Biochemical and Biological Characters of Eimeria

BIOCHEMICAL AND BIOLOGICAL CHARACTERS

David Rollinson, B.Sc.

Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy of the University of London.

Department of Zoology and Applied Entomology, Imperial College Field Station, Ashurst Lodge, Sunninghill, Ascot,

Berkshire. March 1977 TO MY PARENTS Abstract

The use of biochemical techniques for the characterisation of Eimeria spi) has been investigated. Thin layer starch gel electrophoresis has been used to demonstrate the following enzymes in sporulated oocysts of E. tenala : alkaline phosphatase, fructose 1,6-diphosphatase, leucine aminopeptidase, hexose 6-dehydrogenase, glucose 6-phosphate dehydrogenase,dv-glycerophosphate dehydrogenase, isocitrate dehydrogenase (NADP), malate dehydrogenase, malic enzyme, lactate dehydrogenase, 6-phosphogluconate dehydrogenase, adenylate kinase, hexokinase, glucose phosphate isomerase, aspartate aminotransferase, phosphoglucomutase and tetrazolium oxidase.

The electrophoretic mobility of a selection of these enzymes allowed E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella from chickens, E. coecicola, E. intestinalis, E. irresidua, E. magna and E. stiedai from rabbits, E. ninakohlyakimovae, E. ovina and E. weybridgensis from sheep and E. bateri from quail to be differentiated. Intraspecific variation of enzymes has also been demonstrated.

The enzyme type of E. acervulina var. mivati confirmed its position within the E. acervulina complex : genetic studies, utilising as characters enzyme markers and the ability to develop in chick embryos, showed the absence of reproductive isolation between populations of E. acervulina var. mivati and E. acervulina. Similarly, the enzyme type of E. maxima var. indentata confirmed its position within the E. maxima complex : genetic studies, utilising enzyme markers and drug resistance, showed that populations of E. maxima var. indentata and E.maxima were capable of sharing the same gene pool. Consideration has been given to population structure and inheritance of characters. With the exception of leucine aminopeptidase, no variation in the isoenzymes of E. tenella at different stages of development in vitro or in vivo was found.

The buoyant densities of DNA from three Eimeria spp were identical.

No variation was found in LDH and AP isoenzyme patterns in the serum of chickens infected with E. terzena; a decrease in the serum AP levels was demonstrated. An increase in serum LDH activity was noticed in E.stiedai infections of rabbits.

E. terzena (Houghton) was found to develop completely in pheasant and quail embryos and to develop partially in duck embryos and in chicken and quail kidney cell cultures. Latent sporozoites in cell cultures were metabolically active and gave positive reactions for LDH and G6PDH. Sporozoites of. E. terwlia invaded the caeca of quail chicks but did not develop.

Preliminary investigations were made on the induction of tolerance to E. terzena in chickens. Acknowledgements

I am most grateful to Dr. E. U. Canning for her supervision and guidance throughout the course of this study.

Facilities at the Field Station were kindly provided by the Director, Prof. T. R. E. Southwood.

I wish. to thank the following:-

Elizabeth Atkinson (Imperial College), for help with the analysis of LDH in rabbit serum. Mr. P. Bush (Imperial College), for advice on tissue culture techniques and for providing cultures of Aedes pseudoscutellans. Mr. A. F. Batty (Merck Sharp and Dohme Ltd.), for supplying oocysts of the Macster isolate.

Dr. J. Catchpole (Central Veterinary Laboratory, Weybridge), for providing serum from rabbits infected with E. stiedai. Dr. M. L. Chance (Liverpool School of Tropical Medicine), for his collaboration during the buoyant density studies of eimeriine DNA. Toni Davenport (Imperial College), for providing duck eggs and quail and for assistance with the maintenance of animals. Mr. J. K. Lenehan (Imperial College), for advice on polyacrylamide gradient gel electrophoresis.

Dr. P. L. Long and colleagues (Houghton Poultry Research Station), for advice on basic techniques and for the provision of many oocyst cultures.

Dr. E. Michael (May and Baker), for providing oocysts of E. acervulina (Ongar). Dr. C. Parr and Mr. I. Bagster (London Hospital), for an introduction to thin layer starch gel electrophoretic techniques and for the extended loan of an electrophoresis tank. Dr. J. F. Ryley and colleagues (ICI, Pharmaceu- ticals Ltd.), for supplying cultures of rabbit coccidia. Mr. A. J. Spencer (Central Veterinary Laboratory, Weybridge), for providing fertile quail eggs and advice on husbandry methods, and to Dr. R. B. Williams (Wellcome Research Laboratories), for the provision of oocyst cultures.

I am particularly indebted to Dr. L. P. Joyner, Mr. C. C. Norton and colleagues (Central Veterinary Laboratory, Weybridge), for the provision of many of the oocyst cultures used in this study and for their collaboration in experiments involving E. maxima and E. maxima var. indentata.

I am grateful to Lynne Gillespie for showing endless patience whilst learning to decipher my handwriting and for the efficient typing of this thesis.

Finally, my thanks are due to many friends at the Field Station for their interest and kind help during the course of this investigation and to the Medical Research Council for their financial support.

CONTENTS

Page

Abstract ...... 3 Acknowledgements ...... 5 Introduction ...... 14

Introductory Review ...... 16 1. Historial Aspects• • • • • • • • •• .. 16 (A) The Genus Eimeria ...... 16 (B) Coccidia parasitising the domestic fowl .. .. 17

2. Species Characters of Eimeria .. .. Oe .. 19 (A) Morphological Characters ...... 21 i) The Oocyst ...... 21 ii) Endogenous stages .. • • • • . .. 23 (B) Biological Characters .. • • •• .. 24 Pathogenicity • • • • •• .. 24 i) ' ' ii) Prepatent Period .. .. • •• .. 24 iii) Patent Period ...... 26 iv) Reproductive potential .. . .. 26 v) Sporulation time .. • • •• .. 27 vi) Development in culture • • •• .. 28 vii) Drug resistance • . • • • .. 28 viii) Host and site specificity •• .. 29 (C) Immunological Specificity. . .. 29 (D) Concluding Remarks . • • •• .. 32

34 Materials and Methods •• • • 1. Parasites 34 34 2. Experimental Birds • • •• •• 3. Bird Infections • • • • • • .. 37 (A) Isolation Procedures .. ... 37 (B) Standardisation of Dose and Inoculation procedures 38 (C) Single Oocyst • • .. .. • • .. 38 (D) Abnormal Infection Routes • • • . .. 38 .. 39 4. Cortisone Treatment • • '' ' 5. Avian Embryos • • • • . • • . .. 39

6. Avian Embryo Infections • • • • • • .. 39 • ▪

8 Page

41 7. Isolation and Harvesting of Oocysts • • • • • • • •

(A) Collection of oocysts from faeces OS 00 00 41

41 (B) Collection of oocysts from caeca .. 66 06

(D) Sporulation ......

(E) Cleaning and sterilising oocysts .. • • • • .. 42

8. Preparation of sporozoites • • • • - .. .. 42

9. Preparation of merozoites • • • • .. • • .. 43

(A) Preparation of merozoites from chicken caeca .• 43

(B) Preparation of merozoites from embryo and

in vitro culture .. • • .. • • • • . . 43

10. Electrophoresis .. .. • • .. • • • • .. 44

(A) Preparation of enzyme samples .. .. • • .. 44

(B) Polyacrylamide gradient gels .. • • • • .. 45

(D) Thin-Layer Starch gels • • .. • • .. .. 46

(E) Enzyme assay solutions • • • • • • . . . 47

11. (A) Deoxyribonucleic acid Isolation .. • • .. . . 62

(B) Analytical Caesium Chloride density gradient

centrifugation • • .. • • .. • • • • 62

.. • • 12. Tissue Culture .. .. • • . . 63 ''

(A) Sterilisation procedures .. • • • • .. . . 63

(B) Monolayer cultures. .. • • • • . . . . 63

(D) Organ slices .. . .. • • • • • .. 64

13. Staining Techniques .. • • • • • • .. • • .. 64

14. Cytochemistry .. • • • .. • • . . 65

15. Measurements .. • • • • • • • • .. • • . . 65

16. Serum samples .. • • • -. . . . 65

PART I : Biochemical characters of Eimeria species

Introduction 69 ' •

(A) The Characterisation of Protozoa by biochemical

criteria • • • • • • • • • • • • 69

General Considerations • • • • • • . . 69 • 1) Sarcodina • • .. • • • • .. 72

2) Mastigophora ...... 0 a . 74

3) Ciliophora ......

4) Sporozoa . • • • • • . ... • g

5) Microsporidia ...... 85

(B) Isoenzymes in relation to parasitic diseases 86

Page

Results • •• 88 1) Identification of enzymes in oocysts of Eimeria species 88 2) The effects of extraction and storage procedures on water soluble proteins • • • • ...... 91 3) Electrophoretic conditions and enzyme migration ..•• 94 4) Enzyme analysis of oocyst cultures of different ages .. 97 5) Electrophoretic analysis of enzymes in different stages

of the life cycle .. • • • • • • • • • • 100

(a) Oocysts ...... • • • • 100

(b) Sporozoites ...... • • • • 101

(c) Merozoites ...... • • • • 101 6) Total protein in oocyst extracts • . • • • • 103 7) Comparative electrophoretic studies of enzymes in eimeriine parasites .. • • ...... 106 (a) Characterisation of Eimeria species of the chicken 106

i) Differences between species • • • • 106

ii) Differences within species • • • • 113

(b) Characterisation of Eimeria species of the rabbit 117 i) Differences between species .. •• • • 117 ii) Differences within species .. • • • • 125 (c)" Characterisation of Eimeria species of the sheep 125 i) Differences between species .. •• • • 125 (d) Identification of an isolate from quail •• 126 8) The use of enzyme markers in genetic studies 128 (A) The E.acervulina complex : • • • • 128 i) Oocyst sizes • • • • • • 128 ii) Enzyme types • • • • • • 128 iii) Ability to grow in embryos 128 • (a)E.acervulinavar.mivati (Houghton) • • 128 (b)E.acervulina (Weybridge) •• • • • • 130 (c) E.acervulina ('M' and Houghton strains) 130 iv) Investigations into the viability of 'contaminating oocysts' enclosed in incubating eggs • • 131 v) The cross between E.acervuZina (Weybridge) and E.acervulimavar.mivati (Houghton) ..• • 131

(B) The E.maxima complex : • • • • • • • • • 136 i) Enzyme type ...... 136 ii) Crosses between E.maxima (Weybridge) and E.maxima var. indentata • • • • 136 (a) Experiment One .. •• . • • . 136 (b) Experiment Two .. • • • • . • • 141 (c) Experiment Three ...... 141 9) Clones of E.tenella • • • - ...... 145 10) Buoyant density of eimeriine DNA 145 11) Isoenzymes in relation to coccidiosis •• • • 146 (a) E.tenelia infections •• • • 146 (b) E. stiedai infections 146

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Discussion •• • • • • • • • • • • • • • • 149 (A) Biochemical considerations .. .. • • .. .. 149 (B) Recognition of Populations of Eimeria by their enzyme type 152 i) Identification of Eimeria spp using biochemical data 152 ii) E.acervuZina complex ...... 155 iii) E. maxima complex • • • • • • • • • • • • 155 (C) Buoyant density of eimeriine DNA ...... 157 (D) Significance and use of biochemical data in the taxonomy of the 158 Eimeria • • .. • • • • • • • • i) The value of electrophoretic data .. .. 158 ii) Assessment of relationships within the genus 160 iii) Speciation .. • • .. 161 " " " iv) Adaptive significance of enzyme variations .. 162 (E) Population structure with particular reference to laboratory cultures ...... • • • . • • 163 ' ' (F) Clones of coccidia ...... 166 (G) Sexual differentiation • • ...... 169 (H) Consequences of the nuclear divisions occurring within the oocyst • • • • • • .. .. 171 ' ' ' ' (I) Isoenzymes in relation to coccidiosis ...... 175

PART II : Biological Characters of Eimeria species

Introduction .. • • .. .. • • • • 177 Specificity of Eimeria species • • • • • • • 177 1. Host Specificity .. • • • • .. • . • • 177 2. Attempts to breakdown the specificity of Eimeria spp 181 3. Specificity of Eimeria spp of birds in vitro and embryo culture • • • • 183 " " ' ' • •

Results ...... 186 1. Development of Eimeriaspp in chicken embryos .. •• 186 2. Development of E.tenella (Houghton) in avian embryos 186 Development of 3. Eimeriaspp in vitro • • 189

(a) Cells cultures • • • • • • • • 189

(b) Cell suspensions • • • • 193 (c) Organ slices 193 4. Attempts to obtain development of E.tenella (Houghton) in Coturnix coturnix japonica .. • • .. 193 5. Site specificity of E.tenella in chicks . . 197

6. Cytochemistry . • • • • • • • 199

Discussion 204

• • References 00 00 . . 0 • 209 Subsidiary matter 0 . 0 0 00 041 00 08 23o 11

LIST OF TABLES

TABLE Page

One: Details of Eimeria cultures used in this study 35

Two: Buffer systems used in thin-layer starch gel electrophoresis . . 48

Three: List of Enzymes and buffer systems •• • • 50

•• 52 Four: Enzyme assay solutions • •

Five: . Incubating media •• . • • . 66 • • • •

Six: Enzymes identified in water soluble extracts of

Eimeria tenella oocysts • • • • • • 89

Seven: Enzymes identified in water soluble extracts of oocysts of Eimeria spp 90

Eight: Measurements of oocysts .of E. acervulina •• •• 129

Nine: Spectrophotometric analysis of AP levels in the serum of two—week-old Ranger cockerels inoculated with 4 1 x 10 oocysts of E. teneZla (Houghton) .. 147

Ten: Biochemical key to certain Eimeria spp from the chicken 153

Eleven: Electrophoretic migration of ten enzymes in Eimeria spp with reference to E. tenella (Houghton) .. 154 12

LIST OF FIGURES

FIGURE Page

One: Inoculation of sporozoites into a 10—day7old chicken 1 0 embryo •. • • • • • . • • . • 4 Two: Variation in the migration of G6PDH in oocysts of E. acervulina strains, attributable to differences in electrophoretic conditions 95

Three: Glucose phosphate isomerase in oocysts of different ages 98

Four: Leucine aminopeptidases in sporulated and unsporulated oocysts of E. tenella • • • • • • 98 Five: Purified second generation merozoites of E. tenella (Houghton) isolated from chicken caeca . 102

Six: Total' proteins of Eimeria spp demonstrated on polyacrylamide gradient gels 104

Seven: Electrophoretic mobility of enzymes in Eimeria spp from the chicken 108

Eight: Glucose phosphate isomerase activity in mixed populations of E. acervulina (Weybridge) and E. tenella (Houghton) 114

Nine: Intraspecific variation of glucose phosphate isomerase and hexokinase in E. tenella 114

Ten: Intraspecific variation of glucose phosphate isomerase in E. acervuZina 116

Eleven: Electrophoretic mobility of enzymes in Eimeria spp from the rabbit 119

Twelve: Electrophoretic mobility of enzymes in Eimeria spp from the sheep .. 122

Thirteen: Intraspecific variation of glucose phosphate isomerase in 127 E. bateri. Oocysts of E. bateri

Fourteen: E. acervulina /E. acervulina var.mivati cross 132

Fifteen: Gludose phosphate isomerase in populations in the

E. acervuZina/E. acervuZina var.mivati cross 1314 13

LIST OF FIGURES (cont.)

FIGURE Page

Sixteen: Experiment One: E. maxima/E.maxima var.indentata cross 137

Seventeen: Experiment Two: E. maxima/E.maxima var.indentata cross 140

Eighteen: Experiment Thlee:E. maxima/E.maxima var.indentata cross 142 Nineteen: LDH IsoenzymeS of serum from a rabbit inoculated with 5 x 103 oocysts of E. stiedai (Weybridge) • 147 Twenty: Inheritance of characters within an oocyst 167

Twenty- Transfer of characters during meiosis 172 one: 7 Twenty E. tenella (Houghton) infection in chorioallantoic two: membrane of chicken embryo .. • • • . 187

Twenty- E. acervulina var.mivati infection in chorioallantoic

three: membrane of chicken embryo .. • • . • • • 188

Twenty- E. tenella (embryo-adapted) infection in chorioallantoic four: membrane of chicken embryo . .188

Twenty- Stages of E.tenella (Houghton) in chorioallantoic

five: membrane of duck embryo . • • • • • • 190

Twenty - Stages of E. tenella (Houghton) in chorioallantoic

six: membrane of quail embryo • • • • • • • • 191

Twenty- Stages of E. tenella (Houghton) in chorioallantoic seven: 192 membrane of pheasant embryo .. • • • •

Twenty- Stages of E. tenella in monolayer chicken kidney cells 194 .eight: Twenty- Schizont of E. tenella (Houghton) in quail kidney cell 195 nine: Thirty: Sporozoites of E. tenella (Houghton) in the caecum of a quail • 195 Thirty- Schizonts of E. tenella (Houghton) in the liver of a one: 19-day-old 'tolerant' chicken 198

Thirty- Trophozoites of E. tenella (Houghton) in chicken kidney two: cells stained for LDH 200

Thirty- Stages of E. tenella (Houghton) in chicken kidney cells three: stained for G6PDH 201

Thirty- SporozoiteS of E.tenella (Houghton) stained for LDH and four: G6PDH 202 14

.Introduction

Species of the sub-ordei- Eimeriina are protozoan parasites commonly referred to as coccidia. The largest family within this group is the Eimeriidae, most species of which are found in the genera Eimeria and Isospora. Coccidia are of particular economic importance as Some of the diseases that they cause, collectively referred to as coccidiosis, are common. in domestic animals, especially poultry, cattle, rabbits, sheep and goats.

The genus Eimeria in terms of number of species and ubi- quity of occurrence must* be considered to be very successful. Although species show variation in their morphological and biological characters and, in general, can be treated as being immunologically distinct, differences between them are not always easy to detect. Furthermore, there is a growing need, both in laboratory and field situations, to recognise intraspecific populations of these disease organisms. An additional method of characterisation would be advantageous, especially as the validity and usefulness of some of the species characters presently used in identification have been questioned by laboratory investigations.

As biochemical criteria have been used successfully for the recognition of populations of other Protozoa, it was thought desirable to establish whether similar methods would be suitable for the differentiation of Eimeria spp; this problem forms the basis of the first part of the thesis. Consideration is given to aspects such as identification, population structure, the significance of molecular variation, the constancy of biochemical characters and their use as genetic markers. 15 Outstanding features of the genus Eimeria include the relatively high degree of specificity of its species and the frequency of occurrence of several different species in the same host. Although the host and site specificity of Eimeria spp is well recognised, little is actually known of the mechanisms involved. Experiments were devised utilising cell culture, embryonic tissues and the intact animal, as different levels of organisation, in an attempt to determine some of the factors that operate in the intricate host-parasite relationship. These investigations benefited from the accurate definition of the populations under studyi which was permitted by the biochemical techniques. The results of these experiments are presented and considered in the second part of.the.thesis. 16

INTRODUCTORY REVIEW

1. Historical Aspects (A) The Genus Eimeria

'Further, I examined the bile from three Old rabbits. The bile of the first contained a very few small globules, but very many oval corpuscles ...'

This extract is taken from a translation by Dobell (1922) of a letter dated October 19th, 1664 from Anthony von Leeuwenhoek to the Royal Society. Although no abnor- malities of the liver were recorded in the manuscript, it is very tempting to assume, as did Dobell, that the correct interpretation of the 'oval corpuscles' would be oocysts of Eimeria stiedai. Hence, the first recorded observation of a coccidium can, perhaps, be attributed to this celebrated microscopist. A remarkable feat when one considers that over 170 years were to elapse before the parasite was described.

No mention was made by Linnaeus to parasitic protozoa in the 1758 edition of his "Systema Naturae". According to Dobell (1922) a colour picture of a rabbit's liver, in a rare publication dated 1838 by R. Carswell, is believed to show lesions caused by E. stiedai, but it is to a London physician, T. Hake, that the credit goes for the publication of a description in 1839 by which coccidia can be recognised.

The development of a nomenclature for the coccidia was (and is) a very gradual process. Owing mainly to the entanglement of these parasites with other protozoa and even helminths, confusion abounds in the early reports. Generic names such as Psorospermium and Monocystis, which had their origins in different protozoan groups, were at one time applied to the genus. There was also a reluc- tance to acknowledge that the various endogenous stages 17

and the oocyst were part of the same life cycle. Levine (1973a, 1973b) has presented a well documented account of the emergence of the genus Eimeria in the literature.

Investigations in the latter half of the nineteenth century began to unravel the life cycle. The endogenous cycle of Gregarina falciformis in the mouse was described by Eimer (1870). This species was later named Eimeria falciformis by Schneider (1875) and designated the type of the new genus.

In the following years a great number of Eimeria species have been described. Pe116rdy (1964) recognised between seven and eight hundred. Calculations by Levine (1963) revealed that Eimeria spp had been desdribed from only 1.2% of the world's chordates and 5.7% of the world's mammals. Speculating on possible numbers, Levine (1962) thought there might actually be 45,000 species belonging to the genus.

(B) Coccidia parasitising the domestic fowl

The first recorded observation of Eimeria parasitising chickens would appear to be that of Rivolta and Silvestrini (1873), quoted by Tyzzer (1929), who gave an account of sporulation in the oocysts. Wenyon (1926) states that these authors wrote of the oocysts as Psorospermium avium. In a review of the nomenclature, Tyzzer (1929) points out that P. avium was not used until 1878 by Rivolta who applied it to a species of Isospora : a conclusion also formed by Reichenow (1921).

Lacking knowledge of the different stages in the life cycle, Rivolta (1878) named intracellular stages found in a variety of birds, including chickens, Gregarina avium intestinalis. The oldest name applied to the caecal coccidium of the chicken is Coccidium teneawn (Raillet and Lucet 1891); this was corrected to Eimeria tenella by Raillet in 1913.

Many early authors made the common mistake of attri- 18

buting infections in different host species to one species of parasite. Hadley (1911) presented a complete account of the life history, morphology and biology of E. avium basing his observations on material procured from chickens, turkeys, ducks, geese and other birds. In studies of E. avium in partridge and pheasant Verwey (1926) concluded that the variation in form could be attributed to the development of the parasite indifferent hosts. On the basis of oocyst morphology and cross infection experiments, Johnson (1923, 1924) suggested that the parasites of the turkey were distinct from those of the chicken. The view that more than one species parasitising chickens existed was put forward by Reichenow (1921), Johnson (1924) and Wenyon (1926).

Admirable work by Tyzzer (1929) led to the naming of a further three species of Eimeria in chickens: E.acervulina, E. mitis and E. maxima. Using several different characters, including immunospecificity, Johnsdn (1930) distinguished E. necatrix and E. praecox. Then Levine (1938, 1942) recognised E. hagani and E. brunetti respectively, and Edgar and Seibold (1964) named E. mivati.

Yakimoff and Rastegareff (1931) also described E. beachi, E. johnsoni and E. tyzzeri from chickens, solely on observations of the oocyst, but they have not been accepted as valid species (Tyzzer et al. 1932).

Other coccidia which have been reported as occurring naturally in chickens include Cryptosporidium parvum (Tyzzer 1929) and Mmyonella gallinae (Ray 1945). Scholtyseck (1954) described Isospora gallinae ; similarities of the oocyst to Isospora lacazei have caused speculation concerning its validity (Kheysin 1972). 19

2. Species Characters of Eimeria

Taxonomy is an indispensable science. It is necessary that organisms are defined both for recognition when they occur in nature and for the correct interpretation and communication of observations in the laboratory.

A 'species character' is a general term which refers to any attribute of a species that differentiates it from other species. If such a character is to be of diagnostic •value, then it must be reasonably constant. . There are a number of criteria which are used for differentiating species of Eimeria. Joyner and Long (1974) discussed the specific characters of Eimeria spp, paying particular attention to the coccidia of the domestic fowl. They stressed that a single criterion is insufficient for differentiation.

The eimeriine parasites of many animals have been poorly described. Many descriptions of new species are based only on oocyst morphology and the animal from which the sample was obtained (see Clark and Colwell 1974, Wacha and Christiansen 1976). Levine(1973a) pointed out that, of the ninety-five named species from ruminants, the endogenous stages were known for only fifteen and the sites of parasitisation within the host for seventeen. Understand- ably, the parasites of economic significance have been the subject of the most intensive study and a knowledge of these species has led to generalisations pertaining to the genus as a whole. It should not, however, be assumed that characters used for their differentiation will have the same significance for Eimeria spp from fish, reptiles or other less well known groups.

Problems exist in distinguishing populations of individual species of Eimeria. The concept of the fixed species is held no more and there is an increasing aware- ness that a species may exhibit a range of characters. Intraspecific distinctions are becoming apparent as more characters are observed from a growing number of isolates. 20

The intention of this brief review is to describe the characters used to distinguish species of Eimeria, emphasis being given to the degree of variation and the extent to which such variation might be environmentally induced.

A number of terms are currently used for the description of intraspecific groups, e.g. strains, substrains, variants, lines, pure lines. Unfortunately, they have not always been applied in the same sense. The terms as used in this thesis are defined below:

Isolate: a sample obtained from a natural infection at a particular time from a defined loc- ality, hence may contain more than one species.

Strain: a heterogenous group consisting of all the parasites belonging to a single species obtained from an isolate; laboratory strains are usually derived by single oocyst infections and maintained by serial passage.

Line: a line is derived from a strain after experimental manipulation such as exposure to drugs or embryo passage.

Variant: a term which indicates variation within the species. Recently introduced for distinct groups which did not warrant specific status (Long 1973a,Long et al. 1974).

Terminology is presently under consideration by the Coccidiosis Discussion Group in Britain. 21

(A) Morphological Characters i) The Oocyst

The oocyst is the form of the parasite encountered in the external environment. The classification of the coccidia relies heavily upon the morphology of this stage. Hence, the genus Eimeria is characterised by possessing oocysts with four sporocysts each with two sporozoites. The assignment of an oocyst to a species depends on the critical appraisal of a number of different characters. The most important of these are size, colour, shape, surface, shape of sporocysts and the presence or absence of a polar cap, micropyle and various granules and resid- ual bodies.

Tyzzer (1929) realised that the lack of agreement in the dimensions given for E. avium was due to the inclusion of more than one species in the description. He also pointed out that variation occurred within each species and questioned the differentiation of species solely by oocyst size. A thorough investigation was mad?. by Jones (1932) on the natural range of variation among organisms developing from a single oocyst, of the influence of the age and breed of the host and of the duration and severity of infection. She concluded that variation in size occurred with single oocyst infections and, that in the case of E. acervulina,00cysts resulting from an infection with a single oocyst were markedly larger than those from a massive infection. Not only was there a difference between oocysts produced at the same time of patency in individual birds, but there was also a variation between oocysts produced in the same bird on successive days after infection.

Working with E. tenella, Fish (1931) provided evidence for a progressive change in oocyst size during patency. In studies of E. brunetii, Becker et al. (1955), emphasised the tremendous overall range in the length and width of oocysts on different days of infection and from different birds. Investigations have established an increase in 22

oocyst size as the patent period progresses for E. magna (Kheysin 1947a), E. coecicola (Kheysin 1947b), E. intestinalis (Kheysin 1957), E. necatrix (Becker et al. 1956), E. falciformis (Cordero del Campillo 1959) and E. seperata (Duszynski 1971).

The variability of oocyst shape within a species may depend on factors which affect the development. of the macrogametes in the host cells. Kheysin (1972) reported that the appearance of broadly oval and short oocysts of E. intestinalis, as opposed to the usual pear shape, was associated with a heavy infection in which several macrogametes developed within the same host cell. Decrease in oocyst size was also noticed when the inoculation dos- age was increased (Kheysin 1972). In a study of coccidia of lambs, Catchpole et al. (1975), observed that variation in the morphology of the oocysts was related to the size of the inoculum and the stage of patency. Kogan (1962, 1965, quoted by Kheysin 1972) reported that the host's diet may influence the size of the oocysts of E. necatrix; larger oocysts were produced in chickens on a protein diet than by those on a grain diet.

Oocysts of E. acervuZina var. mivati formed in the chorioallantoic membrane of chicken embryos differed in shape from those produced in the normal site (Long 1973a). (The name E. acervulina var. mivati is now preferred to E. mivati, Long 1973a). Jeffers (1975) noticed a decrease in oocyst size in a line of E. tenella selected for precociousness. However, if the oocysts were harvested at the normal time there was no difference in mean size from a control strain. It was suggested that the reduction in oocyst dimensions was not a direct result of selection for precociousness, but rather a function of the time permitted for the growth of the macrogametes.

Evidence for subspecific differences in oocyst morphology is scarce. Joyner (1969) reported that oocysts of two strains of E. acervulina differed very slightly in length. As the difference did not exceed lium, he stressed the doubtful biological significance of this finding. 23

Long et al. (1974) noticed that oocysts of E. praecox var. ceylonensis were slightly larger than those of E. praecox.

ii) Endogenous Stages

The diagnosis of coccidia is often facilitated by examination of the endogenous stages. Characters which are of value for identification include: the size of sporozoites, and the location, time of development and morphological features, including size, of the asexual generations and gametocytes.

Reports on the ultrastructure of coccidia, reviewed by Scholtyseck (1973), suggest that Eimeria sppare quite uniform in their fine structure. Differences have been observed in the mode of formation of merozoites: in many species, the merozoite rudiments appear to originate at the surface of schizonts as typified by E. tenetla (McLaren 1969), whereas in others such as E. callospermophili (Roberts et al. 1970) merozoites originate in thc•interior of the schizont.

Observations in tissue culture have confirmed the predetermined nature of the life cycle, although evidence suggests that the characters of the endogenous stages are not inflexible. Long (1972a) noticed that large second generation schizonts, normally characteristic of E. tenella, were replaced by smaller schizonts confined to epithelial cells in a line selected for growth in avian embryos. Temperature has been shown to affect development times of infections in chicken embryos (Long 1972b). The times of maturation of the second generation schizonts of two strains of E. tenello have been found to differ (Long 1970a).

