Administered chrysanthemum flower oil attenuates hyperuricemia: mechanism of action as revealed by DNA microarray analysis.

We applied Chrysanthemum flower oil (CFO) to a hyperuricemia model by feeding rats a hyperuricemia-inducing diet (HID) and investigated its effect on serum uric acid (SUA) levels and its mode of action. CFO is the oily fraction that contains polyphenols derived from chrysanthemum flowers. Oral administration of CFO to HID-fed rats significantly decreased their SUA levels. It also inhibited xanthine oxidase activities in the liver and increased urine uric acid levels. The effects of CFO on the renal gene expressions that accompanied the induction of hyperuricemia were comprehensively confirmed by DNA microarray analysis. The analysis showed up-regulation of those genes for uric acid excretion by CFO administration. These results suggest that CFO suppresses the increase in SUA levels via two mechanisms: suppression of uric acid production by inhibition of xanthine oxidase in the liver and acceleration of its excretion by up-regulation of uric acid transporter genes in the kidney.

Arthroscopic, histological and MRI analyses of cartilage repair after a minimally invasive method of transplantation of allogeneic synovial mesenchymal stromal cells into cartilage defects in pigs.

BACKGROUND AIMS: Transplantation of synovial mesenchymal stromal cells (MSCs) may induce repair of cartilage defects. We transplanted synovial MSCs into cartilage defects using a simple method and investigated its usefulness and repair process in a pig model. METHODS: The chondrogenic potential of the porcine MSCs was compared in vitro. Cartilage defects were created in both knees of seven pigs, and divided into MSCs treated and non-treated control knees. Synovial MSCs were injected into the defect, and the knee was kept immobilized for 10 min before wound closure. To visualize the actual delivery and adhesion of the cells, fluorescence-labeled synovial MSCs from transgenic green fluorescent protein (GFP) pig were injected into the defect in a subgroup of two pigs. In these two animals, the wounds were closed before MSCs were injected and observed for 10 min under arthroscopic control. The defects were analyzed sequentially arthroscopically, histologically and by magnetic resonance imaging (MRI) for 3 months. RESULTS: Synovial MSCs had a higher chondrogenic potential in vitro than the other MSCs examined. Arthroscopic observations showed adhesion of synovial MSCs and membrane formation on the cartilage defects before cartilage repair. Quantification analyses for arthroscopy, histology and MRI revealed a better outcome in the MSC-treated knees than in the non-treated control knees. CONCLUSIONS: Leaving a synovial MSC suspension in cartilage defects for 10 min made it possible for cells to adhere in the defect in a porcine cartilage defect model. The cartilage defect was first covered with membrane, then the cartilage matrix emerged after transplantation of synovial MSCs.

Phenolics from Glycyrrhiza glabra roots and their PPAR-gamma ligand-binding activity.

Bioassay-guided fractionation of the EtOH extract of licorice (Glycyrrhiza glabra roots), using a GAL-4-PPAR-gamma chimera assay method, resulted in the isolation of 39 phenolics, including 10 new compounds (1-10). The structures of the new compounds were determined by analysis of their spectroscopic data. Among the isolated compounds, 5'-formylglabridin (5), (2R,3R)-3,4',7-trihydroxy-3'-prenylflavane (7), echinatin, (3R)-2',3',7-trihydroxy-4'-methoxyisoflavan, kanzonol X, kanzonol W, shinpterocarpin, licoflavanone A, glabrol, shinflavanone, gancaonin L, and glabrone all exhibited significant PPAR-gamma ligand-binding activity. The activity of these compounds at a sample concentration of 10microg/mL was three times more potent than that of 0.5microM troglitazone.

Mannequin simulation improves the confidence of medical students performing tube thoracostomy: a prospective, controlled trial.

This study was undertaken to determine the educational benefits of mannequin simulation for performance of tube thoracostomy in a porcine model by medical students. Thirty medical students were randomized into two groups; the first performed tube thoracostomy on a mannequin simulator and then a porcine model; the second used only the porcine model. Performance measures included completion of subtasks, time to perform the procedure, a global score assigned by faculty raters, and a self-evaluation survey. Subtask completion rate was similar in both groups (P > 0.05). Mean time to perform the procedure was 9.8 minutes (+/- 0.9, simulator), and 9.3 minutes (+/- 1.0, nonsimulator, P > 0.05). Global scores were 12.3 (+/- 1.3, simulator) and 11.0 (+/- 1.4, non-simulator, P > 0.05). Self-evaluation of confidence (1 = "very", 7 = "not at all") showed the simulator group was significantly more confident (3.4 +/- 0.42) than the nonsimulator group (4.7 +/- 0.49, P < 0.05). All students met basic competencies to perform tube thoracostomy. The simulator group felt significantly more confident to subsequently perform the procedure on a patient, whereas performance was not statistically significantly different for the two groups. Further trials may be needed to delineate the optimal role for these simulators in teaching tube thoracostomy.

