Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins.     

Effects of Lysogeny of Shiga Toxin 2-Encoding Bacteriophages on Pulsed-Field Gel Electrophoresis Fragment Pattern of Escherichia coli K-12

Escherichia coli K-12 lysogens of three different Shiga toxin 2 (Stx2)-encoding bacteriophages were examined for variability in their pulsed-field gel electrophoresis (PFGE) fragment patterns. The PFGE fragment patterns could be classified into three types (i.e., PFGE types B, C, and D). For the PFGE type D, a 255-kbp fragment present in the original K-12 strain was apparently shifted by the size of Stx 2-encoding phage genomic DNA (ca. 65 kbp) to the position at 320 kbp. In contrast, the types B and C showed the above fragment shift plus further 6- and 10-fragment differences, respectively, from the original K-12 strain. The evidence suggests that even a single genetic event like lysogeny can cause marked genotypic modification of the host strain. Received: 21 June 2002 / Accepted: 5 July 2002     

Quantification of Shiga Toxin-Converting Bacteriophages in Wastewater and in Fecal Samples by Real-Time Quantitative PCR

Shiga toxin-converting bacteriophages (Stx phages) are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Stx phages are released from their bacterial hosts after lytic induction and remain free in the environment. Samples were analyzed for the presence of free Stx phages by an experimental approach based on the use of real-time quantitative PCR (qPCR), which enables stx to be detected in the DNA from the viral fraction of each sample. A total of 150 samples, including urban raw sewage samples, wastewater samples with fecal contamination from cattle, pigs, and poultry, and fecal samples from humans and diverse animals, were used in this study. Stx phages were detected in 70.0% of urban sewage samples (10 to 10³ gene copies [GC] per ml) and in 94.0% of animal wastewater samples of several origins (10 to 10¹⁰ GC per ml). Eighty-nine percent of cattle fecal samples were positive for Stx phages (10 to 10⁵ GC per g of sample), as were 31.8% of other fecal samples of various origins (10 to 10⁴ GC per g of sample). The stx₂ genes and stx₂ variants were detected in the viral fraction of some of the samples after sequencing of stx₂ fragments amplified by conventional PCR. The occurrence and abundance of Stx phages in the extraintestinal environment confirm the role of Stx phages as a reservoir of stx in the environment.Shiga toxin-producing Escherichia coli (STEC) is associated with diarrhea, hemorrhagic enterocolitis, and hemolytic-uremic syndrome in humans (46). Escherichia coli serotype O157:H7 is the main cause of these diseases, although other serotypes of E. coli and other enterobacteria species have been described (36). These E. coli serotypes produce at least two immunologically distinct Shiga toxins, called Stx1 and Stx2. In addition to these, several variations of these toxins have been reported in recent years, showing differences in virulence and distribution in the host populations examined (48, 51). Shiga toxin genes are carried by temperate bacteriophages (19, 35). Stx-encoding bacteriophages investigated to date consist of double-stranded DNA and have lambdoid genetic structures (19, 27, 32, 37, 47). The induction and regulation of these phages are directly involved in the production of toxin and, therefore, in the pathogenicity of the strains (8, 50). Stx phages are efficient vectors for the transfer of toxin genes, being able to convert nonpathogenic bacterial hosts into Stx-producing strains by transduction of stx, as has been demonstrated under various conditions (1, 4, 27, 28, 41, 49).Most of the reported outbreaks of STEC infections are associated with cattle products (10, 17), with the consumption of contaminated foods (10, 34), and with several waterborne infections (30). Stx phages are present within fecally contaminated aquatic environments (9, 28, 30, 32, 45). Moreover, a high percentage of STEC strains present in extraintestinal environments carry inducible Stx phages (14, 30).As individuals infected with STEC strains shed large quantities of Stx phages in feces, Stx phages should be prevalent in the environment, as are other viruses transmitted by the fecal-oral route (5, 11) or bacteriophages infecting bacteria present in the intestinal tract (16, 23). Moreover, those STEC strains isolated from food and animals carry inducible Stx phages (24, 27, 42). The virulence profiles of STEC strains isolated from food also suggest the presence of inducible Stx phages (10).Stx phages in sewage have been detected by nested PCR (28, 29, 31). However, to quantify them, the most probable number (MPN) method was applied, which allows only a rough estimate of the amount of Stx phages present in the sample. To assess the number of Stx phages accurately, real-time quantitative PCR (qPCR) technology is a useful tool. This technology is both sensitive and specific, and it gives accurate quantitative results (25). Comparison with a standard enables the number of copies of stx to be quantified, which can then be translated into the number of Stx phage particles.Little is known about the prevalence of phages carrying stx in fecal samples. The data available on the numbers of these phages in fecally contaminated water samples were only roughly estimated. The first step to evaluate the role of Stx phages in the environment as lateral gene transfer vectors is to know the extent of these viruses in the environment. The aim of this study is to report quantitative data on the abundance of Stx phages in urban sewage samples, in wastewater samples from cattle, pigs, and poultry, and in diverse fecal samples, calculated by means of a methodology based on qPCR.     