McDougald and Jeffers (1976) suggested that the number of asexual generations in the life cycle is under genetic control, by using a line of E. tenaZa selected for precociousness in which they recognised gametocytes in vitro after a single asexual generation. In contrast, Long and Rose (1970) observed extended oocyst production, presumably due to additional asexual generations, in 24

betamethasone-treated chicks infected with E. acervulina var. mivati. This suggests an environmental influence on the number of asexual generations.

(B) Biological Characters

i) Pathogenicity

The pathological effects, if any, of the majority of species are not known. Coccidiosis is often thought of as a.disease of naturally gregarious species or of animals under domestication. It is when suitable hosts are brought together in groups and when transmission is enhanced that coccidia cause serious disease. Certain pathological peculiarities of an infection help to identify the organism responsible. The lesions caused by Eimeria spp are attributable either to the maturing asexual generations, as with E. tertalas or to the development of the sexual stages, as in E. maxima infections. McLaughlin (1973) listed 5 parameters of use for evaluating the pathogenesis of coccidial infections: these were mortality, effect on weight gain, lesion production, physiological changes and oocyst production. Comparison of pathological data is hindered by the numerous environmental factors which influence the expression of these characteristics. The pathology and pathogenicity of coccidial infections have been reviewed by Long (1973b). Differences in the pathogenicity of strains of a species have been noted by Joyner (1969) for two strains of E. acervulina, by Joyner and Norton (1969) and Long (1970a)for two strains of E. tertella and by Doran et al. (1974) for three strains of E. tenella. A decrease in the pathogenicity of E. tertel/a as been obtained in a line selected by serial passage in embryos (Long 1972a)and in a line selected for precociousness (Jeffers 1975).

ii) Prepatent Period

The length of the prepatent period depends on the number and timing of the asexual generations prior to the formation of gametocytes. The prepatent period is relatively constant for each species, but there is often an overlap 25

between the species in any one host. Hence, Joyner and Long (1974) considered the character to be of little diagnostic value for species in the chicken; E. praecox was the only species identifiable by this means. The character has, however, been included in a recent diagnostic chart (Reid 1973).

Variations in the prepatent period have been used in separating E. weybridgensis and E. crandallis from domestic sheep (Norton et aZ. 1974). The mean prepatent periods were found to be twenty-six and fifteen days respectively.

The time taken before the onset of oocyst production is not always precise. Considerable variation has been reported for the prepatent period of certain cattle coccidia, as shown by the six to eleven days for E. alabcmensis and eight to twenty-eight days for E. zuernii (Davis et aZ. 1955). E. subepithelialis of carp exhibits a latent phase related to seasonal conditions (Marinsdek 1965): carp fry uhich become infected with this coccidium during the spring do not shed oocysts until the spring of the following year. Zmerzlaya (1965, reported by Kheysin, 1972) reduced the prepatent period of E. carpelli , also of carp, from seventeen to seven days by altering the water temperature in which the experimentally infected fish were kept. Long (1972b)showed that E. tenella developed more rapidly in embryos incubated at 41°C than at 38°C. An increase in the length of the prepatent period may occur when the host has been partially immunised as shown by Henry (1932) with E. caviae infections of guinea pigs and Rommel (1969) with E. polita and E. scabra infections of pigs.

When a large number of oocysts was used as the inoculum as opposed to a single oocyst, the prepatent period was reduced in E. magna and E. irresidua infections and to a lesser extent in E. intestinalis, E. media, and E. perforans infections (Kheysin 1972).

Tyzzer et aZ. (1932) found differences in the prepatent period of E. acervulina in birds which had been infected with 26

oocysts harvested either at the end or the beginning of oocyst production. Jeffers (1975), by continually selec- ting for precociousness produced a population of E. tenella with a markedly reduced prepatent period. The prepatent period can be extended by the use of coccidiostatic drugs which act by inhibiting the development of the parasite. On withdrawal of these drugs development continues to patency. (Reid et al. 1969).

iii) Patent period

The severity of the infection probably has the greatest influence over the patent period. In birds which have been partially immunised the patent period of the challenge infection is invariably shorter (Rose 1973). Long and Rose (1970) demonstrated that corticosteroid treatment of chickens,prior to and during the primary infection,sub- stantially lengthened the patent period of E. acervuZina var.mivati, oocyst production continuing up to the fif- . tieth day. The patent Period will be extended if oocysts are retained in the intestinal tissues and released gradually after endogenous development has ceased. Recent evidence has suggested that it is possible for endogenous stages to be present in immune birds in the absence of oocyst production, since immune birds treated with corticosteroids produced oocysts (Long and Millard, 1976a). A similar situation appears to exist with Toxoplasma infections: asymptomatic toxoplasmosis was reactiv ated by the inoculation of corticosteroids (Frenkel 1973), and by the feeding of Isospora felis to cats with latent toxoplasmosis (Dubey, 1976).

iv) Reproductive potential

The theoretical number of oocysts that each species can produce per oocyst is dependent on the number of merozoites arising from the asexual generations and on the number of fertilised macrogametes. Six factors were suggested by Brackett and Bliznick (1952) which might affect the number of oocysts produced by a coccidial infection. 27

These were:-

1) The inherent potential of the parasite to reproduce in a susceptible host. 2) The immunity developed by the host. 3) A 'crowding' factor in heavy infections of the same species. 4) Competition with other Eimeria spp or other infectious agents. 5) Nutrition of the host. 6) Strain differences of the host.

Evidence to support some of these hypothetical factors was reviewed by Williams (1973) who added variations in the infectivity of oocysts, variations in the numbers of sporozoites or merozoites which reach susceptible cells, variations in the proportion of fertilised macrogametes and the age of the host as factors which might affect the reproductive potential.

Vetterling et al. (1973) compared the Weybridge, Beltsville and Winsconsin strains of E. tenella and found that the Winsconsin strain produced the most oocysts in chickens, although the Beltsville strain produced more in cell culture. Oocyst production was similar in the Weybridge and Houghton strains of E. tenella (Joyner and Norton 1969) but differed between two strains of E. acervu- line (Joyner 1969). Dikovskaya (1974) reported considerable differences in the reproductive ability of thirteen strains of E. tenella. v) Sporulation time

Estimates of the period required for sporulation can be made only on freshly discharged oocysts. Sporulation is dependent on temperature, humidity and oxygen concentration. The oocysts of some species of Eimeria, including those parasitic in fish (Pellerdy 1965), have the ability to sporulate in the tissues of the host. The time taken for 28

sporulation is occasionally cited as an additional characteristic of the species but the conditions for sporulation should be clearly stated.

vi) Development in culture

Results of attempts to obtain development of Eimeria spp in avian embryos and cell culture have been reviewed by Doran (1973). Although variations in culture technique hinder comparison of observations from different laboratories,.interesting differences exist in the ability to develop in culture not only between species but also between populations within the species.

Comparisons of three strains of E. tenella in vitro accentuated the fact that strain variations existed; differences were found in the number of sporozoites that penetrated cells and in oocyst production at six, seven and eight days (Doran et al. 1974). Studies with E. acer- na var.diminuta (Long 1974a)showed that its ability to develop in chicken embryos was similar to that of E. acer- vuZina var.mivati but differed from the Houghton strain of E. acervulina. Lines of E. tenella and E. acervulina var.mivati produced•by repeated passage in chicken embryos showed characters which differed from their respective parent strains (Long 1974b, 1973c). vii) Drug resistance

Drug resistance studies have been restricted almost exclusively to the coccidia of poultry.

Deaths from coccidiosis have virtually been eliminated in medicated birds but,despite extensive drug use, coccidial populations are still present. Jeffers (1974) reported the occurrence of coccidia in 91.9% of 1,145 litter samples from the major broiler producing areas of the United States; tests of 201 randomly chosen E. tenella isolates showed that their drug sensitivity had been reduced by field exposure to anticoccidials. Numerous reports of drug resistance, both naturally occurring and experi- 29

mentally induced, have been reported; details can be found in the reviews by Cuckler et al. (1969), Joyner (1970) and Ryley and Betts (1973). Populations within a species can be differentiated using drug resistance as a character as shown by the work of Norton and Joyner (1975), who developed five drug-resistant lines of E. maxima in the laboratory, which could only be distinguished by drug sensitivity.

The foundations for investigations of the genetic transfer of drug resistance were laid by Ball (1966.) and McLoughlin (1970), although they themselves obtained no evidence for transference of resistance. Recently, both Jeffers (1974) working with E. teritella and Joyner and Norton (1975) working with E. maxima have succeeded in producing crosses from lines differing in their response to anticoccidial drugs. An additional marker, that of precociousness has also been used by Jeffers (in press) to demonstrate the transfer of drug resistance.

viii) Host and Site Specificity

Eimeria spp exhibit marked host specificity, which is a particularly valuable aid for species recognition. The site of development of the different stages of the life cycle, the type of cell parasitised and even the position within the cell provide additional information for diagnosis.

The ways in which host and site specificity may break down under normal and experimental conditions is the subject of a more detailed consideration in Part Two.

Antigenic dissimilarity is a useful criterion by which to distinguish Eimeria species.

Tyzzer et al. (1932) observed that immunity to Execatrix did not protect to any extent against an E.terwa2 infection 30

and vice versa. Immunological specificity has been used as an additional distinguishing character in description of species: Moore and Brown (1951,1952) and Moore et al. (1954) recognised E. adenoides, E. innocua and E. subrotunda from the turkey: Edgar and Seibold (1964) identified E. mivati from the domestic fowl and Norton et al. (1974.) confirmed the distinction of E. weybridgensis from E.ovina in lambs.

The suggestion that cross protection might operate with some species was proposed by Rose and Long (1962) who found that E. terena infections in birds which had been immunised against E. necatrix were not as severe as in unprotected birds, as shown by lesion scoring. Rose (1967a) used oocyst production to substantiate this idea and, using the same two species, showed that solidly immune hosts challenged with the heterologous species produced only 50% of the oocysts expected from non-immunised controls. Par- tial cross protection has also been demonstrated between two other species from the domestic fowl, E. maxima and E. brunetti (Rose 1967b) and between two species from the pig) E. scabra and E. polita (Rommel 1970). In contrast, Hein (1971), after using a variety of immunising and challenge infections, concluded that birds which were resistant to E. maxima were fully susceptible to E.brunetti.

An unexpected relationship has recently been reported by Rose (1975). She found that oocyst production of E. acervulina was consistently higher in birds previously infected with E. maxima and to a lesser extent the reverse was true. She also noted that similar results had previously been obtained for birds which had been immunised with an embryo adapted E. acervulina var.mivati and subsequently inoculated with E. acervulina (Long 1973a) and in those which were immunised with E. acervulina and subsequently given E. praecox (Joyner and Long 1974).

Joyner (1969) reported immunological differences in two strains of E. acervulina. Oocyst production was observed in birds solidly immune to one strain when challenged 31

with the other. Cross infection experiments with eight strains of E. tenella isolated from different zones of the USSR showed that some of them differed immunologically (Dikovskaya 1974), although two laboratory strains of E. tenella were found to give complete cross protection (Joyner and Norton 1969). Chicks previously immunised with a parent strain of E. maxima were completely cross protected against 5 drug-resistant lines derived from it (Joyner and Norton 1975). Good cross protection has also been obtained between lines of E. tenella and E. acervulina var. mivati, passaged in embryos, and their respective parent strains (Long 1972a, 1972c).

Long (1973a)lon the basis of cross protection and other tests,concluded that E. mivati was conspecific with E. acervulina and proposed the name E. acervulina var -. mivati. Cross protection studies have also been of great help in elucidating the relationships of coccidia isolated from the jungle fowl with those from the domestic fowl. Long et al. (1974) found that an infection of one strain of jungle fowl origin protected against homologous challenge and against E. praecox, from the domestic fowl, but did not protect against E. acervulina ; the name E. praecox var. ceylonensis was proposed for the jungle fowl strain. Simi- larly, the close relationship of E. acervuZinavar.diminuta, from jungle fowl, to E. acervulina and E. maximavar.indentata l from jungle fowl, to E. maxima was confirmed by cross protection tests (Long 1974a).

Examination of two British strains of E. maxima (Long, 1974a) showed that cross protection was not complete when small numbers of oocysts were used for the immunising dose. He quotes a personal communication from Hein who found that after three consecutive immunising doses were given, immunity was incomplete against challenge with heterologous strains of E. maxima, although complete protection was foundl after a single light infection, against homologous challenge. These studies indicated that oocyst production was the best available criterion for revealing differences between the strains and again emphasised the importance of detailing the exact experimental conditions. 32

Reid et al. (1961) identified the species to which birds had previously been exposed by determining the immunity which the birds possessed. As cross protection is rarely absolute and immunological specificity requires very care- ful laboratory study, the results of such immunity challenge techniques can no longer be upheld.

(D) Concluding remarks

Field isolates frequently contain more than one species of coccidium. Eimeria spp were found in 81% of 100 bovine faecal samples and two or more species were present in 77.7% of the samples (Skander 1973). In a survey of lambs, Catchpole et al. (1975) reported that, out of 465 faecal samples, 95.5% were positive for coccidia and 65% contained four to six species. Initial identifioation must rely on oocyst morphology, on the host from which the sample was obtained and, when possible, post mortem examination. If distinct oocyst characters are lacking, identification can be a problem.. The determination of other characters requires the separation of the species from an isolate, usually by single oocyst infections; a procedure which is laborious and which, unfortunately, imposes a strict selection.

Laboratory investigations have revealed that many of the characters which are used to distinguish species of Eimeria show variation. Morphological and biological characters are often limited by the need to determine the nature of the parasite, as expressed by its genome, in standard and controlled environmental conditions; hence the importance of accurately describing and standardising the experimental method.

Populations within the species, both strains isolated from the field and experimentally derived lines make the present methods of recognition and identification inadequate. Joyner and Long (1974) concluded that quantitative cross immunity tests were the most satisfactory means available for the differentiation of species of Eimeria parasitising the domestic fowl. Indeed, such tests are proving 33 of value for the recognition of intraspecific populations. Immunological relationships are, however, intricate and complex; tests are by necessity elaborate and are not yet feasible for differentiation of the coccidia of many other animals. Part One of this thesis reports the results of investigations into the use of biochemical criteria for the recognition of eimeriine populations. 34

MATERIALS AND METHODS

1. Parasites

Oocysts of Eimeria spp obtained from either chickens, chicken embryos, quail, sheep or rabbits, were kindly donated by a number of different laboratories. The parasites, their source and the passage number on arrival, if known, are listed in Table 1.

Cultures were stored at 4°C and suspended in 2-2.5% ) until required. potassium dichromate (K2Cr207

2. Experimental Birds

Day old coccidia-free cockerels, either Ranger or Apollo, were obtained from Ross Poultry Limited, Andover. •Birds were kept in 'wire floor cages (2' x 1'6" x 1') under a lamp which piovided constant heat, 34-36°C directly underneath, and light for the first 14 days. Chick starter mash No. 508, obtained from British Oil and Cake Mills Limited (B.O.C.M.) or when this was unavailable chick starter mash, as supplied by Attlee_ and Co., Dorking, was presented twice a day; water was provided ad libitum. Both feeds were free of anticoccidials. The nature and size of the food containers ensured the constant presence of food for at least the first week. Chicks hatched in the laboratory incubators were reared in the same manner.

Breeding colonies of Japanese Quail, Coturnix coturnix japonica were kept on loose litter in metal cages (3' x 2' x 1'6"). The temperature was maintained at 27°C and the light was operated on a 12 hour cycle. Young birds were kept in smaller wire floor cages (12" x 12" x 8"). Laying birds were fed on 'Farmgate', layers mash (B.O.C.M.), non-experimental young birds on chick starter crumbs (B.O.C.M.) or turkey starter crumbs (B.O.C.M.) and experimental young birds on chick starter 35

TABLE ONE

Details of Eimeria cultures used in this study:

Species Strain Line Number on Supplied arrival by

• E.tenella Houghton HPRS Houghton Embryo-adapted 67,70,127 HPRS Weybridge 21 WL Weybridge 227 CVLW E.necatrix Weybridge 48 CVLW " (W44)* 9,11 WL E.brunetti Weybridge 53 CVLW " (w4o)* 7 WL E.acervulina Houghton HPRS var.mivati E.acervulina Houghton HPRS Ongar 6 WL Ongar MB Weybridge 17,20 WL Weybridge 63 CVLW imi 16 CVLW F.maxima var. CVLW indentata statyl-resistant 5 CVLW '.maxima Weybridge (W74) 4 WL Weybridge 85,86,87,88,89 CVLW 90,91,99 CVLW Weybridge statyl-resistant 16 CVLW robenidine-resistant 20 CVLW clopidol-resistant 27 CVLW sulphaquinoxaline-resistant 32 CVLW robenidine-dependent 17 CVLW Houghton 9,10 CVLW Norwich 6 CVLW 36

TABLE ONE (continued)

Species Strain Line Number on Supplied arrival by

E.ovina CVLW E.weybridgen- 18 CVLW sis E.ninakohly- 6 CVLW akimovae E.stiedai ICI E.magna ICI • Weybridge W15 CVLW E.coecicola CVLW' E.intestina- Zis CVLW E.bateri Ascot I Ascot CVLW Weybridge CVLW ? Macster Field MSD Isolate

Suppliers of oocysts:

HPRS - Houghton Poultry Research Sation, Houghton CVLW - Central Veterinary Laboratory, Weybridge WL Wellcome Research Laboratories, Berkhamsted ICI ICI (Pharmaceuticals) Ltd., Macclesfield MB May and Baker, Ongar MSD Merck Sharp and Dohme Ltd., Veterinary Laboratories, Hoddesdon isolated by the author

Figures in parenthesis refer to the passage number of the original culture in that particular laboratory. 37 mash, No. 508 (B.O.C.M.). Strains which have been reared include: Pharoe and Perkolin and the resulting hybrid from a cross between these two.

3. Bird Infections (A) Isolation Procedures

Infected and uninfected birds were kept in separate thermostatically heated rooms, some of which were equipped with filtered air supplies. Animal rooms were thoroughly washed down and fumigated for up to four days with ammonia. Large cages were meticulously cleaned and subjected to methyl bromide (20-40 mg/1 for three days). Smaller cages, food and water hoppers were sterilised at 15 lbs/sq.in. Samples of faeces from uninfected birds awaiting experimentation were routinely checked for oocysts every three to four days and always before experimental infection. The greatest hazard threatening the isolation system was thought to be the transfer of oocysts from rooms containing infected birds to those housing uninfected birds. Consequently, animal house personnel were briefed accordingly.

All experimental birds other than those used for routine passage of E.terie/L2 were cared for solely by the author, access being restricted to the isolation rooms. Before entering these rooms, shoes and clothing were changed and new rubber gloves were used for any procedures. Con- trol uninfected birds were present on all occasions to monitor any accidental infection. Makeshift isolators, housing individual birds,were constructed by surrounding cages (14" x 12" x 9") with heavy duty polythene. Air, which was not filtered, could enter by the food hoppers. Food and water were autoclaved before use and supplied ad libitum. 38

(B) Standardisation of Dose and Inoculation Procedure

The number of oocysts to be administered was determined with the use of an haemocytometer. Suitable dilutions were made to adjust the concentration of oocysts to the required dose (Long and Rowell 1958). The desired number of oocysts in 0.25 ml of H2O was placed at the back of the throat, the bird being released only when the suspension had been swallowed. Oocysts were inoculated directly into the crop of experimental quail with the aid of a catheter tube attached to the syringe.

Single oocysts were obtained by serial dilution following the method of Becker (1934). A tiny drop, containing a few oocysts, was placed in a small plastic well and examined. A micropipette was used to pick out a single oocyst which was deposited into another well. If micro- scopic examination confirmed the presence of only'one oocyst, the oocyst was again transferred and its presence checked. A clean pipette was used for each manipulation. Finally, the oocyst was ejected from a siliconised pipette far back on to the tongue of a bird. The pipette was examined and washed out to ensure that the oocyst had been delivered. Similar procedures were carried out with single sporocysts. At the beginning of the patent period, birds were killed and the appropriate part of alimentary canal was scraped and examined for oocysts.

(D) Abnormal Infection Routes

Attempts to infect birds by ways other than the normal oral route were made by inoculating either oocysts, sporozoites or merozoites, intramuscularly into the thigh, intravenously into a wing vein or intraperitoneally. Penicillin and streptomycin, 2000 units/ml and 2000µg/ml respectively,were also administered. 39

4. Cortisone treatment

The cortisone derivative used was betamethasone (supplied as Betsolan by Glaxo Laboratories Ltd., Greenford). Intramuscular injections of 0.2 mg for 21 day old quail and 0.5 mg for 14 day old chickens were administered 4 days before infection and on alternate days, until 4 days after infection (5 injections altogether).

5. Avian Embryos

White Apollo and light brown Ranger, fertilised chicken eggs were obtained from Ross Poultry Limited, Andover. Lincolnshire pheasantries, Boston, supplied fertilised eggs of the ring-necked pheasant, Phasianus colchicus torquatos. Khaki Cambell ducks (Kortlang, Ashford) reared by T. Davenport, Imperial College, were the source of duck eggs. Quail eggs were collected daily from the breeding colony previously described and stored temporarily at room temperature.

Prior to infection all eggs were incubated in a humid atmosphere at 39°C and occasionally lightly sprayed with water. Duck, quail and pheasant eggs were turned up to five times daily and chicken eggs once.

6. Avian embryo infections

Eggs were supported with the air sac uppermost and can- dled to determine whether development was proceeding normally. A small hole was made in the shell in a position away from major blood vessels as shown in Figure 1. Pheasant eggs proved to be too dense for adequate observation of the blood vessels. The pigmentation on the quail eggs could be removed with wire wool (Rauscher et aZ. 1962) to facilitate examination. Sporozoites were inoculated between the 9th and 11th day of incubation in chicken embryos; on the 7th day in quail, 9th in pheasant and 10th in duck embryos. The required number of sporozoites, in 0.05 ml phosphate buffered 1% saline for the larger eggs and in 0.02 ml for the smaller, was injected into the allantoic cavity. Each embryo also received 2000 units of penicillin and 2000)Lg of strep- 40

Figure One

Inoculation of sporozoites into a 10-day- old chicken embryo 141

tomycin. The shells were swabbed with 70% alcohol and the hole sealed with collodin (BDH Chemicals Limited, Poole). Chick and duck embryos were incubated at 40-41°C, quail and pheasant at 39-40°C. Infected eggs and controls were maintained in an upright position for the duration of the experiment; uninfected embryos were turned normally.

7. Isolation and Harvesting of Oocysts

(A) Collection of Oocysts from Faeces

Faecal samples were collected usually on the first and second days of patency in trays half-filled with 2% K2Cr2O7. The resulting suspension was further diluted and stirred vigorously for at least four hours to disrupt the larger particles completely. It was then strained' through muslin. The sediment was re-suspended, strained again and then discarded. The faecal suspension was allowed to stand overnight, after which the supernatant was drawn off leaving a deposit of small particles and oocysts; the latter were recovered by salt flotation. A very approximate estimate of oocyst numbers in the original sample was obtained by centrifuging 3 mls of the faecal solution. The debris was thoroughly mixed with 25 ml of saturated salt solution, the oocysts were floated up and re-suspended in a total volume of 2 ml and counted.

(B) Collection of Oocysts from Caeca (E. tenella Infec- tions)

Caeca and caecal contents, removed from chickens killed on the seventh day of infection were cut up into small portions. Water was added and the tissues were homogen- ised to a slurry. The suspenS'ion was made up to approximately 10% sodium hypochlorite (12-14% available chlorine) and cooled in an ice bath for 15 minutes. After an equal volume of water had been added, the suspension was filtered through muslin and centrifuged (1500 x g) to deposit the oocysts which were cleaned in distilled water. Unspor- ulated oocysts were obtained by carrying out all procedures at 5°C. 42

Infected embryos were cut longitudinally and carefully prised open. Urate deposits, desquamated epithelium, blood and oocysts, and the chorioallantoic membrane were retained. Oocysts were harvested using the method of Long (1972a)and the modifications of Shirley (1975) with the exception that the chorioallantoic membrane was cut up and also treated. The total number of oocysts recovered from batches of embryos was estimated by counting in an haemocytometer.

(D) Sporulation

Unsporulated oocysts were suspended in 2% K2Cr2O7 in conical flasks. The culture was kept at 27°C and continually aerated for 48 hours. Alternatively, when few oocystS were available, sporulation was carried out in Petri dishes, the sediment being periodically disturbed by the suction produced by a syringe.

(E) Cleaning and Sterilising Oocysts

Oocyst deposits washed clean of K2Cr2O7 were suspended in a 30% sodium hypochlorite solution in an ice bath for 15 minutes. Sterile distilled water was overlayed and the suspension centrifuged at approximately 1500 x g for 10 minutes. Oocysts were then extracted from the hypochlorite/water interface and washed. This procedure resulted in pure suspensions of sterile oocysts; the treatment is known to alter the outer layer of the oocyst wall (Nyberg and Knapp, 1970).

8. Preparation of Sporozoites

The technique for obtaining sporozoites was essentially that of Long (1970a). Sporocysts were released from oocysts by rapid shaking with 0.5 mm glass beads on a Whirlimixer (Jencons). The sporocyst suspension was incubated at 41°C in 0.25% trypsin (1:250 powder, Difco) and 0.5% Bacto bile salt (Difco) made up in phosphate buffered 1% NaC1 at pH 7.6. When examination revealed that the sporozoites had excysted, the incubation mix- 43

ture was centrifuged very briefly to remove larger debris (100 x g for 15 seconds). The sporozoites were then spun down and washed in phosphate buffered 1% NaC1, pH 7.0.

Sporozoites were further purified by passing through a glass bead column (Wagenbach 1969) formed in a 10 ml syringe. Centrifugation of sporozoites in 50% lymphoprep (sodium metrizoate/fic011 solution, Nyegaard and Co.) in 0.9% saline resulted in a practically pure suspension of sporozoites being retained in the supernatent.

9. Preparation of Merozoites

(A) Preparation of Merozoites from Chicken Caeca

The procedure for harvesting merozoites has been developed by Stotish and Wang (1975).

Caeca obtained from birds, 4i days after being inoculated with 1 x 106 oocysts of E.tenella (Houghton), were cut open and the contents discarded. The tissue was cut into small sections and rinsed thoroughly in phosphate buffered 1% saline. The pieces were placed in a conical flask containing 10 vols. of the incubation medium, consisting of 120 mM NaCl, 20 mM Tris-HC1 (pH 7.4), 3 mM K2HPO4, 1 mM CaC12 and 1 mg/ml Bovine serum albumen with the addition of 2 mg/ml trypsin (1:250 powder, Difco) and 0.2 mg/ml hyaluronidase (460 units/mg Sigma). The suspension was continually stirred and incubated at 41°C for 20 minutes, filtered through muslin, centrifuged and washed. The deposit was re-suspended in 1 vol. of phosphate buffered 1% saline and shaken vigorously in an attempt to disrupt cells containing large schizonts. Merozoites were purified with the use of lymphoprep but otherwise as described by Stotish and Wang (1975).

(B) Preparation of Merozoites from embryo and in vitro Culture

Surviving embryos, inoculated with either 1 x 106 sporozoites of E.tenala (Houghton) or with 5 x 105 sporozoites of an embryo adapted line of E.tenella, 1414

4i to 5 days previously, were sacrificed. Allantoic fluid, chorioallantoic membrane, sloughed off epithelium and any obvious deposits were treated with the incubation medium described for caecal tissue. Mero- zoites were isolated and purified in a similar manner.

Isolation of mainly immature second generation schizonts of E.terlella was achieved by dissecting out the large foci apparent on the chorioallantoic membrane, 4 days after inoculation of sporozoites.

Medium from infected monolayer cultures in prescription bottles or Leighton tubes was pooled together and centrifuged; any deposit was retained. The incubation medium,described for caecal tissue,was introduced on to the cell layer and the bottles were gently agitated for 20 minutes at 41°C. Merozoites were purified as previously described with the omission of the density centrifugation.

10. Electrophoresis (A) Preparation of Enzyme Samples Purified sporozoites and merozoites were disrupted in an equal volume of 5% Triton X-100 (Sigma) in Tris-HCl with enzyme stabilisers, dithiothreitol, E-aminocaproic acid and EDTA, each to a concentration of 1.0 mM (Kilgour and Godfrey 1973), pH 7.0.

Packed purified oocysts, in an equal volume of distilled water with enzyme stabilisers, were mechanically homogenated in an ice bath using glass beads and a Whirli- mixer. For the demonstration of malate dehydrogenase and malic enzyme the homogenate was further disinte- grated by ultrasonication. A MSE 150W Ultrasonic disintigrator with an exponential probe (3mm tip) was used to produce waves of 9 microns in amplitude for intermittent periods of 15 seconds. The samples were sonicated for a total of 2 minutes in an ice bath.

Tissue samples were finely diced, homogenated in distilled water with 0.1 ml glass grinders (Jencons) and subjected to a total of 2 minutes ultrasonication 45

(amplitude 9 microns).

The crude enzyme extracts in small polypropylene capsules (TAAB) suspended in 15 ml tubes, were centrifuged at 30,000 x g for 40 minutes at 4°C in a MSE High Speed 18 centrifuge. Occasionally, samples were centrifuged at maximum speed in a bench centrifuge. The supernatant was either used immediately, kept temporarily at 4°C, or stored at -20°C in 5 )..1.1 microcaps (Drummond).

When activity was expected to be particularly weak, Lyphogel (Gelman, Lancing) was used to concentrate the samples to 1/3 - 1/2 of their original volume; alternatively samples were freeze-dried.

(B) Polyacrylamide Gradient Gels

The electrophoretic equipment, gradient gels and sample separators were supplied by Universal Scientific Limited, London. The gels were pre-run for 1 hour at 200V in order to remove contaminants. The tank buffer consisted of Tris (10.75 g/l), EDTA (0.413 g/l) and Boric Acid (5.04 g/1); the pH was 8.3. The sample for analysis was mixed with one volume of 0.5 M sucrose in water containing 0.5 - 1% bromophenol blue. The sucrose prevented upward diffusion and dilution of the sample. 10)J1 microcaps were used for application of the enzyme preparations which were allowed to run into the gel for 1 hour. The sample spacers werethen removed and electrophoresis continued for a further period of 16 - 18 hours at 200V at 4°C. On completion of the electrophoretic run, the gels were taken from the tank and the glass side supports were removed to facilitate staining.

