Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples

Aim:  To evaluate commercial DNA extraction kits for their ability to isolate DNA from Yersinia pestis suspensions and spiked environmental samples.
Methods and Results:  Five commercially available DNA extraction kits were evaluated: the ChargeSwitch gDNA Mini Bacteria Kit, the IT 1-2-3 Sample DNA Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit and the UltraClean Microbial DNA Isolation Kit. The extraction methods were performed upon six Y. pestis strains and spiked environmental specimens, including three swab types and one powder type. Taqman real-time PCR analysis revealed that the use of the MasterPure kit resulted in DNA with the most consistently positive results and the lowest limit of detection from Y. pestis suspensions and spiked environmental samples.
Conclusion:  Comparative evaluations of the five commercial DNA extraction methods indicated that the MasterPure kit was superior for the isolation of PCR-amplifiable DNA from Y. pestis suspensions and spiked environmental samples.
Significance and Impact of the Study:  The results of this study can assist diagnostic laboratories with selecting the best extraction method for processing environmental specimens for subsequent detection of Y. pestis by real-time PCR.     

Cellular fatty acids as chemical markers for differentiation of Yersinia pestis and Yersinia pseudotuberculosis

Aims:  Gas chromatography (GC) was utilized to investigate the cellular fatty acids (CFAs) composition of 141 Yersinia pestis isolates from different plague foci of China, and 20 Yersinia pseudotuberculosis strains as well.
Methods and Results:  The whole cell fatty acid methyl esters (FAMEs) were obtained by saponification, methylation and extraction followed with analysis using a standardized Microbial Identification System (MIS). Y. pestis and Y. pseudotuberculosis strains are quite similar in major CFA profiles, which include 16:0, 17:0 cyclo, 3-OH-14:0, 16:1ω7c and 18:1ω7c, accounting for more than 80% of the total CFAs.
Conclusions:  Yersinia pestis could be easily differentiated from Y. pseudotuberculosis by plotting the ratios of some CFA pairs, i.e.,14:0/18:0 vs 18:1ω7c/18:0, 3-OH-14:0/18:0 vs 18:1ω7c/18:0, 16:1ω7c/18:0 vs 18:1ω7c/18:0, 12:0/18:0 vs 18:1ω7c/18:0 and 12:0 ALDE/18:0 vs 16:1ω7c/18:0 fatty acids.
Significance and Impact of the Study:  In the present study, the normalized Sherlock MIS and Sherlock standard libraries were used to analyse the fatty acid composition of different strains of Y. pestis and Y. pseudotuberculosis . Meanwhile, ratios of certain CFA components were found to serve as chemical markers for differentiating the two closely related bacteria that are difficult to be differentiated by simply comparing CFA profiles based on other researches.     

Dose-response time modelling for highly pathogenic avian influenza A (H5N1) virus infection

Aims: To develop time‐dependent dose–response models for highly pathogenic avian influenza A (HPAI) of the H5N1 subtype virus. Methods and Results: A total of four candidate time‐dependent dose–response models were fitted to four survival data sets for animals (mice or ferrets) exposed to graded doses of HPAI H5N1 virus using the maximum‐likelihood estimation. A beta‐Poisson dose–response model with the N₅₀ parameter modified by an exponential‐inverse‐power time dependency or an exponential dose–response model with the k parameter modified by an exponential‐inverse time dependency provided a statistically adequate fit to the observed survival data. Conclusions: We have successfully developed the time‐dependent dose–response models to describe the mortality of animals exposed to an HPAI H5N1 virus. The developed model describes the mortality over time and represents observed experimental responses accurately. Significance and Impact of the Study: This is the first study describing time‐dependent dose–response models for HPAI H5N1 virus. The developed models will be a useful tool for estimating the mortality of HPAI H5N1 virus, which may depend on time postexposure, for the preparation of a future influenza pandemic caused by this lethal virus.     

Survival capacity in water of Arcobacter species under different temperature conditions

Aims:  To assess the survival capacity in vitro of arcobacters in water at temperatures applied in the food industry.
Methods and Results:  Four strains of each Arcobacter species were inoculated in potable water and water with 1% organic material and stored at 4, 7, 20, 52, 56 and 60°C. Samples were taken at known time points and the numbers of bacteria were determined on Arcobacter -selective medium. All Arcobacter species remained viable for a temperature-dependent period of time, although Arcobacter butzleri displayed a significant longer survival and heat resistance . No significant intraspecies differences were detected, resulting in no definite identification of origin or strain dependency. The survival period for all species was prolonged in the presence of the organic material only for the low temperatures.
Conclusions:  The present study demonstrates that water can act as a reservoir and as a potential source of Arcobacter contamination to humans and animals.
Significance and Impact of the Study:  This study assessed for the first time the survival of all human-related Arcobacter species in water. Particularly A. butzleri showed to be the most robust species with regard to temperature which is interesting as that species is often found in human clinical specimens.     

