[Changes in the heart of neonatal rats after cryptosporidial gastroenteritis of different degrees of severity].

Disturbances at the childhood age increase risk of the appearance of cardiovascular diseases decades later. The nature of this interconnection called ontogenetic programming is not completely understood. Valuable sources of knowledge about mechanisms of ontogenetic programming are data of interspecies study of biology of the body life cycles and of heart physiological capabilities. Taken into account the interspecies differences, these data allow finding the correct direction of experimental investigations. Results of studies of almost 100 homoiothermal species have shown the slow growth and a high loading on the heart at postnatal development to decrease its aerobic capability in adults. Basing on these data, we suggested that the neonatal gastroenteritis causing tachyarrhythmia, malabsorption, and the growth deceleration might lead to pathological changes in the heart. Our task was to evaluate the effect of cryptosporidial gastroenteritis of different degrees of severity on heart of the neonatal rats. By using methods of Real-Time PCR, immunocytochemistry, image analysis, and study of interatrial septum, we have established that a gradual increase of intensity of infestation with Cryptosporidium parvum oocytes causes sharp changes corresponding to the "all or nothing" response. At a weak infestation the interatrial septum was close (like in control), while significant changes in expression of isoforms of heavy chains of alpha- and beta-myosin were absent. At the intermediate and severe infestation, in the interatrial septum the foramen ovale was visualized and there were observed cardiac atrophy and a strong shift of ration of expression of myosin heavy chains toward the low-velocity beta chain. Thus, by disturbing the frequency-strength ratios and causing outflow of resources from the formed heart, the neonatal gastroenteritis produces pathological changes of the organ molecular and anatomical structures. Our results can be interest to evolutionary biologists and physicians, as they show importance of knowledge of evolutionary-comparative investigations for the search for novel risk factors of heart diseases and demonstrate interconnection between gastroenteritis, pathology of interatrial septum, and a change of composition of the main contractile proteins in cardiomyocytes.

[Threshold rat neonatal cardiomyocyte response to gradual cryptosporidial infection severity increase].

Infectious gastroenteritis is one of the common causes of tachyarrythmia, malabsorbtion and growth retardation in children. Our recent studies have indicated that neonatal.cryptosporidial gastroenteritis is associated with long-term cardiomyocyte abnormalities. The aim of the present study was to find out how neonatal cryptosporidiosis of various severities affects cardiac anatomy and cardiomyocyte polyploidization, remodeling and HIF-1α expression. Using real-time PCR, cytometry, immunohistochemistry, image analysis and interatrial septum visual examination, we revealed that gradual increase in cryptosporidial invasion was associated with threshold changes. At weak parasitic infection, interatrial septum was entire and there was no statistically significant change in cardiomyocytes. At moderate and severe infection, all changes in cardiac anatomy and cardiomyocytes were statistically significant and demonstrated approximately similar degree. Compared to control, heart were atrophied and elongated, interatrial septum contained a small window (patentforamrn ovale), and cardiomyocytes lost protein, became elongated, thin and accumulated additional genomes. Also we found HIF-1α mRNA hyperexpression. Notable, the threshold response to gradual stimulus is an important criterion of development programming since such a response is commonly a consequence of abnormal anatomic structure formation and cell differentiation failure. Our results can be interesting for physicians because they indicate that even moderate cryptosporidiosis can be dangerous for neonatal heart and can trigger neonatal programming of cardiovascular pathology. Also, our results for the first time demonstrate the association between gastroenteritis, patent foramen ovale and cardiomyocyte malfunction.

[Rat cardiomyocyte remodeling after neonatal cryptosporidiosis. II. Elongation, excessive polyploidization and HIF-1alpha overexpression].

