Acanthamoeba pearcei N. Sp. (Protozoa: Amoebida) from Sewage Contaminated Sediments

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J. Euk. Microbiol., 42(6), 1995, pp. 702-705 0 I995 by the Society of Protozoologists

Acanthamoeba pearcei N. Sp. (Protozoa: Amoebida) from Sewage Contaminated Sediments THOMAS A. NERAD,*.' THOMAS K. SAWYER,** EARL J. LEWIS*** and SHAWN M. MCLAUGHLIN*** *American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, **Rescon Associates. Box 206 Turtle Cove, Royal Oak, Maryland 21662, and ***NationalMarine Fisheries Service, U.S. Department of Commerce, Oxford, Maryland 21654

ABSTRACT. Seabottom sediments from a discontinued Philadelphia-Camden40-Mile ocean sewage disposal site were cultured for cyst-forming free-living amoebae. Barge delivered wastes were discharged at the site from 1973 until 1980 when the site was closed. One station at the southeast margin of the site was sampled at a depth of approximately 50 m, twice in 1978 and once in 1982, 1983 and 1984. Sediment from the 1978 collection yielded Acanthamoeba polyphaga, Vahlkampfia sp., and an unknown amoeba with stellate endocysts similar to those of A. astronyxis. Trophozoites and cysts of the isolate were typical of those described for the genus Acanthamoeba. Biochemical tests employing enzyme electrophoresis and morphological studies on live and stained specimens showed that the isolate was distinct from other well-described species within the family Acanthamoebidae Sawyer & Griffin, 1975. Supplementary key words. Acanthamoebidae,cysts, free-living amoebae, Vahlkampfia.

T

HE discharge of sewage wastes into coastal waters or the open ocean often leads to the closure of designated areas for the harvest of commercially valuable fish and shellfish, or for recreational use. Decisions to open or close such areas often are based on estimates of the numerical abundance of sewage associated enteric bacteria or a recent history of human infection by pathogenic free-living amoebae (PFLA). Singh & Das [19] cultured pathogenic species of Acantharnoeba and Naegleria from municipal wastes in India and such wastes are now recognized as a source for amoebae of concern to human health

PI.

O'Malley et al. [5] and Sawyer et al. [ 181followed the dispersal of fecal bacteria and Acantharnoeba away from the center of an ocean disposal site situated approximately 65 km seaward from the coast of Delaware and Maryland. The site covered an area of approximately 65 km2 and was used for the disposal of bargedelivered wastes from 1973 to 1980 [3]. One site within the area designated station #205 was sampled at a depth of 50 m in 1978 and yielded a culture with cysts that differed from those of other known species of free-living amoebae. Morphological and biochemical studies showed that the new isolate was a unique species of Acanthurnoeba. We propose the name Acantharnoeba pearcei, n. sp., to recognize Dr. Jack Pearce, National Marine Fisheries Service, NOAA, Woods Hole, MA, for his many contributions to environmental research and education. The new species did not grow at 37" C-39" C, and did not kill mice when inoculated by the intranasal route. METHODS Seabottom sediments from the disposal site were taken with a Smith-McIntyre grab twice in 1978 and once in 1981, 1982, 1983 and 1984. Subsamples from the upper 5 cm of sediment were removed with sterile wooden tongue depressors, placed in sterile bags and kept on ice or in a refrigerator until used for culture. The subsamples, each taken in triplicate, were streaked on the surface of six replicate agar plates previously inoculated with bacteria, Klebsiella pneurnoniae subsp. pneurnoniae ATCC 27889, as a food source. Plates were inverted, placed in plastic boxes and incubated at room temperature [141. One isolate from the 1978 collection appeared to be an undescribed species of amoeba and was cloned and characterized on the basis of cyst and trophozoite morphology [6-81. The amoebae were tested for growth at 37" C-39" C, and for pathogenicity in laboratory mice [ 11. Tests were carried out in this temperature range since most pathogenic amoeba species may be cultured within this

