Bovine Central Nervous System Tissue Research Articles

Detection of Bovine Central Nervous System Tissues in Rendered Animal By-Products by One-Step Real-Time Reverse Transcription PCR Assay

Contamination of rendered animal byproducts with central nervous system tissues (CNST) from animals with bovine spongiform encephalopathy is considered one of the vehicles of disease transmission. Removal from the animal feed chain of CNST originated from cattle of a specified age category, species-labeling of rendered meat products, and testing of rendered products for bovine CNST are tasks associated with the epidemiological control of bovine spongiform encephalopathy. A single-step TaqMan real-time reverse transcriptase (RRT) PCR assay was developed and evaluated for specific detection of bovine glial fibrillary acidic protein (GFAP) mRNA, a biomarker of bovine CNST, in rendered animal by-products. An internal amplification control, mammalian b -actin mRNA, was coamplified in the duplex RRT-PCR assay to monitor amplification efficiency, normalize amplification signals, and avoid false-negative results. The functionality of the GFAP mRNA RRT-PCR was assessed through analysis of laboratory-generated binary mixtures of bovine central nervous system (CNS) and muscle tissues treated under various thermal settings imitating industrial conditions. The assay was able to detect as low as 0.05 % (wt/wt) bovine brain tissue in binary mixtures heat treated at 110 to 130°C for 20 to 60 min. Further evaluation of the GFAP mRNA RRT-PCR assay involved samples of industrial rendered products of various species origin and composition obtained from commercial sources and rendering plants. Low amounts of bovine GFAP mRNA were detected in several bovine-rendered products, which was in agreement with declared species composition. An accurate estimation of CNS tissue content in industrial-rendered products was complicated due to a wide range of temperature and time settings in rendering protocols. Nevertheless, the GFAP mRNA RRT-PCR assay may be considered for bovine CNS tissue detection in rendered products in combination with other available tools (for example, animal age verification) in inspection programs.

Effect of pH, temperature and storage time on the stability of bovine myelin basic protein

Abstract Bovine central nervous system tissue (CNT) harboring highest levels of the infectious agent for prion diseases, has been banned from food and feed supplies. Effective detection of CNT in excessively processed meat and feedstuffs requires the assay based on a stable marker in CNT as the analyte. Myelin basic protein (MBP), the major central nervous system (CNS) myelin protein, was reported to be detectable up to 115 °C in the literature. This study further investigated the effects of pH, temperature and storage time on the stability of purified bovine MBP (18.5 kDa) in solutions. Purified MBP dissolved in 10 mM PBS was adjusted to pH 3.0, 7.2 and 10.5, respectively. Sample at each pH was subjected to three heat treatments, 100 °C, 121 °C, and 133 °C for 30 min. The unheated and 100 °C heated samples at pH 7.2 were stored at 4 °C for 19 days to study the storage stability of bovine MBP. The immunoreactivity of all samples was examined using indirect non-competitive enzyme-linked immunosorbent assay and immunoblotting with an anti-MBP monoclonal antibody. The protein degradation was observed by analyzing samples using SDS-PAGE. Results show that bovine MBP was most stable at pH 7.2 while the protein was least stable at pH 10.5. MBP started to degrade after heating at 121 °C for 30 min at pH 7.2. Storage up to 19 days at 4 °C did not significantly affect the immunoreactivity of unheated and heated samples. However, unheated MBP samples started to show degradation from day 8 of storage; while no degradation was observed in 100 °C heated samples over the storage time. This study demonstrated that bovine MBP can be a suitable marker for the detection of bovine CNT in highly processed food and feed supplies.

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.

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.

Detection of Bovine Central Nervous System Tissue as Bovine Spongiform Encephalopathy Risk Material by Real-Time Reverse Transcriptase-PCR in Raw and Cooked Beef Products

There is increasing evidence of the association of the new variant of Creutzfeldt-Jacob disease (nvCJD) in humans with bovine spongiform encephalopathy (BSE) in cattle. Many countries established legislation of banning central nervous system (CNS) tissues, which are regarded as BSE-specified risk materials (SRM), in human food supply because of the potential transmission of BSE to humans. A real-time reverse transcriptase-PCR assay using the bovine glial fibrillary acidic protein (GFAP) mRNA template for the detection of CNS tissues in raw and cooked beef products was developed in this study. The results showed that (1) this method can detect CNS tissues from bovine and ovine origins, but not from porcine and avian ones; (2) GFAP mRNA can only be detected from brain and spinal cords rather than other tissues; (3) the GFAP mRNA was detectable in CNS tissues even after dilution to 0.001%; and (4) the assay was unaffected by heat treatment at 100°C for 30 min or storage at room temperature for 4 days, and at 4°C for at least 15 days.

