Interpretation errors may still represent a limiting factor for diagnosing Cryptosporidium spp. oocysts with the conventional staining techniques. Humans and machines can interact to solve this problem. We developed a new temporary staining protocol associated with a computer program for the diagnosis of Cryptosporidium spp. oocysts in fecal samples. We established 62 different temporary staining conditions by studying 20 experimental protocols. Cryptosporidium spp. oocysts were concentrated using the Three Fecal Test (TF-Test®) technique and confirmed by the Kinyoun method. Next, we built a bank with 299 images containing oocysts. We used segmentation in superpixels to cluster the patches in the images; then, we filtered the objects based on their typical size. Finally, we applied a convolutional neural network as a classifier. The trichrome modified by Melvin and Brooke, at a concentration use of 25%, was the most efficient dye for use in the computerized diagnosis. The algorithms of this new program showed a positive predictive value of 81.3 and 94.1% sensitivity for the detection of Cryptosporidium spp. oocysts. With the combination of the chosen staining protocol and the precision of the computational algorithm, we improved the Ova and Parasite exam (O&P) by contributing in advance toward the automated diagnosis.

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.

Water buffalo (Bubalus bubalis) is important for the economy of Asia, South America and parts of Europe. Coccidiosis is an important cause of neonatal mortality in livestock, including buffalo. Of more than 12 species of Eimeria in buffalo, Eimeria bareillyi is the most pathogenic. There are uncertainties concerning its asexual and sexual development. During a previously reported outbreak of fatal enteritis associated with E. bareillyi in buffaloes in the Netherlands, sections of small intestine were re-evaluated histologically and by transmission electron microscopy (TEM) to seek details of endogenous development. Profuse asexual multiplication occurred in the jejunum and ileum. Light microscopic examination revealed that parasites divided in two (probably endodyogeny) or more organisms. There were two or more generations of morphologically different merozoites; some of these observations were confirmed by TEM. Details of gametogonic development, including oocyst wall formation are provided. Schizogonic and gametogonic development described in the present study can serve as a guide for differential diagnosis of Eimeria species in histological sections of intestines of buffaloes.

Camelids (llama, alpaca, vicunãs, guanacos) are important for the economy of South America and Eimeria infections are an important cause of mortality in camelids. Of the six species of Eimeria in camelids, Eimeria macusaniensis, considered the most pathogenic, is distinctive; its oocysts are the largest among all Eimeria species in animals, its prepatent period is more than 1 month, and its oocysts have been found in mummies from prehistoric times. Although, E. macusaniensis gametogonic stages are found associated with enteritis in naturally infected camelids, the schizogonic stages are unknown and clinical disease has been reported in some camelids with no oocysts in feces. Described herein are morphological details of gametogonic development and oocyst formation of E. macusaniensis in a naturally infected llama (Lama lama), solely infected with this parasite. Microgamonts, macrogamonts and oocysts were located in large (up to 300 µm diameter) parasitophorous vacuoles of enterocytes in the ileum. Schizonts were not found. Review of previous reports suggests that multinucleated microgamonts have been mistaken for schizonts. Gametogonic development described in the present study can serve as a guide for differential diagnosis of Eimeria species in the histological sections of intestines.

Small marine snails and abalone have been identified as high- and low-risk prey items, respectively, for exposure of threatened southern sea otters to Toxoplasma gondii, a zoonotic parasite that can cause fatal encephalitis in animals and humans. While recent work has characterized snails as paratenic hosts for T. gondii, the ability of abalone to vector the parasite has not been evaluated. To further elucidate why abalone predation may be protective against T. gondii exposure, this study aimed to determine whether: (1) abalone are physiologically capable of acquiring T. gondii; and (2) abalone and snails differ in their ability to concentrate and retain the parasite. Abalone were exposed to T. gondii surrogate microspheres for 24 h, and fecal samples were examined for 2 weeks following exposure. Concentration of surrogates was 2–3 orders of magnitude greater in abalone feces than in the spiked seawater, and excretion of surrogates continued for 14 days post-exposure. These results indicate that, physiologically, abalone and snails can equally vector T. gondii as paratenic hosts. Reduced risk of T. gondii infection in abalone-specializing otters may therefore result from abalone's high nutritional value, which implies otters must consume fewer animals to meet their caloric needs.

