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A comparison of conventional microscopy, immunofluorescence microscopy and flow cytometry in the detection of Giardialamblia cysts in beaver fecal samples
Journal of Immunological Methods 202 Ž1997. 27–33
A comparison of conventional microscopy, immunofluorescence microscopy and flow cytometry in the detection of Giardia lamblia cysts in beaver fecal samples Brent R. Dixon a
a,)
, Monique Parenteau b, Charles Martineau b, Jocelyn Fournier
b
Microbiology Research DiÕision, Bureau of Microbial Hazards, Health Protection Branch, Health Canada, Ottawa, Ont., Canada b Animal Resources DiÕision, Health Protection Branch, Health Canada, Ottawa, Ont., Canada Received 13 March 1996; revised 21 October 1996; accepted 21 October 1996
Abstract A variety of domestic and wild animals are considered to be potential sources of giardiasis in humans. As a result, numerous studies have been reported on the prevalence of Giardia lamblia infection in animals. The majority of these surveys have involved various floatation techniques followed by conventional microscopy in order to detect cysts in fecal samples. Immunofluorescence microscopy has become popular in recent years for the detection of G. lamblia cysts in both clinical and environmental samples. This technique can be automated by combining it with flow cytometry. The present study represents a direct comparison of conventional microscopy, immunofluorescence microscopy, and flow cytometry in terms of their relative efficiency in the detection of G. lamblia cysts in beaver fecal samples. As a result of viewer fatigue, or low cyst concentrations, false negatives were common with conventional microscopy, leading to low prevalence estimates. By specifically targeting the cysts, immunofluorescence microscopy provided more reliable results in a shorter time than conventional methods. When flow cytometry was used in combination with immunofluorescence, a larger number of samples could be examined in a relatively short period of time. The results obtained indicated that this technique allowed for more consistent recognition than either conventional or immunofluorescence microscopy of positive samples containing smaller numbers of cysts. Keywords: Giardia; Cyst; Beaver; Microscopy; Flow cytometry; Immunofluorescence
1. Introduction
Abbreviations: PBS, phosphate-buffered saline; SCID, severe combined immunodeficient; FITC, fluorescein isothiocyanate ) Corresponding author. Postal Locator 2204A2, Microbiology Research Division, 4th Flr. West, Banting Research Centre, Tunney’s Pasture, Ottawa, Ont. K1A 0L2, Canada. Tel.: q1 Ž613. 957-0904; Fax: q1 Ž613. 941-0280.
Giardia lamblia Žsyn. G. duodenalis, G. intestinalis ., is one of the most common intestinal parasites in humans throughout the world. An extensive review of stool examination results in the United States revealed a giardiasis prevalence of 7.2% in 1987 and 5.6% in 1991 ŽKappus et al., 1994.. The prevalence
0022-1759r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 6 . 0 0 2 3 9 - 6
28
B.R. Dixon et al.r Journal of Immunological Methods 202 (1997) 27–33
of giardiasis in developing countries is generally much higher ŽMiotti et al., 1986; Islam, 1990; Janoff et al., 1990.. While transmission of giardiasis is predominantly by the fecal–oral route, waterborne and foodborne transmission are also important ŽBoreham et al., 1990; Thompson et al., 1993.. The concept of animals acting as reservoirs for human giardiasis has also been discussed for some time. Presently, there is a great deal of anecdotal evidence to support this view. A large number of mammals, including most domestic animals and pets, may serve as hosts for G. lamblia ŽDavies and Hibler, 1979; Belosevic et al., 1984; Wallis et al., 1984, 1986; Kirkpatrick and Skand, 1985; Gasser et al., 1987; Kiorpes et al., 1987; St. Jean et al., 1987; Lewis, 1988; Kirkpatrick, 1989; Buret et al., 1990; Koudela et al., 1991; Hamlen and Lawrence, 1994; Xiao, 1994.. Beavers and muskrats, in particular, have been implicated as they represent an important source of cysts in raw surface waters. The involvement of these and other wild and domestic animals in the transmission of giardiasis to humans, has generated considerable discussion in recent years ŽBuret and Olson, 1987; Bemrick and Erlandsen, 1988; Erlandsen et al., 1988; Faubert, 1988; Thompson et al., 1988; Kasprzak and Pawlowski, 1989; Buret et al., 1990; Castor and Lindqvist, 1990; Gasser, 1990; Healy, 1990; Thompson et al., 1993.. Most studies on the prevalence of giardiasis in animals have involved floatation procedures for concentrating cysts from feces, followed by conventional microscopy. These techniques are often very tedious and are influenced by viewer fatigue. In recent years, immunofluorescence labeling has become popular for the detection of G. lamblia cysts in both environmental and clinical samples. Cysts are specifically stained using a fluorescein-antibody conjugate and readily identified under blue light using an epifluorescence microscope. A few studies have also described the advantages of flow cytometry and cell sorting, in conjunction with immunofluorescence, in the detection and enumeration of G. lamblia and Cryptosporidium parÕum in water samples and fecal specimens ŽVesey et al., 1993, 1994a,b; Arrowood et al., 1995. and G. lamblia cyst suspensions ŽErlandsen et al., 1988.. The present study compares the relative efficiency of each of these
techniques in the detection of G. lamblia cysts in beaver fecal samples.
