Horst Aspöck

Molecular Characterization of a Non–Babesia divergens Organism Causing Zoonotic Babesiosis in Europe

In Europe, most reported human cases of babesiosis have been attributed, without strong molecular evidence, to infection with the bovine parasite Babesia divergens. We investigated the first known human cases of babesiosis in Italy and Austria, which occurred in two asplenic men. The complete 18S ribosomal RNA (18S rRNA) gene was amplified from specimens of their whole blood by polymerase chain reaction (PCR). With phylogenetic analysis, we compared the DNA sequences of the PCR products with those for other Babesia spp. The DNA sequences were identical for the organism from the two patients. In phylogenetic analysis, the organism clusters with B. odocoilei, a parasite of white-tailed deer; these two organisms form a sister group with B. divergens. This evidence indicates the patients were not infected with B. divergens but with an organism with previously unreported molecular characteristics for the 18S rRNA gene.

Correlations between morphological, molecular biological, and physiological characteristics in clinical and nonclinical isolates of Acanthamoeba spp.

ABSTRACT Eleven Acanthamoeba isolates, obtained fromAcanthamoeba keratitis patients, from contact lens cases of non-Acanthamoeba keratitis patients, from asymptomatic individuals, from necrotic tissue, and from tap water and two reference strains were investigated by morphological, molecular biological, and physiological means in order to discriminate clinically relevant and nonrelevant isolates. All clinically relevant isolates showedAcanthamoeba sp. group II morphology. 18S ribosomal DNA sequencing revealed sequence type T4 to be the most prevalent group among the isolates and also the group recruiting most of the pathogenic strains. Interestingly, within T4 the strains of no clinical relevance clustered together. Moreover, physiological properties appeared to be highly consistent with initial pathogenicity and with sequence clustering. Altogether, the results of our study indicate a correlation between the phylogenetic relationship and pathogenicity.

Phylogenetic relevance of the genital sclerites of Neuropterida (Insecta: Holometabola)

Abstract Segment 9 of male Raphidioptera, comprising tergite, sternite, gonocoxites, gonostyli and gonapophyses, is a benchmark for homologies in the male and female terminalia of the three Neuropterida orders Raphidioptera, Megaloptera and Neuroptera. The segments relating to genitalia are 9, 10 and 11 in males and 7, 8 and 9 in females. Results from holomorphological and recent molecular cladistic analyses of Neuropterida agree in supporting the sister‐group relationships between: (1) the Raphidioptera and the clade Megaloptera + Neuroptera, and (2) the suborder Nevrorthiformia and all other Neuroptera. The main discrepancy between the results of these studies is the nonmonophyly of the suborder Hemerobiiformia in the molecular analysis. The monophyly of the Megaloptera (which has been repeatedly questioned) is further corroborated by a hitherto overlooked ground pattern autapomorphy: the presence of eversible sacs within the complex of the fused gonocoxites 11 in Corydalidae and Sialidae. The recently discovered paired complex of gonocoxites 10 (parameres) in Nipponeurorthus (Nevrorthidae) indicates that the curious apex of sternite 9 of Nevrorthus and Austroneurorthus is the amalgamation of the sclerites of gonocoxites 10 with sternite 9, interpreted as synapomorphic. In the molecular study, the Nevrorthidae, Sisyridae and Osmylidae branch off in consecutive splitting events, a result that is supported by the analysis of male genital sclerites reported here. Extraordinary parallel apomorphies (e.g. excessive enlargement and modification of gonocoxites 10 ending in a thread‐like ‘penisfilum’) in derived representatives of Coniopterygidae, Berothidae, Rhachiberothidae and Mantispidae corroborate the dilarid clade of the morphological analysis and leads us to hypothesize a sister‐group relationship of the Coniopterygidae with the dilarid clade. A re‐interpretation of the tignum of Chrysopidae as gonocoxites 11 means that the structure previously called the gonarcus represents the fused gonocoxites 9. In Hemerobiidae, the corresponding sclerite is consequently also homologized as fused gonocoxites 9. The enlargement of the lateral wings of the gonocoxites in both families is interpreted as a synapomorphy. Excessive enlargement of gonostyli 11 in the Polystoechotid clade and Myrmeleontiformia supports a sister‐group relationship of these two clades. The occurrence of certain serial homologues of female genitalia structures (gonocoxites and gonapophyses), such as the digitiform processus together with the flat appendices in segment 8 of certain Myrmeleontidae, or the wart‐like processus together with the flat circular sclerites in segment 7 of certain Berothidae, as well as the presence of gonocoxites 8 as pseudosternites in certain Nemopteridae and Coniopterygidae, are probably character reversals. The digitiform processus of tergite 9 (pseudogonocoxites) in Rhachiberothidae and Austroberothella (Berothidae) are either independently developed acquisitions with a function in oviposition, or are homologous sclerites, possibly of epipleurite origin.

