Experimental infection of severe combined immunodeficient (SCID) mice with the human microsporidian Trachipleistophora hominis.

Different courses of microsporidiosis, related to the route of infection, were observed in severe combined immunodeficient (SCID) mice inoculated with spores of the human microsporidian Trachipleistophora hominis (Phylum Microspora). After eye contamination by spores the mice became moribund within 7 to 8 weeks, showing severe infection in the conjunctiva and cornea, and lighter infections in the urinary bladder, liver and spleen. The mean survival time of intramuscularly inoculated SCID mice was 12 weeks, when heavy infection was found in muscles around the site of inoculation, and also in several viscera. Subcutaneously inoculated SCID mice developed skin lesions around the inoculation sites, and heavy urinary bladder infection, and died 6 or 7 weeks after inoculation. Intracerebrally inoculated SCID mice became moribund 5 or 6 weeks after inoculation with massive infection in the urinary bladder and liver, but none in the brain. Intraperitoneally inoculated SCID mice survived for 13 weeks and the urinary bladder and liver were the most heavily infected organs. The SCID mice, inoculated perorally and examined after 23 weeks, were uninfected. Infection was not detected in the brain of any of the inoculated SCID mice. Our results show that T. hominis has very little tissue specificity. Peroral infection seems to be ineffective in T. hominis, but eye conta mination or insect bite (as mimicked by injection) are suggested as possible routes of infection under natural conditions.

The human isolate of Brachiola algerae (Phylum Microspora): development in SCID mice and description of its fine structure features.

Ocular, peroral, intraperitoneal, intramuscular, and subcutaneous inoculation of severe combined immunodeficient (SCID) mice with spores of the human isolate (CDC: V404) of Brachiola algerae (syn. Nosema algerae) (Phylum Microspora) revealed that the microsporidium develops in viscera of the immunodeficient mouse host, but only after the ocular administration of spores. It is hypothesized that the physico-chemical milieu of the conjunctiva and cornea helped to adapt the originally 'poikilothermic microsporidian' to the conditions within the homoiothermic organism. Ocular application of spores caused no clinical signs of disease at the application site. However, severe infection in the liver was found 60 days after infection, manifested as hepatosplenomegaly and multifocal miliary necroses and granulomas containing parasites. No microsporidia were found in any other tissues. Transmission electron microscopy revealed characteristic tubulovesicular 'secretory materials' on the plasma membrane of all developmental stages of B. algerae except sporoblasts and spores. These formations increase the parasite surface and allow more efficient metabolic communication of the parasite with the host cell. It is hypothesized that the presence of these structures is a factor helping the parasite to grow in a variety of hosts and tissues. Ultrastructural characters support the likelihood that B. algerae and B. vesicularum are conspecific, and that there exists a relationship between species of the genera Brachiola and Anncaliia.

[Valgus Osteotomy in the Treatment of the Ischemic Necrosis of Femoral Head in Children: Part II - Clinical Evaluation of the Group.]. / Valgizující osteotomie pri lécení následku ischemické postdysplastické nekrózy hlavice kosti stehenní u detí- II. cást: klinické hodnocení souboru.

The authors present long-term results of the surgical treatment of ischemic necrosis of proximal femur resulting from the treatment of the developmental dysplasia. They used their own technique of intertrochanteric valgus osteotomy in 77 hip joints. The surgical technique was published in the first part of the work. It consists in valgus osteotomy which lengthens femoral neck and, consequently also the whole limb. At the same time a fusion of the physis of the greater trochanter was performed and the pathological antetorsion of the femoral neck corrected. Osteotomy was fixed with a special valgus angled blade plate. In cases where dysplastic acetabulum persists the authors supplement the operation with the shelf operation after Bosworth. All patients operated on for the ischemic necrosis of femoral head were originally treated conservatively. Of the operated on group of 77 hip joints, 69 hip joints were classified in type III after Bucholz-Ogden and 8 hip joints in type II (type II B after Thomas et al.). Valgus operation was performed in children at the age of 3 to 15 years. The patients were further followed up on average 15,4 years, the average age upon the termination of the follow-up was 24,7 years. Eight patients of the group underwent a revision surgery by the same technique for inadequate primary correction or for the loss of correction in the course of the further growth of the hip joint. In 4 patients the authors equalized the length of both limbs by shortening the contralateral femur, in 3 patients additional fusion of the physis of the greater trochanter was performed. In case of dysplastic acetabulum the shelf arthroplasty after Bosworth proved to be very suitable. On the basis of their experience and clinical results the authors recommend to perform the surgery at the age between 7 and 10 years as in earlier operations there may occur a loss of correction during the following period and in children operated on later the adaptation of the operated on hip joint decreases. A prerequisite of a good long-term result is a precise preoperative planning of the operation and its exact technical performance. Key words: developmental dysplasia of the hip joint, ischemic necrosis, valgus intertrochanteric osteotomy of femur.