The aim of this study was to contribute to the better understanding of the relative epidemiological importance of different modes of infection with respect to horizontal transmission of Toxoplasma gondii in endemic settings. We investigated the prevalence of salivary IgA against a sporozoite-specific embryogenesis-related protein (TgERP) in a highly endemic area for toxoplasmosis in Brazil in order to pinpoint parasite transmission via oocysts. Prevalence calculated by salivary IgA specific to TgERP was compared to the prevalence calculated by serum IgG against both TgERP and tachyzoites (in conventional serological tests). Prevalence calculated by different serological and salivary parameters varied in the studied age groups. However, for the 15–21 years age group, values for T. gondii prevalence estimated by conventional serological tests and by anti-TgERP salivary IgA were similar; i.e. 68·7% and 66·6% or 66·7%, respectively, using two different cut-off parameters for salivary IgA anti-TgERP. Furthermore, salivary IgA anti-TgERP for this age group presented the highest specificity (93·33%), sensitivity (93·94%), and likelihood (14·09) compared to all the other age groups. These data demonstrate the importance of age for salivary IgA investigation against TgERP to estimate the mode of T. gondii transmission in endemic settings.

Humans and other hosts acquire Toxoplasma gondii infection by ingesting tissue cysts in undercooked meat, or by food or drink contaminated with oocysts. Currently, there is no vaccine to prevent clinical disease due this parasite in humans, although, various T. gondii vaccine candidates are being developed. Mice are generally used to test the protective efficacy of vaccines because they are susceptible, reagents are available to measure immune parameters in mice, and they are easily managed in the laboratory. In the present study, pathogenesis of toxoplasmosis was studied in mice of different strains, including Human Leukocyte Antigen (HLA) transgenic mice infected with different doses of T. gondii strains of different genotypes derived from several countries. Based on many experiments, the decreasing order of infectivity and pathogenicity of oocysts was: C57BL/6 background interferon gamma gene knock out (KO), HLA-A*1101, HLA-A*0201, HLA-B*0702, Swiss Webster, C57/black, and BALB/c. Mice fed as few as 1 oocyst of Type I and several atypical strains died of acute toxoplasmosis within 21 days p.i. Some Type II, and III strains were less virulent. The model developed herein should prove to be extremely useful for testing vaccines because it is possible to accurately quantitate a challenge inoculum, test the response to different strains of T. gondii using the same preparations of oocysts which are stable for up to a year, and to have highly reproducible responses to the infection.

The Regent Honeyeater (Xanthomyza phyrigia) is an endangered Australian bird species. Breeding populations have been established at Australian zoos in support of re-introduction programmes. This species is the host of a new species of Isospora (Apicomplexa). Oocysts are spherical, 25·8 (22·5–28·75) by 23·8 (20–26·25) μm with a colourless to pale yellow smooth wall undergoing rapid exogenous sporulation, 90% sporulated oocysts in 8 h at 20°C. Each oocyst contains 1 polar granule. Sporocysts are ovoid, 18·67 (17–19) by 9·49 (9–10) μm with a flat Stieda body and spherical substieda body devoid of a hyaline body. The asexual stages and sexual phase is within the enterocytes of the duodenum and jejunum. Faeces collected in the morning (AM, n=84) and in the afternoon (PM, n=90) revealed significant diurnal periodicity in oocyst shedding; 21% (18 of 84) of the AM were positive with the mean of 499 oocysts.g−1 compared to the PM with 91% (82 of 90) bird faeces positive with the mean of 129 723 oocysts.g−1. Therefore, parasite checks for these birds should be carried out in the afternoon to obtain an accurate result. The ecological significance of the high parasite burden in captive birds requires further investigation and comparison to the wild counterparts.

Juvenile hedgehogs having insufficient body weight are often brought for overwintering to hedgehog rehabilitation centres. Faecal samples of juvenile hedgehogs and overwintering hedgehogs (n=188) collected prior to releasing them back into the wilderness were examined for the presence of Cryptosporidium coproantigen and oocysts. Altogether 56 (29·8%) submitted samples were positive for coproantigen. Forty-five (39·5%, n=114) of the positive samples originated from newly rescued hedgehogs, while 11 (14·8%, n=74) positive samples were from animals that spent several months at the station. Fifteen samples subjected to PCR-RFLP analysis on the partial 18S rRNA locus suggested the presence of C. parvum. Multilocus sequence typing on partial 60 kDa glycoprotein gene, 18S rRNA, actin gene, 70 kDa heat shock protein gene sequences revealed 3 different subtype families: IIa, IIc and a new, proposed as VIIa subtype family. Cryptosporidium sp. genotype belonging to VIIa subtype family is closely related to C. parvum but is genetically distinct being probably a hedgehog-specific Cryptosporidium sp. genotype with unknown zoonotical potential. Hedgehogs excreting Cryptosporidium oocysts represent a potential source for human infections, but also an anthroponotic nature of the IIc subtype family should be reviewed.

