The role of 'atypical' Brucella in amphibians: are we facing novel emerging pathogens?

AIMS: To discuss together the novel cases of Brucella infections in frogs with the results of published reports to extend our current knowledge on 'atypical' brucellae isolated from amphibians and to discuss the challenges we face on this extraordinary emerging group of pathogens. METHODS AND RESULTS: Since our first description, an additional 14 isolates from four different frog species were collected. Novel isolates and a subset of Brucella isolates previously cultured from African bullfrogs were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), Fourier transform-infrared (FT-IR) spectroscopy and broth microdilution susceptibility testing. MALDI-TOF MS worked very efficiently for an accurate bacterial identification to the genus level. Within the cluster analysis, 'atypical' brucellae grouped distant from Brucella melitensis and were even more separated by FT-IR spectroscopy with respect to their geographical origin. Minimum inhibitory concentrations of 14 antimicrobial substances are provided as baseline data on antimicrobial susceptibility. CONCLUSIONS: The case history of Brucella infections in amphibians reveals a variety of pathologies ranging from localized manifestations to systemic infections. Some isolates seem to be capable of causing high mortality in zoological exhibitions putting higher demands on the management of endangered frog species. There is considerable risk in overlooking and misidentifying 'atypical' Brucella in routine diagnostics. SIGNIFICANCE AND IMPACT OF THE STUDY: Brucella have only recently been described in cold-blooded vertebrates. Their presence in frog species native to Africa, America and Australia indicates a more common occurrence in amphibians than previously thought. This study provides an extensive overview of amphibian brucellae by highlighting the main features of their clinical significance, diagnosis and zoonotic potential.

Serological survey of Bartonella spp., Borrelia burgdorferi, Brucella spp., Coxiella burnetii, Francisella tularensis, Leptospira spp., Echinococcus, Hanta-, TBE- and XMR-virus infection in employees of two forestry enterprises in North Rhine-Westphalia, Germany, 2011-2013.

We initiated a survey to collect basic data on the frequency and regional distribution of various zoonoses in 722 employees of forestry enterprises in the German state of North Rhine-Westphalia (NRW) from 2011 to 2013. Exposures associated with seropositivity were identified to give insight into the possible risk factors for infection with each pathogen. 41.2% of participants were found to be seropositive for anti-Bartonella IgG, 30.6% for anti-Borrelia burgdorferi IgG, 14.2% for anti-Leptospira IgG, 6.5% for anti-Coxiella burnetii IgG, 6.0% for anti-Hantavirus IgG, 4.0% for anti-Francisella tularensis IgG, 3.4% for anti-TBE-virus IgG, 1.7% for anti-Echinococcus IgG, 0.0% for anti-Brucella IgG and anti-XMRV IgG. Participants seropositive for B. burgdorferi were 3.96 times more likely to be professional forestry workers (univariable analysis: OR 3.96; 95% CI 2.60-6.04; p<0.001); and participants seropositive for Hantavirus 3.72 times more likely (univariable analysis: OR 3.72; 95% CI 1.44-9.57; p=0.007). This study found a surprisingly high percentage of participants seropositive for anti-B. henselae IgG and for anti-F. tularensis IgG. The relatively high seroprevalence for anti-Leptospira IgG seen in this study could be related to living conditions rather than to exposure at work. No specific risk for exposure to C. burnetii and Echinococcus was identified, indicating that neither forestry workers nor office workers represent a risk population and that NRW is not a typical endemic area. Forestry workers appear to have higher risk for contact with B. burgdorferi-infected ticks and a regionally diverse risk for acquiring Hantavirus-infection. The regional epidemiology of zoonoses is without question of great importance for public health. Knowledge of the regional risk factors facilitates the development of efficient prevention strategies and the implementation of such prevention measures in a sustainable manner.

Psychological distress and adjustment to disease in patients before and after radical prostatectomy. Results of a prospective multi-centre study.

