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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.

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.

Rapid and reliable detection of the African swine fever virus (ASFV), the causative agent of often fatal African swine fever, in animal feed is critical for implementing timely emergency control measures. We developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the ASFV p72 gene ( , encoding the major capsid protein) compatible with animal feed. Assay performance (referred to as LAMP1 in this study) was evaluated in comparison with a previously published topoisomerase II gene-based LAMP assay (LAMP2) and three p72 gene-based real-time PCR assays [two recommended by the World Organisation for Animal Health (WOAH) and the third one developed and currently used by the U.S. Department of Agriculture]. LAMP1 was the fastest, with positive results obtained in as early as 3.8 min [compared to at least 5.5 min for LAMP2 and 20 cycles for real-time PCRs (~15 min)]. These assays could detect ASFV from 10 to 10 copies using synthetic DNA. LAMP1 detected as low as 10 TCID /mL of ASFV BA71V stock and had a comparable performance to the USDA real-time PCR assay using both inclusivity (36 ASFV synthetic DNAs and isolates) and exclusivity (13 porcine viruses) panels. On applying six different DNA extraction methods to a variety of animal feed sample types (e.g., complete swine feed, soybean meal), variable yet mostly limited assay inhibitions were observed. When swine feed was inoculated with the ASFV BA71V stock at 10 TCID /g, the newly developed LAMP1 assay reliably detected the virus within 7 min, in contrast to at least 20 min by the USDA real-time PCR (29 cycles). Further validation of this novel p72 gene-based LAMP assay in animal feed will pave the way for its adoption as a rapid screening tool in ASFV feed surveillance, as well as potential application in outbreak response and recovery efforts, to safeguard the nation's animal feed supply.

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.

The U.S. Food, Drug, and Cosmetic Act prohibits the distribution of food that is adulterated, and the regulatory mission of the U.S. Food and Drug Administration (FDA) is to enforce this Act. FDA field laboratories have identified the 22 most common pests that contribute to the spread of foodborne disease (the \"Dirty 22\"). The current method of detecting filth and extraneous material (tails, legs, carcasses, etc.) is visual inspection using microscopy. Because microscopy can be time-consuming and may yield inaccurate and/or nonspecific results due to lack of expertise, an alternative method of detecting these adulterants is needed. In this study, we sequenced DNA from the 5′ region of the cytochrome oxidase I gene of these 22 common pests that contribute to the spread of foodborne pathogens. Here, we describe the generation of DNA barcodes for all 22 species. To date, this is the first attempt to develop a sequence-based regulatory database and systematic primer strategy to identify these FDA-targeted species. DNA barcoding can be a powerful tool that can aid the FDA in promoting the protection and safety of the U.S. food supply.

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.

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.

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a \"ruminant\" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.