Impact of Low Bone Mineral Content Index on Cardiovascular Outcomes in Patients With Coronary Artery Disease Undergoing Percutaneous Coronary Intervention.

The prognostic value of bone mineral content (BMC) for the clinical outcomes of patients with coronary artery disease (CAD) remains unknown. The present study evaluated the association between BMC index (BMCI) and cardiovascular events between January 2020 to June 2021, in consecutive patients (n = 257) with CAD undergoing percutaneous coronary intervention (PCI) at the Nagano Municipal Hospital. BMCI was measured using bioelectrical impedance analysis and calculated as the BMC divided by height squared. Patients were classified as low (<0.918) or high BMCI (≥0.918) groups according to the receiver operating characteristics curve analysis for the primary endpoint, major adverse cardiovascular events (MACE), including cardiovascular death, spontaneous myocardial infarction, stroke, and any revascularization. During a median follow-up of 744 days, the low BMCI group (n = 152) had an increased risk of MACE compared with the high group (n = 105) (19.7 vs 6.7%, P = .004). A low BMCI was significantly associated with MACE in the multivariable Cox and the Inverse Probability of Treatment Weighting analyses (hazard ratio: 3.16, 95% confidence interval: 1.15-8.67, P = .025). In conclusion, among patients with CAD undergoing PCI, BMCI was a predictor for cardiovascular events. Further research is required to determine whether medical interventions for BMC can improve patient prognosis.

Talaromyces mellisjaponici sp. nov., a xerophilic species isolated from honey in Japan.

Five isolates of a xerophilic Talaromyces species were obtained from honey in Japan. Molecular phylogenetic analysis based on a combined dataset for four regions (rRNA internal transcribed spacer, ß-tubulin, calmodulin and RNA polymerase II second largest subunit) revealed that the strains formed an independent clade in section Trachyspermi, which is sister to Talaromyces affinitatimellis, Talaromyces basipetosporus and Talaromyces speluncarum. The strains and their relatives have different growth on creatine agar, yeast extract sucrose agar and dichloran 18 % glycerol agar, different branching patterns (mostly monoverticillate or biverticillate, less frequently divaricate or terverticillate), and different sizes and surface structures of conidia. Xerotolerance tests were also conducted using media adjusted to five different sucrose concentrations (0, 20, 40, 60 and 80 %). The colony diameters of the strains were larger than those of T. affinitatimellis, T. basipetosporus and T. speluncarum at each sucrose concentration. Altogether, the obtained morphological, molecular and physiological data allowed the proposal of Talaromyces mellisjaponici sp. nov. for this novel species, with NBRC 116048T as the type strain.

Identification of the Acidification Mechanism of the Optimal pH for RNase He1.

Ribonuclease (RNase) He1 is a small ribonuclease belonging to the RNase T1 family. Most of the RNase T1 family members are active at neutral pH, except for RNase Ms, U2, and He1, which function at an acidic pH. We crystallized and analyzed the structure of RNase He1 and elucidated how the acidic amino residues of the α1ß3- (He1:26-33) and ß67-loops (He1:87-95) affect their optimal pH. In He1, Ms, and U2, the hydrogen bonding network formed by the acidic amino acids in the ß67-loop suggested that the differences in the acidification mechanism of the optimum pH specified the function of these RNases. We found that the amino acid sequence of the ß67-loop was not conserved and contributed to acidification of the optimum pH in different ways. Mutations in the acidic residues in He1 promoted anti-tumor growth activity, which clarified the role of these acidic amino residues in the binding pocket. These findings will enable the identification of additional targets for modifying pH-mediated enzymatic activities.

Effectiveness of endovascular therapy using stents in a patient with celiac artery compression syndrome due to the median arcuate ligament: A case report.