The aim of this prospective multi-centre study was to evaluate the level of psychological distress (PD) and adjustment to disease in patients who underwent radical prostatectomy. Furthermore, the impact of urinary incontinence and erectile dysfunction on PD was assessed. Anxiety, depression and PD were evaluated using the Hospital Anxiety and Depression Scale in 329 prostate cancer patients before surgery as well as 3, 6 and 12 months after surgery. These results were compared with those of a male German general population reference group. Adjustment to disease was assessed using the Perceived Adjustment to Chronic Illness Scale. Patients reported low levels of PD at all points of assessment similar to population norms of age-matched German men. Persistent PD was seen in about 8% of the patients and 20% had PD at least two of the measurement points. Relevant predictors for PD after surgery were urinary symptoms and baseline PD. Adjustment to disease was highest before surgery and had significantly reduced at 3 and 6 months after surgery. In general, men are resilient to the experience of localised prostate cancer and adjust well psychologically after surgery. However, between 8% and 20% of patients could possibly benefit from mental health support.

Haloarchaeal myovirus φCh1 harbours a phase variation system for the production of protein variants with distinct cell surface adhesion specificities.

The φCh1 myovirus, which infects the haloalkaliphilic archaeon Natrialba magadii, contains an invertible region that comprises the convergent open reading frames (ORFs) 34 and 36, which code for the putative tail fibre proteins gp34 and gp36 respectively. The inversion leads to an exchange of the C-termini of these proteins, thereby creating different types of tail fibres. Gene expression experiments revealed that only ORF34 is transcribed, indicating that φCh1 produces tail fibre proteins exclusively from this particular ORF. Only one of the two types of tail fibres encoded by ORF34 is able to bind to Nab. magadii in vitro. This is reflected by the observation that during the early phases of the infection cycle, the lysogenic strain L11 carries its invertible region exclusively in the orientation that produces that specific type of tail fibre. Obviously, Nab. magadii can only be infected by viruses carrying this particular type of tail fibre. By mutational analysis, the binding domain of gp34 was localized to the C-terminal part of the protein, particularly to a galactose-binding domain. The involvement of galactose residues in cell adhesion was supported by the observation that the addition of α-D-galactose to purified gp34 or whole virions prevented their attachment to Nab. magadii.

Utilization of virus φCh1 elements to establish a shuttle vector system for Halo(alkali)philic Archaea via transformation of Natrialba magadii.

In the study described here, we successfully developed a transformation system for halo(alkali)philic members of the Archaea. This transformation system comprises a series of Natrialba magadii/Escherichia coli shuttle vectors based on a modified method to transform halophilic members of the Archaea and genomic elements of the N. magadii virus Ch1. The shuttle vector pRo-5, based on the repH-containing region of Ch1, stably replicated in E. coli and N. magadii and in several halophilic and haloalkaliphilic members of the Archaea not transformable so far. The Ch1 operon ORF53/ORF54 (repH) was essential for pRo-5 replication and was thus identified as the minimal replication origin. The plasmid allowed homologous and heterologous gene expression, as exemplified by the expression of Ch1 ORF3452, which encodes a structural protein, and the reporter gene bgaH of Haloferax lucentense in N. magadii. The new transformation/vector system will facilitate genetic studies within N. magadii and other haloalkaliphilic archaea and will allow the detailed characterization of the gene functions of N. magadii virus Ch1 in their extreme environments.

Molecular characterisation of Brucella species.

The genus Brucella (Mayer and Shaw, 1920) currently consists often species with validly published names. Within most species further differentiation into biovars exists. Genetically, all Brucella species are highly related to each other, exhibiting sequence similarity values of 98% to 100% in aligned regions (core genome). The population structure is clonal. Despite this close genetic relatedness, the various species can be clearly distinguished from each other by application of high-resolution molecular typing tools, in addition to assessment of phenotype and host preference. Accurate species delineation can be achieved by conventional multiplex polymerase chain reaction (PCR), single nucleotide polymorphism (SNP) analysis and multilocus sequence typing (MLST) or multilocus sequence analysis (MLSA). The last is also suitable for phylogenetic reconstructions, owing to the highly clonal evolution of the different species. Highly discriminatory multilocus variable number of tandem repeats (VNTR) analysis (MLVA) allows both species delineation and differentiation of individual isolates and thus represents a perfect first-line toolfor molecular epidemiological studies within outbreak investigations. More recently,whole genome sequencing (WGS)and the resulting global genome-wide SNP analysis have become available. These novel approaches should help in further understanding the evolution, host specificity and pathogenicity of the genus Brucella.

Camelimonas abortus sp. nov., isolated from placental tissue of cattle.