Comparative Survival of Free Shiga Toxin 2-Encoding Phages and Escherichia coli Strains outside the Gut

The behavior outside the gut of seeded Escherichia coli O157:H7, naturally occurring E. coli, somatic coliphages, bacteriophages infecting O157:H7, and Shiga toxin 2 (Stx2)-encoding bacteriophages was studied to determine whether the last persist in the environment more successfully than their host bacteria. The ratios between the numbers of E. coli and those of the different bacteriophages were clearly lower in river water than in sewage of the area, whereas the ratios between the numbers of the different phages were similar. In addition, the numbers of bacteria decreased between 2 and 3 log units in in situ survival experiments performed in river water, whereas the numbers of phages decreased between 1 and 2 log units. Chlorination and pasteurization treatments that reduced by approximately 4 log units the numbers of bacteria reduced by less than 1 log unit the numbers of bacteriophages. Thus, it can be concluded that Stx2-encoding phages persist longer than their host bacteria in the water environment and are more resistant than their host bacteria to chlorination and heat treatment.     

A Polyclonal Antibody Based Immunoassay Detects Seven Subtypes of Shiga Toxin 2 Produced by Escherichia coli in Human and Environmental Samples

Background

Shiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains.

Methods and Findings

Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity.

Conclusions

The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.     

Shiga Toxins, and the Genes Encoding Them, in Fecal Samples from Native Idaho Ungulates

Cattle are a known reservoir of Shiga toxin-producing Escherichia coli. The prevalence and stability of Shiga toxin and/or Shiga toxin genes among native wild ungulates in Idaho were investigated. The frequency of both Shiga genes and toxin was similar to that reported for Idaho cattle (~19%).     

Purification and Characterization of Shiga Toxin 2f,an Immunologically Unrelated Subtype of Shiga Toxin 2

Background

Shiga-like toxin 2 (Stx2) is one of the most important virulence factors in enterohaemorrhagic Escherichia coli (E. coli) strains such as O157H7. Subtypes of Stx2 are diverse with respect to their sequence, toxicity, and distribution. The most diverse Stx2 subtype, Stx2f, is difficult to detect immunologically, but is becoming more frequently associated with human illness.

Methods and Findings

A purification regimen was developed for the purification of Stx2f involving cation exchange, hydrophobic interaction, anion exchange, and gel filtration. The molecular weight of Stx2f B-subunit was approximately 5 kDa, which appeared significantly smaller than that of Stx2a (6 kDa) on a SDS-PAGE gel, although the size of the A subunit was similar to Stx2a (30 kDa). Stx2f was shown to be active in both cell-free and cell-based assays. The 50% cytotoxic dose in Vero cells was 3.4 or 1.7 pg (depending on the assay conditions), about 3–5 times higher than the archetypical Stx2a, while the activity of Stx2f and Stx2a in a cell-free rabbit reticulocyte system was similar. Stx2f bound to both globotriose-lipopolysaccharide (Gb3-LPS) and globotetraose-LPS (Gb4-LPS, mimics for globotriaosylceramide and globotetraosylceramide, respectively), but its ability to bind Gb4-LPS was much stronger than Stx2a. Stx2f was also much more stable at low pH and high temperature compared to Stx2a, suggesting the toxin itself may survive harsher food preparation practices.

Conclusions

Here, we detail the purification, biochemical properties, and toxicity of Stx2f, from an E. coli strain isolated from a feral pigeon. Information obtained in this study will be valuable for characterizing Stx2f and explaining the differences of Stx2a and Stx2f in host specificity and cytotoxicity.     

