Use of Whatman-41 filters in air quality sampling networks (with applications to elemental analysis)

The operation of a 16-site parallel high volume air sampling network with glass fiber filters on one unit and Whatman-41 filters on the other is reported. The network data and data from several other experiments indicate that (1) Sampler-to-sampler and filter-to-filter variabilities are small; (2) hygroscopic affinity of Whatman-41 filters need not introduce errors; and (3) suspended particulate samples from glass fiber filters averaged slightly, but not statistically significantly, higher than from Whatman-41-filters. The results obtained demonstrate the practicability of Whatman-41 filters for air quality monitoring and elemental analysis

Statistical summary of air quality data for metropolitian Cleveland, Ohio, 1967 - 1972: Total suspended particulates, nitrogen dioxide, and sulfur dioxide

Air-quality data for metropolitan Cleveland, Ohio, from 1967 through 1972 were collated and statistically analyzed. Total suspended particulates (TSP) departed from lognormal distribution in 1972. Nitrogen dioxide and sulfur dioxide, departed significantly from lognormal distributions in 1972. In Cleveland the Ohio standards were not met. However, the data indicate a general improvement in air quality. Unusually high precipitation (43% above the average in 1972) may be responsible in lowering these values from the 1971 levels. The mean values of TSP, NO2, and SO2 are 104, 191, and 83 microgram/cu m respectively

Transformation of crown gall resistant and susceptible Vitis genotypes by Agrobacterium vitis

Transformation of crown gall-susceptible and -resistant Vitis genotypes by Agrobacterium vitis strain CG49 was studied using uidA (GUS) in the p35SGUSINT construct. When greenhouse-grown material propagated through tissue culture was inoculated with CG49(p35SGUSINT) in vitro, the highly crown gall-susceptible V. vinifera Cabernet Sauvignon displayed GUS activity on 53 % of inoculated explants vs. 5 % for the resistant V. amurensis and 0 % for the resistant Couderc 3309. Response of Cabernet Sauvignon suggested a strong effect of shoot polarity on transformation. Inoculation of basal vs. apical explant surface in Cabernet Sauvignon indicated transformation in 88 % of basal inoculated explants with no transformation from apical inoculation. Basal inoculations indicated no transformation of V. amurensis and transformation in 10 % of Couderc 3309 explants. Inoculation of intact plants with CG49(p35SGUSINT) produced GUS-positive sites at 56 % of inoculated sites in Cabernet Sauvignon, 10 % of V. amurensis inoculated sites and 9 % of Couderc 3309 inoculated sites. Resistance to crown gall in these genotypes appears to be due to reduced susceptibility to transformation by A. vitis rather than post-transformation phenomena. These studies were complicated by production of GUS-positive spots from in vitro inoculations using wild-type CG49. Resident microorganisms producing b-glucuronidase may proliferate after tissue degradation by A. vitis-induced cell disruption. Use of in vitro internodal explants from tissue culture-propagated vines greatly reduced GUS expression from control CG49 inoculations and these were readily distinguished (by appearance and location) from GUS-positive spots resulting from transformation with uidA

Endophytic Agrobacterium in crown gall-resistant and -susceptible Vitis genotypes

Several methods were used to study endophytic colonization of Vitis genotypes by Agrobacterium vitis (AV). AV was seldom detected except at inoculated sites, indicating little systemic movement of the bacterium under the conditions of these experiments. AV populations at inoculated sites were evaluated for 10 months following inoculation of crown gall-resistant and -susceptible genotypes. Two months after inoculation, V. amurensis selections had significantly smaller populations than V. vinifera (Cabernet Sauvignon) or V. riparia x V. rupestris (C3309). All crown gall-resistant genotypes had significantly lower populations than Cabernet Sauvignon 10 months after inoculation. Examination of vines one year after inoculation indicated that AV populations were much higher at inoculated sites when crown galls developed. However, even when no galls were apparent, Cabernet Sauvignon had significantly higher AV populations than V. amurensis 689 (6-fold higher) and C3309 (70-fold higher). Crown gall-resistant genotypes appear to support lower populations of AV than the crow gall-susceptible Cabernet Sauvignon. Freezing followed by a two-day incubation significantly increased recovery of Agrobacterium using vascular fluid displacement in naturally-infected and artifically-inoculated vines and therefore increased the sensitivity of indexing for AV in grapevines

Inhibition of crown gall induction by Agrobacterium vitis strain F2/5 in grapevine and Ricinus

