Extracellular like-gregarine stages of Cryptosporidium parvum.

The present study confirms the existence of extracellular stages of Cryptosporidiumparvum during in vitro culture on MDCK, HCT 8 and Vero cells as well as alveolar macrophages, by optic, Nomarski and transmission electron microscopy images. Extracellular trophozoite/gamont, stages in syzygy, zygotes and spores with eight sporozoites were seen in the supernatant of the cultures. The first ultrastructural images of extracellular stages of C. parvum are shown in this study. The morphology of these stages, which have characteristics similar to those of some gregarines, support the contention that Cryptosporidium has closer affinity with gregarines. It also supports the necessity of reconsidering the life cycle of Cryptosporidium and the classification within the coccidia.

Sucker-like structures in two strains of Acanthamoeba: scanning electron microscopy study.

Amebostomes are labile sucker-like structures repeatedly observed and described in Naegleria by different authors. Studying the phagocytic action of some Acanthamoeba species on Vero cells, the formation of similar structures to the Naegleria amebostomes was observed, apparently related to the phagocytic activity on cells.

Influence of hydrogen peroxide on acid-fast staining of Cryptosporidium parvum oocysts.

Infections by the protozoan parasite Cryptosporidium parvum are routinely diagnosed by modified Ziehl-Neelsen (acid-fast) staining of faecal preparations despite the counterstaining and ghost-like appearance of some oocysts. Quantitative studies demonstrated that only a small percentage of oocysts excreted by naturally infected newborn calves displayed acid-fast characteristics, but that percentage increased when the time between excretion and sample staining was increased. The treatment of faecal samples with hydrogen peroxide (10 min, 5 vol. final concentration) caused all oocysts to become acid-fast, with up to 40-fold increases in test sensitivity in samples treated and stained within 3 h of excretion. Flow-cytometry analysis of hydrogen peroxide-treated oocysts also demonstrated increased labelling of oocysts by a commercial monoclonal antibody preparation commonly used for diagnosis.

Glycolytic enzyme activities in Cryptosporidium parvum oocysts.

Oocysts of Cryptosporidium parvum were obtained from an experimentally infected newborn goat. After purification, the oocysts were homogenised and the activities of the glycolytic enzymes measured in the different subcellular fractions. All of the activities of the Embden-Meyerhoff pathway were located in the non-sedimentable, cytoplasmic fraction. Under the conditions used, hexokinase activity was below the limits of detection. The pathway is also characterised by the presence of a pyrophosphate-dependent phosphofructokinase and a carbon dioxide-fixing cycle comprising phosphoenolpyruvate carboxylase, malate dehydrogenase and malate dehydrogenase (decarboxylating) activities. The data presented in this paper suggest that the infective stage of this parasite probably relies on substrate-level phosphorylation for energy generation.

Ultrastructural study of Cryptosporidium development in Madin-Darby canine kidney cells.

Transmission electron microscope studies have been made into phases of the life cycle of a bovine isolate of Cryptosporidium cultured in vitro on Madin-Darby canine kidney cells. The cytoplasm of parasitized cells was noticeably altered, including marked vacuolization and the appearance of membrane structures close to the developing parasites. These changes suggest that the protozoan may release cytopathogenic factors.

The effects of IFN-gamma activated mouse peritoneal and alveolar macrophages on Cryptosporidium parvum development.

Mouse peritoneal and alveolar macrophages were interacted in vitro with C. parvum oocysts and cultured in normal medium and in medium with IFN-gamma. The results showed that in vitro activation of macrophages by IFN-gamma limits C. parvum development although the inhibitory effect is not as potent as in other intracellular parasitic protozoa.

In vitro culture of Glugea sp.

Recently the low host specificity of some microsporidians has been demonstrated and it has been indicated that many of these micro-organisms could be transmitted from invertebrates to mammals and adapt to changes in temperature. In this work, we demonstrate the first successful in vitro culture of a fish microsporidia of the genus Glugea on larval cells of the mosquito Aedes albopictus at 28 degrees C, and we show ultrastructural aspects of the different life cycle stages. It was impossible on salmon cells CHSE-214 at 21 degrees C. This study will be valuable for further work in biochemistry and immunology in addition to chemotherapy for microsporidiosis humans and animals.

