Distribution of Alaria spp. mesocercariae in waterfrogs.

The distribution of Alaria-spp.-mesocercariae within the host is relevant for the examination via Alaria spp. mesocercariae migration technique (AMT) regarding predilection sites and may indicate an interaction between parasite and host. Naturally Alaria-exposed frogs of Pelophylax species (n = 13) were examined for systemic distribution and localization-specific parasite density of Alaria spp. mesocercariae. The frogs were necropsied and their body was divided into the following localizations: inner organs, head, torso, forelimbs, and hind limbs. The localizations were analyzed individually and in toto using Alaria spp. mesocercariae migration technique. Our results showed neither statistical differences concerning the number of mesocercariae in the different localizations nor in respect of the rate of positive localizations. Therefore, an accumulation in a particular predilection site seems unlikely. Further research on a representative sample is necessary before final conclusions can be drawn.

Slaughter of pregnant cattle in German abattoirs--current situation and prevalence: a cross-sectional study.

BACKGROUND: The slaughter of pregnant cattle and the fate of the foetuses are relatively new subjects in the field of animal welfare. The Scientific Committee on Veterinary Measures relating to Public Health (SCVPH), however, does not believe this topic to be a critical issue because of the hitherto supposed rare occurrence of this practice. Some previous studies though, contradict this assessment, emphasising its relevance to animal welfare. With regard to the heterogeneous study design of previous investigations, the objective of this study is to evaluate the current situation concerning the slaughter of pregnant cattle in different German abattoirs. Additionally, the prevalence was assessed semi-quantitatively on the basis of a cross-sectional, voluntary and anonymous survey that was conducted amongst senior veterinary students of the University of Leipzig from 2010 until 2013. RESULTS: Of 255 evaluable questionnaires, 157 (63.6%) mention the slaughter of pregnant cattle, corresponding to 76.9% of all visited abattoirs. Slaughter of pregnant cattle is reported often (>10% of females) in 6 (3.8%), frequently (1-10% of females) in 56 (35.7%), and rarely (<1% of females) in 95 (60.5%) of all cases (n = 157) respectively. About 50% of these animals were reported to be in the second or third stage of gestation. 15 (10.6%) of 142 questionnaires providing information about the foetus, state that the foetus showed visible vital signs after the death of the mother, but in one case the foetus was euthanized subsequently. CONCLUSIONS: The results show that the slaughter of pregnant cattle is a common and widespread practice in German abattoirs. The SCVPH's assumption that pregnant cattle are only slaughtered in rare exceptional cases can no longer be maintained. The high proportion of foetuses in the second and third gestational stage must also be considered. In this context the implementation of suitable studies and detailed analysis of the current situation is indispensable to ensure the high standards in animal welfare in Germany and Europe.

Determination of impact parameters and efficiency of 6.8/15 caliber captive bolt guns.

While the morphological appearance of injuries due to powder-actuated captive bolt stunners has been extensively investigated, medicolegal literature contains, except for one work by Nadjem and Pollak (Arch Kriminol 203:91-102), no further investigations into the physical impact characteristics of these sharp-edged circular punching tools. However, basic physical parameters, such as bolt velocity, momentum, kinetic energy, and energy density, play a crucial role in the medicolegal and traumatological assessment of captive bolt stunners and the related injuries. And also, regulatory bodies demand a reliable and repeatable measurement test set-up for the determination of captive bolt stunners' impact characteristics. Therefore, it is the aim of this work to design and describe a test set-up based on one single photoelectric light barrier and to determine the impact parameters for a series of newly developed cal. 6.8/15 stunning devices. We found that bolt velocity ranges from v = 42 to 54 m/s, while momentum ranges from p = 11 to 14 Ns, and kinetic energy reaches values from E = 224 to 369 J. The efficiency of the captive bolt stunner, defined as the ratio of the kinetic energy of the stunner's bolt to the potential energy of industrial blank cartridges, also described in this work for the first time, was found to vary between 36 and 46 %.

Assessing laboratory performance in Trichinella ring trials.

