Studies on the Coccidia of Passerine Birds by M. Anwar A thesis presented for the Degree of Doctor of Philosophy in the Faculty of Science, University of London. Dept. of Zoology & Applied Entomology, Imperial College Field Station, Ashurst Lodge, Sunninghill, Ascot, Berkshire. November, 1964. ABSTRACT 111 out of 113 passerine birds of 13 species were infected with species of Isospora. The variability of oocyst measurements in all 13 species of bird was high indicating the presence of more than one normal distribution. All birds other than jackdaw and hedge sparrow harboured oval and spherical oocysts. The spherical and oval oocysts of greenfinch and sparrow were examined separately and each was found to be markedly heterogeneous. Studies of endogenous stages from greenfinch and sparrow revealed two species: I. lacazei with schizonts separating merozoites without residual cytoplasm, with spindle-shaped microgametes and macrogametes with dense cytoplasm and no obvious plastic granules; I. chloridis n. sp. with schizonts leaving a residuum when separating merozoites, with comma-shaped microgametes and macrogametes with pale cytoplasm and distinct plastic granules. Relating these endogenous studies with the sporulaticn time of 60 - 72 hours, the prepatent period cf 5 days and oocyst morphology it is shown that no additional species were present in greenfinch and sparrow. Thus the heterogeneity in oval and spherical oocyst populations is not explained in terms of species, rather in terms of conditions supplied by host cells. Oocyst output shows a diurnal periodicity, being greatest at 18.00 hours and least between 6.00 and 13.00 hours. Cytochemical studies were carried out for the detection of DNA, RNA, carbohydrates, proteins, lipids and enzyme activity. DNA was detected in all stages except the macrogamet,... RNA was present in all stages in the cytoplasm and nucleus. Three types rf carbohydrate were present: glycogen, a polysaccharide combined with protein End acid mucopolysaccharide. The first two contribute to the oocyst wall. Special accumulationSof protein mere found in the oocyst wall and in the refractile globules of sporozoites. Acid phosphatase activity was detected in the nucleus and cytoplasm, alkaline phosphatase only in the nucleus. ACKNOWLEDGMENTS I should like to express my sincere gratitude to Dr. E.U. Canning for supervising and guiding me throughout the course of this study, without which this work could not have been done. I am indeed grEteful to Professor B.G. Peters for his helpful advice in the statistical work. The author is also indebted to Professor O.W. Richards, F.R.S. for permission to work in his department. Finally my thanks are due to the other members of staff of the Field Station for their kind help during this investigation. TABLE OF CONTENTS Page INTRODUCTION 1 HISTORICAL REVIEW 3 MATERIAL AND METHODS 15 1. Traps • 15 2. Maintenance of Live Birds 17 3. Recovery of Oocysts 18 a.Common salt method... 18 b.Formol-ether method 19 4. Oocyst Measurements 19 a.Pilot sample 19 b.Complete data 20 c.Special comparison of sparrow and greenfinch data....• 20 5. Sporulation of Oocysts 21 6. Estimation of the Number of Oocysts per Gram of Faeces 21 7. Method for Staining Oocysts 22 8. Determination of the Infected Area of Gut 22 9. Cytological and Cytochemical Techniques 23 a.Fixation 23 b.Cytological methods 24 c.Cytochemical methods 24 i.Nucleic acid 24 ii.Polysaccharides 25 iii, Protein 26 Page iv.Staining of DNA, polysaccharides and protein sections by a combined differential technique 26 v.Lipid... 26 vi.Enzyme activity (acid and alkaline phosphatases) . 26 STUDIES ON THE EXOGENOUS PHASES 27 1. Oocyst Measurements 27 a.Pilot sample 27 b.Complete data 30 c.Special comparison of sparrow and greenfinch 32 2. Sporulation of Oocysts 36 3. Morphology of Oocysts 39 a. General observation........, 40 b. The characters of the sporulated oocysts from sparrow, greenfinch and chaffinch 44 i.Spherical oocyst 44 ii.Oval oocyst 47 c. Comparison of the morphological characters of oocysts from other host species with spherical and oval types from sparrow, greenfinch and chaffinch 47 4. Determination of the Prepatent Period of Isospora spp. and a Note on Cross Infection 49 a.Infection of robin 49 b.Super-infection of greenfinch and chaffinch with oocysts 49 5. The Diurnal Output of Oocysts in Greenfinch 50 STUDIES ON THE ENDOGENOUS PHASES... 54 1.Localization of Endogenous Phases 54. 2.Attempts to clean Birds of Naturally acquired Isospora infections 54 Page 3. Double Infection., 57 4. Internal Stages of Isospora lacazei Labbe 1893 58 a.Sporozoites 58 b.First generation schizogony 58 c.Late generation schizogony 60 d.Microgametocyte 63 e, Macrogametocyte 63 5. Internal Stages of Isospora chloridis n.sp. 63 a.Sporozoite 66 b.First generation schizogony 66 c.Late generation schizogony 66 d.Microgametocyte 68 e, Macrogametocyte 68 CYTOCHEMICAL STUDIES ON ISOSPORA SPP. IN GREENFINCH 72 1. Deoxyribonucleic acid (DNA) 72 2.Ribonucleic acid (RNA) 76 3.Polysaccharides 78 4.Protein... 