Experimental helium-beam radiography with a high-energy beam: Water-equivalent thickness calibration and first image-quality results.

PURPOSE: A clinical implementation of ion-beam radiography (iRad) is envisaged to provide a method for on-couch verification of ion-beam treatment plans. The aim of this work is to introduce and evaluate a method for quantitative water-equivalent thickness (WET) measurements for a specific helium-ion imaging system for WETs that are relevant for imaging thicker body parts in the future. METHODS: Helium-beam radiographs (αRads) are measured at the Heidelberg Ion-beam Therapy Center with an initial beam energy of 239.5 MeV/u. An imaging system based on three pairs of thin silicon pixel detectors is used for ion path reconstruction and measuring the energy deposition (dE) of each particle behind the object to be imaged. The dE behind homogeneous plastic blocks is related to their well-known WETs between 280.6 and 312.6 mm with a calibration curve that is created by a fit to measured data points. The quality of the quantitative WET measurements is determined by the uncertainty of the measured WET of a single ion (single-ion WET precision) and the deviation of a measured WET value to the well-known WET (WET accuracy). Subsequently, the fitted calibration curve is applied to an energy deposition radiograph of a phantom with a complex geometry. The spatial resolution (modulation transfer function at 10 % -MTF10% ) and WET accuracy (mean absolute percentage difference-MAPD) of the WET map are determined. RESULTS: In the optimal imaging WET-range from ∼280 to 300 mm, the fitted calibration curve reached a mean single-ion WET precision of 1.55 ± $\,{\pm}\,$ 0.00%. Applying the calibration to an ion radiograph (iRad) of a more complex WET distribution, the spatial resolution was determined to be MTF10% = 0.49 ± $\,{\pm}\,$ 0.03 lp/mm and the WET accuracy was assessed as MAPD to 0.21 %. CONCLUSIONS: Using a beam energy of 239.5 MeV/u and the proposed calibration procedure, quantitative αRads of WETs between ∼280 and 300 mm can be measured and show high potential for clinical use. The proposed approach with the resulting image qualities encourages further investigation toward the clinical application of helium-beam radiography.

Prevalence of disorders in preweaned dairy calves from 731 dairies in Germany: A cross-sectional study.

The objective of this cross-sectional study was to determine the prevalence of disorders in preweaned calves in 3 regions in Germany, exemplary for structural diversity in dairy farming. A farm visit was performed on a single occasion on 731 dairy farms in the northern, eastern, and southern regions of Germany between December 2016 and July 2019. Farms differed in herd size, geographical location, and management. In the northern region, the farms had a median of 90 milking cows and were often run as full-time family businesses, partly with external workers. The eastern region tended to have larger farms (a median of 251 milking cows), which were often large-scale agricultural enterprises with employees. In the southern region, the farms had a median of 39 milking cows and were often traditional family businesses, some of these being part-time businesses. Clinical examinations were performed on 14,164 preweaned dairy calves (median 12 calves per farm) by trained veterinarians. A complete data set was available for 13,656 calves. Almost half (42.0%) of the evaluated calves were classified as being affected by at least 1 of the common calf disorders. Omphalitis (O, 20.9%; n = 2,876) and diarrhea (D, 18.5%; n = 2,670) were the most frequently recorded diagnoses, whereas respiratory diseases (RD) were observed to a lesser extent (8.7%; n = 1,100). A striking feature was the fact that 7.1% (n = 987) of the calves were affected by more than 1 disorder at the same time (multimorbidity, M). The following combinations of disorders were frequently observed: O and D (n = 596), O and RD (n = 164), and D and RD (n = 140). Disorders such as O and D, as well as M, were predominantly observed in calves aged 2 wk. A gradual increase in the frequency of RD was observed with age. For all disorders except RD, male calves were more often affected than females. Omphalitis was predominantly diagnosed in the summer months, whereas RD, D, and M were more common in the fall. We detected several statistically significant differences in the prevalence of clinical signs and disorders in preweaned dairy calves between the 3 exemplary regions. The prevalence of RD was higher in the south (10.8%) than in the north (8.2%) and east (7.4%). In the north (33.2%), O was observed more frequently than in the other regions (east: 18.9%; south: 10.5%), whereas D was found less frequently in the north (13.8%) than in the east (21.6%) and south (20.0%).

