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The fine structure of Leishmania enriettii in the guinea pig
InwrnationolJournalfor Parasifology.1975.Vol. 5. pp. 651-657.Pergarnon Press.
Printedin GreatBritain.
THE FINE STRUCTURE OF LEZSHMANIA IN THE GUINEA PIG M. W. Department
of Dermatology,
ENRIETTII
KANAN
Slade Hospital, The United Oxford Hospitals, Oxford, England (Received 6 September 1974)
M. W. 1975. The fine structure of Leishmania enriettii in the guinea-pig. International Journal for Parasitology 5: 651-657. A study of the fine structure of Leishmania enriettii in the guinea-pig has been presented. There was close similarity to other members of the same genus and the finding of 2-3 axonemes (rhizoplasts) reported previously by other workers in non-dividing protozoa of the same species has not been confirmed. The functions of the main organelles and the morphological differences observed in comparison with those of other species have been reviewed. Abstract-KANAN
INDEX KEY WORDS: Leishmania enriettii; fine structure; rhizoplast; flagellar structure; pellicle; periblast.
MATERIALS
AND METHODS
Several leishmanial nodules were obtained from the nasal mucosa, the scrotum and vulva, produced experimentally in a batch of 7 guinea-pigs inoculated intravenously with L. enriettii (Kanan, 1975). Cubes of 1 mm were obtained from the excised nodules and immediately fixed in 3% glutaraldehyde in 5% sucrose phosphate buffer + 1% calcium chloride for 3 h on ice. The specimens were then washed with the same buffer and post-fixed in 1% osmium tetroxide diluted in above sucrose buffer for 2 h at room temperature. This was followed by dehydration in graded alcohols, treatment with epoxypropane and embedding in Taab araldite. Ultrathin sections were stained with uranyl acetate and lead citrate and viewed in Siemen’s 101 electronmicroscope.
kinetoplast;
axoneme;
RESULTS
INTRODUCTION SEVERAL reports are available on the ultrastructure of both tissue and cultural forms of L. tropica, L. donovani, L. brasiliensis and L. mexicana (Lofgren, 1950; Sanyal & Sen Gupta, 1967; Pyne, 1958; Sen Gupta, Das Gupta & Bhattacharya, 1951; Chang, 1956; Aleman, 1969; Garnham & Bird, 1962). However, the tissue and leptomonad forms of L. enriettii, the causative organism of guinea-pig leishmaniasis have been described in detail by Muniz & Medina (1948) under the ordinary light microscope. They concluded that the parasite showed certain peculiarities not found in other species of the same genus, the principal of these being the occurrence of 2 or 3 axonemes (rhizoplasts) even in a non-dividing protozoon. In view of this the following report on a recent study of the ultrastructure of this protozoon in the guinea-pig is presented.
guinea-pig;
Leishmanial protozoa mainly occurred inside histiocytes of the host in the vicinity of aggregates of host cell mitochondria and some lysosomes, and were clearly contained within single membrane bound phagolysosomes (Fig. 1). The protozoa were oval or roundish and had an average size of 354 pm x 2.6 pm and showed the following morphological features. The pellicle or periblast Each protozoon had a tripartite plasma membrane cover made of an outer smooth layer 25-30 A and an inner similar layer 25-30 A separated from each other by a clear electron transparent space about 35 A thick (Fig. 2). The subpellicular jibrils Under the pellicle and separated from it by a clear cytoplasmic space, a circumferential row of hollow fibrils ran parallel to the long axis of the protozoon (Fig. 2). Their total number varied between 120 and 220 and each fibril had a diameter of 200-210 A and were separated from each other by a regular distance of 200-250 8, depending on the state of fixation. The nucleus This was situated slightly to one side o jthe body centre and had an ill-defined nuclear membrane and its chromatin was aggregated in a segmental fashion at its periphery in close apposition to the nuclear membrane. It usually had 1 karyosome but some of the nuclei had 2 karyosomes (Fig. 2) which joined the marginal chromatin aggregates. The karyosome could be round, oval or irregular in shape and 651
652
M. W. KANAN
I.J.P.
