Improving interinstitutional and intertechnology consistency of pulmonary SBRT by dose prescription to the mean internal target volume dose.

PURPOSE: Dose, fractionation, normalization and the dose profile inside the target volume vary substantially in pulmonary stereotactic body radiotherapy (SBRT) between different institutions and SBRT technologies. Published planning studies have shown large variations of the mean dose in planning target volume (PTV) and gross tumor volume (GTV) or internal target volume (ITV) when dose prescription is performed to the PTV covering isodose. This planning study investigated whether dose prescription to the mean dose of the ITV improves consistency in pulmonary SBRT dose distributions. MATERIALS AND METHODS: This was a multi-institutional planning study by the German Society of Radiation Oncology (DEGRO) working group Radiosurgery and Stereotactic Radiotherapy. CT images and structures of ITV, PTV and all relevant organs at risk (OAR) for two patients with early stage non-small cell lung cancer (NSCLC) were distributed to all participating institutions. Each institute created a treatment plan with the technique commonly used in the institute for lung SBRT. The specified dose fractionation was 3â€¯× 21.5 Gy normalized to the mean ITV dose. Additional dose objectives for target volumes and OAR were provided. RESULTS: In all, 52 plans from 25 institutions were included in this analysis: 8 robotic radiosurgery (RRS), 34 intensity-modulated (MOD), and 10 3D-conformal (3D) radiation therapy plans. The distribution of the mean dose in the PTV did not differ significantly between the two patients (median 56.9 Gy vs 56.6 Gy). There was only a small difference between the techniques, with RRS having the lowest mean PTV dose with a median of 55.9 Gy followed by MOD plans with 56.7 Gy and 3D plans with 57.4 Gy having the highest. For the different organs at risk no significant difference between the techniques could be found. CONCLUSIONS: This planning study pointed out that multiparameter dose prescription including normalization on the mean ITV dose in combination with detailed objectives for the PTV and ITV achieve consistent dose distributions for peripheral lung tumors in combination with an ITV concept between different delivery techniques and across institutions.

Deep amoA amplicon sequencing reveals community partitioning within ammonia-oxidizing bacteria in the environmentally dynamic estuary of the River Elbe.

The community composition of betaproteobacterial ammonia-oxidizing bacteria (ß-AOB) in the River Elbe Estuary was investigated by high throughput sequencing of ammonia monooxygenase subunit A gene (amoA) amplicons. In the course of the seasons surface sediment samples from seven sites along the longitudinal profile of the upper Estuary of the Elbe were investigated. We observed striking shifts of the ß-AOB community composition according to space and time. Members of the Nitrosomonas oligotropha-lineage and the genus Nitrosospira were found to be the dominant ß-AOB within the river transect, investigated. However, continuous shifts of balance between members of both lineages along the longitudinal profile were determined. A noticeable feature was a substantial increase of proportion of Nitrosospira-like sequences in autumn and of sequences affiliated with the Nitrosomonas marina-lineage at downstream sites in spring and summer. Slightly raised relative abundances of sequences affiliated with the Nitrosomonas europaea/Nitrosomonas mobilis-lineage and the Nitrosomonas communis-lineage were found at sampling sites located in the port of Hamburg. Comparisons between environmental parameters and AOB-lineage (ecotype) composition revealed promising clues that processes happening in the fluvial to marine transition zone of the Elbe estuary are reflected by shifts in the relative proportion of ammonia monooxygenase sequence abundance, and hence, we propose ß-AOB as appropriate indicators for environmental dynamics and the ecological condition of the Elbe Estuary.

[Minimally invasive components of the treatment of tibialis posterior insufficiency in adults : Tendoscopy of the Achilles tendon, minimally invasive calcaneus displacement osteotomy and the sinus tarsi spacer]. / Minimal-invasive Komponenten der Therapie der Tibialis-posterior-Insuffizienz des Erwachsenen : Tendoskopie der Achillessehne, minimal-invasive Kalkaneusverschiebeosteotomie und Sinus-tarsi-Spacer.

