Cryptosporidiumcell culture infectivity assay design

Parasitology ◽  
2011 ◽  
Vol 138 (6) ◽  
pp. 671-681 ◽  
Author(s):  
B. J. KING ◽  
A. R. KEEGAN ◽  
B. S. ROBINSON ◽  
P. T. MONIS
Keyword(s):  
Developmental Stages ◽  
Gastrointestinal Disease ◽  
Cell Monolayer ◽  
Data Interpretation ◽  
Host Cells ◽  
Specific Gene ◽  
Factors Affecting ◽  
Number Of Factors ◽  
Life Cycle Development

SUMMARYMembers of the genusCryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture,in vitrogrowth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design ofin vitroculture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

Infection with the intracellular protozoan parasite Theileria parva induces constitutively high levels of NF-kappa B in bovine T lymphocytes

1989 ◽  
Vol 9 (11) ◽  
pp. 4677-4686
Author(s):  
V Ivanov ◽  
B Stein ◽  
I Baumann ◽  
D A Dobbelaere ◽  
P Herrlich ◽  
...  
Keyword(s):  
T Cells ◽  
Phorbol Esters ◽  
Protozoan Parasite ◽  
Lymphoproliferative Disease ◽  
Host Cells ◽  
Specific Gene ◽  
Theileria Parva ◽  
Nf Kappa B ◽  
Intracellular Protozoan Parasite

The intracellular protozoan parasite Theileria parva causes a lymphoproliferative disease of T cells in cattle and uncontrolled lymphocyte proliferation in culture. We have identified and characterized in infected cells the transcriptional activator, NF-kappa B, whose recognition motifs have been identified in several gene enhancers important for lymphocyte-specific gene expression. NF-kappa B is normally constitutively activated in nuclear extracts derived from B cells and can be induced in T cells and nonlymphoid cells by phorbol esters. Theileria-infected lymphocytes contained constitutively high levels of activated NF-kappa B in nuclear fractions and inactive NF-kappa B in cytoplasmic fractions. The inactive cytoplasmic precursor could be activated by treatment of extracts with deoxycholate, which was shown previously to dissociate NF-kappa B from an inhibitor, I kappa B. Treatment of lymphocyte extracts with 3 mM GTP stimulated NF-kappa B binding to its recognition motif in vitro, thereby distinguishing it from a related nuclear factor, H2-TF1. Selective killing of the parasite, which left the host cells intact, resulted in a rapid loss of NF-kappa B from the nuclear fractions and a slower loss from the cytoplasmic fractions. In parasitized cells, NF-kappa B could not be further stimulated by treatment with 12-O-tetradecanoylphorbol-13-acetate whereas in cells treated to remove the parasite, this compound stimulated elevated levels of NF-kappa B. We propose that high levels of activated NF-kappa B are maintained by the presence of the parasite in infected T cells. Similarly, we propose that the high levels of inactive cytoplasmic precursor are a result of increased synthesis due to the presence of the parasite.

FACTORS AFFECTING THE ACTIVITY OF ALLOXAN IN VITRO

1955 ◽  
Vol 33 (5) ◽  
pp. 780-791 ◽  
Author(s):  
S. J. Klebanoff
Keyword(s):  
Oxygen Uptake ◽  
Succinic Dehydrogenase ◽  
Selective Toxicity ◽  
Factors Affecting ◽  
Stable Factor ◽  
Heat Stable ◽  
Number Of Factors ◽  
Liver Homogenates ◽  
Insight Into

The effect of alloxan on the oxygen uptake and the succinic dehydrogenase activity of rat liver homogenates has been studied in an attempt to gain some insight into the activity of alloxan within the cell. The increased oxygen uptake on the addition of alloxan to liver homogenates was found to be non-enzymatic in nature and to depend on some heat stable factor or factors in the preparation. The ability of reduced glutathione to substitute for the homogenate suggests that this substance may be concerned. It is thought that alloxan is reduced to dialuric acid by a number of factors in the homogenate, and that the increased oxygen uptake results from the spontaneous reoxidation of this latter substance. Experiments with dialuric acid tend to confirm this hypothesis. The inactivation of succinic dehydrogenase by alloxan is greatly influenced by the pH of the solution in which the alloxan is dissolved, as well as by the pH of the enzyme preparation, with acidity increasing and alkalinity decreasing the effect. The progressive nature of the inactivation with time is shown to depend upon the lowering of the pH which is brought about by the addition of alloxan to an unbuffered enzyme mixture. The experimental results are discussed in relation to the possible importance of pH in the selective toxicity of alloxan.

