Bank voles (Myodes glareolus) and house mice (Mus musculus musculus; M. m. domesticus) in Europe are each parasitized by their own distinct species of Aspiculuris (Nematoda, Oxyurida).

The molecular phylogeny and morphology of the oxyuroid nematode genus Aspiculuris from voles and house mice has been examined. Worms collected from Myodes glareolus in Poland, Eire and the UK are identified as Aspiculuris tianjinensis, previously known only from China, while worms from Mus musculus from a range of locations in Europe and from laboratory mice, all conformed to the description of Aspiculuris tetraptera. Worms from voles and house mice are not closely related and are not derived from each other, with A. tianjinensis being most closely related to Aspiculuris dinniki from snow voles and to an isolate from Microtus longicaudus in the Nearctic. Both A. tianjinensis and A. tetraptera appear to represent recent radiations within their host groups; in voles, this radiation cannot be more than 2 million years old, while in commensal house mice it is likely to be less than 10,000 years old. The potential of Aspiculuris spp. as markers of host evolution is highlighted.

Effectiveness of water treatment for the removal of Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia are intestinal parasites of humans and of many other species of animals. Water constitutes an important route of transmission for human infections in both developed and developing countries. In Poland, contamination of water sources with oocysts/cysts is not routinely monitored and scientific research in this field is scarce. Our aim was to compare the contamination of surface and treated water and thus the success of water treatment processes. Water samples (n=94) of between 30 l (surface water) to over 1000 l for tap water, were taken in the period of 2008-2009 using specially constructed equipment with cartridge filtration (Filta-Max; IDEXX, USA). Immunofluorescent assay, and nested polymerase chain reaction were used for the detection of parasites. Cryptosporidium oocysts were found in 85% of surface water and in 59% of raw (intake) water samples. Oocysts were also detected in treated water (16%) but were absent in samples of swimming pool water. The highest mean number of Cryptosporidium oocysts [geometric mean (GM)=61/10 l] was found in samples of rinsing water. Giardia cysts were observed in 61% of surface water samples, in 6% of raw water and in 19% of treated water, with the highest number of cysts noted in rinsing water samples (GM=70 cysts/10 l). Our study highlights the frequent occurrence of parasites in surface waters in Poland and the effectiveness of water treatment for the removal of parasites from drinking water.

Differences in the ecology of Bartonella infections of Apodemus flavicollis and Myodes glareolus in a boreal forest.

The epidemiology of Bartonella species infecting Apodemus flavicollis and Myodes glareolus in a forest in Eastern Poland was followed for 2 years using mark-recapture. Infections could be acquired in any month, but prevalence, and probability of infection, peaked in the summer. There were significant differences in the pattern of infections between the two species. Both hosts were primarily infected as juveniles, but the probability of infection was highest for A. flavicollis, which, evidence suggests, experienced longer-lasting infections with a wider range of Bartonella genotypes. There was no evidence of increased host mortality associated with Bartonella, although the infection did affect the probability of recapture. Animals could become re-infected, generally by different Bartonella genotypes. Several longer lasting, poorly resolved infections of A. flavicollis involved more than 1 genotype, and may have resulted from sequential infections. Of 22 Bartonella gltA genotypes collected, only 2 (both B. grahamii) were shared between mice and voles; all others were specific either to A. flavicollis or to M. glareolus, and had their nearest relatives infecting Microtus species in neighbouring fields. This heterogeneity in the patterns of Bartonella infections in wild rodents emphasizes the need to consider variation between both, host species and Bartonella genotypes in ecological and epidemiological studies.

Functional autonomy of monopolar spindle and evidence for oscillatory movement in mitosis.

