The Journal of protozoology最新文献

An indirect immunofluorescence monoclonal antibody assay was found to have higher sensitivity than usual stains for the detection of Pneumocystis carinii, particularly in bronchoalveolar lavage fluids in which there are only a few parasites, as in HIV-patients or in HIV+ patients with prophylaxis or treatment. For patients without any therapy, when different stains give conflicting results, decisions on therapeutic approaches to be used should consider the patient's clinical and biological status. Prospective studies are necessary to evaluate the predictive value of low parasitism in asymptomatic immunosuppressed patients.

We attempted to cultivate Pneumocystis carinii obtained from two bronchoalveolar lavage fluids of AIDS patients with P. carinii pneumonia, in a system wherein cysteine and 2-mercaptoethanol were substituted for the feeder cells. The presence of P. carinii cysts was monitored for 11 days under conditions of continuous culture. Moderate increase in cyst forms was observed until day 11. Further study with this system would be required to determine if the observed increase in cyst numbers is reproducible and whether the cyst form is a response to adverse in vitro conditions or is a manifestation of growth.

Videomicroscopy in combination with differential-contrast optics was used to study fresh preparations of Pneumocystis carinii from immunosuppressed rats. Certain spherical intracystic bodies appeared to move freely within the cyst wall. Flexing type movement was observed in intracystic ellipsoidal forms attached at a common point in the inner margin of the cyst wall. Greater movement was seen in non-attached thinner elongated forms. Possible extracellular trophic forms and movement were also identified. The movement of the morphological forms of P. carinii has been recorded in real time onto videotape. These initial observations suggest P. carinii is capable of movement and additional studies are under way to substantiate this possibility.

Pregnant cows were immunized to produce hyperimmune bovine colostrum (HBC) by intramuscular injection or intramammary infusion (TI) followed by 3 successive TI boosters with Cryptosporidium parvum (Cp) oocyst antigen mixed with Freund's (F) or Ribi (R) adjuvant. Control cows received no Cp. Colostrum from all cows was skimmed of butterfat and tested for specific anti-Cp immunoglobulin isotypes by ELISA. The HBC from Cp-F and Cp-R immunized cows had IgG1 titers exceeding 1:400,000 and 1:800,000, respectively. Some HBC from Cp-F immunized cows was freeze-dried to facilitate storage and some were irradiated at 42.5 kGy to kill potentially contaminating pathogens. Freeze-drying, but not irradiation, reduced IgG1 titers by only one dilution. Neither treatment affected Western blot banding patterns.

Microtubules and microfilaments are highly conserved cytoskeletal polymers hypothesized to play essential biomechanical roles in the unusual gliding motility of Apicomplexan zoites and in their invasion of, and development within, host epithelial cells. We have identified and isolated Cryptosporidium parvum genes encoding the microtubule proteins alpha- and beta-tubulin and the microfilament protein actin by screening a lambda gt11 C. parvum genomic DNA library with degenerate oligonucleotide and heterologous cDNA hybridization probes respectively. The alpha- and beta-tubulin genes have been partially sequenced and the deduced peptide sequences show greatest homology with the tubulins of the related parasites, T. gondii and P. falciparum. The complete nucleic acid sequence of the actin gene predicts a 376 amino acid, 42 kDa protein having 85% sequence identity with the P. falciparum actin I and the human gamma-actin proteins. Each of these cytoskeletal protein genes was demonstrated to be of cryptosporidial origin by Southern analyses of C. parvum chromosomes fractionated by pulsed field gel electrophoresis; the cloned alpha- and beta-tubulin genes hybridized with chromosomes of ca. 1,200 and 1,500 kb respectively and the cloned actin gene also hybridized with a 1,200 kb chromosome.

To examine the potential of antimicrotubule drugs for treating Pneumocystis carinii infections, and to learn more about this unusual organism on a molecular level, we are studying its tubulin genes. A 0.3 kbp fragment of the P. carinii beta-tubulin gene was amplified by the polymerase chain reaction. Sequence analysis of this DNA revealed that P. carinii beta-tubulin is most closely related to those of the fungal molds. Consistent with these results, P. carinii growth in vitro was sensitive to the antifungal benzimidazoles benomyl and carbendazim.

We undertook expression cloning of Pneumocystis carinii antigens to overcome the difficulties encountered in purification of these antigens. Using monoclonal antibodies to the P. carinii gp120 antigen and polyclonal rabbit antiserum to rat-derived P. carinii, we have isolated cDNA clones encoding immunoreactive moieties. A cDNA clone encoding the 3' portion of a 45-55 kDa antigen of rat-derived P. carinii, was the most abundant clone isolated. The peptide encoded by this cDNA has a novel sequence with a repeated motif rich in glutamic acid residues. Affinity-purified antibodies to this peptide reacted with the 45-55 kDa band of rat-derived P. carinii. The fusion protein was recognized by serum antibodies from rats with natural exposure to P. carinii. The production of this recombinant protein should allow more detailed studies of the host-parasite relationship of this important opportunistic infection.

Oocysts shedding was markedly reduced in guinea pigs inoculated intraintestinally with Cryptosporidium wrairi sporozoites that had been incubated with hyperimmune bovine colostrum raised to C. parvum when compared with shedding in guinea pigs inoculated with sporozoites incubated in either non-immune bovine colostrum or buffered saline. However oocyst shedding was apparently not reduced in guinea pigs inoculated by gavage with oocysts of C. wrairi and subsequently treated twice daily per os with hyperimmune bovine colostrum. Similarly, oocyst shedding was apparently not reduced by oral treatment with hyperimmune bovine colostrum when treatment was begun simultaneously with inoculation of C. wrairi oocysts.

Treatment of rats immunosuppressed with dexamethasone and inoculated with Cryptosporidium parvum oocysts were treated with dehydroepiandrosterone. A significant reduction in cryptosporidial activity was observed as determined by oocyst shedding and colonization of host tissue by parasites. Dexamethasone treatment alone resulted in decreases in T-, B- and natural killer (NK) cell responses and antibody production that, with the exception of NK-cell activity, were all reversed after administration of dehydroepiandrosterone.

The polymerase chain reaction (PCR) using published primers and probes has been compared to conventional stains and immunofluorescence for diagnosis of Pneumocystis carinii. We have screened 71 bronchoalveolar lavage (BAL) fluids from HIV-immunosuppressed patients. Of 34 samples negative by conventional stains and immunofluorescence, only one was positive by PCR. Thirty of 35 samples positive by conventional stains and immunofluorescence were also positive by PCR. One BAL sample, negative by conventional stains but positive by immunofluorescence, was negative by PCR. These data are discussed in relation to clinical and therapeutic conditions of the patients.