Trichinosis (Trichinellosis) is a zoonotic disease acquired by eating raw or not adequately processed pork or wild game infected with the larvae of the roundworm genus Trichinella. According to European regulations, animals susceptible to Trichinella have to be examined for infestation. To evaluate the performance of laboratories in Germany, inter-laboratory comparisons known as "ring trials" were introduced by the Federal Institute for Risk Assessment in 2004. The current method of analysis makes use of tolerance zones based on the number of larvae in the sample, but does not permit one to determine if a given lab can detect an infested sample reliably, as required by the quality assurance recommendations of the International Commission on Trichinellosis (ICT). A new way of analysing the ring trial data is presented here, which is based on Bayesian hierarchical models. The model implements the ICT requirement by providing an estimate for the probability that a given lab would fail to detect a sample containing, say, five larvae. When applied to the 87 labs that participated in Germany's 2009 ring trials, it turns out this probability is greater than 10% for 21 of them, although only 10 of these in fact returned a false negative result. Such a new method is required to abide by the ICT requirements and make ring trials effective.

First detection of Alaria alata mesocercariae in wild boars (Sus scrofa Linnaeus, 1758) from Bulgaria.

The trematode Alaria alata, an intestinal parasite of different carnivore species is widely distributed throughout Europe. The mesocercarial stages of Alaria spp. may infect almost all vertebrate species, including humans, and, in particular, omnivorous scavengers such as wild boars serve as paratenic hosts for the parasite. The introduction of the A. alata mesocercariae migration technique (AMT) opened the way to a reliable detection of Alaria spp. mesocercariae in different body tissues of their paratenic hosts. For the first time, it was possible to detect vital A. alata mesocercariae from two Bulgarian wild boars by means of this new method. In addition, polymerase chain reaction (PCR) examination of the respective parasitic DNA allowed the unequivocal species identification of the parasites as A. alata. Isolation and molecular biological identification of the parasite's developmental stages make significant contributions to completion of data on both the distribution of Alaria spp. in stocks of European game and the relationship between different Eurasian Alaria spp. isolates.

First interlaboratory test for the detection of Alaria spp. mesocercariae in meat samples using the Alaria spp. mesocercariae migration technique (AMT).

Emerging and re-emerging zoonotic diseases affect both public and animal health and require the development and contemporary implementation of suitable detection methods. A growing number of findings of the mesocercarial stage of the digenean trematode Alaria alata in game inhabiting wetlands have necessitated the development of a specific detection method. With the Alaria spp. mesocercariae migration technique (AMT), a specific and sensitive detection method is now available. To make the method accessible to the official controls, method validation is necessary. In this context, interlaboratory tests (IT) are a key factor to demonstrate both (1) the suitability of the respective method and (2) the reference materials. In the first IT performed on this issue, 15 laboratories from nine German federal states took part. Every lab received two negative and four positive standard samples each as well as a standardized examination device for AMT, and a standard operating procedure. All participating laboratories showed very good results in terms of qualitative analysis: 96.7 % of the samples were assessed correctly positive or negative. An analysis of the qualitative performance shows that 263 (58.4 %) of 450 mesocercariae that were inserted in the meatballs were identified by the participants, and 5 (33.3 %) out of 15 labs were able to count at least 70 % of the Alaria spp. mesocercariae. A direct comparison with the results of the German Trichinella IT, which were conducted since 2004, shows that the overall sensitivity of the AMT is even higher than that registered for the reference method for Trichinella detection (e.g. 93 % in 2010). Also, in terms of quantitative analysis, AMT stands up to the comparison with the results from the German Trichinella IT. The refinement of the implementation protocol of this innovative, easy-to-use and cost-effective method harbours great potential for further optimization and successful implementation in the official controls.

Alaria alata mesocercariae in raccoons (Procyon lotor) in Germany.

Alaria alata is a trematode of carnivores from Europe. The mesocercarial stage was recently identified in wild boar meat from Europe. Previous histopathologic studies showed the presence of unidentified parasitic cysts within the tongues of raccoons from northern Germany. For identification of the parasite species, tissue samples of 105 raccoons originating from a National Park in northern Germany and from Berlin metropolitan area were collected. Histological examination of cryotome sections of frozen as well as paraffin-embedded tongues were used to identify parasite cysts. These were located in the connective and adipose tissue and in close proximity to small arterioles, suggesting a hematogenous spread of the parasite. Often, cysts were surrounded with mild infiltration by inflammatory cells. Additionally, mesocercariae were isolated from defrosted tongue samples of 11 raccoons. Molecular-biology assays confirmed the parasite species as A. alata. Except for one positive raccoon from Berlin City, all other positive raccoons originated from the sylvan Müritz National park, indicating an abundance of intermediate hosts in this area. Our results show that raccoons can act as paratenic hosts for A. alata and extend the broad host range of this parasite to a species introduced into Germany.