Susceptibility of the Siberian polecat to subcutaneous and oral Yersinia pestis exposure.

To determine if the Siberian polecat (Mustela eversmannii) represents a suitable model for the study of plague pathogenesis and prevention in the black-footed ferret (Mustela nigripes), polecats were exposed to 10(3), 10(7), or 10(10) Yersinia pestis organisms by subcutaneous injection; an additional group was exposed to Y. pestis via ingestion of a plague-killed mouse. Plague killed 88% of polecats exposed to Y. pestis (71% mortality in the 10(3) group, 100% mortality in the 10(7) and 10(10) groups, and 83% mortality in the mouse-fed group). Within the challenged group, mean day of death post-challenge ranged from 3.6 to 7.6 days; all polecats died on or before day 12 post-challenge. Animals receiving the lowest parenteral dose survived significantly longer than those receiving higher parenteral doses. Within challenged animals, mean survival time was lower in those presenting with significant weight loss by day 3, lethargy, and low fecal output; time to onset of lethargy and other signs was also related to risk of dying and/or plague dose. Six polecats developed serum antibody titers to the Y. pestis F1 protein. Three seropositive polecats survived the initial challenge and a subsequent exposure to a plague-killed mouse, while two seropositive animals later died. This study confirms that the Siberian polecat is susceptible to plague and suggests that this species will offer an appropriate surrogate for black-footed ferrets in future plague studies and related vaccine trials.     

Evaluation of ultrafiltration cartridges for a water sampling apparatus

Aims:  To determine the efficiency of various ultrafiltration cartridges (UFC) in concentrating test micro-organisms from drinking water.
Methods and Results:  Replicate drinking water samples from three potable water supplies were dosed with Bacillus anthracis Sterne, Francisella tularensis LVS, Yersinia pestis CO92, bacteriophages MS2 and phi-X174, and Cryptosporidium parvum. The test micro-organisms were dosed together in 100 l of water, which was then recirculated through one of five different UFC until the retentate volume was reduced to c. 500 ml. The micro-organisms were assayed before and after ultrafiltration concentration and per cent recoveries were calculated. There were nine statistically significant differences among pairs of filters out of a possible 180 different combinations of UFC, test micro-organisms, and water types.
Conclusions:  No filter consistently performed better or worse than the others for each test micro-organism in all water samples tested.
Significance and Impact of the Study:  This study provides performance data on the ability of several different UFC to concentrate a panel of test micro-organisms from three sources of potable water. Water utilities and first responders may use these data when selecting UFC for use in emergency response protocols. This study also provides additional data as to the efficacy of ultrafiltration for recovering bacteria, virus-like particles, and protozoan oocysts from water samples.     

Modelling the photosensitization-based inactivation of Bacillus cereus

Aims:  To study and to develop a model for the photo-destruction of the foodborne pathogen Bacillus cereus , initially treated with a precursor of endogenous photosensitizers (5-aminolevulinic acid, ALA).
Materials and methods:  The cells were incubated in the presence of ALA (3 or 7·5 mmol l⁻¹) for incubation times ranging from 2 to 60 min, inoculated onto the surface of LB Agar plates and submitted to light irradiation. The Weibull model was used to describe the survival curves of B. cereus . Quadratic equations were used to describe the effects of ALA concentration and incubation time on the Weibull model parameters.
Results:  ALA-based photosensitization proved to be an effective tool for inactivation of B. cereus . The decrease in viable counts observed after 20 min of irradiation, ranged from 4 to 6 log CFU g⁻¹.
Conclusions:  The developed model proved to be a parsimonious and robust solution to describe the observed data.
Significance and Impact of the Study:  The study demonstrates the effectiveness of photosensitization on B. cereus on agar plates. The model developed may be useful to optimize inactivation treatments by photosensitization.     