Celiac artery compression syndrome (CACS) is a disease caused by celiac artery compression by the median arcuate ligament (MAL), resulting in intestinal ischemic symptoms. However, a clear method for the invasive treatment of CACS has not yet been established because of limited treatment indications. In particular, only a few reports of endovascular therapy (EVT) using stents as the initial invasive treatment are available. Here, we report a case where EVT was performed using a stent in the celiac artery, resulting in good outcomes. A 59-year-old male patient presented to our hospital with postprandial abdominal pain and was diagnosed with MAL-induced CACS since the abdominal contrast computed tomography examination showed stenosis of a celiac artery origin. He was aware of the abdominal pain symptoms; therefore, we decided to treat CACS with EVT as an invasive treatment. A bare metal stent was placed in the celiac artery, whose lumen was well dilated using intravascular ultrasound. Consequently, he no longer felt abdominal pain and had good stent patency after 15 months. Minimally invasive EVT may be considered the first-line treatment for CACS. Learning objective: The efficacy of endovascular therapy (EVT) using stents for the invasive treatment of celiac artery compression syndrome (CACS) resulting from the compression of the median arcuate ligament has not yet been established. Specifically, the efficacy of EVT using stents for CACS is unknown. We can safely perform EVT with stent placement using intravascular ultrasound for maintaining long-term patency. Therefore, minimally invasive EVT may be considered the first-line treatment for CACS.

Eosinophilic granulomatosis with polyangiitis presenting with eosinophilic myocarditis as the only organ involvement.

A 45-year-old man with a history of bronchial asthma had fever and elevated eosinophils on the day of surgery for sinusitis, resulting in cancellation of the surgery. Two days later, he was referred to our department for electrocardiographic abnormalities. We suspected eosinophilic myocarditis (EM) since he presented with fever, left ventricular hypokinesis, and hypertrophy on echocardiography, and eosinophilia with elevated cardiac enzymes. We immediately performed an endomyocardial biopsy that showed eosinophilic infiltration of the myocardium. He was diagnosed with eosinophilic granulomatosis with polyangiitis (EGPA) since he suffered from asthma, eosinophilia, sinusitis, and EM. Methylprednisolone pulse therapy followed by oral prednisolone and intravenous cyclophosphamide pulse therapy decreased his eosinophils to within the normal range, and his symptoms subsequently improved. In EGPA, cardiac involvement is less commonly seen compared to other organ involvement. Moreover, patients with EGPA who have cardiac involvement generally have other organ involvement as well. In this report, the patient had only cardiac involvement as organ damage associated with EGPA, except for asthma and sinusitis in the prodromal phase, making it clear that patients with EGPA could present with cardiac involvement alone. Therefore, it is recommended to thoroughly examine for cardiac involvement in patients with suspected EGPA. Learning objective: We report a case of eosinophilic granulomatosis with polyangiitis (EGPA) presenting with cardiac involvement alone as organ damage, subsequently diagnosed with eosinophilic myocarditis as confirmed by an endomyocardial biopsy. EGPA usually involves other organs in addition to the cardiovascular system; however, patients with EGPA could present with cardiac involvement alone, as in this case. Thus, we should thoroughly investigate for cardiac involvement in patients with suspected EGPA.

Validation and Comparison of the Prognosis Predicting Ability of Inflammation-Based Scores Following Endovascular Treatment for Peripheral Artery Disease.

We assessed the prognostic ability of several inflammation-based scores and compared their long-term outcomes in patients with peripheral artery disease (PAD) following endovascular treatment (EVT). We included 278 patients with PAD who underwent EVT and classified them according to their inflammation-based scores (Glasgow prognostic score [GPS], modified GPS [mGPS], platelet to lymphocyte ratio [PLR], prognostic index [PI], and prognostic nutritional index [PNI]). Major adverse cardiovascular events (MACE) at 5 years were examined, and C-statistics in each measure were calculated to compare their MACE predictive ability. During the follow-up period, 96 patients experienced MACE. Kaplan-Meier analysis showed that higher scores of all measures were associated with a higher MACE incidence. Multivariate Cox proportional hazard analysis showed that GPS 2, mGPS 2, PLR 1, and PNI 1, compared with GPS 0, mGPS 0, PLR 0, and PNI 0, were associated with an increased risk of MACE. C-statistics for MACE for PNI (.683) were greater than those for GPS (.635, P = .021), mGPS (.580, P = .019), PLR (.604, P = .024), and PI (.553, P < .001). PNI is associated with MACE risk and has a better prognosis-predicting ability than other inflammation-scoring models for patients with PAD following EVT.

Successful treatment of recurrent superior vena cava syndrome due to pulmonary adenocarcinoma using intravascular ultrasound.