VOL.
5.
1975
FIG. 2. Ph = phagosomal membrane (short thin arrows). P = periblast or pellicle (thin arrow). Sf = subpellicular fibril (long thin arrow). N = protozoa1 nucleus. LD = lipid droplet. Kp = kinetoplast facing the flagelfar pouchran (F). G == dense granules. M = protozoa1 mitochondrium. Insert (A): Another protozoa1 nucleus with ring shaped karyosome (K). Insert(B): Another nucleus with 2 karyosomes (K) connected to marginal chromatin (Ch).
654
M. W. KANAN
occasionally in the form of a ring (Fig. 2). The major part of the karyoplasm was granular and much less electron dense. The flagellar structure
Like other flagellates of the same genus L. enriettii had a flagellar structure which consisted of a short cylindrical organ, the blepharoplast which measured 0.23 urn across. The latter had 9 closely attached pairs of hollow fibrils but lacked the central pair of fibrils (Fig. 3) which usually characterize the cilium and the flagellum. The rhizoplast started from a small, broad proximal rootlet in the cytoplasm and joined distally an electron-dense narrower plate from which the axoneme proper arose to stop at the outlet of the flagellar pouch (Fig. 4). The flagellar pouch could be clearly seen as an invagination in the plasma membrane of the protozoon and contained multiple small vesicles of unidentified nature. In all the specimens examined with the electron microscope only 1 axoneme (rhizoplast) has been found in every flagellar pouch. The kinetoplast
This appeared as a deeply electron dense crescent shaped band 1 urn x 0.08 urn made of 32-35 microfibrils assembled in a fence-like pattern inside a huge protozoa1 mitochondrium (Figs. 3 & 4). In all sections examined, it always faced the rhizoplast. Other cytoplasmic organelles included a few more mitochondria of smaller size scattered in the cytoplasm. Rough endoplasmic reticulum membranes were quite infrequent and occasionally a few small smooth vesicles in close proximity to the nucleus could be taken to represent the Golgi apparatus (Fig. 2). Many ribosomes occurred singly and in aggregates throughout the cytoplasm of the protozoon. Also some deeply dense granules of various sizes were frequently encountered (Figs. l-4). Occasionally one or two lipid granules were found as well (Fig. 2) and two or three vacuoles contained various elements and granules of unidentified nature (Figs. 3 & 4). DISCUSSION Except for a few subtle differences, there was close similarity between the fine structure of L. enriettii and other members of the same genus. Muniz & Medina (1948) reported the occurrence of 2-3 axonemes (rhizoplasts) in a high percentage of tissue forms of L. enriettii in ordinary paraffin sections stained with Giemsa even in protozoa which showed no sign of division. In the present study this finding has not been confirmed in spite of repeated search in many of ultrathin sections examined with the electron microscope. However, it is difficult to offer a satisfactory explanation for this discrepancy apart from the expected differences in details resolved by
I.J.P. VOL. 5. 1975
the two types of investigation. If this discrepancy proves correct then the possibility arises that the strain of L. enriettii used in the present experiment differs from the one used by them. Although the average size of the organism in the present material appeared larger than that reported for other types of the same genus, its length of 3.54 urn was observed to be smaller than that of 5.2 urn reported by Muniz & Medina (1948), with the ordinary microscope. This may be due to the marked shrinkage in the size of the protozoon as a result of the fixation effect of gluteraldehyde used in the present study. On the other hand, the larger size of L. enriettii compared with that of other leishmaniae may account for the larger number of the circumferential subpellicular fibrils in some tissue forms of the former. The fine structure of the kinetoplate as described here conforms with that reported for other leishmaniae by other workers, but many more microfibrils could be counted in the kinetoplast of L. enriettii than the maximum number of 20 recorded