Culture and filtration methods for obtaining Pneumocystis carinii trophozoites and cysts.

Two methods for acquisition of Pneumocystis carinii (Pc) trophozoites and cysts are reported. One method, the isolation of Pc from infected rat lung, provides large numbers of trophozoites and cysts but retains rat proteins. Ground lung is filtered through a series of Nucleopore filters from 10 to 3 microns; 1 g of rat lung yields an average of 1.1 x 10(9) Pc trophozoites and 1 x 10(7) cysts. The second method, propagation of Pc in culture with human embryonic lung cells on microcarrier beads, provides Pc trophozoites which are relatively free of host lung material. Cultured organisms may be filtered to remove rare culture monolayer cells. Organisms harvested from filtered lung are free from intact host cells and cell nuclei, however, host cell proteins and host DNA remain. Organisms from culture have minimal host contamination.     

Scanning electron microscope observations on the pathological changes of Malpighian tubules in the worker honeybee,Apis mellifera,infected by Malpighamoeba mellificae

A mixed population of cysts, immature cysts, and trophozoites was observed in the lumen of Malpighian tubules of Apis mellifera infected with Malpighamoeba mellificae. The most distinct pathological change in the infected tubules was swelling of the brush border of epithelial cells. In some areas the brush border was broken down into pieces, and in some cases it was completely engulfed by the parasites. The microvilli of the brush border were long and slender in noninfected tubules. Nuclei were not found in the infected tubules, but were frequently observed in the non-infected tubules. Numerous secretion globules were observed in the cytoplasm of epithelial cells of noninfected Malpighian tubules, but not in the cells of infected tubules. Epithelial cells in the infected tubules were destroyed and consumed by the trophozoites of M. mellificae. Trophozoites possessed protrusions and pits on their surfaces; many also had pseudopodia and apparently consumed the host cell by a mechanism of phagocytosis or endocytosis.     

Encystation of Entamoeba invadens IP-1 is induced by lowering the osmotic pressure and depletion of nutrients from the medium

Trophozoites of Entamoeba invadens IP-1 can be induced to encyst in simple solutions composed of semipermeable constituents (buffer, salts, or sugars) provided that their osmotic pressure is in the range of 60-160 mosmol/kg. Optimal yield of mature cysts was obtained when the osmotic pressure of the medium was 110 mosmol/kg. Encystation could be obtained in the absence of serum although higher yields were obtained in its presence. No difference in the yield of mature cysts was found when either dialyzed or full serum was used. High yields of encystation were obtained (greater than 70%) in the presence of 5% serum in solutions of NaCl, KCl, or MgSO4, suggesting that the mechanism of encystation is not induced via sodium or potassium channels. Cysts were obtained in the presence of 72 mM glucose, indicating that depletion of a carbon source is not the only requirement for encystation. A rapid change in the density of the Entamoeba cells was observed upon transfer of trophozoites (density 1.061-1.073 g/ml) from growth medium to the low osmotic pressure encystation solutions. Within the first 2 min their density decreased (to 1.050 g/ml), but it soon increased, reaching within 30 min a density higher than 1.120 g/ml. As the encystation process continued to completion, the density of the cells gradually decreased, the mature cysts reaching a density of 1.049-1.061 g/ml.     

Comparison of pulsed field gel electrophoresis karyotypes of Pneumocystis carinii derived from rat lung, cell culture, and ferret lung

Pulsed field gel electrophoretic karyotypes of Pneumocystis carinii derived from three sources were compared: immunosuppressed virus-free rats transtracheally inoculated with Pneumocystis-infected rat lung; WI-38 cell/Cytodex bead cell cultures inoculated with the same material; and immunosuppressed ferrets which reactivated latent Pneumocystis pneumonia. Karyotypes of DNA from Pneumocystis trophozoites or cysts from rat lung, and trophozoites from cell culture were identical. In contrast, ferret Pneumocystis DNA karyotypes were distinctly different. Rat Pneumocystis gene probes reacted with Southern- transferred rat Pneumocystis DNA but not with ferret Pneumocystis DNA. We concluded that neither the source nor life stage of rat Pneumocystis carinii influenced genomic karyotype, and that rat and ferret Pneumocystis are genetically diverse.     

Ultrastructure of an Unusual Erythrocytic Form of Plasmodium berghei*

SYNOPSIS. Unusual dense forms were discovered in ultrathin sections of Plasmodium berghei-infected rat erythrocytes. These parasites frequently occurred with one or more typical trophozoites in a single blood cell. They appeared darker than both the neighboring trophozoites and the host erythrocyte. Ribosomes were visible in clusters in their compact cytoplasm. The endoplasmic reticulum, when present, had dilated cisternae often containing a material of low density. Large food vacuoles werecommonly seen along with the small vesicles harboring pigment granules. The single large nucleus had dense nucleoplasm. Multilaminated membraned bodies and sausage-shaped vacuoles were, seen in some of the parasites. The exact identity of this form of P. berghei is not known. Its possible significance is discussed with particular reference to the differentiation of gametocytes.     

