Alliance for Food Protection

NON-TECHNICAL SUMMARY: Studies will address (1) a DNA-based method to identify virulent L. monocytogenes isolates, (2) the influence of high fat in foods on the infectious dose of L. monocytogenes, and (3) disinfectants to kill foodborne parasites.

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APPROACH: Studies to develop a microarray chip for identifying L. monocytogenes isolates will focus on designing DNA probes that are genus and species specific and detects specific virulence factors. Studies on the influence of fat content of foods on virulence of L. monocytogenes will evaluate the presence of different fat contents of milk products on the infectious dose of different Listeria isolates in mice. The third study will determine the effect of sodium hypochlorite, hydrogen peroxide, peracetic acid, glutaraldohyde and ammonia on viability of Cryptosporidium and Cyclospora oocysts.
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PROGRESS: 2003/08 TO 2005/07<BR>
A DNA microarray chip was developed that can differentiate and identify Listeria species, including L. monocytogenes, as well as subtype L. monocytogenes isolates. A redundant and hierarchically structured set of oligonucleotide probes (17-37 mer) targeting five genes (16S rRNA, iap, gltA, gltB, inlB) was designed, synthesized and spotted onto epoxy-derivatized glass slides. Target DNA was amplified from purified genomic DNA via asymmetric polymerase chain reaction (PCR). Results from hybridization of the chip with target DNA from 18 L. monocytogenes strains for which serotype and genotype data were available have shown the array's ability to unambiguously serotype and genotype. The addition of 12-mer spacer molecules significantly increased the intensity of hybridization signals. Mice were perorally administered L. monocytogenes in skim milk, Half & Half or whipping cream to determine whether fat content influences the ability of L. monocytogenes to survive passage through the gut and infect the liver or spleen. The number of fecal samples positive for L. monocytogenes increased with increasing dosage of L. monocytogenes for all three milk products of different fat content. The percentage of mouse livers and spleens L. monocytogenes-positive for each dosage when L. monocytogenes was administered in one of the three vehicles was not significantly different. The dosages necessary for eliciting 50% infection (EC50) were 3.6 x 10to4, 2.0 x 10to4 and 1.3 x 10to4 cfu when administered in whipping cream, Half & Half or skim milk, respectively. These EC50s were not significantly different. Hence, infectivity in mice four days after oral exposure to L. monocytogenes does not appear to be influenced by milk fat content of the delivery vehicle at any dose level within a very broad range. Cryptosporidium parvum oocysts were rendered non-infectious by 1 minute exposure to 5% ammonium hydroxide, 5% hydrogen peroxide, or 5% peracetic acid. Sporulation of Cyclospora oocysts was reduced by ca. 50% when treated with 10% ammonium hydroxide for 1 to 30 min; however, all other treatments, including 70% ethanol, 50% gluteraldehyde, 30% hydrogen peroxide, 70% isopropanol, 35% peracetic acid, or 5% sodium hypochlorite, for 1 to 30 minutes reduced sporulation by ca. 25% or less. No patents or inventions resulted from this grant.
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IMPACT: 2003/08 TO 2005/07<BR>
The microarray chip should be useful for rapid identification and subtyping of Listeria. Animal studies with Listeria may help determine if high fat foods are of higher risk for transmitting listeriosis than low fat foods. Practical chemical treatments will be identified for disinfecting foods contaminated with foodborne parasites.