- Email: [email protected]
Plasmodium wenyoni sp. nov., a malaria parasite of a Brazilian snake
277
"tRANSACTIONSOF THE ROYALSOCIETYOF TROPICAL 1VI.EDICINEAND HYGIENE. Vol. 59. No. 3. May, 1965.
COMMUNICATIONS PLASMODIUM WENYONI SP. NOV., A MALARIA PARASITE OF A BRAZILIAN SNAKE BY
P. C. C. GARNHAM
London School of Hygiene and Tropical Medicine A year or two ago, Dr. R. A. Neal handed me an old collection of blood films, containing various parasites. The films had been prepared by the late Dr. C. M. Wenyon from animals which had died in the London Zoological Gardens, and he had made provisional identifications of the organisms present. One slide was labelled "Haemogregarine and Plasmodium, Natterer's snake, No. 79, 1934," and my attention was immediately excited, because the presence of malaria parasites has never been confirmed in snakes, although there are a few doubtful records, which are analysed below. Examination of the film revealed numerous intra-erythrocytic parasites, of which the greater proportion appeared to be gametocytes of a Plasmodium, while a smaller number were asexual forms, including some in division. The staining (Giemsa's stain) fortunately was still excellent, even after storage of the film for 30 years in an unmounted condition. The parasite clearly represents a new species, and I can find no name more suitable than Plasmodium wenyoni to commemorate the great British protozoologist, revered by all who knew him. Wenyon disliked creating new species of protozoans, but the circumstances and the parasite are sufficiently unique to allow me to hope that he would not altogether have disapproved, even though the following description is based upon a single blood film. The blood film also showed numerous haemogregarines, of a common type, in the erythrocytes.
Plasmodium wenyoni sp. nov. The youngest asexual forms are amoeboid bodies (Fig. 1), lying at one extremity of the erythrocyte, which soon displace, to a certain extent, the nuclcus of the host cell. The cytoplasm of the parasite is tenuous and vacuolated; the pigment is inconspicuous and remains so throughout the course of schizogony. Nuclear division begins while the parasite is still quite small (Fig. 2) and continues until about 12-14 daughter nuclei are produced in a schizont of rather ragged outline (Figs. 3-8). The largest forms seen in the film measured 9~ in diameter, but no completely mature segmenters were detected, and the exact number of merozoites is unknown, though it must exceed 12. The gametocytes show the usual dimorphic characters. The immature microgametocyte (Figs. 9 and 10) is a round body with a diffuse nucleus, fine cytoplasm and
278
PlasmodiumroenyonisP. Nov., A MALARIAPARASITEOF A BRAZILIANSNAKE
groups of pigment granules arranged on the periphery. The colour of the pigment in all forms of the parasite is a dark brown. The mature microgametocyte (Fig. 11) occupies a large part of the cytoplasm of the erythrocyte; it is subspherical in shape, and it pushes the nucleus towards one pole. The nucleus of the parasite is still diffuse, and shows darker-staining areas in a pale matrix. The pigment granules remain dispersed. The macrogametocyte also begins as a small round body (Figs. 12 and 13) but the cytoplasm stains more deeply, and the nucleus is more compact. The larger and possibly mature macrogametocytes (Figs. 14 and 15) are irregular in outline and show fissures and projections of the border. The nucleus remains compact, but is differentiated into lighter and darker portions. Up to 24 spherical pigment granules are scattered in the cytoplasm. The male gametocyte measures about 11~ × 8~ and the female, 13~z × 8~t. Both asexual and sexual stages tend to be round, and certainly no true elongated gametocytes were seen or any parasites curving around the host cell nucleus. Mature erythrocytes appear to be selected for invasion by this species, and they are little affected by the presence of the parasite.
Diagnosis
Plasmodium wenyoni sp. nov. is a malaria parasite of Natterer's snake, Thamnodynastes pallidus, and is characterized by the presence of occasionally amoeboid trophozoites, schizonts of ragged outline containing up to 14 nuclei, and larger gametocytes which are subspherical in shape, with an irregular, indented outline. Rather small pigment granules are numerous in the sexual forms; they are spherical, and dark brown in colour.
