Growth and Airborne Transmission of Cell-Sorted Life Cycle Stages of Pneumocystis carinii

Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.     

The Activities of Current Antimalarial Drugs on the Life Cycle Stages of Plasmodium: A Comparative Study with Human and Rodent Parasites

Background

Malaria remains a disease of devastating global impact, killing more than 800,000 people every year—the vast majority being children under the age of 5. While effective therapies are available, if malaria is to be eradicated a broader range of small molecule therapeutics that are able to target the liver and the transmissible sexual stages are required. These new medicines are needed both to meet the challenge of malaria eradication and to circumvent resistance.

Methods and Findings

Little is known about the wider stage-specific activities of current antimalarials that were primarily designed to alleviate symptoms of malaria in the blood stage. To overcome this critical gap, we developed assays to measure activity of antimalarials against all life stages of malaria parasites, using a diverse set of human and nonhuman parasite species, including male gamete production (exflagellation) in Plasmodium falciparum, ookinete development in P. berghei, oocyst development in P. berghei and P. falciparum, and the liver stage of P. yoelii. We then compared 50 current and experimental antimalarials in these assays. We show that endoperoxides such as OZ439, a stable synthetic molecule currently in clinical phase IIa trials, are strong inhibitors of gametocyte maturation/gamete formation and impact sporogony; lumefantrine impairs development in the vector; and NPC-1161B, a new 8-aminoquinoline, inhibits sporogony.

Conclusions

These data enable objective comparisons of the strengths and weaknesses of each chemical class at targeting each stage of the lifecycle. Noting that the activities of many compounds lie within achievable blood concentrations, these results offer an invaluable guide to decisions regarding which drugs to combine in the next-generation of antimalarial drugs. This study might reveal the potential of life-cycle–wide analyses of drugs for other pathogens with complex life cycles. Please see later in the article for the Editors'' Summary     

Evidence for Rosettes as an Unrecognized Stage in the Life Cycle of Leishmania Parasites

ABSTRACT. Leishmania parasites, which afflict 12 million people in 88 countries, exist as promastigotes transmitted by insect vectors and as amastigotes residing in mammalian macrophages. Promastigote cells arranged in rosettes have also been described but universally disregarded as a distinct stage in the life cycle. We present evidence that only rosettes of Leishmania major promastigotes express cell surface poly‐α2,8 N‐acetyl neuraminic acid (PSA) and PSA containing de‐N‐acetyl neuraminic acid (NeuPSA). Expression of rosette‐specific PSA antigens was mosaic, with individual promastigotes expressing PSA, NeuPSA or both. A 50 kDa protein was detected by Western blot analysis of a detergent‐insoluble cell fraction with both PSA and NeuPSA‐reactive antibodies. Frequencies of rosette formation as well as cell surface PSA/NeuPSA expression were temperature dependent. Rosettes also engaged in an unusual swarming behavior, congregating into extended clusters. Distinct structures resembling cellular fusion bodies were formed in and released from rosettes. The results indicate that rosettes are an unrecognized stage in the life cycle of Leishmania. We hypothesize that rosettes initiate mating in Leishmania during which PSA/NeuPSA expression plays an important role. Recognizing rosettes as a distinct form of the Leishmania life cycle opens new possibilities for treatment or prevention of disease and, possibly, in vitro genetic recombination without passage of cells through insect vectors.     

Ultrastructural Observations on Life Cycle Stages of Pneumocystis carinii

An ultrastructural study of life cycle stages of Pneumocystis carinii in infected rat lungs in situ was undertaken utilizing 8 different modes of fixation. Three of the fixatives employed gave good fixation of cysts and intracystic bodies, but for the trophic forms fixation was only fair. Both the trophic forms and intracystic bodies have nuclear pores. The mitochondria of the organism have cristac that appear lamellar. One of the fixation modes revealed a thin, electron-dense layer on the outer surface of the cell wall, a "fuzzy coat" that had not been described previously. This material appears to mediate tight adhesion of trophic forms with other trophic forms, cysts, and with pneumocytcs of the lung alveolus.     

