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Bacteria-like endosymbionts in Blastocystis sp
Intrrnarmnd
Pergamon
RESEARCH
JournolJor Porosimlogy, Vol. 24, No. I, pp. 147-149, 1994 Copyright c 1994 Australian Society for Parasitology Elsevier Science Ltd Pnnted I” Great Britain. All rights reserved 002&7519/94$6.00+ 0.00
NOTE
BACTERIA-LIKE
ENDOSYMBIONTS D. J. STENZEL*
IN BLASTOCYSTIS
SP.
and P. F. L. BOREHAM?
*Analytical Electron Microscopy Facility, Queensland University of Technology, George Street, Brisbane, Queensland 400 1, Australia TQueensland Institute of Medical Research, The Bancroft Centre, 300 Herston Road, Herston, Brisbane, Queensland 4029, Australia (Received 3 1 May 1993; accepted 5 September 1993) D. J. and BOREHAM P. F. L. 1994. Bacteria-like endosymbionts in EIastocysfis sp. International Journalfor Parasitology 24: 147-149. Bacteria-like endosymbionts were found in vacuolar cells and cysts of Blusfocysris sp. from duck and monkey faecal material. The organisms ultrastructurally resembled Gram-negative bacilli, and were present in the nucleus of Bhtocystis sp. from the duck and in the cytoplasm of Blustocystis sp. from the monkey. Based on size and differences in intracellular location, it is probable that these represent two distinct species of organisms. Abstract-STENZEL
INDEX KEY WORDS: Blustocystis; endosymbionts;
ENDOSYMBIONTS have been reported in a range of protozoan cells, and bacterial endosymbionts appear to be common in protozoa (Ball, 1969; Preer & Preer, 1977; 1984). A report by Zierdt & Tan (1976) indicated endosymbionts may occur in Blastocystis hominis, but subsequent studies by numerous workers have not confirmed this, except for the occurrence of virus-like particles in B. lapemi from a sea-snake (Teow, Ho, Ng, Chan, Yap, Chan, Howe, Zaman & Singh, 1992). In this report we describe bacteria-like organisms (BLOs) present in Blustocystis sp. from fresh faecal material from a monkey (Mucacca macacca) housed at the Institute for Medical Research, Kuala Lumpur, Malaysia, and from a domestic duck (Anusplutyrh~~nchos) from the Brisbane area. We have performed extensive ultrastructural examination of Blastocystis over the past 5 years, including B. hominis from humans (Dunn, Boreham & Stenzel, 1989; Stenzel, Boreham & McDougall, 1991) and BIastocystis sp. from a range of birds (Stenzel, Cassidy & Boreham, in press, a) and mammals (see Boreham & Stenzel, 1993; Stenzel, Cassidy & Boreham, in press, b). During these studies, we have only detected BLOs in Blastocystis sp. from one monkey and one duck. Fresh faecal material was collected into 3% glutaraldehyde fixative, and processed for transmission electron microscopy as previously described (Dunn et al., 1989). Ultrathin (50-90 m) sections were cut, contrasted with uranyl acetate and lead
*To whom all correspondence
should be addressed.
ultrastructure; monkey; duck
citrate, and examined with a JEOL 1200EX transmission electron microscope. Bacteria-like organisms were found within the nucleus of Blastocystis sp. from the duck (Figs. la-d) and within the cytoplasm of Blustocystis sp. from the monkey (Figs. le-g). The BLOs were found in vacuolar cells (Figs. la,e) and in cysts (Figs. lb,f) of Blustocystis sp. from both samples. They were not detected in the central vacuole or in other organelles of Blastocystis from either host. The BLOs appeared as bacilli, surrounded by two bilaminar membranes (Figs. ld,g), morphologically resembling Gram-negative bacteria (see Preer & Preer, 1984). Some organisms appeared to be undergoing division. Almost all Blustocystis cells in ultrathin sections contained BLOs, although the numbers present varied. Within the nucleus of cells from the duck sample usually 2 or 3 BLO profiles were seen, but occastionally up to 40 organisms were present (Fig. lc). The BLOs tended to be more prevalent in the monkey Blastocystis, with up to 12 organisms commonly present in the cytoplasm. The size of the BLOs varied between hosts, with those from the duck sample measuring approximately 0.2 m in width by 0.8-1.0 m in length, while those from the monkey sample were larger, approximately 0.4 e in width and up to 2.0 m in length. Inclusion bodies were present in some BLOs from the monkey isolate (Figs. le,g). Morphologically the BLOs from Bfustocystis sp. resemble the bacterial or rickettsial endosymbionts described in other protozoa (see Ball, 1969; Preer & Preer, 1977; 1984). The different location within the host cells, and size variation between BLOs from the 147
D. J. STENZEL and P. F. L. BOREHAM
FIG. 1. Electron micrographs of bacteria-like endosymbionts in Blustocystis sp. from duck (ad) and monkey (e-g). Nu, nucleus; m, mitochondrion; cv, central vacuole; cw, cyst wall, SC, surface coat; I, lipid droplet. (a) Vacuolar cell and (b) cyst showing endosymbionts (indicated by arrows) within the nucelus. (c) A nucleus showing numerous endosymbiotic organisms. (d) High magnification of endosymbiont. (e) Vacuolar cell and (fj cyst showing endosymbionts (indicated by arrows) within the cell cvtonlasm. (a) High magni~cation of endosymbionts (indicated by arrows).