Alaria alata in wild boars (Sus scrofa, Linnaeus, 1758) in the eastern parts of Germany.

Over the last decade, incidental findings of Alaria alata in stocks of German wild boar during the official Trichinella inspection have been increased. As early as 2006, the German Federal Institute for Risk Assessment pointed out the possible health risk to the consumer posed by this trematode. However, at that time, reliable data concerning the prevalence of the parasite in German wild boars and feral pigs were lacking especially because no appropriate detection method was available. The development of the A. alata mesocercariae technique (Riehn et al., Parasitol Res 107(1):213-220, 2010) now makes it possible to close the remaining gaps in knowledge in this field. Over a 2-year period, 286 retained samples of fresh meat from wild boars originating from different hunting areas in Brandenburg and Saxony-Anhalt, which were tested negative for A. alata during the official Trichinella inspection in the competent veterinary inspection offices, were reexamined with the A. alata mesocercariae migration technique (AMT). In 33 out of 286 retained meat samples (11.5%) with a preliminary negative report, the trematode was demonstrated during the follow-up examination using AMT. This result especially in connection with the highly heterogeneous distribution of the parasite within the hosts' body (Riehn et al., Parasitol Res 107(1):213-220, 2010; Moehl et al., Parasitol Res 105(1):1-15, 2009) shows clearly that a high number of unreported cases of alariosis in wild boars have to be assumed.

Development of a PCR approach for differentiation of Alaria spp. mesocercariae.

To date classification and differentiation of Alaria spp. are based largely on external characteristics and comparative morphology of adult flukes. The accurate differentiation between various Alaria spp. mesocercariae is indeed difficult because there are only few data on morphological and morphometrical features of the parasite's developmental stages. We established a conventional polymerase chain reaction (PCR) for a molecular-based diagnosis of Alaria alata mesocercariae that can aid in their identification. Twenty Alaria spp. mesocercariae specimens were collected from three different wild boars originating from different areas of eastern Germany. DNA from the prepared isolates was extracted, and a primer pair was selected to amplify a 303-bp region of the A. alata genome. The DNA preparations extracted from the field samples as well as A. alata positive controls were successfully amplified and yielded a single sharp band of the expected size. In all samples, molecular identification was consistent with morphological identification. With our new PCR assay, we present the first approach for identification and characterization of A. alata mesocercariae specimens using molecular methods. This practicable and reproducible protocol can be used for both diagnostic and epidemiological purposes.

GC/MS detection of central nervous tissue as specified BSE risk material in meat products and meat and bone meals: thermal stability of markers in comparison with immunochemistry and RT-PCR.

Methods for the detection of central nervous tissue (CNT) are urgently needed in food control as a means for controlling strict adherence to both food labeling and banning of specified BSE risk material. Here, we report data on heat stability of the CNT markers neuron-specific enolase (NSE) in western blotting, glial fibrillary acidic protein (GFAP) in an enzyme linked immunoassay, mRNA(GFAP) in a real-time PCR assay, and several fatty acids (C22:6, C24:0-OH, C24:1ω9/ω7, C24:1ω9-OH/ω7-OH, and C24:0) in gas chromatography mass spectrometry (GC/MS). The sample matrix, a standard material of emulsion-type sausage with varied contents of CNT (brain), was heat-treated in three studies: (1) routine meat technological heat treatment with low (85 °C, 30 min), medium (115 °C, 30 min), and high (133 °C, 30 min, 3 bar) heating of 72 anonymous samples from a blind trial; (2) heat treatment under experimental conditions (100, 110, …, 200 °C, 45 min); and (3) fractionized heating of central nervous system (up to three times) under moderate routine technological conditions (85, 100, and 115 °C, 30 min). The markers of the immunochemical methods showed a low GFAP or very low NSE temperature stability at medium and high temperature conditions. The real-time PCR assay gave inconsistent, non-quantitative results, which indicated an uncontrollable matrix effect. The relevant GC/MS markers (C24:0-OH, C24:1ω9/ω7, and C24:1ω9-OH/ω7-OH) proved to be extremely stable. Neither meat and bone meal conditions (133 °C) nor experimental heating (up to and above 140 °C) showed any reduction of GC/MS CNT quantification. On the contrary, a slight but significant increase was noted over a certain temperature range (120-140 °C) for most fatty acids, possibly due to an improved extractability of the fatty acids. We conclude that a quantitative approach is highly unreliable when using immunochemical methods; moreover, these methods might be basically prone to false-negative results depending on heat treatment and matrix composition. Therefore, antibodies with higher affinity to heat-treated CNT marker epitopes are needed. Relevant amounts of CNT (≥0.5%) in low- and medium-heated products would still be reliably detectable by the GFAP ELISA, which justifies its use as a screening method in official food control. The results obtained by the real-time PCR assay were contradictory to recently published data, indicating a need for further protocol optimization and collaborative trials. Up to date, the analytical approach using GC/MS is the only valid procedure as pertaining to heat stability and quantitative analysis; consequently, it should be recommended as the reference procedure in official food control for CNT detection in heat-treated meat products.