In vivo pharmacokinetics of ketoprofen after patch application in the Mexican hairless pig.

To evaluate the pharmacokinetics of topical drugs, in vitro permeation studies are performed using sacrificed pig skin or human tissues resected at surgery; however, these methods have their limitations in in vivo pharmacokinetics. This study examined the usefulness of Mexican hairless pigs for in vivo pharmacokinetic study, especially the drug concentration in the tissues. A ketoprofen patch was applied on the back of Mexican hairless pigs for 24 h, followed by sequential collection of blood specimens from 0 to 36 h (n=3). Also, the skin, subcutaneous fat, fascia and muscle from the center of the site of application were excised at 12 h after the application (n=4). Ketoprofen was first detected in the plasma at 8 h, the concentration increasing up to 24 h; the plasma concentration began to decrease after the removal of the ketoprofen patch. Ketoprofen concentrations in the tissues decreased with increasing depth of the tissues, but the values in the deep muscles, being the lowest among the tissues examined, were still higher than those in the plasma. While the data of drug concentration in human tissue are difficult to test, the Mexican hairless pig model appears to be attractive for in vivo pharmacokinetic studies of topically applied ketoprofen.

Clinical analysis of EGFR-positive non-small cell lung cancer patients treated with first-line afatinib: A Nagano Lung Cancer Research Group.

BACKGROUND: In the LUX-Lung 3 and LUX-Lung 6 trials, afatinib improved overall survival in previously untreated patients with EGFR 19del mutated non-small cell lung cancer (NSCLC) compared to chemotherapy. The appropriate management of adverse events and dose reduction of afatinib are important for EGFR-positive NSCLC patients. We conducted a retrospective and observational study of patients treated with first-line afatinib for EGFR-positive NSCLC in Nagano prefecture, Japan, focusing on efficacy and toxicities. METHODS: We retrospectively collected the medical records of NSCLC patients initially treated with afatinib between May 2014 and March 2018. RESULTS: A total of 62 patients with a median age of 67 years and a median body surface area (BSA) of 1.57 m2 were included. The overall response rate was 87.7% and median progression-free survival (PFS) was 15.7 months. The median PFS was similar between standard initial dose (40 mg) and reduced initial doses (30 and 20 mg) (15.7 vs. 14.2 months; P = 0.978). The frequency of dose reduction and the discontinuation rate in the 40 mg daily dose group was higher in patients with BSA < 1.58 m2 (100%) compared to BSA ≥ 1.58 m2 (68.2%) (P = 0.014). The frequency of diarrhea was higher in patients with BSA < 1.58 m2 (93.5%) compared to BSA ≥ 1.58 m2 (71.0%) (P = 0.02). CONCLUSION: In real-world clinical practice, first-line afatinib was well managed and was equally as effective as in previous clinical trials of EGFR-positive NSCLC. BSA is considered a predictive marker for appropriate afatinib dose reduction.

Production of viable cloned miniature pig embryos using oocytes derived from domestic pig ovaries.

For production of viable somatic cell nuclear transferred (SCNT) miniature pig embryos, in vitro condition for controlling the quality of recipient oocytes derived from domestic pig ovaries should be evaluated. In the present study, to get information on optimal in vitro maturation (IVM) condition of oocytes, we investigated the effect of IVM duration of recipient oocytes on subsequent development of SCNT miniature pig embryos, the maturation-promoting factor (MPF) activity in recipient oocytes before and after SCNT, and the occurrence of premature chromosome condensation (PCC) and spindle morphologies of donor nuclei following SCNT. The optimal window of the IVM period in terms of in vitro developmental ability of SCNT embryos was determined to be 36-40 h after the start of IVM. The use of recipient oocytes matured for 36 and 40 h resulted in a high level of MPF activity before and after SCNT, and increased the occurrence of PCC in transferred nuclei compared to the use of oocytes matured for 44 and 52 h. The proportion of abnormal spindle-like structures increased as the IVM period was prolonged. In addition, SCNT embryos constructed from recipient cytoplasts obtained after 40 h of maturation by using fetal fibroblasts of miniature pigs were transferred to surrogate miniature pigs, and developed to full term. These results suggest that recipient oocytes matured for 36 h and 40 h effectively induce PCC with a normal cytoskeletal structure because of a high level of MPF activity; furthermore, the 40-h IVM period improves in vitro development of SCNT embryos to the blastocyst stage, resulting in the production of viable cloned miniature pigs.

