Evaluation of Quality Control Methods for Foot-And-Mouth Disease Vaccines by High-Performance Liquid Chromatography.

Foot-and-mouth disease (FMD) is considered one of the highly contagious viral infections affecting livestock. In Korea, an FMD vaccination policy has been implemented nationwide since 2010 for the prevention and control of FMD. Since the vaccines are imported from various countries, standardized quality control measures are critical. In this study, we aimed to validate a high-performance liquid chromatography (HPLC) device in the Animal and Plant Quarantine Agency lab and identify an appropriate FMD vaccine pretreatment method for HPLC-a simple, reliable, and practical method to measure antigen content. Based on the analyses of specificity, linearity, accuracy, repeatability, intermediate precision, limits of detection, and limits of quantification using FMD standard samples, we validated the method using a standard material. Overall, we confirmed that the HPLC technique is effective for the quantitative assessment of the FMD virus 146S antigen in Korea. Using commercial FMD vaccines, we evaluated three separation methods and identified the method using n-pentanol and trichloroethylene as optimal for HPLC analysis. Our HPLC method was effective for the analytical detection of the antigen content in FMD vaccine, and it may be useful as a reference method for national lot-release testing.

Validation of a high performance liquid chromatography method for quantitation of foot-and-mouth disease virus antigen in vaccines and vaccine manufacturing.

Foot-and-mouth disease (FMD) is an infectious viral disease that affects the main meat and dairy production animals, including cattle, sheep, goats and swine. It is readily transmissible and countries where the disease is present suffer harsh international trade restrictions on livestock products and serious economic losses. Vaccines are important tools to contain outbreaks and maintain the status of free with or without vaccination, as defined by the World Organization for Animal Health (OIE). The efficacy of vaccines is reliant on the content and integrity of inactivated virus particles. The long-established method to quantify the viral content of vaccines along the manufacturing process and in the final product is the 140S sucrose density gradient analysis. This method has been a valuable tool for many decades. However, it requires gradient preparation for each sample, a lengthy ultracentrifugation and a manual UV reading of the gradient, rendering it highly operator dependent and almost impossible to automate. We present a method to quantify FMDV particles in vaccines and intermediate process samples that is based on separation of components by size exclusion high performance liquid chromatography (SE-HPLC) and measurement of virus by absorption at 254 nm. The method has been extensively validated; it is accurate, precise and linear. It is applicable to all FMDV strains and sample materials and has a good concordance with the 140S test. The proposed method uses off the shelf HPLC equipment and columns. It is easily automated for high throughput operation, affording a useful process analytical technology and a novel tool for control of final product by manufacturers and regulatory agencies.

National post-market surveillance assessment of veterinary medicines in Korea during the past decade.

BACKGROUND: Veterinary medicines have been widely used for the prevention and treatment of diseases, growth promotion, and to promote feeding efficacy in livestock. As the veterinary medicine industry has steadily grown, it is crucial to set up a baseline for the quality of medicine as well as the insufficiency or excessiveness of the active ingredients in drug products to ensure the compliance, safety and efficacy of these medicines. Thus, the 10 years data of post-marketing quality control study was summarized to determine the rate and extent of non-compliance of these medicines and to establish baseline data for future quality control measures of veterinary medicine. RESULTS: In this study, 1650 drugs for veterinary use were collected per year from each city and province in Korea and analysed for the quantity of active ingredients according to the "national post-market surveillance (NPMS) system" over the past decade. The NPMS assessment was performed using liquid and gas chromatography, titration, UV/Vis spectrophotometry, and bioassays. A total of 358 cases were deemed noncompliant, with the average noncompliance rate for all medicine types being 2.0%. The average noncompliance rates for antibiotics, biologics and other chemical drugs except antibiotics (OCD) were 1.1%, 1.2%, and 3.0%, respectively. The first leading cause for noncompliant products was insufficient quantity of major ingredients (283 cases), and the second leading cause was the existence of excess amount of active ingredients (60 cases). Tylosin, spiramycin, ampicillin, tetracyclines and penicillins were most frequently found to be noncompliant among antibiotics. Among the OCD, the noncompliance was found commonly in vitamin A. CONCLUSION: The overall trend presented gradually decreasing violation rates, suggesting that the quality of veterinary medicines has improved. Consistent application of the NPMS assessment and the establishment of the Korea Veterinary Good Manufacturing Practice (KVGMP) will help to maintain the good quality of medicine.

Characterization of Salmonella enterica Serovar 4,[5],12:i:- Isolates from Korean Food Animals.

