Electronic microarray assays for avian influenza and Newcastle disease virus.

Microarrays are suitable for multiplexed detection and typing of pathogens. Avian influenza virus (AIV) is currently classified into 16 H (hemagglutinin) and 9 N (neuraminidase) subtypes, whereas Newcastle disease virus (NDV) strains differ in virulence and are broadly classified into high and low pathogenicity types. In this study, three assays for detection and typing of poultry viruses were developed on an automated microarray platform: a multiplex assay for simultaneous detection of AIV and detection and pathotyping of NDV, and two separate assays for differentiating all AIV H and N subtypes. The AIV-NDV multiplex assay detected all strains in a 63 virus panel, and accurately typed all high pathogenicity NDV strains tested. A limit of detection of 10(1)-10(3) TCID(50)/mL and 200-400 EID(50)/mL was obtained for NDV and AIV, respectively. The AIV typing assays accurately typed all 41 AIV strains and a limit of detection of 4-200 EID(50)/mL was obtained. Assay validation showed that the microarray assays were generally comparable to real-time RT-PCR. However, the AIV typing microarray assays detected more positive clinical samples than the AIV matrix real-time RT-PCR, and also provided information regarding the subtype. The AIV-NDV multiplex and AIV H typing microarray assays detected mixed infections and could be useful for detection and typing of AIV and NDV.

Multiplex RT-PCR detection and microarray typing of vesicular disease viruses.

A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6TCID(50)/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.

Detection of N-acyl homoserine lactones using a traI-luxCDABE-based biosensor as a high-throughput screening tool.

BACKGROUND: Bacteria use N-acyl homoserine lactone (AHL) molecules to regulate the expression of genes in a density-dependent manner. Several biosensors have been developed and engineered to detect the presence of all types of AHLs. RESULTS: In this study, we describe the usefulness of a traI-luxCDABE-based biosensor to quickly detect AHLs from previously characterized mutants of Burkholderia cenocepacia and Pseudomonas aeruginosa in both liquid and soft-agar co-culture assays in a high-throughput manner. The technique uses a co-culture system where the strain producing the AHLs is grown simultaneously with the reporter strain. Use of this assay in liquid co-culture allows the measurement of AHL activity in real time over growth. We tested this assay with Burkholderia cenocepacia and Pseudomonas aeruginosa but it should be applicable to a broad range of gram negative species that produce AHLs. CONCLUSION: The co-culture assays described enable the detection of AHL production in both P. aeruginosa and B. cenocepacia and should be applicable to AHL analysis in other bacterial species. The high-throughput adaptation of the liquid co-culture assay could facilitate the screening of large libraries for the identification of mutants or compounds that block the synthesis or activity of AHLs.

Phylogenetic relationships of Campylobacter jejuni based on porA sequences.

Campylobacter porins are the dominant major outer membrane protein (MOMP) of these bacteria. They are composed of hypervariable, surface-exposed, peptide loops and membrane-embedded, conserved peptide regions. Porins are functionally important and may also be useful for molecular subtyping methods but have not yet been well characterized. We therefore sequenced the porA gene from 39 Campylobacter isolates, including multilocus sequence type (MLST) reference strains, isolates from patients with the Guillain-Barré syndrome, other clinical isolates, and serotyping reference strains. These were compared with additional sequences available from GenBank. Three distinct porA lineages were observed after phylogenetic analysis. Both Campylobacter coli and Campylobacter jejuni were found with group 3 porA sequences, and this was the only group showing any evidence of recombination among porA genes. There was no recombination between porA genes from C. jejuni groups 1 and 2, suggesting there may be functional constraints on changes at this locus. Most of the amino acid differences among the three groups were present in surface-exposed loops, and dissimilar substitutions were found when groups 1 and 2 MOMP were compared. Different MOMP sequence groups may have different biological or antigenic properties, which in turn may be associated with survival in different environments, host adaptation, or virulence.

pfs-dependent regulation of autoinducer 2 production in Salmonella enterica serovar Typhimurium.

Bacterial intercellular communication provides a mechanism for signal-dependent regulation of gene expression to promote coordinated population behavior. Salmonella enterica serovar Typhimurium produces a non-homoserine lactone autoinducer in exponential phase as detected by a Vibrio harveyi reporter assay for autoinducer 2 (AI-2) (M. G. Surette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046-7050, 1998). The luxS gene product mediates the production of AI-2 (M. G. Surette, M. B. Miller, and B. L. Bassler, Proc. Natl. Acad. Sci. USA 96:1639-1644, 1999). Environmental cues such as rapid growth, the presence of preferred carbon sources, low pH, and/or high osmolarity were found to influence the production of AI-2 (M. G. Surette and B. L. Bassler, Mol. Microbiol. 31:585-595, 1999). In addition to LuxS, the pfs gene product (Pfs) is required for AI-2 production, as well as S-adenosylhomocysteine (SAH) (S. Schauder, K. Shokat, M. G. Surette, and B. L. Bassler, Mol. Microbiol. 41:463-476, 2001). In bacterial cells, Pfs exhibits both 5'-methylthioadenosine (MTA) and SAH nucleosidase functions. Pfs is involved in methionine metabolism, regulating intracellular MTA and SAH levels (elevated levels of MTA and SAH are potent inhibitors of polyamine synthetases and S-adenosylmethionine dependent methyltransferase reactions, respectively). To further investigate regulation of AI-2 production in Salmonella, we constructed pfs and luxS promoter fusions to a luxCDABE reporter in a low-copy-number vector, allowing an examination of transcription of the genes in the pathway for signal synthesis. Here we report that luxS expression is constitutive but that the transcription of pfs is tightly correlated to AI-2 production in Salmonella serovar Typhimurium 14028. Neither luxS nor pfs expression appears to be regulated by AI-2. These results suggest that AI-2 production is regulated at the level of LuxS substrate availability and not at the level of luxS expression. Our results indicate that AI-2-dependent signaling is a reflection of metabolic state of the cell and not cell density.