Induction of estrus in pubertal miniature gilts.

Genetic engineering of miniature pigs has facilitated the development of numerous biomedical applications, such as xenotransplantation and animal models for human diseases. Manipulation of the estrus is one of the essential techniques for the generation of transgenic offspring. The purpose of the present study was to establish a useful method for induction of the estrus in miniature gilts. A total of 38 pubertal miniature gilts derived from 4 different strains were treated with exogenous gonadotropins. Estrus and ovulatory response were examined after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as 200 IU PMSG and 100 IU hCG, 300 IU PMSG and 150 IU hCG, or 1,500 IU PMSG only, followed by 100, 150 or 750 IU hCG 72 h later, respectively. The optimal protocol was determined to be the combination treatment of 200 IU PMSG and 100 IU hCG followed by 100 IU hCG. The administration of 200 IU PMSG and 100 IU hCG was effective in inducing estrus regardless of the strain, although there was a strain difference in the ovulatory response. These results indicate that treatment with a low-dose combination of PMSG and hCG provides one of the simplest methods for induction of estrus and ovulation in pubertal miniature pigs.

Effects of ingested turmeric oleoresin on glucose and lipid metabolisms in obese diabetic mice: a DNA microarray study.

Turmeric, the rhizome of Curcuma longa L., has a wide range of effects on human health. Turmeric oleoresin, an extract of turmeric, is often used for flavoring and coloring. Curcuminoids and turmeric essential oil are both contained in turmeric oleoresin, and both of these fractions have hypoglycemic effects. In the present study, we comprehensively assessed the effect of turmeric oleoresin on hepatic gene expression in obese diabetic KK-Ay mice using DNA microarray analysis and quantitative real-time polymerase chain reaction (PCR). Female KK-Ay mice aged 6 weeks (n = 6/group) were fed a high-fat diet containing turmeric oleoresin, curcuminoids, and essential oil for 5 weeks. The same diet without any of these fractions was used as a control diet. Ingestion of turmeric oleoresin and essential oil inhibited the development of increased blood glucose and abdominal fat mass, while curcuminoids only inhibited the increase in blood glucose. DNA microarray analysis indicated that turmeric oleoresin ingestion up-regulated the expression of genes related to glycolysis, beta-oxidation, and cholesterol metabolism in the liver of KK-Ay mice, while expression of gluconeogenesis-related genes was down-regulated. Real-time PCR analysis was conducted to assess the contribution of the curcuminoids and essential oil in turmeric oleoresin to the changes in expression of representative genes selected by DNA microarray analysis. This analysis suggested that curcuminoids regulated turmeric oleoresin ingestion-induced expression of glycolysis-related genes and also that curcuminoids and turmeric essential oil acted synergistically to regulate the peroxisomal beta-oxidation-related gene expression induced by turmeric oleoresin ingestion. These changes in gene expression were considered to be the mechanism by which the turmeric oleoresin affected the control of both blood glucose levels and abdominal adipose tissue masses. All of these results suggest that the use of whole turmeric oleoresin is more effective than the use of either curcuminoids or the essential oil alone.

The suppression of fragmentation by stabilization of actin filament in porcine enucleated oocytes.

