Targeting Single-Nucleotide Polymorphisms in the 18S rRNA Gene To Differentiate Cyclospora Species from Eimeria Species by Multiplex PCR

Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3′ end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.     

Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism

Cyclospora cayetanensis is a coccidian parasite which causes severe gastroenteritis in humans. Molecular information on this newly emerging pathogen is scarce. Our objectives were to assess genetic variation within and between human-associated C. cayetanensis and baboon-associated Cyclospora papionis by examining the internal transcribed spacer (ITS) region of the ribosomal RNA operon, and to develop an efficient polymerase chain reaction- (PCR)-based method to distinguish C. cayetanensis from other closely related organisms. For these purposes, we studied C. cayetanensis ITS-1 nucleotide variability in 24 human faecal samples from five geographic locations and C. papionis ITS-1 variability in four baboon faecal samples from Tanzania. In addition, a continuous sequence encompassing ITS-1, 5.8S rDNA and ITS-2 was determined from two C. cayetanensis samples. The results indicate that C. cayetanensis and C. papionis have distinct ITS-1 sequences, but identical 5.8S rDNA sequences. ITS-1 is highly variable within and between samples, but variability does not correlate with geographic origin of the samples. Despite this variability, conserved species-specific ITS-1 sequences were identified and a single-round, C. cayetanensis-specific PCR-based assay with a sensitivity of one to ten oocysts was developed. This consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argues for polyparasitism and simultaneous transmission of multiple strains.     

PCR-restriction fragment length polymorphism method for detection of Cyclospora cayetanensis in environmental waters without microscopic confirmation

We developed an alternative nested-PCR-restriction fragment length polymorphism (RFLP) protocol for the detection of Cyclospora cayetanensis in environmental samples that obviates the need for microscopic examination. The RFLP method, with the restriction enzyme AluI, differentiates the amplified target sequence from C. cayetanensis from those that may cross-react. This new protocol was used to reexamine a subset (121 of 180) of surface water samples. Samples previously positive when the CYCF3E and CYCR4B primers (33) and RFLP with MnlI (20) were used were also PCR positive with the new primers; however, they were RFLP negative. We verified, by sequencing these amplicons, that while two were most likely other Cyclospora species, they were not C. cayetanensis. We can detect as few as one oocyst seeded into an autoclaved pellet flocculated from 10 liters of surface water. This new protocol should be of great use for environmental microbiologists and public health laboratories.     

Highly sensitive and specific PCR assay for reliable detection of Cyclospora cayetanensis oocysts

Multiple outbreaks of food-borne gastroenteritis caused by the coccidian parasite Cyclospora cayetanensis have been reported annually in North America since 1995. Detection of C. cayetanensis contamination typically relies on laborious and subjective microscopic examination of produce washes. Molecular detection methods based on nested PCR, restriction fragment length polymorphism, or multiplex PCR have been developed for C. cayetanensis; however, they have not been adequately validated for use on food products. Further challenges include reliably extracting DNA from coccidian oocysts since their tough outer wall is resistant to lysis and overcoming PCR inhibitors in sample matrices. We describe preliminary validation of a reliable DNA extraction method for C. cayetanensis oocysts and a sensitive and specific novel PCR assay. The sensitivity and repeatability of the developed methods were evaluated by multiple DNA extractions and PCR amplifications using 1,000-, 100-, 10-, or 1-ooycst aliquots of C. cayetanensis oocysts in water or basil wash sediment. Successful PCR amplification was achieved on 15 and 5 replicates extracted from aliquots containing 1,000 oocysts in water and basil wash, respectively. All 45 replicates of the 100-oocyst aliquots in water and 5 in basil wash were amplified successfully, as were 43/45 and 41/45 of the 10- and 1-oocyst aliquots in water and 9/15 and 2/15 in basil wash, respectively. The developed primers showed no cross-reactivity when tested against bacteria, nematodes, and protozoans, including Eimeria, Giardia, and Cryptosporidium. Our results indicate that these methods are specific, can reliably detect a single oocyst, and overcome many of the limitations of microscopic diagnosis.     