Patients with malignant diseases may develop symptoms of superior vena cava syndrome (SVCS) quickly because rapid tumor growth does not allow adequate time to develop collateral blood flow. Therefore, malignant SVCS is a medical emergency associated with neurological or pharyngeal-laryngeal signs. Recently, interventional endovascular treatment (EVT) has achieved acceptable results. We describe the case of a 55-year-old woman with pulmonary adenocarcinoma and laryngeal edema. In the first EVT, bare-metal-stent was implanted into the SVCS with intravascular ultrasound (IVUS) guidance. The IVUS showed insufficient stent-mid expansion. We did not use additional ballooning because of the risk of superior vena cava (SVC) rupture. Three months later, the SVCS recurred. A second EVT was performed, and IVUS imaging suggested tumor ingrowth into the SVC through the stent struts. We considered that the tumor ingrowth could be covered in the SVC using stent-graft. The patient showed no recurrence of SVCS for about 12 months. IVUS-guided implantation of stent for the treatment of malignant SVCS has not been reported. This case report revealed that stent therapy using IVUS for SVCS is useful. Learning objective: Superior vena cava syndrome (SVCS) due to malignancy is not rare. Recently, endovascular treatment for SVCS has achieved acceptable results. However, SVC stenting in SVCS as having primary patency rate varies for each report. Intravascular ultrasound (IVUS) guided implantation of stent for malignant SVCS treatment has not been reported. In this case, we suspected insufficient stent expansion and tumor ingrowth as the possible cause of in-stent restenosis. Therefore, stent therapy using IVUS for malignant SVCS can be helpful.

Aspergillus verrucosus sp. nov., a xerophilic species isolated from house dust and honey in Japan.

Three strains of a xerophilic Aspergillus species were isolated from house dust and honey in Japan. A molecular phylogenetic analysis based on the combined dataset for four regions (internal transcribed spacer rDNA, calmodulin, ß-tubulin, and RNA polymerase II second largest subunit) revealed that the strains formed an independent lineage, sister to Aspergillus halophilicus classified in section Restricti. Morphological comparisons show that the strains differ from A. halophilicus in three aspects: (i) the size of cleistothecia, as well as the surface structure and size of ascospores, (ii) the ability to grow on Harrold's agar and dichloran 18 % glycerol agar, and (iii) the lack of conidiophore formation on potato dextrose agar +20 % NaCl. These strains could be clearly distinguished from all known Aspergillus section Restricti species. Therefore, we consider it to be a novel species and propose the name Aspergillus verrucosus sp. nov. (NBRC 115547T).

Symptomatic and severe pulmonary hypertension due to hyperthyroidism.

Hyperthyroidism has been recognised as the cause of secondary pulmonary hypertension (PH). However, several studies have reported that most PH cases due to hyperthyroidism are asymptomatic and not severe. Here, we report the case of a woman in her 30s with symptomatic and severe PH due to hyperthyroidism. She presented with a 2-month history of dyspnoea on exertion, palpitation and lower-extremity oedema. Echocardiography showed severe PH with estimated pulmonary arterial systolic pressure (ePASP) of 62 mm Hg. The diagnostic work-up showed severe PH due to hyperthyroidism. After treatment for hyperthyroidism, improvement of thyroid function contributed to a decrease in ePASP to 30 mm Hg. Similar to the present case, PH due to hyperthyroidism generally improves with the treatment of hyperthyroidism. Therefore, we must not overlook hyperthyroidism as a cause of PH and diagnose and start treatment for PH-causing hyperthyroidism at an early stage.

The causative agents of fascioliasis in animals and humans: Parthenogenetic Fasciola in Asia and other regions.

