Coprological survey of alimentary tract parasites in dogs from Zambia and evaluation of a coproantigen assay for canine echinococcosis.

Faecal samples were collected from the rectum of 540 domestic dogs from four districts (Lusaka, Katete, Petauke and Luangwa) in Zambia between 2005 and 2006 and prevalences of canine alimentary tract parasites were determined by coprological examination. Thirteen different ova and parasites including strongyle (43.3%), Spirocerca lupi (18.7%), taeniid (13.1%), Toxocara canis (7.6%), Sarcocystis sp.* (7.5%), Isospora sp.* (5.7%), Physaloptera sp.* (4.6%), Capillaria sp.* (2.8%), Dipylidium caninum (2.2%), Mesocestoides sp.* (2.0%), Ascaris sp.* (1.7%), Trichuris vulpis* (0.4%) and Schistosoma mansoni* (0.4%) were detected, Ascaris and Schistosoma probably originating from coprophagy. The species with asterisks and later-described Taenia multiceps are for the first time reported from dogs in Zambia. A coproantigen enzyme-linked immunosorbent assay (CoproAg-ELISA) developed for Echinococcus spp. revealed 43 positive dogs and 37 of these harboured taeniid eggs. From 63 of the 71 taeniid egg-positive samples, eggs and DNA thereof were isolated and subjected to a multiplex polymerase chain reaction for differentiating E. granulosus sensu lato, E. multilocularis and Taenia spp. Amplicons indicative for Taenia spp. were obtained from 60 samples. Sequencing of amplicons spanning part of the mitochondrial cytochrome c oxidase subunit 1 gene, which was possible with 38 samples, revealed 35 infections with T. hydatigena and 3 with T. multiceps. Therefore, the CoproAg-ELISA showed some positives, but concrete evidence for the existence of canine E. granulosus infection could not be established. Comparison of the results of the CoproAg-ELISA and Taenia species identification indicated that the CoproAg-ELISA cross-reacts with patent infections of T. hydatigena (57%) and T. multiceps (33%).

Anti-babesial ellagic acid rhamnosides from the bark of Elaeocarpus parvifolius.

Bioassay-guided investigation of the bark of Elaeocarpus parvifolius led to the isolation of three new ellagic acid derivatives, 4-O-methylellagic acid 3'-alpha-rhamnoside (2), 4-O-methylellagic acid 3'-(3''-O-acetyl)-alpha-rhamnoside (3), and 4-O-methylellagic acid 3'-(4''-O-acetyl)-alpha-rhamnoside (4) in addition to the known ellagic acid derivative, 4-O-methylellagic acid 3'-(2'',3''-di-O-acetyl)-alpha-rhamnoside (1). Their structures were elucidated on the basis of analysis of 1H NMR, 13C NMR, HMQC, HMBC and MS spectroscopic data. Compounds 1-4 were evaluated for their growth-inhibitory effect on Babesia gibsoni in vitro. Compounds 2 and 4 showed very weak activity, while compounds 1 and 3 showed moderate activity, with IC50 values of 28.5 and 52.1 microg/ml, respectively.

Sequence variation of the cytochrome b gene of various human infecting members of the genus Leishmania and their phylogeny.

The Cytochrome b (Cyt b) gene has proved to be useful for identification and classification of many mammals and plants. In order to evaluate the utility of this gene for discrimination of Leishmania parasites as well as for exploring their phylogenetic relationships, we determined the nucleotide sequences of the Cyt b gene from 13 human-infecting Leishmania species (14 strains) from the New and Old Worlds. The Cyt b genes, approximately 1080 base pairs, were found to be A/T rich, and their 5' terminal-editing regions were highly conserved. The nucleotide sequence variation among them was enough to discriminate parasite species; 245 nucleotide positions were polymorphic and 190 positions were parsimony informative. The phylogenetic relationships based on this gene, showed good agreement with the classification of Lainson & Shaw (1987) except for the inclusion of L. (L.) major in the L. (L.) tropica complex and the placement of L. tarentolae in another genus. These data show that the Cyt b gene is useful for phylogenetic study of Leishmania parasites.

