dendrobatidis and other chytridiomycetes.

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 degrees 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.

Study of factors mediating effect of photodynamic therapy on bladder in canine bladder model.

The canine bladder model was employed to study the factors mediating the effect of photodynamic therapy (PDT) on the bladder. The recovery (time taken for the bladder volume to return to pre-PDT value), gross and microscopic findings, implicate both bladder high filling pressure (60 cm H2O) and high light dose as factors mediating the effect of photodynamic therapy on bladder capacity. We recommend that photodynamic therapy to the bladder be performed under a filling pressure of 30 cm H2O, which is physiologic, and whole bladder illumination at a light dose not greater than 30 J/cm2.

Photodynamic therapy in patients with colorectal cancer.

A pilot study was conducted, in which photodynamic therapy (PDT), a technique in which malignant cells are destroyed by light after being previously photosensitized by a chemical compound, was tried in a group of 14 patients with recurrent or residual colorectal cancer in the pelvis. Three of the six patients with unresectable pelvic recurrences experienced a significant relief of pain after PDT. In two of the five patients who had an incomplete resection of their pelvic recurrences, there was also a substantial relief of pelvic pain after surgery and PDT. In one of these patients subsequent biopsies proved the disappearance of the residual pelvic microscopic disease after several sessions of PDT. Three patients had a recurrence from a squamous cell carcinoma primary of the anal canal. All recurrences were amenable to surgical resection. In one of the patients, PDT was used in an attempt to sterilize an area of residual tumor that was located over the left ischial tuberosity. The patient experienced good relief of pain, but died of her disease 7 months after PDT. In the other two patients, PDT was used as an "adjunct" after resection of their recurrences. One of these patients was free of disease and died of an unrelated cause 12 months after PDT. The other is alive and well. This study demonstrated that PDT can be safe and tolerable in patients with pelvic malignancies. PDT is capable of tumor destruction, can be used repeatedly in areas previously exposed to ionizing radiation, and may have a role in the prevention and management of pelvic-perineal recurrences from colorectal cancer.

Whole bladder photodynamic therapy for transitional cell carcinoma of bladder.

Our preliminary studies indicate that the bulb-tip technique for whole bladder photodynamic therapy (PDT) illuminates the entire bladder mucosa and is applicable to the management of superficial transitional cell carcinoma of the bladder. This treatment modality may be an option to patients who are failures to other standard treatments. A randomized clinical study is needed to decide on PDT as a primary treatment of choice for transitional cell carcinoma of the bladder.

Photoradiation therapy in advanced carcinoma of the trachea and bronchus.

Photoradiation therapy is a new technique being investigated for the treatment of solid malignant tumors. In this study, 17 patients with advanced, recurrent, biopsy-proven malignant lesions of the trachea or main-stem bronchus were treated by photoradiation therapy. Patients received hematoporphyrin derivative intravenously three days prior to light therapy. The light was delivered from a fiberoptic fiber attached to the output beam of a dye laser (633 +/- 3 nm). The fiber was passed through the large channel of a bronchoscope (Olympus BF 2T). Of the 17 patients, two had no measurable response to the photoradiation therapy, six had partial necrosis of the tumor, seven patients had a greater than 50 percent reduction in the intraluminal volume of tumor, and two were lost to follow-up. Survival ranged from 5 to 210 days (median survival, 40 days). Complications of the treatment were significant in this group of advanced-stage patients and included excessive secretions, fever, pneumonia, and abscess formation.

Accessory cell and HLA compatibility requirements for the generation of specific in-vitro antibody responses to influenza virus by human blood lymphocytes.

The role of antigen presenting cells in specific in-vitro antibody responses to influenza virus by human lymphocytes was investigated. Demonstration of a requirement for accessory cells necessitated vigorous depletion procedures. Accessory cells were removed by passage of mononuclear cells through a Sephadex G10 column followed by separation of cells bearing antigens defined by the monoclonal antibodies OKM1 and UCHM1 by fluorescence-activated cell sorting or panning. Peripheral blood mononuclear cells treated in this way were unable to respond to influenza virus by antibody production in either round-bottom or flat-bottom culture wells. The response could be reconstituted by either irradiated plastic-adherent cells or G10 non-adherent OKM1/UCHM1 positive cells in both autologous or allogeneic (HLA-DR different) combinations. The absence of HLA restriction was not due to non-specific allogeneic effects and was confirmed using irradiated, antigen-pulsed accessory cells which were extensively washed, incubated overnight and washed again to remove free antigen. These data suggest that the normal T-cell repertoire contains T helper cells specific for antigen plus self and antigen plus allo MHC.

Interstitial photoradiation therapy for primary solid tumors in pet cats and dogs.

Photoradiation therapy, a new method for treatment of solid malignant tumors, depends upon the tumor localization and retention of hematoporphyrin derivative, which is activated in vivo by light in the red region of the spectrum. As currently applied to cutaneous and s.c. lesions, the light dose is limited by both normal tissue reactions and the effective penetration of the light through the tissues. In this report, primary solid malignant lesions in pet cats and dogs have been treated by interstitial photoradiation therapy by applying the activating light from a laser [635 +/- 5 (S. D.) nm] directly into the tumor masses thrugh a 200-micrometer quartz fiber optic. Twelve of 14 lesions (four osteosarcomas, two squamous cell carcinomas, two malignant melanomas, one mast cell sarcoma, one fibrosarcoma, one sebaceous gland sarcoma, and a metastatic prostatic carcinoma) responded to treatment, and three are currently considered permanently controlled at 1 year or more following treatment. This method has not only allowed photoradiation therapy to be applied to some remote lesions but has also nearly eliminated normal tissue effects, thus greatly extending the applicability of this treatment to a wide range of human tumors.

Photoradiation therapy. II. Cure of animal tumors with hematoporphyrin and light.

Exposure of mouse and rat tumors of various types to more than 600 nm light 24 or 48 hours after an injection of hematoporphyrin resulted in a substantial number of long-term cures. Since hematoporphyrin is preferentially retained in tumor tissue, selective tumor destruction could be obtained. Light penetration studies and the high efficiency of this technique indicated its applicability even to certain deep-seated human tumors.