Intraspecific variation in morphology of spiny pollen grains along an altitudinal gradient in an insect-pollinated shrub.

Intraspecific variations in pollen morphological traits are poorly studied. Interspecific variations are often associated with pollination systems and pollinator types. Altitudinal environmental changes, which can influence local pollinator assemblages, provide opportunities to explore differentiation in pollen traits of a single species over short distances. The aim of this study is to examine intraspecific variations in pollen traits of an insect-pollinated shrub, Weigela hortensis (Caprifoliaceae), along an altitudinal gradient. Pollen spine phenotypes (length, number and density), pollen diameter, lipid mass (pollenkitt) around pollen grains, pollen production per flower and pollinator assemblages were compared at four sites at different altitudes. Spine length and the spine length/diameter ratio of pollen grains were greater at higher altitudes but not correlated with flower or plant size. Spine number and density increased as flower size increased, and pollen lipid mass decreased as plant size increased. Bees were the predominant pollinators at low-altitude sites whereas flies, specifically Oligoneura spp. (Acroceridae), increased in relative abundance with increasing altitude. The results of this study suggest that the increase in spine length with altitude was the result of selection favouring longer spines at higher-altitude sites and/or shorter spines at lower-altitude sites. The altitudinal variation in selection pressure on spine length could reflect changes in local pollinator assemblages with altitude.

Do aspermic (parthenogenetic) Fasciola forms have the ability to reproduce their progeny via parthenogenesis?

Across Far East Asia, aspermic Fasciola forms are found endemically. They have abnormal spermatogenesis and oogenesis, and it is presumed that their progeny are produced parthenogenetically and clonally. Because of this, they are also termed parthenogenic Fasciola forms. Currently, there is no evidence that they do indeed reproduce parthenogenetically and clonally. In this study, the multilocus genetic type (MLG) in 12 microsatellite markers of adult flukes and their subsequent progeny larvae were analysed using two laboratory aspermic Fasciola triploid strains. The MLGs of adults and their larvae were identical for all markers evaluated, suggesting that these strains reproduce their progeny clonally. The deviation between theoretical and actual frequency within the larvae genotype of the Fh_6 locus resulted in the inability for self-fertilization within individual adult flukes. These findings strongly suggested that aspermic Fasciola forms reproduce their progeny by means of parthenogenesis, possibly gynogenesis.

Molecular and phylogenetic analyses of the liver amphistome Explanatum explanatum (Creplin, 1847) Fukui, 1929 in ruminants from Bangladesh and Nepal based on nuclear ribosomal ITS2 and mitochondrial nad1 sequences.

Explanatum explanatum flukes, liver amphistomes of ruminants, cause significant economic loss in the livestock industry by inducing severe liver damage. A total of 66 flukes from 26 buffaloes and 7 cattle in four different geographic areas of Bangladesh and 20 flukes from 10 buffaloes in the Chitwan district of Nepal were subjected for analysis. The sequences (442 bp) of the second internal transcribed spacer (ITS2) of ribosomal DNA and the variable fragments (657 bp) of mitochondrial nicotinamide dehydrogenase subunit 1 (nad1) of E. explanatum flukes from Bangladesh and Nepal were analysed. The aim of this study was molecular characterization of the flukes and to elucidate their origin and biogeography. In the ITS2 region, two genotypes were detected among the flukes from Bangladesh, while flukes from Nepal were of only one genotype. Phylogenetic analyses inferred from the nad1 gene revealed that at least four divergent populations (groups I-IV) are distributed in Bangladesh, whereas two divergent populations were found to be distributed in Nepal. Fst values (pairwise fixation index) suggest that Bangladeshi and Nepalese populations of group I to IV are significantly different from each other; but within groups III and IV, the populations from Bangladesh and Nepal were genetically close. This divergence in the nad1 gene indicates that each lineage of E. explanatum from diverse geography was co-adapted during the multiple domestication events of ruminants. This study, for the first time, provides molecular characterization of E. explanatum in Bangladesh and Nepal, and may provide useful information for elucidating its origin and dispersal route in Asia.