Usefulness of sampling with cotton swab for PCR-diagnosis of cutaneous leishmaniasis in the New World.

In this study, we tested the polymerase chain reaction (PCR)-method to diagnose cutaneous leishmaniasis (CL) by taking exudate materials from lesions with cotton swabs, using our previously tested (PCR) panel comprised of Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) mexicana and L. (L.) amazonensis. The objectives of the present study were to improve the sampling method convenient for the patients and to test the usefulness of samples taken with cotton swabs. Sixteen patients were clinically diagnosed to have CL including one case of diffuse cutaneous leishmaniasis (DCL) in Ecuador and the causative Leishmania parasites were identified by PCR. All the 12 samples from CL patients of La Mana, positive for Leishmania DNA, were identified as L. (V.) panamensis, while two from CL of Huigra and one from DCL of San Ignacio were L. (L.) mexicana. In the field condition, taking biopsy material is not only painful but sometimes causes iatrogenic bacterial infections. Considering the sensitivity of the test, and convenient sampling procedure, it may be suggested that collection of exudates using cotton swabs may be a better alternative to biopsy sample for PCR-diagnosis of CL.

Detection of species of the subgenus Leishmania parasites using polymerase chain reaction and southern blotting.

In this study, an attempt was made to identify different Leishmania species by polymerase chain reaction (PCR). Fourteen Leishmania strains from stock were tested by PCR and Southern blotting. A pair of primers were employed that anneal to the kinetoplast DNA sequence conserved among subgenus Leishmania. Of the 14 Leishmania strains used in this study, six showed strong bands of approximately 170 bp, and all the positive strains belonged to the species of the subgenus Leishmania viz., Leishmania (Leishmania) garnhami, L. (L.) amazonensis, L. (L.) pifanoi, L. (L.) mexicana, L. (L.) chagasi, and L. (L.) major All the species belonging to the subgenus Viannia used in this study were negative by PCR. These results suggest that the primer pair may be useful for identification of the species belonging to the subgenus Leishmania of the New World as well as to distinguish subgenus Leishmania from subgenus Viannia.

Usefulness of endoscopic ultrasonographic analysis of variceal hemodynamics for the treatment of esophageal varices.

The correlation of between the endoscopic findings of esophageal varices and endoscopic ultrasound findings of the collaterals outside the esophageal wall in patients with portal hypertension remains unclear. We investigated the relationship between esophageal varices and the collaterals by endoscopy and endoscopic ultrasound. Moreover, we investigated the correlation between the collaterals around the esophagus and recurrence of esophageal varices in patients with portal hypertension who had undergone endoscopic injection sclerotherapy. The collaterals were divided into two groups: 1; those with peri-esophageal collateral veins (peri-ECVs) adjacent to the muscularis externa of the esophagus, and 2; those with para-esophageal collateral veins (para-ECVs) distal to the esophageal wall without contact with the muscularis externa. Peri- and para-ECVs were scored as mild or severe according to the stage of development. According to endoscopy, the varix form was significantly larger in severe peri-ECVs group than in mild peri-ECVs group. In contrast, the varix form did not differ significantly between the mild and severe para-ECVs group. The prevalence of perforating veins increased according to the varix form. With regard to variceal recurrence, in patients with variceal recurrences, EUS findings included a significantly higher incidence of severe-type peri-ECVs, a significantly larger number of perforating veins, and a significantly larger diameter of perforating veins compared with patients without recurrence. Moreover, when EUS found the abnormalities when no endoscopic recurrence was found, the results were the almost same as the findings when EUS was performed at the same time when endoscopic recurrence was found. In conclusion, the presence of severe peri-ECVs and large perforating veins in the esophageal wall strongly correlates with occurrence and recurrence of esophageal varices in patients with portal hypertension. An understanding of these EUS abnormalities on the basis of hemodynamics around the esophagus is thought to be important for management of esophageal varices in patients with portal hypertension.

