Induction of immunity using oral DNA vaccines expressing the measles virus nucleocapsid protein.

In this study, we have examined the feasibility of immunisation against measles with plasmid DNA administered by the oral route. After the oral administration, in two 50 microg doses, of poly(DL-lactide-co-glycolide) (PLGA)-encapsulated DNA expressing measles virus nucleoprotein, increasing titres of N-specific serum IgG antibodies were observed in three of ten C3H/He mice over a period of three months. In comparison, oral vaccination of mice with a replication-defective recombinant adenovirus expressing the same transgene induced serum IgG in all animals tested. We also obtained preliminary indication of adjuvant-like activity of PLGA particles when coadministered intraperitoneally (i.p.) with naked plasmid DNA. These experiments demonstrate that oral delivery of either PLGA-encapsulated plasmid DNA or viral vectored DNA is capable of eliciting strong immune responses in mice. We propose that oral administration of biodegradable microparticles offers a novel strategy for future vaccine design for the safe delivery of DNA to mucosal surfaces.

Detection of mRNA by reverse transcription-PCR as an indicator of viability in Escherichia coli cells.

The relationship between the detection of mRNA and cellular viability in Escherichia coli was investigated in cells killed by heat or ethanol. Reverse transcription-PCR (RT-PCR) methods were developed for detecting mRNA from rpoH, groEL, and tufA genes. mRNA from all three genes was detected immediately after the cells had been killed by heat or ethanol but gradually disappeared with time when dead cells were held at room temperature. In heat-killed cells, some mRNA targets became undetectable after 2 to 16 h, whereas after ethanol treatment, mRNA was still detected after 16 h. In contrast, 16S rRNA was detected by RT-PCR in all samples containing dead cells and did not disappear during a subsequent incubation of 16 h at room temperature. Of the different types of nucleic acid, mRNA is the most promising candidate for an indicator of viability in bacteria, but its persistence in dead cells depends on the inactivating treatment and subsequent holding conditions.

Listeria innocua isolated from a case of ovine meningoencephalitis.

This paper reports a naturally occurring case of meningoencephalitis associated with Listeria innocua in a Polled-Dorset ewe. The ewe was one of a housed group of twenty-five, fed ad lib. on wrapped baled silage. L. innocua was isolated after one week from cold enrichment culture of brain and pituitary tissue. Its identity was confirmed by conventional biochemical tests, API Listeria (BioMerieux UK Ltd), the absence of hly and prfA genes using PCR assay and sequencing two variable regions of 16S rDNA. Histological examination demonstrated lesions of vasculitis and perivascular cuffing in the midbrain which were consistent with listeriosis although limited in distribution and severity.

Comparison of the fluorescent redox dye 5-cyano-2,3-ditolyltetrazolium chloride with p-iodonitrotetrazolium violet to detect metabolic activity in heat-stressed Listeria monocytogenes cells.

The fluorogenic redox indicator 5-cyano-2,3-ditolyltetrazolium chloride (CTC) was compared with the chromogenic p-iodonitrotetrazolium violet (INT) and conventional methods to assess cellular viability. Mild heat treatment was used as well-controlled method for producing non-viable and sub-lethally injured cells. CTC gave an underestimation of the viability of Listeria monocytogenes cells when compared with classical plating methods whereas INT gave an overestimation. However, CTC proved to be a sensitive indicator of uninjured cells. The difference between the total count and the CTC count was equivalent to the injured cell population. The fluorescent formazan formed on reduction of CTC was readily detected with a charge coupled device and cells enumerated automatically using image analysis.

Effect of stress treatments on the detection of Listeria monocytogenes and enterotoxigenic Escherichia coli by the polymerase chain reaction.

The relationship between viability assessed by plate counts and detectability by gene probe-polymerase chain reaction (PCR) techniques was examined with cells of Escherichia coli and Listeria monocytogenes previously exposed to a range of stress treatments. In all cases the organisms were detectable by PCR after plate counts had declined to zero. Treatment with acid or hydrogen peroxide caused loss of PCR soon after viability was lost, but strong PCR signals were obtained from starved or desiccated cells long after cells became non-viable. Exposure to temperatures up to 100 degrees C had little effect on detection by PCR and even autoclaving cells at 121 degrees C for 15 min failed to abolish PCR detection completely. There is thus no simple relationship between viability and detectability by PCR. Detection of pathogens by PCR in environmental monitoring requires additional evidence of viability before risk can be properly assessed.