GC/MS detection of central nervous tissues as specified BSE risk material in meat products: validation by an externally controlled blind trial.

The addition of central nervous tissues (CNT), such as brain and spinal cord, in the manufacturing of meat products is either forbidden--if the material falls under the legal definition of specified risk material (SRM)--or must be labelled on the packed product. To foster official food control, several CNT detection methods were developed, but only fatty acid patterns as detected by gas chromatography/mass spectrometry (GC/MS) allow the further characterization of the detected CNT as to both the animal species and--surprisingly--the age of the animal from which the CNT was derived in accordance with the legal definition. Complementing a previous report in this journal by Lücker et al. 2010 (doi: 10.1007/s00216-010-3956-5) on CNT quantification by GC/MS, we now report results of the validation of this new analytical approach by an externally controlled blind trial elucidating its potential to identify the species and age of the CNT detected. The 72 samples (24 standards of emulsion-type sausage, each heated in three different batches: 75°C, 30 min; 115°C, 25 min; 133°C, 40 min) containing porcine, ovine or bovine muscle tissue and differing amounts of CNT (bovine or ovine brain: 0, 0.1, 0.5, 1.0, 3.0% m/m) were produced externally and provided blind for analyses to our laboratory. In accordance with the previous study, heating had no detectable effect on the GC/MS analysis. Judged by the present sensitivity of this method (cut-off 0.2% CNT), all of the samples containing 0.5% or more CNT (n = 57, 100%) were identified correctly as CNT-positive. The CNT species was identified correctly in 54 samples (94.7%), with three samples of one specific standard (0.5% ovine CNT) falsely classified as bovine CNT. However, the CNT age of these samples was correctly classified (more than 12 months). Overall, 57 samples (100%) were correctly classified as SRM-positive and 15 samples (100%) as SRM-negative. To the best of our knowledge, this is the first time that a legal demand for the (1) detection of traces of a specific tissue in a food matrix, (2) the identification of its taxonomic origin and (3) the classification of its age has been shown to be analytically possible in totally blind samples. The very positive validation results of this externally controlled blind trial recommend the present GC/MS approach for the detection of CNT in meat products as a reference method. However, our results also demonstrate the need for further studies, in particular to increase sensitivity and to conduct ring trials including more than one laboratory.

A novel detection method for Alaria alata mesocercariae in meat.

