Microsporidia and Cyclospora: epidemiology and assessment of risk from the environment.

Two classes of parasites with an environmental stage in their lifestyle have recently emerged as significant gastrointestinal pathogens for humans. Microsporidia represent a group that contains a number of genera related to the genus Cryptosporidium. They are generally transmitted via direct human to human contact, but can survive in water and food, and recently have been found in surface water used as drinking source water. Their most common host range is in patients with clinical AIDS. Limited work to date suggests the group is susceptible to chlorine achievable CxT (concentration x time) values and is coagulated by filtration. Cyclospora cayetanensis is a species of parasite that has caused outbreaks from contaminated food. Its major risk is from the use of inadequately treated water used for irrigation. Cyclospora can infect normal and immunosuppressed hosts. Current information regarding the lifestyle, transmission, and control of both groups of parasites are discussed, with a health risk assessment analysis.

Immunohistochemical detection of Listeria antigens in the placenta in perinatal listeriosis.

Listeria monocytogenes, a worldwide pathogen, causes significant perinatal mortality and morbidity and has been implicated in spontaneous abortions, still-births, premature delivery, and neonatal sepsis, often with meningitis. Maternal symptoms are frequently minimal, and diagnosis is made only if the suspicion is high and diagnostic maternal blood or amniotic fluid cultures are performed. Because cultures are not routinely performed on spontaneously aborted fetuses, many authors feel that the true incidence of the disease may be underestimated. To date, the absence of a test to retrospectively diagnose Listeria infection has contributed to the lack of accurate estimates of the incidence of the disease. Seven cases in which immunohistochemical stains were used to confirm the diagnosis of placental listeriosis are described. All placentas showed the characteristic lesions with severe chorioamnionitis, numerous microabscesses, and focal necrotizing villitis. Immunohistochemical localization of Listeria antigen was made to the amnion (focally in areas with no inflammatory infiltrate), the abscesses, and the areas with villitis. In general, the antigen was extracellular and intracellular, predominantly within macrophages or the amnion epithelium. Listeria antigen was often found where definite identification of the organism was not possible on Brown-Hopps or Warthin-Starry stains. The immunohistochemical technique may therefore show an increase in sensitivity of detection of L monocytogenes compared with routine bacterial stains. Moreover, the ability to retrospectively evaluate placental specimens for evidence of this organism should permit the true incidence of perinatal listeriosis to be determined.

The topology of the kinetoplast DNA network.

Kinetoplast DNA (kDNA) of trypanosomatid parasites is a network of approximately 5000 catenated DNA minicircles and approximately 25 maxicircles. We developed the following strategy to deduce the topological linkage of the minicircles of the Crithidia fasciculata network. First, we used graph theory to provide precise models of possible network structures. Second, on the basis of these models, we predicted the frequencies of minicircle oligomers expected from random network breakage. Third, we determined the fragmentation pattern of kDNA networks as a function of the extent of digestion. Fourth, by comparison of the results with the predictions, we identified the model that best represents the network. We conclude that each minicircle is linked on average to three other minicircles. A honeycomb arrangement probably results, with each minicircle typically at the vertex of a hexagonal cell. This topology has implications for the assembly, structure, and function of kDNA networks.

The absence of supercoiling in kinetoplast DNA minicircles.

Crithidia fasciculata kinetoplast DNA is a mitochondrial DNA composed of 5000 minicircles and approximately 25 maxicircles, all catenated into a giant network. By comparing the linking number of minicircles released from the network by limited sonication with that of control minicircles, we demonstrate that not only does the elaborate catenation of the network not cause supercoiling, but that there is no minicircle supercoiling at all. The absence of catenation-induced supercoiling is explained by our finding [using electron microscopy (EM) and gel electrophoresis] that network minicircles are joined by only one interlock; single interlocking can be accommodated without helix distortion. EM revealed that propidium diiodide supertwists all the network minicircles and thereby condenses the network into a much smaller size while maintaining its planarity. At high dye concentration the network is condensed to a size comparable to that found in vivo. Nevertheless, network minicircles bind less propidium than free minicircles, indicating that catenation into a network restricts the supercoiling of individual rings. These studies show that the mitochondrion of trypanosomatids may be a unique niche in nature where a covalently-closed circular DNA is not supercoiled. This absence of supercoiling may be a major factor in promoting the formation of the network.

A knotted free minicircle in kinetoplast DNA.

Kinetoplast DNA, the mitochondrial DNA of trypanosomes, is a network containing thousands of minicircles that are topologically interlocked. The minicircle replication intermediates are free molecules that have been released from the network. We report here that one form of free minicircles is a trefoil knot. Identification of this knotted structure is based on its electrophoretic and sedimentation properties, its response to treatments with restriction enzymes or topoisomerase II, and its appearance by electron microscopy. Except for its topology, the knotted minicircle closely resembles a previously described replication intermediate with a unique gap in the newly synthesized L strand.

A highly bent fragment of Crithidia fasciculata kinetoplast DNA.

Kinetoplast DNA minicircles from Crithidia fasciculata contain a single major region of bent helix. Restriction fragments containing this bent helix have electrophoretic behavior on polyacrylamide gels which is much more anomalous than that of previously studied bent fragments. Therefore, the C. fasciculata fragments probably have a more extreme curvature. Sequencing part of a cloned minicircle revealed an unusual structure for the bent region. In a sequence of 200 bases, the bent region contains 18 runs of 4-6 As with 16 of these runs in the same strand. In some parts of this sequence the A runs are regularly spaced with a periodicity of about 10 base pairs. This spacing is nearly in phase with the twist of the DNA helix. This same sequence arrangement has been observed in other bent fragments, but the number of A runs is much greater in this C. fasciculata sequence. It is likely that there are small bends associated with each A run which, because of their periodic spacing, add up to produce substantial curvature in this molecule. In addition to having highly anomalous electrophoretic behavior, the fragment has unusual circular dichroism spectra. Its spectrum in the absence of ethanol is that of B DNA, but ethanol in the concentration range of 51-71% (w/w) induces changes to forms which are different from those of any well characterized DNA structure. The C. fasciculata bent helix is neither cleaved by S1 nuclease nor modified by bromoacetaldehyde under conditions in which other unusual DNA structures (such as cruciforms or B-Z junctions) are susceptible to attack by these reagents. Finally, a two-dimensional agarose gel analysis of a family of topoisomers of a plasmid containing the bent helix revealed no supercoil-induced relaxation.

Visualization of the bent helix in kinetoplast DNA by electron microscopy.

Kinetoplast DNA minicircles from the trypanosomatid Crithidia fasciculata contain a segment of approximately 200 bp which is probably more highly bent than any other DNA previously studied. Electron microscopy (EM) of relaxed minicircles (2.5 kb) revealed 200-300 bp loops within the larger circles, and the loops could also be detected on full-length linear molecules. Examination by EM of a 219 bp cloned fragment which contains the bent helix revealed that up to 70% of the molecules appeared circular whether or not the ends were cohesive. In contrast, a 207 bp fragment from pBR322 showed no circles and the fragments in general appeared much straighter than the kinetoplast fragments. Treatment of the 219 bp bent kinetoplast fragment with the drug distamycin caused a striking reduction in curvature.