Cell-associated Fraction Research Articles

Organomercurial Lyase (MerB)-Mediated Demethylation Decreases Bacterial Methylmercury Resistance in the Absence of Mercuric Reductase (MerA).

The mer operon encodes enzymes that transform and detoxify methylmercury (MeHg) and/or inorganic mercury [Hg(II)]. Organomercurial lyase (MerB) and mercuric reductase (MerA) can act sequentially to demethylate MeHg to Hg(II) and reduce Hg(II) to volatile elemental mercury (Hg0) that can escape from the cell, conferring resistance to MeHg and Hg(II). Most identified mer operons encode either MerA and MerB in tandem or MerA alone; however, microbial genomes were recently identified that encode only MerB. However, the effects of potentially producing intracellular Hg(II) via demethylation of MeHg by MerB, independent of a mechanism to further detoxify or sequester the metal, are not well understood. Here, we investigated MeHg biotransformation in Escherichia coli strains engineered to express MerA and MerB, together or separately, and characterized cell viability and Hg detoxification kinetics when these strains were grown in the presence of MeHg. Strains expressing only MerB are capable of demethylating MeHg to Hg(II). Compared to strains that express both MerA and MerB, strains expressing only MerB exhibit a lower MIC with MeHg exposure, which parallels a redistribution of Hg from the cell-associated fraction to the culture medium, consistent with cell lysis occurring. The data support a model whereby intracellular production of Hg(II), in the absence of reduction or other forms of demobilization, results in a greater cytotoxicity than the parent MeHg compound. Collectively, these results suggest that in the context of MeHg detoxification, MerB must be accompanied by an additional mechanism(s) to reduce, sequester, or redistribute generated Hg(II). IMPORTANCE Mercury is a globally distributed pollutant that poses a risk to wildlife and human health. The toxicity of mercury is influenced largely by microbially mediated biotransformation between its organic (methylmercury) and inorganic [Hg(II) and Hg0] forms. Here, we show in a relevant cellular context that the organomercurial lyase (MerB) enzyme is capable of MeHg demethylation without subsequent mercuric reductase (MerA)-mediated reduction of Hg(II). Demethylation of MeHg without subsequent Hg(II) reduction results in a greater cytotoxicity and increased cell lysis. Microbes carrying MerB alone have recently been identified but have yet to be characterized. Our results demonstrate that mer operons encoding MerB but not MerA put the cell at a disadvantage in the context of MeHg exposure, unless subsequent mechanisms of reduction or Hg(II) sequestration exist. These findings may help uncover the existence of alternative mechanisms of Hg(II) detoxification in addition to revealing the drivers of mer operon evolution.

Three Novel Bacteria Associated with Two Centric Diatom Species from the Mediterranean Sea, Thalassiosira rotula and Skeletonema marinoi.

Diatoms are a successful group of microalgae at the base of the marine food web. For hundreds of millions of years, they have shared common habitats with bacteria, which favored the onset of interactions at different levels, potentially driving the synthesis of biologically active molecules. To unveil their presence, we sequenced the genomes of bacteria associated with the centric diatom Thalassiosira rotula from the Gulf of Naples. Annotation of the metagenome and its analysis allowed the reconstruction of three bacterial genomes that belong to currently undescribed species. Their investigation showed the existence of novel gene clusters coding for new polyketide molecules, antibiotics, antibiotic-resistance genes and an ectoine production pathway. Real-time PCR was used to investigate the association of these bacteria with three different diatom clones and revealed their preference for T. rotula FE80 and Skeletonema marinoi FE7, but not S. marinoi FE60 from the North Adriatic Sea. Additionally, we demonstrate that although all three bacteria could be detected in the culture supernatant (free-living), their number is up to 45 times higher in the cell associated fraction, suggesting a close association between these bacteria and their host. We demonstrate that axenic cultures of T. rotula are unable to grow in medium with low salinity (<28 ppt NaCl) whereas xenic cultures can tolerate up to 40 ppt NaCl with concomitant ectoine production, likely by the associated bacteria.

Measles virus exits human airway epithelia within dislodged metabolically active infectious centers.