Protocols for in vitro excystation of oocysts of Cryptosporidium parvum, including different chemical pre-incubation steps, were compared to examine some of the biochemical triggers involved in excystation and to define an in vitro excystation protocol of a reproducibly high efficiency. Pre-incubation steps which increased the permeability of the oocysts were found to enhance excystation dynamics and pre-treatment of oocysts with saliva was found to decrease the permeability and reduce excystation. Although excystation was maximal after incubation for 4 h, sporozoites tended to lyse over this period, and maximum sporozoite recovery occurred after 30 min. The results obtained are discussed in relation to excystation protocols adopted by different research groups and a number of recommendations are given for in vitro excystation of C. parvum oocysts.

The genetic structure of a population of the malaria parasite Plasmodium falciparum has been examined in a village in Tanzania. Seventeen alleles of the merozoite surface protein MSP-1 and 23 of MSP-2 were detected by the polymerase chain reaction (PCR) among the blood parasites of the inhabitants. Most infections contained mixtures of genetically distinct parasite clones. PCR was then used to examine individual P. falciparum oocysts, the products of fertilization events, in wild-caught mosquitoes. Forty-five out of 71 oocysts were heterozygous for one or both genes, showing that crossing between clones was taking place frequently, following uptake of mixtures of gametocytes by the mosquitoes. The frequency of heterozygous forms showed that random mating events probably occurred within mosquito bloodmeals between gametes belonging to different parasite clones.

Two clones of the human malaria parasite Plasmodium falciparum, denoted 3D7 and HB3, were grown in vitro under conditions permitting the development of gametocytes. The two clones differ in their allelic forms of two antigen genes MSP1 and MSP2. The alleles can be distinguished as size differences of polymerase chain reaction (PCR) amplified fragments of repetitive regions of each gene. Mosquitoes (Anopheles stephensi) were fed on a mixture of these gametocytes. A total of 128 oocysts was isolated from the midguts of infected mosquitoes from 9 crossing experiments between the clones. DNA extracted from these oocysts was amplified by PCR. Oocysts which contained both alleles of each gene (MSP1 and MSP2) had developed from heterozygotes produced by cross-fertilization events between 3D7 and HB3 gametes. The remaining oocysts contained single alleles of each gene, in parent clone combinations, and these had developed from homozygotes formed by self-fertilizations. The results suggest that gametes in the original mixture fed to mosquitoes had undergone random mating.

Monophyly of all 11 valid Eimeria species from rabbits (Oryctolagus cuniculus Linnaeus, 1758) was revealed based on nuclear 18S rDNA sequence data. This finding implies that these species, which vary considerably in terms of their morphology and biology, diversified on a single host or several closely related species. Phylogenetic analysis divided rabbit Eimeria species into 2 sister lineages, corresponding to the presence/absence of the oocyst residuum. Other morphological or biological traits (oocyst shape and size, presence/absence of oocyst inner structures, pathogenicity, infection site, pre-patent and patent periods, sporulation time, and number of asexual generations) do not explicitly correlate with the phylogeny of rabbit coccidia.

We used the fluorescent dye monochlorobimane (MCB) which binds glutathione (GSH) to localize between 2 and 6 distinctly labelled nuclear and cytoplasmic GSH foci in recently excreted and aged, intact Cryptosporidium parvum oocysts and sporozoites. Buthionine sulfoximine (BSO), a potent and specific inhibitor of GSH, was used to determine whether GSH is synthesized in BSO-treated C. parvum oocysts, by labelling treated oocysts with MCB. Both visual and electronic quantifications were performed. At 5 mM BSO, a significant inhibition of MCB fluorescence, reflecting reduced MCB uptake, was observed in GSH-depleted oocysts (mean±S.D. 35±3·7) compared with controls (3·3±1·2, P=0). This clear reduction occurred only in viable oocysts. 1 mM BSO-treated oocysts exhibited weak or no MCB fluorescence, although they were viable (excluded propidium iodide, PI)), and intact and contained sporozoites by differential interference contrast microscopy (DIC). MCB was used in conjunction with PI to determine C. parvum oocyst viability. Oocysts labelled with MCB/PI or 4′6-diamidino-2-phenyl indole (DAPI)/PI produced comparable labelling patterns. Viable oocysts were labelled with MCB or DAPI whereas dead oocysts were labelled with PI only. The localization of GSH in viable, intact oocysts and excysted sporozoites and UV light-irradiated oocysts and sporozoites revealed no changes in MCB uptake at levels up to 40 mJ.cm−2 irradiation. Although GSH can be detected following MCB localization in both the nucleus and cytoplasm of sporozoites, and can be specifically depleted by BSO treatment, MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts.

