Dorothy E. Farrell

Validation of a polymerase chain reaction method for the detection of rendered bovine-derived materials in feedstuffs.

This study validated a polymerase chain reaction-based method for the detection of a specific bovine mitochondrial gene derived from rendered bovine tissues and admixed with complete animal feed. Four laboratories participated in this effort: one state laboratory and three Food and Drug Administration (FDA) laboratories, including one FDA field laboratory. The protocol used a statistical approach of 90% probability, with a 95% confidence interval for determining acceptable rates of false-positive and false-negative samples. Each participating laboratory analyzed 30 samples of feed each containing 0, 0.125, and 2.0% bovine meat and bone meal (BMBM), for a total of 90 feed samples. The samples were randomized such that the analysts were unaware of the true identity of the test samples. The results demonstrated that all laboratories met the acceptance criteria established for this protocol. The overall rates of false-negative results were 0.83% (1/120) at the level of 0.125% BMBM and 1.67% (2/120) at the level of 2% BMBM. The overall rate of false-negative results for all levels of BMBM was 1.25% (3/240). The rate for false-positive results was 0.83%.

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 Medicines 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 Agencys 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.

Evaluation of Two Commercial Lateral-Flow Test Kits for Detection of Animal Proteins in Animal Feed

Performance characteristics were evaluated for two lateral-flow test kits, Reveal for Ruminant in Feed (Neogen Corporation) and FeedChek (Strategic Diagnostics Inc.), designed to detect ruminant or terrestrial animal proteins in feeds. The stringent acceptance criteria used were developed by the Center for Veterinary Medicine Office of Research to identify test kits with comparable selectivity and sensitivity to microscopy and PCR assay, the analytical methods used by the U.S. Food and Drug Administration (FDA). Guidelines were developed for evaluating the selectivity, sensitivity, ruggedness, and specificity of these kits. These guidelines further stated that ruggedness and specificity testing would be performed only after a test passed both the selectivity and sensitivity assessments. Acceptance criteria for determining success were developed using a statistical approach requiring 90% probability of achieving the correct response, within a 95% confidence interval. A minimum detection level of 0.1% bovine meat and bone meal, consistent with the sensitivity of the methods used by the FDA, was required. 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 the same feed that contained 0.025, 0.05, 0.1, 0.25, 0.5, 1, or 2% bovine meat and bone meal. The Reveal test passed the selectivity assessment but failed the sensitivity assessment, detecting only samples fortified at the 2% level and then only 17 to 33% of those samples, when read according to the label directions. The FeedChek test passed the sensitivity assessment but failed the selectivity assessment, with rates for false-positive results ranging from 34 to 38%, depending on the user. The sensitivity of the Reveal test was affected by the concentration of trace minerals present in the feed; concentrations toward the high end of the normal range prevented the detection of true positive feed samples containing bovine meat and bone meal. Better sensitivity assessments were obtained when lamb meal was used either alone or in combination with bovine meat and bone meal. The FeedChek test was not affected by the concentration of trace minerals or by the type of animal meal used. These results indicate that neither of the two tests is adequate for routine regulatory use.

Evaluation of a Rapid PCR-Based Method for the Detection of Animal Material

A rapid PCR-based analytical method for detection of animal-derived materials in complete feed was developed. Using a commercially available DNA forensic kit for the extraction of DNA from animal feed, a sensitive method was developed that was capable of detecting as little as 0.03% bovine meat and bone meal in complete feed in under 8 h of total assay time. The reduction in assay time was accomplished by reducing the DNA extraction time to 2 h and using the simpler cleanup procedure of the kit. Assay sensitivity can be increased to 0.006% by increasing the DNA extraction time to an overnight incubation of approximately 16 h. Examination of dairy feed samples containing either bovine meat and bone meal, porcine meat and bone meal, or lamb meal at a level of 0.1% (wt/wt basis) suggested that this method may be suitable for regulatory uses. The adoption of this commercially available kit for use with animal feeds yields an assay that is quicker and simpler to perform than a previously validated assay for the detection of animal proteins in animal feed.

