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. were infected with coccidia. He studied the life cycle of the parasite and described some internal stages. He noted the schizont containing 8 to 12 merozoites which varied in size from 8 to 12 y. Hadley (1910) reported that

64 per cent. of English sparrows were infected with coccidia and believed

that the same species were responsible for the conditions of black head in

turkeys and white diarrhoea in chickens. In the same year Cole and Hadley 5. after further study again concluded that black head in turkeys was caused by the same coccidia that were present in sparrows.

Claassen (1923) studied I. lacazei in siskin (Carduelis spinus) and reported two types of schizogony, one type producing merozoites and leaving a residual body, and the other separating merozoites without a residual body. Wenyon (1926) gave the size of the schizonts as about 10 p, producing 8 to 12 merozoites. The merozoites measured 2 to 3 y in breadth by 8 to 12 in length. The microgametocytes %hen fully developed measured p 28 y by 21 y and contained a large number of microgametes from 2 to 4 microns in size. Hosoda (19284 reported Isospora lacazei in Passer montanus satur— atus. In his description of the life cycle he mentioned three types of merozoites; small, intermediate and large size. He did not give any further information about the schizogony stages, but in his illustration he showed a large residual body after formation of the merozoites. He gave the size of the microgametes as 2.25 to 3 p by 0.5 p. He noted that the plastic granules appear in macrogametocyte in the central zone around the

nucleus. Chakravarty and Kar (1944) in India studied the life history of

I. lacazei in Passer domesticus indicus, and Motpastes haemorrhus bengalensis.

They gave the size of the mature schizont which was without residual body as

8.2 to 12.5 p and the size of the merozoites as 6.2 by 2.1 y. The micro— gametocytes measured 8.2 to 12 p, and the microgametes were comma—shaped with a pointed end. The mature macrogamete measured 11 to 17.6 p.

Boughton (1929) studied Isospora in the English sparrow, and found the incidence of infection to be as high as 66 per cent. In 1930 he 6.

Atulkfof introduced biometric cone to in hiOmeasurements of oocysts. He found

considerable variation in the size of oocysts in English sparrows, and also

that there was significant variation in size of oocysts in a single host.

Large oocysts were passed at the beginning of an infection decreasing in size

es the infection progressed. Correlated with the appearance of the smaller

oocyst was an increase in oocyst production. Even so he was unable to

draw a definite conclusion regarding the existence of one or more species

of Isospora in sparrows.

In 1932 Boughton studied the diurnal periodicity of oocyst

production in sparrows and found the heaviest output of oocysts was between

3 p.m. and 8 p.m. under ordinary summer daylight conditions. By keeping

the birds for a minimum of four days under artificial illumination at night

and in darkness during the day the time of heaviest oocyst output could be

changed. Starvation during the morning or afternoon did not change the cycle of oocyst production. In 1933 Boughton completed his studies on the

output of oocysts. He confirmed that the peak production of oocysts was

between 3 p.m. and 8 p.m. Alternating a six hour photoperiod with six hours

of darkness did not change the oocyst production. On this occasion at least

99 per cent. of the sparrows examined were infected and oocysts were always

present in at least one dropping in any 24 hour period. According to

Boughton, Atchley and Eskridge (1935) the interval between peak of oocyst

production could be shortened or lengthened by altering the photoperiods.

$chwalbach (1960) examined 191 sparrows and found that.00cyst

output reached a peak at dusk Olen the birds prepared to perch ,tek. night. 7.

In 1937 Boughton, by superinfecting sparrows over a natural infection, determined a prepatent period for I. lacazei of approximately ten days.

Hall (1933) gave the sporulation time of I. lacazei as 84 hours.

He further reported that the prepatent period varied from 4 to 7 days with an average of 6 days. He gave a figure of 30 days for the patent period based on his observations on one bird only. His measurements of oocyst from four sparrows worm similar to those of Boughton (1930).

In 1933 Galli-Valerio reported a new species of Isospora from

Nucifraga caryocatactes and named it I. nucifragae. This being the first new species in passerines reported since the descriptions of I. lacazei and

I. passerum.

Since this time a total of 26 coccidial species have been described from passerines and these are listed in Table I, though the complete host list for the species is not given. The principal authors were Yakimoff and his co-workers (1936-1938) describing four species of Isospora and one species of Eimeria from European birds, Chakravarty and Kar (1944a and 1947) and later Misra (1947) and Ray, Shivnani, Oommen, and Bhaskaran (1952) who described 16 species of different genera of coccidia from Indian birds. In addition Isospera volki was described by Boughton (1937a) from Parotia lawesi lawesi and ten other birds and Isospora perronciti was described by Carpano

(1937) from Pyrrhula europaea and other birds in Egypt. The question of specific identity in Isospora was taken up again by Scholtyseck (1954). He found two sizes of oocysts in the house sparrow in Germany and assigned 8.

Table I. Species of Coccidia reported frum Passerines.

No. Species of Coccidia 1, Author and Date Host

1 Isospora lecazti Labb6 1893 Carduelis carduelis Synonyms Alauda arvensis la I. rivoltae Labb6 1893 Fringilla coelebs Lanius collurio lb I. communis-passerum Sj3bring 1697 Lanius collurio 2 I. nucifragae Galli-Valerio 1933 Nucifraga caryocatactes 3 I. rocha-limae Yakimoff and Pica pica Gcusseff 1936 4 I. monedulae Yakimoff and Corvus monedula collaris Matschoulsky 1936 5 I. volki Boughton 1937a Patrotia lawesi lawesi 6 I. perronciti Carpano 1937 Pyrrhula europaea 7 I. rodhaini Yakimoff and Corvus spp. Matschoulsky 1938 8 I. fringillae Yakimoff and Fringilla coelebs Gousseff 1938 9 I. muniae Chakravarty and Munia malacca malacca Kar 1944a 10 I. ginginiana Chakravarty and Acridotheres ginginianus Kar 1944a 11 I. temenuchii Chakravarty and Temenuchus pagodarum Kar 1944a 12 I. ginginiana Chakravarty and Acridotheres tristis var. tristis Kar 1947 tristis 13 I. upupae Chakravarty and Upupa epops orientalis Kar 1947 14 I. sturniae Chakravarty end Sturnia malabarica Kar 1947 malabarica 15 I. zosteropis Chakravarty and Zosterops palpebrosa Kar 1947 palpebrosa 16 I. corviae Ray et al. 1952 Corvus macrorlynchus intermedius 17 I. cryptolophae Ray et al. 1952 Seicercus xanthoschistos syn. I. seicercusae ft tt It 18 I. garrulae Ray et al. 1952 Garrula lineatus lineatus 19 I. garrulusae Ray et al. 1952 Garrulus glandarius bisipecularis 20 I. •arusae Ray et al. 1952 Parus dichrous syn. I. lophophniae It ii II 21 I. passerum Scholtyseck 1954 Passer domesticus 22 Eimeria balozeti Yakimoff and Sturnus vulgaris Gousseff 1938a 23 E. malaccae Chakravarty and Munia malacca malacca Kar 1944e 24 E. lucknowensis Misra 1947 Motacilla alba 25 Dorisiella hareni Chakravarty and Munia malacca malacca Kar 1944a 26 Wenyonella mackinnoni Misra 1947 Motacilla alba 9. separate names to them. The larger species he identified as I. lacazei, the smaller species was referred to I. passarum, though through lack of sufficient evidence he concluded his paper by referring them to a single species I. lacazei. Later he again felt able to identify the two species in other birds, I. lacazei in 40 species of birds from several orders and

I. passerum from two crows (Corvus corone corone, C. frugilegus) as well as sparrows (Scholtyseck, 1956).

The question of pathogenicity was discussed by Przygodda and Scholtyseck

(1961) who reported the incidence of Isospora higher in adults than nestling birds and found that the infection was not fatal in nestling$.

Levine and Mohan (1960) observed oocysts of Isospora in faeces of six cattle in Illinois. By comparison they identified these as I. lacazei and indicated that the cattle had picked up the infection during feeding and that the oocysts had passed unchanged through the gut.

Labbe (1896) and Hadley (1911) were among the first workers to make what could be termed a cytochemical observation or interpretation on the macrogametocytes of coccidia. Labe :Joserved that the cytoplasm of coccidia was basophilic. Hadley described "tir.o different sized plastic food granules" in the cytoplasm of macrogametes of Eimeria avium, but gave no further information.

Reichenow (1921) believed that the granules in macrogametes consisted mainly of glycogen and Hosoda (1928) found the "plastic granules" of 10.

E. avium to stain intensely with haematoxylin. Giovannola (1934) found glycogen in insignificant amounts in sporozoites within the oocysts, in schizonts, microgametocytes and young macrogametocytes of Eimeria falciformis and E. stiedae. The application of modern cytochemical methods in an investigation of the morphology of the developmental stages of coccidia, was first undertaken by Cheissin (1935) who used Best's carmine to test for glycogen in Eimeria species.

Since then the distribution cf deoxyribonucleic acid (DNA), ribonucleic acid (RNA), carbohydrates, proteins and lipids has been studied in several species of coccidia mostly of the genus Eimeria and some information is available about the presence of alkaline and acid phosphatase systems.

Most workers agree on the presence of DNA as detected by the

Feulgen reaction in the nuclei of all stages of coccidia except those of the macrogametocytes. This was found by Cheissin (1940 and 1959) for

Eimeria spp. in rabbits, who further observed that the greater part of the microgamete was composed of DNA. His findings were supported by Pattillo and Becker (1955) for Eimeria brunetti and Eimeria acervulina, and Pattillo

(1957) for E. tenella and E. necatrix. Rarely a weak Feulgen positive reaction has been reported for the macrogamete as in Eimeria maxima (Horton—

Smith and Long, 1963).

Ray and Gill (1955) showed that the nucleus of the unsporulated zygote was, like that of the macrogamete, devoid of detectable DNA but that the sporozoite nuclei were Feulgen positive and concluded that the DNA in 11. the sporozoites was formed de novo during the process of sporulation.

Ribonucleic acid (RNA) has been detected in the cytoplasm and karyosomes of several species of coccidia. Roskin and Ginsburg (1944) reported that the cytoplasm and karyosome of the unsporulated oocyst of

E. stiedae stained strongly with pyronin in the methyl—green pyronin method. Pattillo and Becker (1955) found RNA in the nucleus and cytoplasm of all stages of E. brunetti and E. acervulina. Pattillo (1957) found the same for E. tenella and E. necatrix, and Cheissin (1957) for E. magna.

Unusual results were reported by Ray and Gill (1955) who found the macrogametocyte "plastic granules" gave a reaction for RNA with toluidine blue whereas the results of other workers indicate their composition of carbohydrate. Ray and Gill also found the oocyst wall reacted weakly for RNA to toluidine blue.

Carbohydrates of various kinds have been detected in the cytoplasm of coccidia, sometimes as diffuse storage products, otherwise as discreet granules. Edgar, Herrick and Fraser (1944) employing the iodine test detected glycogen in the macrogametocyte of E. tenella, small amounts in the sporozoites but none in the asexual stages, microgametf-cyte or micro— gametes. Cheissin (1935) used Best's carmine and failed to detect glycogen in rabbit Eimeria spp., while Lillie (1947) first used the periodic acid—

Schiff (PAS) technique with controls pre—treated with diastase to detect glycogen, finding it present in the cytoplasm of the oocyst of E. stiedae.

Other carbohydrates have been characterized by predigesting with suitable enzymes before PAS. 12.

In general glycogen was found in considerable quantity in the macrogametocyte and diminished during development of the sporozoites.

Gill and Ray (1954) reported glycogen from E. tenella using PAS with controls pre-treated with saliva. They found insignificant amounts in the sporozoitos and the strongest reaction in the macrogametocytes.

Pattillo and Becker (1955) working with E. brunetti and E. acervulina found that glycogen was present in quantity as small granules in the mature macrogametocyte and zygote, but was mostly used up during the process of sporulation. The PAS positive "plastic granules" found to be composed of muco-protein were present during the development of the macrogamete and contributed to the formation of the oocyst wall which was also PAS positive.

Asexual stages in general were PAS negative but three quarters of the merozoites of E. acervulina contained glycogen. Pattillo (1957) made a similar study of E. tenella and E. necatrix and found that considerably more glycogen was present in the asexual stages than in those of the less pathogenic species studied by Pattillo and Becker (1955). Cheissin (1959) found glycogen accumulated in almost all stages of rabbit Eimeria, but an insignificant amount was found in the microgametocytes. Horton-Smith and

Long (1963) obtained positive PAS results in mature schizonts and merozoites of E. necatrix and E. tenella, and characterized the carbohydrate as glycogen by pre-treating control sections with C,I.-amylase.

Using different fixatives Long and Rootes (1959) and Long, Rootes and Horton-Smith (1961) studied the "plastic granules" in macrogametocytes of E. tenella, E. necatrix, E. acervulina and E. maxima with the PAS 13. reaction. They came to the conclusion that the preservation of the "plast- ic granules" in paraffin sections depended on the fixative used before paraffin embedding. Jennings (1961) obtained a positive reaction by these granules with PAS in E. tenella, E. maxima, E. acervuline. and

E. necatrix and he concluded that the fixative has no effect on the preservation of "plastic granules".

Gill and Ray (1954a) studied the mucopolysaccharide content in

E. tenella and found the highest concentration in the mature second generation schizonts. Young macrogametocytes contained small amounts of mucopolysaccharides but mature macrogametes contained more especially the "plastic granules". Pattillo (1957) found acid mucopolysaccharide in all developmental stages of E. tenella and E. necatrix after staining with toluidine blue and pre-treatment with enzymes at low pH. These results were in contrast to those of Cheissin (1959) and Pattillo and

Becker (1955).

Protein has been studied in different stages of Eimeria usually by the mercuric bromphenol blue method. Pattillo and Becker (1955) showed that in addition to the protein demonstrable by tests for DNA and

RNA in E. brunetti and E. acervulina, the refractile globules of the sporozoites reacted with mercuric bromphenol blue, indicating protein and the "plastic granules" reacted both with this and PAS, indicating that they were composed of mucoprotein or neutral mucopolysaccharide or glyco- protein. The same results were obtained with E. tenella and E. necatrix by Pattillo (1957) and Horton-Smith and Long (1963). 14.

Lipids have been demonstrated in the cytoplasm of coccidia by means of Sudan dyes. Pattillo and Becker (1955) detected lipid material

in the macrogametocytes of E. brunetti and E. acervulina. In the mature

oocyst this material persisted as large globules in the spore and in small

quantity in the refractile globules of the spor2zoites. Lipids were not

found by these workers in the asexual stages, nor by Horton-Smith and Long

(1963) in asexual stages of E. maxima, E. tenella and E. necatrix but were

found in the second generation schizonts of E. tenella by Pattillo (1957).

Cheissin (1959) also found lipids in macrogametocytes and oocysts of E.

magna, E. intestinalis and E. media and Dasgupta (1960) found them in unsporulated oocysts of E. stiedae.

There is little information available concerning enzyme activity

in coccidia and host tissue. Gill and Ray (1954b) studied acid phosphatase

and reported the presence of this enzyme in the karyosomes of all stages

of E. tenella. They found alkaline phosphatase in the nuclei of sporo-

zoites, schizonts, gametocytes and oocysts. Furthermore they found that

the presence of the parasite reduced alkaline phosphatase activity in the host tissue (Ray and Gill, 1954). Cheissin (1959) reported the activity

of alkaline and acid phosphatase in all stages of the life cycle of rabbit

coccidia. He observed the strongest reaction in the karyosomes of the

growing macrogametes and a lesser reaction in the nucleoplasm. Dasgupta

(1961) detected alkaline phosphatase in the karyosome of the macrogameto-

cytes and nuclei of merozoites in E. stiedae. 15.

MATERIALS AND METHODS

1. TRAPS

Two types of cage-trap were constructed to trap the wild birds used in this study. One was made out of wood and the other of wire and half inch poultry netting. In the wooden trap,measuring 48 X 48 X 37 inches (Fig. I), the birds entered through three doors in search of bait

and in the round wire trap, measuring 49 inches in diameter with a height

of 35 inches (Fig. 2), they entered through the funnel. Eoth types were

successful in the capture of many different species of bird. Bait

consisted of a mixture of wheat and bread crumbs. Water was also provided.

The birds caught in these traps were transferred to cages, in a room at a temperature of 23°C. + 3°. These cages measured 24 X 12 x 18

inches. The cages had metal trays which were changeable.

Using the keys of Witherby, Jourdain, Ticehurst and Tucker (1947),

Peterson, Mountfort and Hollom (1956) and Benson (1962) all birds were

identified to species and classified.

In addition six canaries, bought from pet shops, were used in

these experiments. Some unprotected wild birds were also provided by

Sunninghill Park authorities.

The complete list of birds examined is given in Table II. 16.

Fig. 1. Cubical Trap with Three Doors.

Fig. 2. Cylin,f.rical Trap Tith Funnel. 17.

Table II. List of Birds examined for Coccidia.

Host Common Name No. of birds No.of birds examined infected

Passer domesticus House sparrow 38 38

Chloris chloris Greenfinch 28 28

Erithacus rubecula Robin 10 9

Corvus monedula Jackdaw 10 10

Turdus merula Blackbird 4 4

Sitta europaea Nuthatch 5 5

Turdus viscivorus Mistle thrush 2 2

Fringilla coelebs Chaffinch 4 4

Parus major Great tit 2 2

Corvus corone Carrion crow 1 1

Prunella modularis Hedge sparrow or 1 1 . Dunnock Sturnus vulgaris Starling 2 1

Serinus canarius Canary 6 6

Total 113 111

2. MAINTENANCE OF LIVE BIRDS

The cages were thoroughly washed on alternate days. In order to kill the occysts present, a Bunsen burner flame was passed over the inner surface of the cage. The trays of the cages were replaced daily with clean ones. 18.

Some of the birds could be kept in captivity for a week or more

but the starling, robin, blackbird and nuthatch rarely lived more than a

few days. The food which was supplied for the birds differed according

to the habits of birds. Sparrow, greenfinch and chaffinch received grain,

seed and proprietary fowl crumbs. Nuthatch and great tit received nuts

and others received insect larvae and worms.

3. RECOVERY OF 00CYSTS

Apart from the direct examination of faeces in water which was used when the infection rate was high, the following concentration

techniques were used to recover oocysts from faeces.

a. Common Salt Method

This concentration method was introduced by Faust, Sawit2,

Tobie, Odom, Peres and Lincicame (1939) and was found to be suitable for

the present purposes.

The faecal material was mixed with water in a bottle containing

glass—beads, shaken thoroughly to break up the faeces, then filtered

through a wire sieve (40 mesh to an inch) and centrifuged. The deposit

was centrifuged with a saturated solution of common salt, which having a high specific gravity allowed the oocysts to float up to the surface. The

oocysts were removed by placing a coverslip on the brim of the centrifuge

tube. 19. b. Formol—Ether Method

This technique, developed by Ridley and Hawgood (1956), was used for the faeces of birds which feed on worms and insects. The faecal material was diluted with 10 per cent. formol—saline and strained through a wire sieve into a centrifuge tube. Then it was mixed with ether in a ratio of 6 : 1 and centrifuged. The sediment was treated with a saturated solution of common salt as before and oocysts floated up.

4. 00CYST MEASUREMENTS

All the measurements in this thesis were made with an eyepiece micrometer, each division of which represented 3.32 y. Readings were made to the nearest tenth of a division, which was felt to be the limit

of discrimination. Since all measurements were made by the writer it

was considered that operator bias, if any, was constant in all cases. a. Pilot Sample

Not knowing in advance which dimensions might most usefully

separate oocysts from various sources, the following biometrical tests were

applied to a pilot sample of 100 oocysts from each of two species of bird.