Retrospective epidemyological studies evidence that infant diseases leave survivors with an increased susceptibility to cardiovascular diseases in later life. At the same time, the mechanisms of this link remain poorly understood. Based on medical statistics reporting that infectious gastroenteritis is the most common cause of maladies in babies, infants and children, we analysed the effects of moderate cryptosporidial gastroenteritis on the heart and ventricular cardiomyocyte remodelling in rats of the first month of life. The disease was challenged by a worldwide human protozoic pathogen Cryptosporidium parvum (Apicomplexa, Sporozoa). The main symptoms manifested in the growth retardation moderate diarrhea. Using real-time PCR, cytophotometry, confocal microscopy and image analysis, we indicated that cryptosporidiosis was associated, with the atrophy heart and the elongation, narrowing, protein content decrease and hyperpolyploidization of cardiomyocytes and the moderate overexpression of hypoxia inducible factor 1alpha (HIF-1alpha) mRNA. Cardiomyocyte shape remodeling and heart atrophy presented in all age groups. The severity of these changes, hovewer, declined gradually from younger to older groups. In contrast, hyperpolyploidization and HIF-1alpha mRNA overexpression were registered mainly among animals aged between 6 and 13 days, and were barely detected and non-significant in older age groups. In the rat the time period covering 6-13 days after birth is known to coincide with the intensive cardiomyocyte polyploidization and the switch from proliferation to hypertrophy. Thus, our data indicate that neonatal cryptosporidiosis may be potential cardiovascular diseases risk factor and that one of the critical time windows for the growing heart covers the time period when cardiomyocyte undergo polyploidization.

[Cardiomyocyte myosin heavy chain composition change after cryptosporidial gastroenteritis].

Cardiovascular diseases are the most common case of human death in developed countries. Thus, the discovering of their new risk factors is of primary importance. Based on epidemiology studies, vertebrate life-history traits comparison and cross-species cardiomyocyte transcriptome analysis, we suggest that one of these factors could be infectious gastroenteritis. This disease outflows recourses from cardio-vascular system and triggers pathological stimuli, like tachyarrhythmia, inflammation, malapsorption and energy depletion thereby disturbing cardiomyocyte metabolism and function. To test this hypothesis, we challenged gastroenteritis in neonatal rats with widespread human parasite Cryptosporidium parvum (Apicomplexa, Sporozoa). The results obtained by the methods of immunocytochemistry, quantitative morphometry and real-time PCR, indicate that moderate cryptosporidiosis lasting four days induces dramatic shift in myosin isoform expression ration toward isoform beta (with low ATPase activity) both at mRNA (by 1.7-4.5 folds) and protein (by 2.5-6 folds) levels. Antithetical manner of this shift and coherence between changes in mRNA and protein suggest that cryptosporidiosis affects all main steps of a complex myosin heavy chain regulatory network. Since the overexpression of myosin heavy chain beta (showing several times lower ATPase activity than myosin heavy chain alfa) is a generally accepted marker of human cardiac failure, we can consider cryptosporidial gastroenteritis as a new risk factor of cardiac contractile ability impairment. Our data can be interesting for clinicians.

[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.

[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.

[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.

[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.

[Oocyst structure and problem of coccidian taxonomy]. / Struktura ootsist i voprosy sistematiki koktsidii.

A comparative ultrastructural study was made of both thin- and thick-walled oocysts of Cryptosporidium parvum. According to the authors' findings, all the oocysts in C. parvum should be considered as thin-walled, since their walls have been composed of a single membrane or of two, closely apposed membranes without any additional substance in between. Despite the presence of two types of wall-forming bodies (WFB) in the maturing macrogamete or zygote, there is no evidence of their involvement in oocyst wall formation. In this concern, the function and destiny of WFB in C. parvum oocysts still remain obscure. Similar structure of the oocysts wall was reported elsewhere for thin-walled oocysts of fish coccidia of the genera Goussia and Eimeria. In C. parvum, the "thick-walled" oocysts differ from oocysts with thin walls in the availability in the former of a single sporocyst. The sporocyst wall consists of two unequal layers: a thin outer layer and a thicker inner one, in which a characteristic suture line is occasionally seen. By this feature the thick-walled oocysts of C. parvum bear similarities with oocysts of the cyst-forming coccidia (Cystoisospora, Toxoplasma, Sarcocystis) and of the genus Goussia: in all these the valves making up the sporocyst wall are joint just along the suture line. The literary and the authors' own data make it possible to suppose that the suture detected in C. parvum oocysts is located in the sporocyst wall, joining its valves, rather than in the oocyst wall proper, known to be composed of one or two, closely apposed unit membranes. Again, the availability of a suture (or sutures) in the sporocyst hardly provides enough reason to relate C. parvum with either cyst-forming, or fish coccidia, since this structure itself may be of a convergency character, rather than of systematic value. This may be substantiated, at least in part, by the authors' previous findings (Beyer, Sidorenko, 1984) of a similar structure, originally referred to as a "slit channel", in the intraerythrocytic capsule around gamont stage of haemogregarines--the adeleid coccidia of the genus Karyolysus. The suture-like structure could have originated in the evolution independently in different groups of parasitic protozoa to serve eventually as a suitable mechanism for immediate separation of elements involved in protective formation harbouring different developmental stages, including, for example, sporozoites in the eimeriid coccidia, or gamonts in the adeleid coccidia.

