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Current molecular techniques for the diagnosis of listeriosis
Milk and milk products serve as important source of many disease producing microbes including Listeria monocytogenes, which is a Gram-positive, motile, psychotropic bacterium, and is the principal cause of listeriosis in humans and in a wide variety of animals including birds. The disease occurs in sporadic as well as in epidemic form, following the ingestion of food contaminated by this organism. In the world, it is becoming an important food-borne bacterial disease, with low incidence but high case fatality rate. L. monocytogenes primarily affects older, pregnant women, newborns, and adults with weakened immune systems; and it has been recovered from the soil, dust, water, sewage, decaying vegetation, etc. Raw or inadequately pasteurized milk (or milk contaminated post-pasteurization), soft cheeses, ice cream and other dairy products are important sources of L. monocytogenes in humans. The disease has two forms, one febrile gastroenteritis and other inva-sive systemic disease. The control of Listeria in foods relies largely on a HACCP approach and the establishment of effective critical control points in the process. As milk and milk products are important vehicles of L. monocytogenes and clear risk factors, it is emphasized that people susceptible for acquiring listeriosis should not consume unpasteurized milk and milk products.
Background
Immunomagnetic separation (IMS) and immunoassays are widely used for pathogen detection. However, novel technology platforms with highly selective antibodies are essential to improve detection sensitivity, specificity and performance. In this study, monoclonal antibodies (MAbs) against Internalin A (InlA) and p30 were generated and used on paramagnetic beads of varying diameters for concentration, as well as on fiber-optic sensor for detection.
Results
Anti-InlA MAb-2D12 (IgG2a subclass) was specific for Listeria monocytogenes and L. ivanovii, and p30-specific MAb-3F8 (IgM) was specific for the genus Listeria. At all bacterial concentrations (103–108 CFU/mL) tested in the IMS assay; the 1-μm diameter MyOne beads had significantly higher capture efficiency (P < 0.05) than the 2.8-μm diameter M-280 beads with both antibodies. The highest capture efficiency for MyOne-2D12 (49.2% for 105 CFU/mL) was significantly higher (P < 0.05) than that of MyOne-3F8 (16.6 %) and Dynabeads anti-Listeria antibody (9 %). Furthermore, capture efficiency for MyOne-2D12 was highly specific for L. monocytogenes and L. ivanovii. Subsequently, we captured L. monocytogenes by MyOne-2D12 and MyOne-3F8 from hotdogs inoculated with mono- or co-cultures of L. monocytogenes and L. innocua (10–40 CFU/g), enriched for 18 h and detected by fiber-optic sensor and confirmed by plating, light-scattering, and qPCR assays. The detection limit for L. monocytogenes and L. ivanovii by the fiber-optic immunosensor was 3 × 102 CFU/mL using MAb-2D12 as capture and reporter antibody. Selective media plating, light-scattering, and qPCR assays confirmed the IMS and fiber-optic results.
Conclusions
IMS coupled with a fiber-optic sensor using anti-InlA MAb is highly specific for L. monocytogenes and L. ivanovii and enabled detection of these pathogens at low levels from buffer or food.
Listeria monocytogenes is a bacterial pathogen that can invade the central nervous system (CNS), causing meningoencephalitis and brain abscesses.
The diagnosis of CNS listeriosis, based on the isolation of the bacteria in the cerebrospinal fluid (CSF), can be difficult
because of previous antibiotic treatment and a low number of bacteria in the CSF. To improve the sensitivity of microbiological
diagnosis, we have developed a real-time PCR assay for detecting and quantifying L. monocytogenes DNA in the CSF. The designed primers specifically amplify the L. monocytogenes hly gene, which encodes listeriolysin O, a pore-forming cytolysin. The PCR assay for the hly gene (PCR-hly) provides reproducible quantitative results over a wide dynamic range of concentrations and was highly sensitive
while detecting a single gene copy/ml. By assaying a large panel of bacterial species, including species secreting pore-forming
cytolysin, we determined the specificity of the PCR-hly, which exclusively detects the L. monocytogenes DNA. We then analyzed 214 CSF samples from patients suspected of having CNS listeriosis. PCR-hly was positive in all cases
in which L. monocytogenes was isolated by culture. Positive PCR-hly of the CSF was also obtained for five additional, clinically confirmed cases of
CNS listeriosis for which bacterial cultures were negative presumably due to previous treatment with antibiotics. As a complement
to classical bacteriological CSF culture, our designed real-time PCR-hly assay proved to be valuable by enhancing the rapidity
and the accuracy of the diagnosis of CNS infection by L. monocytogenes. In addition, the quantitative results provided may, in some instances, be useful for the follow-up of patients under treatment.
