Vitellogenesis in the cestode <Emphasis Type="Italic">Atractolytocestus huronensis</Emphasis> Anthony, 1958 (Caryophyllidea: Lytocestidae)

Vitellogenesis in the parthenogenetic caryophyllidean tapeworm Atractolytocestus huronensis Anthony, 1958, parasitizing the carp Cyprinus carpio L., has been examined using light (LM) and transmission electron microscopy and cytochemical staining with periodic acid–thiosemicarbazide–silver proteinate for glycogen. It was found that formation of vitellocytes shares the same basic patterns in lower tapeworms with parthenogenetic and normal reproduction. Each vitelline follicle of A. huronensis consists of vitellocytes at various stages of development and an interstitial tissue. Projections of the interstitial tissue surround each vitellocyte and extend as a cytoplasmic sheath on the follicular periphery. In contrary to other Eucestoda, interstitial tissue of A. huronensis includes numerous electron-dense vesicles of various sizes. Maturing and mature vitellocytes contain vitelline material in the form of single small shell globules, which may gradually fuse and give rise to the large shell globule clusters. Shell globule clusters are of two types in A. huronensis. Additional vitelline material is represented by single “lamellar” granules and glycogen in the cytoplasm of mature vitellocytes. The ultrastructural features of vitellogenesis and/or vitellocyte in A. huronensis are compared with that in other caryophyllideans and/or Neodermata.     

Molecular characterization of <Emphasis Type="Italic">Atractolytocestus sagittatus</Emphasis> (Cestoda: Caryophyllidea), monozoic parasite of common carp,and its differentiation from the invasive species <Emphasis Type="Italic">Atractolytocestus huronensis</Emphasis>

Sequence structure of complete internal transcribed spacer 1 and 2 (ITS1 and ITS2) of the ribosomal DNA region and partial mitochondrial cytochrome c oxidase subunit I (cox1) gene sequences were studied in the monozoic tapeworm Atractolytocestus sagittatus (Kulakovskaya et Akhmerov, 1965) (Cestoda: Caryophyllidea), a parasite of common carp (Cyprinus carpio carpio L.). Intraindividual sequence diversity was observed in both ribosomal spacers. In ITS1, a total number of 19 recombinant clones yielded eight different sequence types (pairwise sequence identity, 99.7–100%) which, however, did not resemble the structure typical for divergent intragenomic ITS copies (paralogues). Polymorphism was displayed by several single nucleotide mutations present exclusively in single clones, but variation in the number of short repetitive motifs was not observed. In ITS2, a total of 21 recombinant clones yielded ten different sequence types (pairwise sequence identity, 97.5–100%). They were mostly characterized by a varying number of (TCGT)_n repeats resulting in assortment of ITS2 sequences into two sequence variants, which reflected the structure specific for ITS paralogues. The third DNA region analysed, mitochondrial cox1 gene (669 bp) was detected to be 100% identical in all studied A. sagittatus individuals. Comparison of molecular data on A. sagittatus with those on Atractolytocestus huronensis Anthony, 1958, an invasive parasite of common carp, has shown that interspecific differences significantly exceeded intraspecific variation in both ribosomal spacers (81.4–82.5% in ITS1, 74.4–75.2% in ITS2) as well as in mitochondrial cox1, which confirms validity of both congeneric tapeworms parasitic in the same fish host.     

Classical and molecular cytogenetics of <Emphasis Type="Italic">Khawia sinensis</Emphasis> (Cestoda: Caryophyllidea), invasive parasite of carp, <Emphasis Type="Italic">Cyprinus carpio</Emphasis>

Chromosomes of the invasive tapeworm Khawia sinensis (Caryophyllidea), the specific parasite of common carp, were analyzed by means of conventional Giemsa staining and using fluorescent DAPI and YOYO-1 dyes, silver staining, and fluorescent in situ hybridization (FISH) with 18S rDNA probe. The karyotype is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16, n = 3m + 5t, TCL = 42.54 μm). Constitutive heterochromatin was located at pericentromeric regions of all pairs, except for the largest metacentric pair (no. 1), which possessed no DAPI-positive band. FISH with rDNA probe revealed that both homologues of chromosome pair no. 6 carry a cluster of ribosomal arrays, which were located interstitially close to the centromere. Present results are compared with previous cytogenetic data on Khawia spp., and comments are made on the karyotypes with respect to their phylogenetic links.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Selective activity of extracts of <Emphasis Type="Italic">Margaritaria discoidea</Emphasis> and <Emphasis Type="Italic">Homalium africanum</Emphasis> on <Emphasis Type="Italic">Onchocerca ochengi</Emphasis>

Background  

The current treatment of onchocerciasis relies on the use of ivermectin which is only microfilaricidal and for which resistant parasite strains of veterinary importance are increasingly being detected. In the search for novel filaricides and alternative medicines, we investigated the selective activity of crude extracts of Margaritaria discoidea and Homalium africanum on Onchocerca ochengi, a model parasite for O. volvulus. These plants are used to treat the disease in North West Cameroon.     