Histidine 20, the crucial proximal axial heme ligand of bacterial heme oxygenase Hmu O from Corynebacterium diphtheriae.

The hemin complex of Hmu O, a 24-kDa soluble heme degradation enzyme in Corynebacterium diphtheriae, is coordinated axially to a neutral imidazole of a proximal histidine residue in Hmu O. To identify which of the eight histidines in Hmu O is the proximal heme ligand, we have constructed and expressed the plasmids for eight His --> Ala Hmu O mutants. Reconstituted with hemin, the active site structures and enzymatic activity of these mutants have been examined by EPR, resonance Raman, and optical absorption spectroscopy. EPR of the NO-bound ferrous heme-Hmu O mutant complexes reveals His(20) as the proximal heme ligand in Hmu O, and this is confirmed by resonance Raman results from the ligand-free ferrous heme-H20A. All eight His --> Ala mutants bind hemin stoichiometrically, proving that none of the histidines is essential for hemin-Hmu O formation. However, His(20) is crucial to Hmu O catalysis. Its absence by point mutation has inhibited the conversion of hemin to biliverdin. The ferric heme-H20A complex is pentacoordinate. Resonance Raman of the CO-bound ferrous heme-H20A corroborates this and reveals an Fe-C-O bending mode, delta(Fe-C-O), the first reported for a pentacoordinate CO-bound hemeprotein. The appearance of delta(Fe-C-O) in C. diphtheriae Hmu O H20A but not mammalian HO-1 mutant H25A indicates that the heme environment between the two heme oxygenases is different.

Rapid screening with a recombinant antigen (rK39) for diagnosis of visceral leishmaniasis using dipstick.

OBJECTIVE: To evaluate the diagnostic value of the recombinant antigen of 39 amino acid repeats encoded by a kinesin-like gene of Leishmania changasi (rK39) in serodiagnosis of visceral leishmaniasis (VL). METHODS: In Kashi, Xinjiang, 13 VL patients with splenomegaly and bone marrow aspirate culture positive were subjected to dipstick assay. A drop of whole blood or serum from patient was placed at the absorbing pad at the bottom of the dipstick. Flooding of the bottom protein with buffer allows serum proteins to migrate upwards, producing the positive band and Western blot analysis of rK39 subsequently performed with the sera collected. RESULTS: The end-point titers of anti-rK39 antibodies of these sera were determined by ELISA and found to fall within the range of 10(-2) to 10(-4), which were consistent with the intensity of their reaction with rK39 in dipstick assay. The positive sera could also recognize the specific rK39 band as analyzed by Western blot analysis. CONCLUSION: The rK39 dipstick assay is more rapid, specific, sensitive and less invasive than the conventional methods of diagnosis for VL in the areas of low endemicity.

Heme degradation as catalyzed by a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae.

Hmu O, a heme degradation enzyme in the pathogen Corynebacterium diphtheriae, catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. A bacterial expression system using a synthetic gene coding for the 215-amino acid, full-length Hmu O has been constructed. Expressed at very high levels in Escherichia coli BL21, the enzyme binds hemin stoichiometrically to form a hexacoordinate high spin hemin-Hmu O complex. When ascorbic acid is used as the electron donor, Hmu O converts hemin to biliverdin with alpha-hydroxyhemin and verdoheme as intermediates. The overall conversion rate to biliverdin is approximately 4-fold slower than that by rat heme oxygenase (HO) isoform 1. Reaction of the hemin-Hmu O complex with hydrogen peroxide yields a verdoheme species, the recovery of which is much less compared with rat HO-1. Reaction of the hemin complex with meta-chloroperbenzoic acid generates a ferryl oxo species. Thus, the catalytic intermediate species and the nature of the active form in the first oxygenation step of Hmu O appear to be similar to those of the mammalian HO. However, the considerably slow catalytic rate and low level of verdoheme recovery in the hydrogen peroxide reaction suggest that the active-site structure of Hmu O is different from that of its mammalian counterpart.

