In vitro interactions of the Asian freshwater clam (Corbicula fluminea) hemocytes and Cryptosporidium parvum oocysts. Applied and Environmental Microbiology 63: 2910-2912

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In vitro interactions of the Asian freshwater clam (Corbicula fluminea) hemocytes and Cryptosporidium parvum oocysts. Applied and Environmental Microbiology 63: 2910-2912
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   A  PPLIED AND  E NVIRONMENTAL   M ICROBIOLOGY ,0099-2240/97/$04.00  0July 1997, p. 2910–2912 Vol. 63, No. 7Copyright © 1997, American Society for Microbiology In Vitro Interactions of Asian Freshwater Clam( Corbicula fluminea ) Hemocytes and  Cryptosporidium parvum  Oocysts THADDEUS K. GRACZYK, 1,2 * RONALD FAYER, 3 MICHAEL R. CRANFIELD, 2,4  AND  DAVID BRUCE CONN 5  Department of Molecular Microbiology and Immunology, School of Hygiene and Public Health, 1  and Division of Comparative Medicine, School of Medicine, 4  Johns Hopkins University, Baltimore, Maryland 21205; Medical Department, The Baltimore Zoo, Druid Hill Park, Baltimore, Maryland 21217  2  ; Immunity and Disease Prevention Laboratory, Livestock and Poultry Science Institute, Agricultural Research Service,U.S. Department of Agriculture, Beltsville, Maryland 20705 3  ; and School of Mathematical and Natural Sciences, Berry College, Mount Berry, Georgia 30149 5 Received 3 February 1997/Accepted 9 April 1997 Corbicula fluminea  hemocytes phagocytosed infectious oocysts of   Cryptosporidium parvum  in vitro. After 15,30, 60, 90, and 120 min of incubation, averages of 35.8, 58.0, 69.7, 77.7, and 81.6% of the oocysts werephagocytosed by 24.3, 70.0, 78.5, 87.3, and 93.0% of the hemocytes, respectively. A single clam can retain byphagocytosis an average of 1.84  10 6 oocysts per ml of hemolymph.  C. fluminea  bivalves can serve as biologicalindicators of contamination of wastewaters and agricultural drainages with  Cryptosporidium. Cryptosporidiosis is a zoonotic diarrhoeal disease caused byan intestinal protozoan,  Cryptosporidium parvum . The infec-tious stage, the oocyst, is transmitted via water. The pathogenrepresents a global public health threat, having caused massive waterborne epidemics (10). As many as 5,500 oocysts per literin agricultural drainage have been reported (14). The Asianfreshwater clam ( Corbicula fluminea ) is well adapted to life inunstable and unpredictable habitats and is highly successful in waters receiving agricultural and industrial pollution and urban waste (13). For the past 15 years, this benthic species hasserved as a bioindicator of water pollution (9, 12, 15, 17).  C. fluminea  is a suspension feeder able to filter unicellular algae,bacteria, and detrital particles 1.5 to 10.0  m in diameter. Theclams inhabit agricultural drainage systems and wastewaters(13) known to carry high numbers of   Cryptosporidium  oocysts(14). The purpose of the present study was to describe in vitrointeractions between hemocytes of   C. fluminea  and  C. parvum oocysts.The oocysts of   C. parvum  (strain AUCP-1) were obtained asdescribed previously (2). Oocyst infectivity was determined ina mouse infectivity bioassay (1), and a phosphate-buffered sa-line (PBS) suspension of 1.13  10 7 oocysts/ml was prepared.Hemolymph (0.1 to 0.5 ml) was aspirated (16) from 14 wild-collected  C. fluminea  clams (0.8- to 2.0-cm shell length). Thehemolymph was pooled, and the hemocyte concentration wasdetermined (8). Fifty microliters of undiluted hemolymph wasplaced in each of 30 wells (12-mm diameter) on slides of theMER  IF   LUOR Cryptosporidium/Giardia  test (Meridian Diag-nostic, Inc., Cincinnati, Ohio). The hemocytes were allowed toadhere for 30 min in a humidified chamber at 21°C. Twentyhemocyte monolayers each received 10   l of suspension with1.13    10 5 oocysts, and 