Establishment of a laboratory colony of taiga tick Ixodes persulcatus for tick-borne pathogen transmission studies

Ixodes persulcatus Schulze (I. persulcatus) is distributed in Russia and Far East Asia including Japan, and has been implicated as the vector of several human pathogens. In particular, I. persulcatus acts as the only tick vector for human lyme borreliosis in Japan. In order to elucidate the mechanism of transmission of I. persulcatus-borne pathogens, we developed a laboratory colony of I. persulcatus. Ticks were fed on Syrian hamster and engorged ticks that had dropped off the animals were collected and maintained to allow them to molt. Tick rearing was performed in incubator at 20 degrees C with 95% relative humidity and 12-hour light/dark photo-period regimen. We found out that adult females fed for 8 +/- 2 days and had a pre-oviposition period lasting for 7 +/- 2 days. The minimum egg incubation period was 1 month with the hatched larvae feeding for 3 +/- 1 days and molting to nymphs 3-4 months thereafter. Meanwhile, the nymphs fed for 4 +/- 1 days and molted to adult 2-3 months thereafter. For future analysis of gene expression profiles in I. persulcatus, we cloned and sequenced the actin gene (a housekeeping gene), and found that it is 92.7% to 98.6% homologous to the published sequences of related ixodid ticks. This laboratory colony of I. persulcatus will facilitate investigations on the role of tick-derived molecules on the transmission of I. persulcatus-borne pathogens and will be important for identification of potential anti-tick vaccine and acaricide target molecules.     

Role for des-acyl ghrelin in the responsiveness of plasma hormones and metabolites to ghrelin in Holstein steers

Gastric-derived peptide hormone ghrelin is known for its potent growth hormone (GH) stimulatory effects. The acyl-modification on N-terminal Ser(3) residue is reported to be important to stimulate the ghrelin receptor, GH secretagogue-receptor type1a (GHS-R1a). However, major portion of circulating ghrelin lacks in acylation, and some biological properties of des-acyl ghrelin have been reported in monogastric animals. In the present study, the responsiveness of plasma hormones and metabolites to ghrelin in steers was characterized, and role for des-acyl ghrelin in these changes was investigated. The repeated intravenous administrations of bovine ghrelin (1.0 microg/kg BW) every 2h for 8h to Holstein steers significantly increased the plasma acylated ghrelin, total ghrelin, GH, insulin and NEFA levels. The GH responses in peak values and area under the curves (AUCs) were attenuated by repeated injections of ghrelin, however, the responses of plasma total ghrelin were similar. Plasma insulin AUC decreased after fourth injection of ghrelin while plasma NEFA AUCs gradually increased by repeated injections of ghrelin. Pretreatment of des-acyl ghrelin (10.0 microg/kg BW) 5 min prior to the single injection of ghrelin (1.0 microg/kg BW) did not affect the ghrelin-induced hormonal changes. Moreover, the responses of plasma GH to bovine and porcine ghrelin, which differ in C-terminal amino acid residues, were similar in calves. These data show that (1) GH release was attenuated by repeated administration of ghrelin, (2) ghrelin regulates glucose and fatty acid metabolism probably via different pathway, and (3) des-acyl ghrelin is unlikely the antagonist for ghrelin to induce endocrine effects in Holstein steers.     

Genomic analyses of bovine viral diarrhea viruses isolated from cattle imported into Japan between 1991 and 2005

Thirty-one isolates of bovine viral diarrhea virus (BVDV) isolated within the past 15 years from imported cattle by the Japanese Animal Quarantine Service (AQS) were used in this study in which a 5'-untranslated region of each isolate was genetically analyzed. Twenty-six of the 31 isolates were classified as BVDV1 and the remainder as BVDV2. Phylogenetic analysis of the RT-PCR fragments amplified from the isolates showed the presence of viruses belonging to the BVDV1a, BVDV1b, BVDV1c, unclassified BVDV1 genotypes, and BVDV2. From the cattle of Australian origin, 16 of 17 isolates were classified as BVDV1c. This result was in agreement with a report showing that BVDV1c was a predominant subgenotype in Australia. From the cattle of North American origin, BVDV1 and BVDV2 species were both found. BVDV2 from the North American cattle was identified as the same cluster as the BVDV 890 strain, which is the prototype of BVDV2. These results suggest that the BVDVs isolated from exported cattle at the AQS reflect the predominant genotypes of BVDVs found in the exporting countries. The unclassified BVDV1 genotype of Chinese origin was in the same cluster as the ZM-95 strain, which was isolated from pigs in China. In this study, the genomic properties of 31 isolates of BVDV collected in the AQS were investigated. We concluded that isolates are genetically heterogeneous but geographically restricted. The information obtained from this report will be useful when carrying out epidemiological surveys of BVDV isolated in Japan.     

