Brachiola algerae (Vavra et Undeen, 1970) Lowman, Takvorian et Cali, 2000, originally isolated from a mosquito, has been maintained in rabbit kidney cells at 29°C in our laboratory. This culture system has made it possible to study detailed aspects of its development, including spore activation, polar tube extrusion, and the transfer of the infective sporoplasm. Employing techniques to ultrastructurally process and observe parasite activity in situ without disturbance of the cultures has provided details of the early developmental activities of B. algerae during timed intervals ranging from 5 min to 48 h. Activated and non-activated spores could be differentiated by morphological changes including the position and arrangement of the polar filament and its internal structure. The majority of spores extruded polar tubes and associated sporoplasms within 5 min post inoculation (p.i.). The multilayered interlaced network (MIN) was present in extracellular sporoplasms and appeared morphologically similar to those observed in germination buffer. Sporoplasms, observed inside host cells were ovoid, contained diplokaryotic nuclei, vesicles reminiscent of the MIN remnants, and their plasmalemma was already electron-dense with the "blister-like" structures, typical of B. algerae. By 15 min p.i., the first indication of parasite cell commitment to division was the presence of chromatin condensation within the diplokaryotic nuclei, cytoplasmic vesicular remnants of the MIN were still present in some parasites, and early signs of appendage formation were present. At 30 min p.i., cell division was observed, appendages became more apparent, and some MIN remnants were still present. By two hours p.i., the appendages became more elaborate and branching, and often connected parasite cells to each other. In addition to multiplication of the organisms, changes in parasite morphology from small oval cells to larger elongated "more typical" parasite cells were observed from 5 h through 36 h p.i. Multiplication of proliferative organisms continued and sporogony was well underway by 48 h p.i., producing sporonts and sporoblasts, but not spores. The observation of early or new infections in cell cultures 12-48 h p.i., suggests that there may also exist a population of spores that do not immediately discharge, but remain viable for some period of time. In addition, phagocytized spores were observed with extruded polar tubes in both the host cytoplasm and the extracellular space, suggesting another means of sporoplasm survival. and Finally, extracellular discharged sporoplasms tightly abutted to the host plasmalemma, appeared to be in the process of being incorporated into the host cytoplasm by phagocytosis and/or endocytosis. These observations support the possibility of additional methods of microsporidian entry into host cells and will be discussed.
We compared graft outcome between two types of a novel
composite three-layer carp-collagen-coated vascular graft in
low-flow conditions in a sheep model. Collagen in group A
underwent more cycles of purification than in group B in order to
increase the ratio between collagen and residual fat. The grafts
were implanted end-to-side in both carotid arteries in sheep
(14 grafts in 7 sheep in group A, 18 grafts in 9 sheep in group B)
and artificially stenosed on the right side. The flow in the grafts
in group A decreased from 297±118 ml/min to 158±159 ml/min
(p=0.041) after placement of the artificial stenosis in group A,
and from 330±164ml/min to 97±29 ml/min (p=0.0052) in
group B (p=0.27 between the groups). From the five surviving
animals in group A, both grafts occluded in one animal 3 and 14
days after implantation. In group B, from the six surviving
animals, only one graft on the left side remained patent
(p=0.0017). Histology showed degradation of the intimal layer in
the center with endothelization from the periphery in group A
and formation of thick fibrous intimal layer in group B. We
conclude that the ratio between collagen and lipid content in the
novel three-layer graft plays a critical role in its patency and
structural changes in vivo.