The Journal of protozoology最新文献

Pulmonary lesions seen in autopsies of leprosy patients were initially thought to involve microsporidial infection. After immunohistochemical studies, it was concluded that the infectious microorganism was Cryptococcus neoformans.

Pneumocystis carinii is an important cause of pneumonia in immunocompromised human patients. The organism is also found as a saprophyte in the lungs of many species of animals. Animal models have been used as a source of P. carinii organisms for study of the disease. The rat model has been especially useful. Initially, the infection was latent in most colonies, and P. carinii pneumonia readily developed when animals were immunosuppressed. Today, many barrier raised rodent colonies are free of adventitious viruses, bacteria, Mycoplasma sp., and parasites, including P. carinii. Variability is now seen in the rat model. The use of cultured organisms to experimentally infect rats and mice prior to immunosuppression has met the need for some investigators, however, latent-infected, barrier-raised and isolator-raised rodents are still required. Colonies specifically infected with P. carinii can provide latent-infected animals and are better protected from potentially interfering organisms than barrier-raised animals. The development of these colonies is feasible as investigators and animal producers work together to define and develop this resource.

A mouse model for Pneumocystis carinii has now been established in several strains of mice: C3Heb/FeJ, C3HeN, Balb/c, DBA/2N and athymic. In lieu of using invasive methods for initiating P. carinii infections, mice infected with P. carinii (seed mice) transmitted the disease to mice without latent infection via short term co-habitation. Acute infections in recipient mice developed approximately 5-6 wk after C3Heb/FeJ seeds were removed, while control unseeded litter-mates remained uninfected. This approach allows investigators to consistently transmit P. carinii to mice and to select the strain of mouse desired for use in a particular study.

Oligonucleotide primers were prepared from a clone (B12) which has been shown to be a repetitive sequence in the rat P. carinii genome. Polymerase chain reaction was employed to amplify both rat and human P. carinii DNA. The detection limit of the assay was approximately 600 ng of total nucleic acid. Amplification products from both the rat and human isolates (ca. 780 bp) were characterized by denaturing gradient gel electrophoresis after digestion with Sau3A. No amplification products were obtained when DNA from the following potential pulmonary pathogens were used in identical reactions: Aspergillus fumigatus, Cryptococcus neoformans, Candida albicans, Mycobacterium avium-intracellulare and cytomegalovirus. In a blind study using the B12 primers, P. carinii DNA was successfully amplified in clinical samples which were positive by direct immunofluorescence assay (IFA) as well as in some specimens not identified by direct IFA.

The numerous infections of microsporidia which have been diagnosed in patients with AIDS have revealed the potential of these organisms for establishing themselves when the immune status of the host is compromised. Two species of Encephalitozoon, E. cuniculi and E. hellem, have been diagnosed in man, the former infecting a variety of tissues, the latter restricted to the corneal and conjunctival epithelia. These species are morphologically indistinguishable even at the ultrastructural level but can be separated biochemically. Two human sera were found to react with equal intensity in the ELISA on spores of E. cuniculi and E. hellem purified from in vitro cultures, and gave similar binding patterns in Western blots on SDS-PAGE protein profiles of the two species. This has raised questions about the identity of Encephalitozoon infections diagnosed previously in man. The diagnosis of Enterocytozoon bieneusi, which infects the intestinal enterocytes of AIDS patients and is associated with chronic diarrhoea, requires observation of smears or sections of biopsies or specialist observation of stool preparations. In vitro cultures, which would facilitate the raising of specific antisera, have proved difficult to establish. In vitro and in vivo systems for assaying drugs for microsporidia have revealed that albendazole has a marked effect on parasite numbers and morphology but does not eliminate infection, which resurges when drug pressure is removed.

Pentamidine isethionate inhibits growth of Saccharomyces cerevisiae. Inhibition requires lower drug concentrations under respiratory than fermentative growth conditions. Pentamidine-resistant strains bear heritable resistance mutations. Tested mutations are dominant to wild type sensitivity. These mutations may identify cellular targets of pentamidine and potential mechanisms of fungal pentamidine resistance.

Ultrastructural studies were done on developmental stages of Enterocytozoon bieneusi obtained from HIV seropositive patients suffering from diarrhea. The presence of elaborate multilamellar structures suggest that they give rise to various membrane systems needed for rapid production of disseminating stages.

Tomato lectin, which binds oligosaccharides of N-acetyl-D-glucosamine, and an antiserum against macromolecular chitin were used to probe sections of human and murine lungs infected with Pneumocystis carinii. By light, fluorescence and electron microscopy, lectin and antiserum binding patterns indicated that both human and murine strains of P. carinii express chitin at all identifiable stages of their life cycles. Light microscopic autoradiographs of murine P. carinii cultured in vitro with 3H-glucosamine revealed dense incorporation of the radiolabel into the cell walls in a pattern analogous to those of the antiserum and lectin binding studies. These investigations offer further evidence that chitin is an integral part of the cell wall of P. carinii trophozoites and cysts.

An in vitro model of Cryptosporidium parvum infection was developed utilizing an adherent human intestinal epithelial cell line HT29.74. The efficacy of potential immunologic therapy in the form of Cryptosporidium-specific hyperimmune bovine colostrum was evaluated for the ability to inhibit in vitro infection. Oocysts were purified from stool of chronically infected AIDS patients. Hyperimmune colostrum obtained from cows immunized with Cryptosporidium and nonimmune conventional colostrum were evaluated. oocysts (10(5)-10(6)) were pre-incubated with either hyperimmune colostrum, conventional colostrum, or saline as control, for 15 min at room temperature than applied to a 70% confluent monolayer of HT29.74 cells. Cryptosporidium schizonts were identified and counted per 1,000 HT29.74 cells under oil immersion after 24 h. In the presence of hyperimmune colostrum, parasite infection was inhibited by 82% (p less than 0.001), and the presence of conventional colostrum, infection was inhibited by 67% (p less than 0.001). Treatment with the soluble fraction of hyperimmune colostrum resulted in 69% inhibition (p less than 0.001) compared to the soluble fraction of conventional colostrum which resulted in only 17% inhibition (p = NS). In vitro Cryptosporidium parvum infection of the differentiated human enterocyte cell line HT29.74 is a viable method for screening immunologic therapies. Hyperimmune bovine colostrum was highly inhibitory of Cryptosporidium infection in vitro and its soluble fraction remained significantly inhibitory while the soluble fraction of conventional colostrum did not.

Amplification of DNA by the polymerase chain reaction (PCR) offers a highly sensitive and specific method for detecting DNA sequences in biological samples. We applied this technology to develop an assay for the P. carinii dihydrofolate reductase (DHFR) gene. This assay was found to be sensitive enough to detect as little as 1 organism-'equivalent' of DHFR DNA. In rats with experimentally-induced P. carinii pneumonia, DHFR DNA amplification demonstrated the presence of pulmonary P. carinii 2 wk prior to the onset of histopathological changes. When rat serum was analyzed by PCR, serum P. carinii DNA was found in 5 of 14 experimental rats. Finally, P. carinii DNA was detected in the serum of 7 of 18 patients (39%) with AIDS and active P. carinii pneumonia. These results suggest that circulating serum P. carinii DNA can be detected frequently in the course of pulmonary infection and may represent a blood-borne phase of infection. The PCR detection of P. carinii DNA provides a useful tool to study the natural history of P. carinii infection and may offer a non-invasive diagnostic procedure in some patients with P. carinii pneumonia.