The oscillations of chromosomes associated with a single spindle pole in monocentric and bipolar spindles were analysed by time-lapse cinematography in mitosis of primary cultures of lung epithelium from the newt Taricha granulosa. Chromosomes oscillate toward and away from the pole in all stages of mitosis including anaphase. The duration, velocity, and amplitude of such oscillations are the same in all stages of mitosis. The movement away from the pole in monocentric spindle is rapid enough to suggest the existence of a previously unrecognized active component in chromosome movement, presumably resulting from a pushing action of the kinetochore fiber. During prometaphase oscillations, chromosomes may approach the pole even more closely than at the end of anaphase. Together, these observations demonstrate that a monopolar spindle is sufficient to generate the forces for chromosome transport, both toward and away from the pole. The coordination of the aster/centrosome migration in prophase with the development of the kinetochore fibers determines the course of mitosis. After the breaking of the nuclear envelope in normal mitosis, aster/centrosome separation is normally followed by the rapid formation of bipolar chromosomal fibers. There are two aberrant extremes that may result from a failure in coordination between these processes: (a) A monocentric spindle will arise when aster separation does not occur, and (b) an anaphaselike prometaphase will result if the aster/centrosomal complexes are already well-separated and bipolar chromosomal fibers do not form. In the latter case, the two monopolar prometaphase half-spindles migrate apart, each containing a random number of two chromatid (metaphase) monopolar-oriented chromosomes. This random segregation of prometaphase chromosome displays many features of a standard anaphase and may be followed by a false cleavage. The process of polar separation during prometaphase occurs without any visible interzonal structures. Aster/centrosomes and monopolar spindles migrate autonomously by an unknown mechanism. There are, however, firm but transitory connections between the aster center and the kinetochores as demonstrated by the occasional synchrony of centrosome-kinetochore movement. The data suggest that aster motility is important in the progress of both prometaphase and anaphase in normal mitosis.

Heat-induced reversible hexagonal packing of spindle microtubules.

Epithelial cells cultured from the lung of the Northwest rough-skinned newt (Taricha granulosa granulosa) were subjected to brief (10-15 min) elevated temperature shocks of 33 degrees-36 degrees C during metaphase. Electron microscope studies on these cells reveal that the spindle microtubules (Mts) are differentially stable to heat treatment. The great majority of nonkinetochore Mts are destroyed within the first few minutes of the shock while kinetochore and adjacent Mts rearrange to form hexagonal closely packed structures before disassembling, the latter occurring only after prolonged heat treatment. The significance and theoretical implications of the formation of hexagonal closely packed Mt structures and of the differential stability of spindle Mts to heating are discussed. The data suggest the existence of one or more heat-sensitive structural component(s) which maintain the individual minimum spacing seen between spindle Mts. To our knowledge, this is the first reported instance of the experimental rearrangement of kinetochore Mts into reversible, hexagonal closely packed bundles.

Reorganization of microtubules in endosperm cells and cell fragments of the higher plant Haemanthus in vivo.

The reorganization of the microtubular meshwork was studied in intact Haemanthus endosperm cells and cell fragments (cytoplasts). This higher plant tissue is devoid of a known microtubule organizating organelle. Observations on living cells were correlated with microtubule arrangements visualized with the immunogold method. In small fragments, reorganization did not proceed. In medium and large sized fragments, microtubular converging centers formed first. Then these converging centers reorganized into either closed bushy microtubular spiral or chromosome-free cytoplasmic spindles/phragmoplasts. Therefore, the final shape of organized microtubular structures, including spindle shaped, was determined by the initial size of the cell fragments and could be achieved without chromosomes or centrioles. Converging centers elongate due to the formation of additional structures resembling microtubular fir trees. These structures were observed at the pole of the microtubular converging center in anucleate fragments, accessory phragmoplasts in nucleated cells, and in the polar region of the mitotic spindle during anaphase. Therefore, during anaphase pronounced assembly of new microtubules occurs at the polar region of acentriolar spindles. Moreover, statistical analysis demonstrated that during the first two-thirds of anaphase, when chromosomes move with an approximately constant speed, kinetochore fibers shorten, while the length of the kinetochore fiber complex remains constant due to the simultaneous elongation of their integral parts (microtubular fir trees). The half-spindle shortens only during the last one-third of anaphase. These data contradict the presently prevailing view that chromosome-to-pole movements in acentriolar spindles of higher plants are concurrent with the shortening of the half-spindle, the self-reorganizing property of higher plant microtubules (tubulin) in vivo. It may be specific for cells without centrosomes and may be superimposed also on other microtubule-related processes.

Action of taxol on mitosis: modification of microtubule arrangements and function of the mitotic spindle in Haemanthus endosperm.