Transduction of Porcine Enteropathogenic Escherichia coli with a Derivative of a Shiga Toxin 2-Encoding Bacteriophage in a Porcine Ligated Ileal Loop System

In this study, we have investigated the ability of detoxified Shiga toxin (Stx)-converting bacteriophages Φ3538 (Δstx₂::cat) (H. Schmidt et al., Appl. Environ. Microbiol. 65:3855-3861, 1999) and H-19B::Tn10d-bla (D. W. Acheson et al., Infect. Immun. 66:4496-4498, 1998) to lysogenize enteropathogenic Escherichia coli (EPEC) strains in vivo. We were able to transduce the porcine EPEC strain 1390 (O45) with Φ3538 (Δstx₂::cat) in porcine ligated ileal loops but not the human EPEC prototype strain E2348/69 (O127). Neither strain 1390 nor strain E2348/69 was lysogenized under these in vivo conditions when E. coli K-12 containing H-19B::Tn10d-bla was used as the stx1 phage donor. The repeated success in the in vivo transduction of an Stx2-encoding phage to a porcine EPEC strain in pig loops was in contrast to failures in the in vitro trials with these and other EPEC strains. These results indicate that in vivo conditions are more effective for transduction of Stx2-encoding phages than in vitro conditions.     

R-Plasmid Transfer to and from Escherichia coli Strains Isolated from Human Fecal Samples

Strains of Escherichia coli recently isolated from human feces were examined for the frequency with which they accept an R factor (R1) from a derepressed fi⁺ strain of E. coli K-12 and transfer it to fecal and laboratory strains. Colicins produced by some of the isolates rapidly killed the other half of the mating pair; therefore, conjugation was conducted by a membrane filtration procedure whereby this effect was minimized. The majority of fecal E. coli isolates accepted the R factor at lower frequencies than K-12 F⁻, varying from 10⁻² per donor cell to undetectable levels. The frequencies with which certain fecal recipients received the R-plasmid were increased when its R⁺ transconjugant was either cured of the R1-plasmid and remated with the fi⁺ strain or backcrossed into the parental strain. The former suggests the loss of an incompatibility plasmid, and the latter suggests the modification of the R1-plasmid deoxyribonucleic acid (DNA). In general, the fecal R⁺E. coli transconjugants were less effective donors for K-12 F⁻ and heterologous fecal strains than was the fi⁺ K-12 strain, whereas the single strain of Citrobacter freundii examined was generally more competent. Passage of the R1-plasmid to strains of salmonellae reached mating frequencies of 10⁻¹ per donor cell when the recipient was a Salmonella typhi previously cured of its resident R-plasmid. However, two recently isolated strains of Salmonella accepted the R1-plasmid from E. coli K-12 R⁺ or the R⁺E. coli transconjugants at frequencies of 5 × 10⁻⁷ or less.     

Action of Shiga Toxin Type-2 and Subtilase Cytotoxin on Human Microvascular Endothelial Cells

The hemolytic uremic syndrome (HUS) associated with diarrhea is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB) was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC) has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF) and platelet/endothelial cell adhesion molecule 1 (PECAM-1). HGEC also expressed the globotriaosylceramide (Gb3) receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 −a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis.     

Abundance in Sewage of Bacteriophages That Infect Escherichia coli O157:H7 and That Carry the Shiga Toxin 2 Gene

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.     

Failure of Manganese to Protect from Shiga Toxin

Shiga toxin (Stx), the main virulence factor of Shiga toxin producing Escherichia coli, is a major public health threat, causing hemorrhagic colitis and hemolytic uremic syndrome. Currently, there are no approved therapeutics for these infections; however manganese has been reported to provide protection from the Stx1 variant isolated from Shigella dysenteriae (Stx1-S) both in vitro and in vivo. We investigated the efficacy of manganese protection from Stx1-S and the more potent Stx2a isoform, using experimental systems well-established for studying Stx: in vitro responses of Vero monkey kidney cells, and in vivo toxicity to CD-1 outbred mice. Manganese treatment at the reported therapeutic concentration was toxic to Vero cells in culture and to CD-1 mice. At lower manganese concentrations that were better tolerated, we observed no protection from Stx1-S or Stx2a toxicity. The ability of manganese to prevent the effects of Stx may be particular to certain cell lines, mouse strains, or may only be manifested at high, potentially toxic manganese concentrations.     

Transduction of Enteric Escherichia coli Isolates with a Derivative of Shiga Toxin 2-Encoding Bacteriophage φ3538 Isolated from Escherichia coli O157:H7

We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric Escherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx₂ gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage 3538(Δstx₂::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and E. coli from the physiological stool microflora were infected with 3538(Δstx₂::cat), and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC strain, none of the E. coli strains supported the formation of plaques when used as indicators for 3538(Δstx₂::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of 3538(Δstx₂::cat) that formed plaques on a lawn of E. coli laboratory strain C600. The results of our study demonstrate that 3538(Δstx₂::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains.     