Epidemic myalgia and myositis associated with human parechovirus type 3 infections occur not only in adults but also in children: findings in Yamagata, Japan, 2014.

We previously reported an association between human parechovirus type 3 (HPeV3) and epidemic myalgia with myositis in adults during summers in which an HPeV3 outbreak occurred in children. However, this disease association has not yet been reported elsewhere. We have since continued our surveillance to accumulate data on this disease association and to confirm whether myalgia occurs in children as well as adults. Between June and August 2014, we collected 380 specimens from children with infectious diseases. We also collected clinical specimens from two adult and three paediatric patients suspected of myalgia. We then performed virus isolation and reverse-transcription-PCR using the collected specimens. We detected HPeV3 in 26 children with infectious diseases, which we regarded as indicating an outbreak. We also confirmed HPeV3 infection in all patients suspected of myalgia. In particular the symptoms in two boys, complaining of myalgia and fever, closely matched the criteria for adult myalgia. Based on our findings from 2008, 2011 and 2014, we again urge that clinical consideration be given to the relationship between myalgia and HPeV3 infections during HPeV3 outbreaks in children. Furthermore, our observations from 2014 suggest that epidemic myalgia and myositis occur not only in adults but also in children.

Human metapneumovirus infection among family members.

The transmission of human metapneumovirus (hMPV) among family members is not well understood. We identified 15 families in which multiple members were diagnosed with hMPV infection by real-time PCR in 2008 and 2010. Index patients ranged in age from 2 years to 11 years (median 5 years), and all 15 index cases were children who attended primary school, kindergarten, or nursery school. Contact patients ranged in age from 2 months to 46 years (median 6 years). Excluding five adult cases, contact patients were significantly younger than index patients (P = 0·0389). Of the 12 contact children, seven (58%) were infants who were taken care of at home. The serial interval between the onset of symptoms in an index patient and the onset of symptoms in a contact patient was estimated to be 5 days. These results suggest that the control of school-based outbreaks is important for preventing hMPV infection in infants.

Molecular identification of Fasciola spp. (Digenea: Platyhelminthes) in cattle from Vietnam.

Fasciola spp. were collected from naturally infected cattle at a local abattoir of Khanh Hoa province, Vietnam, for morphological and genetic investigations. Microscopic examination detected no sperm cells in the seminal vesicles, suggesting a parthenogenetic reproduction of the flukes. Analyses of sequences from the first and second internal transcribed spacers (ITS1 and ITS2) of the ribosomal RNA revealed that 13 out of 16 isolates were of Fasciola gigantica type, whereas three isolates presented a hybrid sequence from F. gigantica and Fasciola hepatica. Interestingly, all the mitochondrial sequences (partial COI and NDI) were of F. gigantica type, suggesting that the maternal lineage of the hybrid form is from F. gigantica. No intra-sequence variation was detected.

Hybridization experiments indicate incomplete reproductive isolating mechanism between Fasciola hepatica and Fasciola gigantica.

Experiments on hybridization between Fasciola hepatica and Fasciola gigantica were carried out to clarify whether a reproductive isolating mechanism appears between the two Fasciola species. Molecular evidence for hybridization was based on the DNA sequence of the internal transcribed spacer 1 (ITS1) region in nuclear ribosomal DNA, which differs between the species. The results suggested that there were not pre-mating but post-mating isolating mechanisms between the two species. However, viable adults of the hybrids F1 and F2 were produced from both parental F. hepatica and F. gigantica. The hybrids inherited phenotypic characteristics such as ratio of body length and width and infectivity to rats from parental Fasciola hepatica and F. gigantica. These findings suggest that reproductive isolation is incomplete between Fasciola hepatica and F. gigantica. Adults of the hybrids F1 and F2 were completely different in mode of reproduction from aspermic Fasciola forms that occur in Asia and seem to be offspring originated from hybridization between F. hepatica and F. gigantica and to reproduce parthenogenetically.

Prevalence of intestinal parasites and genotyping of Giardia intestinalis in pet shop puppies in east Japan.

