ABSTRACT
A statistical analysis of clinical, nutritional, and immunological data gathered in a previous study suggest that nutritional factors, and in particular, iron status, appeared to be of significance in mounting an effective immune response to Cryptosporidium infection in young children. The primary protective mechanism seemed to be cell-mediated; humoral immunity was intact in all the study subjects, however, CMI was initially impaired but improved over six weeks.
Subject(s)
Cryptosporidiosis/immunology , Infant Nutritional Physiological Phenomena , Nutritional Status , Animals , Antibodies, Protozoan/analysis , Cryptosporidiosis/blood , Cryptosporidium/immunology , Duodenum , Feces/chemistry , Female , Humans , Immunity, Cellular , Immunoglobulins/analysis , Infant , Intestinal Secretions/immunology , Iron/blood , Male , Philippines , Regression AnalysisABSTRACT
The objective of this project was to demonstrate detection of Cryptosporidium parvum DNA in fixed, paraffin-embedded tissue using the polymerase chain reaction (PCR). DNA was purified from six samples of fixed, paraffin-embedded tissue that were histologically positive for C. parvum and used in the PCR. Previously developed oligonucleotide primers specific for C. parvum were used to amplify a 452-base target sequence, and a 20-base synthetic probe labeled with digoxigenin-11-dUTP was used to detect the amplification product by chemiluminescence. All six samples were positive by PCR; negative controls showed no amplification or hybridization. This approach could provide a sensitive and specific method for detection of parasite material in fixed, paraffin-embedded tissue samples, and prove to be of significant value in retrospective studies of archival material.
Subject(s)
Cryptosporidiosis/diagnosis , Cryptosporidium parvum/isolation & purification , DNA, Protozoan/analysis , Polymerase Chain Reaction , Animals , Base Sequence , Cryptosporidium parvum/genetics , DNA, Protozoan/chemistry , Evaluation Studies as Topic , Gallbladder/parasitology , Humans , Intestines/parasitology , Liver/parasitology , Molecular Sequence Data , Nucleic Acid Hybridization , Oligonucleotide Probes/chemistry , Paraffin Embedding , Retrospective StudiesABSTRACT
The objective of this project was to construct specific and sensitive molecular probes and amplification primers for Cryptosporidium parvum that could be used in diagnosis, retrospective tissue studies, and in epidemiologic surveys. Whole genomic DNA was extracted from oocysts of C. parvum purified from human and bovine feces. A genomic library was constructed in plasmid pUC18 and propagated in Escherichia coli DH5 alpha. Transformants were screened by colony hybridization and autoradiography. The 2.3-kilobase segment in plasmid pHC1, a clone specific for C. parvum, was sequenced by the Sanger method. Computer analysis gave a G+C content of 35%. A 400-base region (bases 470-870) was selected as an amplification target because it contained a unique restriction endonuclease site that could serve as a useful marker. Primers of 26 nucleotides each were synthesized. Sensitive and specific amplification of the target sequence was demonstrated both by ethidium bromide staining of agarose and acrylamide gels, and by hybridization with chemiluminescence-labeled synthetic oligonucleotide probes.
Subject(s)
Cryptosporidiosis/parasitology , Cryptosporidium parvum/genetics , DNA, Protozoan/isolation & purification , Animals , Base Sequence , Cryptosporidium parvum/isolation & purification , DNA Probes , Molecular Sequence Data , Oligonucleotide Probes , Polymerase Chain ReactionABSTRACT
An ELISA was used to measure the Cryptosporidium-specific IgA, IgG, and IgM antibody levels in serum, stool, and duodenal fluid of 15 Filipino children. Antibody levels were measured on admission to the hospital, 1 week later, and at a 6 week follow-up examination. Delayed type hypersensitivity skin tests were used to assay cell mediated immunity (CMI), iron status was measured by serum iron tests and total iron binding capacity, and the degree of malnutrition was determined by clinical examination. Antibody response to Cryptosporidium was qualitatively and quantitatively strong and maintained over time. All subjects showed impaired CMI early with some reconstitution after 6 weeks. All subjects showed some degree of malnutrition and/or depleted iron status.
Subject(s)
Antibodies, Protozoan/biosynthesis , Coccidia/immunology , Cryptosporidiosis/immunology , Cryptosporidium/immunology , Animals , Cryptosporidiosis/complications , Cryptosporidiosis/epidemiology , Duodenum/immunology , Enzyme-Linked Immunosorbent Assay , Feces/analysis , Female , Follow-Up Studies , Humans , Hypersensitivity, Delayed , Immunoglobulin A/biosynthesis , Immunoglobulin G/biosynthesis , Immunoglobulin M/biosynthesis , Infant , Iron/blood , Longitudinal Studies , Male , Nutrition Disorders/complications , Philippines/epidemiology , PrevalenceABSTRACT
A panel of 4 monoclonal antibodies specific for Eimeria tenella, the causative agent of cecal coccidiosis of birds in the genus Gallus, was produced by standard techniques. The indirect fluorescent antibody (IFA) test demonstrated specificity of these 4 antibodies for the microgametocytes. Hybridoma TIA3B9 secreted a monoclonal antibody of subisotype IgG2b that was used throughout the course of this study. Immunologic potency of this antibody was demonstrated by in vitro experiments that revealed a greater than 50% reduction in oocyst production, indicating an apparent inhibition of fertilization.