Short communication: Design and validation of a hygiene score for calves.

The objective of this study was the establishment and validation of a scoring system for calf dirtiness. Defined areas of the belly, side, and rear of the calves were scored according to the percentage of dirtiness: score 1 ranges from 0 to 10% of the area (no or little soiling), score 2 from over 10 to 30% (medium soiling), and score 3 applies to soiling of more than 30% of the area (heavy soiling). The scores of the individual regions were summed up to yield the calf hygiene score (CHS) ranging from 3 to 9. The validation of the CHS was performed by 5 veterinarians on 42 calves. It was validated for its inter- and intra-observer-reliability and against a standard method created by image processing of photographs of calves. The agreement between the observers and the standard method was weak to moderate with kappa values from 0.58 to 0.67. Inter- and intra-observer reliability resulted in a moderate to strong agreement with 29 of 36 kappa values between 0.60 and 0.89. The CHS was thus successfully validated as reliable and could be a useful tool for herd health management.

Technical note: Automatic evaluation of infrared thermal images by computerized active shape modeling of bovine udders challenged with Escherichia coli.

Mastitis causes substantial economic losses and animal suffering in the dairy industry. The trend toward larger herd sizes complicates the monitoring of udder health in individual animals. Infrared thermography has successfully been used for early mastitis detection. However, manual thermogram analysis is time consuming and requires a skilled examiner, and automated image processing has not been tested. The aim of this study was to determine whether automatic evaluation of thermograms showed results comparable to those of manual evaluation of thermograms. Five healthy cows underwent an intramammary challenge with Escherichia coli to induce clinical mastitis. Multiple udder thermograms were taken every 2 h for 24 h before and after the challenge, resulting in 4,143 images in total. All images were evaluated using image recognition software (automatically) and a polygon tool (manually) to calculate the average and maximum surface temperatures. Because of the slightly different regions of interest, temperatures ascertained from the thermograms using the automatic method were consistently lower than those ascertained using the manual method. However, average udder surface temperatures evaluated using both methods were strongly correlated (r = 0.98 in the left hindquarter, and r = 0.99 in the right hindquarter) and showed maximum temperature peaks at the same time, 13 and 15 h after intramammary challenge. In the receiver operating characteristic analysis, both methods provided good results for sensitivity and specificity in detecting clinical E. coli-induced mastitis at different threshold values. For automatically evaluated maximum right hindquarter temperature, sensitivity was 93.75% and specificity was 94.96%, and for manually evaluated maximum right hindquarter temperature, sensitivity was 93.75% and specificity was 96.40%. Thus, automatic thermogram evaluation is a promising tool for automated mastitis detection.

[Tinea faciei caused by Nannizzia persicolor : An underdiagnosed dermatophyte?] / Tinea faciei durch Nannizzia persicolor : Ein unterdiagnostizierter Dermatophyt?

We report on a tinea faciei caused by Nannizzia (N.) persicolor. The 4­year-old boy had probably been infected by a guinea pig. Unambiguous infections caused by N. persicolor are rarely seen in Germany; however, this zoophilic and geophilic dermatophyte may only be rarely identified due to its resemblance to Trichophyton (T.) mentagrophytes. Therefore, the diagnostic attributes of N. persicolor and its differentiation from T. mentagrophytes are described. Particularly in case of contact with rodents, N. persicolor should be kept in mind.

Fatal proteinuric kidney disease in a 30-month-old German Fleckvieh heifer caused by unilateral focal segmental glomerulosclerosis subsequent to a non-functional counterpart kidney.