Structural and functional analysis of the LaMDR1 multidrug resistance gene in Leishmania amazonensis.

We determined primary sequences of the LaMDR1 gene in Leishmania amazonensis, a protozoan parasite that causes cutaneous leishmaniasis. The longest open reading frame encodes 1341 amino acids for a protein consisting of two similar halves, each containing six putative transmembrane domains and one ATP-binding domain. The protein has no potential N-glycosylation sites at the extracellular region. The LaMDR1 protein was 91 and 78% identical to the closely related ldmdr1 in L. donovani and lemdr1 in L. enriettii, respectively, revealing less conservation in the C-terminal than in the N-terminal transmembrane domains. Transfection of LaMDR1 conferred a multidrug resistance phenotype to wild-type promastigotes, which exhibited a significant level of resistance to vinblastine, doxorubicin, and actinomycin D, but not to puromycin and colchicine. This drug specificity of LaMDR1 was overlapping with but distinct from that of ldmdr1, suggesting functional diversity of MDR1 proteins among different Leishmania species.

Non-ulcerative cutaneous lesion in immunodeficient mice with Leishmania amazonensis infection.

Cutaneous leishmaniasis begins as papules or nodules at the site of promastigote inoculation. The next key pathogenic event in this disease is the formation of an ulcer at this site. Leishmania infection in immunodeficient mice, however, showed non-ulcerative cutaneous lesions suggesting the involvement of the immune system in ulcer formation. Severe combined immunodeficient (SCID), recombination-activating gene 2 knockout (RAG-2-/-), and immunocompetent mice were inoculated subcutaneously with cultured L. amazonensis promastigotes. Macroscopic nodules appeared at the inoculation site within 2 weeks of infection in all the mice and gradually extended to the surrounding skin tissue. Although nodules of immunocompetent mice ulcerated within 6 weeks, immunodeficient mice did not form ulcers even after 25 weeks of inoculation. These results strongly suggest the importance of functional T and B cells in ulcer formation of cutaneous leishmaniasis and are consistent with clinical features of non-ulcerative cutaneous leishmaniasis in some AIDS patients. The present study also indicates that the L. amazonensis-infected immunodeficient mouse model might be suitable for studying the mechanisms of ulcer formation in cutaneous leishmaniasis.

Rhabdomyolysis and aggravation of arthritis in a rheumatoid arthritis patient as a result of sepsis due to Staphylococcus aureus infection of a rheumatoid nodule; a catastrophic outcome.

A 63-year-old man with rheumatoid arthritis presented with rhabdomyolysis and intractable arthritis of acute onset. He was diagnosed to have sepsis due to Staphylococcus aureus infection through of an ulcerated rheumatoid nodule. Staphylococcus aureus isolated from pus in the ulcerated rheumatoid nodule and a blood sample obtained from the heart post-mortem produced the toxic shock syndrome toxin-1 (TSST-1). The TSST-1 and/or unmethylated CpG motifs in the oligonucleotides present in a bacterium, Staphylococcus aureus in this case, might be implicated in the induction of rhabdomyolysis and intractable arthritis.

Full-circular surface acoustic wave excitation for high resolution acoustic microscopy using spherical lens and time gate technology.

With a fixed gate width under the condition where the focus of an acoustic lens was set inside the sample, we varied signal taking-in time. Discrimination was made between differences in time required for an ultrasonic signal reflected from the sample to reach the acoustic lens. This process also enabled three types of images to be obtained separately: the surface reflection wave image, a combination of images based on the interference of the surface reflection wave with surface acoustic waves, and the surface acoustic wave image. Thus it was presumed that this process also would reveal the causes of image contrast and allow an easy interpretation of images. Furthermore, the image resolution was improved, because the surface acoustic wave image was drawn by an ultrasonic beam produced by full-circular surface acoustic wave excitation propagating toward the center converging concentrically; the theoretical resolution was 0.4 times the value of the surface acoustic wave wavelength lambda(R) and independent of the defocus value of the acoustic lens. Several kinds of samples were observed with this method. The results showed that the new method permitted observation of the internal structures of samples while offering new knowledge through the data reflecting the ultrasonic wave damping and scatter drawn on the display.