This note reports an unusual case of a high number of oocysts found by dissecting the midgut of Anopheles dirus Peyton & Harrison from Laos, where dissections have not been carried out for many decades. In dissected samples, the number of oocysts ranged from 1 to 250 and tests carried out on the heads and thoraces of A. dirus using the ELISA method were positive for Plasmodium falciparum.

Transmission electron microscopy was used to study Toxoplasma gondii infections in the small intestines of Swiss-Webster mice at 2–48 h post-feeding of oocysts (p.f.). Sporozoites passed through intestinal epithelial cells (enterocytes and goblet cells) and infected all cells except red blood cells in the lamina propria. Parasites in intestinal epithelial cells or in cells in the lamina propria were located within a single type of parasitophorous vacuole, which contained exocytosed electron-dense material and well-developed tubulovesicular membranous networks. Sporozoites did not infect intraepithelial lymphocytes (IELs), but at 48 h p.f. IELs had become infected with tachyzoites arising from those that had developed in the lamina propria. At 48 h p.f., the lamina propria contained numerous tachyzoites, much cellular debris, and few intact cells. The intestinal epithelium exhibited limited cytopathological changes except for villar fusion, slight vacuolation, and cell separation at the bases of enterocytes.

Interactions between parasite genotypes sharing a host are poorly understood, but have important consequences for the epidemiology and evolution of the parasite. In mixed-genotype malaria infections, patterns of asexual replication and transmission favoured by natural selection may be different from those in single-genotype infections. The infectivity to mosquitoes of mixed-genotype and single-genotype infections were compared using 2 clones of Plasmodium chabaudi inoculated into mice either together or alone. Mice given mixed-clone infections received the sum of the inocula given to the single-clone controls. Mosquitoes were fed on the mice and the numbers of oocysts which developed were counted to assess transmission intensity. For 3 combinations of starting inocula and feed days, mixed-clone infections produced more oocysts per mosquito than the sum of the 2 single-clone infections. This effect was correlated with an increase in gametocyte density, but was less clearly related to asexual infection parameters. The results show that interactions between clones in mixed-clone infections can profoundly affect transmission.

Rodent and avian malaria parasites have been reported to have an adverse affect upon the reproductive fitness of mosquitoes. In order to determine whether fecundity reduction occurs in Anopheles gambiae s. l. infected with human malaria a study of wild-caught mosquitoes was undertaken in the Muheza district of north east Tanzania. Fully engorged, indoor resting females were collected daily for 4 months and maintained for 5 days. A sporozoite rate of 11·5% was detected for the whole collection and of those females alive on day 6 an additional 17·5% were infected with oocysts alone. Oocyst, but not sporozoite, infection resulted in a 17·5% reduction in egg production. Fecundity reduction was not caused by a reduction in bloodmeal size in infected females and no size difference was detected between oocyst-infected and uninfected females although sporozoite-positive females were significantly larger. Comparisons in parity between uninfected and infected groups indicate that infection does not affect survival beyond the first gonotrophic cycle as no changes in survivorship occurred as a result of sporozoite infection.

Preponderance of Musca spp. and their accessibility to goat feeds and faeces containing Eimeria oocysts prompted an investigation into the role of these flies in the dissemination and/or transmission of goat eoccidiosis. Seventy per cent of the dissected 300 Musca spp. caught in the goat pens where faeces containing Eimeria oocysts were widely scattered, possessed the oocysts in their digestive organs, with the highest number found in the gut. When Musca spp. were induced to feed on faecal solution containing Eimeria oocysts, oocysts were recovered from the flies through regurgitation during subsequent feeding on glucose solution. At low concentrations of 300 and 500 oocysts per g faeces, substantial numbers of oocysts were recovered from the flies only up to 1/2 hr after feeding on the infected faeces; at higher concentrations, oocysts were recovered up to 2 hr. Results of other experiments also showed that the nurriber of flies in which oocysts were found and the number of oocysts recovered were highest when the flies were dissected immediately and 1/2 hr after exposure to infected faeces. The circumstances under which Musca spp, could sustain eoccidiosis and coccidiosis in a goat herd are discussed.