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Using a combination of morphological and molecular data, we describe a new apicomplexan parasite, Isospora svecica sp. n., from the white-spotted bluethroat, Luscinia svecica cyanecula, from the Czech Republic. Oocysts were found in its intestinal tract. Sporulation was exogenous and took 1-3 days. The oocysts were slightly ellipsoidal, of average size 26.17 × 20.33 μm, with a smooth bilayered wall. Micropyle, oocyst residuum, and polar granules were absent. Sporocysts were bottle-shaped, of an average size of 18.82 × 8.82 μm, with a thin, colourless wall. A conspicuous knob-like Stieda body was present. Substieda body was barely visible. Sporocyst residuum was present in the form of granules of various sizes. Sporozoites were banana-shaped and contained large anterior and small posterior refractile bodies. Partial DNA sequences of three genes were obtained from oocysts of Isospora svecica sp. n., being most closely related to other isosporans described from passerines. Little is known about the parasites of the avian family Muscicapidae, including coccidia, a highly prevalent parasitic protist group in all vertebrate classes. Only six species of the genus Isospora have so far been described in Muscicapidae, together with several "Isospora sp." that in fact most likely represent Isospora lacazei. The newly described Isospora svecica sp. n. differs morphologically from other coccidia reported from muscicapid birds, and represents the first coccidian species described from Luscinia svecica.

Background: The gnateaters Conopophaga spp. are insectivorous passerines commonly observed in high and humid forests, where they remain lodged in thin branches and, sometimes, they fly to the ground to catch insects. The insectivorous feeding habit is related to low prevalence and density of coccidians in passerines; however, several coccidian species are recorded for families of insectivorous passerines., Purpose: This study aimed to examine the feces from gnateaters Conopophaga spp. captured in the municipality of Barra Mansa and in the Itatiaia National Park, State of Rio de Janeiro, Southeastern Brazil, to determine what coccidian parasites were present., Methods: Nine gnateaters were captured with mist nets. Coccidian oocysts were recovered from the fecal samples by flotation in Sheather's saturated solution. Morphological observations, line drawings, photomicrographs and measurements were made in optical microscopy and digitally edited. The molecular analysis included the study of the sequence of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene, with phylogenetic reconstructions based on the neighbor-joining and maximum likelihood analysis., Results: Four Conopophaga spp. were positive for oocysts. An Isospora sp. considered as new to science is described and identified from Conopophaga melanops (Vieillot, 1818) and Conopophaga lineata (Wied, 1831). Isospora borbai n. sp. has oocysts that are subspheroidal, 17-22 × 15-22 (20.2 × 19.1) µm, with rough, bilayered wall, c.1.7 μm thick. Micropyle present, but without micropyle cap. Oocyst residuum absent, but one or two polar granules are present. Sporocysts are ellipsoidal, 12-15 × 8-11 (14.1 × 9.1) µm. The Stieda body is knob-like to half-moon-shaped and sub-Stieda body is rounded. Sporocyst residuum is present, composed of scattered spherules of different sizes. Sporozoites are vermiform with refractile body and nucleus. Molecular analysis at the cox1 gene exhibited similarity greater than 99% with Isospora spp. isolates from other Neotropical passerine birds., Conclusion: Based on the morphological and molecular features, I. borbai is considered as new to science and the first coccidian species recorded from Conopophagidae.

Cystoisospora (Isospora) belli is a coccidian parasite of humans. It can cause serious digestive disorders involving infection of intestines, biliary tract and gallbladder, especially in those with depressed immunity. It has a direct fecal-oral transmission cycle. After ingestion of sporulated oocysts, the parasite multiplies asexually and sexually within host epithelial cells, resulting in unsporulated oocysts that are excreted in feces. The details of asexual and sexual stages are not known and certain inclusions in epithelial cells in biopsy samples have been erroneously identified recently as C. belli. Here, we provide details of developmental stages of C. belli in two patients, in duodenal biopsy of one and biliary epithelium of the other. Immature and mature asexual stages (schizonts/meronts) were seen in epithelial cells. The merozoites were seen singly, in pairs and in groups in single parasitophorous vacuole (pv) in host cytoplasm. Immature and mature meronts were seen together in the same pv; up to eight nuclei were seen in meronts that retained elongated crescent shape; round multinucleated schizonts, seen in other coccidians, were not found. Meronts were up to 25 µm long and contained up to ten merozoites that were 8-11 µm long. The merozoites and meronts contained PAS-positive granules. Microgamonts (male) contained up to 30 nuclei that were arranged at the periphery and had condensed chromatin; 1-3 PAS-positive, eosinophilic, residual bodies were left when microgametes were formed. The microgametes were 4 µm long and PAS-negative. All stages of macrogamonts, including oocysts were PAS-positive. The detailed description of the life cycle stages of C. belli reported here should facilitate in histopathologic diagnosis of this parasite.

Coprological analysis of a sample from one free-living hedgehog was done with the use of a direct flotation method with additional incubation of fecal material. The study revealed three types of eggs and oocysts in the feces. The most commonly diagnosed were oocysts of Isospora rastegaievae (543/3g), while oocysts of Monocystis sp. (267/3g) and eggs of Aonchotheca/Eucoleus spp. (52/3g) were seen less often. This is the first report of coccidia I. rastegaievae (Apicomplexa: Eimeriida) and acephaline gregarine Monocystis sp. (Apicomplexa: Eugregarinida) infection in a hedgehog in Poland.

The New World tyrant-flycatcher (Tyrannidae) Attila rufus (Vieillot, 1819) is commonly known as grey-hooded attila or 'capitão-de-saíra' in Brazil (Sick 1997; CBRO 2014). This species has a wide distribution and their population trends appear to be stable; therefore, it is least concern according to IUCN (2015) criteria.