The current study examined the prevalence of intestinal parasites and genotypes of Giardia intestinalis in puppies from nine pet shops in east Japan. Fresh fecal samples from 1794 puppies (≦3 months old) were collected on one occasion. Giardia spp. was examined for specific coproantigen using ELISA kit (SNAP®Giardia, IDEXX Laboratories, Inc., USA). Other intestinal parasites were detected microscopically using the formalin-ethyl acetate sedimentation technique. Genotyping was determined for the random 29 stool samples identified as Giardia spp. positive using PCR and direct sequencing of the glutamate dehydrogenase (gdh) gene. Overall prevalence of protozoan Giardia spp. and Cystoisospora spp. revealed 23.4% and 11.3%, respectively. Prevalence of ascarids, Strongyloides spp. and hookworms were recorded 1.8%, 1.1% and 0.1%, respectively. Protozoan Giardia spp. and Cystoisospora spp., thus, represent important pathogens among pet shop puppies. All genotyped G. intestinalis isolates were belonged to assemblage C or D, identified as dog-specific genotypes. Zoonotic assemblage A and B were not demonstrated. The result suggests that the risk of zoonotic transmission of G. intestinalis from pet shops puppies to humans may be quite low in Japan.

Cross-antigenicity among EV71 strains from different genogroups isolated in Yamagata, Japan, between 1990 and 2007.

We isolated and identified six subgenogroups (B2, B4, B5, C1, C2, and C4) of enterovirus 71 (EV71) between 1990 and 2007 in Yamagata, Japan. We measured neutralizing antibody (NT Ab) titers against those subgenogroup strains and the BrCr reference strain for antigenic analysis. Serological analysis of 83 residents in Yamagata in 2004 showed that differences in the NT Ab titer of each individual against the different subgenogroups were mostly within 4-fold. Furthermore, sera from guinea pigs, immunized with the B2 and C1 strains indicated cross-antigenicity among the seven different subgenogroups. In conclusion, our results showed that cross-antigenicity exists among EV71 strains from different subgenogroups circulating in the community through genomic evolution. Our results also suggest that eliciting neutralizing antibodies against one genotype is likely to confer cross-neutralization against other genotypes.

Bone formation in rat calvaria ceases within a limited period regardless of completion of defect repair.

A bone defect that is not repaired with bone completely is designated a non-union defect or a critical-size defect. The biological mechanism that regulates the process of bone repair of the critical-size defect remains unknown. The present study was designed to investigate bone repair in a critical-size defect compared with that in a smaller or non-critical-size defect. Our original standardized rat calvarial bone defect model was used for the experiment. The rate of bone formation was examined with X-ray morphometry and the bone production of osteoblasts and osteocytes was assessed by molecular histology with in situ hybridization for type I collagen and osteocalcin. Formation of repaired bone ceased within 24 weeks in both critical- and non-critical-size defects i.e. regardless of completion of the defect repair. The results suggested that osteoblasts and osteocytes cease bone formation, and the differentiation of osteoblast progenitors declines in 24 weeks. Also, bone repair proceeds from the periosteum on both sides of the parietal bone but not from the surface of the bony edge around the original defect. The results could provide useful information for clinical research on bone repair.

Outbreak of human metapneumovirus detected by use of the Vero E6 cell line in isolates collected in Yamagata, Japan, in 2004 and 2005.

A number of epidemiological studies have shown human metapneumovirus (hMPV) to be one of the most important viral agents associated with acute respiratory infections in humans. However, due to the difficulty in growing the virus, all epidemiological studies of hMPV infection have been performed on the basis of the molecular method. Thus, the development of a cell line suitable for the isolation of hMPV from clinical specimens is a crucial step for further research. Using the Vero E6 cell line, which could be stably maintained for 1 month without passage or medium change, we succeeded in isolating 79 strains from 4,112 specimens obtained in Yamagata, Japan, in 2004 and 2005. The total isolation rate was 1.9% (79/4,112). The monthly distribution revealed that hMPV infections occurred between February and April in 2004 and throughout most of the year in 2005. Phylogenetic analysis indicated that subgenogroup B2 was predominant in 2004, whereas three subgenogroups, A2, B1, and B2, had cocirculated in 2005. Although multiple subgenogroups cocirculated in 2005, each individual subgenogroup strain was found to predominate at specific sites. An infectivity assay of hMPV strains also indicated that the infection efficiency in Vero E6 cells was better than that in LLC-MK2 cells. Finally, we found that Vero E6 cells are useful for the isolation of hMPVs and that this utility might aid further research into hMPVs beyond the epidemiological data shown in this study.