scholarly journals Theileria equi claudin like apicomplexan microneme protein contains neutralization-sensitive epitopes and interacts with components of the equine erythrocyte membrane skeleton

2021 ◽  
Author(s):  
Cynthia Onzere ◽  
Lindsay Fry ◽  
Richard Bishop ◽  
Marta Silva ◽  
Reginaldo Bastos ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Vaccine Development ◽  
Membrane Skeleton ◽  
Host Cells ◽  
Effective Vaccine ◽  
Theileria Equi ◽  
Anion Exchange Protein ◽  
Microneme Protein

Abstract Theileria equi (T. equi) is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and β- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.

scholarly journals Effect of the age of the pupal holotissue on the nutritional quality of artificial diets for Trichogramma spp. (Hymenoptera: Trichogrammatidae)

2000 ◽  
Vol 29 (3) ◽  
pp. 555-563 ◽  
Author(s):  
Fernando L. Cônsoli ◽  
José R.P. Parra
Keyword(s):  
Nutritional Quality ◽  
Developmental Stages ◽  
Egg Yolk ◽  
Artificial Diets ◽  
Age Classes ◽  
Factors Affecting ◽  
Fetal Serum ◽  
Bovine Fetal Serum

The nutritional quality of artificial diets composed of pupal holotissues of Diatraea saccharalis (Fabr.) (Lep.: Crambidae) from three different age classes (1-2, 3-4 and 5-6 days old) were tested for rearing Trichogramma galloi Zucchi and T. pretiosum Riley (Hym.: Trichogrammatidae) in vitro. Pupal holotissues were added to egg yolk, bovine fetal serum, lactoalbumine hydrolysate and preservatives, and offered to the parasitoids into artificial eggs. The quality of these diets in supporting the development of both parasitoids was evaluated by assessing the acceptance and parasitism of the artificial host eggs, larval and pupal survival, size, parasitism capacity and longevity of the in vitro-reared females, and the presence of deformed adults. Diets composed of 5-6 d old pupal holotissues did not support the larval development of both parasitoids as well as reduced the acceptance and/or parasitism of artificial eggs by T. galloi and T. pretiosum. The factors affecting the nutritional quality of pupal holotissues collected from different developmental stages are discussed.

scholarly journals Infection with the intracellular protozoan parasite Theileria parva induces constitutively high levels of NF-kappa B in bovine T lymphocytes.

1989 ◽  
Vol 9 (11) ◽  
pp. 4677-4686 ◽  
Author(s):  
V Ivanov ◽  
B Stein ◽  
I Baumann ◽  
D A Dobbelaere ◽  
P Herrlich ◽  
...  
Keyword(s):  
T Cells ◽  
Phorbol Esters ◽  
Protozoan Parasite ◽  
Lymphoproliferative Disease ◽  
Host Cells ◽  
Specific Gene ◽  
Theileria Parva ◽  
Nf Kappa B ◽  
Intracellular Protozoan Parasite

The intracellular protozoan parasite Theileria parva causes a lymphoproliferative disease of T cells in cattle and uncontrolled lymphocyte proliferation in culture. We have identified and characterized in infected cells the transcriptional activator, NF-kappa B, whose recognition motifs have been identified in several gene enhancers important for lymphocyte-specific gene expression. NF-kappa B is normally constitutively activated in nuclear extracts derived from B cells and can be induced in T cells and nonlymphoid cells by phorbol esters. Theileria-infected lymphocytes contained constitutively high levels of activated NF-kappa B in nuclear fractions and inactive NF-kappa B in cytoplasmic fractions. The inactive cytoplasmic precursor could be activated by treatment of extracts with deoxycholate, which was shown previously to dissociate NF-kappa B from an inhibitor, I kappa B. Treatment of lymphocyte extracts with 3 mM GTP stimulated NF-kappa B binding to its recognition motif in vitro, thereby distinguishing it from a related nuclear factor, H2-TF1. Selective killing of the parasite, which left the host cells intact, resulted in a rapid loss of NF-kappa B from the nuclear fractions and a slower loss from the cytoplasmic fractions. In parasitized cells, NF-kappa B could not be further stimulated by treatment with 12-O-tetradecanoylphorbol-13-acetate whereas in cells treated to remove the parasite, this compound stimulated elevated levels of NF-kappa B. We propose that high levels of activated NF-kappa B are maintained by the presence of the parasite in infected T cells. Similarly, we propose that the high levels of inactive cytoplasmic precursor are a result of increased synthesis due to the presence of the parasite.

