Rapid testing and quantification of Salmonella in ileocaecal lymph nodes of Austrian pigs slaughtered for consumption.

Traditionally, quantitative microbial risk assessment (QMRA) is based on culture-dependent technologies. However, molecular quantification could forge additional, detailed information. A prerequisite of quantitative real-time PCR in animal science is a tissue preparation method where large volumes of tissue material can be reduced and particularly target cells can be concentrated. An easy-to-use sample preparation method for food (Matrix-Lysis) was recently adapted to tissues and now permits quantification of target cells from up to 5 g of organic matrix. The aim of this study was to examine the suitability of Matrix-Lysis for quantification of Salmonella in porcine ileocaecal lymph nodes (ICLNs). After demonstrating constant recovery rates, ICLNs from 540 pigs were examined for Salmonella spp. with Matrix-Lysis. Samples were also analysed using ISO 6579:2002, a combined enrichment/qPCR method and a lateral flow test. It could be shown that qPCR coupled with Matrix-Lysis can contribute to QMRA in food safety by enabling reproducible quantitative data, even at low contamination rates.

Fluorescence spectroscopy of the retina from scrapie-infected mice.

Recently, we have proposed that the fluorescence spectra of sheep retina can be well correlated with the presence or absence of scrapie. Scrapie is the most widespread TSE (transmissible spongiform encephalopathy) affecting sheep and goats worldwide. Mice eyes have been previously reported as a model system to study age-related accumulation of lipofuscin, which has been investigated by monitoring the increasing fluorescence with age covering its entire life span. The current work aims at developing mice retina as a convenient model system to diagnose scrapie and other fatal TSE diseases in animals such as sheep and cows. The objective of the research reported here was to determine whether the spectral features are conserved between two different species namely mice and sheep, and whether an appropriate small animal model system could be identified for diagnosis of scrapie based on the fluorescence intensity in retina. The results were consistent with the previous reports on fluorescence studies of healthy and scrapie-infected retina of sheep. The fluorescence from the retinas of scrapie-infected sheep was significantly more intense and showed more heterogeneity than that from the retinas of uninfected mice. Although the structural characteristics of fluorescence spectra of scrapie-infected sheep and mice eyes are slightly different, more importantly, murine retinas reflect the enhancement of fluorescence intensity upon infecting the mice with scrapie, which is consistent with the observations in sheep eyes.

A comparison of the fluorescence spectra of murine and bovine central nervous system and other tissues.

We describe a comparison of the fluorescence spectra of bovine tissues with murine tissues in order to determine whether spectral features are conserved and whether an appropriate and practical laboratory small animal model system could be identified to be used for investigation of tissue- and age-related fluorescence signal patterns. Recently it has been shown that spectral signatures of lipofuscin have enabled the detection of bovine central nervous system (CNS) tissue in meat products with high sensitivity (Schönenbrücher, H., Adhikary, R., Mukherjee, P., Casey, T.A., Rasmussen, M.A., Maistrovich, F.D., Hamir, A.N., Kehrli, M.J., Richt, J., Petrich, J.W. [2008] J Agric Food Chem56, 6220-6226). We report that brain and spinal cord of mice provide fluorescence spectra similar to those of bovine brain and spinal cord. It is concluded that murine CNS tissue is an appropriate model system for bovine CNS tissue for the development of fluorometric CNS detection assays.

Foodproof Salmonella Detection Kit. Performance Tested Method 120301.

The foodproof Salmonella Detection Kit was previously validated in the Performance Tested Methods program for the detection of Salmonella species in a variety of foods, including milk powder, egg powder, coconut, cocoa powder, chicken breast, minced meat, sliced sausage, sausage, smoked fish, pasta, white pepper, cumin, dough, wet pet food, dry pet food, ice cream, watermelon, sliced cabbage, food dye, and milk chocolate. The method was shown to be equivalent to the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) and the U.S. Department of Agriculture, Food Safety and Inspection Service's Microbiology Laboratory Guidebook reference culture procedures. In the first Emergency Response Validation (ERV) extension study, peanut butter was inoculated with S. enterica. ser Typhimurium. For the low inoculation level (1.08 CFU/25 g), a Chi-square value of 2.25 indicated that there was no significant performance difference between the foodproof Salmonella Detection Kit and the FDA-BAM reference method. For high-level inoculation (11.5 CFU/25 g) and uninoculated control, there was 100% agreement between the methods. In the second ERV extension study, peanut butter was inoculated with S. enterica. ser Typhimurium. For both inoculation levels (0.1 and 0.5 CFU/25 g by most probable number), Chi-square values of 0 indicated that there was no significant performance difference between foodproof Salmonella Detection Kit and the FDA-BAM reference method.