Ten oocysts from ten sparrows and ten greenfinches were compared, using

length (L), width (W), shape as measured by the ratio VW and size. The

volume of an oocyst might be expected to be proportional to LV12, and this

value has been used as a measure of size, together with the simpler 20.

expression LW.

An analysis of variance has been computed using these five values

(L, W, L/W, LW and LW2) as shown in Table A.2 (pp. xiii—xiv in the Appendix),

The five means from each individual bird and from the ten birds

of each species are shown in Table A.3 (p. xv).

b. Complete Data.

The measurements of oocysts are shown in Table A.4 (pp. xvi—

xxviii). Dimensions in eyepiece units are shown in groups of four

bearing the mean value for each group (e.g. the lengths 4.9 to 5.3 are

grouped under the mean value 5.1, and the first table shows that 16

oocysts fell within that range). From the nature of the date, there

are no cases in which W exceeds L.

The main diagonal deals with oocysts having practically the same

L and W dimension, and these are taken as being spherical. The adjacent

diagonal is taken as subspherical and all the remainder as oval. Each

table shows the total number and percentage of oocysts classified under

these three headings. Marginal frequency totals for L and W are shown

in each table, and were used for computing the statistics in Table IV

(page 31).

c. Special Comparison of Sparrow and Greenfinch Data.

Because material was freely available and the birds were readily handled and because interest centred in the published work on I. lacazei

in sparrow (Boughton, 1930 and Scholtyseck, 1954), a more detailed 21. comparison of sparrow with greenfinch data was carried out. Accordingly, the length and width data shown in the margins of Table A.4 (pp. xvi—xxviii) were compared with normal curves calculated from the mean and standard deviation. The results, Table A.5 (pp. xxx—xxxi), indicated that the data were heterogeneous and the main diagonal in fable A.4 showed a high proportion of spherical oocysts. It was therefore decided to examine spherical and oval oocysts separately by the same methods omitting the

subspherical as of doubtful classification. These data are in Table A.6

(pp. xxxii—xxxiv).

5. SPORULATION OF 00CYSTS

The faeces deposited during a three—hour period were collected

and the oocysts sporulated. The sporulation times of oocysts from different individuals of the same species and from different species were

compared. The faecal material was treated with a 2.5 per cent. aqueous

solution of potassium dichromate, using an equivalent quantity of faeces

from each bird, and was incubated at 25°C.

6. ESTIMATION OF THE NUMBER OF 00CYSTS PER GRAM OF FAECES

The oocysts per gram of faeces were counted by the technique of

Stoll (1923) and Gordon and Whitlock (1939) modified by Parfitt (1958), using a McMaster slide and a saturated solution of common salt. The

number of oocysts per gram of faeces was estimated by the following 22. formula: ax 100 x 15 bx c

a = number of oocyst counted in the McMaster slide.

b = weight of faeces.

c = volume of saturated solution of common salt which

was mixed with faeces.

7. METHOD FOR STAINING 00CYSTS

Fresh oocysts were stained by the Crouch and Becker method (1931), in which the oocysts were cleaned from material by washing and centri- fugation and were treated with warm glacial acetic acid. They were then stained with a 0.1 per cent. solution of Janus Green for ten minutes, were washed and then stained with a concentrated solution of eosin for five minutes and washed again.

8. DETERMINATION OF THE INFECTED AREA OF GUT

To find the portion of intestine parasitized, the following method adopted by Bray (1958), was used.

Faecal specimens were taken from the lower part of the intestine, beginning with the rectum and progressing forwards until on examination no oocysts were found. The part of gut immediately posterior to the negative 23. site was collected and preserved in order to study the endogenous phases.

9. CYTOLOGICAL AND CYTOCHEMICAL TECHNIQUES

In order to prepare the tissue for cytological and cytochemical study, the infected parts of the intestine were cut into 3 mm. pieces and were prepared for sectioning. a. Fixation

Several fixatives were tried, and no single fixative was found to be suitable for all purposes. Formol-sublimate was found to be the most successful and was the principal fixative used in cytological study.

The fixing agents used for preservation of different substances in the parasite were Carnoy's fluid for nucleic acid, protein and glycogen, Serra's fixative modified by Brachet 1953 (alcohol, formalin and acetic acid

6 : 3 : 1) for RNA, Hellyls fluid for carbohydrate and nucleic acid, and

Bouin for acid mucopclysaccharide. After fixation, the tissues were dehydrated with alcohol, cleared and embedded in paraffin for sectioning.

In enzymic studies the intestine was fixed by the method of Gomori

(1952) in chilled acetone, dehydrated in alcohol and absolute ethanol-ether, hardened in celloidin followed by chloroform, cleared in benzene and embedded in low melting point paraffin wax (MP 49 - 52°C.).

For the detection of lipid, Baker's formaldehyde calcium fixative was used (Baker, 1944, 1949). Tissue prepared in this way was embedded in 24. gelatin for frozen sectioning. Tissues were cut transversely at 6 F for routine cytological and cytochemical examination. b. Cytological Methods

In order to study the internal phases of coccidio, the sections of tissues were stained either in Ehrlich's haematoxylin or Heidenhain's iron haematoxylin. These stains were used for studying the morphology of the different stages of the parasite and as a preliminary study before the cytochemical investigations. c. Cytochemical Methods

Cytological stains being non-specific with regard to the nature of the parasite cell components, cytochemical methods were employed.

For studying the different substances in the parasite, the following techniques were used: i. Nucleic Acid

Staining of ribonucleic acid (RNA) and deoxyribonucleic acid

(DNA) was carried out using the methyl-green pyronin Y staining method of

Kurnick (1955). In this method RNA stained bright red and DNA bluish- green. Pre-treatment of slides in ribonuclease (1 mg per ml distilled water) at 37°C. for one hour was carried out as a control selectively to remove RNA components (Brachet, 1953).

The best result followed fixation in Serra's fluid and Carnoy's fluid. The Feulgen reaction was used as a more specific method of 25. recognizing sites of deoxyribonucleic acid (Gomori, 1952; Danielli, 1953).

The Schiff's reagent employed was prepared by the method outlined by de

Tomasi (1936). Sections were treated with N-HC1 at 60°C. before staining in Schiff's reagent. Using Carnoy's fluid, Helly's fluid or formol

sublimate as fixatives, the time of hydrolysis in HC1 was 8 minutes.

For confirmation, some control sections were incubated in distilled

water instead of N-HC1 and were treated in Schiff's reagent.

ii. Polysaccharides

The periodic acid-Schiff technique (PAS) developed by McManus

(1946) was used for the location of polysaccharides. To characterize

further, tha PAS positive material, control sections were treated in

various ways.

For the determination of aldehyde grouping$,sections were treated

with Schiff's reagent as in the PlIS technique but without previous periodic acid treatment (Dogitsh, 1963).

Glycogen was detected by incubating the control slide in 1% diastase in phosphate buffer pH -Z0 at 37°C. for two hours followed by the

PAS technique. Glycogen was also tested for by the Best's Carmine method

as given in Pearse (196p).

For identification of acid mucopolysaccharides, the Alcian Blue

method of Steedman (1950) was adopted. For further investigation the

control slides were incubated in hyaluronidase (phosphate buffer pH 6.8)

at 37°C. for one hour. 26. iii. Protein

Protein was identified by the mercuric bromphenol blue (HgBPb)

method adopted by Mazia, Brewer and Alfert (1953) modified by Bonhag

(1955) and the Naphthol-Yellow-S method of Deitch (1953). Positive

sites stained blue and yellow respectively.

iv. Staining of DNA, polysaccharides and protein sections a

combined differential technique.

In this method, which was developed by Himes and Moriber (1956),

Azure A - Schiff's reagent was used to detect DNA, followed by PAS to

locate polysaccharides and finally 0.02% solution of Naphthol-Yellow-S

in 1% acetic acid for proteins.

In this method nucleic acid stained blue, polysaccharides red and proteins yellow.

v. Lipid

Frozen sections were employed for detecting the presence of

lipid. The sections were treated by Sudan Black B, in 70A alcohol

(Baker, 1944 and 1949) and were mounted in glycerine.

vi.Enzyme activity (acid and alkaline phosphatases)

Acid and alkaline phosphatase were detected in internal stages

by Gomori's method (1952) using material prepared by the method mentioned

earlier.

Activation of the enzyme was carried out by incubating the

sections in a solution containing sodium beta-glycerophosphate at 37°C.

(0.05 M-acetate buffer pH 5 for acid phosphatase and 24 sodium diethyl

barbiturate for alkaline phosphatase). The site of enzyme activity was

noted by black precipitation in both cases. 27.

STUDIES ON THE EXOGENOUS PHASES

As many coccidia have been described from the oocyst stage alone, a particular study of the exogenous stages of Isospora from passerines was made.

Studies of 1) measurements of oocysts passed in faeces from captured birds, 2) the sporulation times under similar conditions of oocysts from different birds and 3) the morphological characters of the wall and contents of the oocysts, were collected in an effort to determine which of the oocyst characters are valuable in specific determination.

1. 00CYST MEASUREMENTS

Measurements were made of oocysts collected from the faeces of newly captured birds. The number of oocysts measured varied from 25 to

120 per bird. In order to ensure that oocysts recovered from the captured birds were actually parasites and had not simply passed through the gut after ingestion with food material, the faeces were mixed with 2.5 per cent. potassium dichromate and were kept until the oocysts sporulated, the oocysts were then compared with the oocysts from faeces obtained on the day of capture. a. Pilot Sample

A pilot sample was made first on ten sparrows and ten greenfinches by obtaining ten oocysts from each bird. Five measurements, namely, 28.

length (L), width (w), shape as in the ratio L/W and size LW2 and LW were made, so as to obtain the most suitable value to be used as a standard

for measuring oocysts. The results of these biometrical measurements

are shown in Table A.2 (pp. xiii-xiv). These results as shown by the

variance ratio (F) and their associated probabilities (P) are as follows:

(i) The line (a) of each analysis shows that (apart from L/W) the four

"dimensions" are almost exactly equivalent in their capacity for discrimin-

ation. Hence, LW and LW2 would be of no greater value than L and W. A point of interest is that, owing to a thorough-going mixture of spherical

and oval oocysts there is no significant difference in the average shape

as measured by the ratio L/W.

(ii) Line (h) of each analysis indicates (for all five dimensions) that

there is considerable heterogeneity in the cocysts from individual birds

of each species.

(iii) The mean standard deviation and variability of the five values

from each species of bird ere shown in Table III (page 2g). The coeffic-

ient of variance in both species is high, indicating the existence of more than one normal distribution in sparrow and greenfinch. The heterogeneity of oocysts in sparrows seems to be greater than that in

greenfinches. The ratio L/W indicates that there is no significant

difference in the shape of oocysts from both species, even though there

is variation in length and width between the species.

The conclusion from the pilot sample is that in later measure- 2 ments there is no need to consider further the values LW and LW as

29.

Table III. Pilot sample. (200 oocysts) Measures of size and shape (see appendix Tables A.1 and A.2)

Sparrow (1) mean ± st.error (S) st.deviation V = 100 S

L (y) 23.9 + 0.322 3.22 13.5

(y) 22.2 + 0.313 3.13 14.1 L/W 1.070 ± 0.0073 0.073 6.80

LW 538 ± 14.3 143 26.5

LW2* 12400 + 490 4903 39.5

Greenfinch

(WD mean + st.error (S) st.deviation V = 100 S In L (y) 26.7 —+ 0.21 2.10 7.86 W (y) 25.2 + 0.203 2.03 8.05 L/W 1.060 + 0.0065 0.065 6.13 # LW` 677 _+ 10.09 100.9 14.9 LV2A 17200 + 389 3897 22.6

* Adjusted for change of scale to microns. 30. measures of size, though the ratio L/W may still be of use as a measure of shape. b. Complete Data

The distribution of length (L), width (w) and L/W values for 5660 oocysts measured from 13 species of birds are summarized in Table

IV (page 31). These distributions are given in the form of mean values, standard error, standard deviation and coefficient of variance (V%) for the oocysts from each host species. Variabilities (V%) in all species of birds are high indicating the presence of more than one normal distribution.

The oocysts measured from jackdaw, showed that almost all are spherical, as is indicated by the ratio LA measurements. Hedge—sparrow shows the highest L/W ratio indicating that oocysts from this bird are almost oval in shape. The ratio of other species of birds varied between those of these two suggesting the presence of both spherical and oval oocysts. Table A.4 (pp. xvi—xxviii) arranged as correlation tables relating to L and W by frequency groups, shows a high variation in the proportion of spherical and oval cysts among the host species. An inspection of marginal L and W totals indicates strongly that there is a bimodal distribution at least in some hosts. This indicates a possibility that there is a mixture of varieties.

A more detailed examination of the data in the case of sparrows and greenfinches was carried out because material was freely available and 31.

Table IV. Distribution of length and width for all oocysts measurements

HOSTS Number LENGTH WIDTH _RATIO of oocysts measured Mean (y) S (y) V% Mean (II) S (0 V% L/W n 7 ' 3-c'

Sparrow 2097 23.90+0.059 2.70 11.297 22.50+0.056 2.55 11.333 1.06 Green- 1014 25.60+0.071 2.26 8.828 24.10+0.066 2.09 8.672 1.06 finch

Chaffinch 173 23.80+0.232 3.05 12.815 22.00+0.220 2.90 13.182 1.08

Nuthatch 195 23.35+0.169 2.36 10.107 22.75+0.145 2.02 8.879 1.03

Great Tit 65 27.15+0.300 2.41 8.877 25.00+0.420 2.22 8.880 1.09

Mistle 93 20.20+0.167 1.61 7.970 19.75+3.148 1.43 7.240 1.02 Thrush

Blackbird 114 20.15+0.195 2.08 10.323 18.50+0.150 1.60 8.649 1.09

Jackdaw 1029 19.85+0.064 2.07 10.428 19.80+0.064 2.04 10.303 1.002 Starling 100 21.20+0.158 1.58 7.45 20.90+0.1578 1.578 7.55 1.014 Canary 150 21.10+0.122 1.49 7.,)62 23.80+0.117 1.44 6.923 1.014 Robin 430 19.00+3.123 2.56 13.474 17.70+0.073 1.52 8.586 1.07 Hedge- 100 21.30+0.153 1.53 7.18 17.80+0.131 1.31 7.36 1.20 Sparrow Carrion 100 23.30+0.300 3.03 12.875 22.75+0.277 2.77 12.176 1.02 Crow

Total 5660 32.

the oocysts were of similar structure in the tl.“) birds. c. Special Comparison of Sparrow and Greenfinch

The marginal totals of Table A.4 (pp. xvi-xxviii) have been used

to fit a single Normal curve to the frequency data. This yields the expected frequency (E) which can be compared with those observed (0) by means of the chi-squared test.

V 2 = S (0 - E)2 E

The point of this test in this situation is that high values of chi-squared would discredit the hypothesis that the observed data formed a single normal distribution curve.

Table A.5 (pp. xxx-xxxi) and graph I (p. 33) analyse the length and width of 2097 oocysts from sparrows and 1014 from greenfinches. All

the chi-squared values are extremely high showing that the data are markedly heterogeneous in both hosts. The proportions of spherical and oval cysts shown in Table A.4 reveal a mixture of the two types of oocysts

in both host species. It, therefore, seemed desirable to deal separately

with two shapes in Table A.6 (pp. xxxii xxxiv) and graphs II and III

(pp. 34-35). The diameter (L) was measured in the case of spherical oocysts and length (L) and width (1) were measured in the case of oval oocysts.

The chi-squared values of Table A.6 are all very high as before.

33

GRAPH I

Dimensions of oocysts in sparrows and greenfinches compared with normal distributions

Sparrow a-length b- width 600- 600 —

500 500 -

400 400- . le o 300- 0'iv 300 L.

200 200-

100• 100-

- -I 0 I 2 S standard deviation 2b 2'5 30p 20 25 30p Greenfinch

400 c-length 400 d-width

300 300

200

10 100

-3 -2 -I 0 1 2 3 S -3 -2- -I 0 I 2 3 S standard deviation 20 25 30 p 20 25 30 p 3

GRAPH II

Dimensions of spherical rattjajparrows and greefinches compared with normal distributions

Sparrow

35-

30-

25

10

5-

0 -I 0 1 S standard deviation io 25 30 p

Greenfinch

35

30

25

0 -I 0 $ standard deviation

20 25 30 11 35

GRAPH 11 I pimensions of oval oocysts in sparrows and greenfinches compared with normal distributions Sparrow a -length 35 b-width

30

25

20

15

10

-2 -I 0 I 2 S standard deviation 20 25 30 F. Greenfinch c-length d - width 45

40

35 35

30 30

2 25

20

15 15-

10 All 10 -

5- 5 0 C C -2 -I 0 I -3 -2 -I 0 I 2 3 S standard deviation 20 25 30 20 25 30 36.

It might have been anticipated that the spherical oocyst data, or the oval oocyst data, or both, might have an approximately Normal distribution and that the high chi-squared values of Table A.5 were due to a bimodal mixture of the two. This however is clearly not the case: both spherical and oval oocysts, from both sparrow and greenfinch, are markedly heterogeneous.

It should be noted that in graph I actual frequencies are shown whereas in graphs II and III the frequencies are relative, giving curves of equal area so as to facilitate comparison.

2. SPORULATION OF 00CYSTS

The sporulation time of oocysts is one of the criteria normally used in the identification of species of coccidia. This could be useful if standard conditions were applied to enable a comparison of time to be made. Unfortunately this has not often been the case.

No cases were observed in which sporulated oocysts were passed in the faeces of a bird. In the freshly passed oocyst, the zygote almost fills the cavity within the cyst wall (Plate I, Nos. 1 & 5), but within a few hours the mass of protoplasm contracts away from the wall leaving a clear space. The zygote, then, divides into two rounded bodies, the sporoblasts

(Plate I, Nos. 3 & 7). Each spherical sporoblast elongates slightly and secretes around itself a refractile envelope forming the sporocyst. The protoplasm inside the sporocyst divides and produces four sporozoites (Plate

I, Nos. 4 & 8). The oocyst is regarded as having completed sporulation when Explanation of Plate I

Sporulation of I. lacazei (Nos. 1-4) and I. chloridis (Nos. 5-8) at

25°C., fresh preparation. (Camera lucida drawing).

1. Freshly passed oocyst.

2. Oocyst after 16-24 hours.

3. Oocyst after 36-48 hours.

4. Sporulated oocyst after 60-72 hours.

5. Freshly passed oocyst.

6. Oocyst after 16-24 hours.

7. Oocyst after 36-46 hours.

8. Sporulated oocyst after 60-72 hours. ro N

Tr

co

'0

O Ul I 38. the sporozoites are visible as separate bodies within the sporocysts.

When a pure line coccidial infection is available the most accurate estimate of sporulation time is that at which 50 per cent. of oocysts are sporulated. Sporulation time is sometimes taken as minimum time for oocyst development, i.e. when a single oocyst is seen sporulated and sometimes when 50 per cent. of oocysts are sporulated.

In mixed infections the minimum time (one oocyst sporulated) cannot be used. Occasionally a sample is obtained when less than 50 per cent. of oocysts complete sporulation and such samples complicate the sporulation time for 50 per cent. of the oocysts as a standard. Here the sporulation time was taken as that when the majority of oocysts were sporulated. The sporulation times of oocysts from seven host species were compared and uniformity was applied to the following data: amount of faeces, quantity of 2.5 per cent. potassium dichromate used to cover the faeces, size of petri dish used for sporulation and temperature.

The sporulation time of oocysts from the seven host species are given in Table V, the times given in columns 1-7 are for samples taken from different birds. Only one sample was sporulated from hedge-sparrow but a sample was taken from each of seven jackdaws.

Similar results were obtained from sparrow, greenfinch and chaff- inch where the sporulation time varied between 60 and 72 hours. The time for oocysts from nuthatch was rather longer at 72-84 hours, rather shorter from canary and jackdaw at 48 - 60 hours and very short from the 39.

