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This study sought to determine if proteins associated with pain in humans could be measured using a swine in vitro model of inflammation. This would constitute the first step towards using them as surrogate endpoints to help support effectiveness indications for investigational new animal drugs to control pain in swine. Swine whole blood samples were cultured in vitro with E. coli derived-lipopolysaccharide (LPS) or without LPS for 24 h. Supernatants from these cultures were collected to determine the concentration of proteins associated with pain and whether the levels were altered in response to LPS-induced inflammation. Bradykinin protein levels steadily increased over time due to LPS stimulation and returned to 0 h levels after 6 h of culture. Corticotrophin-releasing factor protein levels were not affected by LPS. Substance-P protein trended towards increasing concentrations after LPS stimulation, following a time-concentration profile similar to that observed with bradykinin. These results suggest that 2 biomarkers may be useful as surrogate endpoints for evaluation of pain.

Non-steroidal anti-inflammatory drugs (NSAID) are a family of chemicals that function to reduce pain, fever, and inflammation, and they are commonly used in people and animals for this purpose. Currently there are no NSAIDs approved for the management of inflammation in swine due to a lack of validated animal models and suitable biomarkers to assess efficacy. A previous in vitro study examining biomarkers of inflammation identified fourteen genes that were significantly altered in response to Escherichia coli lipopolysaccharide (LPS)-induced inflammation. In the present study, five of those fourteen genes were tested in vivo to determine if the same effects observed in vitro were also observed in vivo . Plasma levels of prostaglandin E 2 (PGE 2 ), an essential mediator of fever and inflammation, were also determined. Two groups of swine were stimulated with LPS with the second group also treated with flunixin meglumine. Blood was collected at 0, 1, 3, 6, 8, 24, and 48 h post LPS-stimulation. The RNA was extracted from the blood and quantitative real-time-PCR (qRT-PCR) was utilized to determine the expression patterns of CD1, CD4, serum amyloid A2 (SAA2), Caspase 1, and monocyte chemoattractant protein 1 (MCP-1). The LPS-stimulated animals demonstrated a statistically significant alteration in expression of SAA2 and CD1 at 3 h post-stimulation. Flunixin meglumine treated animals’ demonstrated reduced expression of CD1 in comparison to the LPS-stimulated swine at 24 and 48 h post LPS-stimulation. Flunixin meglumine treated animals exhibited reduced expression of SAA2 at 48 h post-stimulation compared to LPS-stimulated swine. Swine treated with LPS demonstrated statistically significant increases in plasma PGE 2 at 1 h post-stimulation. Swine treated with flunixin meglumine had no increase in plasma PGE 2 levels at any time. These results demonstrate that PGE 2 production, along with two out of five genes (SAA2 and CD1) have the potential to serve as early biomarkers of inflammation as well as indicators of NSAID efficacy.

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.

Myers, M. J., Scott, M. L., Deaver, C. M., Farrell, D. E., Yancy, H. F. Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs. J. vet. Pharmacol. Therap.33, 1–8. The impact of nonsteroidal anti-inflammatory drugs (NSAID) on prostaglandin E2 (PGE2) production and cyclooxygenase 2 (COX-2) mRNA expression in bovine whole blood (WB) cultures stimulated by lipopolysaccharide (LPS) was determined, using the blood from six Holstein dairy cattle in various stages of lactation. Peak production of PGE2 occurred 24 h after LPS stimulation but did not result in detectable concentrations of thromboxane B2 (TXB2). The NSAID indomethacin, aspirin, flunixin meglumine, and 4-[5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzene sulfonamide (PTPBS; celecoxib analogue), along with dexamethasone, were all equally effective in reducing the concentration of PGE2 in the bovine WB culture supernatants. Bradykinin exhibited peak supernatant concentrations 1 h after LPS stimulation. Dexamethasone and the NSAID used in this study were equally effective at inhibiting bradykinin production. Peak induction of COX-2 mRNA occurred 3 h post-LPS stimulation. However, neither dexamethasone nor any of the NSAID used in this study altered COX-2 mRNA concentrations. In contrast, aspirin, flunixin meglumine, and PTPBS reduced tumor necrosis factor-alpha (TNFα) mRNA concentration. These results demonstrate that bovine blood cells respond to NSAID therapy like other mammalian cells with respect to inhibition of PGE2 production and suppression of TNF mRNA induction, but do not inhibit induction of COX-2 mRNA.

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.