Encystation of Entamoeba invadens IP-1 Is Induced by Lowering the Osmotic Pressure and Depletion of Nutrients from the Medium1

ABSTRACT. Trophozoites of Entamoeba invadens IP-1 can be induced to encyst in simple solutions composed of semipermeable constituents (buffer, salts, or sugars) provided that their osmotic pressure is in the range of 60–160 mosmol/kg. Optimal yield of mature cysts was obtained when the osmotic pressure of the medium was 110 mosmol/kg. Encystation could be obtained in the absence of serum although higher yields were obtained in its presence. No difference in the yield of mature cysts was found when either dialyzed or full serum was used. High yields of encystation were obtained (>70%) in the presence of 5% serum in solutions of NaCl, KCl, or MgSO₄, suggesting that the mechanism of encystation is not induced via sodium or potassium channels. Cysts were obtained in the presence of 72 mM glucose, indicating that depletion of a carbon source is not the only requirement for encystation. A rapid change in the density of the Entamoeba cells was observed upon transfer of trophozoites (density 1.061–1.073 g/ml) from growth medium to the low osmotic pressure encystation solutions. Within the first 2 min their density decreased (to 1.050 g/ml), but it soon increased, reaching within 30 min a density higher than 1.120 g/ml. As the encystation process continued to completion, the density of the cells gradually decreased, the mature cysts reaching a density of 1.049–1.061 g/ml.     

Fractionation of Sporogonial Stages of the Microsporidian Encephalitozoon cuniculi by Percoll® Gradients

Microsporidia are obligate intracellular parasites that are increasingly recognized as a cause of opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi has been identified in humans with AIDS and infects a wide range of mammalian hosts. Little is known about the metabolic processes that regulate growth and replication of microsporidia. Examination of the individual stages of development will facilitate such studies and reveal possible targets for drug therapy. The purpose of this study was to fractionate and purify stages of the microsporidian life cycle. Encephalitozoon cuniculi were cultured in RK-13 cells. The tissue supernatants containing multiple parasite stages, empty microsporidial husks and host cell debris were collected, washed, and subjected to differential centrifugation in 80% stock isotonic Percoll. Transmission electron microscopy and SDS-polyacrylamide gel electrophoresis were used to compare the content and purity of each fraction. Mature spores formed a band at a density of approximately 1.138 g/ml. Sporoblasts were found at densities between 1.102 g/ml and 1.119 g/ml. A mixture of sporonts, sporoblasts, microsporidial husks, and cell debris remained at the top of the gradient and additional centrifugation in 30% and 50% Percoll resulted in separation of these stages. These results represent the first step toward fractionating stages of microsporidia infecting humans.     

Interplay between the parasite Amoebophrya sp. (Alveolata) and the cyst formation of the red tide dinoflagellate Scrippsiella trochoidea

Syndiniales (Alveolata) are marine parasites of a wide range of hosts, from unicellular organisms to Metazoa. Many Syndiniales obligatorily kill their hosts to accomplish their life cycle. This is the case for Amoebophrya spp. infecting dinoflagellates. However, several dinoflagellate species known to be infected by these parasites produce diploid resting cysts as part of their life history. These resting cysts may survive several seasons in the sediment before germinating. How these parasites survive during the dormancy of their host remained an open question. We successfully established infections by Amoebophrya sp. in the red tide dinoflagellate Scrippsiella trochoidea. This host strain was homothallic and able to continuously produce typical calcified cysts covered by calcareous spines. Presence of the parasite significantly speeded up the host cyst production, and cysts produced were the only cells to resist infections. However, some of them were clearly infected, probably earlier in their formation. After 10 months, cysts produced in presence of the parasite were able to germinate and new infective cycles of the parasite were rapidly observed. Thus, a very novel relationship for protists is demonstrated, one in which parasite and host simultaneously enter dormancy, emerging months later to propagate both species.     

Toxoplasma gondii: purification of trophozoites propagated in cell culture.

A new procedure is described for the purification of trophozoites from the virulent RH strain of Toxoplasma gondii propagated in baby hamster kidney (BHK-21) cell cultures. The culture medium containing host cell debris and trophozoites was filtered through glass-wool filtering fiber, which removed most host cell material. The filtrate containing trophozoites was centrifuged, and the trophozoite pellet was resuspended and washed in phosphate-buffered saline. An average of about 75% of the original number of trophozoites was recovered. No loss of trophozoite viability was observed as determined by the rate of host cell culture monolayer destruction. The amount of host cell material contamination in the final trophozoite fraction was negligible as determined by measuring radioactivity in the trophozoite fraction after cofiltration with noninfected host cell material which had been prelabeled with radioactive precursors.     