Thermal tolerance experiments help establish survival probabilities for tilapia, a group of potentially invasive aquatic species

1. Estimating the probability of establishment is an important step toward managing ecological risk posed by natural or intentional introductions. Species introductions in certain geographic areas may pose less of a threat when environmental conditions are unlikely to promote long-term establishment. Species commonly referred to as 'tilapia' have become widespread in certain areas of the southeastern United States, yet concerns remain regarding their potential spread to other areas.
2. We created a model to predict the survival of tilapia in Georgia (U.S.A.) based on individual 'thermal experience'. Laboratory experiments were conducted to measure the duration of survival of two strains of tilapia, Oreochromis nilotica and O. nilotica  ×  O. aureus hybrids, exposed to a variety of temperature regimes with three minimum temperatures and two rates of temperature decline.
3. We used Mayfield hierarchical logistic regression (MHLR) to describe the daily probability of survival as a function of the rate of temperature decline, average sustained temperature, tilapia strain and mass–length ratio.
4. Tilapia generally survived constant minimum temperatures over 12 °C but survival rate was mediated by the rate of decline, mass–length ratio and strain. For every 1 °C increase in minimum temperature, tilapia were 2.76 times more likely to survive. Fish exposed to rapid temperature decline were less likely to survive than those exposed to slowly decreasing temperature. More robust fish (higher mass–length ratio) were more likely to survive, and strain had a minimal effect despite being supported by the best MHLR model.
5. Our model can be used to estimate survival probability for tilapia under known surface water temperature regimes. Using MLHR in conjunction with experimental tolerance data may be useful for estimating the likelihood of establishment of potentially invasive species.     

Independent acquisition and insertion into different chromosomal locations of the same pathogenicity island in Yersinia pestis and Yersinia pseudotuberculosis

We show that Yersinia pestis and pesticin-sensitive isolates of Y. pseudotuberculosis possess a common 34 kbp DNA region that has all the hallmarks of a pathogenicity island and is inserted into different asparaginyl tRNA genes at different chromosomal locations in each species. This pathogenicity island (YP-HPI) is marked by IS 100 , has a G + C content different from its host, is flanked by 24 bp direct repeats, encodes a putative, P4-like integrase and contains the iron uptake virulence genes from the pgm locus of Y. pestis . These findings indicate independent horizontal acquisition of this island by Y. pestis and Y. pseudotuberculosis . The two YP-HPI locations and their possession of an integrase gene support a model of site-specific integration of the YP-HPI into these bacteria.     

Response of Listeria monocytogenes to liquid smoke

Aims:  To investigate the effect of liquid smoke on growth, survival, proteomic pattern and haemolytic potential of Listeria monocytogenes.
Methods and Results:  Growth and survival curves were recorded in brain–heart infusion broth supplemented with three concentrations of liquid smoke. L. monocytogenes growth was inhibited in the presence of 15 μg ml⁻¹ phenol while a rapid decrease in cell viability occurred in the presence of 30 μg ml⁻¹ phenol. The proteome of L. monocytogenes cytosoluble proteins was slightly modified after 2-h incubation with 30 μg ml⁻¹ phenol but no protein already characterized in response to other known stresses was induced, except the protease ClpP. Liquid smoke inhibited the haemolytic potential without affecting hly gene expression, showing a potential inhibition of protein activity or stability.
Conclusions:  The presence of liquid smoke in a rich medium strongly affected growth and survival of L. monocytogenes . Brief smoke stress affected the metabolic pathways and inhibited the haemolytic activity of L. monocytogenes .
Significance and Impact of Study:  This study is a first step in the investigation of the influence of a smoked product on L. monocytogenes strains.     

Protective effect of salicylates against hydrogen peroxide stress in yeast

Aims:  To investigate the effects of salicylates in Saccharomyces cerevisiae exposed to oxidative stress induced by hydrogen peroxide (H₂O₂).
Methods and Results:  Saccharomyces cerevisiae was cultured through to the postlogarithmic phase of growth. Stress was induced by the addition of 1·5 mmol l⁻¹ H₂O₂ for 1 h, while N-acetyl-l-cysteine (NAC) and glutathione (GSSG) were used as control agents that affect the redox balance. Sodium salicylate, at 0·01–10 mmol l⁻¹or acetylsalicylic acid, at 0·02–2·5 mmol l⁻¹ was administered at various times before hydrogen peroxide stress. Both agents conferred resistance to a subsequent hydrogen peroxide stress, similarly to the induction of the adaptive response observed upon pretreatment with NAC and GSSG. Sodium salicylate was more potent as a short-term, but not as a long-term pretreatment agent, compared to acetylsalicylic acid.
Conclusions:  Pharmacological pretreatment with salicylates resulted in dose related increases in cell survival, indicating the induction of the protective response in yeast.
Significance and Impact of the study:  The possible role of salicylates in the modulation of the hydrogen peroxide stress response in eukaryotic cells address questions on the effects of these commonly used therapeutic agents in a number of disorders exhibiting an oxidative stress component.     

