[Neonatal gastroenteritis triggers long-term cardiomyocyte atrophy, remodeling and irreversible hyperpolyploidization].

Growth retardation, inflammation and cardiac overload in early childhood are linked with hypertension and infarction in adults. This link was termed as developmental programming. Exact mechanisms and critical time frames for development of the heart are still unknown. To elucidate these questions, we developed a model of moderate cryptosporidial gastroenteritis triggering main programming factors. Sliding the time point of infection day by day (from day 4 to day 18), we tested complete rat neonatal period. Also, we repeated all experiments 30 days after infection. Using methods of cytometry, immunocytochemistry and confocal microscopy, we compared sensitivity of ventricular cardiomyocyte shape, protein content and ploidy. Our data indicated that gastroenteritis lasting four days triggered cardiomyocyte atrophy, almost doubling cell length to width ratio, and premature and excessive polyploidization. Surprisingly, nucleus and cytoplasm reacted to the disease differently. Cardiomyocytes accumulated genomes only when the disease covered the time period between 6 and 14 days after birth, when cells substitute proliferative growth with hypertrophy. Contractile proteins and cell shape on the contrary, showed high sensitivity in the course of complete neonatal period. After restoration, ploidy did not regress, whereas cell shape and protein content revealed moderate restoration. Taking into account that somatic polyploidy is irreversible and that it alters global gene expression pattern, we may suggest that genome duplication is one of the instruments of developmental programming and that gastroenteritis is one if the triggers of this programming.

[The ultrastructural changes of Sarcocystis ovifelis infested sheep tongue myofibrils].

Structural changes were observed in filaments of Sarcocystis ovifelis infected sheep tongue myofibrils. In sarcocysts containing myofibrils, actin filaments and Z-disks, myosin filaments and M-line were seen destroyed. Protein bridges, uniting actin and myosin filaments into a joint complex (net), eventually become not visible, and as a result separate Z-disks and free filaments appear. Fibrils, referred to as leptomeric, have been first revealed between protrusions of the sarcocyst surface apparatus. These are striated filaments with periodic 100 nm striation of dark and light bands, made of thin and short 120-200 nm long filaments 5 nm in diameter. The genesis of leptomeric fibrils still remains obscure. In sarcocysts infected myofibrils these may be involved in metabolite transportation to the intercellular space and back.

[Intestinal cryptosporidiosis at an early age and its negative consequences].

This review calls the attention of physicians, primarily pediatricians, to cryptosporidiosis, a still little known intestinal infection caused by the protozoan pathogen Cryptosporidium parvum (Coccidia, Sporozoa). By using 10--14-day rats as a model, the authors have first provided evidence that even 4-day intestinal cryptosporidiosis may trigger obvious negative changes in the liver and heart, i.e. in the organs where the parasite does not develop. In the infected rats, growth retardation was registered, in addition to liver hypertrophy and partial heart atrophy, and growth retardation. Light and electron microscopies, absorption and fluorescence cytometry, quantitative morphometry, and image analysis were applied. In both hepatocytes and cardiomyocytes, the polyploid cell fraction was seen much increased, with the occurrence of 4c, 8c, and even 16c nuclei. Besides, in the hepatocytes, the amount of glycogen decreased whereas the level of protein increased, along with enhanced nucleolar activity in the nuclei. Unlike, the cardiomyocytes of the infected rats were characterized by protein decrease, in addition to almost two-fold cell body elongation. This is the first documented evidence for serious pathological changes in the extraintestinal organs, caused by the intestinal pathogen C. parvum. Within the first 4 days of infection, both the liver and heart of the host seem to work under stress. It is plausible that on modulating liver and heart ploidy, the intestinal parasitic infection (cryptosporidiosis) may bring about functional impairments of these organs, untypical of early age, leading eventually to long-term consequences in further life of formerly infected individuals.

[The structural and functional analysis of the surface apparatus in four species of Sarcocystis (Sporozoa, Apicomplexa)].