We have studied the effect of taxol on mitosis in Haemanthus endosperm. Immuno-Gold Stain (IGS), a new immunocytochemical method (17), was used to visualize microtubules (MTs) in the light microscope. Observations on MT arrangements were correlated with studies in vivo. Chromosome movements are affected in all stages of mitosis which progresses over at least 10(4) range of taxol concentrations. The three most characteristic effects on MTs are: (a) enhancement of the lateral associations between MTs, seen especially during the reorganization of the polar region of the spindle, (b) promotion of MT assembly, leading to the formation of additional MTs in the spindle and MT arrays in the cytoplasm, and (c) an increase in MT stability, demonstrated in their increased cold resistance. In this report, the emphasis is on the primary, immediate effects, occurring in the first 30 min of taxol action. Effects are detected after a few mins, are reversible, and are concentration/time dependent. The spindle and phragmoplast are remarkably modified due to the enhancement of lateral associations of MTs and the formation of abundant nonkinetochore and polar, asterlike MTs. The equatorial region of the interzone in anaphase may be entirely depleted of MTs, and the spindle may break perpendicular to the spindle axis. Mitosis is completed in these conditions, providing evidence for the motile autonomy of each half-spindle. Trailing chromosome arms in anaphase are often stretched and broken. Chromosome fragments are transported away from the polar regions, i.e., in the direction opposite to that expected (5, 6). This supplies the first direct evidence of pushing by elongating MTs in an anastral higher plant spindle. These observations draw attention to the relation between the lateral association of MT ends to assembly/disassembly and to the role of such an interaction in spindle function and organization.

The force for poleward chromosome motion in Haemanthus cells acts along the length of the chromosome during metaphase but only at the kinetochore during anaphase.

The force for poleward chromosome motion during mitosis is thought to act, in all higher organisms, exclusively through the kinetochore. We have used time-lapse. video-enhanced, differential interference contrast light microscopy to determine the behavior of kinetochore-free "acentric" chromosome fragments and "monocentric" chromosomes containing one kinetochore, created at various stages of mitosis in living higher plant (Haemanthus) cells by laser microsurgery. Acentric fragments and monocentric chromosomes generated during spindle formation and metaphase both moved towards the closest spindle pole at a rate (approximately 1.0 microm/min) similar to the poleward motion of anaphase chromosomes. This poleward transport of chromosome fragments ceased near the onset of anaphase and was replaced. near midanaphase, by another force that now transported the fragments to the spindle equator at 1.5-2.0 microm/min. These fragments then remained near the spindle midzone until phragmoplast development, at which time they were again transported randomly poleward but now at approximately 3 microm/min. This behavior of acentric chromosome fragments on anastral plant spindles differs from that reported for the astral spindles of vertebrate cells, and demonstrates that in forming plant spindles, a force for poleward chromosome motion is generated independent of the kinetochore. The data further suggest that the three stages of non-kinetochore chromosome transport we observed are all mediated by the spindle microtubules. Finally, our findings reveal that there are fundamental differences between the transport properties of forming mitotic spindles in plants and vertebrates.

UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.

Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation.

Structure and organization of the living mitotic spindle of Haemanthus endosperm.

New details of mitotic spindle structures in the endosperm of Haemanthus katherinae (Bak) have been demonstrated by differential interference microscopy. Spindle fibers are clearly seen in the living spindle extending from the kinetochores to the polar region. Individual spindle fibers consist of a bundle of smaller filaments which diverge slightly from the kinetochore and intermingle with filaments from other spindle fibers as they approach the polar region. The degree of intermingling increases during metaphase and anaphase. The chromosomes stop moving when the spindle fibers are still 5 to 10microns long; then the fibers disappear. These observations explain some aspects of spindle movements which were difficult to reconcile with earlier concepts of spindle organization.

Between-year variation and spatial dynamics of Cryptosporidium spp. and Giardia spp. infections in naturally infected rodent populations.

Prevalence and abundance of Cryptosporidium spp. and Giardia spp. infections were studied over the 8-year period in 3 species of rodents in N.E. Poland (bank vole Myodes glareolus-1523; yellow-necked mouse Apodemus flavicollis- 638; common vole Microtus arvalis- 419). Prevalence was 53.8, 28.1 and 62.3% respectively for Cryptosporidium spp. and 58.3, 24.4 and 74.2% respectively for Giardia spp. Prevalence and abundance of infection varied markedly across 8 years of the study with 1998 and 2002 being years of higher prevalence and abundance, following changes in the densities of host species. The distribution of intestinal protozoa in forest rodents did not vary in the 3 isolated sites during the 4-year study. In the case of Cryptosporidium, fewer older animals carried infection and infections of the oldest bank and common voles were relatively milder. In the case of Giardia in yellow-necked mice, infections were more common in older age classes (2 and 3). The two species showed significant co-occurrence and in animals carrying both species there was a strong significant positive correlation between abundance of infection with each. These data are discussed in relation to the parasite genotypes identified in this region and in respect of the role of various ecological factors in shaping of intestinal protozoa communities.