To develop improved automated subtyping approaches forListeria monocytogenes, we characterized the discriminatory power of different restriction enzymes for ribotyping. When 15 different restriction
enzymes were used for automated ribotyping of 16 selected L. monocytogenes isolates, the restriction enzymesEcoRI, PvuII, and XhoI showed high discriminatory ability (Simpson's index of discrimination > 0.900) and produced complete and reproducible restriction
cut patterns. These three enzymes were thus evaluated for their ability to differentiate among isolates representing the two
major serotype 4b epidemic clones, those having ribotype reference pattern DUP-1038 (51 isolates) and those having pattern
DUP-1042 (20 isolates). Among these isolates, PvuII provided the highest discrimination for a single enzyme (nine different subtypes; index of discrimination = 0.518). A combination
of PvuII and XhoI showed the highest discriminatory ability (index of discrimination = 0.590) for these isolates. A group of 44 DUP-1038 isolates
and a group of 12 DUP-1042 isolates were identical to each other even when the combined data for all three enzymes were used.
We conclude that automated ribotyping using different enzymes allows improved discrimination of L. monocytogenes isolates, including epidemic serotype 4b strains. We furthermore confirm that most of the isolates representing the genotypes
linked to the two major epidemic L. monocytogenes clonal groups form two genetically homogeneous groups.
Listeria monocytogenes is an opportunistic intracellular pathogen that has become an important cause of human foodborne infections worldwide. Given its close relationship to other Listeria species and its tendency to produce non-specific clinical symptoms, the availability of rapid, sensitive and specific diagnostic tests for the differentiation of L. monocytogenes from other Listeria species is helpful for selecting appropriate treatment regimens. In addition, with L. monocytogenes comprising a diversity of strains of varying pathogenicity, the ability to precisely track the strains involved in listeriosis outbreaks and speedily determine their pathogenic potential is critical for the control and prevention of further occurrences of this deadly disease. Extensive research in recent decades has revealed significant insights regarding the molecular mechanisms of L. monocytogenes infection. This in turn has facilitated the development of laboratory procedures for enhanced detection and identification of L. monocytogenes, and has also contributed to the implementation of improved control and prevention strategies against listeriosis. The purpose of this review is to summarize recent progress in the species-specific identification, subtyping and virulence determination of L. monocytogenes strains, and to discuss future research needs pertaining to these important areas of listeriosis.
The multifaceted properties of Listeria monocytogenes allow the microorganism to grow and multiply in various food matrices even under adverse conditions. Therefore methods are needed to detect and trace this pathogen along the entire food supply network. Analytical methods have to fulfill several needs and also should meet the requirements of governmental, scientific and industrial parties. Among these demands, the level of detection based on genus and/or species or even strain specific information is of high practical significance to the food manufacturer. Hence, the release of sufficiently resolved information should be integrated into risk analysis and elucidation of contamination routes. This review aims at providing a current overview of methods for detecting, isolating and subtyping L. monocytogenes in various matrices, taking into account recent studies indicating the different drawbacks and advantages of commonly applied methods.
As part of our long-term objective of assessing risk for Listeria monocytogenes and Salmonella spp. in dairy herds, we carried out a cross-sectional study to determine the prevalence of the two organisms. The study population consisted of a sample of dairy herds enrolled in the Quality Milk Promotion Services at Cornell during the period of April 1998 to March 1999. The sample was stratified by geographical region to assure representation. Four hundred and four dairy farms were enrolled in the study. In-line milk filters were collected from each farm for bacteriological examination of L. monocytogenes and Salmonella spp. Four hypothesized risk factors were evaluated for their association with the likelihood of the presence of each of the two organisms using logistic regression analysis. Listeria monocytogenes was isolated from 51 (12.6%) of the milk filters. We found region-specific differences in the rate of farms with positive milk filters for this pathogen. Salmonella spp. were isolated from 6 (1.5%) milk filters. One isolate was confirmed as Salmonella enterica Serotype Typhimurium DT 104. There was no significant association between any of the hypothetical risk factors and the likelihood of Salmonella spp. isolation. Our study demonstrated that both L. monocytogenes and Salmonella spp. were prevalent in milk filters in New York dairy herds and that Salmonella was isolated at a significantly lower rate then L. monocytogenes.
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