Antiviral biflavonoids from <Emphasis Type="Italic">Radix Wikstroemiae</Emphasis> (<Emphasis Type="Italic">Liaogewanggen</Emphasis>)

Background  

Radix Wikstroemiae is a common Chinese herbal medicine. The ethyl acetate fraction of the ethanolic extract of W. indica possesses potent in vitro antiviral activity against respiratory syncytial virus (RSV). This study aims to identify the antiviral components of the active fraction.     

The importance of <Emphasis Type="Italic">Helicobacter pylori tnpA</Emphasis>, <Emphasis Type="Italic">tnpB</Emphasis>, and <Emphasis Type="Italic">cagA</Emphasis> genes in various gastrointestinal diseases

The cag (cytotoxin-associated gene) pathogenicity island (cagPAI) is one of the major virulence determinants of Helicobacter pylori (H. pylori). The purpose of this study was to investigate the association of the three genes (tnpA, tnpB, and cagA) in H. pylori isolated from Azerbaijani patients with the different gastrointestinal disease. A total of 362 gastric biopsies were collected from hospitals of Tabriz University of Medical Sciences, and were cultured on Brucella agar. The tnpA, tnpB, and cagA genes were detected by PCR. Of the total 264 H. pylori isolates, tnpA, tnpB, and cagA genes were detected in 120 (45.5%), 56 (21.2%) and 172 (65.2%), respectively. A significant association between tnpA and tnpB genes and clinical outcomes were found (P < 0.05). The cagA status was not related to clinical outcomes in our subjects. The predominant genotype among cag-PAI is the cagA. The prevalence of tnpA, tnpB, and cagA genes are high in patients with gastric cancer, and a significant association is revealed between tnpA and tnpB with gastric cancer.     

Massive presence of <Emphasis Type="Italic">Echinococcus granulosus</Emphasis> (Cestoda,Taeniidae) cysts in a wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) from Spain

We report a heavy infestation of a free-living wild boar sow from Spain with Echinococcus granulosus cysts and state its molecular characterization. We found >65 hydatid cysts in the thoracic and abdominal cavities of the sow. Parasites were routinely processed for their identification and histopathology and DNA molecular characterization of the E. granulosus cysts were carried out. The polymerase chain reaction results confirmed the E. granulosus identity of the cysts and restriction fragment length polymorphism and sequencing revealed its G1 genotype. Our results suggest that wild boar could be involved in the epidemiology of E. granulosus, particularly considering that large amounts of carcass remains are available to dogs and wolves during the hunting season. The recent population increase of the wild boar in Spain and the DNA confirmation that the wild boar isolate shared identical sequences to the sheep strain emphasize the importance of the reported finding in public health.     

Association analysis of <Emphasis Type="Italic">SLC22A4</Emphasis>, <Emphasis Type="Italic">SLC22A5</Emphasis> and <Emphasis Type="Italic">DLG5</Emphasis> in Japanese patients with Crohn disease

Crohn disease (CD) is an inflammatory bowel disease characterized by chronic transmural, segmental, and typically granulomatous inflammation of the gut. Recently, two novel candidate gene loci associated with CD, SLC22A4 and SLC22A5 on chromosome 5 known as IBD5 and DLG5 on chromosome 10, were identified through association analysis of Caucasian CD patients. We validated these candidate genes in Japanese patients with CD and found a weak but possible association with both SLC22A4 (P=0.028) and DLG5 (P=0.023). However, the reported genetic variants that were indicated to be causative in the Caucasian population were completely absent in or were not associated with Japanese CD patients. These findings imply significant differences in genetic background with CD susceptibility among different ethnic groups and further indicate some difficulty of population-based studies.     

