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.

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.

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.

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.

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.

Inflammatory mediator production in swine following endotoxin challenge with or without co-administration of dexamethasone.

The inflammatory response in swine challenged with lipopolysaccharide (LPS) has only been partially characterized. As swine are increasingly used in biomedical research, it is important to determine if they respond to endotoxin challenge in a manner similar to other model systems. Accordingly, 24 Poland China x Landrace barrows were treated with saline, LPS, dexamethasone, or LPS and dexamethasone, with six animals in each treatment group. The kinetics of TNFalpha, IL-1beta, IL-6, IL-8, IL-10, nitric oxide (nitrate/nitrite), and neopterin production in swine plasma were examined at 1, 3, 6, 9, and 24 h after acute LPS challenge. Lipopolysaccharide increased plasma TNFalpha levels, which peaked 1 h post-challenge. Dexamethasone decreased LPS-induced TNFalpha by approximately 60%. Plasma IL-6 levels peaked 3 h post-LPS challenge, returning to basal levels by 9 h. Swine given both LPS and dexamethasone had minimal IL-6 levels. Control and dexamethasone-only treated animals never exhibited systemic TNFalpha or IL-6 levels. Lipopolysaccharide increased plasma IL-10 1 h after challenge. Dexamethasone did not alter plasma IL-10 levels in LPS-challenged swine. Interleukin-1beta was constitutively present in plasma and was not altered by any combination of treatments. Plasma IL-8 was not observed in any treatment group. Plasma nitrate/nitrite levels were maximal 24 h post-challenge. Dexamethasone treatment prevented increases in plasma nitrate/nitrite levels in LPS-treated animals. Lipopolysaccharide induced levels of neopterin; dexamethasone served to further increase plasma neopterin levels in LPS-challenged animals. The discordant regulation of inflammatory mediators suggests that the immunological responses by swine to LPS are distinct from the responses seen in rodent and human studies.

Characterization of a polymerase chain reaction-based approach for the simultaneous detection of multiple animal-derived materials in animal feed.

In this study, a polymerase chain reaction (PCR) primer set capable of amplifying a mitochondrial DNA segment of multiple species (cattle, sheep, goats, deer, and elk) whose rendered remains are prohibited from being fed to ruminants was characterized. However, the primer set also amplifies DNA derived from the rendered remains of pigs and horses, which are exempt from the feed ban. PCR amplicons derived from pig DNA have a restriction endonuclease site recognized by Hinf1, while the horse DNA-derived amplicon has a unique restriction endonuclease site recognized by HypCH4III. This "universal" PCR primer produced an amplicon with DNA extracted from dairy feed containing either bovine meat and bone meal or pig blood meal. Enzymatic digestion of the PCR amplicons from these feed samples with Hinf1 resulted in cleavage products only from samples containing pig blood meal. However, Hinf1 digestion of these amplicons was not complete. Further analysis of the pig blood meal with primers specific for bovine or porcine DNA demonstrated the presence of both bovine- and porcine-derived DNA. Enzymatic digestion confirmed these findings. Additional testing was conducted with dry dog food samples labeled as containing either lamb, chicken, turkey, or chicken and fish. The universal PCR primer produced an amplicon only for the dog food containing lamb meal. This paper is the first to describe a simplified approach for the detection of the prohibited species of concern in the feed ban.

Development of a polymerase chain reaction-based method to identify species-specific components in dog food.

