Effect of increasing levels of wasted date palm in concentrate diet on reproductive performance of Ouled Djellal breeding rams during flushing period.

AIM: The aim of the study was to assess the effect of two levels of wasted date (WD) by replacing commercial concentrate on the reproductive performance of Ouled Djellal (OD) rams. MATERIALS AND METHODS: Eighteen mature (2-year-old) OD rams were equally allocated to three groups and fed during 11 weeks with one of three different experimental diets, that contained 0% (0 WD), 50% (50 WD), or 75% (75 WD) of WDs in concentrate diet. Live body weight (LBW), body condition scoring (BCS), scrotal circumference (SC), testicular weight (TW), sperm production and quality, plasma testosterone concentration (T), and sexual behavior (reaction time and number of mounts with ejaculation) were regularly recorded from every ram. RESULTS: LBW, SC, and TW changed significantly among diet groups and during the experimental period (p<0.001), the highest averages were recorded in (0 WD) group. LBW, BCS, SC, TW, semen volume, and percentage of the positive hypo-osmotic swelling test were (p<0.001) positively influenced by flushing period. Nevertheless, sperm concentration showed a significant (p<0.001) decrease at day 30, followed by a return to the initial values afterward. There were no differences (p>0.05) between diet groups for plasma testosterone concentration and semen attributes, except that (50 WD) group expressed the lowest overall value of semen concentration. Furthermore, neither time nor diet affected (p>0.05) sperm motility and reproductive behavior parameters. CONCLUSIONS: It is possible to introduce WD as unconventional local feeding resources in flushing diet of breeding rams without disturbing their reproductive performance.

[Spontaneous pneumomediastinum: A rare complication of dermatomyositis]. / Pneumomédiastin spontané : une complication rare de la dermatomyosite.

Dermatomyositis is a rare connective tissue disease of unknown origin, including inflammatory myopathy and cutaneous manifestations. Several pulmonary complications associated to dermatomyositis were described; especially interstitial lung disease. Some rare and particular pulmonary complications were reported in the literature such as pneumodiastinum and pneumothorax. We are describing here, a case report about a female patient, who presented with dermatomyositis associated to pneumomediastinum as a severe and lethal complication without pneumothorax. It is a novel observation depicting this severe and rare complication. Brutal dyspnea and cervical subcutaneous crackling are alarming signs that should make practitioners think about this complication.

Contamination of broiler turkey farms by Salmonella spp. in Morocco: prevalence, antimicrobial resistance and associated risk factors. / Contamination des élevages de dinde de chair par Salmonella spp. au Maroc : prévalence, antibiorésistances et facteurs de risque associés.

The authors present a study that estimates the prevalence, sensitivity to antibiotics and distribution of Salmonella spp. serotypes in 20 broiler turkey farm buildings in the north-west of Morocco. Each farm was inspected three times for this purpose; one batch of ten pools of five droppings per farm was sampled on each visit (n = 600) for analysis. The high isolation rate observed for Salmonella spp. (35%) and the serotypes isolated were alarming. The authors found 62 Salmonella-positive isolates and identified nine serotypes: S. Kentucky (21 isolates, 33.8%), S. Parkroyal (10 isolates, 16.3%), S. Agona (7 isolates, 11.3%), S. Saintpaul (6 isolates, 9.6%), S. Typhimurium (5 isolates, 8%), S. Enteritidis and S. Heidelberg (4 isolates each, 6.4%), S. Newport (3 isolates, 4.8%) and S. Ruzizi (2 isolates, 3.2%). The Salmonella spp. antimicrobial resistance results showed that 93.5% (58/62) of the strains were resistant to at least one antibiotic. Multi-resistant strains (resistant to three or more antibiotics) accounted for 80.64% of the strains isolated. The percentage with resistance to ceftazidime (third-generation cephalosporin), ceftriaxone and cefotaxime was lower at 4.8%. Three strains of S. Agona with extended-spectrum beta-lactamase were detected, with a high level of resistance to ceftriaxone and a minimum inhibitory concentration of 16 µg/ml. The variables associated with contamination are linked to: the cleanout period (p = 0.037); antibiotic treatment (p = 0.001); infection of turkey poults at placement (p = 0.002); manure storage (p = 0.003); keeping sick turkeys in the turkey house (p = 0.009); the season (p = 0.001); and the age of the turkeys at the time of sampling (p = 0.01).

