Monitoring the Long-Distance Transport of Fusarium graminearum from Field-Scale Sources of Inoculum

The fungus Fusarium graminearum causes Fusarium head blight (FHB) of wheat. Little is known about dispersal of the fungus from field-scale sources of inoculum. We monitored the movement of a clonal isolate of F. graminearum from a 3,716 m2 (0.372 ha) source of inoculum over two field seasons. Ground-based collection devices were placed at distances of 0 (in the source), 100, 250, 500, 750, and 1,000 m from the center of the clonal sources of inoculum. Three polymorphic microsatellites were used to identify the released clone from 1,027 isolates (790 in 2011 and 237 in 2012) of the fungus. Results demonstrated that the recovery of the released clone decreased at greater distances from the source. The majority (87%, 152/175 in 2011; 77%, 74/96 in 2012) of the released clone was recaptured during the night (1900 to 0700). The released clone was recovered up to 750 m from the source. Recovery of the released clone followed a logistic regression model and was significant (P < 0.041 for all slope term scenarios) as a function of distance from the source of inoculum. This work offers a means to experimentally determine the dispersal kernel of a plant pathogen, and could be integrated into management strategies for FHB.

Templated Assembly by Selective Removal for Size-Selective Sorting of Biological Materials

This work presents the size-selective sorting of single biological cells using the assembly process known as Templated Assembly by Selective Removal (TASR). We have demonstrated experimentally, for the first time, the selective self assembly and sorting of single SF9 cells (a clonal isolate, derived from Spodoptera frugiperda (Fall Armyworm) IPLB-Sf21-AE cells) into patterned hemispherical sites on rigid assembly templates using TASR. TASR-based size-selective assembly of biological systems represents a potentially valuable tool, with potential for implementation in several biological applications, such as cell sorting for medical research or diagnostics, or isolation of single cells for the study of biological or mechanical behavior.

Deletion of BBA64, BBA65, and BBA66 Loci Does Not Alter the Infectivity of Borrelia burgdorferi in the Murine Model of Lyme Disease

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to highly disparate environmental signals encountered in its tick vector versus vertebrate hosts. Whole-genome transcriptional profile analysis of B. burgdorferi, propagated in vitro under mammalian-host-specific conditions, revealed significant upregulation of several linear plasmid 54 (lp54)-encoded open reading frames (ORFs). Among these ORFs, BBA64, BBA65, and BBA66 have been shown to be upregulated in response to multiple mammalian-host-specific signals. Recently, we determined that there was no significant difference in the ability of BBA64− mutant to infect C3H/HeN mice compared to its isogenic control strains, suggesting that B. burgdorferi might utilize multiple, functionally related determinants to establish infection. We further generated BBA65− and BBA66− single mutants in a noninfectious, lp25− clonal isolate of B. burgdorferi strain B31 (ML23) and complemented them with the minimal region of lp25 (BBE22) required for restoring the infectivity. In addition, we generated a BBA64− BBA65− BBA66− triple mutant using an infectious, clonal isolate of B. burgdorferi strain B31 (5A11) that has all of the infection-associated plasmids. There were no significant differences in the ability to isolate viable spirochetes from different tissues of C3H/HeN mice infected via intradermal needle inoculation with either the individual single mutants or the triple mutant compared to their respective isogenic parental strains at days 21 and 62 postinfection. These observations suggest that B. burgdorferi can establish infection in the absence of expression of BBA64, BBA65, and BBA66 in the murine model of Lyme disease.

Genomic sequence of a clonal isolate of the vaccinia virus Lister strain employed for smallpox vaccination in France and its comparison to other orthopoxviruses

Since 1980 there has been global eradication of smallpox due to the success of the vaccination programme using vaccinia virus (VACV). During the eradication period, distinct VACV strains circulated, the Lister strain being the most commonly employed in Europe. Analysis of the safety of smallpox vaccines has suggested that they display significant heterogeneity. To gain a more detailed understanding of the diversity of VACV strains it is important to determine their genomic sequences. Although the sequences of three isolates of the Japanese Lister original strain (VACV-LO) are available, no analysis of the relationship of any Lister sequence compared to other VACV genomes has been reported. Here, we describe the sequence of a representative clonal isolate of the Lister vaccine (VACV-List) used to inoculate the French population. The coding capacity of VACV-List was compared to other VACV strains. The 201 open reading frames (ORFs) were annotated in the VACV-List genome based on protein size, genomic localization and prior characterization of many ORFs. Eleven ORFs were recognized as pseudogenes as they were truncated or fragmented counterparts of larger ORFs in other orthopoxviruses (OPVs). The VACV-List genome also contains several ORFs that have not been annotated in other VACVs but were found in other OPVs. VACV-List and VACV-LO displayed a high level of nucleotide sequence similarity. Compared to the Copenhagen strain of VACV, the VACV-List sequence diverged in three main regions, one of them corresponding to a substitution in VACV-List with coxpox virus GRI-90 strain ORFs, suggestive of prior genetic exchanges. These studies highlight the heterogeneity between VACV strains and provide a basis to better understand differences in safety and efficacy of smallpox vaccines.