A thorough understanding of the mechanism underlying fragmentation would contribute to the improvement of the developmental ability of reconstructed embryos after nuclear transfer. We conducted the present study to elucidate the influence of the nuclear transfer method on fragmentation of enucleated oocytes and the relationship between change in actin filament distribution and fragmentation. In Experiment 1, we examined activation rates of in vitro matured oocytes. These were 12.9% in maturation alone, 75.7% in electrical stimulation, and 57.9% in ethanol/cycloheximide treatment. In Experiment 2, we observed a higher rate of fragmentation (P < 0.05) in cultured oocytes that had been enucleated and electrically stimulated than in oocytes subjected to the other treatments (maturation alone, enucleation alone and enucleation plus ethanol/cycloheximide activation). In Experiment 3, we stained enucleated and electrically stimulated oocytes with rhodamine/phalloidin dye to show discontinuous distributions in the ooplasm of treated oocytes; oocytes in the other treatment groups showed homogenous distributions of actin filaments (AFs). In Experiment 4, we added cytochalasin B, an inhibitor of AF polymerization, to the culture medium, which prevented fragmentation of enucleated plus electrically stimulated oocytes (cytochalasin B, [+] 0.0%, [-] 60.7% at 24 h after treatment, P < 0.05). In Experiment 5, we investigated the relationship between fragmentation and alteration in AF distribution in enucleated plus electrically stimulated oocytes. At 0 h of culture, enucleated plus electrically stimulated oocytes showed discontinuous distributions of AFs, while nontreated oocytes showed homogenous AF distributions. At 24 and 48 h of culture, fragmentation proceeded in enucleated plus electrically stimulated oocytes and the discontinuous AF distribution diminished with time. In Experiment 6, we added hyaluronic acid (HA) to the culture medium, which suppressed fragmentation of enucleated plus electrically stimulated oocytes (HA, [+] 28.5%, [-] 66.4% at 24 h after treatment, P < 0.05). The results suggest that electrical stimulation induces a change in the AF distribution of oocytes, resulting in fragmentation, and that the addition of HA to the culture media is effective for the suppression of fragmentation.

[Radiation recall pneumonitis induced by Gefitinib (Iressa): a case report].

Gefitinib is a newly developed molecular-target drug with selective inhibitory activity for tyrosine kinase of the epidermal growth factor receptor and has an encouraging effect on non-small cell lung cancer in an advanced stage. The adverse drug reactions including diarrhea, skin eruptions and liver dysfunction have been considered mild. However, cases of severe acute lung injuries were reported after approval of the drug in Japan in July, 2002. We report a case of recurrent large cell carcinoma of the lung in a 73-year-old man who suffered from radiation recall pneumonitis induced by Gefitinib. Two months after radiation therapy to the mediastinal and right hilar lesions was completed, he started to take Gefitinib at a dose of 250 mg/day. Six weeks later, he complained acutely of a dry cough, slight fever and effort dyspnea, and his chest CT demonstrated ground-glass opacity corresponding to the previous radiation field. In administering Gefitinib, as well as other cytotoxic drugs, meticulous monitoring for acute lung injury and radiation recall reaction is required.

Cryo-preserved porcine kidneys are feasible for teaching and training renal biopsy: "the bento kidney".

BACKGROUND: The use of patients as the primary teaching modality for learning procedures is being questioned. While there have been advancements in the technology used for performing needle biopsies in both native and transplanted kidneys, there has been little advancement in teaching and training tools. We have developed a portable ex-vivo kidney, the Bento Kidney, using cryo-preserved porcine kidneys for teaching this procedure. METHODS: The kidney is thawed, perfused by a pump, covered with skin for realistic haptic feedback, and then used with existing biopsy technology to teach the technique. RESULTS: Thirty porcine kidneys were used in this pilot research, and nine were shipped to physicians at a distant facility. Renal biopsy was then performed using a core biopsy needle and ultrasound guidance. There was some leakage of fluid from all kidneys noted. All trainees felt that the model was realistic, and judged at a mean score of 8.7 (SD 0.8) on a scale of 1 (not useful) to 10 (very useful). CONCLUSIONS: This feasibility study demonstrates that cryo-preserved porcine kidneys can be successfully used to teach and train renal biopsy techniques, and provides haptic feedback as well as realistic real-time ultrasound images. Further large scale studies are needed to demonstrate value from the educational point of view for nephrology and transplantation.

The effects of fasting and general anesthesia on serum chemistries in KCG miniature pigs.