Effect of Recombinant Growth Hormone and Chromium Picolinate on Cytokine Production and Growth Performance in Swine

The effect of dietary chromium picolinate (CrP) and recombinant porcine growth hormone, somatotropin (rPST) administration on growth performance and cytokine production in Landrace-Poland China gilts was determined using a 2 by 2 treatment array. Treatments were: (1) control (basal diet), (2) CrP-supplemented diet (basal diet + 300 micrograms Cr3+/kg diet as CrP), (3) rPST (100 pg/kg body weight/day), and (4) rPST+CrP. CrP-supplemented diets were fed beginning at 20 kg body weight through 90 kg. Administration of rPST was begun at 60 kg weight and continued through 90 kg. All rPST treated pigs demonstrated improvements in growth performance versus controls. Pigs given CrP-supplemented diets showed no differences in growth performance. At 90 kg, pigs were challenged with endotoxin (lipopolysaccharide, 0.2 microgram/kg i.v.). Blood samples were collected at 0, 1, and 3 h postchallenge. Plasma IL-6 levels increased from 23 U/ml at time 0 to 1,927 U/ml at 3 h for control swine. Swine from the CrP treatment group had IL-6 levels of 8,130 U/ml at 3 h post-LPS. There were no differences in plasma IL-6 from pigs in the rPST and rPST+CrP treatment groups compared to the controls. Endotoxin challenge had no effect on either blood glucose levels or induction of TNF-alpha in any treatment group. PBMC from CrP-treated animals produced more IL-2 than peripheral blood mononuclear cells from all other groups.

Challenge differentially affects cytokine production and metabolic status of growing and finishing swine.

Growing (35 kg body weight) and finishing (85 kg body weight) swine challenged with endotoxin (Escherichia coli O55:B5) at a dose of either 2 or 20 microg/kg produced tumor necrosis factor (TNF)alpha in a dose-response relationship as measured by bioassay. Peak TNFalpha plasma levels were observed 1-2 hr post-challenge, returning to basal values 4 hr post-challenge. However, both an enzyme-linked immunosorbent assay specific for swine TNFalpha and total human TNFalpha demonstrated no dose-response relationship; peak plasma levels of immunoreactive TNFalpha were also observed 1-2 hr post-challenge. Maximal plasma interleukin-6 levels occurred 1-2 hr post-challenge and remained elevated through 8 hr post-challenge; there was no effect of lipopolysaccharide dose or metabolic status. Although the metabolic status of the animals also affected glucose levels, with growing animals exhibiting greater sensitivity compared with finishing animals, endotoxin-induced decreases in blood glucose levels were primarily dose-dependent. In contrast, changes in plasma urea nitrogen and free fatty acid (FFA) levels were strictly related to the metabolic status. Urea nitrogen levels were unchanged in growing swine, whereas they were increased in finishing swine and remained elevated 24 hr post-challenge. FFA levels in growing and finishing swine increased 3-6 hr post-challenge. FFA levels returned to basal values for finishing swine 24 hr post challenge, but in growing swine remained elevated 24 hr post-challenge. Plasma aspartate transaminase levels were increased through 24 hr post-challenge; animals given a dose of 20 microg/kg exhibited the greatest increase. Similarly, swine challenged with a dose of 20 microg/kg also exhibited the greatest increase in levels of conjugated bilirubin; there was no effect on unconjugated (free) bilirubin. These results demonstrate that endotoxin challenge of swine result in a pattern of changes that are dependent on both the dose of endotoxin used and the metabolic status of the animal examined.

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.

Inflammatory cytokines, pleuropneumonia infection and the effect of dexamethasone.

Objectives:Actinobacillus pleuropneumoniae causes an often fatal infection of swine due to pleuropneumonia. To determine if inflammatory cytokines are associated with A. pleuropneumoniae-induced pneumonia, infected and noninfected animals were concomitantly administered saline or dexamethasone. Methods: Twenty-four swine were treated with saline, A. pleuropneumoniae, dexamethasone, or A. pleuropneumoniae and dexamethasone (n = 6). The plasma levels of TNF-α, IL-1β, IL-6, IL-8, and IL-10 were examined through time of necropsy (72 h). Gross pathology and histopathology was performed on all animals. Results: Dexamethasone had no effect on A. pleuropneumoniae-induced increases in lung/body weight ratios. Gross pathology of the infected pigs included fibrinous pleuropneumonia with necrosis and hemorrhage in a focal to a multifocal pattern. Histopathology of infected pig lungs revealed necrotizing extensive, fibrinopurulent pneumonia with edema and fibrinopurulent pleuritis. Plasma IL-6 levels were elevated in A. pleuropneumoniae-infected animals beginning 6 h after infection. Dexamethasone treatment did not alter A. pleuropneumoniae-induced plasma IL-6 levels. A. pleuropneumoniae infection did not elicit plasma levels of TNF-α, IL-1β, IL-8, or IL-10. Conclusion: These results suggest that the pneumonia caused by A. pleuropneumoniae infection is not due to the release of systemic inflammatory cytokines.

