Pork Loin Treated with High Hydrostatic Pressure as a Food Processing Technology: Subacute Toxicity of the Freeze-Dried Powder and Cytotoxicity of the Methanol Extracts.

High hydrostatic pressure (HP) treatment is used in food processing owing to its sterilization effect. Meat or meat products are sterilized and become tender by HP processing. Therefore, the variety of HP-processed meat products has increased worldwide. However, little is known about the safety of HP-processed meat products. The aim of this study was to determine the effects of HP processing and HP combined with 0.4 M sodium carbonate treatment (HP-Na) on pork loins and to evaluate the subacute toxicity and cytotoxicity of these processing methods. In an in vivo study, we performed 90- and 180-day feeding tests in mice and did not detect any adverse effects in HP-processed and HP-Na-processed pork loins. In addition, we evaluated the cytotoxicity of HP-processed meats, and did not observe any obvious toxicity associated with pork loin extracts in vitro. These results suggest that HP is not associated with risk factors during processing.

Synergistic effects of high hydrostatic pressure, mild heating, and amino acids on germination and inactivation of Clostridium sporogenes spores.

The synergistic effects of high hydrostatic pressure (HHP), mild heating, and amino acids on the germination of Clostridium sporogenes spores were examined by determining the number of surviving spores that returned to vegetative growth after pasteurization following these treatments. Pressurization at 200 MPa at a temperature higher than 40°C and treatment with some of the 19 l-amino acids at 10 mM or higher synergistically facilitated germination. When one of these factors was omitted, the level of germination was insignificant. Pressures of 100 and 400 MPa were less effective than 200 MPa. The spores were effectively inactivated by between 1.8 and 4.8 logs by pasteurization at 80°C after pressurization at 200 MPa at 45°C for 120 min with one of the amino acids with moderate hydrophobicity, such as Leu, Phe, Cys Met, Ala, Gly, or Ser. However, other amino acids showed poor inactivation effects of less than 0.9 logs. Spores in solutions containing 80 mM of either Leu, Phe, Cys, Met, Ala, Gly, or Ser were successfully inactivated by pasteurization by more than 5.4 logs after pressurization at 200 MPa at 70°C for 15 to 120 min. Ala and Met reduced the spore viability by 2.8 and 1.8 logs, respectively, by pasteurization at a concentration of 1 mM under 200 MPa at 70°C. These results indicate that germination of the spores is facilitated by a combination of high hydrostatic pressure, mild heating, and amino acids.

Genome comparison and phylogenetic analysis of Orientia tsutsugamushi strains.

Orientia tsutsugamushi (OT) is an obligate intracellular bacterium belonging to the family Rickettsiaceae and is the causative agent of scrub typhus, or Tsutsugamushi disease. The complete genome sequences of two OT strains (Boryong and Ikeda) have recently been determined. In the present study, we performed a fine genome sequence comparison of these strains. Our results indicate that although the core gene set of the family Rickettsiaceae is highly conserved between the two strains, a common set of repetitive sequences have been explosively amplified in both genomes. These amplified repetitive sequences have induced extensive genome shuffling and duplications and deletions of many genes. On the basis of the results of the genome sequence comparison, we selected 11 housekeeping genes and carried out multilocus sequence analysis of OT strains using the nucleotide sequences of these genes. This analysis revealed for the first time the phylogenetic relationships of representative OT strains. Furthermore, the results suggest the presence of an OT lineage with higher potential for virulence, which may explain the clinical and epidemiological differences between 'classic' and 'new' types of Tsutsugamushi disease in Japan.

Change of thermal inactivation of Clostridium botulinum spores during rice cooking.

Cooked and packed rice (CaPR), a popular rice product in Japan, is distributed with oxygen-absorbing agents and without refrigeration. When the final product was inoculated with spores of several strains of proteolytic Clostridium botulinum at a dose of 10(3) spores per g (2 x 10(5) spores per package) and incubated at 30 degrees C, the bacteria grew and produced neurotoxins in 40 days. To simulate more realistic cases of contamination, the same dose of spores was inoculated before the cooking process. When cooked at 100 degrees C for 30 min, a small number of spores survived and the toxins were detected in some of the samples after incubation for 180 days. However, when cooked at 100 degrees C for 15 min immediately followed by 105 degrees C for 15 min, neither survivors nor the toxins were detected during incubation for 270 days after cooking. Even when inoculated with 10(5) spores per g of one of the most heat-resistant strains, 213B, viable spores were not detected after cooking. The inactivation by these heating conditions in different media indicated that the spores were inactivated >1,000-fold more in rice suspension than in cooked meat medium or phosphate buffer. It was therefore suggested that rice contains component(s) that facilitates thermal inactivation of C. botulinum.

The Whole-genome sequencing of the obligate intracellular bacterium Orientia tsutsugamushi revealed massive gene amplification during reductive genome evolution.

Scrub typhus ('Tsutsugamushi' disease in Japanese) is a mite-borne infectious disease. The causative agent is Orientia tsutsugamushi, an obligate intracellular bacterium belonging to the family Rickettsiaceae of the subdivision alpha-Proteobacteria. In this study, we determined the complete genome sequence of O. tsutsugamushi strain Ikeda, which comprises a single chromosome of 2 008 987 bp and contains 1967 protein coding sequences (CDSs). The chromosome is much larger than those of other members of Rickettsiaceae, and 46.7% of the sequence was occupied by repetitive sequences derived from an integrative and conjugative element, 10 types of transposable elements, and seven types of short repeats of unknown origins. The massive amplification and degradation of these elements have generated a huge number of repeated genes (1196 CDSs, categorized into 85 families), many of which are pseudogenes (766 CDSs), and also induced intensive genome shuffling. By comparing the gene content with those of other family members of Rickettsiacea, we identified the core gene set of the family Rickettsiaceae and found that, while much more extensive gene loss has taken place among the housekeeping genes of Orientia than those of Rickettsia, O. tsutsugamushi has acquired a large number of foreign genes. The O. tsutsugamushi genome sequence is thus a prominent example of the high plasticity of bacterial genomes, and provides the genetic basis for a better understanding of the biology of O. tsutsugamushi and the pathogenesis of 'Tsutsugamushi' disease.

