Detecting Trichinella infections using inverse microscopy and an improved larval counting technique.

Several methods for the detection of Trichinella in meat are legally prescribed in regulation (EC) No 2075/2005, which prescribes the magnetic stirrer method for pooled sample digestion (MSM) as the reference method. However, the MSM's multistage protocol requires several preparatory steps that seem to be accountable for the loss of larvae. Here we present a modified MSM (mMSM) based on: (1) an inversion of the optical path using inverse microscopy; and (2) a modified larval counting basin (mLCB, 'Trichoview'). This enables one to examine samples of up to 40 ml and reduces the examination area from 72 to 10.3 cm2. Preparatory steps that might cause the loss of Trichinella larvae are eliminated from the new protocol. Correspondingly, the overall analytical time is reduced. In a direct and blinded comparison using 60 digest samples containing spiked vital Trichinella larvae (1-90 L1), both methods performed well for both small and large numbers of L1. However, 1278 of 1285 L1 (99.4%) were detected using the mMSM, while MSM recovered only 1225 L1 (95.3%). The improvement stems largely from samples with small numbers of L1: in all samples spiked with fewer than 10 L1, the recovery rate of mMSM was 100% compared to only 93% with MSM. Our data suggest that the use of the mMSM can improve the recovery rate by about 4% and therefore reduce the chances of a false-negative result in a sample containing 5 larvae by a factor of about 4.

Sequential pressurized liquid extraction to determine brain-originating fatty acids in meat products as markers in bovine spongiform encephalopathy risk assessment studies.

A new approach using sequential pressurized liquid extraction described recently [J. Poerschmann, R. Carlson, J. Chromatogr. A, 1127 (2006) 18-25] was applied to determine lipid markers originating from central nervous system (CNS) tissue of cows in heat-processed sausages. These studies are very important in quality control as well as risk assessment studies in the face of the bovine spongiform encephalopathy (BSE) crisis. Diagnostic CNS lipid markers, which should not be present in meat products without CNS addition, were recognized on complete transesterification as polar 2-hydroxy-fatty acids (2OH-24:0, 2OH-24:1, 2OH-22:0, 2OH-18:0, shorthand designation) as well as odd-numbered non-branched fatty acids beyond C(22). An array of other fatty acids including lignoceric acid (24:0), nervonic acid (24:1), arachidonic acid (20:4), and polyunsaturated nC(22)-surrogates are strongly related to CNS lipids, but occur as traces in meat products without CNS addition as well, thus reducing their value as diagnostic markers. Samples including meat products without CNS addition, meat with 3% CNS addition, as well as pure CNS homogenates, were subjected to sequential PLE (pressurized liquid extraction) consisting of two steps: n-hexane/acetone 9:1 (v/v) extraction at 50 degrees C to remove neutral lipids, followed by chloroform/methanol 1:4 (v/v) extraction at 110 degrees C to isolate polar CNS lipids (two 10 min PLE cycles each). To enhance the fractionation efficiency, cyanopropyl modified silica as well as chemically not modified silica sorbent was used at the outlet of the PLE cartridge to retard polar lipids in the first extraction step. This method proved superior to widely distributed exhaustive lipid extraction followed by solid-phase extraction (SPE) using silica regarding lipid recoveries and clear-cut boundaries between lipid classes. Methodological studies showed that the alcoholysis using trimethylchlorosilane/methanol (1:9, v/v) is an excellent method for the complete transesterification of lipids and quantitative formation of methyl esters.

[Brain emboli in the lungs of cattle]. / ZNS-Emboli in der Rinderlunge.

There is no information whether the BSE agent is introduced into the human food chain through contamination of the lungs of cattle with central nervous system tissue (CNS). Studies in the United Kingdom and in the USA showed that CNS tissue could contaminate the lungs after using pneumatic powered air injection stunners (e.g. "The Knocker") or after pithing. Thus, pithing was forbidden in the European Union since January 2001. In German abattoirs conventional cartridge-fired stunners (e.g. model by Schermer) are usually applied. Pithing was used up to December 2000 in approx. 75% of the German abattoirs. In the present study 323 lungs of cattle were analysed for CNS. The lungs were derived from cattle exclusive stunned by use of the knocker from Schermer. 60% of the lungs contained emboli which were tested with immuno chemistry as well as immuno histochemistry to detect CNS. Two of 108 pooled samples showed a faint immuno reaction in the anti-NSE and anti-GFAP immunoblot. Further two particles showed a faint reaction for NSE and GFAP in immuno histochemistry, thus suggesting the presence of CNS. Even though CNS tissue could not be shown in the histological investigation, we used our findings to estimate the worst case scenario for human BSE exposure risk (HER) by lung contaminated by CNS emboli. The content of CNS in the samples was estimated to be about 0.11% when the respective immuno reactions were calibrated against standards containing known brain concentrations. Under the assumption that only one lung in the pooled samples was contaminated with BSE-infected central nervous tissue, the HER was calculated to reach a maximum of 2.2 x 10(-5) CoID50/consumer after consumption of a sausage with a portion of 10% lung. The results of our study suggest that the contamination of the lung with CNS after using a conventional cartridge-fired stunner cannot be excluded, however, the incidence appears to be very low. In addition, presumed CNS emboli, if at all, are microscopically small. Furthermore the incidence of BSE in Germany is very low and lungs of cattle are usually not consumed. Thus we can judge the potential for human oral exposure after consumption of lungs of cattle which were stunned in Germany to be extremely low. A final assessment, however, is impossible as there is no knowledge about the minimum infectious dose for humans.