Table V. Sporulations of Isosp3ra spp. from seven different host species at 25°C. Sporulation Times in Hours Host Weight of Quantity of species faeces (gm) Potassium Number of Observations Dichromate 1 2 3 4 5 6 7

Sparrow 0.5 15 60 60 72 60 72 72 -

Greenfinch If II 72 60 60 72 72 60 -

Chaffinch u it 72 60 72 - - - -

Nuthatch It II 84 72 - - - - -

Jackdaw ft II 48 48 60 46 60 48 48

Canary II ft 60 60 48 60 - - _

Hedge-sparrow it II 24 - - - - -

hedge sparrow at 24 hours.

These results are later related to differences in oocyst structure,

3. MORPHOLOGY OF 00CYSTS

The sporulated oocysts from all the twelve species of birds examined revealed that the birds harboured but a single coccidian genus, namely Isospora. Whereas greenfinch, sparrow and chaffinch harboured apparently identical oocysts of two types, structural differences could be detected between these and oocysts from other host species. Plate II,

Nos. 1-13 illustrates the main features of the sporulated oocysts from the twelve host species examined. 40. a. General Observations

A micropyle was not observed in any of the oocysts. Vith one exception the oocyst walls were double-contoured, colourless and smooth.

In one robin an additional type (Plate II, No. 12; Plate III, No.7) with rough brownish-coloured, double-contoured walls was present as well as oocysts with smooth walls. In no case was an nocystic residual body present but in all the oocysts one or more polar granules were present,

some ovoid (e.g. Plate II, Nos. 5 and 8), some splinter-like (e.g. Plate II,

Nos. 1 and 10).

The sporocysts were pear-shaped with an apical cap behind the Stiedabody except in oocysts from mistle thrush and the rough-walled type

from robin (Plate II, Nos. 6 and 12). The sporocyst contents consisted of

sporozoites and a residuum. In most cases the contents were enclosed within a membrane but this was absent in the oocysts from jackdaw and hedge

sparrow (Plate II, Nos. 8 and 13). The sporocystic residuum was of three

types: in cysts from sparrow, greenfinch and chaffinch there were fine granules scattered about the sporozoites as well as a compact mass (Plate

II, Nos. 1-3); in those of nuthatch and the rough-walled type from robin only the fine granules were present (Plate II, Nos. 4 and 12); in all other

cases only the compact mass was present (Plate II, Nos. 5-11 and 13).

The sporozoites of all oocyst types were elongate, pointed at

the anterior end and contained a central nucleus and refractile globules at both ends. In the case of the rough—walled oocyst from robin, Explanation of Plate II.

Sporulated oocysts from different species of bird; fresh preparation.

(Camera lucida drawing)

1. Spherical oocyst from sparrow and greenfinch.

2. Oval oocyst from sparrow, greenfinch and chaffinch.

3. Oocyst from chaffinch (spherical).

4. " nuthatch.

5. It It great tit.

6. mistie thrush.

7. It It blackbird.

B. It II jackdaw.

9. It II starling.

10. canary.

11. It robin.

12. 11 11

13. " hedge-sparrow.

4.1

1 2 3

4 5 6

7 8 9 to

11 12 13

PLATE II Explanation of Plate III

Microphotographs of oocysts from different species of birds; fresh

preparation. (Nos. 1-6 x 360 and No. 7 x 760).

1. An oocyst from sparrow releasing sporocyst after application of

pressure.

2. Fully sporulated oocyst from sparrow.

3. canary.

4. nuthatch.

5 and 6. Oocysts inside the host cell, found in the faeces of robin.

7. Sporulated oocyst from robin. High magnification showing rough oocyst

wall.

42.

1

M %pit

•

4 5 6

7

PLATE III 4-3

Table VI. Characters of Sporulated Isosporan Oocysts from Different Species of Passerine Birds.

Host Oocyst Wall Polar.Granules Sporocyst SporOzoite Sporulation . Time Shape Sporocystic Sporocystic Measurement • Residue Membrane Mean (u) ' Range Size (u)

Nuthatch Double contoured, One to three Pear-shaped Present Present 17.3x10.6 15.0-19.0x Elongated with 84 hrs. snoothI colourless splinter-shaped (fine granules) 9.2-13.2 one pointed end 1.32 u thick present - 8.5x3.6 u Great Tit Double contoured, Two to more H 11 Present n 18.5x10.5 17.5-21.0x Elongated with _ snoothIcolourless ovoid present (compact mass) 9;5-12.0 one pointed end 1.2 u thick 10.0x3.0 u Mistle thrush Double contoured, One to three n 11 Present 11 16.2x9.0 14.8-17.8x Elongated with _ snooth,colourless ovoid present (compact mass) 8.0-10.5 - one pointed end ' 1.0 u thick 9.0x3.0 u

It It 11 Blackbird Double contoured, One to two Present 12.5x8.0 11.5-16.0x Elongated with _ snooth,colourless elongate present (compact mass) 7.0-10.5 one pointed end 1.0 u thick 8.3x2.5 u

Jackdaw Double contoured, One to three 11 H Present Absent 14.0x8.0 12.5-16.0x Elongated with 48 hrs. snooth,colourless ovoid present (compact mass) 6.8-9.0 • one pointed end 0.7 u 8.0x3.2 u

Starling Double contoured, One to two n n Present Present 14.0x8.5 12.2-16.2x Elongated with _ snooth,colourless elongate present (compact mass) 7.3-9.4 one pointed end 1.0 u 8.0x3.0 u

Canary Double contoured, One to three tt H Present - n 15.2x9.0 13.7-17.5x Elongated with 60 hrs. snoothIcolourless splinter-shaped (compact mass) 8.5-11.0 one pointed end 1.2 u present 8.0x3.0 u smooth Double contoured, Two or more of 11 Present H 12.5x8.0 10.4-14.2x Elongated with - wall snooth,colourless elongate present (compact mass) 6.7-9.5 one pointed end 0.9 u •. 8.0x2.7 u Robin El 11 . rough Double. contoured, Two or more n Present 12.5x8.2 11.0-16.0x Elongated - wall brown colour,out- splinter-shaped (fine granules) 7.8-9.5 7.0x2.0 u line rough. 1.3 u present

Hedge-sparrow Double contoured, One to two n n Present Absent 14.0x9.0 12.5-16.5x Elongated with 24 hrs. smooth,colourless ovoid present (compact mass) 8.0-11.0 one pointed end 1.3 u 8.0x2.0 u 44. the refractile globules were nyt observed due to the lack of material.

The oocysts from sparrow and greenfinch have been studied biometrically and by measurement alone can be characterized as oval and spherical. Oocysts from chaffinch also falling into oval and spherical types are apparently identical morphologically with those from greenfinch and sparrow. These oocysts ar,-: described in detail and the characters of the remaining oocyst types are summarized in Table VI (p. a3). b. The Characters of the Sporulatod Oocysts from Sparrow, Greenfinch and

Chaffinch

Spherical and oval oocysts can be distinguished in the three hosts (e.g. Fig. 3) and are described separately. Measurements of these oocysts are given in Table VII. The oocysts from greenfinch have a greater mean size than oocysts from sparrow and chaffinch. The oocyst walls of the oval oocysts are slightly thinner than those of the spherical cysts. i. Spherical Oocyst. (Plate I Not,. 1-4, Plate II Nos. 1 and 3, Plate III No. 2).

The oocyst wall i.s double contoured, smooth, colourless and in

varies from 1.0 p (greenfinch and chaffinch) to 1.04 y (sparrow).

A micropyle and oocystic residual body are absent but there are one to three splinter—like polar granules present. The two pyriform sporocysts each have a Stieda body present with an apical cap at the pointa end. 45.

Fig. 3. Oocysts from Greenfinch. x 360. 46.

Table VII. Measurements of sporulated oocysts and their contents from

sparrow, greenfinch and chaffinch.

Host and the Oocyst Thickness Sporocyst Size of of oocyst shape of oocyst Mean (y) Range size wall (p) Mean (y) Range size sporozoite (J) (v)

spherical 22.9 16.6-30.0 1.04 16.6x10.6 15-19x 9.4x3.0 cyst 9.5-12.0 sparrow ii oval cyst 25.1x22.0 17.25-33.2 0.9 15.0x9.4 14.5-17.2 17.5x2.5 x16.6-30.0 x8.5-11.0

(spherical 24.7 20.0-30.0 1.0 16.8x10.8 I5-19.25x 9.6x3.2 green- cyst 9.2-12.5 finch oval cyst 27.0x23.4 22.6-33.2x 0.86 16.0x9.6 14.5-18.2 8.0x3.0 16.6-30.0 x8.7-12.2

(spherical 22.7 17.3-30.0 1.0 16.2x10.0 14.0-18.5 9.0x3.0 Chaf- cyst x8.5-11.0 finch oval cyst 24.0x21.7 17.3-31.5x 0.9 14.75x9.2 13.5-18.5 7.5x2.5 16.6-23.3 x8.3-11.5

The sporocystic residuum is present both as a compact mass and as fine scattered granules. The sporozoit&s, which are not arranged in any particular order within the sporocyst, are elongate, pointed at the anterior end and blunt at the posterior with refractile globules present at both ends. The sporocyst-

ic residuum and sporozoites are enclosed in a membrane within the sporocyst.

The process of sporulation is illustrated in Plate I Nos. 1-4. At

25°C. the contraction of the oocyst contents begins a few hours after passing 47.

from the host to the exterior. The division of the zygote (Plate I No.2) begins at 16-24 hours and rounded sporoblasts are formed by 36-48 hours (Plate I No.3). The development o± the sporoblasts into sporocysts with sporozoites is completed within 62-72 hours.

ii. Oval Oocyst. (Plate I Nos, 5-8, Plate II No.2). The oocyst wail is somewhat thinner than that of the spherical cyst, measuring 0.86 p. (greenfinch) and 0.9 )1 (sparrow and chaffinch). The polar granules here are ovoid instead of splinter-like and two or more

are present. From all three hosts the sporocysts are mailer than those from the spherical cysts even when the oocyst containing them are larger. The remaining morphological characters are identical with those of the spherical cysts. Sporulation takes place within the same time, 62-72

hours, and the process is identical.

c. Comparison of the Morphological Characters of Oocysts from Other Host Species with Spherical and Oval Types from Sparrow. Greenfinch

and Chaffinch

Oocysts recovered from all but jackdaw and hedge sparrow ranged from spherical to oval in shape. The splinter-like polar granules are taken to characterize the spherical type and the ovoid polar granules to characterize the oval type from sparrow, greenfinch and chaffinch. The oocysts from nuthatch are without the compact mass of

sporocyst residuum but otherwise resemble the spherical type. The sporulation time is longer at 84 hours. Those from great tit are without the scattered granules of the sporocyst residuum but otherwise resemble the oval type. Those from mistle thrush are without apical cap of the Stieda body and the scattered granules of the sporocyst residuum but otherwise resemble the oval type. Those from blackbird, starling and smooth walled type from robin have elongate polar granules and lack the scattered granules of sporocyst residuum. Those from jackdaw lack the scattered granules of the sporocyst residuum and membrane enclosing the sporocyst contents. They were almost all spherical but have ovoid polar granules. The sporulation time is

48 hours. Those from canary lack the scattered granules of the sporocyst residuum but otherwise resemble the spherical type. The sporulation time is 60 hours. The rough walled type from robin have a double contoured brownish rough wall. The polar granules are splinter-like, the compact mass of the sporocyst residuum is absent as well as the apical cap of sporocyst

Stieda body. The oocysts from hedge sparrow are almost all oval in shape.

The polar granules are oval. The scattered granules of the sporocyst residuum are absent as is the membrane round the sporocyst contents. The sporulation time is only 24 hours. 49.

4, DETERMINATION OF THE PREPATENT PERIOD OF ISOSPORA SP?. AND A NOTE ON CROSS INFECTION

All birds except one robin and one starling were infected with Isospora when captured. The starling died the day after capture and could not be used for further experiment. The robin remained alive for 20 days and was the only bird free of infection available for infection experiments.

a. Infection of Robin

No oocysts were passed by this robin as determined by daily examination of faeces for ten days. The bird was then given a heavy dose of sporulated oocysts previously collected from another robin. These oocysts were a mixture of the smooth-walled type and the rough-walled type (Plate II Nos. 11 & 12, Plate III Nos. 5, 6, 7). The faeces were examined daily and the first oocysts were recovered four days after infection. All were of the smooth-walled type. The bird died on the 20th day after capture without passing rough-walled oocysts. This latter

infection was assumed not to have taken and the prepatent period of the species with smooth-walled oocysts was estimated at 96 hours.

b. Super-Infection of Greenfinch and Chaffinch with Oocysts

As further uninfected birds were unobtainable, attempts to assess the prepatent period of Isosoora species were made by imposing a massive dose of oocysts over an existing light infection and noting when a

considerable rise in oocyst output took place. 50.

A. Two greenfinches received 2 x 105 oocysts collected from another green—

finch and sporulated.

B. One greenfinch and one chaffinch received 2.5 x 105 oocysts collected

from sparrow.

In both experiments the infective oocysts were a mixture of spherical and oval types (Plate II Nis. 1 & 2).

The output of oocysts was recorded for five days before and daily after super—infection. The results are recorded in Graph IV

(p. 51). The greenfinch which received oocysts from sparrow became clean two days after super—infection and remained clean for five days.

It then received a further dose of oocysts from sparrow and passed oocysts after five days. In the other three birds there was a noticeable rise in oocyst output five days after super—infection.

The prepatent periods of the species of Isospora with oval and spherical oocysts are five days and cross infection between hosts is possible.

This result supports the evidence from biometric and morphological studies of the exogenous stages that the same two species of Isospora infect sparrow, greenfinch and chaffinch.

5. THE DIURNAL OUTPUT OF 00CYSTS IN GREENFINCH

The faeces of three greenfinches were examined to determine the pattern of oocyst output over a 24 hour period. The experiment conducted at the end of May involved the hourly collection of faeces and examination Explanation of Graph IV

Daily output of oocysts from greenfinch and chaffinch super- infected with oocysts from greenfinch and sparrow.

B.1. Solid line = output of oocysts from greenfinch super-infected with sporulated oocysts collected from sparrow. B.2. Dotted line = output of oocysts from greenfinch super-infected with sporulated oocysts collected from greenfinch. B.3. Broken line with dots = output of oocysts from chaffinch super- infected with sporulated oocysts collected from sparrow. B.4. Broken line . output of oocysts from greenfinch super-infected with sporulated oocysts collected from greenfinch.

All birds super-infected on day 5, B.1, received a second dose on day 13.

Rise of oocyst output showed by black spot on day 10 (B.2, B.3, and B.4) and day 18 indicates a prepatent period of 5 days in each case. Number of oocysts ( logarithmic scale)

c) 70 > I 52. for oocyst numbers over a 24 hour period. Graph V (P. 53) shows the results of the experiment with one greenfinch. The number of oocysts passed (solid line) is shown in comparison with the weight of faeces passed

(dotted line).

Quite clearly there is a peak period of production at 18.00 hours 7 when 10 oocysts were passed, whereas between 6.00 and 13.00 hours very few oocysts were passed, varying between 0 and 103 oocysts. The number of oocysts passed per hour bore no relation to the amount of faeces passed.

Similar results of peak and off peak periods were obtained for all three greenfinches, though the actual number of oocysts passed varied from bird to bird.

Number of oocysts logarithmic scale I-- ,o tr) 0 nt L_ 0 0 _ 0.... 0 'o 0.. .c ci L. 6.:3 O 0,------0— v.4, 4a. ...c,,, 0 ,), . 0...... ______► gzoto w• g 2• in 0---- 0 r ...... ------w 5 go ------0 0 ------Or 'I' 0.::. : 0 tri E .6 -o------..... _ ...._0. ... I o P. .E. in IT .17b a. «0 L. < to" D L.. 170 tr,, CC >. N0...... 0,...... , 0 OP Lc \ (.D 8 \ i a.3 o -o" 411 .. ... ••• .... to 0 In •• •" 1 Iner ....- '' 4.'. Os 1-- ...:...... ___ -43:_ ._ - .s LI- 0n) ------_ %." ------t --- to I I to_ 9 o co::. I I 1 _ ------* in Let 0 cv 0 ( D ) saDaot 246!am 54.

STUDIES ON THE ENDOGENOUS PHASES

The internal stages of Isospora spp. were studied in greenfinches.

These birds were caught in quite large numbers and lent themselves to

further study as they could be kept in captivity for periods up to two months, and could be subjected to a certain amount of handling and

experimental procedure without dying. A comparison was made of these

stages with those found in sparrows.

1. LOCALIZATION OF ENDOGENOUS PHASES

The parasites attack only the area of small intestine immediately posterior to the gizzard. All the asexual stages were confined to the first

3 cm of the duodenum and even the sexual stages were very rarely found beyond this limited area. The first asexual stages are localized in a smaller area of the intestine near the gizzard. They attack the epithelial cells of the villi.

2. ATTEMPTS TO CLEAN BIRDS OF NATURALLY ACQUIRED ISOSPORA INFECTIONS

All of the greenfinches examined were found to be infected when captured. To rid these birds of the naturally acquired infection, they were (a) prevented from ingesting further sporulated oocysts, and (b) were given sulphamezathine to remove the existing internal stages and prevented from ingesting sporulated oocysts.

A. In the method recommended by Wasielewski (1904) to prevent birds 55.

from ingesting further sporulated oocysts, the birds are transferred to

clean cages every 24 hours. The efficacy of this method was tested on

two greenfinches. The faeces passed in 24 hours were collected and the number of oocysts per gram calculated. One bird died after 28 days and

at the end passed nearly the same number of oocysts per day as at the

beginning of the experiment. The second greenfinch remained alive for

34.days and the daily output of oocysts per gram of faeces is shown in

Graph VI (p. 56). Again nearly the same number of oocysts was passed

throughout the experimental period.

In a similar experiment with two other greenfinches, the cages were changed every 12 hours instead of every 24 hours and the output of oocysts was calculated for the 12 hour periods. One bird died after 11 days the other after 22 days and oocysts were being passed at approximately

the same level at the end as at the beginning of the experiments. A continuous asexual reproduction could account for the persistence of the infection when sporulated oocysts were withheld.

B. Sulphamezathine is a well known chemotherapeutic agent used in preventing coccidiosis in poultry. The dose recommended for poultry

(1 ml sulphamezathine in 75 ml water) was fatal to canaries so a half strength dose was adopted.

Two canaries and three greenfinches each received 1 ml sulphamez— athine in 150 ml water for two successive days every five days. The birds were isolated and the cages were changed every 24 hours.

The number of oocysts per gram of faeces was estimated at 3200 56

GRAPH VI

Daily output of Isospora oocysts per gramme of faeces of Greenfinches ) le sca ic hm it r a 0

(log 0 0-0 ts s oocy f o ber m Nu

12 16 20 24 28 32 34 Days 57.

per gram (both canaries) 20,000 per gram, 12,000 per gram and 16,000 per gram (greenfinches). The canaries survived the drug treatment but after 30 days showed no significant drop in oocyst output. The greenfinches showed a drop in oocyst output to the 1,000 oocysts per gram level but died after 12, 14 and 13 days respectively. Further experiment with the drug should prove useful.

3. DOUBLE INFECTION

As clean birds were not available for infection to study the early schizogonic phases, the first generation schizonts were distinguished from 5 the later schizonts by super-imposing a massive infection of 2 x 10 oocysts on a natural infection. These oocysts were mixed oval and spherical types from greenfinches. The birds were sacrificed at intervals during the prepatent period previously estimated at 5 days and the infection examined in sections. Two types of schizont were found which were absent or very rare in the naturally infected birds. They were taken to be the first generation schizonts of two species of Isospora. In naturally acquired infections of all but two greenfinches, a single type of schizont, microgametocyte and macrogametocyte were present. These stages were identified as Isospora lacazei, and because they were present in abundance in birds passing mainly spherical oocysts these oocysts were taken to be the exogenous phases of this species and were linked with one of the first generation types. In the remaining two greenfinches in addition to I. lacazei, asexual and sexual stages of a quite different type 58. were present. These and the other type of first generation schizont were taken to be stages of a separate species of Isospora for which the name Isospora chloridic n. sp. is proposed. These birds were passing abundant oval oocysts which were taken to be the corresponding exogenous phases. Both species are described below.

