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Am. J. Trop. Med. Hyg., 68(2), 2003, pp. 228-232
Copyright © 2003 by The American Society of Tropical Medicine and Hygiene

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DETECTION OF CRYPTOSPORIDIUM PARVUM AND GIARDIA LAMBLIA CARRIED BY SYNANTHROPIC FLIES BY COMBINED FLUORESCENT IN SITU HYBRIDIZATION AND A MONOCLONAL ANTIBODY

THADDEUS K. GRACZYK, BARBARA H. GRIMES, RONALD KNIGHT, ALEXANDRE J. DA SILVA, NORMAN J. PIENIAZEK, AND DUNCAN A. VEAL
The W. Harry Feinstone Department of Molecular Microbiology and Immunology, and Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland; Division of Environmental Health, On-Site Wastewater Section, Raleigh, North Carolina; Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Fluorometrics Research Team, Department of Biologic Sciences, Macquarie University, Sydney, New South Wales, Australia


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Wild-caught synanthropic flies were tested for the presence of Cryptosporidium parvum and Giardia lamblia on their exoskeletons and in their digestive tracks by fluorescent in situ hybridization and fluorescein isothiocyanate (FITC)–conjugated monoclonal antibody (MAb) against Cryptosporidium and Giardia cell wall epitopes. The levels of C. parvum were positively correlated with the levels of G. lamblia, indicating a common source of contamination. The majority of oocysts and cysts were potentially viable (C. parvum = 80% and G. lamblia = 69%). More G. lamblia cysts occurred on the exoskeleton of the flies than within the digestive tracts; the opposite relationship was observed for C. parvum. No genotype other than C. parvum G2 was found to be associated with flies. Because filth flies carry viable C. parvum oocysts and G. lamblia cysts acquired naturally from unhygienic sources, they can be involved in the epidemiology of cryptosporidiosis and giardiasis. Fluorescent oligonucleotide probes used together with FITC–conjugated MAb represent a convenient and cost-effective technique for rapid and specific identification of human-infectious species of Cryptosporidium and Giardia mechanically transported by flies, and for the assessment of the viability of these pathogens.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cryptosporidium parvum is an anthropozoonotic protozoan parasite that significantly contributes to mortality of people with various immune system impairments.1 Diarrheal disease is initiated by a microscopic stage, the oocyst, and a single oocyst can cause infection in immunosuppressed individuals.2 The 50% infectious dose (ID50) of geographically diverse isolates of C. parvum for immunocompetent people ranges from 9 to 1,042 oocysts.3 Giardia lamblia is another anthropozoonotic protozoan parasite that is transmitted via microscopic cysts and causes diarrheal disease worldwide.4 This species has also been referred to as G. duodenalis or G. intestinalis.5 Detection of C. parvum and G. lamblia in environmental samples is commonly achieved by fluorescein isothiocyanate (FITC)–conjugated monoclonal antibody (MAb) (immunofluorescent assay [IFA]).6 However, these assays are not specific to C. parvum and other species of Cryptosporidium can produce a positive IFA reaction.7

Refuse and promiscuous-landing synanthropic filth flies are transport hosts for a variety of pathogens of public health importance, as reviewed recently.8 Outbreaks of foodborne diarrheal diseases demonstrate distinct seasonal patterns which coincide with seasonal increases in abundance of synanthropic flies during warm months of the year.9,10 Interestingly, the first complete reports on foodborne cases of cryptosporidiosis for the United States provided by the Foodborne Disease Active Surveillance Network (FoodNet) demonstrated a strong seasonal trend.11,12 The involvement of flies in mechanical transmission of C. parvum in experimental settings has been recently reported,13–15 but it not known if wild-caught filth flies carry this pathogen. Molecular data demonstrated that C. parvum can be carried by flies for at least three weeks after elimination of the contamination source.15 Although G. lamblia cysts have been reported in synanthropic flies, the viability of these cysts was not determined.16

Fluorescent in situ hybridization (FISH) uses fluorescently labeled oligonucleotide probes targeted to species-specific sequences of 18S rRNA.17 Since rRNA has a short half-life and is only present in high copy numbers in viable organisms, FISH can differentiate between potentially viable and non-viable cells.18–21 The FISH technique has been developed for specific detection of C. parvum and G. lamblia.19–21