Age and comorbidity as risk factors for vocal cord paralysis associated with tracheal intubation.

BACKGROUND: Vocal cord paralysis after tracheal intubation may be attributed to ageing and comorbidity. However, the relationship between patient characteristics and the risk of vocal cord paralysis is unknown. METHODS: We prospectively analysed data representing 31 241 consecutive surgery patients who underwent tracheal intubation to determine whether duration of intubation, age, sex, and cardiovascular, cerebrovascular, and metabolic diseases were risk factors for vocal cord paralysis associated with intubation. Patients with vocal cord paralysis from any other causes were excluded. RESULTS: Twenty-four (0.077%) suffered vocal cord paralysis (left, 16 patients; right, 8 patients). The risk was increased when intubation lasted 3-6 h (odds ratio, 2.0; 95% confidence interval, 1.1-5.6; P = 0.002) or 6 h or more (odds ratio, 14.5; 95% confidence interval, 5.2-40.9; P < 0.0001). The risk was increased in patients aged 50-69 (odds ratio, 3.6; 95% confidence interval, 1.2-11.1; P = 0.02) and 70 yr or above (odds ratio, 3.9; 95% confidence interval, 1.2-12.8; P = 0.02). The risk was increased with diabetes mellitus (odds ratio, 2.5; 95% confidence interval, 1.1-7.3; P = 0.03) and hypertension (odds ratio, 2.1; 95% confidence interval, 1.1-6.0; P = 0.03). CONCLUSIONS: The risk of vocal cord paralysis was increased three-fold in patients aged 50 or above, two-fold in patients intubated 3-6 h, 15-fold in patients intubated 6 h or more, and two-fold in patients with a history of diabetes mellitus or hypertension. Our results are informative for informed consent, patient counselling, and intubation decision-making.

Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003.

Phylogenetic analysis of 45 enterovirus 71 (EV71) isolates for 6 years in Yamagata, Japan, clarified that the annual outbreak of hand-foot-and-mouth disease was due to four genetically distinct subgenogroups, including a novel "B5." Our results suggest that the importation of EV71 from surrounding countries has had a major epidemiological impact on the local community used in our study.

Genetic characterization of parthenogenic Fasciola sp. in Japan on the basis of the sequences of ribosomal and mitochondrial DNA.

Accurate identification of aspermic Fasciola forms in Japan remains difficult because of their morphological variations. In order to characterize the forms genetically, nucleotide sequences of ribosomal internal transcribed spacer (ITS1 and ITS2) and mitochondrial cytochrome c oxidase I (COI) and NADH dehydrogenase I (NDI) genes in 34 liver flukes from 16 prefectures in Japan were analysed. Two major forms represented by Fsp 1 and Fsp 2 had sequences identical to or closely resembling those of F. hepatica and F. gigantica, respectively, in all the 4 DNA markers and were mainly distributed in northern and eastern-western parts of Japan, respectively. Fsp 1 and Fsp 2 would have been introduced into Japan with infected cattle of 2 distinct lineages via the Korean Peninsula and spread through limited parts of Japan (northern and eastern-western parts) together with the movement of each cattle lineage. The Japanese form (Fsp 1/2), which showed heterozygosity in ribosomal DNA and Fsp 2 haplotype in mitochondrial DNA, may have originated in interspecific cross hybridization between paternal F. hepatica and maternal F. gigantica.

Opposing effects of oestradiol and progesterone on intracellular pathways and activation processes in the oxidative stress induced activation of cultured rat hepatic stellate cells.