INTRODUCTION: A case of secondary focal segmental glomerulosclerosis (FSGS) in a heifer is presented. A 30-month-old female German Fleckvieh heifer showed deterioration of the general condition, a poor nutritional status, proteinuria, hypoalbuminemia, and renal azotemia. Pathologically, it was diagnosed with unilateral hydronephrosis, and contralateral renal fibrosis with numerous cysts. Histologically, the fibrotic kidney showed FSGS, hyaline reabsorption droplets in proximal tubular epithelial cells, interstitial fibrosis, and tubulointerstitial inflammation. Apart from that, thrombotic microangiopathy (TMA) was seen in few renal arteries and meningeal arterioles. Pathogenesis of FSGS secondary to unilateral renal parenchymal loss (hydronephrosis) and TMA is discussed.

Identification of Arcanobacterium hippocoleae by MALDI-TOF MS analysis and by various genotypical properties.

In the present study an Arcanobacterium hippocoleae strain isolated from a uterus swab of an apparently healthy mare could be identified by phenotypic properties, by MALDI-TOF MS analysis and genotypically by investigating the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region and the genes encoding the ß subunit of bacterial RNA polymerase (rpoB), elongation factor tu (tuf) and glyceraldehyde 3-phosphate dehydrogenase (gap). The presented data are one of the few reports about the species A. hippocoleae and might help to elucidate the role this species plays in infections of horses.

Hepatic Lipodystrophy in Galloway Calves.

Hepatic lipodystrophy in Galloway calves is a fatal liver disease affecting a small proportion of the Galloway breed described in different parts of Europe and North America during the past decades. The clinical findings include a diversity of neurological signs. Clinical pathology findings frequently indicate hepatobiliary disease. Postmortem examination reveals an enlarged, pale yellow, and firm liver. Histologic lesions include hepatic fibrosis, hepatic lipidosis, and bile duct hyperplasia. To date, the etiopathogenesis remains obscure. Infectious causes, intoxications, and a hereditary origin have been considered. We describe hepatic lipodystrophy in Galloway calves from an extensively farmed cow-calf operation in southern Germany. Main clinical findings in 6 calves were consistent with hepatic encephalopathy. Clinical pathology findings in 5 of 6 tested animals revealed increased concentration of total bilirubin (maximum value [MV], 54 µmol/l; reference range [RR], <8.5 µmol/l), direct bilirubin (MV, 20 µmol/l; RR, <3.4 µmol/l), increased activity of gamma glutamyl transferase (MV, 162 U/l; RR, <36 U/l) and glutamate dehydrogenase (MV, 420 U/l; RR, <16 U/l). In addition, activity of glutathione peroxidase was decreased in all tested ( n = 5) animals (MV, 61 U/g hemoglobin [Hb]; RR, >250 U/g Hb). Postmortem examination in 6 calves revealed a firm, diffusely enlarged yellow liver with a finely nodular surface. Histologic lesions included hepatic fibrosis, hepatic lipidosis, and bile duct hyperplasia. Our findings add to the existing data on hepatic lipodystrophy in the Galloway breed and outline a protocol to aid in the diagnosis of this disorder.

Investigations on the possible impact of a glyphosate-containing herbicide on ruminal metabolism and bacteria in vitro by means of the 'Rumen Simulation Technique'.