4. INTERNAL STAGES OF ISOSPORA LACAZEI LABBE 1893

The stages of I. lacazei within the epithelial cells of the villi are illustrated in Plate IV Nos. 1-18. a. Sporozoites The sporozoites were observed after excystation from oocysts. They were examined fresh and after fixation and staining. They are elongate, pointed anteriorly and blunt posteriorly, with refractile granules at both ends. The nucleus is situated centrally (Plate IV, No. 1). No sporozoites were found in the elongate stage within host cells and after rounding off they cannot be distinguished from the uninucleate stages of schizogony and gametogony. b. First Generation SchizoRonv The youngest stages observed were small rounded schizonts with pale cytoplasm and six nuclei found 48 hours after superinfection (Plate IV, No.2). The nuclei of the schizonts consisted of a deeply staining karyosome surrounded by a clear halo within the nuclear membrane. This schizont undergoes further nuclear division, followed by separation of the Explanation of Plate IV

Camera lucida drawings showing the endogenous phases of I. lacazei (Labbe, 1893). (No.1, drawn from a fresh preparation, Nos. 2-18 drawn from a section of small intestine stained with Heidenhain's Haematoxylin).

1. Sporozoites from the ruptured oocysts. 2. First generation schizont in the host cell. 3. Developing first generation schizont, separating merozoites. 4. Mature first generation schizont containing 10 merozoites. 5. Merozoite rounded off within the host cell. 6-9. Developmental stages of the late generation schizont. 10.Nearly mature late generation schizont, separating merozoites. 11.Mature late generation schizont containing 16 merozoites. 12-13. Development of microgametocyte. 14. Mature microgametocyte containing microgametes. 15-16. Development of macrogametocyte. 17.Mature macrogametocyte. 18.Oocyst within the host cell. 59

2 4 5 3 6

7 8 9 I0 12

18 13 14 Is 16 17

0,N PLATE IV 60. cytoplasm around the nuclei (Plate IV, No.3) Fig. 4a. The mature first generation schizont measuring 7.5 x 7.0y contains 10 - 12 sausage-shaped merozoites (Plate IV, No.4) and Fig. 4, a and b. This stage can be seen up to 72 hours after superinfection, but is scarce after this. They were rarely identified in naturally infected birds. These stages invariably develop in a position superior to the host cell nuclei, lying between the nuclei and brush border of the epithelial cell. c. Late Generation Schizogonv The youngest stages observed were small rounded uninucleate bodies, lying in a position inferior to the host cell nucleus (Plate IV, Nos. 5,6). Growth and nuclear division produces the late generation schizonts (Plate

IV, Nos. 7, 8), The nuclei are similar to those of the first generation schizonts. About 16 nuclei are produced before separation of the merozoites

(Plate IV, Nos. 9, 10) and Fig. 5. The mature late generation schizonts measured 16.0 x 10.0 )4.

These stages contain 14 - 16 crescent-shaped merozoites. The merozoites measure 5.2 x 1.6 ,y,t, each with a centrally placed nucleus. This type of schizont was found in natural infections and was particularly abundant in

the birds in which the majority of oocysts were spherical. 61.

Fig. 4. Section of small intestine of greenfinch showing: a and b. First generation schizonts of I, lacazei; c. First generation schizont of I, chloridis. d. Residual body of first generation schizont of I. chloridis.

(Heidenhain's Haematoxylin) x WO. 62.

Fig. 5. Sections of small intestine of greenfinch showing the late generation schizonts of I. lacazei:

a. Separated merozoites within schizont. b. Schizont showing 15 merozoites.

(Heidenhain's Haematoxylin) x 760. 63.

Like the late generations of schizonts the sexual stages develop behind the nuclei of the host cells. The merozoites round off after penetrating the host cell and at this stage the developing gametocytes are similar to the young schizonts. They develop into macrogametocytes with a single nucleus, and microgametocytes with many nuclei which are considerably smaller than those of the schizonts. d. Microgametocvte The microgametocyte undergoes repeated nuclear division without cytoplasmic separation (Plate IV, Nos. 12-14). The nuclei of the mature microgametocyte are considerably smaller than those of the schizonts. They finally separate at the surface as spindle-shaped microgametes leaving a scattered residual cytoplasmic body (Fig. 6a), The mature microgametocyte measures 15.0 x 12.5)1. e, Macrogametocvte The macrogametocyte remains uninucleate and enlarges in the host cell (Plate IV, Nos. 15-17), until it reaches its mature size of 16,0 x 13.0 )1. The nucleus contains a large karyosome surrounded by a clear area. The cytoplasm which is strongly basophilic shows no sign of the large

"plastic granules" so characteristic of the macrogametocytes of most coccidia (Fig. 7).

5. INTERNAL STAGES OF ISOSPORA CHLORIDIS N. SP.

The developmental stages of I. chloridis also takes place within epithelial cells of the viii. The complete cycle is shown in Plate V, 64.

Fig. 6. Section of small intestine of greenfinch showing: a. Microgametocyte of I. lacazei; b. Microg=etocyte of I. chloridis.

(Heidenhain's Haematoxylin) x 760. 65.

Fig. 7. Section of small intestine of greenfinch showing the macrogametocyte of I. lacazei:

(Heidenhain's Haematoxylin) x 800. 66.

Nos. 1-17. Schizogony and gametogony of this species take place superior to host cell nuclei behind the brush border of the epithelial cell. a. Sporozoite ex The sporozoites were found after encystation from the oocyst under pressure and care was taken to observe the actual sporozoites emerging from an oval as opposed to a spherical ooeysti the free sporozoites and sporo- zoites within the oocyst were compared after staining of the oocyst by the Janus green method of Crouch and Becker (1931). The sporozoitest,ferc not obviously different from that of S. lacazei except that those of I. chloridis were smaller than those of I. lacazei (Plate V, No.1). The stage immediately after penetration of the host cell by the sporozoite was not observed and in any case would be difficult to distinguish from later uninucleate stages. b, First Generation Schizogony The youngest schizonts observed were small, round and with 5 - 6 nuclei. The mature form has a maximum of 8 nuclei. Cytoplasmic division begins on one side of the body (Plate V, No.2) separating the merozoites like bananas in a bunch leaving a mass of residual cytoplasm on one side (Plate V, No.4). The merozoites in cross-section lying free of the residual cytoplasm are seen in Plate V, No. 3 and Fig. 4c. The mature schizont measures 8.5 x 7.5 ).1 and schizogony is campleted within 72 to 84 hours. c. Late Generation Schizogony After rupturing the host cell, the merozoites of the first gener- ation attack new host cells to continue the asexual cycle. The merozoites Explanation of Plate V

Camera lucida drawings showing the endogenous phases of I, chioridis n.sp. (No. 1, drawn from a fresh preparation, Nos. 2-17, drawn from the section of small intestine stained with Heidenhain's Haematoxylin).

1. Sporozoites from the oocyst. 2-3. First generation schizont in the host cell.

4. Mature first generation schizont, separating 6 merozoites. 5. Merozoites rounded off within the host cell. 6-7. Developmental stages of the late generation schizont. 8. Mature late generation schizont containing 24 merozoites. 9-12. Development of microgametocyte. 13. Mature microgametocyte containing microgametes. 14-15. Development of macrogametocyte. 16.Mature macrogametocyte showing "plastic granules". 17.Oocyst within host cell.

67

ftb

4 2 3 5 6

9 7 8 I0 12

14 17 13 15 16

PLATE V 68. round off (Plate V, No.5) and undergo nuclear division the nuclei taking up a peripheral position (Plate V, Nos. 6, 7). The final nuclear division produces 20 - 24 nuclei in a body measuring 17.0 x 14.0 j. Cytoplasmic division separates the merozoites from the surfaceleaving a residual body centrally, the whole body being rosette like (Plate V, No.8). The merozoites are broad with a pointed end (Fig. 8) and measure 4.0 x 1.7r.

The sexual stages of this species are also found in the position superior to the host cell nucleus. The early developmental stages of the microgametocytes and macrogametocytes are rounded uninucleate bodies indistinguishable from the uninucleate bodies of the asexual cycles. d. Microgametocyte The microgametocytes undergo nuclear division which is not directly followed by the cytoplasmic division, characteristic of schizogony. The nuclei are eventually separated at the surface as comma-shaped micro- gametes (Plate V, No. 13, Fig. 6b) quite distinct from the spindle shape gametes of I. lacazei. Further the residual cytoplasm is left as a compact single mass instead of the diffuse residuum of I. lacazei. The microgame- tocyte measures 15.0 x 12,0 }.1. e. Macrogametocyte.

The macrogametocyte remains uninucleate during growth. At a very early stage the plastic granules, characteristic of most coccidia 69.

•

.

•

1

• I

Fig; 8. Section of small intestine of greenfinch showing the late generation schizont of I. chloridis, with residual body.

(Heidenhain's Haematoxylin) x 800. 70.

Fig. 9, Section of small intestine of greenfinch showing the macrogame-

tocyte of I. chloridis.

(Heidenhain's Haematoxylin) x 800. 71. macrogametes are laid down. They appear peripherally in the cytoplasm and increase in size and number during the development of the macrogametocyte (Plate V, Nos. 14-16). The mature macrogamete, measuring 17.0 x 13.0 u has pale cytoplasm. The nuclear membrane is indistinct and it is difficult to distinguish the extra karyosomal area of the nucleus from the cytoplasm (Fig. 9). The lack of plastic granules and distinct nucleus of I. lacazei are in complete contrast. 72.

CYTOCHEMICAL STUDIES ON ISOSPORA SIT, IN GREENFINCH

The positive results of most cytochemical tests for specific cell constituents are more clearly seen in the absence of counterstaining, which would render the cell outline more visible. Thus to aid the identification of parasitic stages in sections tested cytochemically, alternate strips of paraffin ribbons containing the sections were mounted on separate slides and stained with conventional cytological stains especially haematoxylin. The sections were compared. Unless specifically stated to the contrary the results of the fee cytochemical tests apply to I. lacazei which was studied for, presence of deoxyribonucleic acid, ribonucleic acid, polysaccharides, protein, lipid and enzyme activity. Material of I. chloridis was not available for a complete study and only the testsfor DNA, RNA and polysaccharide were undertaken.

1. DEOXYRIBONUCLEIC ACID (DNA)

Schiff's reagent following hydrochloric acid hydrolysis gave consistently good results in locating DNA which showed up as a characteristic magenta colour (Fig. 10). Control slides were incubated in distilled water at 60°C. for a time equal to the acid hydrolysis of the test slides. In this case the aldehyde groups were not produced and the magenta colour did not appear. DNA was shown to be present in the nuclei of all stages except the macrogametocytes. In all asexual stages DNA was restricted to an intranuclear ring 73.

b—

Fig. 10. Section of small intestine of greenfinch after Feulgen reaction

showing the site of DNA in: a. Microgametocyte of I. lacazei;

b. Microgemetocyte of I. chloridis.

x 800. 74. and was found thus in the first generation and late generation merozoites of both species and in all uninucleate stages (Plate VI, Nos. 1, 2, 4) 14., 15). The nuclei of the developing microgametocytes (Plate VI, Nos. 9, 10, 11) were similar in their DNA content to those of the schizonts and these stages could be distinguished only by the number not the structure of their nuclei. However mature microgametocytes showed an increased DNA content, each entire nucleus being Foulgen positive. The two species were easily distinguished at this stage, I. lacazei nuclei being spindle-shaped (Plate

VI, No. 13 and Fig. 10a) and I. chloridis nuclei comma-shaped (Plate VI, No. 17 and Fig. 10b). Further practically the entire microgametes appeared to be composed of DNA as little or no cytoplasm would be detected round the individual nuclei after separation from the residual body. No DNA was detected in the nucleus of the macrogametocyte. That the nucleus was Feulgen negative does not imply that DNA is absent but indicates rather that the Feulgen reaction is not sufficiently sensitive to detect the very small amount present. Sites of DNA are also located by methyl green in the methyl green pyronin. Y test, If RNA is removed by pre-treatment with ribonuclease the green staining DNA is rendered clear. This test for DNA was used only in support of the findings by the Feulgen reaction. The sites of DNA indicated were identical with those shown by the Feulgen test and since it is less sensitive it also failed to show DNA in the macrogamete. Explanation of Plate VI

Cytochemical studies on the internal stages of Isospora spp. in greenfinch. (Camera lucida drawings) (Figs. 1-13, I. lacazei; Figs. 14-17, I. chioridis).

1. First generation schizont after Feulgen reaction, showing DNA in nuclei. 2. Late generation merozoite after Feulgen reaction. 3, Late generation morozoite after Methyl green Pyronin Y, showing baso- philia caused by RNA. 4. Rounded uninucleate body in host cell after Feulgen reaction. 5. Rounded uninucleate body in host cell after Methyl green Pyronin Y, showing basophilia removable by RNase. 6-8. Different stages of developing macrogametocyte stained with Methyl green Pyronin Y, showing basophilia removable by RNase. 9, Developing microgametocyte after being stained with Methyl green Pyronin Y, showing DNA in nucleicisaring and RNA granules. 10.Developing microgametocyte after Feulgen reaction showing DNA. 11.Immature microgametocyte after Feulgen reaction showing DNA. 12.Microgametocyte viler Methyl green Pyronin Y, showing RNA. 13.Microgametocyte after Feulgen reaction showing DNA in microgametes. 14.First generation schizont with residual body after Feulgen reaction, showing DNA. 15, Late generation merozoite from schizont with residual body after Feulgen reaction, showing DNA. 16.Late generation schizont with residual body after Methyl green Pyronin Y, showing RNA. 17.Microgametocyte after Feulgen reaction, showing DNA in microgametes.

75

...... :•ta:1 O

4 2 3 5

1 6

f°°00 i i czs 0000 00 1

9 I0

7 8

-0-ass -0 00 os /0 0 o 0 ,0 00; :0 0- 0 04 ,000 0.0 . 0 00 0 0: so 0 .00 0t" °,•0. 14 o O 13

II 12

`,9• itt964 "

15 49 I , a

17 16

IO)J

PLATE VI 76.

2. RIBONUCLEIC ACID (RNA)

Sections stained with methyl green pyronin Y revealed the presence of ribonucleic acid. RNA is chiefly responsible for basophilia in the cell and in addition to staining with basic dyes (e.g. haematoxylin) it reacts specifically with pyronin Y to produce a light red colour (Fig. 11). Control sections were pre-treated with ribonuclease to remove the RNA and did not react with pyronin Y. All stages showed RNA in the cytoplasm and most stages had an additional store of RNA in the nucleus forming the karyosome. In general the cytoplasm of the mature form of any stage was more strongly basophilic than that of the young form. Thus whereas the small uninucle ate stage which could develop into schizont, macro- or microgametocyte contained little RNA'in the cytoplasm (Plate VI, No.5) the merozoites and mature gametocytes were strongly basophilic (Plate VI, Nos. 3, 7, 8, 12, 16). The schizonts of I. chioridis (Plate VI, No. 16) contained RNA in the residuum as well as in the merozoites. The macrogametocytes showed irregular accumulationsof RNA dotted in generally basophilic cytoplasm (Plate VI, Nos. 7, 8). The nuclei of the merozoites and macrogametocytes (Plate VI, Nos. 3, 16, 6-8) contained karyosomes composed of RNA and in the macrogametocytes RNA could also be detected in the nucleoplasm. The nuclei of early microgametocytes (Plate VI, No.9) showed no RNA within the

DNA ring and the nuclei of the microgametes were composed only of DNA. 77.

Fig. 11. Section 'of snail intestine of greenfinch after methyl green

Pyronin Y staining, showing the site of RNA in the macrogametocyte of I. lacazei. x 800. 78.

3. POLYSACCHARIDES

Polysaccharides are laid down as reserves in the cytoplasm. They can be detected by the highly sensitive periodic acid Schiff (PAS) technique, which being non-specific shows up all polysaccharides as a deep red colour (Fig. 12). Polysaccharides may be further characterized. Incubation of the slides with diastase was used as a selective method for the digestion of glycogen. Any PAS positive material left after treatment with diastase was not glycogen (Fig. 13). Best's Carmine is a specific stain for glycogen and slides thus stained were compared with the PAS slides. I. lacazei was examined for acid mucopolysaccharides which were detected with Alcian Blue. Sections were not available of I. chloridis for this test. PAS positive material identified as glycogen by digestion of control sections with diastase, was detected in the merozoites and schizonts of the first and late generations of both species (Plate VII, Nos. 1, 2, 3,

4). It occurred as fine granules in the cytoplasm not great in quantity and in I. chloridis was present in the residuum as well as the merozoites. Similarly PAS positive material identifiable as glycogen by its complete removal with diastase was found as granules in the cytoplasm of the microgametocytes (Plate VII, No. 8). This was left in the residuum and did not contribute a source of energy to the microgametes.

Glycogen was present throughout the development of the macrogame- tocytes (Plate VII, Nos. 5-7, Fig. 12), though there was a considerable Fig. 12. Section of snail intestine of greenfinch after PAS showing the

presence of polysaccharides in the macrogametocytes of I, lacazei.

X 800. Fig. 13. Section of small intestine of greenfinch, after PAS preceded by

diastase digestion, showing the presence of granules not removable

by enzyme treatment in the mature macrogametocyte and newly

formed oocyst of I. lacazoi.

x BOO. Explanation of Plate VII

Cytochemical studies of internal stages of Isospora spp. in greenfinch.

(Camera lucida drawings). (Figs. 2 and 4, I, chloridis; other figs. I. lacazei).

1. First generation schizont after PAS; showing the presence of glycogen in the cytoplasm of merozoites, 2. First generation schizont after PAS; showing the presence of glycogen in the cytoplasm of the merozoites and the residual body. 3. Late generation merozoite after PAS; showing the site of glycogen in

the cytoplasm.

4. Late generation schizont with residual body after PAS; showing the presence of glycogen in the cytoplasm of the merozoites and the

residual body. 5-7, Developing stages of macrogametocytes after PAS; glycogen showing as accumulations in the cytoplasm. 8. Mature microgametocyte after PAS; indicating the site of glycogen.

9, Newly formed oocyst after PAS; showing glycogen in the cytoplasm and the oocyst wall.

10,Newly formed oocyst after PAS; preceded by diastase digestion, showing the presence of granules not removable with enzyme. 11. Late generation merozoite after Alcian Blue; showing the presence of

acid mucopolysaccharide in the cytoplasm. 12-14. Developing stages of macrogametocytes stained with Alcian Blue;

showing the sites of acid mucopolysaccharide in the cytoplasm. 15. Mature microgametocyte after Alcian Blue; showing the presence of

acid mucopolysaccharide.