Les auteurs présentent une étude réalisée dans le but d'estimer la prévalence, la sensibilité aux antibiotiques et la distribution des sérotypes de Salmonella spp. dans vingt bâtiments d'élevage de dindes de chair, situés dans le nord-ouest du Maroc. Trois visites par élevage ont été effectuées dans ce cadre ; un lot de dix pools de cinq fientes par élevage a été prélevé à chaque visite (n = 600) en vue d'analyse. Le taux d'isolement de Salmonella est important (35 %) et les sérotypes isolés sont alarmants. Au total, les auteurs ont identifié et dénombré 62 souches de Salmonella réparties en neuf sérotypes, à savoir : 21 S. Kentucky (33,8 %), 10 S. Parkroyal (16,3 %), 6 S. Saintpaul (9,6 %), 7 S. Agona (11,3 %), 5 S. Typhimurium (8 %), 4 S. Enteritidis (6,4 %), 4 S. Heidelberg (6,4 %), 3 S. Newport (4,8 %) et 2 S. Ruzizi (3,2 %). Les résultats d'antibiorésistance des souches de Salmonella spp. ont montré que 93,50 % (58/62) des souches sont résistantes à au moins un antibiotique. Les souches multirésistantes (résistantes à trois antibiotiques ou plus), quant à elles, constituent 80,64 % des souches isolées. Le pourcentage de résistances à la ceftazidime (céphalosporine de troisième génération), à la ceftriaxone et à la céfotaxime était plus faible, évalué à 4,80 %. Trois souches de S. Agona à bêta-lactamase à spectre étendu ont été détectées, présentant un haut niveau de résistance à la ceftriaxone et une concentration minimale inhibitrice de 16 µg/ml. Les variables associées à cette contamination sont liées à la durée du vide sanitaire (p = 0,037), au traitement par antibiotiques (p = 0,001) et à la contamination des dindonneaux dès la mise en place (p = 0,002), au stockage du fumier (p = 0,003), au maintien des dindes malades dans le bâtiment d'élevage (p = 0,009), à la saison d'élevage (p = 0,001) et à l'âge des dindes au moment du prélèvement (p = 0,01).

Los autores presentan un estudio encaminado a determinar la prevalencia, sensibilidad a los antibióticos y distribución de los serotipos de Salmonella spp. en veinte explotaciones de cría de pavos asaderos del noroeste de Marruecos. Como parte del estudio se realizaron tres visitas a cada granja, y en cada ocasión se obtuvo un lote de diez muestras compuestas de una mezcla de cinco heces por explotación (n = 600) para someterlo a análisis. El índice de aislamiento de salmonelas es tan importante (un 35%) como preocupantes resultan los serotipos aislados. Los autores detectaron y contaron en total 62 cepas de Salmonella repartidas en nueve serotipos: 21 S. Kentucky (33,8%), 10 S. Parkroyal (16,3%), 6 S. Saintpaul (9,6%), 7 S. Agona (11,3%), 5 S. Typhimurium (8%), 4 S. Enteritidis (6,4%), 4 S. Heidelberg (6,4%), 3 S. Newport (4,8%) y 2 S. Ruzizi (3,2%). Los resultados de los antibiogramas de estas cepas de salmonelas ponen de relieve que un 93,50% (58/62) de ellas son resistentes a por lo menos un antibiótico. Las cepas multirresistentes (resistentes a tres o más antibióticos) representan un 80,64% de las cepas aisladas. El porcentaje de resistencias a la ceftazidima (cefalosporina de tercera generación), la ceftriaxona y la cefotaxima resultaba más bajo, de un 4,80%. Se detectaron tres cepas de S. Agona productoras de betalactamasas de espectro extendido que presentaban un elevado nivel de resistencia a la ceftriaxona y una concentración inhibidora mínima de 16 µg/ml. Las variables asociadas a esta contaminación guardan relación con la duración del vacío sanitario (p = 0,037), el tratamiento con antibióticos (p = 0,001) y la contaminación de los polluelos desde el momento de la instalación (p = 0,002), el almacenamiento de estiércol (p = 0,003), la permanencia de los pavos enfermos en los locales de cría (p = 0,009), la temporada de cría (p = 0,001) y la edad de los pavos en el momento de obtener las muestras (p = 0,01).

