Efficacy and limitations of an ATP-based monitoring system.

Monitoring of sanitation is an essential function of laboratory animal facilities. The purpose of the current study was to assess the ability of an ATP-based system to detect microbes and organic contaminants. Serial dilutions of Escherichia coli, Staphylococcus aureus, Toxocara canis eggs, Toxoplasma gondii tachyzoites, epithelial cells, and rodent blood, urine, and feces were analyzed according to the manufacturer's recommendations. The limit of E. coli detection was 10(4) organisms; sonication of E. coli significantly improved detection, indicating incomplete bacterial lysis in the detection system. Detection of S. aureus was significantly greater than that of E. coli with a limit of detection of 10(2); sonication did not alter results. In contrast, detection of T. canis, T. gondii, RBC, and epithelial cells was robust and ranged from 2 T. canis eggs to 10 epithelial cells. Urine was weakly detected, with a limit of detection at 1:10 dilution. Detection of all cell types except epithelia had a strong linear correlation to total cell number. In addition, our data demonstrate that the efficacy of the detection system can be affected adversely by residual disinfectants and that sample-bearing swabs are stable for more than 7 h after swabbing. These data demonstrate that this ATP based system sensitively detects pure cells and organic contaminants with a strong degree of linear predictability. A limitation of the system is its inability to detect gram-negative bacteria efficiently because of incomplete cell lysis.

Detection of pathogenic leptospires by real-time quantitative PCR.

Definitive diagnosis of leptospirosis has traditionally depended upon the isolation of leptospires from clinical specimens or the demonstration of seroconversion in paired acute and convalescent serum samples. Both of these approaches require expertise not routinely available in clinical laboratories and usually result in delayed diagnosis. Conventional PCR assays have been developed, but all have limitations which have restricted their widespread use. In order to overcome these limitations, a real-time PCR assay was developed using a 423 bp target on the lipL32 gene, which is conserved among pathogenic serovars of LEPTOSPIRA: Reactions were monitored by SYBR green fluorescence and melting curve analysis. Representative serovars from 16 species of Leptospira and over 40 species of other bacteria and fungi were tested. Positive results were obtained with all pathogenic leptospiral serovars, with the exception of Leptospira fainei serovar Hurstbridge. The analytical sensitivity of this assay was 3 genome equivalents per reaction; approximately 10 genome equivalents were detectable in human urine. Leptospiral DNA was amplified from blood containing EDTA or citrate anticoagulants, but heparin, sodium polyanetholesulfonate and saponin were inhibitory. The assay successfully detected leptospiral DNA from serum and urine samples of patients with leptospirosis. This assay has the potential to facilitate rapid, sensitive diagnosis of acute leptospirosis.

Neuronal survival following remote adenovirus gene delivery.

OBJECT: Virus-mediated central nervous system gene delivery is a promising means of treating traumatized tissue or degenerative diseases. In the present study, the authors examined gene expression and neuronal survival in the spinal cord after sciatic nerve administration of an adenovirus vector expressing a LacZ reporter gene. METHODS: The time course of adenovirus gene expression, DNA fragmentation, and neuronal density were quantified in rat lumbar spinal cord by staining for beta-galactosidase (beta-Gal), terminal deoxynucleotidyl transferase, and cresyl violet after microinjection of either saline or the reporter virus into rat sciatic nerve. The expression of beta-Gal following remote vector delivery peaked at 7 days and declined thereafter but was not accompanied by neuronal cell death, as measured by DNA fragmentation. No significant difference in spinal motor neuron density was detected between virus-treated and control rats at any time point examined. Although the spinal cords removed from rats treated with cyclosporine prior to adenovirus injection contained substantially more neurons staining for beta-Gal at 7 days (67% of total neurons), the decay in the number of stained neurons was not paralleled by a decline in motor neuron density. CONCLUSIONS: The authors conclude that remote gene expression is suppressed by a noncytolytic process.