81

1 2 3

4

5 6

7 8

9 II 12

13

14 101.1 15

PLATE VII 82. increase in the quantity in the mature form and on the whole the individual granules were larger as the gametocyte increased.in size. Strangely the glycogen present in the newly encysted zygote (oocyst) was all in the form of very fine granules, indicating that a sudden change took place in the glycogen content when fertilisation occurred. The oocyst wall itself was strongly PAS positive with a glycogen component (Plate VII, No. 9). I. chloridis was not examined further for polysaccharides. Sections of I. lacazei were digested with diastase and subjected to the PAS technique (Fig. 13). Glycogen was removed but a less intense PAS positive reaction still occurred in both cytoplasm and wall (Plate VII, No. 10). In the wall the PAS positive sites were in the form of small flat plates and in the cytoplasm as fine granules. This material was not present in stages other than mature macrogmnetes and oocysts. Sections of I. lacazei were also stained with Alcian Blue when granules of acid mucopolysaccharide were found present in all asexual and sexual stages. The nature of these granules was confirmed by pre-treatment of sections with hyaluronidase to break the linkages of hyaluronic acid so that no blue colouration was obtained with Alcian Blue (Plate VII, Nos. 11- 15). The granules (Fig. 14) were quite large and irregular in shape and obviously did not correspond to the fine granules remaining after diastase treatment as above. When sections are pro-treated with diastase only fine small granules of polysaccharide are shown to be present by the PAS technique, acid mucopolysaccharide$remain after diastase treatment but will not be Fig. 14. Section of small intestine of greenfinch after staining with

Alcian Blue indicating acid mucopolysaccharide in the cytoplasm

of macrogametocyte of I, lacazei.

x SOO, 34.. visible as they are PAS negative. It can be concluded that at least three types of polysaccharides are present, large granules of glycogen, large granules of acid mucopolysaccharide, both present in all stages and an unidentified polysaccharide in the form of small granules present in macro- gametocytes and oocyst only.

4. PROTEIN

As proteinsare omnipresent components of all tissues a general protein test is of limited value in histochemistry. However tests have been used in this study to show special accumulationSof protein, as for instance in the oocyst wall. The mercuric bromphenol blue test (Hg BPB) indicates protein by a blue colouration. In the colour photograph (Fig. 15) the blue colour is largely altered to brown because of the quality of light used in..the photo- micrograph. Naphthol yellow - S shows the protein as a yellowish colour used (Fig. 16b), The latter can begin conjunction with PAS which shows carbo- hydrate red (Fig. 16a) and Azur A which shows DNA blue green (Fig. 16c). The detection of protein was carried out in the late generation merozoites, growing macrogametocytes and oocysts of I. lacazei. Protein is demonstrable in both nucleus and cytoplasm and coincides with the regionsof nucleic acid as shown by the test for RNA with additional accumulationsat the periphery of the mature macrogametocyte. Protein in the merozoite is shown in Plate VIII, No. 2, in the immature macrogametocytee in Plate VIII, Nos. 3 and 4, and in the mature macrogametocytes in Plate Fig. 15. Section of small intestine of greenfinch stained with Mercuric Bromphenol Blue, showing the location of protein in macrogameto- cyte of I. lacazei. x 800. 86.

Fig, 16. Section of small intestine of greenfinch after triple stain

showing:

a. Polysaccharides in the macrogametocyte of I. lacazei.

b. Protein in the macroametocyte of I. lacazei.

c. DNA in the schizont of I. lacazei.

x 800. Explanation of Plate VIII

Cytochemical studies of I. lacazei in greenfinch, (Camera lucida drawings).

1. Sporozoite stained with mercuric bromphenol blue, showing protein localization.

2. Late generation merozoite showing the sites of protein, after staining with mercuric bromphenol blue. 3-5. Developing stages of macrogametocytos stained with mercuric bromphenol blue, showing the sites of protein. 6. Newly formed oocyst showing the presence of protein in wall and also in contents, after staining with mercuric bramphenol blue. 7, Oocyst inside the host cell after mercuric bromphenol blue test, showing the sites of protein. 8. Sporocyst stained with mercuric bromphenol blue, showing the site of protein. 9, Oocyst from the faeces stained with mercuric bramphenol blue, showing the presence of protein in wall and contents. 10.Late generation merozoite stained with Sudan Black B, showing lipid globules in the cytoplasm. 11.Macrogametocyte stained with Sudan Black B, showing lipid globules distributed in the cytoplasm and around the nucleus. 12.Sporozoite stained with Sudan Black B, showing the lipid globules at both ends. 13.Macrogametocyte showing the presence of acid phosphatase in the cytoplasm and the nucleus. 1L.. Macrogametocyte showing the site of alkaline phosphatase inside the nucleus. 15. Late generation merozoite showing the site of alkaline phosphatase in the nucleus.

• • • • • 0 tr) • • • • • cr. • •

• • • * • • s • • 01) • • • •• • • • • • • • . • • • • -

co

•=0 ,c) 88.

VIII, No. 5.

In the newly formed oocyst,protein occurs as flat plates forming the oocyst wall (Plate VIII, No. 6). These are similar to but larger than the plates reacting with PAS after digestion with diastase (Plate VII, No. 10). In older intracellular oocysts the wall reacts uniformly for protein (Plate VIII, No. 7). It seems that the oocyst wall when newly laid down has a muco- protein component seen as small plates and a protein component as large plates. The plates merge with one another to form a thinner but uniform oocyst wall.

5. LIPID

Sections fixed in Baker's formaldehyde calcium were cut on a rocking microtame at-IE9C. Sudan Black B was used to stain lipid material which appeared as black globules. Of the endogenous phases only the merozoites and macrogametocytes of I. lacazei were tested. Lipid was sparse in the merozoites (Plate VIII, No. 10) occurring mainly in the cyto- plasm at the two poles, with little round the central nucleus. In the macrogametocyte lipid reserves were abundant with especially large globules in the vicinity of the nuclear membrane (Plate VIII, No. 11).

6. ENZYME ACTIVITY

Two enzymes, acid and alkaline phosphatase were tested for and located in I. lacazei. 89.

Using Gomori's method the presence of acid phosphatase is shown by a brownish-black precipitate and alkaline phosphatase by a definite black colour.

Acid phosphatase was detected in growing macrogametocytes where the site of enzyme activity appeared to be in both nucleus and cytoplasm.

In the cytoplasm the precipitate occurred as snail rings and in the nucleus there was a strong reaction on the membrane and a diffuse intranuclear reaction (Plate VIII, No, 13).

Alkaline phosphatase was detected in the macrogmnotocyte and late generation merozoites. In the macrogametocyte the only site of alkaline phosphatase activity was within the nuclear membrane appearing as an eccent- ric dark globule (Plate VIII, No. 14), probably corresponding to the karyosome. In the merozoites there was also a dark spot within the nucleus

(Plate VIII, No. 15) but as the nuclear membrane was not visible, it was impossible to say if the activity extended beyond the karyosome. 90.

CYTOCHEMICAL STUDIES ON THE EXOGENOUS PHASES

Same information was already obtained ,about the protein and polysaccharide content of the endogenous oocyst and cytochemical studies of exogenous oocyst were limited to detection of protein and lipid. Two problems arose in the study of oocysts recovered from faeces: firstly, the difficulty in recognizing the two different species of Isosoora, and secondly, the poor penetration of fixative through the oocyst wall. Satisfactory results were not obtained with Helly's fluid and paraffin sections which were recommended by Pattillo and Becker (1955). The cytochemical study of the oocyst's contents was carried out after breaking the hard wall of the oocyst under pressure. The oocyst wall gave a uniform reaction for protein to mercuric bromphenol blue (Plate VIII, No. 9), as did the late endogenous oocyst (Plate VIII, No. 7). Unsporulated exogenous oocystsgave a less intense reaction for protein than did ' the endogenous oocyst but this may be explained by the escape of some cytoplasm from the exogenous oocyst as it was crushed for fixation or by the poor penetration of stain. In the sporocyst emptied of its sporozoites (Plate VIII, No. 8), protein was detected in the wall, in the SUeda body and as accumulations within - these laWrixere probably fragments of residual body. In the sporozoites apart from the general cytoplasm, there was an intense reaction for protein in the refractile globules and in the nucleus (Plate VIII, No.1).

The refractile globules were the only lipid reserves in the exogenous stages (Plate VIII, No. 12). 91.

DISCUSSION

In the present study Isospora infections were detected in 111 out of 113 passerine birds by recovery of oocysts from their faeces. This high

incidence is in agreement with the results of Boughton (1933), who found

that over 99 per cent. of the English sparrows he examined were infected,

but contrasts with the work of other authors, for instance, Scholtyseck

(1956) who found 37 per cent. infection in 684 passerines belonging to

40 species. These low incidences may be attributable to the method of

examination.

Present experiments on the output of oocysts in greenfinches have

shown that there is a diurnal periodicity in oocyst production. In any

24 hour period, between 6.00 and 13.00 hours, oocysts would be difficult to detect. Thus examination of dead birds or of single droppings from live

birds could lead to an erroneously negative diagnosis. Examination should

be made of droppings from the peak period of oocyst production (18.00 hours in greenfinches) or, of samples collected at intervals during the 24 hour

period.

Because of the difficulty of diagnosing coccidial species, when

two or more species closely resembling one another occur in the same host, whenever possible descriptions of species should be based on pure line infections and the characters of the endogenous phases should be linked with the oocyst characters.

Tyzzer (1928) recommended the criteria for isolating and describ— ing new species as the length of prepatent period, sporulation time, host specificity, characteristic distribution in the host, cross immunity, 92. pathogenicity, morphology, studies and the relation of parasite to the host

cell. The very condition, that makes Tyzzer's recommendFtions difficult

to follow, obtained in this study - namely that clean hosts were not

available for pure line infections. For such cases the description of

the cocyst must be complete, based on measurements and details of morphology,

as recommended by Levine (1961). The shape of the oocyst appears to be reasonably constant for a given species (Davies, Joyner and Kendall, 1963),

thus the shape index L/W is a useful characteristic.

In all, there have been recorded from passerine birds 26 species of coccidia of which 21 belong to the genus Isospora. In many of these

cases the specific determination was based on oocyst measurements with

incomplete descriptions of oocyst contents.

The results of this study clearly suggest that oocyst measurements

alone are insufficient to separate species.

In all but jackdaw (spherical oocysts) and hedge sparrow (oval

oocysts) a mixture of oval and spherical oocysts was present in the passer-

ines examined, but there was variation in the proportion of spherical and oval oocysts in the different host species. The suggestion of a bimodal distribution in these hosts indicated the possibility of two forms represented by the oval and spherical oocysts respectively.

However, a further examination of the greenfinch and sparrow data by application of the X? test separately to measurements of spherical and oval oocysts revealed that in each case more than one mode was present -

that two or even three populations of both spherical and oval oocysts

existed. 93.

Examination of morphological characters showed that only two types of oocyst existed in greenfinch, chaffinch and sparrows oval and spherical. These could be distinguished by the thickness of the oocyst wall, shape of polar granules and.% the size of the sporocysts in addition to their shape. The sporulation times were identical. Examination of the internal stages again revealed two species only.

It was further observed that both types of oocysts passed by greenfinches are significantly larger than those passed by sparrows. By cross—infection experiments it was found that both hosts harbour the same two species. Thus the size of the oocyst is influenced by the host, possibly the size of the host cell influences the speed of growth and final size of the macrogamete or the physiological requirements of the parasites may be better provided by the greenfinch host.

Other explanations for the heterogeneity of oocyst measurements may be found in previous results. Jones (1932),werking with E. acervulina and E. maxima, found great variation in the size of oocysts and attributed this to the crowding of the parasite and to the age and inherent differences in the host birds. Becker, Zimmermann and Pattillo (1955), working with

E. brunetti, found that in the later stages of the patent period the oocysts were significantly larger. Boughton (1930), in a biometrical study of

I. lacazei in sparrows, found that the occysts became progressively smaller as the sexual phases of the cycle continued. In the present study the age of the hosts was net known, nor the duration infection nor the infective dose and if these factors influence oocyst size then the heterogeneous 94. distribution observed here is to be expected. Scholtyseck (1954) fell into a trap when he described two populations of oocysts from sparrows as separate species, I. lacazei and I. passerum, when their morphological characters were identical.

Analysis of variance of present data showed firstly that between individual birds of a species there was considerable heterogeneity in all five oocyst dimensions taken, including shape LA, and secondly that there was no significant difference in the average shape (L/6V) of oocysts from the two host species examined. The first is explained by individual birds harbouring oocysts of the two shapes in different proportions. In the second case, because of the thoroughgoing mixture of oval and spherical opcysts,the proportion of oval and spherical oocysts from one group of ten birds was the same as the proportion in the other. Thus the average shape was the same in the two host species.

Though two species of Isospora were evidently present in green— finch, sparrow and chaffinch, the identification proved difficult because, as already mentioned the descriptions of the 26 species of coccidia from passerines are mostly inadequate and based on oocyst measurements only.

Even when no differences are apparent in exogenous phases of coccidia, e.g. Eimeria adenoeides, E. meleagridis and E. meleagrimitis in turkey, which Clarkson (1960) found to have the same sporulation time and structure of oocyst contents, differences can be found in the endogenous phases. Because there are only slight differences in the contents of the spherical and oval oocysts of passerines, some workers (Hosoda, 1928a; 95.

Henry, 1932 and Chakravarty and Kar, 1944) have taken them to belong to

the same species. But in spite of this )ocyst similarity and the overlap-. in shape in the subspherical range, the oval and spherical oocysts correspond to schizogonic and gametocytic stages which are specifically different.

Isospora lacazei was the first species to be described from passerines. Labb6 (1893) recorded it under the name of Diplospora lacazii

from a number of host species but gave only a sketchy description of the oocysts which were round and contained pyriform sporocysts each with four sporozoites. In 1896 he synonymized D. rivoltae (oocysts 16 — 18 p) with

D. lacazei (oocysts 23 — 25 it) as he found intermediates, extended the host list and changed the spelling to lacazei. This is the correct spelling as it honours Felix Joseph Henri de Lacaze—Duthiers and lacazii is clearly a lapsus

Later authors identified the species from a variety of hosts, a few giving some description of the endogenous stages but as many of the observations were contradictory it is unlikely that all the parasites reported under this name were the same as that seen by Labbe.

The spherical oocysts from greenfinch, chaffinch and sparrow agree with Labb6's description but differ in some respects from all the other descriptions. Though a considerable number of workers have identified

I. lacazei from passerines only a few of these have described morphological details to expand Labb4Is description.

Sj5bring (1897) found Isospora in eleven species of bird in Sweden.

From one of these hosts,Lanius colluriJ, hG described I. passerum which 96, accords with I. lacazei except in that 4 - 6 sporozoites were described from each sporocyst. Later workers have discounted this observation and synonymized it with I. lacazei. Further to Labb6is description the presence of the oocyst polar granules and the Stieda body of the sporocysts were mentioned. If he were mistaken in his observation of the number of sporozoites these oocysts differ from the spherical ones in the present study only in the shape and colour of the polar granules, which were ovoid and greenish, and in the presence of the sporocystic membrane.

Claassen (1923) described I. lacazei from the siskin (Carduelis spines), with oocysts which were smaller (16 - 17 p) but with a sporulation time in agreement with the present study.

Hosoda (1928a) included oval and suLspherical oocysts from Passer montanus saturates in I. lacazei. Structurally the oocysts were similar to the present ones except that they did not have polar granules, but did havedsimiler sporulation time.

Henry (1932.) als-) included oval and subspherical oocysts in her description of I. lacazei. She indicated the existence of a micropyle, though this was inconspicuous, and recorded the sporulation time as only

24 hours. These points differentiate her 3ocysts from the present ones.

Hall (1933) recorded a sporulation time of 84 hours for I. lacazei whereas Chakravarty and Kar (1944) gave 4 - 5 days. They gave no mention of the sporocystic residuum but described a micropyle in the spherical oocysts of I. lacazei from Passer domesticus indicus and Uotpastes haenorr- hus bengiliensise 97.

Scholtyseck (1954) describes structurally similar oocysts of two

sizes from Passer domesticus and names the larger (30 — 39.u) I. lacazei and

the smaller (18 — 30)u) I. passerum. The dangers of separating species on measurement alone have been pointed out. In any case the smaller oocysts correspond with the size range of I. lacazei and the use of I. passerum is not permissible as it has already passed into synonymy with

I. lacazei, having previously been used by Sj'5bring. Scholtyseck shows the Stieda body just as a thickening of the sporocyst wall not as a cap.

Levine and Mohan (1960) have given the fullest account to date of

I. lacazei oocysts from Passer domesticus. Even so there are differences between these and the present ones. No vacuoles were described in the sporozoites and the compact mass of the spoxocyst residuum was not mentioned.

However these authors mentioned for the first time the membrane enclosing the sporozoites and residuum together within the cocyst, a feature observed in the present study.

Because the descriptions are inadequate to separate them, at present it is necessary to leave all the forms observed by these authors in the species I. lacazoi. If future workers concentrate on full descriptions of exogenous and endogenous stages from single host species, some of these forms may be separated off as distinct species. In this work the spherical oocysts from sparrow, greenfinch and chaffinch are assigned to I. lacazei and a full account of the endogenous stages is given.

The other species with oval oocysts from these hosts is considered new and is named I. chioridis n. sp. Of the 21 species of Iscspora 98. described from passerines, nearly all are separated as species on measure— ments alone and others have such inadequate descriptions that it is imposs— ible to compare them. The present species has been compared with I. fringillae from chaffinches (Yakimoff and Gousseff, 1938) but certain morphological differences — e.g. the absence of polar granules in I. fringillae — distinguish it. Further the description of I. fringillae was sketchy and it is not possible fully to compare it. A new specific name was necessary for the species with oval oocysts in the present study.

Neither Isospora rivoltae (first used by Labb4 for oocysts in passerines and later synonymized by him with I. lacazei) nor I. passerum made synonymous with I. lacazei by Itenyon (1926) can be used. AS the internal stages were studied in the greenfinch (Chloris chloris) the name Isospora chloridis has been used.

The study of the internal phases in greenfinches revealed that schizonts and gametocytes of two types were recognisable supporting the previous finding of two separate oocyst shapes. The first stage schizonts were discovered by superimposing a massive infection over a natural infect— ion which itself revealed the second generation schizonts and gametocytes.

As both species I. lacazei and I. chloridis were present in one host it was difficult to determine to which species the schizonts and gametocytes belonged. Tyzzer (1929) suggested that pure line infections should be established to study coccidiel life cycle. In this study clean birds were not available and pure lines were impossible.

The only method of linking the two sets of endogenous stages with 99. the spherical and oval oocysts was to correlate the frequency of the type of oocyst with the internal stages. The internal stages as illustrated in Plate IV were most frequent when the output of spherical oocysts

(I. lacazei) was high. The internal stages as illustrated in Plate V were only found in two greenfinches and these were passing abundant oval oocysts

(i. chioridis).

In all previous work where internal stages of Isospora have been described in passerines, the species has been identified as I. lacazei.

Vasielewski (1904), Wenyon (1926) and Chakraverty and Kar (1944) all describe schizonts whiCh separate merozoites without residual cytoplasm.

Claassen (1923) describes two types of schizont, one with and the other without residual cytoplasm and Hosoda (1928e) describes only schizonts with residual cytoplasm. As also the dimensions given for these types of schizont differ, it is unlikely that all belong to one species. Little detail is given about the gametocytes but Hosoda who describes only schizonts with residual cytoplasm mentions "plastic granules" in the macrogametes.

In the present work, in the majority of greenfinches only schizonts without residual cytoplasm, microgametocytes with spindle—shaped microgametes and macrogametes with dense cytoplasm and no obvious plastic granules were present. These were linked with the spherical oocysts and were identified as I. lacazei. In the remaining two greenfinches, in addition to the stages of I. lacazei were schizonts with residual cytoplasm, microgam,tocytes with comma—shaped microgametes and macrogametos with pale 100. cytoplasm and obvious plastic granules. These were identified as I. chloridis. It would appear that the species studied by Hosoda (1928a) was not I. lacazei but I. chloridis.

Only I. lacazei and I. chloridis have here been studied endo— genously as well as exogenously. A number of other forms of Isospora have been examined by measurements and morphological observations on the oocysts.

The oocysts found in different host species showed that considerable variation occurred in measurements and oocystic and sporocystic characters.

Whether or not these characters can be used to distinguish them as separate species needs further investigation.

