Comparative genomics reveals Cyclospora cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens.

BACKGROUND: Cyclospora cayetanensis is an apicomplexan that causes diarrhea in humans. The investigation of foodborne outbreaks of cyclosporiasis has been hampered by a lack of genetic data and poor understanding of pathogen biology. In this study we sequenced the genome of C. cayetanensis and inferred its metabolism and invasion components based on comparative genomic analysis. RESULTS: The genome organization, metabolic capabilities and potential invasion mechanism of C. cayetanensis are very similar to those of Eimeria tenella. Propanoyl-CoA degradation, GPI anchor biosynthesis, and N-glycosylation are some apparent metabolic differences between C. cayetanensis and E. tenella. Unlike Eimeria spp., there are no active LTR-retrotransposons identified in C. cayetanensis. The similar repertoire of host cell invasion-related proteins possessed by all coccidia suggests that C. cayetanensis has an invasion process similar to the one in T. gondii and E. tenella. However, the significant reduction in the number of identifiable rhoptry protein kinases, phosphatases and serine protease inhibitors indicates that monoxenous coccidia, especially C. cayetanensis, have limited capabilities or use a different system to regulate host cell nuclear activities. C. cayetanensis does not possess any cluster of genes encoding the TA4-type SAG surface antigens seen in E. tenella, and may use a different family of surface antigens in initial host cell interactions. CONCLUSIONS: Our findings indicate that C. cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens. Amino acid metabolism and post-translation modifications of proteins are some major differences between C. cayetanensis and other apicomplexans. The whole genome sequence data of C. cayetanensis improve our understanding of the biology and evolution of this major foodborne pathogen and facilitate the development of intervention measures and advanced diagnostic tools.

Co-infection with Cyclospora cayetanensis and Salmonella typhi in a patient with HIV infection and chronic diarrhoea.

A 45-year-old-Haitian male patient with fever, abdominal cramping, chronic diarrhoea and weight loss of about 3 kg was investigated. Stool examination revealed Salmonella typhi and Cyclospora cayetanensis. The HIV test was positive with a CD4 count of 130 cells/mm(3). We provided the first report of co-infection Cyclospora cayetanensis and Salmonella typhi in a HIV patient with chronic diarrhoea. The patient was treated with oral ciprofloxacin, 500 mg, twice daily for two weeks, with a good clinical outcome.

[Research progress on the biology and epidemiology of Cyclospora cayetanensis].

Cyclosporiasis is one of the emerging parasitic diseases. Cyclospora cayetanensis is so far the only species infecting humans in the Cyclospora genus. This paper reviews mainly the biological characteristics of C. cayetanensis and the current epidemiology status of human infection.

[Cyclospora cayetanensis: biology, environmental distribution and transfer]. / Cyclospora cayetanensis: biología, distribución ambiental y transferencia.

Cyclospora cayetanensis is an apicomplexan protozoan that has emerged as an important pathogen causing endemic or epidemic diarrheal disease worldwide. In industrialized countries, the parasite has been recognized as the causative agent of several outbreaks of diarrheal illness mostly associated with produce imported from endemic areas. In developing countries, human cyclosporosis is widely distributed. Infection rates from 0% to 41.6% have been described in the general population. However, the epidemiology, biology, and ecology of C. cayetanensis are not fully understood. The life cycle is not completely characterized, although it appears to require a single human host to be accomplished. The role of animals as natural reservoirs of the parasite remains to be determined. Little information is available concerning the environmental distribution and vehicles of transmission of C. cayetanensis. Contaminated water, foods or soil can be vehicles of spread of the parasite. The significant uncertainties that remain in the knowledge of C. cayetanensis highlight the need for continuing research in several areas, including its basic biology and environmental distribution.

[Importance of contact with soil in the transmission of cyclosporosis]. / Importancia del contacto con la tierra en la transmisión de la ciclosporosis.