BACKGROUND: Oxidative stress, including the generation of reactive oxygen species (ROS), is involved in hepatofibrogenesis. The authors' previous studies have shown that oestradiol suppresses hepatic fibrosis in animal models and attenuates the activation of cultured rat hepatic stellate cells (HSCs), which possess oestrogen receptor subtype beta and are also activated by ROS. AIMS: To define the mechanisms by which female sex hormones play an antifibrogenic role in activated HSCs, the effects of oestradiol and progesterone on ROS generation processes and intracellular pathways, leading to the activation of HSCs undergoing oxidative stress, was examined. METHODS: HSCs, isolated from rats, were cultured for 7 days with oestradiol or progesterone for 24 hours as pretreatment, and oxidative stress was then induced by exposure to low doses of hydrogen peroxide for another 24 hours. RESULTS: Oestradiol inhibited ROS generation and antioxidant enzyme loss via the suppression of NADH/NADPH oxidase activity, and attenuated hydrogen peroxide induced transforming growth factor-beta1 (TGF-beta1) expression, HSC proliferation and transformation, and the activation of mitogen activated protein kinase (MAPK) pathways and transcription factors. Progesterone exerted a stimulatory effect through the progesterone receptor on the induction of ROS generation processes and intracellular pathways, resulting in TGF-beta1 expression and HSC activation, and fibrogenic effects were inhibited by oestradiol. CONCLUSION: These findings show for the first time that oestradiol inhibits the activation of transcription factors by suppressing ROS generation processes and the MAPK pathways, and inactivates the downstream transcription processes involved in TGF-beta1 expression and HSC activation, whereas progesterone acts in opposition to the favourable effects of oestradiol and its effects are blocked by oestradiol.

The first record of Dirofilaria immitis infection in a Humboldt penguin, Spheniscus humboldti.

Dirofilaria immitis infection is an important parastic disease in many mammals, especially canids, but has not been reported in bird hosts. Filarial worms were isolated from the lumen of the right atrium of the heart and the connective tissue of the lung of a captive female Humboldt penguin, Spheniscus humboldti, that died at a zoo in Japan. One of these worms was observed morphologically and identified as D. immitis by features such as 4 pairs of cephalic papillae, 1 pair of cervical papillae, esophagus divided into 2 regions, 4 pairs of pre-anal papillae, 5 pairs of post-anal papillae, and unequal spicules. In addition, the partial DNA sequence (234 bp) of mitochondrial CO / gene of the filarial worm was identical to that of D. immitis. This is the first report of D. immitis infection in a bird.

Epidemics of two Victoria and Yamagata influenza B lineages in Yamagata, Japan.

We attempted to predict epidemics of influenza B, focusing on B/Victoria/2/87-like (V) and B/Yamagata/16/88-like (Y) lineages, in Yamagata, Japan. We collected 9624 nasopharyngeal swabs for virus isolation from patients with respiratory infections between 1996 and 2003 and 237 sera for seroepidemiological analysis by haemagglutination-inhibition test in 2001. We isolated 424 V-lineage and 246 Y-lineage viruses during the study period. Three herald viruses in the 2000--2001 season enabled us to predict a V-lineage epidemic in the following season. However, another V-lineage epidemic occurred in the 2002--2003 season, although we caught four herald Y-lineage viruses, whose antigenic drift was suggested by seroepidemiological study, at the end of the previous season. Since the epidemiology of the two influenza B lineages remains unclear, a careful watch should be kept on these lineages in order to provide effective public-health strategies against future epidemics.

[Endobronchial brachytherapy for intra-luminal recurrence after lung cancer resection].

A 63-year-old man who had squamous cell carcinoma in left lung was received left lower lobectomy with lymph node dissection (pT3N0M0). Twenty months after surgery, the patient showed bloody sputum and bronchofiberscopy revealed intra-luminal recurrence on trachea. Endobronchial brachytherapy in combination with external beam radiotherapy was selected and complete remission was achieved. After the brachytherapy, bronchitis was observed and was healed 23 months after the therapy.