In general, as noted by Levine (1961), Isospora species tend not to be host specific and several host species may be infected with the same species of Isospora. It is a mistake, therefore, to identify new species of Isospora merely because they have been found in a new host.

•Unfortunately early descriptions of Isospora species from passerines are insufficiently detailed for the forms observed in this study to be identified with them.

All except jackdaw and hedge sparrow harboured both oval and spherical oocysts but variations in oocyst contents were noted as follows, which differentiated them from I. lacazei, I. chloridis end from each other.

' 1. The oocysts found in nuthatch differ from all the others in having a long sporulation time of 81 hours and a sporocyst residuum lacking the compact mass.

2. The oocysts from jackdaw and canary are similar in their sporulation time but a membrane enclosing both the sporozoites and residuum

is present in the oocysts from canary and absent in those from jackdaw.

3. The oocysts from canary, starling, great tit and blackbird and one type (smooth wall) from robin were similar in their structure.

All lacked a sporocyst residuum in the form of fine granules, though a compact mass was present. There were size variations between hosts but those from starling and canary were of the same order. 4. Oocysts from mistle thrush lacked the apical cap of the

Stieda body.

5. Oocysts from hedge sparrow were almost all oval, The sporulation time was only 24 hours; the sporocysts lacked a membrane round hie the sporozoites andAresiduum of fine granules was also absent. There do seem sufficient characters here to distinguish this as a separate species.

Further work is required.

6. Some oocysts recovered from the robin had a rough brownish wall which gave it an appearance entirely different from all other oocysts seen. However, it was found in only one bird and an attempt to transmit it to another robin failed. It may therefore not be a true parasite of the robin — the oocysts could have passed unchanged through the robin gut.

Further work is required.

More work is required do all these types before conclusions may be drawn as to their relationships.

The cytochemical studies have given information about the distribution of nucleic acids, proteins, carbohydrates, lipids and two 14%

enzymes in the different stages of the life cycle of Isospora. Because

material of I. chloridis was not available for tests other than for nucleic

acids and glycogen the two species cannot be compared for their chemical

content. Inferences may be drawn regarding the function of the substances

in the different stages of the parasite.

Both I. lacazci and I. chloridis were tested for DNA and similar

results were obtained, namely that DNA was present in all stages except

the macrogametes. Negative results for DNA were obtained ''ith .the Feulgen reaction and methyl green in the methyl green pyronin Y test. This agrees

with the results of previous investigators except those of Horton-Smith

and Long (1963) who obtained a weak reaction for DNA in the macrogametes of

Eimeria maxima. It is usual to find only small quantities of DNA in nuclei

in the resting state while DNA accumulates at the onset of nuclear division.

The nucleus of the coccidial macrogamete remains undivided throughout its

growth and maturation and only after fertilisation does the nucleus undergo division. It is unlikely that DNA is completely absent in the macrogamete nucleus. Serra (1947) failed to demonstrate DNA in the oocyte of the

snail and he concluded that the DNA was present in amounts below the sensit- ivity of the Feulgen test. Alfert (1950) studying oogenesis and cleavage

in the mouse obtained the same result. The macrogamete of E. mexima is

of large size even for a coccidium and Horton-Smith and Long (1963) suggest-

ed that its large nucleus contains sufficient DNA to be demonstrable with

the Feulgen reaction. Nuclei of smaller coccidial species may contain

DNA in a quantity just below the limit of sensitivity of this test. 103.

All asexual stages of I. lacazei and I. chloridis contained

DNA in the form of a ring at the periphery of the nuclei. The micro— gametes consisted almost entirely of DNA. Thus the amount of DNA in

Isospora stages coincides with the nuclear activity. Dividing stages, schizonts and microgametocytos have active nuclei with appreciable DNA whereas the growing macrogametos have resting nuclei with little DNA.

It is well known that strongly basophilic cells are active in protein synthesis (Loewy and Siekevitz, 1963). Basophilia is imparted to the cell by the presence of RNA. RNA was found present in all stages of I. lacazei, where its distribution closely paralleled that of protein.

This may be explained by the fact that RNA plays a central role in protein synthesis. Thus RNA was detected throughout the cytoplasm, and in the karyosome, where it is thought to be stored prior to its utilization by the cytoplasmic ribosomes for protein synthesis. A certain amount of protein synthesis may also occur in the karyosome.

Of the three carbohydrates detected, glycogen was the most widespread. It was found in varying quantity in all asexual and sexual stages of I. lacazei and I. chloridis. Glycogen has been determined as an important source of energy in the parasite (Edgar et al. 1944, Cheissin,

1959). Thus the quantity accumulated in the cytoplasm of the parasite is related to its metabolic and physical activity. The absence of glycogen in the microgamete may be correlated with the fact that the gamete requires little energy for movement as it merely penetrates a nearby macrogamete — the absence of this reserve, in fact, suggests that the microgamete is 104. incapable of extended "searching" for the macrogamete. Glycogen present in the cytoplasm of the microgametocyte is required to provide energy for the nuclear divisions and separation of the microgametes and any reserve remaining after this, is left behind in the residuum.

Glycogen was present in the growing schizonts and in the separated merozoites, and in the schizont residuum of I. chloridis. As in the microgametocytes, the glycogen in the schizont provides a source of energy for nuclear and cytoplasmic division. The merozoites after release from the parent schizont leave the host cell and actively seek a new cell, for which migration a source of energy is required. The glycogen present in the schizont residuum of I. chloridis represents only that glycogen left in the schizont cytoplasm after separation of the morozoites and does not imply active metabolism or movement on the part of the residuum.

The mature macrogamete was richest in its reserve of glycogen.

This reserve was laid down throughout the development of the macrogamete and was seen in the mature gamete as close-packed coarse granules. After fertilisation, formation of the •:-,cyst and expulsion from the host cell, the zygote receives no further nutriment from without, the energy for the entire process of sporulation, nuclear and cytoplasmic division and cyto- plasmic differentiation, comes from the reserves of the macrogamete. It is interesting to note that immediately after fertilisation the coarse granules of glycogen were immediately converted to fine granules. Thus

the development of the zygote starts immediately, while still within the 105. host cell. The oocyst wall was itself strongly PAS positive with a glycogen component.

One point of particular interest concerns the "plastic granules" of the macrogametocyte. These granules, so characteristic of many coccidia, were not detectable in I. lacazei but were easily discernable in I. chloridis. Previous workers (Pattillo and Becker, 19553 Horton—

Smith and Long, 1963) have reported that the plastic granules are composed of a protein—carbohydrate complex and that they play a part in the form— ation of the oocyst wall. In I. lacazei the PAS technique after incubat— ion of sections in diastase to remove glycogen, showed the presence of fine granules of polysaccharide distributed throughout the cytoplasm. As protein has an equally wide distribution in the cell it is possible that the protein—carbohydrate complex is evenly distributed in the cytoplasm and not accumulated in large granules.

Following fertilisation of the macrogamete the oocyst wall is formed and using the same technique as above, i.e. PAS after diastase incubation, polysaccharide was detectable in th(; wall as flat plates.

Mercuric bromphenol blue indicated plates of protein in the newly formed oocyst wall but a uniform distribution of protein in the mature cyst wall.

These results suggest that the oocyst wall is formed from a protein— carbohydrate complex present in the cytoplasm of the macrogamete together with a glycogen component. Further, the oocyst is laid down first as flat plates at the periphery of the zygote and these are later joined up as more protein is added. 106.

Acid mucopolysaccharide was found in the cytoplasm of the macro— gamete and late generation merozoites of I. lacazei. It was also sparsely distributed in the cytoplasmic residuum of the microgametocyte. Previous results are variable with respect to the presence of this substance.

Cheissin (1959) and Pattillo and Becker (1955) did not find acid mucopoly— saccharide in any stage of rabbit coccidia and E. brunetti and E. acervulina respectively but Gill and Ray (1954a) working with E. tenella and Pettillo

(1957) with E. tenella and E. necatrix demonstrated it in these species.

It seems possible, therefore, that there is variation between species, some producing acid mucopolysaccharide, others not.

There is, as yet, no complete explanation of the function of this substance in tissues or cells. It may be involved in water retention as was suggested by Rogers (1961). If this function is acceptable as a possibility in coccidia, acid mucopolysaccharides in the parasites may be involved in a form of osmotic control of the exchange of fluid between host cell end parasite.

Claude (1949) states that lipids are usually regarded as storage substances which can be drawn upon to produce a supply of energy. Thus those stages of the parasite which are most active and have a high metabolic rate will contain some stored lipids. Like the carbohydrate reserves the lipids were found in merozoites and macrogametes of I. lacazei, the former being capable of locomotion and the latter when fertilised being involved in the development of the sporozoites.

Although a little work on enzyme activity in coccidial parasites 107. has been done, the sites of activity have not been fully demonstrated and it is thus difficult to relate the occurrence of phosphatases to their function.

According to Danielli (1946) the presence of alkaline phosphatase in the cell nucleus is probably related to the synthesis of nucleic acid. Sullivan (1950) working with Colpidium campylum reported alkaline phosphat— ase activity in the perinuclear area and suggested that it might be connected with the synthesis of ribonucleic acid. vicklund (1948) also suggested that the activity of the phosphatase was in some way connected with an increase in nucleic acid synthesis.

Alkaline phosphatase activity in this study was only demonstrated in the nucleus, probably in the karyosome.

There is, as yet, no agreement on the function of the phosphatases.

In contrast to Danielli, as above, Mugerd (1951) held the opinion that they were concerned with cell growth. Thus acid phosphatase, which was detected in both nucleus and cytoplasm in this study, may be concerned with carbohydrate or protein metabolism. 108.

SUMMARY

111 out of 113 passerine birds of 13 species were found harbouring coccidia of the genus Isospora.

Studies of oocyst measurements revealed that oval and spherical oocysts were present in all but hedge sparrow (only oval) end jackdaw

(only spherical). The variabilities of length and width values in all species of bird were high indicating the presence of more than one normal distribution.

A more detailed examination of greenfinch and sparrow data was undertaken by fitting a single normal curve to the frequency data by means of the chi-squared test. High values of chi-squared revealed a hetero- geneous population. Further the chi-squared test was applied to oval and spherical oocysts separately and still both oval and spherical cysts were found to be heterogeneous. These studies of oocyst measurements were related to studies of oocyst morphology and endogenous stages to see if the mixed population of oocysts were of specific rank.

Studies of oocyst morphology revealed that in greenfinch, sparrow and chaffinch there were two types which could he distinguished by shape oval or spherical, thickness of oocyst wall, size of sporocysts, and shape of polar granules.

Studies of the endogenous phases in greenfinch confirmed the existence of two species, I. lacazei with spherical oocysts and I. chlorid- is n. sp. with oval oocysts. I. lacazei has first and second generation 109. schizonts which separate merozoites without leaving a residual body, the microgametes are spindle—shaped and thy; macrogametes have dense cytoplasm and no obvious "plastic granules". I. chioridis has first and second generation schizonts each leaving a spherical residuum after separating the merozoites, comma—shaped microgametes and macrogametes with pale cytoplasm and distinct "plastic granules".

The populations of spherical end oval oocysts revealed by biometric studies, therefore,do not correspond to species but are explained only in terms of conditions imposed on developing macrogametes by the host cells.

Morphological studies on oocysts from the remaining host species revealed an oocyst type from each of nuthatch, jackdaw, hedge sparrow, and robin and a further type was found in canary, starling, great tit, blackbird and robin. In some cases sporulation times were calculated also for these oocysts. The author considers that studies on endogenous phases should be undertaken before attempting to classify these types as species.

A study of the output of oocysts in greenfinches revealed a diurnal periodicity vdth a peak output at 18.00 hours and a minimum output between 6.00 and 13.00 hours. The number of oocysts passed per hour bore no relation to the amount of faeces passed.

In attempts to clean greenfinches of existing natural infections of I. lacazei and I. chioridis infective oocysts were withheld from birds by changing cages every twenty four hours and every twelve hours, both periods being less than the calculated sporulation times of 63 — 72 hours. 110.

There was very little decrease in the output of oocysts in four experiments when birds were examined for 11 days, 22 days, 28 days and 31 days. It was concluded that asexual reproduction was continuous providing gametocytes over a long period.

Experiments with sulphamezathine as a therapeutic agent for treatment of Isospora infections indicated some effect in greenfinches but a balance between toxicity to parasite and toxicity to host was not achieved.

As clean birds were not available prepatent periods were calcelated by superimposing a heavy dose of Isospora oocysts on existing natural infections. I. lacazei and I. chloridis were shown to be cross infective between greenfinch, chaffinch and sparrow. The prepatent periods were five days for both species. The prepatent period of a form from robins was four days.

Cytochemical investigations were carried out to detect nucleic acids, carbohydrates, proteins, lipids and certain enzymes, namely acid and alkaline phesphatase.mainly in I. lacazei.

In the asexual stages DNA was located in an intranuclear ring.

The microgametes consisted almost entirely of DNA but none was detectable in the macrogamete.

RNA was found in the cytoplasm of all stages, rather more in the mature than immature stages. It was also present in the karyosome of the nucleus and in the mecrogamete nucleoplasm.

Three types of polysaccharide were detected. Glycogen was found in small quantity in the schizonts, merozoitc-A And microgAmetocyte8. The macrogamete had abundant glycogen in the form of coarse granules. After

fertilisation these were broken down into fine granules and glycogen was

detected in the oocyst wall. An unidentified polysaccharide, not glycogen,

was detected in the macrogamete cytoplasm and in the oocyst wall as flat

plates. A comparison of the distribution of this polysaccharide and of protein indicated that this was a protein/polysaccharide complex correspond—

ing to the "plastic granules" observed in other coccidia. Acid mucopoly—

saccharide was present in all stages.

Apart from the protein generally distributed in the cell special

accumulations were found in the oocyst wall, here combined with polysacchar—

ide and in the refractile globules of the sporozoites. These latter

also contained lipid. A small amount of lipid was found in the merozoites

and as globules near the nucleus of the macrogamete.

Acid phosphatase was detected in the cytoplasm of macrogametes,

and on the nuclear membrane and diffusely in the nucleoplasm. Alkaline phosphatase was found only within the nucleus, probably in the karyosome. 112.

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APPENDIX

Table Al.. Pilot Sample (200 oomists).

Measurements of oocysts in eyepiece units for 10 sparrows and 10 green-

finches using 10 oocysts per bird.

L = Length of oocyst.

W = Width of oocyst.

B = Total for 10 oocysts per bird.

S = Total for 10 birds per species.

X = Total for 2 species (200 oocysts).

(These data are analysed in Table A.2) ii.

To.b1e At 1 Cont.

SPARROW

7.0 6.n I 1.14 I 53.04 I 360.67 ; 0.0 7.2 1.11 57.60 j 414.72 I 7.2 7.0 j 1.03 50.40' 352.QO ( 8.0 7.0 i 1.14 56.00 392.00 ~ 7.2 7.0 1.03 50.40 352.80 ! 8.2 C.O 1.02 65.60 524.80 ! 7.6 6.4 1.10 40.64 3ll.30 i 8.0 Jit.o1_14 56.00 392.00 ! Ii B.O ,7.6 1.05 60.00 462.0B ---~~---Ga~ 71.0 I 10.09--- --562.44--l-·W;62~05--·! \ ; I ____._.1

GREENFINCH :.... - ----T------T !____l __ L ___ rl,_ W t_I/W_-+--~---+---~----' t ' I . ! I i 7. 5 I 7.0 '1.07 I 52.50 i 367. 50 j - 9.0 1 7•5 1.20 167.50 506.25 I 0.0 0.0 1.00 I 64.00 512.00 I 8.0! 7.0 1.14 - 56.00 392.00 1 0.0 7.5 1.06 i 60.00 450.00 . I 8.0 l 0.0 1.00 64. 00 512.00 7.5 I 7.5 1.00 56. 25 421.87 I 9.0 I 0.0 1.12 72.00 576.00 8.0 ,7.0 ,1.14 56.00 392.00 I --1, --~~-W- 7.0 _ I 1.14 56.00 392.00 ! Bll l B1,O I 74.5 : 10.B7 604.25 4521.62 _.,~ _ ... __--'- ___---J.- __ ---L Table A.1 Cont.

SPARROW

L W L/W LW LW2

7.8 7.6 1.02 59.28 490.53 9.2 9.0 1.02 82.80 745.20 7.8 7.2 1.08 56.12 404.35 7.8 7.8 1.00 60.84 474.55 8,2 8.0 1.02 65.60 524.80 9.2 8.4 1,08 77.28 649.15 8.6 8.2 1.04 70.52 578.26 7.6 7.4 1.02 56.24 416.18 8.6 8.0 1.07 68.80 550.40 8.0 8,0 1.00 64,00 512.00 B 2 82.8 79.6 10.35 661.48 5305.42

GREENFINCH

L W L/W LW LW2

8.0 7.0 1,14 56.00 392.00 8.0 8.0 1,00 64.00 512.00 8.5 7.0 1.21 59.50 416.50 6.5 6.5 1.00 42,25 274.62 8.0 7,o 1.14 56.00 392.0o 8.5 7.5 1.13 63.75 478.12 9.3 7.5 1.24 69.75 523.12 8.0 8.0 1.00 64.00 512.00 8.0 7.0 1.14 56.00 392.00 8.0 7.0 1.14 56.00 392.00 B 12 80.8 72,5 11.14 587.25 4284.36 -__ iv.

Table A.1 Cont.

SPARROW

L W L/W LW LW2

7.0 6.0 1.16 42.00 252.00 7.4 7.4 1.00 54.76 405.22 9.0 8.0 1.12 72.00 576.00 7.3 7.2 1.0$ 56.16 404.35 7.2 6.8 1.06 48.96 332.93 7.0 7.0 1,00 49.00 343.00 7.4 7.0 1.05 51.80 362.60 7.0 7.0 1.00 49.00 343.00 7.5 7.5 1.00 56.25 421.87 7.o 7.0 1.00 49.00 343.00 B 3 74.3 70.9 10.47 523.93 3783.97

GREENFINCH

L W L/W LW LW2

8.0 8.0 1,00 64.00 512.00 9.0 7.5 1.20 67.50 506.25 9.0 9.0 1.00 81.00 729.00 9.5 9.0 1.05 85.50 769.50 8.0 8.0 1.00 64.00 512,00 8.5 8.0 1.06 68.00 544.00 8.0 8.0 1.00 64.0o 512.0o 9.0 (3.0 1,12 72.00 576.00 8.0 7.0 1.14 56.00 392.00 8.2 8.0 1.02 65.60 524.80

B13 85.2 80.5 10.59 687.60 5577.55 v.

Table A.1 Cont.

SPARROW

L W L/W LW LW2

. 6.0 5.6 1,07 33.60 188.16 7.0 6.6 1.06 46.20 304.92 7.2 7.2 1.00 51.84 373.25 7.2 6.6 1.09 47.52 313.63 7.8 7.0 1.11 54.60 332.20 7.0 7.0 1.00 49,00 343.00 6.0 6.0 1.00 36.00 216.00 7.0 6.6 1.06 46.20 304.92 7.0 6.6 1.06 46.20 304,92 6.2 6.0 1.03 37.20 223,20 B 4 68.4 65.2 10.48 448,36 2954.20

GREENFINCH

L W L/W LW LW2

8.0 8.o 1.00 64.00 512.0o 8.o 7,0 1.14 56,00 392.00 7.5 7.o 1.07 52.5o 367.50 8.2 8.o 1.02 65.60 524.80 7.5 7.5 1.00 56.25 421.87 8.0 7.5 1.06 60.00 450.0o 7.2 7.0 1.02 50.40 352,80 8.0 7.5 1.06 60.00 450.00 7.5 7.5 1.00 56.25 421.87 7.0 7.0 1.00 49.00 343.00 76.9 74.0 570.00 B14 10.37 4235.84 vi.