Distomum musculorum suis (DMS), the mesocercarial stage of the trematode Alaria alata, can cause severe damages within their hosts, and since several reports about cases of human larval alariosis have been published, it became apparent that infected game animals and in particular wild boars are a potential source of infection for both humans and animals. A final statement concerning the health risks for consumers could not be given due to the lack of information about both the prevalence of DMS and the suitability of Trichinella inspection methods to detect this parasite in wild boar meat. Our studies concentrate on (1) the verification of suitability of the official digestion methods for Trichinella spp. for DMS detection in wild boars, (2) development, optimization, and validation of methods, and (3) the distribution of the parasites within their paratenic hosts. A total of 868 individual samples/digests from 48 wild boars were analyzed by the reference method for Trichinella detection in meat samples according to regulation (EC) No. 2075/2005. In addition to the official protocol, a method modification with Pankreatin(c) and bile acid was applied for analysis of adipose tissue samples (n = 89). On the basis of our results, a new detection method based on a larvae migration technique was developed and used for detection of DMS in 574 single samples. Furthermore, the distribution patterns of DMS in wild boars in a total of 1377 single sample migrations/digestions from 35 positive animals were analyzed by application of all three methods. The official digestion method for Trichinella spp. in wild boars meat is inapplicable for the detection of A. alata mesocercariae as it shows shortcomings in both digestion and sampling. A direct comparison between the newly developed A. alata mesocercariae migration technique and the official digestion method for Trichinella spp. based on 574 single samples from 18 animals clearly shows that the sensitivity to detect A. alata developmental stages in tissues of wild boars of the new method is nearly 60% higher compared with the magnetic stirrer method for pooled sample digestion as laid down in regulation (EC) No. 2075/2005. Among other advantages, this method offers a simple, highly applicable, fast, and cost effective way to detect DMS in wild boars which is already applicable in routine veterinary inspection.

Suitability of antigens PGP 9.5 and neurofilament light as marker proteins for detection of neuronal tissue in processed meat products.

The enforcement of rules for food labeling and quantitative ingredient declaration presupposes appropriate test systems. Additionally, central nervous system (CNS) tissue of ruminants is classified as specified risk material for the transmission of prion diseases, and its detection is needed to support the specified risk material ban. Existing antibody-based test systems are hampered by relatively high limits of detection and susceptibility to food processing conditions. For that reason we tested a broad panel of commercially available monoclonal antibodies to identify marker antigens appropriate for the development of a sensitive test system. Western blot analysis using organ-specific samples from cow, pig, and chicken and differently processed meat products containing defined amounts of CNS tissue revealed neurofilament light (NF-L) and protein gene product 9.5 (PGP 9.5) as suitable antigens for the organ-specific and sensitive detection of porcine and bovine CNS tissue. None of the tested PGP 9.5 antibodies displayed cross-reactivity to chicken tissues. Both antigens could be detected in moderately (F(10)121.1 = 0.84) and strongly (F(10)121.1 = 4.01) heated processed meat products containing 5% (NF-L) or 0.2% (PGP 9.5) CNS tissue, respectively. Further, two monoclonal antibodies (clones 13C4 and 31A3) directed against PGP 9.5 were used for the development of a sandwich enzyme-linked immunosorbent assay. The limits of detection of the enzyme-linked immunosorbent assay were approximately 2% added CNS tissue in fresh processed meat products and approximately 0.5% for strongly heated processed meat products (F(10)121.1 = 4.01). In conclusion this test system constitutes a valuable supplementation to existing procedures, which could improve enforcement of food safety regulations.

Biology of Alaria spp. and human exposition risk to Alaria mesocercariae-a review.

Recent incidental background findings of Alaria alata mesocercariae ["Distomum muscularis suis," Duncker, 1896] in meat of wild boars during official Trichinella inspection initiated a re-assessment of the potential human health risk as posed by this parasite. The present review of the literature on Alaria biology shows that the human exposition risk should no longer be accepted to be negligible, as it demonstrates a general lack of knowledge in relevant areas of Alaria biology confounding any risk analysis. Sound risk assessment needs future studies which should concentrate on the most pressing questions of (1) the optimization and/or development of methods for reliable Alaria mesocercariae detection, (2) the distribution of the mesocercariae within their paratenic hosts, i.e., identification of potential predilection sites, particularly in wild boars, and (3) their prevalence in sylvatic populations of animals with respect to their introduction into the human food chain. Further, the degree and possibly also the species specificity of Alaria mesocercariae tenacity within the paratenic hosts and respective meat as pertaining to food technological treatments need to be elucidated. While these questions remain unanswered, it is an incontrovertible fact that Alaria mesocercariae have a potentially high human pathogenicity by both occupational and alimentary exposition.

Species and age determination of central nervous system tissue by fatty acid patterns.