Research two hosts, indicate that they probably represent two distinct species of organisms (see Preer & Preer, 1984). There was no evidence of degradation or digestion of the BLOs within the Bfastocystis cells, suggesting that they were true endosymbionts. We have no evidence that Blastocystis is phagocytic, except for the rare amoeboid form (Dunn et al., 1989). There was no morphological evidence of pathogenic effects on Blastocystis cells, which appeared ultrastructurally typical for their host type (see Boreham & Stenzel, 1993). A previous sample, collected 15 months earlier, from the same monkey did not reveal endosymbionts. Faecal samples from other monkeys housed in the same enclosure contained Blastocystis sp. without endosymbionts. The report by Zierdt & Tan (1976) described spherical and rod-shaped endosymbionts, which were designated “alpha”, and were present in the central vacuole of 8 isolates of B. hominis in culture. The structures were surrounded by a cell membrane but no cell wall, and were only present in large cells during axenization of the cultures. It is possible that these were not endosymbionts, but cytoplasmic extensions into the central vacuole (see Boreham & Stenzel, 1993). The significance of endosymbiotic organisms in protists is not fully known. These organisms represent an additional form of genetic information which can be transferred to progeny, and which may be capable of altering characteristics of the host cell. Endosymbionts have been suggested to influence morphology, physiology and pathology of some parasitic protists (Preer & Preer, 1977; 1984). It has been indicated that endosymbionts alter the surface membrane chemistry of Crithidia oncopelti (see Dwyer L Chang, 1976), the metabolism of Paramecium aurelia and Crithidia oncopelti (see Lee, Lee & Weis, 1985), division in Euplotes aediculatus (see Heckmann, 1975), and toxin production (see Preer & Preer, 1977). Effects on the response of parasites to chemotherapeutic drugs, and on infectivity and virulence have also been suggested (see Nemanic, Owen, Stevens & Mueller, 1979). At least in some instances, the bacterial endosymbiont is capable of providing some nutritional requirements of the host cell, and subsequently altering host specificity (Lee et al., 1985). Bacteria-like endosymbionts are not common in Blastocystis spp., and their effects on the host cell remain unknown. Hence, caution needs to be taken when performing biochemical or cytochemical analyses of Blastocystis containing endosymbionts, as their presence may significantly affect the results. Acknowledgements-This
work was supported in part by a
Note
149
Research Encouragement Award from the Queensland University of Technology. The authors wish to thank Dr Karen Lai Pen Foon, Institute for Medical Research, Kuala Lumpur, Malaysia for providing monkey faecal material, and Mr Matthew Cassidy for technical assistance.