The purpose of the present study was to determine if wild-caught synanthropic filth flies carry anthropozoonotic pathogens such as C. parvum and G. lamblia and, if so, to determine whether pathogens carried by flies are potentially viable. An additional purpose was to test the suitability and to optimize the protocol of dual labeling, FISH and FITC-conjugated MAb, of C. parvum and G. lamblia recovered from non-biting flies.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Wild fly capture, identification, and processing. Non-biting synanthropic flies were captured in August 2001 at three locations near Raleigh, North Carolina: 1) a North Carolina State University (NCSU) dairy unit, 2) an NCSU waste-processing facility, and 3) grocery store trash dumpsters near Lake Wheeler. The air temperatures ranged from 26°C to 33°C, and the relative humidity ranged from 95% to 100%, as recorded on line by the North Carolina Agricultural Research Station Weather and Climate Network. Flies were captured with a sweep net over surfaces where flies were landing. Flies were placed into labeled plastic bags, transported to the laboratory in a cooler with ice packs, and stored at 4°C until processed.15 Flies were identified to the family taxon level using morphologic criteria9,22 and surface-eluted as described previously.15 After elution, the flies were homogenized in batches of 10 specimens in an Eppendorf tube (VWR, Piscataway, NJ) with a plastic grinding pin fitting the bottom of the tube,23 and the homogenate (approximately 1 mL) was transferred to a 15-mL tube. The eluting fluid24 was added to the homogenate to obtain a volume of 15 mL and decanted after debris sedimented to the bottom, resulting in a volume of approximately 13 mL. The surface eluants (15 mL) and decanted fluids (approximately 13 mL) were separately processed by the cellulose acetate membrane (25-mm diameter, 3.0-µm pore size; Millipore, Bedford, MA) (CAM)-filter dissolution method.24 Processing of C. parvum oocysts by the CAM-filter dissolution method does not alter their infectivity as tested in vivo by mouse bioassay,25 although in vitro testing indicates that oocyst infectivity decreases.26 Surface elution ensures recovery of particles derived from the exoskeleton of the fly, and homogenization releases the particles within the digestive tract.15 The resulting pellets were stored in 200 µL of 95% ethanol at 4°C18 until analyzed by FISH and FITC-conjugated MAb.

Oligonucleotide probes. A C. parvum-specific oligonucleotide probe, i.e., Cry 1, (5'-CGG TTA TCC ATG TAA GTA AAG-3') was used.19 The probe targets the positions between 138 and 160 on the C. parvum 18S rRNA27 and does not hybridize with oocysts of C. baileyi and C. muris.19 Oocysts of C. parvum with fluorescing internal structures after hybridization tested viable and oocysts without fluoresce were dead.18,19 Two probes specific for G. lamblia (as tested against G. microti, G. ardea, and G. muris) were used; Giar-4 (5'-CGG CGG GGG GCC AAC TAC-3'), and Giar-6 (5'-CGG GGC TGC CGC GGC GCG-3')20 were used for all samples. Giardia lamblia cysts producing a positive FISH reaction tested viable.20 All probes were synthesized by the DNA Analysis Facility of the Johns Hopkins University (Baltimore, MD) in 1.0-µM scale, purified by high-performance liquid chromatography, and labeled with a single molecule of a fluorochrome (hexachlorinated 6-carboxyfluorescein).

Monoclonal antibodies. An FITC-conjugated combination of MAb against the cell wall antigens of Cryptosporidium and Giardia from the MERIFLUORTM Cryptosporidium/Giardia test kit (Meridian Diagnostic, Inc., Cincinnati, OH) was used.

Testing of oligonucleotide probes and MAb. Cryptosporidium parvum and G. lamblia were obtained from experimentally infected calves, and C. serpentis was obtained a clinically infected snake (Pituophis melanoleucus).28 Cryptosporidium serpentis oocysts were used because the Cry 1 probe has not been tested with this species.19 Cysts and oocysts were purified by cesium chloride gradient centrifugation29 and stored at 4°C in phosphate-buffered saline (PBS) pH 7.4. Cryptosporidium parvum and C. serpentis oocysts were subjected to treatments with 70% formic acid, 70% acetic acid, and 5% dodecyltrimethyl ammonium bromide (DTAB) for 30 min at 80°C to increase oocyst wall permeability.19 Giardia lamblia cysts were not subjected to wall permeabilization treatment because this does not effect the intensity of FISH.20 In addition C. parvum and C. serpentis oocysts and G. lamblia cysts were subjected to pretreatment with acetone and ethanol as used in the CAM-filter dissolution method.24 The oligonucleotide probes, i.e., Cry 1, Giar-4, and Giar-6, and FITC-conjugated MAb were tested jointly for their reactivity with C. parvum, and C. serpentis oocysts and G. lamblia cysts under the previously described conditions.19,20

