scholarly journals Method Detection Limits of PCR and Immunofluorescence Assay for Cryptosporidium parvum in Soil

1998 ◽  
Vol 64 (6) ◽  
pp. 2281-2283 ◽  
Author(s):  
Mark J. Walker ◽  
Carlo Montemagno ◽  
Juliet C. Bryant ◽  
William C. Ghiorse
Keyword(s):  
Cryptosporidium Parvum ◽  
Detection Limits ◽  
Immunofluorescence Assay ◽  
Pcr Analysis ◽  
Quantitative Nature

We determined and compared the method detection limits (MDLα) of a PCR and an immunofluorescence assay (IFA) for detection of Cryptosporidium parvum oocysts in soils. Based on the MDLα and the quantitative nature and stability of the IFA, PCR analysis is not a useful screening step for soil studies of oocyst transport.

scholarly journals Implication of Potential Differential Roles of the Two Phosphoglucomutase Isoforms in the Protozoan Parasite Cryptosporidium parvum

Pathogens ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 21
Author(s):  
Jiawen Nie ◽  
Jigang Yin ◽  
Dongqiang Wang ◽  
Chenchen Wang ◽  
Guan Zhu
Keyword(s):  
Enzyme Activity ◽  
Recombinant Proteins ◽  
Cryptosporidium Parvum ◽  
Plasma Membranes ◽  
Parasitophorous Vacuole ◽  
Protozoan Parasite ◽  
Immunofluorescence Assay ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane ◽  
Intracellular Parasites

Phosphoglucomutase 1 (PGM1) catalyzes the conversion between glucose-1-phosphate and glucose-6-phosphate in the glycolysis/glucogenesis pathway. PGM1s are typically cytosolic enzymes in organisms lacking chloroplasts. However, the protozoan Cryptosporidium parasites possess two tandemly duplicated PGM1 genes evolved by a gene duplication after their split from other apicomplexans. Moreover, the downstream PGM1 isoform contains an N-terminal signal peptide, predicting a non-cytosolic location. Here we expressed recombinant proteins of the two PGM1 isoforms from the zoonotic Cryptosporidium parvum, namely CpPGM1A and CpPGM1B, and confirmed their enzyme activity. Both isoforms followed Michaelis–Menten kinetics towards glucose-1-phosphate (Km = 0.17 and 0.13 mM, Vmax = 7.30 and 2.76 μmol/min/mg, respectively). CpPGM1A and CpPGM1B genes were expressed in oocysts, sporozoites and intracellular parasites at a similar pattern of expression, however CpPGM1A was expressed at much higher levels than CpPGM1B. Immunofluorescence assay showed that CpPGM1A was present in the cytosol of sporozoites, however this was enriched towards the plasma membranes in the intracellular parasites; whereas CpPGM1B was mainly present under sporozoite pellicle, although relocated to the parasitophorous vacuole membrane in the intracellular development. These observations indicated that CpPGM1A played a house-keeping function, while CpPGM1B played a different biological role that remains to be defined by future investigations.

scholarly journals Detection of Cryptosporidium parvum in Horses: Thresholds of Acid-Fast Stain, Immunofluorescence Assay, and Flow Cytometry

1999 ◽  
Vol 37 (2) ◽  
pp. 457-460 ◽  
Author(s):  
D. J. Cole ◽  
K. Snowden ◽  
N. D. Cohen ◽  
R. Smith
Keyword(s):  
Flow Cytometry ◽  
Cryptosporidium Parvum ◽  
Immunofluorescence Assay ◽  
Immunofluorescent Antibody ◽  
Acid Fast Staining

Feces collected from three asymptomatic horses and seeded withCryptosporidium parvum oocysts (101 to 106/g of feces) were evaluated by acid-fast staining (AF), an immunofluorescent antibody (IFA) technique, and flow cytometry. The thresholds of detection were 5 × 105 oocysts/g of feces for the IFA and AF techniques and 5 × 104oocysts/g for flow cytometry.

scholarly journals Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy

2006 ◽  
Vol 73 (3) ◽  
pp. 947-955 ◽  
Author(s):  
B. H. Al-Adhami ◽  
R. A. B. Nichols ◽  
J. R. Kusel ◽  
J. O'Grady ◽  
H. V. Smith
Keyword(s):  
Cryptosporidium Parvum ◽  
Immunofluorescence Microscopy ◽  
Uv Light ◽  
Fluorescein Isothiocyanate ◽  
Immunofluorescence Assay ◽  
Cryptosporidium Hominis ◽  
Specific Fluorescence ◽  
Thymine Dimers ◽  
Texas Red

ABSTRACT To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ�cm−2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ�cm−2. With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ�cm−2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ�cm−2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.