Brain in human nutrition and variant Creutzfeldt-Jakob disease risk (vCJD): detection of brain in retail liver sausages using cholesterol and neuron specific enolase (NSE) as markers.

No information is available about the consumption of brain via meat products. With respect to the new variant of Creutzfeldt-Jakob disease (vCJD) and the presumed food-borne transmission of bovine spongiform encephalopathy (BSE) to humans, a preliminary survey for brain and/or spinal cord (tissues of the central nervous system, CNS) was conducted. We applied a previously developed integrated procedure using cholesterol and neuron specific enolase (NSE) as markers. Quantification of cholesterol had to be backed up by NSE immunochemistry in order to account for low specificity and relatively high variances. Out of 126 high-quality finely graded liver sausages, five samples (4 %) showed positive NSE immunoresponses. In four of these samples a transgression of the normal maximum cholesterol content was obtained. The identification of such a considerable number of CNS-positive sausages indicates that brain consumption is not as rare as previously assumed. Overall, the present integrated method could be successfully applied for the detection of CNS in heat-treated meat products. Its routine application in official food control would deter illegal practice and thus help to control transmissible spongiform encephalopathies.

Identification of central nervous system tissue in retail meat products.

A procedure to detect tissues from the central nervous system that involved quantification of cholesterol and immunochemical detection of neuron-specific enolase and glial fibrillary acidic protein was used to analyze 402 samples of heat-treated meat products from various food outlets in Germany. The cholesterol content of 16 samples (4.0%) indicated the possible presence of central nervous system tissue because the levels exceeded the normal maximum cholesterol content of cooked sausages. In 7 of these 16 heat-treated meat products, immunoblotting of both neuron-specific enolase and glial fibrillary acidic protein confirmed the presence of CNS tissue. Repeated sampling by veterinary officials and analysis by both cholesterol quantification and immunoblotting confirmed these findings. Whereas all of the control samples (with and without added central nervous system tissue) were correctly classified by both cholesterol quantification and immunoblotting, negative results of immunoblotting must be carefully interpreted in the case of intensively heat-treated meat products. Thus, studies have yet to establish an increase in sensitivity of immunoblotting of neuron-specific enolase and glial fibrillary acidic protein. However, the detection of illegal use of central nervous system tissue in heat-treated retail meat products demonstrates the need for suitable analytical methods to control transmissible encephalopathies and to enforce labeling laws.

Determination of analytical limits in solid sampling ETAAS: a new approach towards the characterization of analytical quality in rapid methods.

In the present study the lower analytical limits of solid sampling electrothermal atomization atomic absorption spectrometry (SS-ETAAS) were characterized by means of blank measurements and--for the first time--by means of the calibration curve method, where a calibration near the range of these limits (limit of decision, detection and quantification) was performed. The limit of decision as derived from blank measurements was calculated according to the 3sigma-criterion to be 0.003 and 0.019 ng for Cd and Pb, respectively. For Pb and Cd a roughly three-fold increase of these limits was observed when the calibration method according to DIN 32 645 was applied. When solid reference material was used, only a slight increase could be observed. The analytical limits were 2 to 20 times lower than reported for sample decomposition methods. The blank measurement and conventional calibration curve method, however, do not account for factors relating to solid sampling such as sample mass and matrix. Therefore, the calibration curve model was applied to data derived from comparisons between direct solid sampling ETAAS and a compound reference method (ETAAS following sample homogenization and digestion). The observed analytical limits were not found to be substantially increased if enough samples with low element contents were available for calibration. Coupling of the calibration curve model with the comparison of methods included real test samples and thus the relevant maximum sample mass and analyte content in the range of the lower analytical limits. As validation procedures frequently include comparisons of methods, the present approach might prove to be of some general interest for the characterization of analytical quality in rapid methods.

Development of an integrated procedure for the detection of central nervous tissue in meat products using cholesterol and neuron-specific enolase as markers.

The emergence of a new variant of Creutzfeldt-Jakob disease during the bovine spongiform encephalopathy epidemic has focused attention on the use of tissue from the central nervous system (CNS) in food. So far, the banning of CNS tissue could not be effectively controlled because procedures for detection were missing. With regard to preventive health protection and labeling law enforcement, we have developed an integrated procedure for the detection of CNS tissue in meat products. Herein, we show that antigenic characteristics of neuron-specific enolase (NSE) quantitatively survive technological treatment including severe homogenization and pressure heating. Using both poly- and monoclonal antibodies against NSE in the Western blot, bovine and porcine brain could be detected in sausages, albeit with varying sensitivity (1 to 4%). Sensitivity was increased after reduction of fat content (30 to 40%) of the samples by means of a soxhlet extraction. This made possible the detection of brain addition as low as 0.25% when using monoclonal antibodies. Immunohistology showed distribution of CNS tissue in heat-treated meat products to be homogeneous. Immunoreaction was not found to be bound to morphologically intact histological or cytological structures; however, it proved to be highly specific. The quantification of cholesterol provides a low-cost screening method for the rapid identification of meat products, suspicious with regard to CNS tissue addition. Cholesterol content increased by 26 mg per 100 g of fresh substance for each percentage of brain added to internally produced reference material. Using three different approaches (internal reference material, raw material, and field samples), a provisional cutoff point of normal cholesterol content was calculated for emulsion-type cooked sausages to be 115 mg/100 g (P < 0.05).