Two new frog species from the Litoria rubella species group from eastern Australia.

The bleating tree frog (Litoria dentata) is one of the more prominent pelodryadid frogs of eastern Australia by virtue of its extremely loud, piercing, male advertisement call. A member of the Litoria rubella species group, L. dentata has a broad latitudinal distribution and is widespread from coastal and subcoastal lowlands through to montane areas. A recent mitochondrial DNA analysis showed a deep phylogeographic break between populations of L. dentata on the mid-north coast of New South Wales. Here we extended the mitochondrial survey with more geographically comprehensive sampling and tested the systematic implications of our findings with nuclear genome wide single-nucleotide polymorphism, morphological and male advertisement call datasets. While similar in appearance and in male advertisement call, our integrative analysis demonstrates the presence of three species which replace each other in a north-south series. We redescribe Litoria dentata, which is restricted to coastal north-eastern New South Wales, and formally describe Litoria balatus sp. nov., from south-eastern Queensland, and Litoria quiritatus sp. nov., from the mid-coast of New South Wales to north-eastern Victoria.

Evidence of a salt refuge: chytrid infection loads are suppressed in hosts exposed to salt.

With the incidence of emerging infectious diseases on the rise, it is becoming increasingly important to identify refuge areas that protect hosts from pathogens and therefore prevent population declines. For the chytrid fungus Batrachochytrium dendrobatidis, temperature and humidity refuge areas for amphibian hosts exist but are difficult to manipulate. Other environmental features that may affect the outcome of infection include water quality, drying regimes, abundance of alternate hosts and isolation from other hosts. We identified relationships between water bodies with these features and infection levels in the free-living hosts inhabiting them. Where significant relationships were identified, we used a series of controlled experiments to test for causation. Infection loads were negatively correlated with the salt concentration of the aquatic habitat and the degree of water level fluctuation and positively correlated with fish abundance. However, only the relationship with salt was confirmed experimentally. Free-living hosts inhabiting water bodies with mean salinities of up to 3.5 ppt had lower infection loads than those exposed to less salt. The experiment confirmed that exposure to sodium chloride concentrations >2 ppt significantly reduced host infection loads compared to no exposure (0 ppt). These results suggest that the exposure of amphibians to salt concentrations found naturally in lentic habitats may be responsible for the persistence of some susceptible species in the presence of B. dendrobatidis. By manipulating the salinity of water bodies, it may be possible to create refuges for declining amphibians, thus allowing them to be reintroduced to their former ranges.

Comparison of modelling techniques for milk-production forecasting.

The objective of this study was to assess the suitability of 3 different modeling techniques for the prediction of total daily herd milk yield from a herd of 140 lactating pasture-based dairy cows over varying forecast horizons. A nonlinear auto-regressive model with exogenous input, a static artificial neural network, and a multiple linear regression model were developed using 3 yr of historical milk-production data. The models predicted the total daily herd milk yield over a full season using a 305-d forecast horizon and 50-, 30-, and 10-d moving piecewise horizons to test the accuracy of the models over long- and short-term periods. All 3 models predicted the daily production levels for a full lactation of 305 d with a percentage root mean square error (RMSE) of ≤ 12.03%. However, the nonlinear auto-regressive model with exogenous input was capable of increasing its prediction accuracy as the horizon was shortened from 305 to 50, 30, and 10 d [RMSE (%)=8.59, 8.1, 6.77, 5.84], whereas the static artificial neural network [RMSE (%)=12.03, 12.15, 11.74, 10.7] and the multiple linear regression model [RMSE (%)=10.62, 10.68, 10.62, 10.54] were not able to reduce their forecast error over the same horizons to the same extent. For this particular application the nonlinear auto-regressive model with exogenous input can be presented as a more accurate alternative to conventional regression modeling techniques, especially for short-term milk-yield predictions.

Efficacy of SYBR 14/propidium iodide viability stain for the amphibian chytrid fungus Batrachochytrium dendrobatidis.

