Haile F. Yancy

Potential Use of DNA Barcodes in Regulatory Science : Applications of the Regulatory Fish Encyclopedia

The use of a DNA-based identification system (DNA barcoding) founded on the mitochondrial gene cytochrome c oxidase subunit I (COI) was investigated for updating the U.S. Food and Drug Administration Regulatory Fish Encyclopedia (RFE; http://www.cfsan.fda.gov/-frf/rfe0.html). The RFE is a compilation of data used to identify fish species. It was compiled to help regulators identify species substitution that could result in potential adverse health consequences or could be a source of economic fraud. For each of many aquatic species commonly sold in the United States, the RFE includes high-resolution photographs of whole fish and their marketed product forms and species-specific biochemical patterns for authenticated fish species. These patterns currently include data from isoelectric focusing studies. In this article, we describe the generation of DNA barcodes for 172 individual authenticated fish representing 72 species from 27 families contained in the RFE. These barcode sequences can be used as an additional identification resource. In a blind study, 60 unknown fish muscle samples were barcoded, and the results were compared with the RFE barcode reference library. All 60 samples were correctly identified to species based on the barcoding data. Our study indicates that DNA barcoding can be a powerful tool for species identification and has broad potential applications.

Public health response to puffer fish (Tetrodotoxin) poisoning from mislabeled product.

Tetrodotoxin is a neurotoxin that occurs in select species of the family Tetraodontidae (puffer fish). It causes paralysis and potentially death if ingested in sufficient quantities. In 2007, two individuals developed symptoms consistent with tetrodotoxin poisoning after ingesting home-cooked puffer fish purchased in Chicago. Both the Chicago retailer and the California supplier denied having sold or imported puffer fish but claimed the product was monkfish. However, genetic analysis and visual inspection determined that the ingested fish and others from the implicated lot retrieved from the supplier belonged to the family Tetraodontidae. Tetrodotoxin was detected at high levels in both remnants of the ingested meal and fish retrieved from the implicated lot. The investigation led to a voluntary recall of monkfish distributed by the supplier in three states and placement of the supplier on the U.S. Food and Drug Administrations Import Alert for species misbranding. This case of tetrodotoxin poisoning highlights the need for continued stringent regulation of puffer fish importation by the U.S. Food and Drug Administration, education of the public regarding the dangers of puffer fish consumption, and raising awareness among medical providers of the diagnosis and management of foodborne toxin ingestions and the need for reporting to public health agencies.

Effects of live attenuated and killed Salmonella vaccine on T-lymphocyte mediated immunity in laying hens.

The impact of live and killed Salmonella vaccines on cell-mediated immunity (CMI) was investigated in 18- and 32-week-old White Leghorn chickens, by assessing splenic lymphocyte proliferation, expression of IL-2 mRNA in concanavalin A (Con A) stimulated cells and flow cytometric analysis of cell subpopulations. Con A and Salmonella enteritidis (SE) flagella induced proliferation of splenocytes were enhanced in the 18- and 32-week-old chickens treated with live vaccine, compared to the corresponding control chickens. Among the killed vaccine treated birds, Con A-mediated response was higher in the 18-week-old chickens compared to the corresponding control birds. Increased proliferation was accompanied by increased CD4 and reduced CD8 and gammadelta T-lymphocytes in the 18-week-old live vaccine treated chickens. Relative expression of IL-2 mRNA in Con A-stimulated splenocytes from 18-week-old birds was not affected by vaccine treatment. Overall, live vaccine was more effective in increasing the lymphocyte proliferation to Con A as well as SE antigen. This enhanced CMI may prove beneficial in protecting chickens against SE infection.

Immunophenotype and gene expression profile of mesenchymal stem cells derived from canine adipose tissue and bone marrow.

Veterinary adult stem cell therapy is an emerging area of basic and clinical research. Like their human counterparts, veterinary mesenchymal stem cells (MSCs) offer many potential therapeutic benefits. The characterization of canine-derived MSCs, however, is poorly defined compared to human MSCs. Furthermore, little consensus exists regarding the expression of canine MSC cell surface markers. To address this issue, this study investigated characteristics of cultured canine MSCs derived from both adipose tissue and bone marrow. The canine MSCs were obtained from donors of various breeds and ages. A panel of cell surface markers for canine MSCs was selected based on current human and canine literature and the availability of canine-reactive antibodies. Using flow cytometry, canine MSCs were defined to be CD90(+)CD44(+)MHC I(+)CD14(-)CD29(-)CD34(-)MHC II(-). Canine MSCs were further characterized using real-time RT-PCR as CD105(+)CD73(+)CD14(+)CD29(+)MHC II(+)CD45(-) at the mRNA level. Among these markers, canine MSCs differed from canine peripheral blood mononuclear cells (PBMCs) by the absence of CD45 expression at the mRNA level. A novel high-throughput canine-specific PCR array was developed and used to identify changes in the gene expression profiles of canine MSCs. Genes including PTPRC, TNF, β2M, TGFβ1, and PDGFRβ, were identified as unique to canine MSCs as compared to canine PBMCs. Our findings will facilitate characterization of canine MSCs for use in research and clinical trials. Moreover, the high-throughput PCR array is a novel tool for characterizing canine MSCs isolated from different tissues and potentially from different laboratories.

