Extracellular development of erythrocytic stages ofPlasmodium falciparum

Continuous culture of the erythrocytic cycle ofPlasmodium falciparum within human erythrocytes maintained under appropriate conditionsin vitro has facilitated a large body of fundamental investigations with this important pathogen. It has revealed complex relationships between the developing intracellular parasite and its host erythrocyte. In pursuit of further understanding of these relationships, we have now developed, and here describe, methods that support extracellular development of the erythrocytic stages ofP. falciparum. Since only 1 to 2% of an inoculum of free merozoites can develop extracellularly through the entire schizogonic cycle to again form merozoites, the method does not support continuous extracellular culture. The work shows that the parasitophorous membrane and vacuole are not essential. Development of the parasites beyond the ring stage does however require constituents of the erythrocyte membrane.Abbreviations CPD citrate-phosphate dextrose - KIS KI medium with 10% human serum - RP-C RP medium without bicarbonate or serum - RPS RP medium with bicarbonate and serum     

Trichomonas vaginalis: strain differences in adhesion to plastic and virulence in vitro and in vivo

Trichomonas vaginalis isolated from clinical cases were grown axenically in TYI-S-33 medium. Various strains of this flagellate showed different adhesion characteristics in medium containing bovine serum or Cohn fractions thereof. These were not inversely related to the time in axenic culture. The adsorption of serum components to the parasites was assessed in relationship to adhesion but in many cases was probably not adhesion-related. All strains tested for virulence in vitro were almost equally cytotoxic for HeLa tissue cultures. However, in infectivity trials, one of these strains exhibited the highest adhesion capability and proved to be the most virulent for mice, and another (cloned) strain with the poorest adhesion capability failed to cause infection. Other strains exhibited lessened virulence following their extended axenic cultivation. It therefore appears that the in vivo pathogenic potential of the parasites growing in axenic culture is inherently strain-dependent. The findings suggest that although adhesion in whole serum-containing medium is sufficient to differentiate between variousTrichomonas isolates, it is insufficiently sensitive to correlate adhesion with virulence. It apparently is important to identify the adhesion-mediating factor(s) in serum or in its Cohn fractions IV-1 and IV-4 and to use it (them) to elucidate the possible correlation between the parasite's capacity to adhere and its pathogenicity.     

Influence of media supplements on growth and survival ofCampylobacter pylori

Experiments were designed to determine the role of heme and the importance of other factors in the growth ofCampylobacter pylori. Campylobacter pylori strains were tested for their ability to synthesize porphyrin, for their ability to grow and be maintained on basal medium and basal medium supplemented with blood or blood products, and for the influence of bovine serum albumin and catalase on viability. Results indicated thatCampylobacter pylori does not require heme as a source of porphyrin. Growth ofCampylobacter pylori could not be sustained on media containing starch or hemoglobin, but was sustained on media containing erythrocytes, serum, bovine serum albumin or catalase. The ability to grow on media containing bovine serum albumin and catalase suggests that protection from toxic fatty acids and the prevention of toxic product formation may be important factors in the growth and survival ofCampylobacter pylori in vitro. Both bovine serum albumin and catalase combined provide the minimum requirements which allow the spectrum ofCampylobacter pylori present in a single culture to grow on blood-free media.     

A new compact and cell dense continuous culture system

A new compact and cell dense continuous culture system was developed. The system consisted of a culture bag, which had two compartments separated by a semipermeable membrane, and an external rotator. Cells were cultured in the inner compartment with medium containing serum or the necessary growth factors. The outer compartment was filled with medium without serum or growth factors. The concentration of cells in culture was normally in the order of 10(7). Due to the large number of cells, sufficient carbon dioxide was produced so that use of a CO2 incubator was unnecessary. The medium in the outer part was frequently changed, but no medium changes were necessary in the inner bag. Using this system, a concentrated yield of antibody as well as a large number of cells could be obtained.     