Measles virus (MeV) is the most contagious human virus. Unlike most respiratory viruses, MeV does not directly infect epithelial cells upon entry in a new host. MeV traverses the epithelium within immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for ~3–5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may dislodge while epithelial function is preserved. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In dislodged infectious centers, ciliary beating persisted, and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, which models the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center dislodging. 5-ethynyl-2’-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the dislodged infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeV’s strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host.

Comparative Integrated Omics Analysis of the Hfq Regulon in Bordetella pertussis.

Bordetella pertussis is a Gram-negative strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Previously, we have shown that RNA chaperone Hfq is required for virulence of B. pertussis. Furthermore, microarray analysis revealed that a large number of genes are affected by the lack of Hfq. This study represents the first attempt to characterize the Hfq regulon in bacterial pathogen using an integrative omics approach. Gene expression profiles were analyzed by RNA-seq and protein amounts in cell-associated and cell-free fractions were determined by LC-MS/MS technique. Comparative analysis of transcriptomic and proteomic data revealed solid correlation (r2 = 0.4) considering the role of Hfq in post-transcriptional control of gene expression. Importantly, our study confirms and further enlightens the role of Hfq in pathogenicity of B. pertussis as it shows that Δhfq strain displays strongly impaired secretion of substrates of Type III secretion system (T3SS) and substantially reduced resistance to serum killing. On the other hand, significantly increased production of proteins implicated in transport of important metabolites and essential nutrients observed in the mutant seems to compensate for the physiological defect introduced by the deletion of the hfq gene.

Annexin A2 associates to feline calicivirus RNA in the replication complexes from infected cells and participates in an efficient viral replication

Cellular proteins have been identified to participate in calicivirus replication in association with viral proteins and/or viral RNAs. By mass spectrometry from pull-down assays, we identified several cellular proteins bound to the feline calicivirus (FCV) genomic RNA; among them the lipid raft-associated scaffold protein Annexin (Anx) A2. AnxA2 colocalizes with FCV NS6/7 protein and with the dsRNA in infected cells; moreover, it was found associated with the viral RNA in the membrane fraction corresponding to the replication complexes (RCs), suggesting its role during FCV replication. AnxA2-knockdown from CrFK cells prior to infection with FCV caused a delay in the cytopathic effect, a strong reduction of viral non-structural proteins and dsRNA production, and a decrease of FCV yield in both cell-associated and supernatant fractions. Taken together, these results indicate that AnxA2 associates to the genomic RNA of FCV and is required for an efficient FCV replication.

Evaluation of an Anti-HER2 Nanobody Labeled with 225Ac for Targeted α-Particle Therapy of Cancer.

Human epidermal growth factor receptor type 2 (HER2) is overexpressed in numerous carcinomas. Nanobodies (Nbs) are the smallest antibody-derived fragments with beneficial characteristics for molecular imaging and radionuclide therapy. Therefore, HER2-targeting nanobodies could offer a valuable platform for radioimmunotherapy, especially when labeled with α-particle emitters, which provide highly lethal and localized radiation to targeted cells with minimal exposure to surrounding healthy tissues. In this study, the anti-HER2 2Rs15d-nanobody was conjugated with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ( p-SCN-Bn-DOTA) and radiolabeled with an α-emitter 225Ac with a high yield (>90%) and a radiochemical purity above 95%. The 225Ac-DOTA-Nb binding affinity was 4.12 ± 0.47 nM with an immunoreactive fraction above 80%. Binding to low HER2-expressing MDA-MB-231 cells was negligible, whereas HER2-overexpressing SKOV-3 cells could be blocked with an excess of unlabeled nanobody, confirming the specificity of binding. Noncompeting binding to HER2 was observed in the presence of an excess of trastuzumab. The cell-associated fraction of 225Ac-DOTA-Nb was 34.72 ± 16.66% over 24 h. In vitro, the radioconjugate was toxic in an HER2-mediated and dose-dependent manner, resulting in IC50 values of 10.2 and 322.1 kBq/mL for 225Ac-DOTA-Nb and the 225Ac-DOTA control, respectively, on SKOV-3 cells, and 282.2 kBq/mL for 225Ac-DOTA-Nb on MDA-MB-231 cells. Ex vivo biodistribution studies, performed in mice bearing subcutaneous HER2-overexpressing and low HER2-expressing tumors, showed a fast uptake in SKOV-3 tumors compared to MDA-MB-231 (4.01 ± 1.58% ID/g vs 0.49 ± 0.20% ID/g after 2 h), resulting also in high tumor-to-normal tissue ratios. In addition, coinjection of 225Ac-DOTA-Nb with Gelofusine reduced kidney retention by 70%. This study shows that 225Ac-DOTA-Nb is a promising new radioconjugate for targeted α-particle therapy and supports its further development.

