The relationship between concentration of a dual marker strain of Salmonella Typhimurium in bovine faeces and its probability of detection by immunomagnetic separation and culture

AIMS: To modify a strain of Salmonella serotype Typhimurium to express unique marker traits and then define how the concentration of the marker in bovine faeces affects the probability of its detection by culture preceded by immunomagnetic separation (IMS). METHODS AND RESULTS: DNA encoding for the production of green fluorescent protein (gfp) and resistance to kanamycin was inserted into the bacterial chromosome of Salm. Typhimurium. Transposon insertion was demonstrated by Southern blot hybridization. Varying amounts of one electroporant (gfpSal-1) were inoculated into suspensions of bovine faeces and attempts made to isolate gfpSal-1 using a protocol based on pre-enrichment incubation, IMS and enrichment in selective media. Isolates of gfpSal-1 were differentiated from wild strains of Salmonella using fluorescence under u.v. light and expression of kanamycin resistance. A logistic and Gompertz function each derived from the dose-response data partially explained the observations with the fit of the Gompertz function judged to be superior. The 10, 50 and 90% limits of detection from the Gompertz function were estimated to be 1.92, 2.03 and 2.27 CFU g(-1) respectively. CONCLUSIONS: Reliance on the traditional concept of 'limit of detection' could introduce unacceptable errors in the interpretation of test findings when the concentration of Salm. Typhimurium in bovine faeces (pooled or individual) is below ca 3 CFU g(-1) of faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The dose-response curve can be used to aid the design of protocols for detecting Salmonella in individual and pooled faecal specimens. The experiments demonstrate that both reporter genes in tandem are useful for studying the performance of culture-based methods for detecting pathogens in faeces.     

Induction of structural and numerical changes of chromosome, centrosome abnormality, multipolar spindles and multipolar division in cultured Chinese hamster V79 cells by exposure to a trivalent dimethylarsenic compound

Dimethylarsine iodide (DMI) was used as a model compound of trivalent dimethylarsenicals [DMA(III)], and the biological effects were extensively investigated in cultured Chinese hamster V79 cells. When the cytotoxic effects of DMA(III) were compared with those of inorganic arsenite and dimethylarsinic acid [DMA(V)], DMA(III) was about 10,000 times more potent than DMA(V), and it was even 10 times more toxic than arsenite. Depletion of cell glutathione (GSH) did not influence the cytotoxic effects of DMA(III), whereas it enhanced the cytotoxicity of arsenite. Chromosome structural aberrations, such as gaps, breaks and pulverizations, and numerical changes, such as aneuploidy, hyper- and hypo-tetraploidy, were induced by DMA(III) in a concentration-dependent manner. Mitotic index increased 9-12h after the addition of DMA(III), and then declined. By contrast, the incidence of multinucleated cells increased conversely with the decrease in mitotic index at and after 24h of exposure. The mitotic cell-specific abnormality of centrosome integrity and multipolar spindles were induced by DMA(III) in a time- and concentration-dependent manner. Moreover, DMA(III) caused abnormal cytokinesis (multipolar division) at concentrations that were effective in causing centrosome abnormality, multipolar spindles and aneuploidy. These results showed that DMA(III) was genotoxic on cultured mammalian cells. Results also suggest that DMA(III)-induced multipolar spindles and multipolar division may be associated with the induction of aneuploidy. In addition, the centrosome may be a primary target for cell death via multinucleated cells.     

Paternal reciprocal translocation t(11;16)(p13;q24.3) in a Silver-Russel syndrome patient

We describe a 7-month-old male child with Silver-Russel syndrome (SRS) phenotype, presented with two major clinical features: low birth weight, short stature, and minor features, such as macrocephaly, clinodactyly, essential for the diagnosis of SRS. Routine cytogenetic studies with GTG-banding showed 46,XY,t(11;16)(p13;q24.3). Fluorescence in situ hybridisation (FISH) with single copy probes BAC (11p13) and PAC (16q24.3), showed a reciprocal translocation. Chromosomal analysis of the mother was normal and the phenotypically normal father had apparently identical translocation t(11;16)(p13;q24.3). The disruption of growth factor genes at 11p and 16q breakpoint regions due to reciprocal translocation in the father might have caused SRS phenotype in the child.     

Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability

Using the pUT-miniTn5 vector system developed by the laboratory of K.N. Timmis, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosomes of Pseudomonas aeruginosa and Burkholderia cepacia; Vitreoscilla hemoglobin (VHb) was expressed at 8.8 and 0.8 nmol/g wet weight of cells in the respective engineered strains. The vgb-bearing P. aeruginosa outgrew wild-type P. aeruginosa and degraded benzoic acid faster than the latter strain at both normal and low aeration. In contrast, the vgb-bearing B. cepacia strain had a growth advantage over the wild-type strain at ca. 90 ppm, but not at ca. 120 ppm 2,4-dinitrotoluene (DNT); no difference in DNT degradation was seen between the two strains at either normal or low aeration. The results demonstrate the practicality of enhancing bioremediation with vgb stably integrated into the chromosome, but also suggest that a minimal level of VHb expression is required for its beneficial effects to be seen. Journal of Industrial Microbiology & Biotechnology (2001) 27, 27–33. Received 20 October 2000/ Accepted in revised form 04 May 2001     

Purification of plasmid DNA by an integrated operation comprising tangential flow filtration and nitrocellulose adsorption

There is an increasing interest in the development of scaleable and reproducible plasmid DNA purification protocols for vaccine and gene therapy. The use of an integrated unit operation, comprising tangential flow microfiltration coupled with the adsorption of contaminants onto nitrocellulose membranes as a single processing step was examined in this work. Experiments were performed using a custom-built tangential flow microfiltration rig (membrane area=12.5 cm(2)). Tangential flow filtration-adsorption of E. coli lysates containing a plasmid product removed most solids (>75%) and decreased chromosomal DNA contamination by 75% w/w. Total plasmid DNA concentration and supercoiled content of the permeate were virtually identical to those of the feed, indicating a recovery yield of 100% (transmission equal to 1). Results were similar for E. coli lysates containing either a 6.9 kb or a 20 kb plasmid. Significant reductions in RNA, endotoxin, and protein levels were also observed. The reproducibility and potential for scale up of this integrated filtration-adsorption operation makes it at attractive option for intermediate- to large-scale pharmaceutical-grade plasmid processing.     

Chiasma Repatterning Across a Chromosomal Hybrid Zone Between Chromosomal Races of Mus Musculus Domesticus

Chiasma number and distribution were analysed in male house mice from a karyotypic hybrid zone between the CD race (2n=22) and the standard race (2n=40) located in central Italy. Chiasma repatterning occurs across the transect. The overall trend produces a diminution of chiasmata in the mice with CD chromosomal background. The progressive reduction of chiasmata indicates that genes could pass from one race to another in an asymmetrical way: from metacentric races to the standard population.     

Metazoan motor models: kinesin superfamily in C. elegans

In eukaryotic cells members of the kinesin family mediate intracellular transport by carrying cellular cargo on microtubule tracks. The nematode Caenorhabditis elegans genome encodes 21 members of the kinesin family, which show significant homology to their mammalian orthologs. Based on motor domain sequence homology and placement of the motor domain in the protein, the C. elegans kinesins have been placed in eight distinct groups; members of which participate in embryonic development, protein transport, synaptic membrane vesicles movement and in the axonal growth. Among 21 kinesins, at least 11 play a central role in spindle movement and chromosomal segregation. Understanding the function of C. elegans kinesins and related proteins may help navigate through the intricacies of intracellular traffic in a simple animal.     

E. coli chromosomal DNA in a transgene locus created by microprojectile bombardment in tobacco

Transgenic Research -