In vitro multiplication of Cryptosporidium parvum in mouse peritoneal macrophages.

Cryptosporidium parvum of bovine origin was developed in vitro in unsensitized mouse peritoneal macrophages. Macrophages growing in RPMI medium were infected with sporozoites or with oocysts, and after staining infections were studied by light microscopy. A high parasitic index was obtained with multiple infections occurring commonly. This is a simple method for the study of Cryptosporidium biology, and for in vitro assays of pharmacological activity.

Respiratory cryptosporidiosis in a bovine.

Intestinal cryptosporidiosis has been extensively studied in young calves, but respiratory invasion by Cryptosporidium in these animals has, surprisingly, not been investigated. In the present study the parasite was observed in lung tissue of a calf, using light and electron microscopy. This demonstrates that Cryptosporidium can develop in the bronchial epithelium in bovines.

Growth of an Acanthamoeba isolate on a gram-negative bacterium, probably Pasteurella haemolytica.

A gram-negative bacterium, probably Pasteurella haemolytica, was found to support the growth of Acanthamoeba spp. This provides a useful means for initial isolation of Acanthamoeba spp. and for culturing these parasites to high cell densities.

Anti-oxidant enzymes in Cryptosporidium parvum oocysts.

Oocysts of Cryptosporidium parvum showed relatively low levels of SOD activity. The SOD which had a pI of 4.8 and an approximate molecular weight of 35 kDa appeared to be iron dependent. Catalase, glutathione transferase, glutathione reductase and glutathione peroxidase activity could not be detected, nor could trypanothione reductase. No NADH or NADPH oxidase activity could be detected, nor could peroxidase activity be demonstrated using o-dianisidine, guaiacol, NADPH or NADH as co-substrates. However, an NADPH-dependent H2O2 scavenging system was detected in the insoluble fraction.

Ultrastructural details of Cryptosporidium parvum development in calf intestine.

Cryptosporidium parvum and C. muris appear to be different species found in calves, with different oocysts size and distribution on the gastrointestinal tract. This work presents new images of C. parvum ultrastructure in calf intestine, mainly its development in nonmicrovillous cells and the presence of microtubular structures in the membrane enveloping the macrogamonts and immature oocysts.

Antigen incorporation on Cryptosporidium parvum oocyst walls.

Cryptosporidium parvum oocysts are the infective stages responsible for transmission and survival of the organism in the environment. In the present work we show that the oocyst wall, far from being a static structure, is able to incorporate antigens by a mechanism involving vesicle fusion with the wall, and the incorporation of the antigen to the outer oocyst wall. Using immunoelectron microscopy we show that the antigen recognized by a monoclonal antibody used for diagnosis of cryptosporidiosis (Merifluor(R), Meridian Diagnostic Inc.) could be found associated with vesicles in the space between the sporozoites and the oocysts wall, and incorporated to the outer oocyst wall by an unknown mechanism.

The different behavior of diphtheria toxin, modeccin and ricin in HeLa cells infected with Trypanosoma cruzi.

We have studied the action of diphtheria toxin, modeccin and ricin on HeLa cells infected by Trypanosoma cruzi. Parasitized HeLa cells were resistant to diphtheria toxin and modeccin, whereas non-parasitized cells from the same cultures and control cultures showed cytopathological alterations. Protein synthesis, assayed by the incorporation of labelled methionine, diminished in toxin-treated control cultures but remained unaltered in the infected ones, compared to synthesis by untreated infected cells. Ricin, on the other hand, is a toxin that enters the cytoplasm by endocytosis. It has greater cytopathological effects in parasitized cells than in non-parasitized ones from the same cultures or uninfected control cells. Protein synthesis was inhibited in infected cultures treated with ricin.