REFERENCES BALL G. H. 1969. Organisms living on and in protozoa. In: Research in Protozoology, Vol. 3 (Edited by CHEN T. T.), pp. 565-718. Pergamon Press, London. BOREHAM P. F. L. & STENZEL D. J. 1993. Blastocystis in Humans and Animals: Morphology, Biology and Epizootiology. Advances in Parasitology 32: I-70. DUNN L. A., BOREHAM P. F. L. & STENZEL D. J. 1989. Ultrastructural variation of Blastocystis hominis stocks in culture. International Journal for Parasitology 19: 43- 56. DWYER D. M. & CHANG K.-P. 1976. Surface membrane carbohydrate alterations of a flagellated protozoan mediated by bacterial endosymbiotes. Proceedings of the National Academy of Sciences of the United States of America 73: 852-856. HECKMANN K. 1975. Omikron, ein essentieller Endosymbiont von Euplotes aediculatus. Journalof Protozoology 22: 97-104. LEE J. J., LEE M. J. & WEIS D. S. 1985. Possible adaptive value of endosymbionts to their protozoan hosts. Journal of Protozoology 32: 38(t382. NEMANIC P. C., OWEN R. L., STEVENSD. P. &MUELLER J. C. 1979. Ultrastructural observations on giardiasis in a mouse model. II. Endosymbiosis and organelle distribution in Giardia muris and Giardia lamblia. Journal of Infectious Diseases 140: 222-228. PREER L. B. & PREER J. R. 1977. Inheritance of infectious elements. In: Cell Biology: a Comprehensive Treatise, Vol. 1 (Edited by GOLDSTEIN L. & PRESCOTT D. M.), pp. 319373. Academic Press, New York. PREER J. R. & PREER L. B. 1984. Endosymbionts. In: Bergey‘s Manualof Systematic Bacteriology, Vol. 1 (Edited by KRIEG N. R. & HOLY J. G.), pp. 795-942. Williams & Williams, Baltimore. STENZEL D. J., BOREHAM P. F. L. & MCDOUGALL R. 1991. Ultrastructure of Blastocystis hominis in human stool samples. International Journal for Parasitology 21: 807812. STENZEL D. J., CASSIDY M. F. & BOREHAM P. F. L. (in press a) Morphology of Blastocystis sp. from domestic birds. Parasitology Research. STENZEL D. J., CASSIDY M. F. & BOREHAM P. F. L. (in press b) Morphology of Blastocystis sp. isolated from circus animals. International Journal for Parasitology. TEOW W. L., Ho L. C., NG G. C., CHAN Y. C., YAP E. H., CHAN P. P., HOWE J., ZAMAN V. & SINGH M. 1992. Viruslike particles in a Elastocystis species from the sea-snake, Lapemis hardwickii. International Journalfor Parasitology 22: 1029-1032. Z~ERDT C. H. &TAN H. 1976. Endosymbiosis in Blastocystis hominis. Experimental Parasitology 39: 422430.
Pergamon
RESEARCH
JournolJor Porosimlogy, Vol. 24, No. I, pp. 147-149, 1994 Copyright c 1994 Australian Society for Parasitology Elsevier Science Ltd Pnnted I” Great Britain. All rights reserved 002&7519/94$6.00+ 0.00
NOTE
BACTERIA-LIKE
ENDOSYMBIONTS D. J. STENZEL*
IN BLASTOCYSTIS
SP.
and P. F. L. BOREHAM?
*Analytical Electron Microscopy Facility, Queensland University of Technology, George Street, Brisbane, Queensland 400 1, Australia TQueensland Institute of Medical Research, The Bancroft Centre, 300 Herston Road, Herston, Brisbane, Queensland 4029, Australia (Received 3 1 May 1993; accepted 5 September 1993) D. J. and BOREHAM P. F. L. 1994. Bacteria-like endosymbionts in EIastocysfis sp. International Journalfor Parasitology 24: 147-149. Bacteria-like endosymbionts were found in vacuolar cells and cysts of Blusfocysris sp. from duck and monkey faecal material. The organisms ultrastructurally resembled Gram-negative bacilli, and were present in the nucleus of Bhtocystis sp. from the duck and in the cytoplasm of Blustocystis sp. from the monkey. Based on size and differences in intracellular location, it is probable that these represent two distinct species of organisms. Abstract-STENZEL
INDEX KEY WORDS: Blustocystis; endosymbionts;
ENDOSYMBIONTS have been reported in a range of protozoan cells, and bacterial endosymbionts appear to be common in protozoa (Ball, 1969; Preer & Preer, 1977; 1984). A report by Zierdt & Tan (1976) indicated endosymbionts may occur in Blastocystis hominis, but subsequent studies by numerous workers have not confirmed this, except for the occurrence of virus-like particles in B. lapemi from a sea-snake (Teow, Ho, Ng, Chan, Yap, Chan, Howe, Zaman & Singh, 1992). In this report we describe bacteria-like organisms (BLOs) present in Blustocystis sp. from fresh faecal material from a monkey (Mucacca macacca) housed at the Institute for Medical Research, Kuala Lumpur, Malaysia, and from a domestic duck (Anusplutyrh~~nchos) from the Brisbane area. We have performed extensive ultrastructural examination of Blastocystis over the past 5 years, including B. hominis from humans (Dunn, Boreham & Stenzel, 1989; Stenzel, Boreham & McDougall, 1991) and BIastocystis sp. from a range of birds (Stenzel, Cassidy & Boreham, in press, a) and mammals (see Boreham & Stenzel, 1993; Stenzel, Cassidy & Boreham, in press, b). During these studies, we have only detected BLOs in Blastocystis sp. from one monkey and one duck. Fresh faecal material was collected into 3% glutaraldehyde fixative, and processed for transmission electron microscopy as previously described (Dunn et al., 1989). Ultrathin (50-90 m) sections were cut, contrasted with uranyl acetate and lead
*To whom all correspondence
should be addressed.
