Vermamoeba vermiformis resides in water-based heater-cooler units and can enhance Mycobacterium chimaera survival after chlorine exposure.

BACKGROUND: Mycobacterium chimaera colonizes water-based heater-cooler units (HCUs), from which it can spread to patients during surgery. Vermamoeba vermiformis is a free-living waterborne amoeba, which was consistently present within HCUs. AIM: To determine whether these amoebae can be involved in the persistent presence of M. chimaera. METHODS: An in-vitro disinfection model. FINDINGS: Increased survival of M. chimaera was observed after chlorine exposure in the presence of V. vermiformis. Confocal microscopy demonstrated the intracellular presence of M. chimaera in V. vermiformis. CONCLUSION: In this way, V. vermiformis can contribute to the persistent presence of M. chimaera in HCUs. Cleaning and disinfection protocols should take this phenomenon into account.

Acanthamoeba castellanii interferes with adequate chlorine disinfection of multidrug-resistant Pseudomonas aeruginosa.

Verona-Integron-encoded-Metallo-ß-lactamase-positive Pseudomonas aeruginosa (VIM-PA) is a cause of hard-to-treat nosocomial infections, and can colonize hospital water networks alongside Acanthamoeba. We developed an in-vitro disinfection model to examine whether Acanthamoeba castellanii can harbour VIM-PA intracellularly, allowing VIM-PA to evade being killed by currently used hospital disinfectants. We observed that A. castellanii presence resulted in significantly increased survival of VIM-PA after exposure to chlorine for 30 s or for 2 min. This undesirable effect was not observed after disinfection by 70% alcohol or 24% acetic acid. Confocal microscopy confirmed the presence of VIM-PA within A. castellanii pseudocysts. Our data indicate that A. castellanii contributes to persistent VIM-PA colonization of water systems after chlorine treatment.

Haemostatic changes in urogenital schistosomiasis haematobium: a case-control study in Gabonese schoolchildren.

In many tropical areas schistosomiasis is a major health problem causing hepatosplenic, intestinal or urogenital complaints. Hepatosplenic schistosomiasis mansoni is also characterized by blood coagulation abnormalities. Liver pathology plays a role in the development of haemostatic changes and the parasitic infection may directly affect coagulation. However, these contributing factors cannot be studied separately in hepatosplenic schistosomiasis infections. This pilot study provides insight in haemostatic changes in urinary schistosomiasis by studying coagulation parameters in schistosomiasis haematobium-infected Gabonese schoolchildren. Selection on urinary schistosomiasis patients without hepatosplenic complaints allows for the investigation of the direct effects of the parasite on haemostasis. Levels of von Willebrand Factor (VWF) antigen, active VWF and osteoprotegerin were elevated, indicating inflammation-mediated endothelial activation. In contrast to hepatosplenic schistosomiasis, thrombin-antithrombin complex and D-dimer levels were not affected. Despite its small sample size, this study clearly indicates that Schistosoma haematobium directly alters the activation status of the endothelium, without initiation of coagulation.

Adaptation to life on land at high O2 via transition from ferredoxin-to NADH-dependent redox balance.

Pyruvate : ferredoxin oxidoreductase (PFO) and iron only hydrogenase ([Fe]-HYD) are common enzymes among eukaryotic microbes that inhabit anaerobic niches. Their function is to maintain redox balance by donating electrons from food oxidation via ferredoxin (Fd) to protons, generating H2 as a waste product. Operating in series, they constitute a soluble electron transport chain of one-electron transfers between FeS clusters. They fulfil the same function-redox balance-served by two electron-transfers in the NADH- and O2-dependent respiratory chains of mitochondria. Although they possess O2-sensitive FeS clusters, PFO, Fd and [Fe]-HYD are also present among numerous algae that produce O2. The evolutionary persistence of these enzymes among eukaryotic aerobes is traditionally explained as adaptation to facultative anaerobic growth. Here, we show that algae express enzymes of anaerobic energy metabolism at ambient O2 levels (21% v/v), Chlamydomonas reinhardtii expresses them with diurnal regulation. High O2 environments arose on Earth only approximately 450 million years ago. Gene presence/absence and gene expression data indicate that during the transition to high O2 environments and terrestrialization, diverse algal lineages retained enzymes of Fd-dependent one-electron-based redox balance, while the land plant and land animal lineages underwent irreversible specialization to redox balance involving the O2-insensitive two-electron carrier NADH.

Performance of the commercially available SERION ELISA classic Echinococcus IgG test for the detection of cystic echinococcosis in clinical practice.

Diagnosis of cystic echinococcosis (CE) is at present mainly based on imaging techniques. Serology has a complementary role, partly due to the small number of standardized and commercially available assays. Therefore we examined the clinical performance of the SERION ELISA classic Echinococcus IgG test. Using 10 U/ml as a cut-off point, and serum samples from 50 CE patients and 105 healthy controls, the sensitivity and specificity were 98.0% and 96.2%, respectively. If patients with other infectious diseases were used as negative controls, the specificity decreased to 76.9%, which causes poor positive predictive values. However, if results between 10 and 15 U/ml are classified as indecisive, the specificity of positive results (≥15 U/ml) increased to 92.5% without greatly affecting the sensitivity (92.0%). Using this approach in combination with imaging studies, the SERION ELISA classic Echinococcosis IgG test can be a useful aid in the diagnosis of CE.