Minimally invasive medializing calcaneal osteotomy, the implantation of a tarsi spacer and the tendoscopy of the posterior tibial tendon have been established as treatment options for tibialis posterior insufficiency grade II. The minimally invasive medializing calcaneal osteotomy allows a correction of the hindfoot valgus like an open procedure with a significantly lower risk of wound healing problems. It has gained increasing popularity within recent years. There is also good evidence for arthroereisis, with the use of the sinus tarsi spacer being primarily an addition to calcaneus sliding osteotomy. The possibilities for tendoscopy of the posterior tibial tendon are limited to debridement and synovectomy. Various papers report minimally invasive alternatives to the strayer procedure for a shortened gastrocnemius muscle. The proximal lengthening of the medial head of the gastrocnemius muscle is particularly popular as a soft tissue-sparing, less traumatic procedure. So far, no reports of a minimally invasive cotton osteotomy have been found in the literature.

[Malignant acanthosis nigricans associated with cholangiocarcinoma]. / Acanthosis nigricans malin révélant un cholangiocarcinome.

INTRODUCTION: Acanthosis nigricans is a dermatosis characterized by the presence of a hyperpigmented, velvety cutaneous thickening in the flexural areas, especially axillary and inguinal fossas, and lateral faces of the neck. AN is usually a benign condition but can sometimes reveal an internal malignancy corresponds to a cutaneous paraneoplasic syndrome. Literature shows a predominant association with gastric adenocarcinoma. Here, we report a rare association between AN and cholangiocarcinoma. CASE REPORT: We report a 43-year-old woman who presented an extensive AN associated to a tripe palms syndrome and florid cutaneous papillomatosis. She consulted in dermatology because of the itchiness of the lesions as well as for esthetics reasons. Complementary investigations enabled to diagnose a cholangiocarcinoma without visceral metastasis and she was treated by tumor resection and chemotherapy. Consequently, a slight improvement of the skin condition and the disappearance of pruritus were observed. CONCLUSION: AN should be considered as cutaneous sign either of malignancy or endocrinopathy and therefore requires further investigations. The existence of extensive lesions, pruritus, tripe palms syndrome, florid cutaneous papillomatosis or mucous lesions, associated to an AN is a sign of malignancy should be investigated urgently the early diagnosis of which can lead to a better prognosis.

[Fractures of the lateral process of the talus-snowboarder's ankle]. / Frakturen des Processus lateralis tali ­ "snowboarder's ankle".

Fractures of the lateral tubercle of the talus (PLT) are rare. With the increasing popularity of the trend sport snowboarding, the incidence of PLT fractures has increased. The most common classification of PLT fractures is the Hawkins classification. The aim of this review was to raise awareness for the injury and discuss the current evidence. A literature search revealed eight studies, each including at least seven patients. Six out of the eight studies were descriptive, retrospective case series without predefined treatment concepts. These resulted in only moderate treatment outcomes. Due to the low number of patients, the lack of computed tomography (CT) or magnetic resonance imaging (MRI) and inconsistent treatment approaches, these studies do not allow to draw conclusions on a treatment concept for PLT fractures. The other two studies validated existing treatment regimens. Overall, surgical treatment of dislocated fractures and conservative treatment of non-dislocated fractures was carried out with satisfactory results. The outcome of conservative treatment of dislocated factures remains unclear. A reason for the inconsistent treatment results could be the observed concomitant injuries, including dislocation of the tendons of the peroneus muscles (46%), calcaneal chondral injuries (48%) and subluxation of the subtalar joint (7%). Based on the limited evidence available, the authors recommend the application of CT and MRI for PLT fractures to assess concomitant injuries, which are the primary indication for surgery. Dislocated type I and II fractures (>2 mm) should be treated operatively, type III and non-dislocated type I and II fractures can be treated conservatively by immobilization and partial weight-bearing for 6 weeks.

[Minimally invasive cheilectomy]. / Minimalinvasive Cheilektomie.