The comparable ultrastructural analysis of the sarcocyst surface apparatus (SSA) was made for four species of Sarcocystis: Sarcocystis muris, S. fusiformis, S. medusiformis, and Sarcocystis sp. from buffalo heart muscles. In all these species, SSA contains a surface membrane, overmembrane complex with glycocalyx, and submembrane complex made of two glycoprotein SSA primembrane layers. SSA makes numerous primary vesicle-like protrusions and pits in between. Some vesicles containing two layers, PM1 and PM2, are pinching off from the totally formed protrusions. Then these vesicles are directed into infected host cell to participate in its degradation. In the SSA pits neither over-, nor submembrane complex is present, the pits being made of the surface membrane only. It is important that fibrillar structures penetrate through the SSA membrane into pits from the host cell. Besides, SSA forms secondary protrusions with different structures in various species of Sarcocystis. They increase the sarcocyst surface and transport different substances along intermediate filaments from the SSA pits membrane to the sarcocyst body. At the same time, deep invaginations are found in the SSA of old sarcocysts. We thought that these structures increased the sarcocyst surface and thus promote to intensify metabolism. This study-defined presence of membranous vesicles in secondary protrusions. According to their structure and localization, the membranous vesicles may be involved in the building of the sarcocyst surface membrane.

[Structural and functional characterization of cyst cells in Sarcocystis sp. (Sporozoa, Apicomplexa)]. / Osobennosti stroeniia i funktsional'nye kharakteristiki kletok v tsistakh sarkosporidii.

By means of light and electron microscopy, the structural pattern of muscle cysts (sarcocysts) was examined for the four species of the genus Sarcocystis: S. muris (from murine skeletal muscles), Sarcocystis sp. and S. fusiformis (from, respectively, heart and skeletal muscles of buffalo), and S. ovifelis (from ovine tong muscles). The orderly fashion of the interior of the cyst is attained by partitition of its space into numerous compartments with the involvement of the intermediate filaments. These, in their turn, are bound to each other by thin filaments to make eventually a common filamentous net. The net limits separate groups of cells referred to as cyst zoites. The common net of filaments and microtubules (when present) may be regarded not only as the organizer of the cyst interior cytoskeleton, but also as the main mechanism of substance transportation in various directions: from the host cell to the sarcocyst, and within or outside the cyst. The role of dedifferentiation, proliferation and differentiation processes is suggested in the establishment of the fixed sequence of events throughout the unidirectional development of cyst cells and their interaction, from precystic meronts to cyst merozoites (gamonts). Special attention is paid to metrocyte morphogenesis and functioning. In the present work, metrocytes subjected to apoptosis were recognized. It is suggested that phenomenon of programmed cell death in metrocytes may be associated with the control of cell number in mature and ageing sarcocysts.

[Cell response of rat liver parenchyma to the infection by the intestinal protozoan pathogen Cryptosporidium parvum (Sporozoa, Coccidia)]. / Reaktsiia kletok parenkhimy pecheni krys na zarazheniie kishechnym protozoinym patogenom Cryptosporidium parvum (Sporozoa, Coccidia).

In the present work, the authors' previous studies of a "distant action", exerted by an intestinal pathogen (Cryptosporidium parvum) on the liver of experimentally infected baby rats, were extended to include shifts in the quantity of glycogen, protein and nuclear DNA in the host liver at different degrees of infection. One of the outcomes of this work is the discovery of a very quick response of hepatocytes and a high sensitivity of rat liver to parasitic invasion even at a weak intensity of infection. 85-90 h after oocyst feeding to rats, glycogen quantity in their livers was 2.5 times lower that in the control. This suggests that the infected host liver worked under energetic starvation conditions. The proposed coefficients of general infection (I) and infection with intracellular stages (F) made it possible to distinguish between the total abundance of parasites in the host intestine during the whole period of infection, and the number of feeding intracellular stages available by the moment of autopsy. The glycogen amount in rat hepatocytes does not depend on I, and negatively correlates with F. Unlike, the protein content in hepatocytes positively correlates with I, being independent of F. Despite the obvious deficiency of amino acids in the infected rats, as a consequence of cryptosporidiosis-induced malabsorption, the protein synthesis in their hepatocytes was not at all inhibited but, on the contrary, much activated. This is a most characteristic feature of the distant action of C. parvum on the liver of parasitized host. With C. parvum infection, the share of polyploid hepatocytes does not correlate with either I, or F. However, compared to the control, the mean values of relative numbers of polyploid cells in weakly, moderately, and heavily infected animals (according to I values) were higher by 20, 100 and 100%, respectively. In hepatocyte nuclei of C. parvum infected rats, the total area of nucleoli increases almost by 30%. The above changes are discussed in terms of both the liver compensatory response to the existing pathology (diarrhea), and the host-parasite relationships. Studies into the distant action of an intestinal pathogen (C. parvum) on non-intestinal organs (liver) of the infected host may be qualified as a new and original approach to pathogenesis of protozoan infections (coccidioses sensu lato), to which young host specimens are known to be most susceptible.