The severe combined immunodeficient mouse as a definitive host for Sarcocystis muris.

Peroral and intraperitoneal inoculation of severe combined immunodeficient (SCID) mice with cystozoites of three coccidia of the genus Sarcocystis (Protozoa, Apicomplexa; S. dispersa, Sarcocystis sp., and S. muris) revealed that after peroral administration, only S. muris could develop in the immunodeficient mouse host. The cystozoites of S. muris transformed into gamonts and, after fertilization, performed sporulation with the production of infectious sporocysts in the small intestine of the SCID mice. Impaired immunity is probably responsible for the unusual behavior of S. muris (which is normally the heteroxenous mouse-cat parasite) in the SCID mice. We hypothesize that the phylogenetic distance between the intermediate and final hosts is the reason why cystozoites of the two other Sarcocystis species tested (S. dispersa with a mouse-owl cycle and Sarcocystis sp. with a murine rodent-snake cycle) could not develop when inoculated into SCID mice.

Nosema portugal, N. SP., isolated from gypsy moths (Lymantria dispar L.) collected in portugal

A microsporidium Nosema portugal n. sp. was isolated from gypsy moths, Lymantria dispar L, collected near Lisbon, Portugal, in 1985. The life cycle includes two sequential developmental cycles, a primary and a secondary cycle. The primary cycle occurs in midgut epithelial cells, where primary spores are produced within 48 h. The primary spores immediately extrude their polar filaments, presumably to infect other cells. In the target tissues (salivary glands and fat body) the secondary development cycle is followed by the formation of environmental spores. Primary spores were also sometimes present in target tissues. Fresh unfixed and unstained primary spores have a large posterior vacuole and measured 4.8 x 2.7 &mgr;m. Ultrastructurally, they have 5-8 polar filament coils, a large posterior vacuole, abundant endoplasmic reticulum, and were binucleate. Mature unfixed and unstained environmental spores were highly refractive and the posterior vacuole and nuclei could not be seen through the spore coat. Fresh environmental spores measured 4.5 x 1.9 &mgr;m. Ultrastructurally, environmental spores were binucleate, with a typical polaroplast, 10-11 isofilar polar filament coils, and a series of 4-6 thin polar filament-like tubules situated at the posterior end of the row of typical polar filament coils. The ssu rRNA sequences strongly suggest that this species is more closely related to the Vairimorpha subgroup within the Nosema/Vairimorpha clade than to the Nosema subgroup. Copyright 1999 Academic Press.

Protein Metabolism in Lecithotrophic Larvae (Gastropoda: Haliotis rufescens).

Rates of protein depletion, synthesis, and turnover were measured in larvae of the abalone Haliotis rufescens as an approach to understanding macromolecular metabolism during lecithotrophic development. Protein content decreased linearly during development to metamorphic competence, with 34% of the initial protein in eggs depleted during the 8-day larval life span. Fractional rates of protein synthesis (percentage of total body-protein synthesized per day) decreased during development, from 40% (1-day-old trochophore larva) to 14% (7-day-old veliger larva). Separation of proteins by one-dimensional gel electrophoresis showed that protein pools in larvae are dominated by two high-molecular-weight protein classes (88 and 121 kDa). When the proteins of 1- and 3-day-old larvae were labeled with a mixture of 35S-methionine and cysteine, the pattern on two-dimensional gels showed that the turnover process (protein synthesis and degradation) involved hundreds of different proteins. The energy gained from loss of protein could account for 20% of the protein turnover rates for trochophore larvae and 79% of the lower turnover costs for late-stage veligers. Lecithotrophic larvae of H. rufescens maintained high biosynthetic activities, with up to 40% of their whole-body protein being turned over each day. Such dynamic processes during development of nonfeeding larvae would contribute significantly to maintenance metabolism.