Salmonella enterica subspecies enterica serovar 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium, has emerged as one of the most common serotypes related to human salmonellosis. In this study, the 22 isolates of S. 4,[5],12:i:- from food animals were identified by a specific multiplex polymerase chain reaction between 2009 and 2012. The isolation rate of S. 4,[5],12:i:- accounted for 1.7% (22/1271) of Salmonella spp. isolates from food animal origins: more specifically, 7.6% (18/235) from pigs and 0.6% (4/686) from chickens. The predominant S. 4,[5],12:i:- isolates in Korea belonged to phage type DT193 (12/22) with ampicillin-streptomycin-sulfonamide-tetracycline (ASSuT) resistance pattern (9/22). The XbaI-pulsed-field gel electrophoresis (PFGE) analysis revealed 11 different pulsotypes, and the major X-1 pattern was shared by 8 isolates. The isolates belonging to pattern X-1 were further subdivided into three BlnI-PFGE patterns and four variable-number tandem-repeat analysis (MLVA) allele combinations. The combining of MLVA and PFGE data could be valuable in characterizing highly clonal strains and discriminating their epidemiological relationship.

Enhancing effects of serum-rich and cytokine-supplemented culture conditions on developing blastocysts and deriving porcine parthenogenetic embryonic stem cells.

The present study was conducted to develop an effective method for establishment of porcine parthenogenetic embryonic stem cells (ppESCs) from parthenogenetically activated oocyte-derived blastocysts. The addition of 10% fetal bovine serum (FBS) to the medium on the 3rd day of oocyte culturing improved the development of blastocysts, attachment of inner cell masses (ICMs) onto feeder cells, and formation of primitive ppESC colonies. ICM attachment was further enhanced by basic fibroblast growth factor, stem cell factor, and leukemia inhibitory factor. From these attached ICMs, seven ppESC lines were established. ppESC pluripotency was verified by strong enzymatic alkaline phosphatase activity and the expression of pluripotent markers OCT3/4, Nanog, and SSEA4. Moreover, the ppESCs were induced to form an embryoid body and teratoma. Differentiation into three germ layers (ectoderm, mesoderm, and endoderm) was confirmed by the expression of specific markers for the layers and histological analysis. In conclusion, data from the present study suggested that our modified culture conditions using FBS and cytokines are highly useful for improving the generation of pluripotent ppESCs.

Determination of phthalates in raw bovine milk by gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS) and dietary intakes.

Low levels of phthalates, including di(2-ethylhexyl) phthalate (DEHP), in raw bovine milk were determined using gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). A fast and convenient process of sample treatment combined with TOF-MS analysis (medium resolution >5000), yielded good recoveries (85-125%) and low limits of detection (<0.002 mg kg(-1)). The most commonly used phthalate, DEHP, was found in 15 out of 30 samples monitored in this study. DEHP concentrations in raw milk ranged from not detected to 0.154 mg kg(-1), and the mean concentration was 0.057 mg kg(-1). The dietary intake of DEHP was about 0.004 mg kg(-1) body weight day(-1) if a child (24 months, 13 kg body weight) drinks 1 L day(-1) of milk that contains the mean concentration of DEHP found in raw milk. The estimated dietary intake corresponded to 8% of the European Union tolerable daily intake (TDI) of 0.05 mg kg(-1) body weight day(-1). Dimethyl phthalate (DMP) and di-n-butyl phthalate (DBP) were found from two and 20 samples, respectively, at low levels. Diethyl phthalate (DEP), butylbenzyl phthalate (BBP), and di-n-octyl phthalate (DnOP) were not found in any of the samples.

Relationship of PCDD/Fs congener profiles between beef and raw milk in South Korea.

The relationship between profiles of residual PCDD/Fs in beef and raw milk was examined by measuring concentrations and detected frequencies. Unrelated samples of beef and raw milk were collected from nine regions in South Korea. Congener-specific profiles of PCDD/Fs in beef and raw milk were very similar. PCDFs, particularly 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7,8-HxCDF, and 2,3,4,6,7,8-HxCDF, were dominant congeners in both beef and raw milk suggesting that sources of contamination may not significantly differ nationwide. The profiles of PCDD/Fs in domestic beef and raw milk in this study were closer to the profiles of emission from metal industries although Korea imports over 75% of feedingstuffs. The ratios of PCDF/PCDD in TEQ concentration were more than 5 and 15 in beef and raw milk, respectively. The mean concentrations of PCDD/Fs in 60 samples of beef and 60 samples of raw milk were 0.80 pg WHO-TEQ/g fat and 0.65 pg WHO-TEQ/g fat, respectively. The residual profiles of PCDD/Fs in raw milk resembled that in beef although the congener profiles might change throughout the food chain. This indicated that monitoring of dioxins in milk could provide information for contamination of milk itself or other associated food.