I

range. Isoenzyme profiles for propionyl esterase (PE), leucine aminopeptidase (LA) and acid phosphatase (AP) were determined as described in an earlier publication [4]. Morphological features of live trophozoites and cysts were determined by phase contrast microscopy, and fixed specimens were studied after staining with nuclear red [6]. RESULTS The sediment sample that yielded A. pearcei was taken at the southeast margin of a disposal site at 38" 19.6' N and 74" 18.0' W. Water depth was 50 m and sediment consisted ofdark brown sand mixed with pebbles and shell fragments. The site was a shallow basin where sediment accumulated rather than dispersed as a result of current and tidal activity. Sediment from the first collection in April, 1978 was culture positive for A. polyphagu, A. pearcei and an unidentified species of Vahlkarnpjia. The same site yielded A. polyphagu again in 198 1 but was negative in 1982, 1983 and 1984. Most Probable Numbers (MPN) were 5 1 for total coliforms and 35 for fecal coliforms in 1978, but sediments were negative thereafter (unpubl. data). Acantharnoeba pearcei trophozoites (n = 25) measured 25.042.5 pm long (2 = 33.1) by 17.5-25.0 wide (X = 22.1) alive, and 22.5-39.0 long (2 = 27.9) by 12.5-17.5 (2 = 14.7) after fixation with Nissenbaum's solution and staining with nuclear red. Trophozoites were variable in shape, longer than wide in locomotion, but often wider than long while feeding. Measurements were based only on locomotive forms. Locomotive forms became progressively distorted or contracted in the presence of heat or light from the microscope. Stained specimens sometimes were deformed and filled with deeply stained digestive vacuoles. Cysts were spherical with a smooth ectocyst and deeply scalloped endocysts with 5-7, rarely 8-9, blunt arms or rays. Live cysts (n = 25) measured 17.5-25.0 km (K = 20.5) in diameter. Morphological features of the cysts showed that A. pearcei may be placed within Acantharnoeba Group 1 proposed by Pussard & Pons [9], for species with stellate endocysts. Comparative Table 1. Comparative measurements of cysts of Acanthamoeba pearcei, n. sp., A. comandoni [ 131,A. astronyxis [ 131 and A. gr@ni [lo] (in microns).' Species

702

x

Strain

20.5 ATCC 50435 19.2 ATCC 30137 21 .O-30.0 25.6 ATCC 30135 14.0-25.0 19.8 ATCC 30731 a Measurements based on 25 cysts each-grown on freshwater agar.

A. pearcei A. astronyxis A. comandoni A. nrifini

To whom correspondence should be addressed.

Diameter (range)

17.5-2 5 .O

16.0-28.0

NERAD ET AL.-NEW SPECIES OF ACANTHAMOEBA

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Fig. 1-5. Trophozoites, and cysts ofAcanthamoebapearcei, n. sp. 1. Live trophozoites. Note variations in shape, x 600.2-3. Live trophozoites. Note broad triangular shape o f locomotive forms and filose posterior uroid, x 1600. 4-5. Living cysts. Note stellate endocyst with blunt tips and variable number o f arms, x 1600.

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J. EUK. MICROBIOL., VOL. 42, NO. 6, NOVEMBER-DECEMBER 1995

measurements for A. pearcei and several other species within Group 1 are given in Table 1. Representative features of the amoebae and cysts are shown in Figs. 1-5. Acanthamoeba pearcei, n. sp. (Figs. 1-5) Diagnosis. Trophozoites typical of the genus Acanthamoeba Volkonsky, 1931, family Acanthamoebidae Sawyer & Griffin, 1977, order Acanthopodina Page, 1976, class Lobosa Carpenter, 186I , phylum Rhizopoda Von Siebold, 1845. Locomotive forms 25.0-42.5 pm long (X = 33.1) by 17.5-25.0 pm wide (X = 22.1) and feeding forms approximately as wide as long. Amoebae contract in presence of light or heat from the microscope altering typical features of locomotive and feeding forms. Fixed and stained specimens approximately 22.5-39.0 pm long (mean 27.9) by 12.5-17.5 pm wide (R = 14.7); nucleus 3.5-4.0 pm (R = 3.5). Living cysts 17.5-25.0 pm in diameter (X = 20.5). Ectocyst smooth without depressions at tips of arms or rays of the endocyst. Endocysts with 3-7 rays or arms, rarely 8-9, that are deeply scalloped with blunt or rounded tips. Acanthamoeba pearcei, strain ATCC 50435 did not grow at 37” C-39” C, and was not pathogenic to laboratory mice via the intranasal route. Type locality. Sewage contaminated ocean sediments from the northeast Atlantic Ocean, 65 km offshore from Maryland and Delaware, USA, approximately 38” 19.6’ N, 74” 18.0’ W. Type Strain, Deposited at the American Type Culture Collection, Rockville, Maryland 20852, Accession no. ATCC 50435. Isoenzyme profiles: zymograms for propionyl esterase (PE), leucine aminopeptidase (LA) and acid phosphatase (AP) by enzyme electrophoresis were distinct for A. pearcei (ATCC 50435) and morphologically similar A. astronyxis (ATCC 30 137). The PE zymogram for A. pearcei had seven bands compared to five bands for A. astronyxis with only one band shared by both species. The zymograms for LA had a single band with that of A . pearcei being more anodal than that of A. astronyxis. The AP zymogram for A. pearcei had one discrete anodal band and two cathodal bands in contrast to six anodal and two cathodal bands for A, astronyxis. The single anodal band for A . pearcez appeared to be shared with one of the two anodal bands for A. antronyxis. Twenty-one bands were expressed by the two strains, but only four (19%) were shared.