Type locality and host Brazil. From a single specimen of Thamnodynastes pallidus, which died in the Zoological Gardens, London, after 4 months in captivity.
Discussion The first "malaria parasite" of snakes was described by BOUET(1909) from specimens of Naja sp. or Sepedon sp. collected on the Ivory Coast and in Senegal No forms in division were seen; nevertheless, Bouet, for convenience, named the parasite Plasmodium mesnili, though it clearly belonged to the genus Haemoproteus, as was pointed out by WEsYoN (1926) in reference to his own material from the Sudan. Marcel and Andr6 LEGER (1914) collected a specimen of Sepedon haemachates from Haut S~n~gal, Niger, in which numerous gametocytes of the so-called P. n~snili were detected. Later, MAc~m (1919) discovered a heavy infection of "malaria parasites" in a Naja nigricollis caught in Accra. He described schizogonic stages in which a dumb-bellshaped figure was said to become binucleate, and to separate into two merozoites. The gametocytes were large, oval or elongate bodies, with scattered pigment. The figures shown in Macfie's coloured plate can all be better interpreted as stages in the development of gametocytes, rather than this most improbable type of schizogony or binary fission.
6
13
7
14
8
15
All figures are from camera Zucidu drawings of Giemsa stained parasites x 1800 FIG. 1. Trophozoite. FIGS. Schizonts.schizont. FIG. 2.3-8. Binucleate FIGS. 9, 10. Immature microgametocytes.
FIG. 11. Mature microgametocyte. FIGS. 12, 13. Immature macrogametocytes
14, 15.
Macrogametocytes.
P. c. c. ~ A R N H A M
279
The frequent occurrence of two or more parasites in a single cell might easily have led Macfie astray, and this parasite again should be identified as Haemoproteus mesnili. Since the time of these original observations, pigmented parasites have occasionally been reported in snakes, but in general, workers have followed WENCON (1926) in classifying them as haemoproteids. However, DE M~LLO and DA FONSEC~ (1958) described Plasrnodium passeritae in the Indian snake, Passerita mycterizans, on the basis of the presence of pigmented schizonts in the erythrocytes. They described minute amoeboid parasites, and schizonts, containing 2, 3 or 4 chromatin masses, often in the form of a piroplasm-like Maltese cross, but gametocytes were absent. The infection was scanty and was first thought to be due to a piroplasm, until the small dots of brownish pigment, scattered in the cytoplasm, were observed. Mrs. de Mello, widow of the late Colonel J. de Mello, informed the writer that the blood films of this animal no longer exist, and it is impossible to arrive at a firm conclusion about the identity of this parasite, which from the illustrations looks unlike a species of Plasmodium. The apparent rarity of species of Plasmodium in snakes is in striking contrast to their frequency in lizards, in which 23 species are known to occur. Snakes are by no means immune to other arthropod-borne infections, and haemogregarines, trypanosomes and haemoproteids are often found. These groups of parasites, particularly the haemogregarines, however, are transmitted by ticks and mites, and perhaps snakes are not bitten by mosquitoes, the natural vectors of Plasmodium. The occurrence of Plasmodium wenyoni in a snake from Brazil is not altogether unexpected, as there is probably more lizard malaria in the New World tropics and sub-tropics than anywhere in the world. The possibility that P. wenyoni might be an infection of saurian malaria in an abnormal host (the snake) has to be considered. There is a certain similarity between P. wenyoni and the South American species, P. tropiduri, P. pifanoi and P. torrealbai, but the gametocytes of the new parasite are sufficiently distinctive to allow the species to be easily differentiated.
Summary 1. A new species of Plasmodium (P. wenyoni) is described from an old blood film of a Natterer's snake (T. pallidus) from Brazil. 2. This is probably the first record of the presence of genuine malaria parasites in snakes.
References BOUET,G. (1909). C.R. Soc. Biol., Paris, 66, 43. LEGER,i . & LEGER,A. (1914). Bull. Soc. Path. exot., 7, 488. MACFIE, J. W. S. (1919). Ann. trop. J~fed. Parasit., 13, 23. DE MELLO, I. F. & DA FONS~CA, L. (1938). Proc. Indian Acad. Sci., 7, 13, 47. WENYON, C. M. (1926). Protozoology, Vol. 2. London: Bailli~re TindaU & Cox.