The disc polyacrylamide electrophoresis system was a simplified version of that devised by Ornstein (1964) and Davies (1964) and similar to that used by Gardener et al. (1974) for leishmaniae. This entailed a single running gel with neither spacer nor sample gel. The polyacrylamide gels were composed of either 5% or 71% 46 Cyanogum 41 (BDH Ltd.) made up in 0.25M Tris-HC1 (pH 8.9). The electrode buffer was 0.05M Tris-glycine (pH 8.3). Thoroughly cleaned tubes were used at all times to simplify the removal of the gels. One volume of 0.5M sucrose in water containing 0.5 - 1% bromophenol blue was added to each sample to give a total of 141. At the beginning. of an experiment a current of 1 mA per tube was generated until the bromophenol blue could be detected in the gel. The current was then increased to 2 mA/tube or 4 mA/tube and electrophoresis was continued for 30 to 90 minutes at 4°C. The run was terminated when the tracker dye, marking the buffer front, reached a predetermined point at the anodic end of the gel. The gels were then removed from the glass tubes and stained accordingly.

(D) Thin-Layer Starch Gels

The thin-layer starch gel apparatus was essentially that described by Wraxall and Culliford (1968), who devised a mit.lro-technique for the enzyme typing of blood stains. Glass plates lmm thick with a border of glass also 1mm thick were made to give a total gel mould of approximately 21 cm x 14 cm x 1 mm. One electrophoretic tank was kindly provided by Dr. C. Parr, London Hospital, and two others were made with only slight modifications.

Samples were run on a 10.87% hydrolysed starch gel. Previously boiled cotton threads, soaked in the desired extract were inserted into slots cut into the gel. Using 1 cm slots,seven samples could be run on each gel; an increase in this number could be achieved by reducing the slot width. If enzyme activity was weak, up to three threads could be placed all together. The glass plates were laid on a sheet of cellophane to separate them from water-cooled aluminium alloy bases. In practically all experiments a constant voltage of 250V was maintained. The duration of the electrophoresis was between two and four hours. 47 Buffer systems which have been used are detailed-in Table 2 and the particular buffers used for each enzyme in Table 3.

(E) Enzyme Assay Solutions Disc gels were placed into plastic tubes and stained indiv- idually. The staining solution was applied directly to the exposed surface of the polyacrylamide gradient and thin layer starch gels. To minimize diffusion, especially in enzyme-coupled reactions, filter paper or occasionally 1% agar containing the enzyme assay solution were used as an overlay on the gel. Incubation took place at 37°C.

Coomassie Brilliant Blue R250, 0.25g in 100 ml of meth- anol-water-acetic acid (5,5,1 by vol.), was used as a . general protein stain (1 to 4 hours). Destaining was carried out by soaking for prolonged periods in 7% acetic acid.

Enzyme assay solutions are detailed in Table 4. Chemicals were purchased from Sigma Chemical Company, London. Shaw and Prasad (1970) was the major source of reference.

A technique was devised to convert the ultra-violet light method for the demonstration of ASAT and ALAT (Kilgour and Godfrey 1973) to a direct staining method. The principle of the method depends on enzyme coupling and the detection of the oxidation of NADH to NAD. MDH is included in the staining mixture to convert oxalacetate to malate in the ASAT assay and LDH is included to convert pyruvate to lactate in the ALAT assay. The sites of the correspondig oxidation of NADH to NAD, indicative of the initial enzyme reaction, can be detected by ultraviolet light. It was found, however, that the addition of the terazolium salt, MTT, and PMS, after the 1 hr incubation with the assay solution, resulted, almost immediately, in the zones of transferase activity showing up as unstained areas on a blue background. The tetrazolium salt had been reduced by the NADH except in those areas of the gel where the conversion to NAD had taken place. Similar methods were used for DHR and PK.

Controls consisted of the assay solution without the sub- - strate. When the specificity of an enzyme was in doubt, one TABLE TliO

Buffer Systems used in Thin Layer Starch Gel Electrophoresis

Electrode Components Gel Components Buffer Buffer pH pH Number (per litre) (per litre)

1 0.214M Phosphate 29.1g.K2HPO4 7.0 1.06g K2HPO4 7.0 0.027M Citrate 5.7g Citric Acid 0.254g Citric Acid 1.86g BoricAcid 2 0.3M Borate 18.55g Boric Acid 8.0 8.5 2.0g NaOH 0.48g NaOH electrode buffer 3 0.2M Phosphate 460 ml 0.2M NaH2PO42H2O 5.8 50 ml of 5.8 40 ml 0.2M Na2HPO4 4 0.378M Tris 45.8g Tris 6.0 33.3m1 of electrode buffer 6.0 0.165M Citrate 34.2g Citric Acid 5 0.15M Tris- 18.16g Tris 9.0 1.81g Tris 9.0 Citric Acid

6 0.14M Tris 16.35g Tris 7.0 66.7m1 of electrode buffer 7.0 0.043M Citric Acid 9.04g Citric Acid TABLE TWO (cont.)

Buffer Systems used in Thin Layer Starch Gel Electrophoresis

Buffer Buffer Electrode Components Gel Components pH Number (per litre) p (per litre)

7. 0.004M Na2EDTA 1.44g EDTA 200m1 of electrode buffer , 0.10M Borate 6.18g Boric Acid 8.6 8.6 0.18M Tris 21.80g Tris 8 0.0546M Tris 6.61g Tris 0.12g Tris 0.245M Boric Acid 15.17g Boric Acid 7. 5 1.79g Boric Acid 7.5 9 0.2M Phosphate 255m1 0.2M NaH PO H 0 2 4 2 6.8 65m1 of electrode buffer 7.0 245m1 0.2M Na2HP04'7H20 50

TABLE THREE

List of Enzymes and Buffer Systems

Abbrevi- Buffer Enzyme ation System

1) Esterase ('non specific') 2,8 2) AcP Acid phosphatase 6,9 Orthophosphoric monoester phosphohydrolase 3) AP Alkaline phosphatase 5,6,7,8* Orthophosphoric monoester phosphohydrolase 4) FDP Fructose.1, 6- diphosphatase 1,2,3,6 Fructose 1, 6- diphosphate D-glyceraldehyde -3- phosphate lyase 5) LAP Leucine Aminopeptidase 2,5,7 L-Leucyl-peptide hydrolase 6) H6DH Hexose 6- dehydrogenase 4,6 7) G6PDH Glucose 6- phosphate dehydrogenase 2,5,6,8 D-Glucose -6- phosphate: NADP oxireductase 8) GDH Glutamate dehydrogenase 3,6 L-Glutamate: NAD(P) oxireductase (deaminating)

9) 0.(.-GPDH 0(-Glycerophosphate dehydrogenase 1,6 off-Glycerol -3- phosphate: (acceptor) oxireductase 10) I DH Isocitrate dehydrogenase 1,6,8 threo -Ds- Isocitrate: NAD oxireductase- (decarboxylating) threo -Ds- Isocitrate: NADP oxireductase- (decarboxylating) 11) MDH Malate dehydrogenase 1,4,6,8 L-Malate: NAD oxireductase (decarboxylating) 12) ME Malic enzyme 1,6,8 L-Malate: NADP oxireductase (decarboxylating)

13) LDH Lactate dehydrogenase 1,2,6,7,9 DL-Lactate: NAD oxireductase

14) 6PGDH 6-Phosphogluconate dehydrogenase 5,6,7,9 6- Phospho -D- gluconate: NADP oxireductase 15) SDH Succinate dehydrogenase Succinate: (acceptor) oxireductase 51

TABLE THREE (cont.)

List of Enzymes and Buffer Systems

Abbrevi- Buffer Lnzyme ation System

16) AK Adenylate kinase 6,7 ATP: AMP phosphotransferase 17) HK Hexokinase 6,7 ATP: D-hexose 6-phosphotransferase 18) PK Pyruvate kinase 6 ATP: pyruvate phosphotransferase 19) GPI Glucose phosphate isomerase 2,3,9 D-Glucose -6-phosphate ketol-isomerase 20) ASAT Aspartate aminotransferase 5 L-Aspartate: 2-oxoglutarate aminotransferase 21) ALAT -Alanine aminotransfer.ase 5 L-Alanine: 2-oxoglutarate aminotransferase 22) TAT Tyrosine aminotransferase L-Tyrosine: 2-oxoglutarate aminotransferase 23) PGM Phosphoglucomutase 6,7 °(-D- Glucose-1, 6-diphosphate:v(- D-glucose -1- phosphate phosphotransferase 24) DHR Dihydrofolate reductase 5 5,6,7,8,- Tetrahydrofolate: NADP oxireductase 25) TO Tetrazolium oxidase 7

* Buffer Nos. underlined indicate most commonly used buffer TABLE FCUR

Enzyme Assay Sclutions

Enzyme Substrate Coenzyme + other additives Buffer

1) Esterase 1 ml , rock, 0 naphthyl 5 mg, Fast Blue RR 4 m1,0.5M Tris-HCI,pH 7.0 acetate 4 ml, H2O

2) AcP 10 mg Nack- naphthyl 5 mg, Black K Salt 8 m1,0.05M Acetate,pH 5.0 phosphate

3) AP 6 mg Na B - naphthyl 10 mg Fast Blue RR 5 m1,0.5M Tris-HC1,pH 8.5 phosphate 10 mg MgSO4 7H20 5 ml H2O

4) FDP 6 mg Fructose 1,6- 6 mg NADP 2 m1,0.5M Tris-HC1,pH 7.5 phosphate 1 mg PMS 5 mg NBT • 40 ut PGI 10 ut G6PDH 5 mg MgC1 2 5 ml H2O TABLE FOUR (zont.) Enzyme Assay S;lutions

Enzyme Substrate Coenzyme t other additives Buffer

5) LAP 4 mg L - leucyl 0- 5 mg Black K Salt 5 ml 0.02 M Tri-maleate naphthylamide 5 ml H2O pH 6.0 , 5 mg MgC1 2

6) H6DH 1 ml 1M Galactose phosphate 6 mg NADP 2 ml 0.5M Tris-HC.1, pH 7.0 6 mg NBT 1 mg PMS 5 ml H2O

7) G6PDH 10 mg Glucose 6- phosphate 6 mg NADP 8 ml 0.3M Tris-HCl, pH 8.0 4 mg NBT 1 mg PMS

8) GDH 1 ml 1M Na Glutamate pH 7.0 5 mg NAD 5 ml 0.05M phosphate, pH 7.0 6 mg NBT 1 mg PMS • 5 ml H2O TABLE FOUR (cont.)

Enzyme Assay Solutions

Enzyme Substrate Coenzyme + other additives Buffer

9) ot.GPDH 8 mg Na o(-glycerophosphate 5 mg NAD 2 ml 0.5M Tris-HC1,pH 7.0 6 mg NBT 1 mg PMS 6 ml H2O

10) IDH 10 mg Na isocitrate 5 mg NAD or 5 mg NADP 8 ml 0.25M Tris-HC1,pH 8.0 2 mg ADP 6 mg NBT 1 mg PMS 0.5 ml 0.25M MnC1 2

11) MDH 1 ml 1M Na L-malate, 5 mg NAD 8 ml 0.25M Tris-HC1,pH 8.0 pH 7.0 6 mg NBT 1 mg PMS TABLE FOUR (cont.)

i Enzyme Assay Solutions

Enzyme Substrate Coenzyme + other additives Buffer.

12) ME 1 ml 1M Na L-malate 5 mg NADP 8 ml 0.25M Tris-HCI,pH 8.0 pH 7.0 6 mg NBT 1 mg PMS

13) LDH 1 ml 1M Na DL-lactate 5 mg NAD 2 ml 0.5M Tris-HC1,pH 7.0 pH 7.0 3 mg NBT 1 mg PMS 6 mi H2O •

14) 6PGDH 10 mg Na 6-phosphogluconate 5 mg NADP 2 ml 0.5M Tris-HC1,pH 7.0 3 6 mg NBT 1 mg PMS • 6 ml H2O TABLE FOUR (cont.)

Enzyme Assay Solutions

Enzyme Substrate Coenzyme + other additives Buffer

15) SDH 1 ml 0.1M Sodium succinate 5 mg NAD 5 ml 0.05 K2HPO4 ,pH 7.0 2 mg ATP 4 mg NBT 1 mg PMS 1 ml EDTA

16) AK 10 mg Glucose 5 mg NADP 2 ml 0.5M Tris-HC1,pH 7.0 40 ut Hexokinase 10 ut G6PDH 2 mg ADP •

4 mg MgCl2 • 4 mg NBT 1 mg PMS ' 6 ml H2O TABLE FO'JR (cont.)

Enzyme Assay Solutions

Enzyme Substrate Coenzyme + other additives Buffer

17) HK 10 mg Glucose 5 mg NADP 2 ml 0.5M Tris-HC1,pH 7.0 4 mg ATP 4 mg MgC1 2 5 mg NBT 1 mg PMS 10 ut G6PDH 6 ml H2O

18) PK 5 mg Na3phosphenol 5 mg ADP . 7 ml 0.433M Glycine, pyruvate 5 mg NADH pH 9.0 ca. 20 ut LDH 4 mg MgC1 2 After 1 hr incubation 1 mg PMS 4 mg NBT TABLE FOUR (.::ont.)

Enzyme Assay Solutions

Enzyme Substrate Coenzyme + other additives Buffer

19) GPI 6 mg Fructose 6-phosphate 5 mg NADP 5 mi 0.3M Tris-HC1,pH 8.0 20 ut G6PDH 6 mg NBT

. 1 mg PMS 3 ml H2O

, 20) ASAT 10 mg L-aspartic acid 4 mg NADH 5 ml 0.1M phosphate, 100 ut MDH pH 7.4 0.5 ml O. 1Mck-ketoglutarate After 1 hr incubation 2 mg MTT 1 mg PMS TABLE FOUR (cont.)

Enzyme Assay Solutions

Enzyme Substrate Coenzyme + other additives Buffer

21) ALAT 10 mg L-alanine It mg NADH 5 ml 0.1M phosphate, 50 ut LDH pH 7.4 0.5 ml 0.1MfN-ketoglutarate After 1 hr incubation 2 mg MTT 1 mg PMS

22) TAT 10 mg Tyrosine 5 mg NAD 10 ml 0.5M Tris-HCl, 2 mg Pyridoxal phosphate pH 8.0 6 mg NBT 1 mg PMS 10 ut GDH

TABLE FOUR (c.,ont.)

Enzyme Assay Solutions

Enzyme Substrate Coenzyme + other additives Buffer

23) PGM 10 'mg Glucosel-phosphate 6 mg NADP .2 ml 0.5M Tris-HCl 20 ut G6PDH pH 8.0 1 mg PMS 4 mg NBT 4 mg MgC1 2 6 ml H2O

24) DHR 1.5 mg Dihydrofolic acid 5 mg NADPH 5 ml 0.1M phosphate 4 mg MgCl2 pH 7.4 After 1 hr incubation 2 mg MTT 1 mg PMS

25) TO None 6 mg NBT 5 ml 0.5M Tris-HCl 1 mg PMS pH 7.0 TABLE FOUR (cont.)

Enzyme Assay Solutions

Abbreviations:

NBT - Nitroblue tetrazolium MTT - MTT tetrazolium PMS - Phenasine methosulphate NAD - Nicotinamide adenine dinucleotide NADP - Nicotinamide adenine dinucleotide phosphate 62

gel was simultaneously stained for the enzymes in question..

11. (A) Deoxyribonucleic Acid Isolation

Preparation of deoxyribonucleic acid (DNA) was carried out in a similar manner to that described for leishmaniae (Chance et al. 1974).

Samples of sporulated oocysts, at least 5 x 108, were homo- genised in 0.1 SSC (0.015M NaC1, 0.0015M Na Citrate), using glass beads and mechanical agitation. The resulting suspension was drawn off and centrifuged to remove the larger debris. To every 3 ml of the supernatent was added 2 ml of lysis solution (6% aminosalicylate, 0.5% sodium dodecyl sarcosinate N.L.97 and 1% NaC1). After this mixture had been incubated at 37°C for 30 minutes with 1 mg/ml predig- ested pronase,an equal volume of the phenol m-cresol solution, as described by Kirby (1965), was added. The complete mixture was shaken vigorously for 5 minutes and then centrifuged to separate the phases at approximately 800 x'g for 15 minutes. The aqueous layer was removed and the DNA recovered from this by precipitation with 2 vols. of ethanol. The DNA strands were carefully wound around a sealed glass pipette, dissolved in SSC and reprecipitated with ethanol; this procedure was repeated two or three times. DNA was also prepared from purified sporozoites (approximately 1 x 109) disrupted in the lysate solution.

(B) Analytical Caesium Chloride Density Gradient Centri- fugation

Dr. M. Chance of the Liverpool School of Tropical Medicine performed all operations concerned with the density gradient centrifugation. 1 - 2 pg of DNA were centrifuged together with 1pg of Escherichia coli DNA as marker at 45,000 r.p.m. in an MSE analytical ultracentrifuge for 18 hours. Microdensitometer tracings of the photographs were made using a Joyce Loebl chromoscan. The buoyant densities were determined according to Vinograd and Hearst (1962). 63

12. Tissue Culture

(A) Sterilisation Procedures Glassware, filter holders and instruments were cleaned in 2% Decon 75 solution (Decon Laboratories Ltd.). Particu- larly dirty glassware was treated with chromic acid. Cover- slips were washed in two changes of absolute ethanol and one of ether.

Sterilisation by moist heat was carried out in an autoclave at 15 lbs sq.in. for 20 minutes. Materials were wrapped •in Alcan Foil and solutions held in bottles with caps which were left loose during autoclaving. Dry heat was used for glass beads, pipettes and coverslips; this entailed temperatures over 160°C for at least 2 hours. Solutions, which could not be subjected to high temperatures, were sterilised by filtration through 0.22 m millepore filters and prefil- ters in Swinnex 25 filter holders. A sterile cabinet, swabbed out with 70% alcohol and irradiated with ultraviolet light, was used for temporary storage of sterilised materials. All experimental manipulations were performed in a laminar flow cabinet using aseptic technique.

(B) Monolayer Cultures Primary cultures of chicken and quail kidney cells were obtained using methods similar to those described by Doran (1970,71a). The medium for growing cells before inoculation of the parasite was Doran's medium consisting of: 80% Hanks balanced salt solution (HBSS), 10% lactalbumen hydrolysate (LAH 2.0% solution in HBSS) and 10% foetal calf serum. Phenol red indicator was contained in the medium and the pH was adjusted to 7.0 - 7.2 with sodium bicarbonate. Pen- icillin and streptomycin were added to concentrations of 100 units/ml and 100tAg/m1 respectively.

Cells were grown in Leighton tubes on 9 x 35 mm coverslips, 2 on 24 mm coverslips in plastic Petri dishes (Sterilin Limited) kept in sandwich boxes gassed with a 5% CO2 -95% air mixture, and on the sides of prescription bottles (200,100,60 ml).

When the cell layers were confluent, usually after three days, they were inoculated with sporozoites, normally at 64 5 a dose of 2 x 10 sporozoites per ml of culture medium. Cultures were washed after 4 hours and fresh medium added. If cell growth was appearing to be too rapid, the serum content was reduced to 5%.

Chicken kidney cells and cell aggregates obtained as for monolayer cultures were placed in 100 ml conical flasks 5 with 30 ml of Doran's medium at a concentration of 1 x 10 per ml. The cells were incubated at 41°C and kept in suspension by the use of a magnetic stirrer. Sporozoites 5 were added, 1 x 10 per ml of culture medium:and the flasks sealed in an atmosphere of 5% CO2. Cells were removed and examined at intervals.

(D) Organ Slices

The alimentary canal of chick embryos) sacrificed at 20 days., was removed and slices no greater than 1 mm in thickness were taken from the duodenum, small intestine and•caecum. The slices were washed in phosphate buffered saline and placed in individual droplets of Doran's medium containing 6 either 1 x 106 sporozoites of E. tenella or 20 x 10 second generation merozoites of E. tenella and incubated at 41°C for 4 hours. The slices were then washed and placed on a 0.251pm millipore filter which was suspended above the medium in a Petri dish by a stainless steel grid. Contact was maintained with the medium, minimal essential medium (MEM) with 10% foetal calf serum, by small wicks made of 0.25p .m millipore filters.

Similar experiments were conducted with intact slices of kidney from two-week-old chickens and slices of alimentary canal obtained from fifteen-day-old quail embryos.

13. Staining Techniques

Impression smears and monolayer cultures on coverslips were air-dried and fixed momentarily in methyl alcohol, stained with 10% Giemsa stain in phosphate buffer at pH 7.2 for 30 - 45 minutes, rinsed in tap water and mounted•in green euparol. 65 Tissues for sectioning were cut into portions not greater than 5 mm2 and fixed in Carnoy's fixative or neutral buffered formal-saline. Dehydration took place in a graded series of ethanol mixtures. Cedarwood oil was occasionally used as a clearing agent and tissues were embedded in para- ffin wax or paraplast. Sections were stained with Ehrlich's haematoxylin as described by Clerk (1973) or by the Giemsa- collophonium technique (Bray and Garnham 1962).

14. Cytochemistry

Sporozoites, merozoites and developmental stages in tissue culture were tested for lactate dehydrogenase, succinic dehydrogenase, glucose 6-phosphate dehydrogenase, aspartate aminotransferase and leucine aminopeptidase. Purified merozoites and sporozoites,which were smeared on to glass slides coated with glycerin albumen, and cells grown on coverslips in Leighton tubes were either incubated as unfixed preparations in the staining solution or were prefixed in cold acetone or glutaraldehyde, and then incubated at 37°C. Staining solutions were basically as detailed by Pearse (1972) and are listed in Table 5. After incubation, slides were washed in phosphate buffered 1% saline, counter- stained in Orange G and mounted in glycerine jelly. Obser- vations were made immediately as the preparations were ephemeral. Rigorous controls were conducted, with and without substrate or coenzyme and by the use of heat-treated preparations.

15. Measurements

General measurements were made directly using a micrometer eye piece. For comparative purposes, measurements were determined from enlarged prints taken at known magnifica- tions with a Wild Photomicroscope.

16. Serum Samples

Blood obtained from the wing of a young bird was transferred to cool tubes and allowed to coagulate for 1 hour at 4°C. After centrifugation the serum was drawn off, the sample was used immediately or stored temporarily at -20oC. Serum from rabbits infected with E. stiedai was 66

TABLE FIVE

Incubating Media

Enzyme Substrate Incubating Solution

LDH 13.2 ml 1M Na 1M DL-Lactate NAD 2 mg (pH 7.0) NBT 1 mg PBSA (pH 7.4) 2 ml

G6PDH 2 mg Glucose 6-phosphate NADP 2 mg NBT 1 mg 0.1M NaCn 0.1 ml 0.05M MgCl2 0.5 ml PBSA (pH 7.4) 1.5 ml

SDH 1.0 ml 2.5M Na Succinate 0.05M MgC1 0.5 ml 2 2 0.1M NaCN 0.1 ml PBSA•(pH 7.4) 1.5 ml NBT 1 mg

LAP 4 mg L-leucyl -B naph- Fast Blue B salt 2 mg thylamide 0.1M Acetate buffer 2 ml 1% NaCl 1 ml 0.1M NaCN 0.1 ml After 30 min. incubation 0.1M cupric sulphate -30 secs

ASAT 4 mg L-aspartic acid 0.01M*1

The determination of serum alkaline phosphatase levels was carried out as detailed in Sigma technical bulletin No. 104 using a Beckman spectrophotometer. Electrophor- etic analysis of serum samples was carried out as described for extracts of parasites. 68

PART I

Biochemical characters of Eimeria species 69

INTRODUCTION

A) The Characterisation of Protozoa by Biochemical Criteria

General Considerations

It is well established that animal species show a range of variation for most of their phenotypic characteristics; variation which has both a genetic and an environmental component. Protozoa are no exception; members of a single species may vary in both morphological and physiological characters. In almost all well studied species, races or strains have been detected.

Traditional means of identifying protozoa have relied on morphological features. Such methods are inadequate where distinct morphological characters are absent or where variation is such that a broad phenotypic overlap exists between related groups. During recent years, novel taxonomic methods have been developed,which have greatly facilitated the analysis of genetic variability and population structure in many groups of organisms (see Wright 1974). Of particular importance has been the utilisation of biochemical and biophysical techniques, which has enabled the taxonomist to investigate subtle differences occurring at the molecular level. Indeed, direct studies on the genetic material, deoxyribonucleic acid (DNA), are also providing valuable taxonomic data.

One of the most rewarding areas of research has been that ,concerned with the examination of functionally related molecules, such as the isoenzymes of a given enzyme in different individuals. As it now seems certain that the sequence of nucleotides that make up a structural gene is translated with a high degree of accuracy into a sequence of amino acids making up a polypeptide chain, examination of polypeptides provides indirect information about DNA. In general, it can be assumed that any change in the base sequence of a DNA molecule will be reflected in a substitution, deletion or 70

addition of an amino acid in the polypeptide coded by the gene in which that alteration occurred. Techniques for analy- sing the amino acid sequences of proteins are at.present difficult and time-consuming. However, striking differences have been shown to exist in functionally related proteins isolated from different sources, as exemplified by studies on haemoglo- bins (Zuckerkandl and Pauling 1962, quoted by Stephen 1974). A method of characterising proteins which is sensitive to certain single amino acid alterations and allows reasonably rapid examination of a large number of samples is gel electrophoresis.

The principle of this method of separating proteins depends upon passing an electric current through an electrophoretic medium containing the proteins and upon the possession of varying electrical charges by the proteins to be separated. A polypeptide has a net negative or-positive charge, depending on the balance of charges conferred upon it by its amino acid composition and on the folding-of the molecule. At a given pH, the net -electrical charge is dependent upon the number of exposed amino and carboxyl groups which are ionised. At its isoeleLL±1c point, a protein possesses the same number of charged carboxyl and amino groups and is neutral. The greater the charge on a molecule the faster it will move in an electric field in the direction of the electrode having the opposite charge. Not all differences in the genetic material are detected by this technique. Nucleotide changes may occur without altering the amino acid sequence of the polypeptide and many amino acid changes may occur without altering the net charge of the molecule. Shaw (1970) estimated that only 30% of the possible nucleotide substitutions code for amino acids with different charges.

As various chemical and physical conditions may alter the charge or chemical properties of a protein, thus changing the migration of the molecule during electrophoresis, protein extraction and storage procedures should be standardised. Variations of isoenzyme and protein patterns have been reported after freezing and thawing of samples (Smith 1968), after ultrasonic treatment (Dubbs 1966), after the use of Triton X-100 (Momen et al. 1975) and after lyophilisation, refrigeration and incu- 71

bation of extracts (Ruff et aZ. 1971).

Besides utilising the net electrical charge of a protein, separation can also be affected by a molecular sieving in the gel. This is particularly the case with gradient pore acrylamide gels. With this method proteins are forced to migrate until they reach their 'pore limit' at which point they can travel no further. Because the leading edge of each zone always encounters more resistance than its trailing edge, the bands become sharper as the run progresses.

In a short review of the literature, Bourns (1974) emphasized the apparent dearth of information concerning the application of biochemical and serological methods in taxonomic studies of Protozoa and parasitic helminths. The methods of 'molecular taxonomy' are, however, providing the protozoologist with valuable information. The following • review illustrates the important contribution that such methods have already made to the understanding of certain groups of Protozoa. 72

I. Sarcodina

Kates and Goldstein (1964) made a comparison of the characteristics of proteins present in strains of Amoeba proteus, Amoeba discoides and Chaos chaos . No differences were found in the protein composition of the strains of Amoeba, although Chaos chaos was clearly different from the others. On strictly biochemical grounds, they concluded that A. proteus and A. discoides should not be considered as different species.

Although little taxonomic significance was attached to their findings, Visvesvera and Balamuth (1975) noted variation in the electrophoretic patterns of the soluble and particulate fractions of a pathogenic Acanthamoeba (Lilly strain) and the non pathogenic Acanthamoeba castell- ani (Singh strain). The presence of carboxypeptidases was established in non-pathogenic strains of Entamoeba histolytica and Acanthamoeba but could not be demonstrated in pathogenic •E. histolytica (Jarumilinta and Maegraith 1961).

Analysis of the electrophoretic properties of glucose phosphate isomerase (GPI) revealed differences between Entamoeba species (Montvalo and Reeves 1968). The electrophoretic mobilities of the GPI in the cultures examined fell into three groups: a fast migrating enzyme characterising atypical E. histolytica strains and two E. moshkowskii strains, an intermediately migrating enzyme found in typical E. histolytica and a slow migrating enzyme yielded by E. invadens and E. terrapinae. The uniform mobility of GPI in typical E. histolytica strains was striking as the strains were of diverse geographic origin and had dates of isolation ranging from 1924 to 1959. The classification of cultures of amoebae based on the properties of this single enzyme was identical to that previously proposed by Reeves et al. (1967) based on studies of glucokinase, but the use of three additional enzymes, L-malate: NADP oxireductase, phosphoglucomutase and NADP diaphorase produced evidence of differences between the strains of typical E. histolytica but not 73

between four cultures of E. invadens and E. terrapinae (Reeves and Biscoff 1968). Although the partially purified malic enzyme of E. invadens was determined to be a single species of protein by zone sedimentation gel filtration and gel electrophoresis, three isoenzymes were demonstrated by isoelectric focusing (Buro and Weller 1974). This technique might well prove useful for comparative purposes.

Gelderman et aZ. (1971a) reported that quantitative and qualitative differences found in the DNA of morphologically similar E. histolytica, E. histolytica-like amoebae and E. moshkowskii were sufficient for them to be designated as different species. In a further study, differences in DNA base compositions were found in strains of amoeba which had previously been thought to belong to the same species (Gelderman et aZ. 1971b). Valid distinctions could also be made when the buoyant densities of, DNA from axenically grown E. histolytica strains were compared (Reeves et al. 1971).