in L. donovani by Sanyal & Sen Gupta (1967). Recent views on the origin and nature of the kinetoplast do not agree with the original suggestion of Swezy’s (1916) and later by Trager 8c Rudzinska (1964) that it was derived from the nucleus. Steinert & Van Assel (1967) demonstrated that the kinetoplast in two of the Trypanosomatidae was derived from a mitochondrial genome which was shown to be exceptionally sensitive to acriflavine, like the extrachromosomal genetic factor of yeast. The DNA nature of the kinetoplast was originally suggested by a positive Feulgen stain obtained under the ordinary light microscope by Bresslau & Scremin (1924). Gras& & Pyne (1965) reviewed the ultrastructure of the kinetoplast in different flagellates which clearly indicated a remarkable evolution in the inframicroscopic organization of this organelle and suggested an evolutionary origin from the mitochondria. This conception confirmed previous similar observations made by Clark&Wallace (1960). Moreover, Nass & Nass (1963a, 6) demonstrated microfilaments within normal mitochondria in cells of the chick embryo and suggested that these represented DNA. Grass6 & Pyne (1965) also thought that the kinetoplastic DNA and hence the mitochondrial DNA were quite different from the nuclear DNA in their macromolecular organization and in their behaviour during cell division as viewed in the electron-micrographs. Function-wise it has been postulated by Sanyal & Sen Gupta (1967) that the close relation of the kinetoplast to the route of the axoneme probably facilitates flagellar movement by directing the cytoplasmic current into the hollow fibrils of the flagellum or by mediating in some other way the transfer of energ; ;or such movL.ment. As to the function of the subpellicular fibrils Adler (1964) has pointed out that they probably enable slight flexion and extension of the body of the leptonomad, which, with the movement of the
I.J.P.
VOL.
5.
1975
Ultrastructure
of L. enriettii
655
656
M.
W.
KANAN
I.J.P. VOL. 5. 1975
Ultrastructure of L. enriettii
I.I.P. VOL.5. 1975
flagellum help the steering of the flagellate. On the other hand, there is no evidence to support the suggestion of Sanyal & Sen Gupta (1967) that the fibrils of the rhizoplast might have developed from the subpellicular fibrils. One of the interesting findings in the present study was the polarization of host cell mitochondria by the protozoon which was also alluded to by other workers in other species of leishmania. This phenomenon probably indicates the vast energy requirement by the parasitized histiocyte during the process of phagocytosis. This disagrees with the speculation of Sanyal & Sen Gupta (1967) that the mitochondria in the host cell serve a nutritional function for the intracellular organism. Acknowledgements-The
author is on an M.R.C. grant No. G973/712T. His thanks are due to Professor G.
Weddell of the Human Anatomy Dept., Oxford University, who provided facilities for electronmicroscopy, Dr. David Bradley of the Pathology Dept., Radcliffe Infirmary, Oxford, who provided the strain of L. enriettii and Miss Patricia Van Diest, who is also included in the same grant for technical assistance. The author is also grateful to Dr. T. .I. Ryan, Consultant Dermatologist, Dept. of Dermatology, Oxford, for his enthusiastic help and advice. REFERENCES ADLER S. 1964. Leishmania. In Advances in Parasitology (Edited by DAWES B.), Vol. 2, pp. 35-96. Academic
Press, New York. of cultured Leishmania Parasitology 24: 259-264.
ALEMANC. 1969. Finestructure brasiliensis. Experimental
BRESSLAUE. & SCREMINL. 1924. Die Kerne der Trypanosomen und ihr Verhalten fur Nuclealreaktion. Archiv. fir Protistenkunde
48: 509-515.
of Leishmania donovani. Journal of Parasitology 42: 126-136.
CHANG P. C. H. 1956. The ultrastructure
CLARK T. B. & WALLACEF. G. 1960. A comparative study of kinetoplast ultrastructure in the Trypanosomatidae. Journal of Parasitology 7: 115-124.
657
GARNHAMP. C. C. & BIRD R. G. 1962. A preliminary study of the finestructure of Leishmania mexicana as seen under the electron microscope. Science Reports Institute Super Sanita 2: 83-88.