OBJECTIVES: To determine whether there is a relationship between species-specific mitochondrial DNA (mtDNA), especially canine and feline mtDNA, and detectable amounts of pentobarbital in previously analyzed dog food samples. SAMPLE POPULATION: 31 dog food samples previously analyzed for pentobarbital (limit of detection, 1 microg/kg). PROCEDURE: Polymerase chain reaction (PCR) analysis was performed on dog food samples by use of PCR primers specific for either canine, feline, equine, bovine, porcine, ovine, or poultry mtDNA. RESULTS: PCR amplicons specific for feline or canine mtDNA at a 0.007% (70 microg/g [wt/wt basis]) or 0.0007% (7 microg/g) level, respectively, were not found in the 31 dog food samples. Most of the 31 dog food samples had a PCR amplicon on PCR analysis when a PCR primer set capable of simultaneously detecting mtDNA of cows, pigs, sheep, goats, deer, elk, and horses was used. Results of PCR analysis by use of primers specific for bovine, swine, sheep and goat, or horse mtDNA revealed amplicons specific for bovine or swine mtDNA only in 27 of the 31 samples. Analysis of the remaining 4 samples failed to yield amplicons for any mammalian mtDNA. Pentobarbital was detected in 2 of these 4 samples. Results of PCR analysis correlated with the stated ingredient list for most, but not all samples. CONCLUSIONS AND CLINICAL RELEVANCE: Because canine and feline mtDNA were not found in a set of retail dog food samples, these results indicate that the source of pentobarbital in dog food is something other than proteins from rendered pet remains.

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.

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-alpha, IL-1beta, 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-alpha, IL-1beta, 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.

The effect of endotoxin and dexamethasone on enrofloxacin pharmacokinetic parameters in swine.

The impact of Escherichia coli-derived lipopolysaccharide (LPS) on the pharmacokinetic parameters of enrofloxacin in swine was assessed to determine whether this model would substitute for a pleuropneumonia infection model for pharmacokinetic evaluation of drugs. All animals received a single i.v. dose of enrofloxacin (5 mg/kg). Half the animals also received dexamethasone (0.5 mg/kg) to determine the impact of inflammation on any changes in enrofloxacin pharmacokinetics, as most of the effects of LPS are due to elaboration of inflammatory mediators. Administration of LPS alone (2.0 microg/kg) was associated with a decrease in clearance of enrofloxacin. Volume of distribution at steady state was increased in the dexamethasone-treated animals. The terminal elimination half-life of enrofloxacin was significantly increased in the LPS group. Dexamethasone administration, either alone or in combination with LPS challenge, increased the volume of distribution both at steady state and during the elimination phase. Lipopolysaccharide challenge did not affect the volume of distribution. Lipopolysaccharide challenge did not affect urinary excretion of enrofloxacin but did increase the urinary excretion of its principal metabolite, ciprofloxacin. However, the increased excretion did not begin until 24 h after administration of enrofloxacin. Because these pharamcokinetic results are different from those obtained with the pleuropneumonia model using the bacteria Actinobacillus pleuropneumoniae, the results of this study demonstrate that LPS is not a generic substitute for infection for the pharmacokinetic evaluation of drugs.

Influence of porcine Actinobacillus pleuropneumoniae infection and dexamethasone on the pharmacokinetic parameters of enrofloxacin.

The impact of Actinobacillus pleuropneumoniae (APP) infection in swine on the pharmacokinetic parameters of enrofloxacin were determined. Twenty-four animals were used in a 2 x 2 factorial of treatment groups (six animals per group) to determine the impact of APP-induced inflammation and the anti-inflammatory drug dexamethasone on enrofloxacin pharmacokinetic parameters. All animals received enrofloxacin as a single intravenous dose (5 mg/kg). Administration of dexamethasone was associated with an increase in clearance of enrofloxacin Clearance of enrofloxacin was not affected by APP. Volume of distribution at steady state was significantly increased in the dexamethasone-treated pigs. Volume of distribution at steady state was decreased by APP infection. Dexamethasone significantly increased the terminal elimination half-life of enrofloxacin. APP infection decreased the terminal elimination half-life of enrofloxacin in the infected pigs. Infection and dexamethasone significantly decreased the urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios. This study shows that APP infection does affect plasma pharmacokinetic parameters. Dexamethasone and APP infection may reduce renal clearance of enrofloxacin with a compensatory increase in intestinal clearance. Neither infection nor dexamethasone altered the metabolism of enrofloxacin to ciprofloxacin, the principal metabolite of enrofloxacin.