Cyclospora cayetanensis is an emergent pathogen that causes endemic and epidemic diarrhea worldwide. The epidemiology of the infection is not well known. Transmission of the parasite occurs through an environmental vehicle. In industrialized countries, cyclosporosis has been most often associated with either food-borne outbreaks or traveller's diarrhea. In developing countries, infection has been linked with contaminated water or food, contact with animals or soils, and variables related with socioeconomic status. In a Venezuelan community, a strong correlation between environments conducive to fecal contamination and infection was observed, suggesting that direct contact with contaminated soil may be an important route of transmission in areas with substandard housing developments, and poverty a predisposing factor for cyclosporosis.

Enhanced concentration and isolation of Cyclospora cayetanensis oocysts from human fecal samples.

Cyclospora cayetanensis is the causative agent of cyclosporiasis, an emerging infectious disease. We present a new method for the purification of C. cayetanensis oocysts from feces using a modified detachment solution and Renocal-sucrose gradient sedimentation. This method yields oocysts free from adherent fecal debris and amenable to processing using flow cytometry.

[Study on the expression of immune function in cases infected by Cyclospora cayetanensis].

In Cyclospora cayetanensis oocyst-positive patients, T cell subsets in peripheral mononuclear cell and membrane interleukin-2 receptor (mIL-2R) were detected with the method of biotin-streptavidin (BSA) and soluble interleukin-2 receptor (sIL-2R) as well as special IgG, IgM in serum was detected by ELISA. Results showed that there is a significant difference between the infected and uninfected individuals.

Cyclosporiasis in an infant.

This report describes cyclosporiasis in a seven month old infant who presented with incessant crying and refusal of feeds. The routine modified ZN stained smears showed the oocysts of Cyclospora when all other tests failed to reveal enteric pathogens. The need for the clinical laboratory to screen faeces samples for all possible pathogens in a given clinical situation needs to be emphasized.

Effects of temperature and different food matrices on Cyclospora cayetanensis oocyst sporulation.

Effects of temperature on the sporulation of the parasite Cyclospora cayetanensis were studied in 2 food substrates, dairy and basil. Unsporulated Cyclospora oocysts were subjected to freezing and heating conditions for time periods ranging from 15 min to 1 wk. Oocysts were then removed from the food substrates and placed in 2.5% potassium dichromate for 2 wk to allow viable unsporulated oocysts to differentiate and fully sporulate, and to determine the percentage sporulation as an indicator of viability. Sporulation occurred when oocysts resuspended in dairy substrates were stored within 24 hr at -15 C. When oocysts were placed in water or basil, sporulation occurred after incubation for up to 2 days at -20 C, and up to 4 days at 37 C. Few oocysts sporulated when incubated for 1 hr at 50 C. Sporulation was not observed in basil leaves or water at -70 C, 70 C, and 100 C. Sporulation was not affected when incubated at 4 C and 23 C for up to 1 wk, which was the duration of the experiment in both of the tested substrates.

Detection of Cyclospora cayetanensis oocysts in human fecal specimens by flow cytometry.

A diagnosis of cyclosporiasis typically involves stool examinations for the presence of Cyclospora oocysts by means of microscopy. In recent years, flow cytometry has been gaining in popularity as a novel method of detecting pathogens in environmental and clinical samples. The present study is an evaluation of a flow cytometric method for the detection and enumeration of Cyclospora oocysts in human fecal specimens associated with food-borne outbreaks of cyclosporiasis in Ontario, Canada. Flow cytometry results were generally very comparable to the original microscopy results for these specimens, in terms of both presence or absence of oocysts and relative oocyst concentrations. Of the 34 fecal specimens confirmed positive for Cyclospora by microscopy, 32 were also found positive by flow cytometry, and 2 others were considered equivocal. Of the eight fecal specimens reported to be negative by microscopy, two were found positive by flow cytometry and five others were considered equivocal. These two flow cytometry-positive samples and one of the equivocal samples were confirmed by microscopic reexamination, suggesting that flow cytometry may be more sensitive than microscopy. While the sample preparation time for flow cytometry is similar to or slightly longer than that for microscopy, the actual analysis time is much shorter. Further, because flow cytometry is largely automated, an analyst's levels of fatigue and expertise will not influence results. Flow cytometry appears to be a useful alternative to microscopy for the screening of large numbers of stool specimens for Cyclospora oocysts, such as in an outbreak situation.