Colonic short-chain fatty acids inhibit encystation of Entamoeba invadens

Entamoeba parasites multiply as trophozoites in the layer of mucus that overlies the colonic epithelium. In response to stimuli that are not understood, trophozoites stop multiplying and differentiate into cysts that are released to infect another host. In the colon, Entamoeba trophozoites are exposed to the large variety of biochemicals that are carried into or are produced within this organ. The normal bacterial population of the colon releases large amounts of short-chain fatty acids (SCFAs). These compounds have effects on the growth, differentiation and repair of the colonic epithelium that correlate with de-creased activity of a Class I/II histone deacetylase (HDAC). We found that the formation of cysts, but not the growth of trophozoite-stage Entamoeba invadens parasites, was inhibited by physiologic concentrations of SCFAs. Variable levels of cyst formation did occur if SCFA concentrations were lowered. Specific inhibitors of Class I/II-type HDACs also prevented encystation, and trophozoites exposed to these compounds had increased levels of acetylation of histone H4 and other nuclear proteins. These results suggest that production of the infectious cyst stage of Entamoeba parasites is regulated in part by the levels of SCFAs made by the bacterial population of the colon.     

Separation and concentration of schizonts of Plasmodium falciparum by Percoll gradients

A technique for the separation of schizonts of Plasmodium falciparum is described. The different stages of the asexual cell cycle of the parasite were positioned according to their density in a continuous gradient of Percoll. Young trophozoites coincided with erythrocytes in a broad band corresponding to densities from 1.075 to 1.100 g/ml, whereas schizonts were concentrated at a density approximating 1.062 g/ml. The viability of the parasites was unimpaired by this procedure. Young trophozoites and schizonts continued their normal life cycle when cultured after the separation procedure. The percentage of recovery was high, reaching 80% of the initial quantity. Possible applications of the technique are discussed.     

Separation and Concentration of Schizonts of Plasmodium falciparum by Percoll Gradients1

A technique for the separation of schizonts of Plasmodium falciparum is described. The different stages of the asexual cell cycle of the parasite were positioned according to their density in a continuous gradient of Percoll. Young trophozoites coincided with erythrocytes in a broad band corresponding to densities from 1.075 to 1.100 g/ml, whereas schizonts were concentrated at a density approximating 1.062 g/ml. The viability of the parasites was unimpaired by this procedure. Young trophozoites and schizonts continued their normal life cycle when cultured after the separation procedure. The percentage of recovery was high, reaching 80% of the initial quantity. Possible applications of the technique are discussed.     

Interaction between Karyolysus sp. and rock lizard liver cells during hibernation]

During the rock lizard hibernation, no nuclear division occurs in the exoerythrocytar trophozoites of Karyolysus sp., found in hepatocytes or Kupffer's cells. In addition, organelles of the apical complex are seen persisting in these trophozoites, unlike the situation routinely observed in the majority of other intracellular sporozoans. Thus, the parasites under study can be compared with hypnozoites of other coccidia. The material being examined from natural rather than experimental conditions, during lizards' hibernation, the dynamics of host-parasite interrelations can be followed that involves the appearance in the infected cell of autophagous vacuoles, changes in mitochondrial structure and pattern of endoplasmic reticulum, the connection of the system of lamellar channels with the space of the parasitophorous vacuole. The results of the present observations may suggest, first, that during the host hibernation, exoerythrocytar trophozoites of Karyolysus being intracellular in their spatial distribution, are able to obtain nutrients from the outer, extracellular space, through the system of lamellar channels; second, that these channels can represent intercellular connections, which makes one consider the exoerythrocytar trophozoites as intercellular rather than intracellular parasites.     

Comparison of levels of inactivation of two isolates of Giardia lamblia cysts by UV light

The effects of 254-nm UV irradiation on two human isolates (WB and H3) of Giardia lamblia cysts were assessed using a collimated beam protocol and a Mongolian gerbil model. The levels of infection of cysts in the gerbils were assessed based on the presence of cysts in feces and the presence and activity of trophozoites in the small intestine of inoculated gerbils. The results suggest that there were differences in the infectivities of the WB and H3 isolates, as well as in susceptibilities of the parasites to UV light. Without UV exposure, gerbils were more readily infected by isolate H3 cysts. After UV exposure of the cysts, however, the gerbils were more susceptible to isolate WB cysts.     

Binding of fluorescein-labelled lectins on trophozoites and cysts of 3 strains of Toxoplasma gondii

The presence of carbohydrates on the surface of trophozoites and cysts of Toxoplasma gondii was investigated using 26 fluorescein labelled lectins. Three strains of parasites at different phases were tested; intra-cellular, intra-cystic and free trophozoites did not stain. However Soja hispida lectin intensly bind on cyst wall. The fluorescence could be eliminated by preincubation with certain carbohydrates, but not with trophozoite soluble antigen. These results suggest to the authors that cyst wall may be of host rather than of parasite origin.     