Coccidian parasites are of major importance in animal production, public health and food safety. The most frequently used representative in basic research on this group is Toxoplasma gondii. Although this parasite is well investigated there is no adequate in vitro model for its sexual development available and knowledge on this important life cycle phase is therefore scarce. The use of Isosporasuis, a sister taxon to T. gondii and the causative agent of piglet coccidiosis, could provide a solution for this. In the present study an in vitro model for neonatal porcine coccidiosis in cells representative for the in vivo situation in the piglet gut was developed and evaluated. The parasite development was investigated by light and transmission electron microscopy and optimum culture conditions were evaluated. Intestinal porcine epithelial cells (IPEC-J2) adequately representing the natural host cells supported the development of all endogenous life cycle stages of I. suis, including gametocytes and oocysts. A concentration of 5% fetal calf serum in the culture medium led to highest gametocyte densities on day 12 post infection. Low infection doses (≤1 sporozoite for 100 host cells) were best for oocyst and gametocyte development. The presented system can also be used for immunostaining with established antibodies developed against T. gondii (in our case, anti-TgIMC3 antibodies directed against the inner membrane complex 3). The complete life cycle of I. suis in a cell line representing the natural host cell type and species provides a unique model among coccidian parasites and can be used to address a wide range of topics, especially with regard to the sexual development of coccidia.

In this study, 51 piglets originating from five different sows were included in the investigations. The animal source of all sows had a history of Clostridium perfringens type A (β2) infection. The piglets of three sows (n = 31) were experimentally infected with Isospora suis within the first 4 h after birth and were randomly assigned to the treatment group or the sham-dosing group. The piglets of the two remaining sows (n = 20) served as I. suis-uninfected controls. Twelve hours post-infection, the animals in the treatment group (n = 15) were treated with toltrazuril (20 mg/kg BW, Baycox® 5% suspension). During an observation period of 14 days faecal consistency, faecal oocyst counts, faecal germ counts, mortality, body weight development and clinical status were recorded. One piglet per study group and litter was necropsied, and intestinal tissue samples were taken for histopathological investigations and in situ hybridisation on study days (SDs) 3 and 14. I. suis-infected but untreated piglets showed clinical disease resulting in liquefaction of faeces and decreased body weight development. In 59.2% of the observations, I. suis-infected but untreated piglets showed abnormal faecal consistencies whereas only 12.0% or respectively 4.4% of the faecal samples had a pasty consistency in the I. suis-infected-treated or in the control animals. The mean body weight at the end of the study was 3.37 kg in the I. suis-infected but untreated piglets while the average body weight in the I. suis-infected-treated animals was calculated as 4.42 kg and the control animal's mean body weight was 4.45 kg. Moreover, mortality, occurring between SDs 8 and 14, in this study group was 38.5% (n = 5), with 30.8% (n = 4) died from necrotic enteritis. In contrast, no piglets died in the I. suis-uninfected control group or in the toltrazuril-treated study group. The results of this study corroborate the hypothesis that simultaneous infection with I. suis and C. perfringens type A soon after birth leads to distinct interactions between the two pathogens and result in an increase in clinical disease, mortality and metabolically active C. perfringens type A.

Four of 17 cirl buntings (Emberiza cirlus) involved in a trial translocation in 2004 for conservation purposes died and were examined postmortem. Two of the cirl buntings showed intestinal and hepatic lesions, including necrotising enteritis, consistent with isosporoid coccidiosis, and a third had an intestinal infestation of isosporoid coccidia. Sporulated oocysts from faecal samples from the birds were identified as Isospora normanlevinei, a parasite previously detected in cirl bunting populations in continental Europe. In a subsequent translocation of 75 cirl buntings from Devon to Cornwall in 2006, each brood of birds was placed in strict quarantine at low stocking density, with improved hygienic precautions and detailed health surveillance, and each bird was treated prophylactically with toltrazuril in an attempt to control the disease but not eliminate the I normanlevinei parasites. Seventy-two of the 75 birds were successfully reared and released, and there were no apparent clinical or pathological signs of isosporoid coccidiosis in any bird. I normanlevinei was detected in the released population, an indication that it had been successfully conserved.

Summary: Highly purified antigen and appropriate controls are essential for antigen-specific immunoassays. In the case of Isospora suis, the causative agent of neonatal porcine coccidiosis, the only current source of antigen is oocysts isolated from faeces. The aim of this study was to develop a procedure for high-grade purification of I. suis oocysts from piglet faeces to obtain both antigen and representative controls suitable for in vitro re-stimulation of lymphocytes. This was achieved by use of filtration, density-gradient centrifugation and fluorescence-activated cell sorting (FACS). The feasibility for immunological studies was demonstrated with IFN-gamma ELISPOT assays after in vitro re-stimulation of lymphocytes from previously infected swine using the obtained antigen. The developed method allowed the production of highly purified antigen and representative controls from faeces with an oocyst recovery rate of 14%. Regarding the application of the obtained material it could be shown that lymphocytes from I. suis-infected pigs react in an antigen-specific manner in terms of an in vitro recall response by the production of IFN-gamma. This demonstrates the suitability of the developed method for the production of antigen and controls for sensitive immunological readout systems. Moreover, the detected specific IFN-gamma response encourages further functional studies on the cellular immune response to I. suis.

Isospora suis is an important parasitic infection in intensive pig production worldwide, responsible for significant economic losses. In this study the efficacy of toltrazuril treatment against isosporosis was evaluated, under field conditions and throughout the nursing period, in reducing (i) the mean time to onset of diarrhoea and oocyst excretion, (ii) the odds of diarrhoea and, (iii) the odds and level of oocyst excretion, adjusting for the heterogeneity of I. suis infection among litters and across time. In a 300-sow farrow-to-finish commercial operation, twenty-five litters were randomly allocated to receive toltrazuril (thirteen litters) or no treatment (twelve litters). The course of infection was followed in all piglets by coprological examination from day 6 after farrowing until weaning. Parametric shared frailty models, generalised linear mixed models and a two-part random effects model were used in the analyses. Treated piglets had longer mean time to onset of oocyst excretion, lower odds of excreting oocysts and lower mean amount of excreted oocysts on any day during the nursing period. Diarrhoea was less likely to occur in treated piglets. Variance partition coefficients revealed that almost half of the variation in the odds of oocyst excretion and diarrhoea was ascribed to unknown or unmeasured factors that operate at higher than the piglet levels of aggregation. Thus, beyond toltrazuril treatment, control of isosporosis in commercial pig farms can be improved by identification and quantification of these factors., ((c) 2010 Elsevier B.V. All rights reserved.)