Diagnosis of kala-azar by nested PCR based on amplification of the Leishmania mini-exon gene.

To diagnose visceral leishmaniasis (kala-azar), we have developed a nested PCR method based on amplification of the mini-exon gene, which is unique and tandomly repeated in the Leishmania genome. Nested PCR was sufficiently sensitive for the detection of DNA in an amount equivalent to a single Leishmania parasite or less. We examined the usefulness of this PCR method using bone marrow aspirates and buffy coat cells collected from kala-azar patients who had or had not received chemotherapy in northwest China. We obtained PCR positivity for all of the parasitologically positive bone marrow samples from the patients. Some ambiguities with the primary PCR results were eliminated by the subsequent nested PCR. The buffy coat samples from 7 of 12 patients with splenomegaly were positive by the nested PCR, although only 2 of them were positive for parasites by culture. However, buffy coat samples from nine children, whose splenomegaly has been reduced and clinically cured by antimony treatment, were all negative. Thus, this nested PCR method represents a new tool for the diagnosis of kala-azar with patient blood samples instead of bone marrow or spleen aspirates obtained by more invasive procedures.

[Transsphenoidal surgery and gamma-knife radiosurgery for a treatment of recurrent craniopharyngioma with moyamoya vessels].

A recurrent craniopharyngioma associated with moyamoya vessels was successfully treated by partial removal of the tumor via the transsphenoidal approach followed by gamma-knife radiosurgery. This 19-year-old man was first treated by partial tumor removal and radiotherapy (54Gy) at the age of 6 years. Growth hormone and human chorionic gonadotropin were given from the ages of 13 to 18 years. At ag 17 years, follow-up magnetic resonance imaging (MRI) revealed regrowth of the tumor. At the age of 19 years, he was readmitted for treatment of the enlarging remnant tumor. Neurological examination revealed bilateral blindness. MRI showed marked suprasellar, sphenoidal and bilateral cavernous sinus extension of the tumor. Angiography revealed stenosis of the right internal carotid artery and the M1 and A1 segments of the right cerebral arteries, as well as occlusion of the C3 segment of the left internal carotid artery. There were vault and ethmoidal moyamoya vessels. The patient underwent tumor removal via the transsphenoidal approach, instead of craniotomy, to avoid injury to the transdural anastomosis. The intrasellar solid tumor was partially removed. The tumor was then irradiated by the gamma knife. MRI 15 months after the treatment showed marked reduction of the tumor. The pathogenesis of the moyamoya phenomenon and the choice of the treatment in this patient are discussed.

Leishmania mini-exon genes for molecular epidemiology of leishmaniasis in China and Ecuador.

The mini-exon gene is unique and is tandemly repeated in the Leishmania genome. The transcribed region is highly conserved, but the non-transcribed spacer region is distinct in length and in sequence among different Leishmania species. The usefulness of PCR amplification of the Leishmania mini-exon gene was examined for molecular epidemiology of visceral and cutaneous leishmaniasis. We previously described a PCR method for amplification of the mini-exon gene and obtained positive amplification in bone marrow aspirates of patients with visceral leishmaniasis in China. In this study, we have cloned and sequenced two PCR products from the patients. The sequences of two products revealed 100% identity and showed more similarity to the mini-exon gene of L. donovani Indian strain than those of L. donovani complex in Africa and South America. We also applied this PCR method to the diagnosis of cutaneous leishmaniasis. We obtained positive PCR amplification in skin biopsy materials taken from patients with cutaneous leishmaniasis in Ecuador. Since this PCR amplification is simple and requires only a pair of primers to detect all Leishmania species distributed in Ecuador, the method may be a useful tool for the detection of parasites, not only from patients, but also from sandflies and reservoir animals in this area of endemicity.