2. Materials and methods 2.1. Sample collection Between November 1992 and February 1993, 94 beavers were trapped in rural and urban regions around the city of Ottawa, Ontario, for the purpose of population control. Fecal material was obtained by making an incision through the abdominal wall and colon. Feces from each animal were then transferred to specimen bottles containing an equal volume of 10% buffered formalin, and labeled according to location and date of collection. 2.2. Cyst concentration Fecal samples were further diluted with 10% buffered formalin to prepare suspensions which could be drawn up in a Pasteur pipet. G. lamblia cysts were then concentrated by sucrose gradient floatation as described by Buret et al. Ž1990. with the following modifications. Approximately 0.5 ml of diluted feces were layered over 5 ml 1 M sucrose Žlightly stained with methylene blue to help distinguish the layers. in a 15-ml polystyrene, conical centrifuge tube. The tube was then centrifuged at 400 = g for 5 min and the supernatant above the sucrose layer was transferred to a clean tube and centrifuged as before. The supernatant was discarded and the pellet was resuspended in 0.5 ml of phosphate-buffered saline ŽPBS. pH 7.4, containing 0.01% Žvrv. Triton X-100 ŽSigma, St. Louis, MO.. 2.3. Control preparation A positive control cyst suspension was prepared from the feces of severe combined immunodeficient ŽSCID. mice inoculated with a mouse fecal suspension of Giardia muris kindly provided by Dr. M.E. Olson, University of Calgary, Calgary, Alberta. Mice were housed in pairs in polycarbonate shoebox cages. Fecal pellets deposited through a wire grid bottom over 2-day periods were collected from the base pan. The feces were transferred to a sterile container in
B.R. Dixon et al.r Journal of Immunological Methods 202 (1997) 27–33
which 25 ml of PBS containing 0.05% Žvrv. Tween 80 Žfresh control. or 25 ml of 10% buffered formalin Žfixed control. was added. Fresh and fixed negative control suspensions were prepared in the same manner from the feces of uninfected SCID mice. The care of mice used in this study was in accordance with the guidelines of the Health Protection Branch Animal Care Committee. 2.4. ConÕentional microscopy Following sucrose floatation, one drop of beaver fecal suspension was transferred to a microscope slide with a coverslip. A single control slide was prepared in the same manner from the fresh positive control suspension for each batch of samples examined. Each slide was then examined at 400 = magnification. A total of 94 beaver fecal samples were tested. The presence or absence of G. lamblia cysts was recorded for each sample. 2.5. Immunofluorescence microscopy Following sucrose floatation, 20 ml of beaver fecal suspension was transferred to a microscope slide and air dried. Slides were then fixed for 5 min in acetone and air dried. 25 ml of Giardia-cel IF Test reagent Žfluorescein-labeled anti-Giardia monoclonal antibody. ŽCellabs, distributed by Wellmark Diagnostics Ltd., Guelph, Ontario. was added to each slide. Slides were incubated in an humidity chamber for 30 min at 378C. Finally, slides were gently washed by irrigation in PBS, excess moisture was removed, and a drop of supplied mounting fluid was added along with a coverslip. Slides were examined immediately or stored for a few hours in an humidity chamber at 48C. A single control slide was prepared in the same manner from the fresh positive control suspension for each batch of samples examined. Each slide was examined at 400 = magnification using an epifluorescence microscope with filter system for fluorescein isothiocyanate ŽFITC.. A total of 94 beaver fecal samples were tested. The presence or absence of G. lamblia cysts was recorded for each sample. 2.6. Flow cytometry Following sucrose floatation, 200 ml of beaver fecal suspension was transferred to each of two
29
tubes. 25 ml of Giardia-cel IF Test reagent ŽCellabs. was added to one of the tubes while 25 ml of PBS was added to the other tube Žautofluorescence control.. Following a 30-min incubation at 378C, the contents of both tubes were washed with 2 ml of PBS and centrifuged at 400 = g for 5 min. Pellets were then resuspended in 0.5 ml of PBS and stored at 48C until analysis by flow cytometry. Positive and negative controls Žboth fresh and fixed. were similarly prepared for each batch of samples examined. A flow cytometer ŽFACSCAN, Becton Dickinson, Mississauga, Ontario. equipped with an argon-ion laser operating at 488 nm, and Lysys II support software, were used for the acquisition of cysts and the analysis. Calibration of the FACSCAN was performed using Calibrite beads ŽBecton Dickinson, Mississauga, Ontario. as recommended by the supplier. A total of 10 000 particles Žungated. were acquired for each of the beaver fecal samples tested, with the exception of 6 samples in which fewer particles could be acquired due to their unusual consistency. Dual parameter histograms Žright angle light scatter vs. fluorescence. were plotted for each sample. Whenever a sample was found to be positive for G. lamblia cysts, a drop from the tube was transferred to a microscope slide and examined on an epifluorescence microscope for confirmation. To confirm the reliability of the flow cytometry results, Table 1 Presence or absence of Giardia lamblia cysts in beaver fecal samples Ž ns94. using three different detection methods a Beaver I.D. number
Conventional microscopy
Immunofluorescence microscopy
Flow cytometry
64 62 58 27 102 127 76 53 103 55 108 109 90 79
y y q y y q y q q q q q y y
y y q y q q q q q q q y q y
q q q q q q q q q q q q q q
a
Only samples found to be positive with one or more methods are represented.