GRASSEP. P. & PYNEC. K. 1965. Sur l’origine et l’homologie du cin&oplaste. In Progress in Profozoology. Excerpta Medica, Amsterdam. KANAN M. W. 1975. Mucocutaneous leishmaniasis in guinea-pigs inoculated intravenously with Leishmania enriettii. British Journal of Dermatology
92:
663-673,
LOFGRENR. 1950. The structure of Leishmania tropica as revealed by phase contrast and electron microscopy. Journal of Parasitology 60: 617-625.
MUNIZ J. & MEDINAH. 1948. Leishmaniose tegumentar do cobaio. Hospital Rio de Janeiro 33: 7-25. NASS M. M. K. & NASS S. 1963a. Intramitochondrial fibers with DNA characteristics. I. Fixation and electron staining reactions. Journal of Cell Biology 19: 593-611.
NASS S. Kc NASS M. M. K. 19636. Intramitochondrial fibers with DNA characteristics. II. Enzymatic and hvdrolytic treatments. Journal of Cell Biolopv _. 19:
6i 3-629. PYNE C. K. 1958. Electron microscopic investigations on the leptomonad form of Leishmania donovani. Experimental Cell Research 14: 388-397.
SANYALA. B. & SEN GUPTA P. C. 1967. Fine structure of Leishmania in dermal leishmanoid. Transactions of the Royal Society of Tropical Medicine and Hygiene 61: 211-216. SEN GUPTA P. C., DAS GUPTA N. N. & BHATTAC~ARYA
D. L. 1951. Electron and photomicrographic studies of the flagellate form of Leishmania donovani. Nature 167: 1063-1064.
STEINERTM. & VAN ANGELS. 1967. The loss of kinetoplastic DNA in two species of Trypanosomatidae treated with acriflavin. Journal of Cell Biology 34: 489-503.
SWEZY 0. 1916. The kinetonucleus of flagellates of the binuclear theory of Hartmann. University of California Publications in Zoology 16: 185-240. TRAGER W. & RUDZINSKAM. A. 1964. The riboflavin requirement and the effects of acriflavin on the fine structure of the kinetoplast of Leishmania tarenfolae. Journal of Protozoology 11: 133-145.
Printedin GreatBritain.
THE FINE STRUCTURE OF LEZSHMANIA IN THE GUINEA PIG M. W. Department
of Dermatology,
ENRIETTII
KANAN
Slade Hospital, The United Oxford Hospitals, Oxford, England (Received 6 September 1974)
M. W. 1975. The fine structure of Leishmania enriettii in the guinea-pig. International Journal for Parasitology 5: 651-657. A study of the fine structure of Leishmania enriettii in the guinea-pig has been presented. There was close similarity to other members of the same genus and the finding of 2-3 axonemes (rhizoplasts) reported previously by other workers in non-dividing protozoa of the same species has not been confirmed. The functions of the main organelles and the morphological differences observed in comparison with those of other species have been reviewed. Abstract-KANAN
INDEX KEY WORDS: Leishmania enriettii; fine structure; rhizoplast; flagellar structure; pellicle; periblast.
MATERIALS
AND METHODS
Several leishmanial nodules were obtained from the nasal mucosa, the scrotum and vulva, produced experimentally in a batch of 7 guinea-pigs inoculated intravenously with L. enriettii (Kanan, 1975). Cubes of 1 mm were obtained from the excised nodules and immediately fixed in 3% glutaraldehyde in 5% sucrose phosphate buffer + 1% calcium chloride for 3 h on ice. The specimens were then washed with the same buffer and post-fixed in 1% osmium tetroxide diluted in above sucrose buffer for 2 h at room temperature. This was followed by dehydration in graded alcohols, treatment with epoxypropane and embedding in Taab araldite. Ultrathin sections were stained with uranyl acetate and lead citrate and viewed in Siemen’s 101 electronmicroscope.