Characterization of late acyltransferase genes of Yersinia pestis and their role in temperature-dependent lipid A variation

Yersinia pestis is an important human pathogen that is maintained in flea-rodent enzootic cycles in many parts of the world. During its life cycle, Y. pestis senses host-specific environmental cues such as temperature and regulates gene expression appropriately to adapt to the insect or mammalian host. For example, Y. pestis synthesizes different forms of lipid A when grown at temperatures corresponding to the in vivo environments of the mammalian host and the flea vector. At 37 degrees C, tetra-acylated lipid A is the major form; but at 26 degrees C or below, hexa-acylated lipid A predominates. In this study, we show that the Y. pestis msbB (lpxM) and lpxP homologs encode the acyltransferases that add C12 and C(16:1) groups, respectively, to lipid IV(A) to generate the hexa-acylated form, and that their expression is upregulated at 21 degrees C in vitro and in the flea midgut. A Y. pestis deltamsbB deltalpxP double mutant that did not produce hexa-acylated lipid A was more sensitive to cecropin A, but not to polymyxin B. This mutant was able to infect and block fleas as well as the parental wild-type strain, indicating that the low-temperature-dependent change to hexa-acylated lipid A synthesis is not required for survival in the flea gut.     

Transmission dynamics of an iridescent virus in an experimental mosquito population: the role of host density

Abstract.  1. The transmission of insect pathogens cannot be adequately described by direct linear functions of host and pathogen density due to heterogeneity generated from behavioural or physiological traits, or from the spatial distribution of pathogen particles. Invertebrate iridescent viruses (IIVs) can cause patent and lethal infection or a covert sub-lethal infection in insects. Aedes aegypti larvae were exposed to suspensions of IIV type 6 at two densities. High larval density increased the prevalence of aggression resulting in potentially fatal wounding.
2. The overall prevalence of infection (patent + covert) was positively influenced by host density and increased with exposure time in both densities. The survival time of patently infected insects was extended by ≈ 5 days compared with non-infected insects.
3. Maximum likelihood models based on the binomial distribution were fitted to empirical results. A model incorporating heterogeneity in host susceptibility by inclusion of a pathogen-free refuge was a significantly better fit to data than an all-susceptible model, indicating that transmission is non-linear. The transmission coefficient ( υ ) did not differ with host density whereas the faction of the population that occupied the pathogen-free refuge (Π_R) was significantly reduced at high host density compared with the low density treatment.
4. The transmission of free-living infective stages of an IIV in Ae. aegypti larvae is non-linear, probably because of density-related changes in the frequency of aggressive encounters between hosts. This alters host susceptibility to infection and effectively reduces the proportion of hosts that occupy the pathogen-free refuge.     

IL-17 contributes to cell-mediated defense against pulmonary Yersinia pestis infection

Pneumonic plague is one of the world's most deadly infectious diseases. The causative bacterium, Yersinia pestis, has the potential to be exploited as a biological weapon, and no vaccine is available. Vaccinating B cell-deficient mice with D27-pLpxL, a live attenuated Y. pestis strain, induces cell-mediated protection against lethal pulmonary Y. pestis challenge. In this article, we demonstrate that prime/boost vaccination with D27-pLpxL confers better protection than prime-only vaccination. The improved survival does not result from enhanced bacterial clearance but is associated with increased levels of IL-17 mRNA and protein in the lungs of challenged mice. The boost also increases pulmonary numbers of IL-17-producing CD4 T cells. Interestingly, most of these cells simultaneously produce canonical type 1 and type 17 cytokines; most produce IL-17 and TNF-α, and many produce IL-17, TNF-α, and IFN-γ. Neutralizing IL-17 counteracts the improved survival associated with prime/boost vaccination without significantly impacting bacterial burden. Thus, IL-17 appears to mediate the enhanced protection conferred by booster immunization. Although neutralizing IL-17 significantly reduces neutrophil recruitment to the lungs of mice challenged with Y. pestis, this impact is equally evident in mice that receive one or two immunizations with D27-pLpxL, suggesting it cannot suffice to account for the improved survival that results from booster immunization. We conclude that IL-17 plays a yet to be identified role in host defense that enhances protection against pulmonary Y. pestis challenge, and we suggest that pneumonic plague vaccines should aim to induce mixed type 1 and type 17 cellular responses.     