[Formation and diversity of parasitophorous vacuoles in parasitic protozoa. The Coccidia (Sporozoa, Apicomplexa)]. / Puti formirovaniia parazitofornykh vakuolei i ikh raznoobrazie u paraziticheskikh prosteishikh. Koktsidii (Sporozoa, Apicomplexa).

Data on parasitophorous vacuole (PV) formation in host cells (HC) harbouring different intracellular protozoan parasites have been reviewed and critically analysed, with special reference to the main representatives of the Coccidia. The vacuole membrane (PVM) is the interface between host and parasite, playing a role in nutrient acquisition by the parasite from the HC. The PV phenomenon is regarded as a generalized HC response to the introduction of alien bodies (microorganisms), which eventually reflects the evolutionary established host-parasite relationships at cellular, subcellular and molecular levels. Special attention has been paid to the existing morpho-functional diversity of the PVs within the same genera and species of parasites, and even at different stages of the parasite life cycle. The PVM is generally considered to derive from the HC plasmalemma, whose biochemical composition undergoes significant changes as the intravacuolar parasite grows. The original HC proteins are selectively excluded from the PVM, while those of the parasite are incorporated. As the result, the changed PVM becomes not fusigenic for HC lysosomes. For Toxoplasma gondii and other cyst-forming coccidia (Isospora, Sarcocystis), a definite correlation has been noticed between the extent of rhoptry and dense granule secrets released by a zoite during HC internalization, on the one hand, and the pattern of the PV that forms, on the other one. In T. gondii, tachyzoites, known to discharge abundant secrets, commonly force the development of PVs limited with a single unit membrane and equipped with a tubulovesicular network in the lumen. Unlike, bradyzoites known to be deficient in secretory materials trigger the formation of PVs with a three-membrane lining composed of the changed invaginated plasmalemma in addition to two membranes of endoplasmic reticulum. The two different types of PV harbour, respectively, exoenteric and enteric stages of T. gondii, the latter being confined to the cat intestine only. Unlike, all endogenous stages of the classic intestinal coccidia (Eimeria spp.) develop within PVs limited with a single membrane, with some invaginations extending into the PV lumen. Unusual PV patterns are characteristic of the extracytoplasmic eimerian coccidia (Cryptosporidium, Epieimeria) and adeleid haemogreagarines (Karyolysus). In cyst-forming coccidia, the PVM is actively involved in tissue cyst wall formation, thus protecting the encysted parasites from recognition by the host immune system. All this strongly suggests that the PV is far from being an indifferent membraneous vesicle containing a parasite, but represents a metabolically active compartment in infected cells. Since all the coccidia are obligate intracellular parasites, the mode of their intimate interaction with the HC, largely accomplished via the PV and its membrane, is vital for their survival as biological species.

[The role of the sarcocyst surface apparatus in utilizing the host cell muscle structures]. / Rol' poverkhnostnogo apparata myshechnykh tsist sarkosporidii v protsesse utilizatsii struktur myshechnoi kletki khoziaina.