Water quality in turkey farms in Khemisset (Morocco) and potential risk factors. / Qualité des eaux en élevage de dinde à Khémisset (Maroc) et hypothèses sur les facteurs de risques.

The objectives of this study were to assess the microbiological and physical/chemical quality of water in broiler turkey farms in the province of Khemisset (north-western Morocco) and, based on a questionnaire, to ascertain potential risk factors for contamination of drinking water with faecal coliforms. A total of 80 samples were collected and analysed in 20 farms (four from each farm). At the main inlet to the water line at the entrance to each turkey house, 100% of the samples were of unacceptable quality in terms of faecal coliforms, Escherichia coli, faecal streptococci, sulphitereducing anaerobes and enterococci. A significant reduction in microbiological contamination of the water line (p < 0.05) was observed on Day 60. While more than 90% of the samples were of satisfactory quality in terms of pH, nitrites, conductivity, nitrates and iron, only 35% were satisfactory in terms of total hardness and only 20% met quality standards for ammonium content. The factors affecting levels of contamination with faecal coliforms were water chlorination (p = 0.065; odds ratio = 14; 90% confidence interval [CI] = 1.14-71), cleaning and disinfection (p = 0.028; odds ratio = 14; 95% CI = 1.25-156.6) and antibiotic treatment (p = 0.001; odds ratio = 6; 95% CI = 2.1-35.2). To improve water quality in poultry farms, farmers are advised to protect wells from contamination and to install water purification units (pre-oxidation, coagulation, flocculation, disinfection). In addition, turkey houses and rearing equipment should be rigorously cleaned and disinfected between each batch of birds.

Cette étude a pour objectifs d'évaluer la qualité microbiologique et physico-chimique de l'eau des élevages de dinde de chair dans la province de Khémisset (nord-ouest du Maroc) et de formuler, au moyen d'un questionnaire, certaines hypothèses sur les facteurs de risque potentiels associés à la contamination de l'eau d'abreuvement par les coliformes fécaux. Au total, 80 échantillons ont été prélevés et analysés dans 20 élevages (quatre de chaque élevage). Au sas de la ligne d'abreuvement, 100 % des échantillons étaient de qualité inacceptable en ce qui concerne les coliformes fécaux, Escherichia coli, les streptocoques fécaux, les anaérobies sulfito-réducteurs et les entérocoques. Une réduction significative de la contamination microbiologique a été observée en bout de la ligne d'abreuvement (p < 0,05) au jour 60. Plus de 90 % des échantillons étaient de qualité satisfaisante pour ce qui concerne le pH, les nitrites, la conductivité, les nitrates et le fer ; en revanche, seulement 35 % et 20 % d'échantillons étaient conformes pour ce qui concerne la dureté totale et la présence d'ammonium, respectivement. Les facteurs associés à la contamination par les coliformes fécaux étaient la chloration de l'eau (p = 0,065 ; rapport des cotes = 14 ; intervalle de confiance [IC] à 90 % = 1,14-71), le nettoyage et la désinfection (p = 0,028 ; rapport des cotes = 14 ; IC à 95 % = 1,25-156,6) et le traitement par antibiotiques (p = 0,001 ; rapport des cotes = 6 ; IC à 95 % = 2,1-35,2). Afin d'améliorer la qualité de l'eau dans les élevages avicoles, il est recommandé aux éleveurs de protéger les puits contre la contamination et d'installer des stations de potabilisation de l'eau (pré-oxydation, coagulation, floculation, désinfection). Il convient également de procéder à un nettoyage et une désinfection rigoureux des bâtiments et du matériel d'élevage à la fin de chaque bande.

