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11.
Xenorhabdus strains from entomopathogenic nematodes isolated from United Kingdom soils by using the insect bait entrapment method were characterized by partial sequencing of the 16S rRNA gene, four housekeeping genes (asd, ompR, recA, and serC) and the flagellin gene (fliC). Most strains (191/197) were found to have genes with greatest similarity to those of Xenorhabdus bovienii, and the remaining six strains had genes most similar to those of Xenorhabdus nematophila. Generally, 16S rRNA sequences and the sequence types based on housekeeping genes were in agreement, with a few notable exceptions. Statistical analysis implied that recombination had occurred at the serC locus and that moderate amounts of interallele recombination had also taken place. Surprisingly, the fliC locus contained a highly variable central region, even though insects lack an adaptive immune response, which is thought to drive flagellar variation in pathogens of higher organisms. All the X. nematophila strains exhibited a consistent pattern of insecticidal activity, and all contained the insecticidal toxin genes xptA1A2B1C1, which were present on a pathogenicity island (PAI). The PAIs were similar among the X. nematophila strains, except for partial deletions of a peptide synthetase gene and the presence of insertion sequences. Comparison of the PAI locus with that of X. bovienii suggested that the PAI integrated into the genome first and then acquired the xpt genes. The independent mobility of xpt genes was further supported by the presence of xpt genes in X. bovienii strain I73 on a type 2 transposon structure and by the variable patterns of insecticidal activity in X. bovienii isolates, even among closely related strains.  相似文献   
12.
Campylobacter jejuni can be isolated from different animal hosts. Various studies have used multilocus sequence typing to look for associations between particular clones of C. jejuni and specific hosts. Here, we describe the isolation of a novel clone (sequence type 3704 [ST-3704]) of C. jejuni associated with the bank vole (Myodes glareolus).Campylobacter jejuni is one of the most common causes of gastroenteritis in humans, with food (primarily chicken) believed to be the main vehicle for infection (8). Although high prevalences are found in livestock, C. jejuni has also been isolated from wildlife, including wild birds and wild mammals, and from the farm environment (1, 2, 4, 7, 11, 13). Multilocus sequence typing (MLST) has been used to study the distribution of specific clones among isolates from different hosts and the environment (1, 2, 3, 7, 10, 15, 16, 17). Such molecular epidemiological studies have provided evidence for some host-associated genotypes. For example, some MLST clonal complexes are associated with cattle (sequence type 61 [ST-61] and ST-42 complexes), whereas others are associated with wildlife, such as rabbits and wild birds, and environmental samples (ST-45, ST-177, ST-677, ST-682, and ST-952 complexes) (2, 4, 13). In addition, previously unreported sequence types have been identified in wildlife and environmental samples (4). In this study, we describe the identification of a novel strain of C. jejuni that represents a new sequence type and that is restricted primarily to one wildlife host, namely, bank voles (Myodes glareolus), from which it was isolated over a relatively wide geographic area and time period.We undertook longitudinal studies of feces collected from the sympatric wild rodents bank voles and wood mice (Apodemus sylvaticus) in a private West Cheshire (United Kingdom) woodland habitat. Feces collected from both species were analyzed for the presence of Campylobacter spp. during two sampling periods (May to July 2001 and January 2003). In 2004 (summer/autumn) and 2005 (winter/spring), cross-sectional surveys were conducted on 6 farms (5 dairy and 1 beef farm) in South Cheshire (approximately 30 km away) to investigate the role of wildlife as reservoirs of zoonotic enteric pathogens for livestock. Farms were sampled in summer/autumn on one occasion and in winter/spring on a second occasion; feces were collected from cattle, from wild mammals opportunistically, and from live-trapped rodents. For the isolation of Campylobacter spp., approximately 0.2 ml of fecal homogenate (1:1 feces in brain heart infusion broth) was added to campylobacter enrichment broth containing 5% (vol/vol) lysed horse blood (Southern Group Labs, Corby, United Kingdom), and samples were incubated at 37°C for 24 h under microaerobic conditions in a variable-atmosphere incubator (Don Whitley Scientific Ltd., Shipley, United Kingdom) before being