[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.

[One more biological characteristic of coccidia in the genus Cryptosporidium (Sporozoa: Apicocomplexa)]. / Ob eshche odnoi biologicheskoi osobennosti koktsidii roda Cryptosporidium (Sporoza, Apicomplexa).

Suckling rats were used as a model host for our previous EM studies of the endogenous development of Cryptosporidium parvum isolated from spontaneously infected calves (Beier e. a., 1990; Beier, Sidorenko, 1990). In the course of repeated infections it was noticed that the oocysts discharged by the recipient host (rat) were obviously larger than those in the donor's (calf's) isolates. Keeping in mind the presumable taxonomic significance of coccidian oocysts as a constant and rather reliable tool for species discrimination we made a comparative quantitative and statistical analysis of the mean values of oocysts of C. parvum, originating from several sources (table 2): a random human isolate (N 1), several random isolates from spontaneously infected calves (N 2-5), isolates from calves (N 6-8) used as the infecting material for suckling rats, and fecal samples from the experimentally infected rats (N 9-15). The results obtained have shown that the oocysts discharged by rats (N 9-15) were larger that those of calf origin (N 6-8), with the differences being statistically significant with 95% confidence. Besides, within the same host (rat) at least two oocyst groups were distinguished (N 9-11 and 12-15, resp.) whose differences in mean values also appeared statistically significant. The larger oocysts displayed differences in morphology bearing distinct walls which were never observed either in the donor isolates or in the smaller population of the recipient oocysts. The established differences in oocyst dimensions lay presumably within the frames of the normal reaction of C. parvum and other Cryptosporidium species, due to biological peculiarities of unusual life cycles of these unique coccidia: their homoxenous (i.e. confined to one host body only) development is combined with polyxeny (i.e. a wide host specificity that involves the number of host species representing different environmental conditions). The parasite's ability to change its functional morphology and size may appear some kind of preadaption to the number of varying conditions met by these polyxenous coccidia. This and other relevant assumptions are discussed in the paper.

[Electron microscopic research on cryptosporidia. III. Parasite-host relations]. / Elektronno-mikroskopicheskoe issledovanie kriptosporidii. III. Parazito-khoziainnye otnosheniia.

A study was made of the host-parasite relationship with Cryptosporidium parvum (Apicomplexa, Sporozoa), which parasitizes the intestine of newborn rats experimentally infected with oocysts isolated from C. parvum-infected calves. The endogenous development of the parasite occurs extracytoplasmically in the microvillar compartment of the enterocytes. The formation of the extracytoplasmic parasitophorous vacuole (PV), like that surrounding the endogenous stages of C. parvum, is regarded as one of the possible and evolutionary established ways for the intracellular parasite to escape from the host cell lysosomal digestion. Special attention is paid to the attachment zone of C. parvum, where a multimembranous organelle is formed serving eventually as a feeder organelle. No other specialized cytostome, similar to the micropore of other coccidia, has been so far revealed in the growing stages of Cryptosporidium. The characteristic ultrastructural organization of the endogenous stages of C. parvum and of other Cryptosporidium species so far investigated, along with the peculiar structure of the cryptosporidia-surrounding PV, to say nothing of some other distinctive features--all this makes it possible to distinguish between the genus Cryptosporidium and other coccidian genera, and warrants the separation of the former into a separate family Cryptosporidiidae Léger, 1911. Unlike, the addition to this family, besides Cryptosporidium Tyzzer, 1910, of another genus, Epieimeria Dykova, 1981, on the ground of the "epicellular" localization of both the genera claimed by Levine (1984), seems hardly correct, due to the totally different patterns of ultrastructural organization and host-parasite relationship recently reported for Epieimeria anguillae by Molnar and Baska (1986).

[The detection of human cryptosporidiosis in Leningrad]. / Obnaruzhenie kriptosporidioza cheloveka v Leningrade.