The amphibian chytrid fungus Batrachochytrium dendrobatidis is a recently described pathogen that has been implicated as a causal agent in the global decline in amphibians. Research into its biology and epidemiology has frequently involved in vitro experimentation. However, this research is currently limited by the inability to differentiate between viable and inviable zoospores. Stains are frequently used to determine cell viability, and this study tested a 2-colour fluorescence assay for the detection and quantification of viable B. dendrobatidis zoospores. The results show that the nucleic acid stains SYBR 14 and propidium iodide are effective in distinguishing live from dead zoospores, and a protocol has been optimized for their use. This viability assay provides an efficient and reliable tool that will have applications in B. dendrobatidis challenge and amphibian exposure experiments.

Co-morbidity in a cystic fibrosis population attending a regional clinic.

Pulmonary disease remains the major cause of morbidity in patients with cystic fibrosis (CF). However, of 115 patients attending a regional CF clinic we noted 16 cases (14%) with co-morbid conditions. Of this group, 4 of 115 patients (3.5%) had renal problems including both structural and functional defects and 4 (3.5%) had neurological disorders, 3 of which were types of epilepsy. Notably, 3 of 115 patients (2.6%) had different forms of neoplasia, all of which required significant surgical and/or chemotherapeutic intervention. There is now increasing evidence of the association between digestive tract malignancy and CF, which further complicates management of these already complex cases.

Optimisation of handling, activation and assessment procedures for Bufo marinus spermatozoa.

In the present study, we investigated handling, activation and assessment procedures for cane toad (Bufo marinus) spermatozoa. Optimisation of these techniques will facilitate the maintenance of sperm viability during cryopreservation and during in vitro fertilisation (IVF) techniques in reproduction technologies for endangered species. Spermatozoa were taken from testicular macerates and assessed using plasma membrane integrity assays (live/dead stains) and quantitative scores of motility parameters. In the assessment of sperm viability using live/dead stains, there were small but significant differences in the percentage of sperm from cryopreserved samples staining positive with propidium iodide, Hoechst H33258 and Trypan blue; these differences were not large and all stains performed acceptably. Spermatozoa were activated by dilution of testicular macerates in water at one of two dilution ratios (1 : 6 or 1 : 20) with or without 0.1-5.0 mM theophylline. Sperm plasma membrane integrity (unstained spermatozoa) was unaffected by either dilution ratio (osmolarity) or theophylline concentration. However, sperm motility was significantly affected by osmolarity and theophylline concentration. The stimulation of sperm motility increased with higher theophylline concentrations and these strongly interacted with lower osmolarities through a higher dilution ratio of sperm macerates with water. Spermatozoa were exposed to increasing centrifugation forces to determine tolerance to physical stresses encountered during washing procedures. Forces between 50 and 800 g were associated with a significant reduction in motility (mean 56 +/- 3% decreasing to 27 +/- 3%), but did not affect staining. In conclusion, centrifugation should be minimised in anuran sperm washing procedures; osmotic shock associated with higher dilution ratios reduces the capacity of anuran sperm to achieve high percentages of motile sperm, leading to a likely trade-off between dilution required for activation and sperm motility to optimise IVF fertilisation rates; and optimal conditions for sperm motility after activation occur at lower dilutions of suspensions with 5.0 mM theophylline. The present study has improved protocols for the handling of anuran sperm during pre- and post-cryopreservation procedures.

Sarcoidosis and MIF gene polymorphism: a case-control study in an Irish population.

