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

Abstract

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 ...

A reduced-cost approach for analyzing fresh produce for contamination with Cryptosporidium oocysts and/or Giardia cysts

“…In this case, the concerns include that the recovery efficiencies achieved by Cook et al (2006aCook et al ( , 2007 were not duplicated in independent or semiindependent studies (e.g. Amoros, Alonso, & Cuesta, 2010;Rzezutka et al, 2010); even in the validation studies in which 'expert' labs participated, although recovery efficiencies for Cryptosporidium from raspberries were similar to those of the developing lab, the recovery efficiencies of Cryptosporidium from lettuce were significantly lower (Cook et al, 2006b) and also misidentification of oocysts was a problem, despite the participating labs being described as 'expert'.…”

“…ISO 15553, 2006;US EPA 1623, 2005. Initial work in developing a Standard Method for analyzing fresh produce for contamination has largely focused upon these two parasites, and the methods have been based broadly on the water method, with elution from the surfaces of the fresh produce as the initial steps (Cook et al, 2006a(Cook et al, , 2006b(Cook et al, , 2007Robertson & Gjerde, 2000. In brief, these methods depend on an elution step, followed by concentration based on centrifugation and immunomagnetic separation (IMS), and detection by immunofluorescent antibody testing (IFAT).…”

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

“…By incorporating IMS following centrifugation, Cook et al (22) obtained an average recovery of 46.0% Ϯ 19% for artificially cyst-contaminated leafy green products. A method developed by Robertson and Gjerde (9) involving two washing procedures by rotating drum and sonication, followed by centrifugation and IMS, resulted in an average recovery of 67%.…”

“…While the conventional concentration and separation method for foods used in the present study made use of centrifugation and filters, other methods, such as that of Cook et al (22), incorporate immunomagnetic separation (IMS), which also specifically targets G. duodenalis cysts. By incorporating IMS following centrifugation, Cook et al (22) obtained an average recovery of 46.0% Ϯ 19% for artificially cyst-contaminated leafy green products.…”

Spinacia oleracea L. Leaf Stomata Harboring Cryptosporidium parvum Oocysts: a Potential Threat to Food Safety

“…However, a growing shift in consumer dietary habits toward fresh and organically grown produce correlates with an increased occurrence of food-borne outbreaks of Cryptosporidium infection (3,4,10,20,30,33). Irrigation waters have been suggested to be among the major routes of Cryptosporidium contamination of fresh produce (6,8,29,32,36). For instance, 36% of waters used to irrigate crops traditionally eaten raw in the United States and Central America tested positive for Cryptosporidium parvum oocysts (36).…”