Some ultrastructural data on Microsporidium ceylonensis, a cause of corneal microsporidiosis.

Sections of corneal tissue infected with Microsporidium ceylonensis were restained or processed for electron microscopy. Confirmation was obtained that the parasite develops in macrophages and that spores are uninucleate. New information is provided that sporoblasts and spores develop synchronously within a membrane in the host cell, spores have an anisofilar polar tube of 6-10 wide coils and 2-3 narrow coils and details are given of the spore wall and internal organisation. The parasite was compared on the one hand with Encephalitozoon, which exhibits asynchronous intravacuolar development of merogonic and sporogonic stages and has spores with isofilar polar tubes and on the other hand with species reported from mammals, of which the sporogonic stages develop synchronously within sporophorous vesicles and the spores have anisofilar polar tubes. Even so, a generic emplacement could not be established. Attention is drawn to the similarities between M. ceylonensis and Nosema sp. described from the cornea of a woman in Botswana.

Microsporidia of the genus Trachipleistophora--causative agents of human microsporidiosis: description of Trachipleistophora anthropophthera n. sp. (Protozoa: Microsporidia).

Trachipleistophora anthropophthera n. sp., was found at autopsy in the brain of one and in the brain, kidneys, pancreas, thyroid, parathyroid, heart, liver, spleen, lymph nodes, and bone marrow of a second patient with AIDS. The parasite is similar to the recently described T. hominis Hollister, Canning, Weidner, Field, Kench and Marriott, 1996, in having isolated nuclei, meronts with a thick layer of electron dense material on the outer face of their plasmalemma and sporogony during which spores are formed inside a thick-walled sporophorous vesicle. In contrast to T. hominis, this species is dimorphic as it forms two kinds of sporophorous vesicles and spores: Type I--round to oval polysporous sporophorous vesicle, 7-10 microns in size, usually with eight spores (3.7 x 2.0 microns), thick endospores, subterminal anchoring disc and anisofilar polar filaments forming seven thicker and two thinner terminal coils. This type of sporophorous vesicle is associated with 25-30 nm filaments extending into the host cell cytoplasm. Type II--smaller, bisporous sporophorous vesicle (4-5 x 2.2-2.5 microns) with two, nearly round, thin-walled spores, 2.2-2.5 x 1.8-2.0 microns in size, having 4-5 isofilar coils. No outside filamentous elements are associated with the bisporous sporophorous vesicle. Both types of sporophorous vesicles were common in the infected brain tissue and could be found within the same cell. The newly described species, together with T. hominis and previously reported Pleistophora-like parasites from human muscle, likely represent a group of closely related human microsporidia.

Molecular phylogenetic relatedness of Frenkelia spp. (Protozoa, Apicomplexa) to Sarcocystis falcatula Stiles 1893: is the genus Sarcocystis paraphyletic?

The coccidians Frenkelia microti and F. glareoli (Apicomplexa: Sarcocystidae) form tissue cysts in the brain of small rodents (intermediate hosts) while oocysts are formed in the intestine of final hosts, buzzards of the genus Buteo. The inclusion of the small subunit ribosomal RNA gene sequences (SSU rRNA) of both Frenkelia species into the SSU rRNA trees of other, tissue cyst-forming coccidia strongly supports paraphyly of the genus Sarcocystis. Frenkelia spp. exhibit close relatedness to Sarcocystis falcatula Stiles 1893, a bird-opossum parasite, recognized under its junior synonym S. neurona Dubey et al. 1991, as the causative agent of equine protozoan myeloencephalitis on the American continent. As the definition of the genus Frenkelia is based on a plesiomorphic character (affinity to the neural tissue) of supposedly low phylogenetic value, the synonymization of the genus Frenkelia with Sarcocystis is proposed. This renders the genus Sarcocystis monophyletic.