Fluorescent in situ hybridization and FITC-conjugated MAb. All FISH and FITC-conjugated MAb testing applied to all material recovered from flies, i.e., surface eluant and decanted fluid pellets, was carried out in Eppendorf tubes in a total volume of 100 µL of hybridization buffer21 at 48°C for one hour.20 The concentration of each oligonucleotide probe was 1 mM and MAb was diluted 1:1. After incubation, the tubes were centrifuged twice at (8,000 x g for two minutes at 4°C and the pellets were resuspended in 100 µL of PBS. Five 20-µL samples were transferred onto five lysine-coated wells (5-mm diameter) on a teflon-coated glass slide (Carlson Scientific, Inc., Peotone, IL) and air-dried. The wells were examined with an Olympus (Melville, NY) BH2-RFL epifluorescent microscope with a dry 60x objective and a BP-490 exciter filter. The number of potentially viable C. parvum and dead, i.e., empty, oocysts (oocyst shells) was counted. A similar enumeration procedure was applied to G. lamblia cysts with the modification that small amounts of diffuse fluorescing cysts were also scored as dead. Infectious C. parvum oocysts and G. lamblia cysts obtained from experimentally infected calves were used as positive controls. Negative controls consisted of snap-frozen and thawed C. parvum oocysts and G. lamblia cysts.

Extraction of C. parvum DNA, amplification by a polymerase chain reaction (PCR), and sequencing of the amplified fragments. The DNA was extracted from 20 (seven each from sites 1 and 2 and six from site 3) 150-µL aliquots of eluants containing fly-derived debris using proteinase K digestion and glass bead disruption of the oocysts as described elsewhere.15,30 The PCR amplification using primers for the Cryptosporidium oocyst wall protein (COWP) and small subunit (SSU) rRNA genes31,32 followed our previously described protocols.15 Sequencing analysis was performed based on a published protocol33 with some modifications. Briefly, amplified products from all PCRs were purified using the Strataprep PCR purification kit (Stratagene, La Jolla, CA), according to the manufacturer’s instructions. Sequencing reactions were analyzed on the Perkins Elmer (Wellesley, MA) ABI 3100 automatic DNA sequencer, and the sequences were assembled using SeqMan II (DNASTAR Inc., Madison, WI).

Statistical analysis. The numbers of C. parvum oocysts and G. lamblia cysts recovered by the CAM-filter dissolution method was adjusted for the method recovery efficiency of 78.8%.24 Statistical analysis was carried out with Statistix 4.1 (Analytical Software, St. Paul, MN). The numbers of C. parvum oocysts and G. lamblia cysts recovered from flies were tested by Wilk-Shapiro ranking plots to determine whether their distribution conformed to a normal distribution and if so, a parametric test, i.e., two-sample t test, was used. The degree of linear association was assessed using Pearson’s correlation coefficient (R). Statistical significance was considered to be a P < 0.05.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fly trapping. The numbers of flies representing families Muscidae, Calliphoridae, Lauxaniidae, and Anthomyiidae were 246, 105, 25, and 25, respectively (Figure 1Go). Although representatives of all families were found at each site, Muscidae dominated at the dairy (66% of all flies) and waste facility (72% of all flies) sites (Figure 1Go).



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  		    FIGURE 1. Results of trapping in August 2001 of wild synanthropic filth flies at a North Carolina State University (NCSU) dairy unit (site 1), an NCSU waste-processing facility (site 2), and grocery store trash dumpsters near Lake Wheeler (site 3).

 
Testing of oligonucleotide probes. Cryptosporidium serpentis oocysts did not produce a positive FISH result in any of the trials. Treatments of C. parvum oocysts with 70% formic acid, 70% acetic acid, and 5% DTAB resulted in significantly brighter fluorescence in comparison with untreated oocysts. However, fluorescence intensity of treated oocysts was similar to the intensity of fluorescence observed for C. parvum oocysts pretreated with acetone and ethanol. Pretreatment with acetone and ethanol did not increase fluorescence intensity of G. lamblia cysts.