Table A.1 Cont.

SPARROW

L W L/W LW LW2

5.2 5.0 1.04 26.00 130.00 6.0 5.6 1,07 33.60 188.16 M 8.2 1.07 72.16 591.71 6.2 6.0 1.03 37.20 223.20 5.8 5.2 1.11 30.16 156.83 5.6 5,4 1.03 30.24 163.30 8.2 6.4 1.28 52.48 335.87 8.2 8.0 1.02 65,60 524,80 6.0 6.0 1.00 36.00 216.00 5.6 5.0 1.11 28.00 140.00 B 1 65.6 5 60,8 10,76 411.44 2669.87

GREENFINCH

L W L/W LW LW2

8.5 8.5 1,00 72,25 614.12 7.5 7,0 1,07 52.50 367.50 8.0 7.2 1.11 57.60 414.72 7.5 7.5 1.00 56.25 421,87 8.5 7.5 1.13 63.75 478.12 8.0 7.5 1.06 60.00 450.00 8.0 7,5 1.06 60.00 450.00 8.5 8.5 1.00 72.25 614.12 8;0 7.5 1,06 60.00 450.00 9.0 9.0 1.00 81.00 729.00

B15 81.5 77.7 10.49 635.60 4989.45 vii.

Table A.1 Cont.

SPARROW

L W L/W LW

7.0 5.8 1.25 40.60 235.48 6.0 5.2 1.15 31.20 162.24 6.0 5.0 1.20 30.00 150.00 6.5 5.5 1.18 35.35 194.42 6.0 5.0 1.20 30.00 150.00 6.0 6.o 1.00 36.0o 216.00 6,0 5.5 1.09 33.00 181.50 5.5 5.0 1.10 27.50 137.50 7.o 5.8 1.26 40.60 235.48 6.8 5.5 1.23 37.40 205.70

B6 62.8 54.3 11..66 I 341.65 1868.32 j

GREENFINCH

L W L/W LW LW2

9.0 7.8 1.15 70.20 547.56 8.0 7.0 1.14 56.00 392.00 8.5 7.0 1.21 59.50 416.50 7.0 7,0 1.00 49.00 343.00 7.5 6.5 1.15 48.85 317.52 3.2 7.0 1.17 57.40 401.80 7.5 7.0 1.07 52.50 367.50 8.2 7.5 1.09 62.50 468.75 8.2 7.8 1.05 63.96 498.88 7.3 7.0 1.04 51.10 357.70

B16 79.4 71.6 11.07 571.01 4131.21 viii.

Table A.l C~.

SPARROW I -, ___ +_2' __ '------"1'~J .J- -L/W... ---- I I 7.B 7.2 1.00 !--5:~--+-~::-1 . I 8.0 7.0 1.25 61.60 431.20 j . 7.2 7.0 1.02 50.40 352.00 I 7.6 7.6 1.00 57.76 430.98 6.4 6.0 1.06 38.40 230,40 I 7.2 7.2 1.00 51.04 373.25 I 8.0 7.0 1.14 56.00 392,00 I 7.0 7.0 1.00 49.00 343.00 6.2 6.0 1.03 20 7.0 1.02 62.4037.20 },223.406.72 I' 1----;;--t 7~> 69.0 10.60 520.76 ". 3675.90-! I _. __~ ______~ _____~ __ ,_--,-_ __._.... • ____-J..

GREENFINCH

; L I w ---+-----.1-.--'--"-'

7.0 6.0 1.03 47.60 323.68 7.5 7.0 1.07 52.50 367.50 8.0 7.5 1.06 60.00 450.00 9.0 13.0 1.12 72.00 576.00 7.5 6.5 1.15 40.75 316.87 6.5 6.5 1.00 42.25 274.62 0.5 8.5 1.00 72.25 614.12 6.5 6.5 1.00 42.25 274.62 8.2 7.0 1.17 57.40 401.00 0,0 n.o 1.00 64.00 512.00 ---.--- 10.60 559.00 4lll.21 l---_B_1_7_-t __76_,_7 __ ~3_ ix.

Table A.1 Cont.

L W L/W LW LW2

6.0 6.0 1.00 36.00 216.00 6.0 6.0 1.00 36.00 216.00

• 6.0 6.0 1.00 36.00 216.00 6.2 5.4 1.14 33.48 180.79 6.2 6.0 1.03 37.20 223.20 6.4 5.2 1.23 33.28 173.06 6.0 5.4 1.11 32.40 174.96 6.6 6.0 1.10 39.60 237.60 6.2 5.0 1.23 31.00 155.00 5.8 5.4 1.07 31.32 169.13 61.4 56.4 10.91 346.28 1961.74

GREENFINCH

L iv LA LW LW2

D 0 t 9.0 1.00 81.00 729.00 71 O • 1 C 8.5 1.00 72.25 614.12 •

CO 8.5 1.00 72.25 614.12 1 CP • CO 8.5 1.00 72.25 614.12 • fa 0 % 8.0 1.12 72.00 576.00 • O 0 8.2 1.09 83.80 687.16 • CO • 0 8.0 1.00 64.00 512.00

D P •

O 8.5 1.00 72.25 614.12 O • • 0 C 8.0 1.00 64.00 512.00

CO C 0 8.0 1.00 64.00 512.00

B 85.0 83.2 10.21 717.80 5984.64 18 X.

Table A.1 Cont.

SPARROW

L W L/W LW LW2

8.0 7.5 1.06 60.00 450.00 9.0 7.0 1.28 63.00 441.00 6.8 6.0 1.13 40.80 244.80 8.0 7.2 1.11 57.60 414.72 9.0 8.0 1.12 72.00 576.00 8.0 8.0 1.00 64.00 512.00 7.5 6.5 1.15 48.85 317.52 8.5 7.5 1.13 63.75 478.12 7.5 7.0 1.07 52.50 367.50 7.0 6.5 1.07 45.50 295.75 B 79.3 71.2 11.12 568.00 4097.41 9

GREENFINCH

L Vd L/W LW LW2

7.0 7.0 1.00 49.00 343.00 7.0 7.0 1.00 49.00 343.00 7.2 7.2 1.00 51.84 373.25 7.5 7.0 1.07 52.50 367.50 7.5 7.5 1.00 56.2:J 421.87 7.5 7.5 1.00 56.25 421.87 7.5 7.5 1.00 56.25 421.87 7.8 7.8 1.00 60.86 474.70 7.8 7.5 1.04 58.50 438.75 7.2 6.5 1.10 46.80 304.20 74.0 72.5 10.21 537.25 3910.01 1319 xi.

Table A.1 Cont.

SPARROW

L W L/W LW LW2

8.6 8.0 1.07 68.80 550.40 7.0 6.8 1.03 47.60 323.68 7.2 6.4 1.12 46.08 294.91 6.4 6.4 1.00 40.96 262.14 6.4 6.0 1.06 38.40 230.40 7.0 6.6 1.06 46.20 304.92 7.0 6.8 1.03 47.60 323.68 6.6 6.6 1.00 43.56 287.50 8.8 7.8 1.12 68.64 535.39 7.0 6.6 1.06 46.20 404.92 72.0 68.0 10.55 494.04 3517.94 B10

GREENFINCH

L W L/W LW LW2

8.0 8.0 1.00 64.00 512.00 8.5 8.0 1.06 68.00 544.00 8.0 8.0 1.00 64.00 512.00 8.0 7.5 1.06 60.00 450.00 9.0 8.5 1.05 76.50 650.25 8.5 8.0 1.06 68.00 544.00 9.0 8.0 1.12 72.00 576.00 8.0 8.0 1.00 64.00 512.00 8.5 8.0 1.06 68.00 544.00 9.0 8.0 1.12 72.00 i 576.00 T 676.50 5420.25 B20 84.5 80.0 10.53

Table A.I Cont. Bird Totals.

SPARROW

L W LA LW LW2

81 78.2 71.8 10.89 562.44 4062.05 82 82.8 79.6 10.35 661.48 5305.42 B3 74.3 70.9 10.47 528.93 3783.97 B4 68.4 65.2 10.48 448.36 2954.20 B5 65.6 60.8 10.76 411.44 2669.87 B6 62.8 54.3 11.66 341.65 1868.32 87 74.2 69.8 10.60 520.76 3675.90 88 61.4 56.4 10.91 346.28 1961.74 B9 79.3 71.2 11.12 568.00 4097.41 B10 72.0 68.0 10.55 494.04 3517.94

Si 719.0 668.0 .07.79 4883.38 33896.82

GREENFINCH

L W LA LW LW2

811 81.0 74.5 10.87 604.25 4521.62 B12 80.8 72.5 11.14 587.25 4284.36 B13 85.2 80.5 10.59 687.60 5577.55 814 76.9 74.0 10.37 570.00 4235.84 815 81.5 77.7 10.49 635.60 4989.45 B16 79.4 71.6 11.07 571.01 4111.21 B17 76.7 72.3 10.60 559.00 4111.21 818 85.0 83.2 10.21 717.80 5984.64 B19 74.0 72.5 10.21 537.25 3910.01 B20 84.5 80.3 10.53 676.50 5420.25

S2 805.0 758.8 106.08 6146.26 47146.14

X 1524.0 1426.8 213.87 11029.64 81042.96 xiii.

Table A.2. Pilot Sample (2)0 oocysts).

Analyses of Variance for L, W, L/W, LW and LN2 in eyepiece units

Each total sum of squares is subdivided into three components as follows:

(a) Between host species. (b) Between birds per species. (c) Within birds per species.

Each analysis of variance is set out as follows:

F. Sum of Deg. of Mean Variance P. Squares Freedom Square Ratio Probability

(a) %(S2)/100—X2/200 1 (h) Z (B2)/lo- I(52)/no 18 (c) E (x2) - (132)/10 180

Total: Z:(x2) — X2/200 199 xiv.

Table A.2 Cont. EYEPIECE UNITS

L. S. of S. D.F. M. Squ. F. P.

a 36.98 1 36.98 a/b 11.10 < 1% b 59.99 18 3.333 b/c 8.24 <0.1% c 72.77 180 0.4043

Total 169.74 199

W. S. of S. D.F. M. Squ. F. P.

a 41.22 1 41.22 a/b 10.71 'c 14 b 69.31 18 3.850 b/c 12.39 :0.1,:, c 55.92 180 0.3106 Total 166.45 199

L/W S. of S. D.F. M. Squ. F. P.

a 0.0146 1 0.0146 a/b 1.13 b 0.2335 18 0.01297 b/c 3.23 <0.1% c 0.7219 180 0.00401

Total 0.9700 199

LW S. of S. D.E. M. Squ. F. P.

a 7974.33 1 7974.33 Vb 11.229 (1% b 12782.67 18 710.148 b/c 10.533 <,0.1% c 12136.23 180 67.4235

Total 32893.23 199

LW2 S. of S. D.F. M. Squ. F. P.

a 877812.50 1 877812.50 a/b 10.75543 'c1% b 1469082.40 18 81615.68 b/c 10.254875 .0.1% c 1432569.77 180 7958.720

Total 3779464.67 199

xv.

Table A.3. Pilot Sample (200 oocysts).

Means for birds and hosts (microns).

SPARROW GREENFINCH

B.1 26.00 23.80 B.11 26.90 24.70 3.2 27.50 26.40 8.12 26.80 24.00 13.3 24.70 23.50 8.13 28.30 26.70 B.4 22.70 21.65 8.14 25.50 24.60 3.5 21.80 20.20 B.15 27.00 25.80 B.6 20.85 18.30 8.16 26.35 23.80 13.7 24.60 23.20 B.17 25.50 24.70 B.8 20.43 18.70 B.18 28.25 27.60 33.9 26.30 23.65 8.19 24.60 24.10 13.10 24.00 22.60 13.20 28.n0 26.60

Total 23.90 22.20 26.70 25.26 xvi.

Table A.4. Length and width of Isospora oocysts shown as frequency tables, (eyepiece units). (For explanation see page 20 ).

SPARROW

5.1 5.5 5.9 6.3 6.7 7.1 1 7.5 7.9 8.3 2.7 9.1 9.5 19.9 f.W W ' I

f 5.1 16 10 25 15 4 4 1 74 ..] 5.5 17 41 24 17 10 2 111 5.9 108 111 73 52 3 7 1 355 ----;-- 6.3 93 49 96 11 § 6 260 4-- t 6.7 85 137 30 18 2 272 7.1 1 274 88 179 35 14 2 592 7.5 67 77 23 13 6 2 188 F-- 7.9 106 66 25 16 213 8.3 9 3 81 2 22 8.7 • 3 1 2 6 3 1 4 if .1„ ! 16 27 174 243 228 573 199 394 140 60 36 6 I 1 : 2097

No.

Spherical oocysts (Main diagonal) 781 37.244

Subspherical (Second It 583 27.802

Oval It (Remainder) 733 34.954 Total: 2097 100 xvii.

Table A.4 Cont.

GREENFINCH

\ 1 I \\,,,, 5.1 5.5 5.9 16.3 '.).7 7.1 7.5 7.9 8.3 8.7 9.1 9.5 9.9 f.W _\ . ___ 1_5.1 1 1 F--- 5.5 1 l 1 3

5.9 3 6 14 2 2 30

6.3 10 6 18 8 7 1 50 1.-- 6.7 50 51 27 23 1 4 1 157

164 56 | 94 37 17 3, 371 7.1 __ 7.5 71 68 3 15 5 1 163 7 1 7.9 96 14 40 19 169

8.3 5 6 6 17

8.7 25 11 1 r 37 15 1 16 9.1 __—_ ___ f.1, 4 14 63 247 164 291 60 107 60 3 1 1 1014

No.

Spherical oocysts (Main diagonal) 439 43.294

Subspherical " (Second 217 21.400

Oval it (Remainder) 358 35.306

Total: 1014 100 Table A.4 cont.

-6gAFFINCH

1 \L 5.1 5.5 5.9 6.3 6.7 7.1 7.5 7.9 8.3 8.7 9.1 9.5 9.9 f.lk IN \ I 5.1 2 2 3 11 5.5 1 6 1 8 5.9 5 12 5 11 33 i 6.3 1 2 16 l 26 1 6.7 5 14 28 7.1 26 5 5 1 l l 39 7.5 3 4 3 l 11 7.9 7' 1 8.3 l ,____ _ . 8.7 2 4 6

9.1 2 1 3 f.l. 2 3 14 21 13 69 15 12 2 8 11 3 173

No.

Spherical oocysts (Main diagonal) 54 31.214 Subspherical " (Second " ) 50 28.902 Oval " (Remainder) 69 39.884 Total: 173 100 Table A.4 Cont.

NUTHATCH --Tr. 5.1 5.5 5.9 6.3 6.7 7.1 7.5 7.9 8.3 8.7 9.1 f.W W L

5.1 1 ..r. ... 1 5.5 2 3 5 5.9 5 2 1 1 9 6.3 43 1 4 1 49 6.7 21 7 1 1 1 31 7.1 49 7 11 1 1 69 7.5 3 4 1 8 7.9 13 6 1 20 8.3 2 1 3 f.L 1 2 I 8 45 23 61 11 30 11 I- 2 1 ! 195

No. Spherical oocysts (Main diagonal) 139 71.282

Subspherical " (Second it ) 31 15.897 Oval " (Remainder) 25 12.821 Total: 195 100 XX.

Table A.4 Cont.

GREAT TIT

5.9 6.3 6.7 7.1 7.5 7.9 8.3 8.7 9.1 9.5 f.W Id --1-- 5.9 1 1 1 3 6.3 6.7 1 2 2 1 6 7.1 1 5 6 2 1 15 7.5 1 4 1 5 415_ 7.9 8 1 5 2 1 17 8.3 2 1 1 4 8.7 2 1 3 , ___ ; 9.1 2 2 f.L 1 1 4 9 19 6 12I--11 2 65

No. Spherical oocysts (Main diagonal) 18 27.692 Subspherical " (Second 13 20.00 Oval " (Remainder) 34 52.308 Total: 65 100 Table A.4 Cont.

MISTLE THRUSH

1 L 4.7 5.1 5.5 5.9 6.3 6.7 7.1 7.5 f.tu I 4.7 1

5.1 2 1 3

5.5 9 2 2 1 14

5.9 47 4 3 54 i 6.3 10 1 11

6.7 5 2 7

7.1 2 2 7.5 1 J 1 1 f.1, 3 1 9 i 50 12 11 7 1 93

No.

Spherical oocysts (Main diagonal) 76 81.720

Subspherical " (Second il ) 6 6.452

3val " (Remainder) 11 11.828

Total: 93 100 Table A.4 Cont.

BLACKBIRD

1 \\\L\ 3.9 4.3 4.7 5.1 15.5 5.9 6.3 16.7 17.17.5 f.W W 1

3•9 1 1

4.3

4.7 1 3 1 1 6

5.1 9 3 9 6 1 28 ---- 5.5 6 14 1 5 1 27

5.9 17 12 7 5 41

6.3 4 3 1 8

6.7 2 1 3

f.L 1 1 12 10 41 23 13 11 2 114

No. 2. Spherical oocysts (Main diagonal) 38 33.333

Subspherical " (Second It ) 34 29.825 Oval " (Remainder) 42 36.842

Total: 114 100

Table A.4 Cont.

JACKDAW

3.9 4.3 4.7 5.1 5.5 5.9 6.3 6.7 17.1 7.5 7.9 f.W VA, T 3.9 1 1 2

4.3 3 3 -,- 4.7 13 1 1 15 4 -, ------5.1 150 1 3 1 2 1 158

5.5 97 1 1 99

5.9 446 1 2 1 450

6.3 78 1 79

6.7 105 16 121 102 102 _____7.14 _ f.1.. 1 3 13 151 99 45 80 108 123 1 1029

No. 1. Spherical occysts (Main diagonal) 995 96.696

Subspherical II (Second 1 ) 18 1.749

Oval II (Remainder) 16 1.555

Total: 1029 100 xxiv.

Table A.4 Cont.

STARLING

L 5.5 5.9 6.3 6.7 7.1 7.5 f.IN _ _...... \VII\ 5.5 4 4 I 5.9 29 5 1 35 6.3 18 4 22 6.7. 15 2 17 7.1 17 1 18 f.L 4 33 23 19 20 1 100 L

No. Spherical oocysts (Main diagonal) 83 83

Subspherical 11 (Second 11 ) 16 16

Oval 11 (Remainder) 1 1 Total: 100 100 xxv.

Table A.4 Cont.

CANARY

L 5.1 5.5 5.9 6.3 6.7 7.1 f.W

5.1 2 1 1 4 r----- 5.5 3 3 5.9 42 6.3 44 1 45 11111 10 52 36 6.7 III i 36 7.1 10 10 f.I.. 2 3 43 45 36 21 150

No.

Spherical oocysts (Main diagonal) 137 91.333

Subspherical 11 (Second

Oval (Remainder) 13 8.667

Total: 150 100 Table A.4 Cont.

ROBIN

N, I --.* N. 3.9 4.3 4.7 5.1 5.5 15.9 6.3 16.7 17.1 7.5 17.9 8.3 8.7 f.tY W \\ 3.9 2 4 l 7 4.3 5 2 5 l 13 4.7 13 9 1 2 1 23 5.1 106 33 26 10 8 5 1 189 5.5 64 25 3 6 6 1 2 107

5.9 40 7 10_ 17 7 3 — 84 6.3 2 1 3 6.7 1 l 2

7.1 1 1 2 f.L 2 9 13 120 98 92 24 25 30 9 6 1 2 430

No. Spherical oocysts (Main diagonal) 231 53.721

Subspherical " (Second I I 80 18.635 Oval " (Remainder) 119 27.674 Total: 430 100

Table A.4 Cont.