kinetoplast;
axoneme;
RESULTS
INTRODUCTION SEVERAL reports are available on the ultrastructure of both tissue and cultural forms of L. tropica, L. donovani, L. brasiliensis and L. mexicana (Lofgren, 1950; Sanyal & Sen Gupta, 1967; Pyne, 1958; Sen Gupta, Das Gupta & Bhattacharya, 1951; Chang, 1956; Aleman, 1969; Garnham & Bird, 1962). However, the tissue and leptomonad forms of L. enriettii, the causative organism of guinea-pig leishmaniasis have been described in detail by Muniz & Medina (1948) under the ordinary light microscope. They concluded that the parasite showed certain peculiarities not found in other species of the same genus, the principal of these being the occurrence of 2 or 3 axonemes (rhizoplasts) even in a non-dividing protozoon. In view of this the following report on a recent study of the ultrastructure of this protozoon in the guinea-pig is presented.
guinea-pig;
Leishmanial protozoa mainly occurred inside histiocytes of the host in the vicinity of aggregates of host cell mitochondria and some lysosomes, and were clearly contained within single membrane bound phagolysosomes (Fig. 1). The protozoa were oval or roundish and had an average size of 354 pm x 2.6 pm and showed the following morphological features. The pellicle or periblast Each protozoon had a tripartite plasma membrane cover made of an outer smooth layer 25-30 A and an inner similar layer 25-30 A separated from each other by a clear electron transparent space about 35 A thick (Fig. 2). The subpellicular jibrils Under the pellicle and separated from it by a clear cytoplasmic space, a circumferential row of hollow fibrils ran parallel to the long axis of the protozoon (Fig. 2). Their total number varied between 120 and 220 and each fibril had a diameter of 200-210 A and were separated from each other by a regular distance of 200-250 8, depending on the state of fixation. The nucleus This was situated slightly to one side o jthe body centre and had an ill-defined nuclear membrane and its chromatin was aggregated in a segmental fashion at its periphery in close apposition to the nuclear membrane. It usually had 1 karyosome but some of the nuclei had 2 karyosomes (Fig. 2) which joined the marginal chromatin aggregates. The karyosome could be round, oval or irregular in shape and 651
652
M. W. KANAN
I.J.P.
VOL.
5.
1975
FIG. 2. Ph = phagosomal membrane (short thin arrows). P = periblast or pellicle (thin arrow). Sf = subpellicular fibril (long thin arrow). N = protozoa1 nucleus. LD = lipid droplet. Kp = kinetoplast facing the flagelfar pouchran (F). G == dense granules. M = protozoa1 mitochondrium. Insert (A): Another protozoa1 nucleus with ring shaped karyosome (K). Insert(B): Another nucleus with 2 karyosomes (K) connected to marginal chromatin (Ch).
654
M. W. KANAN
occasionally in the form of a ring (Fig. 2). The major part of the karyoplasm was granular and much less electron dense. The flagellar structure
Like other flagellates of the same genus L. enriettii had a flagellar structure which consisted of a short cylindrical organ, the blepharoplast which measured 0.23 urn across. The latter had 9 closely attached pairs of hollow fibrils but lacked the central pair of fibrils (Fig. 3) which usually characterize the cilium and the flagellum. The rhizoplast started from a small, broad proximal rootlet in the cytoplasm and joined distally an electron-dense narrower plate from which the axoneme proper arose to stop at the outlet of the flagellar pouch (Fig. 4). The flagellar pouch could be clearly seen as an invagination in the plasma membrane of the protozoon and contained multiple small vesicles of unidentified nature. In all the specimens examined with the electron microscope only 1 axoneme (rhizoplast) has been found in every flagellar pouch. The kinetoplast
This appeared as a deeply electron dense crescent shaped band 1 urn x 0.08 urn made of 32-35 microfibrils assembled in a fence-like pattern inside a huge protozoa1 mitochondrium (Figs. 3 & 4). In all sections examined, it always faced the rhizoplast. Other cytoplasmic organelles included a few more mitochondria of smaller size scattered in the cytoplasm. Rough endoplasmic reticulum membranes were quite infrequent and occasionally a few small smooth vesicles in close proximity to the nucleus could be taken to represent the Golgi apparatus (Fig. 2). Many ribosomes occurred singly and in aggregates throughout the cytoplasm of the protozoon. Also some deeply dense granules of various sizes were frequently encountered (Figs. l-4). Occasionally one or two lipid granules were found as well (Fig. 2) and two or three vacuoles contained various elements and granules of