Morphological characterization and <Emphasis Type="Italic">HSP70</Emphasis>-, <Emphasis Type="Italic">IGS</Emphasis>-based phylogenetic analysis of two microsporidian parasites isolated from <Emphasis Type="Italic">Antheraea pernyi</Emphasis>

Two microsporidian isolates were extracted from single infected egg-laying tussah silk moth (Antheraea pernyi) in Liaoning Province, China. The microsporidia were subsequently grown in silk moth larvae, isolated, and subjected to morphological characterization (by light and transmission electron microscopy) and phylogenetic analysis (based on conserved genes). One type of spore was long-axis-oval in shape, measuring 4.71 × 1.95 μm, and the other type was short-axis-oval, measuring 3.64 × 2.17 μm. These dimensions were markedly different from those reported in the spores of the common microsporidia infecting A. pernyi, namely, Nosema pernyi (4.36 × 1.49 μm). A neighbor-joining phylogenetic tree based on HSP70 indicated that these microsporidia belonged to Nosema species and were closely related with Nosema bombycis and Nosema ceranae. Furthermore, in the phylogenetic tree based on the intergenic spacer (IGS) region, the long-axis-oval isolates were closely related and tended to form a clade away from the short-axis-oval isolates and N. pernyi isolates. The microsporidia isolated from A. pernyi clustered in one group. Nosema bombycis, Nosema spodopterae, and Endoreticulatus spp. appeared to be genetically distant from N. pernyi. The two isolates from A. pernyi fell in the Nosema group, but their spores differed from those of the spores of the common A. pernyi parasite N. pernyi, both in morphological and genetic aspects. The two isolates were designated Nosema sp. Ap (L) and Nosema sp. Ap (S). IGS was found to be informative in ascertaining phylogenetic relationships among species, and even closely related strains, of microsporidia.     

Larvicidal activity of silver nanoparticles synthesized using <Emphasis Type="Italic">Plumeria rubra</Emphasis> plant latex against <Emphasis Type="Italic">Aedes aegypti</Emphasis> and <Emphasis Type="Italic">Anopheles stephensi</Emphasis>

In the present study activity of silver nanoparticles (AgNPs) synthesized using Plumeria rubra plant latex against second and fourth larval instar of Aedes aegypti and Anopheles stephensi was determined. Range of concentrations of synthesized AgNps (10, 5, 2.5, 1.25, 0.625, 0.3125 ppm) and aqueous crude latex (1,000, 500, 250, 125, 62.50, 31.25 ppm) were tested against larvae of A. aegypti and A. Stephensi. The synthesized AgNps from P. rubra latex were highly toxic than crude latex extract in both mosquito species. The LC₅₀ values for second and fourth larval instars after 24 h of crude latex exposure were 1.49, 1.82 ppm against A. aegypti and 1.10, 1.74 ppm against A. stephensi respectively. These figures were 181.67, 287.49 ppm against A. aegypti and 143.69, 170.58 ppm against A. stephensi respectively for crude latex extract. The mortality rates were positively correlated with the concentration of AgNPs. The characterization studies of synthesized AgNPs by UV–Vis spectrophotometry, transmission electron microscopy (TEM), Particle size analysis (PSA) and zeta potential confirmed the spherical shape and size (32–200 nm) of silver nanoparticles alongwith stability. Toxicity studies carried out against non-target fish species Poecilia reticulata, the most common organism in the habitats of A. aegypti and A. stephensi showed no toxicity at LC₅₀ and LC₉₀ doses of the AgNPs. This is the first report on mosquito larvicidal activity of latex synthesized nanoparticles.     

The life cycle of <Emphasis Type="Italic">Stephanoprora aylacostoma</Emphasis> n.sp. (Digenea: Echinostomatidae), parasite of the threatened snail <Emphasis Type="Italic">Aylacostoma chloroticum</Emphasis> (Prosobranchia,Thiaridae), in Argentina

A new species, Stephanoprora aylacostoma is described and its life cycle was resolved experimentally. The prosobranch snail Aylacostoma chloroticum Hylton Scott, collected in the Yacyretá dam, Province of Misiones, Argentina, was found naturally infected with large-tailed cercariae possessing a prepharyngeal body and corpuscles in the excretory system but lacking collar spines. Metacercariae, which encysted on the gills of experimentally infected fishes Cnesterodon decemmaculatus (Jenyns) and Poecilia reticulata (Peters) (Poecilidae), developed collar spines after 10 days. Tetragonopterid fishes Moenckhausia dichroura (Kner), Astyanax erythropterus (Holmberg) and Hyphesobrycon serpae (Durbin in Eigenmann) were found infected naturally. Sexually mature adults were recovered from domestic chicks at day 7 post-exposure. Eggs shed in chick faeces developed to miracidia within 13–15 days; sporocysts were found on the gills of snails. The new species differs from other species of the genus in its larger eggs, in the smaller, slender body and smaller collar spines of the adult and in the morphological and biological features of the larval stages.     