Comparison of Levels of Inactivation of Two Isolates of Giardia lamblia Cysts by UV Light

The effects of 254-nm UV irradiation on two human isolates (WB and H3) of Giardia lamblia cysts were assessed using a collimated beam protocol and a Mongolian gerbil model. The levels of infection of cysts in the gerbils were assessed based on the presence of cysts in feces and the presence and activity of trophozoites in the small intestine of inoculated gerbils. The results suggest that there were differences in the infectivities of the WB and H3 isolates, as well as in susceptibilities of the parasites to UV light. Without UV exposure, gerbils were more readily infected by isolate H3 cysts. After UV exposure of the cysts, however, the gerbils were more susceptible to isolate WB cysts.     

Concentration and enzyme content of in vitro-cultured Babesia bigemina-infected erythrocytes

Clones of in vitro-cultured Babesia bigemina-infected erythrocytes were concentrated by several density gradient procedures. The density range of infected erythrocytes containing pairs of parasites was 1.077 to 1.089 g/ml, whereas the density range of infected erythrocytes containing single parasites was 1.092 to 1.100 g/ml. Three enzymes--lactate dehydrogenase, glucose-phosphate isomerase, and glutamate dehydrogenase--were found associated with infected erythrocytes. The parasite-specific enzyme and/or isoenzymes were shown to have different mobility patterns in starch gel electrophoresis from those found in the normal bovine erythrocytes. The enzyme 6-phosphogluconate dehydrogenase was not detected as a parasite-specific enzyme in B. bigemina-infected erythrocytes.     

Assessment of human natural killer and lymphokine-activated killer cell cytotoxicity against Toxoplasma gondii trophozoites and brain cysts

Because previous work has suggested that NK cells may be important in host resistance against the intracellular parasite Toxoplasma gondii we examined whether human NK cells and lymphokine-activated killer (LAK) cells have activity against trophozoites and cysts of this organism in vitro. A method to radiolabel Toxoplasma trophozoites with 51Cr was developed and direct cytotoxic activity was determined by using modifications of the standard 51Cr release assay. Viability of 51Cr-labeled trophozoites assessed by both methylene blue staining and trypan blue exclusion was greater than 90%. Significantly more 51Cr was released by anti-Toxoplasma antibody and C than by antibody in the absence of C. Incubation of trophozoites with freshly isolated human NK cells or NK cells activated with either rIL-2 or rIFN-alpha did not result in significant release of 51Cr (specific lysis was 0 to 2.3%). In contrast, the average specific lysis of radiolabeled trophozoites by LAK cells was significant (specific lysis was 7.8% +/- 1.1, p less than 0.01). In a series of separate experiments, preincubation of radiolabeled trophozoites with heat-inactivated normal or Toxoplasma antibody-positive human serum increased the cytotoxicity of LAK cells from a mean specific lysis of 15% +/- 4.5 to 39% +/- 8.5, respectively (p less than 0.05), as assessed by 51Cr release. Because previous work has shown that radioisotope release from parasites may be nonspecific, separate experiments were performed to determine the cytotoxicity of LAK cells against antibody-coated trophozoites by using ethidium bromide-acridine orange staining to assess effector cell damage. LAK cells had a mean specific lysis of 51% against antibody-coated trophozoites by ethidium bromide-acridine orange staining. Preincubation with heat-inactivated Toxoplasma-antibody positive human serum did not increase activity of rIL-2-activated NK cells against 51CR-labeled trophozoites. Neither human NK cells (freshly isolated or activated by rIL-2 or rIFN-alpha) nor LAK cells were cytotoxic for purified preparations of cysts of Toxoplasma isolated from the brains of chronically infected mice.     

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.     

Concentration and Enzyme Content of In Vitro-Cultured Babesia bigemina-Infected Erythrocytes1

ABSTRACT. Clones of in vitro-cultured Babesia bigemina-infected erythrocytes were concentrated by several density gradient procedures. The density range of infected erythrocytes containing pairs of parasites was 1.077 to 1.089 g/ml, whereas the density range of infected erythrocytes containing single parasites was 1.092 to 1.100 g/ml. Three enzymes-lactate dehydrogenase, glucose-phosphate isomerase, and glutamate dehydrogenase-were found associated with infected erythrocytes. The parasite-specific enzyme and/or isoenzymes were shown to have different mobility patterns in starch gel electrophoresis from those found in the normal bovine erythrocytes. The enzyme 6-phosphogluconate dehydrogenase was not detected as a parasite-specific enzyme in B. bigemina-infected erythrocytes.