A method of ultrasonic 3-D computed velocimetry.

A method of ultrasonic three-dimensional (3-D) vector velocimetry, which is derived by extending the previously proposed two-dimensional (2-D) vector velocimetry, is presented. In the proposed method, the three vector components of velocity to be measured are defined as the velocity in the beam axial direction, and angle velocities in two transverse directions. To derive the three components of velocity, signals detected by a 2-D array transducer are first 2-D Fourier transformed in the spatial domain parallel to the 2-D array transducer and then are one-dimensional (1-D) Fourier transformed in the time domain. The advantage of the proposed method is that it uses a linear signal processing, so it can simultaneously measure individual velocities of multiple scatterers. Computer simulations clearly demonstrate the effectiveness of the proposed method.

Histidine-132 does not stabilize a distal water ligand and is not an important residue for the enzyme activity in heme oxygenase-1.

Heme oxygenase is a key enzyme in the oxygen-dependent heme catabolism pathway. In order to clarify the role of highly conserved His132 in heme oxygenase isoform-1, we have prepared 30 kDa truncated rat heme oxygenase mutants in which His132 has been replaced by Ala, Gly, and Ser. The expressed recombinant mutant proteins were isolated in inclusion bodies and were recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 gel filtration column chromatography, as it eluted out of the column at the void volume. The gel filtration-purified heme oxygenase mutants have spectroscopic and enzymatic properties identical to those of wild type. The hemin complex of the H132A mutant exhibits a transition between a high-spin acid form and a low-spin alkaline form with a pKa value of 7.6 identical to that in the wild-type complex. These results demonstrate that His132 in heme oxygenase does not link to the coordinated water molecule and is not an essential residue for the enzyme activity. These results are in accordance with our previous preliminary results [Ito-Maki, M., Ishikawa, K., Mansfield Matera, K., Sato, M., Ikeda-Saito, M., & Yoshida, T. (1995) Arch. Biochem. Biophys. 317, 253-258] but contradict a recent report that His132 is the distal base linked to the coordinated water molecule and an important residue for enzyme catalysis [Wilks, A., Ortiz de Montellano, P. R., Sun, J., & Loehr, T. M. (1996) Biochemistry 35, 930-936]. Prolonged storage of the solubilized fraction from the inclusion bodies of the mutants, H132S in particular, results in an increase in the void volume fraction with a concomitant decrease of the 30 kDa fraction. We infer that His132 plays a structural role in stabilization of the heme oxygenase protein.

Detection of Trypanosoma congolense and T. brucei subspecies in cattle in Zambia by polymerase chain reaction from blood collected on a filter paper.

To facilitate epidemiology studies of African trypanosomiasis in cattle in Zambia, we adapted a polymerase chain reaction (PCR) method using blood spotted on filter papers. For easy preparation of template DNA from the dried blood, we adapted a simple DNA extraction method using Chelex-100, an anion-exchange resin. Using primers directed for repetitive nuclear DNA sequences, species-specific DNA amplifications were detected from the blood of rats infected with Zambian isolates of T. congolense and T. brucei subspecies. The method was sensitive enough to detect a single trypanosome for both species. In the Eastern Province of Zambia, 240 cattle were examined for motile flagellates in the buffy coat by the microhematocrit method, and 100 of them were positive for the test. These 100 animals were further examined by thin blood smears and PCR for species identification. The thin blood smear revealed 62 and 14 animals with T. congolense and T. brucei subspecies infection, respectively, whereas the PCR detected 73 of the former and 38 of the latter species. These results indicate that dried blood spots on filter papers are a useful source of DNA for detection of African trypanosomes by PCR.