OBJECTIVES: Treatment of hallux rigidus by minimally invasive resection of the dorsal osteophytes, synovectomy and resection of the dorsal part of the metatarsal head. INDICATIONS: Hallux rigidus grades II and III CONTRAINDICATIONS: End-stage osteoarthritis of the first metatarsophalangeal joint with beginning ankylosis. SURGICAL TECHNIQUE: Osteophytes around the metatarsophalangeal joint are removed using a 1 cm incision dorsomedial, approximately 3 cm proximal of the joint space. The dorsal third of the metatarsal head is resected with a burr to improve dorsiflexion. The extent of bone resection is checked with an image intensifier. Loose bone fragments removed with a rangeur. An arthroscopy can be performed to check the completeness of bone resection, the irrigation of the joint and, if needed, to extend the synovectomy. POSTOPERATIVE MANAGEMENT: Removal of the sutures after 2 weeks. Depending on pain, the patient can change from the postoperative shoe to a normal soft, comfortable and wide shoe after 1-2 weeks. Nonsteroidal drugs can be prescribed as needed. Active and passive mobilization of the metatarsophalangeal joint is also recommended. RESULTS: The technique allows a soft-tissue-preserving resection of the osteophytes and a partial resection of the metatarsal head. The main advantages are limited soft-tissue trauma and rapid rehabilitation. In all, 21 women and 17 men with hallux rigidus stages II and III (Vanore) underwent surgery. Minimum follow-up was 12 months. In 1 patient, injury of the extensor hallucis longus tendon was observed. Two patients underwent revision surgery. One patient was converted to a metatarsophalangeal fusion, while another patient received a resection arthroplasty. At the latest follow-up, the AOFAS (American Orthopaedic Foot & Ankle Society) score averaged 88.7 points.

Hydroxylamine released by nitrifying microorganisms is a precursor for HONO emission from drying soils.

Nitrous acid (HONO) is an important precursor of the hydroxyl radical (OH), the atmosphere´s primary oxidant. An unknown strong daytime source of HONO is required to explain measurements in ambient air. Emissions from soils are one of the potential sources. Ammonia-oxidizing bacteria (AOB) have been identified as possible producers of these HONO soil emissions. However, the mechanisms for production and release of HONO in soils are not fully understood. In this study, we used a dynamic soil-chamber system to provide direct evidence that gaseous emissions from nitrifying pure cultures contain hydroxylamine (NH2OH), which is subsequently converted to HONO in a heterogeneous reaction with water vapor on glass bead surfaces. In addition to different AOB species, we found release of HONO also in ammonia-oxidizing archaea (AOA), suggesting that these globally abundant microbes may also contribute to the formation of atmospheric HONO and consequently OH. Since biogenic NH2OH is formed by diverse organisms, such as AOB, AOA, methane-oxidizing bacteria, heterotrophic nitrifiers, and fungi, we argue that HONO emission from soil is not restricted to the nitrifying bacteria, but is also promoted by nitrifying members of the domains Archaea and Eukarya.

[Endoprosthesis failure in the ankle joint : Histopathological diagnostics and classification]. / Endoprothesenversagen im oberen Sprunggelenk : Histopathologische Diagnostik und Klassifikation.

INTRODUCTION: Endoprostheses of the ankle joint show higher revision rates of 3.29 revisions per 100 component years. The aims of this study were the application and modification of the consensus classification of the synovia-like interface membrane (SLIM) for periprosthetic failure of the ankle joint, the etiological clarification of periprosthetic pseudocysts and a detailed measurement of proliferative activity (Ki67) in the region of osteolysis. MATERIAL AND METHOD: Tissue samples from 159 patients were examined according to the criteria of the standardized consensus classification. Of these, 117 cases were derived from periprosthetic membranes of the ankle. The control group included 42 tissue specimens from the hip and knee joints. Particle identification and characterization were carried out using the particle algorithm. An immunohistochemical examination with Ki67 proliferation was performed in all cases of ankle pseudocysts and 19 control cases. RESULTS: The consensus classification of SLIM is transferrable to endoprosthetic failure of the ankle joint. Periprosthetic pseudocysts with the histopathological characteristics of the appropriate SLIM subtype were detectable in 39 cases of ankle joint endoprostheses (33.3%). The mean value of the Ki67 index was 14% and showed an increased proliferation rate in periprosthetic pseudocysts of the ankle (p-value 0.02037). CONCLUSION: In periprosthetic pseudocysts an above average higher detection rate of type 1 SLIM induced by abrasion (51.3%) with an increased Ki67 proliferation fraction (p-value 0.02037) was found, which can be interpreted as local destructive intraosseus synovialitis. This can be the reason for formation of pseudocystic osteolysis caused by high mechanical stress in ankle endoprostheses. A simplified diagnostic classification scoring system of dysfunctional endoprostheses of the ankle is proposed for collation of periprosthetic pseudocysts, ossifications and the Ki67 proliferation fraction.