unidentified nature (Figs. 3 & 4). DISCUSSION Except for a few subtle differences, there was close similarity between the fine structure of L. enriettii and other members of the same genus. Muniz & Medina (1948) reported the occurrence of 2-3 axonemes (rhizoplasts) in a high percentage of tissue forms of L. enriettii in ordinary paraffin sections stained with Giemsa even in protozoa which showed no sign of division. In the present study this finding has not been confirmed in spite of repeated search in many of ultrathin sections examined with the electron microscope. However, it is difficult to offer a satisfactory explanation for this discrepancy apart from the expected differences in details resolved by
I.J.P. VOL. 5. 1975
the two types of investigation. If this discrepancy proves correct then the possibility arises that the strain of L. enriettii used in the present experiment differs from the one used by them. Although the average size of the organism in the present material appeared larger than that reported for other types of the same genus, its length of 3.54 urn was observed to be smaller than that of 5.2 urn reported by Muniz & Medina (1948), with the ordinary microscope. This may be due to the marked shrinkage in the size of the protozoon as a result of the fixation effect of gluteraldehyde used in the present study. On the other hand, the larger size of L. enriettii compared with that of other leishmaniae may account for the larger number of the circumferential subpellicular fibrils in some tissue forms of the former. The fine structure of the kinetoplate as described here conforms with that reported for other leishmaniae by other workers, but many more microfibrils could be counted in the kinetoplast of L. enriettii than the maximum number of 20 recorded in L. donovani by Sanyal & Sen Gupta (1967). Recent views on the origin and nature of the kinetoplast do not agree with the original suggestion of Swezy’s (1916) and later by Trager 8c Rudzinska (1964) that it was derived from the nucleus. Steinert & Van Assel (1967) demonstrated that the kinetoplast in two of the Trypanosomatidae was derived from a mitochondrial genome which was shown to be exceptionally sensitive to acriflavine, like the extrachromosomal genetic factor of yeast. The DNA nature of the kinetoplast was originally suggested by a positive Feulgen stain obtained under the ordinary light microscope by Bresslau & Scremin (1924). Gras& & Pyne (1965) reviewed the ultrastructure of the kinetoplast in different flagellates which clearly indicated a remarkable evolution in the inframicroscopic organization of this organelle and suggested an evolutionary origin from the mitochondria. This conception confirmed previous similar observations made by Clark&Wallace (1960). Moreover, Nass & Nass (1963a, 6) demonstrated microfilaments within normal mitochondria in cells of the chick embryo and suggested that these represented DNA. Grass6 & Pyne (1965) also thought that the kinetoplastic DNA and hence the mitochondrial DNA were quite different from the nuclear DNA in their macromolecular organization and in their behaviour during cell division as viewed in the electron-micrographs. Function-wise it has been postulated by Sanyal & Sen Gupta (1967) that the close relation of the kinetoplast to the route of the axoneme probably facilitates flagellar movement by directing the cytoplasmic current into the hollow fibrils of the flagellum or by mediating in some other way the transfer of energ; ;or such movL.ment. As to the function of the subpellicular fibrils Adler (1964) has pointed out that they probably enable slight flexion and extension of the body of the leptonomad, which, with the movement of the
I.J.P.
VOL.
5.
1975
Ultrastructure
of L. enriettii
655
656
M.
W.
KANAN
I.J.P. VOL. 5. 1975
Ultrastructure of L. enriettii
I.I.P. VOL.5. 1975
flagellum help the steering of the flagellate. On the other hand, there is no evidence to support the suggestion of Sanyal & Sen Gupta (1967) that the fibrils of the rhizoplast might have developed from the subpellicular fibrils. One of the interesting findings in the present study was the polarization of host cell mitochondria by the protozoon which was also alluded to by other workers in other species of leishmania. This phenomenon probably indicates the vast energy requirement by the parasitized histiocyte during the process of phagocytosis. This disagrees with the speculation of Sanyal & Sen Gupta (1967) that the mitochondria in the host cell serve a nutritional function for the intracellular organism. Acknowledgements-The
author is on an M.R.C. grant No. G973/712T. His thanks are due to Professor G.