AIMS: This study was performed in a well-established in vitro model to investigate whether the application of a glyphosate-containing herbicide might affect the bacterial communities and some biochemical parameters in a cow's rumen. METHODS AND RESULTS: The test item was applied in two concentrations (high and low) for 5 days. In a second trial, fermentation vessels were inoculated with Clostridium sporogenes before the high dose was applied. Effluents were analysed by biochemical, microbiological and genetic methods. A marginal increase in short-chain fatty acid production and a reduction in NH3 -N were observed. There were minor and rather equivocal changes in the composition of ruminal bacteria but no indications of a shift towards a more frequent abundance of pathogenic Clostridia species. Clostridium sporogenes counts declined consistently. CONCLUSIONS: No adverse effects of the herbicide on ruminal metabolism or composition of the bacterial communities could be detected. In particular, there was no evidence of a suspected stimulation of Clostridia growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibiotic activity of glyphosate resulting in microbial imbalances has been postulated. In this exploratory study, however, intraruminal application of concentrations reflecting potential exposure of dairy cows or beef cattle did not exhibit significant effects on bacterial communities in a complex in vitro system. The low number of replicates (n = 3/dose) may leave some uncertainty.

A controlled trial on the effect of feeding dietary chestnut extract and glycerol monolaurate on liver function in newborn calves.

Beginning in the fall of 2010, an increasing and alarming number of cases of calves suffering from liver dystrophy were reported in the south of Germany. An epidemiological investigation was carried out by the authors between November 2010 and July 2011, leading to the implication of a commercial dietary supplement as the potential cause for this outbreak. The components of this product were first tested in a cell culture model and two of them (dietary chestnut extract and glycerol monolaurate) showed a cytotoxic effect. The objective of this study was therefore to evaluate the effect of supplemental feeding of both components alone or in combination on liver function in newborn calves on a commercial dairy farm. Ten calves were enrolled in each of the three treatment groups and the control group (group O) following a blocked design. Treatment consisted of supplementation with chestnut extract at 0.02% of birth body mass (BM) (group C), supplementation with glycerol monolaurate at 0.006% of BM (group G) or a combined treatment (group CG) for five consecutive days. The effect of treatments on liver function was evaluated clinically and by measurement of glutamate dehydrogenase (GLDH) and aspartate aminotransferase (AST) activities as well as the determination of the concentrations of glucose, L-lactate and total bilirubin in serum. There was a significant increase in GLDH and AST activities and a significant decrease in glucose concentration in treatment groups C and CG compared with the control group (p ≤ 0.035), whereas no difference was shown for group G. Survival was significantly decreased in groups C (p = 0.029) and CG (p = 0.001) compared with both group G and the control group. These results suggest that dietary chestnut extract in an amount of 0.02% of BM alone or in combination has a toxic effect on liver function in newborn calves.

Infection-induced anaemia: a cross-sectional study of 14,636 German travellers aged 20-49 years.

BACKGROUND: Anaemia is a frequently diagnosed condition which can develop as a consequence of numerous factors, including infectious diseases (IDs). Travelling, especially in sub-/tropical regions, leads to an elevated risk of contracting IDs. The aim of our study was to assess the epidemiological significance of IDs in inducing anaemia among a large cohort of returned travellers. METHODS: This was a cross-sectional study in which data on 17,009 returned travellers aged 20-49 years who consulted the travel medicine clinic of the University of Munich between 1999 and 2011 were retrieved and analysed. RESULTS: Of the returned travellers, 8.3 % (6.0 % of males/10.4 % of females) were diagnosed with anaemia. The prevalence of anaemia was significantly elevated among patients of African (21.4/28.3 %) and Asian (11.6/15.7 %) origin. When the study population was restricted to the 14,636 travellers of German origin, 7.1 % of the returned travellers (4.6/9.6 %) were diagnosed with anaemia. The prevalence was significantly elevated among patients who travelled for >30 days (5.7 of males/10.6 % of females) and for male travellers visiting friends and relatives (7.7 %). However, these correlations were confounded by malaria. The prevalence of anaemia was significantly elevated only among returned travellers diagnosed with malaria (36.1 of males/26.9 % of females) and with symptomatic intestinal Entamoeba histolytica infections (30.0/33.3 %). CONCLUSION: Following the exclusion of confounding by malaria from the statistical analysis, the prevalence of anaemia was found to be significantly elevated among patients of African and Asian origin, and among patients of German origin who had travelled for >30 days, it could be mainly attributable to chronic, long-lasting causes. Although more than 550 travel-associated IDs were assessed in our study, only symptomatic intestinal Entamoeba histolytica infections and, to an even larger extent, malaria were determined to be of epidemiological significance for inducing anaemia among travellers.

