Towards standard methods for the detection of Cryptosporidium parvum on lettuce and raspberries. Part 1: Development and optimization of methods

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We report a method for detecting Giardia duodenalis cysts on lettuce, which we subsequently use to examine salad products for the presence of Giardia cysts and Cryptosporidium oocysts. The method is based on four basic steps: extraction of cysts from the foodstuffs, concentration of the extract and separation of the cysts from food materials, staining of the cysts to allow their visualization, and identification of cysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Cyst staining is also performed using proprietary reagents. The method recovered 46.0% ؎ 19.0% (n ‫؍‬ 30) of artificially contaminating cysts in 30 g of lettuce. We tested the method on a variety of commercially available natural foods, which we also seeded with a commercially available internal control, immediately prior to concentration of the extract. Recoveries of the Texas Red-stained Giardia cyst and Cryptosporidium oocyst internal controls were 36.5% ؎ 14.3% and 36.2% ؎ 19.7% (n ‫؍‬ 20), respectively. One natural food sample of organic watercress, spinach, and rocket salad contained one Giardia cyst 50 g ؊1 of sample as an indigenous surface contaminant.With the increasing concern about transmission of pathogenic microorganisms by foods, there is a need to control the entire food chain, from primary producer to consumer. This can be accomplished through screening and certification programs, which apply highly sensitive and cost-effective methods for detecting food-borne pathogens. These will require the use of detection methods which are robust, reproducible, and practical.The food industry is becoming increasingly aware of the potential for contamination of foodstuffs with the transmissive stages of Giardia duodenalis (9,15,17,20). Fresh produce, in particular, as it is consumed with minimal preparation, is a potential vehicle of transmission, and G. duodenalis cysts have been detected on produce in several countries (1,12,13,14). Contaminated irrigation water, especially, appears to constitute a major route of contamination of fresh produce (3, 21). Practical and reliable detection methods for monitoring foodstuffs will aid the prevention of parasitic disease outbreaks associated with contaminated food (8).The procedure developed could be used by analytical laboratories that may be asked to analyze lettuce or salad products for more than one parasite. The simultaneous elution and enumeration of Cryptosporidium oocysts and Giardia cysts from the same sample using a single extractant is the most convenient option, given that combination immunomagnetic separation (IMS) kits for both Cryptosporidium and Giardia are available commercially. Previously published methods that use IMS (12, 13, 14) used commercial kits that were developed for concentrating cysts and oocysts from water concentrates, not food matrices. These do not maximize cyst and oocyst recoveries from foods. In order to maximize cyst and oocyst recoveries from foods, the choice of ...

Section: Resultsmentioning

confidence: 99%

“…Extraction of cysts was performed according to the method of Cook et al (4). The sample was placed in a filtered stomacher bag (Seward, London, United Kingdom).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Development of a Method for Detection of Giardia duodenalis Cysts on Lettuce and for Simultaneous Analysis of Salad Products for the Presence of Giardia Cysts and Cryptosporidium Oocysts

Cook¹,

Nichols

Wilkinson³

et al. 2007

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“…Samples with higher numbers of food particles make identification and enumeration of protozoan parasites difficult and contribute to lower recoveries (9,10). Food particles are capable of inhibiting the formation of the antigen-antibody complex necessary in immunofluorescence detection methods, by obstructing or chemically altering the epitope region where binding occurs (9,11).…”

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confidence: 99%

Enhancing the Detection of Giardia duodenalis Cysts in Foods by Inertial Microfluidic Separation

Ganz

Clime

Farber

et al. 2015

The sensitivity and specificity of current Giardia cyst detection methods for foods are largely determined by the effectiveness of the elution, separation, and concentration methods used. The aim of these methods is to produce a final suspension with an adequate concentration of Giardia cysts for detection and a low concentration of interfering food debris. In the present study, a microfluidic device, which makes use of inertial separation, was designed and fabricated for the separation of Giardia cysts. A cyclical pumping platform and protocol was developed to concentrate 10-ml suspensions down to less than 1 ml. Tests involving Giardia duodenalis cysts and 1.90-m microbeads in pure suspensions demonstrated the specificity of the microfluidic chip for cysts over smaller nonspecific particles. As the suspension cycled through the chip, a large number of beads were removed (70%) and the majority of the cysts were concentrated (82%). Subsequently, the microfluidic inertial separation chip was integrated into a method for the detection of G. duodenalis cysts from lettuce samples. The method greatly reduced the concentration of background debris in the final suspensions (10-fold reduction) in comparison to that obtained by a conventional method. The method also recovered an average of 68.4% of cysts from 25-g lettuce samples and had a limit of detection (LOD) of 38 cysts. While the recovery of cysts by inertial separation was slightly lower, and the LOD slightly higher, than with the conventional method, the sample analysis time was greatly reduced, as there were far fewer background food particles interfering with the detection of cysts by immunofluorescence microscopy. Giardia duodenalis is an enteric protozoan parasite which infects a wide range of hosts, including humans and a variety of domestic and wild mammals. It is the most commonly identified intestinal parasite worldwide (1), with an estimated 2.8 ϫ 10 8 human cases of giardiasis annually (2). G. duodenalis prevalence is much higher in children than adults and in developing than developed countries (3). Transmission of giardiasis involves the ingestion of the environmentally robust cyst life stage, either by direct contact or indirectly through cyst-contaminated water or food. It is currently estimated that 7% of giardiasis cases in the United States are foodborne (4). Foods that have previously been reported to be contaminated with G. duodenalis cysts include fresh fruits and vegetables, shellfish, and, more recently, meats (5). Fresh fruits and vegetables contaminated with G. duodenalis cysts are of particular public health concern, as they are generally consumed raw and often originate in developing countries with lower standards of water quality and hygiene. An overview by Robertson (6) of the foodborne outbreaks which occurred between the years 1984 and 2012 identified a total of nine giardiasis outbreaks worldwide. A summary of the reported incidence of Giardia spp. in fresh produce was tabulated by Dixon (7) and included 38 studies, with prevalence...

“…To determine the suitability of the developed primers and DNA extraction method for sensitive and specific detection of Cyclospora oocysts spiked in produce washes, 30 g of fresh basil leaves (grown in-house) was processed by stomaching in 200 ml 1 M glycine buffer (pH 5.5) as described previously (2). Five aliquots from freshly prepared 1,000-and 100-oocyst stocks and 15 (five samples tested on three separate occasions) aliquots from prepared 10-or 1-oocyst stocks (quantified as described above) were spiked into 50 l of the concentrated basil wash sediment, and DNA was extracted from the spiked samples and amplified by PCR as described above.…”

Section: Methodsmentioning

confidence: 99%

Highly Sensitive and Specific PCR Assay for Reliable Detection of Cyclospora cayetanensis Oocysts

Lalonde

Gajadhar

2008

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.