Temporal and between-site variation in helminth communities of bank voles ( Myodes glareolus) from N.E. Poland. 1. Regional fauna and component community levels.

Helminth infections were studied in bank voles (Myodes glareolus) from 3 woodland sites in N.E. Poland in the late summers of 1999 and 2002, to assess the temporal stability of derived statistics describing the regional helminth fauna and component community structure, and spatial influence on the latter. Regional helminth fauna changed dramatically between the two years, primarily due to a fall in the abundance of Syphacia petrusewiczi but was partially compensated for by an increase in Mesocestoides lineatus and Cladotaenia globifera. It was dominated by nematodes overall, but more so in 1999 than in 2002 when larval cestodes were more frequent. Most derived parameters for component community structure varied considerably between sites and the two surveys, the hierarchical order for sites not being maintained between surveys. They were susceptible to the disproportionate influence of three relatively rare, unpredictable species with the greatest overall aggregated distribution among hosts. Jaccard's similarity index was less influenced by the rare species, showing greater stability between sites and across years. In conclusion, temporal variation confounded any site-specific characteristics of the summary measures quantified in this study and their usefulness is therefore restricted to the years in which the surveys were conducted.

Temporal and between-site variation in helminth communities of bank voles ( Myodes glareolus) from N.E. Poland. 2. The infracommunity level.

The relative importance of temporal and spatial effects was assessed in helminth communities of bank voles (Myodes glareolus) in 3 woodland sites in N.E. Poland in the late summers of 1999 and 2002. Among common species the rank order of sites in relation to prevalence and abundance of infection was maintained between surveys. Site effects accounted for most of the deviance (in statistical models), and time was less important, so the exact location from which voles were sampled was of critical importance. The only exception was Syphacia petrusewiczi. In contrast, for derived measures such as species richness and diversity, most deviance was accounted for by host age, and the interaction between site and year was significant, implying that rank order of sites changed between years. Temporal effects on derived measures were generated primarily by a combination of relatively small changes in prevalence and abundance of the common, rather than the rare, species between the years of the study. In the medium-term, therefore, helminth communities of bank voles in N.E. Poland had a stable core, suggesting a substantial strong element of predictability.

Cryptosporidium parvum: the course of Cryptosporidium parvum infection in C57BL/6 mice co-infected with the nematode Heligmosomoides bakeri.

The effects of Heligmosomoides bakeri infection on the course of a concurrent Cryptosporidium parvum infection were studied in C57BL/6 mice. Mice were initially infected with 80 L(3) of H. bakeri and then challenged with 10(4) oocysts of C. parvum, administered during the patent period of the nematode infection (28 day post H. bakeri infection). The number of C. parvum oocysts excreted in the feces and the number of adult H. bakeri in the small intestine were monitored during the experiment. Concurrent H. bakeri infection resulted in a prolonged course of infection with C. parvum. The intensities of both parasite infections were higher in co-infections. We also investigated the cellular immune response at 14 and 42 days post infection C. parvum. During infection with C. parvum there was an increase in production of IFN-gamma and IL-12 but co-infection with H. bakeri inhibited IFN-gamma secretion. The present study is the first to demonstrate that infection with H. bakeri markedly exacerbates the intensity of a concurrent C. parvum infection in laboratory mice and also affects immune effectors mechanisms in co-infection with H. bakeri.

Drugs with colchicine-like effects that specifically disassemble plant but not animal microtubules.

Mitosis is arrested in Haemanthus endosperm by amiprophos-methyl and oryzalin at a concentration of 100 nM, and anaphase chromosome movements are modified at 10 nM. Prolonged exposure to these drugs results in a classical c-mitosis. Anaphase chromosome movement is arrested within less than 30 seconds without any detectable change of MT arrangement, as shown by the immunogold staining method. In the next phase of amiprophos-methyl and oryzalin action, however, all non-kinetochore MTs, and especially those most sensitive to these drugs, polar microtubules, which form abundantly during anaphase, disassemble. Kinetochore microtubules form tight bundles that are very resistant to further drug action and often elongate before they finally disassemble. Because these drugs inhibit MT assembly in vitro in a concentration-dependent manner, we conclude that MT assembly is required for chromosome movements in anaphase and that the elongation of polar MTs is necessary for the progress of anaphase. No effect of the drugs on mitosis was detected, even at the saturated level, in the tissue culture of the frog Xenopus.