Los autores describen un estudio destinado a evaluar la calidad microbiológica y fisicoquímica del agua de explotaciones productoras de pavos de engorde de la provincia de Khemisset (noroeste de Marruecos) y a formular, mediante un cuestionario, ciertas hipótesis sobre los posibles factores de riesgo asociados a la contaminación del agua de bebida por coliformes fecales. En total se tomaron y analizaron 80 muestras de 20 explotaciones. En la esclusa de entrada del agua en la línea de bebederos, el 100% de las muestras era de calidad inaceptable por lo que respecta a la presencia de coliformes fecales, Escherichia coli, estreptococos fecales, bacterias anaerobias sulfitorreductoras y enterococos. A la salida de la línea de bebederos se observaba una reducción significativa de la contaminación microbiológica (p < 0,05) en el día 60. Más del 90% de las muestras eran de calidad satisfactoria por lo que respecta a pH, nitritos, conductividad, nitratos y hierro. En cambio, solo un 35% y un 20% de las muestras satisfacían los criterios relativos a la dureza total y la presencia de amonio, respectivamente. Los factores asociados a la contaminación por coliformes fecales eran la cloración del agua (p = 0,065; razón de probabilidades = 14; intervalo de confianza [IC] al 90% = 1,14-71), la limpieza y desinfección (p = 0,028; razón de probabilidades = 14; IC al 95% = 1,25-156,6) y el tratamiento con antibióticos (p = 0,001; razón de probabilidades = 6; IC al 95% = 2,1-35,2). Con el fin de mejorar la calidad del agua en las explotaciones avícolas se recomienda a los productores que protejan los pozos de la contaminación e instalen potabilizadoras de agua (preoxidación, coagulación, floculación y desinfección). Asimismo, conviene proceder a una limpieza y una desinfección rigurosas de los locales y el material de cría al final de cada banda.

Efficient degradation of methabenzthiazuron photoinduced by decatungstate anion in water: kinetics and mechanistic studies.

This study concerns the elimination of methabenzthiazuron (MBTU) photocatalysed by sodium decatungstate salts W10O32(4-)·(DTA) in aqueous solution under irradiation at 365 nm. Ninety percentage of MBTU (10(-4) M) is mineralised in the presence of the photocatalyst (2×10(-4) M) after 7 d under exposure and the formation of nitrate, sulphate and ammonium confirmed this phenomenon. In aerated conditions, the photodegradation rate of MBTU clearly increased in the presence of DTA by a factor of 40 when compared to direct photolysis with ΦMBTU=2.5×10(-2) and t1/2 (MBTU)=1.4 h. Oxygen appeared essential since 2 times inhibition of MBTU disappearance and the photocatalytic cycle interrupt were observed in the absence of oxygen. The degradation mechanism has been elucidated through the photoproducts identification by LC-ESI-MS analysis. Two processes were implied in the degradation: electron transfer and H atom abstraction reactions both involving W10O32(4-∗) excited state species. In the primary steps of the degradation, the aromatic ring hydroxylation was observed by electron transfer leading to OH-MBTU isomers and H atom abstraction reaction gave benzthiazuron and a supposed demethylated product. Secondary oxidations permitted the hydroxylation of both products.

H2O2, signal, substrate, mutagen and chemorepellent from physiology to biochemistry and disease.

The history of the study by our group of the generation, the role and the effects of H2O2 in the thyroid, is summarized. The relations with thyroid diseases are discussed: myxedematous cretinism, thyroiditis, thyroid cancer, congenital hypothyroiddism, are discussed. A new role of H2O2 in the chemorepulsion of bacteria is proposed.

Potential role of Noxes in the protection of mucosae: H(2)O(2) as a bacterial repellent.