Parthenogenetic Fasciola is the causative agent of fascioliasis in animals and humans and is widely distributed in Asian countries, such as Japan, South Korea, China, Vietnam, Thailand, the Philippines, Myanmar, Bangladesh, Nepal, and India. Parthenogenetic Fasciola geographically originated from central and eastern China, where it exists between the habitats of Fasciola hepatica and Fasciola gigantica; it likely appeared thousands of years ago following hybridization between F. hepatica and F. gigantica. Parthenogenetic Fasciola consists of diploids and triploids that possess nuclear genome of both F. hepatica and F. gigantica and mitochondrial genome of either F. hepatica or F. gigantica. Maternal parents of parthenogenetic Fasciola are either F. hepatica having Fh-C4 haplotype or F. gigantica having Fg-C2 haplotype in mitochondrial NADH dehydrogenase subunit 1 (ND1) nucleotide sequences. Parthenogenetic Fasciola flukes with the Fh-C4 haplotype have spread from China to South Korea and Japan, whereas the flukes with the Fg-C2 haplotype have not only spread to Korea and Japan but also southward to Vietnam, Thailand, the Philippines, Myanmar, Bangladesh, Nepal, and India. Parthenogenetic Fasciola can be distinguished from F. hepatica and F. gigantica using combinational DNA sequence analysis of nuclear phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold) along with mitochondrial ND1 markers. The establishment of parthenogenetic Fasciola is expected as follows: parthenogenetic diploids with the Fh-C4 and Fg-C2 haplotypes first appeared based on single or multiple interspecific hybridization events; subsequently, parthenogenetic triploids emerged via backcross events between the maternal parthenogenetic diploid and either paternal bisexual F. hepatica or F. gigantica. Parthenogenetic Fasciola diploids and triploids then survived for thousands of years by clonal parthenogenetic reproduction, and generated descendants with ND1 haplotypes, which were derived from the Fh-C4 and Fg-C2 due to nucleotide substitution. Thus, the emergence of parthenogenetic Fasciola may be due to extremely uncommon and accidental events. Parthenogenetic Fasciola should be treated as a new asexual hybrid species.

Molecular analyses confirm the coexistence of Fasciola gigantica and parthenogenetic Fasciola in the Philippines.

Fasciola flukes collected from domestic buffalos and cattle in the Philippines were confirmed as Fasciola gigantica and parthenogenetic Fasciola based on DNA analyses of nuclear pepck and pold genes, and the mitochondrial ND1 gene. This study is the first to elucidate that F. gigantica and parthenogenetic Fasciola coexist in the Philippines with prevalences of 90.6% and 9.4%, respectively. The F. gigantica population showed a high genetic diversity with 25 ND1 haplotypes, suggesting that F. gigantica has existed in the Philippines for a long time. In contrast, parthenogenetic Fasciola flukes showed a single ND1 haplotype (Fsp-ND1-P1), which was identical to the founder haplotype, Fg-C2 of parthenogenetic Fasciola in China. These results indicate that parthenogenetic Fasciola in the Philippines is a recently introduced population from a neighboring continent.

Impact of Frailty and Age on Clinical Outcomes in Patients Who Underwent Endovascular Therapy.

PURPOSE: Information on the relationship between frailty and the outcome of endovascular therapy (EVT) in elderly patients with lower extremity peripheral artery disease (PAD) is scarce. This study aimed to reveal the impact of frailty on the prognosis of super-elderly patients who underwent EVT. MATERIALS AND METHODS: From August 2015 to August 2016, 335 consecutive patients who underwent EVT were enrolled in the I-PAD registry from 7 institutes in Nagano prefecture. Among them, we categorized 323 patients into 4 groups according to age and the presence or absence of frailty as follows: elderly with frailty (age ≥ 75, Clinical Frailty Scale [CFS] ≥ 5), elderly without frailty (age ≥ 75, CFS ≤ 4), young with frailty (age < 75, CFS ≥ 5), and young without frailty (age < 75, CFS ≤ 4); we analyzed them accordingly. The primary endpoints were major adverse cardiovascular and limb events (MACLE), defined as a composite of cardiovascular death, myocardial infarction, stroke, admission for heart failure, major amputation, and revascularization. The secondary endpoint was cardiovascular death. RESULTS: The median follow-up period was 2.7 years. In the elderly with frailty, elderly without frailty, young with frailty, and young without frailty groups, the freedom rates from MACLE were 34.9%, 55.7%, 35.4%, and 63.0%, respectively (p<0.001) and from all-cause death were 43.5%, 73.4%, 50.7%, and 90.9%, respectively (p<0.001). The freedom rates from MACLE were significantly higher among elderly patients with frailty than among young patients without frailty (55.7% vs 35.4%, p=0.01). In multivariate analysis, frailty was independently associated with MACLE incidence. CONCLUSION: Frailty as defined by CFS might be a predictor of MACLE incidence in patients with PAD who underwent EVT. By considering treatment indications for patients with PAD by focusing on frailty rather than age, we may examine whether EVT policies are appropriate and manage patient and caregiver expectations for potential improvement in functional outcomes. Further studies are expected to investigate whether changes in frailty after EVT change prognosis.

