Direct observation of pressure-induced amorphization of methane/ethane hydrates using Raman and infrared spectroscopy.

The pressure-induced amorphization (PIA) of ice and clathrate hydrates occurs at temperatures significantly below their melting and decomposition points. The PIA of type I clathrate hydrates containing methane and ethane as guest molecules was investigated using Raman and infrared (IR) spectroscopy. With isothermal compression at 100 K, methane hydrate (MH) underwent PIA at 2-3.5 GPa, whereas ethane hydrate (EH) underwent PIA at 4.0-5.5 GPa. The type I clathrate structure consists of small (512) and large (51262) cages. The Raman results revealed that the collapsed small and large cages in the amorphous forms of MH and EH were not distinguishable. The collapsed cages, including the methane and ethane molecules, were similar to the small and large cages, respectively. Their water networks were folded or expanded during the PIA process so that the cavity sizes of the collapsed cages were compatible with those of the guest molecules. Peaks in the IR spectra of crystalline MH assignable to the ro-vibrational transition of methane in large cages were observed in the C-H stretching wavenumber region below 40 K. The ro-vibrational IR band disappeared after amorphization, suggesting that the rotational motion of the methane molecule in the large cage was frozen by the collapse, as reported in previous dielectric spectroscopic and simulation studies. This study contributes to a better understanding of the changes in the local structure around guest molecules during PIA and the dynamics of the guest molecules.

Toxocara tanuki larval distribution in mice and the infectivity of tissue larvae.

Toxocara tanuki is a common large roundworm in raccoon dogs. Experimental infection studies of T. tanuki in mice were conducted to clarify the distribution and infectivity of larvae in tissue. Groups of BALB/c and C57BL/6 mice (n = 5 mice/group) were each inoculated with 1000 embryonated T. tanuki eggs and necropsied at 7, 31, 91, and 182 days post inoculation (dpi). The number of larvae in the central nervous system, heart, lungs, kidneys, spleen, gastrointestinal tract, liver, and carcass was examined. Larvae obtained from the aforementioned mice on different days of the necropsy were orally inoculated into four groups of ICR mice (n = 6 mice/group) that were then necropsied at 21 dpi. Larvae were recovered from all mice. In the BALB/c and C57BL/6 mice, most of the larvae (> 88.7%) were recovered from the liver and the remainder from other tissues. The total number of larvae recovered from C57BL/6 mice was significantly higher than that from BALB/c mice, but no difference in the relative larval distribution within the viscera between the two mouse strains was observed. The mean recovery percentage of larvae from ICR mice infected with 182-day-old tissue larvae was 3.3%. Our findings showed that T. tanuki larvae migrated predominantly to the liver of mice and that the larvae maintained their infectivity for at least half a year.

Detection of larvae of Toxocara cati and T. tanuki from the muscles of free-ranging layer farm chickens.

Although raw or undercooked livestock meat or viscera has been suggested to be a source of human toxocariasis, there have been few reports on the prevalence of Toxocara larvae in the tissue of livestock animals. To investigate the presence of Toxocara larvae in chickens, we examined 50 culled chickens from a commercial layer farm. The liver, breast meat, and thigh meat were separated individually and artificially digested to examine for the presence of larvae. Nematode larvae were detected in 2 out of 50 chickens. One larva was detected from the breast meat, and it was molecularly identified as Toxocara tanuki. The other from the thigh meat of another chicken was molecularly identified as Toxocara cati. The present study demonstrated for the first time that T. tanuki larvae do infect chickens in the natural environment. The fact that Toxocara spp. larvae were found in muscles of farm chickens suggests that consumption of raw or undercooked chicken meat may present a risk for human toxocariasis.

Toxocara cati larval migration to mouse fetuses through transplacental infection.

To examine the Toxocara cati larval migration in mouse fetuses through vertical transmission, 7 pregnant mice were orally inoculated with 5000 embryonated eggs at day 4 and again with the same dose at day 9 of gestation (total 10,000 eggs / mouse). Seven non-pregnant mice of the same age were also inoculated likewise. All mice were necropsied 12 days after the first inoculation with eggs. The carcass, viscera, uterus, placenta and fetuses were individually removed and digested with pepsin for larval counts. Larvae were recovered from all pregnant and non-pregnant mice. The majority of the larvae were recovered from the carcass, and some from the viscera, uterus, placenta and fetuses. There were no differences between the pregnant and non-pregnant mice for the number of larvae in the carcass, viscera and uterus. Of a total of 103 fetuses from the 7 pregnant mice, larvae were recovered from 74 fetuses (71.8 %). The mean number of larvae recovered per infected fetus was 2.6 (n = 103), and the maximum number of larvae recovered in a fetus was 9. This study showed that most mouse fetuses were infected with T. cati larvae through intrauterine transmission, although the number of larvae was low. Since no significant difference in the number of larvae recovered from the pregnant and non-pregnant mice was observed, it is suggested that pregnancy does not directly affect the vertical transmission of T. cati larvae.

Tolerance to low temperatures of Toxocara cati larvae in chicken muscle tissue.

Infectivity of Toxocara cati larvae in muscle tissue of chickens after storage at 4°C and -25°C was assessed in a mouse bioassay to provide information on the risk of meat-borne toxocarosis. Muscle tissue samples of 30-day old T. cati infections were stored at 4°C for 14 and 28 days and at -25°C for 12, 24 and 48 h, whereafter, larvae were released by digestion. For each experimental group, the released larvae were inoculated in six mice. After 15 days, mice were euthanized and larval burden was assessed by digestion. In the control group (no storage of the infected chicken meat), 47.9% of the inoculated larvae established in mice, whereas storage of meat at 4°C for 14 days or 28 days reduced the recovery to 24.1% or 3.3%, respectively. Muscle larvae exposed to -25°C for 12, 24 or 48h did not establish in the mice. The observation that larvae retain infective after refrigeration at exposure in 4°C for 28 days, emphasize the zoonotic potential of poultry meat as a causative agent of human toxocarosis.

Enantioselective and highly sensitive determination of carvedilol in human plasma and whole blood after administration of the racemate using normal-phase high-performance liquid chromatography.

A highly sensitive HPLC method for enantioselective determination of carvedilol in human whole blood and plasma was developed. Carvedilol and S-carazolol as an internal standard extracted from whole blood or plasma were separated using an enantioselective separation column (Chiralpak AD column; 2.0 diameter x 250 mm) without any chiral derivatizations. The mobile phase was hexane:isopropanol:diethylamine (78:22:1, v/v). The excitation and emission wavelengths were set at 284 and 343 nm, respectively. The limits of quantification for the S(-)- and R(+)-carvedilol enantiomers in plasma and blood were both 0.5 ng/ml. Intra- and inter-day variations were less than 5.9%. As an application of the assay, concentrations of carvedilol enantiomer in plasma and blood samples from 15 patients treated with carvedilol for congestive heart failure were determined.