Cryptosporidium: Oocyst production and hybridoma generation for examining colostrum and monoclonal antibody roles in cryptosporidial infections.

Persistent Link:
http://hdl.handle.net/10150/184335
Title:
Cryptosporidium: Oocyst production and hybridoma generation for examining colostrum and monoclonal antibody roles in cryptosporidial infections.
Author:
Arrowood, Michael James.
Issue Date:
1988
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Techniques for the large-scale isolation of Cryptosporidium oocysts and sporozoites, obtained from the feces of experimentally infected Holstein calves, were developed employing discontinuous sucrose gradients and isopycnic Percoll gradients. The three step oocyst recovery method utilized two sequential discontinuous sucrose gradients followed by one Percoll gradient. Recovered oocysts were essentially free of debris and bacteria and represented 34% of the original oocyst suspension. Sporozoites were recovered from excystation mixtures on a single Percoll gradient. Sixty-three percent of the original sporozoites were recovered with 2.2% contamination by intact oocysts and virtually no oocyst walls. Eight anti-oocyst hybridomas were derived from oocyst-immunized mice: five from BALB/c mice and three from RBF/Dn mice. The monoclonal antibody (Mab) OW3 reacted specifically with C. parvum oocysts in immunofluorescent assays (IFA) and was shown to be superior to conventional stains for detecting oocysts in fecal smears from infected individuals. Sixteen anti-sporozoite hybridomas were derived from sporozoite-immunized BALB/c mice. The Mabs appeared to react with cell surface and cytoplasmic antigens by IFA. Two anti-sporozoite Mabs (C8C5, C6B6) reacted with a 20 kDa sporozoite antigen in western blots while the Mab C4A1 reacted with multiple antigens in western blots. These three Mabs (C8C5, C6B6, C4A1) were examined for potential modulation of cryptosporidial infections in vivo by oral Mab administration to oocyst-inoculated neonatal mice. The role for colostrum and breast milk in controlling cryptosporidial infections was examined by immunizing mouse dams and experimentally infecting their neonatal offspring. Colostrum and Mab-treated neonatal mice were sacrificed four days post infection. No difference in infection rates was observed among the treatment groups. Suckling mice treated daily with orally administered mixtures of Mabs (purified or ascitic fluid) showed significantly reduced parasite loads compared to control mice at four days post infection. In vitro cultivation of C. parvum was successful through asexual stages in human fetal lung, bovine turbinate and murine L929 cells. Parasite numbers that developed in the cell cultures varied from infection run to infection run.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Cryptosporidium.; Protozoan diseases -- Treatment.; Colostrum.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Microbiology and Immunology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Sterling, Charles R.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleCryptosporidium: Oocyst production and hybridoma generation for examining colostrum and monoclonal antibody roles in cryptosporidial infections.en_US
dc.creatorArrowood, Michael James.en_US
dc.contributor.authorArrowood, Michael James.en_US
dc.date.issued1988en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractTechniques for the large-scale isolation of Cryptosporidium oocysts and sporozoites, obtained from the feces of experimentally infected Holstein calves, were developed employing discontinuous sucrose gradients and isopycnic Percoll gradients. The three step oocyst recovery method utilized two sequential discontinuous sucrose gradients followed by one Percoll gradient. Recovered oocysts were essentially free of debris and bacteria and represented 34% of the original oocyst suspension. Sporozoites were recovered from excystation mixtures on a single Percoll gradient. Sixty-three percent of the original sporozoites were recovered with 2.2% contamination by intact oocysts and virtually no oocyst walls. Eight anti-oocyst hybridomas were derived from oocyst-immunized mice: five from BALB/c mice and three from RBF/Dn mice. The monoclonal antibody (Mab) OW3 reacted specifically with C. parvum oocysts in immunofluorescent assays (IFA) and was shown to be superior to conventional stains for detecting oocysts in fecal smears from infected individuals. Sixteen anti-sporozoite hybridomas were derived from sporozoite-immunized BALB/c mice. The Mabs appeared to react with cell surface and cytoplasmic antigens by IFA. Two anti-sporozoite Mabs (C8C5, C6B6) reacted with a 20 kDa sporozoite antigen in western blots while the Mab C4A1 reacted with multiple antigens in western blots. These three Mabs (C8C5, C6B6, C4A1) were examined for potential modulation of cryptosporidial infections in vivo by oral Mab administration to oocyst-inoculated neonatal mice. The role for colostrum and breast milk in controlling cryptosporidial infections was examined by immunizing mouse dams and experimentally infecting their neonatal offspring. Colostrum and Mab-treated neonatal mice were sacrificed four days post infection. No difference in infection rates was observed among the treatment groups. Suckling mice treated daily with orally administered mixtures of Mabs (purified or ascitic fluid) showed significantly reduced parasite loads compared to control mice at four days post infection. In vitro cultivation of C. parvum was successful through asexual stages in human fetal lung, bovine turbinate and murine L929 cells. Parasite numbers that developed in the cell cultures varied from infection run to infection run.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectCryptosporidium.en_US
dc.subjectProtozoan diseases -- Treatment.en_US
dc.subjectColostrum.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorSterling, Charles R.en_US
dc.contributor.committeememberOlson, George B.en_US
dc.contributor.committeememberJoens, Lynn A.en_US
dc.contributor.committeememberGerba, Charles P.en_US
dc.contributor.committeememberSammons, David W.en_US
dc.identifier.proquest8814207en_US
dc.identifier.oclc701107429en_US
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