Inflammation-based assessment for the risk stratification of mortality in patients with heart failure.

The Glasgow Prognostic Score (GPS) has been established as a useful resource to evaluate inflammation and malnutrition and predict prognosis in several cancers. However, its prognostic significance in patients with heart failure (HF) is not well established. To investigate the association between the GPS and mortality in patients with HF, we assessed 870 patients who were 20 years old and more and had been admitted for acute decompensated HF. The GPS ranged from 0 to 2 points as previously reported. Over the 18-month follow-up (follow-up rate, 83.9%), 143 patients died. Increasing GPS was associated with higher HF severity assessed by New York Heart Association functional class and B-type natriuretic peptide (BNP) levels. Kaplan-Meier analysis showed significant associations for mortality and increased GPS. In multivariate analysis, compared to the GPS 0 group, the GPS 2 group was associated with high mortality (hazard ratio 2.92, 95% confidence interval 1.77-4.81, p < 0.001) after adjustment for age, sex, blood pressure, HF history, HF severity, hemoglobin, renal function, sodium, BNP, left ventricular ejection fraction, and anti-HF medications. In conclusion, high GPS was significantly associated with worse prognosis in patients with HF. Inflammation-based assessment by the GPS may enable simple evaluation of HF severity and prognosis.

Body composition and mortality in patients undergoing endovascular treatment for peripheral artery disease.

An inverse correlation between body mass index and mortality in patients with peripheral artery disease (PAD) has been reported. However, little information is available regarding the impact of body composition on the clinical outcomes in patients with PAD. This study evaluated the relationships between the lean body mass index (LBMI), body fat % (BF%), and mortality and major amputation rate in patients with PAD. We evaluated 320 patients with PAD after endovascular treatment (EVT) enrolled from August 2015 to July 2016 and divided them into low and high LBMI and BF% groups based on their median values (17.47 kg/m2 and 22.07%, respectively). We assessed 3-year mortality and major amputation for the following patient groups: Low LBMI/Low BF%, Low LBMI/High BF%, High LBMI/Low BF%, and High LBMI/High BF%. During the median 3.1-year follow-up period, 70 (21.9%) patients died and 9 (2.9%) patients experienced major amputation. The survival rate was lower in the Low LBMI than in the High LBMI group, and was not significantly different between the Low and High BF% groups. Survival rates were lowest in the Low LBMI/Low BF% group (57.5%) and highest in the High LBMI/High BF% group (94.4%). There were no significant differences in major amputation rate between the Low LBMI and High LBMI groups, and between the Low BF% and High BF% groups. The Low LBMI and Low BF% groups were associated with an increased risk of mortality after adjustment for age, sex, frailty and conventional risk factors [hazard ratio (HR): 4.02; 95% confidence interval (CI) 2.10-7.70; p < 0.001 and HR: 4.48; 95% CI 1.58-12.68, p = 0.005, respectively], for age, sex, hemodialysis, and prior cerebral cardiovascular disease (HR: 3.63; 95% CI 1.93-6.82; p < 0.001 and HR: 4.03; 95% CI 1.43-11.42, p = 0.009, respectively) and for age, sex, and laboratory date (HR: 3.97; 95% CI 1.88-8.37; p < 0.001 and HR: 3.31; 95% CI 1.15-9.53, p = 0.026, respectively). In conclusion, Low LBMI and Low BF% were associated with poor prognosis in patients undergoing EVT for PAD, and mortality was the lowest in the High LBMI/High BF% group compared with other body composition groups.

Detection and molecular characteristics of Pyelosomum cochlear (Digenea: Pronocephalidae) in the urinary bladder of the green sea turtle (Chelonia mydas) in the Northwest Pacific Ocean.