Investigators are obligated to optimize the perioperative care of experimental animals, but little is known about the effects of anesthesia and surgery on serum chemistries in KCG pigs. The objective of this study was to examine the influence of fasting and surgery under general anesthesia on 27 serum chemistries in KCG miniature pigs to improve management. Crossbred KCG minipigs were used at a mean of 12.3 mo of age (range, 8.6 to 14.9) and 33.4 kg of body weight (range, 24.0 to 40.2). Serum chemistries were evaluated at the start and end of a 24 h fasting period in fasted animals (n = 6). No significant differences were observed between the starting and postfasting studies. Partial hemilaminectomy of the lumbar spine was carried out in 2 groups of animals. Those given sevoflurane anesthesia (n = 7) had significant decreases in serum albumin, potassium, inorganic phosphorus, gamma-glutamyltransferase peptidase, cholinesterase, and glucose postoperatively compared with preoperative values. Animals given isoflurane (n = 7) anesthesia had significantly decreased total protein, albumin, triglyceride, phospholipids, sodium, potassium, calcium, alanine aminotransferase, alkaline phoshatase and glucose after surgery compared with levels before surgery. In a separate experiment (n = 7), serum glucose and insulin also decreased during the postoperative period after isoflurane anesthesia. These results demonstrate that select serum electrolytes, glucose, and insulin of KCG miniature pigs are altered after general anesthesia. Investigators must be aware of the effects of anesthetic agents on experimental animals to provide optimal care and for interpretation of experimental data.

Oral administration of lactic acid bacteria isolated from traditional South Asian fermented milk 'dahi' inhibits the development of atopic dermatitis in NC/Nga mice.

We investigated the suppressive effect of lactic acid bacteria (LAB) isolated from traditional South Asian fermented milk 'dahi' on the development of atopic dermatitis (AD) using NC/Nga AD model mice. In the initial evaluation, we confirmed the effect of LAB on serum total IgE using ovalbumin (OVA)-induced type 1 allergy model mice. Forty-one bacterial strains isolated from dahi were evaluated for their ability to induce interleukin (IL)-12 production and suppress IL-4 production in splenocytes obtained from OVA-sensitized mice. Of the 41 strains tested, Lactobacillus delbrueckii subsp. lactis R-037 exhibited the greatest IL-12 induction, suggesting that it is a potent Th1 inducer. Oral administration of heat-treated R-037 significantly suppressed the elevation of serum total IgE in OVA-induced type 1 allergy model mice. In NC/Nga AD model mice, oral administration of heat-treated R-037 reduced inflammatory auricular thickness and alleviated the AD clinical score although the effect on serum total IgE level was unclear. Histopathological findings showed a tendency toward improvement of inflammation. Hyperkeratosis in particular showed improvement in dermatitis skin lesions. These results suggest that oral administration of R-037 may alleviate AD.

Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source.

Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

Inflammation-based prognostic score, prior to neoadjuvant chemoradiotherapy, predicts postoperative outcome in patients with esophageal squamous cell carcinoma.

BACKGROUND: Recent studies have revealed that Glasgow prognostic score (GPS), an inflammation-based prognostic score, is associated with poor outcome in a variety of tumors. However, few studies have investigated whether GPS measured prior to neoadjuvant chemoradiotherapy (nCRT) is useful for postoperative prognosis of patients with advanced esophageal squamous cell carcinoma (ESCC). METHODS: GPS was calculated on the basis of admission data as follows: patients with both an elevated C-reactive protein (>10 mg/L) and hypoalbuminaemia (<35 g/L) were allocated a GPS score of 2. Patients in whom only 1 of these biochemical abnormalities was present were allocated a GPS score of 1, and patients with a normal C-reactive protein and albumin were allocated a score of 0. All patients underwent radical en-bloc resection 3-4 weeks after nCRT. RESULTS: A total of 48 patients with clinical TNM stage II/III were enrolled. Univariate analyses revealed that there were significant differences in cancer-specific survival in relation to grade of response to nCRT (P = .004), lymph node status (P = .0065), lymphatic invasion (P = .0002), venous invasion (P = .0001), pathological TNM classification (P = .015), and GPS (P < .0001). GPS classification showed a close relationship with lymphatic invasion, venous invasion, and number of lymph node (P = .0292, .0473, and .0485, respectively). GPS was found to be the only independent predictor of cancer-specific survival (odds ratio, 0.17; 95% confidence interval, 0.06-0.52; P = .0019). CONCLUSIONS: GPS, measured prior to nCRT, is an independent novel predictor of postoperative outcome in patients with advanced ESCC.

Suppression by licorice flavonoids of abdominal fat accumulation and body weight gain in high-fat diet-induced obese C57BL/6J mice.