Duox proteins are members of the NADPH oxidase (Nox) family and are responsible for hydrogen peroxide (H(2)O(2)) production by various tissue types including bronchial and intestinal mucosae. The antimicrobial killing role of H(2)O(2) in leukocytes and macrophages is generally considered as the paradigm of its function. We investigated here the positive role of H(2)O(2) in the prevention of cellular invasion by Salmonella. We show that H(2)O(2), under conditions that preserved bacterial growth, has a repellent effect on Salmonella motility on agar plates. In addition, H(2)O(2) produced by PCCl3, a rat thyroid cell line, reduces bacterial invasion of the cells by around 40%. To test whether the observed phenotype is attributable to H(2)O(2) production, we constructed a CHO stable cell line expressing Duox2 protein at the cell surface (CHO-D2). The transfected cells produce a high amount of H(2)O(2). Upon infection with Salmonella, the invasion of CHO-D2 cells was reduced by up to 60%. In both PCCl3 and CHO expressing Duox2 cells, normal invasion was restored upon incubation with catalase. Our data suggest that H(2)O(2) at reduced concentrations acts as a repellent for bacteria, keeping them away from cells, a situation that could naturally prevent mucosal cells infection in vivo.

Bacteriocin activity by Lactobacillus curvatus CWBI-B28 to inactivate Listeria monocytogenes in cold-smoked salmon during 4 degrees C storage.

The inhibition effectiveness of a bacteriocin produced by Lactobacillus curvatus CWBI-B28 against Listeria monocytogenes was investigated in cold-smoked salmon during storage at 4 degrees C. Three bacteriocin-based strategies for the control of L. monocytogenes in foods (i.e., producing bacteriocin in situ, spraying with partially purified bacteriocin, and packaging in bacteriocin-coated plastic film), plus a newly developed method that uses cell-adsorbed bacteriocin (i.e., a suspension of producer cells on which maximum bacteriocin has been immobilized by pH adjustments), were assessed. Although all the approaches inactivated L. monocytogenes in cold-smoked salmon, various efficacy levels were observed. The behavior of L. monocytogenes was similar in samples treated with either partially purified bacteriocin or in situ bacteriocin production. In both of these cases, the counts of the pathogen declined to below the detectable limit of 0.7 log CFU/cm2 within the first week, but a approximately 0.95- and 1.3-log increase, respectively, occurred after day 14. The bioactive packaging film resulted in a slower inactivation of the pathogen but prevented any subsequent increase in the CFU throughout 22 days of storage at 4 degrees C. Application of the cell-adsorbed bacteriocin was shown to be the most effective means, as it resulted in a complete inactivation of the pathogen within 3 days, and no increase in Listeria counts occurred up to 22 days.

Screening of SLC26A4 (PDS) gene in Pendred's syndrome: a large spectrum of mutations in France and phenotypic heterogeneity.

Sensorineural hearing defect and goiter are common features of Pendred's syndrome. The clinical diagnosis of Pendred's syndrome remains difficult because of the lack of sensitivity and specificity of the thyroid signs. The identification of PDS as the causative gene allowed molecular screening and enabled a re-evaluation of the syndrome to identify potential diagnostic characteristics. This report presents the clinical and genotypic findings of 30 French families, for whom a diagnosis of Pendred's syndrome had been made. Twenty-seven families had at least one mutated allele. Twenty-eight different mutations were identified, 11 of which had never been previously reported. The main clinical characteristics were: early hearing loss, fluctuation in terms of during deafness evolution, and the presence of an enlarged vestibular aqueduct.

Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton.

Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram-negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. Electron microscopy (EM) indicates that the TTSSs of Shigella flexneri are composed of: (1) an external needle; (2) a transmembrane domain; and (3) a cytoplasmic bulb. EM analysis of purified and negatively stained parts 1, 2 and a portion of 3 of the TTSS, together termed the "needle complex" (NC), produced an average image at 17 A resolution in which a base, an outer ring and a needle, inserted through the ring into the base, could be discerned. This analysis and cryoEM images of NCs indicated that the needle and base contain a central 2-3 nm canal. Five major NC components, MxiD, MxiG, MxiJ, MxiH and MxiI, were identified by N-terminal sequencing. MxiG and MxiJ are predicted to be inner membrane proteins and presumably form the base. MxiD is predicted to be an outer membrane protein and to form the outer ring. MxiH and MxiI are small hydrophilic proteins. Mutants lacking either of these proteins formed needleless secretons and were unable to secrete Ipa proteins. As MxiH was present in NCs in large molar excess, we propose that it is the major needle component. MxiI may cap at the external needle tip.