"tRANSACTIONSOF THE ROYALSOCIETYOF TROPICAL 1VI.EDICINEAND HYGIENE. Vol. 59. No. 3. May, 1965.
COMMUNICATIONS PLASMODIUM WENYONI SP. NOV., A MALARIA PARASITE OF A BRAZILIAN SNAKE BY
P. C. C. GARNHAM
London School of Hygiene and Tropical Medicine A year or two ago, Dr. R. A. Neal handed me an old collection of blood films, containing various parasites. The films had been prepared by the late Dr. C. M. Wenyon from animals which had died in the London Zoological Gardens, and he had made provisional identifications of the organisms present. One slide was labelled "Haemogregarine and Plasmodium, Natterer's snake, No. 79, 1934," and my attention was immediately excited, because the presence of malaria parasites has never been confirmed in snakes, although there are a few doubtful records, which are analysed below. Examination of the film revealed numerous intra-erythrocytic parasites, of which the greater proportion appeared to be gametocytes of a Plasmodium, while a smaller number were asexual forms, including some in division. The staining (Giemsa's stain) fortunately was still excellent, even after storage of the film for 30 years in an unmounted condition. The parasite clearly represents a new species, and I can find no name more suitable than Plasmodium wenyoni to commemorate the great British protozoologist, revered by all who knew him. Wenyon disliked creating new species of protozoans, but the circumstances and the parasite are sufficiently unique to allow me to hope that he would not altogether have disapproved, even though the following description is based upon a single blood film. The blood film also showed numerous haemogregarines, of a common type, in the erythrocytes.
Plasmodium wenyoni sp. nov. The youngest asexual forms are amoeboid bodies (Fig. 1), lying at one extremity of the erythrocyte, which soon displace, to a certain extent, the nuclcus of the host cell. The cytoplasm of the parasite is tenuous and vacuolated; the pigment is inconspicuous and remains so throughout the course of schizogony. Nuclear division begins while the parasite is still quite small (Fig. 2) and continues until about 12-14 daughter nuclei are produced in a schizont of rather ragged outline (Figs. 3-8). The largest forms seen in the film measured 9~ in diameter, but no completely mature segmenters were detected, and the exact number of merozoites is unknown, though it must exceed 12. The gametocytes show the usual dimorphic characters. The immature microgametocyte (Figs. 9 and 10) is a round body with a diffuse nucleus, fine cytoplasm and
278
PlasmodiumroenyonisP. Nov., A MALARIAPARASITEOF A BRAZILIANSNAKE
groups of pigment granules arranged on the periphery. The colour of the pigment in all forms of the parasite is a dark brown. The mature microgametocyte (Fig. 11) occupies a large part of the cytoplasm of the erythrocyte; it is subspherical in shape, and it pushes the nucleus towards one pole. The nucleus of the parasite is still diffuse, and shows darker-staining areas in a pale matrix. The pigment granules remain dispersed. The macrogametocyte also begins as a small round body (Figs. 12 and 13) but the cytoplasm stains more deeply, and the nucleus is more compact. The larger and possibly mature macrogametocytes (Figs. 14 and 15) are irregular in outline and show fissures and projections of the border. The nucleus remains compact, but is differentiated into lighter and darker portions. Up to 24 spherical pigment granules are scattered in the cytoplasm. The male gametocyte measures about 11~ × 8~ and the female, 13~z × 8~t. Both asexual and sexual stages tend to be round, and certainly no true elongated gametocytes were seen or any parasites curving around the host cell nucleus. Mature erythrocytes appear to be selected for invasion by this species, and they are little affected by the presence of the parasite.
Diagnosis
Plasmodium wenyoni sp. nov. is a malaria parasite of Natterer's snake, Thamnodynastes pallidus, and is characterized by the presence of occasionally amoeboid trophozoites, schizonts of ragged outline containing up to 14 nuclei, and larger gametocytes which are subspherical in shape, with an irregular, indented outline. Rather small pigment granules are numerous in the sexual forms; they are spherical, and dark brown in colour.
Type locality and host Brazil. From a single specimen of Thamnodynastes pallidus, which died in the Zoological Gardens, London, after 4 months in captivity.