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B.R. Dixon et al.r Journal of Immunological Methods 202 (1997) 27–33
a positive beaver fecal sample and a positive control sample were analysed on a Coulter Epics-Elite cell sorter. Cysts were physically separated from fecal debris and were examined on an epifluorescence microscope.
3. Results Of the 94 beaver fecal samples examined by conventional microscopy, 7 were positive for G. lamblia cysts and 87 were negative ŽTable 1.. Several of the samples were questionable due to overlapping debris or poor resolution. As they could not be
confirmed, such samples were considered negative. Positive control slides contained large numbers of cysts. Using immunofluorescence microscopy, cysts appeared as bright green oval-shaped bodies, making them readily apparent even in concentrated fecal debris. Of the 94 samples examined, 9 were positive and 85 were negative ŽTable 1.. Positive control slides contained large numbers of cysts. Of the 94 samples tested by flow cytometry, 14 were positive and 80 were negative ŽTable 1.. The dual parameter histograms for positive and negative beaver samples are shown in Fig. 1 along with their respective autofluorescence controls. The lack of
Fig. 1. Dual parameter histograms of beaver fecal suspensions generated by immunofluorescence flow cytometry. Ž A. Positive beaver sample stained with Giardia-cel IF test reagent Žleft., and unstained Žautofluorescence control. Žright.. Ž B . Negative beaver sample stained with Giardia-cel IF test reagent Žleft., and unstained Žautofluorescence control. Žright..
B.R. Dixon et al.r Journal of Immunological Methods 202 (1997) 27–33
particles in the predetermined gate in autofluorescence histograms indicated that autofluorescent debris had no influence on the results. While large numbers of cysts were present in positive control samples, fewer were present in positive beaver samples. No cysts were seen in either the negative controls or negative beaver samples. While fixed control cyst suspensions remained stable for many months, fresh suspensions could only be used with flow cytometry for a few days. After this period, when fresh cysts were stained, they demonstrated a markedly lower fluorescence intensity than they did initially. The presence of G. lamblia cysts was confirmed using immunofluorescence microscopy for each of the 14 positive samples. In addition, concentrated cyst suspensions were obtained following cell sorting on a positive beaver fecal sample and on a positive control sample, confirming that gated particles represented on histograms were cysts. It often took over 30 min to check a slide for the presence of cysts by conventional microscopy, and viewer fatigue was clearly a factor. Similarly, careful examination of a slide using immunofluorescence took from 15 to 30 min to complete. Analysis by flow cytometry, however, took less than 4 min per sample.
4. Discussion The present study involves a direct comparison of three techniques in terms of their effectiveness in detecting G. lamblia cysts in beaver fecal samples. Conventional microscopy was found to be a very tedious, time-consuming and unreliable means of determining the presence of cysts. Unless there were a large number of cysts present, they were difficult to observe among the fecal debris. When a cyst was located, its identity was often difficult to verify as nuclei and other internal structures were not always visible. As a result, a number of false negatives were recorded. A considerable improvement in the detection of cysts was achieved by concentrating cysts and staining with fluorescein-labeled anti-Giardia antibodies. With immunofluorescence microscopy, cysts were readily identified among fecal debris. However, this
31
technique was still relatively time-consuming, particularly if there were few or no cysts present on the slide. Flow cytometry in combination with immunofluorescence was a rapid and very effective procedure for the detection of G. lamblia cysts in fecal samples, and provided a great improvement over microscopical techniques. By analysing fecal material according to right angle light scatter and intensity of fluorescence, cysts could be readily differentiated from debris. Positive samples could then be easily confirmed using an epifluorescence microscope. An examination of the suspensions obtained from the cell sorter further confirmed that particles within the gate ŽFig. 1A. were, in fact, G. lamblia cysts. Fixed control samples could be prepared and used for many months with no apparent loss in the ability of the cysts to bind with the antibody over time, as was seen with the fresh controls. As the stability of these fixed controls allowed for more consistency in the separation of cysts from fecal debris using flow cytometry, they are recommended over fresh controls. While the time required to prepare the samples for flow cytometry was similar to that of the microscopical techniques, the analysis was very rapid. As a result, many more samples could be examined in a day. More importantly, there was no viewer fatigue or observer variation involved, allowing for more consistent and reliable results. Whenever discrepancies occurred among the results of the three methods, the positive result was always found with flow cytometry, indicating that it may recognize, with more consistency, samples containing smaller numbers of cysts. Based on the number of positive results, immunofluorescence microscopy appeared to be second best in this regard. In general, when a relatively large number of cysts were present, they were not difficult to detect using any of the methods, but when fewer cysts were present, they were only detected using immunofluorescence microscopy or flow cytometry. In only one case were cysts observed with conventional microscopy and flow cytometry, but not with immunofluorescence microscopy. On the basis of the flow cytometry results, the prevalence of G. lamblia infection in beavers in the study area was 14.9%. This figure is similar to those