Isospora suis is a coccidian parasite infecting piglets soon after birth. While the gross epidemiology of I. suis is well known, little knowledge exists on the ecology of the oocysts. To study the development and survival of oocysts of I. suis under controlled laboratory conditions, known numbers of oocysts ( approximately 200 in each of 4 replicates) were exposed to all combinations of 4 relative humidities (53-100% RH) and 3 temperatures (20 degrees , 25 degrees , 30 degrees C). Determination of viability was based on morphological and fluorescent properties of the oocyst as well as on the permeability of the oocyst wall characterized by inclusion/exclusion of the fluorescent dye propidium iodide. The viability of the oocysts was studied over time by fluorescence and light microscopy until <5% of the oocysts were considered to be viable. The sporulation rate increased with temperature, however, the infective sporocyst stage was reached within 24h at all temperatures, while RH did not seem to affect sporulation. Results show a rapid reduction in viable oocysts exposed to high temperatures (25 degrees C and 30 degrees C) in combination with low relative humidities (53% RH and 62% RH), at which conditions oocysts died within 24h. Viability was higher when oocysts were exposed to higher relative humidities (75% RH and 100% RH) as well as a lower temperature (20 degrees C). However, even at 75% RH the oocysts died within 24-60 h at 30 degrees C to 20 degrees C, respectively, while the most favourable condition appeared to be 100% RH and 25 degrees C at which condition the percentage of viable oocysts decreased from 100% to 17% in 96 h. The results indicate that it may be possible to reduce the infection pressure of I. suis in modern sow herds by changing the environmental conditions and/or the management within the farrowing pens, and thereby increase animal welfare without relying on the use of routine medication.

The aim of this study was to investigate the intra-litter infection dynamics of Isospora suis under natural conditions, and to study any association between parasite transmission and the contamination level of the farrowing pen by applying different interventions in order to reduce the transmission of I. suis infection within the litter. The study was divided in 2 trials including in total 22 litters (254 piglets). The first trial included 4 litters (where standard procedures practiced routinely on the farm piglets were applied) and the piglets were followed coprologically from farrowing until 2 weeks after weaning. The sows of those litters were also examined at various intervals before and after farrowing. The second trial included the application of 3 different management procedures: (A) standard farm hygiene and management procedures, (B) standard farm hygiene and management procedures+the first piglets found to excrete I. suis oocysts in each pen were removed from the pen, and (C) reduced cleaning. Each procedure was studied in 2 litters. This was replicated 3 times to yield a total of 18 litters. The results suggested that (i) the sow does not play an important role in transmission of I. suis in the farrowing pen; (ii) in natural infections, both the age of the piglet age at onset of oocyst excretion and the oocyst excretion patterns may vary considerably; (iii) the course of oocyst excretion or development of diarrhoea is related to the time of initial infection and (iii) piglets, which are heavy at birth, are more prone to acquire I. suis infection. Moreover, it was demonstrated that cleaning could be an effective means of restricting the spread of the parasite within the litter and thus the development of diarrhoea.

Faecal samples were collected from four 8 days old snow bunting nestlings from one nest in Ny-Alesund, Spitsbergen, in summer 2006. After sporulation, samples were examined for coccidian parasites using flotation centrifuging. We found isosporan oocysts in three birds, intensity of infection varied between individuals from 35 to 6,000 oocysts per defecation. All oocysts belonged to one species, which is described here as a new species. The spherical or subspherical oocysts (Fig. 1) have a brownish, smooth, relatively thin (about 1.1 microm) bilayered wall. Average size of sporulated oocysts was 26.2 +/- 0.13 x 23.6 +/- 0.16 microm (24.1-28.4 x 21.5-26.9; n = 10) with a shape index (length/width) of 1.11 +/- 0.01 (1.01-1.29). The sporulated oocysts have no micropyle or residuum but enclose one large (3.3 x 2.8 microm) ring-formed polar granule. The sporocysts are ovoidal, slightly pointed at the end opposite the Stieda body, 18.2 +/- 0.06 x 9.9 +/- 0.03 microm (17.1-19.0 x 9.0-10.8; n = 14), shape index 1.85 +/- 0.008 (1.70-1.99). The Stieda body has a prominent knob-like cap and a well-visible round substieda body. Sporocysts contain compact sporocyst residuum composed of small, uniform granules and sporozoits with usually three large refractile bodies and a smaller nucleus. The prepatent period is less than 8 days. This is the first description of an avian isosporan parasite that succeeds transmission while in the High Arctic.

Isospora michaelbakeri is one of the Isospora species most commonly found in the wild field, which can cause severe infection and mortality in young sparrows. In this study, we selected I. michaelbakeri (Chung Hsing strain) as a pathogen to orally inoculate russet sparrows (Passer rutilans), spotted munia (Lonchura punctulata), canary (Serinus canaria), Java sparrows (Padda oryzivora), chicken (Gallus domesticus), ducks (Anas platyrhynchos) and BALB/c mice. The results indicated that I. michaelbakeri infected only russet sparrows. Infected sparrows displayed lethargy, muscular weakness and fluffy feathers, followed by rapid death. Liver and spleen enlargement was seen in the infected birds. Schizonts were identified in thin smears from the venous blood, enlarged livers and spleens. Histopathological examination revealed schizonts and merozoites from the liver and spleen of infected russet sparrows, but not from other species experimentally inoculated with I. michaelbakeri in the present study.