Cyclospora cayetanensis: a review of an emerging parasitic coccidian

Cyclospora cayetanensis is a sporulating parasitic protozoan that infects the upper small intestinal tract. It has been identified as both a food and waterborne pathogen endemic in many developing countries. It is an important agent of Traveller's Diarrohea in developed countries and was responsible for numerous foodborne outbreaks in the United States and Canada in the late 1990s. Like Cryptosporidium, infection has been associated with a variety of sequelae such as Guillain-Barré syndrome, reactive arthritis syndrome (formally Reiter syndrome) and acalculous cholecystitis.There has been much debate as to where to place C. cayetanensis taxonomically due to its homology with Eimeria species. To date, the only genomic DNA sequences available are the ribosomal DNA of C. cayetanensis and three other species; within these a high degree of homology has been observed. This homology and the lack of sequence data from other Cyclospora species have hindered identification methods.     

Cyclospora cayetanensis: biology, environmental distribution and transfer

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.     

Co-infection of Giardia intestinalis and Cyclospora cayetanensis in an immunocompetent patient with prolonged diarrhea: case report

Cyclospora cayetanensis is an agent of emerging infectious disease, and a recognized cause of diarrhea in some patients. Also, the flagellated protozoan, Giardia intestinalis, induces a diarrheal illness of the small intestine. Cases of cyclosporiasis are frequently missed, primarily due to the fact that the parasite can be quite difficult to detect in human fecal samples, despite an increasing amount of data regarding this parasite. On the other hand, G. intestinalis can be readily recognized via the microscopic visualization of its trophozoite or cyst forms in stained preparations or unstained wet mounts. In this report, we describe an uncommon case of co-infection with G. intestinalis and C. cayetanensis in an immunocompetent patient with prolonged diarrhea, living in a non-tropical region of Turkey.     

Detection and differentiation of coccidian oocysts by real-time PCR and melting curve analysis

Rapid and reliable detection and identification of coccidian oocysts are essential for animal health and foodborne disease outbreak investigations. Traditional microscopy and morphological techniques can identify large and unique oocysts, but they are often subjective and require parasitological expertise. The objective of this study was to develop a real-time quantitative PCR (qPCR) assay using melting curve analysis (MCA) to detect, differentiate, and identify DNA from coccidian species of animal health, zoonotic, and food safety concern. A universal coccidia primer cocktail was designed and employed to amplify DNA from Cryptosporidium parvum, Toxoplasma gondii, Cyclospora cayetanensis, and several species of Eimeria, Sarcocystis, and Isospora using qPCR with SYBR Green detection. MCA was performed following amplification, and melting temperatures (T(m)) were determined for each species based on multiple replicates. A standard curve was constructed from DNA of serial dilutions of T. gondii oocysts to estimate assay sensitivity. The qPCR assay consistently detected DNA from as few as 10 T. gondii oocysts. T(m) data analysis showed that C. cayetanensis, C. parvum, Cryptosporidium muris, T. gondii, Eimeria bovis, Eimeria acervulina, Isospora suis, and Sarcocystis cruzi could each be identified by unique melting curves and could be differentiated based on T(m). DNA of coccidian oocysts in fecal, food, or clinical diagnostic samples could be sensitively detected, reliably differentiated, and identified using qPCR with MCA. This assay may also be used to detect other life-cycle stages of coccidia in tissues, fluids, and other matrices. MCA studies on multiple isolates of each species will further validate the assay and support its application as a routine parasitology screening tool.     

Cryptosporidium parvum and Cyclospora cayetanensis: a review of laboratory methods for detection of these waterborne parasites

