Unexpected thermal stability of two enveloped megaviruses, Emiliania huxleyi virus and African swine fever virus, as measured by viability PCR.

BACKGROUND: The particle structure of Emiliania huxleyi virus (EhV), an algal infecting member of nucleocytoplasmic large DNA viruses (NCLDVs), contains an outer lipid membrane envelope similar to that found in animal viruses such as African swine fever virus (ASFV). Despite both being enveloped NCLDVs, EhV and ASFV are known for their stability outside their host environment. METHOD: Here we report for the first time, the application of a viability qPCR (V-qPCR) method to describe the unprecedented and similar virion thermal stability of both EhV and ASFV. This result contradicts the cell culture-based assay method that suggests that virus "infectivity" is lost in a matter of seconds (for EhV) and minutes (for ASFV) at temperature greater than 50 °C. Confocal microscopy and analytical flow cytometry methods was used to validate the V-qPCR data for EhV. RESULTS: We observed that both EhV and ASFV particles has unprecedented thermal tolerances. These two NCLDVs are exceptions to the rule that having an enveloped virion anatomy is a predicted weakness, as is often observed in enveloped RNA viruses (i.e., the viruses causing Porcine Reproductive and Respiratory Syndrome (PRRS), COVID-19, Ebola, or seasonal influenza). Using the V-qPCR method, we confirm that no PRRSV particles were detectable after 20 min of exposure to temperatures up to 100 °C. We also show that the EhV particles that remain after 50 °C 20 min exposure was in fact still infectious only after the three blind passages in bioassay experiments. CONCLUSIONS: This study raises the possibility that ASFV is not always eliminated or contained after applying time and temperature inactivation treatments in current decontamination or biosecurity protocols. This observation has practical implications for industries involved in animal health and food security. Finally, we propose that EhV could be used as a surrogate for ASFV under certain circumstances.

Impact of ABCB1 genotype in Collies on the pharmacokinetics of R- and S-fexofenadine.

Thirty-two Collies were used to determine the impact of ABCB1 genotype and phenotype on the plasma pharmacokinetics of fexofenadine's (Fex) R- and S-enantiomers after bolus Fex administration, as human P-gp exhibits stereoselectivity. Each Collie's ABCB1 genotype and ivermectin (IVM) sensitivity (phenotype) was determined prior to study enrolment. Wild-type (WT) Collies had lower plasma concentrations of the individual enantiomers as compared to heterozygous IVM nonsensitive (HNS), heterozygous IVM-sensitive (HS) and homozygous mutant (MUT) Collies. Based on pairwise statistical comparison, WT Collies had statistically significantly lower (AUC0-last ) and peak (Cmax ) values compared to HS, HNS and MUT Collies. Tmax was not influenced by genotype/phenotype. Inter-individual variability in PK metrics tended to be largest for WT Collies. Although the influence of genotype/phenotype on Fex PK occurred with the individual isomers, impairment of S-Fex absorption, particularly in the MUT dogs, exceeded that associated with R-Fex. Since Fex elimination occurs primarily via biliary excretion via a transporter other than P-glycoprotein, and based upon our understanding of Fex absorption kinetics, we attributed these differences primarily to the absorption portion of the profile. These differences are expressed in a stereo-specific manner. These results demonstrate the potential negative impact on estimates of drug effectiveness and toxicity, especially for P-gp substrates that do not exhibit Central Nervous System toxicities.

Influence of ABCB1 Genotype in Collies on the Pharmacokinetics and Pharmacodynamics of Loperamide in a Dose-Escalation Study.

Thirty-three Collies (14 male and 19 female) were used in a dose-escalation study to determine the impact of ABCB1 genotype on loperamide pharmacokinetics (PK) and pharmacodynamics (PD). Loperamide was orally administered in four ascending doses (0.01, 0.05, 0.1, or 0.2 mg/kg) over a 4-wk period to fasted Collies. Comparisons were made within each dose to genotype, phenotype, and whether Collies received three (3D) or four (4D) loperamide doses. The 3D and 4D groupings had statistically significant differences in systemic drug exposure (defined by the area under the concentration-versus-time profile estimated from time zero to the last quantifiable drug concentration, AUC0-last). In contrast, statistical differences in AUC0-last only occurred in the comparison between wild-type (WT) Collies versus homozygous mutant (Mut) Collies administered 0.1 mg/kg. Statistical differences in the proportionality relationship were observed when comparing 3D to 4D Collies, and the WT to Mut Collies. Intersubject variability in drug exposure tended to be twice as high between Mut and WT Collies. Associations were observed between systemic drug exposure and ataxia or depression but not between systemic drug exposure and mydriasis or salivation. Thus, Collies expressing the greatest sensitivity to CNS-associated effects of loperamide (Mut) tended to have higher drug exposure compared with those less sensitive to the adverse effects of loperamide. Genotype and phenotype only partially explained differences in loperamide PK and PD, suggesting this relationship may not be straightforward and that other factors need to be considered. Accordingly, the PD and PK of one P-glycoprotein substrate only partially predicted the likelihood of adverse responses to unrelated substrates.

