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

THADDEUS K. GRACZYK 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

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BARBARA H. GRIMES 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

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RONALD KNIGHT 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

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ALEXANDRE J. DA SILVA 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

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NORMAN J. PIENIAZEK 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

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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

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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.

Author Notes

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@jhsph.edu.
  • 1

    Fayer R, Speer CA, Dubey JP, 1997. The general biology of Cryptosporidium. Fayer R, ed. Cryptosporidium and Cryptosporidiosis. Boca Raton, FL: CRC Press, 1–42.

  • 2

    Rose JB, 1997. Environmental ecology of Cryptosporidium and public health implications. Annu Rev Public Hlth 18 :135–161.

  • 3

    Okhuysen PC, Chappell CL, Crabb JH, Sterling CR, DuPont HL, 1999. Virulence of three distinct Cryptosporidium parvum isolates for healthy adults. J Infect Dis 180 :1275–1281.

    • Search Google Scholar
    • Export Citation
  • 4

    Wolfe MS, 1992. Giardiasis. Clin Microbiol Rev 5 :93–100.

  • 5

    Lu SQ, Baruch AC, Adam RD, 1998. Molecular comparison of Giardia lamblia isolates. Int J Parasitol 28 :1341–1345.

  • 6

    Graczyk TK, Fayer R, Cranfield MR, 1998. Zoonotic transmission of Cryptosporidium parvum: implications for waterborne cryptosporidiosis. Parasitol Today 13 :348–351.

    • Search Google Scholar
    • Export Citation
  • 7

    Graczyk TK, Cranfield MR, Fayer R, 1996. Evaluation of commercial enzyme immunoassay (EIA) and immunofluorescent antibody (IFA) test kits for detection of Cryptosporidium oocysts of species other than Cryptosporidium parvum.Am J Trop Med Hyg 54 :274–279.

    • Search Google Scholar
    • Export Citation
  • 8

    Graczyk TK, Knight R, Gilman RH, Cranfield MR, 2001. The role of non-biting flies in the epidemiology of human infectious diseases. Microbes Infect 3 :231–235.

    • Search Google Scholar
    • Export Citation
  • 9

    Greenberg B, 1973. Flies and Diseases, Biology and Disease Transmission. Princeton, NJ: Princeton University Press.

  • 10

    Ebeling W, 1978. Urban Entomology. Davis, CA: University of California Press.

  • 11

    Dietz V, Vugis D, Nelson R, Wicklund J, Nadle J, McCombs KG, Reddy S, 2000. Active, multisite, laboratory-based surveillance for Cryptosporidium parvum.Am J Trop Med Hyg 62 :368–372.

    • Search Google Scholar
    • Export Citation
  • 12

    Wallace DJ, van Gilder T, Shalow S, Fiorentino T, Segler SD, Smith KE, Shiferar B, Etzel R, Garthright WE, Angulo FJ, 2000. Incidence of foodborne illness reported by the foodborne diseases active surveillance network (FoodNet)- 1997. FoodNet Working Group. J Food Prot 63 :807–809.

    • Search Google Scholar
    • Export Citation
  • 13

    Graczyk TK, Cranfield MR, Fayer R, Bixler H, 1999. House flies (Musca domestica) as transport hosts of Cryptosporidium parvum.Am J Trop Med Hyg 61 :500–504.

    • Search Google Scholar
    • Export Citation
  • 14

    Graczyk TK, Fayer R, Cranfield MR, Mhangami-Ruwende B, Knight R, Trout JM, Bixler H, 1999. Filth flies are transport hosts of Cryptosporidium parvum.Emerg Infect Dis 5 :726–727.

    • Search Google Scholar
    • Export Citation
  • 15

    Graczyk TK, Fayer R, Knight R, Mhangami-Ruwende B, Trout JM, DaSilva AJ, Pieniazek NJ, 2000. Mechanical transport and transmission of Cryptosporidium parvum oocysts by wild filth flies. Am J Trop Med Hyg 63 :178–183.

    • Search Google Scholar
    • Export Citation
  • 16

    Doiz O, Clavel A, Morales S, Varea M, Castillo FJ, Rubio C, Gomez-Lus R, 2000. House fly (Musca domestica) as a transport vector for Giardia lamblia.Folia Parasitol 47 :330–331.

    • Search Google Scholar
    • Export Citation
  • 17

    Amann RL, Ludwig W, Scleifer KH, 1995. Phylogenetic identification and in-situ detection of individual microbial cells without cultivation. Microbiol Rev 59 :143–169.

    • Search Google Scholar
    • Export Citation
  • 18

    Vesey G, Ashbolt N, Wallner G, Dorsch M, Williams KL, Veal DA, 1995. Assessing Cryptosporidium parvum oocyst viability with fluorescent in-situ hybridization using ribosomal RNA probes and flow cytometry. Betts WB, Casemore D, Fricker C, Smith H, Watkins J, eds. Protozoan Parasites and Water. Cambridge, United Kingdom: Royal Society of Chemistry, 133–138.

  • 19

    Vesey G, Ashbolt N, Fricker EJ, Deere D, William KL, Veal DA, Dorsch M, 1998. The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidium parvum oocysts. J Appl Microbiol 85 :429–440.

    • Search Google Scholar
    • Export Citation
  • 20

    Dorsch MR, Veal DA, 2001. Oligonucleotide probes for specific detection of Giardia lamblia cysts by fluorescent in situ hybridization. J Appl Microbiol 90 :836–842.