A gram-negative, rod-shaped, non-spore-forming bacterium, isolated from placental tissue of a cow, was investigated for its taxonomic position. On the basis of 16S rRNA gene sequence similarities, strain UK34/07-5(T) was shown to belong to the class Alphaproteobacteria, closely related to the type strain of Camelimonas lactis (96.0 % sequence similarity). The polyamine pattern showed the major compound spermidine and moderate amounts of putrescine. The major quinone was ubiquinone Q-10. The polar lipid profile was composed of the major compounds phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine and moderate amounts of diphosphatidylglycerol, three unidentified aminolipids and an unidentified phospholipid. The profile of major fatty acids, consisting of C(19 : 0) cyclo ω8c and C(18 : 1)ω7c, with C(18 : 0) 3-OH as the hydroxylated fatty acid, was very similar to that of C. lactis M 2040(T). The results of DNA-DNA hybridization and physiological and biochemical tests allowed both genotypic and phenotypic differentiation of the isolate from C. lactis. The relatively low 16S rRNA gene sequence similarity of 96.0 % to C. lactis M 2040(T) and marked differences in the polar lipid profiles as well as the results of physiological tests and the DNA-DNA hybridization data support the creation of a novel species, for which the name Camelimonas abortus sp. nov. is proposed, with the type strain UK34/07-5(T) ( = CIP 110303(T)  = CCUG 61094(T)  = DSM 24741(T)  = CCM 7941(T)).

Reduced soluble receptor for advanced glycation end-products (sRAGE) scavenger capacity precedes pre-eclampsia in Type 1 diabetes.

OBJECTIVE: Increased advanced glycation end-products (AGEs) and their soluble receptors (sRAGE) have been implicated in the pathogenesis of pre-eclampsia (PE). However, this association has not been elucidated in pregnancies complicated by diabetes. We aimed to investigate the serum levels of these factors in pregnant women with Type 1 diabetes mellitus (T1DM), a condition associated with a four-fold increase in PE. DESIGN: Prospective study in women with T1DM at 12.2 ± 1.9, 21.6 ± 1.5 and 31.5 ± 1.7 weeks of gestation [mean ± standard deviation (SD); no overlap] before PE onset. SETTING: Antenatal clinics. POPULATION: Pregnant women with T1DM (n = 118; 26 developed PE) and healthy nondiabetic pregnant controls (n = 21). METHODS: Maternal serum levels of sRAGE (total circulating pool), N(ε)-(carboxymethyl)lysine (CML), hydroimidazolone (methylglyoxal-modified proteins) and total AGEs were measured by immunoassays. MAIN OUTCOME MEASURES: Serum sRAGE and AGEs in pregnant women with T1DM who subsequently developed PE (DM PE+) versus those who remained normotensive (DM PE-). RESULTS: In DM PE+ versus DM PE-, sRAGE was significantly lower in the first and second trimesters, prior to the clinical manifestation of PE (P < 0.05). Further, reflecting the net sRAGE scavenger capacity, sRAGE:hydroimidazolone was significantly lower in the second trimester (P < 0.05) and sRAGE:AGE and sRAGE:CML tended to be lower in the first trimester (P < 0.1) in women with T1DM who subsequently developed PE versus those who did not. These conclusions persisted after adjusting for prandial status, glycated haemoglobin (HbA1c), duration of diabetes, parity and mean arterial pressure as covariates. CONCLUSIONS: In the early stages of pregnancy, lower circulating sRAGE levels, and the ratio of sRAGE to AGEs, may be associated with the subsequent development of PE in women with T1DM.

Muscarinic and beta-adrenergic regulation of heart rate, force of contraction and calcium current is preserved in mice lacking endothelial nitric oxide synthase.

Nitric oxide (NO) is an ubiquitous signaling molecule produced from L-arginine by NO synthase (NOS). In the vasculature, NO mediates parasympathetic endothelium-dependent vasodilation. NO may also mediate the parasympathetic control of myocardial function. This is supported by the observations that NOS3, the endothelial constitutive NOS, is expressed in normal cardiac myocytes from rodents and human, and NOS and/or guanylyl cyclase inhibitors antagonize the effect of muscarinic agonists on heart rate, atrio-ventricular conduction, contractility and L-type calcium current. Here we examine the autonomic regulation of the heart in genetically engineered mice deficient in NOS3 (NOS3-KO). We show that the chronotropic and inotropic responses to both beta-adrenergic and muscarinic agonists were unaltered in isolated cardiac tissue preparations from NOS3-KO mice, although these mice have a defective parasympathetic regulation of vascular tone. Similarly, beta-adrenergic stimulation and muscarinic inhibition of the calcium current did not differ in cardiac myocytes from NOS3-KO mice and those from wild-type mice. RT-PCR did not demonstrate upregulation of other NOS isoforms. Similarly, Gi/Go proteins and muscarinic receptor density were unaltered. These data refute the idea that NOS3 is obligatory for the normal autonomic control of cardiac muscle function.