Cell-free DNA: A Neglected Source for Antibiotic Resistance Genes Spreading from WWTPs.

Cell-associated ARGs in wastewater treatment plants (WWTPs) has been concerned, however, cell-free ARGs in WWTPs was rarely studied. In this study, the abundances of four representative ARGs, sulII, tetC, blaPSE-1, and ermB, in a large municipal WWTP were investigated in both cell-associated and cell-free fractions. Cell-associated ARGs was the dominant ARGs fraction in the raw wastewater. After biological treatment, sludge settling, membrane filtration, and disinfection, cell-associated ARGs were substantially reduced, though the ratios of ARG/16S rRNA gene were increased with disinfection. Cell-free ARGs persisted in the WWTP with a removal of 0.36 log to 2.68 logs, which was much lower than the removal of cell-associated ARGs (3.21 logs to 4.14 logs). Therefore, the abundance ratio of cell-free ARGs to cell-associated ARGs increased from 0.04-1.59% to 2.00-1895.08% along the treatment processes. After 25-day-storage, cell-free ARGs in both biological effluent and disinfection effluent increased by 0.14 log to 1.99 logs and 0.12 log to 1.77 logs respectively, reflecting the persistence and low decay rate of cell-free ARGs in the discharge water. Therefore, cell-free ARGs might be a kind of important but previously neglected pollutant from WWTPs, which added potential risks to the effluent receiving environments.

Glucansucrases produced by fructophilic lactic acid bacteria Lactobacillus kunkeei H3 and H25 isolated from honeybees.

Twenty fructophilic isolates from the stomachs of honeybee Apis mellifera ligustica from the region of Plovdiv, Bulgaria were obtained. Fructophilic isolates H3 and H25 showed formation of mucous colonies during cultivation on medium with sucrose, suggesting exopolysaccharide synthesis. The sequencing analysis of 16S rRNA identified isolates H3 and H25 as fructophilic lactic acid bacteria Lactobacillus kunkeei. The in situ analysis and periodic acid-Schiff's staining, showed that Lb. kunkeei H3 and H25 produce extracellular glucansucrases with molecular weight of about 300 kDa. In the cell-associated fractions, additional glucansucrase is detected with molecular weight of about 180 kDa. The content of α-(1 → 6) linkages in the glucans synthesized with extracellular glucansucrases from H3 and H25 after dextranase hydrolysis was significantly lower than this one of the classical dextran - about 35 and 62%, respectively. These results suggest a more branched structure of the studied polymers.

Identification of pathogenic factors potentially involved in Staphylococcus aureus keratitis using proteomics

Staphylococcus is a leading cause of microbial keratitis, characterized by destruction of the cornea by bacterial exoproteins and host-associated factors. The aim of this study was to compare extracellular and cell-associated proteins produced by two different isolates of S. aureus, a virulent clinical isolate (Staph 38) and a laboratory strain (Staphylococcus aureus 8325-4) of weaker virulence in the mouse keratitis model. Proteins were analyzed using 2D polyacrylamide gel electrophoresis and identified by subsequent mass spectrometry. Activity of staphylococcal adhesins was assessed by allowing strains to bind to various proteins adsorbed onto polymethylmethacrylate squares. Thirteen proteins in the extracellular fraction and eight proteins in the cell-associated fractions after bacterial growth were produced in increased amounts in the clinical isolate Staph 38. Four of these proteins were S. aureus virulence factor adhesins, fibronectin binding protein A, staphopain, glyceraldehyde-3-phosphate dehydrogenase 2 and extracellular adherence protein. The clinical isolate Staph 38 adhered to a greater extent to all mammalian proteins tested, indicating the potential of the adhesins to be active on its surface. Other proteins with increased expression in Staph 38 included potential moonlighting proteins and proteins involved in transcription or translation. This is the first demonstration of the proteome of S. aureus isolates from keratitis. These results indicate that the virulent clinical isolate produces more potentially important virulence factors compared to the less virulent laboratory strain and these may be associated with the ability of a S. aureus strain to cause more severe keratitis.