[Valgus osteotomy in the treatment of sequelae of ischemic post-dysplastic necrosis of femoral head in children. Part I: surgical technique.]. / Valgizující osteotomie pri lécení následku ischemické postdysplastické nekrózy hlavice stehenní kosti u detí: I. cást - operacní technika.

The authors deal with the surgical technique of the reconstruction of coxa vara in children. They present valgus osteotomy on the Pauwels principle with the simultaneous apophyseodesis of the growth plate of greater trochanter, lengthening of fermoral neck fixed by 120 degrees angled blade plate for adolescents allowing a parallel lengthening of the lower limb. The main indication for this technique is postdysplastic coxa vara and congenital coxa vara in children. However, it may be used also in adults. The benefits of the method consist in the possibility of exact planning of the surgery. Key words: valgus osteotomy, reconstruction, DDH, ischemic necrosis of the femoral head.

Staining of microsporidian spores by optical brighteners with remarks on the use of brighteners for the diagnosis of AIDS associated human microsporidioses.

Conditions for the effective fluorescence labelling of microsporidian spores by optical brighteners, based on the presence of chitin in the spore wall, are described. Spores of Vairimorpha ephestiae, V. necatrix, V. plodiae, Nosema bombycis, N. apis, N. algerae, Encephalitozoon cuniculi and Enterocytozoon bieneusi were examined. The degree of binding of Calcofluor White M2R (CFW) to untreated spores depends on the conditions and time of storage and the degree of bacterial contamination of the spore sample. Unpurified spores, stored in water are unreliable as control material for the estimation of CFW labelling. However, spores subjected to alkaline treatment by NaOH before CFW application are visualized with ease under all experimental conditions by their bright, not quenching fluorescence in shortwave light (approximately 350 nm) in CFW dilutions of 10(-4) or even lower. Similar improvement in labelling is achieved by exposing spores to CFW dissolved in 0.5-1N NaOH. As well as Calcofluor White M2R other optical brighteners (e.g. Uvitex 2B, Ciba-Geigy or Rylux BA, Ostacolor) can be used for labelling of spores.

Bacillidium cyclopis Vávra, 1962 - Description of the ultrastructural cytology and transfer to the genus Mrazekia Léger and Hesse, 1916 (Microspora, Mrazekiidae).

The ultrastructural cytology of the microsporidium Bacillidium cyclopisVávra, 1962, a parasite of the copepod Acantho cyclop s americanus in Czechoslovakia, is described, with emphasis on the mature spore. All life cycle stages are diplokaryotic. The three-layered spore wall has a stratified exospore. The polaroplast has two distinctly separated lamellar parts where the lamellae are folded concentrically around the anterior part of the polar filament. Anterior lamellae are narrow and closely packed; posterior lamellae are wider and more loosely arranged. The polar filament has an anterior straight and wide section, the "manubrium", where the posterior third is widened in a characteristic manner, and a final section, arranged in 2-3 narrow, isofilar coils. The attachment of the polar filament is more or less oblique in relation to the long axis of the spore. The ejected filament leaves the spore in a distinct angle, and the narrow part is characteristically and regularly coiled. Glycoconjugates are present in the layers of the anchoring disc, in one of the external layers of the manubroid part of the polar filament, and in two distinct layers of the coils. The diplokaryon partially encloses the manubrium in a mantle-like fashion. B. cyclopis is compared to re-examined preparations of Mrazekia argoisiiLéger and Hesse, 1916. These two species are the only Bacillidium-like microsporidia of Crustacea, and they are distinguished from all the other Bacillidium-like microsporidia by their swollen posterior end of the manubrium. This character, which is easily seen also using light microscopy, is considered diagnostic for the genus Mrazekia, and B. cyclopis is transferred to this genus. Mrazekiidae Léger and Hesse, 1922 is the valid family name, with Bacillidiidae Larsson, 1986 as a junior synonym.