Immune responses in vitro. II. Mixed leukocyte interaction in a protein-free medium

Addition of mercaptoethanol to the culture medium has permitted a mixed leukocyte interaction (MLI) to be achieved with mouse spleen cells in a chemically defined protein-free medium. Results which confirm those obtained in culture medium supplemented with heterologous sera are: 1) the maximum incorporation of isotope occurred between 60 and 72 h of culture; 2) the index of stimulation was 3–15; 3) the number of responder and stimulator cells required for maximum incorporation was 3 x 10⁶ – 4 x 10⁶ and 4) immunization resulted in a higher level of incorporation that occurred earlier than the maximum response of normal cells. Other results obtained confirm previous reports which are in apparent conflict: i.e., 1) stimulation of parental strains (P₁) by F₁ hybrids (P₁ x P₂) may be either equal to or one-half that induced by the other parental strains (P₂); and 2) F₁ hybrids may or may not be stimulated by the parental strains. These latter two results were found to be dependent on the strains used. Other advantages of the system are discussed in terms of the elimination of artifacts that occur in medium containing serum and the usefulness in examination of molecular events occurring early in immune reactions.     

Human urine stimulates in vitro growth of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> and <Emphasis Type="Italic">Trypanosoma rangeli</Emphasis>

Previous studies conducted in Leishmania led us to test the hypothesis that addition of human urine (HU) to the Liver Infusion Tryptose (LIT) medium would stimulate the in vitro growth of Trypanosoma cruzi and Trypanosoma rangeli strains. Herein, we show that the addition of 3% HU to LIT medium (LIT-HU3) significantly stimulated the growth of all the T. rangeli strains studied when compared with the parasite growth in conventional LIT medium (p < 0.05), and it was equivalent to the growth observed in LIT supplemented with fetal calf serum (FCS) in two parasite strains. Four out of the six T. cruzi strains analyzed showed a significant increase in parasite multiplication in LIT-HU3 (p < 0.05). However, two parasite strains presented good growth in both LIT and LIT-HU, suggesting differences in the parasite’s ability to grow in vitro. Furthermore, we have not observed differences in T. cruzi growth in LIT-HU3 and LIT supplemented with heat-denatured HU and in the metacyclogenesis of parasite strains cultured in LIT-HU3. These results allow concluding that the addition of HU to LIT medium stimulates the in vitro growth of T. rangeli and T. cruzi and can replace FCS as a supplement in culture medium.     

In vitro cultivation of fields isolates of Plasmodium falciparum in Mali

Malaria immunology, molecular biology and pathogenicity studies often require the adaptation of Plasmodium falciparum field isolates to continuous in vitro cultivation. For this purpose we have established propagation protocols of asexual erythrocytic stages of P. falciparum samples from malaria patients or asymptomatic carriers in Mali. The parasites were grown in standard culture medium supplemented by human serum and in a culture medium without human serum but supplemented by AlbuMax 1. The candle jar environment and tissue culture flasks gassed with 5% CO2, 5% O2 and 90% N2 obtained from a portable gas mixer were used. Protocols for parasite cultivation in a resource-poor setting were developed. These protocols were successfully applied to fresh isolates in Mali as well as to blood samples frozen in liquid nitrogen and shipped to a laboratory in U.S.A.     

Establishment of a Phagocytic Cell Line from Bombina orientalis

Continuous serum-free culture of Bok-2 cells was generated from primary culture of the tail bud stage embryos of the toad, Bombina orientalis. Bok-2 cells can be maintained in modified L-15 serum-free medium prepared by mixing L-15 medium, lactalbumin enzymatic hydrolysate, sucrose and sodium bicarbonate. Bok-2 cells have an ameboid behavior and morphology. When Bok-2 and viable Candida albicans were co-cultured, Bok-2 cells showed an immune response characterized by chemotaxis, phagocytosis and partial clearing activity of Candida cells and colonies. And Bok-2 cells also showed phagocytosis of latex beads without serum treatment and displayed numerous pseudopodia, membrane evagination for phagocytosis and phagosome activity. On the basis of these results, Bok-2 cells were identified as professional phagocytes.     

Supplementation Effect of Early Pregnancy Factor-Positive Serum into Bovine In Vitro Fertilization Culture Medium