[Percutaneous calcaneal sliding osteotomy]. / Die perkutane Kalkaneusverschiebeosteotomie.

OBJECTIVES: Correction of calcaneal malalignment as part of a hindfoot correction procedure. INDICATIONS: Varus and valgus malalignment of the calcaneus, increased calcaneal pitch. CONTRAINDICATIONS: Osteoarthritis of the subtalar joint. Fixed and symptomatic deformities of the subtalar joint. SURGICAL TECHNIQUE: After having identified and marked the desired planes of the osteotomy under image intensifier, a percutaneous v­shaped calcaneal osteotomy is performed. The osteotomy allows 3­dimensional correction of the calcaneus by defining the planes of the osteotomy. The procedure allows correction of varus and valgus deformities, as well as a change of the calcaneal pitch. The osteotomy is fixed by percutaneous screws. POSTOPERATIVE MANAGEMENT: Postoperative care includes a 6-week period of partial weight bearing with 10 kg. The ankle joint should be mobilized. After x­ray control of sufficient bone healing, weight bearing can be increased stepwise over another 4­week period up to full body weight. A full length orthotic is recommended for at least 12 months with heel cup and good medial support. RESULTS: The procedure allows correction of calcaneal deformities with preservation of soft tissue, normally as part of a hindfoot correction, e. g., in posterior tibial tendon insufficiency, varus deformities or total ankle replacement. In the literature and in our patients, the rate of injuries of the neurovascular bundle was not increased compared to open surgery. The average calcaneal shift was 1 cm, when necessary an additional correction was realized by rotation of the tuber calcanei.

[Tendoscopy of the Achilles tendon. Indications, technique and results]. / Tendoskopie der Achillessehne. Indikation, Technik und Ergebnisse.

BACKGROUND: Endoscopic surgical techniques are nowadays standard procedures in medicine. The advantages of these minimally invasive techniques compared to open techniques are a smaller access route with reduced tissue damage, reduced scarring and often faster postoperative mobilization. Tendoscopy can be used to treat pathologies of tendons as well as of the surrounding tissues. OBJECTIVES: This article presents the advantages of endoscopic treatment of the Achilles tendon compared to open procedures as well as the chances and limitations of tendoscopy. MATERIAL AND METHODS: Surgical instructions for endoscopy of the Achilles tendon are presented and a review of the literature is given. RESULTS: The literature review showed excellent results for pathologies of the paratenon and Achilles tendinitis. Compared to open surgery there was a significantly lower rate of wound healing problems. All articles reported a high reduction of pain level with an early return to sports activities. Limitations of the procedure are extensive intratendinous pathologies and alterations of tendon insertion sites. CONCLUSION: Tendoscopy of the Achilles tendon is a safe but sometimes challenging minimally invasive technique for the treatment of paratendinopathy.

Prenatal radiation exposure: dose calculation.