Camelimonas lactis gen. nov., sp. nov., isolated from the milk of camels.

Three strains of Gram-negative, rod-shaped, non-spore-forming bacteria (M 2040(T), M 1973 and M 1878-SK2), isolated from milk of camels at a camel-milk production farm in the United Arab Emirates, were investigated for their taxonomic allocation. On the basis of 16S rRNA gene sequence similarities, all three strains were shown to belong to the Alphaproteobacteria and were most closely related to Chelatococcus asaccharovorans and Chelatococcus daeguensis (95.1 and 95.2 % sequence similarity to the respective type strains). meso-Diaminopimelic acid was detected as the characteristic peptidoglycan diamino acid. The predominant compound in the polyamine pattern was spermidine, and sym-homospermidine was not detectable. The quinone system was ubiquinone Q-10. The polar lipid profile included the major compounds phosphatidylcholine and diphosphatidylglycerol and moderate amounts of phosphatidylethanolamine, phosphatidylglycerol, an unidentified glycolipid and two unidentified aminolipids. Minor lipids were also detected. The major fatty acid profile, consisting of C19 :0 cyclo ω8c and C18:1 ω7c, with C18 :03-OH as the major hydroxylated fatty acid, was similar to those of the genus Chelatococcus. The results of DNA-DNA hybridization experiments and physiological and biochemical tests allowed both genotypic and phenotypic differentiation of the isolates from described Chelatococcus species. Isolates M 2040(T), M 1973 and M 1878-SK2 were closely related on the basis of DNA-DNA reassociation and therefore represent a single novel species. In summary, low 16S rRNA gene sequence similarities of 95 % with Chelatococcus asaccharovorans and marked differences in polar lipid profiles as well as in polyamine patterns support the description of a novel genus and species to accommodate these strains, for which the name Camelimonas lactis gen. nov., sp. nov. is proposed. The type strain of Camelimonas lactis is M 2040(T) (=CCUG 58638(T) =CCM 7696(T)).

Monitoring the quality of frozen-thawed venous segments using bioimpedance spectroscopy.

OBJECTIVE: Storage at temperatures as low as -80 °C and below (cryopreservation) is considered a method for long-term preservation of cells and tissues, and especially blood vessel segments, which are to be used for clinical operations such as transplantation. However, the freezing and thawing processes themselves can induce injuries to the cells and tissue by damaging the structure and consequently functionality of the cryopreserved tissue. In addition, the level of damage is dependent on the rate of cooling and warming used during the freezing-thawing process. Current methods for monitoring the viability and integrity of cells and tissues after going through the freezing-thawing cycle are usually invasive and destructive to the cells and tissues. Therefore, employing monitoring methods which are not destructive to the cryopreserved tissues, such as bioimpedance measurement techniques, is necessary. In this study we aimed to design a bioimpedance measurement setup to detect changes in venous segments after freezing-thawing cycles in a noninvasive manner. APPROACH: A bioimpedance spectroscopy measurement technique with a two-electrode setup was employed to monitor ovine jugular vein segments after each cycle during a process of seven freezing-thawing cycles. MAIN RESULTS: The results demonstrated changes in the impedance spectra of the measured venous segments after each freezing-thawing cycle. SIGNIFICANCE: This indicates that bioimpedance spectroscopy has the potential to be developed into a novel method for non-invasive and non-destructive monitoring of the viability of complex tissue after cryopreservation.

The synthetic liver X receptor agonist GW3965 reduces tissue factor production and inflammatory responses in human islets in vitro.

