Detection of Salmonella sp. in Dermanyssus gallinae using an FTA filter-based polymerase chain reaction.

Salmonella spp. bacteria are responsible for some of the most important zoonoses worldwide. Because Dermanyssus gallinae (DeGeer) (Acari: Dermanyssidae) has been recently reported to be an experimental vector of Salmonella Enteritidis, it would be of benefit to evaluate the presence of this bacterium in mites. A molecular detection tool associating a simple filter-based DNA preparation with a specific 16S rDNA Salmonella sp. polymerase chain reaction (PCR) amplification was described. The limit of detection with this method was 2 x 10(4) bacteria per mite. To adapt this technique for large-scale studies, two sizes of mite pools were tested and a preliminary investigation was carried out on mites from 16 currently or previously contaminated farms. Mites sampled from one farm of each type were positive for Salmonella, suggesting that Dermanyssus could act as a reservoir between flocks. In further investigations, it will be necessary to carry out a large-scale study to assess the role of D. gallinae in the epidemiology of avian salmonellosis.

Comparison of the VIDAS system, FTA filter-based PCR and culture on SM ID for detecting Salmonella in Dermanyssus gallinae.

AIMS: To compare different analytical methods for detecting Salmonella in Dermanyssus gallinae. METHODS AND RESULTS: The detection limit of three Salmonella detection methods [Vitek immunodiagnostic assay (VIDAS) Salmonella immuno-concentration/immunoassay, FTA filter-based PCR, and Salmonella detection and identification medium (SM ID) preceded by a pre-enrichment step] was evaluated by crushing mites in serial dilutions of pure cultures of Salmonella enterica ssp. Enterica serotype Enteritidis. Each method was then compared for its ability to detect Salmonella in artificially contaminated mites. In 105 mites artificially engorged with Salm. Enteritidis-contaminated blood, Salmonella was isolated from 68 samples of the samples cultured on SM ID and tests were positive for Salmonella using FTA filter-based PCR and VIDAS in 77 and 65 samples, respectively. Using SM ID as our reference method, specificities and sensitivities were 97% and 94% and 73% and 98.5% for VIDAS and PCR, respectively. CONCLUSIONS: Each method allowed the detection of Salmonella in contaminated mites and is usable for screening mites. PCR is more sensitive but less specific than VIDAS for detecting Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the VIDAS has been used to detect pathogens in vectors. The development of analytical methods for Salmonella detection in mites is a necessary step in the study of the role of D. gallinae as a vector of salmonellae and to check the contamination of D. gallinae in poultry facilities.

Molecular cloning and characterization of aldehyde oxidases in Arabidopsis thaliana.

Using degenerate primers designed by deduced amino acid sequences of known aldehyde oxidases (AO) from maize and bovine, two independent cDNA fragments were amplified by reverse transcription-polymerase chain reaction (PCR). The two corresponding full-length cDNAs (atAO-1 and atAO-2; 4,484 and 4,228 bp long, respectively) were cloned by screening the Arabidopsis cDNA library followed by rapid amplification of cDNA end-PCR. These cDNAs are highly homologous at both the nucleotide and amino acid sequence levels, and the deduced amino acid sequences showed high similarity with those of maize and tomato AOs. They contain consensus sequences for two iron-sulfur centers and a molybdenum cofactor (MoCo)-binding domain. In addition, another cDNA having a sequence similar to that of the cDNAs was screened (atAO-3; 3,049 bp), and a putative AO gene (AC002376) was reported on chromosome 1, which (atAO-4) was distinct from, but very similar to, the above three AOs. atAO-1, 2, 3, and 4 were physically mapped on chromosomes 5, 3, 2 and 1, respectively. These data indicate that there is an AO multigene family in Arabidopsis. atAO-1 protein was shown to be highly similar to one of the maize AOs in respect to a region thought to be involved in determination of substrate specificity, suggesting that they might encode a similar type of AO, which could efficiently oxidize indole-3-acetaldehyde to indole-3-acetic acid (IAA). atAO-1 and atAO-2 genes were expressed at higher levels in lower hypocotyls and roots of the wild-type seedlings, while atAO-3 was slightly higher in cotyledons and upper hypocotyls. The expression of atAO-1 was more abundant in the seedlings of an IAA overproducing mutant (superroot1; sur1) than in those of wild type. atAO-2 and atAO-3 transcripts were rather evenly distributed in these seedlings. A possible involvement of atAO genes in phytohormone biosynthesis in Arabidopsis is discussed.