ultrastructure; monkey; duck
citrate, and examined with a JEOL 1200EX transmission electron microscope. Bacteria-like organisms were found within the nucleus of Blastocystis sp. from the duck (Figs. la-d) and within the cytoplasm of Blustocystis sp. from the monkey (Figs. le-g). The BLOs were found in vacuolar cells (Figs. la,e) and in cysts (Figs. lb,f) of Blustocystis sp. from both samples. They were not detected in the central vacuole or in other organelles of Blastocystis from either host. The BLOs appeared as bacilli, surrounded by two bilaminar membranes (Figs. ld,g), morphologically resembling Gram-negative bacteria (see Preer & Preer, 1984). Some organisms appeared to be undergoing division. Almost all Blustocystis cells in ultrathin sections contained BLOs, although the numbers present varied. Within the nucleus of cells from the duck sample usually 2 or 3 BLO profiles were seen, but occastionally up to 40 organisms were present (Fig. lc). The BLOs tended to be more prevalent in the monkey Blastocystis, with up to 12 organisms commonly present in the cytoplasm. The size of the BLOs varied between hosts, with those from the duck sample measuring approximately 0.2 m in width by 0.8-1.0 m in length, while those from the monkey sample were larger, approximately 0.4 e in width and up to 2.0 m in length. Inclusion bodies were present in some BLOs from the monkey isolate (Figs. le,g). Morphologically the BLOs from Bfustocystis sp. resemble the bacterial or rickettsial endosymbionts described in other protozoa (see Ball, 1969; Preer & Preer, 1977; 1984). The different location within the host cells, and size variation between BLOs from the 147
D. J. STENZEL and P. F. L. BOREHAM
FIG. 1. Electron micrographs of bacteria-like endosymbionts in Blustocystis sp. from duck (ad) and monkey (e-g). Nu, nucleus; m, mitochondrion; cv, central vacuole; cw, cyst wall, SC, surface coat; I, lipid droplet. (a) Vacuolar cell and (b) cyst showing endosymbionts (indicated by arrows) within the nucelus. (c) A nucleus showing numerous endosymbiotic organisms. (d) High magnification of endosymbiont. (e) Vacuolar cell and (fj cyst showing endosymbionts (indicated by arrows) within the cell cvtonlasm. (a) High magni~cation of endosymbionts (indicated by arrows).