The banning of specified risk materials (SRMs) from food chain is one of the most important measures to protect the consumer against any exposition with the agent of transmissible spongiform encephalopathies (TSEs). In order to control the SRM-ban, suitable methods for the detection of SRMs have to be developed. In this study homogenized samples of central nervous system (CNS) tissue from cattle (n=38), sheep (n=38) and pig (n=40) were analyzed by gas chromatography-mass spectrometry (GC-MS). The analysis was focused on the identification of fatty acid ratios for species differentiation and age estimation. We found a novel fatty acid ratio (C24:1(n-9)/(n-7))/(2OH-C24:1(n-9)/(n-7) for species differentiation. For the first time, we used multivariate analysis for species differentiation, based on fatty acid ratios. It showed an excellent predictive ability (Q_(cum)(2))Q_(cum)(2)=0.842. For age estimation in cattle CNS we discovered a novel fatty acid ratio 2OH-C25:0/2OH-C24:1(n-7). The ratio is strongly correlated with age (coefficient of correlation (r)=0.935, Spearman-Rho). The corresponding regression analysis which allows the prediction of age by this ratio was acceptable (coefficient of determination (R(2))=0.831).

Transmissible spongiform encephalopathies: assessment of exposure risk in the histological working environment using GC-MS detection of fatty acids as marker for central nervous tissues.

To elucidate the risk of occupational exposure to the agent of transmissible spongiform encephalopathies (TSE) in the histological working environment, we assessed the principal suitability of three analytical methods for the detection of tissues of the central nervous system (CNT). We tested a neuron-specific enolase (NSE) Western blot, a glial fibrillary acidic protein (GFAP) ELISA and the GC-MS detection of some CNT typical fatty acids (FAs): omega9-tetracosenic acid, omega7-tetracosenic acid, lignoceric acid, and cerebronic acid. Histological sample processing (formalin fixation, dehydration, paraffin embedding) affected both of the immunochemical approaches considerably. The NSE Western blot produced negative results without exception. The results for the GFAP ELISA were better but still far too insensitive. Thus, both methods were judged to be unsuitable in their present form without major analytical adjustment. GC-MS sensitivity remained unaffected by the formalin fixation process. Sensitivity was reduced in the course of the final dehydration step using xylene in the histological sample processing. However, this reduction was found to be rather moderate (range 42-59%) when compared to the immunochemical methods. Overall, we judged GC-MS to be a promising analytical approach for the assessment of a potential TSE exposure risk via airborne CNT particles in the histological working environment. All the FAs we tested showed very low but detectable baseline contents. Thus, cut-off values must be used in the present GC-MS approach. The most suitable FA turned out to be omega9-tetracosenic acid due to the greatest difference between its content in histological CNT samples and the respective cut-off value (689:1). Preliminary results by GC-MS monitoring of CNT via omega9-tetracosenic acid (and other FAs) on filters of routinely used vacuum cleaners and on filters after air sampling indicate that the airborne CNT/TSE exposure risk in the histological laboratory is minor if existing at all. However, further in depth studies will have to validate our preliminary findings and assess these results in the light of possible future data on human oral and/or pulmonary TSE susceptibility.

Trichinella detection: identification and statistical evaluation of sources of error in the magnetic stirrer method for pooled sample digestion.

Proficiency testing (PT) is the use of inter-laboratory comparisons to determine the performance of individual laboratories for specific tests or measurements, and to monitor a laboratory's performance. Participation in proficiency testing provides laboratories with an objective means of assessing and demonstrating the reliability of the data they are producing. To ensure the reliability of Trichinella detection and meat hygiene within the European Union and afford optimal protection to the consumer, PT is conducted under the direction of the European National Reference Laboratories for Trichinella. Evaluation of data from the national PT showed that lab-internal shortcomings are frequent. These shortcomings are specifically related to: (1) improper sample collection and preparation; (2) incorrect transposition and application of the protocol as laid down in Annex I, Chapter I, Nr. 3 (a-g) of the Commission Regulation (EC) No. 2075/2005; (3) insufficient sedimentation times; and (4) improper equipment.(e.g. Prost and Nowakowski, 1990; Rossi and Pozio, 2008; Forbes and Gajadhar, 1999; Rossi and Pozio, 2008). To test the hypothesis that both method based errors as well as internal lab errors can influence the accuracy and precision of the magnetic stirrer method for pooled sample digestion (MSM), we initiated a study to evaluate the analytical uncertainty of the MSM. Results presented here are based on: (i) data from PT in Germany (2008, 2009, and 2010); (ii) within-lab performance conducting high volumes of MSM; (iii) larval recovery experiments; and (iv) statistical evaluation of data resulting from these procedures. Quantitative data from the PT show that on average only 60% of Trichinella larvae were detected. Even laboratories that showed relatively good performance (>80% larva recovery, no false negative or false positive results), frequently reported samples with an unexpectedly low larval count (loss of >2 larvae). In our own laboratory, high numbers of repeated analyses of standards and re-analyses of residual fluids indicated that these outliers could be described by a binomial distribution based on a laboratory-specific Trichinella-detection probability. Results of recovery experiments indicate that only a part of the total larval losses can be attributed to lab-internal shortcomings inasmuch as a significant number of L1 could be isolated from the residual and washing fluids.