Balling gun-induced trauma in cattle: clinical presentation, diagnosis and prevention.

Pharyngeal trauma in cattle can occur during the administration of oral medication using a balling gun. The number of cases of severe complications due to bolus application that have been referred to our hospital has increased from nil between 1996 and 2008 to three or four per year. In our experience, reports by bovine veterinarians of patients with severe and often fatal pharyngeal trauma, which were not referred to the clinic, have become more common in recent years as well. The incidence of this complication is likely to be higher than this number of referrals suggests. Diagnosis without the help of imaging techniques, such as radiography and endoscopy, may be difficult, especially in cases where exploration of the pharynx cannot be carried out, or is unable to confirm the absence or presence of a lesion. Prognosis is often poor in cases where perforation has been confirmed. Boluses are increasingly administered by the owners or farm personnel without the supervision of a veterinarian. In order to prevent losses due to balling gun-induced injuries, the veterinarian plays a crucial role in giving advice to his clients. Five cases of cattle suffering from varying degrees of balling gun-induced trauma are presented, and consideration is given to incorrect application techniques.

Attendance at iron deficiency anaemia clinic: audit of outcomes 5 years on.

AIM: To determine the long-term outcomes of patients in whom no initial cause for their anaemia is established. METHOD: Six hundred and thirty-nine patients presenting to an iron deficiency anaemia pathway were prospectively entered onto a database. Initial assessment included haematological review, coeliac screen, oesophagogastroduodenoscopy and colonic imaging as per British Society of Gastroenterology guidelines. A 5-year audit of outcomes was undertaken using patient medical records and hospital laboratory databases. RESULTS: A diagnosis was found initially in 266 (42%) patients, with 124 (19%) having a gastrointestinal (GI) luminal neoplasm, 54 (8%) of which were malignant. Twelve patients had a nonluminal or non-GI malignancy. Eighty-eight (14%) had benign upper GI bleeding and 23 (4%) had coeliac disease. One hundred and forty-three (22%) did not have confirmed iron deficiency anaemia on review of haematinics. Complete records were available for 595 (93%) patients at 5 years. Of the 373 patients in whom a cause was not initially diagnosed, 6 (2%) were ultimately diagnosed with a GI luminal malignancy and 18 (5%) with a nonluminal or non-GI malignancy. There was no difference in the incidence of malignancies between those with or without confirmed iron deficiency. CONCLUSIONS: Most patients in whom no cause was found at initial investigation resolve on oral iron supplements. Patients with normal ferritin values had as high an incidence of GI malignancies as those with low values and should be investigated. In the over 50s if the anaemia remains after a course of iron further investigation is recommended as there is a significant incidence of both GI and non-GI pathology.

[High incidence of jaundice in young calves in Southern Germany]. / Häufung von Ikterus bei jungen Kälbern in Süddeutschland.