Differential cytokine mRNA expression in swine whole blood and peripheral blood mononuclear cell cultures.

The kinetics of interleukin-2 (IL-2), IL-6, IL-8 and IL-10 gene expression in concanavalin A (Con A)-activated whole blood (WB) and peripheral blood mononuclear cell (PBMC) cultures were examined using reverse transcriptase-polymerase chain reaction (RT-PCR). Unstimulated PBMC or WB cultures failed to show increases in basal cytokine PCR amplicon levels for any cytokine examined. PBMC cultures demonstrated peak expression of IL-2, IL-6, IL-8 and IL-10 mRNA levels at 12, 24, 24 and 6h, respectively. WB cultures exhibited peak IL-2, IL-6, IL-8 and IL-10 mRNA levels at 24, 12, 6 and 24h, respectively. PBMC cultures consistently exhibited higher levels of IL-2 mRNA at all times examined than did WB cultures. WB cultures consistently had higher levels of IL-6 mRNA than PBMC cultures. IL-8 and IL-10 protein levels in PBMC cultures were first detected 12h after stimulation and continued to increase in concentration through 48h. In WB cultures, IL-8 and IL-10 protein levels were first noted at 12 and 6h, respectively. WB culture IL-8 and IL-10 levels quickly reached equilibrium after being detected and remained at levels lower than those noted in PBMC cultures. These results show WB cultures represent an approach with reduced cost and time when compared to traditional cell culture and isolation methods. It may also produce an in vitro test system that more closely resembles in vivo conditions.

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.

Interlaboratory evaluation of a real-time multiplex polymerase chain reaction method for identification of salmon and trout species in commercial products.

This interlaboratory study evaluated a real-time multiplex polymerase chain reaction (PCR) method for identification of salmon and trout species in a range of commercial products in North America. Eighty salmon and trout products were tested with this method by three independent laboratories. Samples were collected in the United States and Canada, and only the collecting institution was aware of the species declaration. Following analysis with real-time PCR, all three laboratories were able to identify species in 79 of the 80 products, with 100% agreement on species assignment. A low level of fraud was detected, with only four products (5%) found to be substituted or mixtures of two species. The results for two of the fraudulent products were confirmed with alternate methods, but the other two products were heavily processed and could not be verified with methods other than real-time PCR. Overall, the results of this study show the usefulness and versatility of this real-time PCR method for the identification of commercial salmon and trout species.

Validation of a PCR-Based Method for the Detection of Various Rendered Materials in Feedstuffs Using a Forensic DNA Extraction Kit

A method trial was initiated to validate the use of a commercial DNA forensic kit to extract DNA from animal feed as part of a PCR-based method. Four different PCR primer pairs (one bovine pair, one porcine pair, one ovine primer pair, and one multispecies pair) were also evaluated. Each laboratory was required to analyze a total of 120 dairy feed samples either not fortified (control, true negative) or fortified with bovine meat and bone meal, porcine meat and bone meal (PMBM), or lamb meal. Feeds were fortified with the animal meals at a concentration of 0.1% (wt/wt). Ten laboratories participated in this trial, and each laboratory was required to evaluate two different primer pairs, i.e., each PCR primer pair was evaluated by five different laboratories. The method was considered to be validated for a given animal source when three or more laboratories achieved at least 97% accuracy (29 correct of 30 samples for 96.7% accuracy, rounded up to 97%) in detecting the fortified samples for that source. Using this criterion, the method was validated for the bovine primer because three laboratories met the criterion, with an average accuracy of 98.9%. The average false-positive rate was 3.0% in these laboratories. A fourth laboratory was 80% accurate in identifying the samples fortified with bovine meat and bone meal. A fifth laboratory was not able to consistently extract the DNA from the feed samples and did not achieve the criterion for accuracy for either the bovine or multispecies PCR primers. For the porcine primers, the method was validated, with four laboratories meeting the criterion for accuracy with an average accuracy of 99.2%. The fifth laboratory had a 93.3% accuracy outcome for the porcine primer. Collectively, these five laboratories had a 1.3% false-positive rate for the porcine primer. No laboratory was able to meet the criterion for accuracy with the ovine primers, most likely because of problems with the synthesis of the primer pair; none of the positive control DNA samples could be detected with the ovine primers. The multispecies primer pair was validated in three laboratories for use with bovine meat and bone meal and lamb meal but not with PMBM. The three laboratories had an average accuracy of 98.9% for bovine meat and bone meal, 97.8% for lamb meal, and 63.3% for PMBM. When examined on an individual laboratory basis, one of these four laboratories could not identify a single feed sample containing PMBM by using the multispecies primer, whereas the other laboratory identified only one PMBM-fortified sample, suggesting that the limit of detection for PMBM with this primer pair is around 0.1% (wt/wt). The results of this study demonstrated that the DNA forensic kit can be used to extract DNA from animal feed, which can then be used for PCR analysis to detect animal-derived protein present in the feed sample.

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.