Isospora carliae sp. n. is described from the blue-throated rainbow skink Carlia rhomboidalis (Peters), from Daintree Forest, North Queensland, Australia. Oocysts are ellipsoidal, 16.8-21.0 x 12.6-15.4 microm in size, with their two sporocysts, 9.0-14.0 x 7.0-9.24 microm in size, positioned along the wide axis. Sporozoites contain a distinct refractile body and are accompanied by a residuum. All endogenous development occurs within the host-cell nucleus. Nuclei are sometimes invaded by several merozoites, but only infections by a single parasite persist. Nuclei lodging meronts, mature microgamonts and premature macrogamonts have an elongate shape. Some meronts exhibit a membrane-bound cytoplasmic inclusion that contains many micronemes.

Three new species of coccidia are described from Marble-throated skink Marmorosphax tricolor from New Caledonia, namely, Isospora bocagei sp. n., Acroeimeria rouxi sp. n., and Choleoeimeria sadlieri sp.n. All species differ markedly from other eimerian coccidia described from scincid hosts. Isospora marmorosphaxi develops extra-nuclearly in small intestine. A. rouxi develops epicitoplasmatically in small intestine. C. sadlieri affects the gall bladder mucosa. Generic affiliation of Eimeria-like coccidia from reptiles is discussed and all taxa (with adequate information on endogenous development available) from scincid hosts are revised and placed into genera Acroeimeria and Choleoeimeria.

Two hundred and twenty-four anurans of 6 species (47 adults and 16 tadpoles of Rana blairi, 35 R. catesbeiana, 31 Hyla chrysoscelis, 30 adults and 46 tadpoles of Pseudacris triseriata triseriata, 11 Bufo woodhousii, and 8 Acris crepitans) from Pawnee Lake, Lancaster County, Nebraska, were surveyed for coccidian parasites during March 2001 to May 2002. Of these, 23 of 30 (77%) adults and 4 of 46 (9%) tadpoles of P. t. triseriata shed oocysts of Isospora cogginsi n. sp. Oocysts of I. cogginsi were ovoid, 19.3 x 15.1 (18-23 x 11-20) microm, with a thin, smooth, colorless, single-layered wall, with no micropyle or oocyst residuum. Sporocysts were ovoid, 13.3 x 9.9 (11-15 x 9-13) microm, with a thin, colorless, smooth wall, and Stieda body absent. Sporocyst residuum was present, 5.5 x 5.3 (4-7 x 4-7) microm, consisting of numerous granules. Histological examination of frogs and tadpoles infected with the new species revealed endogenous stages including mature meronts, developing microgamonts, mature microgametes, mature macrogamonts, and young unsporulated oocysts located in the cytoplasm of the epithelial cells of the small intestine. Concurrently, 2 adult P. t. triseriata shed oocysts of Eimeria streckeri. Oocysts of E. streckeri were spherical, 15.7 x 15.4 (14-17 x 14-19) microm, with a thin, smooth, single-layered, colorless wall with an oocyst residuum composed of numerous granules surrounding a large vacuolated area, with a previously undescribed globularlike body present within the vacuole, and no micropyle. Sporocysts were ovoid, 9.1 x 6.1 (7-10 x 5-7) microm, with a thin, colorless, smooth wall with a Stieda body and sporocyst residuum. Our results are the first to document infection of adult and tadpole stages of frogs of the same species with the same species of coccidian, indicating that adult frogs may contaminate breeding ponds with oocysts during their breeding season and infect tadpoles directly by the ingestion of sporulated oocysts.

Between January and March 2001, eight 4- to 8-wk-old camels (Camelus dromedarius) from 2 farms from Dubai area of the United Arab Emirates were submitted for necropsy examination. The camels had diarrhea of 2-5 days duration. Grossly, a severe diphtheroid-to-hemorrhagic colitis was seen in all animals. Gamonts, unsporulated oocysts, sporulating oocysts, and fully sporulated oocysts were present in the intestinal epithelium and the lamina propria. Fully sporulated oocysts contained 2 sporocysts and 4 sporozoites in each sporocyst. Oocysts from fecal samples resembled oocysts of Isospora orlovi. This is the first report of an isosporan parasite associated with hemorrhagic enteritis in the large intestine of any animal.

Four new species of Isospora are described from Australian geckoes. Isospora gehyrae n. sp. from Gehyra cf. variegata in South Australia have 18.5-22.5 x 17.5-20.0 microm oöcysts with 10.0-12.5 x 7.5-9.0 microm sporocysts; endogenous stages develop in the host cell cytoplasm. Of the two species found in Heteronotia binoei from northern Queensland, Isospora cytoheteronotis n. sp., with oöcysts of 20.0-26.0 x 17.5-25.0 microm and sporocysts of 10.0-13.5 x 7.5-11.5 microm, undergoes endogenous development in its host cell cytoplasm, whereas I. nucleoheteronotis n. sp., with oöcysts of 17.5-22.5 x 17.5-21.5 microm and sporocysts of 9.0-12.5 x 6.5-10.0 microm, develops in the host cell nucleus. I. oedurae n. sp. from Oedura rhombifer in northern Queensland has oöcysts of 22.5-25.0 x 22.5-24.0 microm and sporocysts of 12.5-14.0 x 7.5-11.5 microm, and undergoes endogenous development in its host cell nuclei.

Parasites from swine faeces were examined for autofluorescence. Oocysts of Eimeria polita, E. scabra and Isospora suis, cysts of Balantidium coli and eggs of Oesophagostomum dentatum, Strongyloides ransomi and Trichuris suis (but not those of Ascaris suum) emitted light after excitation with UV light. I. suis oocyst counts in McMaster chambers utilising autofluorescence were compared to those from conventional bright field microscopy. Similarly, faecal smears containing I. suis were examined using the same techniques. Autofluorescence was superior to bright field microscopy in detecting oocysts after flotation and was highly significantly more sensitive when direct smears were examined.