Cryptosporidium and Cyclospora are obligate, intracellular, coccidian protozoan parasites that infest the gastrointestinal tract of humans and animals causing severe diarrhea illness. In this paper, we present an overview of the conventional and more novel techniques that are currently available to detect Cryptosporidium and Cyclospora in water. Conventional techniques and new immunological and genetic/molecular methods make it possible to assess the occurrence, prevalence, virulence (to a lesser extent), viability, levels, and sources of waterborne protozoa. Concentration, purification, and detection are the three key steps in all methods that have been approved for routine monitoring of waterborne oocysts. These steps have been optimized to such an extent that low levels of naturally occurring Cryptosporidium oocysts can be efficiently recovered from water. The filtration systems developed in the US and Europe trap oocysts more effectively and are part of the standard methodologies for environmental monitoring of Cryptosporidium oocysts in source and treated water. Purification techniques such as immunomagnetic separation and flow cytometry with fluorescent activated cell sorting impart high capture efficiency and selective separation of oocysts from sample debris. Monoclonal antibodies with higher avidity and specificity to oocysts in water concentrates have significantly improved the detection and enumeration steps.To date, PCR-based detection methods allow us to differentiate the human pathogenic Cryptosporidium parasites from those that do not infect humans, and to track the source of oocyst contamination in the environment. Cell culture techniques are now used to examine oocyst viability. While fewer studies have focused on Cyclospora cayetanensis, the parasite has been successfully detected in drinking water and wastewater using current methods to recover Cryptosporidium oocysts. More research is needed for monitoring of Cyclospora in the environment. Meanwhile, molecular methods (e.g. molecular markers such as intervening transcribed spacer regions), which can identify different genotypes of C. cayetanensis, show good promise for detection of this emerging coccidian parasite in water.     

Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay

Cyclospora cayetanensis, a coccidian parasite, with a fecal-oral life cycle, has become recognized worldwide as an emerging human pathogen. Clinical manifestations include prolonged gastroenteritis. While most cases of infection with C. cayetanensis in the United States have been associated with foodborne transmission, waterborne transmission has also been implicated. We report on the development and application of a real-time, quantitative polymerase chain reaction assay for the detection of C. cayetanensis oocysts, which is the first reported use of this technique for this organism. Both a species-specific primer set and dual fluorescent-labeled C. cayetanensis hybridization probe were designed using the inherent genetic uniqueness of the 18S ribosomal gene sequence of C. cayetanensis. The real-time polymerase chain reaction assay has been optimized to specifically detect the DNA from as few as 1 oocyst of C. cayetanensis per 5 microl reaction volume.     

Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus

We used PCR to test various protocols and define a technique for DNA extraction directly from chicken-shed stool samples for the identification of Eimeria species that parasitize birds. It was possible to extract and amplify DNA of seven Eimeria species from field stool samples, using both protocols tested; extractions made with phenol/chloroform protocols gave the best results. The primers were specific and sensitive, allowing amplification of samples containing as few as 20 oocysts, both in individual and in a multiplex PCR. Individualized PCR with the phenol/chloroform DNA extraction protocol detected a larger number of Eimeria species. Molecular diagnosis was found to be practical and precise, and can be used for monitoring and epidemiological studies of Eimeria.     

Evaluation of Cryptosporidium parvum genotyping techniques.

We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Cryptosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, and C. serpentis), two Eimeria species (E. neischulzi and E. papillata), and Giardia duodenalis were used to evaluate the specificity of primers. Furthermore, the sensitivity of the genotyping primers was tested by using genomic DNA isolated from known numbers of oocysts obtained from a genotype 2 C. parvum isolate. PCR amplification was repeated at least three times with all of the primer pairs. Of the 11 protocols studied, 10 amplified C. parvum genotypes 1 and 2, and the expected fragment sizes were obtained. Our results indicate that two species-differentiating protocols are not Cryptosporidium specific, as the primers used in these protocols also amplified the DNA of Eimeria species. The sensitivity studies revealed that two nested PCR-restriction fragment length polymorphism (RFLP) protocols based on the small-subunit rRNA and dihydrofolate reductase genes are more sensitive than single-round PCR or PCR-RFLP protocols.     

The beta-tubulin gene of Babesia and Theileria parasites is an informative marker for species discrimination