84 5.Lipid 88 6.Enzyme Activity 88 CYTOCHEMICAL STUDIES ON THE EXOGENOUS PHASES 90 DISCUSSION 91 SUMMARY 108 REFERENCES 112 APPENDIX i-xxxiv 1. INTRODUCTION The coccidia are protozoan parasites which inhabit the epithelial lining of the intestine and associated organs of several groups of invert- ebrates and vertebrates, including man.. The life cycle comprises an intracellular or endogenous phase within the host, and an exogenous phase which is completed in the oocyst, that is voided in the faeces. Coccidia are found in a wide variety of vertebrates, and in passerine birds the commonest genus is Isospora. The work on coccidia of passerine birds has been restricted to studies of the oocyst except for a few incomplete descriptions of endogenous phases (Hosoda, 1928a etq.). Thus little is known about the morphology, and almost nothing about the cytochemistry of the intracellular phases. This is in contrast to the great deal of knowledge about the life cycle and cytochemistry of the other genera of coccidia. It is not at all surprising, therefore, that the specific identification of coccidia in passerine birds is uncertain because all the descriptions have been based on the oocyst. Thus while some authors (e.g. Yakimoff et al. 1936, 38) believe that the genus Isospora in passerines is represented by several species parasitizing different and widely separated hosts, others (e.g. Scholtyseck, 1954) hold the view that several that have been described as distinct species belong to one, namely Isospora lacazei Labb6 1893. This controversy can only be resolved when more is known of the life cycle and cytochemistry of parasites, and the following work was undertaken with a view to contributing some information towards this end. 2. The objectives of this work have been to study the species of Isospora occurring in passerine birds collected at the Imperial College Field Station in Berkshire with particular emphasis on: 1. The incidence of parasites and study of oocyst production. 2. A description of the endogenous phases of the life cycle together with cytochemical observations. 3. The correlation of these findings with specific descriptions that are current in the literature. 3. HISTORICAL REVIEW The earliest description of coccidia from passerine birds is that of Rivolta (1869). Rivolta noted the division of oocyst contents into two masses, but he did not distinguish Eimeria from Isospora, and grouped them as Psorospermium avium. Isospora in passerine birds was first described by Labb6 (1893) under the name of Diplospora lacazii, from the European goldfinch (Carduelis carduelis), European sky lark (Alauda arvensis) and other unnamed birds in France. Oocyst diameters were given as 23 - 25 y. In the same paper Labbe (1893) described Diplospora rivoltae with oocysts measuring 16 - 18 y, as a distinct species from the red-backed shrike (Lanius collurio), chaffinch (Fringilla coelebs), continental blue tit (Parus caeruleus) and other, unnamed, birds in France. Labbe (1896) re-investigated the problem and changed the spelling of the specific name to D. lacazei. This spelling is in conformity with the new International Code, but Henry (1932) after discussing the question of nomenclature wrongly adopted the spelling I. lacazii, as originally used by ',abbe, and this form was also used by Becker (1934) and Boughton,Boughton & Volk (1938). Labbe (1896) was able to demonstrate its presence in a wide variety of birds, which he listed. As he found oocysts of a range of sizes intermediate between those of D. lacazei and D. rivoltae, he concluded that only one species D. lacazei occurs in passerine birds. However he did not in this paper give measurements. 4. Sjobring (1897) reported Isospora from eleven species of birds in Sweden, of which nine belong to the Passeriformes. He described the oocysts as being spherical, sometimes oval, with a double-contoured wall. He illustrated the oocyst with five sporozoites in each sporocyst. There was no micropyle and no oocystic residual body. Sj5bring used two names for this coccidium. At the beginning of his paper he called it Isospora passerum, and later Isospora communis-passerum. He remarked, however, that except for the number of sporozoites, there was little difference between his coccidium and Labbe's Diplospora lacazei and D. rivoltae. Wenyon (1926) reviewing the coccidia, noted the morphological similarities between I. passerum and I. lacazei, and considered the former to be synonymous with the latter. This supported Labbe's view that there is only one species of Isospora in passerine birds. Laveran (1898) gave some descriptions of the endogenous phases. He was the first author to describe the microgametes of Isospora in the lark. According to his report the length of the microgametes was between 2 and 3 p, and 30 of them were grouped around a residual body. Wasielewski (1904) in Germany examined 500 newly caught birds and found that 20 per cent.