Studies on the DNA of Acanthamoeba spp revealed two components (Adam et al. 1969). The renaturation characteristics of the minor component suggested that it was of mitochon- drial origin. The buoyant densities of both the major and the minor components were found to differ between A. casteZZ-- ani, A. palestinensis, A. poZyphaga and A. astromjxis. 74

2. Mastigophora

Investigations on the activities and isoenzymes of malate and lactate dehydrogenases by Bayne and Roberts (1969) showed that the bands of lactate dehydrogenase of Trypano- soma equiperdum, demonstrated after electrophoresis, did not correspond in position to those of T. conorhini. Immune inhibition studies of trypanosomal aminotransferases also suggested that species-characteristic isoenzymes might exist (Godfrey and Kilgour 1972). The usefulness of isoenzymes as an extra taxonomic tool for the identification of trypanosomes was confirmed by Kilgour and Godfrey (1973). They found that isoenzymes of two aminotransferases produced consistently different electrophoretic patterns which could be used, not only to distinguish between species of trypanosomes, but also subspecifically between rat adapted and ruminant-infective isolates of Trypanosoma vivax. These findings were extended by.a study of alanine and aspartate aminotransferases in samples of T. vivax collected from naturally infected Nigerian cattle (Kilgour et aZ. 1975). Three distinct isoenzymes were demonstrated which were found to be stable in experimental animals. The migration patterns of the two aminotransferases were also shown to be of use in distinguishing between culture forms of T. rangeli, T. cruzi and T. Zewisi (Toye 1974a) and between various isolates of T. cruzi (Toye 1974b).

Bagster and Parr (1973) found that T. lewisi, T. congolense, T. vivax and T. brucei brucei differed from each other quite clearly in the electrophoretic behaviour of three more enzymes: glucose 6-phosphate dehydrogenase, phosphoglucose isomerase and malic enzyme. Differences in the mobility of malic enzyme in two strains of T. brucei brucei were also noticed. Parr and Taylor (1974) reported the use of isoenzymes of phosphoglucomutase for the identification of trypanosomes.

The measurement of enzyme ratios as a means of differentiating trypanosomes was suggested by Parr and Godfre\ (1973) and Godfrey and Kilgour (1973). Differences in the alanine aminotransferase: aspartate aminotransferase ratios between 75

.1% brucei brucei and T. brucei rhodesiense were found by Steiger et al. (1974), who also emphasised the need for further work to explore the use of such methods for the differentiation of closely related haemoflagellate species.

Trypanosoma mega was found to possess both a larger cell volume and a greater DNA content than T. lewisi (Lopez and Melton 1975).

Research into many aspects of leishmaniasis has been hindered by the difficulties of distinguishing between species and strains. However, biochemical techniques are now also proving of great value for the characterisation of leishmaniae.

Two forms of malic dehydrogenase (MDH) which differed markedly in their physical and kinetic properties were demonstrated in Leishmania tarentolae grown in vitro ( Krassner 1968). A preliminary investigation of disc polyacrylamide electrophoresis of MDH for the identification of Leishmania isolates was made by Gardener and Howells (1972). Later, the examination of a large number of isolates revealed numerous MDH variants (Gardener et al. 1974). Using thin layer starch gel electrophoresis,Kilgour et al.(1974) found a similar degree of variation in alanine and aspartate aminotransferases in Leishmania strains. These enzymes had been demonstrated earlier in L. tarentolae in which they gave one discrete zone of activity (Fair and Krassner 1971). The electrophoretic mobility of phosphatases, esterases and general proteins have also been used for characterising these parasites (Ebert 1974 a,b, 1973).

Other genera of flagellates have received less attention. Isoenzymes of malate dehydrogenase have been reported in Trichomonas vaginalis (Brugerolle 1973). An isoenzyme of malic enzyme was detected in heterotrophic cultures of Euglena gracilis, but was found to be repressed by photosynthesis (Karn and Hudock 1973). Recently, Dooris and McGhee (1976) used electrophoretic characters for differentiating Crithidia hczrmosa and C. facciculata. 76

An extensive study of the DNA of flagellates representing the major genera and sub-genera of the order Kinetoplastida was made by Newton and Burnett (1972). The possession of nuclear and kinetoplast DNA in this group, both free to vary in base compositon, increases the variability that may be seen between related organisms. In general, it was found that organisms which were believed to be closely related possessed similar DNA base compositions. The range of variation of the kinetoplast DNA was considerably less than that of the nuclear DNA. The determination of the DNA buoyant density has been particularly useful for the study of the systematics of the genus Leishmania (Chance 1972,Chance et al. 1973,Chance et ca. 1974). In most instances, the results agreed with those obtained by a study on the electrophoretic mobility of MDH (Gardener et al. 1974); strains possessing distinctive DNAs generally had their characteristic MDH type. Schnur and Chance (1976) made a comparison of four methods used for the characterisation of African leishmanial strains: buoyant densities of nuclear and kine- toplastic DNA; electrophoretic variation of MDH; sero- typing by the differential precipitation of leishmanial excreted factors with homologous and heterologous anti- sera; and determination of amastigote dissemination patterns in Syrian hamsters following the intrasplenic inoculation of cultured promastigotes. They concluded that there was reasonable agreement between all the methods and that the characters used for' differentiating strains did not vary independently.

A suggestion was.made by Laurent and Steinert (1970) that the contour length of the circular kinetoplast DNA molecules might be species specific. Chance et al. (1974) examined the isolated kinetoplast DNA by electron microscopy and found no differences in minicircle contour length between different Leishmania isolates, despite the considerable diversity of kinetoplast DNA buoyant densities within the genus.

In the phytoflagellate orders Euglenida and Volvocida are found some colourless, exclusively heterotrophic forms, 77

3. Ciliophora

The techniques of Hunter and Markert (1957) of histochemical identification of enzymes, which had been electrophoretically resolved, were used by Allen (1959) on the esterases of Tetrahymena. She found that variety I of T. pyriformis contained two classes of esterases, an eserine sensitive class and an eserine insensitive class. The former class was reported to show strain differences. A further report by Allen (1960) detailed the manner in which the eserine types were inherited within a clone during vegetative reproduction and after conjugation within the strain.

Seventeen bands with acid phoshatase activity were separated by starch gel electrophoresis from extracts of different genotypes of a variety of T. pyriformis (Allen et al., 1963). These bands were characterised by several different methods involving genetic analysis, chemical characterisation, varying growth conditions and distribution in various cell fractions. Some of the members of the group of acid phosphatases showed genetic variation. Studies of the patterns found in certain heterozygotes suggested that 'hybrid enzymes' may be formed by the association between the products of two alleles in heterozygotes. Tetra- hymena vorar strain V2S undergoes a distinctive morphogen- etic change from a small-mouthed saprozoically feeding form to a large-mouthed carnivorous form. Stamler (1974) studied isoenzyme changes during the transformation process and found an alteration of the acid phosphatase banding pattern specific to transforming cells. Variation in lactate dehydrogenase isoenzymes was found to occur during the population cycle in T. pyriformis suggesting a dependence of enzyme expression on environmental conditions (Corbett 1973).

The use of enzyme variation both as a taxonomic tool and as a means of studying genetic variation in Tetrahymena was further established by Allen (1965), Borden et al. (1973a, 1973b). In a very comprehensive study, Borden et al. (1973c) examined the electrophoretic mobility patterns on starch gel of eight enzymes in forty-three classical strains of Tetrahymena pyriformis. 78

Of particular importance was the observation that some strains, designated as identical but obtained from different sources, had little resemblance to one another in their isoenzyme compositions. Other strains designated as different by their contributors often had very similar and identical isoenzyme patterns. In conclusion:these workers proposed that the present strain designations be abandoned and reassessed. For this purpose, they compiled 5 'phenotypic' sets (phenosets), based on their electrophoretic data. Nielsen and Andronis (1975) studied the mobility patterns of 5 enzymes in strains of Tetrahymena pyriformis using polyacrylamide disc gel electrophoresis and reaffirmed the proposal of strain designations on a basis of 'phenosets'.

Parcriecium aurelia is also a complex which has been sub- divided into a number of breeding groups, formerly called syngens. The isoenzyme variation within and between such groups has been examined. Tait (1970) observed the electrophoretic mobility of five enzymes in extracts of this ciliate on starch gel. Although few differences were found in stocks from the same syngen, only two out of fourteen syngens of Paramecium aurelia were indistinguishable. Intra- syngenic variation was also found to be rare in the esterases of these organisms, although the syngens differed in a complex way from each other suggesting that several gene differences were involved (Allen et al. 1971, Allen and Gibson 1971a). This differs from the extensive variation of esterases and phosphatases within the breeding units of T. pyriformis (Allen and Weremiuk 1971). Rowe et al. (1971) pointed out the contribution of bacteria to the esterase zymograms of P. aumlia.

The base compositon of DNA mole.cules from different syngens of Paramecium avrelia were found to be similar, although isolated observations of other workers had not indicated such a similarity (Allen and Gibson 1971b). It is interesting to note that the density of anyParaneciumDNAvaried depending upon whether the cells were grown in the presence of bacteria or in axenic medium. The same authors reported variation in the base composition of the breeding groups of T. pyriformis. 79 which are morphologically very similar to the green forms capable of photosynthesis. Substantial differences were found in the DNA buoyant densities between Euglena gracilis and its heterotrophic counterpart Astasia Zonga (Mandel 1967). Less difference was noted when the DNAs of Chlamydomonas and Polytoma obtusum were compared. But P.obtusum, P.uvella and P.agilis differed quite markedly in the base compositon of their DNA (Mandel 1967). ,

Mandel and Honigberg (1964) reported differences between the DNA base compositions of Trichomonas gallinae and T. vaginalis. They also found that the ratios of DNA:RNA differed between two strains of T. gallinae, strains which could not be separated by their DNA base composition. 80

4. Sporozoa

The sporozoa include intracellular parasites of the genera Plasmodium and Eimeria. As the intimate relationship between host and parasite is difficult to analyse, little is known of the precise metabolic requirements of these parasites. Electrophoresis has proved to be a useful method for distinguishing parasite-specific enzymes from those of host origin. Studies on malate dehydrogenases in P.lophurag and P. berghei and their host cells, the duck and mouse erythrocyte respectively, revealed differences in electrophoretic and catalytic properties (Sherman 1966). Other enzymes which have been identified by electrophoretic techniques include: lactate dehydrogenase in P. lophurae and P. berghei (Sherman 1961, 1962), glutamate dehydrogenase in P. lophurae (Sherman et al. 1971), pyruvate kinase in P. lowwelesi and P. berghei (Oels'hlegel et al. 1975) and lactacte dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, catalase, esterase and aspartate aminotransferase in P.berghei(Tsukamoto 1974).

The first study in which variations in the electrophoretic mobility of enzymes were used to characterise malaria parasites was that of Carter (1970). He reported differences in the electrophoretic migration of glucose phosphate isomerase (GPI), 6-phosphogluconate dehydrogenase (6PGDH), adenylate kinase-(AK) and malate dehydrogenase (MDH) in extracts of P. berghei berghei, P. berghei yoelii, P.berghei killicki and a Nigerian strain of P.berghei. Further studies on strains of P. berghei and on strains of four subspecies of P. vinckei revealed considerable variation in the electrophoretic migration of a selection of enzymes (Carter 1973). In general, the results substantiated the classical taxonomy of these parasites and provided further information on the . relationships between the subspecies, Using enzyme type as a diagnostic feature, in addition to morphological characters, Carter and Walliker (1975) divided the rodent parasite originally described as P. chabaudi into two distinct species, P. atiabaudi and P. vinckei petteri. Momen et al. (1975) reported that MDH could not be detected by electrophoretic means in erythrocytic stages of certain strains of P. berghei but was present in others. They suggested that this supported the 81

view of Killick-Kendrick (1974) that the "berghei group" should be divided into two species.

The differences in electrophoretic mobility of enzymes have been shown to be precise and stable characteristics of the malaria parasites (Walliker et a/.1971). A survey of the electrophoretic form of GPI, LDH, MDH and 6PGDH was made in a selection of strains of human and simian malaria parasites (Carter and Voller 1973). Genetic differences between parasites were clearly demonstrated and the findings indicated that variation was probably widespread within species of Plasmodium. Carter and McGregor (1973) made a study of electrophoretic forms of enzymes in P. falciparum in samples of blood from Gambian women and children. Variation was found in the three enzymes examined (LDH, GPI and 6PGDH) and the frequency of the variant forms of each enzyme was established. From statistical considerations of the distribution of the enzyme forms among the samples, it was estimated that the samples contained an average of about two parasite clones each. Isolates from a number of different localities, in Eastern and Western tropical Africa, were examined to determine the distribution of enzyme variants across the continent, to test the hypothesis that genetic divisions exist between populations of P..falciparum (Carter and Voller 1975). The frequencies of the different forms of GPI, 6PGDH and LDH were found not to differ greatly in populations from the East to the West coast. While not disproving the existence of genetic divisions between P. falciparum populations, the distribution of the variations provided no evidence for such an idea.

Investigations have been made to detect isoenzymic changes during the life cycle of Plasmodium. Carter (1972) found that in P.berghei yoelii and the Nigerian P.berghei (both now assigned to P.yoelii,Killidk-Kendrick 1974) the GPI proteins of the oocyst and the blood parasites were identical. Using polyacrylamide gel electrophoresis,Howells and Maxwell (1973a) demonstrated that the malarial oocyst possessed isoenzymes of NAD and NADP dependent isocitrate dehydrogenases which were distinguishable from those of the mid-gut of Anopholes stephansi. These enzymes could not be detected in intraery- 82 throcytic forms of chloroquine sensitive or resistant strains of P. berghei (Howells and Maxwell 1973b).

The availability of techniques for differentiating strains of P. berghei facilitated genetic studies on the malaria parasites. By making crosses between lines which differed in both enzyme type and drug sensitivity, parasites were obtained which exhibited recombinant characters (Walliker et al. 1971). Further investigations showed that a conventional genetic analysis was possible (Walliker et al. 1973). The cloning of the products of a cross between two distinct lines produced the expected recombinant classes and lines with the parental characteristics. Controls showed that the recombinant forms had arisen by the cross-fertilisation of gametes in the mosquitoes. Oxbrow (1973) carried out experiments to find a cross-immunity marker in P. berghei and to assess the use of such a character in genetic studies with drug resistance and isoenzyme markers. He showed that the genetic factors controlling the ability of the parasites to survive in hosts immune to P.berghei werefrequently involved in recombination between parasites. A similar process was postulated for p. falciparum which would provide a mechanism for the production of large numbers of antigenic variants.

Genetic studies have been extended to P.chabaudi (Walliker et al. 1975). Two lines of P.chabaudidiffering in three characters were crossed. The results showed that recombination had occurred between each of the three characters. Although only a small number of clones were examined, no evidence of linkage was found between the three markers: a drug-resistant character segregated independently from two enzyme markers. The genetic basis of chloroquine resistance in P.chabaudi has been investigated by Rosario (1976) using two lines which differed in drug sensitivity and their electrophoretic form of 6PGDH and LDH. The results showed that the chloroquine resistance, which developed in P.chabaudi, was a stable character inherited in simple Mendelian fashion and underwent genetic recombination with other markers.

In a cross between a virulent line and a mild line ofP.yoe1ii yoelii, enzyme and drug sensitivity differences between the 83 two lines were used as additional genetic markers (Walliker et al. 1976). It was found that the principle determinant of virulence had a simple pattern of inheritance which could be most easily explained as being due to a single gene.

Analytical ultracentrifugation of isolated DNA showed that all subspecies of the P. berghei complex have the same buoyant density but are distinct from P.vinckei and P. chabaudi (Chance and Warhurst 1973). The DNA relationships were further examined by DNA-DNA hybridisation studies using the technique of thermal elution of preformed hybrids from a hydroxyapatite column. The results showed that, in terms of the base sequence homology, P.berghei berghei strain K173 was widely separated from the subspecies P.berghei yoelii 17X andP.berghei N67, and that there was only a distant relationship with P.vinckei and P. chabaudi.

Surprisingly little is know about the biochemistry of eimeriine parasites considering that large numbers of purified oocysts can be obtained for biochemical studies. L -lactate dehydrogenase purified from unsporulated oocysts of E.stiedai produced one band of enzyme activity after electrophoresis on polyacrylamide gel (Frandsen and Cooper 1972). Other enzymes which have been extracted from oocysts of Eimeria include glucose 6-phosphate dehydrogenase (Frandsen and Ennis 1974) and fructose 1,6-diphosphate aldolase (Mitchell et a1.1976) inE.stiedaildihydrofolate reductase (Wang et al.1975a) and amylopectin phosphorylase (Wang et al. 1975b) in E.terzella. Three leucine aminopeptidases were found by Wang (1974) in extracts of unsporulated oocysts of E.tenella after polyacrylamide gel electrophoresis. App- arently,these enzymes gradually disappeared during sporulation and were replaced by a new leucine aminopeptidase located only in the cytoplasm surrounding sporocysts. Shirley (1975) presented preliminary results on the enzyme variation found in different species and strains of Eimeria from the domestic fowl. He found that the electrophoretic mobility of LDH, GPI,6PGDH and G6PDH allowed the identification of each of the six species examined. 84

In an attempt to produce additional criteria for the identification of species of piroplasms infecting cattle and rodents, Gourlay et aZ.(1970) investigated the use of disc electrophoresis of soluble proteins. Distinct zymograms were obtained for Babesia rodhaini, B.microti and Nuttalliamusculi. Momen (1975) suggested that the variation in the mobility of enzymes as shown by electrophoresis might prove to be a significant aid in taxonomic studies. He found variation in the migration patterns of LDH, GPI and GDH in B.rodhaini, B. hylomisci and B. microti. Differences in the DNA buoyant densities provided further evidence that B.rodhaini and B.microti are distinct species (Momen and Chance 1976). B. rodhaini and B.hylomisci possessed DNA with similar buoyant densities; the base compositon of Anthemosoma garnhami indicated that this parasite was not closely related to either rodentBabesia or Plasmodium. 85

5. Microsporidia

Fowler and Reeves (1974a) examined hydrophobic extracts from microsporidian isolates. They found that the patterns obtained were highly reproducible and suggested their use for identification. There findings did not wholly support the present classification. Some species of Nosema shared the same pattern as a species of TheZohania,whereas two other species of Nose-:awere found to differ. Similarly, Fowler and Reeves (1974b) examined hydrophilic extracts of spores of Nosema sp and Nosema trichopZusiae. Unlike the hydrOphobic extracts, the electrophoretic patterns obtained for hydrophilic extracts appeared to be dependent on four factors: the genus of host used for propagation; the temperature the hosts were reared at; the length of time the spores had been stored and the spore incubation period. However, they concluded that the use of major protein bands could provide characters useful in the separation and identification of microsporidian isolates. No overlap of major bands was observed in the comparison of Nosema sp and Nosema trichopZusiae, indicating that these isolates were not closely related.

In a comparison of two observed forms of an isolate then considered to represent a mixed infection of Nosema necatrix and Thelohania diazoma, Fowler and Reeves (1974c) used electrophoretic analysis of spore proteins as evidence for the idea that the isolate was in fact N.necatrix,a dimorphic microsporidian. 86

B) Isoenzymes in relation to parasitic diseases

Numerous observations have now been published in relation to the usefulness of isoenzyme studies in human pathology (see Latner and Skillen 1968). Isoenzymes have been of particular use for diagnosis; the greatest success has been obtained with serum LDH for detecting disease of the heart and liver. Diagnosis depends upon the possession of characteristic isoenzymes by tissues; when damage occurs to a tissue, isoenzymes are released into the serum which can be detected by electrophoretic analysis.

Despite many investigations on serum enzyme levels in parasitised animals, studies on isoenzyme changes are rare. KuCera and Weiser(1975) investigated changes in activities of LDH isoenzymes in the gut and fat body of Barathra brassicae and GaZleria mellonella infected by the microsporidia Nosema plodiae and Pleistophora schubergi. Two of the four isoenzymes in the fat body of both insects infected by N. T.lodiae increased in activity by the fifth day of infection. Akinyemi (1975) obL-:,ved an increase in the levels of alcohol dehydrogenase (ADH) in the livers of rats infected with T. cruzi, T. lewisi, T. congolense and T. rhodiense ADH zymograms indicated that only certain isoenzymes were elevated and that differences occurred in the mobilities of ADH in the livers of infected animals. Whether the trypanosomes themselves contained high levels of this enzyme was not considered. Despite widespread tissue necrosis, no changes in serum LDH activity or isoenzyme patterns were observed in the serum of rabbits infected with T. gambiense (Deihl and Risby 1974). The differences in MDH between asca- rid species and the tissues of their natural hosts led Zee and Zink am (1975) to suggest that electrophoretic analyses might prove a useful method for detecting extra-intestinal forms of parasitism, Analyses of the isoenzyme patterns of LDH in plasma of patients suffering from either Plasmodium falciparum or P. vivax indicated that severe haemolysis of the host red blood corpuscles was responsible for LDH levels (Tsukamotoet al. 1975). No variation in serum 'non-specific' esterases, LAP or AP activity levels or isoenzyme patterns were found associated with the malaria infection. 87

Coccidiosis is often associated with destruction and damage of tissues; particularly those of the gut. Investiga- tions were made to determine whether changes in the serum enzymes of the host could be related to pathology. As plasma AP in the domestic fowl has been shown to be of intestinal and liver origin (Bide 1969); special attention was given to this enzyme. 88

RESULTS

1) Identification of enzymes in oocysts of Eimeria spp

The enzymes which have been identified in sporulated oocysts of E.tenella(Houghton) and their relative activities, estimated by visual assessment of the degree of staining in the gel, are detailed in Table 6. The enzymes which have been demonstrated to occur in the oocysts of other Eimeria spp are shown in Table 7.

During the examination of enzymes, utilising the phenasine- tetrazolium technique, in addition to the blue bands marking the site of the isoenzymes of the enzyme under investigation, distinct light areas were occasionally noticed in the starch. These light bands appeared to be constant for a particular sample. The bands could be demonstrated, although not quite as obviously in gels stained with PMS and MTT and exposed to light, and were considered to be sites of activity of the enzyme tetrazolium oxidase.

Attempts to demonstrate the following enzymes in sporulated oocysts of E.tenella (Houghton) and E.acervulina (Weybridge) failed: 'non-specific' esterases (opCB-naphthyl acetate as substrate), ACP, GDH, ALAT, SDH, TAT and IDH (NAD). No activity could be demonstrated for GDH, SDH and TAT. Results obtained for ALAT and IDH (NAD) were inconsistent and any activity which was demonstrated was diffuse. Although no discrete bands were formed,a suggestion of esterase and ACP activity was recorded after disc gel electrophoresis.

Two attempts were made to demonstrate DHR and PK in sporulated oocysts of E.tenella(Houghton) but no activity was observed.

No formation of formazan deposits was observed in controls without substrate and coenzymes. 89

TABLE SIX

Enzymes identified in water soluble extracts of Eimeria tenella oocysts

Enzyme Activity

AP FDP LAP H6DH G6PDH ckGPDH IDH (NADP) MDH (NAD) ME (NADP) LDH 6PGDH AK GPI ASAT HK PGM TO 90

TABLE SEVEN

Enzymes identified in water soluble extracts of oocysts of Eimeria spp

vae

kimo lis

a a ens la i H i in t hly lina t idua

la t U . idg da l ico ko vu na br r u tl) t.) .0 tes ie t .6 ina CI s irres coec in mag tene n ..0 brune wey ace E Z 0. E. E. E. E. E. E. E. E. E. rzi E. W rzi 4.1

AP + + + + + FDP + + + + + LAP + + + + + + + + + + + H60:: + + + G6PDH + + + + + + + + + + + + + d`--GPDH + + + + + IDH + + + MDH + + + + + ME + + + LDH + + + + + + + + + + + + + + 6PGDH + + + + + + + + + + + + AK + + + + + GPI + + + + + + + + + + + + + + ASAT + + + + + + + + + + + + + HK + + + + + + + + + + + PGM + + + + + + + + + + + TO ‘- + + + + + + + 91

2} The effects of extraction and storage procedures on water soluble proteins

Sporulated oocysts of E.tenella (Houghton) were treated in the ways detailed below. Three enzymes, LDH, GPI and ME, were analysed on thin-layer starch gels. The effect of each treatment on the migration patterns and activity of the enzymes, as shown by the degree of staining, was recorded.

Treatment 1

Oocysts were suspended in an equal volume of one of the following:

(a) Phosphate buffered 1% saline (pH 7.4) (b) Distilled water (c) Gel buffer to be used in electrophoresis (d) 0.01 M Tris-HCl (pH 7.0) plus enzyme stabilisers (dithiothreitol, e-aminocaproic acid and EDTA each to a concentration of 1.0 mM)

(e) 5% Triton X-100 in d

The preparations were disrupted with glass beads. After centrifugation,the supernatants were analysed immediately.

Treatment 2

Disruption of oocysts suspended in 0.01M Tris-HCl (pH 7.0) plus enzyme stabilisers was achieved by one of the following methods:

(a) Grinding in a 0.1 ml glass minihomogeniser (b) Addition of glass beads and violent agitation

The preparations were centrifuged and the supernatants analysed immediately. 92

Treatment 3

Oocysts were suspended in 0.01 M Tris-HC1 (pH 7.0) plus enzyme stabilisers and disrupted with glass beads. The homogenates were further treated by:

(a) Incubation at 37°C for 1 hour (b) Sonication (total of 2 minutes, amplitude 9 microns)

The preparations were centrifuged and the supernatants analysed immediately.

Treatment 4

Oocysts were suspended in 0.01 M Tris-HC1 (pH 7.0) plus enzyme stabilisers and disrupted with glass beads. After sonication and centrifugation,the supernatant was either analysed immediately or:

(a) Lyophilised and stored in sealed containers at -20°C

(b) Reduced with Lyphogel (c) Stored at 4°C (d) Stored at -20°C.

No differences were observed in either the activity or the mobility of the enzymes that could be accounted for by the kind of solution in which the oocysts had been disrupted.

The addition of glass beads to oocyst suspensions followed by violent agitation was the most satisfactory method of homogenisation. Equally good oocyst disruption and subsequent enzyme activities were found in samples prepared using a minihomogeniser but the final volume of sample obtained was much less. Attempts to disrupt oocysts by ultrasonication were unsuccessful: many oocysts remained intact and consequently no enzyme activity was demonstrated in these samples.

ME was not demonstrated in homogenates which were not sonicated after the initial disruption. When the mechanical 93 disruption of oocysts was followed by ultrasonication, ME was demonstrated as a weak band of activity in samples sonicated for one minute and as a distinct zone of activity in samples which had been sonicated for two minutes. The banding pattern of GPI was altered by sonication: although the position of the main band remained unchanged, four or five sub-bands (sec- ondary bands), which decreased in activity towards the anode, were observed in sonicated. samples, as opposed to one band in fresh unsonicated samples. LDH showed as a single band of activity in sonicated and unsonicated samples.

Incubation of the extracts at 37°C for one hour did not alter the mobility or the activity of the enzymes.

Samples maintained good but decreasing LDH and GPI activity when stored up to two days at 4°C and up Lo 7 days at -20°C. In stored preparations, the original single band of GPI activity was replaced by numerous sub-bands. ME activity was lost very rapidly and in order to demonstrate this enzyme fresh samples were always required.

Freeze-dried preparations, in general, showed a marked • decrease in activity when compared with fresh preparations. Direct comparisons were hindered, however, by the difficulty of reconstituting the dried extracts to the equivalent volume. Bands attributable to ME were not detected in lyophilised samples. LDH and GPI retained reasonable activity in freeze- dried preparations after storage for one year four months. Lyophogel reduced the sample size by approximately one third of the original volume. The concentrated extract showed a corresponding increase in enzyme activity; the mobility of the enzymes was not altered. 94

3) Electrophoretic conditions and enzyme migration

The use of particular buffers for specific enzyme systems in coccidia was developed by trial and error. The variants of an enzyme were not always separated by a single system of electrophoresis. PGM in extracts of oocysts of E. maxima (Weybridge) and E. maxima var. indentata migrated similar distances using Buffer 5 (0.15 M Tris-Citrate, pH 9.0), but differences in the mobilities of the enzymes were detected using Buffer 6 (0.14 M pH 7.0). Figure 2, a,b,c, shows slight variations in the mobilities of G6PDH in extracts of various strains of E. acervulina which were attributable to differences in the buffers used and the time allowed for the electrophoretic run. For comparative studies, each enzyme was routinely tested under several electrophoretic conditions in order to distinguish all of the variants.

Differences between enzymes were not always easy to detect using gradient pore acrylamide gels. G6PDH and LDH in extracts of E. maxima (strains) and E. tertefla (Houghton), which were separable by thin-layer starch gel electrophoresis, banded in the same positions on gradient pore gels. This suggested that the molecules were of a similar shape but possessed charge differences. In contrast, the variants of the enzyme GPI in strains of E. acervuZina, as shown by thin-layer starch gel, migrated different distances on the gradient pore gels, which suggested differences in molecular shape.

The tight banding of the proteins allowed individual gels to be stained for a number of different enzymes. Analysis of extracts of oocysts of E. tertalaand E. maxima showed that GPI migrated further than LDH which migrated further than G6PDH, indicating that GPI is the smallest molecule. 95

Figure Two

Variation in the migration of G6PDH in oocysts of E. acervulina strains, attributable to differences in electrophoretic conditions

a)Buffer 7 (0.1 M Borate, 0.18 M Tris 0.004 M Na2EDTA) for 4 hours b) Buffer 5 (0.15 M Tris) for 21 hours c)Buffer 7 for 5i hours

A - E. acervuZina (Houghton) B - E. acervulina var. mivati (Houghton) C - E. acervulina (M) D - E. acervuZina (Ongar) E - E. acervulina (Weybridge) 96

Figure 2a

A B C D E

Figure 2b

A B C

Figure 2c

4) Enzyme analysis of oocyst cultures of different ages

Sporulated oocysts are frequently stored at 4°C in 2% or 2.5% K2Cr2O7. Extracts of oocysts which had been stored in this manner for different periods of time were analysed by thin- layer starch gel electrophoresis to determine whether any change in the mobility or activity 'of the enzymes LDH, G6PDH, MDH and GPI could be attributed to the age of the oocysts. The parasites and the time in storage are detailed below. The oocysts were derived from the same stock cultures; the oldest and youngest samples were separated by four generations.

