Reclassification of Herbaspirillum putei as a later heterotypic synonym of Herbaspirillum huttiense, with the description of H. huttiense subsp. huttiense subsp. nov. and H. huttiense subsp. putei subsp. nov., comb. nov., and description of Herbaspirillum aquaticum sp. nov.

Resequencing of the 16S rRNA gene of the type strain of Herbaspirillum putei Ding and Yokota 2004 revealed 99.9 % sequence similarity to that of the type strain of Herbaspirillum huttiense (Leifson 1962) Ding and Yokota 2004. This high phylogenetic relatedness of H. putei and H. huttiense was confirmed by the results of DNA-DNA hybridization between H. huttiense DSM 10281(T) and H. putei ATCC BAA-806(T) (reassociation value 96 %). Therefore, it is proposed to reclassify the type strain of H. putei as a strain of H. huttiense. However, the genome of the type strain of H. putei is about 0.9 Mb larger than that of the H. huttiense type strain. This results in a decrease in the reassociation value in the reciprocal DNA-DNA hybridization to 72 %, a level slightly above the threshold for delineating bacterial species. These data and distinctive phenotypic characteristics indicate that the name Herbaspirillum putei is a later heterotypic synonym of Herbaspirillum huttiense and permit the description of two novel subspecies, Herbaspirillum huttiense subsp. huttiense subsp. nov. (type strain ATCC 14670(T) =JCM 21423(T) =DSM 10281(T)) and Herbaspirillum huttiense subsp. putei subsp. nov., comb. nov. (type strain 7-2(T) =JCM 21495(T) =ATCC BAA-806(T)). Three bacterial strains, IEH 4430(T), IEH 4515 and IEH 8757, isolated from water were found to be the closest relatives of these strains. Strain IEH 8757 was classified as a strain of H. huttiense subsp. putei. Studies of genotypic and phenotypic features of strains IEH 4430(T) and IEH 4515 showed that the strains represent a novel species, which is most closely related to H. huttiense and for which the name Herbaspirillum aquaticum sp. nov. is proposed (type strain IEH 4430(T) =DSM 21191(T) =ATCC BAA-1628(T)).

Detection of central nervous system tissue on meat and carcass-splitting band saw blade surfaces using modified fluorescent glial fibrillary acidic protein enzyme-linked immunosorbent assay sampling and extraction procedures.

This study was conducted to determine optimal buffer pH, extraction procedure, and temperature for detecting central nervous system (CNS) tissue on meat surfaces and on carcass-splitting band saw blades using swab sampling. Glial fibrillary acidic protein (GFAP) is restricted to CNS tissue and has been used as a marker for CNS tissue presence in meat products. Sample preparation, extraction procedure, and extraction temperature of glial fibrillary acidic protein fluorescent enzyme-linked immunosorbent assay (GFAP F-ELISA) were modified to detect CNS tissue on meat surfaces and on carcass-splitting band saw blades. Maximum GFAP recovery was observed with an extraction buffer pH of 7.4. Extracting samples at room temperature by vortexing for 30 s in 1 ml of extraction buffer (phosphate-buffered saline [pH 7.4] plus 0.05% sodium dodecyl sulfate) consistently provided detection of GFAP on meat surfaces contaminated with 500 microg of spinal cord suspension per 50 cm2 and on carcass-splitting band saw blades contaminated with 20 microg of spinal cord suspension per 50 cm2. Recovery of GFAP was not affected by storing samples overnight at 4 degrees C. The current studies demonstrate the effectiveness of modified sampling procedures and preparations, sample extraction buffer pH, and extraction temperatures. These modifications introduced to the original F-ELISA sampling protocol result in asensitive and repeatable assay for detection of CNS tissue on meat surfaces and on carcass-splitting band saw blades.

Alternative cutting methods to minimize transfer of nervous system tissue during steak preparation from bone-in short loins.

Fresh beef products, such as steaks, may become contaminated with potential specified risk materials (SRMs), such as central nervous system tissue, during the fabrication of bone-in loin subprimals. The objective of this study was to evaluate current and alternative cutting methods that could be used to minimize the transfer of nervous system tissue (NST) tissue during preparation of steaks from bone-in short loins. Bone-in short loins were cut according to three methods. (i) Cutting method I-The vertebral column bones were removed prior to cutting the loin into steaks from the medial (vertebral column) to lateral (flank) side. (ii) Cutting method II--The loin was cut into steaks from the vertebral column side to the flank side prior to removal of the vertebral column bones. (iii) Cutting method III--The loin was cut into steaks from the flank side to the vertebral column side prior to removal of the vertebral column bones. Results indicated that surface areas along the vertebral column cutting line had detectable (0.10 and 0.22% NST/100 cm2) and, thus, higher potential SRM contamination than resulting steak surfaces or the cutting blade. Overall, there were no detectable (<0.10% NST/100 cm2) differences in NST contamination of steaks produced by the three cutting methods. Immunohistochemical evaluation of areas on excised and ground steak surfaces indicated that regardless of cutting method, there was generally "no" to "moderate" staining, suggesting that detectable (0.137 to 0.201% NST) contamination from these samples was most likely due to peripheral nerve detection. These results imply that steaks may be cut from bone-in short loins prior to removal of the vertebral column bones without affecting the transfer of NST to resulting steaks at concentrations <0.10% NST/100 cm2.

Comparison of immunochemical (enzyme-linked immunosorbent assay) and immunohistochemical methods for the detection of central nervous system tissue in meat products.

Three methods are widely used in the United States to detect the presence of central nervous system (CNS) tissue in meat products: the fluorescent enzyme-linked immunosorbent assay (F-ELISA), developed in this laboratory, the colorimetric Ridascreen Risk Material 10/5 ELISA (R-ELISA), and the U.S. Department of Agriculture, Food Safety and Inspection Service immunohistochemical (IHC) procedure. These assays are based on the immunological detection of glial fibrillary acidic protein (GFAP), a neural antigen largely restricted to the CNS. The objective of the current study was to compare the sensitivity and repeatability of these tests for detecting the presence of neural tissue in meat. Ground beef spiked with 0.05 to 0.5% of bovine brain, spinal cord (SC), or dorsal root ganglia, as well as advanced meat recovery samples, were evaluated by each of the three GFAP detection procedures. Interassay coefficients of variation for the F-ELISA GFAP standards were 7 to 25%, and intra-assay variation due to sampling and extraction of spiked ground beef was 7 to 13% for SC and 8 to 14% for brain (n = 10). The F-ELISA was the most sensitive of the methods tested, capable of detecting 0.3 ng GFAP standard per well and the presence of 0.05% brain and SC in meat. The R-ELISA standards produced highly variable results (up to 36% variation) and, as a result, none of these standards were different from zero (n = 26). The R-ELISA resulted in high sample variation in SC-spiked ground beef samples (coefficients of variation were 23 to 50%) and did not detect the presence of brain contamination. After modification of the R-ELISA sampling and extraction methods, SC-spiked sample variation was reduced to 16 to 20%, and sensitivity was improved from 0.3 to 0.2% SC, although brain tissue was still not detected. The IHC analysis of CNS-adulterated ground beef had a sensitivity of 0.2% SC and 0.05% brain, with false-negative rates of 10 to 20% at and above the stated sensitivities. None of the methods examined detected dorsal root ganglia contamination. The F-ELISA detected the presence of CNS contamination in 20% of the advanced meat recovery samples, compared to 3.5 to 5% for the R-ELISA and 2% for IHC. This study suggests that the F-ELISA is much more sensitive and repeatable than either the R-ELISA or the IHC procedure method for the detection of CNS tissue in meat products.