Survival of a surrogate African swine fever virus-like algal virus in feed matrices using a 23-day commercial United States truck transport model.

African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA viruses (NCLDVs) and is stable in a variety of environments, including animal feed ingredients as shown in previous laboratory experiments and simulations. Emiliania huxleyi virus (EhV) is another member of the NCLDVs, which has a restricted host range limited to a species of marine algae called Emiliania huxleyi. This algal NCLDV has many similar morphological and physical characteristics to ASFV thereby making it a safe surrogate, with results that are applicable to ASFV and suitable for use in real-world experiments. Here we inoculated conventional soybean meal (SBMC), organic soybean meal (SBMO), and swine complete feed (CF) matrices with EhV strain 86 (EhV-86) at a concentration of 6.6 × 107 virus g-1, and then transported these samples in the trailer of a commercial transport vehicle for 23 days across 10,183 km covering 29 states in various regions of the United States. Upon return, samples were evaluated for virus presence and viability using a previously validated viability qPCR (V-qPCR) method. Results showed that EhV-86 was detected in all matrices and no degradation in EhV-86 viability was observed after the 23-day transportation event. Additionally, sampling sensitivity (we recorded unexpected increases, as high as 49% in one matrix, when virus was recovered at the end of the sampling period) rather than virus degradation best explains the variation of virus quantity observed after the 23-day transport simulation. These results demonstrate for the first time that ASFV-like NCLDVs can retain viability in swine feed matrices during long-term transport across the continental United States.

A single-laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance.

The U.S. Food and Drug Administration is responsible for ensuring that the nation's food supply is safe and accurately labeled. This task is particularly challenging in the case of seafood where a large variety of species are marketed, most of this commodity is imported, and processed product is difficult to identify using traditional morphological methods. Reliable species identification is critical for both foodborne illness investigations and for prevention of deceptive practices, such as those where species are intentionally mislabeled to circumvent import restrictions or for resale as species of higher value. New methods that allow accurate and rapid species identifications are needed, but any new methods to be used for regulatory compliance must be both standardized and adequately validated. "DNA barcoding" is a process by which species discriminations are achieved through the use of short, standardized gene fragments. For animals, a fragment (655 base pairs starting near the 5' end) of the cytochrome c oxidase subunit 1 mitochondrial gene has been shown to provide reliable species level discrimination in most cases. We provide here a protocol with single-laboratory validation for the generation of DNA barcodes suitable for the identification of seafood products, specifically fish, in a manner that is suitable for FDA regulatory use.

Real-time PCR assay for the detection of pufferfish products.

An assay was developed for the rapid detection of products containing tissues from potentially toxic pufferfish (family Tetraodontidae), as part of the U.S. Food and Drug Administration Center for Veterinary Medicine and Center for Food Safety and Applied Nutrition's charter to protect human health. In this study, we developed a TaqMan assay derived from DNA barcode data (650 bp starting at the 5' end of the mitochondrial cytochrome c oxidase I gene) for the specific detection of pufferfish. The method requires only 1 h of total run time, a significant improvement over current methods, which can require 24 to 96 h for completion. The probes were tested against 105 species of fish and were able to detect 20 species of pufferfish; no cross-reactivity was shown with 85 species of nonpufferfish, including 20 related species from the same order (Tetraodontiformes). These results demonstrate that this assay is suitable for the rapid and specific detection of pufferfish and that it could be a useful regulatory tool to protect human health.

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.

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 Administration's 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.

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.

Metastatic progression and gene expression between breast cancer cell lines from African American and Caucasian women.