ly identifiable solely on the basis of their morphological features. Pussard and Pons [9] proposed that each species be placed into one of three groups on the basis of cyst morphology in order to facilitate preliminary identification. Group 1 was proposed for species with distinctly scalloped endocysts, including A. astronyxis, A. comandoni, and A . echinulata. Acanthamoeba pearcei clearly belongs to Group 1 , most closely resembling A. astronyxis. The principle characteristics for distinguishing between the two are the features of their cysts and their distinctly different zymograms. The new species, A. pearcei, is the fifth species originally isolated from marine or brackish waters. The first, A. grifini, was found in Long Island Sound, New York [lo], the second, A. hatchetti, from Baltimore Harbor, MD [ 151,the third, A. jacobsi, from the discontinued New York 12-Mile nearshore waste disposal site [16], and the fourth, A. stevensoni, from contaminated shellfish beds near Staten Island, New York [ 171. Acanthamoeba hatchetti and A. stevensoni were highly pathogenic to mice, and A . jacobsi mildly pathogenic. Acanthamoeba grifini and A. pearcei were not pathogenic under the conditions tested and neither grew when incubated at 39”C-40”C. Recently described species of Acanthamoeba cultured from coastal sediments suggest that sewage wastes entering brackish or marine ecosytems have not been fully appreciated as a source of potentially pathogenic free-living amoebae. As interest in “opportunistic” amphizoic amoebae progresses, it is likely that other “new” species will be discovered that are identifiable only at the molecular or cellular level. Enzyme electrophoresis, however, remains a reliable biochemical tool for confirming or rejecting initial identifications based on morphological criteria.

DISCUSSION Free-living cyst-forming amoebae within the genus Acanthamoeba are reliable indicators of the persistence or dispersal of sewage wastes in rivers, bays, coastal waters and the open ocean [l 1, 12, 181. O’Malley et al. [ 5 ] and Sawyer et al. [18] recovered several species of Acanthamoeba from 55% of the sediment samples taken within an 8 km radius of the center of an ocean waste disposal site located approximately 65 km from shore [3]. Sediments at the site were negative for fecal coliform bacteria within 1 year after the cessation of waste disposal but not for Acanthamoeba until after 3 years (unpublished data). Bacteriological data alone did not explain whether enteric bacteria had been flushed from the seabottom by current and tidal activity, or whether they were present in a viable but nonculturable condition. We did not recover A. pearcei from the 19811984 sediment samples nor were they positive for fecal coliform bacteria during this post dumping period. Our results suggest that sewage contaminants on the seabottom were flushed or dispersed into the open ocean by current and tidal activity. We believe that a combination of enteric bacteria and cyst-forming protozoans provides a better means for monitoring health of marine and estuarine ecosystems than does the use of non-spore forming bacteria alone. There are several species ofAcanthamoeba, that are not readi-