PROBLEM: Early pregnancy factor (EPF) has been detected in pregnant bovine serum, and its activity appeared from 24 to 48 hr after insemination. However, in bovine in vitro fertilization (IVF), an EPF-like substance(s) had been detected in the culture medium of fertilized ovum. Therefore, we think that EPF and EPF-like substance(s) are very important materials for the development of the embryo. In this study, we examined the development of the embryo when fertilized bovine ova were cultured with IVF culture medium supplemented with EPF-positive or -negative serum. METHOD OF STUDY: EPF activity of each serum (fetal calf serum [FCS], calf serum [CS], estrus bovine serum, and pregnant bovine serum) was assessed by the bovine-rosette inhibition test. The sera were supplemented in TCM-199 culture medium, and IVF bovine ova were cultured with the media for 6 or 7 days, respectively. The culture media of each group were evaluated for EPF activity by the bovine-rosette inhibition test 48 hr after IVF. The cleavage rate of each group was calculated at 48 hr, and 6 or 7 days after IVF. The culture medium of cumulus cells was also tested for EPF activity. RESULTS: Only the pregnant bovine sera were EPF positive. All the culture media 48 hr after IVF became EPF positive. Additionally, the culture medium of cumulus cells did not have EPF activity. There was no significant difference in the cleavage rate of the EPF-positive and - negative sera 48 hr after IVF. However, the cleavage rate of EPF-positive sera tended to be higher than the negative sera. In contrast, the blastocyst development rates of EPF-positive sera were significantly higher than those of the negative sera 6 to 7 days after IVF (P < 0.05). CONCLUSIONS: The data suggest that an EPF-like substance(s) may be secreted from the in vitro fertilized bovine ovum but not from the cumulus cell, and that the EPF in the pregnant serum may accelerate the development of the bovine embryo in IVF.     

Improved techniques for the in vitro cultivation ofEimeria tenella in primary chick kidney cells

Primary kidney cells of 1- to 4-week-old chickens (PCKC) grown in Flexiperm chambers or culture flasks were infected with ultrapure sporozoites of twoEimeria tenella strains. For the 24-h parasite-free adaptation phase of the PCKC culture, Williams E medium plus 10% foetal calf serum (FCS) was used, and for the subsequent parasite-containing 168-h maintenance phase, we used medium 199 plus 2.5% FCS. Monolayers established during that time enabled the routine development of all schizont generations as well as, in general, young oocysts. The parasite stages propagated in FLEX were rendered visible by modified PAS-AO staining. Sporulated oocysts differed in length and width from those recovered after their passage through chickens. These results show thatE. tenella can reliably be reproduced from sporozoites to oocysts in PCKC cultures. However, the yield of oocysts was generally low, indicating that mass production of oocysts is achieved only by passaging sporulated oocysts through chickens.     

Routine large-scale production of monoclonal antibodies in a protein-free culture medium

A defined culture medium and cultivation technique are described which allow the long-term production on a large scale of monoclonal antibodies and myeloma proteins without the use of serum, serum proteins, or protein-containing liposomes. The high initial purity of the culture supernatants obtained with protein-free medium facilitates the purification of the monoclonal antibodies, the immunoglobulin (Ig) purity of which is limited only by the genetic homogeneity of the cells. Successful growth of 8 hybridoma and 2 myeloma lines has been achieved. SDS-PAGE analysis revealed that the levels of Ig production in the protein-free medium were comparable to those achieved with serum-supplemented media and with media supplemented with purified serum proteins. Stability of Ig production during continuous cultivation in the protein-free medium over an extended period was studied for a myeloma and a hybridoma line, both of which produced high levels (50–200 μg/ml) of Ig for periods of 11 and 22 weeks, respectively.     

Infectivity for mice of Trypanosoma cruzi I and II strains isolated from different hosts

In this paper, the infectivity for mice of Trypanosoma cruzi I and II strains isolated from sylvatic animals, triatomines, and humans is determined using fresh blood examination, hemoculture, culture of macerated organs, and polymerase chain reaction (PCR). Six strains were considered to have low infectivity (9.1–18.2%), five medium (27.3–45.4%), and one high (100.0%). Infectivity of T. cruzi strains isolated from sylvatic animals was significantly higher than that of strains isolated from humans and triatomines (p=0.0141). No significant difference was observed between the infectivity of T. cruzi I and II strains. The parasite was detected by fresh blood examination in one strain, by hemoculture and culture of macerated organs in four strains, and by PCR in all strains. We conclude that the infectivity is related to the host from which the strains were isolated, but the infectivity is not related to the genetic group of the parasite. We also conclude that the majority of the strains studied have low and medium infectivity for mice, and that PCR is an important tool to detect T. cruzi in strains with this biological characteristic.     