A fragment of the beta-tubulin gene was polymerase chain reaction (PCR) amplified from genomic DNAs of Babesia bovis, Babesia bigemina, Babesia divergens, Babesia major, Babesia caballi, Babesia equi, Babesia microti, Theileria annulata and Theileria sergenti. Single amplification products were obtained for each of these species, but the size of the amplicons varied from 310 to 460 bp. Sequence analysis revealed that this variation is due to the presence of a single intron, which ranged from 20 to 170 bp. The extensive genetic variability at the beta-tubulin locus has been exploited to develop two types of species identification assays. The first assay can be used on samples containing mostly parasite DNA, like those prepared from infected erythrocytes. Following PCR amplification, the species identification is obtained directly from the size of the products (for Babesia species infecting human or horse) or using a simple PCR-restriction fragment length polymorphism (RFLP) protocol (for Babesia species infecting cattle). The second assay can be used on samples prepared from whole blood, that contain both parasite and host DNAs. In this case, due to the strong conservation of the beta-tubulin gene, co-amplification of a gene fragment from the host DNA was observed. A nested PCR assay was developed for the specific amplification of parasite DNA, using a primer designed to span the exon-intron boundary. Direct identification of Babesia species infecting human and horse is again obtained after the electrophoretic separation of the amplification products, while for Babesia and Theileria species infecting cattle, differentiation is based on a nested PCR-RFLP protocol. These methods may be used for the simultaneous identification of horses and cattle carrying multiple parasites by means of a single PCR or using the PCR-RFLP protocol.     

Survey for Cyclospora cayetanensis in domestic animals in an endemic area in Haiti.

From January 1997 through July 1998, we examined stool samples from 327 domestic animals, including pigs, cattle, horses, goats, dogs, cats, guinea pigs, chicken, ducks, turkeys, and pigeons in Leogane, Haiti, for the presence of Cyclospora cayetanensis infection. No coccidian oocysts morphologically compatible with C. cayetanensis were detected in any of the animal samples, despite their living in, or near, households with infected individuals. These results suggest that domestic animals are not reservoir hosts for C. cayetanensis and that in this endemic area, humans are the only natural host for this parasite.     

Detection of four important Eimeria species by multiplex PCR in a single assay

The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1–5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl.     

Construction of PCR primers to detect and distinguish Eimeria spp. in northern bobwhites and a survey of Eimeria on gamebird farms in the United States

Coccidiosis is an important disease in captive gamebirds, including northern bobwhites (Colinusvirginianus). Three Eimeria species, Eimeria lettyae, Eimeria dispersa, and Eimeria colini, have been described in bobwhites. Distinguishing the various Eimeria spp. is often problematic because of similarity in oocyst morphology and site of infection and thus requires live bird infections to distinguish between the coccidian species. To aid in identification and diagnosis, PCR specific primers were generated against the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene using sequences obtained from coccidian-positive samples collected from diagnostic cases or litter from captive bobwhites. Three distinct Eimeria spp. were detected. Species-specific primers were constructed and used to survey the prevalence of the species in 31 samples collected from 13 states. The primers survey results identified E. lettyae, E. dispersa, and Eimeria sp. in 20 (64.5%), 22 (71%), and 29 (93.5%) of the samples, respectively. Mixed infections were common: 13 (41.9%) samples had 3 Eimeria spp., 14 (45.2%) had 2 spp., and 4 (12.9%) samples had only 1 species. The species were widely distributed over the area sampled and were not associated with the age of the flock.     

Cephalosporium maydis is a distinct species in the Gaeumannomyces-Harpophora species complex

Cephalosporium maydis is an important plant pathogen whose phylogenetic position relative to other fungi has not been established clearly. We compared strains of C. maydis, strains from several other plant-pathogenic Cephalosporium spp. and several possible relatives within the Gaeumannomyces-Harpophora species complex, to which C. maydis has been suggested to belong based on previous preliminary DNA sequence analyses. DNA sequences of the nuclear genes encoding the rDNA ITS region, β-tubulin, histone H3, and MAT-2 support the hypothesis that C. maydis is a distinct taxon within the Gaeumannomyces-Harpophora species complex. Based on amplified fragment length polymorphism (AFLP) profiles, C. maydis also is distinct from the other tested species of Cephalosporium, Phialophora sensu lato and members of Gaeumannomyces-Harpophora species complex, which supports its classification as Harpophora maydis. Oligonucleotide primers for H. maydis were developed that can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen. These diagnostic PCR primers will aid the detection of H. maydis in diseased maize because this fungus can be difficult to detect and isolate, and the movement of authentic cultures may be limited by quarantine restrictions.     