Ingestion of Cryptosporidium oocysts by Caenorhabditis elegans.

Cryptosporidium parvum has been associated with outbreaks of human illness by consumption of contaminated water, fresh fruits, and vegetables. Free-living nematodes may play a role in pathogen transmission in the environment. Caenorhabditis elegans is a free-living soil nematode that has been extensively studied and serves as a good model to study possible transmission of C. parvum oocysts that may come into contact with produce before harvest. The objective of this study was to determine whether C. elegans could serve as a potential mechanical vector for transport of infectious C. parvum and Cyclospora cayetanensis in agricultural settings and whether C. elegans could ingest, excrete, and protect oocysts from desiccation. Seventy to 85% of worms ingested between 0 and 500 oocysts after 1 and 2 hr incubation with oocysts. Most of the nematodes ingested between 101 and 200 oocysts after 2 hr. Intact oocysts and empty shells were excreted by nematodes. Infectivity was determined by the neonatal assay with different treatments of worms (intact or homogenized) or oocysts or both. Adult C. elegans containing C. parvum kept in water were infective for mice. In conclusion, C. elegans adults can ingest and excrete C. parvum oocysts. Caenorhabditis elegans containing C. parvum oocysts can infect mice but does not seem to protect oocysts from extreme desiccation at 23 C incubation of a day or longer. Cyclospora oocysts were not ingested by C. elegans. The role of free-living nematodes in produce contamination needs to be further examined.

Cyclospora cayetanensis, a food- and waterborne coccidian parasite.

Food- and waterborne coccidia including Cryptosporidium parvum, Cyclospora cayetanensis, Sarcocystis hominis and Sarcocystis suihominis, and Isospora belli are cyst-forming apicomplexan protozoa that cause intracellular infections, predominantly in the epithelial cells of the intestine. They are transmitted by oocysts from person-to-person by the fecal-oral route or via contaminated water or food. The most common symptom of infection is diarrhea, however, asymptomatic infections occur. Infections are associated with intestinal inflammation, with pathological lesions such as villus blunting, and abnormal function such as malabsorption. Mild-to-moderate, self-limiting diarrhea is common in healthy individuals ingesting infective stages of these organisms. However, patients with immune dysfunction can have severe intestinal injury and prolonged diarrhea. Diagnosis in many cases is made by a microscopic examination of the stool, and the use of appropriate staining techniques, but more recently molecular methods for detection are used increasingly. Effective antimicrobial treatment for prolonged infection in immunocompromised patients is available for most of these infections. These gastrointestinal coccidial pathogens have important similarities in epidemiology, disease pathogenesis, clinical manifestations, diagnosis, and treatment. Although there are many other cyst-forming coccidia of public health, veterinary and/or economic importance, discussion in this chapter will be limited to C. cayetanensis, as an important example of the group. Aspects of the biology, epidemiology, diagnosis, disease, treatment and control are considered. This parasite is considered to be an emerging pathogen. From 1990 to 2000, there were 11 foodborne outbreaks of cyclosporosis in North America that affected at least 3600 people. There are many outstanding questions regarding this parasite and under-reporting is common because general diagnostic methods for intestinal parasites are inadequate for detection of Cyclospora.

Effects of pesticides on sporulation of Cyclospora cayetanensis and viability of Cryptosporidium parvum.

Disease outbreaks caused by the coccidian parasite Cyclospora cayetanensis in food have been linked to consumption of raspberries that may have been contaminated through exposure to water mixed with insecticides and fungicides that may have been sprayed onto the berries. Three different fungicides (captan 50% W.P., benomyl 50% W.P., and zineb 75% W.P.) and two different insecticides (malathion 25% W.P. and diazinon 4E 47.5%) were evaluated at five different concentrations and for exposure times of 30 min to 1 week. Sporulation of C. cayetanensis did not decrease with use of any of the pesticides from time periods of 30 min to 24 h at all concentrations. Sporulation percentage was reduced with the fungicide benomyl at 1 week of exposure. The growth of the parasite Cryptosporidium parvum was also evaluated using captan 50% W.P., benomyl 50% W.P., and diazinon 4E 47.5%. Oocyst infectivity was reduced only after 7 days of exposure. These results indicate that these pesticides used at recommended concentration levels do not affect the sporulation of Cyclospora.