A New Shiga Toxin 2 Variant (Stx2f) from Escherichia coli Isolated from Pigeons

We have isolated Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from the feces of feral pigeons which contained a new Stx2 variant gene designated stx_2f. This gene is most similar to sltIIva of patient E. coli O128:B12 isolate H.I.8. Stx2f reacted only weakly with commercial immunoassays. The prevalence of STEC organisms carrying the stx_2f gene in pigeon droppings was 12.5%. The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx2 variants and raises the question whether horizontal gene transfer to E. coli pathogenic to humans may occur.     

Metabolic Signatures of Cultured Human Adipocytes from Metabolically Healthy versus Unhealthy Obese Individuals

Background and Aims

Among obese subjects, metabolically healthy and unhealthy obesity (MHO/MUHO) can be differentiated: the latter is characterized by whole-body insulin resistance, hepatic steatosis, and subclinical inflammation. Aim of this study was, to identify adipocyte-specific metabolic signatures and functional biomarkers for MHO versus MUHO.

Methods

10 insulin-resistant (IR) vs. 10 insulin-sensitive (IS) non-diabetic morbidly obese (BMI >40 kg/m²) Caucasians were matched for gender, age, BMI, and percentage of body fat. From subcutaneous fat biopsies, primary preadipocytes were isolated and differentiated to adipocytes in vitro. About 280 metabolites were investigated by a targeted metabolomic approach intracellularly, extracellularly, and in plasma.

Results/Interpretation

Among others, aspartate was reduced intracellularly to one third (p = 0.0039) in IR adipocytes, pointing to a relative depletion of citric acid cycle metabolites or reduced aspartate uptake in MUHO. Other amino acids, already known to correlate with diabetes and/or obesity, were identified to differ between MUHO''s and MHO''s adipocytes, namely glutamine, histidine, and spermidine. Most species of phosphatidylcholines (PCs) were lower in MUHO''s extracellular milieu, though simultaneously elevated intracellularly, e.g., PC aa C32∶3, pointing to increased PC synthesis and/or reduced PC release. Furthermore, altered arachidonic acid (AA) metabolism was found: 15(S)-HETE (15-hydroxy-eicosatetraenoic acid; 0 vs. 120pM; p = 0.0014), AA (1.5-fold; p = 0.0055) and docosahexaenoic acid (DHA, C22∶6; 2-fold; p = 0.0033) were higher in MUHO. This emphasizes a direct contribution of adipocytes to local adipose tissue inflammation. Elevated DHA, as an inhibitor of prostaglandin synthesis, might be a hint for counter-regulatory mechanisms in MUHO.

Conclusion/Interpretation

We identified adipocyte-inherent metabolic alterations discriminating between MHO and MUHO.     

Incidence of Vibrio parahaemolyticus Bacteriophages and Other Vibrio Bacteriophages in Marine Samples

The distribution of the aerobic spirochetes Leptospira in surface waters, soil, and aquatic animals was investigated. Isolates from water and soil exhibited physiological characteristics common to members of the "biflexa complex," none were capable of infecting experimental animals, and leptospires could not be isolated from the eight genera of aquatic animals examined. The isolation frequencies from surface waters were: stream, 100%; lake, 65%; spring, 28%; bog lake, 5%; and marsh, 0%. With the exception of the stream, more isolations were obtained from the soil adjacent to the water than from the water. Leptospires were most frequently associated with soils of high moisture and organic matter content.     

Geldanamycin Enhances Retrograde Transport of Shiga Toxin in HEp-2 Cells

The heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) has been shown to alter endosomal sorting, diverting cargo destined for the recycling pathway into the lysosomal pathway. Here we investigated whether GA also affects the sorting of cargo into the retrograde pathway from endosomes to the Golgi apparatus. As a model cargo we used the bacterial toxin Shiga toxin, which exploits the retrograde pathway as an entry route to the cytosol. Indeed, GA treatment of HEp-2 cells strongly increased the Shiga toxin transport to the Golgi apparatus. The enhanced Golgi transport was not due to increased endocytic uptake of the toxin or perturbed recycling, suggesting that GA selectively enhances endosomal sorting into the retrograde pathway. Moreover, GA activated p38 and both inhibitors of p38 or its substrate MK2 partially counteracted the GA-induced increase in Shiga toxin transport. Thus, our data suggest that GA-induced p38 and MK2 activation participate in the increased Shiga toxin transport to the Golgi apparatus.     