Isospora suis and Eimeria are frequent coccidian parasites of pigs. The unsporulated oocysts of Eimeria species and of I. suis are difficult to differentiate. Therefore, a species-specific PCR was developed. PCR products were amplified from Eimeria polita, Eimeria porci, and Eimeria scabra using primers from the conserved 18S rRNA regions and were subsequently sequenced. Based on variable sequence regions, primers were constructed for the differentiation of the three Eimeria species and I. suis. Using a combination of PCRs detecting one or two species, all four coccidian species were detected (theoretical lower detection level: DNA content of 250 oocysts of each Eimeria species or 25 oocysts of Isospora in 1microl) and differentiated. The PCR-based differentiation of the above mentioned species provides a useful alternative to microscopy.

Parasitological examination of European otter originating from Extremadura, Spain revealed the presence of a new isosporan species. Oöcysts of Isospora lutrae n. sp. are spherical to subspherical, 31.2 (27.5-32) x 29.6 (28-31) microm and have a smooth wall c. 1 microm thick. Sporocysts are ellipsoidal, 18.2 (17-19) x 14.4 (14-16) microm and lack Stieda and substieda bodies. A spherical sporocyst residuum is present, consisting of granules scattered among the sporozoites. Sporozoites are spindle-shaped, 12.4 x 2.5 microm and have anterior and posterior refractile bodies. Based on its unique morphologic structure and host, I. lutrae is considered to be new.

Fifty-seven Hirundo rubicola savignii swallows were collected from Damietta, Tanta, Dakahlyia and Sharkia Provinces, Egypt. They were examined for coccidian parasites. The percentage of infection with Isospora stages was 12.3%. After diagnosis it was noted that the parasites belong to a new species. The unsporulated oocysts were spherical, measuring 25.7-31.9 microm in diameter with a mean of 28.3 microm. A micropyle, polar granule and oocyst residuum were absent. Sporocysts appeared lemon-shaped and measured 19.6-24.5 microm x 12.5-17.5 microm with a mean of 22.9 x 14.4 microm. Stieda body and the sporocyst's residual body were clearly visible. Sporozoites measured 11-14.2 x 4.4-5.1 microm with a mean of 11.4 x 4.7 microm. Sporulation time was 72 h at room temperature (25 degrees C). Endogenous stages including schizogony and gamogony were detected in epithelial cells of the duodenum, jejunum and ileum of the host. Schizogony consisted of two generations. Mature first generation schizonts reached up to 11 microm in diameter and produced merozoites measuring 3.5 x 1.7 microm. Mature second generation schizonts measured 17.2 x 12.3 microm and produced merozoites measuring 11.8 x 2.2 microm. Gamogonic stages were differentiated into microgamonts and macrogamonts. Mature microgamonts were spherical and measured 23.2 microm in diameter, producing curved microgametes measuring 4.5 x 0.7 microm. The ovoid macrogametes measured 19.6 x 14.7 microm and were characterized by a large nucleus and nucleolus. Early, more or less spherical, oocysts were detected inside the intestinal epithelial cells and in the intestinal lumen. They measured 19.6 microm in diameter. The sporont measured 17.2 microm in diameter. Cytochemical studies on schizogony, gamogony and oocysts were accomplished and showed distribution of polysaccharides and composition of the oocyst wall.

A new species of isosporan parasite is described from the faecal contents of the buff-throated foliage-gleaner Automolus ochrolaemus and the olive-backed foliage-gleaner A. infuscatus collected in the rainforests of eastern Ecuador. Sporulated oöcysts are subspherical to ovoidal, 23.4 x 21.3 (18-28 x 17-24) microns, colourless, with a smooth, double-layered wall with the inner layer darker and thinner; the shape-index (length/width) is 1.1 (1-1.22). Oöcysts contain one polar granule, but lack a micropyle and an oöcyst residuum. Sporocysts are ovoidal, 15.4 x 9.9 (14-17 x 8-11) microns, with a smooth single-layered wall and a small nipple-like Stieda body attached to a small, inconspicuous, acentric substieda body. Sporozoites are vermiform with one prominent posterior, refractile body (c. 4 x 5 microns), and a centrally located nucleus of equal size. Sporozoites are randomly arranged in the sporocysts with a subspherical sporocyst residuum composed of coarse granules.

Redescriptions are given of the mature oocysts of Isospora ameivae Carini, 1932, from the teiid lizard Ameiva ameiva, and Isospora hemidactyli Carini,1936 from the gecko Hemidactylus mabouia, in north Brazil. The endogenous stages of the two parasites in the small intestine are described. Those of I. ameivae are intracytoplasmic, whereas those of I. hemidactyli are intranuclear.

Cryptosporidium parvum and Isospora belli oocysts stained with carbol-fuchsin, as in a modified Ziehl Neelsen technique, fluoresce bright red under green light (546nm). Cryptosporidium oocysts tend to fluoresce more brightly the less intensely stained they appear under transmitted light; this is not the case with Isospora. Fuchsin-stained Cyclospora cayetanensis oocysts fluoresce rather dimly, but those not taking the dye retain their typical autofluorescence. Cryptosporidium and Isospora oocysts are also autofluorescent, appearing violet under u.v. light (365 nm), and green under violet (405 nm) and blue-violet light (436 nm). Their autofluorescence does not survive the staining procedure.

A survey of Isospora suis performed in 177 faecal samples from 30 swine farms detected thin wall type I. suis oocysts in seven samples. This type of oocyst measuring 23.9 by 20.7 microns had a retracted thin wall similar to that of the genus Sarcocystis. This type of oocysts, isolated from four different faecal samples, was inoculated in four-five-days-old piglets free of contamination in order to verify the life cycle and pathogenicity of the species. The pigs were kept in individual metal cages and fed with cow milk. Daily faecal collections and examinations were performed until the 21st day after infection. MacMaster and Sheather's methods were used for oocyst counting and identification. Infected piglets produced yellowish-pasty diarrhoea with slight dehydration. The prepatent and patent periods were respectively from 6 to 9 and 3 to 10 days after infection. Oocyst elimination was interrupted on the 10th and 11th days after infection with biphasic cycles. Thin and thick wall oocysts were detected in the same faecal samples. Thin walls were not observed in unsporulated oocytes. The observations suggest that this type of oocysts could appear in specific strains which occur in the later stages of their development. These oocysts seem to be responsible for clinical and pathogenic signs of neonatal isosporosis in pigs.