Research two hosts, indicate that they probably represent two distinct species of organisms (see Preer & Preer, 1984). There was no evidence of degradation or digestion of the BLOs within the Bfastocystis cells, suggesting that they were true endosymbionts. We have no evidence that Blastocystis is phagocytic, except for the rare amoeboid form (Dunn et al., 1989). There was no morphological evidence of pathogenic effects on Blastocystis cells, which appeared ultrastructurally typical for their host type (see Boreham & Stenzel, 1993). A previous sample, collected 15 months earlier, from the same monkey did not reveal endosymbionts. Faecal samples from other monkeys housed in the same enclosure contained Blastocystis sp. without endosymbionts. The report by Zierdt & Tan (1976) described spherical and rod-shaped endosymbionts, which were designated “alpha”, and were present in the central vacuole of 8 isolates of B. hominis in culture. The structures were surrounded by a cell membrane but no cell wall, and were only present in large cells during axenization of the cultures. It is possible that these were not endosymbionts, but cytoplasmic extensions into the central vacuole (see Boreham & Stenzel, 1993). The significance of endosymbiotic organisms in protists is not fully known. These organisms represent an additional form of genetic information which can be transferred to progeny, and which may be capable of altering characteristics of the host cell. Endosymbionts have been suggested to influence morphology, physiology and pathology of some parasitic protists (Preer & Preer, 1977; 1984). It has been indicated that endosymbionts alter the surface membrane chemistry of Crithidia oncopelti (see Dwyer L Chang, 1976), the metabolism of Paramecium aurelia and Crithidia oncopelti (see Lee, Lee & Weis, 1985), division in Euplotes aediculatus (see Heckmann, 1975), and toxin production (see Preer & Preer, 1977). Effects on the response of parasites to chemotherapeutic drugs, and on infectivity and virulence have also been suggested (see Nemanic, Owen, Stevens & Mueller, 1979). At least in some instances, the bacterial endosymbiont is capable of providing some nutritional requirements of the host cell, and subsequently altering host specificity (Lee et al., 1985). Bacteria-like endosymbionts are not common in Blastocystis spp., and their effects on the host cell remain unknown. Hence, caution needs to be taken when performing biochemical or cytochemical analyses of Blastocystis containing endosymbionts, as their presence may significantly affect the results. Acknowledgements-This
work was supported in part by a
Note
149
Research Encouragement Award from the Queensland University of Technology. The authors wish to thank Dr Karen Lai Pen Foon, Institute for Medical Research, Kuala Lumpur, Malaysia for providing monkey faecal material, and Mr Matthew Cassidy for technical assistance.
REFERENCES BALL G. H. 1969. Organisms living on and in protozoa. In: Research in Protozoology, Vol. 3 (Edited by CHEN T. T.), pp. 565-718. Pergamon Press, London. BOREHAM P. F. L. & STENZEL D. J. 1993. Blastocystis in Humans and Animals: Morphology, Biology and Epizootiology. Advances in Parasitology 32: I-70. DUNN L. A., BOREHAM P. F. L. & STENZEL D. J. 1989. Ultrastructural variation of Blastocystis hominis stocks in culture. International Journal for Parasitology 19: 43- 56. DWYER D. M. & CHANG K.-P. 1976. Surface membrane carbohydrate alterations of a flagellated protozoan mediated by bacterial endosymbiotes. Proceedings of the National Academy of Sciences of the United States of America 73: 852-856. HECKMANN K. 1975. Omikron, ein essentieller Endosymbiont von Euplotes aediculatus. Journalof Protozoology 22: 97-104. LEE J. J., LEE M. J. & WEIS D. S. 1985. Possible adaptive value of endosymbionts to their protozoan hosts. Journal of Protozoology 32: 38(t382. NEMANIC P. C., OWEN R. L., STEVENSD. P. &MUELLER J. C. 1979. Ultrastructural observations on giardiasis in a mouse model. II. Endosymbiosis and organelle distribution in Giardia muris and Giardia lamblia. Journal of Infectious Diseases 140: 222-228. PREER L. B. & PREER J. R. 1977. Inheritance of infectious elements. In: Cell Biology: a Comprehensive Treatise, Vol. 1 (Edited by GOLDSTEIN L. & PRESCOTT D. M.), pp. 319373. Academic Press, New York. PREER J. R. & PREER L. B. 1984. Endosymbionts. In: Bergey‘s Manualof Systematic Bacteriology, Vol. 1 (Edited by KRIEG N. R. & HOLY J. G.), pp. 795-942. Williams & Williams, Baltimore. STENZEL D. J., BOREHAM P. F. L. & MCDOUGALL R. 1991. Ultrastructure of Blastocystis hominis in human stool samples. International Journal for Parasitology 21: 807812. STENZEL D. J., CASSIDY M. F. & BOREHAM P. F. L. (in press a) Morphology of Blastocystis sp. from domestic birds. Parasitology Research. STENZEL D. J., CASSIDY M. F. & BOREHAM P. F. L. (in press b) Morphology of Blastocystis sp. isolated from circus animals. International Journal for Parasitology. TEOW W. L., Ho L. C., NG G. C., CHAN Y. C., YAP E. H., CHAN P. P., HOWE J., ZAMAN V. & SINGH M. 1992. Viruslike particles in a Elastocystis species from the sea-snake, Lapemis hardwickii. International Journalfor Parasitology 22: 1029-1032. Z~ERDT C. H. &TAN H. 1976. Endosymbiosis in Blastocystis hominis. Experimental Parasitology 39: 422430.