A Molecular Tool for Rapid Detection and Traceability of Cyclospora cayetanensis in Fresh Berries and Berry Farm Soils.
ABSTRACT: Due to recent outbreaks of cyclosporiasis associated with consumption of fresh berries, producers are demanding modern microbiological tools for the rapid and accurate identification of the human pathogen Cyclospora cayetanensis in berries and environmental samples. The aim of the present work was to develop a molecular tool based on a PCR approach for the rapid and accurate detection of C. cayetanensis. A nested PCR assay was validated for the amplification of a 294 bp size region of the 18S rRNA gene from C. cayetanensis. The limit of detection for the nested PCR assay was validated using 48 berry samples spiked with ~0, 10, 100, and 1000 oocyst per gram of sample. With this assay, it was possible to detect as few as 1 oocyst per gram of berry, in a 50 g sample. Sanger DNA sequencing and phylogenetic analysis were carried out to confirm the presence of C. cayetanensis in berry (n = 17) and soil (n = 5) samples. The phylogenetic analysis revealed that the C. cayetanensis sequences obtained from Mexico clustered within a group recovered from China, Peru, Guatemala-Haiti, and Japan. The PCR protocol designed in the present study could be an important tool for the rapid and accurate detection of this human pathogen in environmental and food samples.
Project description: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.
Project description: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.
Project description:Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.
Project description:Berries and vegetables are potential transmission vehicles for eggs of pathogenic parasites, such as Echinococcus spp. We developed a SYBR Green based semi-quantitative real-time PCR (qPCR) method for detection of Echinococcus multilocularis and Echinococcus canadensis DNA from berry samples. A set of primers based on the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene was designed and evaluated. To assess the efficacy of the assay, we spiked bilberries (Vaccinium myrtillus) with a known amount of E. multilocularis eggs. The detection limit for the assay using the NAD1_88 primer set was 4.37?×?10-5?ng/?l of E. multilocularis DNA. Under artificial contamination of berries, 50 E. multilocularis eggs were reliably detected in 250?g of bilberries. Analytical sensitivity of the assay was determined to be 100% with three eggs. As an application of the assay, 21 bilberry samples from Finnish market places and 21 bilberry samples from Estonia were examined. Previously described sieving and DNA extraction methods were used, and the samples were analyzed for E. multilocularis and E. canadensis DNA using semi-quantitative real-time PCR and a melting curve analysis of the amplified products. Echinococcus DNA was not detected in any of the commercial berry samples. This easy and fast method can be used for an efficient detection of E. multilocularis and E. canadensis in bilberries or other berries, and it is applicable also for fruits and vegetables.
Project description:This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAMTM, HEXTM, Cy5TM, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×103 copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.
Project description:Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.
Project description:This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler's diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×103 oocysts for C. parvum, >1×104 cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.
Project description:BACKGROUND:Toxoplasma gondii is the third most important contributor to health burden caused by food-borne illness. Ingestion of tissue cysts from undercooked meat is an important source of horizontal transmission to humans. However, there is an increasing awareness of the consumption of fresh fruit and vegetables, as a possible source for oocyst transmission, since this stage of the parasite can persist and remain infective in soil and water for long time. Herein, we outline findings related with detection of T. gondii oocysts in vegetables and berry fruits, which are usually raw consumed. The procedure includes the estimation of the number of oocysts. METHODS:Food samples were collected from local producers and supermarket suppliers. Toxoplasma gondii oocysts were concentrated after washing the samples by applying high resolution water filtration and immunomagnetic separation (method 1623.1: EPA 816-R-12-001-Jan 2012), in order to (i) remove potential Cryptosporidium spp. oocysts and Giardia spp. cysts present in the samples; and (ii) select T. gondii oocysts. Toxoplasma gondii oocyst detection and an estimation of their numbers was performed by conventional PCR and real time qPCR, using specific primers for a 183-bp sequence of the T. gondii repetitive DNA region. All PCR-positive DNA samples were purified and sequenced. Restriction enzyme digestion with EcoRV endonuclease confirmed the presence of the T. gondii DNA fragment. In addition, the presence of the parasite was observed by fluorescent microscopy, taking advantage of the oocysts autofluorescence under UV light. RESULTS:Forty percent of the analysed samples (95% CI: 25.5-56.5%) presented the expected PCR and digested DNA fragments. These fragments were confirmed by sequencing. Microscopic autofluorescence supported the presence of T. gondii-like oocysts. The estimated mean (±?SE) oocyst concentration was 23.5?±?12.1 oocysts/g, with a range of 0.6-179.9 oocysts/g. CONCLUSIONS:Our findings provide relevant evidence of contamination of fresh vegetables and berry fruits with T. gondii oocysts.
Project description: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.
Project description:Cyclospora cayetanensis is a coccidian parasite that is associated with foodborne outbreaks of gastrointestinal illnesses. Raspberries have been implicated as a vehicle of infection in some of these outbreaks. Most of the molecular techniques used for the detection of parasites commonly use the 18s rRNA as a target gene, which is highly conserved. The conserved nature of the 18s rRNA gene among coccidia means that there is potential for cross-reactivity from primers intended to target this gene in C. cayetanensis with the same gene in related coccidia. This provides an additional challenge in developing a specific detection method. The aim of this study is to develop a new, more specific assay to detect C. cayetanensis in berry fruits. This new assay, targeting the internal transcribed spacer 1 (ITS-1) region, was tested on three different berry matrices: raspberries, blueberries, and strawberries. The new assay showed good efficiency (102%), linearity (r 2 = 0.999), repeatability (standard deviation of Cq 0.2 (95% CI: 0.2, 0.3) and specificity for Cyclospora, with no cross-reactivity with related coccidia (Toxoplasma gondii, Eimeria mitis, Cystoisospora canis, and Cryptosporidium parvum) when tested in vitro. The method development was initially conducted using Cyclospora DNA only. After it was confirmed to have an acceptable performance, the method was evaluated using the oocysts of C. cayetanensis. The method was also improved by incorporating an internal control as a duplex in order to monitor PCR inhibition due to sample matrix components. The duplex assay also showed a good efficiency (100%) and linearity (r 2 = 0.99). The results showed that the new assay has potential for standard use in food testing laboratories. Furthermore, results regarding important factors related to assay robustness are discussed.