The unborn child requires special protection. In this context, the indication for an X-ray examination is to be checked critically. If thereupon radiation of the lower abdomen including the uterus cannot be avoided, the examination should be postponed until the end of pregnancy or alternative examination techniques should be considered. Under certain circumstances, either accidental or in unavoidable cases after a thorough risk assessment, radiation exposure of the unborn may take place. In some of these cases an expert radiation hygiene consultation may be required. This consultation should comprise the expected risks for the unborn while not perturbing the mother or the involved medical staff. For the risk assessment in case of an in-utero x-ray exposition deterministic damages with a defined threshold dose are distinguished from stochastic damages without a definable threshold dose. The occurrence of deterministic damages depends on the dose and the developmental stage of the unborn at the time of radiation. To calculate the risks of an in-utero radiation exposure a three-stage concept is commonly applied. Depending on the amount of radiation, the radiation dose is either estimated, roughly calculated using standard tables or, in critical cases, accurately calculated based on the individual event. The complexity of the calculation thereby increases from stage to stage. An estimation based on stage one is easily feasible whereas calculations based on stages two and especially three are more complex and often necessitate execution by specialists. This article demonstrates in detail the risks for the unborn child pertaining to its developmental phase and explains the three-stage concept as an evaluation scheme. It should be noted, that all risk estimations are subject to considerable uncertainties. • Radiation exposure of the unborn child can result in both deterministic as well as stochastic damage und hitherto should be avoided or reduced to a minimum.• In case of radiation a suitable hygiene consultation may be necessary.• For risk assessment a three-stage concept is applied, which, depending on the radiation exposure, estimates or calculates the dose for the unborn child.• The radiologist plays a crucial role as a competent advisor and provider of reliable expert information. Citation Format: • Scharwächter C, Röser A, Schwartz CA et al. Prenatal Radiation Exposure: Dose Calculation. Fortschr Röntgenstr 2015; 187: 338 - 346.

Reconstruction of focal cartilage defects in the talus with miniarthrotomy and collagen matrix.

SURGICAL PRINCIPAL AND OBJECTIVE: Treatment of focal cartilage defects (traumatic or osteochondrosis dissecans) of the talus using a collagen matrix. The goal is to stabilize the superclot formed after microfracturing to accommodate cartilage repair. The procedure can be carried out via miniarthrotomy, without medial malleolus osteotomy. INDICATIONS: International Cartilage Repair Society (ICRS) grade III and IV focal cartilage defects of the talus > 1.5 cm(2). CONTRAINDICATIONS: Generalized osteoarthritis, inflammatory joint disease, gout, neuroarthropathy. SURGICAL TECHNIQUE: Miniarthrotomy to open the ankle joint. Debridement of unstable cartilage and necrotic bone, curettage of cysts. Filling of the bone defects with autologous cancellous bone. Sealing of reconstructed bone with fibrin glue and attachment of a collagen matrix shaped to precisely fit the defect. POSTOPERATIVE REGIMEN: Immobilization for 48 h. Partial weight bearing of 10 kg for 6 weeks, with continuous passive motion. Increasing weight bearing from 7 weeks onwards. RESULTS: Follow-up of at least 30 months in 14 patients showed improvement in the Score of the American Orthopedic Foot and Ankle Society (AOFAS) from 50 to 89 points, with equal mobility on both sides of the upper ankle joint.

Influence of p-cresol on the proteome of the autotrophic nitrifying bacterium Nitrosomonas eutropha C91.

In this study, the effect of the organic micropollutant and known inhibitor of nitrification, p-cresol, was investigated on the metabolism of the ammonia oxidizing bacteria (AOB) Nitrosomonas eutropha C91 using MS-based quantitative proteomics. Several studies have demonstrated that AOB are capable of biotransforming a wide variety of aromatic compounds making them suitable candidates for bioremediation, yet the underlying molecular mechanisms are poorly described. The effect of two different concentrations of the aromatic micropollutant p-cresol (1 and 10 mg L(-1)) on the metabolism of N. eutropha C91, relative to a p-cresol absent control, was investigated. Though the rate of nitrification in N. eutropha C91 appeared essentially unaffected at both concentrations of p-cresol relative to the control, the expressional pattern of the proteins of N. eutropha C91 changed significantly. The presence of p-cresol resulted in the repressed expression of several key proteins related to N-metabolism, seemingly impairing energy production in N. eutropha C91, contradicting the observed unaltered rates of nitrification. However, the expression of proteins of the TCA cycle and proteins related to xenobiotic degradation, including a p-cresol dehydrogenase, was found to be stimulated by the presence of p-cresol. This indicates that N. eutropha C91 is capable of degrading p-cresol and that it assimilates degradation intermediates into the TCA cycle. The results reveal a pathway for p-cresol degradation and subsequent entry point in the TCA cycle in N. eutropha C91. The obtained data indicate that mixotrophy, rather than cometabolism, is the major mechanism behind p-cresol degradation in N. eutropha C91.