Labeling of fly-recovered material based on FISH and FITC-conjugated MAb. Potentially viable G. lamblia cysts were clearly differentiated from nonviable and non-G. lamblia cysts by color as a result of FISH and labeling with the MAb (Figure 2Go). Nonviable cysts were represented by 1) shells with apparently structurally damaged walls (Figure 2CGo and 2Go) intact cells with a very small amount of internal structures with diffused appearance (Figure 2BGo). In comparison, potentially viable intact cysts were filled out completely with cytoplasm without the gap between the internal structures and the wall (Figure 2AGo).



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  		    Figure 2. Fluorescent in situ hybridization and fluorescein isothiocyanate–conjugated monoclonal antibody images of Giardia lamblia cysts (A, B, and C) and Cryptosporidium parvum oocysts (D) recovered from wild-caught synanthropic filth flies. A, Viable G. lamblia cyst. B, nonviable G. lamblia cyst. C, G. lamblia cyst shell (non-viable cysts) with structural wall damage (arrow). D, Viable C. parvum oocyst (arrow) and oocyst shells (nonviable oocysts) (arrowheads). Note the structural damage to the oocyst wall and a small gap between the oocyst wall and the internal structures of the viable oocyst. Scale bars = 7 µm in A, B, and C and 4 µm in D.

 
The FISH procedure and labeling with the MAb clearly differentiated potentially viable C. parvum oocysts from non-viable and non-C. parvum oocysts (Figure 2Go). Oocysts labeled by FISH and the MAb were predominantly intact, showed a small gap between the oocyst wall and internal structures, and in most of them the sporozoites were visible (Figure 2Go). In comparison, dead oocysts frequently showed discernable damage to their walls (Figure 2Go). Rarely, FISH-positive potentially viable oocysts had noticeable ruptures in their walls, which was clearly revealed by staining with the MAb (Figure 2Go).

No FISH-negative, MAb-negative or FISH-positive, MAb-negative pathogens were observed. Fluorescence of other particles in the samples was not observed except for weak autofluorescence of nonstructural debris.

The numbers of fly-derived potentially viable and nonviable C. parvum oocysts and G. lamblia cysts are shown in Table 1Go. The numbers of C. parvum showed a positive correlation with the numbers of G. lamblia (R = 0.99, P < 0.03, by Pearson’s correlations), indicating a common source of contamination. In general, more G. lamblia were found than C. parvum, and more viable pathogens were found than nonviable pathogens (Table 1Go). Viable C. parvum oocysts accounted for 80% of all oocysts, and more than 69% of G. lamblia cysts were also viable (Table 1Go). Significantly more G. lamblia cysts were found on fly exoskeleton than within the digestive tracts (t = 32.3, P < 0.03, by two-sample t test); the opposite relationship was observed with C. parvum (Table 1Go). There was a significant positive correlation (R = 0.73, P < 0.01, by Pearson’s correlations) between viable and nonviable pathogens identified in the digestive tracts of flies collected from all three sites for both parasite species.


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TABLE 1
Cryptosporidium parvum oocysts and Giardia lamblia cysts carried by wild-caught synanthropic filth flies and detected by combined fluorescent in situ hybridization and fluorescein isothiocyanate–conjugated monoclonal antibody*
 
Polymerase chain reaction and genotyping of C. parvum. The PCR amplicons were obtained with DNA extracted from all oocyst isolates by applying three primer pairs for amplification fragments of the COWP and SSU rRNA genes. All fly eluate DNA samples were tested with each primer and all amplicons were sequenced. Sequencing analysis of these amplicons has shown that all isolates were uniformly C. parvum G2 (Genotype 2) in both loci, the genotype commonly found in humans and animals.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that wild synanthropic flies carry potentially viable anthropozoonotic pathogens (C. parvum and G. lamblia) naturally acquired from unhygienic sources such as feces, manure, toilets, abattoirs, trash, carcasses, or sewage. Thus, flies can be involved in giardiasis or cryptosporidiosis via deposition of these pathogens on visited surfaces, i.e., food items or raw, pre-processed food products. Since mechanical transfer of pathogens by flies can be achieved through defecation, regurgitation, or mechanical dislodgement,8,9 the proportion of viable versus dead cysts is of epidemiologic importance. The troubling finding of the present study is that the vast majority of pathogens were viable; thus, capable of infection. Epidemiologic involvement of non-biting flies in cryptosporidiosis and giardiasis is difficult to prove because cases of diarrheal diseases resulting from fly visitations will be classified as foodborne. Foodborne cases and outbreaks of cryptosporidiosis and giardiasis have been extensively documented.11,12,34