10 monolayers that received 10   l of PBS served as unexposed controls. The incubation of six mono-layers (four oocyst-exposed and two control monolayers) wasstopped at 15, 30, 60, 90, and 120 min. Following incubation,the wells were washed three times with PBS and incubated for10 min with 1.0% glutaraldehyde, air dried, and fixed withmethanol. A minimum of 100 hemocytes were examined forevidence of phagocytosis with the aid of bright-field and phase-contrast microscopes with a 40   objective. Three hemocytemonolayers from each time point (two oocyst-exposed and onecontrol monolayer) were acid-fast stained (AFS), and the threemonolayers were stained with the immunofluorescent antibodyof the MER  IF   LUOR  test. The immunofluorescence-stainedslides were examined, and the fluorescence was scored (6, 7).The number of nonphagocytosed oocysts was determined forthe entire well area using a 40  objective, and the number of phagocytosed oocysts (per hemocyte) was determined at amagnification of   100 for at least 100 phagocytes.The mean hemocyte concentration of the pooled  C. fluminea hemolymph was 4.50    10 5 cells/ml; individual hemocytemonolayers contained an average of 2.25    10 4 cells. Theproportion of hemocytes to oocysts in the assay was approxi-mately 1/5. Phagocytosed oocysts were clearly visible by bright-field and phase-contrast microscopy at 15-, 30-, and 60-mintime points and became less visible at 90- and 120-min timepoints. In AFS preparations, the phagocytosed oocysts dis-played nonuniform, bright red coloration with characteristicblack granules (Fig. 1). Viewed by fluorescence microscopy,nonphagocytosed oocysts and the hemocyte-internalized oo-cysts at the 15-min time point had the highest (5  ) brightapple-green fluorescence, and it significantly decreased overtime (Spearman rank correlations;  R  0.97,  P   0.01) (Ta-ble 1). The number of hemocytes showing phagocytosis in-creased significantly over time (  R    0.93,  P     0.03). Thenumbers of nonphagocytosed oocysts significantly decreased(  R    0.88,  P     0.05), whereas the mean number of phago-cytosed oocysts per cell significantly increased (  R  0.90,  P   0.04) (Table 1). After 15, 30, 60, 90, and 120 min of incubation,averages of 35.8, 58.0, 69.7, 77.7, and 81.6% of the oocysts werephagocytosed by 24.3, 70.0, 78.5, 87.3, and 93.0% of the he-mocytes, respectively. The kinetics of phagocytosis was given * Corresponding author. Mailing address: Johns Hopkins Univer-sity, School of Hygiene and Public Health, Department of MolecularMicrobiology and Immunology, 615 North Wolfe St., Baltimore, MD21205. Phone: (410) 614-4984. Fax: (410) 955-0105. E-mail: tgraczyk@phnet.sph.jhu.edu.2910  by a binomial time-dependent curve,  y  24.8  1.1  x  5.0  3  x 2 , where  y  is the percent phagocytosed oocysts and  x  is the inter-action time (in minutes).The present study is the first report of interaction betweenhemocytes of a freshwater benthic bivalve,  C. fluminea , and  C. parvum  oocysts.  C. fluminea  hemocytes have been shown to becapable of erythrophagocytosis (16), and  Corbicula japonica hemocytes have been chemotactically attracted to  Vibrio para- haemolyticus  and  Escherichia coli  (11). In vitro phagocytosis of  C. parvum  oocysts by Eastern oyster ( Crassostrea virginica )hemocytes has been demonstrated in vitro and in vivo (3, 4, 8).We conclude that waterborne oocysts of   Cryptosporidium  arefiltered from the water by  C. fluminea  and are then phagocy-tosed by hemocytes. As various environmental factors mayaffect the recovery and phagocytosis of particles by freshwaterbenthic bivalves (13), the kinetics of phagocytosis demon-strated in vitro may differ in vivo. C. fluminea  from agricultural drainage systems has beenused as a bioindicator for agricultural pollutants, contami-nants, and