The fine structure of the endogenous stages of five species of Isospora of lacertile hosts is reviewed and compared. All species were intranuclear and were the following: I. deserti, from Agama pallida from Israel; I. cannoni, from Diporiphora australis from Queensland, Australia; and new, yet undescribed species from Hemidactylus turcicus from Israel. Heteronota binoei from Queensland, and Carlia rhomboidalis from Queensland. In young infections the intranuclear parasitophorous vacuole (PV) maintained at one point a junction zone with the nucleolemma. Finding couples of trophozoites sharing the same PV suggests a binary division (or endodyogeny?) prior to merogony. There was some overall conformity in structure among the respective stages of the different species, particularly of the gamonts. Some interspecific differences were, however, evident in the texture of the wall-forming bodies. The wall formation followed the scheme described for avian and mammalian eimeriid coccidia. The smaller size Isospora from C. rhomboidalis exhibited fine structural peculiarities and affected its host nucleus differently. More conspicuous peculiarities were evident in the differentiation process of I. cannoni meronts, involving a formation of centrally positioned large inclusions, a mitochondrial plaque-like organelle and large uniquely structured vesicular mitochondria.

Development from inoculated sporozoites to unsporulated oocysts of Isospora suis Biester, 1934 is described in a swine testicular (ST) cell line. Sporozoites penetrated ST cells within 1 hr postinoculation (PI). Development was initially by endodyogeny to produce binucleate type I meronts and type I merozoites. Division by endodyogeny continued during the 13-day observation period and type I merozoites were the developmental stages most abundant at observation periods >3 days PI. Mutinucleate type II meronts and type II merozoites were first observed 7 days PI. Gamonts and oocysts were present 12 days PI. Oocysts did not sporulate in vitro. The ultrastructural features of stages were similar to those that occur in the pig host.

Protozoan parasites were the most frequently identified etiologic agents in waterborne disease outbreak from 1991 to 1994. The waterborne parasites Giardia lamblia, Naegleria fowleri, Acanthamoeba spp., Entamoeba histolytica, Cryptosporidium parvum, Cyclospora cayetanesis, Isospora belli, and the microsporidia are reviewed. For each parasite, the review includes history, life cycle, incidence, symptoms, and therapy. Clinical detection methods are compared, and emerging technologies are discussed. Information on the association of these parasites with waterborne outbreaks is reviewed. Current information on protozoan parasites identified as etiological agents in waterborne outbreaks is discussed. Water industry issues related to recent disease outbreaks are examined in the context of water quality testing regulations for G. lamblia and those proposed for C. parvum. The review identifies the limitations of the American Society of Testing and Materials water-testing method for these parasites. An overview of federal regulations affecting the water industry and laboratories that test for water quality is also provided. The article highlights the importance of the clinical laboratory as a frontline defense for the detection of infectious organisms. The review points to the need for clinical laboratories, physicians, and public health personnel to cooperatively plan and assess the challenge of meeting this potential public health threat.

Coccidial parasites of the genus Isospora cause intestinal disease in several mammalian host species. These protozoal parasites have asexual and sexual stages within intestinal cells of their hosts and produce an environmentally resistant cyst stage, the oocyst. Infections are acquired by the ingestion of infective (sporulated) oocysts in contaminated food or water. Some species of mammalian Isospora have evolved the ability to use paratenic (transport) hosts. In these cases, infections can be acquired by ingestion of an infected paratenic host. Human intestinal isosporiasis is caused by Isospora belli. Symptoms of I. belli infection in immunocompetent patients include diarrhea, steatorrhea, headache, fever, malaise, abdominal pain, vomiting, dehydration, and weight loss, blood is not usually present in the feces. The disease is often chronic, with parasites present in the feces or biopsy specimens for several months to years. Recurrences are common, Symptoms are more severe in AIDS patients, with the diarrhea being more watery. Extraintestinal stages of I. belli have been observed in AIDS patients but not immunocompetent patients. Treatment of I. belli infection with trimethoprim-sulfamethoxazole usually results in a rapid clinical response. Maintenance treatment with trimethoprim-sulfamethoxazole is needed because relapses often occur once treatment is stopped.

Meat samples were collected from the oesophagus and tongue of 38 camels slaughtered at the main abattoir of Al-Ahsa city, Saudi Arabia. Five cats and three dogs, conventionally reared and coccidia-free, were caged individually in steel cages. Camel meat was pooled, minced and fed to four cats and two dogs. One cat and one dog were not fed meat and were kept as noninfected controls. Faecal samples from infected and control animals were examined daily for a period of 2 months after feeding the meat. Three cats fed camel meat passed in their faeces oocysts of Toxoplasma gondii, Isospora felis and Isospora rivolta. The fourth cat passed only T. gondii and I. felis oocysts. One of the dogs fed camel meat passed oocysts of Isospora canis, Hammondia heydorni and Sarcocystis cameli sporocysts. The second dog excreted only S. cameli sporocysts.

A review article discussing the taxonomy, parasite life cycle and laboratory diagnosis of Isospora belli is presented. The pathology, epidemiology, clinical findings and therapy of human intestinal infection with Isospora belli are also addressed.