LITERATURE CITED

ACKNOWLEDGMENTS The authors gratefully acknowledge Dr. Donald Leaf (retired), and Marria O’Malley, Environmental Protection Agency Region 111, for the opportunity to participate in the PhiladelphiaCamden 40-Mile Site study during 1974-1 984. Cpt. Jack Gaines (retired), of the Federal Drug Administration, N. Kingston, Rhode Island, kindly provided the Most Probable Number data for coliform bacteria from unpublished surveys. We also appreciate the interest and support of Dr. Aaron Rosenfield (retired), former Director, National Marine Fisheries Service, U.S. Dept. of Commerce, during the course of this study. 1. Daggett, P.-M., Sawyer, T. K. &Nerad, T. A. 1982. Distribution and possible interrelationships of pathogenic and nonpathogenic Acanthamoeba from aquatic environments. Microb. Ecol., 8:37 1-386. 2. Martinez, A. J. 1985. Free-living Amoebas: Natural History, Prevention, Diagnosis, Pathology and Treatment of Disease. CRC Press, Boca Raton, Florida. 3. Muir, W. C. 1983. History of ocean disposal in the mid-Atlantic bight. In: Duedall, I. W., Ketchum, B. H., Park, P. K. & Kester, D. R. (ed.), Wastes in the Ocean, Vol. 1: Industrial and Sewage Wastes in the Ocean. Wiley Interscience, New York, Pp. 273-31 1. 4. Nerad, T. A. & Daggett, P.-M. 1979. Starch gel electrophoresis: an effective method for separation of pathogenic and nonpathogenic Naegleria species. J. Protozool., 26:6 13-6 15. 5. O’Malley, M. L., Lear, D. W., Adams, W. N., Gaines, J., Sawyer, T. K. & Lewis, E. J. 1982. Microbial contamination of continental shelf sediments by wastewater. J. Water Pollut. Cntrl. Fed., 54: 131 11317. 6. Page, F. C. 1967. Re-definition of the genus Acanthamoeba with descriptions of three species. J . Protozool., 14:709-724. 7. Page, F. C. 1976. An Illustrated Guide to Freshwater and Soil Amoebae. Freshwater Biological Association Scientific Publ. No. 34, Ambleside, Cumbria, England. 8. Page, F. C. 1988. A New Key to Freshwater and Soil Gymnamoebae with Instructions for Culture. Freshwater Biological Association. The Feny House, Ambleside, Cumbria, England.

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9. Pussard, M. & Pons, R. 1977. Morphologie de la pario kystique et taxonomie du genre Acanthamoeba (Protozoa: Amoebidae). Protistologica, 13:5 57-5 98. 10. Sawyer, T. K. 1971. Acanthamoeba grtfini, a new species of marine amoeba. J . Protozool., 18:650-654. 1 1. Sawyer, T. K. 1980. Marine amoebae from clean and stressed bottom sediments of the Atlantic Ocean and Gulf of Mexico. J. Protozool., 27: 13-32. 12. Sawyer, T. K. 1992. Distribution of microbial agents in marine ecosystems as a consequence of sewage-disposal practices. In: Rosenfield, A. & Mann, R.(ed.), Dispersal of Living Organisms into Aquatic Ecosystems. Maryland Sea Grant College, Univ. Md. Systems, College Park, Maryland 20742, Publ. UM-SC-TS-92-04. 13. Sawyer, T. K. & Griffin, J. L. 1971. Acanthamoeba comandoni and A . astronyxis: taxonomic characteristics of mitotic nuclei, “centrosomes” and cysts. J. Protozool., 18:382-388. 14. Sawyer, T. K. & Bodammer, S. M. 1983. Marine amoebae (Protozoa: Sarcodina) as indicators of healthy or impacted sediments in the New York Bight apex. In: Duedall, I. W., Ketchum, B. H., Park, P. K. & Kester, D. E. (ed.), Wastes in the Ocean, Vol. 1: Industrial and Sewage Wastes in the Ocean, Wiley Interscience, New York, Pp. 337352.

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15. Sawyer, T. K., Visvesvara, G. S. & Harke, B. A. 1977. Pathogenic amoebae from brackish and ocean sediments, with a description of Acanthamoeba hatchefti, n. sp. Science, 196:1324-1325. 16. Sawyer, T. K., Nerad, T. A. & Visvesvara, G. S. 1992. Acanthamoeba jacobsi, sp. n. (Protozoa: Acanthamoebidae) from sewage contaminated ocean sediments. Proc. Helminthol. SOC.Wash,, 59:223226. 17. Sawyer, T. K., Nerad, T. A., Lewis, E. J. & Mchughlin, S. M. 1993. Acanthamoeba stevensoni sp. n. (Protozoa: Amoebida) from sewage contaminated shellfish beds in Raritan Bay, New York. J. Euk. Microbiol., 40:742-746. 18. Sawyer, T. K., Lewis, E. J., Galasso, M., Lear, D. W., O’Malley, M. L., Adams, W. N. & Gaines, J. 1982. Pathogenic amoebae in ocean sediments near wastewater sludge disposal sites. J. Water Pollut. Cntrol. Fed., 54:1318-1323. 19. Singh, B. N. & Das, S. R. 1972. Occurrence of pathogenic Naegleria aerobia, Hartmannella culbertsoni and H. rhysodes in sewage sludge samples of Lucknow. Current Sci. (India), 41:277-28 1.