Parasite Strain Time in storage (nearest month)

E. maxima Weybridge 7 Weybridge 9 Weybridge 16

E. tenella Houghton 0 Houghton 1 Houghton 2 Houghton 8 Houghton 15

In extracts from oocysts 15 and 16 months old very weak activity was exhibited for LDH and G6PDH, although no differences were noted in these enzymes in samples from oocysts stored for up to nine months. MDH activity was weak in samples over two months old and was not detected in oocysts which had been stored for 15 and 16 months. , GPI activity was found in all the extracts. This enzyme was isolated as one discrete band of activity in extracts of fresh oocysts and of those less than 2 months old. The older cultures, eight months and above, yielded an enzyme which resulted in a diffuse zone of staining incorporating numerous sub- bands, as shown in Figure 3. The effect was similar to that produced by storage of the enzyme preparations. 98

Figure Three Glucose phosphate isomerase in oocysts of different ages

A - E. maxima 7 months B - E. tenella 8 months C - E. tenella 15 months Electrophoretic conditions: Buffer - 9 Time - 4 hrs

Figure Four Leucine aminopeptidases in sporulated and unsporulated oocysts of E. tenella

A - Unsporulated oocysts E.tenella (Houghton) LAP-1, LAP-3 B - Sporulated oocysts E.tenala(Houghton) LAP-2, LAP-3 C - Sporulated oocysts E.tenaL2(Weybridge) LAP-2,variationofLAP-23 Electrophoretic conditions: Buffer - 2 Time - 3 hrs 99

Figure 3

Figure 4

1 2 14.

A 100

- General observations of other enzymes emphasised that activity was more pronounced in oocysts which had been recently harvested from chickens.

Oocysts from sheep and rabbits did not appear to be influenced by storage as quickly as those from chickens. Good enzyme activity was found in extracts of oocysts stored for over twelve months. Oocysts of E.ovina gave reasonable activity for ASAT, LDH, PGM, G6PDH and GPI after thirty-six months in storage.

5) Electrophoretic analysis of enzymes in different stages of the life cycle

a) Oocysts The following enzymes in extracts of sporulated and unsporulated oocysts of E.Ile11(2(Houghton) were compared: LDH,IDH (NADP), G6PDH, H6DH, MDH, 6PGDH, ME, GPI, PGM, ASAT and LAP.

The activity of enzymes in extracts of unsporuisted oocysts tended to be greater than that found in sporulated oocysts; . this was particularly so for MDH. An exception was ASAT which was weaker in unsporulated oocysts.

The only isoenzyme difference found between the two forms was that shown by LAP, all the other enzymes produced identical banding patterns. The fastest migrating enzyme showing LAP activity, LAP-1, of the unsporulated oocystswas not demonstrated in sporulated oocysts which possessed LAP-2. Common to both oocyst types was LAP-3 (Figure 4). Occasionally, a very weak, slow band, LAP-4, was noticed in unsporulated oocysts.

. The enzymes, LDH, GPI, G6PDH and PGM were identical in the different forms of the oocysts of E.acervuZina (Weybridge) and of E. maxima (Weybridge). 101 b) Sporozoites and Sporocysts

Purified sporozoites and sporocysts of E.tenella(Houghton) produced enzyme patterns of LDH, G6PDH, MDH, GPI and PGM typical of oocysts. The only consistently demonstrable band of LAP activity was LAP 3. c) Merozoites

Fairly pure samples of second generation merozoites of E.tenella (Houghton) were isolated from the caecal tissue of 7 infected chickens (Figure 5). Average yields of 5 x 10 merozoites were obtained from three to four-week-old birds which had received 1 x 105 oocysts 4i to 5 days previously. The enzymes LDH, GPI, MDH, PGM and G6PDH in extracts of merozoites could not be distinguished from the same enzymes in oocysts. LAP activity was not demonstrated.

6 Chick embryos which had been inoculated with 1 x 10 sporozoites of E.tenella (Houghton) yielded an average of 2 x 106 merozoites per embryo. Larger numbers were obtained using an embryo adapted line of E.tenelZa (Houghton), (passage Nos.: 70,71,72,73 after the initial infection of embryos), 5 when 3 x 10 sporozoites resulted in a yield of approximately 4 x 106 merozoites per embryo. The migration patterns of LDH, GPI, G6PDH, in extracts of merozoites produced in embryos) were identical to those obtained for the same enzymes in merozoites produced in the caeca of birds.

7 At least 4 x 10 merozoites were required to produce a sufficiently concentrated sample for electrophoresis. Only rarely were second generation merozoites obtained from monolayer chicken kidney cell cultures in sufficient quantities for enzyme analysis. Merozoites harvested from culture were stored in Doran's medium at -20°C until sufficient numbers had been acquired. Great variation occurred in the numbers harvested. Kidney cell cultures harvested at 110 to 120 hours 5 post-infection yielded a maximum of 8 x 10 per 60 ml prescription bottle. On two occasions only were extracts of merozoites 102

Figure Five Purified second generation merozoites of E.tenella (Houghton) isolated from chicken caeca

- 10 1-IM

•-••■•■••••

■■• V. 103

produced in vivo run against sporulated oocysts; no variation occurred in the electrophoretic mobility of GPI, LDH or G6PDH.

Attempts to harvest 1st generation merozoites from monolayer kidney cell cultures were unsuccessful.

Enzyme activity of parasite origin was not detected in caecal tissues or chorioallantoic membranes containing immature second generation schizonts.

6) Total protein in oocyst extracts

Starch gel electrophoresis was unsuitable for the demonstration of general proteins. The bands tended to be diffuse and the gel was difficult to destain.

Disc and gradient pore acrylamide gels gave better resolution of bands. Slab gradient gels were used in preference to disc gels which' were found difficult to align for comparative purposes.

Zymograms of total protein, in extracts of sporulated and unsporulated oocysts of E.1t6men2(Houghton), produced on polyacrylamide gradient gels showed differences between the two stages (Figure 5a).

Homogenates of sporulated oocysts of E.?7'zella (Weybridge and Houghton) ,E.acervulina (Houghton) and E. maxima (Houghton, Norwich, Weybridge and Robenidine-dependent) were compared. Twelve bands were recognised on disc gels but up to 19 were demonstrated on gradient gels. The position of the majority of the bands was found to be common to all samples. Distinct differences were found between the species but no reproducible variation could be detected between populations within a species. Typical results are depicted in Figures 6a, 6b. 104

Figure Six Total proteins of Eimeria spp demonstrated on polyacrylamide gradient gels

a) A - Unsporulated oocysts of E.tenella (Houghton) B - Sporulated oocysts of E.tenella (Houghton) C - Sporulated oocysts of E.acervulina (Houghton)

b) Strains of E.maxima showing no detectable variation in total protein bands separated on polyacrylamide gradient gels

A - E.tenella (Houghton) B - E.maxima (Houghton) C - E.maxima (Norwich) D - E.maxima (Robenidene -dependent) E - E.maxima (Weybridge) 105

Figure 6a

IIla

ABC A BC

Figure 6b

ABC DEE 106

7) Comparative electrophoretic studies of enzymes in eimeriine parasites

a) Characterisation of Eimeria species of the chicken

(i) Differences between species

Enzymes in sporulated oocysts of E.tenelZa(Houghton), necatrix (Weybridge), E. acervulina (Weybridge), E. brunetti (Weybridge) and E.maxima (Weybridge) were compared by thin layer starch gel electrophoresis. No samples of E.hagani, E.mitis or E. praecox were obtained. The zymograms obtained for AP, FDP, LAP, G6PDH, o(GPDH, LDH, 6PGDH, ASAT, GPI, HK, PGM, AK and TO are presented diagramatically in Figure 7 a - m. Little separation of the enzymes AP, FDP,o(GPDH and AK was achieved, but the distinct differences found in the mobilities of the other enzymes allowed each_species to be distinguished readily by its enzyme type.

LAP: Two zones of activity were demonstrated in all of the sporulated oocysts. Both bands showed characteristic migration for the species. The order of migration, starting with the species which possessed the most anodal migrating enzyme, was E. necatrix, E. tenella, E. acervulina, E. brunetti and E.maxima (Fig. 7c).

G6PDH: One broad band of activity was demonstrated in all of the species. The order of migration was E. acervulina, E. maxima, E. brunetti followed by E necatrix and E.tenelZa which could not be distinguished. (Fig. 7d)

LDH: One band of activity was demonstrated in all of the species. The order of migration was E. brunetti, E. necatrix, E. tenella, E. acervulina and E. maxima (Fig. 7f). Little variation was found between E.necatrix and E.tenella and between E. acervulina andE. maxima. 107

6PGDH: One band of activity was demonstrated in all of the species. Small, but characteristic, variations in the mobilities of the enzymes were found. The order of migration was E. acervulina, E. brunetti, E. tenelLz, E. maxima and E.necatrix. (Fig. 7g).

ASAT: An anodal and cathodal band of activity were demonstrated. The order of migration was E. maxima,, E. brunetti, E. acervulina followed by E.necatrix and E. tenena. The latter two were separated by the cathodal band. (Fig. 7h).

GPI: One main band of activity was found.in each species, occasionally anodal sub-bands were present. The order of migration was E. brunetti, E. maxima, E. necatrix, E. tenella and E. acervuZina. (Fig. 7i).

HK: One main band of activity followed by a minor band was demonstrated in each species. The order of migration was E. acervulina, E. necatrix, E. brunetti, E. tenella and E. maxima. (Fig• 7J)-

PGM: One main band of activity followed by a minor band was demonstrated. Occasionally, anodal sub bands were present. Based on the major band of activity, the order of migration was E. brunetti, E. maxima, E. necatrix, E. acervulina and E. tenella. (Fig. 7k).

TO: One band of TO activity was demonstrated in each species. The order of migration was E. acervulina, E. necatrix, E. tenella and E. maxima. The TO in oocysts of E. brunetti was not determined. (Fig. 7m).

Due to the difficulties involved in manipulating small 5 numbers of oocysts,a minimum of 5 x 10 oocysts was needed to produce sufficient extract for three or four enzyme runs. 5 However, when a sample of 4.5 x 10 oocysts of E.tertella was 4 mixed with 5 x 10 oocysts of E. acervulina, both species could be detected after electrophoresis of the resulting extract, as shown in Fig. 8. When a species contributed less than 1/10th of the total oocyst count it was not detected.

109

Figure 7c Enzyme: LA~ Buffer - 2 Time - 3 hrs

.J- Vl/II I

~ -- A B c D E

Figure 7d Enzyme: G6PDH Buffer - 8 Time - 4 hrs

A B c D E 110

Figure 7e Enzyme: d-... GPDH Buffer - 6 Time - 4 hrs + lZm· rzm rzm VIIII

-A- -B- ·D E

Figure 7f Enzyme: LDH Buffer - 1 Time - 4 hrs

fZZZZ) + IZZZZJ IZZZZJ

A B c D E

Figure 7g Enzyme: 6PGDH Buffer - 6 Time - 4 hrs

A B c D E 1 11

Figure 7h Enzyme: ASAT Buffer - 5 + Time - 2 hrs

un A B c D E

Figure 7i Enzyme: GPI Buffer - 9 Time - 4 hrs

+ VII!I

A B C D E

Figure 7j Enzyme: HK Buffer - 7 rz::::z::I:D Time - 4 hrs fZll] CI:I:z:::J UIZJ + o::::z:::J I777l ' 1 1 J a CliZZI

, 1 1 Il Vll 1 -- A B C D E 112

Figure 7k Enzyme: PGM Buffer - 6 Time - 4 hrs

A B c o E Figure 71 Enzyme: AK Buffer - 6 Time - 4 hrs rzm +

-B- -c- -0- E

Figure 7m Enzyme: TO Buffer - 7 Time - 4 hrs

+ Il Z Z 1 III III

A c o E 113

The ability to identify the species in a mixed population is useful, especially as field isolates are rarely monospecific. a) (ii) Differences within species

Having established the degree of variation at the species level, investigations were made on the enzymes of strains of coccidia of the chicken, many of which had undergone routine laboratory passage, and on lines which had been selected by passage in the presence of drugs or by passage in chicken embryos.

Two strains of E. tenella, the Houghton and the Weybridge, were found to possess fourteen enzymes with identical mobilities. The enzymes were AP, FDP, H6DH, G6PDH,ckGPDH, IDH, MDH, LDH, 6PGDH, AK, ASAT, HK, PGM, TO. Differences were found in LAP (Fig. 4) and GPI (Fig. 9). An embryc-adapted line of E. tenella (Houghton), which had been passaged seventy-three times in chicken embryos, was identical in its enzyme type to E. tenella (Houghton), with the exception of HK which was slower in mobility (Fig. 9b). E. tenella (Houghton) passaged once in chicken embryos retained the same HK pattern as the parent population (Fig. 9b). The Macester isolate contained oocysts which had four enzymes, G6PDH, LDH, 6PGDH, GPI, in common with E. tenella (Houghton).

No differences were found in cultures of E. necatrix (Weybridge) E. brunetti (Weybridge), E. acervulina (Weybridge) or E. maxima (Weybridge) which had been donated by different laboratories.

The Weybridge strain of E. maxima could not be distinguished from the Houghton and Norwich strains. The following enzymes had identical mobilities: AP, FDP, LAP, H6DH, G6PDH,O(GPDH, IDH, MDH, LDH, 6PGDH, AK, GPI, ASAT, HK and PGM. Similarly, no variation was encountered in the enzyme complement of five drug-resistant lines of E. maxima (robenidine-resistant, robenidine-dependent, methyl benzoquate (statyl), clopidol and sulphaquinoxaline-resistant). Differences in the banding patterns of PGM in the methyl- benzoquate and robenidine-resistant lines from the parent . E. maxima (Weybridge) population were achieved on two occasions, but this was not confirmed when further extracts were analysed. 114

Figure Eight Glucose phosphate isomerase activity in mixed populations of E. acervulina (Weybridge) and E. tenella (Houghton)

A - E. acervulina 5 x 105 5 B - E. acervulina 2.5 x 10 E. tenella 2.5 x 10.5 5 C - E. acervulina 1 x 10 E. tenella 4.0 x 105 4 D - E. acervulina 5 x 10 E. tenella 4.5 x 105 5 E E. tenella 5 x 10

Figure Nine (a) Intraspecific variation of glucose phosphate isomerase in E. tenella

A - Embryo-adapted, E. tenella B - E. tenella (Houghton) C - E. tenella (Weybridge)

Buffer - 9 Time - 4 hrs

Figure 9 (b) Intraspecific variation of hexokinase in E. tenella

A - Embryo-adapted, E. tenella B - E. tenella (Houghton) C - E. .tenella (Houghton) after one passage in chicken embryos

Buffer - 7 Time - 4 hrs 115

Figure Eight

Figure Nine (a) 0

Figure 9 (b)

A y

A 116

Figure Ten Intraspecifc variation of glucose phosphate isomerase in E. acervulina

A - E acervulina var mivati (Houghton) B - E . acervuZina (Houghton) C - E .acervuZina (M) D - E acervulina (Weybridge)

Electrophoretic conditions: Buffer - 9 Time - 6 hrs

Alleses■—.

A 117

Further investigations involving greater resolution of the enzyme are required. A methyl benzoquate-resistant line of E. maxima var. indentata did not differ from a sensitive population as shown by the mobilities of AP, LAP, G6PDH, LDH, 6PGDH, GPI, HK and PGM, which, with the exception of PGM, were also identical to those of E. maxima strains. The slower mobility of PGM distinguished E. maxima var. indentata from E. maxima (Fig. 16, b,c).

E. acervulina var. mivati (Houghton) and the Ongar, Weybridge, Houghton and M strains of E. acervulina had fourteen enzymes in common: AP, FDP, LAP,H6DH, GPDH, IDH, MDH, LDH, 6PGDH, AK, ASAT, HK, PGM and TO. They could be distinguished by the mobility of GPI as depicted in Figure 10. E. acervulina, Houghton and 'M' strains, and E. acervulina var. mivati possessed a faster migrating enzyme than the Ongar and Weybridge strains of E. acervulina. Variation in G6PDH was also demonstrated, but the separation was not adequate for reliable identification (Fig. 2).

7) b) Characterisation of Eimeria species of the rabbit

(i) Differences between species

E. coecicola, E. intestinalis, E. magna and E. stiedai (Weybridge strains) were distinguished from each other by differences in the mobilities of LAP, G6PDH, LDH, 6PGDH, GPI, ASAT, HK and TO as shown in Figure 11 a - h. E. irresidua was characterised by the electrophoretic migration of LDH, GPI, HK and PGM as depicted in Figure 11 c,e,g and i.

LAP: Two zones of activity were demonstrated in all of the sporulated oocysts. Little variation occurred in tha mobilities of the enzymes. The order of migration (starting from the anode) was E. magna, E. stiedai, E.coecicola and E. intestinalis (Fig. 11a).

G6PDH: One band of activity was demonstrated in each species. The order of migration was E. stiedai, followed by E. coecicolaa E. magna which could not be distinguished, and E. intestinalis (Fig.11b).. 118

LDH: One band of activity was demonstrated for each species. The order of migration was E. intestinalis, E. stiedai, E. coecicola, E. magna and E. irresidua (Fig. 11c).

6PGDH: One band of activity was demonstrated for each species. The order of migration was E. stiedai, E. magna, E. intestinalis and E. coecicola (Fig. 11d).

GPI: One main band of activity was demonstrated in each species; occasionally, anodal sub-bands were demonstrated. The order of migration was E. irresidua, E. magna, E. intestinalis, E. stiedai and E. coecicola (Fig. 11e).

ASAT: One main band of activity was demonstrated in each species; cathodal bands were not consistent. The order of migration was E. intestinalis, E. stiedai, E. coecicola and E. magna (Fig. 11f).

HK: One main band of activity followed by a minor band was demontrated in each species. The order of migration was E. magna, E. coecicola, and E.stiedai. Under the conditions used, E.irresidua migrated towards the cathode (Fig. 11g).

TO: One band of activity was demonstrated in each species. The order of migration was E. intestinalis, E. magna, E. coecicola and E. stiedai (Fig. 11h).

PGM: One main band of activity and a second minor band (anodal) were found in E. irresidua and E. stiedai. The order of migration was E.s'tiedai followed by E. irresidua (Fig. 11i). 119

Figure Eleven Electrophoretic mobility of enzymes in Eimeria species from the rabbi t

F E. coecicola (Weybridge) G E. intestinalis (Weybridge) H E. irresidua (Weybridge) l E. magna (Weybridge) J E. stiedai (Weybridge) ..

Figure lIa Enzyme: LAP Buffer - 2 Time - 3 hrs +

F G l J

Figure lIb Enzyme: G6PDH Buffer - 8 Time - 4 hrs +fZJ

F G l J 120 Figure Ile Enzyme: LDH Buffer - l Time - 4 hrs

F G H l J

Figure lld Enzyme: 6PGDH Buffer - 6 Time - 4 hrs

fZZZl +~

F G l J

Figure Ile Enzyme: GPI Buffer - 9 Time - 4 hrs

+ vn [Zln

~ VlZïi fZZZJ F G H l J 121 Figure llf Enzyme: ASAT Buffer - 5 Time - 2 hrs

F G l J I-2 ( l - 2 E. magna (I CI)

Figure llg Enzyme: HK Buffer - 6 Time - 4 hrs

lZZ2J +fm

F H l J

122

Figure llh Enzyme: TO Buffer - 7 Time - 2 hrs

+ A

F G I J Figure lli Enzyme: PGM Buffer - 7 Time - 4 hrs UA1

+ A

H J

Figure Twelve Electrophoretic mobility of enzymes .in Eimeria species from the sheep

K - E. ninakohlyakimovae L - E. ovina M E. weybridgensis 123 Figure 12a Enzyme.. G6PDH Buffer - 8 Time - 4 hrs

K L M Figure 12b Enzyme: LDH Buffer - 1 Time - 4 hrs

~K- --- L M Figure 12c Enzyme: GPI Buffer - 9 Time - 4 hrs +

L M

Figure 12d Enzyme: ASAT + Buffer - 5 Time - 2 hrs

--- K L M 124

Figure 12e Enzyme: PGM

Buffer - 6 V/IJ Time - 4 hrs ILLLJ

K L M

Figure 12f Enzyme: LAP

Buffer - 5 IZZZl Time - 3.20 hrs + lZZZl flZZ]

K M

Figure 12g Enzyme: H6DH

Buffer - 9 Time - 4 hrs lZZ2l +

K M 125 b) (ii) Differences within species

Two strains of E.stiedai, Weybridge and ICI, could not be distinguished by the migration patterns of LAP, G6PDH, LDH, 6PGDH, GPI, ASAT and HK. The Weybridge and ICI strains of E. magna differed only in the mobility of ASAT, the enzyme of the Weybridge strain having a slower mobility (Fig. 11f).

7) c) Characterisation of Eimeria species of the sheep (i) Differences between species

E. ninakohZyakimovae, E. ovina and E. weybridgensis were distinguished from each other by differences in the mobilities of G6PDH, LDH, GPI, ASAT and PGM, as shown in Fig. 12, a - e. LAP and 6PGDH in extracts of oocysts of E. ninakohlyakimovae and E. weybridgensis also showed variation.

G6PDH: One band of activity was demonstrated in each species. The order of migration starting from the anode was E. weybridgensis, closely followed by E. ovina and E. ninakohlyakimovae, which- could not be distinguished-(Fig. 12a).

LDH: One band of activity was found in each species. The order of migration was E. ninakohlyakimovae, E. weybridgensis and E. ovina (Fig. 12b).

GPI: One main band of activity and a number of anodal sub-bands were demonstrated in each species. The order of migration was E. weybridgensis, E. ovina and E. ninakohlyakimovae (Fig. 12c).

- ASAT: One main band of activity was demonstrated in each species. The order of migration was E. ninakohZyakimovae, E. weybridgensis, E. ovina (Fig. 12d) . 126

PGM: Two bands of activity were found in E. ovina and E. weybridgensis; three bands were apparent in E. ninakohZyakimovae. The order of migration was E. weybridgensis, E. ninakohZyakimovae and E. ovina (Fig. 12e).

LAP: Two main bands of activity were demonstrated. No differences were found between E. ninakohZy- akimovae and E. weybridgensis (Fig. 12 f ).

H6DH: One band of activity was demonstrated. The order of migration was E. weybridgensis followed by E. ninakohZyakimovae (Fig. 12g).

7) (d) Identification of an isolate from quail, Coturnix coturnix japonica

Oocysts found in faecal samples of Japanese quail, Coturnix coturnix japonica were used to infect four-week-old coccidia-free quail. The enzyme type of the oocysts produced in these birds bore no resemblance to any of the Eimeria spp of the chicken which had been examined. The morphology of the oocysts, the prepatent period. of 4i days and the site of oocyst production in the duodenum and small intestine suggested that E. bateri, Bhatia, Pondey and Pande (1965), was present in the population. A direct electrophoretic comparison of the enzymes, LAP, G6PDH, LDH, 6PGDH, ASAT, GPI, HK and PGM of the isolate with a Weybridge strain of E. bateri showed that all but GPI had identical mobilities. The GPI of the Weybridge strain had a much faster mobility (Fig. 13a).

The oocyst population appeared dimorphic, large and small oocysts being present (Fig. 13b). This has been noted before by Shah and Johnson (1971) and Norton and Peirce (1971) for E. bateri. The pattern of the enzymes studied confirmed that it was likely that the oocysts belonged to a single species, E.bateri.

127

Figure Thirteen (a) Intraspecific variation of glucose phosphate isomerase in E. bateri

A - E. bateri (Ascot) B - E. bateri (Weybridge) Buffer - 9 Time - 4 hrs

Un A

Figure 13 (b) An unsporulated, a sporulating and a sporulated oocyst of E. bateri (Ascot) 10 vim 1 f

• 128

8) The use of enzyme markers in genetic studies

A) The Eimeria acervulina complex

The close relationship between E. acervulina and E. mivati has been investigated by Long (1973a) who suggested, on biological and immunological evidence, that E. mivati was not sufficiently different to warrant separate specific status and proposed the name E. acervulina var. mivati. An experiment was designed to establish whether E. acervulina var. mivati (Houghton) and E. acervulina (Weybridge) were reproductively isolated or whether they could share the same gene pool:

(i) Oocyst sizes

Very little difference occurred between the sizes of the oocysts in various populations of E. acervulina as shown in Table 8.

(ii) Enzyme types

The electrophoretic data presented in 7a (ii) reported that the Ongar, Weybridge, Houghton and M strain of E. acervulina and the Houghton strain of E. acervulinavar. mivati had fourteen enzymes in common. An enzyme that separated the group was GPI. E. acervulina, Ongar and Weybridge strains, possessed GPI-2, whereas E. acervulina, Houghton and M strains, and E. acervulina var.mivati., Houghton strain, possessed GPI-1, a faster migrating enzyme.

(iii) Ability to grow in chicken embryos

The strains could be told apart by their 'ability to passage in embryos.

(a) E. acervulina var.. mivati (Houghton) 4 Inoculation of 5 x 10 sporozoites of E. acervulina var.. mivati (Houghton) into the allantoic cavity of 9-to 11- day-old chicken embryos resulted in the production of oocysts which could be harvested and sporulated 5 to 5i days later. Approximately 5 x 105 oocysts were recovered 129

TABLE EIGHT

Measurements of oocysts of E. acervulina

Strain or Line Length km Width Am Shape Index

E.acervulina (Weybridge) 18.21 (1.0) 15.08 (1.12) 1.21 E.acervulina (Houghton) 17.59 (2.14) 14.02 (1.11) 1.25 E.acervuZina (Ongar) 17.83 (2.01) 14.49 (0.99) 1.23 E.acervuZina (M) 18.75 (1.96) 14.9 (1.02) 1.26 E.acervulina var. mivati 18.30 (2.30) 14.19 (1.14) 1.29 (Houghton) E.acervulina/E.acervulinax 16.83 (1.56) 13.98 (1.42) 1.20 var. mivati (F1) E.acervulina/E.acervulinax 17.66 (1.63) 14.83 (1.51) 1.19 var. mivati- single oocyst (F2) E.acervulina/E.acervulinax 17.67 (1.22) 13.63 (1.06) 1.30 var. mivati- single oocyst (F2) E.acervulina/E.acervulinax 16.94 (1.74) 15.29 (1.88) 1.11 var. mivati- (F2) 1 ep E.acervulina var. mivatix 17.59 (1.60) 15.43 (1.06) 1.14 1 ep E.acervulina (Weybridge)x 15.96 (1.53) 13.38 (0.94) 1.19 1 ep 1 cp

KEY: figures in parentheses = standard deviation, 100 observations 1 ep = one passage in eggs 1 cp = one passage in chicks x = measurements made by direct observation 130

per embryo. The mobility of GPI in extracts of oocysts harvested from embryos was found to be identical to the parent population i.e. GPI-1.

(b) E. acervulina (Weybridge)

Attempts to passage E. acervuZina (Weybridge) in chicken 5 embryos, by the inoculation of. 1 x 10 sporozoites into 7 the allantoic cavity, failed. When 1 x 10 sporozoites were used as the inoculum,a few oocysts were detected 5i days later in urate deposits. On the first occasion, when this small quantity was harvested, the oocysts did not sporulate. In two further attempts, using the same concentration, a small number of oocysts capable of sporulation were harvested. These oocysts were passaged through chickens to provide sufficient material for enzyme analysis and to test the ability of this line to grow in chicken embryos. No alteration of the GPI had occurred. The only detectable enzyme band was GPI-2 characteristic of the parent population. No improvement in the ability to grow in embryos was found; oocyst production was noticed only in 3 out of 5 embryos which had received 7 1 x 10 sporozoites and in 1 embryo out of 5 which had received 1 x 106 sporozoites. No oocysts were detected 5 4 in embryos which had received 1 x 10 or 1 x 10 sporozoites.

The 'M' strain and Houghton strain of E. acervulina could not be separated by electrophoretic techniques from E. acervulina var.mivati. Attempts were made to determine whether these populations possessed the ability to grow in chicken embryos.

Sporozoites of both strains did not produce infections 6 in chicken embryos at concentrations up to 1 x 10 . However, oocysts of the 'M' strain had been stored for 6 months at 4°C which might have affected subsequent development. 131

(iv) Investigations into the viability of 'contaminating' oocysts enclosed in incubating eggs

Although sporozoites were purified before inoculation in to embryos, when exceptionally large numbers were used (1 x 107), some contamination with intact oocysts undoubtedly occurred. It was necessary to determine whether sporulated oocysts could retain their viability when enclosed in incubating eggs: Two batches of sporulated oocysts of E. tenella (Houghton) were prepared. One was kept at 41 °C in K2Cr2O7 for 6 days, the other was inoculated into the allantoic cavity of 10-day-old chicken embryos and retrieved after 6 days. No infection was 6 subsequently produced in 10 chickens when up to 1 x 10 oocysts of either batch were administered orally.

When this experiment was repeated, using oocysts which had been incubated at 39° C in K2Cr2O7 for only 4 days, 2 birds out of 5 which received a massive dose (unknown number) showed caecal haemorrhage. No infections occurred in control birds which had not been administered with oocysts.

The possibility of 'contaminating' viable oocysts being carried over with unsporulated oocysts harvested from embryos should not be excluded. If contaminating oocysts were present • in the harvested urate deposits, they would be detectable in their sporulated condition.

(v) The cross between E. acervulina (Weybridge) and E. acervulina var.mivati (Houghton)

Populations of E. acervuZina (Weybridge) and E. acervuZinavar. mivati (Houghton) differed in two stable characters: the mobility of their GPI and the ability to grow in chicken embryos when 4 5 x 10 sporozoites were used as the inoculum. Figure 14 details the cross that was made between the two populations.