Singlepath Salmonella. Performance Tested Method 060401.

Singlepath Salmonella is an immunochromatographic (lateral flow) assay for the presumptive qualitative detection of Salmonella spp. in food. A previous AOAC Performance Tested Method study evaluated Singlepath Salmonella as an effective method for the detection of Salmonella spp. in the following selected foods: dried skimmed milk, black pepper, dried pet food, desiccated coconut, cooked peeled frozen prawns, raw ground beef, and raw ground turkey. In this Emergency Response Validation extension, creamy peanut butter was inoculated with S. enterica. ser. Typhimurium. For low contamination level (1.08 CFU/25 g), a Chi-square value of 0.5 indicated that there was no significant difference between Singlepath Salmonella and the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) reference method. For high-level and uninoculated control there was 100% agreement between the methods.

Monitoring the accumulation of lipofuscin in aging murine eyes by fluorescence spectroscopy.

The integrated fluorescence of murine eyes is collected as a function of age. This fluorescence is attributed to pigments generally referred to as lipofuscin and is observed to increase with age. No difference in fluorescence intensity is observed between the eyes of males or females. This work provides a benchmark for further studies that are planned in order to use such signatures as markers of central nervous system (CNS) tissue or even of diseased CNS tissue and provides a basis for determining the age of a healthy animal.

Comparative studies of a real-time PCR method and three enzyme-linked immunosorbent assays for the detection of central nervous system tissues in meat products.

The removal of certain central nervous system (CNS) tissues (part of the bovine spongiform encephalopathy risk material) from the food chain is one of the highest priority tasks associated with avoiding contamination of the human food chain with the agent of bovine spongiform encephalopathy. A recently developed real-time PCR assay and three commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of CNS tissues in minced meat and three types of heat-treated sausages were evaluated. Bovine brain was used for spiking of internal reference material, and its detectability was examined during storage times of 12 months (for frozen minced meat and liver sausage) and 24 months (for sausages treated with medium and high heat). The real-time PCR method and both ELISA kits detected 0.1% CNS tissue in frozen minced meat and 0.1 or 1% CNS tissue in heat-treated meat products. The detectability of the amplified mRNA target region with the PCR assay was similar to the detectability of antigen by the ELISAs. Because the real-time PCR method also can be used to distinguish cattle, ovine, and caprine CNS tissues from porcine CNS tissues, it seems to be suitable as a routine diagnostic test for the sensitive and specific detection of CNS tissues in meat and meat products.

Fluorescence-based method, exploiting lipofuscin, for real-time detection of central nervous system tissues on bovine carcasses.

The removal of central nervous system (CNS) tissues as part of bovine spongiform encephalopathy (BSE) risk material is one of the highest priority tasks to avoid contamination of the human food chain with BSE. No currently available method enables the real-time detection of possible CNS tissue contamination on carcasses during slaughter. The fluorescent pigment lipofuscin is a heterogeneous, high-molecular weight material that has been shown to be enriched in high concentrations in neuronal tissues. In this study, lipofuscin fluorescence was investigated as a marker for real-time detection of CNS contamination. Front-faced fluorescence spectra of brain and spinal cord samples from 11 cattle gave identical, reproducible fluorescence signal patterns with high intensities. The specificity of these spectra was assessed by investigating 13 different non-CNS tissues enabling the differentiation of brain and spinal cord by signal intensity and structure of the spectra, respectively. Small quantities of bovine spinal cord were reliably detected in the presence of raw bovine skeletal muscle, fat, and vertebrae. The presented data are a fundamental basis for the development of a prototype device allowing real-time monitoring of CNS tissue contamination on bovine carcasses and meat cuts.

Detection of central nervous system tissues in meat products: validation and standardization of a real-time PCR-based detection system.

Several phenotypic as well as genotypic methods have been published describing the detection of central nervous system (CNS) tissues that are part of the bovine spongiform encephalopathy (BSE) risk material in food products. However, none of these methods is able to differentiate between CNS tissue of the banned ruminant species and tissues of other animal species. A quantitative and species-specific real-time RT-PCR method has been developed that enables the reliable identification of CNS tissues in meat and meat products. This method is based on a messenger (m)RNA assay that uses bovine, ovine and caprine glial fibrillary acidic protein (GFAP) encoding gene sequences as markers. The in-house validation studies evaluated the tissue specificity of up to 15 bovine tissues and the standardization of absolute as well as relative quantitative measurement. The specific amplification of spinal cord and brain tissue GFAP cDNA has been shown previously. In addition, two commercially available ELISA kits were used for the comparative analysis of artificially contaminated minced meat. Small quantities of bovine brain that had been stored over the recommended period of 14 days were examined. The real-time PCR method proved to be suitable for the detection of 0.1% CNS tissue. No false negative results were observed. The quantitative detection of GFAP mRNA using real-time RT-PCR seems a suitable tool in routine diagnostic testing that assesses the illegal use of CNS tissue in meat and meat products. The stability of the selected target region of the GFAP mRNA also allows the detection of CNS tissues after the meat has been processed.