IpgD, a protein secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE and implicated in entry focus formation.

Invasion of epithelial cells by Shigella flexneri involves entry and intercellular dissemination. Entry of bacteria into non-phagocytic cells requires the IpaA-D proteins that are secreted by the Mxi-Spa type III secretion machinery. Type III secretion systems are found in several Gram-negative pathogens and serve to inject bacterial effector proteins directly into the cytoplasm of host cells. In this study, we have analysed the IpgD protein of S. flexneri, the gene of which is located on the virulence plasmid at the 5' end of the mxi-spa locus. We have shown that IpgD (i) is stored in the bacterial cytoplasm in association with a specific chaperone, IpgE; (ii) is secreted by the Mxi-Spa type III secretion system in amounts similar to those of the IpaA-D proteins; (iii) is associated with IpaA in the extracellular medium; and (iv) is involved in the modulation of the host cell response after contact of the bacterium with epithelial cells. This suggests that IpgD is an effector that might be injected into host cells to manipulate cellular processes during infection.

The development of a FACS-based strategy for the isolation of Shigella flexneri mutants that are deficient in intercellular spread.

In the disease course of bacillary dysentery, pathogenic Shigella flexneri invade colonic epithelial cells and spread both within and between host cells. The ability to spread intercellularly allows the organism to infect an entire epithelial layer without significant contact with the extracellular milieu. Using fluorescence activated cell sorter (FACS)-based technology, we developed a rapid and powerful selection strategy for the isolation of S. flexneri mutants that are unable to spread from cell to cell. The majority of mutants identified using this strategy harbour mutations that affect the structure of their lipopolysaccharide or the ability of the bacteria to move intracellularly via actin-based motility; both factors have previously been shown to be essential for cell-to-cell spread. However, using a modified strategy that eliminated both of these types of mutants, we identified several mutants that provide us with evidence that bacterial proteins of the type III secretion system, which are essential for bacterial entry into host cells, also play a role in cell-to-cell spread.

Intracellular growth and metacyclogenesis defects in Trypanosoma cruzi carrying a targeted deletion of a Tc52 protein-encoding allele.

We have identified previously a Trypanosoma cruzi gene encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactions. Furthermore, we have reported that Tc52 also played a role in T. cruzi-associated immunosuppression observed during Chagas' disease. In an effort to understand further the biological role of Tc52, we used a gene-targeted deletion strategy to create T. cruzi mutants. Although T. cruzi tolerates deletion of one wild-type Tc52 allele, deletion of both genes is a lethal event, indicating that at least one active Tc52 gene is required for parasite survival. Monoallelic disruption of Tc52 (Tc52+/-) resulted in the production of T. cruzi lines that express less Tc52 mRNA and produced lower amounts of Tc52 protein compared with wild-type cells. In axenic cultures, growth rates of epimastigote forms bearing an interrupted allele were not different from those of wild-type parasites. Furthermore, monoallelic disruption of the Tc52 gene did not modify the growth rate of epimastigotes or their sensitivity to inhibition by benznidazole and nifurtimox, the two drugs used to treat Chagasic patients. Moreover, the antimonial drug SbIII, which is known, at least in Leishmania parasites, to be conjugated to a thiol and extruded by an ATP-coupled pump, had a similar effect on wild-type and mutant parasites, being equally sensitive. Hence, parasite drug sensitivity was also observed in clones overexpressing the Tc52 protein as well as in those carrying an antisense plasmid construct. Surprisingly, a significant impairment of the ability of epimastigotes carrying a Tc52 single gene replacement or antisense construct to differentiate into metacyclic trypomastigotes and to proliferate in vitro and in vivo was observed, whereas no significant enhancement of these biological properties was seen in the case of parasites that overexpress Tc52 protein. Moreover, functional complementation of Tc52+/- single mutant or selection of antisense revertant clones demonstrated that the phenotype observed is a direct consequence of Tc52 gene manipulation. Taken together, these results may suggest that Tc52 could participate among other factors in the phenotypic expression of T. cruzi virulence.

The outer membrane component, YscC, of the Yop secretion machinery of Yersinia enterocolitica forms a ring-shaped multimeric complex.