Microtubule dynamics determine chromosome lagging and transport of acentric fragments.

The general direction of transport of spindle inclusions including acentric chromosome fragments during mitosis in endosperm of the higher plants Haemanthus is predictable and stage-dependent. Their segregation is random and they are usually eliminated from the spindle. This transport is superimposed on normal chromosome segregation. Thus, there are 2 superimposed mitotic transports: one which distributes kinetochores and the other which distributes spindle inclusions. The functional relation of these 2 transports to each other is not well understood. However, due to this 'non-kinetochore transport,' fragments may persist a few consecutive divisions before being permanently eliminated from the nucleus. Malfunction of kinetochores of any chromosome, resulting in the loss of their anchorage within the spindle, subjects them to 'non-kinetochore' transport and nearly certain, permanent elimination from the spindle. Additionally, experimental evidence presented here demonstrates that rapid polymerization (elongation) of microtubules may desynchronize anaphase and cause lagging of whole chromosomes. This may be one more, previously unconsidered, factor which may cause the malfunction of the kinetochore fiber and consequent elimination of one or a few chromosomes from the spindle.

Preliminary molecular characterization of Cryptosporidium parvum isolates of wildlife rodents from Poland.

Isolates of Cryptosporidium were collected from 3 species of woodland and field rodents (Clethrionomys glareolus, Microtus arvalis, and Apodemus flavicollis) and were characterized by polymerase chain reaction amplification and sequencing of fragments of the oocyst wall protein (COWP) gene and of the 18S ribosomal RNA gene. Sequence analysis of these markers revealed that the animals were infected with C. parvum, and that the genotype involved was almost identical to the mouse genotype previously described from Mus musculus. Thus, small rodents should be considered as an important reservoir of C. parvum genotypes closely related to the zoonotic genotype 2 and potentially hazardous to humans.

Calves as a potential reservoir of Cryptosporidium parvum and Giardia sp.

Studies on cryptosporidiosis and giardiosis were carried out between March and April 1997 on 75 calves from 9 selected farms of Wielkopolska macroregion. Faecal specimens from calves, 3-13 days old, were screened for oocysts of C. parvum using Ziehl-Neelsen staining and both for oocysts of C. parvum and cysts of Giardia sp. using direct immunofluorescent (MerIFluor Cryptosporidium/Giardia) assay. The oocysts of C. parvum assessed by Ziehl-Neelsen staining were revealed on 6 (67%) of 9 farms examined. The prevalence of infection ranged from 20-88%, and in some farms intensity of oocyst shedding was very high. However, in 35 calves assessed for mixed infections of C. parvum and Giardia sp., oocysts of Cryptosporidium were found in 18 (51%) calves and cysts of Giardia sp. were detected in 5 (14%) of 35 calves. Only in one calf was found coinfection with both parasites. The intensity of Giardia sp. infection was extremely low. Histological examination of the gut sections from immunosuppressed BALB/c mice experimentally infected with C. parvum isolates from calves revealed endogenous stages of C. parvum on the brush border of the ileum. The high prevalence and intensive shedding of cryptosporidial oocysts by calves in farms examined in this study suggests that naturally infected calves may be significant reservoirs for C. parvum infections in man and wild animals.

[The ecology of Cryptosporidium parvum infection in small rodent populations]. / Srodowiskowe uwarunkowania zarazen Cryptosporidium parvum w populacjach drobnych gryzoni.

The prevalence and abundance of Cryptosporidium parvum were studied over a three year period (1997-1999) in three species of rodents sampled from forest and abandoned fields in the Mazury Lake District, Poland. The overall prevalence was consistently higher in voles compared with Apodemus flavicollis (70.6% in Clethrionomys glareolus, 73.0% in Microtus arvalis and 27.8% in A. flavicollis). The prevalence and abundance of infection also varied across the 3 years of the study with 1998 being the year of higher prevalence and abundance of the parasite. Fewer older animals carried the infection, and their infections were relatively mild. We found no consistent pattern of seasonal changes despite the significance of seasonal differences. Host sex did not influence either the prevalence or abundance of infection with C. parvum. A great proportion of recaptured voles developed chronic infections between consecutive trapping sessions and only a small number of animals recovered. However, yellow-necked mice seem to be much more resistant to infection that became self-limiting. Our results firmly establish that the common woodland and grassland wild rodents in the Mazury Lake District constitute a significant and hazardous reservoir of C. parvum for animals and humans.