scholarly journals Differentiation between Human and Animal Isolates of Cryptosporidium parvum Using rDNA Sequencing and Direct PCR Analysis

1997 ◽  
Vol 83 (5) ◽  
pp. 825 ◽  
Author(s):  
Una M. Morgan ◽  
Clare C. Constantine ◽  
David A. Forbes ◽  
R. C. Andrew Thompson
Keyword(s):  
Cryptosporidium Parvum ◽  
Pcr Analysis ◽  
Direct Pcr ◽  
Rdna Sequencing ◽  
Animal Isolates

Characterization and immunolocalization of an oocyst wall antigen ofCryptosporidium parvum(Protozoa: Apicomplexa)

Parasitology ◽  
1991 ◽  
Vol 103 (2) ◽  
pp. 171-177 ◽  
Author(s):  
A. Bonnin ◽  
J. F. Dubremetz ◽  
P. Camerlynck
Keyword(s):  
Monoclonal Antibody ◽  
Immunoelectron Microscopy ◽  
Cryptosporidium Parvum ◽  
Parasitophorous Vacuole ◽  
Periodate Oxidation ◽  
Immunofluorescence Assay ◽  
Minor Components ◽  
Oocyst Wall ◽  
Gold Particles ◽  
Electron Lucent

A monoclonal antibody (OW-IGO) raised against purified excysted oocysts ofCryptosporidium parvumreacted in an immunofluorescence assay with the oocyst wall. The corresponding antigen was localized by immunoelectron microscopy in fibrillous material present in the parasitophorous vacuole of developing macrogametes and in the wall of both single and double layered sporulating oocysts. Gold particles were also detected over electron-lucent vesicles of the macrogametes by immunoelectron microscopy. On Western blotting ofC. parvumoocyst extracts, major bands at 250 and 40 kDa and several minor components were recognized by Mab OW-IGO. Almost complete abolition of Western blot reactivity occurred after periodate oxidation of oocyst antigen, suggesting that monoclonal antibody OW-IGO reacts with a carbohydrate epitope. Taken together, our results suggest that a fibrillous glycoproteinic material is released in the parasitophorous vacuole from electron-lucent vesicles during gametogenesis, and later condensed in the oocyst wall.

scholarly journals Role of CpSUB1, a Subtilisin-Like Protease, in Cryptosporidium parvum Infection In Vitro

2009 ◽  
Vol 8 (4) ◽  
pp. 470-477 ◽  
Author(s):  
Jane W. Wanyiri ◽  
Patsharaporn Techasintana ◽  
Roberta M. O'Connor ◽  
Michael J. Blackman ◽  
Kami Kim ◽  
...  
Keyword(s):  
Serine Protease ◽  
Cryptosporidium Parvum ◽  
Protease Activity ◽  
Diarrheal Disease ◽  
Genomic Sequence ◽  
Host Cells ◽  
Pcr Analysis ◽  
Apical Region ◽  
Gene Encoding

ABSTRACTThe apicomplexan parasiteCryptosporidiumis a significant cause of diarrheal disease worldwide. Previously, we reported that aCryptosporidium parvumsubtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity inC. parvumlysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in theC. parvumgenome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA fromC. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of ∼64 kDa and ∼48 kDa, forC. parvumlysates and proteins “shed” during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 byC. parvumlysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity inC. parvumand for processing of gp40/15. Importantly, the recombinant prodomain inhibitedC. parvuminfection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.

scholarly journals Role of Immunoglobulin A Monoclonal Antibodies against P23 in Controlling Murine Cryptosporidium parvum Infection

1998 ◽  
Vol 66 (9) ◽  
pp. 4469-4473 ◽  
Author(s):  
F. Javier Enriquez ◽  
Michael W. Riggs
Keyword(s):  
Monoclonal Antibodies ◽  
Cryptosporidium Parvum ◽  
Matched Control ◽  
Intestinal Parasites ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoglobulin A ◽  
Passive Immunization ◽  
Immunofluorescence Assay