inoculated onto campylobacter blood-free medium containing cefoperazone and amphotericin. These plates were incubated for up to 96 h at 37°C under microaerobic conditions before being examined for the presence of colonies characteristic of Campylobacter spp. All media were obtained from LabM (IDG), Bury, United Kingdom. Suspect isolates were presumptively identified as C. jejuni by Gram staining, no growth in air, and hippurate hydrolysis (6, 18) tests. For further assignment to species, cell lysates were prepared from isolates and subjected to a number of genus- and species-specific PCR assays (5, 14, 19). The whole genome sequence was obtained from bank vole strain C414 by shotgun sequencing.Of the samples obtained from woodland rodents, 23% (10/43) of bank vole samples collected from May to July 2001 and 51% (38/75) collected in January 2003 were positive for Campylobacter spp., whereas Campylobacter spp. were not recovered from any wood mouse samples (40 samples collected from May to July 2001 and 31 samples collected in January 2003). For the farm rodents (samples collected from September 2004 to April 2005), a total of 655 wood mice and 194 bank voles were sampled. In total, 18% (34/194) of bank voles were positive for Campylobacter spp., compared to 1% (6/655) of wood mice (Table (Table1).1). In total, 151 isolates were identified as Campylobacter spp. by using a genus-level 16S rRNA gene PCR assay (14). However, of all of these rodent isolates, only three isolates from wood mice from the farm survey could be identified as C. jejuni by species-specific PCR assays. The remaining rodent isolates from both the woodland and the farm study did not give any amplicons using species-specific PCR assays (5, 14, 19).

TABLE 1.

Number of samples from rodents captured in the woodland and farm studies, as well as from cattle in the farm study, positive for Campylobacter jejuni and specifically for ST-3704
Animal speciesBacterial species or strainNo. of positive samples/total no. of samples
Woodland study
Farm study (2004-2005)
May-July 2001January 2003
Bank volesC. jejuni10/4338/7534/194
C. jejuni ST-370410/4338/7534/194
Wood miceC. jejuni0/400/316/655
C. jejuni ST-3704NAaNA3/655
CattleC. jejuniNANA12/497
C. jejuni ST-3704NANA1/497
Open in a separate windowaNA, not applicable.Two of these PCR assays targeted the hipO gene (14, 19) and used the same gene sequence (GenBank accession no. z36940) for design of the primers, and this gene shares only 90% homology with the hipO gene of bank vole strain C414 (data not shown). Furthermore, the other PCR assay used to identify C. jejuni targeted the ceuE gene (5), and the ceuE gene of C414 shares only 92% homology with the ceuE gene of NCTC11168 (data not shown). The primer binding sites were also divergent for these targets in C414 (5, 19) or were not present at all (14). By use of the method of Karenlampi et al. (9), which involves PCR amplification and sequencing of a groEL fragment, all of the bank vole isolates from the woodland study and one bank vole isolate from the farm study were found to have the same sequence. This sequence (C414, GenBank accession number HQ213856) was aligned with partial groEL sequences from other Campylobacter isolates, and phylogenetic trees were inferred from this alignment by using algorithms within the MEGA4 software package (12). In these trees, C414 clustered with C. jejuni strains and not with C. coli strains (Fig. (Fig.1).1). Hence, both hippurate hydrolysis (18) testing and partial groEL sequence analysis suggested that the bank vole-associated isolates were C. jejuni.Open in a separate windowFIG. 1.Evolutionary relationships of strain C414, C. jejuni, C. coli, and C. lari. The evolutionary history was inferred using the minimum evolution (ME) method. The bootstrap consensus tree was inferred from 500 replicates. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those for the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method and are reported in units of the number of base substitutions per site. The ME tree was searched using a close-neighbor-interchange (CNI) algorithm at a search level of 3. A neighbor-joining algorithm was used to generate the initial tree. Codon positions included were first plus second plus third plus noncoding positions. There were a total of 540 positions in the final data set. Phylogenetic analyses were conducted in MEGA4 (12).MLST analysis was carried out on 41 bank vole isolates from the woodland study and 34 bank vole isolates and 6 wood mouse isolates from the farm studies, with each isolate representative of a single sample, using the method of Dingle et al. (3). For each of the seven loci, a