The YscC protein of Yersinia enterocolitica is essential for the secretion of anti-host factors, called Yops, into the extracellular environment. It belongs to a family of outer membrane proteins, collectively designated secretins, that participate in a variety of transport processes. YscC has been shown to exist as a stable oligomeric complex in the outer membrane. The production of the YscC complex is regulated by temperature and is reduced in strains carrying mutations in the yscN-U operon or in the virG gene. The VirG lipoprotein was shown to be required for efficient targeting of the complex to the outer membrane. Electron microscopy revealed that purified YscC complexes form ring-shaped structures of approximately 20 nm with an apparent central pore. Because of the architecture of the multimer, YscC appears to represent a novel type of channel-forming proteins in the bacterial outer membrane.

Bronchoalveolar lavage in adult sickle cell patients with acute chest syndrome: value for diagnostic assessment of fat embolism.

Fat embolism of necrotic bone marrow could be a frequent cause of acute chest syndrome (ACS) in sickle cell syndromes (SC), as suggested by postmortem findings. To check this hypothesis in living patients, we evaluated the presence of fatty macrophages recovered by bronchoalveolar lavage (BAL) in ACS. We investigated 20 consecutive cases of ACS by BAL, and identification of alveolar cells containing fat droplets was performed using oil red O (ORO), a specific neutral fat stain. The specificity of the method was determined on control groups, including eight SC patients without acute chest syndrome and 15 non-SC patients. A cut-off of > 5% of alveolar macrophages containing fat droplets was determined from the control groups to assess the diagnosis of fat embolism. In 12 ACS episodes, BAL exhibited > 5% of fatty macrophages, ranging from 10% to 100% (median value 46.5%). In 11 cases, fat embolism was associated with proven (n = 8) or probable (n = 3) bone marrow infraction, which mostly predated ACS. Eight ACS episodes were associated with a low percentage (< or = 5%) of fatty alveolar macrophages and could be related to a cause other than fat embolism in six episodes, such as sepsis, in-situ thrombosis, or rib infarcts generating hypoventilation. This study supports the diagnostic yield of BAL for fat embolism, which can be incriminated in 60% of cases of ACS in this adult population.

Mutational analysis of the Yersinia enterocolitica virC operon: characterization of yscE, F, G, I, J, K required for Yop secretion and yscH encoding YopR.

Pathogenic yersiniae secrete the Yop anti-host proteins using a type-III secretion pathway. The components of the secretion machinery are encoded by three loci on the pYV plasmid: virA, virB, and virC. In this paper we describe the characterization of eight non-polar mutants of the virC locus, constructed by allelic exchange. The yscE, FG, I, J and K mutants were defective in Yop secretion and independent of Ca2+ (CI) for their growth at 37 degrees C. Substitution of the 12 N-terminal amino-acid residues of YscF impaired secretion of YopB and YopD only and led also to a CI phenotype. The culture supernatant of the yscH mutant contained all the Yops except the 18 kDa YopR. Complementation experiments and an immunoblot analysis confirmed that YopR is encoded by the yscH gene. The LD50 for the mouse of the yscH mutant was 10-fold higher than that of the parental strain indicating that YopR is involved in pathogenesis. The phenotype of the yscM mutant was similar to that of the wild-type strain. However, overproduction of YscM from a multicopy plasmid in wild-type Yersinia enterocolitica prevented Yop secretion and synthesis. A hybrid YopH-LacZ' protein, encoded by a gene transcribed from the lac promoter, was secreted by a strain overexpressing YscM, showing that the secretion machinery was still functional. These results indicate that YscM plays a role in the feedback inhibition of Yop synthesis when secretion is compromised by acting as a negative regulator of Yop synthesis.

VirG, a Yersinia enterocolitica lipoprotein involved in Ca2+ dependency, is related to exsB of Pseudomonas aeruginosa.