The identification of free-living environmental isolates of amoebae from Bulgaria

A survey was carried out in Bulgaria to determine the presence of free-living amoebae (FLA) from environmental sources. In 171 (61.1%) of 280 samples, isolates of Acanthamoeba with group II or III morphology, as well as Hartmannella spp. were recovered. Five isolates named “6” (artificial lake), Ep (lake), G2 (soil), R4* (river) and PK (spring water)—all exhibiting a highly efficient proliferation in axenic cultures—were subsequently cloned and subjected to molecular analyses for identification and genotyping In accordance with morphological findings, PCR-based analyses identified four isolates (6, Ep, G2, R4*) belonging to the genus Acanthamoeba. Confirmation of these findings was obtained by phylogenetic analysis using partial sequencing of the 18S rDNA (ASA.S1) Acanthamoeba-gene. Comparison of these sequences with corresponding regions from other Acanthamoeba strains available from GenBank sorted all four isolates into the sequence type group T4 that contains most of the pathogenic Acanthamoeba strains already identified. The fifth isolate (PK) exhibited morphological characteristics matching those of Hartmannella, and scored negative in the Naegleria fowleri and Acanthamoeba PCRs.

Cytotoxic Activities of Alkylphosphocholines against Clinical Isolates of Acanthamoeba spp.

ABSTRACT Free-living amoebae of the genus Acanthamoeba are causing serious chronic conditions such as destructive keratitis in contact lens wearers or granulomatous amoebic encephalitis in individuals with compromised immune systems. Both are characterized by the lack of availability of sufficiently effective and uncomplicated, manageable treatments. Hexadecylphosphocholine (miltefosine) is licensed for use as a topical antineoplastic agent, but it is also active in vitro against several protozoan parasites, and it was applied very successfully for the treatment of human visceral leishmaniasis. The aim of our study was to evaluate the efficacy of hexadecylphosphocholine and other alkylphosphocholines (APCs) against Acanthamoeba spp. The in vitro activities of eight different APCs against three Acanthamoeba strains of various pathogenicities were determined. All substances showed at least amoebostatic effects, and some of them disrupted the amoebae, as shown by the release of cytoplasmic enzyme activity. Hexadecylphosphocholine exhibited the highest degree of cytotoxicity against trophozoites, resulting in complete cell death at a concentration as low as 40 μM, and also displayed significant cysticidal activity. Hexadecylphosphocholine may be a promising new candidate for the topical treatment of Acanthamoeba keratitis and, conceivably, even for the oral treatment of granulomatous amoebic encephalitis.

The evolutionary history of holometabolous insects inferred from transcriptome-based phylogeny and comprehensive morphological data

BackgroundDespite considerable progress in systematics, a comprehensive scenario of the evolution of phenotypic characters in the mega-diverse Holometabola based on a solid phylogenetic hypothesis was still missing. We addressed this issue by de novo sequencing transcriptome libraries of representatives of all orders of holometabolan insects (13 species in total) and by using a previously published extensive morphological dataset. We tested competing phylogenetic hypotheses by analyzing various specifically designed sets of amino acid sequence data, using maximum likelihood (ML) based tree inference and Four-cluster Likelihood Mapping (FcLM). By maximum parsimony-based mapping of the morphological data on the phylogenetic relationships we traced evolutionary transformations at the phenotypic level and reconstructed the groundplan of Holometabola and of selected subgroups.ResultsIn our analysis of the amino acid sequence data of 1,343 single-copy orthologous genes, Hymenoptera are placed as sister group to all remaining holometabolan orders, i.e., to a clade Aparaglossata, comprising two monophyletic subunits Mecopterida (Amphiesmenoptera + Antliophora) and Neuropteroidea (Neuropterida + Coleopterida). The monophyly of Coleopterida (Coleoptera and Strepsiptera) remains ambiguous in the analyses of the transcriptome data, but appears likely based on the morphological data. Highly supported relationships within Neuropterida and Antliophora are Raphidioptera + (Neuroptera + monophyletic Megaloptera), and Diptera + (Siphonaptera + Mecoptera). ML tree inference and FcLM yielded largely congruent results. However, FcLM, which was applied here for the first time to large phylogenomic supermatrices, displayed additional signal in the datasets that was not identified in the ML trees.ConclusionsOur phylogenetic results imply that an orthognathous larva belongs to the groundplan of Holometabola, with compound eyes and well-developed thoracic legs, externally feeding on plants or fungi. Ancestral larvae of Aparaglossata were prognathous, equipped with single larval eyes (stemmata), and possibly agile and predacious. Ancestral holometabolan adults likely resembled in their morphology the groundplan of adult neopteran insects. Within Aparaglossata, the adult’s flight apparatus and ovipositor underwent strong modifications. We show that the combination of well-resolved phylogenies obtained by phylogenomic analyses and well-documented extensive morphological datasets is an appropriate basis for reconstructing complex morphological transformations and for the inference of evolutionary histories.