32
B.R. Dixon et al.r Journal of Immunological Methods 202 (1997) 27–33
reported in other prevalence studies on beavers in North America ŽDavies and Hibler, 1979; Frost et al., 1982; Wallis et al., 1986; Isaac-Renton et al., 1987; Erlandsen et al., 1988, 1990; Roach et al., 1993.. One problem inherent in prevalence studies on wild animals is that G. lamblia cysts are often shed intermittently, as they are in beavers ŽDavies and Hibler, 1979; Wallis et al., 1986., and an examination of a single fecal sample from each animal may result in false negatives and a low prevalence estimation. Flow cytometry with immunofluorescence labeling appears to be ideal for the rapid screening of large numbers of fecal samples for G. lamblia cysts. In addition to prevalence studies in animals, preliminary results from this laboratory indicate that this procedure may also be useful for screening human clinical specimens in outbreak situations. While not all clinical laboratories will have direct access to a flow cytometer and epifluorescence microscope, they are routinely used in hospitals, universities and larger laboratories for immunological work, and the present study describes another important use for this equipment. Work is presently underway in this laboratory to extend this procedure to the simultaneous detection of other intestinal parasites including Cryptosporidium parÕum and Entamoeba histolytica. Acknowledgements The authors are very grateful to Nicole Beausoleil, Sandra Stall and Michel Sanche, Animal Resources Division, Health Protection Branch, Health Canada, for the inoculation and monitoring of the mice and the regular collection of feces. The authors would also like to express their deep appreciation to Pierre Chabiaque, National Capital Commission, Ottawa, Ontario, for his dedicated efforts in the trapping of beavers and collection of fecal samples. References Arrowood, M.J., Hurd, M.R. and Mead, J.R. Ž1995. A new method for evaluating experimental cryptosporidial parasite loads using immunofluorescent flow cytometry. J. Parasitol. 81, 404. Belosevic, M., Faubert, G.M., Guy, R. and MacLean, J.D. Ž1984.
Observations on natural and experimental infections with Giardia isolated from cats. Can. J. Comp. Med. 48, 241. Bemrick, W.J. and Erlandsen, S.L. Ž1988. Giardiasis – is it really a zoonosis? Parasitol. Today 4, 69. Boreham, P.F.L., Upcroft, J.A. and Upcroft, P. Ž1990. Changing approaches to the study of Giardia epidemiology: 1681–2000. Int. J. Parasitol. 20, 479. Buret, A. and Olson, M.E. Ž1987. Giardiasis in laboratory animals. CALASrACTAL 1987 Edmonton, Alberta, p. 35. Buret, A., denHollander, N., Wallis, P.M., Befus, D. and Olson, M.E. Ž1990. Zoonotic potential of giardiasis in domestic ruminants. J. Infect. Dis. 162, 231. Castor, S.B. and Lindqvist, K.B. Ž1990. Canine giardiasis in Sweden: no evidence of infectivity to man. Trans. Roy. Soc. Trop. Med. Hyg. 84, 249. Davies, R.B. and Hibler, C.P. Ž1979. Animal reservoirs and cross-species transmission of Giardia. In: W. Jakubowski and J.C. Hoff ŽEds.., Waterborne Transmission of Giardiasis. Environmental Protection Agency, Cincinnati, Ohio, p. 104. Erlandsen, S.L., Sherlock, L.A., Januschka, M., Schupp, D.G., Schaefer, F.W., Jakubowski, W. and Bemrick, W.J. Ž1988. Cross-species transmission of Giardia spp. : inoculation of beavers and muskrats with cysts of human, beaver, mouse, and muskrat origin. Appl. Environ. Microbiol. 54, 2777. Erlandsen, S.L., Sherlock, L.A., Bemrick, W.J., Ghobrial, H. and Jakubowski, W. Ž1990. Prevalence of Giardia spp. in beaver and muskrat populations in northeastern states and Minnesota: detection of intestinal trophozoites at necropsy provides greater sensitivity than detection of cysts in fecal samples. Appl. Environ. Microbiol. 56, 31. Faubert, G.M. Ž1988. Evidence that giardiasis is a zoonosis. Parasitol. Today 4, 66. Frost, F., Harter, L., Plan, B., Fukutaki, K. and Holman, B. Ž1982. Giardiasis in Washington State. U.S. Environmental Protection Agency, Cincinnati, Ohio, EPA-600r1-82-016, 80 pp. Gasser, R.B. Ž1990. Is giardiasis a zoonosis? Austral. Vet. J. 67, 456. Gasser, R.B., Eckert, J. and Rohrer, L. Ž1987. Isolation of Giardia from Swiss cattle and cultivation of trophozoites in vitro. Parasitol. Res. 73, 182. Hamlen, H.J. and Lawrence, J.M. Ž1994. Giardiasis in laboratoryhoused squirrel monkeys: a retrospective study. Lab. Animal Sci. 44, 235. Healy, G.R. Ž1990. Giardiasis in perspective: the evidence of animals as a source of human Giardia infections. In: E.A. Meyer ŽEd.., Giardiasis. Elsevier, New York, p. 305. Isaac-Renton, J.L., Moricz, M.M. and Proctor, E.M. Ž1987. A Giardia survey of fur-bearing water mammals in British Columbia, Canada. J. Environ. Health 50, 80. Islam, A. Ž1990. Giardiasis in developing countries. In: E.A. Meyer ŽEd.., Giardiasis. Elsevier, New York, p. 235. Janoff, E.N., Taylor, D.N., Echeverria, P., Glode, M.P. and Blaser, M.J. Ž1990. Serum antibodies to Giardia lamblia by age in populations in Colorado and Thailand. West. J. Med. 152, 253. Kappus, K.D., Lundgren, R.G., Juranek, D.D., Roberts, J.M. and Spencer, H.C. Ž1994. Intestinal parasitism in the United States:
B.R. Dixon et al.r Journal of Immunological Methods 202 (1997) 27–33 update on a continuing problem. Am. J. Trop. Med. Hyg. 50, 705. Kasprzak, W. and Pawlowski, Z. Ž1989. Zoonotic aspects of giardiasis: a review. Vet. Parasitol. 32, 101. Kiorpes, A.L., Kirkpatrick, C.E. and Bowman, D.D. Ž1987. Isolation of Giardia from a llama and from sheep. J. Vet. Res. 51, 277. Kirkpatrick, C.E. Ž1989. Giardiasis in large animals. The Compendium – Food Animal 11, 80. Kirkpatrick, C.E. and Skand, D.L. Ž1985. Giardiasis in a horse. J. Am. Vet. Med. Assoc. 187, 163. Koudela, B., Nohynkova, J., Pakandl, M. and Kulda, ´ ´ E., Vıtovec, ´ J. Ž1991.. Giardia infection in pigs: detection and in vitro isolation of trophozoites of the Giardia intestinalis group. Parasitology 102, 163. Lewis, P.D. Ž1988. Prevalence of Giardia sp. in dogs from Alberta. In: P.M. Wallis and B.R. Hammond ŽEds.., Advances in Giardia Research. University of Calgary Press, Calgary, Alberta, p. 61. Miotti, P.G., Gilman, R.H., Santosham, M., Ryder, R.W. and Yolken, R.H. Ž1986. Age-related rate of seropositivity of antibody to Giardia lamblia in four diverse populations. J. Clin. Microbiol. 24, 972. Roach, P.D., Olson, M.E., Whitley, G. and Wallis, P.M. Ž1993. Waterborne Giardia cysts and Cryptosporidium oocysts in the Yukon, Canada. Appl. Environ. Microbiol. 59, 67. St. Jean, G., Couture, Y., Dubreuil, P., Frechette, J.L. Ž1987. ´
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Diagnosis of Giardia infection in 14 calves. J. Am. Vet. Med. Assoc. 191, 831. Thompson, R.C.A., Meloni, B.P. and Lymbery, A.J. Ž1988. Humans and cats have genetically-identical forms of Giardia: evidence of a zoonotic relationship. Med. J. Aust. 148, 207. Thompson, R.C.A., Reynoldson, J.A. and Mendis, A.H.W. Ž1993. Giardia and giardiasis. Adv. Parasitol. 32, 71. Vesey, G., Slade, J.S., Byrne, M., Shepherd, K., Dennis, P.J. and Fricker, C.R. Ž1993. Routine monitoring of Cryptosporidium oocysts in water using flow cytometry. J. Appl. Bacteriol. 75, 87. Vesey, G., Hutton, P., Champion, A., Ashbolt, N., Williams, K.L., Warton, A. and Veal, D. Ž1994a. Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water. Cytometry 16, 1. Vesey, G., Narai, J., Ashbolt, N., Williams, K. and Veal, D. Ž1994b. Detection of specific microorganisms in environmental samples using flow cytometry. Methods Cell Biol. 42, 489. Wallis, P.M., Buchanan-Mappin, J.M., Faubert, G.M. and Belosevic, M. Ž1984. Reservoirs of Giardia spp. in southwestern Alberta. J. Wildlife Dis. 20, 279. Wallis, P.M., Zammuto, R.M. and Buchanan-Mappin, J.M. Ž1986. Cysts of Giardia spp. in mammals and surface waters in southwestern Alberta. J. Wildlife Dis. 22, 115. Xiao, L. Ž1994. Giardia infection in farm animals. Parasitol. Today 10, 436.
A comparison of conventional microscopy, immunofluorescence microscopy and flow cytometry in the detection of Giardia lamblia cysts in beaver fecal samples Brent R. Dixon a
a,)
, Monique Parenteau b, Charles Martineau b, Jocelyn Fournier
b
Microbiology Research DiÕision, Bureau of Microbial Hazards, Health Protection Branch, Health Canada, Ottawa, Ont., Canada b Animal Resources DiÕision, Health Protection Branch, Health Canada, Ottawa, Ont., Canada Received 13 March 1996; revised 21 October 1996; accepted 21 October 1996
Abstract A variety of domestic and wild animals are considered to be potential sources of giardiasis in humans. As a result, numerous studies have been reported on the prevalence of Giardia lamblia infection in animals. The majority of these surveys have involved various floatation techniques followed by conventional microscopy in order to detect cysts in fecal samples. Immunofluorescence microscopy has become popular in recent years for the detection of G. lamblia cysts in both clinical and environmental samples. This technique can be automated by combining it with flow cytometry. The present study represents a direct comparison of conventional microscopy, immunofluorescence microscopy, and flow cytometry in terms of their relative efficiency in the detection of G. lamblia cysts in beaver fecal samples. As a result of viewer fatigue, or low cyst concentrations, false negatives were common with conventional microscopy, leading to low prevalence estimates. By specifically targeting the cysts, immunofluorescence microscopy provided more reliable results in a shorter time than conventional methods. When flow cytometry was used in combination with immunofluorescence, a larger number of samples could be examined in a relatively short period of time. The results obtained indicated that this technique allowed for more consistent recognition than either conventional or immunofluorescence microscopy of positive samples containing smaller numbers of cysts. Keywords: Giardia; Cyst; Beaver; Microscopy; Flow cytometry; Immunofluorescence
1. Introduction
Abbreviations: PBS, phosphate-buffered saline; SCID, severe combined immunodeficient; FITC, fluorescein isothiocyanate ) Corresponding author. Postal Locator 2204A2, Microbiology Research Division, 4th Flr. West, Banting Research Centre, Tunney’s Pasture, Ottawa, Ont. K1A 0L2, Canada. Tel.: q1 Ž613. 957-0904; Fax: q1 Ž613. 941-0280.