Heart Ratios at Different Stages in the Life Cycle of Lampreys

Heart ratios (heart weight/body weight. ×100) have been obtained for freshly killed and preserved (10% formalin) specimens of the lampreys Petromyzon marinus, Lampetra fluviatilis and L. planeri. Values for comparable stages were significantly higher in the preserved material, probably as a result of retention and fixation of blood in the heart chambers. The regression slope for the logarithmic relationship between heart and body weights was close to one in all stages of the three species investigated. A marked increase was observed in the heart ratio during metamorphosis, apparently attributable to an increase in the number and density of myocardial fibres. Heart ratios in the lamprey were generally higher than for most other fishes, with values of 0.1 and 0.3 being obtained for example in the case of freshly killed ammocoete and sexually mature adult stages of L. fluviatilis.     

Endogenous Stages of the Life Cycle of Isospora rivolta in the Dog

SYNOPSIS. Coccidia-free beagle puppies were experimentally infected with a cloned culture of Isospora rivolta oocysts. The endogenous stages were found in the posterior 1/2 of the small intestine, and rarely in the cecum and colon. Maximum numbers of all stages occurred just anterior to the ileocecal valve. Endogenous stages were found in the distal third of the villi, predominantly parasitizing subepithelial cells of the lamina propria; however, stages were occasionally present in epithelial cells. The number of asexual generations could not be determined from their structure, but evidence based on oocyst production suggested that there were at least 2 asexual generations. The schizonts were 17–24 by 12–25 μ and contained 4–24 merozoites, the most common number being 4 or 8. Schizonts with mature merozoites were found as early as 72 hr, but were present in maximum numbers at 96 hr. Merozoites had slender curved bodies and were 10.5–13.4 by 2.3–3.0 μ. Mature gamonts were found by 144 hr. Mature microgametocytes were 13.4 by 8.7 μ and contained 50–70 microgametes. Microgametes had slightly curved tapering bodies (5.8–6.4 by 0.6 μ) with 2 posteriorly directed flagella 11–14 μ long. Mature macrogametes had reticular cytoplasm and a uniformly large nucleus and nucleolus.
The prepatent period was 142–146 hr. The patent period was 13–23 days with an average of 19 days.     

Pattern of Replication of a Colicin Factor During the Cell Cycle of Escherichia coli

The ColBM, trp, lac episome was transferred to a lacZ derivative of Escherichia coli B/r, and the manner of replication of this colicinogenic factor was followed through the cell cycle. The results suggest a pattern of replication not connected with any particular stage in the cell cycle.     

Changes in the Carotenoid Pattern During the Synchronous Life Cycle of Scenedesmus

The wild type (WT) of Scenedesmus obliquus and a mutant lacking chlorophyll b and the light-harvesting complexes (WT-LHC₁) were synchronized by a light-dark regime. Both cultures contained the same type of carotenoids. However, concentrations and patterns of carotenoids were different during their synchronous life cycles. The concentration of total carotenoids followed more or less that of chlorophyll. The WT contained more carotenoids per cell mass, but slightly less per chlorophyll. It is discussed that part of the carotenoids of the mutant, lacking the peripheral antenna of PSII, might be located in the chlorophyll b-less apoprotein or in an enlarged core antenna of PSII. During the life cycle of Scenedesmus the carotenes are initially synthesized and most of the α-carotene is immediately oxidized to lutein which is inserted in the antennae systems of PSII and PSI. The further oxidation of lutein to loroxanthin seems to depend on both the change from dark to light, and on stages of the life cycle itself. Although the major part of β-carotene appears to be inserted in the reaction centers, a fraction of the total pool is rapidly converted to violaxanthin, following the onset of illumination. The conversion may serve to protect against photooxidation. Further conversion of violaxanthin to neoxanthin occurs to a greater extent in the mutant, WT-LHC₁. The results demonstrate (1) the close connection between the carotenoid pattern and the modulation of the photosynthetic apparatus during the life cycle of Scenedesmus and (2) the flexibility of the organism in compensating for the absence of the light-harvesting complexes of photosystems II by adjusting the carotenoid distribution.     