Radical Surgical Therapy of Abdominal Cystic Hydatid Disease: Factors of Recurrence

Abstract. A series of 74 consecutive patients (48 women, 26 men) were operated for abdominal hydatid disease between June 1949 and December 1995. The patients ranged in age from 15 to 81 years (median 49 years). In 69 cases only the liver was affected; two patients had concomitant extrahepatic disease (one spleen, one spleen and lung), and 3 had cysts in the spleen only. Cysts were multiple in 11 patients and calcified in 24. Conservative surgical procedures were used for 22 cysts in 20 patients [open partial (n= 3), open total (n= 6), closed total cystectomy (n= 9), marsupialization (n= 2), drainage (n= 2)] and radical surgical procedures for 72 cysts in 54 patients [pericystectomy (n= 41), wedge liver resection or hemihepatectomy (n= 25), splenectomy (n= 5), radical resection of a lung cyst (n= 1)]. Altogether 37 patients (50%) were given perioperative antihelmintic chemotherapy with mebendazole (18 patients) or albendazole (19 patients). Operative mortality rates were 5.0% after conservative surgery and 1.8% after radical surgery. Morbidity rates were 25.0% following conservative surgery and 24.1% following radical surgery. Antihelmintic therapy was well tolerated by all but five patients. All side effects were entirely reversible. Among the 74 patients, 60 (81.0%) were available for long-term follow-up (median 7.2 years; range 2.0–47.0 years). Recurrence of disease was seen in 9 of 60 patients at an interval of 3 months to 20 years from the first operation. The rate of recurrence was significantly lower after radical surgical procedures (p= 0.03) and after closed removal of the cyst (p= 0.04).

Purification of egg yolk immunoglobulins: A two-step procedure using hydrophobic interaction chromatography and gel filtration

A two-step chromatographic procedure was developed for the isolation and purification of hen IgY antibodies from egg yolk. The antibodies were completely separated from vitellin and lipids by hydrophobic interaction chromatography followed by gel filtration. Almost no residual yolk proteins, no immunoglobulin aggregates, and no antibody fragments could be detected in the final extract. Moreover, the method described, guarantees the recovery of antibodies of undiminished activity. Although the final yield is somewhat lower than that obtained by an isolation method consisting of two precipitation steps with polyethylene glycol and alcohol respectively, the procedure described is particularly recommended when highly purified antibody preparations are needed.

Successful Treatment of Disseminated Acanthamoeba sp. Infection with Miltefosine

We report on an HIV-negative but immunocompromised patient with disseminated acanthamoebiasis, granulomatous amoebic encephalitis, and underlying miliary tuberculosis and tuberculous meningitis. The patient responded favorably to treatment with miltefosine, an alkylphosphocholine. The patient remained well with no signs of infection 2 years after treatment cessation.

Echinococcus granulosus strain differentiation based on sequence heterogeneity in mitochondrial genes of cytochrome c oxidase-1 and NADH dehydrogenase-1

Genetic analyses of Echinococcus granulosus isolates from different intermediate host species have demonstrated substantial levels of variation for some genotype (strain) clusters. To determine the range of genetic variability within and between genotypes we amplified and cloned partial cox1 and nadh1 genes from 16 isolates of E. granulosus from 4 continents. Furthermore, we sequenced different clones from a PCR product to analyse the intra-individual genetic variance. The findings showed a moderate degree of variance within single isolates and a significant degree of variance between the cluster of genotypes G1-G3 (sheep, Tasmanian sheep and buffalo strain), genotypes G4 (horse strain) and G5 (cattle strain) and the cluster of the genotypes G6 (camel strain) and G7 (pig strain). The variance of up to 2.2% within genotypes was relatively low compared with that of 4.3-15.7% among genotypes. The present results indicate that a re-examination of the classification of 5 genotypes of Echinococcus is warranted. Hence, our data highly support a re-evaluation of the taxonomy of the clades G1-G3, G4, G5, G6/7 and G8 (cervid strain) within the genus Echinococcus.