Chickens which received oocysts of both E. acervulina var. mivati (GPI-1) andE. acervulina (GPI-2) produced oocysts, Fl, which were characterised by both GPI-1 and GPI-2 as shown in Figure 15a. The enzyme extracts used for this illustrated plate had been

132

Figure Fourteen

E. acervulina / E. acervulina var. mivati (cross)

E.acervulina var . mivati (Houghton) E. acervuZin (Weybridge) P1

GPI-2

EGG PASSAGE 5x104 sporozoites)

1 oocysts no oocysts produced produced GPI-1

4 1.5 x 10 oocysts E..acervulina var. mivati 4 1 . 5 x 10 oocysts E.acervulina

CHICKEN PASSAGE

I oocysts produced Fl GPI-1 and GPI-2

EGG PASSAGE (5x104 sporozoites)

I oocysts produced F2 GPI-1 and GPI-2 133

stored for three days at 4°C to demonstrate that the banding patterns were due entirely to the different populations and not to any deterioration of the enzyme. Sporozoites, excysted from the Fl oocysts, produced successful infections when inoculated at concentrations of 5 x 104 into the allantoic cavity of chicken embryos. Analysis of the GPI in the oocysts (F2) from this infection revealed not only a very marked GPI-1, but also a weaker GPI-2 band as shown in Figure 15b.

To test whether the presence of an E. acervulinavarmivati infection could influence or enhance the ability of E. acervulina (Weybridge) to develop in embryos, sporozoites were obtained from an artificially mixed population of E. acervulina var. mivati and E. acervulina (Weybridge) and inoculated into embryos at concentrations of 1 x 105 and 1 x 106. The only GPI type detected in the resulting oocyst populations was GPI-1, characteristic of E. acervulina var.mivati.

Thirteen birds were orally inoculated with single oocysts from the Fl produced by the E. acervulina var.mivati/E. acervulina cross. Five days later, the birds were killed and scrapings of the upper part of the alimentary canal revealed that five birds had become infected. The oocysts from each bird were treated separately, sporulated and used to infect batches of four chickens in an attempt to produce sufficient oocysts for electrophoretic analysis. Three of the resulting oocyst cultures were found to have an enzyme type similar to E. acervulina (Weybridge) and one to E. acervulina var.mivati (Houghton). One population possessed both enzyme forms GPI-1 and GPI-2.

Sporozoites of the latter population were used to infect embryos at concentrations of 5 x 104. A very few oocysts were noticed in the urate deposits of the embryos;, insufficient oocysts were produced for enzyme analysis. 134 Figure Fifteen

(a) Glucose phosphate isomerase in populations in the E . acervulina/E . acervulina var, mivati cross

A - E. acervulina (Weybridge) P1 GPI-2 B - E. acervulina/E .acervu lira var . Fl mivati GPI-1, GPI-2 C - E. acervulina var, mivati P1 GPI-1 Buffer - 9 Time - 4 hrs

Figure 15 (b) Glucose phosphate isomerase in the F2 population resulting from the passage of the E. acervulina/E . acervuZina var . mivati cross in chicken embryos

D - E. acervulina/E . acervulina v ar . mivati GPI-2, GPI-1 After passage in eggs Buffer - 9 Time - 5 hrs 135

Figure 15a

GPI-1

GPI -2

INIMMINIMOD A B C

Figure 15b

GPI-1

G P1-2

D 136

8) B) The E. maxima complex

E. indentata was originally isolated from the Ceylon Jungle Fowl, Gallus lafayetti, and named by Fernando and Remmler (1973). Further investigations by Long (1974a) showed that the oocysts were similar to those of E. maxima; the parasite reproduced well in chickens, was pathogenic and showed marked cross-protection against two strains of E. maxima isolated in Britain. The name E. maxima var. indentata was suggested for the parasite (Long 1974a). Experiments were designed in conjunction with the Central Veterinary Laboratory, Weybridge, utilising resistance to methyl benzoquate (statyl) and PGM type as genetic markers, to establish whether E. maxima and E. maxima var. indentata could share the same gene pool.

(i) Enzyme types

The results of the electrophoretic analysis of the enzymes of E. maxima var. indentata, presented earlier (7 (a),ii), strongly suggested that the parasite belongs to the E. maxima complex. PGM was the only enzyme out of 8 which showed variation; the PGM of E. maxima var. indentata, PGM-2, was distinguished from that of E. maxima (Weybridge), PGM-1, by its slower migration (Fig. 16, a,b). No enzyme differences were detected between sensitive and drug resistant lines of E. maxima var. indentata. Similarly, no enzyme differences had arisen during fourteen successive passages of E. maxima (Weybridge) or during its acquisition of drug resistance.

(ii) Crosses between E. maxima (Weybridge) and E. maxima var . indentata

All experimental procedures, other than the preparation of oocysts and enzyme analysis,were carried out by Mr. C.C. Norton and colleagues at the Central Veterinary Laboratory.

(a) Experiment One

The experimental details and the enzyme types of each population are outlined in Figure 16 a. Oocysts of a

137

Figure Sixteen (a) EXPERIMENT ONE

E.maxima (Weybridge) - E.maxima var.indentata Statyl-sensitive Statyl-resistant PGM-1 PGM-2

250 oocysts 250 oocysts per bird per bird

1000 oocysts per bird

5, 21-day-old 5, 21i-day-old chickens chickens 80 ppm statyl Plain food

Fl No oocysts oocysts (PGM-1, PGM-2)

1000

10, 35-day-old chickens 80 ppm statyl 1 F2 oocysts (PGM-1, PGM-2) 138

Figure Sixteen (b) (c) Phosphoglucomutase in populations from Experiment One

A - E.maxima var. indentata P1 Statyl-resistant PGM-2 B - E.maxima/E.maximavar.indentata F2 Statyl-resistant PGM-1, PGM-2 C - E. maxima P1 Statyl-sensitive PGM-1 Buffer - 6 Time - 4 hrs 139 Figure 16b

A B

Figure 16c

P1 F2 P1

A 140

Figure Seventeen (a) EXPERIMENT TWO

P1 (Weybridge) E.maxima E.maxima var. indentata Statyl-resistant Statyl-sensitive PGM-1 PGM-2

250 oocysts 250 oocysts per bird per bird 1000 oocysts per bird

5,21-day-old 5,21-day-old chickens chickens Plain food 80 ppm statyl

V Fl oocysts no oocysts (PGM- )

1000

10,28-day-old chickens 80 ppm statyl

F2 oocysts (PGM-1) 141

statyl sensitive line of E. maxima (Weybridge), which was completely inhibited in birds receiving 80 ppm statyl, and oocysts of a statyl-resistant line of E. maxima var. indentata were inoculated simultaneously into birds. Each bird received a total of 500 oocysts; 250 of E. maxima and 250 of E. maxima var, indentata. The birds were reared on plain food. Fl oocysts, isolated from the faeces of these birds, were characterised by both PGM-1 and PGM-2. The F1 oocysts were used to infect a second batch of birds which were on a medicated diet. The F2 oocysts resulting from this infection were collected and analysed. Both PGM-1 and PGM-2 were demonstrated in the extracts, as illustrated in Figure 16 b,c. The proportion of the population characterised by PGM-1 was small, as judged by the degree of staining in the gel, consequently the band is only vaguely discernible in the photograph.

(b) Experiment Two

The experimental details and the enzyme type of each population are outlined in Figure 17 a. Oortvsts of a statyl-resistant line of E. maxima and oocysts of a statyl-sensitive line of E. maxima var. indentata, which was completely inhibited in birds receiving 80 ppm statyl, were inoculated simultaneously into birds. Each bird received a total of 500 oocysts, 250 of E.maxima and 250 of E. maxima .var.indentata. The birds were reared on plain . food. Fl oocysts, isolated from the faeces of these birds, were characterised only by PGM-1. The Fl oocysts were used to infect a second batch of birds on a medicated diet. The F2 oocysts resulting from this infection were collected and analysed. PGM-1 was demonstrated in the extracts.

The experimental details and the enzyme type of each population are outlined in Figure 18 a. This Experiment differed from Experiment Two only in the proportions of the parent populations in the initial inoculum. Each bird received 500 oocysts, 100 oocysts of E. maxima and 400 oocysts of E. maxima var. indentata. 142

Figure Eighteen (a) EXPERIMENT THREE

P1 (Weybridge) var. E.maxima E.maxima indentata Statyl-resistant Statyl-sensitive

PGM-1 PGM-2

100 oocysts 400 oocysts 1000 oocysts per bird per bird per bird

18, 26-day-old 5, 21-day-old chickens chickens Plain food 80 ppm statyl

Fl. oocysts no oocysts (PGM-1, PGM-2)

1000

10, 27-day-old chickens 80 ppm statyl

F2 oocysts (PGM-1, PGM-2) 1 43

Figure Eighteen (b) (c) Phosphoglucomutase in populations from Experiment Three

A - E. maxima P1 Statyl-resistant PGM-1 B - E. maxima/E. maxima var. indentata F2 Statyl-resistant PGM-1,PGM-2 C - E. maxima var.indentata P1 Statyl-sensitive PGM-2 Buffer - 6 Time - 4 hrs 144 Figure 18b

A B C A

Figure 18c

P1 F2 P1 145

The Fl oocysts were characterised by PGM-1 and PGM-2. Both enzymes were detected in extracts of the F2 oocysts; as shown in Figure 18 b,c. The proportion of the population characterised by PGM-2 was small, as judged by the degree of staining in the gel.

9) Clones of E. tenella

Fifteen birds were each infected with a single sporocyst of E. tenella (Houghton). After 7 days, the birds were killed and the caeca removed. Scrapings of the caecal wall were examined for oocysts, which were found in a caecum of one bird, but in no others.

Tissue containing individual second generation schizonts of E. tenella (Houghton) dissected from the chorioallantoic membrane of an infected chicken embryo was gently disrupted in a glass homogeniser and inoculated into Leighton tubes containing monolayer kidney cell cultures. Coverslips were removed at intervals and examined for parasites. No parasites were found.

10) Buoyant density of eimeriine DNA

8 Oocyst samples of 5 x 10 were used to provide sufficient DNA for analysis. Sporozoite samples of 4 x 109 also yielded adequate amounts of DNA, although these preparations were not analysed. DNAs isolated from sporulated oocysts of E.tenella (Houghton and Weybridge), E. acervulina (Weybridge) and E. coecicola were compared. Ultracentrifugation revealed that the buoyant densities of the samples were identical at 1.711 g/cm3. Although only a limited number of runs were made, the accuracy of the method is such that reasonable confidence may be placed on the third decimal place (Chance' et al. 1974). From the relationship p = 1.660 + 0.098 (GO), Schildkraut et al. 1962, all three Eimeriaspp can be considered to have a GC content of around 527, 146

11) Isoenzymes in relation to coccidiosis

(a) E. tenella infections

Thirty, 4i-week-old chickens were inoculated with 1 x 105 oocysts of E.tenella (Houghton). Serum samples were obtained from 4 birds on days 1, 3, 5, 7 and 9 post-infection. Samples were collected at the same time each day, before the morning feed. No differences were found in the isoenzyme patterns of AP or LDH in the serum of infected birds compared with that of uninfected birds. Five isoenzymes were demonstrated for LDH, two for AP. A decrease in the activity of the enzymes, as judged by the intensity of staining, was noticed on the 5th day of infection.

The drop in AP activity in the sera of infected birds was confirmed by spectrophotometry (Table 9 ). A decrease in activity was apparent on day three of the infection. A reduction in the total proteins was also noticed, by analysis on polyacrylamide gradient gels, in the sera of bird° on the 5th day of infection. No alteration in the position of protein ' bands was observed.

(b) E. stiedai infections

LDH in serum collected from two rabbits infected with 5000 oocysts of E. stiedai, Weybridge and ICI strains respectively, was analysed by disc gel electrophoresis. The serum samples were kindly donated by Dr. J. Catchpole, Central Veterinary Laboratory, Weybridge. Changes in the LDH isoenzymes during the course of an infection were indicated. Unfortunately, • the samples differed slightly in the degree of lysis of red blood corpuscles, but the haemoglobin content of the samples did not appear to correlate with the LDH zymograms. Figure 19 shows the results which were similar for both infections. On the 14th day after inoculation of oocysts,a marked increase in total LDH occurred; the bands LDH-4 and LDH-5 were more obvious. No variation was found at any other time of infection, despite prodigious oocyst production between the 18th and 28th day. 1 47

Figure Nineteen LDH isoenzymes of serum from a rabbit 3 inoculated with 5 x 10 oocysts of E.stiedai (Weybridge)

A - Day of inoculation B - 7 days after inoculation C - 14 days after inoculation D - 21 days after inoculation

Table Nine Spectrophotometric analysis of AP levels in the serum of two-week-old Ranger cockerels 4 • inoculated with 1 x 10 oocysts of E.tenella (Houghton)

N.B. One Sigma Unit of phosphatase will liberate 1).1M of p-Nitrophenol per hour. 148

Figure Nineteen

mow 4111 L D H -1 -2 low - 3 OOP 011011 oft -4 - 5

A C D

Table Nine

Day Post- Units of Serum Alkaline Mean inoculation Phosphatase per ml. Sigma Units

7.1, 8.6, 6.75, 6.5, 8.35 7.25 Controls 7.35, 6.4, 6.55, 7.7

1 4.65, 9.55, 7.35, 6.9 7.10 2 6.2, 7.9, 8.0, 6.85 7.21 3 5.25, 4.25, 3.55, 4.75 4.45 4 5.0, 3.9, 3.6, 6.45 4.74 5 4.75, 3.7, 3.75, 3.75 3.99 6 5.05 6.25, 6.6, 4.25 5.54 7 3.1, 3.5, 2.4, 3.4 3.1 8 6.75, 4.25, 5.2, 3.55 4.94 9 4.0, 3.8, 3.65, 3.3 3.69 10 3.7, 5.75, 6.9, 7.5 5.96 149

DISCUSSION

A) Biochemical considerations

Metabolic studies on Eimeria species have been confined to the non-parasitic stages of the life cycle, particularly to the processes of sporulation and excystation. Studies on the parasitic stages have been restricted to the demonstration and location of individual enzymes by histochemical methods. Ryley (1973) surveyed the histochemical tests carried out by a number of authors on a variety of coccidia. The present investigation has confirmed and extended the list of enzymes which have been identified in Eimeria spp.

The inability to demonstrate an enzyme in an organism by electrophoretic methods is not sufficient evidence to deny its presence. There are a number of factors which can account for a negative staining reaction after electrophoresis:

1) The enzyme was not extracted into solution in sufficient concentration. 2) The activity of the enzyme was reduced or lost during sample preparation or during electrophoresis. 3) The enzyme was not concentrated within a narrow zone on the gel, probably due to the use of an unsuitable buffer. 4) The enzyme assay solution was not optimal.

Hence, although AP has been demonstrated histochemically to occur in all stages of E.stiedai (Frandsen 1968) and in E. acervulina and E.necatrix (Michael and Hodges 1973) it has not been demonstrated by electrophoretic techniques.

Enzymes which have previously been shown to occur in stages of Eimeria and which have been detected after electrophoresis include AP, LAP, G6PDH, LDH,C(GPDH and FDP. The enzymes ASAT and IDH demonstrated in this study have previously been identified in Toxoplasma gondii, but not in species of Eimeria. Other enzymes which have been demonstrated electrophoretically, namely MDH, ME, H6DH, AK, GPI, HK, PGM and TO, had not previously been 150

demonstrated in coccidia. Shirley (1975) has also demonstrated, by electrophoretic techniques LDH, GPI, G6PDH and PGDH in stages of Eimeria spp of the chicken.

The presence of an enzyme is not sufficient evidence to assume the presence of a particular metabolic pathway. Beyer (1965, 1970), after observing that the intensities of reactions obtained foro(GPDH and SDH in rabbit,coccidia and E. tertelL2 varied during the life cycle, concluded that there was a switch of metabolic pathways. The intracellular stages, such as the developing schizont and macrogamete, apparently survived solely by glycolysis, whereas merozoites andfertilised macrogametes were both capable of an oxidative metabolism. SDH has, however, been demonstrated in developing schizonts of E.necatrix (Michael and Hodges, 1973) and micro- and macrogametocytes of E. stiedai (Frandsen 1968). The present observations of MDH in unsporulated and sporulated oocysts, sporozoites and merozoites and of IDH in unsporulated and sporulated oocysts provides further evidence that enzymes of a conventional TCA cycle are present in these stages. However, SDH could not be identified in extracts of oocysts of E. terwlla.

The differing electrophoretic mobility of parasite MDH to that of the host indicates that the parasite synthesises its own enzyme. The greater activity of MDH demonstrated in extracts of unsporulated as opposed to sporulated oocysts correlates with the higher respiration rates during sporulation (Ryley 1973). Since MDH and ME were more easily demonstrated after ultrasonication of extracts it could be that these enzymes are membrane bound, possibly associated with mitochon- dria.

.Enzymes typically associated with glycolysis were abundant in all of the stages examined. Strong activity was also found for G6PDH and 6PGDH, enzymes typically associated with the pentose phosphate pathway.

The rapid multiplication of eimeriine parasites suggests that they are capable of rapid protein synthesis. Transamina- tion is often involved in amino acid synthesis. Although attempts were made to identify ALAT, ASAT and TAT in oocysts, 151

only. the ASAT assay (glutamate —4 oxalacetate transaminase) gave a positive result.

Exopeptidase enzymes, which split off terminal amino groups of polypeptides, occur as multiple enzymes in many parasites (Von Brand 1973). Such a multiple enzyme, LAP, was identified in oocysts of Eimeria spp. Isoenzymes of LAP were found to vary between sporulated and unsporulated oocysts of E. tenella but the enzyme types did not correlate precisely with those reported by Wang (1974). Unsporulated oocysts were found to possess LAP-1, LAP-3 and a weak LAP-4 and sporulated oocysts possessed LAP-2 and LAP-3, whereas Wang (1974) found LAP-1, 2 and 3 in unsporulated oocysts and a different LAP, LAP-5 in sporulated oocysts. These differences may be accounted for by strain variation similar to that found between the Houghton and Weybridge strains of E.tenella or to the isolation and demonstration procedures used. The physiological functions of this enzyme require closer study. It is possible, for example, that LAP -1 has a role in sporulation and LAP -2 is required for excystation. LAP-3, the only isoenzymeform found in sporozoites may also be concerned with excystation or perhaps cell penetration.

Differences in structural protein and possibly differences in the biochemical activities between unsporulated and sporulated oocysts were indicated by the zymograms of total proteins obtained for the two stages.

It might be expected that metabolic differences occurring between the exogenous oocysts and the parasitic stages within the host would be reflected by their isoenzymes. However, those enzymes which were demonstrated in both sporozoites and merozoites could not be distinguished from those of the oocyst. 152

B) Recognition of populations of Eimeria by their enzyme type

i) Identification of Eimeria species using biochemical data

Three ways of utilising the electrophoretic data for the identification of unknown samples were considered.

The clearly defined differences between the enzymes of the Eimeria spp of chickens allowed biochemical keys, as shown in Table 10, to be constructed with reference to a previously characterised Eimeria sp. By comparing the mobilities of an enzyme of an unknown sample to the known reference marker,a stepwise progression through the key enables the identification of the species. Unfortunately, a key such as this has an inherent rigidity which does not allow for intraspecific variation of the enzymes.

Alternative methods rely on the comparison of a selection of enzymes of an unknown to those of a known species. A direct comparison, where the sample to be identified is analysed alongside extracts of species which are believed to be present, is the most satisfactory method. Although it is possible to use one strain, such as -E.teruala(Houghton) as a reference marker for all comparisons (Table 11), such a method can only cater for monospecific samples and gives a poor assessment of intraspecific variation.

When a general enzyme type was constructed for each species, all the populations so far examined showed 80% or greater similarity to their species type. This means that no greater variation than two out of ten enzymes has been found between intraspecific populations. In assessing the specific status of an unknown sample by the direct comparison method it is possible to allow for intraspecific variation. When more populations have been examined it should be possible to define, with greater confidence, the degree of enzyme similarity that is required for designation to a particular species. The present figure of 80% could well be too large. 1 53

TABLE TEN

Biochemical key to certain Eimeria spp from the chicken

Reference marker: E. tenella (Houghton)

1) Lactate dehydrogenase: Faster in mobility - E.brunetti

Lactate dehydrogenase: Same or slower in mobility - 2

2) Glucose phosphate isomerase: Same or faster in mobility - 3 Glucose phosphate isomerase: Slower in mobility - E.acervulina

3) Hexokinase: Faster in mobility - E.necatrix

Hexokinase: Slower in mobility - E.maxima

Hexokinase: Same mobility - E.tenella 154

TABLE ELEVEN

Electrophoretic migration of ten enzymes in Eimeria spp with reference to E.tenella (Houghton)

Enzyme E.acervulina E.brunetti E.maxima E.necatrix E.tenella 0

.AP - 0• 0 - 0

FDP - - - - 0

LAP - - - + G6PDH + + + 0 0 0

c4GPDH + 0 0 0 0

LDH - + - 0 0

6PGDH + + - - 0

GPI - +. + + 0 HI( + . + - + 0

PGM + + + +

= greater migration 0 = similar migration = lesser migration 155

Although the large biochemical differences between-species of Eimeria demonstrated by electrophoretic techniques have been shown to be of great value in identifying and describing members of different species, ideally the enzyme characterisation should not be used in isolation but in conjunction with other known characters.

For valid comparisons of results obtained- in different laboratories it is essential that electrophoretic techniques are standardised. Variations found in the migration of enzymes due to different electrophoretic conditions necessitates the use of similar experimental procedures.

ii) E. acervulina complex

Four strains of E.acervulina and the Houghton strain of E.acervulina var.mivati had 14 enzymes in common, only GPI and G6PDH showed variation. This provided exceptionally strong evidence for believing that all 5 populations belonged to the same species.

E. acervulina (Weybridge) and E. acervulina var.. mivati (Houghton) differed in two stable genetic characters: the mobility of GPI and the ability to grow in chicken embryos when 5 x 104 sporozoites were used as the inoculum. The appearance of GPI-2 in the F2 oocysts harvested from embryos demonstrated that there had been a transfer of genetic material between the two parent populations. This provides further proof for the suggestion of Long (1973a) that E.acervulina var.mivati, at least the Houghton isolate, is conspecific with E. acervulina.

iii) E. maxima complex

The results of electrophoretic analysis suggested that E. maxima var. indentata belonged to the E.maxima complex.

The results of the cross in Experiments One and Three indicated that either the drug-resistant factor(s) or the PGM type or both of these characters had been transferred between populations of E. maxima var. indentata and E. maxima (Weybridge), as organisms with a new combination of characters 156

were formed. It can therefore be concluded that E. maxima (Weybridge) and E. maxima var. indentata do not behave as distinct species as they are not reproductively isolated and are capable of sharing the same gene pool.

In Experiment Two, only one enzyme, PGM-1, was detected in the Fl generation. This was puzzling; presumably the production of E.nirixima type oocysts was far greater than that of E. maxima var. indentata type, so much so that PGM-2 was not detected in extracts of the mixed Fl population. The cross in Experiment Three was designed to favour a greater production of oocysts of E. maxima var. indentata than E: maxims (Weybridge). Four times as many oocysts of E. maxima var. indentata were used in the inoculum. Even so, the degree of staining of PGM in the extracts of the Fl oocysts suggested a preponderance of E. maxima.

The phenomenon of drug resistance appearing in populations of parasites which have not previously been exposed to the drug is of obvious practical importance. 157

C) Buoyant density of eimeriine DNA

The similarity found in the buoyant densities of DNA from E.tenella (Houghton and Weybridge),E. acervulina (Weybridge) and E.coecicola was surprising. The amount of variation shown to exist between species by electrophoretic techniques would suggest substantial differences in the DNA. The preparation of samples followed very closely schedules which have proved successful for other Protozoa. It seems unlikely,therefore, that the procedures adopted for isolation of the DNA molecules would have been inadequate, although the effects of the physical disruption of the oocysts on the buoyant density of the isolated DNA was not determined, since DNA isolated from excysted sporozoites was not analysed.

Similarity in base composition does not necessitate a close relationship (Chance et aZ. 1974). It is probabl6 that E. tenellaandE.acervulinaare quite distant systematically from E. coecicola even though their DNA buoyant densities were similar. If confirmation is obtained that the DNA buoancy is uniform within the genus Eimeria, although unexpected by cr,mr,,rison with other Protozoa, then it may prove useful for assessing the relationships between eimeriine genera.

The only other recorded value for the buoyant density of 3 DNA isolated from a species of Eimeria is 1.682 g/cm for E.t6mella(Wang and Stotish 1975) which reflects .a GC content of 22% as opposed to 52% reported in this study. (The figure of 41% stated by Wang and Stotish (1975) is incorrect according to a personal communication from Wang). The discrepancy in the results is difficult to explain and is one which cannot be accounted for by strain differences. Further studies are clearly necessary. 158

D) The significance and use of biochemical data in the taxonomy of the Eimeria

i) The value of electrophoretic data

There are theoretical advantages in using electrophoretic data over the more conventional criteria used for characteri- , sation of eimeriine populations.

Electrophoretic analysis of enzymes reveals very precise information on the genetic composition of organisms. Differ- ences in the migration of enzymes reflect discrete differences in DNA and a known amount of genetic variation is examined. The genetic basis of morphological or physiological traits is seldom known and consequently difficulties may arise in assessing the relative importance of such traits in species characterisation. Whereas, each enzyme studied can be accorded equal value it is not always possible to decide on the 'weighting' or merits of other kinds of characters: for example, whether the shape of an oocyst is of greater diagnostic value than the size of a sporozoite. Consequently, many characters and the conclusions drawn from their observation may be based on a great deal more genetic information than others.

The different phenotypic characters manifested by eimeriine populations when subjected to different environmental conditions . may obscure similar genotypes. In contrast, the present studies indicate thatin Eimeria spp the expression of genetic information at the enzyme level is stable. No variation in the isoenzymes of E.tenella(Houghton) were detected when the merozoites were produced in the caeca of chickens, the chorioallantoic membrane of chicken embryos or in kidney cells in culture. However, examples are available from studies on other groups of organisms that suggest that environmental factors may influence isoenzyme production. Variation was found by Edwards et al.(1971) in the isoenzymes of acetylcholinesterase in Nippostrongylus brasiliensis which could be correlated with the effects of host immunity. A dependence of enzyme expression on environmental conditions has also been proposed by Corbett (1973) who found variations in LDH during the population cycle of Tetrahymena pyriformis. 159

It is possible that variations in isoenzyme expression, not as yet detected for Eimeria spp, could occur as a result of different stimuli from the environment. Even if this were so, the enzyme complement is probably far less likely to show the equivalent degree of variation as the gross phenotypic characters and is, therefore, a particularly useful species character.

Electrophoretic characters, with the exception of LAP, have been found to be remarkably constant throughout the life cycle of E.ti?.rtella. At the biochemical level, as judged by the electrophoretic mobility of enzymes, sporulated and unsporulated oocysts, sporozoites and second generation merozoites were identical, whereas at the morphological level these stages bear little resemblance to one another. Although this observation is not of immediate practical use for studies on Eimeria spp, it may well prove to be of importance in determining the relationships of certain stages within other genera of coccidia. Hence, where asexual stages of an organism occur in one host and sexual stages in another, as in the case of Toxoplasma gondii, Sarcocystis spp and Frenkelia spp, it should be possible to identify the stages of a single species by the enzyme type.

A further advantage of electrophoretic analysis is that the observations are purely objective: the identification of bands of enzyme activity in gels is precise and straightforward. Subjectivity may occasionally enter into interpretation of morphological data.

The reliability of electrophoretic studies on genetic variation increases with the number of enzymes that are examined. As investigations tend to be biased towards a particular group of enzymes, those which are water soluble and can be visualised by histochemical methods, the variation found in the small number of enzymes examined might not be typical of the genome as a whole. 160 ii)Assessment of relationships within the genus

All the Eimeria spp compared showed between 70% and 100% variation in their enzymes which must reflect major differences in their genes. Considering that only a fraction of the differences which occur at the genetic level are detectable by electrophoresis and that there is a possibility of identical enzyme migration by chance, then the species examined must be well separated from each other. The systematic value of electrophoretic data diminishes as the similarity value decreases. Thus when no enzymes are held in common (100% variation) the method ceases to be of value. As the data obtained for enzyme variation of Eimeria spp from chickens, rabbits and sheep suggested a great deal of divergence at the species level, it is doubtful whether enzyme electrophoresis will contribute much information of systematic value at the level of the genus. However, certain enzymes, such as ok.GPDH and FDP, appear a little more conservative than others, although no enzyme was found which was monomorphic for all of the species examined. If a large enough set of conservative enzymes could be found, a comparison of the enzymes between different genera might be of use in the evaluation of relationships.

Similarities have been noted between Eimeria spp of the same host. E.necatrix (Weybridge) and E. tenella (Houghton and Weybridge) were found to have similar migrations for G6PDH,o(GPDH and LDH. This is interesting as the species also have certain biological characters in common. The sexual generations of E.necatrix occur in the caeca of chickens, the site where E.tenella completes its whole development. Horton-Smith and Long (1965) showed that E. necatrix will undergo its whole life cycle in the caeca. Some cross protection exists between the two species (Rose 1967a). In bothE.ivnella andE.necatrix.infe-ctions first generation schizogony occurs in the crypts of LieberkUhn and second generation schizonts develop away from the epithelium in fibrocytes of the tunica propria where they cause the disruption of blood vessels (Long 1973b).

Similarities in the mobilities of enzymes could, however, also be attributed to chance. The mobility of LDH and G6PDH shared by E. coecicola and E. magna would not be expected from the 161 known biological characters of the species.