Analysis of Borrelia burgdorferivlsE Gene Expression and Recombination in the Tick Vector

ABSTRACT Expression and recombination of the antigenic variationvlsE gene of the Lyme disease spirochete Borrelia burgdorferi were analyzed in the tick vector. To assessvlsE expression, Ixodes scapularis nymphs infected with the B. burgdorferi strain B31 were fed on mice for 48 or 96 h or to repletion and then crushed and acetone fixed either immediately thereafter (ticks collected at the two earlier time points) or 4 days after repletion. Unfed nymphs also were examined. At all of the time points investigated, spirochetes were able to bind a rabbit antibody raised against the conserved invariable region 6 of VlsE, as assessed by indirect immunofluorescence, but not preimmune serum from the same rabbit. This same antibody also bound to B31 spirochetes cultivated in vitro. Intensity of fluorescence appeared highest in cultured spirochetes, followed by spirochetes present in unfed ticks. Only a dim fluorescent signal was observed on spirochetes at the 48 and 96 h time points and at day 4 postrepletion. Expression of vlsE in vitro was affected by a rise in pH from 7.0 to 8.0 at 34°C. Hence, vlsE expression appears to be sensitive to environmental cues of the type found in theB. burgdorferi natural history. To assessvlsE recombination, nymphs were capillary fed theB. burgdorferi B31 clonal isolate 5A3. Ticks thus infected were either left to rest for 4 weeks (Group I) or fed to repletion on a mouse (Group II). The contents of each tick from both groups were cultured and 10 B. burgdorferi clones from the spirochetal isolate of each tick were obtained. ThevlsE cassettes from several of these clones were amplified by PCR and sequenced. Regardless of whether the isolate was derived from Group I or Group II ticks, no changes were observed in thevlsE sequence. In contrast, vlsEcassettes amplified from B. burgdorferi clones derived from a mouse that was infected with B31-5A3 capillary-fed nymphs showed considerable recombination. It follows that vlsErecombination does not occur in the tick vector.

Temperature-sensitive expression of all-Torpedo and Torpedo-rat hybrid AChR in mammalian muscle cells.

When the four subunits of the Torpedo californica nicotinic acetylcholine receptor (AChR) are expressed in mammalian fibroblasts, they properly assembly into alpha 2 beta gamma delta pentamers only at temperatures lower than 37 degrees C (Claudio, T., W. N. Green, D. S. Hartman, D. Hayden, H. L. Paulson, F. J. Sigworth, S. M. Sine, and A. Swedlund. 1987. Science (Wash. DC). 238:1688-1694). Experiments here with rat L6 myoblast cell lines indicate that this temperature sensitivity is not specific to fibroblasts, but is intrinsic to Torpedo subunits. A clonal isolate of L6 cells cotransfected with the four Torpedo subunit cDNAs synthesizes the exogenous AChR subunits at 37 degrees and 26 degrees C, but expresses Torpedo AChR complexes only at the lower temperature. When Torpedo alpha alone is expressed in L6 myotubes, hybrid AChRs are formed, again only at temperatures below 37 degrees C. These hybrid AChRs can contain either two Torpedo alpha subunits or one each of rat and Torpedo alpha, proving that the two alpha subunits in an AChR pentamer need not derive from the same polysome. Further analysis of hybrid and all-Torpedo AChR established that there is no internally sequestered pool of AChR at the nonpermissive temperature, and that the AChR, once formed, is thermostable. Two lines of experimentation with alpha subunits expressed in fibroblasts indicate that alpha polypeptides exhibit different conformations at 26 degrees and 37 degrees C, favoring the hypothesis that the temperature-sensitive step occurs before assembly and reflects, at least in part, misfolding of subunits: at 37 degrees C, there is a reduction in the fraction of alpha subunits that (a) bind the AChR antagonist alpha-bungarotoxin with high affinity; and (b) bind a monoclonal antibody that recognizes correctly folded and/or assembled alpha subunit.