toxicants (9, 12, 15, 17) but not for  Cryptosporidium .Ecological and physiological features make this benthic clamattractive for biomonitoring of water contamination with  Cryp-tosporidium . This bivalve is abundant in wastewaters and has a wide geographical range (13), which would allow direct com-parison of water contamination at different sites within North America. The species is resistant to environmental variationsand habitat disturbances induced by adverse weather condi-tions which facilitate contamination with  Cryptosporidium  (14).The clam is collectible through the year and can be readily heldin field enclosures (13). The morphology of the AFS phagocy-tosed oocysts did not change over 2 h. This indicates the use-fulness of a low-cost and time-efficient AFS procedure whichcan be applied for screening of   C. fluminea  hemocytes. C. fluminea  has the highest filtration rate of all freshwaterbivalves (up to 2.50 liters/hr) and is capable of developingdense populations (up to 3,750 clams/m 2 ) (13). In the PotomacRiver, phytoplankton density declined up to 75% after waterpassed over the  C. fluminea  beds (13). In the Chowan River,the entire water column overlying  C. fluminea  beds has beenfiltered every 1.0 to 1.6 days (13). In the Trinity River, whichaverages 0.25 m in depth, the entire water volume overlying  C. fluminea  beds was filtered every 16 min (13).  C. fluminea  de- velops particularly high-density beds in agricultural drainages FIG. 1. Photomicrograph showing migrating hemocyte of the Asian freshwater clam ( C. fluminea ) with a phagocytosed  C. parvum  (strain AUCP-1) oocyst (arrow)and free, nonphagocytosed oocysts (arrowheads). Bar  10  m. TABLE 1. Results of interactions between  C. fluminea  hemocytesand  C. parvum  AUCP-1 oocysts in an in vitro slidephagocytosis assay  a Interactiontime (min)No. of nonphagocytosedoocysts  b No. of phagocytosedoocysts/cell(mean  SD)  c Mean fluores-cence score  d Range Mean  SD 15 69–111 87.0  9.9 1.8  0.3 5  30 31–57 45.0  6.1 3.1  0.9 4  60 27–41 32.5  4.1 3.4  1.1 3  90 17–30 22.8  3.5 3.9  1.4 3  120 13–22 15.4  3.2 4.1  1.7 1   a Data for each time point were derived from four replicates in which hemo-cyte monolayers of 2.25  10 4 cells each received 1.13  10 5 oocysts.  b Oocysts adhering to the glass. Values were determined by a 10-min exami-nation with a 40  objective.  c Determined with a 100  objective for 100 cells.  d Hemocyte monolayers were processed with the MER  IF   LUOR Cryptospo- ridium/Giardia  test for direct immunofluorescence, which was scored on a scalefrom 1  to 5  , where 5  is the brightest. V OL  . 63, 1997 FRESHWATER CLAMS AND  CRYPTOSPORIDIUM   OOCYSTS 2911   which carry high loads of   Cryptosporidium  oocysts, in certainlocations as high as 5,500 oocysts/liter (14). The clam, becauseof its important ecological functional role in reducing theamount of seston, has significant epidemiological andepizootiological importance in recovery of   C. parvum  and re-duction of the oocyst load. At a concentration of 4.50    10 5  /ml, the hemocytes wereable to phagocytose on average 81.6% of the  C. parvum  oo-cysts. By extrapolation, each milliliter of   C. fluminea  hemo-lymph could internalize 1.84  10 6 oocysts. A single medium-size clam (13) might retain in its tissue, by hemocyteinternalization alone, an average of 3.68    10 6  waterborneoocysts.In the United States,  Corbicula  clams, which are not com-mercially offered for human consumption, are collected fromthe wild and consumed raw by minority ethnic groups (5).Thus,  C. fluminea  clams may play an epidemiological role infood-borne cryptosporidiosis. The study was supported by the Maryland Zoological Society andthe AKC Fund of New York. REFERENCES 1.  Fayer, R.  1994. Effect of high temperature on infectivity of   Cryptosporidium parvum  oocysts in water. Appl. Environ. Microbiol.  