Zoonotic potential of Enterocytozoon bieneusi.

The reservoirs and the modes of transmission of the most frequent microsporidial species in humans, Enterocytozoon bieneusi, are still unknown. We have examined fecal samples of 26 humans and 350 animals from 37 species to find 18 samples containing this parasite from humans, cats, pigs, cattle, and a llama. Genotypic characterization of the internal transcribed spacer of the rRNA gene resulted in 14 different genotypes, 6 of them previously undescribed. Phylogenetic analysis revealed the lack of a transmission barrier between E. bieneusi from humans and animals (cats, pigs, and cattle). Thus, E. bieneusi appears to be a zoonotic pathogen.

Close genotypic relationship between Enterocytozoon bieneusi from humans and pigs and first detection in cattle.

The reservoirs and the routes of transmission of Enterocytozoon bieneusi are still unknown. In humans, it is the most commonly found microsporidial species. It has also been found repeatedly in pigs, too. The first detection of E. bieneusi in cattle is reported herein. Two distinct genotypes were characterized and compared with 4 other genotypes from humans, 6 from pigs, and 1 from a cat. From these 13 E. bieneusi genotypes known to date, 25 polymorphic sites could be identified in the internal transcribed spacer of the rRNA gene. The spectrum of polymorphisms within and between each of the 4 host species indicates a close relationship between E. bieneusi strains from humans and pigs, whereas those from cattle are more distantly related. The data suggest the absence of a transmission barrier between pigs and humans for this pathogen.

Significance of ahpC promoter mutations for the prediction of isoniazid resistance in Mycobacterium tuberculosis.

To determine the value of ahpC promoter mutations for the rapid prediction of isoniazid resistance, this genomic region was characterized in 50 isoniazid-resistant and 12 isoniazid-sensitive Mycobacterium tuberculosis isolates. Of the resistant isolates, 12 had ahpC promoter mutations, but only one possessed both an ahpC promoter mutation and a katG codon 315 substitution, although the latter was found in the majority (54%) of the isoniazid-resistant isolates investigated. This investigation presents empirical evidence that the central portion of the ahpC promoter is the most valuable genetic locus to complement katG codon 315 characterizations in order to increase the sensitivity of molecular tests for the prediction of isoniazid resistance.

Strain differentiation in microsporidia.

Microsporidia are obligate intracellular, spore-forming protozoa and are regarded as newly emerging pathogens . Enterocytozoon spp. as well as Encephalitozoon spp. are recognized as major aetiological agents in chronic diarrhoea of immnunocompromised patients. The detection and differentiation of strains within microsporidial species is a prerequisite for the elucidation of their hitherto unknown reservoirs and their mode of transmission . In Enterocytozoon bieneusi, the most prevalent human-pathogenic microsporidium, 6 different genotypes of the internal transcribed spacer (ITS) of the rRNA gene are known so far, with 12 polymorphic sites . This pathogen has infrequently been detected in 2 animal hosts only, pigs and rhesus macaques, and only the genotype of the latter has also been found in a human patient, too. Encephalitozoon cuniculi has a wider confirmed spectrum of animal hosts, but only one polymorphic site is known in the ITS, differing in 3 different numbers of a tetranucleotide repeat. Therefore, further genomic targets may have to be characterized, too. Few data are available on strain differentiation in Encephalitozoon intestinalis and E. hellem.

Limited range of genetic variation in Echinococcus multilocularis.

DNA sequencing of 1.3 kb of rDNA containing both internal transcribed spacers (ITS1, ITS2) and adjoining rRNA coding regions in each of 11 Echinococcus multilocularis isolates from Germany, Japan, and Alaska resulted in identical nucleotide sequences except for a single polymorphic locus 54 bp upstream of the 3' end of the 18S coding region, separating Eurasian isolates from an Alaskan isolate. The same base substitution was found in each of 2 additional isolates from Alaska. The distribution of the resulting genotypes with regard to their origin is highly significant (>99.9%) and corresponds to the traditional subspecies Echinococcus multilocularis multilocularis and Echinococcus multilocularis sibiricensis.

The role of endogenous noradrenaline in the beta-blocker withdrawal phenomenon--studies with cultured heart cells.

An in vitro model to evaluate the role of endogenous noradrenaline in the beta-blocker withdrawal phenomenon is described: Beating chicken heart muscle cells (5000 beta 1-adrenoceptors/cell) and heart nonmuscle cells (3000 beta 2-adrenoceptors/cell) were cultured in serum-free, hormone-supplemented medium. Basal state, subtype selective down-regulation of beta-adrenoceptors by endogenous noradrenaline (decrease in receptor number, beta 1 more than beta 2) was simulated by addition of noradrenaline to the culture medium; chronic beta-blockade was simulated by exposure of the cells for 3 days to various beta-blockers (propranolol, no ISA; timolol, slight ISA; pindolol, strong ISA). Beta-blocker withdrawal phenomenon--increased response in isoproterenol-induced cAMP production and positive inotropy--is correlated with the increase in the number of beta-adrenoceptors after withdrawal of the drugs. Propranolol induces a withdrawal phenomenon at every degree of noradrenaline-induced basal state down-regulation of beta-adrenoceptors; in contrast, a withdrawal phenomenon by pindolol is only seen at a higher degree of beta-adrenoceptor down-regulation. In the presence of physiological noradrenaline concentrations pindolol affects beta-adrenoceptor subtypes in a qualitatively different manner: the number of beta 1-adrenoceptors is increased, the number of beta 2-adrenoceptors is decreased. This finding demonstrates that the intrinsic sympathomimetic activity of nonselective beta-blockers can manifest itself only if the receptors are not strongly down-regulated. As beta 2-adrenoceptors are present in a much less down-regulated state than beta 1, ISA mainly acts on beta 2-adrenoceptor subtype, thus, presenting a beta 2-"pseudo-selectivity" of ISA.

Endogenous noradrenaline and beta-adrenoceptor regulation in cultured heart cells.

a) Cultured heart muscle cells from chicken embryos and neonatal rats possess about 5000 beta 1-adrenoceptors/cell, heart non muscle cells from these species about 3000 beta 2-adrenoceptors/cell. Agonist binding in heart muscle cells results in cAMP formation and in a positive inotropic effect. b) Beta 1- and beta 2-adrenoceptors of heart cells are downregulated to different degrees in the presence of physiological noradrenaline concentrations in the culture medium. c) Using chicken heart cells in culture, a cellular model of the beta-blocker withdrawal phenomenon in the heart has been established, elucidating the modifying action of endogenous catecholamines and the role of ISA (partial agonist activity) in this phenomenon and classifying the so-called "beta 2-pseudo-selectivity" of the ISA action. d) In the absence of added catecholamines, (-)propranolol exposure of the cells for 3 days increases the number of beta-adrenoceptors of chicken heart muscle cells up to 170% of control, while in rat heart muscle cells the receptor number remains unchanged. The mechanism of this "basal" receptor downregulation in chicken heart muscle cells is unclear. The data presented demonstrate the usefulness of heart cell cultures in the study of beta-adrenoceptor regulation of the heart.