AIMS/HYPOTHESIS: Optimising islet culture conditions may be one strategy for reducing islet loss prior to, and immediately after, islet transplantation. Liver X receptor (LXR) agonism has previously been shown to increase insulin release from pancreatic islets and reduce inflammation in leucocytes. Our aim was to investigate whether the synthetic LXR agonist GW3965 could modulate the inflammatory status of human pancreatic islets. METHODS: Levels of pro-inflammatory cytokines and tissue factor in isolated human islets were determined by TaqMan low density array and/or real-time quantitative RT-PCR (mRNA levels) and enzyme immunoassay (EIA) (protein levels). Islet viability was measured using intracellular ATP content, ADP/ATP ratio, mitochondrial dehydrogenase activity (XTT assay) and insulin secretion in a dynamic glucose-challenge model. Apoptosis was determined by EIA measurement of histone-DNA complexes present in cytoplasm and by assaying caspase-3/-7 activity. RESULTS: Treatment of LPS-stimulated human islets with the synthetic LXR agonist GW3965 (1 micromol/l) for 24 h reduced mRNA and protein levels of selected pro-inflammatory cytokines (IL-8, monocyte chemotactic protein-1 and tissue factor). Moreover, GW3965 had no adverse effect on insulin secretion, islet viability or apoptosis. No excess of lipid accumulation could be detected with the dosage and exposure time used. CONCLUSIONS/INTERPRETATION: LXR activation suppresses inflammation in human islets in vitro without adverse effects on islet viability. Short-term moderate activation of LXR prior to islet transplantation may represent a possible strategy for improving post-transplant islet survival.

Anti-angiogenic factors and pre-eclampsia in type 1 diabetic women.

AIMS/HYPOTHESIS: Elevated anti-angiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt1), a soluble form of vascular endothelial growth factor receptor, and endoglin, a co-receptor for TGFbeta1, confer high risk of pre-eclampsia in healthy pregnant women. In this multicentre prospective study, we determined levels of these and related factors in pregnant women with type 1 diabetes, a condition associated with a fourfold increase in pre-eclampsia. METHODS: Maternal serum sFlt1, endoglin, placental growth factor (PlGF) and pigment epithelial derived factor were measured in 151 type 1 diabetic and 24 healthy non-diabetic women at each trimester and at term. RESULTS: Approximately 22% of the diabetic women developed pre-eclampsia, primarily after their third trimester visit. In women with pre-eclampsia (diabetic pre-eclampsia, n = 26) vs those without hypertensive complications (diabetic normotensive, n = 95), significant changes in angiogenic factors were observed, predominantly in the early third trimester and prior to clinical manifestation of pre-eclampsia. Serum sFlt1 levels were increased approximately twofold in type 1 diabetic pre-eclampsia vs type 1 diabetic normotensive women at the third trimester visit (p < 0.05) and the normal rise of PlGF during pregnancy was blunted (p < 0.05). Among type 1 diabetic women, third trimester sFlt1 and PlGF were inversely related (r(2) = 42%, p < 0.0001). Endoglin levels were increased significantly in the diabetic group as a whole vs the non-diabetic group (p < 0.0001). CONCLUSIONS/INTERPRETATION: Higher sFlt1 levels, a blunted PlGF rise and an elevated sFlt1/PlGF ratio are predictive of pre-eclampsia in pregnant women with type 1 diabetes. Elevated endoglin levels in women with type 1 diabetes may confer a predisposition to pre-eclampsia and may contribute to the high incidence of pre-eclampsia in this patient group.

Functional ethanol tolerance in Drosophila.

In humans, repeated alcohol consumption leads to the development of tolerance, manifested as a reduced physiological and behavioral response to a particular dose of alcohol. Here we show that adult Drosophila develop tolerance to the sedating and motor-impairing effects of ethanol with kinetics of acquisition and dissipation that mimic those seen in mammals. Importantly, this tolerance is not caused by changes in ethanol absorption or metabolism. Rather, the development of tolerance requires the functional and structural integrity of specific central brain regions. Mutants unable to synthesize the catecholamine octopamine are also impaired in their ability to develop tolerance. Taken together, these data show that Drosophila is a suitable model system in which to study the molecular and neuroanatomical bases of ethanol tolerance.

Mapping quantitative trait Loci for left-sided displacement of the abomasum in German Holstein dairy cows.