The participation of the sarcocyst surface apparatus (SSA) of two sarcosporidian species, Sarcocystis muris and S. ovifelis (Coccidia, Sporozoa, Apicomplexa), in degradation of disrupted host cell substances was investigated. After degradation, these substances are transported through the membrane of the SSA to the sarcocyst ground substance (GS), but this process cannot be regarded as endocytosis. At first, the transported substances were found in SSA pits in the form of fibrillar structures. Later on, these were seen as twisted up granules. In some cases, such granules restore their fibrillar shape, penetrate through the SSA membrane and appear in the sarcocyst GS. In other cases, the small granules may be released from SSA pits directly to the sarcocyst GS. Besides, two SSA primembrane layers were seen to disappear during the transportation of host cell substances. In addition, multimembrane structures (membranous whorls) were first demonstrated between the plasmalemma and inner membrane complex of the zoite pellicle. Multimembrane structures were found, in addition, in the zoite cytoplasm in connection with micronemes. These structures resembling chloroplast granae of thylakoids may presumably fill the gap in membrane pool of the SSA contributing to its renewal.

[The structure of the surface apparatus in sarcocysts of Sarcosporidia in the persistence process]. / Strukturnaia organizatsiia poverkhnostnogo apparata sarkosporidii v protsesse persistirovaniia.

The structure of the sarcocyst surface apparatus (SSA) was investigated for two sarcosporidian species: Sarcocystis muris (non-pathogenic) and S. fusiformis (pathogenic). The surface membrane, being the main SSA subsystem, makes numerous vesicle-like protrusions with different ultrastructural patterns. This made it possible to distinguish between four and three types of these protrusions in S. fusiformis and S. muris, respectively. Vesicles of similar structure, pinched off from the fully formed protrusions, were classified, correspondingly, in the same four and three different types. A presumable functional role of both protrusions and membrane-coated vesicles in pathogenicity of different sarcosporidian species is proposed. The vesicles pinched off from corresponding protrusions may be involved in transporting certain substance complexes from the sarcocyst to the harbouring host cell. In addition, another way of substance transporting was observed, when the cystic substances, not surrounded with any membrane coating, are thrown from open protrusions directly into the immediate cytoplasm of the host cell.

[Morphofunctional changes in hepatocytes during the early postnatal development of rats experimentally infected with the intestinal protozoan pathogen, Cryptosporidium parvum (Coccidia, Sporozoa)]. / Morfofunktsional'nye izmeneniia pecheni v rannem postnatal'nom razvitii u krys, éksperimental'no zarazhennykh kishechnym protozoinym patogenom Cryptosporidium parvum (Coccidia, Sporozoa).

Morphofunctional changes in hepatocytes of 10-14-day old rats were followed in norm and after experimental infection with different doses of oocysts of Cryptosporidium parvum. The liver index (ratio between the liver and body masses) varied with the intensity of invasion on the background of slowing down up to the total cessation of animal growth rates, and all this obviously pointed to severe pathology. In the infected rats, some cytological indices were shifted compared to the norm: protein amount and the average number of genomes per hepatocyte were seen to increase, the normal ratio between cells with different ploidy levels being violated. The particular correlation analysis was employed to distinguish between the ontogenetic (animal growth related) and pathologic (related to the infection intensity) polyploidization and hypertrophy in hepatocytes. In 10-14-day old rats, the former is affected primarily by the increase in the share of multinuclear hepatocytes, whereas the latter is accomplished by the increase in the number of cells with polyploid nuclei (4c and 4c x 2 cells). In the heavily infected rats, the ontogenetic polyploidy was almost totally suppressed due, presumably, to their growth rate inhibition, the rise in hepatocyte ploidy resulting form the obvious pathological changes in the liver. In the infected rats, the ontogenetic hypertrophy of hepatic parenchymatous cells was not manifested, and the observed protein accumulation in hepatocytes also resulted from the pathological changes in the liver. It is obvious that changes in cell hypertrophy (protein content) may serve as a more susceptible tool that readily perceives the host's stress experienced due to the parasitic infection (cryptosporidiosis), than cell ploidy: the levels of the respective responses of these two parameters differing by 4 times. However, due to the known reversible nature of hypertrophy, it cannot be used for the aims of a long-term prediction about the future mode of liver functioning in the animal that survived cryptosporidiosis. Unlike, such a parameter as frequencies of hepatocytes with different ploidy levels is much more useful in this respect.