The genus Pyelosomum consists of parasitic flukes occurring primarily in marine turtles; Pyelosomum cochlear Looss 1899 is the only species of this genus that parasitizes the urinary bladder. In this study, we detected flukes in the urinary bladders of 20 of 88 green sea turtles (Chelonia mydas) harvested in the Ogasawara Islands, in the Northwest Pacific Ocean. We identified the flukes as P. cochlear based on detailed morphological observations and comparisons of morphometric measurements of the species reported previously. Nucleotide sequences of nuclear ribosomal 18S and 28S regions and the mitochondrial cytochrome c oxidase subunit 1 (COI) region were determined for the flukes. The 18S and 28S phylogenetic trees revealed that the species of the superfamily Pronocephaloidea, including P. cochlear, constituted a single clade, but the species of the family Pronocephalidae did not constitute a single taxon. These findings suggest that Pronocephalidae is a paraphyletic group. The COI sequences of P. cochlear exhibited high genetic diversity, suggesting that they would be useful markers to understand the genetic structure of the parasite and its evolutionary relationship with the host turtle populations. This is the first study to provide the nucleotide sequences of Pyelosomum species; these data will be available for further molecular studies of this genus and its related taxa.

Detection and molecular characteristics of Rhytidodoides sp. (Digenea: Rhytidodidae) from the gall bladder of green sea turtles (Chelonia mydas) in the Ogasawara Islands, Japan.

Trematodes of the genus Rhytidodoides are parasitic in marine turtles. Of the already known species, Rhytidodoides similis Price, 1939, occurs especially in the gall bladder. In this study, we surveyed 73 green sea turtles (Chelonia mydas) in the Ogasawara Islands, Japan, and detected Rhytidodoides sp. from the gall bladders of 18 turtles. A detailed morphological analysis revealed that the forebody of Rhytidodoides sp. differed slightly in shape from that of R. similis. There has been no information on DNA sequences of the family Rhytidodidae. A molecular phylogeny based on 28S rDNA sequences of Rhytidodoides sp. and related taxa suggested that the Rhytidodidae is sister to the other families of Echinostomatoidea. The intraspecific diversity of Rhytidodoides sp. was examined by using DNA sequences of mitochondrial cytochrome c oxidase subunit 1 gene (COI). The population genetic features of the COI haplotypes demonstrated that Rhytidodoides sp. is highly diverse in the Ogasawara Islands. The DNA sequences determined in this study will contribute to the species identification of congeners and the taxonomic reconsideration of the Echinostomatoidea.

Predictive Value of Abdominal Fat Distribution on Coronary Artery Disease Severity Stratified by Computed Tomography-Derived SYNTAX Score.

This study aimed to evaluate the association between abdominal fat distribution (AFD) and coronary artery disease (CAD) complexities using the computed tomography (CT)-derived SYNTAX score (CT-SXscore). Coronary computed tomographic angiography (CCTA) was performed in patients with suspected CAD. Plain abdominal CT was performed to measure visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) areas. To assess AFD, VAT/SAT (V/S) ratios were calculated. The CT-SXscore was calculated in patients with significant stenoses assessed by CCTA. Of 942 enrolled patients, 310 (32.9%) had 1 or more significant stenoses. The CT-SXscore showed a positive correlation with the V/S ratio (r = 0.33, p < 0.001). In the multivariate regression analysis, the V/S ratio was the only independent predictor for CAD severity based on the CT-SXscore (ß = 0.25; t = 4.14; p < 0.001), even though the absolute SAT and VAT areas showed no relationship to the CT-SXscore. Regarding the 4 CAD-patient groups divided according to their median VAT and SAT areas, the CT-SXscore was significantly higher for the high VAT/low SAT group than for any other group (19.6 ± 11.5 vs 13.3 ± 9.6 in the low VAT/low SAT, 10.1 ± 8.5 in the low VAT/high SAT, and 12.2 ± 8.7 in the high VAT/high SAT groups; p < 0.001 for all). In conclusion, it was found that the V/S ratio is a useful index for predicting CAD severity and that AFD may be a more important risk factor for CAD than the absolute amount of each abdominal fat.

Development of a multiplex PCR method for discriminating between Heterakis gallinarum, H. beramporia, and H. indica parasites of poultry.