A whole-genome scan using an affected paternal half-sib design was utilized to detect quantitative trait loci (QTL) for left-sided displaced abomasum (LDA) in German Holsteins. A total of 360 animals from 14 paternal half-sib families were genotyped, for a total of 306 polymorphic microsatellites. For a whole-genome scan, 221 markers were equally distributed over all 29 bovine autosomes, with an average distance of 13.7 cM. For fine-mapping, a total of 85 additional microsatellites were used. We identified genome-wide significant QTL on Bos taurus autosome (BTA) 1 (54.6 to 58.3 cM) and on BTA3 (5.9 cM). Furthermore, 3 chromosome-wide significant QTL were located on bovine chromosomes 21, 23, and 24. In addition, we found 11 QTL that cosegregated in grandsire families but that were not significant in the across-family analysis. These QTL were located on BTA5, 6, 10, 12, 15, 16, 17, 19, 23, and 26. This study is the first report on QTL for LDA and is a first step toward identifying single nucleotide polymorphisms for LDA-QTL.

Involvement of endothelium-derived relaxing factor in the pressure control of renin secretion from isolated perfused kidney.

Using isolated rat kidneys perfused at controlled pressure, we examined a potential role of endothelium-derived relaxing factor (EDRF) in the pressure control of renin secretion. We found that stimulation of EDRF release by acetylcholine (1 mumol/liter) increased mean perfusate flow rates from 15.0 +/- 0.5 to 18.0 +/- 0.5 ml/min per g and average renin secretion rates from 3.5 +/- 0.5 to 16.0 +/- 2.0 ng angiotensin I/h per min per g at a perfusion pressure of 100 mmHg (mean +/- SEM, n = 6). Those effects of acetylcholine were significantly reduced during inhibition of EDRF formation with NG-nitro-L-arginine (100 mumol/liter), but they were not affected with the cyclooxygenase inhibitor indomethacin (10 mumol/liter). Lowering of the perfusion pressure from 100 mmHg to 40 mmHg resulted in an increase of average renin secretion rates from 3.5 +/- 0.5 to 79 +/- 12 ng AngI/h per min per g under control conditions (n = 8), and to 171 +/- 20 ng AngI/h per min per g in the presence of 10 mumol/liter acetylcholine (n = 3). The rise of renin secretion in response to a reduction of the renal artery pressure was markedly attenuated with inhibitors of EDRF formation such as NG-nitro-L-arginine (100 mumol/liter) and related compounds. During inhibition of EDRF formation, addition of sodium nitroprusside (10 mumol/liter) increased mean perfusate flow rates from 12.0 +/- 0.5 to 23.0 +/- 2.0 ml/min per g and average renin secretion rates from 2.0 +/- 0.5 to 18.0 +/- 1.5 ng AngI/h per min per g at 100 mmHg (n = 5). Lowering of the perfusion pressure from 100 mmHg to 40 mmHg under those conditions increased average renin secretion rates to 220 +/- 14 ng AngI/h per min per g (n = 5). Taken together, our findings suggest that EDRF and related activators of soluble guanylate cyclase stimulate renin secretion from isolated kidneys, predominantly at lower perfusion pressure. Moreover, pressure control of renin secretion appears to require the tonical stimulation by intrarenal EDRF.

Erythropoietin in polycystic kidneys.

Erythropoietin (EPO) formation in kidneys of 18 patients with autosomal dominant polycystic kidney disease (ADPKD) was investigated. In 12 patients on hemodialysis and in 6 patients with preterminal renal failure serum, EPO was 29 +/- 7 and 16 +/- 1.5 mU/ml and hemoglobin concentrations were 11.0 +/- 0.6 and 12.7 +/- 1.2 g/dl, respectively. Cyst fluid from a total of 357 renal cysts was obtained by either in vivo aspiration or immediately after nephrectomy. The cysts contained variable concentrations of bioactive EPO from undectable values up to 3.2 U/ml. A pronounced enrichment of EPO was observed in cysts with sodium concentrations greater than 100 mmol/liter, suggesting an association with proximal tubular malformations. The EPO concentrations in the cysts were neither correlated with the protein concentration nor with the oxygen pressure of the cyst fluid. Using a cDNA probe for human EPO, mRNA for EPO was localized in stroma cells of the cyst walls by an in situ hybridization technique. Our findings suggest that single interstitial cells juxtaposed to proximal tubular cysts may produce EPO independent of the oxygen pressure inside the cysts, which ameliorates the anemia during end-stage polycystic kidney disease.

Brucellosis of the common vole (Microtus arvalis).