Discussion The first "malaria parasite" of snakes was described by BOUET(1909) from specimens of Naja sp. or Sepedon sp. collected on the Ivory Coast and in Senegal No forms in division were seen; nevertheless, Bouet, for convenience, named the parasite Plasmodium mesnili, though it clearly belonged to the genus Haemoproteus, as was pointed out by WEsYoN (1926) in reference to his own material from the Sudan. Marcel and Andr6 LEGER (1914) collected a specimen of Sepedon haemachates from Haut S~n~gal, Niger, in which numerous gametocytes of the so-called P. n~snili were detected. Later, MAc~m (1919) discovered a heavy infection of "malaria parasites" in a Naja nigricollis caught in Accra. He described schizogonic stages in which a dumb-bellshaped figure was said to become binucleate, and to separate into two merozoites. The gametocytes were large, oval or elongate bodies, with scattered pigment. The figures shown in Macfie's coloured plate can all be better interpreted as stages in the development of gametocytes, rather than this most improbable type of schizogony or binary fission.
6
13
7
14
8
15
All figures are from camera Zucidu drawings of Giemsa stained parasites x 1800 FIG. 1. Trophozoite. FIGS. Schizonts.schizont. FIG. 2.3-8. Binucleate FIGS. 9, 10. Immature microgametocytes.
FIG. 11. Mature microgametocyte. FIGS. 12, 13. Immature macrogametocytes
14, 15.
Macrogametocytes.
P. c. c. ~ A R N H A M
279
The frequent occurrence of two or more parasites in a single cell might easily have led Macfie astray, and this parasite again should be identified as Haemoproteus mesnili. Since the time of these original observations, pigmented parasites have occasionally been reported in snakes, but in general, workers have followed WENCON (1926) in classifying them as haemoproteids. However, DE M~LLO and DA FONSEC~ (1958) described Plasrnodium passeritae in the Indian snake, Passerita mycterizans, on the basis of the presence of pigmented schizonts in the erythrocytes. They described minute amoeboid parasites, and schizonts, containing 2, 3 or 4 chromatin masses, often in the form of a piroplasm-like Maltese cross, but gametocytes were absent. The infection was scanty and was first thought to be due to a piroplasm, until the small dots of brownish pigment, scattered in the cytoplasm, were observed. Mrs. de Mello, widow of the late Colonel J. de Mello, informed the writer that the blood films of this animal no longer exist, and it is impossible to arrive at a firm conclusion about the identity of this parasite, which from the illustrations looks unlike a species of Plasmodium. The apparent rarity of species of Plasmodium in snakes is in striking contrast to their frequency in lizards, in which 23 species are known to occur. Snakes are by no means immune to other arthropod-borne infections, and haemogregarines, trypanosomes and haemoproteids are often found. These groups of parasites, particularly the haemogregarines, however, are transmitted by ticks and mites, and perhaps snakes are not bitten by mosquitoes, the natural vectors of Plasmodium. The occurrence of Plasmodium wenyoni in a snake from Brazil is not altogether unexpected, as there is probably more lizard malaria in the New World tropics and sub-tropics than anywhere in the world. The possibility that P. wenyoni might be an infection of saurian malaria in an abnormal host (the snake) has to be considered. There is a certain similarity between P. wenyoni and the South American species, P. tropiduri, P. pifanoi and P. torrealbai, but the gametocytes of the new parasite are sufficiently distinctive to allow the species to be easily differentiated.
Summary 1. A new species of Plasmodium (P. wenyoni) is described from an old blood film of a Natterer's snake (T. pallidus) from Brazil. 2. This is probably the first record of the presence of genuine malaria parasites in snakes.
References BOUET,G. (1909). C.R. Soc. Biol., Paris, 66, 43. LEGER,i . & LEGER,A. (1914). Bull. Soc. Path. exot., 7, 488. MACFIE, J. W. S. (1919). Ann. trop. J~fed. Parasit., 13, 23. DE MELLO, I. F. & DA FONS~CA, L. (1938). Proc. Indian Acad. Sci., 7, 13, 47. WENYON, C. M. (1926). Protozoology, Vol. 2. London: Bailli~re TindaU & Cox.