[Effect of developing Sarcocystis muris tissue cysts on ultrastructural change of murine muscle fibers]. / Vliianie razvivaiushchikhsia tkanevykh tsist Sarcocystis muris na izmenenie ul'trastruktury myshechnogo volokna myshi.

A study was made of the influence exerted by developing sarcocysts of Sarcocystis muris on the ultrastructural organization of muscle fibres, both harbouring the sarcocysts (HSM) and sarcocyst-free (SFM), from skeletal muscles of experimentally infected mice. Muscle fibres of non-infected mice of the same age served as a control. Mice were sacrificed 6 months following feeding S. muris oocysts (or sporocysts). The developing sarcocysts seriously destroyed HSM: their myofilaments were no hold in register, cross-bridges almost entirely disappeared, M-lines and Z-disks looked as broken structures. The majority of actin myofilaments were arranged along myosin myofilaments as discrete units. The host cell sarcoplasm was packed with numerous vacuoles of different form and size. Compared to muscle fibres in the control, SFM of infected mice also displayed an obvious ultrastructural alteration. On the periphery of SFM, some destroyed sarcomeres with swollen myofilaments were noticed whose cross-bridges were totally lacking. In other extreme areas myosin and actin myofilaments were disintegrated into thin straightened filaments 2.0-2.5 nm in diameter. It is supposed that HSM and SFM of the infected mice may experience different kinds of influence on the part of the developing intracellular parasite (sarcocyst). And it dos not seem unlikely that various biologically active substances, produced by the parasite, may be vesicle transported to SFN through the endomysium space.

[Intracellular parasitism and sarcocystosis]. / Vnutrikletochnyi parazitizm i problema sarkotsistoza.

The obligatory heterogenous tissue cyst-forming coccidia of the genus Sarcystosis are regarded as an excellent example of specific coexistence of two organisms, i.e., the host and parasite. These parasitic protozoans are known as causative agents of the chronic, often life-threatening disease, sarcocystosis, which still cannot be effectively controlled. In Sarcocystis, the entire phase of asexual multiplication was transferred to the intermediate host. Of special interest is the parasite's ability to persist in this host at the stage of tissue cyst or sarcocyst. This is a giant meront, in which unidirectional development proceeds starting from a little differentiated metrocyte, through intermediate cells, and towards highly differentiated cyst merozoites (gamonts) unable to further divide. The life span of the sarcocyst depends, to a great extent, on self-regulation within the cyst itself and on relations between the cyst and its immediate environment. A totally new field of research into Sarcocystis was initiated by the discovery that the intracellular parasite damages both cyst harboring and intact muscle cells, apart from the adjacent connective and nervous tissue. The previously unknown cytopathological effects of sarcocysts have been described and characterized. The changes observed within and outside the sarcocysts have been analyzed in terms of general biological processes: proliferation, differentiation, and programmed cell death.

[Further comment on the coccidian nature of cryptosporidia (Sporozoa: Apicomplexa)]. / Eshche raz o koktsidiinoi prirode kriptosporidii (Sporozoa: Apicomplexa).

