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Bacterial DNA activates mouse macrophages, B cells, and dendritic cells in a TLR9-dependent manner. Although short ssCpG-containing phosphodiester oligonucleotides (PO-ODN) can mimic the action of bacterial DNA on macrophages, they are much less immunostimulatory than Escherichia coli DNA. In this study we have assessed the structural differences between E. coli DNA and PO-ODN, which may explain the high activity of bacterial DNA on macrophages. DNA length was found to be the most important variable. Double-strandedness was not responsible for the increased activity of long DNA. DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) methylation of E. coli DNA did not enhance macrophage NO production. The presence of two CpG motifs on one molecule only marginally improved activity at low concentration, suggesting that ligand-mediated TLR9 cross-linking was not involved. The major contribution was from DNA length. Synthetic ODN >44 nt attained the same levels of activity as bacterial DNA. The response of macrophages to CpG DNA requires endocytic uptake. The length dependence of the CpG ODN response was found to correlate with the presence in macrophages of a length-dependent uptake process for DNA. This transport system was absent from B cells and fibroblasts.

The complete sequence of two plasmids, pHS-Tet (5.1 kb) and pHS-Rec (9.5 kb), isolated from Haemophilus parasuis strain HS1543 has been obtained. Plasmid pHS-Tet contains four open reading frames including a tet(B) tetracycline resistance gene which unusually did not have an associated tetR repressor gene. From a total of 45 H. parasuis isolates surveyed (15 international reference strains, 15 field isolates selected for their genetic diversity, and 15 recent Australian field isolates), 2 tetracycline-resistant field isolates (HS226 and HS1859) were identified. Analysis of three additional isolates from the same disease outbreak as strain HS1859 revealed a further tetracycline-resistant H. parasuis strain (HS1857, serovar 8) and a tetracycline-resistant Actinobacillus pleuropneumoniae strain (HS1861). An approximately 10.6-kb plasmid was identified in field isolate HS226 and outbreak strains HS1857, HS1859, and HS1861. Southern hybridization analysis of these plasmids showed that the Tet B determinant was present, and restriction digest comparisons suggest that these plasmids are related. This is believed to be the first report of native H. parasuis plasmids and Tet B-mediated tetracycline resistance in this microorganism.Haemophilus parasuis is the causative agent of Glässer's disease in swine. Common symptoms of this disease include anorexia, pyrexia, and lameness, though some pigs may die suddenly during acute outbreaks (19).The use of antibiotics in animal production as treatment or prophylaxis against common infections or at subtherapeutic levels in feed to promote growth is under increasing scrutiny (11). Tetracycline has a long history of use in the swine industry (17), and its use generates a strong selective pressure that has resulted in the exchange of tetracycline resistance genes associated with plasmids or transposons within and between bacterial species (13). Tetracycline resistance determinants Tet B, Tet H, and Tet M have previously been found in other members of the Pasteurellaceae (7, 9). Tetracycline-resistant isolates of H. parasuis have previously been reported in Austria (15) and Denmark (1) although the mechanism of this resistance has not been elucidated.In this study, we have identified and sequenced two native H. parasuis plasmids, one of which encoded the Tet B tetracycline resistance determinant. A survey of tetracycline resistance was undertaken in 45 H. parasuis strains, identifying tet(B) plasmidmediated tetracycline resistance in two additional H. parasuis strains. One of these tetracycline-resistant field isolates was isolated from a disease outbreak involving both H. parasuis and Actinobacillus pleuropneumoniae. Tetracycline resistance determinant tet(B) was identified in an A. pleuropneumonie and a further H. parasuis isolate from this outbreak. MATERIALS AND METHODSBacteria. Escherichia coli strains were cultured in Luria-Bertani medium (LB) with appropriate antibiotics. Strain XL1-Blue MR (Stratagene) was used in electroporations using plasmid pHS-Tet, and strain TST-1 (E. co...

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in travelers to countries where the disease is endemic and causes a major disease burden in the indigenous population, particularly children. We describe here the generation and preclinical characterization of candidate strains of ETEC which are intended to provide the basis of a live attenuated oral vaccine to prevent this disease. It has been shown previously that a spontaneously arising toxin-negative variant ETEC strain, E1392/75-2A, could confer 75% protection against challenge when administered to volunteers. Unfortunately this strain induced mild diarrhea in 15% of recipients. To eliminate the unacceptable reactogenicity of strain E1392/75-2A, it was further attenuated by introducing three different combinations of defined deletion mutations into the chromosome. A mouse intranasal model of immunization was developed and used to show that all of the strains were immunogenic. Immune responses against colonization factor antigens (CFAs) were particularly strong when the bacterial inocula were grown on "CFA agar," which induces strong expression of these antigens. Two of the strains were selected for a phase I dose escalation safety study with healthy adult volunteers. Freshly grown organisms were harvested from CFA agar plates and administered to volunteers as a suspension containing from 5 ؋ 10 7 to 5 ؋ 10 9 CFU. The vaccine was well tolerated at all doses and induced significant immune responses in all recipients at the highest dose of either strain. The results provide the basis for further clinical evaluation of these vaccine candidates.Enterotoxigenic Escherichia coli (ETEC) is a common cause of dehydrating diarrhea in developing countries and may be life threatening, particularly in weanling infants. In addition, ETEC is the predominant cause of travelers' diarrhea in adults from the developed world visiting regions where ETEC infection is endemic (29). In developing countries, the incidence of ETEC infections leading to clinical disease decreases with age, indicating that immunity to ETEC infection can be acquired and suggesting that an approach to ETEC vaccination involving a live attenuated vaccine may prove successful. In contrast, adults from industrialized countries who visit areas of endemicity are highly susceptible to ETEC infections. ETEC diarrhea is caused by colonization of the small intestine by enterotoxigenic strains of E. coli and subsequent elaboration of enterotoxins. Two types of enterotoxins have been identified in ETEC strains. The heat-labile toxin (LT) is highly homologous in structure to the cholera toxin, a multisubunit protein of the form AB 5 . The A subunit is the active component of the toxin and functions to increase the activity of adenylate cyclase. This is delivered into host cells by the B subunits, which bind to gangliosides on the cell surface. The heat-stable toxin (ST) is a small (19-amino-acid) nonimmunogenic polypeptide that has guanylate cyclase-stimulating activity. In addition, it has been ...

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