Construction and characteristics of artificial consortia of Scenedesmus obliquus-bacteria for S. obliquus growth and lipid production

In this work, artificial consortia of Scenedesmus obliquus and selected bacterial strains were constructed to investigate the effects of bacteria on microalgal biomass and lipid production, as well as their inter-relationship in a photoautotrophic system. Microalgal growth was enhanced when co-cultivated with single selected bacterial strains, among which Acidovorax facilis represented the largest biomass concentration increase of 24.8%, compared to the axenic culture of S. obliquus. The consortium system also enhanced microalgal lipid content, lipid productivity, and the proportion of saturated fatty acids and oleic acid. Moreover, the selected bacterial strains adhered directly to the S. obliquus cell surface, which was necessary for the establishment of significant interaction. Co-cultivation of S. obliquus and bacterial strains resulted in changes in both the concentration and composition of proteins and polysaccharides dissolved in the culture supernatant and cell-associated (bound) fractions, which partially contributed to the observed differences in microalgal biomass and lipid production.

Cellular Protein WDR11 Interacts with Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication.

It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wild-type and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment. While the TGN has been proposed to be the major site of HSV-1 secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both our understanding of basic cellular biology as well as how this protein is co-opted by HSV.

Extracellular proteases are released by ciliates in defined seawater microcosms

The biodegradation of proteins in seawater requires various proteases which are commonly thought to be mainly derived from heterotrophic bacteria. We, however, found that protists showed a high protease activity and continuously produced trypsin-type enzymes. The free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium was isolated and used for microcosm incubation with different concentrations of killed bacteria as food for 10 days. The results showed that the co-existence of the ciliate with its associated bacterium produced a significant protease activity in both cell-associated and cell-free fractions while that in the associated bacterium only microcosm was negligible. The protease profiles are different between cell-associated and cell-free fractions, and a trypsin-type enzyme hydrolyzing Boc-Val-Leu-Lys-MCA was detected throughout the period in the presence of ciliates. This suggests that ciliates release proteases into the surrounding environment which could play a role in protein digestion outside cells. It has been previously suggested that bacteria are the major transformers in seawater. We here present additional data which indicates that protists, or at least ciliates with their specific enzymes, are a potential player in organic matter degradation in water columns.

Screening and characterization of extracellular polysaccharides produced by Leuconostoc kimchii isolated from traditional fermented pulque beverage.

We report the screening and characterization of EPS produced by LAB identified as Leuconostoc kimchii isolated from pulque, a traditional Mexican fermented, non-distilled alcoholic beverage produced by the fermentation of the sap extracted from several (Agave) maguey species. EPS-producing LAB constitutes an abundant bacterial group relative to total LAB present in sap and during fermentation, however, only two EPS-producing colony phenotypes (EPSA and EPSB, respectively) were detected and isolated concluding that despite the high number of polymer-producing LAB their phenotypic diversity is low. Scanning electron microcopy analysis during EPS-producing conditions revealed that both types of EPS form a uniform porous structure surrounding the bacterial cells. The structural characterization of the soluble and cell-associated EPS fractions of each polymer by enzymatic and acid hydrolysis, as by 1D- and 2D-NMR, showed that polymers produced by the soluble and cell-associated fractions of EPSA strain are dextrans consisting of a linear backbone of linked α-(1→6) Glcp in the main chain with α-(1→2) and α-(1→3)-linked branches. The polymer produced by the soluble fraction of EPSB strain was identified as a class 1 dextran with a linear backbone containing consecutive α-(1→6)-linked D-glucopyranosyl units with few α-(1→3)-linked branches, whereas the cell-associated EPS is a polymer mixture consisting of a levan composed of linear chains of (2→6)-linked β-D-fructofuranosyl residues with β-(2→6) connections, and a class 1 dextran. According to our knowledge this is the first report of dextrans and a levan including their structural characterization produced by L. kimchii isolated from a traditional fermented source.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-583) contains supplementary material, which is available to authorized users.

Quantitative detection of epstein-barr virus DNA in cerebrospinal fluid and blood samples of patients with relapsing-remitting multiple sclerosis.