Giardia lamblia Žsyn. G. duodenalis, G. intestinalis ., is one of the most common intestinal parasites in humans throughout the world. An extensive review of stool examination results in the United States revealed a giardiasis prevalence of 7.2% in 1987 and 5.6% in 1991 ŽKappus et al., 1994.. The prevalence
0022-1759r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 6 . 0 0 2 3 9 - 6
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B.R. Dixon et al.r Journal of Immunological Methods 202 (1997) 27–33
of giardiasis in developing countries is generally much higher ŽMiotti et al., 1986; Islam, 1990; Janoff et al., 1990.. While transmission of giardiasis is predominantly by the fecal–oral route, waterborne and foodborne transmission are also important ŽBoreham et al., 1990; Thompson et al., 1993.. The concept of animals acting as reservoirs for human giardiasis has also been discussed for some time. Presently, there is a great deal of anecdotal evidence to support this view. A large number of mammals, including most domestic animals and pets, may serve as hosts for G. lamblia ŽDavies and Hibler, 1979; Belosevic et al., 1984; Wallis et al., 1984, 1986; Kirkpatrick and Skand, 1985; Gasser et al., 1987; Kiorpes et al., 1987; St. Jean et al., 1987; Lewis, 1988; Kirkpatrick, 1989; Buret et al., 1990; Koudela et al., 1991; Hamlen and Lawrence, 1994; Xiao, 1994.. Beavers and muskrats, in particular, have been implicated as they represent an important source of cysts in raw surface waters. The involvement of these and other wild and domestic animals in the transmission of giardiasis to humans, has generated considerable discussion in recent years ŽBuret and Olson, 1987; Bemrick and Erlandsen, 1988; Erlandsen et al., 1988; Faubert, 1988; Thompson et al., 1988; Kasprzak and Pawlowski, 1989; Buret et al., 1990; Castor and Lindqvist, 1990; Gasser, 1990; Healy, 1990; Thompson et al., 1993.. Most studies on the prevalence of giardiasis in animals have involved floatation procedures for concentrating cysts from feces, followed by conventional microscopy. These techniques are often very tedious and are influenced by viewer fatigue. In recent years, immunofluorescence labeling has become popular for the detection of G. lamblia cysts in both environmental and clinical samples. Cysts are specifically stained using a fluorescein-antibody conjugate and readily identified under blue light using an epifluorescence microscope. A few studies have also described the advantages of flow cytometry and cell sorting, in conjunction with immunofluorescence, in the detection and enumeration of G. lamblia and Cryptosporidium parÕum in water samples and fecal specimens ŽVesey et al., 1993, 1994a,b; Arrowood et al., 1995. and G. lamblia cyst suspensions ŽErlandsen et al., 1988.. The present study compares the relative efficiency of each of these
techniques in the detection of G. lamblia cysts in beaver fecal samples.