Seroprevalence of Coxiella burnetii infections among cats in different living environments

The seroprevalence of Coxiella burnetii infection among pet cats in Japan and Korea and stray cats in Japan was investigated by an indirect fluorescent antibody technique and PCR test. Forty-four (14.2%) of 310 pet cats in Japan were seropositive, as were 15 (41.7%) of 36 stray cats in Japan and 10 (8.6%) of 116 pet cats in Korea. The antibody positive rate in stray cats was significantly higher than that in pet cats, but there was no correlation between the rates in Japanese and Korean pet cats. In this study, the prevalence of C. burnetii infection among cats in different living environments was found and it is difficult to deny that stray cats would be one of the important sources of infection for human Q fever.     

A novel gene encoding a thrombin inhibitory protein in a cDNA library from Haemaphysalis longicornis salivary gland

A novel thrombin inhibitory protein coding gene was identified from a cDNA library derived from salivary gland of partially-fed Haemaphysalis longicornis (hard tick). The gene encoded a 93-amino acid protein, designated chimadanin, which had a signal peptide sequence and was predicted to be a secretory protein. It showed no similarity to any other previously identified proteins or conserved domain sequences. The gene was expressed during blood feeding and suggested to be expressed mainly in the salivary gland. The predicted mature region of chimadanin was expressed in Escherichia coli and characteristics of the recombinant chimadanin were determined. The activated partial thromboplastin time and the prothrombin time in sheep plasma were significantly prolonged by chimadanin in a dose dependent manner. Amidolytic activity of thrombin was also inhibited by chimadanin in a dose dependent manner and it suggested that chimadanin was an anticoagulant with thrombin inhibitory activity. This newly identified thrombin inhibitor may play an important role in tick blood feeding.     

Visualizing gas molecules interacting with supported nanoparticulate catalysts at reaction conditions

Understanding how molecules can restructure the surfaces of heterogeneous catalysts under reaction conditions requires methods that can visualize atoms in real space and time. We applied a newly developed aberration-corrected environmental transmission electron microscopy to show that adsorbed carbon monoxide (CO) molecules caused the {100} facets of a gold nanoparticle to reconstruct during CO oxidation at room temperature. The CO molecules adsorbed at the on-top sites of gold atoms in the reconstructed surface, and the energetic favorability of this reconstructed structure was confirmed by ab initio calculations and image simulations. This atomic-scale visualizing method can be applied to help elucidate reaction mechanisms in heterogeneous catalysis.     

Effects of fasting and refeeding on plasma concentrations of leptin, ghrelin, insulin, growth hormone and metabolites in swine

The effects of nutritional status, such as fasting and refeeding, on leptin and ghrelin secretion in swine were examined. The swine (n = 4) were fasted for 54 h and plasma hormone levels were measured before, during and after fasting. Plasma leptin and insulin concentrations began to decrease 12 and 6 h into the fasting period, respectively (P < 0.05), and maintained a low level for the remaining period of fasting. Plasma leptin and insulin returned to the pre‐fasting value 6 and 12 h after refeeding, respectively. Plasma ghrelin concentrations showed a nocturnal periodicity during the fasting period; it increased nocturnally at 36 and 42 h into the fasting period (P < 0.05). Plasma growth hormone levels did not show any remarkable changes during the fasting. Plasma glucose levels showed a modest fall during fasting and significantly decreased (P < 0.05) at 24 h into the fasting period, returning to pre‐fasting levels after refeeding. Plasma nonesterified fatty acid levels increased (P < 0.05) at 12 h into the fasting period and returned to the pre‐fasting level 6 h after refeeding. These results indicate that plasma leptin, insulin and ghrelin play an important role in maintaining energy homeostasis in swine. The plasma ghrelin did not continuously increase, but showed nocturnal periodicity during fasting. This may suggest that ghrelin is also involved in physiological processes other than energy homeostasis.