new allele was identified (aspA227, glnA297, gltA253, glyA338, pgm-424, tkt-337, and uncA250), generating a new sequence type, ST-3704, for all of the bank vole isolates tested (100% C. jejuni-positive samples). ST-3704 was also identified in 3 wood mouse isolates (3/6 samples), with the remaining isolates representing known sequence types (ST-61 and ST-583). Other C. jejuni isolates (n = 16) from the farm cross-sectional studies were also subjected to MLST analysis, and 15 of these belonged to known sequence types (ST-45, ST-61, ST-257, and ST-403); however, one calf isolate (1/12 C. jejuni-positive samples from cattle) was identified as ST-3704 (Table (Table1).1). Furthermore, more recently (September 2008), we isolated C. jejuni ST-3704, but no other sequence type, from the F1 progeny of a captive colony of bank voles. These animals were fed an artificial diet, which suggests that this clone can be maintained in captive bred animals with no environmental exposure and is therefore strongly associated with the bank vole host. A selection of ST-3704 bank vole isolates (n = 76) from both the woodland and the farm study, the three ST-3704 wood mouse isolates, and other Campylobacter isolates from cattle were subjected to genotyping using macrorestriction pulsed-field gel electrophoresis (PFGE) and analyzed as described previously (11). Remarkably, all 79 of the ST-3704 isolates examined shared similar macrorestriction patterns (<3 bands different) following digestion with SmaI, suggesting that they represent a single clone circulating largely within bank vole populations. Sample macrorestriction patterns for isolates from bank voles from different sources are shown in Fig. Fig.2,2, which also includes examples of other C. jejuni isolates whose banding patterns are more typical of those normally observed for C. jejuni and for C. fetus isolates from cattle.Open in a separate windowFIG. 2.SmaI PFGE analysis of C. jejuni ST-3704, C. jejuni isolates belonging to other sequence types, and C. fetus isolates from bank voles (BV), wood mice (WM), and cows from four Cheshire farms (F1, F2, F3, and F4). Lane λ, lambda ladder PFGE marker (New England Biolabs, Hitchin, United Kingdom).Thus, we report the identification of a new strain of C. jejuni, ST-3704, isolated from different bank vole populations over a relatively wide geographic area with respect to the home range of bank voles, as well as in a captive colony containing F1 individuals, over a period of 7 years. Representatives of this strain could not easily be identified to the species level by using current PCR assays. Isolates of the clone are indistinguishable by MLST and share similar PFGE patterns. Although we have also detected ST-3704 rarely among samples from other animal hosts, including those sharing the same habitat, we isolated ST-3704 largely from the bank vole. Our observations suggest that ST-3704 represents a novel clone of C. jejuni associated with the bank vole host.  相似文献   
13.
14.
How old is your fold?   总被引:1,自引:0,他引:1  
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15.
Donation of human tissue for transplant and research has historically been facilitated within the hospital mortuary. In 2006 NHSBT Tissue Services opened the Dedicated Donation Facility [DDF], the first facility in the UK dedicated to the donation of tissues under strictly controlled conditions. Nine family members who had agreed and experienced the transfer of their deceased relative to the DDF for tissue donation participated in a service evaluation applying qualitative data collection methods and framework analysis. The evaluation aimed to: understand the decision-making process of family members who agreed to their deceased relative being moved to the DDR for tissue donation; identify any concerns that family members had; gather the views of family members regarding the 'service' provided to them by NHSBT Tissue Services. Family members were unaware of the possibility of tissue donation. The process of reasoning behind both agreeing to tissue donation and movement of the deceased to the DDF by family members was fundamentally, 'the benefit to others' that tissue donation would bring, and fulfilling the wishes of the deceased [when known]. Family decision making was facilitated by: (i) a positive rapport with the requester, (ii) satisfaction with the information provided to the family about what would happen, and (iii) trust in that what was being said would happen. Family members were satisfied with the service provided to them by Tissue Services and confident in agreeing to the transfer of their deceased relative to the dedicated facility for tissue donation.  相似文献   
16.