The presence of Epstein-Barr Virus (EBV) DNA in cerebrospinal fluid (CSF) and peripheral blood (PB) samples collected from 55 patients with clinical and radiologically-active relapsing-remitting MS (RRMS) and 51 subjects with other neurological diseases was determined using standardized commercially available kits for viral nucleic acid extraction and quantitative EBV DNA detection. Both cell-free and cell-associated CSF and PB fractions were analyzed, to distinguish latent from lytic EBV infection. EBV DNA was detected in 5.5% and 18.2% of cell-free and cell-associated CSF fractions of patients with RRMS as compared to 7.8% and 7.8% of controls; plasma and peripheral blood mononuclear cells (PBMC) positivity rates were 7.3% and 47.3% versus 5.8% and 31.4%, respectively. No significant difference in median EBV viral loads of positive samples was found between RRMS and control patients in all tested samples. Absence of statistically significant differences in EBV positivity rates between RRMS and control patients, despite the use of highly sensitive standardized methods, points to the lack of association between EBV and MS disease activity.

Molecular and Functional Characterization of an Invertase Secreted by Ashbya gossypii

The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity toward sucrose was approximately 10 times higher than toward raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-μm-plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii.

The Enterocyte-Associated Intestinal Microbiota of Breast-Fed Infants and Adults Responds Differently to a TNF-α-Mediated Pro-Inflammatory Stimulus

Co-evolved as an integral component of our immune system, the gut microbiota provides specific immunological services at different ages, supporting the immune education during our infancy and sustaining a well-balanced immunological homeostasis during the course of our life. In order to figure out whether this involves differences in the microbial groups primarily interacting with the host immune system, we developed a non-invasive HT29 cell-based minimal model to fingerprint the enterocyte-associated microbiota fraction in infants and adults. After depicting the fecal microbial community of 12 breast-fed infants and 6 adults by 16S rDNA amplicon pools 454 pyrosequencing, their respective HT29 cell-associated gut microbiota fractions were characterized by the universal phylogenetic array platform HTF-Microbi.Array, both in the presence and absence of a tumor necrosis factor-alpha (TNF-α)-mediated pro-inflammatory stimulus. Our data revealed remarkable differences between the enterocyte-associated microbiota fractions in breast-fed infants and adults, being dominated by Bifidobacterium and Enterobacteriaceae the first and Bacteroides-Prevotella and Clostridium clusters IV and XIVa the second. While in adults TNF-α resulted in a profound impairment of the structure of the enterocyte-associated microbiota fraction, in infants it remained unaffected. Differently from the adult-type gut microbial community, the infant-type microbiota is structured to cope with inflammation, being co-evolved to prime the early immune response by means of transient inflammatory signals from gut microorganisms.

Partition of perfluoroalkyl substances (PFASs) in whole blood and plasma, assessed in maternal and umbilical cord samples from inhabitants of arctic Russia and Uzbekistan

Perfluoroalkyl substances (PFASs) are ubiquitous in the environment world-wide. Our overall objective was to assess the exposure to PFASs experienced by delivering women and their new-borns in the industrial city of Norilsk (arctic Russia) and the rural Aral Sea region of Uzbekistan, with the secondary objective of evaluating the distribution of PFASs between blood cell and plasma fractions. Six PFASs were detected in every sample from Norilsk city with the plasma concentration sequence of: PFOS≫PFOA>PFNA>FOSA>PFHxS>PFUnDA. In the Uzbekistani samples, only PFOS was reported above the MDL (0.08ng/mL). The median plasma concentrations of PFOS of 11.0ng/mL for the Norilsk mothers was comparable to that reported for western countries, while that for Uzbekistan was considerably lower (0.23ng/mL). Apparent increases in the maternal-cord concentration ratios for both whole blood and plasma were evident with the length of the carbon chain for both the carboxylate and the sulfonate PFASs. The median value of this ratio for FOSA in plasma was the lowest, while that for whole blood was the highest. Other than for FOSA, the observed plasma–whole blood concentration ratios for maternal and umbilical cord blood were consistent with a priori calculations using appropriate packed cell and plasma volumes for neonates and pregnant women at term. Clearly FOSA favored whole blood, and acid–base equilibrium calculations suggested that the resonance-stabilized sulfonamidate ion resides in the blood cell fraction. Thus for PFASs and related compounds with pKa values with magnitudes comparable to physiological pH, it is pertinent to measure the cell-associated fraction (separately or as whole blood). Our study illustrates that consideration of both the physico-chemical properties of the contaminants and the physiological attributes of blood matrices were helpful in the interpretation of our findings.