Received 12-2-94, 6-20-95; Accepted 7-5-95.

J. Euk. Microbiol. 42(6), 1995, pp. 705-708 0 1995 by the Society of Protozoologists

Antisporozoite Antibodies with Protective and Nonprotective Activities: In Vitro and In Vivo Correlations Using Plasmodium gallinaceum, an Avian Model ADELINA D. RAMIREZ,’ ELIANA M. M. ROCHA**.’and ANTONIANA U. KRETTLI**,2

*Nticleo Universitario Rafael Rangel. Universidad de Los Andes, Trujillo, Venezuela, and, **Centro de Pesquisas Re& Rachou / FIOCR UZ and Department of Parasitology, Federal University of Minas Gerais. Av. August0 de Lima, 1715, CEP 30190-002, Belo Horizonte, MG, Brazil ABSTRACT. A correlation was observed between in vivo and in vitro activity of six monoclonal antibodies (mAb) against the major circumsporozoite protein of the avian malaria Plasmodium gallinaceum as follows. (I) Two mAb were protective, totally abrogating sporozoite infectivity to chicks, its natural host, in vivo; they caused 100% inhibition of sporozoite invasion (ISI) in vitro to SL-29 chicken fibroblasts and intense IS1 to cultured chicken macrophages, as well as inhibited the exoerythrocytic development of sporozoites taken up by macrophages, the initial cell host of P.gallinaceum sporozoites. (2) Two mAb were partially protective in that they reduced sporozoite infectivity to chicks, caused partial IS1 to SL-29 and macrophage cells and partial inhibition to the exoerythrocyticdevelopment of sporozoites in macrophages in vitro. (3) Two mAb were totally inactive in vivo although they both bound to the sporozoite antigens as detected by indirect immunofluorescence, western blot, and ELISA; they both failed to induce IS1 or inhibit the exoerythrocytic development in macrophages. The possible participation of macrophages as the initial cell type involved in sporozoite destruction in the presence of anti-circumsporozoite antibodies is discussed. Supplementary key words, Macrophages, malaria, monoclonal antibodies, protective immunity, sporozoites.

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REVIOUS attempts t o select proteins a s vaccine candidates i n malaria h a v e been based on their preferential recognition by sera from i m m u n e individuals in endemic areas or by protective monoclonal antibodies directed against t h e circumsporozoite (CS) protein [S, lo]. T h e CS protein covers the surface of sporozoites, is shed when cross-linked by antibodies, mediates parasite adherence and hepatocyte invasion [4]. Volunteers vaccinated with synthetic o r recombinant CS repeats of Plasm o d i u m falciparum (NANP)n exhibited only partial protection. They developed an antibody response that correlated with the dose ofvaccine used [ 1,5],but the overall titers ofantisporozoite

antibodies were low, thus new vaccines based on multiple antigen peptides which include T and B cell epitopes and deliver larger doses of the immunogen(s) are being developed [lo]. In an attempt t o elucidate further the role of antibodies in protection we have produced and previously studied several monoclonal antibodies (mAb) against the major CS antigen on the sporozoites of Plasmodium gallinaceum, a n avian malaria parasite [6, 121. We now describe different i n vitro biological activities of some of these mAb which correlate directly with their in vivo ability t o neutralize parasite infectivity to chickens, its natural host.

I Current address: Department of Pathology, Federal University of Alagoas, Macei6, Alagoas, Brazil. To whom correspondence should be addressed. Abbreviations: CS, circumsporozoite; CSP, circumsporozoite precipitation; EEF, exoerythrocytic forms; IFI, indirect immunofluorescence; IPA, immunoperoxidase assay; ISI, inhibition of sporozoite invasion; mAb, monoclonal antibodies; WB, western blot.

MATERIALS A N D METHODS Sporozoite isolation. Sporozoites were isolated from salivary glands of Aedes fluviatilis mosquitoes, experimentally infected a s described [ I l l and used t o infect cell monolayers a n d to induce chicken infections i n the presence o f m A b or immune sera.

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