The biology and ecology of synanthropic filth flies suggest that their potential for mechanical transmission of C. parvum and G. lamblia is high. The females can live up to a month,9 lay up to six egg batches,9 and produce up to 12 generations in the summer9 and several generations during winter (while indoors).9 Filth flies can travel up to 20 miles,35 and their movement is oriented toward unsanitary sites.9,35

The results of the FISH applications were largely in accordance with those obtained previously for C. parvum and G. lamblia.19–21 The present study used FITC-conjugated MAb together with three fluorescently labeled oligonucleotide probes specific for both pathogen species. Incorporation of FITC-conjugated MAb into the FISH protocol allows (irrespectively of the FISH outcome) observation of the external morphology of pathogens and assessment of any structural damage to their walls. Thus, even pathogens that are FISH positive and theoretically viable19,20 can be nonviable based on cell wall damage. An advantage of our dual approach is shown in Figure 2DGo, in which all three C. parvum oocysts produced positive IFA reactions when tested according to the current U.S. drinking water regulations.36,37 However, only one of these oocysts posed a potential public health risk, which was demonstrated exclusively by the combination of the FISH technique with staining with an MAb. Additionally, the dual use of oligonucleotide probes and mAb eliminates the need for labeling the probes with multiple molecules of a fluorochrome.19

The FISH technique has multiple major advantages (intensively discussed previously)19,20 over the PCR and FITC-conjugated MAb, i.e., IFA, used alone. The most important advantage of the FISH technique and the fluorescent MAb used in combination in the present study was the assessment of viability of even a single pathogenic cell. Such resolution is not available, or extremely impractical, in any other technique. For example, a recently developed and highly sensitive reverse transcriptase–PCR for detection of RNA showed that the lowest number of C. parvum oocysts that can be assessed for their viability was 103.38 An additional practical feature of the combined protocol is elimination of the need for an extra step for permeabilization of oocyst walls,19 since permeabilization is achieved through the procedural step of the CAM-filter dissolution method.24

When one considers the advantages of the FISH technique for identification of viable C. parvum and G. lamblia, it is surprising that this technique has not been more widely implemented into screening of a variety of environmental samples. Both pathogens can be transmitted via water and they are of serious concern (particularly C. parvum) to the drinking water industry37,39 which prompted the application of the FISH technique for water testing.19 However, because of autofluorescent unicellular algae that produce a high background level, the fluorescence of FISH-stained C. parvum oocysts was not bright enough to enable their detection in environmental water concentrates.19 Since only weak autofluorescence of nonstructural debris was observed in the present study, the FISH procedure is a suitable, i.e., convenient and cost-effective, technique for identification of C. parvum and G. lamblia transported by insect vectors.

Received December 27, 2001. Accepted for publication May 9, 2002.

Acknowledgments: We thank D. E. Griffin (Johns Hopkins University, Baltimore, MD) and J. Trout (United States Department of Agriculture, Beltsville, MD) for facilitating this study, and D. Norris (Johns Hopkins University, Baltimore, MD) for his comments on the manuscript. We also thank B. Engber (North Carolina Department of Environment and Natural Resources, Raleigh, NC) for his consultation on capturing of wild flies and selection of capture sites.

Financial support: The study was supported by the U.S. Environmental Protection Agency (Washington, DC) (grant no. R824995), the Faculty Innovation Fund (grant no. H040-951-0189), The Center for A Livable Future (Baltimore, MD) (grant no. H040-951-0180), and the AKC Fund (New York, NY) (grant no. H630-951-2002).

Authors’ addresses: Thaddeus K. Graczyk and Ronald Knight, The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205. Barbara H. Grimes, Division of Environmental Health, On-Site Wastewater Section, 1642 Mail Service Center, Raleigh, NC, Telephone: 919-715-0141, Fax: 919-715-3227. Alexandre J. Da Silva and Norman J. Pieniazek, Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341, Telephone: 770-488-4073, Fax: 770-488-4108. Duncan A. Veal, Fluorometrics Research Team, Department of Biologic Sciences, Macquarie University, Sydney, New South Wales 2109, Australia, Telephone: 61-2-9850-8185, Fax: 61-2-9850-8253.

Reprint requests: Thaddeus K. Graczyk, The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205, Telephone: 410-614-4984, Fax: 410-955-0105, E-mail: tgraczyk{at}jhsph.edu.


REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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