The high-pathogenicity island of Yersinia pseudotuberculosis can be inserted into any of the three chromosomal asn tRNA genes

Pathogenicity islands (PAIs) have been identified in several bacterial species. A PAI called high-pathogenicity island (HPI) and carrying genes involved in iron acquisition (yersiniabactin system) has been previously identified in Yersinia enterocolitica and Yersinia pestis . In this study, the HPI of the third species of Yersinia pathogenic for humans, Y. pseudotuberculosis , has been characterized. We demonstrate that the HPI of strain IP32637 has a physical and genetic map identical to that of Y. pestis . A gene homologous to the bacteriophage P4 integrase gene is located downstream of the asn tRNA locus that borders the HPI of strain IP32637. This int gene is at the same position on the HPI of all three pathogenic Yersinia species. However, in contrast to Y. pestis 6/69, the HPI of Y. pseudotuberculosis IP32637 is not invariably adjacent to the pigmentation segment and can be inserted at a distance ≥ 190 kb from this segment. Also, in contrast to Y. pestis and Y. enterocolitica , the HPI of Y. pseudotuberculosis IP32637 can precisely excise from the chromosome, and, strikingly, it can be found inserted in any of the three asn tRNA loci present on the chromosome of this species, one of which is adjacent to the pigmentation segment. The pigmentation segment, which is present in Y. pestis but not in Y. enterocolitica , is also present and well conserved in all strains of Y. pseudotuberculosis studied. In contrast, the presence and size of the HPIs vary depending on the serotype of the strain: an entire HPI is found in strains of serotypes I only, a HPI with a 9 kb truncation in its left-hand part that carries the IS 100 sequence and the psn and ybtE genes characterizes the strains of serotype III, and no HPI is found in strains of serotypes II, IV and V.     

Neither the devil nor the deep blue sea: larval mortality factors fail to explain the abundance and distribution of Tischeria ekebladella

Abstract.  1. The distribution, abundance and population dynamics of herbivorous insects may be affected by trophic interactions, by abiotic influences, or by intra-specific processes. Relatively little is known about how trophic influences vary across space. Here, we investigate spatial variation in mortality in the oak-feeding leaf miner Tischeria ekebladella as attributable to individual causal agents.
2. Leaf miners were experimentally introduced on 67 trees on an island 5 km² in area in south-western Finland. On each tree, some larvae were protected by a muslin bag, others by a glue barrier around the branch and some left exposed.
3. In the bagged transplants, 78.4% of larvae survived, compared with only 9.6% in the other two treatments. Most of the mortality was because of airborne agents: mortality on branches sheltered by a glue barrier was as high as on fully exposed branch tips.
4. We consider mortality caused by parasitoid wasps to be the main source of larval death and the primary factor driving general patterns of survival. The effects of bird predation and premature leaf abscission were negligible.
5. We detected spatial aggregation in larval survival and parasitism rates at the level of individual trees, but not across the landscape.
6. Spatial variation in overall leaf miner survival, parasitism and leaf abscission does not suffice to explain patterns of incidence and abundance of wild T. ekebladella on experimental trees. Rather, we identify metapopulation dynamics as a likely determinant of the spatial distribution of T. ekebladella in the landscape.     

Rapid cold-hardening in a Karoo beetle, Afrinus sp.

Abstract.  In the insect rapid cold-hardening response, survival at subzero temperatures is greatly improved by a brief pre-exposure at a milder temperature. It is predicted that insects with minimal cold tolerance capabilities living in variable environments should use rapid cold-hardening to survive sudden cold snaps. This is tested in Afrinus sp., a beetle that lives in an exposed habitat on rock outcrops in the Karoo Desert, South Africa, where microclimate temperatures drop infrequently to below freezing. Afrinus sp. shows a significant rapid cold-hardening response: survival of a 2-h exposure to −6.5 °C is much improved after pre-exposure to −2 °C, to 0 °C with a 2-h return to the rearing temperature, and to 40 °C, but not after pre-exposure to 0 °C. Little is known about the mechanism of the rapid cold-hardening response, although the data suggest that rapid cold-hardening may be mediated via several different mechanisms.