Analysis of parasites by electrorotation.

AIMS: The application of the AC electrokinetic technique of electrorotation for studying eukaryotic parasite transmission stages is reviewed. Electrorotation is a noninvasive technique that utilizes electrically energized microelectrode structures within micro-fluidic chambers to probe the physiological structure of micro-organisms. Application of the technique to the transmission life cycle stages of three separate genera of protozoan parasites, Cryptosporidium, Giardia and Cyclospora, and one nematode genus Ascaris, each of significant public health importance, is described. METHODS AND RESULTS: Standard electrorotation apparatus, consisting of micro-fabricated electrodes in a fluidic chip, quadrature sinusoidal signal generator, microscope and image capture system, was used to study each organism. Spectra of cellular rotation rate were recorded as a function of applied electric field frequency and compared with standardized biological tests, where appropriate, to illustrate the effectiveness and versatility of the electrorotation technique. CONCLUSIONS: Electrorotational determination of the viability of individual G. intestinalis cysts, Cryptosporidium parvum and Cyclospora cayetanensis oocysts has been achieved. The sporulation state of Cyclospora cayetanensis oocysts was also readily determined, as was the fertilization state of A. suum ova. SIGNIFICANCE AND IMPACT OF THE STUDY: Electrorotation is a simple, noninvasive and versatile analytical technique suited to a wide range of particle types and capable of incorporation into integrated Lab-on-a-chip devices.

Cyclospora spp.

PURPOSE OF REVIEW: Infection with Cyclospora cayetanensis continues to pose many questions, both in endemic populations and in travelers and food-borne outbreaks. The present review discusses existing knowledge but focuses more on what is yet to be learned about this infection. RECENT FINDINGS: Information on the parasite in endemic settings continues to be gathered, and similarities to and differences from other intestinal coccidia, especially Cryptosporidium spp., are becoming clearer. Food-borne outbreaks in North America continue despite efforts to identify and limit importation of particular items, such as berries, at certain times of the year. Study of Cyclospora spp. found in east African primates has shed some light on human infection but raises many new questions regarding the biology of the organism. SUMMARY: Despite new information being gathered regarding Cyclospora spp., including infection rates in various age and population groups, significant gaps remain in our knowledge of such basic issues as the factors that influence infectivity, seasonality, mode of food contamination, and geographic distribution. These gaps highlight the need for continued study on a variety of fronts, including surveillance, and clinical and basic biology.

Experimental studies on cyclosporiosis.

Swiss albino mice get Cyclospora infection after orally inoculated with sporulated oocysts. Two weeks post inoculation, most of them passed numerous acid fast immature oocysts in their stools. One week later, light microscopic examination of their intestinal H & E stained sections revealed parasitic stages in a supranuclear location within enterocytes. They were most prominent in the mucosal villi. Lamina propria was expanded by an inflammatory infiltrate. The combined parasitological and histopathological present studies helped in distinguishing Cyclospora which is often confused with Cryptosporidia in stool samples and with Isospora in intestinal sections. Electron microscopy demonstrated both sexual and asexual developmental stages of Cyclospora in ultrathin infected sections. Therefore, Cyclospora species require only a single host to complete its entire life cycle

Microsporidia and Cyclospora: epidemiology and assessment of risk from the environment.

Two classes of parasites with an environmental stage in their lifestyle have recently emerged as significant gastrointestinal pathogens for humans. Microsporidia represent a group that contains a number of genera related to the genus Cryptosporidium. They are generally transmitted via direct human to human contact, but can survive in water and food, and recently have been found in surface water used as drinking source water. Their most common host range is in patients with clinical AIDS. Limited work to date suggests the group is susceptible to chlorine achievable CxT (concentration x time) values and is coagulated by filtration. Cyclospora cayetanensis is a species of parasite that has caused outbreaks from contaminated food. Its major risk is from the use of inadequately treated water used for irrigation. Cyclospora can infect normal and immunosuppressed hosts. Current information regarding the lifestyle, transmission, and control of both groups of parasites are discussed, with a health risk assessment analysis.