Evaluation of PCDD/Fs characterization in animal feed and feed additives.

Safety control of feed and feed additives is necessary to have safe food of animal origin. Based on media reports, nine incidents regarding dioxins and/or PCBs contaminations occurred worldwide during the last decade. Korea is a country which imports feed and feed additives. In this study, various kinds of feed and feed additives were analyzed to monitor the contamination level of dioxins. The level of PCDD/Fs in fish oil was the highest with a concentration of 23.33ngkg(-1), which is equivalent to a toxicological concentration of 4.68ngWHO-TEQ/kg. Feed from animals origin such as chicken meal, animal fat, fish meal, fish oil, and shell powder showed relatively higher concentrations of PCDD/Fs. Feed from plants origin, minerals, and additives ranged from non-detects for bit pulp and ethoxyquin to 8.28ngkg(-1) for dl-methionine. From a toxicological point of view, the highest concentration in vitamins was 0.08ngWHO-TEQ/kg among the feed additives. 2,3,4,7,8-PeCDF was the dominant congener in samples of fish oil, fish meal, and shell powder. Animal fat showed that the pattern of PCDD/Fs depends on the sources of contamination. A sample of animal fat showed 1,2,3,4,7,8-HxCDF and the other sample showed 1,2,3,4,7,8-HxCDD as a primary congener. Generally, low levels of PCDDs were detected in feed additives. Patterns of PCDD/Fs in choline chloride were different with that in choline chloride from an incident in Europe in 2000.

Background levels and dietary intake of PCDD/Fs in domestic and imported meat in South Korea.

A survey was conducted in South Korea to determine residual levels and dietary intake of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) from meat. Altogether 119 domestic and 164 imported samples of beef, pork, and chicken were examined. The mean levels of PCDD/Fs in upper bound were 0.21, 0.22, and 0.04pg WHO-TEQ/g fat for beef, pork, and chicken, respectively. The low level of PCDD/Fs in chicken probably resulted from the low fat content in the samples used. The samples were separated into domestic and imported products in order to investigate the sources of contamination. PCDFs were the more dominant congeners in domestic beef and were similar to those found from emission of incineration. However, the congener profiles of domestic beef and incineration were difficult to compare because the environmental fate and animal metabolism were involved. The upper bound dietary intake of PCDD/Fs from beef, pork and chicken was calculated to be 0.04pg/WHO-TEQ/kgbw/day. The combined consumption of beef, pork, and chicken was found to be 84.8g per day for a person weighing 60kg and represented 5.7% of their total daily food intake.

Evaluation of two fast and easy methods for pesticide residue analysis in fatty food matrixes.

Two rapid methods of sample preparation and analysis of fatty foods (e.g., milk, eggs, and avocado) were evaluated and compared for 32 pesticide residues representing a wide range of physicochemical properties. One method, dubbed the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for pesticide residue analysis, entailed extraction of 15 g sample with 15 mL acetonitrile (MeCN) containing 1% acetic acid followed by addition of 6 g anhydrous magnesium sulfate and 1.5 g sodium acetate. After centrifugation, 1 mL of the buffered MeCN extract underwent a cleanup step (in a technique known as dispersive solid-phase extraction) using 50 mg each of C18 and primary secondary amine sorbents plus 150 mg MgSO4. The second method incorporated a form of matrix solid-phase dispersion (MSPD), in which 0.5 g sample plus 2 g C18 and 2 g anhydrous sodium sulfate was mixed in a mortar and pestle and added above a 2 g Florisil column on a vacuum manifold. Then, 5 x 2 mL MeCN was used to elute the pesticide analytes from the sample into a collection tube, and the extract was concentrated to 0.5 mL by evaporation. Extracts in both methods were analyzed concurrently by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry. The recoveries of semi-polar and polar pesticides were typically 100% in both methods (except that basic pesticides, such as thiabendazole and imazalil, were not recovered in the MSPD method), but recovery of nonpolar pesticides decreased as fat content of the sample increased. This trend was more pronounced in the QuEChERS method, in which case the most lipophilic analyte tested, hexachlorobenzene, gave 27 +/- 1% recovery (n=6) in avocado (15% fat) with a<10 ng/g limit of quantitation.