Trypanosoma congolense: appearance and distribution of variable antigen types during metacyclic differentiation in vitro.

Differentiation of epimastigotes and production of infective metacyclic forms of Trypanosoma congolense were examined in a culture system which enabled manipulation of the population density of insect forms. Scanning electron microscopy of cultures revealed the attachment sites of epimastigotes in detail, showing them to be attached as 'clusters' or 'bundles' and having associated fibrillar structures. Dividing epimastigotes were observed either within individual bundles or in association with two bundles. Metacyclic forms were detected by an immunofluorescence antibody test (IFAT) using metacyclic variable-antigen type (M-VAT) specific monoclonal antibodies, by day 7 after seeding cultures. Trypanosomes expressing M-VATs appeared singly in bundles, observed by both IFAT and an immunogold labelling method. Statistical analysis using Poisson calculations suggested that, in general, the distribution of metacyclics expressing individual M-VATs was random throughout cultures.

Ultrastructural studies on the intracellular fate of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) and Chlamydia trachomatis (strain lymphogranuloma venereum 434): modulation of intracellular events and relationship with endocytic mechanism.

Previous observations on the highly infectious LGV strain 434 of Chlamydia trachomatis and the guinea pig inclusion conjunctivitis (GPIC) strain of C. psittaci (which requires centrifugation of inocula with host cell monolayers for maximum infectivity) indicated that infectivity differences were expressed, not at entry, but at an intracellular stage affecting multiplication. Centrifugation increased the potential of internalized chlamydiae to undergo productive infection. Here, analysis of the intracellular fate of chlamydiae by ultrastructural methods indicates that strain GPIC exhibits two patterns of behaviour depending on the mode of inoculation. Strain GPIC showed limited entry, with 47% of intracellular organisms becoming associated with thorotrast-labelled lysosomes, following static incubation with monolayers. In contrast, with centrifugation, entry was not limited and association with lysosomes was reduced to 12%; strain 434 behaved similarly but independently of the mode of inoculation. The different results for strain GPIC correlated with distinct entry mechanisms. Entry during static incubation was unimpaired either by treatment with cytochalasin D or by temperature reduction to 20 degrees C, suggesting that it was pinocytic. Entry during centrifugation was markedly impaired by both treatments, suggesting that it was phagocytic. The data lead to two novel conclusions: first, that chlamydiae can apparently enter cells by both pinocytic and phagocytic mechanisms; second, that the entry mechanism influences intracellular fate. It is suggested that entry mechanism is linked to selection of the vesicle membrane forming around the internalizing chlamydiae. This, in turn, may influence both intracellular translocation and subsequent inhibition or promotion of multiplication of the internalized parasite.

Trypanosoma congolense: interactions between trypanosomes expressing different metacyclic variable antigen types in vitro and in vivo.

In Trypanosome congolense, the surface antigens expressed by cultured metacyclic forms are a limited and serodeme-specific subset of variable antigen types (VATs). Experiments were carried out in mammalian-form (MF) cultures, comprising dividing trypanosomes which express metacyclic VATs (M-VATs) maintained in vitro with a mammalian cell feeder layer, or in mice following infection with cultured metacyclic populations. Selective neutralization experiments were performed by incubating populations of trypanosomes with one or a combination of McAbs to remove their respective M-VATs. Deletion of one M-VAT from the population had no effect on expression and growth of other unrelated M-VATs. Deleted M-VATs were detected in very small proportions of the total population 2-7 days following neutralization. Subsequent incubation of neutralized cultures resulted in growth of deleted M-VATs which, in most cases, reached pre-neutralization levels. It appears, therefore, that in conditions where expression of M-VATs on dividing trypanosomes occurs, removal of individual M-VATs results in their re-expression within the population.