Stool specimens from 127 adults with gastrointestinal disorders, 357 persons engaged in a livestock farming in the district of Leningrad were examined for Cryptosporidium oocysts with the Ziehl-Neelson routine. Five positive cases were established among the latter cohort. Besides, the study conducted at the hospitals of Leningrad that enrolled 373 children aged under 7 years revealed oocysts in 10 of them. Two of the latters had high temperature (39-40 degrees C) during 3 and 5 days, respectively. Common clinical symptoms of cryptosporidiosis were vomiting, profuse diarrhea and weakness. Accompanied with the normalization of the stool the mean recovery time was 7.4 days. One child recovered 15 days after the disease onset. For the first time human cryptobiosis was detected in the USSR (Leningrad). Its sources and the routes of transmission still remain unknown.

[An electron microscopic study of Cryptosporidium. II. The stages of gametogenesis and sporogony in Cryptosporidium parvum]. / Elektronno-mikroskopicheskoe issledovanie kriptosporidii. II. Stadii gametogeneza i sporogonii kriptosporidii Cryptosporidium parvum.

The ultrastructure of stages of gametogony and sporogony of C. parvum from the intestine of experimentally infected suckling rats was studied by transmission electron microscope. Unlike merogony, in which the whole cytoplasm of the mother meront is used up for the merozoite formation, during microgametogony the large residual mass of gamonts remains in contact with the feeder organelle even after microgamete outbudding. Unlike other coccidia, during the microgametogenesis in C. parvum, the nuclear substance of the daughter nuclei is not separated into osmiophilic (containing the condensed chromatin) and achromatinic parts. The gamete outbudding in C. parvum is accompanied by evagination of the pellicle of the mother gamont whose cytoplasm displays some slit-like canals that seem to sequester the daughter nuclei with some portion of the surrounding cytoplasm. The flagella-free microgametes of C. parvum resemble somatic cells, rather than male sexual cells of other coccidia. The study of thick-walled oocysts of C. parvum made it possible to suggest that the fragile wall of the oocyst proper may be easily destroyed in the course of processing of the material to look eventually as a ghost of electron lucent substance in the parasitophorous vacuole, whereas the structures revealed on the electronograms may presumably represent the outer and inner layers of the sporocyst. If so, the suture described elsewhere in the cryptosporidial oocysts, is to be considered as belonging to the sporocyst wall rather than to the oocyst wall, i.e. likely as in other investigated coccidia. However, the question on the mode of sporozoite excystment in the thin-walled oocysts of C. parvum still remains obscure.

[Electron microscopic research on Cryptosporidium. I. The asexual stages in the development of Cryptosporidium parvum]. / Elektronno-mikroskopicheskoe issledovanie kriptosporidii. I. Bespolye stadii razvitiia Cryptosporidium parvum.

Ultrastructural studies were conducted on asexual developmental stages of C. parvum in the ileal fragment of the intestine of 10-11 day old rats experimentally infected with oocysts isolated from calf feces. A young trophozoite is covered with the typical trimembranous apicomplexan pellicle. As the parasite grows, the inner complex of its apical pellicle, facing the host enterocyte, is seen to reduce up to a unit membrane to make a complex multimembranous "feeding organelle" which is in contact with a thick electron dense band bordering the host-parasite interface. It looks likely that no micropores or any other feeding structures exist in the parasite. Unlike, the opposite body part of the trophozoite, facing the lumen of the intestine, preserves its trimembranous pellicle. Two merozoite generations were followed. In addition to numerous ribosomes, rhoptries, micronemes, and trimembranous pellicle, subpellicular microtubules were observed in the segmenting merozoites. The merogony follows the pattern of ectomeric schizogony. However, no details of nuclear division were detected. The whole cytoplasm of the mother meront is completely used up for the merozoite formation without any residual mass to be left.

[Electron microscopic study of the cyst of the sarcosporidian, Sarcocystis ovifelis]. / Elektronno-mikroskopicheskoe izuchenie tsisty sarkosporidii Sarcocystis ovifelis.

Cysts of S. ovifelis, examined from the sheep oesophagus muscles have been shown to be covered by a cyst wall made of a primary and a secondary envelopes. Within the cyst, three morphologically different cell types are distinguished: metrocytes, merozoites, and interstitial cells. The latter have been first discovered for the genus Sarcocystis, in addition to earlier literature evidence of their availability in cysts of the genus Frenkelia.