Heterakis gallinarum, H. beramporia, and H. indica are common nematodes in gallinaceous poultry in Asian countries, and the infections occasionally lead to declining health of the hosts. These three Heterakis spp. can be identified by the morphological characteristics of the male worms; however, the female worms and eggs cannot be identified because they have no reliable morphological characteristics for discrimination. In addition, H. gallinarum is a well-known vector of fetal protozoan Histomonas meleagridis, making the discrimination between these three Heterakis species important in basic and clinical veterinary parasitology. We analyzed nuclear ribosomal 18S-ITS1-5.8S-ITS2-28S DNA sequences of these three Heterakis species. The 18S, 5.8S, and 28S DNA sequences had very high homology between the species; however, the ITS1 and ITS2 sequence similarity was 68.5 %-93.2 %. H. gallinarum, H. beramporia, and H. indica were divided into separate clades in the ITS1 and ITS2-concatenated phylogenetic tree. Therefore, to develop a multiplex PCR method for discriminating between the three Heterakis species, we designed species-specific reverse primers within the ITS2 region as follows: H. gallinarum-specific HgI2-R, H. beramporia-specific HbI2-R5, and H. indica-specific HiI2-R. The multiplex PCR amplified 396-bp, 272-bp, and 482-bp fragments specific to H. gallinarum, H. beramporia, and H. indica DNA, respectively, and did not amplify the fragments using other chicken nematode DNAs such as Ascaridia galli, Oxyspirura mansoni, Dispharynx nasuta, and Cheilospirura hamulosa. These results suggest that the multiplex PCR would serve as a useful tool for identifying and diagnosing infections of H. gallinarum, H. beramporia, and H. indica in poultry.

Prevalence of Nosema species infections in Apis cerana japonica and Apis mellifera honeybees in the Tohoku region of Japan.

We investigated here, the prevalence of Nosema microsporidia infections in the honeybees, Apis cerana japonica and Apis mellifera, in the Tohoku region of Japan. We detected Nosema ceranae DNA in 14 (2.8%) of 509 A. cerana japonica and in 34 (21.9%) of 155 A. mellifera honeybees from Aomori, Iwate, Akita, Yamagata, and Fukushima prefectures. Nosema apis DNA was undetectable in A. cerana japonica and A. mellifera. The unidentifiable Nosema species that genetically differed from N. apis, N. ceranae, and N. neumanni in terms of small subunit (SSU) rDNA, large subunit rDNA, and internal transcribed spacer sequences was identified in 105 (20.6%) of 509 A. cerana japonica and in 1 (0.6%) of 155 A. mellifera honeybees, and from Iwate prefecture. A phylogenetic tree based on SSU rDNA sequences showed that the Nosema sp. belonged to the same clade as N. thomsoni detected in moth and solitary bees in North America and N. pieriae found in cabbage butterfly in Turkey, which have not hitherto been detected in honeybees. The morphological characteristics of the spores should be analyzed to enable species identification of the Nosema sp.

Genetic diversity and population structure analyses based on microsatellite DNA of parthenogenetic Fasciola flukes obtained from cattle and sika deer in Japan.

Understanding the population structure of Fasciola flukes in domestic and wild animals is important for determining the extent of cross-infection between them. Although the parthenogenetic Fasciola flukes in Japan have been shown to comprise five genetic types based on the ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1) regions, these genetic regions are not suitable for analyzing their population structure. In the present study, the genetic diversity and population structure of the parthenogenetic Fasciola flukes in Japan were studied using microsatellite DNA, ITS1, and nad1 regions. A total of 144 parthenogenetic Fasciola flukes, obtained from cattle and sika deer in 16 localities, were individually analyzed using PCR-RFLP for ITS1, PCR-direct sequence analysis for nad1, and post-labeling PCR and capillary electrophoresis for microsatellite DNA regions. The flukes showed higher genetic diversity in the microsatellite DNA regions than ITS1 and nad1. The population structures of parthenogenetic Fasciola flukes were unclear, however, it was suggested that the flukes are more diverse populations. We hypothesized that their distribution throughout Japan is closely related to livestock movement dependent on human activity. Moreover, it is considered that cross-infection of the flukes between cattle and sika deer possibly has occurred in the past.