2. Materials and methods 2.1. Sample collection Between November 1992 and February 1993, 94 beavers were trapped in rural and urban regions around the city of Ottawa, Ontario, for the purpose of population control. Fecal material was obtained by making an incision through the abdominal wall and colon. Feces from each animal were then transferred to specimen bottles containing an equal volume of 10% buffered formalin, and labeled according to location and date of collection. 2.2. Cyst concentration Fecal samples were further diluted with 10% buffered formalin to prepare suspensions which could be drawn up in a Pasteur pipet. G. lamblia cysts were then concentrated by sucrose gradient floatation as described by Buret et al. Ž1990. with the following modifications. Approximately 0.5 ml of diluted feces were layered over 5 ml 1 M sucrose Žlightly stained with methylene blue to help distinguish the layers. in a 15-ml polystyrene, conical centrifuge tube. The tube was then centrifuged at 400 = g for 5 min and the supernatant above the sucrose layer was transferred to a clean tube and centrifuged as before. The supernatant was discarded and the pellet was resuspended in 0.5 ml of phosphate-buffered saline ŽPBS. pH 7.4, containing 0.01% Žvrv. Triton X-100 ŽSigma, St. Louis, MO.. 2.3. Control preparation A positive control cyst suspension was prepared from the feces of severe combined immunodeficient ŽSCID. mice inoculated with a mouse fecal suspension of Giardia muris kindly provided by Dr. M.E. Olson, University of Calgary, Calgary, Alberta. Mice were housed in pairs in polycarbonate shoebox cages. Fecal pellets deposited through a wire grid bottom over 2-day periods were collected from the base pan. The feces were transferred to a sterile container in
B.R. Dixon et al.r Journal of Immunological Methods 202 (1997) 27–33
which 25 ml of PBS containing 0.05% Žvrv. Tween 80 Žfresh control. or 25 ml of 10% buffered formalin Žfixed control. was added. Fresh and fixed negative control suspensions were prepared in the same manner from the feces of uninfected SCID mice. The care of mice used in this study was in accordance with the guidelines of the Health Protection Branch Animal Care Committee. 2.4. ConÕentional microscopy Following sucrose floatation, one drop of beaver fecal suspension was transferred to a microscope slide with a coverslip. A single control slide was prepared in the same manner from the fresh positive control suspension for each batch of samples examined. Each slide was then examined at 400 = magnification. A total of 94 beaver fecal samples were tested. The presence or absence of G. lamblia cysts was recorded for each sample. 2.5. Immunofluorescence microscopy Following sucrose floatation, 20 ml of beaver fecal suspension was transferred to a microscope slide and air dried. Slides were then fixed for 5 min in acetone and air dried. 25 ml of Giardia-cel IF Test reagent Žfluorescein-labeled anti-Giardia monoclonal antibody. ŽCellabs, distributed by Wellmark Diagnostics Ltd., Guelph, Ontario. was added to each slide. Slides were incubated in an humidity chamber for 30 min at 378C. Finally, slides were gently washed by irrigation in PBS, excess moisture was removed, and a drop of supplied mounting fluid was added along with a coverslip. Slides were examined immediately or stored for a few hours in an humidity chamber at 48C. A single control slide was prepared in the same manner from the fresh positive control suspension for each batch of samples examined. Each slide was examined at 400 = magnification using an epifluorescence microscope with filter system for fluorescein isothiocyanate ŽFITC.. A total of 94 beaver fecal samples were tested. The presence or absence of G. lamblia cysts was recorded for each sample. 2.6. Flow cytometry Following sucrose floatation, 200 ml of beaver fecal suspension was transferred to each of two
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tubes. 25 ml of Giardia-cel IF Test reagent ŽCellabs. was added to one of the tubes while 25 ml of PBS was added to the other tube Žautofluorescence control.. Following a 30-min incubation at 378C, the contents of both tubes were washed with 2 ml of PBS and centrifuged at 400 = g for 5 min. Pellets were then resuspended in 0.5 ml of PBS and stored at 48C until analysis by flow cytometry. Positive and negative controls Žboth fresh and fixed. were similarly prepared for each batch of samples examined. A flow cytometer ŽFACSCAN, Becton Dickinson, Mississauga, Ontario. equipped with an argon-ion laser operating at 488 nm, and Lysys II support software, were used for the acquisition of cysts and the analysis. Calibration of the FACSCAN was performed using Calibrite beads ŽBecton Dickinson, Mississauga, Ontario. as recommended by the supplier. A total of 10 000 particles Žungated. were acquired for each of the beaver fecal samples tested, with the exception of 6 samples in which fewer particles could be acquired due to their unusual consistency. Dual parameter histograms Žright angle light scatter vs. fluorescence. were plotted for each sample. Whenever a sample was found to be positive for G. lamblia cysts, a drop from the tube was transferred to a microscope slide and examined on an epifluorescence microscope for confirmation. To confirm the reliability of the flow cytometry results, Table 1 Presence or absence of Giardia lamblia cysts in beaver fecal samples Ž ns94. using three different detection methods a Beaver I.D. number
Conventional microscopy
Immunofluorescence microscopy
Flow cytometry
64 62 58 27 102 127 76 53 103 55 108 109 90 79
y y q y y q y q q q q q y y
y y q y q q q q q q q y q y
q q q q q q q q q q q q q q
a
Only samples found to be positive with one or more methods are represented.
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a positive beaver fecal sample and a positive control sample were analysed on a Coulter Epics-Elite cell sorter. Cysts were physically separated from fecal debris and were examined on an epifluorescence microscope.
3. Results Of the 94 beaver fecal samples examined by conventional microscopy, 7 were positive for G. lamblia cysts and 87 were negative ŽTable 1.. Several of the samples were questionable due to overlapping debris or poor resolution. As they could not be
confirmed, such samples were considered negative. Positive control slides contained large numbers of cysts. Using immunofluorescence microscopy, cysts appeared as bright green oval-shaped bodies, making them readily apparent even in concentrated fecal debris. Of the 94 samples examined, 9 were positive and 85 were negative ŽTable 1.. Positive control slides contained large numbers of cysts. Of the 94 samples tested by flow cytometry, 14 were positive and 80 were negative ŽTable 1.. The dual parameter histograms for positive and negative beaver samples are shown in Fig. 1 along with their respective autofluorescence controls. The lack of
Fig. 1. Dual parameter histograms of beaver fecal suspensions generated by immunofluorescence flow cytometry. Ž A. Positive beaver sample stained with Giardia-cel IF test reagent Žleft., and unstained Žautofluorescence control. Žright.. Ž B . Negative beaver sample stained with Giardia-cel IF test reagent Žleft., and unstained Žautofluorescence control. Žright..