The coccidian nature of the genus Cryptosporidium was undoubtedly accepted by Tyzzer who was the first to describe this sporozoan parasite in 1907. Electron microscopic studies made in 70-90s demonstrated the intracellular, although extracytoplasmic localization of Cryptosporidium spp. The pattern of Cryptosporidium life cycle fits well that of other intestinal homogeneous coccidian genera of the suborder Eimeriina: macro- and microgamonts develop independently, a microgamont gives rise to numerous male gametes, oocysts serving for parasite's spreading in the environment. Along with these characters, Cryptosporidium spp. demonstrate some secondary peculiarities (an endogenous phase of development in microvilli of epithelial surfaces, two morphofunctional types of oocysts, the smallest number of sporozoites per oocyst, a multi-membraneous "feeder" organelle etc.), which may be due presumably to their early acquisition of specialization in the course of evolution. The recent studies based on molecular sequence data (18S rRNA) applied to 8 eimeriid and isosporid coccidian genera (Morrison, Ellis, 1997), suggested that the subclass Coccidia (class, according to Morrison and Ellis) be considered monophylic if Cryptosporidium were excluded, and this genus was regarded as the sister group to the rest of the Apicomplexa, or as the sister to the suborder (class) Hematozoa within the Apicomplexa. Either of these placements of Cryptosporidium definitely conflicts with both the generally accepted taxonomic scheme by Levine (1982) and the phenotypically based phylogeny of the phylum Apicomplexa (Barta e. a., 1990). The author's opinion is that the differences between the examined eimeriid and isosporid coccidia, on the one hand, and Cryptosporidium, on the other hand, provided by molecular sequence data, may testify primarily to the well known morphofunctional dissimilarities between the compared organisms, rather than cast doubt on the coccidian nature of Cryptosporidium. Again, these data can hardly prove that Cryptosporidium does not belong to the coccidia. Thus, the modern molecular sequence data, despite their obvious scientific value, would make sense for phylogeny estimation only, if they are critically analysed and considered in combination with results of the relevant basic research.

[One of the possible ways of propagation of the unicellular parasites Sarcocystis muris in the musculature of the intermediate host]. / Odin iz vozmozhnykh putei rasprostraneniia odnokletochnykh parazitov Sarcocystis muris v muskulature promezhutochnogo khozaina.

20 laboratory mice (Mus musculus) were fed each a single dose of 20,000 Sarcocystis muris sporocysts to be then sacrificed 1, 2.5, 4, 6 and 10 months following infection (p.i.). A visual infection of the murine corps demonstrated that the number of sarcocysts per animal increased regularly as the time of infection was progressing, being eventually higher after 6 and 10 months p.i. than within 1-4 months p.i. This phenomenon was poorly understood from the knowledge that the tissue cysts (sarcocysts) are able to increase in size, rather than in number, and that the original number of sarcocysts largely depends on the number of precystic merozoites available. In our experiments, each of 20 mice was fed an equal number of sporocysts, and thus the number of precystic merozoites ought to be expected also more or less equal. EM investigation of murine skeletal muscles 6 and 10 months p.i. revealed, along with numerous normal sarcocysts, the presence of some separate, individual zoites, both within and outside the muscle fibre, in the endomysium. Besides, a colony of zoites, living freely without any visible common wall, was detected within a muscle fibre adjacent to another one, containing a sarcocyst of normal structure. These zoites may have originated from one or more sarcocysts, whose cyst walls were spontaneously broken, time after another, and thus led the cyst cells go out. These discharged zoites could either perish, being enzymatically degraded, or penetrate the neighbouring muscle fibres to proceed their further development. The colony making zoites were confined to two cell types only: the intermediate cells and the merozoites (gamonts). No metrocytes were recognized due, presumably, to inability of these little differentiated cells, devoid of penetrative organelles, to invade the host muscle cell. The colony of zoites turned out to be a developing population of live cells, able to destroy progressively the harbouring muscle fibre, except its basal membrane and sarcolemma. It does not seem unlikely that the outer coverings of the infected cell could be transformed eventually into a cyst wall to make, thus, a new sarcocyst. The above phenomena have never been found in murine muscles earlier than 6 months p.i. Although these facts are few and far between, they may prompt a possible mechanism of sarcocyst increase in number, in the intermediate host with age, even without any additional sporocyst contamination.

[The localization and functional significance of arylsulfatases in parasitic sarcosporidian protozoa in the tissue-cyst life cycle phase]. / Lokalizatsiia i funktsional'noe znachenie arilsul'fataz u paraziticheskikh prosteishikh sarkosporidii v faze tkanevoi tsisty zhizhnennogo tsikla.