Chlorine sensitivity of feline calicivirus, a norovirus surrogate.

The sensitivity to free chlorine of feline calicivirus (FCV), a norovirus surrogate, was examined relative to chlorine demand. When a crude suspension of FCV was treated with a sodium hypochlorite solution containing 10 microg/ml free chlorine, the extent of the decrease of viral infectivity clearly depended on the volume of the reaction mixture. The apparent sensitivity of FCV to free chlorine increased with the reduction of host cell debris, indicating that chlorine demand must be minimized to know the true sensitivity of the virus. We therefore partially purified the viruses from the host cell components and found that the infectivity of FCV was reduced by more than log 4.6 by 5 min of treatment with 300 ng/ml free chlorine.

Survival of two Orientia tsutsugamushi bacterial strains that infect mouse macrophages with varying degrees of virulence.

Orientia tsutsugamushi, an intracellular parasitic bacterium, comprises numerous strains of differing virulence. When BALB/c mice were infected intraperitoneally with this pathogen, a virulent strain known as Karp was found to multiply in the intraperitoneal macrophages and kill the mouse. In contrast, an avirulent strain, Kuroki, was shown to invade macrophages but be eliminated from the cells, allowing mouse survival. O. tsutsugamushi invades its host cell cytoplasm through phagocytosis and disruption of phagosomal membranes but some bacteria are then killed by phago-lysosomes within 1h of infection. Microscopic observations could not differentiate the Karp and Kuroki strains during entry and subsequent cell killing by phago-lysosomes. However, the Kuroki cells failed to divide and were markedly deformed following cytoplasmic invasion at several days post-infection. These findings suggest that macrophages have a mechanism to eliminate O. tsutsugamushi in the cytoplasm, if the invading bacteria escape phagosomal clearance, and that it is this mechanism that Kuroki does not survive. Additionally, significant levels of nitric oxide (NO) are produced in macrophages by Kuroki, but not by Karp. An NO synthase inhibitor, however, does not increase the growth of Kuroki, suggesting that NO is induced in a strain-dependent manner but does not effect proliferation.

A new member of the trombiculid mite family Neotrombicula nagayoi (Acari: Trombiculidae) induces human dermatitis.

We present the first definitive evidence that the mite Neotrombicula nagayoi bites humans under natural conditions in Japan. Initially, bites resulted in mild pruritus without pain. However, skin reactions increased gradually year by year with severe pruritus with pain being reported by the victim after being bitten repeatedly. Six species of trombiculid mites comprising three genera were isolated from soil samples collected from August to October in both 2001 and 2002 at a study site where a man was bitten by N. nagayoi. The dominant species was L. intermedium (72.4%) followed by L. pallidum (8.3%) and N. nagayoi (8.1%). N. nagayoi was found only in August and September. We did not detect the pathogen Orientia tsutsugamushi in any of the unfed larvae, including N. nagayoi, collected from the soil samples.

Mite vectors (Acari: Trombiculidae) of scrub typhus in a new endemic area in northern Kyoto, Japan.

Between 1983 and 1999, 27 human cases of scrub typhus (two fatal) occurred in the Nodagawa River basin of northern Kyoto, Japan, an area where no cases had been previously reported. Antibody screening of infected patients' sera showed that nine of 15 patients had high titers against the Gilliam type of Orientia tsutsugamushi (Hayashi). To determine the vector mite transmitting the disease, we studied rodent and chigger populations in and near a rice field alongside the Nodagawa River between 1996 and 1999. The most common rodent species was Microtus montebelli (Milne-Edwards), representing 73.3% (33/45) of the population. The mite index (average number of mites per infested host) was highest (190.8) in Leptotrombidium pallidum Nagayo, Mitamura & Tamiya parasitizing on M. montebelli, followed by Leptotrombidium intermedium (Nagayo, Mitamura & Tamiya) (174.9) on the same host species. Orientia tsutsugamushi was isolated from 60.5% (23/38) of rodents and from 71.2% (37/52) of pools of engorged L. pallidum. The Gilliam type of O. tsutsugamushi was most prevalent in rodents, and in engorged L. pallidum and it was the only type recovered from 10 isolates inoculated into L 929 cells for indirect immunofluorescence examination. Orientia tsutsugamushi infected 14.3% (181/1263) and 14.8% (306/2066) of engorged and unfed L. pallidum larvae, respectively, and was also detected in 0.055% (2/3634) of unfed L. intermedium, although previous studies suggest that this mite rarely bites humans. These results show that L. pallidum is the primary vector species of scrub typhus in this new endemic area in Japan.

Phylogenetic characterization of Orientia tsutsugamushi isolated in Taiwan according to the sequence homologies of 56-kDa type-specific antigen genes.

Fourteen strains of Orientia tsutsugamushi isolated in Taiwan were characterized by sequencing 56-kDa type-specific antigen genes and patterns of restriction fragment length polymorphism (RFLP) predicted by a computer program. The strains showed high varieties in sequence homologies and were classified to 10 types by predicted patterns of RFLP. Furthermore, all Taiwan strains were not identical in typing with strains analyzed previously. These results suggest that there are various types of O. tsutsugamushi in Taiwan that are different from those distributed in other countries.