B.R. Dixon et al.r Journal of Immunological Methods 202 (1997) 27–33
particles in the predetermined gate in autofluorescence histograms indicated that autofluorescent debris had no influence on the results. While large numbers of cysts were present in positive control samples, fewer were present in positive beaver samples. No cysts were seen in either the negative controls or negative beaver samples. While fixed control cyst suspensions remained stable for many months, fresh suspensions could only be used with flow cytometry for a few days. After this period, when fresh cysts were stained, they demonstrated a markedly lower fluorescence intensity than they did initially. The presence of G. lamblia cysts was confirmed using immunofluorescence microscopy for each of the 14 positive samples. In addition, concentrated cyst suspensions were obtained following cell sorting on a positive beaver fecal sample and on a positive control sample, confirming that gated particles represented on histograms were cysts. It often took over 30 min to check a slide for the presence of cysts by conventional microscopy, and viewer fatigue was clearly a factor. Similarly, careful examination of a slide using immunofluorescence took from 15 to 30 min to complete. Analysis by flow cytometry, however, took less than 4 min per sample.
4. Discussion The present study involves a direct comparison of three techniques in terms of their effectiveness in detecting G. lamblia cysts in beaver fecal samples. Conventional microscopy was found to be a very tedious, time-consuming and unreliable means of determining the presence of cysts. Unless there were a large number of cysts present, they were difficult to observe among the fecal debris. When a cyst was located, its identity was often difficult to verify as nuclei and other internal structures were not always visible. As a result, a number of false negatives were recorded. A considerable improvement in the detection of cysts was achieved by concentrating cysts and staining with fluorescein-labeled anti-Giardia antibodies. With immunofluorescence microscopy, cysts were readily identified among fecal debris. However, this
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technique was still relatively time-consuming, particularly if there were few or no cysts present on the slide. Flow cytometry in combination with immunofluorescence was a rapid and very effective procedure for the detection of G. lamblia cysts in fecal samples, and provided a great improvement over microscopical techniques. By analysing fecal material according to right angle light scatter and intensity of fluorescence, cysts could be readily differentiated from debris. Positive samples could then be easily confirmed using an epifluorescence microscope. An examination of the suspensions obtained from the cell sorter further confirmed that particles within the gate ŽFig. 1A. were, in fact, G. lamblia cysts. Fixed control samples could be prepared and used for many months with no apparent loss in the ability of the cysts to bind with the antibody over time, as was seen with the fresh controls. As the stability of these fixed controls allowed for more consistency in the separation of cysts from fecal debris using flow cytometry, they are recommended over fresh controls. While the time required to prepare the samples for flow cytometry was similar to that of the microscopical techniques, the analysis was very rapid. As a result, many more samples could be examined in a day. More importantly, there was no viewer fatigue or observer variation involved, allowing for more consistent and reliable results. Whenever discrepancies occurred among the results of the three methods, the positive result was always found with flow cytometry, indicating that it may recognize, with more consistency, samples containing smaller numbers of cysts. Based on the number of positive results, immunofluorescence microscopy appeared to be second best in this regard. In general, when a relatively large number of cysts were present, they were not difficult to detect using any of the methods, but when fewer cysts were present, they were only detected using immunofluorescence microscopy or flow cytometry. In only one case were cysts observed with conventional microscopy and flow cytometry, but not with immunofluorescence microscopy. On the basis of the flow cytometry results, the prevalence of G. lamblia infection in beavers in the study area was 14.9%. This figure is similar to those
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reported in other prevalence studies on beavers in North America ŽDavies and Hibler, 1979; Frost et al., 1982; Wallis et al., 1986; Isaac-Renton et al., 1987; Erlandsen et al., 1988, 1990; Roach et al., 1993.. One problem inherent in prevalence studies on wild animals is that G. lamblia cysts are often shed intermittently, as they are in beavers ŽDavies and Hibler, 1979; Wallis et al., 1986., and an examination of a single fecal sample from each animal may result in false negatives and a low prevalence estimation. Flow cytometry with immunofluorescence labeling appears to be ideal for the rapid screening of large numbers of fecal samples for G. lamblia cysts. In addition to prevalence studies in animals, preliminary results from this laboratory indicate that this procedure may also be useful for screening human clinical specimens in outbreak situations. While not all clinical laboratories will have direct access to a flow cytometer and epifluorescence microscope, they are routinely used in hospitals, universities and larger laboratories for immunological work, and the present study describes another important use for this equipment. Work is presently underway in this laboratory to extend this procedure to the simultaneous detection of other intestinal parasites including Cryptosporidium parÕum and Entamoeba histolytica. Acknowledgements The authors are very grateful to Nicole Beausoleil, Sandra Stall and Michel Sanche, Animal Resources Division, Health Protection Branch, Health Canada, for the inoculation and monitoring of the mice and the regular collection of feces. The authors would also like to express their deep appreciation to Pierre Chabiaque, National Capital Commission, Ottawa, Ontario, for his dedicated efforts in the trapping of beavers and collection of fecal samples. References Arrowood, M.J., Hurd, M.R. and Mead, J.R. Ž1995. A new method for evaluating experimental cryptosporidial parasite loads using immunofluorescent flow cytometry. J. Parasitol. 81, 404. Belosevic, M., Faubert, G.M., Guy, R. and MacLean, J.D. Ž1984.
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