HONO emissions from soil bacteria as a major source of atmospheric reactive nitrogen.

Abiotic release of nitrous acid (HONO) in equilibrium with soil nitrite (NO2(-)) was suggested as an important contributor to the missing source of atmospheric HONO and hydroxyl radicals (OH). The role of total soil-derived HONO in the biogeochemical and atmospheric nitrogen cycles, however, has remained unknown. In laboratory experiments, we found that for nonacidic soils from arid and arable areas, reactive nitrogen emitted as HONO is comparable with emissions of nitric oxide (NO). We show that ammonia-oxidizing bacteria can directly release HONO in quantities larger than expected from the acid-base and Henry's law equilibria of the aqueous phase in soil. This component of the nitrogen cycle constitutes an additional loss term for fixed nitrogen in soils and a source for reactive nitrogen in the atmosphere.

[Lateral ligament injuries of the ankle joint]. / Die Verletzung des lateralen Kapsel-Band-Apparats des Sprunggelenks.

Lateral ligament injuries are the most common sports injury and have a high incidence even in non-sportive activities. Although lateral ligament injuries are very common there is still a controversial debate on the best management. The diagnosis is based on clinical examination and X-ray images help to rule out fractures. Further imaging, especially magnetic resonance imaging (MRI) is used to diagnose associated injuries. According to the recommendations of the various scientific societies the primary therapy of lateral ligament injuries is conservative. Chronic ankle instability develops in 10-20 % of patients and the instability can be a result of sensomotoric deficits or insufficient healing of the lateral ligament complex. If the patient does not respond to an intensive rehabilitation program an operative reconstruction of the lateral ligaments has to be considered. Most of the procedures currently performed are anatomical reconstructions due to better long-term results compared to tenodesis procedures.

[Technical aspects in the provision of orthopedic shoes for athletes]. / Orthopädieschuhtechnische Versorgung beim Sportler.

From the technical point of view different groups must be distinguished in the provision of orthopedic shoes for athletes. The one group encompasses athletes who are provided with insoles in the hope of improved comfort or better performance. The other group includes athletes with diseases or injuries of the lower limbs for whom the provision of appropriate shoes and insoles makes it possible for them to participate again actively in their chosen sport. In such cases one has to differentiate between the purely physical disorder of the foot and biomechanical disorders. For the first group the correct fitting of the shoe is the most important factor whereas in the second group attempts are made to correct and/or compensate for the deficit by means of insoles and wedges as well as supporting or bedding elements. The particular challenge for all such measures is to achieve an as small as possible impairment on the sport activity or, in the ideal cases, even to support it.

[Fusion of the first tarsometatarsal joint using a plantar tension band osteosynthesis]. / Die Arthrodese des Tarsometatarsal-I-Gelenks mit einer plantaren Zuggurtungsosteosynthese.

OBJECTIVE: Treatment of hallux valgus in patients with a pathology of the first metatarsocuneiform (MC) joint by a fusion of the first MC fixed by a plantar plate. The plantar plate has biomechanical advantages and has good soft tissue coverage by the M. abductor hallucis. INDICATIONS: Instability or degenerative arthritis of the first MC joint in patients with hallux valgus. CONTRAINDICATIONS: Short first metatarsal. SURGICAL TECHNIQUE: Bone-saving resection of the first MC joint. Arthrodesis using a compression screw and a plantar interlocking plate. Distal soft tissue procedure and resection of the exostosis. POSTOPERATIVE MANAGEMENT: For 6 weeks, a long sole, post-operative shoe with weight bearing as pain allows. Mobilization of the first metatarsophalangeal joint when the wound healing is assured. Full weight bearing after 6-8 weeks in a normal shoe, when the bone healing is completed on the x-rays. No sports with high demands on the foot for 12 weeks. Orthotics only in cases with persisted pain or associated pathology. RESULTS: In a case control study including 72 patients, a significantly lower rate of nonunion and soft tissue problems, compared to dorsal or medial plate positioning, was observed.