Collaborative trial for validation of a real-time reverse transcriptase-polymerase chain reaction assay for detection of central nervous system tissues as bovine spongiform encephalopathy risk material: part 1.

A collaborative trial was conducted to evaluate a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of central nervous system (CNS) tissues in meat products (e.g., sausages). The method is based on the detection of ruminant glial fibrillary acidic protein (GFAP) mRNA by applying real-time RT-PCR. The assay was evaluated through a multicenter trial involving 12 participating laboratories that received coded cDNA obtained from 3 different types of sausages. The participants used 5 different real-time detection systems. The results obtained in this validation revealed that this real-time RT-PCR assay performed well in the different laboratories with a detection limit of at least 0.1% CNS in those test materials that contained strongly heat-treated samples (sausages cooked at 120 degrees C) and the medium heat-treated samples (sausages cooked at 80 degrees C). The detection limit of liver sausages was determined to be 0.2% of CNS. Neither the samples with no CNS additive nor the bovine DNA and the negative control containing 100% swine brain gave any positive signals. The presented results indicate that the real-time RT-PCR assay was just as reproducible between laboratories, as repeatable within a laboratory, could reliably be used for detection of bovine spongiform encephalopathy risk material in meat and meat products, and signify that it may be used with confidence in any laboratory.

[From farm to fork--Mycobacterium avium ssp. paratuberculosis (MAP) as zoonotic agent?]. / From farm to fork--Mycobacterium avium ssp. paratuberculosis (MAP) als Zoonoseerreger?

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of the paratuberculosis (Para Tb) in ruminants. In addition, this pathogen has been suspected to be implicated in the pathogenesis of Morbus Crohn disease (MC), causing chronic inflammatory intestine changes of humans. The participation of MAP in this illness is discussed intensively and has very contradictory opinions. On the one hand several times succeeded in proving MAP DNA in changed human tissues as well as, in recent time, the bacteria has been isolated from patient's blood. On the other hand there are many publications which support the opposite opinion. In critical evaluation of already available data, therefore the hypothesis can be formulated that MAP could possibly take part in the MC of humans. The reliable verification of this hypothesis will only be possible, if the diagnostic procedures can be refined upon the substantial deficit concerning the sensitivity and/or specificity of the diagnostic methods. In addition, till now there is lack of optimized statistically case control studies. The conceivable transmission of the bacteria to humans by the direct animal contact has been considered as possible vector, furthermore, MAP has been detected in pasteurised milk and other food of animal origin. The prevalence data, usually estimated by ELISA for milk cattle stock show over 80% prevalence in many counties of the Federal Republic of Germany with an individual case prevalence ranging between 1% and 17% in different stocks. Comparable data are present also from other countries as well as for small ruminants. MAP has been concerned as a global problem, moreover the high spreading rate of MAP in wild animal populations as well as the considerable ability of the bacteria to survive in different stages of the infectious- and contamination-cycle, which might hardly be broken through. Thus it requires intensive research efforts for the development of the methodical diagnostic process as basis for valid epidemiological investigations of animals, humans and food.

Novel molecular method for detection of bovine-specific central nervous system tissues as bovine spongiform encephalopathy risk material in meat and meat products.

The emergence of a new variant of Creutzfeldt-Jacob disease during the bovine spongiform encephalopathy epidemic has focused attention on the use of tissues from the central nervous system (CNS) in food. For efficient consumer protection, European legislation prohibits several bovine tissues, encompassing mainly the central nervous system, from the food chain. A quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was designed to identify bovine spongiform encephalopathy risk material in meat and meat products. This was based on an mRNA assay that used bovine, ovine, and caprine glial fibrillary acidic protein (GFAP) encoding gene sequences as a marker. The real-time RT-PCR assay allowed the detection of bovine, ovine, or caprine CNS tissues in meat and meat products. Bovine brain at a concentration of 0.01% yielded a positive PCR reaction. The real-time RT-PCR assay included a housekeeping gene as an endogenous control. The detection was not affected by heat treatment of the meat products. The quantitative real-time RT-PCR detection of GFAP mRNA appeared to be useful as a routine diagnostic test for the detection of illegal use of CNS tissues in meat and meat products. The stability of the specific region of GFAP mRNA also allows the detection of CNS tissues after meat processing steps. The use of organ- and species-specific subunits of mRNA might be a promising approach for the detection of other banned tissues.