Comparisons of protein zymograms may be of use for systematic purposes; the sharing of major protein bands may well extend to the generic level. iii) Speciation

The conditions necessary for speciation in sexually repro- ducing parasitic Protozoa must be similar to those required by free-living organisms. Essentially some form of physical isolation of populations must exist which allows genetic divergence to proceed to such a point that should the populations re-encounter one another they will be reproductively isolated. It has been shown that the genetic divergence of E. acervulina var. mivati (Houghton) from E. acervulina is small; by the use of appropriate markers it has been demonstrated that the populations can share the same gene pool. Similarly, there would seem little doubt that E. maxima -var.indentata belongs to the E. maxima complex.

The geographical range of a parasite is limited by the distribution of its host. Spatial isolation of.the sexual stages within the host might in turn contribute to the genetic divergence responsible for reproductive isolation. The divergence of E. maxima var. indentata from E. maxima is probably a result of the total or partial isolation existing between the jungle fowl and the domestic fowl.

The variations between the strains of E. acervulina and E. acervulina var. mivati are an indication of differences occurring between the populations of this species in the domestic fowl. The extent to which there is communication between populations in the field is unknown. Whether true polymorphisms exist where two or more distinct forms of a species are capable of occurring in the same habitat at the same time is also not known. 162

iv) Adaptive significance of enzyme variations

Characters which have been selected for in a population are not necessarily associated with major gene differences or differences which can be detected by the resolution of electrophoretic techniques. Drug resistant populations of E. maxima, for example, could not be separated by observations on the mobility of their enzymes and yet were known to be clearly different in their resistance to various drugs.

Enzyme differences that do occur are not necessarily associated with any selective advantage that the population might have. Hence, although an embryo-adapted line of E. tenella (Houghton) differed from the parent strain of E. tenella in the mobility of HK, the isoenzyme of the embryo-adapted form might not be of any physiological significance or contribute in any way to the ability of that line to grow in the chorioallantoic membrane of embryos. Similarly, E. acervulina var.mivati (Houghton) possesses a different GPI tp E. acervulina (Weybridge and Ongar) and will grow in chicken embryos, whereas the other strains Will not or will do so poorly; the Houghton and M strains of E. acervulina have the same GPI type as E. acervulina var.mivati but do not possess the same ability .to grow in embryos.

Due primarily to the large amount of molecular variation revealed by electrophoresis in natural animal populations, a controversy exists in population biology concerned with the processes responsible for maintaining high levels of genetic polymorphism. It has been suggested that most of the variation is adaptively neutral, an argument convincingly put by Kimura (1969a, 1969b), whereas many workers feel that some form of balancing selection must be operating (e.g. Ayala et aZ. 1972). Lewontin (1974) has presented a balanced and detailed consideration of the problem. The dispute is not concerned with the accepted fact that evolutionary change at the morphological or physiological level is a result of the process of natural selection operating through adaptive changes in DNA; it is whether all or most of the evolutionary change in DNA is due to natural selections. 163

It seems feasible that random genetic changes at the molecular level may take place which in no way affect the fitness of the organism. For example, if a mutation occurred in the gene coding for hexokinase, in the embryo-adapted line of E. tenella, producing a single amino acid alteration at a place in the molecule which was not associated with the active site, the physiological function of the enzyme might not be disturbed, but the difference in the molecule might be detected by electrophoresis. Such a mutation could be considered to be a neutral mutation and would be neither at a selective advantage nor dis- advantage and could become passively fixed in the population. The words of King and Jukes (1969) help to clarify the situation: "Evolutionary change is not imposed upon DNA from without, it arises from within. Natural selection is the editor rather than the composer of the genetic message. One thing the editor does not do is to remove changes which it is unable to perceive."

E) Population structure with particular reference to laboratory cultures

The enzyme variation found below the species level in Eimeria spp was small in comparison with other groups of parasitic Protozoa, (e.g. Carter 1973, Kilgour and Godfrey 1973). A direct comparison is perhaps unjustified as the amount of divergence within any group is also a reflection, in part, of the status of the taxonomic categories which may well differ. Enzyme analyses of a large number of eimeriine field isolates are required to determine the degree of intraspecifc variation in natural populations.

The oocyst cultures examined have been remarkably homogenous: only in samples which were believed to contain more than one population was more than one enzyme type found. This may, • possibly, have been due to the insensitivity of the techniques used, but as it has been shown that oocysts forming not more than one tenth of the total population can be detected, enzyme variants would have to be present in very small numbers in order not to contribute to the resulting zymogram. Laboratory procedures of deriving oocyst cultures from single or small numbers of .00cysts will, obviously, tend to reduce the genetic variation 1614 and lead to the production of monomorphic populations.

The life cycle of eimeriine parasites is perhaps conducive to the production of homogeneity. The possibility of inbreeding which would favour the increase of identical genotypes within the population is strong. Natural infections are likely to result from the ingestion of a small number of oocysts, probably oocysts which h6.ve been produced at the same time in the same animal. The multiplication of one particular genotype during the asexual stages of schizogony, the close proximity of gametocytes produced by genetically identical merozoites, and the limited time and restricted position at which the gametocytes'are found, are all likely to contribute towards the homogeneity of the population.

Genetic heterogeneity in a population produces individuals that have less than maximal fitness; this reduction in fitness has been called genetic load. It does, however, give a species an advantage in that it is more likely to be able to respond to a changing environment and to occupy various environmental niches. The intracellular environment of Eimeri7, spp is presumably relatively static and the parasites are particularly specific in the sites in which they live: the parasites might be considered to have a small genetic load. Small levels of variability are consistent with the findings of Bryant (1974) that strong correlations exist between degrees of heterogeneity and environmental variability for several invertebrate species.

However, the fact that populations can occasionally be selected for drug resistance or for an ability to grow in• chicken embryos suggests that heterogeneity exists; the factors, which are needed to overcome such selection screens, being found only in a small proportion of the individuals making up the population. Successive exposures to the particular screen continually select the desired characters, which might not be apparent after one exposure. For example, after only one passage in chicken embryos, followed by one in chicken, a population of E. acervulina (Weybridge) showed no improvement in its ability to produce oocysts in chicken embryos.

•Another possible mechanism by which the ability to overcome 165 a selective screen may arise is by spontaneous mutation. Such mutations might provide the factors, such as an alternative metabolic pathway, which are necessary for survival during the selection pressure. A single step mutant of Toxoplasma gondii that was fifty-fold more resistant to adenine arabinoside was isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguan- idine (Pfefferkorn, personal communication). Similar mutagenesis yielded temperature-sensitive mutants of T.gondii which had lost the ability to form plaques in culture at 40°C, but grew well at 33°C (Pfefferkorn and Pfefferkorn, in press).

Once a population has developed drug resistance it appears that the characteristic is stable. No loss in the resistance of E.tenellato glycarbylamide occurred when passaged nine times in the absence of drugs. (Gardiner and McLoughlin 1963) or to quinoline when passaged ten times (Ryley and Betts 1973). Presumably, as no competitive genotypes were introduced, the characters persisted in the population. Ball (1966) showed that drug resistance characters can be lost from a heterogenous population: when a small number of glycarbylamide-resistant parasites were introduced into a host with a larger inoculum of normal .E. tenella, the resistant forms diminished during passage through chickens. 1 66

F) Clones of coccidia

Unfortunately methods of obtaining genetically pure lines (clones) of coccidia are very much in their infancy. There is now little doubt that the first zygotic division is the place where meiosis occurs (Canning and Anwar 1968, Canning and Morgan 1975). Thus the parasites are haploid throughout their life cycle except immediately after fusion of the gametes when a diploid zygote is formed.

The first nuclear division after fertilisation is unusual in being a single phase reduction division (Canning and Anwar 1968). This is followed by two mitotic divisions giving rise to four sporocysts each with two sporozoites. If the micro- and macrogamete differed in their genotypes and all the products of the post-fertilisation divisions are retained, then two genetically distinct populations could be produced by one oocyst. This is diagramatically shown in Figure 20: utilising GPI as a character with the gametes differing in the form of the enzyme which they possess.

An attempt was made to demonstrate this and at the same time to produce the definitive proof that E. acervuZina var.mivati (Houghton) was a member of the E. acervulina complex: single oocyst infections were produced of the Fl population of a cross between E.acervulina (Weybridge) GPI-2, and E. acervulina var. mivati (Houghton) GPI-1. Inconclusive evidence was obtained as only 5 populations were established, three characterised by GPI-2, one by GPI-1, and one by both GPI-1 and GPI-2.

Further experiments are obviously required, which should include 'cloned' parent populations and controls using single oocyst infections of an artificially mixed culture. A comprehensive study would be a formidable task, especially as only a small percentage of the Fl population would show the mixed character. The suggestion that mixed populations can arise from one oocyst might help to explain the morphological heterogeneity found in single oocyst infections. (The standard deviations of the means of measurements of oocysts derived from single oocyst infections was comparable to those found for normal infections, as shown in Table 10). FIGURE TWENTY

Inheritance of characters within an oocyst

GPI-1 GPI-2

GAMETES MICROGAMETE MACROGAMETE (n) (n)

FERTILIZATION

ZYGOTE (2n)

MEIOSIS (one step)

GPI-1 GPI-2 (n) (n) z mitosis mitosis SPOROBLASTS GPI-1 GPI-1 GPI-2 GPI-2

SPOROZOITES GPI-1 GPI-1 GPI-1 GPI-1 GPI-2 GPI-2 GPI-2 GPI-2 (n) (n) (n) (n) (n) (n) (n) (n) . 168

Certain strains are known to differ immunologically (e.g. Joyner 1969). If any of the enzymes studied are directly responsible for initiating an immunological response, i.e. if they act as antigens, then a close correlation might exist between the degree of enzyme variation and the degree of cross protection between strains. One oocyst possessing a mixture of characters from two parent strains might produce a population which could give immunological protection against both strains.

A clone can only be established with certainty by using single sporozoites, single sporocysts or any of haploid stages in the life cycle. The finding of oocysts in a caecum of one chicken out of fifteen which were inoculated with single sporocysts of E.tenella (Houghton) does not hold much promise for this method as an efficient cloning mechanism. Walliker (1976) attempted single sporozoite infections of P. chabaudi in mice; only one out of ninety-four animals developed an infection in the blood. The ease at which Toxoplasma gondii grows in culture does allow a reliable cloning method and improvement of culture techniques may well facilitate cloning of Eimeria spp. Recently, Shirley and Millard (1976) recovered oocysts capable of sporulation from the allantoic cavity of five chicken embryos which had been inoculated with a single sporozoite of an embryo adapted line of E.tenella (Houghton). 169

G) Sexual differentiation

The progeny of a single haploid trophozoite of Toxoplasma gondii grown in culture has been found to be genetically capable of differentiating into either micro- or macrogametocytes when a cat infected with cloned T.gondii yielded viable oocysts (Pfefferkorn, personal communication). Similarly, Bishop (1958) found that gametocytes of toth sexes were able to develop in clones derived from single trophozoites of Plasmodium gallinaceum in chickens and during the genetic investigations of Walliker (1976), oocysts were produced in mosquitoes fed on thicket rats infected with cloned P. yoelii. Haberkorn (1970a) described the complete development of E. falciformis in mice following the administration of single merozoites, and Shirley and Millard (1976) obtained both macro- and microgametocytes in embryos inoculated with single sporozoites of E.tenella. If a sporozoite or merozoite or its equivalent is capable of giving rise to both micro- and macrogametocytes, the haploid cell must be bisexual and it is necessary to consider mechanisms which could be responsible for sexual differentiation.

Grell (1953) found that when a sporozoite of Eucoccidium•dinophili a coccidium lacking schizogony, entered a host, it invariabl:: developed into a macrogametocyte and he suggested that some factor emanating from the macrogamete stimulated the development of microgametes. Other investigations have suggested that sexual characters are differentiated at least in the generation prior to the formation of gametocytes. Canning (1961), on the basis of polysaccharide content and the possession of nucleoli differentiated two types of merozoites of Barrouxia schneideri which gave rise either to micro- or macrogametocytes. Similar nuclear differences were observed in Adelea ovata (Canning 1973). Klimes et aZ. (1972) distinguished the sexes of E.tenella on the basis of PAS staining and retraced the origin of micro- and macrogametocytes to the merozoites and schizonts of the last generation. Morgan (1973) placed the sexual differentiation at an earlier stage. He found that sporocysts were either PAS+ve or PAS-ve and suggested that the sex of the stages was already determined in the sporocysts. 170

Observations in tissue culture of both sexes in one host cell would seem to preclude the possibility of environmental influence on sexual differentiation at the onset of gametocyte formation (Klimes et al.1972), although influences of the host cell in vivo cannot be studied so readily.

Although deviations from the normal course of mitotic processes have been shown to have important consequences in sex determination, the examples are rare. Cleveland (1949) described in Trichonympha, a hypermastigid flagellate, a highly aberrant type of mitosis, which had the character of a sex differentiating division, but this also depended on the segreg- ation of sex genes which took place at meiosis. Also in species of two families of Diptera differential mitotic divisions occur which are characterised by the elimination of chromosomes and determine sexual differentiation (Bacci 1965). This phenomenon is unlikely to occur in the haploid eimeriines.

Until an external stimulus can be identified which is responsible for the expression of the sex determinants and until it is known at which stage of the life cycle such a stimulus acts, the explanation of sex differentiation will be difficult. 171

II) Consequences of the nuclear divisions occurring within the oocyst

Investigations on E.maxima and E. Ila by Canning and Anwar (1968) revealed that the zygotic meiosis occurs without doubling of DNA to produce two haploid nuclei. The division appears to be a single one-step reduction division which occurs without the formation of chiasmata and centromeres. Thus, exchange of genetic material between chromosomes by crossing over cannot occur. Individual variation is achieved solely by the random- isation of chromosomes; the haploid nuclei produced as a result of the meiotic division may contain a mixture of the original complement of chromosomes in the micro- and macrogamete. Two important consequences of the absence of crossing over are:

(1) Favourable gene combinations will be kept together on the same chromosome (which might favour homogeneity).

(2) The assortment of characters will be limited and confined to the different permutations of chromosomes which produce viable organisms_

Genetic studies should provide further evidence on this latter point by establishing the linkage that exists between different characters. The reassortment of characters will be very limited if no crossing over occurs, especially if the haploid number of chromosomes is small. The haploid number of 5 has been reported for E.maxima (Canning and Anwar 1968, Schol- tyseck 1963).

The present results have shown that in E.acervulina the GPI locus and the factors responsible for the ability to grow in eggs assort independently, as do the PGM locus and factors responsible for resistance to methyl benzoquate in E. maxima. If the loci for the characters which were studied had been on the same chromosome and no crossing over occurred, it would not have been possible to demonstrate the transfer of characters as illustrated in Figure 21a.

Figure Twenty-One Transfer of characters during meiosis

a) Characters on Same Chromosome 1 - PGM-1 2 - PGM-2 GAMETES X - methyl benzoquate- resistant X !I 1 - - methyl benzoquate- 1 1 sensitive 1 1 1

1 Possible 2 Arrangement of JJ 1 2I IX Chromosomes I 1 1 1 I 1 1 1 I I 1 1 1 I 1 I

2 1X I 1 • 1 1 )1 1 • 1 1 I I

1 1 1 1

GENOTYPES

Figure Twenty-One

b) Characters on Different Chromosomes

GAMETES 1 2 1 Ix i i i I 1 I I

1 Possible 2 1 Arrange- ment of X C hromo- somes

I I I I

2 1

2 1 1 1 1 2 i 1 1 1 I

1 I

IX) I L)I L )1 1 X )1 1 X ) 1 1 I 1 )

GENOTYPES 174

. If the two loci were on different chromosomes and linkage did not occur, all combinations of the characters would be possible (Fig. 21b).

The practical significance of this can be realised if two drug resistant characters are considered. Theoretically, it would not be possible,when two drugs act on different sites of the same chromosome,for populations resistant to both drugs to be produced by recombination when crosses occurred between lines resistant to only one. Hence, a population resistant to methyl benzoquate could not passively acquire resistance to any drug acting on the products of the same chromosome. Resistance could only be acquired independently by mutation. An indication that this might be so, or at least that certain characters might be linked on the same chromosome comes from the work of Joyner and Norton (1975). They found that resistance factors could be transferred between a line of E.maxima resistant to clopidol and a line resistant to sulphaquinoxaline and between a line resistant to methyl benzoquate and a line resistant to sulphaquinoxaline. However, resistance factors could not be transferred between a line resistant to methyl benzoquate and another resistant to clopidol. As methyl benzoquate and clopidol belong to different classes of anticoccidial compounds it is unlikely that they would share the same site of action, although there is no reason whey they should not act on the same chromosome. No other evidence of possible linkage between characters has been reported for either Eimeria or Plasmodium spp.

Further genetic investigations are needed to help elucidate the nuclear processes occurring within the oocyst. 175

I) Isoenzymes in relation to coccidiosis

Decreases in serum AP during E.tenelZa infections have previously been reported by Chute et al.(1969) and Arnastauskiene and Kadyle (1973). The present study showed a decrease in activity on the third day of infection and could perhaps be correlated with the release,of the first generation merozoites; the greatest loss of serum proteins being associated with the extensive tissue damage caused to the caecal wall by the large second generation schizonts. No serum isoenzyme changes were found in E.tenella infections which were indicative of tissue damage caused by .coccidia. The elevation of LDH and the more obvious LDH-5 - LDH-4 bands in the serum of rabbits infected with E.stiedai requires further investigation, especially as LDH-5 is characteristic of liver tissue, the site of E. stiedai infections. Smith and McShan (1949) concluded that stages of E. sti edai elicited an increase in the SDH activity of the livers of rabbits 10 and 15 days after infection. The high level of activity decreased as the infection approached the 20th day.

If serum isoenzymes could be found which would serve as indicators of intestinal damage, then it should be possible to produce a method for monitoring the severity of an infection. Such methods would be useful in detecting the effects of anticoccidial compounds against the parasites. Biochemical methods, based on the alteration of glutamic-oxaloacetic transaminase levels in serum, have been suggested as a means of detecting antihelmintic activity against liver fluke (Campbell and Barry, 1970). 176

PART II

Biological Characters of Eimeria species 177

Introduction

Specificity of Eimeria species

Marquardt (1973) pointed out that coccidia have as high a degree of specificity as any group of infectious agents. Species of Eimeria are committed to particular niches: Not only are these parasites restricted to a narrow range of hosts, they are confined to particular organs within the host, to certain cells within the organ and even to specific sites within the cell.

During the evolution of the host parasite relationship, characters of the parasite are selected for which best suit it for a particular habitat. Specialisation, in turn, imposes a limit on distribution, due to the increasing genetic commitment to one specific environment. Other hosts or other sites within the host, if encountered, prove to be in someway hostile or unsuitable for the satisfactory development of the parasite. Although numerous investigations have been attracted to the problems of specificity, the factors which are involved in confining the parasite to specific niches are still largely unknown.

1) Host specificity

Most experimental work on host specificity has been directed towards assessing the host range of eimeriine parasites of veterinary importance. In general, it has been found that species of Eimeria will infect a limited number of hosts; where a larger range of host species have been reported they tend to be closely related and usually belong to the same genus.

Assumptions on the host range of a species, using evidence from natural infections, have been based primarily on the identification of oocysts and hence on the morphology of oocysts. Generally, oocysts of identical morphology from closely related species have been considered to be the same species. 178

Further insight into the possible relationships of eimeriine isolates can be gained by a consideration of the• biology of the hosts. In a study of coccidia of sandhill cranes, Grus canadensis, Courtney et aZ.(1975), found two species of Eimeria, E. gruis and E. reichenowi. Both species were found in Florida sandhill cranes, G.c. pratensis, greater sandhiilcranes, G.c. tabida from Florida and Arizona and also in lesser sandhill cranes, G.c. canadensis, from Texas. The findings were substantiated by the fact that there was opportunity for transfer of coccidia between the three subspecies. Greater sandhill cranes and Florida sandhill cranes occur in the same wintering grounds and the greater and lesser sandhill cranes have breeding grounds that overlap in central Canada.

The need for studies on the endogenous development and cross transmission capabilities of eimeriine parasites, before reaching conclusions on host ranges,was emphasised by Wacha and Christiansen (1974) in a study of parasites from N. American snakes. On the basis of oocyst structure a broad host range was found, similar oocysts being found in systematically closely related snakes. The rigidity of host specificity in Eimeria spp from fish has also been questioned (Molnar and Haneck 1974). Vetterling and Widmer (1968) des- scribed E.cascabeli from.the rattle snakes, Crotalus viridis viridis of N. Colorado and C.v. heMaii, of S. Colorado. Attempts to transfer the parasite to sidewinders, C. cerastes laterorepens, and garter snakes, Thamnophis satwitis, were unsuccessful.

Most cross transmission experiments have relied upon the introduction of oocysts from a known source into an 'abnormal' host: the presence or absence of oocysts in the faeces, at a later date, being an indication of the suitability of the animal as a host of the parasite. It is imperative in such experiments that the history of the experimental animals is known, that the oocyst cultures used are precisely defined -and that the animals are maintained free of extraneous infection. The inability to reproduce the results of certain earlier investigations have tended to invalidate them.

Levine and Ivens (1965) reviewed the cross infection experiments performed with Eimeria spp of rodents. 179

They reported that 8 out of 9 attempts to transfer Eimeria between species of the same genus were successful, but none of 47 attempts to transfer parasites to species in different genera succeeded. After reviewing the literature pertaining to the Eimeria spp from ruminants, Levine and Ivens (1970) concluded that transmission of species between members of different tribes probably does not occur, although they left open the question of whether transmission between the domestic goat and the domestic sheep takes place.

Todd et al. (1968) showed that E. bilamellata could be transmitted among at least four species of the ground squirrel, Spermophilus. E.callospermophili was found to infect six species of ground squirrel as well as the closely related prairie dog, Cynomys Zeucurus (Todd and Hammond 1968a). Similarly, Todd and Hammond (1968b) reported the occurrence of E. larimerensis in five species of SpermophiZus as well as in Cynomys Zeucurus.- Joseph (1969) reported that most of the Eimeria spp described from the grey squirrel, Sciurus caroZinensis, either occurred naturally in the fox squirrel, S. niger rufiventer, or were transmissible to it.

A marked lack of host specificity was found for E. chinchillae, originally described from the chinchilla, Chinchilla laniger. De Vos (1970) succeeded in transmitting this parasite to animals representing seven different genera.

E. contorta isolated from a laboratory colony of rats, Rattus norvegicus was transmitted to mice, Mus musculus, and multimammate rats, Akstomys nantensis (Haberkorn 1971a).E. seperata from the rat was able to establish and complete its life cycle in the mouse, an 'abnormal' host (Mayberry and Marquardt 1973).

E. nieschulzi from rats was reported to penetrate mouse intestinal epithelium but did not undergo schizogony (Marquardt 1966). Complete schizogony of E. falciformis from mice occurred in rats but no gametocytes were found (Haberkorn 1970a). Sporozoites of this parasite did not appear to divide after entering epithelial cells in the caeca and upper colon of chickens. However, E. tenella from the chicken formed some schizonts in intestines of mice (Haberkorn 1970b). 180

Coccidia from rabbits are believed to occur naturally and experimentally in more than one host genus (Duszynski and Marquardt 1969).

In a study of coccidiosis in kangaroo rats, Doran (1953) found that E. mohavensis occurred naturally in Dipodomys pana- mintinus mohavensis but not in the sympatric D. merriani merriani. However, in the laboratory E. mohavensis could be transmitted to D. merriani merriani and, in fact, produced more oocysts than in the normal host.

McLoughlin (1969) summarised the results of cross transmission studies with coccidia from chickens and turkeys. If the findings of earlier workers, that have not been repeated, are attributed to extraneous infections then the majority of species can be considered to be specific for a single host.

After inoculation of large numbers of oocysts of E. colchici from pheasants into turkeys, Norton (1967) observed that a light infection was produced. The parasite was not well suited to the alternative host, and could not be maintained indefin- itely.

In the description of three new species of coccidia from the Canada goose, Branta canadensis, Farr (1953) also noted that the domestic goose, Anser anser anser, served as one experimental host for all three species. Levine (1953) recorded E. brantae from B.c. hutchinsi and B.c. parvipes. Other examples of the host range of Eimeria spp from geese are given by Pe116rdy (1965).

Fernando and Remmler (1973a,b) described 6 new species from the Ceylon Jungle fowl Gallus lafayetti solely on the basis of oocyst morphology. Many of the species completed their life cycle in the domestic fowl and are now believed to be variants of the Eimeria spp named from the domestic fowl (Long et al. 1974; Long 1974a).

Vetterling (1976) attempted to transmit E.tenella from the domestic fowl, Gallus gallus, to other genera of gallinaceous birds. Sporozoites were found in the lamina propria of some birds of five species at 4 hr post-inoculation, but no stages 181 were found thereafter except in the Gallus gallus and the chukar, AZectoris graeca. Oocyst production occurred only in the domestic fowl.

Few direct studies have been made to detect the influence of genetic variation of the host on the host-parasite relationship. Rosenberg (1941) found differences between five breeds ofc young chickens in their susceptibility to E.tendla infections. Working with the same parasite, Champion (1954) considered that selective breeding was effective in establishing lines of chickens resistant to E. tenella. Long (1968) examined five strains of chickens and found that they differed in their resistance to four species of coccidia. However, Vetterling (1976) found no variation in the susceptibility of six breeds of chicken to E.tenella as judged by oocyst production.

The genetic background of the mouse, an unnatural host, was found to be crucial in determining whether E.seperata from rats completed its life cycle (Mayberry et al. 1975; Marquardt 1976); the development of the parasite took place in some strains of mice but not in others.

2) Attempts to breakdown the specificity of Eimeria species The administration of corticosteroid drugs have been shown to increase the susceptibility of 'abnormal' hosts to infection with coccidia. E. meleagrimitis from the turkey was found to infect chickens medicated with dexamethasone, although E. tenella inoculations did not produce an infection in medicated turkey poults (McLoughlin 1969). Todd et al.(1971) reported the occurrence of second generation schizonts of E. vermiformis from the mouse in dexamethasone-treated rats six days after infection. No sexual or extra-intestinal stages were detected in tissues from rats, although one treated rat did pass a few oocysts after eleven days. The oocysts failed to sporulate and could not be identified. In a later report, Todd and Lepp (1972) described completion of the life cycle of E. vermiformis in dexamethasone-treated rats.

The distribution of E. acervulina var. mivati in the intestines of birds treated with corticosteroids was found to be much 182

wider than that in controls (Long and Rose 1970). Long (1970b) found that E.telwIlacould develop in the livers of dexamethasone'- treated chicks.

Leathem (1969) reported that E.tenella a parasite which develops in the caeca of domestic fowl, infected the small intestine of caecectomised and normal birds. By introducing the sporozoites of E. necatrix,` the asexual generations of which normally occur in the chicken intestine, directly into the caeca, Horton-Smith and Long (1965) found that the whole life cycle was completed in that organ.SiMilarly,E. acervulinavar.mivati was shown to be capable of development in the caeca (Horton- Smith and Long 1966) and by using very large numbers of sporozoites, Joyner and Norton (1972) obtained the development of E. acervulina in the caeca. Neither E.praecox.norE.maxima developed in this site but infection of the small intestine did occur when sporozoites were introduced via the caecum (Long and Millard 1976b).

Davies and Joyner (1962) found that the oocysts of four species of Eimeria from the fowl could pxcyst when inoculated intraperitoneally, intramuscularly and intravenously and that ' development occurred in the sites normally invaded by the coccidia. Sharma (1964) also observed.normal development after intravenous injections of oocysts of seven Eimeria spp from fowl. Mice inoculated intravenously, intraperitoneally, sub- cutaneously and intramuscularly with oocy -ts of E. falciformis developed normal infections (Haberkorn 1970a). Attempts to infect calves by the intraperitoneal route failed (Fitzgerald 1965).

Pe116rdy (1969a) found that intravenous inoculation of sporozoites, oocysts and merozoites of E.stiedai resulted in development in the normal site, the liver of rabbits. Repeated injections of excysted E.stiedai into the femoral muscles of rats, chickens, guinea fowls and mice produced no infections (Pellerdy 1969b). 183

In further attempts to break through the specificity of coccidia, Pe116rdy (1969b) tested the susceptibility of rats to E. stiedai after splenectomy, hydrocortisone treatment, irradiation and ethionine feeding; no evidence of development was found.

3) Specificity of avian Eimeria spp in vitro and embryo culture

Doran (1973) presented an extensive review of work conducted on the growth of coccidia in cultured cells. He observed that a wide variety of cells from seventeen different species of animals had been used in attempts to obtain development of Eimeria and Isospora and also that development of Eimeria proceeded further when cell cultures from the natural host were used. In general, the sexual generations appear to have more specific requirements than the asexual generations.

Only E. tenella has been shown to complete its whole life cycle in cultured cells (Doran 1970). E. necatrix produced schizonts in culture (Doran 1971b) as did E.brunetti (Ryley and Wilson 1972) and an embryo adapted E. acervulinavar.mivati (Long, 1973c).

Doran (1971c) compared the development of E.tenella in nonembryonic cell cultures prepared from the kidneys of the chicken, Chinese pheasant, chukar partridge and turkey. The life cycle was completed in both chicken and pheasant cells but proceeded only to gametocytes in partridge cells and to mature second generation schizonts in turkey cells. Develop- ment in cells from unnatural hosts was delayed; oocysts were found on the sixth day in chicken cells but not until the ninth day in pheasant cells. In a more detailed study, Doran and Augustine (1973) obtained the complete life cycle of E. tenelia in primary cultures of kidney cells from two to three week old chickens, guinea fowl, partridges, pheasants, quail and turkeys. Hence development occurred in representatives of three families, Phasianidae, Nunididae and Meleagridae. Development was better, however, in chicken cells; stages appeared earlier and were larger and oocyst production was far greater than in cells from unnatural hosts. 184

Shibalova (1970) obtained the whole endogenous development of E.tenella in both cultured chicken and quail embryonic fibro- blasts. E.brunetti developed as far as second generation schizonts in both cell types.