Detection of Bovine Central Nervous System Tissue in Liver Sausages Using a Reverse Transcriptase PCR Technique and a Commercial Enzyme-Linked Immunosorbent Assay

The suitability of a reverse transcriptase (RT) PCR assay was evaluated for the detection of bovine central nervous system (CNS) tissue specifically in liver sausages. Because of its emulsifying effect, CNS tissue was frequently added to this kind of meat product in the past. On standard samples, the RT-PCR technique reliably detected a concentration of 0.25% bovine CNS tissue in liver sausages stored for up to 28 days. Following the successful application of RT-PCR for the detection of bovine CNS tissue in these specially prepared samples, a field study was performed with a total of 258 liver sausages purchased in retail markets. All sausages were tested with both an RT-PCR assay and a commercial enzyme-linked immunosorbent assay (ELISA) kit. Nine (3.5%) of the retail liver sausage samples were positive for CNS tissue in the ELISA, but none were positive for this tissue in the RT-PCR assay. All positive ELISA results indicated the presence of 0.23 to 0.30% CNS tissue. Recent studies have indicated that the RT-PCR assay is not as sensitive for porcine CNS tissue as for bovine CNS tissue, which this assay can detect at 0.25%. Although the ELISA is not species specific, the CNS tissue detected by the ELISA is assumed to stem from a nonbovine species. The RT-PCR technique is a sensitive tool for the detection of bovine CNS tissues in a problematic matrix such as liver sausage. ELISA screening followed by a species-specific RT-PCR assay for bovine CNS tissue is a practical approach for monitoring meat products for compliance with European food regulations.

Detection of beef contaminated by bovine central nervous system tissue by enzyme immunoassay

To study influencing factors of detection of bovine central nervous system (CNS) tissue contaminated beef by enzyme immunoassay (EIA), and the method was applied to the detection of imported beef and domestic beef of China. Raw beef homogenates containing different concentrations of raw CNS tissue and the same samples which were heated were detected after different time by RIDASCREEN(r) Risk Material 10/5 and RIDASCREEN(r) Probennahme- zubehor Sampling tools kits. PBS suspension and sample dilution buffer (SDB) suspension of bovine brain tissue with the same concentration of the standard were detected. Beef from USA and domestic market of China were then detected by the kits. The kits could detect both raw and heated CNS tissue in the products with high sensitivity. The absorbance values (AV) increased with the concentrations of CNS in samples. Heating and increasing of time could decrease the absorbance values of the samples which contain CNS tissue. The AV of the PBS suspension of bovine brain tissue was higher than the SDB suspension and the AV of both were higher than the AV of standard of the same concentration. No CNS tissue was detected from all imported beef. No CNS tissue was detected in all samples from domestic market of China except for foxtail. The EIA method has high sensitivity for detection of bovine CNS tissue contaminated beef with the glial fibrillary acidic protein (GFAP) as accurate target substance. Heating and increasing of time can lead to decreasing of the AV of samples. Improper slaughter process can lead to contamination of bovine products by bovine CNS tissue.

Assessment and Modification of a Western Blot Assay for Detection of Central Nervous System Tissue in Meat Products in the United States

Health hazards associated with meat contaminated by the bovine spongiform encephalopathy agent have led to the development of tests for the presence of this agent. The objective of this study was to optimize a neuron-specific enolase Western blot assay for use in the United States. We compared the original test with a modified protocol to evaluate the detection limit for the presence of central nervous system (CNS) tissue in experimentally inoculated samples and compared and evaluated the utility of these tests for detecting CNS tissue in retail sausages. Sensitivity and specificity of the original and modified protocols were evaluated using the kappa statistic to assess agreement between the results of the two protocols. The original protocol resulted in 100% specificity and 92% sensitivity for raw samples and 92% specificity and 72% sensitivity for cooked samples. The modified protocol resulted in 92% specificity and 89% sensitivity for raw samples and 83% specificity and 75% sensitivity for cooked samples. The kappa statistic for protocol comparison was 0.94 for raw samples and 0.74 for cooked samples. Both protocols correctly identified CNS tissue in positive controls for each replicate. Although the Western blot technique should be considered for screening for the presence of bovine CNS tissue in meat samples, the techniques should be further optimized to address problems of low sensitivity. A test with higher sensitivity is needed to protect consumers from food safety threats associated with bovine CNS tissue.

Reverse Transcription–Polymerase Chain Reaction Assay for Species-Specific Detection of Bovine Central Nervous System Tissue in Meat and Meat Products

This paper reports the development of a reverse transcription–polymerase chain reaction (RT-PCR) assay coupled with restriction fragment length polymorphism (RFLP) analysis to specifically detect the glial fibrillary acidic protein (GFAP) mRNA of bovine central nervous system (CNS) tissue in minced meat and meat products. RNA extracted from bovine brain tissue and brain tissue from other mammals yielded a 168-bp CNS-specific signal after RT-PCR. The species specificity of the assay can be obtained by subsequent RFLP analysis of the amplified RT-PCR product. To determine the tissue specificity of the RT-PCR assay, various bovine tissues were analyzed. Bovine GFAP mRNA was detected in the brain and spinal cord, and these results are consistent with the reported large amounts of detectable protein in these tissues. Additionally, GFAP mRNA was present in skeletal muscle tissue samples and in some heart muscle tissue samples, but heat treatment of samples prior to extraction resulted in the loss of the non-CNS signals. To evaluate the stability and detectability of bovine GFAP mRNA in comminuted meat and in cooked meat products, mixtures containing bovine brain homogenate at concentrations of 0.5 to 5% were prepared and analyzed. The examination of minced meat with added bovine brain homogenate revealed GFAP mRNA RT-PCR signal stability for at least 7 days at a storage temperature of 4°C. In cooked meat products bovine GFAP mRNA signal was detectable for at least 35 days. Bovine brain homogenate at a concentration of 0.5% was successfully detected in all of the experiments conducted, and no false-negative results were obtained. It is concluded that bovine GFAP mRNA can serve as a sensitive and specific marker for bovine CNS tissue in minced meat and in pasteurized meat products.