Macrophage migration inhibitory factor is a key pro-inflammatory mediator. A 5-CATT repeat functional polymorphism within the promoter of the gene was previously associated with the lowest promoter activity. It was hypothesised that patients exhibiting a 5-CATT allele would have a less aggressive inflammatory response with an associated less severe clinical phenotype in sarcoidosis. Irish Caucasian sarcoidosis patients (n = 173) followed up for 1-39 yrs and a control group (n = 166) were genotyped for the CATT repeat polymorphism. Disease severity at the time of diagnosis and at the time of elaboration of the present study was assessed by the presence of thoracic and extrathoracic symptoms, erythema nodosum, radiographic interstitial changes (chest radiograph score equal to stage II or greater, or high-resolution computed tomography confirmed), pulmonary function tests, steroid use, erythrocyte sedimentation rate, C-reactive protein and angiotensin-converting enzyme levels. In the Irish population studied, no evidence was found of a significant association between either sarcoidosis susceptibility and disease severity and the 5-CATT repeat functional polymorphism in the macrophage migration inhibitory gene. The present study found no significant association between the 5-CATT repeat macrophage migration inhibitory factor gene polymorphism and sarcoidosis, and did not support the overriding role for macrophage migration inhibitory factor in driving sarcoidosis pathogenesis.

Effect of sperm concentration, medium osmolality and oocyte storage on artificial fertilisation success in a myobatrachid frog (Limnodynastes tasmaniensis).

The present study optimised artificial fertilisation and oocyte storage conditions in Limnodynastes tasmaniensis (Myobatrachidae). Data on general reproductive biology, the effect of sperm motility and concentration, medium osmolality and oocyte storage on artificial fertilisation success are presented. Egg number was most strongly correlated with bodyweight (r = 0.819). Sperm yield was correlated with testes weight (r = 0.827), which was strongly correlated with snout-vent length (r = 0.772). Optimal artificial fertilisation occurred in 0-7 mOsm kg(-1) amphibian Ringer, similar to ranid, bufonid and hylid species. High fertilisation rates were achieved using spermatozoa with little forwards progressive motility at comparatively low concentrations (3 x10(4) sperm cells mL(-1)) and with no relationship between percentage sperm motility and fertilisation success (correlation of fertilisation rate with sperm motility after activation: r = -0.145). Oocytes stored in 5 mOsm kg(-1) solutions showed no significant decline in fertilisability after 2 h, showing that swelling of the jelly surrounding the eggs does not prevent sperm from fusing with the oocyte in this species. Fertilisability of oocytes was extended to > 4 h in medium to high osmolality solutions (124-271 mOsm kg(-1)). These data allow for the future use of L. tasmaniensis in developing assisted reproductive technology protocols for foam-nesting myobatrachid species, many of which are now threatened with extinction in the wild.

Managing exacerbations of COPD: room for improvement.

Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are a major cause of hospital admissions. Because of the consequent morbidity, mortality and burden on hospital resources, COPD management guidelines have been formulated. We reviewed 62 consecutive patients with AECOPD admitted from September 1st to December 18th 2000 in St. Vincents University Hospital, Ireland, including 3 months follow-up data, to evaluate the quality of care and in particular to assess the care of such patients by respiratory and non-respiratory physicians. There was a frequent failure to objectively confirm the diagnosis of COPD by spirometry (completed in 39 of the 51 patients who, at admission, had been previously labelled with COPD (76%), and in 53 out of 62 patients (85%) at the end of the study period), or to estimate severity by quantifying the FEV1 as a percentage of the normal predicted range (estimated in only 21 of the 39 patients who had spirometry previously performed (53%)). Those patients managed with input from respiratory physicians were more likely to have their diagnosis of COPD confirmed with spirometry (p < 0.05). They were also more likely to have out-patient follow-up arranged at discharge (p < 0.05). There was a trend towards the more frequent prescribing of oxygen to hypoxic patients in "respiratory" than in "non-respiratory" managed cases (p = 0.182) and a shorter hospital stay (0.1 < p < 0.5). 4 out of 11 severely hypoxaemic patients at admission (PO2 < 7.3kPa) were not screened at discharge for possible long term oxygen therapy (36%). 20 patients received combination antibiotic therapy with no infiltrate on CXR (32%). Pulmonary rehabilitation was offered to 12 patients (19%). 5 out of 18 current smokers had documented smoking cessation advice (28%) and none received smoking cessation pharmacotherapy. Finally we noted that the Hospital In-Patient Enquiry (HIPE) data and casualty department admission books were frequently misleading or medical records unlocatable (in 30 out of 92 cases (33%)). We conclude that the management of AECOPD at St. Vincent's University Hospital is frequently suboptimal, and may be managed better with respiratory physician involvement. In particular, there could be more frequent spirometric confirmation of the diagnosis of COPD, better screening for long term oxygen therapy and more conservative use of antibiotics. Audit is complicated by difficulty accessing relevant data.