OBJECTIVE: Between September, 2010, and August, 2011, a series of cases of jaundice of unknown origin in young calves was detected in a number of farms in Southern Germany. This paper describes the syndrome on the basis of 57 cases, and the approach taken to discover the cause. MATERIAL AND METHODS: The clinical course of the disease is described in 19 patients. Using a case definition (calves aged 1-3 weeks, total serum bilirubin > 20 µmol/l and/or serum glutamate dehydrogenase [GLDH] activity >50U/l and/or autopsy findings with striking liver pathology [jaundice, liver dystrophy, cirrhosis]), 36 farms were included in an epidemiological survey. In a feeding trial, two batches of a dietary supplement feed, previously used in diseased calves on farms, were fed at the dosage recommendations of the manufacturer to four clinically healthy calves over 5days. Four other calves served as controls. The calves were clinically monitored daily, and blood samples were investigated using clinical chemistry and haematology. RESULTS: Clinical examination revealed behavioural alterations (weakness, tonic-clonic seizures and bawling just before death), recumbency, jaundice and discolouration of faeces. In less severe cases without clinical signs, there was an increase in serum bilirubin concentration and/or GLDH activity. In the epidemiological survey of affected farms, the feeding of a diet supplement feed was registered in 54 of 57 cases. The feeding of two batches of that diet supplement feed to four clinically healthy calves resulted in a significant (p<0.05) increase in bilirubin and lactate concentrations, as well as the GLDH activity in serum, but without serious impairment of the general condition, whereas in control calves, no comparable changes were observed. CONCLUSION: The results of the epidemiological survey and the feeding trial suggest a causal involvement of a dietary supplement feed. The toxic principle is unknown. CLINICAL RELEVANCE: Knowledge of the clinical picture and the probable feed-related context is important to detect this disease early. The suspected dietary supplement feed has been taken off the market, but with other products similar problems may arise.

First description of Trueperella (Arcanobacterium) bernardiae of animal origin.

In the present study a Trueperella (Arcanobacterium) bernardiae strain isolated from an anal swab of a three-day-old piglet could be identified phenotypically, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing the 16S rDNA, the 16S-23S rDNA intergenic spacer region (ISR) and by sequencing the superoxide dismutase A encoding gene sodA. The present study gives the first information about the presence of T. (A.) bernardiae in specimen of animals.

Interleukin 12B gene polymorphism and apparent resistance to hepatitis C virus infection.

Cellular immunity with interferon gamma production could have a role in protection from hepatitis C virus (HCV). Interleukin (IL)-12 is a key cytokine in promoting such anti-viral T helper 1 (Th1) responses. We hypothesized that a genetic background able to promote cellular responses may be associated with apparent protection from infection and have investigated the distribution of the functional 1188A/C polymorphism of IL-12B in HCV exposed but uninfected cases. The frequency of the high IL-12-producing C allele was determined by restriction enzyme genotyping in 76 exposed-uninfected individuals and 105 healthy controls. Overall, the C allele was found in 27.6% of exposed-uninfected cases compared with 16.7% of healthy controls [chi(2) = 6.3, P = 0.02, odds ratio (OR) = 1.9, 95% confidence interval (CI) = 1.1-3.2]. CC genotype was found in 10.5% of exposed-uninfected cases compared with 0.9% controls (chi(2) = 9.3, P = 0.01, OR = 12, 95% CI = 1.5-100). Individuals at high risk of HCV infection yet who remain uninfected may be resistant in some way to infection. In our cohort of exposed-uninfected cases a genetic background of enhanced IL-12 production was associated with apparent resistance to HCV infection. This lends support to a central role for cellular immune responses in protecting from infection.

Application of the glutaraldehyde test in cattle.

In a cohort study involving 62 cows from an experimental farm, the kinetics of the glutaraldehyde test (GAT) according to Sandholm (1974) was examined by testing samples of EDTA blood, lithium heparinate blood, serum, and plasma taken at various intervals. Total protein was measured in serum, and fibrinogen was determined in plasma. Gamma globulin was measured by electrophoresis. All glutaraldehyde tests were performed in duplicates, and the relation of the two results was used as measurement of precision. Optimal cut-off of the GAT time was determined as the zenith of the sum of sensitivity and specificity of various intervals for detecting combinations of gamma globulin and fibrinogen levels above 32 g/l. Precision was the best in EDTA blood. The relation between coagulation time and gamma globulin plus fibrinogen is best described by an exponential curve. The maximum value for the sum of sensitivity and specificity was found at 7 and 8 min. Seven days was the shortest interval observed between a negative test result (>15 min) and a strongly positive test result (<3 min). Twenty-one days was the shortest interval observed between a strongly positive test result and a negative test result. EDTA blood should be used for the GAT. A cut-off of 8 min yields the highest efficiency. Test results must be viewed in light of clinical findings.