The hydrolytic enzymes arylsulphatases (AS) were detected in developing tissue cysts of Sarcocystis ovifelis, using two methods: the Goldfischer lead technique, with two different pH values-5.5 (AS-A) and 4.2 (AS-B), and the Hopsu-Havu barium technique (Gayer, 1974). The enzymatic activity was identified by the presence of an electron dense finely granulated precipitation. In cyst cells, lead sulphate precipitation was spotted only in the inner membrane complex (IC) of the pellicle, whereas barium sulphate marked, in addition, the plasma membrane. Besides, AS activity was detected in the endoplasmic reticulum, Golgi complex, lysosomes and micronemes of cyst cells. Of interest is the finding of AS in the outer membrane of IC and matrix of pellicular evaginations. In the cyst ground substance (CGS) of S. ovifelis AS activity is confined to the membrane and matrix of transport vesicles, originating from cyst cell pellicle evaginations. These cystic vesicles carry enzymes from the places of their synthesis, in the cyst cells, to the tissue cyst periphery near the cyst wall. In the CGS, the obvious precipitations of lead and barium sulphate, respectively, are seen around some cyst cells being in the state of destroying due to natural death, and around so-called apoptotic-like bodies made from the destroyed cells. AS activity is seen both in the cyst wall and in vesicles separating from the wall ("wall vesicles") that find eventually their way in the cytoplasm of infected muscle cells, the granulation being observed around destroyed organelles of such cells. The investigated dynamics of AS movement, by means of the transport cystic and wall vesicles, extends general knowledge of the distant metabolic interaction between cells of the host and the parasite in tissue cysts of Sarcocystis spp.

[The effect of coccidia of the genus Sarcocystis on the morphofunctional organization of the skeletal musculature in experimentally infected mice. I. The muscle fiber]. / Vliianie koktsidii roda Sarcocystis na morfofunktsional'nuiu organizatsiiu skeletnoi muskulatury éksperimental'no zarazhennykh myshei. I. Myshechnoe volokno.

At the ultrastructural level, the cellular response of skeletal muscles on developing Sarcocystis muris sarcocysts has been followed in mice at different times after sporocyst feeding, i.e. in 1, 2.5, 6 and 10 months, resp. The developing cyst creates a progressive degeneration of the infected muscle cell that involves organelle disorganization and formation of numerous vacuoles in the cytoplasm as a consequence of cell edema. Products of the host cell degradation, shaped as fibrillar-granular structures, are seen to find their way to the cyst wall outgrowings, where they become denser and on being covered with membranes appear eventually in the sarcocyst ground substance. Later on, the membranes around the granules disappear. In the course of its development, the sarcocyst totally destroys not only the harbouring muscle cell and the nearest connective tissue elements of the endomysium, but also the previously intact neighbouring cells. The involvement of some proteolytic enzymes in this process is suggested.

[The effect of coccidia of the genus Sarcocystis on the morphofunctional organization of the skeletal musculature in experimentally infected mice. II. The connective tissue elements of the endomysium]. / Vliianie koktsidii roda Sarcocystis na morfofunktsional'nuiu organizatsiiu skeletnoi muskulatury éksperimental'no zarazhennykh myshei. II. Soedinitel'notkannye élementy éndomiziia.

As an extension of the previous communication (Radchenko, et al., 1996), a study was made of the response of connective tissue elements, surrounding the Sarcocystis muris infected muscle fibers. As earlier, the S. muris sarcocysts were examined in mice 1, 2.5, 6, and 10 months after sporocyst feeding. Within the first 2.5 months after infection, marked accumulations of lymphocyte-like cells and collagen fibres are observed in the endomysium, and simultaneously the activity of capillary endothelial cells is seen to enhance due to the appearance of much more micropinocytotic vesicles, compared to the uninfected control. All this may be qualified as the host organism protective reaction to the parasitic infection. 6 and 10 months after infection, not only collagen fibres, but also some other fibrillar structures of the endomysium undergo degradation, the damage of capillary endothelial cells starting from breaking the outer membrane (in 6 months) and terminating in lysing the whole cell (in 10 months). Besides, structural abnormalities were noticed in the axon endings.