Pathogenic yersiniae require Ca2+ for growth at 37 degrees C. They harbor closely related plasmids of about 70 kb that are essential for virulence. At 37 degrees C and in the absence of Ca2+ ions, these plasmids cause a decrease in growth rate and the release of large amounts of proteins called Yops. Here we describe the virG gene of Yersinia enterocolitica; virG is located just upstream of the virF gene, which encodes the transcriptional activator of some plasmid virulence factors. Analysis of the VirG amino acid sequence suggested that virG encodes a lipoprotein, which was confirmed by [3H]palmitate labeling of VirG-PhoA fusion proteins. A nonpolar virG mutant was constructed and found to be Ca2+ independent for growth at 37 degrees C but to still secrete Yops. This phenotype was complemented by the introduction of a plasmid harboring an intact virG gene. VirG was found to be homologous to ExsB, a protein encoded by a Pseudomonas aeruginosa gene located in the locus controlling exoenzyme S synthesis. Interestingly, the exsA gene, located just downstream of exsB, is also homologous to virF.

MxiG, a membrane protein required for secretion of Shigella spp. Ipa invasins: involvement in entry into epithelial cells and in intercellular dissemination.

Entry of Shigella flexneri into epithelial cells involves secretory proteins, the Ipa proteins, and their dedicated secretion apparatus, the Mxi-Spa translocon, which is encoded by the mxi and spa operons. We have characterized the mxiG gene that is located at the proximal part of the mxi operon. Inactivation of mxiG abolished lpa secretion, which indicates that MxiG is an essential component of the Mxi-Spa translocon. Immunoblotting analysis of membrane fractions suggests that the 42 kDa MxiG protein is associated with both the inner and outer membranes. Taking advantage of the complementation of the mxiG mutant by a plasmid carrying a wild-type copy of mxiG (which restored Ipa secretion, entry into HeLa cells, and cell-to-cell spread) we mutagenized the mxiG gene carried by the complementing plasmid to replace the RGD motif of MxiG by RAD. This mutation (mxiG*), which had no effect on the stability of the protein, did not affect Ipa secretion in vitro or entry into HeLa cells, but impaired intercellular dissemination. Therefore, MxiG and possibly proteins secreted by the Mxi-Spa translocation are involved not only in entry but also in spread of Shigella between epithelial cells.

YscU, a Yersinia enterocolitica inner membrane protein involved in Yop secretion.

Pathogenic yersiniae secrete antihost Yop proteins by a recently discovered secretion pathway which is also encountered in several animal and plant pathogens. The components of the export machinery are encoded by the virA (lcrA), virB (lcrB), and virC (lcrC) loci of the 70-kb pYV plasmid. In the present paper we describe yscU, the last gene of the virB locus. We determined the DNA sequence and mutated the gene on the pYV plasmid. After inactivation of yscU, the mutant strain was unable to secrete Yop proteins. The topology of YscU was investigated by the analysis of YscU-PhoA translational fusions generated by TnphoA transposition. This showed that the 40.3-kDa yscU product contains four transmembrane segments anchoring a large cytoplasmic carboxyl-terminal domain to the inner membrane. YscU is related to Spa40 from Shigella flexneri, to SpaS from Salmonella typhimurium, to FlhB from Bacillus subtilis, and to HrpN from Pseudomonas solanacearum.

YscN, the putative energizer of the Yersinia Yop secretion machinery.

Pathogenic yersiniae secrete a set of 11 antihost proteins called Yops. Yop secretion appears as the archetype of the type III secretion pathway. Several components of this machinery are encoded by the virA (lcrA) and virC (lcrC) loci of the 70-kb pYV plasmid. In this paper, we describe yscN, another gene involved in this pathway. It is the first gene of the virB locus. It encodes a 47.8-kDa protein similar to the catalytic subunits of F0F1 and related ATPases, as well as to products of other genes presumed to be involved in a type III secretion pathway. YscN contains the two consensus nucleotide-binding motifs (boxes A and B) described by Walker et al. (J. E. Walker, M. Saraste, M. J. Runswick, and N. J. Gay, EMBO J. 1:945-951, 1982). We engineered a pYV mutant encoding a modified YscN protein lacking box A. This mutant, impaired in Yop secretion, can be complemented in trans by a cloned yscN gene. We conclude that YscN is a component of the Yop secretion machinery using ATP. We hypothesize that it is either the energizer of this machinery or a part of it.