Weddell of the Human Anatomy Dept., Oxford University, who provided facilities for electronmicroscopy, Dr. David Bradley of the Pathology Dept., Radcliffe Infirmary, Oxford, who provided the strain of L. enriettii and Miss Patricia Van Diest, who is also included in the same grant for technical assistance. The author is also grateful to Dr. T. .I. Ryan, Consultant Dermatologist, Dept. of Dermatology, Oxford, for his enthusiastic help and advice. REFERENCES ADLER S. 1964. Leishmania. In Advances in Parasitology (Edited by DAWES B.), Vol. 2, pp. 35-96. Academic
Press, New York. of cultured Leishmania Parasitology 24: 259-264.
ALEMANC. 1969. Finestructure brasiliensis. Experimental
BRESSLAUE. & SCREMINL. 1924. Die Kerne der Trypanosomen und ihr Verhalten fur Nuclealreaktion. Archiv. fir Protistenkunde
48: 509-515.
of Leishmania donovani. Journal of Parasitology 42: 126-136.
CHANG P. C. H. 1956. The ultrastructure
CLARK T. B. & WALLACEF. G. 1960. A comparative study of kinetoplast ultrastructure in the Trypanosomatidae. Journal of Parasitology 7: 115-124.
657
GARNHAMP. C. C. & BIRD R. G. 1962. A preliminary study of the finestructure of Leishmania mexicana as seen under the electron microscope. Science Reports Institute Super Sanita 2: 83-88.
GRASSEP. P. & PYNEC. K. 1965. Sur l’origine et l’homologie du cin&oplaste. In Progress in Profozoology. Excerpta Medica, Amsterdam. KANAN M. W. 1975. Mucocutaneous leishmaniasis in guinea-pigs inoculated intravenously with Leishmania enriettii. British Journal of Dermatology
92:
663-673,
LOFGRENR. 1950. The structure of Leishmania tropica as revealed by phase contrast and electron microscopy. Journal of Parasitology 60: 617-625.
MUNIZ J. & MEDINAH. 1948. Leishmaniose tegumentar do cobaio. Hospital Rio de Janeiro 33: 7-25. NASS M. M. K. & NASS S. 1963a. Intramitochondrial fibers with DNA characteristics. I. Fixation and electron staining reactions. Journal of Cell Biology 19: 593-611.
NASS S. Kc NASS M. M. K. 19636. Intramitochondrial fibers with DNA characteristics. II. Enzymatic and hvdrolytic treatments. Journal of Cell Biolopv _. 19:
6i 3-629. PYNE C. K. 1958. Electron microscopic investigations on the leptomonad form of Leishmania donovani. Experimental Cell Research 14: 388-397.
SANYALA. B. & SEN GUPTA P. C. 1967. Fine structure of Leishmania in dermal leishmanoid. Transactions of the Royal Society of Tropical Medicine and Hygiene 61: 211-216. SEN GUPTA P. C., DAS GUPTA N. N. & BHATTAC~ARYA
D. L. 1951. Electron and photomicrographic studies of the flagellate form of Leishmania donovani. Nature 167: 1063-1064.
STEINERTM. & VAN ANGELS. 1967. The loss of kinetoplastic DNA in two species of Trypanosomatidae treated with acriflavin. Journal of Cell Biology 34: 489-503.
SWEZY 0. 1916. The kinetonucleus of flagellates of the binuclear theory of Hartmann. University of California Publications in Zoology 16: 185-240. TRAGER W. & RUDZINSKAM. A. 1964. The riboflavin requirement and the effects of acriflavin on the fine structure of the kinetoplast of Leishmania tarenfolae. Journal of Protozoology 11: 133-145.