Improved isolation of salmonellae from faeces using a semisolid rappaport-vassiliadis medium

A modified semisolid Rappaport-Vassiliadis (MSRV) enrichment medium was evaluated as an alternative to Rappaport-Vassiliadis (RV) broth for culture of salmonellae from faeces. Faeces from 1544 subjects were cultured using MSRV medium, selective agars, and RV and selenite F enrichment broths. Of the 183 strains ofSalmonella spp. isolated, 88 % were recovered on MSRV medium, whilst only 59 % were recovered using RV broth. When MSRV medium was combined with direct culture and selenite enrichment, 98.9 % of salmonellae were recovered. The MSRV culture was found to be easy to read, and in most cases confirmation of organisms asSalmonella spp. could be made 24 hours after receipt of the faecal specimen.     

Cell cycle traverse in AHH-1 tk +/− human lymphoblastoid cells exposed to the chromosomal mutagen,m-amsa

AHH-l tk +/− cells were exposed to the chemotherapeutic agent, m-amsa, both in complete medium and in medium without serum, subcultured in complete medium, and the effect on the traverse of the cell cycle determined by flow cytometric analysis of bromodeoxyuridine (BrdUrd)-labeled DNA. After exposure to m-amsa (day 0), the percentage of S-phase cells increased significantly (P < 0.0017) with increasing concentration. Cells also accumulated in G2/M as evidenced by the significant (P < 0.0026), concentration-dependent increase in the percentage of cells detected within this phase. Serum deprivation during exposure resulted in significantly (P = 0.024) more cells in S-phase than in cultures exposed to m-amsa in complete medium. After three days in culture, a significant (P = 0.0001) accumulation of cells in G2/M was present; the percentage of cells in G2/M did not differ significantly (P = 0.148) in cultures exposed to m-amsa in complete medium or in serum-free medium. However, a significant (P < 0.001) loss of S-phase cells was found in cultures exposed without serum. At day 7, no significant concentration effects were detected (G0/G1, P = 0.6026; S-phase, P = 0.9773; G2/M, P = 0.8401). These results demonstrate that exposure to m-amsa perturbs the traverse of the cell cycle, initially by inhibiting the completion of S-phase and followed by an accumulation of cells in G2/M. In addition, exposure to m-amsa under conditions of serum deprivation results in an increased percentage of cells in the initial S-phase after exposure, the loss of S-phase cells from the culture after three days, and the appearance of a subdiploid peak, consistent with cells undergoing apoptosis. © 1996 Wiley-Liss, Inc.     

Staphylococcus aureus exopolysaccharide in vivo demonstrated by immunomagnetic separation and electron microscopy.

Staphylococcus aureus strains were separated from mastitis milk samples without cultivation by using monodisperse magnetic polymer particles coated with polyclonal antiserum against an encapsulated S. aureus strain. Exopolysaccharide was verified by transmission electron microscopy and the serum soft-agar culture technique. Capsular polysaccharide was found on virtually all clinical isolates. Surface protein A and S. aureus-specific cell wall components were masked when the strains were cultured on an exopolysaccharide-promoting medium. Masking of surface determinants was dependent on their concentration on the bacterial surface as well as on exopolysaccharide abundance. The polysaccharide layer on in vivo bacteria was reduced markedly after just one transfer from milk to blood agar plates but was reexpressed after culturing was done on a capsule-generating medium.     

Effects of temperature and white sucker (Catostomus commersoni) serum supplement on the in vitro multiplication of Cryptobia catostomi in cell-free culture medium

 Cryptobia catostomi, a non-pathogenic haemoflagellate of white suckers (Catostomus commersoni) multiplied rapidly in modified TDL-15 medium supplemented with 10% fetal bovine serum and 1% heat-inactivated white sucker serum (WSS) at 10°C and 18°C. The numbers of C. catostomi counted were significantly higher in cultures incubated at 10°C than in those incubated at 18°C beginning at 3 weeks post-incubation. The culture forms (from the eighth subculture at 9 months after isolation) were morphologically similar to blood forms and were infective to laboratory-raised white suckers. The parasite survived for about 4 weeks in the medium without WSS at 18°C. The present study indicates that WSS supplement supports the in vitro multiplication of C. catostomi and that its multiplication is temperature-dependent. Received: 24 May 1995 / Accepted: 18 October 1995     

Experiments to produce cysts in cultures of Histomonas meleagridis—the agent of histomonosis in poultry