60: 2732–2735.2.  Fayer, R., and W. Ellis.  1993. Paromomycin is effective as prophylaxis forcryptosporidiosis in dairy calves. J. Parasitol.  79: 771–774.3.  Fayer, R., T. K. Graczyk, C. A. Farley, E. J. Lewis, and J. M. Trout.  Thepotential role of waterfowl and oysters in the complex epidemiology of  Cryptosporidium parvum . J. Am. Water Works Assoc., in press.4.  Fayer, R., C. A. Farley, E. J. Lewis, J. M. Trout, and T. K. Graczyk.  1997.Potential role of the Eastern oyster,  Crassostrea virginica , in the epidemiol-ogy of   Cryptosporidium parvum . Appl. Environ. Microbiol.  63: 2086–2088.5.  Fried, B., and S. Emili.  1987. Experimental infection of   Corbicula fluminea (Bivalvia: Corbiculidae) with  Echinostoma revolutum  cercariae. J. Parasitol. 73: 655–656.6.  Graczyk, T. K., M. R. Cranfield, and R. Fayer.  1995. A comparative assess-ment of direct fluorescence antibody, modified acid fast stain, and sucroseflotation techniques for detection of   Cryptosporidium serpentis  oocysts insnake fecal specimens. J. Zoo Wildl. Med.  26: 396–402.7.  Graczyk, T. K., M. R. Cranfield, and R. Fayer.  1996. Evaluation of commer-cial enzyme immunoassay (EIA) and immunofluorescence antibody (IFA)test kits for detection of   Cryptosporidium  oocysts of species other than  Cryp-tosporidium parvum . Am. J. Trop. Med. Hyg.  54: 274–279.8.  Graczyk, T. K., R. Fayer, E. J. Lewis, C. A. Farley, and J. M. Trout.  In vitrointeractions between hemocytes of the Eastern oyster,  Crassostrea virginica Gmelin, 1791 and  Cryptosporidium parvum  oocysts. J. Parasitol., in press.9.  Kira, S., Y. Nogami, K. Taketa, and H. Hayatsu.  1996. Comparison of techniques for monitoring water-borne polycyclic mutagens: efficiency of blue rayon, Sep-Pak C18, and a biota,  Corbicula , in concentrating benzo-(a)pyrene in a model water system. Bull. Environ. Contam. Toxicol.  57: 278–283.10.  Kramer, M. H., B. L. Herwaldt, G. F. Craun, R. L. Calderon, and D. D. Juranek.  1996. Surveillance for waterborne disease outbreaks—UnitedStates, 1993–1994. Morbid. Mortal. Weekly Rep.  45: 1–33.11.  Kumazawa, N. H., and N. Morimoto.  1992. Chemotactic activity of hemo-cytes derived from a brackish-water clam,  Corbicula japonica , to  Vibrio para- haemolyticus  and  Escherichia coli  strains. J. Vet. Med. Sci.  54: 851–855.12.  Leland, H. V., and B. C. Scudder.  1990. Trace elements in  Corbicula fluminea from the San Joaquin River, California. Sci. Total Environ.  97–98: 641–672.13.  McMahon, R. B.  1991. Mollusca: Bivalvia, p. 315–401.  In  J. H. Thorp and A. P. Covich (ed.), Ecology and classification of North American freshwaterinvertebrates. Academic Press, Inc., and Hartcourt Brace Jovanovich, Pub-lishers, San Diego, Calif.14.  Rose, J. B., J. T. Lisle, and M. LeChevallier.  1997. Waterborne cryptospo-ridiosis: incidence, outbreaks, and treatment strategies, p. 93–109.  In  R.Fayer (ed.),  Cryptosporidium  and cryptosporidiosis. CRC Press, Inc., BocaRaton, Fla.15.  Tatem, H. E.  1986. Bioaccumulation of polychlorinated biphenyls and metalsfrom contaminated sediment by freshwater prawns,  Macrobrachium resen- bergii  and clams,  Corbicula fluminea . Arch. Environ. Contam. Toxicol.  15: 171–183.16.  Tuan, T. L., and T. P. Youshino.  1987. Role of divalent cations in plasmaopsonin-dependent and independent erythrophagocytosis by hemocytes of the Asian clam,  Corbicula fluminea . J. Invertebr. Pathol.  50: 310–319.17.  Winger, P. V., C. Sieckman, T. W. May, and W. W. Johnson.  1984. Residuesof organochlorine insecticides, polychlorinated biphenyls, and heavy metalsin biota from Apalachicola River, Florida, 1978. J. Assoc. Off. Anal. Chem. 67: 325–333. 2912 GRACZYK ET AL. A  PPL  . E NVIRON . M ICROBIOL  .
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