Based on the morphologic characteristics of oocysts of the genus Isospora, we have demonstrated that I. ohioensis is a relatively common Isospora species in dogs which are resident in Chonbuk province, Korea. The prepatent period of I. ohioensis was four days. The size of the unsporulated oocysts in fresh stool specimens was 22.9 x 19.8 microns (R = 1.16). The size of the contracted sporonts was 17.4 x 16.3 microns (R = 1.06). By 96 hours, sporulation is complete and the ratio of length/width was constant relatively. And the sizes of oocysts and sporocysts were 22.8 x 20.5 microns (R = 1.11) and 15.0 x 10.8 microns (R = 1.39), respectively. At this time it is most reliable for the measurements of oocyst sizes of I. ohioensis to provide with the identifiable clues against the others. It is therefore recommended that the clinical fecal specimens suspected of isosporosis should first be incubated and aerated for 96 hours before a definitive parasitological diagnosis can be reached. These and other observations contribute to the understanding of the biological characteristics and laboratory and clinical diagnosis of isosporosis.

A review on infections by Isospora belli and Sarcocystis spp. both in healthy and in AIDS patients is done on the basis of literature and personal data. In this view a special focus is made on isospora belli infection in AIDS because of its high recurrence after successful attack therapy. Consequently the most recent protocols for maintenance and attack therapy in these patients are reported. At the end, concerning ultrastructural pathology, the features of some Isospora belli developing stages are described by means of electron microscopy on duodenal biopsy specimens from a patient.

The ultrastructure of Isospora suis sporozoites, type-1 meronts, and type-1 merozoites was examined, using transmission electron microscopy of infected cultured cells. The ultrastructure of sporozoites and type-1 merozoites was similar. Each possessed trimembranous pellicles, subpellicular microtubules, a conoid, anterior and posterior polar rings, rhoptries, micronemes, a single vesicular nucleus, tubular mitochondria, Golgi complexes, ribosomes endoplasmic reticula, inactive micropores, amylopectin bodies, lipid bodies, dense bodies, and crystalloid bodies. Merozoites were produced by endodyogeny. Ultrastructural events associated with merozoite production by type-1 meronts are described.

I. belli is common in tropical and subtropical environments. Sporadic outbreaks have occurred in mental institutions and in day-care centers in the United States. The infection is common in immunosuppressed patients, particularly those with AIDS living in tropical areas. Transmission probably occurs by the fecal-oral route. Chronic diarrhea is the major clinical manifestation. The pathophysiology of I. belli-induced diarrhea is unknown. Laboratory diagnosis is readily performed with the examination of unstained and stained stool specimens particularly by the modified Ziehl-Nielsen method. I. belli infection responds promptly to trimethoprim-sulfamethoxazole therapy and recurrent disease can be prevented with either trimethoprim-sulfamethoxazole or pyrimethamine-sulfadoxine.

Faecal samples of 56 common house crows (Corvus splendens Vieillot) were collected from the Petaling Jaya and Kelang districts of Selangor, peninsular Malaysia, and examined for coccidia. Intestinal tracts of 8 of the above crows wee histologically examined under light microscopy to determine the site of coccidial infection and the endogenous stages present. Fifty three (94.6%) crows had coccidial oocysts morphologically conforming to only one species of Isospora in their faeces at the time they were examined. The sporulated oocysts were found to be Isospora corviae (Ray et al. 1952) which has been emended to I. corvi. These oocysts are redescribed in greater detail. Corvus splendens is a new host record for I. corvi. Coccidial infection was observed in all the intestinal tracts and generally confined to the anterior two thirds of the intestine. The parasites occurred within intestinal epithelial cells, located usually above the host cell nucleus. Developmental stages of both the asexual and sexual phases were found in the epithelium, and are deemed to be the endogenous stages of I. corvi on the basis of the oocysts recovered from the same crows used for histological study. These stages are described here for the first time. The prevalence of I. corvi, its relationship with the host C. splendens, and its probable transmission from C. macrorhynchus are discussed.

A review is given on recent knowledge of the genera Caryospora, Isospora, Cystoisospora, Hammondia, Toxoplasma, Besnoitia, Sarcocystis and Frenkelia. Caryospora hitherto considered to be monoxenous was found to have an optional intermediate host. Many Isospora species had to be transferred to the genus Cystoisospora because optional intermediate hosts were discovered. The obligatory two-host genus Hammondia could be confirmed to be distinct from the genus Toxoplasma. Even though the life cycles of Besnoitia wallacei and B. darlingi could be elucidated, the mode of transmission of B. besnoiti which is of considerable economic importance is still unknown. Since the discovery of the life cycle of the sarcosporidia in 1972 more than 600 papers have been published on this parasite. At present, 122 Sarcocystis species are named, and of 56 species both the definitive and intermediate hosts are known. It was shown that not only carnivorous and omnivorous mammals but also birds of prey, owls, and reptiles are definitive hosts of Sarcocystis species. the close relationship between the genera Sarcocystis and Frenkelia was confirmed by several investigators. Biology and Sarcocystis are of special interest. In the developmental cycle of most Sarcocystis species there are two schizogonic generations in endothelial cells of blood vessels, and in some species there is an additional asexual multiplication by endodyogeny in white blood cells. Some Sarcocystis species are highly pathogenic in non-immune intermediate hosts. Moreover, the sarcocysts which hitherto have been considered to be apathogenic may impair their hosts. In horses, they may cause myopathy, and in pigs, they have considerable influence on several parameters determining meat quality.

Mononucleated reticulo-endothelial stages of Isospora from Sparrows were studied by T. E. M. during the winter period when the extra intestinal infection is chronic. Results show a very slow evolution from merozoite to trophozoite stage, a transformation of the parasite nucleus and a characteristic reaction of the host cell. These observations are discussed in relation to what is known of hypnozoite and chronic schizogony of Plasmodium.