[Cryptosporidium parvum (Apicomplexa: Sporozoa, Coccidia)--the optimization of a technic for isolating a large number of oocysts]. / Cryptosporidium parvum (Apicomplexa: Sporoza, Coccidia)--optimizatsiia tekhniki polucheniia bol'shoi massy ootsist.

The Waldman e. a. (1986) method of separation of Cryptosporidium spp. oocysts from feces by using a percoll discontinuous density gradient appeared a method of choice for obtaining large numbers of oocysts of C. parvum free of fecal contamination. Feces of 7-12 day old calves, spontaneously infected with C. parvum, were concentrated and purified by the above technique. The purified oocysts were shown to be infectious by inoculation of 6-9 day old rats with an average dose of 20,000 oocysts per animal. The rats shed oocysts after 4 days. At necropsy on day 4 postinoculation, the pattern of endogenous development appeared normal, when examined on frozen sections of fresh tissue, using the Bright cryostat, stained with hematoxylin and eosin. Samples of the clean sediment, presumably containing only oocysts of C. parvum, were smeared and stained with carbol fuchsin after Ziehl-Neelsen, and with gentian violet after Sidorenko (1988). With the latter technique, an intense gentian violet staining screened all the constituents of the smear, except the oocysts, which being "negatively stained" looked as small transparent spheres 4-5 mkm in diameter. But of special interest was the reaction of the smeared organisms with carbol fuchsin. Some organisms stained dark red and had a variable number of dark granules, seemingly on the surface; whereas others stained light reddish, if at all, and appeared as transparent spheres. It does not seem unlikely that the sediment, resulting from the final step of percoll separation, may contain, besides oocysts, some other endogenous stages (meronts, gamonts, thin-walled oocysts) that appeared in the lumen of the intestine because of an intense flow of diarrheal fluid during cryptosporidiosis. Unlike the thick walled oocysts, other endogenous stages are not covered with protective walls and thus fail to absorb acid fast staining. Segmented meronts were obviously observed on the rat fecal smears 96 hours after infection. This observation enables us to propose that newly infected hosts-recipients may obtain, with diarrheal fecal masses of infected donors, not only sporulated oocysts, but also some earlier developmental stages. Merozoites, released from the segmented meronts, could start in the intestine asexual rounds, thus shortening the resulting prepatent period. Fluctuations in prepatent period duration are characteristic of Cryptosporidium spp., and the above observation may be one of its explanations.

[Cellular interactions in the intracellular parasitism of cryptosporidia. I. The effect of Cryptosporidium parvum on the phosphatase activity in the small intestine enterocytes of experimentally infected newborn rat pups]. / Kletochnye vzaimodeistviia pri vnutrikletochnom parazitirovanii kriptosporidii. I. Vliianie kriptosporidii Cryptosporidium parvum na fosfataznuiu aktivnost' v énterotsitakh tonkoi kishki éksperimental'no zarazhennykh novorozhdennykh krysiat.

Cytochemical methods for detection of non-specific phosphatases were employed at the light microscope level for identification of enzymatic activity in the small intestine of new-born rats (6--11 days old), both infected and non-infected with the intestinal coccidium Cryptosporidium parvum. In the new-born rats, the level of alkaline and especially acid phosphatase is originally very low, suggesting their insignificant involvement in digestion processes in suckling animals compared to rats of older age (3 month old). However, a heavy colonization of the brush border of the intestinal villi of the new-born rats with cryptosporidia results in obvious inactivation of phosphatases in the infected enterocytes, in contrast to the neighbouring parasite-free host cells. The general picture of metabolic interaction between cells of a unicellular parasite (C. parvum) and those of its metazoan host (rat) much resembles that observed in the course of Elmeria spp. infection, but differs from that induced by Toxoplasma gondii endogenous stages in the cat intestine. Details of cell interaction with intracellular parasitism need additional studies at the ultrastructural level.