Ultrastructural characters of the spermatozoon of the cestode<Emphasis Type="Italic"> Corallobothrium solidum</Emphasis> Fritsch, 1886 (Cestoda: Proteocephalidea), a parasite of the electric catfish<Emphasis Type="Italic"> Malapterurus electricus</Emphasis>

The fine structure of the mature spermatozoon of the corallobothriine tapeworm Corallobothrium solidum Fritsch, 1886 (Cestoda: Proteocephalidea) from the electric catfish Malapterurus electricus from the Nile River in Egypt was studied by transmission electron microscopy for the first time. The filiform spermatozoon of C. solidum contains two axonemes of unequal length and a typical 9+1 trepaxonematan pattern. A single helicoidal crested body (30–200 nm thick) is localized at the anterior extremity of the gamete. The cortical microtubules line the periphery of the cell, largely parallel to the long axis of the spermatozoon and exhibiting signs of twisting at the beginning of region II. The nucleus, in the form of an electron-dense (largely in gametes of testes) and/or fibrous cord (largely in gametes from male reproductive ducts and seminal vesicle), coils in a spiral through the middle part (region III) of the spermatozoon. The cytoplasm contains electron-dense granules in regions II, III and partly in region IV. The cytoplasm of some spermatozoa exhibits an apparently higher electron-density at the end of the nucleated region (III), and continuously toward the middle part of region IV. The anterior and posterior extremities of the spermatozoa have a single axoneme. The ultrastructural features of the mature spermatozoon of C. solidum mostly coincide with those of the spermatozoon of other proteocephalideans, especially the gangesiine Electrotaenia malopteruri parasitizing the same host.     

Frequent <Emphasis Type="Italic">BRAF</Emphasis><Superscript><Emphasis Type="Italic">V600E</Emphasis></Superscript> and Absence of <Emphasis Type="Italic">TERT</Emphasis> Promoter Mutations Characterize Sporadic Pediatric Papillary Thyroid Carcinomas in Japan

Pediatric papillary thyroid carcinoma (PTC) has unique features but requires further genetic investigation. Moreover, there has been increasing concern about the risk for pediatric PTC in Japan after the Fukushima accident. This study aims to evaluate the frequencies of BRAF and TERT promoter mutations and to examine their significance in non-radiation-associated pediatric PTCs in Japan. We enrolled 81 pediatric PTC patients aged ≤20 years. The control group included 91 adult PTCs from patients >20 years old. BRAF and TERT mutations were analyzed by allele-specific-PCR and/or Sanger sequencing. Compared with adult PTCs, pediatric PTCs exhibited larger tumor size, more frequent lymph node metastasis, and less classical histology. The prevalence of BRAF ^V600E in pediatric PTCs was 54% and significantly lower than that in adults of 85%. In the pediatric PTCs, BRAF ^V600E was positively associated with older age, classical histology, and the lymph node metastasis but independent from other clinicopathological factors. TERT mutations were identified in 13% of adults and in none of the pediatric PTCs. In conclusion, pediatric PTCs are characterized by more advanced clinicopathological features, lower BRAF ^V600E frequency, and absence of TERT mutation. The BRAF ^V600E frequency in this study is similar to the reported BRAF ^V600E frequency in the ultrasonographically screened pediatric PTCs in Fukushima.     

Concurrent <Emphasis Type="Italic">M. tuberculosis</Emphasis>, <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>, and <Emphasis Type="Italic">Candida albicans</Emphasis> infection in liver metastasis of bowel carcinoma

We describe the case of a 62-year-old patient with fever and abdominal pain of the right upper quadrant with a known history of adenocarcinoma of the colon and presence of hepatic metastases. One of the liver lesions underwent aspiration, and pus was sent for microbiological testing. Cultures of the pus were positive for Klebsiella pneumoniae and Candida albicans and polymerase chain reaction for Mycobacterium tuberculosis was concurrently positive; the patient received treatment for all three pathogens and improved clinically. One may consider searching not only for the usual pathogens of liver abscesses (gram-negative bacteria, anaerobic bacteria and gram-positive bacteria), but in special cases might consider pursuing a more detailed search for coexistence of fungi and mycobacteria in patients with cancer.     