Assessment of reproductive status in male echidnas.

This study reports the development and application of techniques to assess the reproductive status of male echidnas. The pattern of testosterone secretion over a 24-h period in five echidnas was documented. Testosterone secretion after injection i.m. of either 1000 IU hCG (n=4) or 4 microg GnRH agonist (n=6) was determined to establish whether this could be used as a practical index of the prevailing steroidogenic capacity of the testes. hCG (1000 IU) was also used to assess seasonal changes in testosterone secretion in six echidnas over a 13-month period. Seasonal changes in testicular volume were examined by transabdominal ultrasonography. Electroejaculation was attempted to monitor seasonal changes in sperm production, which was also determined by spermatorrhea. There was no apparent diurnal pattern of testosterone secretion in echidnas and circulating concentrations of testosterone remained relatively low (maximum 1.2 ng/mL) and stable over 24h. Injection of hCG resulted in an increase (P<0.01; n=4) in testosterone concentration with a peak (2.9+/-0.3 ng/mL) approximately 4h after injection. GnRH also induced an increase (P<0.01; n=6) in circulating testosterone that was apparent after 1h (2.6+/-0.3 ng/mL) and concentrations remained elevated (3.4+/-0.3 ng/mL) for up to 8h after injection. Seasonal changes in testosterone secretion determined after injection of hCG, increased (P=0.03; n=6) from late-autumn, peaked in late-winter, and decreased by early-spring. Testicular volume followed a similar seasonal pattern (P<0.01; n=6) with an increase from late-autumn, peak in winter and a decline in mid-spring. There was no seasonal change in live weight. Electroejaculation was attempted throughout two breeding seasons but no semen was obtained. Spermatorrhoea in the echidna was described for the first time and was subsequently used to assess seasonal sperm production. Spermatozoa were found in the urine from June to September. This study has demonstrated that exogenous hormones can be used to obtain an index of the prevailing steroidogenic capacity of the testes in echidnas, which is not apparent with repetitive non-stimulated samples over 24 h. The assessment of testosterone secretion after injection of trophic hormones provides a valuable and practical procedure for the assessment of reproductive status. Testicular ultrasonography and spermatorrhea are useful in assessing reproductive status and in this study were successfully used to determine seasonal reproduction in captive echidnas.

Detailed phylogeny of ammonia-oxidizing bacteria determined by rDNA sequences and DNA homology values.

A comparison of the phylogeny of 38 isolates of chemolithoautotrophic ammonia-oxidizing bacteria (AOB) based on 16S rRNA gene sequences, 16S-235 rDNA intergenic spacer region (ISR) sequences and species affiliations based on DNA homology values was performed. The organisms studied all belong to the beta-subclass of the Proteobacteria and included representatives of Nitrosomonas, Nitrosococcus and Nitrosospira. The similarity values of the 16S rDNA sequences were high, particularly within the Nitrosospira genus, and based on these sequences it is difficult to determine the phylogenetic position of some AOB. As an alternative and supplement to 16S rRNA gene sequencing, the ISR was sequenced and analysed phylogenetically. Due to considerably lower similarity values, the ISR-based phylogeny gives a better resolution than the phylogeny based on the functional 16S rRNA gene. Since the ISR-based phylogeny of AOB is highly consistent with the 16S rDNA based phylogeny, ISR sequencing appears as a suitable tool for resolving the detailed phylogeny of AOB. The phylogenetic position of two isolates of the former genus 'Nitrosolobus' (now included in the Nitrosospira genus) is not clear. These organisms are close relatives of the former Nitrosospira spp. and 'Nitrosovibrio' spp. (now Nitrosospira), but based on their marginal positions in the phylogenetic trees, DNA-DNA hybridization data and phenotypic characteristics, it is suggested that 'Nitrosolobus' should be a separate genus. DNA homology determination of 11 Nitrosospira isolates revealed two new species of Nitrosospira. The phylogeny of AOB reflected in the trees based on the rDNA sequences is consistent with the species affiliations of AOB by DNA homology values. This observation will probably be important for the interpretation of results from studies of natural diversity of AOB.