The endogenous development of Isospora rivolta (Grassi) was studied in cats fed oocysts, and was compared with the endogenous cycle after feeding them mice infected with I. rivolta. For the mouse-induced cycle, 14 newborn cats were killed 12 to 240 h after having been fed mesenteric lymph nodes and spleens ofmice. Asexual and sexual development occurred throughout the small intestine, in epithelial cells of the villi and glands of Lieberkühn. The number of asexual generations was not determined with certainty, but there were at least 3 structurally different meronts. Type I meronts appeared at 12-48 h postinoculation (HPT). They were 8.5(6-13) x 5.1(3-6) micrometer, contained 2-8 merozoites, and divide by binary division or endodyogeny. Type II meronts were multinucleate merozoite-shaped meronts within a single parasitophorous vacuole. They were found at 48-172 HPI and measured 12.6(9-18) x 9.8(9-13) micrometer. Individual multinucleate merozoite-shaped meronts were 7-13 x 3-5 micrometer in sections and contained 2-30 slender (5.5 x 1.0 micrometer) merozoites. Type III meronts occurred at 72-192 HPI and gamonts at 72-96 HPI. Mature microgamonts measured 11.3(9-15) x 8.0(6-9) micrometer in sections and up to 21.5 x 14 micrometer in smears, and contained up to 70 microgametes. Macrogamonts measured 13.3(11-18) x 9.0(5-13) micrometer in sections and 18 x 16 micrometer in smears, and contained up to 70 microgametes. Macrogamonts measured 13.3(11-18) x 9.0(5-13) micrometer. Sporulation was completed within 24 h at 22-26 C. For the study of the oocyst-induced cycle in cats, 18 newborn cats were killed between 6 and 192 HPI. The endogenous development was essentially similar to the mouse-induced cycle, but merogony and gametogony occurred 12-48 h later than in the latter cycle. Isospora rivolta was pathogenic for newborn but not for weaned cats. Newborn cats fed 10(6) sporocysts or infected mice usually developed diarrhea 3-4 days after inoculation. Microscopically, desquamation of the tips of the villi and cryptitis were seen in the ilium and cecum in association with meronts and gamonts. For the study of the development of I. rivolta in mice, mice were killed from day 1 to 23 months after having been fed 10(5)-10(6) sporocysts, and their tissues were examined for the parasites microscopically, and by feeding to cats. The following conclusions were drawn. (A) Isospora rivolta most freqeuntly invaded the mesenteric lymph nodes ofmice and remained there for 23 months at least. Ii also invaded the spleen, liver, and skeletal muscles of mice. This species could not be passed from mouse to mouse. Sporozoites increased in size from approximately 6.8 x 4.9 micrometer on day 1 to approximately 13.4 x 6.9 micrometer on day 31 postinoculation. Division was not seen. Prepatent period was 4-7 days and patent periods ranged from 2 to several weeks.

The ultrastructural changes occurring during macrogametogenes of Isospora felis were studied in the small intestine of the cat at 8 and 9 days post-infection. The parasites were situated in parasitophorous vacuoles within the host epithelial cells. The early macrogamont could be identified by its ellipsoidal shape and by the presence of a large nucleus with a distinct nucleolus. Throughout the development of the macrogamont the organism remained limited by a pellicle. At an early stage of macrogametogenesis the formation of the wall-forming bodies of types I and II (WFB I and WFB II) was initiated simultaneously. The formation of the WFB I appeared to involve the rough endoplasmic reticulum (rER) and the Golgi bodies, whereas the WFB II developed within cisternae of the rER. Early in the developmental process vacuoles were observed which budded off from the nucleus and formed multimembranous vacuoles. During gametogenesis both lipid globules and polysaccharide granules developed within the cytoplasm. The mature macrogamete was spherical in appearance and was limited by a pellicle. It possessed a large nucleus with a nucleolus and the cytoplasm was filled with numerous WFB I, WFB II, lipid globules and polysaccharide granules. A few multimembranous vacuoles were also present together with canaliculi, mitochondria, Golgi bodies and some rER.

Merozoites of a presumed Isospora species were invested by a close-fitting vacuole of host-cell origin following their entry into mononuclear, phagocytic cells of the evening grosbeak (Hesperiphona vespertina). Here, the merozoites transformed into trophozoites that were bound by a plasmalemma, beneath which lay short segments of the inner membrane complex from the pellicle of the former merozoite. Nuclear division followed and in schizonts of various sizes (probably representing several asexual generations), merozoite buds appeared around the peripheral cytoplasm. Merozoites also were seen forming within the internal cytoplasm of some schizonts. A conical, anterior projection of the nucleus containing microtubules (the centrocone) and an adjacent pair of centrioles often were seen in forming merozoites. Some maturing merozoites lay singly within distended parasitophorous vacuoles in the cytoplasm of the phagocytic cells, whereas in other vacuoles, groups of merozoites were seen. In addition to the well-documented "standard" apicomplexan features, merozoites of the Isospora sp. contained two spheroidal vesicles closely invested by several layers of membrane, which lay anterior to the nucleus near a Golgi complex. The merozoites possessed about 26 pellicular microtubules, three polar rings, two or three rhoptries, and two or more micropores. Two types of merozoites were seen; one, electron-dense and the second, considerably less so. In certain of their ultrastructural features, the merozoites of the Isospora sp. in the grosbeak resemble more closely the bradyzoites of many of the heteroxenous, isosporoid species than the merozoites of the conventional, gut-based species of Isospora and Eimeria.

The endogenous stages of Isospora serini Arogão and Isospora canaria Box are described from experimentally infected canaries, Serinus canarius Linnaeus. Unlike other Coccidia, the first part of the I. serini life cycle takes place in mononuclear phagocytes. Five asexual generations are described from this cell type; 2 additional asexual generations and the sexual stages take place in the intestinal epithelium. Isospora canaria, on the other hand, has a conventional coccidian life cycle in that all of the endogenous stages are in the epithelium of the small intestine, with 3 asexual generations and the sexual generation described in the duodenal epithelium. The 2 species differ in their position relative to the nucleus of the intestinal epithelial cell. Isospora serini is usually on the lumenal side of the nucleus while I. canaria is below the nucleus, toward the basement membrane. The prepatent period is 4-5 days for I. canaria and 9-10 days for I. serini. Patency lasts for 11-13 days in I. canaria infections, but duration of oocyst output is more chronic in I. serini infections, persisting for as long as 231 days. Both species have a diurnal periodicity of oocyst discharge which occurs in late afternoon and evening.