Examination of attachment and survival of Toxoplasma gondii oocysts on raspberries and blueberries

The consumption of Toxoplasma gondii oocysts on fresh produce may be a means of its transmission to humans. Cats shed T. gondii oocysts, which contaminate produce directly or contaminate water sources for agricultural irrigation and pesticide and fertilizer applications. Cyclospora cayetanensis is a related coccidial parasite, and outbreaks of diarrhea caused by C. cayetanensis have been associated with the ingestion of contaminated raspberries. The oocysts of these coccidians are similar in size and shape, indicating that they may attach to and be retained on produce in a similar manner. In the present study the attachment and survival of T. gondii oocysts on 2 structurally different types of berries were examined. Raspberries and blueberries were inoculated individually with 1.0 x 10(1) to 2.0 x 10(4) oocysts of sporulated T. gondii. Berries inoculated with 2.0 x 10(4) oocysts were stored at 4 C for up to 8 wk. Oocyst viability and recovery were analyzed by feeding processed material to mice. Mice fed T. gondii-inoculated berries stored at 4 C for 8 wk developed acute infections. In other experiments mice fed raspberries inoculated with > or = 1.0 x 10(1) oocysts became infected, whereas only mice fed blueberries inoculated with > or = 1.0 x 10(3) oocysts became infected. This study demonstrates that T. gondii oocysts can adhere to berries and can be recovered by bioassays in mice and that raspberries retain more inoculated oocysts than do blueberries. The results suggest that T. gondii may serve as a model for C. cayetanensis in food safety studies.     

Comparison of three microscopic techniques for diagnosis of Cyclospora cayetanensis

Cyclospora cayetanensis oocysts in the feces of humans from Kathmandu, Nepal were identified on the basis of their size and other morphological characteristics. We compared the detection of C. cayetanensis oocysts in the feces using three microscopic techniques such as formalin-ether sedimentation, sucrose centrifugal floatation, and direct smear. Standard procedures were used for the formalin-ether sedimentation and the sucrose centrifugal floatation techniques using 0.5 g of feces, however, the direct smear technique was performed using 10 microl of fecal suspension (0.005 g of feces) and observed under the fluorescent microscope. Of the 403 samples examined, 21 samples were positive for oocysts by all three techniques. Therefore, in these 21 samples, the number of oocysts recovered by the three techniques were compared. The highest number of oocyst was obtained by the sucrose centrifugal floatation technique. In contrast, the formalin-ether sedimentation technique was found to be the least reliable concentration technique for the detection of Cyclospora in human feces. Surprisingly, the direct smear technique was found to be an effective and rapid technique for diagnosis of C. cayetanensis making it a technique of choice for routine epidemiological investigation of the prevalence of this infection in human populations.     

The identification of Cryptosporidium species (protozoa) in Ifsahan, Iran by PCR-RFLP analysis of the 18S rRNA gene

Cryptosporidium is an important protozoan that cause diarrheal illness in humans and animals. Different species of Cryptosporidium have been reported and it is believed that species characteristics are an important factor to be considered in strategic planning for control. We therefore analyzed oocysts from human and animal isolates of Cryptosporidium by PCR-RFLP to determine strain variation in Isfahan. In total, 642 human fecal samples from children under five years of age, immunocompromised patients, and high risk persons and 480 randomly selected rectal specimens of cows and calves in Isfahan were examined. Microscopic examination showed that 4.7% (30/642) of human samples and 6.2% (30/480) of animal samples were infected with Cryptosporidium. After identification of the samples infected with the parasite, oocysts were purified and their DNA was extracted. We used PCR-RFLP analysis of a 1750-bp region of 18S rRNA gene to identify Cryptosporidium species. The human samples were infected with Cryptosporidium parvum II, C. muris, C. wrairi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520951). The cattle samples were identified as C. parvum II, C. muris, C. wrairi, C. serpentis, C. baileyi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520952). Also we found a new genotype infecting both human and cattle samples (GenBank accession numbers: DQ520950). In addition to demonstrating the widespread occurrence of most species of Cryptosporidium, C. parvum, we also observed extensive polymorphism within species. Furthermore, the occurrence of the same species of parasite in both animal and human samples shows the importance of the animal-human cycle.