scholarly journals High Humidity and Age-Dependent Fruit Susceptibility Promote Development of Trichothecium Black Spot on Apple

Plant Disease ◽  
2019 ◽  
Vol 103 (2) ◽  
pp. 259-267 ◽  
Author(s):  
Pengbo Dai ◽  
Xiaofei Liang ◽  
Yajing Wang ◽  
Mark L. Gleason ◽  
Rong Zhang ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Disease Incidence ◽  
Germination Rate ◽  
Black Spot ◽  
Apple Fruit ◽  
High Humidity ◽  
Factors Affecting ◽  
Fruit Maturity ◽  
Fruit Bagging

Fruit bagging is a widely used orchard practice in China. Trichothecium black spot (TBS) is a disease highly associated with the fruit bagging. In this study, we characterized in vitro factors affecting the causal agent, Trichothecium roseum, and TBS development and infection histology on field-bagged apple fruit in situ. Under in vitro conditions, conidial germination required exogenous nutrients, and the germination rate was significantly promoted by high humidity, a condition mimicking the bag microenvironment. Germ tubes penetrated fruit via natural openings including stomata, lenticels, and surface cracks. To determine the chronology of infection by T. roseum, ‘Fuji’ fruit were inoculated in the field at different developmental stages. The earliest infection occurred 60 days after full bloom (dafb), and disease incidence increased as fruit maturity advanced. At harvest time (165 dafb), lesions on more recently inoculated fruit (105 dafb, 150 dafb) were larger than lesions from fruit inoculated on earlier dates. Histological observation showed that infection of younger fruit elicited stronger host lignification responses restricting lesion development. Taken together, our results support the hypothesis that high humidity in sealed bags and increased susceptibility associated with advancing fruit maturity are key factors promoting T. roseum infection and TBS symptom development on bagged apple fruit.

FACTORS AFFECTING THE ACTIVITY OF ALLOXAN IN VITRO

1955 ◽  
Vol 33 (1) ◽  
pp. 780-791
Author(s):  
S. J. Klebanoff
Keyword(s):  
Oxygen Uptake ◽  
Succinic Dehydrogenase ◽  
Selective Toxicity ◽  
Factors Affecting ◽  
Stable Factor ◽  
Heat Stable ◽  
Number Of Factors ◽  
Liver Homogenates ◽  
Insight Into

The effect of alloxan on the oxygen uptake and the succinic dehydrogenase activity of rat liver homogenates has been studied in an attempt to gain some insight into the activity of alloxan within the cell. The increased oxygen uptake on the addition of alloxan to liver homogenates was found to be non-enzymatic in nature and to depend on some heat stable factor or factors in the preparation. The ability of reduced glutathione to substitute for the homogenate suggests that this substance may be concerned. It is thought that alloxan is reduced to dialuric acid by a number of factors in the homogenate, and that the increased oxygen uptake results from the spontaneous reoxidation of this latter substance. Experiments with dialuric acid tend to confirm this hypothesis. The inactivation of succinic dehydrogenase by alloxan is greatly influenced by the pH of the solution in which the alloxan is dissolved, as well as by the pH of the enzyme preparation, with acidity increasing and alkalinity decreasing the effect. The progressive nature of the inactivation with time is shown to depend upon the lowering of the pH which is brought about by the addition of alloxan to an unbuffered enzyme mixture. The experimental results are discussed in relation to the possible importance of pH in the selective toxicity of alloxan.

scholarly journals 291 EFFECT OF DIFFERENT TRANSPORT TEMPERATURES (+4°C, +32°C) ON IN VITRO MATURATION OF OOCYTES COLLECTED FROM CATTLE AND SHEEP OVARIES

