Validation of a PCR-based method for the detection of various rendered materials in feedstuffs using a forensic DNA extraction kit.

A method trial was initiated to validate the use of a commercial DNA forensic kit to extract DNA from animal feed as part of a PCR-based method. Four different PCR primer pairs (one bovine pair, one porcine pair, one ovine primer pair, and one multispecies pair) were also evaluated. Each laboratory was required to analyze a total of 120 dairy feed samples either not fortified (control, true negative) or fortified with bovine meat and bone meal, porcine meat and bone meal (PMBM), or lamb meal. Feeds were fortified with the animal meals at a concentration of 0.1% (wt/wt). Ten laboratories participated in this trial, and each laboratory was required to evaluate two different primer pairs, i.e., each PCR primer pair was evaluated by five different laboratories. The method was considered to be validated for a given animal source when three or more laboratories achieved at least 97% accuracy (29 correct of 30 samples for 96.7% accuracy, rounded up to 97%) in detecting the fortified samples for that source. Using this criterion, the method was validated for the bovine primer because three laboratories met the criterion, with an average accuracy of 98.9%. The average false-positive rate was 3.0% in these laboratories. A fourth laboratory was 80% accurate in identifying the samples fortified with bovine meat and bone meal. A fifth laboratory was not able to consistently extract the DNA from the feed samples and did not achieve the criterion for accuracy for either the bovine or multispecies PCR primers. For the porcine primers, the method was validated, with four laboratories meeting the criterion for accuracy with an average accuracy of 99.2%. The fifth laboratory had a 93.3% accuracy outcome for the porcine primer. Collectively, these five laboratories had a 1.3% false-positive rate for the porcine primer. No laboratory was able to meet the criterion for accuracy with the ovine primers, most likely because of problems with the synthesis of the primer pair; none of the positive control DNA samples could be detected with the ovine primers. The multispecies primer pair was validated in three laboratories for use with bovine meat and bone meal and lamb meal but not with PMBM. The three laboratories had an average accuracy of 98.9% for bovine meat and bone meal, 97.8% for lamb meal, and 63.3% for PMBM. When examined on an individual laboratory basis, one of these four laboratories could not identify a single feed sample containing PMBM by using the multispecies primer, whereas the other laboratory identified only one PMBM-fortified sample, suggesting that the limit of detection for PMBM with this primer pair is around 0.1% (wt/wt). The results of this study demonstrated that the DNA forensic kit can be used to extract DNA from animal feed, which can then be used for PCR analysis to detect animal-derived protein present in the feed sample.

Locomotion in simulated zero gravity: ground reaction forces.

BACKGROUND: Exercise is likely to be an important countermeasure to bone demineralization, which remains a concern for astronauts during long-duration spaceflight. However, loads on the feet during exercise with 1 G equivalent gravity replacement are not known. The purpose of this study was to compare ground reaction forces (GRFs) during over-ground and simulated zero gravity (0 G) locomotion. HYPOTHESIS: It was hypothesized that sufficient gravity replacement loading could be applied to the subjects such that GRF profiles similar to those seen in 1 G would occur during locomotion in a zero-gravity locomotion simulator (ZLS). METHODS: GRFs were measured during overground walking and running, and during locomotion in two restraint harness designs in the ZLS with an initial loading of 1 body weight. Load cells measured the gravity replacement load (GRL) in the ZLS. Joint angles at the hip and knee were also measured by goniometers. RESULTS: Peak forces were greater in overground locomotion than in the ZLS; however, loading rates were greater in the ZLS running conditions than in overground running. The knee joint was more flexed at key times in the support phase during running in the ZLS compared with overground. CONCLUSIONS: Large loads and loading rates can be generated at the feet during simulated 0 G exercise although peak forces during running in the ZLS are less than overground running at the same speed. The refinement of the gravity replacement system to provide a constant 1 G load should be considered.

Effect of oral administration of low doses of pentobarbital on the induction of cytochrome P450 isoforms and cytochrome P450-mediated reactions in immature Beagles.

OBJECTIVE: To determine the effect of oral administration of low doses of pentobarbital on cytochrome P450 (CYP) isoforms and CYP-mediated reactions in immature Beagles. ANIMALS: 42 immature (12-week-old) Beagles. PROCEDURE: Dogs were grouped and treated orally as follows for 8 weeks: low-dose pentobarbital (50 microg/d; 4 males, 4 females), mid-dose pentobarbital (150 microg/d; 4 males, 4 females), high-dose pentobarbital (500 microg/d; 4 males, 4 females), positive-pentobarbital control (10 mg/kg/d; 2 males, 2 females), positive-phenobarbital control (10 mg/kg/d; 2 males, 2 females), and negative control (saline 10.9% NaCl] solution; 5 males, 5 females). Serum biochemical and hematologic values were monitored. On necropsy examination, organ weights were determined, and histologic evaluation of tissue sections of liver, kidney, small intestine, testes, epididymis, and ovaries was performed. Hepatic and intestinal drug-metabolizing enzyme activities were measured, and relative amounts of CYP isoforms were determined by western blot analysis. RESULTS: The amount of a hepatic CYP2A-related isoform in dogs from the high-dose pentobarbital treatment group was twice that of dogs from the negative control group. CYP2C was not detectable in small intestinal mucosa of dogs from the negative control group; measurable amounts of CYP2C were found in dogs from the various (low-, mid-, and high-dose) pentobarbital treatment groups and from positive-pentobarbital and positive phenobarbital control groups. Several CYP-mediated reactions increased in a dose-dependent manner. The lowest calculated effective dose of pentobarbital ranged from 200 to 450 microg/d. CONCLUSIONS AND CLINICAL RELEVANCE: Several CYP isoforms and their associated reactions were induced in dogs by oral administration of low amounts of pentobarbital.

Tibial flexural wave propagation in vivo: potential for bone stress injury risk assessment.

OBJECTIVE: To determine the reliability of tibial flexural wave propagation velocity (FWPV) and the effect of overlying soft-tissues on the result. DESIGN: The velocity of tibial flexural wave propagation was repeatedly measured in healthy subjects. BACKGROUND: The tibia is the most frequently stress-fractured bone of runners and in military training populations. Developing techniques to measure tibial bone strength and, thereby, fracture resistance may be useful in pre-participation examinations in order to identify at-risk individuals. One such method, tibial flexural wave propagation velocity (FWPV) has been reported to be a measure of tibial stiffness. METHODS: The tibial FWPV was measured 80 times per subject (10 trials X 2 setups/day X 4 days) in a sample of 25 young women. Two methods of calculating FWPV were used (Peak method time domain and Phase method frequency domain). Intraclass correlations (ICCs) were calculated. RESULTS: The results demonstrated that the peak method was more reliable then the phase method. The ICCs ranged from 0.81-0.96 for the peak method and from 0.59-0.89 for the phase method. The 95% confidence intervals demonstrated that the FWPV could discriminate between subjects with low, medium, or high velocity values with reasonable accuracy and confidence. The soft-tissue overlying the tibia was not significantly (p = 0.63) correlated with FWPV. CONCLUSION: In summary, it is possible to discriminate between varying levels of tibial FWPV. Furthermore, if FWPV is related to bone stress fracture resistance, then this method may be useful in determining stress fracture risk prior to intensive physical activity. RELEVANCE: The tibia is the most frequently stress-fractured bone of runners and in military training populations. This study explores the feasibility of tibial FWPV as a potential method to assess tibial stress fracture resistance.