A freeze-fracture study of microsporidia (Protozoa: Microspora): II. The extrusion apparatus: Polar filament, polaroplast, posterior vacuole.

Developmental stages (sporoblasts and spores) of five species of microsporidia (Amblyospora bracteata, A. varians, A. weiseri, Nosema apis, Tuzetia sp.), fixed by conventional EM fixatives or by glutaraldehyde supplemented with potassium chloride, were studied by freeze-fracture using ultrathin sections as controls. It was found that the polar filament is formed by coalescence of cytoplasmic vesicles of the Golgi zone. The filament is enveloped by a single cytoplasmic membrane ("filament ensheathing membrane" = FEM) showing more intramembrane particles (IP) on the EF face (in the order of 1000 IP/µm(2)) and less IP (in the order of 500 IP/µm(2)) on the PF face. During maturation of the spore the IP on the FEM progressively disappear. The polaroplast is a stack of cytoplasmic membranes revealing more IP (in the order of 1500 IP/µm(2)) on the PF face and less particles (in the order of 400 IP/µm(2)) on the EF face. When KCl is added to the fixative the polaroplast remains unswollen and its membranes are smooth. In swollen polaroplasts the membranes are modified, probably by mechanical stretching. The polaroplast membranes are not direct outgrowths of the FEM. The posterior vacuole is limited by a single cytoplasmic membrane. No evidence was found supporting the idea that the filament ends in the posterior vacuole.

Breathlessness in healthy subjects at physical load.

The aim of the study was to ascertain the reasons which lead to discontinuance of exercise on the bicycle ergometer in healthy untrained subjects and to assess the dependence of dyspnea on breathing pattern and on ventilation. The physical load was progressively increased to the maximum in 11 volunteers at the age of 21 +/- 1 years. During exercise some cardiovascular and respiratory parameters were measured simultaneously with the degree of dyspnea. Breathlessness was rated by means of a scaling according Borg, where 0 indicates no, 10 maximal dyspnea. Dyspnea was not a reason for termination of maximal exercise, its value being 6 +/- 1.9 in men and 4.5 +/- 2.3 in women at the end of exercise. The reasons for termination of exercise were the sensations of general fatigue and pain in lower the extremities. The degree of dyspnea correlated with the minute ventilation, with the decrease of end-tidal CO2 concentration, with the duration of exercise and some other values. The grading varied among subjects. The mathematical dependence of dyspnea was summarised by two regression equations, one without suppression, the other with suppression of interindividual differences in responses.

[Comparison of the physical fitness of the young population of Czechoslovakia with similar data from other European countries]. / Srovnání telesné zdatnosti nasí mladé populace se stejnými údaji nekterých evropských státu.

In order to analyze the relationship between VO2 max and height, body mass and lean body mass a multi-longitudinal survey was conducted on three different age groups of randomly selected children from a small Czech community. Beginning at the initial ages of 8, 12 and 16 years subjects were subsequently retested three times at two year intervals. At overlapping ages there were no differences in the various age groups between height and VO2 max. By utilizing mean values for the various parameters at specific calendar ages a growth curve was constructed for each sex for the age range 8 to 20 years. The values were compared with longitudinal studies of various countries and no substantial differences were found. When VO2 max was then compared to height, body mass and lean body mass it was apparent that the almost linear relationship with height was the most precise. In addition the children remained, generally speaking, in their same rank order for VO2 max for the three different age groupings.

Ventilation of an additional dead space.

The authors studied the question of the completeness of ventilation of an additional dead space in the form of a tube 3 cm in diameter and with a volume of 600 ml. Seven young volunteers were examined while breathing with and without the tube, seated at rest and during a two-grade exercise load on a bicycle ergometer. The criterion of ventilation of the tube was enlargement of the dead space by 600 ml during breathing through the tube. The functional dead space was always calculated from the tidal volume and the CO2 concentration in mixed expired air and in an end-tidal sample, using the Bohr equation. In every case, the tube was found to be completely ventilated by breathing, both under resting conditions and during exercise. In breathing during the bicycle exercise, the ratio of the functional dead space to tidal volume fell from 0.3 to 0.19 and a similar decrease was recorded in breathing through the tube.