2005 ◽  
Vol 17 (2) ◽  
pp. 296
Author(s):  
O.B. Ozdas ◽  
M. Tas ◽  
U. Cirit ◽  
M. Evecen ◽  
K. Demir ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Developmental Stages ◽  
Chi Square ◽  
Medium Temperature ◽  
Factors Affecting ◽  
Group I ◽  
Significant Difference ◽  
Temperature Group ◽  
C 32

At present, blastocyst rates in embryos obtained from in vitro maturation of oocytes, and their fertilization and culture, is still not at the desired level. One of the most important problems encountered in in vitro culture studies is seen in the maturation period of oocytes until they reach the fertilizable level. Transport time of the ovaries and, in particular, temperature of the transport medium used are among the factors affecting complete maturation. The aim of this study was to determine the effects of different transport temperatures (4°C, 32°C) of sheep and cattle ovaries on the in vitro maturation of oocytes. Two experimental groups were formed in the study. Sheep and cattle ovaries were put into saline solution at 32°C. The ovaries were transported at the same temperature (Group I) or at 4°C following a 10-min incubation at room temperature (Group II), in 2–4 h to the laboratory (n = 6). For each group, oocytes were collected from ovaries using the dissection method and selected oocytes were matured in their own group in 700 μL TCM-199 (supplemented with pyruvate, LH, FCS) for 23 h at a gas atmosphere of 5% CO2, 5% O2, 90% N2 and at 38.8°C. At the end of maturation, oocytes were cleansed from their cumulus oophorus cells and fixed in acetic acid-ethyl alcohol (1:3) for 48 h. The developmental stages until MII of oocytes stained with aceto-orcein were then examined under the phase contrast microscope. The chi-square test was used for statistical analysis (Table 1). While oocytes obtained from sheep ovaries transported at +32°C reached the MII stage at a faster rate compared to those at +4°C (P < 0.001), no statistically significant difference was observed between the maturation to the MII stage of oocytes obtained from cattle ovaries transported at +4°C and +32°C. As a result of this study, while it was established that cattle ovaries could be transported at both +4°C and +32°C and that there was no difference in oocyte maturation, a medium temperature of +4°C was determined to be unsuitable for transporting sheep ovaries. Table 1. Stages of development in sheep and cattle oocytes after 23 h of culture This work was supported by Istanbul University.

Some factors affecting the cultivationin vitroof the erythrocytic stages ofPlasmodium knowlesi

Parasitology ◽  
1969 ◽  
Vol 59 (4) ◽  
pp. 915-924 ◽  
Author(s):  
P. I. Trigg
Keyword(s):  
Technical Assistance ◽  
Plasmodium Knowlesi ◽  
Host Cells ◽  
World Health ◽  
Vitamin B ◽  
Low Oxygen ◽  
New Host ◽  
Factors Affecting ◽  
Oxygen Tensions

A study was made of some of the factors which affect the growth ofPlasmodium knowlesiin blood diluted in the Harvard medium. Some slight enhancement of growth and re-invasion of new host cells was obtained whenP. knowlesiwas incubated in low oxygen tensions. Some growth but no re-invasion was obtained under anaerobic conditions. High oxygen tensions were also inhibitory.The lipids present in the plasma are important for the growth ofP. knowlesi in vitro.Stearic acid and cholesterol are required by the parasite but do not account for all the growth promoting properties of plasma. A requirement for vitamin B12was also demonstrated.I should like to thank Dr F. Hawking and Dr J. Williamson for their advice and for reading through the manuscript, Mr T. J. Scott-Finnigan for technical assistance, Dr K. Mervyn (Glaxo Research Ltd.) for samples of vitamin B12analogues and the Hachmeister Company (Pittsburgh) for a sample of TEM 4T. This work received financial assistance from the World Health Organization.

scholarly journals Theileria equi claudin like apicomplexan microneme protein contains neutralization-sensitive epitopes and interacts with components of the equine erythrocyte membrane skeleton

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Cynthia K. Onzere ◽  
Lindsay M. Fry ◽  
Richard P. Bishop ◽  
Marta G. Silva ◽  
Reginaldo G. Bastos ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Vaccine Development ◽  
Membrane Skeleton ◽  
Host Cells ◽  
Effective Vaccine ◽  
Theileria Equi ◽  
Anion Exchange Protein ◽  
Microneme Protein

AbstractTheileria equi is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and β- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.

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