Identification of 13 Human Microsatellite Markers via Cross-species Amplification of Fecal Samples from Rhinopithecus bieti

Yunnan snub-nosed monkeys (Rhinopithecus bieti) are 1 of 3 snub-nosed monkey species endemic to China. Only ca. 1500 individuals remain in high-altitude forests 3000–4500 m above sea level on the Tibetan Plateau, making them the nonhuman primate living at the highest known elevation. It is one of the most endangered 25 primate species in the world. Proper knowledge of the population genetics and social system of Rhinopithecus bieti will contribute to more appropriate conservation management decisions. Cross-species amplification of human microsatellite loci has facilitated analysis of the population genetics and reproductive strategies of various primate species. We screened 72 human-derived markers to assess their utility in Yunnan snub-nosed monkeys. Thirteen of them produced reliable results and exhibited moderate levels of polymorphism.     

Adenosine Deaminase Enhances the Immunogenicity of Human Dendritic Cells from Healthy and HIV-Infected Individuals

ADA is an enzyme implicated in purine metabolism, and is critical to ensure normal immune function. Its congenital deficit leads to severe combined immunodeficiency (SCID). ADA binding to adenosine receptors on dendritic cell surface enables T-cell costimulation through CD26 crosslinking, which enhances T-cell activation and proliferation. Despite a large body of work on the actions of the ecto-enzyme ADA on T-cell activation, questions arise on whether ADA can also modulate dendritic cell maturation. To this end we investigated the effects of ADA on human monocyte derived dendritic cell biology. Our results show that both the enzymatic and non-enzymatic activities of ADA are implicated in the enhancement of CD80, CD83, CD86, CD40 and CCR7 expression on immature dendritic cells from healthy and HIV-infected individuals. These ADA-mediated increases in CD83 and costimulatory molecule expression is concomitant to an enhanced IL-12, IL-6, TNF-α, CXCL8(IL-8), CCL3(MIP1-α), CCL4(MIP-1β) and CCL5(RANTES) cytokine/chemokine secretion both in healthy and HIV-infected individuals and to an altered apoptotic death in cells from HIV-infected individuals. Consistently, ADA-mediated actions on iDCs are able to enhance allogeneic CD4 and CD8-T-cell proliferation, globally yielding increased iDC immunogenicity. Taken together, these findings suggest that ADA would promote enhanced and correctly polarized T-cell responses in strategies targeting asymptomatic HIV-infected individuals.     

Sunlight Inactivation of Fecal Bacteriophages and Bacteria in Sewage-Polluted Seawater

Sunlight inactivation rates of somatic coliphages, F-specific RNA bacteriophages (F-RNA phages), and fecal coliforms were compared in seven summer and three winter survival experiments. Experiments were conducted outdoors, using 300-liter 2% (vol/vol) sewage-seawater mixtures held in open-top chambers. Dark inactivation rates (k_Ds), measured from exponential survival curves in enclosed (control) chambers, were higher in summer (temperature range: 14 to 20°C) than in winter (temperature range: 8 to 10°C). Winter k_Ds were highest for fecal coliforms and lowest for F-RNA phages but were the same or similar for all three indicators in summer. Sunlight inactivation rates (k_S), as a function of cumulative global solar radiation (insolation), were all higher than the k_Ds with a consistent k_S ranking (from greatest to least) as follows: fecal coliforms, F-RNA phages, and somatic coliphages. Phage inactivation was exponential, but bacterial curves typically exhibited a shoulder. Phages from raw sewage exhibited k_Ss similar to those from waste stabilization pond effluent, but raw sewage fecal coliforms were inactivated faster than pond effluent fecal coliforms. In an experiment which included F-DNA phages and Bacteroides fragilis phages, the k_S ranking (from greatest to least) was as follows: fecal coliforms, F-RNA phages, B. fragilis phages, F-DNA phages, and somatic coliphages. In a 2-day experiment which included enterococci, the initial concentration ranking (from greatest to least: fecal coliforms, enterococci, F-RNA phages, and somatic coliphages) was reversed during sunlight exposure, with only the phages remaining detectable by the end of day 2. Inactivation rates under different optical filters decreased with the increase in spectral cutoff wavelength (50% light transmission) and indicated that F-RNA phages and fecal coliforms are more susceptible than somatic coliphages to longer solar wavelengths, which predominate in seawater. The consistently superior survival of somatic coliphages in our experiments suggests that they warrant further consideration as fecal, and possibly viral, indicators in marine waters.