Lipopolysaccharide (LPS) induced inflammatory changes to differentially expressed miRNAs of the host inflammatory response.

In veterinary medicine, inflammation in swine is evaluated principally by clinical signs. This method is often unreliable when assessing large animal populations because of inconsistent interpretations of clinical observations. This study examined whether changes in miRNA expression can predict the severity of the inflammatory response in swine after administration of Escherichia coli lipopolysaccharide (LPS). Whole blood from swine challenged with LPS at 0.125 µg/kg to 2.0 µg/kg body weight was collected at 0, 1, 3, and 8 h post LPS-challenge. Mature miRNAs were extracted from plasma and quantitative real-time-PCR (qRT-PCR) was used to evaluate the 84 most abundant swine miRNAs found in plasma. The miRNA changes in expression were assessed using the comparative CT Method (ΔΔCT method) for normalization with an exogenous control. The results revealed that expression of ssc-let-7e-5p, ssc-mir-22-3p, and ssc-miR-146a-5p were the most significantly changed miRNA over the time course. At 1 h post-LPS, ssc-let-7e-5p decreased as the LPS dosage levels increased from 0.125 to 1.0 µg/kg. Similarly, as the LPS doses increased from 0.125 to 0.5 µg/kg, ssc-miR-22-3p levels significantly decreased at 1 h post-LPS. In the 2.0 µg/kg LPS, ssc-miR-146a-5p levels increased between 0 and 3 h post-LPS; however, expression was downregulated with a 145 % decrease from 3 to 8 h. The three miRNA biomarkers suggest potentially useful surrogate endpoints for the evaluation of inflammatory and endotoxemia responses in swine.

Molecular mechanism of action responsible for carrageenan-induced inflammatory response.

Carrageenan-induced inflammation has long been used as an in vivo model of local inflammation. We developed an in vitro model of inflammation using primary blood cells to characterize gene induction following carrageenan (λ-CGN) stimulation and identify the signal transduction pathway(s) through which λ-CGN worked, using swine whole blood cultures from Yorkshire barrows. Blood samples were divided into stimulated and unstimulated groups. Unstimulated blood was a control for λ-CGN treated cultures to delineate treatment effects from time-in-culture effects. All cultures were collected and separated into two fractions; supernatant for ELISA analyses and white blood cells for mRNA expression. Lambda (λ)-CGN induced MCP-1 at the proteomic and the genomic levels. Lambda-CGN increased IL-8 protein production but had no impact on serum amyloid A protein levels. Alveolar Macrophage-Derived Neutrophil Chemotactic Factor-II (AMCF-II), a swine-specific member of the IL8/GRO family, showed increased gene expression. TNF-α and IL-6 protein levels were not induced by λ-CGN stimulation. Stimulation of HEK-293 cells co-transfected with a single pattern recognition receptor and the secreted embryonic alkaline phosphatase (SEAP) read-out system demonstrated that λ-CGN signals through the TLR-2 and TLR-4 signal transduction pathways. Using silencing RNA to inhibit TLR6 expression in TLR2 transfected HEK-293 cells indicated that λ-CGN works through the TLR2/6 pathway. Silencing TLR6 expression in TLR4 transfected HEK-293 cells showed that λ-CGN stimulation of this cell line worked through a TLR4/6 heterodimer, as lipopolysaccharide (LPS) induced SEAP production through a TLR4 homodimer. These results demonstrate that although carrageenan can stimulate through TLR4 signaling pathways, it initiates an inflammatory response in these cells that differs from a typical endotoxin effect such as LPS stimulation, in terms of the pathways and gene products altered, suggesting that activation of TLR2/6 and TLR4/6 are the predominant pathways through which carrageenan induces inflammatory responses.

Identification of swine protein biomarkers of inflammation-associated pain.

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.

Detection of rendered meat and bone meals by PCR is dependent on animal species of origin and DNA extraction method.

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.

Development, evaluation, and peer verification of a rapid real-time PCR method for the detection of animal material.

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.

Assessment of two enzyme-linked immunosorbent assay tests marketed for detection of ruminant proteins in finished 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.