Studies on contamination of beef with tissues of the central nervous system (CNS) as pertaining to slaughtering technology and human BSE-exposure risk.

Contamination of beef by tissues of the central nervous system (CNS) due to slaughter technology causes some concern considering the potential health hazard by food borne exposure to the infectious agent of BSE. The present study was designed to quantify the extent of CNS contamination as pertaining to stunning and splitting technology. Of the 726 animals 48 contained a total of 58 emboli-like particles in lungs and/or right ventricles. The incidence of emboli-like particles was found to be slightly higher in animals slaughtered without pithing (5.9%) than in the animals slaughtered with pithing (4.1%). Of the 58 emboli-like particles only two were positive in the anti-NSE western immunoblotting (0.3% of the 726 animals). The immuno reaction of these NSE-positive particles was several orders of magnitude lower as obtained by pure brain material. The microscopical analysis of the two NSE-positive emboli-like particles for presence of CNS-like tissues was negative. Following splitting of carcasses by sawing with and without prior removing the spinal cord we found NSE-positive reactions in 32% and 17% of the samples, respectively. The immuno reaction, however, was predominantly comparable to standard material containing less than 0.5% CNS. Overall the results show that CNS contamination of bovine carcasses cannot be excluded by current slaughter technology. However, the additional human BSE-exposure risk can be judged to be at least minor when considering extent of contamination, dilution effects and BSE-testing.

Detection of tissues of the central nervous system (CNS) as specified risk material (SRM) in meat products by means of gas chromatography-mass spectrometry (GC-MS).

Determination of specified risk material (SRM) in processed meat products was performed by quantification of brain specific fatty acids using gas chromatography-mass spectrometry (GC-MS). Results from SMP (internal standardised meat products) based analyses showed that absolute concentrations of CNS are correlated (r2 = > 0.97) with the contents of the CNS typical fatty acids docosahexaenoic acid (C 22:6), nervonic acid (C 24:1), lignoceric acid (C 24) and cerebronic acid (C 24oh). GC-MS detection limits were measured at 0.01% CNS. The cut off value was calculated at 0.39% (w/w) CNS in SMP. In a controlled blindfold experiment we were able to identify correctly all positive and negative SMP samples, respectively. Our results indicate that GC-MS based SRM detection may serve as a reference method for immunochemical and immunohistochemical determination of SRM in processed meat products.

[Molecular biological detection of tissues of central nervous system in meat products]. / Molekularbiologische Erfassung von Geweben des Zentralen Nervensystems in Fleischerzeugnissen.

In principle, molecular biological methods can be used for the detection of specified risk material (SRM) in meat products. We were able to identify suitable target mRNAs for the marker proteins glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) of tissues of the central nervous system (CNS). The selected primers for GFAP ("GFAP87") and MBP ("MBP51") facilitated the detection of CNS in non-heated and heated standards of emulsion type (and raw) sausages with defined addition of brain by reverse transcription and polymerase chain reaction (RT-PCR). Stability of the mRNA proved to be given over a minimum of 48 hours. First results indicate an even higher stability of the target mRNAs over time and an ample stability against meat technological influences such as storage, temperature, and ripening. RT-PCR with GFAP87 facilitates the detection of CNS of all relevant slaughter animals in meat products. Thus it can be applied in food labeling control. Using RT-PCR and MBP51 as primer we were able to detect selectively the CNS of bovines, ovines and caprines but not CNS of porcines and poultry. Thus we have a second RT-PCR detection procedure for CNS in meat products which, however, yields results equivalent to the legal definition of SRM. The further development of these molecular biological methods would be of considerable importance for routine food control and reduction of human TSE-exposure risk.