HEDGE SPARROW

! , L 4.7 15.1 5.51 5.9 16.3 16.7 7.1 7.5 17.91 t.t, LI I 1 L I 4.7 2 3 1 6 5.1 1 18 23 4 49 5.5 4 11 2 24 5.9 5 9 1 18 6.3 3

6.7 7.1 7.5 7.9 1 24 42 12 19 1 1 100

No.

Spherical oocysts (Main diagonal) — —

Subspherical ii (Second It ) 10 10

Oval f 1 (Remainder) 90 90 Total: 100 100

Table A.4 Cont.

CARRION CROW

5.1 15.5 5.9 6.3 6.7 7.1 7.5 7.9 8.3 8.7 9.1 f.VI W

5.11 5 J.- 1 6 5.5 5 5 5.9 14 2 16 6.3 1 1 6.7 7 3 10 -, 7.1 20 8 29 7.5 17 17 — -1 _ 7.9 161 16 f.L 5 5 15 1 7 20 17 29 1 100

No. Spherical oocysts (Main diagonal) 85 85

Subspherical (Second Oval It (Remainder) 15 15 Total: 160 100

Tables A.5 and A.6.

Tests for Goodness of Fit of data with Normal expectation: chi-squared.

KEY: Variate, in eyepiece units (Width of grouping: 0.4 units) (xa): Deviate. (x-1)/S: Normal deviate. Z: Normal deviate. 100;10::(2): Percentage frequency. E: nZ/t(Z) = Expected actual frequency, 0: Observed actual frequency. 3-,(0-E)2/E.

NOTE: All values of ;X in Tables A.5 and A.6 are very high, indicating that the data can not be taken to represent a single Normal distribution.

XXX.

Table A.5. n = 2097 SPARROW Lenpth = 7.19 S = 0.813

x-5t x5R Z E 0 X.2 S 5.1 -2.09 -2.57 0.0147 15.18 16 0.0443 5.5 -1,69 -2.08 0.0459 47.40 27 8.78 5.9 -1.29 -1.59 0.1127 116,38 174 28.53 6.3 -0.89 -1.09 0.2203 227.50 243 1.056 6.7 -0.49 -0.603 0.3332 344,09 228 39.167 7,1 -0.09 -0.11 0.3965 409.46 573 65.32 7.5 0.31 0.380 0.3712 383.34 199 88.64 7.9 0.71 0.07 0.2732 282.13 394 50.08 8.3 1.11 1.36 0.1582 163.37 140 3.34 8.7 1.51 1.86 0.0707 73.01 60 2.318 9.1 1.91 2.35 0.0252 26.02 36 3.828 9.5 2.31 2.84 0.0071 7.33108 6 7 0.476 9.9 2.71 3.33 0:0017 1,755 9• 1 Total 2.0306 2096.96 2097 291.5793

SPARROW Width n = 2097 Tc = 6.73 S = 0.7694 x2 x x-R x-5-c Z E 0 S

5.1 I -1.63 -2.12 0.04.22 46.40 74 16.42 5.5 -1.23 -1.60 0.1109 121.96 111 0.985 5.9 -0.83 -1.08 0.2227 244.90 1355 49.50 6.3 -0.43 -0.56 0,3410 375,00 260 35.27 6.7 -0.03 -0.04 0.3986 438.36 272 63.13 7.1 0.37 0.48 0.3555 390,96 592 103,38 7.5 0.77 1.00 0,2420 266.14 188 22.94 7.9 1.17 1.52 0.1257 138.29 213 40.36 8.3 1.57 2.04 0.0498 54.77, 22 19.607 6 / 8.7 1.97 2.56 0.0151 16.603.63 / 20.23 4 10 5.17 9.1 2.37 3.08 0.0033 Wp104016 moo 1.9068 2097,01 2097 356.762 Total ____

Table A.5 Cont, n . 1014 GREENFINCH Length, 3E = 7.72 S = 0.681

x x-R x-R Z E 0 3

5.9 -1.82 -2.67 0.0113 6.7 -.. 10 h i 18 7.601 6.3 -1.42 -2.08 0.0459 27.364 .5441 6.7 -1.02 -1.50 0.1295 77.20 63 2.612 7,1 -0.62 -0.91 0,2637 157.20 247 51.3 7.5 -0.22 -0.323 0.3790 225.93 164 16.97 7.9 0.18 0.264 0.3857 229.92 291 16.23 8.3 0.58 0.852 0.2780 165.72 60 67.44 8.7 0.98 1.44 0.1415 84.35 107 6.08 9.1 1.38 2.03 0.0508 30.28) 60 ) 9.5 1.70 2.61 0.0132 7.87; 39.58 3> 64 15.07 9.9 2,18 3.20 0.0024 1.43) 1) Total 1.7010 1014.00 1014 183.303

GREENFINCH Width n = 1014 M = 7.26 S = 0.6301

2 x x--51 Zzal; Z E X S 5.1 -2.16 -3.43 0.0012 0.77 ) 1 5.5 -1.76 -2.79 0.0081 5.21 30.90 3 )34 0.33.1 5.9 -1.36 -2.16 0.0387 24.92 300 6.3 -0.96 -1.52 0.1257 00.94 50 11.827 6.7 -0.56 -0.890 0.2605 172.88 157 1.46 7.1 -0.16 -0,254 0.3867 249.00 371 59.77 7.5 0.24 0.381 0.3712 239.00 163 24.17 7.9 0.64 1.02 0.2371 152.67 169 1.75 8.3 1.04 1.65 0.1023 65.87 17 36.26 8.7 1.44 2.28 0.0297 19.12 37 16.69 9.1 1.84 2.92 0.0056 3.60 16 42.44 Total 1.5748 1013.98 1014 194.678

Table A.6.

n = 781 SPARROW Spherical x = 6.89 S = 0.7273

x-x Z Z 100 E 0 f77) 5.1 -1.79 -2.46 0.0194 1.07 8.37 16 6.95 5.5 -1.39 -1.91 0.0644 3.56 27.79 17 4.19 5.9 -0.99 -1.36 0.1582 8.74 68.27 108 23.12 6.3 -0.59 -0.81 0.2874 15.88 124.03 93 7.76 6.7 -0.19 -0.26 0.3857 21.31 166.45 85 39.86 7.1 0.21 0.29 0.3825 21.14 165.07 274 71.88 7.5 0.61 0.84 0.2803 15.49 120.97 67 24.08 7.9 1.01 1.39 0.1518 8.39 65.51 106 25.03 8.3 1.41 1.94 0.0608 3.36 26.24 11.33 8.7 1.81 2.49 0.0180 0.99 7.77 6 8.29 3 4 0.632 9.1 2.21 3.04 0.0012 0.066 0.52 3 Total 1.8097 99.996 780.99 781 1214.83

GREENFINCH Spherical n = 439 3r = 7.44 S = 0.631

x x...4C ro2 x2i 1 Z Z 100 E 0 tiN., S i:717) +---- i 5.9 -1.54 -2.44 0.0203 1.29 5.67 6.3 -1.14 -1.81 0.0775 4.93 21.65). 27.32 10)13 7.506 6.7 -0.74 -1.17 0.2012 12.80 56.20 50 0.684 7.1 -0.34 -0.54 0.3448 21.94 96.32 164 47.55 7.5 0.06 0.095 0.3975 25.28 110.98 71 14.40 7.9 0.46 0.73 0.3056 19.45 85.37 96 1.32 8.3 0.86 1.36 0.1586 10.07 44.19 5 34.75 8.7 1.26 2.03 0.0540 3.44 15.08 25 6.53 9.1 1.66 2.63 0.0126 0.80 3.52 15 37.44 Total 1.5715 100.00 438.98 439 150.18

Table A.6 Cont. n = 733 SPARROW Oval Length = 7.55 S = 0.075

x x-2 x-3-c Z Z 100 E 0 De s f72) 5.9 -1.65 -1.88 0.0601 3.17 23.21 25 0.138 6.3 -1.25 -1.43 0.1435 6.67 48.91 39 2.008 6.7 -0.85 -0.97 0.2492 11.59 84.94 94 0.966 7.1 -0.45 -0.51 0.3503 16.29 119.40 I162 15.199 7.5 -0.05 -0.06 0.3982 10.52 135.27 44 61.502 7.9 0.35 0.40 0.3683 17.13 125.53 211 58.194 8.3 0.75 0.86 0.2756 12.01 93.94 65 8.915 8.7 1.15 1,31 0.1691 7.86 57.64 54 0.230 9.1 1.55 1.77 0.0033 3.87 20.39 32 0,459 9.5 1.95 2.22 0.0339 1.58 11.5-5 1 5.30 i7 4.502 9.9 2.35 2.68 0.0110 0.51 3.75 1) Total 2.1505 100.00 732.53 733 152.193

GREENFINCH Oval Length n = 358 i = 8.10 S = 0.6429

x x52 x-a Z Z 100 E 0 Xi2 S fri )

6.3 -1.00 2,80 0.0079 0.49 1.76110 04 3ia 0.414 2.10 0.0371 2.31 0,20 . 7 6.7 -1.40 1,21 7.1 -1,00 1.56 0.1102 7.37 26.35 32 -0.60 0.935 0.2589 16.13 57.76 37 7.46 7.5 20.96 7.9 -0.20 0.312 0.3802 23.70 04.03 127 8.3 0.20 0.312 0.3802 23.70 84.83 41 22.65 8.7 0.60 0.935 0.2589 16.13 57.76 76 5.76 0.1182 7.37 26.35" 34) 9.1 1.00 1.56 0.012 9.5 1,40 2.18 0.0371 2.31 0.20 36.39 2)37 9.9 1.80 2.80 0.0079 0.49 1.76 1) Total 1.6046 100.00 357.96 358 58.460

Table A 6 Cont. n = 733 SPARROW Oval Width 7 = 6.567 S = 0.7887 T--- x x-rc X••:5"C Z 100 E 0 )C2 S 5.1 -1.47 -1.86 0.0707 3.64 26,72 48 16,95 5.5 -1,07 -1.36 0.1582 8.15 59.78 53 0.769 5.9 -0.67 -0.85 0.2780 14.33 105.06 136 9.112 6.3 -0.27 -0.34 0.3765 19.40 142.28 118 4.143 6.7 0.13 0.16 0.3939 20.30 148.85 50 65.645 7.1 0.53 0.67 0.3187 16.43 120.44 230 99.662 7.5 0.93 1.18 0.1909 10.25 75.16 44 12.9113 7.9 1.33 1.69 0.0957 4.93 36.16 41 0.648 1.87 13.72), 10) 8.3 1.73 2.19 0.0363 I 8.7 2.13 2,70 0.0104 0.54 3.93?18.56 20.3 1.666 0.0024 0.12 I 0.91) 1) 9.1 2.53 3.21 i Total 1.9397 99.96 733.01 733 211.513

n = 358 GREENFINCH Oval Width = 7.05 S = 0.5925

x x-It x.-1Z Z Z 100 E 0 ;CF S i 1 ,':(Z) --1 5.1 -1.95 -3.29 0.0017 0.115 0.41) 1) 5.5 -1.55 -2.62 0.0129 0,870 3.11118.20 2k27 4.255 5.9 -1.15 -1.94 0.06013 4.101 14.68) 24, 6.3 -0.75 -1.26 0.1804 12.168 43.56 34 2.098 6.7 -0.35 -0.59 0.3352 22.609 80.94 56 7.685 7.1 0.05 0.08 0,3977 26.825 96.03 151 31;466 7.5 0.45 0.76 0.2989 20,161 72.17 24 32.151 7.9 0.85 1.43 0.1435 9.679 34.65 59 17.112 2.907 10.L1 8;3 1.25 2.11 0.0431 -11244 617 2.379 8.7 1.65 2.78 0.0084 0.566 2.03) • lf Total 1.4826 100.00 357.99 358 97.146 Reprinted from EXPERIMENTAL PARASITOLOGY, Volume 14, Number 1, August 1963 Copyright © 1963 by Academic Press Inc. Printed in U. S. A.

EXPERIMENTAL PARASITOLOGY 14, 49-51 (1963)

Mechanical Transmission of Malaria

M. Anwar '

Department of Parasitology, London School of Hygiene and Tropical Medicine, London, England

(Submitted for publication, 29 October 1962) In a series of experiments on mechanical transmission of malarial parasites, using Aedes aegypti as the transmitting agent of Plasmodium gallinaceum in chicks, only one case of positive infection was achieved. When Anopheles stephensi was used to transmit P. berghei in mice, all results were negative. Rhodnius prolixus was used successfully in the transmission of P. berghei: five out of seventeen mice became infected. The greater success with R. prolixus is related partly to the experimental technique and partly to the quantity of blood involved and the digestive habits of the insects.

INTRODUCTION a strain isolated in the Belgian Congo from In contrast to the considerable amount of Thamnomys surdaster and sent to the Lon- information in existence about the mechanical don School of Hygiene and Tropical Medicine, transmission of trypanosomes, spirochetes, where it has been maintained by blood inocu- bacteria, and viruses, very little is known re- lation. garding the mechanical transmission of For the mechanical transmission of P. malaria. It is known that malaria can be gallinaceum 4-9-day-old chicks of the breed transmitted from one human being to another Rhode Island Red crossed with Light Sussex by parenteral injection of trophozoites, but were used, while 3-week-old white mice (Mus the one and only attempt using an insect musculus) served as hosts for P. berghei. agent, that of Mayne (1928) using Aedes The insects which were employed for the thibaulti and Anopheles quadrimaculatus to transmission experiments were Rhodnius pro- transmit tertian malaria, proved negative. A lixus, Anopheles stephensi, and Aedes aegypti series of experiments was designed to deter- var. queenslandensis. The experiments with mine whether mechanical transmission of the mosquitoes were conducted in an insec- malaria by blood sucking insects is possible. tary maintained at a constant temperature of 28°C and a relative humidity of 80%. MATERIALS AND METHODS For each experiment with R. prolixus a Two species of malarial parasites were mouse heavily parasitized with P. berghei was used: Plasmodium gallinaceum Brumpt 1935, fixed on a square of cardboard, side by side a strain obtained from the Wellcome Labora- with a clean mouse. The hair on the back of tories, London, in 1936 and since maintained both mice was shaved and the areas were by blood inoculation through 4-9-day-old- wetted with a piece of moist cotton wool to chicks with occasional mosquito passage; and flatten the remaining hairs against the skin, Plasmodium berghei Vincke and Lips 1948, rendering it easier for the bugs to feed. The bugs were confined in a glass jar by means 1 Present address: Department of Zoology and Applied Entomology, Imperial College, London, of a filter paper perforated by a central hole S.W.7, England. 1 inch in diameter. The mice were arranged 49 50 ANWAR with the shaved part of the back directly over TABLE I the hole so that the bugs had access to the Attempts to Infect Mice and Chicks with skin. The bugs were repeatedly fed first on Plasmodium Species Positive results an infected mouse for 1 minute and then Number immediately on a clean mouse for another Vector and of experi- Actual To of minute, for a period of 30 minutes. This ex- species ments number total periment was performed daily for 7 days Rhodnius prolixus using the same pair of mice. From the eighth P. berghei in mice 17 5 30 day following the cessation of feeding, blood Aedes aegypti samples from the clean mice were taken for P. gallinaceunz in chicks 21 1 5 examination every day for the next 15 days. In all, seventeen experiments were performed Anopheles stephensi P. berghei in mice 28 0 0 in this way. In experiments with mosquitoes approxi- more successful agent in the mechanical mately 400 A. aegypti var. queenslandensis transmission of malarial parasites than the which had been starved for 24 hours were mosquito. allowed to feed on infected and clean chicks. As before, pairs of animals were used, one DISCUSSION heavily infected with P. gallinaceum and the Failure to obtain as large a number of other clean. Both chicks were fixed on positive infections with the mosquitoes as squares of cardboard with their feet and with the bugs is probably related to the fact heads tied down with bandages to prevent that relatively few mosquitoes feed on clean them from disturbing the mosquitoes. The animals directly after an infective meal. The mosquitoes feeding on the two birds were nature of the experiment precludes an ac- disturbed every 30 seconds so that they could curate estimate of the number of mosquitoes alternate their feeding between the two. This passing directly from one to the other, but experiment was conducted for 30 minutes in all probability the greater the number of each day for 4 days, after which the mos- mosquitoes feeding directly on a clean animal quitoes were discarded. A similar experiment after an infective meal, the higher would be was performed using Anopheles stephensi as the number of positive infections obtained. the vector for the transmission of P. berghei Furthermore, the mosquito proboscis is much in mice. Blood samples were taken from the smaller than that of Rhodnius and the clean chicks and mice on the fifth day after amount of blood carried by it is correspond- the first transmission experiment and daily ingly small. Falleroni (1926), in a study of during the next 15 days. the physiological processes associated with parasitism of RESULTS Anopheles maculipennis, draws attention to two different uses of the liquid Plasmodium berghei was successfully trans- sucked by the mosquito. The two constitu- mitted from infected mice to clean mice in ents of its food material, blood and plant five of the seventeen attempts. Plasmodium juices, were consigned to different compart- gallinaceum was transmitted by A. aegypti ments of the gut, the former to the stomach to only one out of twenty-one chicks, but A. and the latter to the esophageal diverticulum. stephensi failed to transmit P. berghei to Blood passes at once into the stomach of the any of the twenty-eight mice used. The com- biting Anopheles, and, as there is no regurgi- plete results are recorded in Table I. tation, the two food elements are isolated and Thus Rhodnius prolixus appears to be a digested separately. Falleroni, aided in his MECHANICAL TRANSMISSION OF MALARIA 51 conclusions by the dissection of various parts quite ready to feed the next day. This, of the mouth apparatus and examination of coupled with the fact that the bugs had much contents, excluded the possibility of direct larger mouth parts, is likely to have resulted transmission of malaria when he found no in greater success in transmission of malaria parasitized erythrocytes remaining in the parasites by Rhodnius. proboscis. In the current experiments six ACKNOWLEDGMENTS slides were examined containing the dissected proboscides of mosquitoes which had just fed I am indeed grateful to Professor P. C. C. Garn- ham, head of the Department of Parasitology, Lon- on an infected chick or mouse; no infected don School of Hygiene and Tropical Medicine, not erythrocytes were found and even uninfected only for his permission to carry out these experi- ones were rare. It would appear in these cases ments but also for his interest and advice in this that either the proboscis failed to penetrate work. to a blood vessel or the blood remained only My thanks are due also to Dr. E. U. Canning of the Zoology Department, Imperial College of Science a very short time in or on the proboscis. and Technology, for correction of the manuscript, Trypanosomes have been transmitted and to Mr. W. Chinery for help during the early mechanically using Mansonia and Aedes on part of the investigation. more than one occasion (Martin et al. 1908; REFERENCES Nieschultz, 1940). However, being extracor- FALLERONI, D. 1926. Notes on the biology of puscular parasites, they do not have to rely Anopheles maculipennis. Rivista di Malariol- on the presence of erythrocytes for success- ogia 5, 551. ful transmission but can move actively in a MARTIN, G., LEBOEUF, A., AND ROUBAITD, E. 1908. thin film of blood plasma on the proboscis. Experiences de transmission du (Nagana) Par The larger number of positive infections les Stomoxes et Par les Moustiques du genre Mansonia. Bulletin de la Societe de Pathologic R. prolixus obtained in the experiments with Exotique et de ses Filiales 1, 355-358. probably resulted from the immediate con- MAYNE, B. 1928. A note on some recent attempts tinuation on a clean mouse of an interrupted to transmit malaria organisms mechanically feed on an infected mouse. As many as fifty through mosquito biting. Indian Journal of bugs fed on the infected mouse, followed by Medical Research 15, 1067-1071. NIESCIIULTZ, 0. 1940. t)ber die mechanische 'Ober- an immediate continuation of feeding on the tragung, von Trypanosoma congolense durch clean animals. Moreover, the bugs never fed Aedes aegypti. Archiv fiir Schips and Tropen- to repletion at any time and therefore were Hygiene, 44, 30-33.