ABSTRACT Cryptosporidium parvum is an important diarrhea-causing protozoan parasite of immunocompetent and immunocompromised hosts. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections with bacteria, viruses, and parasites, but little is known about the role of IgA in the control of C. parvuminfection. We assessed the role of IgA during C. parvum infection in neonatal mice. IgA-secreting hybridomas were developed by using Peyer’s patch lymphocytes from BALB/c mice which had been orally inoculated with viable C. parvumoocysts. Six monoclonal antibodies (MAbs) were selected for further study based on indirect immunofluorescence assay reactivity with sporozoite and merozoite pellicles and the antigen (Ag) deposited on glass substrate by gliding sporozoites. Each MAb was secreted in dimeric form and recognized a 23-kDa sporozoite Ag in Western immunoblots. The Ag recognized comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with P23, a previously defined neutralization-sensitive zoite pellicle Ag. MAbs were evaluated for prophylactic or therapeutic efficacy against C. parvum, singly and in combinations, in neonatal BALB/c mice. A combination of two MAbs given prophylactically prior to and 12 h following oocyst challenge reduced the number of intestinal parasites scored histologically by 21.1% compared to the numbers in mice given an isotype-matched control MAb (P < 0.01). Individual MAbs given therapeutically in nine doses over a 96-h period following oocyst challenge increased efficacy against C. parvuminfection. Four MAbs given therapeutically each reduced intestinal infection 34.4 to 42.2% compared to isotype-matched control MAb-treated mice (P < 0.05). One MAb reduced infection 63.3 and 72.7% in replicate experiments compared to isotype-matched control MAb-treated mice (P < 0.0001). We conclude that IgA MAbs directed to neutralization-sensitive P23 epitopes may have utility in passive immunization against murineC. parvum infection.

scholarly journals Coxiella burnetii Shedding Routes and Antibody Response after Outbreaks of Q Fever-Induced Abortion in Dairy Goat Herds

2008 ◽  
Vol 75 (2) ◽  
pp. 428-433 ◽  
Author(s):  
Elodie Rousset ◽  
Mustapha Berri ◽  
Benoit Durand ◽  
Philippe Dufour ◽  
Myriam Prigent ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Complement Fixation ◽  
Q Fever ◽  
Immunofluorescence Assay ◽  
Dairy Goat ◽  
Pcr Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potential Hazard ◽  
Fecal Shedding ◽  
The Relationship

ABSTRACT Q fever is a zoonosis caused by Coxiella burnetii, a bacterium largely carried by ruminants and shed into milk, vaginal mucus, and feces. The main potential hazard to humans and animals is due to shedding of bacteria that can then persist in the environment and be aerosolized. The purpose of this study was to evaluate shedding after an outbreak of Q fever abortion in goat herds and to assess the relationship with the occurrence of abortions and antibody responses. Aborting and nonaborting goats were monitored by PCR for C. burnetii shedding 15 and 30 days after the abortion episodes. PCR analysis of all samples showed that 70% (n = 50) of the aborting and 53% (n = 70) of the nonaborting goats were positive. C. burnetii was shed into vaginal mucus, feces, and milk of 44%, 21%, and 38%, respectively, of goats that aborted and 27%, 20%, and 31%, respectively, of goats that delivered normally. Statistical comparison of these shedding results did not reveal any difference between these two groups. PCR results obtained for the vaginal and fecal routes were concordant in 81% of cases, whereas those for milk correlated with only 49% of cases with either vaginal or fecal shedding status. Serological analysis, using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and complement fixation tests, showed that at least 24% of the seronegative goats shed bacteria. Positive vaginal and fecal shedding, unlike positive milk shedding, was observed more often in animals that were weakly positive or negative by ELISA or IFA. Two opposite shedding trends were thus apparent for the milk and vaginal-fecal routes. Moreover, this study showed that a nonnegligible proportion of seronegative animals that delivered normally could excrete C. burnetii.

scholarly journals Randomly Amplified Polymorphic DNA PCR Analysis of Bovine Cryptosporidium parvum Strains Isolated from the Watershed of the Red River of the North

1998 ◽  
Vol 64 (6) ◽  
pp. 2262-2265 ◽  
Author(s):  
Kevin V. Shianna ◽  
Russell Rytter ◽  
Jonathan G. Spanier
Keyword(s):  
Cryptosporidium Parvum ◽  
Protozoan Parasite ◽  
Geographic Area ◽  
Red River ◽  
Pcr Analysis ◽  
Randomly Amplified Polymorphic Dna ◽  
Strain Variation ◽  
The North ◽  
Neonatal Calves ◽  
Dna Pcr

ABSTRACT Cryptosporidium parvum is a protozoan parasite that causes the disease cryptosporidiosis in a variety of mammals, including neonatal calves and humans. Millions of oocysts are shed during acute cryptosporidiosis, and zoonotic transmission is inferred, though not proven, to be a general phenomenon. Very little is known about the degree of strain variation exhibited by bovine and human isolates, though such knowledge would enable the amount of bovine-to-human transmission to be more precisely analyzed. This research was initiated to determine whether variations exist among bovine strains isolated from a localized geographic area, the watershed of the Red River of the North. Sixteen strains were isolated and compared to each other and to two human and two calf strains from Australia by randomly amplified polymorphic DNA PCR. A statistical analysis of the data indicated that the isolates belonged to four different groups of strains.

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