Effects of ß-hydroxybutyrate and isoproterenol on lipolysis in isolated adipocytes from periparturient dairy cows and cows with clinical ketosis.

An in vitro model was used to investigate effects of ß-hydroxybutyrate and isoproterenol (ß-adrenergic receptor agonist) on lipolysis in isolated adipocytes from late pregnant and recently calved dairy cows (n=5) and cows with clinical ketosis (n=3). Incubation with 3.0 mmol/L ß-hydroxybutyrate reduced lipolysis in isolated adipocytes. This inhibitory effect was lower in the first lactation week (47%±16%) compared with late pregnancy (71%±6.5%). Incubation with 0.3 µmol/L isoproterenol stimulated lipolysis in isolated adipocytes from periparturient dairy cows. Basal lipolysis resulted in non-esterified fatty acid to glycerol ratios in the incubation media of 2.0±0.23 in prepartum samples, 2.1±0.23 in the first lactation week and 2.2±0.09 in cows with clinical ketosis. ß-Hydroxybutyrate reduced lipolysis by 45%±9.6% in isolated adipocytes from cows with clinical ketosis, indicating that impaired feedback of ß-hydroxybutyrate may not play a role in the disease etiology.

Protein and fat mobilization and associations with serum ß-hydroxybutyrate concentrations in dairy cows.

The objective of this study was to obtain information on variation between dairy cows in muscle and fat tissue mobilization around parturition and to study the association between protein and fat mobilization and serum ß-hydroxybutyrate (BHBA) concentrations (hyperketonemia) in this period. Thirty-four cows kept under similar conditions at a university dairy farm (no experimental treatments) were monitored from 4 wk before until 8 wk after calving. Mobilization of muscle protein was investigated by analysis of plasma 3-methylhistidine concentrations (3-MH, analyzed by a recently developed HPLC tandem mass spectrometry method) and ultrasound measurements of longissimus muscle thickness. Mobilization of fat tissue was monitored by serum nonesterified fatty acid (NEFA) concentrations and ultrasound measurements of backfat thickness. Large variation was observed between cows in onset and duration of periparturient protein and fat mobilization. Plasma 3-MH concentrations and muscle thickness profiles indicated that protein mobilization started, on average, before parturition and continued until approximately wk 4 of lactation. Serum NEFA concentrations and backfat thickness profiles showed that fat mobilization occurred from parturition until the end of the study. Thus, muscle protein mobilization occurred in advance of fat mobilization in most cows from this study. We hypothesized that this might be due to a prepartum amino acid deficiency in the absence of negative energy balance. The incidence of hyperketonemia in this study was 16/34 = 47%. With the exception of 3 cows defined as having severe hyperketonemia, cows with lower 3-MH concentrations had higher serum BHBA concentrations. A possible explanation for this observation might be that higher mobilization of protein around calving might restrict ketone body production due to the higher availability of glucogenic precursors in the period of most severe negative energy balance and highest fat mobilization. The validity of this hypothesis needs to be confirmed, but data from this study indicate that further research on the role of protein mobilization in the etiology of hyperketonemia in dairy cows is needed.

Technical note: quantification of plasma 1- and 3-methylhistidine in dairy cows by high-performance liquid chromatography-tandem mass spectrometry.

To improve monitoring of protein mobilization in dairy cows, we developed and evaluated a method to quantify 1-methylhistidine and 3-methylhistidine in plasma by HPLC-tandem mass spectrometry. The analytical method described is (1) sensitive: both histidine derivates can be detected in the picomole range; (2) accurate: intra- and interassay coefficients of variation were < 5% for all standard solutions of 1-methylhistidine and 3-methylhistidine measured (31 to 500 pmol); (3) specific: 1-methylhistidine is clearly separated from 3-methyl-histidine in plasma samples from dairy cows; and (4) flexible: can be easily adapted to measure other amino acids or compounds containing a primary amine. 1-Methylhistidine is present in plasma of dairy cows at concentrations of 5.0 ± 1.7 µM, similar to concentrations of 3-methylhistidine (4.4 ± 2.4 µM). Analytical separation of both histidine metabolites is essential when plasma 3-methylhistidine is used as indicator for muscle breakdown in dairy cows. Specific quantification of the concentration of 3-methylhistidine in bovine plasma samples by HPLC tandem mass spectrometry can improve monitoring of protein mobilization in dairy cows.

Immunologic activity of schistosomal and bacterial TLR2 ligands in Gabonese children.