Effects of intravenous administration of lipopolysaccharide on cytochrome P450 isoforms and hepatic drug metabolizing enzymes in swine.

OBJECTIVE To investigate effects of bacteria-mediated inflammation on hepatic drug metabolizing enzymes (DMEs) in swine via a lipopolysaccharide (LPS) challenge technique. ANIMALS 22 Poland China-Landrace crossbred barrows. PROCEDURES In experiment 1, 10 market-weight swine were treated with LPS (20 microg/kg, IV [n = 5 swine]) or sham-injected (5) 24 hours before slaughter. In experiment 2, 12 growing and finishing swine were treated with LPS at 2 or 20 microg/kg, IV (n = 3 swine/age group/treatment) 24 hours before slaughter. Hepatic DMEs, cytochrome P450 (CYP) isoforms, and CYP-mediated reactions were measured. RESULTS In experiment 1, LPS administered at 20 microg/kg decreased most hepatic DME components and inhibited enzymatic activities. In experiment 2, both doses reduced protein content in subcellular fractions and inhibited some DME- and CYP-mediated activities. In growing and finishing swine, CYP2A and CYP2B isoforms were not detected after treatment with LPS; the CYP1A2 isoform was eliminated in growing but not in finishing swine. Lipopolysaccharide also reduced CYP2D6 content in growing and finishing swine but increased CYP2E content. Lipopolysaccharide had no effect on swine CYP2C11, CYP2C13, or CYP3A content. The CYP2B-mediated 7-pentoxyresorufin O-dealkylase activity in growing and finishing swine was totally eliminated, and 7-ethoxyresorufin (indicating CYP1A activity) and aniline (mediated by CYP2E) metabolism was decreased. CONCLUSIONS AND CLINICAL RELEVANCE Effect of LPS treatment on swine CYPs appeared to be isoform specific; age-related metabolic status of the swine and the LPS dose modified this effect. Lipopolysaccharide-induced inflammation may affect metabolism of drugs and xenobiotics in swine.

Oxytetracycline-Mediated Alteration of Murine Immunocompetence

The immunomodulatory effect of oxytetracycline (OTC) on murine splenic lymphocytes (MSL), peritoneal exudate macrophage (PEM) functions and antibody production was examined. In vivo exposure to OTC slightly delayed initiation of antibody formation during the primary response. However, OTC exposure had no effect on either the peak time of antibody response or peak antibody titer. OTC also had no significant effect on the secondary antibody response. Mitogen-induced proliferation of MSL cocultured with OTC and pokeweed mitogen, phytohemagglutinin or concanavalin was equivocal. However, allogeneic stimulation of MSL was inhibited at 100 micrograms/ml OTC. There was also a decrease in the number of cells recovered. OTC had no effect on lymphocyte cytotoxicity in cells cultured in vitro. OTC inhibited the cytotoxic response of Corynebacterium parvum-elicited PEM at 10 micrograms/ml (effector:target of 10:1). Low levels of OTC (1-10 micrograms/ml) augmented the cytotoxic response (effector:target of 5:1). The effect of OTC on induction of PEM cytotoxicity was assessed by coculturing thioglycollate-elicited (TG) PEM in vitro with IFN-gamma and endotoxin along with 0-100 micrograms/ml OTC. Induction of cytotoxicity was inhibited at 0.5 microgram/ml. The effect of OTC on TG-PEM antimicrobial activity was assessed by measuring reduction of nitroblue tetrazolium (NBT) and cytochrome C. OTC inhibited the reduction of NBT at 500 micrograms/ml following PMA stimulation of TG-PEM. OTC had no effect on either NBT or cytochrome C reduction following stimulation with opsonized zymosan. These results demonstrate that OTC-mediated immunosuppression is a multifaceted event, with differing sensitivities both between immune cells and between different pathways within the same cell.