We applied licorice flavonoid oil (LFO) to high-fat diet-induced obese C57BL/6J mice and investigated its effect. LFO contains hydrophobic flavonoids obtained from licorice by extraction with ethanol. The oil is a mixture of medium-chain triglycerides, having glabridin, a major flavonoid of licorice, concentrated to 1.2% (w/w). Obese mice were fed on a high-fat diet containing LFO at 0 (control), 0.5%, 1.0%, or 2.0% for 8 weeks. Compared with mice in the control group, those in the 1% and 2% LFO groups efficiently reduced the weight of abdominal white adipose tissues and body weight gain. A histological examination revealed that the adipocytes became smaller and the fatty degenerative state of the hepatocytes was improved in the 2% LFO group. A DNA microarray analysis of the liver showed up-regulation of those genes for beta-oxidation and down-regulation of those for fatty acid synthesis in the 2% LFO group. These findings suggest that LFO prevented and ameliorated diet-induced obesity via the regulation of lipid metabolism-related gene expression in the liver.

Education and research using experimental pigs in a medical school.

Medium-sized animals such as miniature pigs are considered to be important for education and training in medical schools to master the skills required in surgical treatment. Much still remains to be done to establish total management for animal experiments using pigs. Improvement of the effective utilization of pigs is also required from the economical and ethical points of view. We have been providing a support system at a facility for experimental animals in a medical school for 3 years, and herein we introduce our personal experiments as an instructional lecture. Before starting surgical training using live pigs, sufficient education concerning animal ethics and dry laboratory training was completed. Four kinds of miniature pigs have been used as experimental animals; porcine rearing pens have been improved and a postoperative care system has been implemented. Moreover, staff at the center offer a preoperative service of anesthesia for surgical education, training, and research. Chronic experiments have increased to represent 35% and 48% of experiments using pigs in 2003 and 2004, respectively. Experimental pigs have undergone secondary use after being killed to reduce the number of animals used in experiments. Sharing and reuse have allowed effective use of miniature pig tissues and cells for research, and have reduced the number of animals used. We recommend that researchers consider use of our total systems because they can improve the quality of medical education and research and facilitate effective use of tissues and cells by sharing and reuse among different departments.

Molecular investigation of hepatitis E virus infection in domestic and miniature pigs used for medical experiments.

BACKGROUND: Hepatitis E virus (HEV) infection is highly prevalent among domestic pigs in Japan. It has been reported that pig handlers such as farmers and veterinarians are at increased risk of contracting HEV infection. Pigs are regarded as the most acceptable candidate animals for xenotransplantation and, recently, they are being used as experimental animals. METHODS: We investigated the prevalence of IgG class antibodies to swine HEV (anti-HEV) and HEV RNA among 152 2-month-old domestic pigs and 38 miniature pigs of 4 to 10 months of age that had been brought to our center for medical experiments from five swine farms (A-E) in Japan. Serum samples were tested for anti-HEV by in-house enzyme immunoassay, and for HEV RNA by reverse transcription-polymerase chain reaction using primers targeting the open reading frame 2 (ORF2) region. RESULTS: One percent (one of 84), 6% (one of 16), and 38% (20 of 52) of the domestic pigs from farms A, B and C, respectively, had detectable HEV RNA, and the 22 HEV isolates recovered from the viremic pigs were 89.8 to 100% identical to each other in the 412-nucleotide sequence of ORF2 and segregated into three clusters within genotype 3. Although one pig from farm A had detectable HEV RNA reproducibly, the HEV isolate recovered from this pig was up to 100% similar to those recovered from pigs from farm C, and the sera from all 84 pigs from farm A were negative for anti-HEV. These results suggested that farm A is free from HEV infection. As the viremic pig from farm A had been raised for 1 month in a barn at our center before serum sampling, it is most likely that the pig acquired HEV infection in the barn at our center where HEV-viremic pigs from farm C had been reared for several days approximately 3 months earlier. The 38 miniature pigs from farms D and E were negative for both anti-HEV and HEV RNA. In an attempt to further investigate the prevalence of HEV infection, pigs that were being raised in four swine farms (farms A, C, D, and E) were tested for anti-HEV. Although 96 (86%) of the 112 pigs from farm C were positive for anti-HEV, none of the 48 pigs in farm A and 138 miniature pigs in farms D and E was positive for anti-HEV. CONCLUSIONS: These results suggest that three of the five swine farms tested were free from HEV, and that periodic testing for anti-HEV and HEV RNA of pigs used as experimental animals and pigs raised in swine farms from which pigs are purchased, is useful for providing HEV-free pigs to researchers who are engaged in studies using pigs.