17.
Using varied strains of Blakeslea trispora it is shown that, contrary to earlier reports, a limiting role in hormone synthesis in mated cultures cannot be distinctively assigned to either the plus or the minus partner.  相似文献   
18.
Strains of Xenorhabdus nematophilus and Photorhabdus luminescens were genetically marked with kanamycin resistance and the xylE gene to aid theirdetection in water and soil. Following release in river water, cells declined to undetectable levelsin 6 d. In sterile river water, this decline was enhanced with cells detectable for only 2 d. In sterileMilli-Q purified water, the decline was slower than in either sterile or non-sterile river water.Survival in soil was also restricted with cells only detectable for 7 d. These experiments indicatedthat both X. nematophilus and P. luminescens have limited survival orcompetitive abilities in these environments. The faster decline of populations in sterile river waterwas unexpected, and the possible formation of specialized survival stages was investigated. Insterile water, a non-culturable but viable population of cells was detected, indicating that cellsmay survive longer than anticipated in the environment and remain undetectable using standardmicrobiological methods. The implications of this work to the use of these strains in biologicalcontrol and the release of genetically-modified micro-organisms is discussed.  相似文献   
19.
The susceptibility of larvae of the Diamondback moth (DBM), Plutella xylostella to infection by three baculoviruses was evaluated in the laboratory using a microdroplet feeding assay. The viruses tested were a granulovirus (GV), originally isolated in Taiwan from P. xylostella larvae (Px GV-Taiwan); the nucleopolyhedrovirus (NPV) from Galleria mellonella (Gm NPV), and the NPV from Autographa californica (Ac NPV). Neonate P. xylostella larvae were susceptible to infection by all three viruses. In an extensive series of bioassays carried out over a 21-month period, LD 50S for neonate DBM larvae ranged from 1.0-8.9 viral occlusion bodies (OB) for Px GV-Taiwan, and 9.5-30.2 OB for Gm NPV and Ac NPV. LT 50S for the three viruses ranged from 3.8-6.0 days at 27 C, with Gm NPV having a significantly shorter LT 50 than the other two viruses. Second and third instar larvae of P. xylostella were significantly less susceptible to infection by Px GV-Taiwan (LD 50 s ranging from 18-57 OB/larva) than were neonate larvae. Gm NPV also initiated infection in several other lepidopterous pest species that colonize brassica crops. In particular, neonate Crocidolomia binotalis larvae proved highly susceptible to Gm NPV, with mean LD 50 s ranging from 2.1 to 9.3 OB/larva and a mean LT 50 of 4.8 days at a dose of 8.08 OB. Heliothis virescens neonate larvae were also highly susceptible to Gm NPV (LD 50 , 7.1 OB), but Mamestra brassicae larvae were less so (LD 50 , 80-270 OB). The results of the bioassays suggest that Px GV-Taiwan is highly infective and could be developed as a selective microbial pesticide for DBM. While Gm NPV has a higher LD 50 in DBM larvae, its wider host range may be of considerable value in situations where DBM occurs on cruciferous crops together with a complex of other lepidopterous pests.  相似文献   
20.
At present, effective treatment for non-severe malaria is the most important malaria control strategy in Africa. Pyrimethamine-sulfadoxine (PSD) is rapidly becoming the first-line treatment in areas of chloroquine resistance, although the parasite chemoresistance factors that dispose towards clinical failure with PSD are still unclear. Here, Bill Watkins and colleagues analyse the relationship between the pharmacokinetic properties of two treatment combinations (PSD and chlorproguanil-dapsone) in vivo and the respective in vitro isobolograms for parasites with specific drug-resistance patterns. From this relationship, they develop a hypothesis that may explain clinical drug failure and differential efficacy between treatments. The deductions can be tested in field studies to validate or refute the model.  相似文献   
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