Management and response to treatment of Helicobacter pylori gastritis.

Gastritis associated with Helicobacter pylori was present in gastric biopsies from 24/95 (25%) children and adolescents undergoing endoscopy for recurrent abdominal pain and upper gastrointestinal symptoms. H pylori associated gastritis occurred mainly in older children (8-16 years) and was significantly associated with low socioeconomic class and a family history of peptic ulcer disease. Antral nodularity was a common endoscopic finding in H pylori positive children. Eighteen children, all over 5 years of age, were treated with tripotassium dicitratobismuthate (De-Nol) for two months and ampicillin for two weeks. In 12 children follow up gastric biopsies were obtained six weeks after completion of treatment. In 9/12 (75%) children H pylori was eradicated, and gastritis improved.

Immune responses to Helicobacter pylori in children with recurrent abdominal pain.

The systemic immune response to Helicobacter pylori was examined in 69 children with recurrent abdominal pain and upper gastrointestinal symptoms. Twenty one (30%) children were histologically positive for H pylori. Eighteen of the 21 positive subjects and two H pylori negative subjects (one with normal mucosa, one with lymphocytic gastritis) were positive for H pylori IgG antibodies by enzyme linked immunosorbent assay (ELISA) (86% sensitivity, 98% specificity). In children with H pylori associated gastritis, there was a significant positive correlation (p less than 0.05) between IgG antibody titres and patient age. Intra-assay comparison of sera from histologically negative adults with those of histologically negative children showed that the cut off for positivity in the ELISA for adults was greater than that for children. Immunoblotting showed IgG positivity in 20 of the 21 patients with H pylori infection (95% sensitivity). Both ELISA and immunoblotting for IgA and IgM H pylori antibodies had poor discriminatory value for determining infection. Serological detection of H pylori IgG antibodies seems to be valuable in the assessment of children presenting with recurrent abdominal pain and other gastrointestinal symptoms, but assays must first be validated in paediatric populations.

Motor disorders of the oesophagus in gastro-oesophageal reflux.

Mechanisms of gastro-oesophageal reflux were studied by oesophageal manometry and pH monitoring in 33 children: nine controls, 15 with gastro-oesophageal reflux alone, and nine with reflux oesophagitis. A total of 122 episodes of reflux were analysed in detail: 82 (67%) were synchronous with swallowing and 40 (33%) asynchronous. Infants with trivial symptoms had gastro-oesophageal reflux synchronous with swallowing, whereas those with serious symptoms had slower acid clearance and asynchronous reflux. There were significant differences in lower oesophageal sphincter pressure and amplitude of oesophageal contractions between controls and patients with both gastro-oesophageal reflux and reflux oesophagitis. In reflux oesophagitis there was a decrease in lower oesophageal sphincter pressure and the contractions had a bizarre waveform suggesting a neuropathic process.

Campylobacter pylori gastritis.

Campylobacter pylori colonisation of the stomach is strongly associated with type B non-autoimmune gastritis in adults. In a retrospective study of 38 gastric biopsy specimens taken during upper gastrointestinal endoscopy in children attending this hospital we found C pylori in nine (24%). Ten biopsy specimens showed histological evidence of gastritis and C pylori was found in eight.

Budd-Chiari syndrome treated by Senning operation.

Budd-Chiari syndrome was diagnosed in a 13 year old boy who presented with ascites. Angiographic studies showed occlusion at the ostia of the hepatic veins. This was treated surgically by the Senning operation of transcaval dorsocranial resection of the liver and hepatocaval anastomosis. The patient's ascites cleared and he remains well 10 months after surgery.