African American (AA) women have a lower overall incidence of breast cancer than do Caucasian (CAU) women, but a higher overall mortality. Little is known as to why the incidence of breast cancer is lower yet mortality is higher in AA women. Many studies speculate that this is only a socio-economical problem. This investigation suggests the possibility that molecular mechanisms contribute to the increased mortality of AA women with breast cancer. This study investigates the expression of 14 genes which have been shown to play a role in cancer metastasis. Cell lines derived from AA and CAU patients were analyzed to demonstrate alterations in the transcription of genes known to be involved in cancer and the metastatic process. Total RNA was isolated from cell lines and analyzed by RT-PCR analysis. Differential expression of the 14 targeted genes between a spectrum model (6 breast cancer cell lines and 2 non-cancer breast cell lines) and a metastasis model (12 metastatic breast cancer cell lines) were demonstrated. Additionally, an in vitro comparison of the expression established differences in 5 of the 14 biomarker genes between African American and Caucasian breast cell lines. Results from this study indicates that altered expression of the genes Atp1b1, CARD 10, KLF4, Spint2, and Acly may play a role in the aggressive phenotype seen in breast cancer in African American women.

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.

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 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.

Comparative study of matrix metalloproteinase expression between African American and Caucasian Women.

To date there are 26 human matrix metalloproteinases (MMPs) which are classified according to their substrate specificity and structural similarities. The four major subgroups of MMPs are gelatinases, interstitial collagenases, stromelysins, and membrane-type matrix metalloproteinases (MT-MMPs). This study investigates the expression of 26 MMPs, which have been shown to play a role in cancer metastasis. Breast tissues and cell lines derived from African American patients and Caucasian patients were assayed to demonstrate alterations in the transcription of genes primarily responsible for degrading the extracellular matrix (ECM). The expression levels of the extracellular matrix and adhesion molecules were analyzed using the gene array technology. Steady state levels of mRNAs were validated by RT-PCR analysis. Total RNA was isolated from tissue and cell lines and used in the RT-PCR assays. From this data, differential expression of MMPs between 6 breast cancer cell lines and 2 non-cancer breast cell lines was demonstrated. We have performed an in vitro comparison of MMP expression and established differences in 12 MMPs (3, 7, 8, 9, 11-15, 23B, 26, and 28) expression between African American and Caucasian breast cell lines. Thus, evidence indicates that altered expression of MMPs may play a role in the aggressive phenotype seen in African American women.

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.

Identification of potential biomarkers of P-glycoprotein substrate neurotoxicity in transgenic mice expressing the mutated canine ABCB1 gene.

OBJECTIVE: To identify biomarkers of P-glycoprotein (P-gp) substrate neurotoxicity in transgenic mice expressing the mutant canine ABCB1 gene (ABCB1-1Δ). ANIMALS: 8 ABCB1 knock-in and knock-out transgenic mice expressing the ABCB1-1Δ gene and 8 control mice expressing the wild-type canine ABCB1 gene (ABCB1-WT). PROCEDURES: Groups including 2 ABCB1-1Δ mutant mice and 2 ABCB1-WT mice were administered the P-gp substrates ivermectin (10 mg/kg, SC), doramectin (10 mg/kg, SC), moxidectin (10 mg/kg, PO), or digoxin (1.53 mg/kg, SC). A toxicogenomic approach based on DNA microarrays was used to examine whole-genome expression changes in mice administered P-gp substrates. RESULTS: Compared with control ABCB1-WT mice, ABCB1-1Δ mutant mice developed neurotoxic signs including ataxia, lethargy, and tremors similar to those reported for dogs with the ABCB1-1Δ mutation. Microarray analysis revealed that gene expression was altered in ABCB1-1Δ mutant mice, compared with findings for ABCB1-WT mice, following administration of the same P-gp substrates. Gene pathway analysis revealed that genes with a ≥ 2-fold gene expression change were associated with behavior and nervous system development and function. Moreover, 34 genes were altered in the ABCB1-1Δ mutant mice in all 4 drug treatment groups. These genes were also associated with behavior, which was identified as the top-ranked gene network. CONCLUSIONS AND CLINICAL RELEVANCE: These study data have facilitated understanding of the molecular mechanisms of neurotoxicosis in ABCB1-1Δ mutant mice following exposure to various P-gp substrates. Some genes appear to be potential biomarkers of P-gp substrate neurotoxicity that might be used to predict the safety of those drugs in dogs with the ABCB1-1Δ mutation.

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, ß2M, TGFß1, and PDGFRß, 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.