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Objective—To identify biomarkers of P-glycoprotein (P-gp) substrate neurotoxicity in transgenic mice expressing the mutant canine ABCB1 gene (ABCB1-1Δ). Animals—8 ABCB1 knock-in and knock-out transgenic mice expressing the ABCB1-1Δ gene and 8 control mice expressing the wild-type canine ABCB1 gene (ABCB1-WT). Procedures—Groups including 2 ABCB1-1Δ mutant mice and 2 ABCB1-WT mice were administered the P-gp substrates ivermectin (10 mg/kg, SC), doramectin (10 mg/kg, SC), moxidectin (10 mg/kg, PO), or digoxin (1.53 mg/kg, SC). A toxicogenomic approach based on DNA microarrays was used to examine whole-genome expression changes in mice administered P-gp substrates. Results—Compared with control ABCB1-WT mice, ABCB1-1Δ mutant mice developed neurotoxic signs including ataxia, lethargy, and tremors similar to those reported for dogs with the ABCB1-1Δ mutation. Microarray analysis revealed that gene expression was altered in ABCB1-1Δ mutant mice, compared with findings for ABCB1-WT mice, following administration of the same P-gp substrates. Gene pathway analysis revealed that genes with a ≥ 2-fold gene expression change were associated with behavior and nervous system development and function. Moreover, 34 genes were altered in the ABCB1-1Δ mutant mice in all 4 drug treatment groups. These genes were also associated with behavior, which was identified as the top-ranked gene network. Conclusions and Clinical Relevance—These study data have facilitated understanding of the molecular mechanisms of neurotoxicosis in ABCB1-1Δ mutant mice following exposure to various P-gp substrates. Some genes appear to be potential biomarkers of P-gp substrate neurotoxicity that might be used to predict the safety of those drugs in dogs with the ABCB1-1Δ mutation.

There are currently no validated animal models or suitable biomarkers with which to ascertain the efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) in equine, bovine or ovine species during conditions of endotoxemia. This has resulted in approval of only one NSAID, flunixin meglumine, for controlling inflammation due to endotoxemia in bovine and equine animals, while none are approved in ovine animals for this claim. This study aims to investigate biomarkers with which to test efficacy of NSAIDs in these species. To this end, the effects of Escherichia coli lipopolysaccharide (LPS) induced inflammation on gene expression were investigated. Whole blood from each species was cultured and stimulated with LPS, after which RNA was extracted at various times. RNA was analyzed via quantitative RT-PCR (qRT-PCR) to determine differential expression of biomarkers. Results indicated up-regulation of cluster of differentiation 1 (CD1) gene in bovine and serum amyloid A (SAA) gene in ovine cultures. Down-regulation of cluster of differentiation 4 (CD4) gene and Caspase 1 was seen in bovine, and of CD1 in equine cultures. This work demonstrates that LPS stimulation alters expression of these genes in these species. These genes may be useful biomarkers for inflammation which could serve as markers for NSAID efficacy.

The capability of eight commercially available DNA extraction kits to extract bovine DNA originating in meat and bone meal from fortified feed was evaluated. Four different batches of bovine meat and bone meal (BMBM) were used for DNA extraction with the eight commercial DNA extraction kits. Within each kit, there were minimal differences in the batch-to-batch amounts of extracted DNA. There were differences between the kits in the amounts of DNA that could be extracted from the same amount of starting BMBM. These differences did not translate into differences in the amount of amplifiable DNA from BMBM-fortified dairy feed. Using a validated real-time PCR method, the kit yielding the highest amount extractable DNA was completely unable to yield a positive PCR result; one other kit was also unable to produce a positive PCR result from DNA extracted from BMBM-fortified feed. There was a complete lack of a correlation between the amount of bovine DNA isolated from BMBM by a given extraction kit compared with the relative amounts of DNA isolated from fortified animal feed as evidenced by the cycle threshold values generated using the real-time PCR method. These results demonstrate that extraction of DNA from processed animal protein is different for pure ingredients and fortified animal feeds. These results indicate that a method specifically developed using just animal-derived meat and bone meal may not yield a functional assay when used to detect animal tissues in complete animal feed.

The performance characteristics of two enzyme-linked immunosorbent assay (ELISA) test kits, ELISA Technologies' MELISA-Tek test and Tepnel BioSystems' BioKit for (Cooked) Species Identification test, designed to detect ruminant proteins in animal feed, were evaluated. The test kits were evaluated by using acceptance criteria developed by the U.S. Food and Drug Administration's Center for Veterinary Medicine Office of Research for evaluating selectivity, sensitivity, ruggedness, and specificity. The acceptance criteria for determining success used a statistical approach requiring a 90% probability of achieving the correct response within a 95% confidence interval. In practice, this measure requires the test to achieve the correct response 58 times for every 60 samples evaluated, or a 96.7% accuracy rate. A minimum detection level of 0.1% bovine meat and bone meal (BMBM) was required, consistent with the sensitivity of the analytical methods presently used by the U.S. Food and Drug Administration. Selectivity was assessed by testing 60 dairy feed samples that contained no added animal proteins; sensitivity was determined by evaluating 60 samples (per level of fortification) of this same feed that contained 0.025, 0.05, 0.1, 0.25, 0.5, 1, or 2% BMBM. The MELISA-Tek test passed the acceptance set-point criteria for selectivity assessment but failed the sensitivity assessment at all levels except at the 2% level. The MELISA-Tek test came close to passing at the 1% level, detecting true-positive findings at a rate of 93%, but failed at lower levels, in spite of the label claim of 0.5% sensitivity. The BioKit for (Cooked) Species Identification test detected only 2 of 17 samples fortified at the 2% BMBM level and failed to detect any other BMBM-fortified samples. The results of this evaluation indicate that neither test is adequate for regulatory use.

Four real-time PCR assays that can be used with U.S.- and European Union-rendered materials to detect three ruminant species (bovine, caprine, and ovine) and a select set of avians (chicken, goose, and turkey) were developed. This method was evaluated against stringent acceptance criteria previously developed by the U.S. Food and Drug Administration, Center for Veterinary Medicine's Office of Research. Acceptance criteria for determining success used a statistical approach requiring a 90% probability of achieving the correct response, within a 95% confidence interval. A minimum detection level of 0.1% meat and bone meal (MBM) was required, consistent with the sensitivity of the validated PCR-based method currently used by the U.S. Food and Drug Administration as an aid in enforcement of the Agency's feed ban. PCR primer specificity was determined by using a panel of DNA samples derived from 16 different animal species. The method is able to detect 0.1% rendered material in complete feed in less than 1.5 h of total assay time, a significant improvement over the current method, which requires 7 to 8 h for completion. The real-time assay for the detection of animal material passed stringent acceptance criteria for sensitivity, selectivity, and specificity. The method also passed ruggedness, real-time platform, and second analyst trials. Two external laboratories participating in a peer-verification trial demonstrated 100% specificity in identifying bovine MBM, ovine MBM, or caprine meat meal, while exhibiting a 0.6% rate of false positives. These results demonstrated that this method was capable of being used by other laboratories.