Identification of 108 SNPs in <Emphasis Type="Italic">TSC</Emphasis>, <Emphasis Type="Italic">WNK1</Emphasis>, and <Emphasis Type="Italic">WNK4</Emphasis> and their association with hypertension in a Japanese general population

The deletion of thiazide-sensitive Na–Cl cotransporter (TSC, SLC12A3) causes Gitelmans syndrome characterized by low blood pressure, while deletions of the WNK1 (PRKWNK1) and WNK4 (PRKWNK4) genes cause familial hypertension known as pseudohypoaldosteronism type II. Recent studies have revealed that cell surface expression of TSC is regulated by WNK1 and WNK4. We hypothesized that molecular variations in TSC, WNK1, and WNK4 could lead to an increased morbidity of hypertension. We identified 52, 35, and 21 polymorphisms in Japanese hypertensives by sequencing the entire coding regions of TSC, WNK1 and WNK4, respectively. Twenty-one representative polymorphisms were genotyped in 1,818 Japanese individuals (771 subjects with hypertension and 1,047 controls) randomly sampled in Suita city. The results indicated that the systolic blood pressure in men with the CT+TT genotype in WNK4 C14717T was 3.1 mmHg higher than those with the CC genotype (p=0.042) after adjustment with confounding factors such as age, BMI, hyperlipidemia, diabetes mellitus, antihypertensive drug use, smoking, and drinking. Multivariate logistic regression analysis (with adjustment for the same parameters) in men revealed that the odds ratio for the presence of hypertension of the CT+TT genotype in C14717T to the CC genotype was 1.62 (p=0.010, 95% confidence interval, 1.12–2.33). Association of TSC and WNK1 with hypertension was not observed. In conclusion, our study suggests the possible involvement of WNK4 in essential hypertension in a Japanese general population.     

Histochemical and enzyme-histochemical studies of <Emphasis Type="Italic">Oncomelania hupensis</Emphasis> (Gredler, 1881), the intermediate host of <Emphasis Type="Italic">Schistosoma japonicum</Emphasis>

The distribution of glycogen, DNA and histone, and localization of activity of ten enzymes such as glucose-6-phosphatase (G-6-Pase), cytochrome oxidase (CCO), lactate dehydrogenase (LDH), 5′-nucleotidase (5′-NT), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G-6-PDH), alkaline phosphatase (ALP), acid phosphatase (ACP), Mg²⁺-adenosine triphosphatase (Mg²⁺-ATPase), and cholinesterase (CHE) in Oncomelania hupensis (Gredler, 1881) snails, the intermediate host of Schistosoma japonicum, was surveyed using the histochemical and enzyme-histochemical techniques. The results showed that the glycogen with high activity was widely distributed in snails. DNA showed its strongest reaction in testis. The histone was distributed in sites of the radula, parenchyma of penis and ovary. LDH and SDH were widely distributed in snails and all were abundant. Sites of the strongest enzyme activities of G-6-PDH were evident in the reproductive system together with central ganglia. Only the ovary showed a strong enzyme activity of G-6-Pase. Most tissues and organs contained CCO, and the activity of this enzyme was very strong. 5′-NT showed a strong enzyme activity in the ovary and testis. Mg²⁺-ATPase was localized in sites of the liver, stomach and reproductive gland. A strong enzyme activity of ALP appeared in the digestive system. ACP showed a low activity in snails. The central ganglia, nervous stem, liver, branchial duct, epithelia of the head and foot regions showed a very strong enzyme activity of CHE. The findings could provide a theoretical basis for development of highly effective molluscicides with low toxicity to other biota, as well as means for novel snail control strategies.     

Objective facial paralysis grading based on<Emphasis Type="Italic">P</Emphasis><Subscript><Emphasis Type="Italic">face</Emphasis></Subscript> and eigenflow

To provide physicians with an objective and quantitative measurement of single-sided facial paralysis, the paper presents a computer-based approach that is different from the nine existing, subjective and hand-performed international scales, such as House-Brackman. For voluntary expressions of a patient, this approach used P_face, which stems from D_face, to measure the asymmetry between two sides of the face and used eigenflow to measure the expression variations between the patient and normal subjects. The results from P_face and eigenflow were then combined by the support vector machine produce to P_degree. A study of 25 subjects revealed that P_degree could differentiate paralysis states (P_degree≧0) and normal states (P_degree<0), with the ability to grade facial paralysis automatically. Moreover, the P_face of specific facial areas can be used in the supervision of the rehabilitation process.