Diagnostic accuracy of faecal calprotectin estimation in prediction of abnormal small bowel radiology.

BACKGROUND: [corrected] Patients being investigated for symptoms of abdominal pain, diarrhoea and or weight loss often undergo small bowel radiology as part of their diagnostic workup mainly to exclude inflammatory bowel disease. AIM: To assess and compare the utility of a single faecal calprotectin estimation to barium follow through as well as conventional inflammatory markers such as erythrocyte sedimentation rate and C-reactive protein in exclusion of intestinal inflammation. METHODS: Seventy-three consecutive cases undergoing barium follow through for investigation of symptoms of diarrhoea and or abdominal pain with or without weight loss were studied. The control group comprised 25 cases with known active Crohn's disease (positive controls), 26 normal healthy volunteers (negative controls) and 25 cases of irritable bowel syndrome diagnosed by Rome II criteria. Symptoms, erythrocyte sedimentation rate and C-reactive protein were recorded at recruitment and a single stool sample assayed for calprotectin within 7 days prior to or after barium follow through. RESULTS: The median calprotectin value in the active Crohn's group, irritable bowel syndrome group and normal volunteers was 227 microg/g of stool, 19 and 10 microg/g respectively (P < 0.0001). A faecal calprotectin above a cut-off value of 60 microg/g was able to predict all nine cases with an abnormal barium follow through as well as all six cases with a normal barium follow through but with organic intestinal disease. The negative predictive value of a single calprotectin result below 60 microg/g of stool was 100% compared with 91% each for erythrocyte sedimentation rate > 10 mm and C-reactive protein > 6 mg/L and 84% for a combination of erythrocyte sedimentation rate and C-reactive protein in predicting absence of organic intestinal disease. CONCLUSION: A single stool calprotectin value < 60 microg/g of stool obviates the need for further barium radiology of the small bowel, is more accurate than measurement of erythrocyte sedimentation rate or C-reactive protein and effectively excludes Crohn's disease or non-functional gastrointestinal disease.

Role of activator site position and a distal UP-element half-site for sigma factor selectivity at a CRP/H-NS-activated sigma(s)-dependent promoter in Escherichia coli.

Transcription initiation by the stress-associated sigma(S)-containing RNA polymerase holoenzyme (E sigma(S)) in Escherichia coli is often subject to complex regulation that involves multiple additional regulators and histone-like proteins. csiD is a stationary phase-inducible sigma(S)-dependent gene in E. coli that requires activation by cAMP-CRP (bound to a site centred at -68.5 nucleotides upstream of the transcriptional start site) and is positively modulated by the abundant nucleoid-associated proteins H-NS and Lrp. By shifting the CRP box to positions between -80.5 and -60.5, we could demonstrate that: (i) activation is equally helix phase dependent as at classic class I promoters; (ii) E sigma(S) prefers a CRP box location at -68.5/-70.5, whereas E sigma(70) is nearly inactive with such an arrangement; and (iii) with the CRP site moved to -60.5, transcription can be initiated efficiently by both holoenzymes. The csiD promoter region also contains a distal UP-element half-site located downstream of the CRP box, as demonstrated by mutational studies, in which this element was either eliminated or completed to a full UP-element. The UP-element half-site favours E sigma(S)-mediated expression, whereas with the full UP-element, nearly wild-type levels of csiD transcription were observed in the absence of sigma(S). Finally, we show that the two histone-like proteins, H-NS and Lrp, both act by influencing activation by cAMP-CRP, but do so by different mechanisms. In particular, H-NS directly or indirectly increases positional stringency for the CRP binding site. The implications of these findings with respect to sigma factor selectivity, activation mechanisms used by the two holoenzymes and the architecture of sigma(S)-dependent promoters are discussed.