A systemic disease occurred in a wild population of the common vole Microtus arvalis in South Moravia (Czech Republic) during the years 1999-2003. Acute infections were characterized by edema of extremities, occasionally with colliquating abscesses, arthritis, lymphadenitis, perforations of the skin resulting from colliquated abscesses, orchitis, and peritoneal granulomas. From the clinical samples, small Gram-negative coccobacilli were isolated and identified as Ochrobactrum intermedium by API 20NE and colistin sensitivity profiles. However, subsequent rrs (16S rRNA) and recA (recombinase A) gene sequencing analysis of two isolates (CCM 4915=CAPM 6434; CCM 4916=CAPM 6435) identified them as Brucella sp. with sequence identities of 100% to other Brucella spp. Analysis of the omp2a/b genes confirmed the two isolates as Brucella. In AMOS polymerase chain reaction (PCR), a 2000-bp fragment was generated that was not seen in other brucellae. Experimental infection of outbred ICR mice with these isolates resulted in a mortality rate of 50%. Based on the results of the molecular investigations and the mortality observed in experimentally infected mice we conclude that the epizootic was caused by Brucella sp. and not by Ochrobactrum intermedium. The study demonstrates the limitations of commercial biochemical test systems in accurately differentiating among Ochrobactrum and Brucella.

[Urinary tract infections in infants and children -- a consensus on diagnostic, therapy and prophylaxis]. / Harnwegsinfektionen im Säuglings- und Kindesalter. Konsensusempfehlungen zu Diagnostik, Therapie und Prophylaxe.

Urinary tract infections (UTI) are among the most common bacterial infections in infants and children. The early diagnosis of a pyelonephritis and its rapid, calculated antibacterial therapy are decisive for the prognosis. Urogenital anomalies, renal damage and bladder dysfunction may influence the risk of recurrences of UTI and pyelonephritic scarring. Diagnostic strategies therefore should focus on their early recognition. Pediatricians, urologists and infectiologists are cooperating in diagnostic, therapy and prophylaxis of UTI. The aim of the interdisciplinary consensus presented was to work out a concept which may help to manage childhood UTI in daily practice.

[Thiamine and its derivates in cattle blood measured by HPLC in healthy animals, in patients suffering from CCN and in their cohorts]. / Analyse von Vitamin B1 und dessen Derivaten im Rinderblut mittels HPLC-Technik bei gesunden, an CCN erkrankten Tieren und deren Kohorten.

The aim of this project was to develop a routine method to measure thiamine and its derivates in blood, to detect the need of vitamin B1-supply in cattle faster and more precise than until now using the transketolase test. For that, thiamine and its derivates (thiaminediphosphate = TDP,-monophosphate = TMP) were analysed in cattle blood by HPLC technique with post-column derivatization. Moreover, the levels in healthy cattle were compared with those in herds with single CCN cases. EDTA-blood-stored up to 10 days by 6 or 20 degrees C- was the most appropriate substrate. Sensitivitiy and specifity were significantly better than (TT, sum of all fractions): 1.95% and 2.10%; thiamine and TMP: 7.02% and 9.17%. The lowest concentration, which could be measured, was 0.5 ng/ml. 72 samples could be analysed daily. In the blood of 280 healthy calves and cows (group A), 201 clinically unsuspicious animals out of stocks with single cases of CCN (group B) and 12 patients with clinical obvious CCN (group C), thiamine and its phosphates were measured and the means calculated (ng/ml). After this, the thiamine status is characterized by (ng/ml): (A) thiamine 13.5 (4.61-28.8),TDP 51.0 (33.1-82.2), TMP 8.79 (2.23-18.1),TT 73.3 (44.6-114); (B) thiamine 8.73 (0.00-20.0), TDP 43.5 (15.5-75.7),TMP 6.15 (0.00-16.6), TT 59.4 (18.7-96.5); (C) thiamine 3.85 (0.00-1 1.5), TDP 14.5 (0.00-28.3), TMP 1.27 (0.00-4.87), TTP 19.6 (4.92-35.9). So, HPLC with post-column derivatization offers a precise, automatically working method for a fast diagnose of thiamine deficiency. The lowest critical value of TT is 40 ng/ml for healthy animals in the blood. The sample (EDTA-blood) can be posted simply at temperatures up to 20 degrees C. In comparison to other measuring methods, this HPLC-technique enables an early recognition of the risk to come down with CCN in endangered cattle herds.