Effect of essential oils of Zataria multiflora on in vitro rumen fermentation, protozoal population, growth and enzyme activity of anaerobic fungus isolated from Mehraban sheep

This study was conducted to investigate the effects of different doses (0, 150, 300, 450 and 600μg/ml) of Zataria multiflora essential oils (ZMEO) on in vitro gas production, fermentation parameters and protozoal population using rumen inocula from growing lambs fed a high concentrate diet. Gas production (GP) was recorded at 1–144h of incubation. After 144h, the incubation was stopped and inoculants filtered to determine dry matter disappearance (D144). In separate runs, the effects of ZMEO on total protozoal count and species distribution were studied. The main constituents of ZMEO were carvacrol (515g/kg), p-cymene (204g/kg) and thymol (154g/kg). Addition of ZMEO reduced asymptotic GP, GP rate, D144 and dry matter disappearance (P<0.001), while GP lag and half-time were increased (P<0.001). Also GP after 24h of incubation, ammonia production, microbial biomass and true in vitro degradability were decreased, whereas the partitioning factor increased with addition of ZMEO (P<0.001). Total VFA was the highest at 150μg/ml of ZMEO. Inclusion of ZMEO decreased the number of Entodinium spp., Epidinium caudatum and Epidinium ecaudatum (P<0.001). At the doses higher than 150μg/ml of ZMEO Isotricha spp. as well as Dasytricha spp. were completely disappeared.Light microscopy showed that the isolated fungi were similar to Neocalimastix sp. Inclusion of ZMEO entirely inhibited growth and filter paper digestion by isolated fungus. The avicelase and β-1,4-glucosidase activity in both extracellular and cell associated fractions was decreased by ZMEO (P<0.01). It is concluded that the ZMEO has the potential to modulate ruminal fermentation and further in vivo research is needed to determine the optimal doses of ZMEO.

Strategies for improved stability of Peste des Petits Ruminants Vaccine

The main focus of this work was the improvement of the stability of the current PPRV vaccine. First, new formulations based on the Tris buffer were tested, with and without the addition of sucrose and trehalose and compared with the formulation normally used to stabilize the vaccine, the Weybridge medium. The results show a virus half-life of 21h at 37°C and 1 month at 4°C for the Tris/trehalose liquid formulation and, in the lyophilized form, the formulation was able to maintain the viral titer above the 1×104 TCID50/mL (>10 doses/mL) for at least 21 months at 4°C (0.6log lost), 144h at 37°C (0.6log lost) and 120h at 45°C (1log lost).Secondly, a strategy based on culture medium composition manipulation aiming at improving the intrinsic PPRV vaccine stability was also evaluated. The addition of 25mM fructose resulted in a higher virus production (1log increase) with higher stability (2.6-fold increase compared to glucose 25mM) at 37°C. Increased concentrations of NaCl, improved virus release, reducing the cell-associated fraction of the virus produced. Moreover this harvesting strategy is scalable and more suitable for a larger scale production than the freeze/thaw cycles normally used.The information gathered in this work showed that it is possible for the PPRV vaccine to have adequate short-term stability at non-freezing temperatures to support manufacturing, short-term shipping and storage. The identification of a more stable formulation should significantly enhance the utility of the vaccine in the control of a PPRV outbreak.

Galleria mellonella as an infection model for Campylobacter jejuni virulence

Larvae of Galleria mellonella (Greater Wax Moth) have been shown to be susceptible to Campylobacter jejuni infection and our study characterizes this infection model. Following infection with C. jejuni human isolates, bacteria were visible in the haemocoel and gut of challenged larvae, and there was extensive damage to the gut. Bacteria were found in the extracellular and cell-associated fraction in the haemocoel, and it was shown that C. jejuni can survive in insect cells. Finally, we have used the model to screen a further 67 C. jejuni isolates belonging to different MLST types. Isolates belonging to ST257 were the most virulent in the Galleria model, whereas those belonging to ST21 were the least virulent.