Experiments were done with cultured trophozoite stages of different clonal strains (Histomonas meleagridis/Turkey/Austria/2922-C6/04 and H. meleagridis/Chicken/Hungary/5009-C2/05) of H. meleagridis in order to induce a cyst formation as it is known in other intestinal parasites. It was shown that the best multiplication of H. meleagridis occurred at 40°C in a full medium 199, when fetal calf serum and rice starch had been added. Under these conditions, numerous amoebic stages (8–12 μm in diameter) without and a few with flagellum were seen showing regular reproduction rates. When the conditions of culture were experimentally changed—and thus became worse—by decreasing the temperature, by deprivation of the medium from fetal calf serum and/or rice starch, and by changing the osmolarity, the pH, or the MgCl₂ concentration, many of the amoebic stages (containing starch granules) were destroyed, and several had obtained a spherical shape. If the culture conditions became even worse, smaller spherical stages occurred, which had only diameters of 4–7 μm and which appeared more condensed. Both spherical stages did not contain starch granules. All the previously seen stages disappeared constantly. Since a similar decrease of the optimal living conditions also occurs when intestinal or cloacal feces are deposited outside from the bird’s body, the results obtained here may underline the interpretation that some of the formerly amoebic stages are able to become large spherical stages and later small spherical stages. The large spherical stage would be some type of precysts while the smaller ones would represent true cysts.     

Computersimulation von Wachstum und Produktbildung bei Saccharomyces cerevisiae

A mathematical model with three modifications was stated, which describes the diauxic growth of Saccharomyces cerevisiae on aerobic conditions in a glucose-limited medium. The model was investigated by means of an analogcomputer. The first and simplest modification of the model is based on a well-known growth model and includes an additional team for catabolic respression. This 1st model could be fitted without complications to the experimental data of a batch culture, which shows growth, consumption of glucose, and production of ethanol during a first log-phase and growth and consumption of ethanol during a second log-phase. Complementation with the terms necessary for continuous culture conditions gives the 2nd model and, therefore, the possibility to predict the properties of the corresponding continuous culture. The prediction agrees qualitatively with the experimental data. After consideration of additional factors as the inhibition of growth and ethanol production by ethanol a 3rd model allowed to fit the experimental data of a continuous culture.     

Primary culture of lymphoid organ cells and haemocytes of kuruma shrimp,Penaeus japonicus

A primary cell culture system was developed for the cells of lymphoid organ tissue of kuruma shrimp, Penaeus japonicus. Minced tissues of lymphoid organs were seeded and incubated at 30 °C in medium 199 supplemented with 20% foetal bovine serum, a salt mixture and a lactalbumin hydrolysate (0.1 g/l). Fibroblast-like cells and epithelioid-like cells survived for 54 days. Cells did not survive after trypsin, collagenase or hyaluronidase treatment used for cell dissociation. Mitogens (Con A, PHA-P, Pokeweed) and insulin did not enhance cell proliferation. When penaeid rod-shaped DNA virus (PRDV) was inoculated into the lymphoid organ cell culture, a cytopathic effect was observed within 8 days. On the other hand, large granular haemocytes that were fractionated using a Percoll continuous density gradient were not infected with PRDV in vitro within 10 days, which was the longest period of haemocyte maintenance.     

Management of mycoplasma contamination in in vitro culture of Plasmodium falciparum without antibiotic treatment – a preliminary report

The presence of foreign contamination, especially of mycoplasmas, is a major hindrance in long term in vitro cultivation of Plasmodium falciparum and may be a source of false-positive results. Efforts have been made to control mycoplasma contamination by trypsinization of P. falciparum culture. Samples of accidentally contaminated cultures were used for this study. The presence of Mycoplasma orale in contaminated culture was ascertained by a species-specific PCR-based mycoplasma detection kit (Takara; Cat. No.6601). Trypsinization was carried out using trypsin-EDTA and the growth profile of P. falciparum was monitored for more than three weeks post-trypsinization. The studies were carried out with four different P. falciparum strains, various serum supplements and human erythrocytes belonging to different blood groups. It was interesting to observe that, irrespective of the different strains of P. falciparum and the variety of serum supplements and erythrocytes, mycoplasma contamination can successfully be removed from P. falciparum culture by trypsinization. No antibiotic except gentamicin, which is routinely used, was added to the medium. Results of this study indicate that the frequent appearance of mycoplasma in continuous long-term cultures of P. falciparum can be managed by trypsinization.