Schistosomes carry lipid moieties that interact with the immune system. To understand the consequence of interactions in terms of polarizing the cytokine profiles, the effect of two Toll-like receptor-2 (TLR2) activating schistosomal lipid fractions was studied on whole blood from Gabonese children living in a schistosomiasis endemic area. One fraction contained lysophosphatidylserine [monoacylglycerophosphoserine (lysoGPSer)] plus diacylphosphatidylserine [diacylglycerophosphoserine (GPSer)] while the other contained lysoGPSer and only a trace of GPSer. The effect of these schistosomal lipid fractions was compared with the known bacterial TLR2 ligands PAM3CSK4 and MALP-2. PAM3CSK4 and MALP-2 had preferential IL-10-activating capacities, while the fraction containing lysoGPSer plus GPSer had a strong TNF-alpha-inducing capacity. The fraction containing lysoGPSer was neutral with respect to pro- vs. anti-inflammatory effects. When Th1 and Th2 cytokines were analysed, the schistosomal lipid fraction containing lysoGPSer plus GPSer showed a stronger Th2 response compared to PAM3CSK4, MALP-2 and lysoGPSer alone. Therefore, the study indicates that not only TLR2 ligands derived from bacteria or from parasites can generate distinct cytokine profiles but also that the composition of lipid entities reaching the immune system can be important in leading to different immune outcomes. This information may be important for exploitation of immune modulatory molecules.

Cellular and humoral responses to influenza in gabonese children living in rural and semi-urban areas.

BACKGROUND: With the current attention to the pandemic threat of avian influenza viruses, it is recognized that there is little information on influenza in Africa. In addition, the effects of influenza vaccination in African countries could be very different from the effects in regions with less exposure to microorganisms and parasites. METHODS: To monitor the presence of influenza viruses and investigate the immunological responses to influenza vaccination, schoolchildren in semi-urban and rural regions of Gabon were studied. Influenza-specific antibody responses to the 3 strains represented in the vaccine were determined in the serum. Furthermore, cytokine responses were measured after in vitro stimulation of whole blood by influenza antigens, before and after vaccination. RESULTS: Prevaccination titers of antibody against H3N2 were high. At vaccination, the titers of antibody against the 3 influenza strains increased significantly. The anti-H1N1 and anti-B responses after vaccination were weaker in rural schoolchildren than in semi-urban schoolchildren. Influenza-specific cytokine responses were induced within a week, showing significantly lower interferon- gamma and significantly higher interleukin-5 in the children from rural areas. CONCLUSIONS: Prevaccination antibody levels indicated that influenza viruses circulate in Gabon. Altogether, influenza vaccination induces weaker immune responses in a rural population than in a semi-urban population of Gabonese schoolchildren.

Antibody responses to Ascaris-derived proteins and glycolipids: the role of phosphorylcholine.

In addition to proteins, glycolipids can be targets of antibody responses and contribute to host-pathogen interaction. Following the structural analysis of Ascaris lumbricoides-derived glycolipids, the antibody responses of a group of children with no, light and heavy infections were analysed. The role of the phosphorylcholine moiety, present on Ascaris glycoproteins and glycolipids, in antibody reactivity of these infected individuals was determined. Children carrying heavy infections showed highest IgG reactivity to glycolipids compared to lightly or non-infected children. Substantial IgG antibody reactivity to both (glyco)proteins and glycolipids was found to be directed to the phosphorylcholine moiety as determined by either removal of this group or a competition assay. This was most pronounced for glycolipids, where removal of the phosphorylcholine moieties by hydrofluoric acid treatment abrogated IgG antibody reactivity. Measurement of IgG4 and IgE isotypes showed no IgG4 reactivity to Ascaris glycolipids, but raised IgE responses were detected in subjects with light or no Ascaris infections, suggesting that IgE responses to glycolipids may play a role in controlling parasite burden. Differences found in antibody profiles to glycolipids and (glyco)proteins, indicate that these different classes of compounds may have distinct roles in shaping of and interacting with humoral immune responses.

The extraordinary mitochondrion and unusual citric acid cycle in Trypanosoma brucei.

African trypanosomes are parasitic protozoa that cause sleeping sickness and nagana. Trypanosomes are not only of scientific interest because of their clinical importance, but also because these protozoa contain several very unusual biological features, such as their specially adapted mitochondrion and the compartmentalization of glycolytic enzymes in glycosomes. The energy metabolism of Trypanosoma brucei differs significantly from that of their hosts and changes drastically during the life cycle. Despite the presence of all citric acid cycle enzymes in procyclic insect-stage T. brucei, citric acid cycle activity is not used for energy generation. Recent investigations on the influence of substrate availability on the type of energy metabolism showed that absence of glycolytic substrates did not induce a shift from a fermentative metabolism to complete oxidation of substrates. Apparently, insect-stage T. brucei use parts of the citric acid cycle for other purposes than for complete degradation of mitochondrial substrates. Parts of the cycle are suggested to be used for (i) transport of acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, (ii) degradation of proline and glutamate to succinate, (iii) generation of malate, which can then be used for gluconeogenesis. Therefore the citric acid cycle in trypanosomes does not function as a cycle.