Gene Fitness Landscapes of Vibrio cholerae at Important Stages of Its Life Cycle

Vibrio cholerae has evolved to adeptly transition between the human small intestine and aquatic environments, leading to water-borne spread and transmission of the lethal diarrheal disease cholera. Using a host model that mimics the pathology of human cholera, we applied high density transposon mutagenesis combined with massively parallel sequencing (Tn-seq) to determine the fitness contribution of >90% of all non-essential genes of V. cholerae both during host infection and dissemination. Targeted mutagenesis and validation of 35 genes confirmed our results for the selective conditions with a total false positive rate of 4%. We identified 165 genes never before implicated for roles in dissemination that reside within pathways controlling many metabolic, catabolic and protective processes, from which a central role for glycogen metabolism was revealed. We additionally identified 76 new pathogenicity factors and 414 putatively essential genes for V. cholerae growth. Our results provide a comprehensive framework for understanding the biology of V. cholerae as it colonizes the small intestine, elicits profuse secretory diarrhea, and disseminates into the aquatic environment.     

Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle

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Parasites and Pathogens of the Honeybee (Apis mellifera) and Their Influence on Inter-Colonial Transmission

Pathogens and parasites may facilitate their transmission by manipulating host behavior. Honeybee pathogens and pests need to be transferred from one colony to another if they are to maintain themselves in a host population. Inter-colony transmission occurs typically through honeybee workers not returning to their home colony but entering a foreign colony (“drifting”). Pathogens might enhance drifting to enhance transmission to new colonies. We here report on the effects infection by ten honeybee viruses and Nosema spp., and Varroa mite infestation on honeybee drifting. Genotyping of workers collected from colonies allowed us to identify genuine drifted workers as well as source colonies sending out drifters in addition to sink colonies accepting them. We then used network analysis to determine patterns of drifting. Distance between colonies in the apiary was the major factor explaining 79% of drifting. None of the tested viruses or Nosema spp. were associated with the frequency of drifting. Only colony infestation with Varroa was associated with significantly enhanced drifting. More specifically, colonies with high Varroa infestation had a significantly enhanced acceptance of drifters, although they did not send out more drifting workers. Since Varroa-infested colonies show an enhanced attraction of drifting workers, and not only those infected with Varroa and its associated pathogens, infestation by Varroa may also facilitate the uptake of other pests and parasites.     

Nuclear DNA Content Variation in Different Life Cycle Stages of Sugar Kelp,Saccharina latissima

Marine Biotechnology - Ploidy variants can be utilized to increase yield, introduce sterility, and modify specific traits with an economic impact. Despite economic importance of Saccharina species,...     

Nose-Rings and Transmission of Helminth Parasites in Outdoor Pigs

Five growing pigs experimentally infected with low doses of Oesophagostomum dentation, Ascaris suum, and Trichuris suis were turned out with 5 helminth-naïve pigs on each of 3 pastures in June 1996 (Group 1). On one pasture all pigs received nose-rings. After slaughter of Group 1 in October, pasture infectivity was monitored using helminth-naïve, unringed tracer pigs. In 1997, helminth-naïve young pigs were turned out on the contaminated pastures in May (Group 2) and again in August (Group 3). Again all pigs on one pasture received nose-rings. All pigs and pastures were followed parasitologically and reduction in grass cover was monitored. Based on the acquisition of infection by the naïve pigs in Group 1, the estimated minimal embryonation times for eggs deposited on pasture were 23–25 days for O. dentatum, 5–6 weeks for A. suum and 9–10 weeks for T. suis. Results from tracer pigs and grass/soil samples indicated that pasture infectivity was light both years. Free-living stages of O. dentatum did not survive the winter. The nose-rings reduced rooting considerably, resulting in three-fold more grass cover on the nose-ring pasture compared to the control pastures by the end of the experiment. Nevertheless, the nose-rings did not significantly influence parasite transmission.