Cryptosporidium parvum and Giardia lamblia Recovered from Flies on a Cattle Farm and in a Landfill

Abstract

Filth flies associated with a cattle barn and a municipal landfill were tested positive by combined immunofluorescent antibody and fluorescent in situ hybridization for Cryptosporidium parvum and Giardia lamblia on their exoskeletons and in their guts. More pathogens were carried by flies from the cattle barn than from the landfill; 81% of C. parvum and 84% of G. lamblia pathogens were presumptively viable.

Cryptosporidium parvum and Giardia lamblia are zoonotic parasites that cause diarrheal disease worldwide, and C. parvum significantly contributes to the mortality of people with impaired immune systems (7, 25). Fluorescent in situ hybridization (FISH) utilizes fluorescently labeled oligonucleotide probes targeted to species-specific sequences of C. parvum and G. lamblia 18S rRNA (6, 22, 23). Because rRNA has a short half-life and is only present in numerous copies in viable organisms, FISH allows for identification of viable C. parvum and G. lamblia (6, 17, 23). Synanthropic filth flies are transport hosts for a variety of pathogens of public health importance (14). Fly-associated transmission of C. parvum and G. lamblia has been discovered relatively recently (2, 5, 8, 11, 12, 13, 15), but it remains unknown if it is a widespread phenomenon and whether these pathogens are capable of initiating human infections.

Nonbiting flies were captured (15) in September at two locations near Gdansk, Poland: (i) dairy cattle farm Bystra (54^o18′N, 18^o46′E) and (ii) municipal landfill Szadolki (54^o19′N, 18^o33′E). Flies were killed (11), identified to the family taxon level (15), and preserved in 75% ethanol. Flies were surface eluted (15) and homogenized (10), and the eluant and the homogenate were processed by the cellulose acetate membrane filter dissolution method (9) as described previously (15). Elution ensures recovery of particles from the flys' exoskeleton and homogenization from their guts (15). The resulting pellets were processed by the combined immunofluorescent antibody (IFA) specific to Cryptosporidium and Giardia spp. and FISH with oligonucleotide probes specific to C. parvum and G. lamblia as described previously (15). Positive and negative controls were prepared as described previously (15). The numbers of recovered pathogens were adjusted for the efficiency of the cellulose acetate membrane method (78.8%) (9). Statistical analysis was carried out with Statistix 7.0 (Analytical Software, St. Paul, Minn.).

The total numbers of Muscidae, Sarcophagidae, and Calliphoridae flies were 104, 2, and 2, respectively. Only Muscidae flies (n = 100) were caught at the cattle farm. Combined IFA and FISH analyses clearly differentiated between presumptively viable versus nonviable C. parvum oocysts (Fig. 1) and G. lamblia cysts (data not shown). Presumptively viable oocysts were intact and revealed a gap between the wall and internal structures, and the sporozoites were visible (Fig. 1). In comparison, dead oocysts, i.e., oocyst shells, frequently had discernible damage to their walls (Fig. 1). Nonviable G. lamblia cysts were represented by shells with structurally damaged walls or intact cells with a very small amount of internal structures with diffused appearance. In comparison, presumptively viable cysts were filled out completely with cytoplasm without the gap between the internal structures and the wall. In general, more C. parvum than G. lamblia was recovered, and this relationship was statistically significant at the cattle farm (rank sum test; t = 1.22, P < 0.05) (Table 1). Significantly more pathogens per fly were recovered from flies at the cattle farm than from those collected at the landfill (rank sum test; t = 9.31, P < 0.05) (Table 1). Overall, significantly more presumptively viable than nonviable pathogens were carried by flies (rank sum test; t = 15.8, P < 0.01) (Table 1). Presumptively viable C. parvum oocysts accounted for 81% of all oocysts and over 84% of G. lamblia cysts were presumptively viable (Table 1). In general, more C. parvum oocysts were found on flys' exoskeletons than in their guts; this relationship was opposite for G. lamblia (Table 1).

FIG. 1.

Combined IFA and FISH image of C. parvum oocysts recovered from wild-caught filth flies at dairy cattle barn Bystra, near Gdansk, Poland. Viable C. parvum oocyst and oocyst shell, i.e., nonviable oocyst. Scale bar, 5 μm.

TABLE 1.

C. parvum oocysts and G. lamblia cysts recovered from wild-caught synanthropic flies (Gdansk, Poland) and detected by combined IFA and FISH^a

Fly collection site	Pathogen extraction site (no. of fly batches)	Mean no. of pathogen cystic stages per SPP.
		C. parvum			G. lamblia
		Viable	Dead	Overall	Viable	Dead	Overall
1	Exoskeleton (5)	6.3	1.7	8.0	4.8	1.1	5.9
	Gut (5)	9.9	2.0	11.9	1.6	0	1.6
2	Exoskeleton (3)	1.7	0.7	2.4	1.3	0.5	1.8
	Gut (3)	3.5	1.6	5.1	0.5	0.3	0.8
Mean total value		15.7	3.7	19.4	6.1	1.2	7.3

^aSite 1, dairy cattle farm Bystra; site 2, municipal landfill Szadolki. Flies were separated by family and processed in batches of a maximum of 20 specimens.

The present study, together with other studies (2, 5, 8, 13, 15), leaves no doubt that synanthropic flies carry C. parvum and G. lamblia naturally acquired from unhygienic sites, and it confirms findings that these pathogens are viable (15) and infectious (13) while carried by flies. Thus, nonbiting flies can cause human or animal cryptosporidiosis or giardiasis via deposition of these pathogens on visited surfaces, i.e., food. Food-borne cases and outbreaks of cryptosporidiosis and giardiasis have been extensively documented (1, 4, 24), and therefore it is epidemiologically important to assess the involvement of filth flies in transmission of these food-borne infections.

Mechanical transmission of pathogens by flies is intensive because it is achieved through defecation, regurgitation, or mechanical dislodgement (14). Some species of the family Muscidae (predominantly collected in the present study) can live up to a month, lay up to six egg batches, and produce up to 12 generations in the summer in temperate climates and several generations during the winter (while indoors) (16). Muscidae spp. can travel up to 20 miles (21) and orient their movement toward unsanitary sites (16, 21). The mechanisms of pathogen transmission and the biology and ecology of Muscidae spp. (14, 16, 21) indicate a high potential for dissemination of C. parvum and G. lamblia, which can significantly impact human health, although outbreaks are rarely related to flies. These findings emphasize the need for enforcement of various fly control measures at places such as cattle barns and landfills (16).

The biological basis for more intense pathogen transport in the barn is the fact that cattle feces strongly attracts flies for feeding and breeding (13), and feces viscosity enhances the efficiency of fly hairs and bristles in trapping suspended particles and oocysts (14).

FISH has multiple advantages over PCR and IFA for identification of C. parvum and G. lamblia (3, 6, 15); the most important advantage is assessment of viability of even a single pathogen cell, which can be observed in Fig. 1. Such resolution is not available, or extremely impractical, with any other techniques. For example, with recently developed highly sensitive reverse transcriptase-PCR, the lowest number of C. parvum oocysts that can be assessed for viability is 10³ (18). As only weak autofluorescence of nonstructural debris was observed in the present study, FISH is a suitable technique for identification of C. parvum and G. lamblia transported not only by flies but also other insect vectors (19, 20, 26).