    • Search Google Scholar
    • Export Citation
  • 21

    Deere D, Vesey G, Milner M, Williams K, Ashbolt N, Veal DA, 1998. Rapid method for fluorescent in situ ribosomal RNA labelling of Cryptosporidium parvum.J Appl Microbiol 85 :807–818.

    • Search Google Scholar
    • Export Citation
  • 22

    Borror DJ, DeLong MD, Triplehorn CA, 1981. An Introduction to the Study of Insects. Philadelphia: W. B. Saunders.

  • 23

    Graczyk TK, Fayer R, Cranfield MR, Conn DB, 1998. Recovery of waterborne Cryptosporidium parvum oocysts by freshwater benthic clams (Corbicula fluminea). Appl Environ Microbiol 64 :427–430.

    • Search Google Scholar
    • Export Citation
  • 24

    Graczyk TK, Cranfield MR, Fayer R, 1997. Recovery of water-borne oocysts of Cryptosporidium parvum from water samples by the membrane-filter dissolution method. Parasitol Res 83 :121–125.

    • Search Google Scholar
    • Export Citation
  • 25

    Graczyk TK, Fayer R, Cranfield MR, Owens R, 1997. Cryptosporidium parvum oocysts recovered from water by the membrane filter dissolution method retain their infectivity. J Parasitol 83 :111–114.

    • Search Google Scholar
    • Export Citation
  • 26

    Carreno RA, Pokorny NJ, Weir SC, Lee H, Trevors JT, 2001. Decrease in Cryptosporidium parvum oocyst infectivity in vitro using the membrane filter dissolution method for recovering oocysts from water samples. Appl Environ Microbiol 67 :3309–3313.

    • Search Google Scholar
    • Export Citation
  • 27

    Deere D, Vesey G, Ashbolt N, Davies KA, Williams KL, Veal D, 1989. Evaluation of fluorochromes for flow cytometric detection of Cryptosporidium parvum oocysts labelled by fluorescent in situ hybridization. Lett Appl Microbiol 27 :352–356.

    • Search Google Scholar
    • Export Citation
  • 28

    Graczyk TK Cranfield MR, 1998. Experimental transmission of Cryptosporidium oocysts isolates from mammals, birds, and reptiles to captive snakes. Vet Res 29 :187–195.

    • Search Google Scholar
    • Export Citation
  • 29

    Kilani RT, Sekla L, 1987. Purification of Cryptosporidium oocysts and sporozoites by cesium chloride and percoll gradients. Am J Trop Med Hyg 36 :505–508.

    • Search Google Scholar
    • Export Citation
  • 30

    Da Silva AJ, Schwartz DA, Visvesvara GS, deMoura H, Slemenda SB, Pieniazek NJ, 1996. Sensitive PCR diagnosis of infection by Enterocytozoon bieneusi (Microsporidia) using primers based on the region coding for small subunit rRNA. J Clin Microbiol 34 :989–987.

    • Search Google Scholar
    • Export Citation
  • 31

    Spano F, Putignani L, McLauchlin J, Casemore DP, Crisanti A, 1997. PCR-RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum, and between C. parvum isolates of human and animal origin. FEMS Microbiol Lett 150 :209–217.

    • Search Google Scholar
    • Export Citation
  • 32

    Johnson DW, Pieniazek NJ, Griffin DW, Misener L, Rose JB, 1995. Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples. Appl Environ Microbiol 61 :3849–3855.

    • Search Google Scholar
    • Export Citation
  • 33

    Pieniazek NJ, Bornay-Llinares FJ, Slemenda SB, DaSilva AJ, Moura INS, Arrowood AJ, Ditrich O, Addiss DG, 1999. HIV-infected patients harbor four distinct genotypes of Cryptosporidium parvum: implications for diagnosis, epidemiology, and prevention. Emerg Infect Dis 5 :444–449.

    • Search Google Scholar
    • Export Citation
  • 34

    Bean NH, Goulding SJ, Lao C, Angulo EJ, 1996. Surveillance for foodborne disease outbreaks - United States, 1988–1992. MMWR Morb Mort Wkly Rep 45 :1–66.

    • Search Google Scholar
    • Export Citation
  • 35

    Murvosh CM, Thaggard CW, 1966. Ecological studies of the house fly. Ann Entomol Soc Am 59 :533–547.

  • 36

    Rodgers MR, Flanigan DJ, Jakubowski W, 1999. Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. Washington, DC: U.S. Environmental Protection Agency, Office of Water. EPA/821-R-99-001, 24–27.

  • 37

    Rodgers MR, Flanigan DJ, Jakubowski W, 1999. Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. Washington, DC: U.S. Environmental Protection Agency, Office of Water. EPA/821-R-99-006.

  • 38

    Jenkins MC, Trout J, Abrahamsen MS, Higgins J, Fayer R, 2000. Estimating viability of Cryptosporidium parvum oocysts using reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding amyloglucosidase. J Microbiol Methods 34 :97–106.

    • Search Google Scholar
    • Export Citation
  • 39

    Anonymous, 1994. National primary drinking water regulations: Cryptosporidium, Giardia, viruses, disinfection, byproducts, water treatment plant data and other information requirements. Fed Register 59 :669–858.

    • Search Google Scholar
    • Export Citation
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