Biological control measures to prevent or reduce Agrobacterium vitis-caused losses in grapevine cultures are a worldwide increasing challenge. In the present study, tumour development in grapevine (Vitis vinifera L.) was induced in the sensitive cv. Kerner by infection with Agrobacterium vitis strain K306, carrying the p35Sgus-int plasmid with the gus gene as marker for transformation by the wild-type T-DNA. Pre-inoculation with the non-tumorigenic A. vitis strain F2/5 prevented tumour induction by K306(p35gus-int). Strain M1154, a Tn5 mutant of F2/5 in the luxR-like aviR gene, partially reduced the biocontrol efficiency compared to the wild-type F2/5. GUS-labelling by K306gus was poor in grapevine in contrast to A. tumefaciens 281(p35gus-int)-induced tumours in Arabidopsis, indicating plant species-dependent variable gus expression. To use the more reliable direct mRNA expression assay by RTPCR, a new experimental plant/A. vitis system was established with Ricinus communis as model plant. Ricinus/A. vitis galls were available within one week after K306gus inoculation, reached diameters up to 5 cm, and contained more abundant GUS staining. An additional transformation marker, mRNA expression of the T-DNA-located iaaM oncogene, coding auxin synthesis, was apparent only in tumours induced by the wild-type A. vitis strain K306 in the absence of the gus construct, which is under the control of the strong 35S CaMV promoter. F2/5 pre-inoculation suppressed GUS staining and gus mRNA expression. DAPI staining revealed the loss of vital fluorescent cell nuclei in F2/5-inoculated grapevine tissue and thus inhibition of any successful T-DNA transfer into host cell nuclei. Differentiation of typical circular vessels in globular vascular bundles in M1154-pretreated galls suggests interference with plant auxin metabolism. In conclusion, together with successfully establishing a new experimental model system, Ricinus/A. vitis, pre-treatment of host tissue with the non-pathogenic strain F2/5 resulted in preventing the integration and expression of the oncogenic T-DNA of A. vitis strains by locally necrotizing host cell nuclei.

Biological control of Agrobacterium vitis using non-tumorigenic agrobacteria

The potential use of non-tumorigenic agrobacteria for the biological control of grapevine crown gall in Italy was investigated. Four Agrobacterium strains belonging to the species radiobacter and vitis were used to protect the susceptible cv. Malvasia Istriana grafted on the rootstock 420 A. Moreover, the effect of each treatment on grapevine vitality and growth was assessed, including the percentage of marketable vines, as determined by industry standards. Treatments with the antagonists clearly reduced tissue colonization by the pathogen, with a drop of more than 100-fold in pathogen populations in the samples collected at the graft point. Another important effect was the reduction of internal necrosis possibly induced by the high concentration of the nopaline strain CG 49 used in the experiments. According to viticultural and commercial parameters, treatments with the antagonists improved the quality of the vines, with fewer discards and a high percentage of marketable material. Therefore, these antagonists can be considered beneficial for grapevine

Evaluation of a Chlamydia trachomatis-specific, commercial, real-time PCR for use with ocular swabs.

BACKGROUND: Trachoma, the leading infectious cause of blindness worldwide, is caused by conjunctival Chlamydia trachomatis infection. Trachoma is diagnosed clinically by observation of conjunctival inflammation and/or scarring; however, there is evidence that monitoring C. trachomatis infection may be required for elimination programmes. There are many commercial and 'in-house' nucleic acid amplification tests for the detection of C. trachomatis DNA, but the majority have not been validated for use with ocular swabs. This study evaluated a commercial assay, the Fast-Track Vaginal swab kit, using conjunctival samples from trachoma-endemic areas. An objective, biostatistical-based method for binary classification of continuous PCR data was developed, to limit potential user-bias in diagnostic settings. METHODS: The Fast-Track Vaginal swab assay was run on 210 ocular swab samples from Guinea-Bissau and Tanzania. Fit of individual amplification curves to exponential or sigmoid models, derivative and second derivative of the curves and final fluorescence value were examined for utility in thresholding for determining positivity. The results from the Fast-Track Vaginal swab assay were evaluated against a commercial test (Amplicor CT/NG) and a non-commercial test (in-house droplet digital PCR), both of whose performance has previously been evaluated. RESULTS: Significant evidence of exponential amplification (R2 > 0.99) and final fluorescence > 0.15 were combined for thresholding. This objective approach identified a population of positive samples, however there were a subset of samples that amplified towards the end of the cycling protocol (at or later than 35 cycles), which were less clearly defined. The Fast-Track Vaginal swab assay showed good sensitivity against the commercial (95.71) and non-commercial (97.18) tests. Specificity was lower against both (90.00 and 96.55, respectively). CONCLUSIONS: This study defined a simple, automated protocol for binary classification of continuous, real-time qPCR data, for use in an end-point diagnostic test. This method identified a population of positive samples, however, as with manual thresholding, a subset of samples that amplified towards the end of the cycling program were less easily classified. When used with ocular swabs, the Fast-Track Vaginal swab assay had good sensitivity for C. trachomatis detection, but lower specificity than the commercial and non-commercial assays it was evaluated against, possibly leading to false positives

An outbreak of acute haemorrhagic conjunctivitis associated with coxsackievirus A24 variant in The Gambia, West Africa.

OBJECTIVE: An outbreak of acute haemorrhagic conjunctivitis occurred in The Gambia, West Africa in 2011. Affected individuals presented with conjunctival haemorrhages, swelling and ocular discharge. In an effort to identify a causative agent of the disease, ocular swabs were taken from patients during the acute and convalescent phases. Total RNA was extracted from all samples and reverse-transcriptase PCR performed using primers specific for all enteroviruses. Resulting amplicons were sequenced and data compared to known sequences using the BLAST algorithm. RESULTS: Forty-eight swabs were included in the analysis. Of these, 21 acute and 9 convalescent swabs (65% of the total) gave positive PCR results. Sequence analysis of the resulting amplicons indicated 99% sequence identity with coxsackievirus A24 variant identified during independent outbreaks of acute haemorrhagic conjunctivitis around the world and suggest the Gambian outbreak was due to this virus