After inoculating sporozoites into the allantoic cavity of developing chicken embryos, Long (1966) found that E. tenella, E. brunetti and E. acervulina van mivati completed their whole life cycle in the chorioallantoic membrane. These findings have been confirmed by other workers (Itagaki et al. 1972, Jeffers and Wagenbach 1970). Many attempts to infect embryos with E. praecox and E. maxima have failed (Long 1973c) although Shibalova (1970) claimed to have obtained the development of E. praecox and E. necatrix. Infections with E. acervulina only took place when massive numbers of sporozoites were inoculated into the allantoic cavity (Long 1973a).

Initial attempts to infect chicken embryos in sites other than the chorioallantoic membrane were unsuccessful (Long, 1965). Long (1971) later reported the occurrence of developmental stages of E. tenella in the liver of chick embryos. Gametogony was found to occur more freely in dexamethasone-treated embryos.. Long and Millard (1973) reported the development of E. brunetti, E. acervlina var. mivati and E. necatrix in the liver of chick embryos.

Long (1971) attempted to obtain an infection of E. tenella in goose embryos. No development was found in untreated embryos, but in one embryo which had received dexamethasone second generation schizonts were found. No infection occurred when E.tenella was inoculated into the amniotic cavity of chick embryos or into the allantoic cavity of embryonated quail and turkey eggs (Long 1965). Duck and quail embryos were successful hosts for an embryo adapted strain of E. tenella, the whole endogenous cycle being completed in epithelial cells of the chorioallantois (Long and Millard 1975). However, duck and quail embryos could not be infected with pathogenic strains of E. tenella, E. brunetti, E. acervulina or an embryo-adapted strain of E. acervulina var. mivati. Fitzgerald (1970) reported finding gametocytes of E. stiedai in the chorioallantoic membrane of chicken eggs after inoculation of sporozoites, but.was not able 185 to obtain consistent results.

By repeated passage in embryos, lines of E. tenella and E. acervulina var. mivati have been produced which differ in certain characteristics from the parent strains (Long 1972a, 1972b). Judged by oocyst production, both lines show an increasing commitment to development in embryos, whereas development in chickens is poor with a marked decrease in pathogenicity. The lines have retained their original immunogenicity and birds which were exposed to one infection were highly protected to challenge with the parent strain.

The type of cell which a coccidium prefers is constant under natural conditions. The majority of Eimeria spp develop in epithelial cells. E. tertelia appears to be consistent in the type cell it utilises, wherever development occurs. The second generation schizonts develop in cells of mesodermal origin and gametocytes in endothelial cells. In tissue eta- ture it has been noticed that gametocytes developed only in islands of epithelial cells (Bedrnik 1969, Long 1969, Doran 1971a). In the liver of dexamethasone-treated chicken embryos second generation schiionts developed in cells lining the sinusoids and gametocytes in hepatocytes, which are mesodermal and endodermal in origin respectively (Lee and Long 1972). 186

RESULTS

1) Development of Eimeria spp in chicken embryos

The complete development of E. tertel&z(Houghton), an embryo adapted line of E. tenella (Houghton) and E. acervulina var. mivati (Houghton) occurred in the chorioallantoic membrane of chicken embryos after the inoculation of 1 x104 to 5x 104 sporozoites in to the allantoic cavity. Characteristic colonies of large second generation schizonts of E. tenella (Houghton) were found 4i to 5 days after inoculation (Fig. 22a); oocysts were located in the urate deposits at 7 days (Fig.22b). Stages of E.acervulina var.mivati (Houghton) and the embryo adapted line of E. tenella (Houghton) were confined to the epithelial cells (Figs. 23,24). Oocysts of E. acervulinavar.mivati were harvested 5 to 5i days after inoculation of sporozoites.

No development of either the 'M' or Houghton strains of E. acervulina was detected when 1 x 104 and 1 x 106 sporozoites were used as the inoculum. Limited oocyst production of E. acervulina (We.ybridge) occurred after the inoculation of 1 x 107 sporozoites.

4 No evidence of development was obtained after 1 x 10 , 1 x 105 and 1 x 106 sporozoites of E. maxima (Weybridge) were 4 inoculated into the allantoic cavity. Similarly, when 1 x 10 and 1 x 106 sporozoites of E. bateri were used as the inoculum, no stages of the parasite were found in the chorioallantoic membrane and no oocyst production was detected.

2) Development of Eimeria tenella (Houghton) in avian embryos

Sporozoites of E. tenelta(Houghton) were inoculated into the allantoic cavity of duck, quail and pheasant eggs at 5 6 concentrations of 1 x 10 and 1 x 10 . The embryos were sacrificed at appropriate times and examined for parasites.

A very few small groups of immature second generation 187

Figure Twenty-Two (a)E. tenella (Houghton) infection in chorioallantoic membrane of chicken embryo (5 days) 10 p,m

(b) Oocysts of E.Tnella (Houghton) in urate deposits of chicken embryo (7 days). (x 500) 188

Figure Twenty-three

E. acemulina var. mivati infection in chorioallantoic membrane of chicken embryo (3i days). 10 pm

Figure Twenty-four E. tenella (embryo-adapted) infection in chorioallantoic membrane of chicken embryo (6 days).

10 pm

,4 i■ 10 v.

t 189

schizonts were found in duck chorioallantoic membrane after 4 and 41 days (Fig. 25 a,b). Mature schizonts containing merozoites were found after 5 days. The merozoites gave a positive reaction for LDH (Fig. 25 c) and were assumed to be viable. No sexual stages were discovered and no oocysts were detected in urate deposits or scrapings of the chorioallantoic membrane 7, 8 and 9 days after inoculation.

Unsporulated oocysts were obtained from quail embryos inoculated with sporozoites 160 hours previously. Due to the small numbers of oocysts produced and the difficulties encountered trying to harvest them, accurate determinations of the numbers produced per embryo were not made. Oocyst production did appear to be dependent on the number of sporozoites used in the inoculum; fewer oocysts were produced in embryos inocu- 4 lated with 2 x 10 sporozoites than in those inoculated with 5 6 1 x 10 or 1 x 10 . Sporulation rates of 41% and 26% were achieved on two separate occasions. Schizonts were found both within and below the epithelial cells of the chorioallantoic membrane (Fig. 26).

Pheasant embryos were found to be more suitable than duck or quail for the development of E.tenella (Houghton). Col- onies containing up to 10 large second generation schizonts were found at 5 days (Figure 27); large schizonts containing merozoites were also observed at 7 days. Oocysts were found in the urate deposits and scrapings of the chorioallantoic membrane at 7 and 8i days; the number produced was small. A batch of oocysts obtained after 7 days had a sporulation rate of 42%; those obtained at 8i days did not sporulate.

No information concerning the mortality of infected embryos was obtained as the mortality rate of control embryos was high.

3) Development of Eimeria spp in vitro

(a) Cell cultures

E. tenella (Houghton) developed as far as second generation schizonts in monolayer chicken kidney cell cultures (Figure 28); 190

Figure Twenty-five Stages of E.tenella (Houghton) in chorioallantoic membrane of duck embryo

10 pm (a, b) Schizonts (4 days)

(c) Merozoites of E.tenella (Houghton), from ruptured schizonts in chorioallantoic membrane of duck embryo, stained for LDH (5 days)

10 Om 191

Figure Twenty-six Stages of E.tertena (Houghton) in chorioallantoic membrane of quail embryo. (a) Schizonts in disrupted membrane (116 hrs) 10 ym

tr-44 444.0.4 % "•,* • 1. „.

(b) Microgametocyte (160 hrs) 10 um

10 pm 192

Figure Twenty-seven

Stages of E.tenen2(Houghton) in chorioallantoic membrane of pheasant embryo

(a) Mature schizonts (120 hrs) 10 pm

(b) Mature schizonts (144 hrs)

(c) Merozoites (120 hrs) (x 1000) 193 very few gametocytes were produced. Quail kidney cells also supported the development of asexual generations of E. teneaa (Houghton) as shown in Figure 29; no gametocytes were detected.

E. bateri sporozoites invaded both chicken and quail kidney cells but no development other than a slight rounding up of the sporozoites occurred.

E. tenella sporozoites were found within cells of a mosquito cell line (Aedes pseudoscuteLlczns ) 4 hours after inoculation on to cell cultures held at 37°C. The cells did not survive at this temperature.

(b) Cell suspensions

Attempts to use suspensions of chicken kidney cells as an alternative method of culture were unsuccessful; a small number of infected cells were found 4 hours after the addition of sporozoites to the medium but all the cells deteriorated rapidly and no further dev elopment was found.

Organ slices were used in an attempt to study the development of E. teIltella in different tissues. However, tissue slices of caecum, duodenum and small intestine from 20-day-old chick embryos and from 15-day-old quail embryos and slices of kidney ' from 2-week-old chickens rapidly became disorganised. Compara- tive studies could not be made. A few 'macrogamete-like bodies' were seen 1 days after the exposure of•caecal tissue from chick embryos to merozoites, - but no other signs of development were found.

4) Attempts to obtain development of E.tenella (Houghton) in Coturnix coturnix japonica

E. teneaa (Houghton) was found to complete its development in quail embryos and to develop at least as far as second generation schizonts in quail kidney cells in culture. However, the young bird, as judged by oocyst production, was resistant to the parasite; no oocysts were produced by 3-week-old birds 1914

Figure Twenty-eight

Stages of E.tenella (Houghton) in monolayer chicken kidney cells 10 p m a) Intracellular Sporozoites (14 hrs.)

b) Immature schizont (96 hrs.) - note adjacent cell with latent sporozoite 195

Figure Twenty-nine

Schizont of E.tenella (Houghton) in quail kidney cell (108 hrs) 10 p.m

Figure Thirty

Sporozoites of E.tenella (Houghton)in the caecum of a quail (16 hrs) 10 yim 196

6 . orally inoculated with 1 x 10 oocysts of E.tenella (Houghton).

Sporozoites of E.tenella were able to excyst in the quail alimentary canal; 3 hours after the oral inoculation of 6 4 x 10 oocysts into 6-week--old quail, sporozoites were observed in the lower small intestine and caeca; no intact oocysts were found.

Sporozoites were capable of penetrating the caecal tissues; 16 hours after oral inoculation with E. tenella, sporozoites were found both within and below the epithelial cell layer of the villi (Figure 30). No development of the parasite, after the invasion of the caecal tissues, was detected; 48 hours after oocyst inoculation sporozoites were still found within epithelial cells of the caecal villi, they appeared normal. No parasitic stages were found at or after 96 hours.

A 22-day-old quail chick was orally inoculated with approxi- 3 mately 5 x 10 oocysts of E.tenella which had been harvested from quail embryos. The bird did not subsequently suffer from caecal haemorrhage and no oocysts.were produced.

Five 21 day old quail were immunosuppressed with betametha- 5 sone and orally inoculated with 1 x 10 oocysts of E. tenella. No development of the parasite was detected and oocysts were not produced. Similarly no oocyst production occurred in 14- 5 day-old immunosuppressed chickens orally inoculated with 1 x 10 oocysts of E. bateri (Ascot).

6 Intraperitoneal inoculations of 1 x 10 oocysts of E. tenella into normal 21-day-old quail did not produce a detectable infection.

4 After the inoculation of 5. x 10 sporozoites of E. tenella (Houghton) on the 7th day of incubation, quail embryos were allowed to develop normally. Five days later one embryo was sacrificed, parasites were found in the chorioallantoic membrane. The quail chicks were allowed to hatch. Between 10 and 5 15 days of age the chicks were orally inoculated with 1 x 10 oocysts of E.tenella (Houghton). A total of 11 birds were treated in this manner; no development of the parasite was 197

detected.

5) Site specificity of E. tenella in chickens

Thirty-three chicks were hatched from eggs which had been 4 inoculated on the 10th day of incubation with 1 x 10 sporozoites of E.tenella (Houghton). Forty-one control chicks which received only penicillin/streptomycin in saline on the 10th day of incubation were hatched in the same incubator. All the 4 birds were orally inoculated with 4 x 10 oocysts of E.tenella (Houghton) at ten days of age. Differences in mortality between the two groups was slight; a total of 61% (23 out of 33) of the 'tolerant' birds, those which had been exposed to infection as embryos, survived, compared to 68% (28 out of 41) control birds.

Examination of five 'tolerant' and five control birds, which had received 1 x 106 oocysts at 14 days of age, on the 5th day after infection, revealed extensive haemorrhage in the caeca and lower small intestine. Subsequent examination showed second generation schizonts in the lower small intestinal tissues in both control and 'tolerant' groups.

Six 'tolerant' chicks were also inoculated intraperiton- 6 eally and intravenously with 1 x 10 oocysts at 14 days of age. On the fifth day the birds were examined; extensive haemorrhage occurred in the caeca and lower intestine. In the liver of one 'tolerant' bird, which had received an intravenous inoculation, mature schizonts were observed (Fig. 31).

5 Parasites appeared to be confined to the caeca when 1 x 10 oocysts of E.tenella were inoculated intramuscularly, intravenously and intraperitoneally into batches of four normal 14-day- old chicks. Haemorrhage in the small intestine was noticed, 6 at 5 days afterintravenous inoculation of 1 x 10 oocysts into three normal 14-day-old chicks.

6 Intravenous inoculation of 1 x 10 sporozoites resulted in a normal infection in the caeca, but no parasitic stages 6 were obtained when 2 x 10 merozoites were inoculated intravenously or intraperitoneally. 198

Figure Thirty-one

Schizonts of E.ter2(Houghton) in the liver of a 19-day-old 'tolerant' chicken

10 ),_i.m 199

6) Cytochemistry

Parasites in cell monolayers lend themselves to cytochemical investigations far more readily than the parasite in vivo. Attempts were made primarily to determine whether sporozoites, which had been observed in cells in culture for up to 4 days without undergoing development, were metabolically active and whether parasitised cells differed from uninfected cells.

Monolayer chicken kidney cells were treated as unfixed preparations. Enzyme activity was lost in cells fixed in glutaraldehyde and cold acetone.

No activity was detectNifor ASAT or LAP. Positive reactions were given by sporozoites, trophozoites in vitro and merozoites produced in vitro and in vivo for LDH and G6PDH (Figs. 32,33). Discrete formazan granules were formed within cells. Although formazan deposits eventually formed in controls, consisting of infected monolayer kidney cell cultures incubated in the staining solution without the substrate, slides were examined after 25 minutes incubation at which time all controls were negative. Heat treated preparations. always gave a negative result.

LDH was particularly active in both the host cell and intracellular parasite; activity first became apparent in the parasite (Fig. 32b).

A less satisfactory result was obtained for SDH, no clear distinction could be made between the reactions of cells incubated with and without the substrate.

Sporozoites located within cells three days after the infection of the culture were found to give a positive reaction for LDH and G6PDH (Fig. 34).

A large mass, within sporozoites, presumably the refractile body, was stained using the LDH and G6PDH enzyme assay solutions. This structure became rounded and tended to decrease in size after the sporozoites had invaded: occasionally in intracellular sporozoites it was not visible. 200

Figure Thirty-two

Trophozoites of E.tenella (Houghton) in chicken kidney cells stained for LDH

(a) 24 hrs after infection, 30 mins. staining

141 4. 0011714," 00 a sae .ivel) %air 9* • • • 10. me • , s • 1. ?iriale4a0

.41 44 f. •• A; • 1111 •11:• • giV • 70 • r .• 10.- 14,1. 3 .44.0%* ,4 :44 gr 11-. • r •

(b) 48 hrs after infection, 15 mins. staining

(a) Trophozoite of E.terieNa (Houghton) in chicken kidney cell stained for G6PDH

At r.t • Aft.* rt •- 4, ,r .0 0 4 1 . 'tit ,1;111 •I . 61144. . • $ .? ) .0 • 4.41111 ce 4,-.... ' -

(b) Second generation schizont, from caecum of chicken stained for G6PDH 202 Figure Thirty-four

Sporozoites of E.tenella (Houghton) stained for LDH and G6PDH

(a) Recently excysted - LDH

(b) Intracellular sporozoite, 3 days after inoculation onto chicken kidney cell culture - LDH

-11414414. .Y44,14 • •I . 4 ;,,,,,..." it idip NO . • a, , , • 0„,.t ,

No differences in LDH and.G6PDH activity was observed between infected and uninfected cells.

Preliminary results indicated that sporozoites stored at 4°C deteriorated more rapidly in phosphate buffered 1% saline than in HBSS. After three days, 71% of the remaining sporozoites in the saline gave a positive reaction for G6PDH compared to 95% in HBSS. 204

DISCUSSION

Avian embryos and cells in vitro provide environments which are unsuitable for the development of certain eimeriine parasites but which are at least partially satisfactory for others. Characters can be selected for which improve the ability of the parasite to develop in unnatural conditions: lines of E. tenella and E. acervulina var.mivati have been produced which actually develop better in chicken embryos than in chickens (Long 1972a,b). Variations in the abilities of populations within a species to develop in culture, besides emphasising the importance of the parasite genotype, suggest that the factors hindering the development of parasites in culture might be few. Further experimentation with in vitro culture techniques will, probably, help to elucidate the stringent requirements of different Eimeria spp. As yet, no subtle molecular differences between closely related parasites are known which could account for the extraordinary variations in behaviour observed in culture.

Excystation is a non-specific phenomenon (see Marquardt 1973). Sporozoites can be obtained from oocysts in vitro using standardtechniques; excystation of E. tenella occurred in quail an abnormal host.

Sporozoite invasion of cells, at least of E. tenella is non-specific. Sporozoites of this species have entered cells of pheasant, duck, quail and chicken origin. Observations of sporozoites within insect cells suggest a total lack of specificity, although phagocytosis by the cells must not be excluded. Vetterling (1976) found sporozoites in the lamnia propria of caeca of turkey,chukar, peafowl and guinea fowl, four hours after inoculation of oocysts. of E. tenella; Haberkorn (1970b) observed stages of E. tenella in the intestines of mice.

Intracellular sporozoites do not always commence development. Fayer and Hammond (1967) thought that sporozoites of E. bovis survived within cultured bovine cells for 17 days-; 205

sporozoites of E. auburnensis have been observed on the 18th day (Clark and Hammond 1969). Long and Millard(1968) showed that sporozoites of E. tenella in the crypts of LieberkUhn could survive for longer than 60 days in chickens treated with metachlorpindol.

E. bateri was found, in this study, to invade chicken and quail cells in vitro but no development occurred., Sporozoites of E. tenella have been observed within cells in culture four days after their inoculation while normal development of parasites took place in adjacent cells. Cytochemical tests on latent sporozoites in culture indicated that they were metabolically active and hence viable. Sporozoites of E. tenella were observed in the caecal tissues of quail but did not develop; it would be of interest to attempt to infect chickens with caecal homogenates of quail, which had been exposed to E. tenella, to test whether the sporozoites were still capable of producing an infection.

The different behaviour of sporozoites in culture is puzzling and requires explanation. It could be speculated that the energy stores become so depleted in some sporozoites that any future development is hindered. Vetterling and Doran (1969) observed that during a 30 minute period of excystation,at 42.9°C, carbohydrate reserves in the sporozoites of three species of avian coccidia fell by about two thirds. The fall in carbohydrate content of E. acervulina sporozoites was correlated with a decline in infectivity which may have been caused by the inability of the sporozoites to penetrate host cells after excystation. However, intracellular sporozoites are probably capable of utilising the energy resources of the host cell; the G6PDH and LDH activity of latent sporozoites was reasonably strong.

Long (1973c) pointed out that insufficient attention has been directed to determine the most useful source of serum in the culture medium and whether the serum used, usually calf serum, contains a high titer of natural antibodies to Eimeria. It is possible that sporozoites are damaged or altered before penetration so that they are subsequently incapable of development. 206

Differences in the behaviour of sporozoites within an inoculum could be accounted for by the population being heterogenous, only some members possessing the necessary attributes for development. Alternatively, as kidney cell cultures are themselves heterogenous with regard to cell types, it is possible that an invaded host cell may not be suitable for the development of the sporozoite, which may require some stimulus from the cell before it can proceed with development. Merozoites produced in culture also gave a positive reaction for LDH and G6PDH. Although this reflects their viability, as with sporozoites, it is not necessarily an indication of their capability for future development. Doran (1974) did not obtain development of culture-produced merozoites when he introduced them into fresh cultures.

The complete development of E. tenella (Houghton) in pheasant and quail embryos and the development at least as far as second generation schizonts in duck embryos indicates that host specificity in embryos is not as rigid as in young birds. Var- iations in the development of the parasite in the embryos of the different species appear to reflect the systematic positions of the birds to the domestic fowl. Of the birds examined the pheasant is considered the closest ancestral relative (Vetterling 1976) and it was in the pheasant embryo that development was most successful. The quail, pheasant and domestic fowl all belong to the sub-family Phasianinae, whereas the duck, in the embryo of which only limited asexual development was detected, belongs to a different order, the Anseriformes. Despite the development of E. tenella (Houghton) in embryos and cell cultures of Coturnix cotumix japonica, the rigid barr- ier of the young birds to this parasite could not be broken. It seemed reasonable to assume that the differences in the susceptibility of quail chicks and embryos to infection was a reflection of their immunological status. However, attempts to.alter the immunological response of the quail, by corticosteroid treatment and by the induction of tolerance. did not result in an infection. It may be that the immunological response was not sufficiently altered in these experiments. Whether the gross physical conditions within the quail caeca, such as temperature and pH were vastly different from those 207

within the chicken was not determined.

Immune tolerance is a term used to indicate a state in which an individual is unable to respond to specific antigen. Toler- ance to a large variety of antigens can be induced when the antigens are injected neonatally. Immunological tolerance has been induced, in chicken embryos to certain bacterial and viral antigens (Burton 1954; Friedman and Gaby 1960; Hanson and Cannefox 1967; Rubin et al. 1962 and Solomon 1968). It was suggested by Jeffers and Wagenbach (1970) that inoculation of embryonated eggs induced partial tolerance to E. tenella. If immunological tolerance could boinduced,a breakdown of site specificity might be expected. Chicks which had been immunosuppressed with dexamethasone were found to harbour E. tenella infections in their livers (Long 1970b); development of this parasite has also been reported in the liver of chick embryos (Long 1971). The present findings were inconclusive;

After inoculation with oocysts of E.tenella no differences were found in mortality between normal chicks and chicks which had been exposed to the parasite as embryos but, in the liver of one 'tolerant' bird second generation schizonts were found. Although the apparent absence of a cellular reaction around the parasites suggested a much reduced immunological response, further investigations would be necessary before definitive statements could be made. In a more comprehensive survey, Jeffers and Wagenbach (1970) found that mortality was greater in the chicks which had been exposed to the developing parasite. To establish whether immunological tolerance can -be induced, antibody production should be monitored. Specific antibodies are known to be produced in normal chickens infected with E. tertella (see Rose 1973; Movsesijan et al. 1975) ; if birds were tolerant to the parasite the antibody levels would be lower and the birds would be expected to develop little immunity.

Tyzzer (1929) reported that E..mtella infection of the chicken is normally limited to the caeca but may occasionally involve parts of the small and large intestine adjacent to the caecal junction. Leathem (1969) re-examined the tissue specificity of E.tenella and made studies of the parasite in caecectomised chickens; he confirmed the involvement of the 208 small and large intestines. In the presentstudyl using a strain accurately defined by biochemical methods as E. tenella, second generation schizonts, which caused tissue damage and haemorrhage,were located in the lower regions of the ileum of birds which received large doses of oocysts. Whether stages of the parasite occur in the small intestinal tissues after smaller inoculations and remain undetected is not known. It is a frequent observation that large infections of Eimeria spp result in the spreading of the organisms over a greater length of the intestine (Marquardt 1973): the reasons that the parasites do not normally develop over such a wide range is obscure.

That site specificity is strong is further indicated by the results of parenteral inoculations: the parasites being found in the same site as if the birds had been exposed by mouth. Although the mechanisms by which the parasites reach their normal site are not known,observations by Haberkorn (1971 b) on E. falciformis indicated that the oocysts were trapped in the liver and reached the small intestine via the bile duct. El-Kasby and Sykes (1973) reported the role of macrophages in the excystation of Eimeria acervulina after parenteral infection. It has also been suggested that macrophages are involved in the normal transport of sporozoites of E. necatrix and E. tenella from the epithelium of the villi to the glandular epithelia of the digestive tract (Van Doornink and Becker 1957; Pattillo 1959; Challey and Burns 1959).

The questions posed by the specificity shown by Eimeria spp are intriguing; only by the accumulation of observations on the intricate host-parasite relationship will some of the answers be .found. Neither the parasite nor the host should be considered in isolation; the host-parasite relationships that have evolved are based on numerous interactions. 209

REFERENCES

ADAM, K.M.G., BLEWETT, D.A. and FLAMM, W.G. 1969. The DNA of Acanthamoeba spp; a method for extraction and its characterisation. J.Protozool. 16 (1) : 6-12.

AKINYEMI, J.A. 1975. Rat liver alcohol dehydrogenase isoenzymes: influence of infection with Trypanosoma. Z. Parasitenkde 46 : 125-131.

ALLEN, S.L. 1959. Strain differences in the esterases of Tetrahymena. Anat. Record. 134 : 524-525.

ALLEN, S.L. 1960. Inherited variation in the esterases of Tetrahymena. Genetics. 45 : 1051-1070.

ALLEN, S.L. 1965. Genetic control of enzymes in Tetrahymena.Brookhaven Symposia in Biology. 18 : 27-54.

ALLEN, S.L., BYRNE, B.C. and CRANLITE, D.L. 1971. Intersyngenic variations in the esterases of bacterised Paramecium aurelia.Biochem. Genet. 5 : 135-150.

ALLEN, S.L. and GIBSON, I. 1971a. Intersyngenic variationsin the esterases of axenic stocks of Paramecium aurelia. Biochem. Genet. 5 : 161-181.

ALLEN, S.L. and GIBSON, I. 1971b. The purification of DNA from the genomes of Paramecium aurelia and Tetrahymena pyriformis. J. Protozool. 18 (3) : 518-525.

ALLEN, S.L.,MISCH, C.S. and MORRISON, B.M. 1963. Variations in the electrophoretically separated acid phosphatases of Tetrahymena. J. Histochem. Cytochem. 11 : 706-719.

ALLEN, S.L. and WEREMIUK, S.L. 1971. Intersyngenic variations in the esterases and acid phosphatases of Tetrahymena pyriformis. Biochem. Genet. 5 : 119.

ARNASTAUSKEINE, T.V. and KADYTE, B.A. 1974. Activity of alkaline phosphatases in the blood and intestines of chickens with experimental cecal coccidiosis. Proc. 3rd Int. Cong. Parasit. Munich. 3 : 1467.

AYALA, F.J., POWELL, J.R., TRACEY, M.L., MOURAO, C.A. and PERES-SALAS,S. 1972. Enzyme variability in the Drosophila willistoni group IV. Genic variation in natural populations of Drosophila willistoni. Genetics. 70 : 113-119.

BACCHI, G. 1965, Sex Determination. Pergamon Press Ltd.

BAGSTER, I.A. and PARR, C.W. 1973. Trypanosome identification by electrophoresis of soluble enzymes. Nature Lond. 244 : 364-366.

BALL, S.J. 1966. The development of resistance to glycarbylamide and 2-chloro-4-nitro-benzomide in Eimeria tenella in chicks. Parasit- ology. 56 : 25-37. 210

BAYNE, R.A. and ROBERTS, J.F. 1969. Activities and isozymes of malate and lactate dehydrogenases in culture and bloodstream from trypanosomes. Comp. Biochem and Physiol. 29 : 731-741.

BECKER, E.R. 1934. Infection with a single coccidian oocyst and its significance. Am. Nat. 68 : 571-574

BECKER, E.R., JESSEN, R.J.,PATTILLO, W.H. and VAN DOORNINCK, W.H. 1956. A biometrical study of the oocyst of Eimeria necatrix, a parasite of the common fowl. J. Protozool. 3 : 126-131.

BECKER, E.R., ZIMMERMANN, W.J. and PATTILLO,W.H. 1955. A biometrical study of the oocyst of Eimeria brunetti, a parasite of the common fowl. J. Protozool. 2 : 1455-150.

BEDRNIK, P. 1969. Some results and problems of cultivation of Eimeria tenella in tissue cultures. Acta. Vet. (Brno). 38 : 31-35.

BEYER, T.V. 1965. Analysis of the metabolism of different developmental stages of rabbit intestinal coccidia. Progress in Protozoology: p. 159. 2nd Int. Cong. Protozool., London. Int. Cong. Series No.91.

BEYER, T.V. 1970. Coccidia of domestic animals: some metabolic peculiarities of particular stages of the life cycle. Proc. 2nd Int. Cong. Parasit. J. Parasit. 56 : 28_

BIDE, R.W. and DORWOOD, W. J. 1970. Plasma alkaline phosphatase in the Fowl: changes with starvation. Poult.Sci. 49 : 708-713.

BISHOP, A. 1958. An analysis of the development of resistance to meta- chloridine in clones of Plasmodium gallinaceum. Parasitology 48 : 210-234.

BORDEN, D., MILLER, E.T., NANNEY, D.L. and WHITT, G.S. 1973a. The inheritance of enzyme variants for tyrosine aminotransferase, NADP- dependent malate dehydrogenase, NADP-dependent isocitrate dehydrogenase and tetrazolium oxidase in Tetrahymena pyriformis, Syngen 1. Genetics, 74 : 595-603.

BORDEN, D., WHITT, G.S. and NANNEY, D.L. 1973b. Isozymic heterogeneity in Tetrahymena strains. Science 181 : 279-280.

BORDEN, D., WHITT, G.S. and NANNEY, D.L. 1973c. Electrophoretic characterisation of classical Tetrahymena pyriformis strains. J. Protozool. 20 : 693-700.

BOURNS, T.K.R. 1974. Protozoa and parasitic helminths. In Biochemical and Immunological Taxonomy of Animals, ed. C.A. Wright : 387-395. London: Academic Press.

BRACKETT, S. and BLIZNICK, A. 1952. The reproductive potential of five species of coccidia of the chicken as demonstrated by oocyst production. J. Parasit. 38 : 133-139.

BRAY, R. S. and GARNHAM, P.C.C. 1962. The Giemsa collophonium method for staining Protozoa in tissue sections. Indian J. Malar. 16 : 153-155.

BRUGEROLLE, G. and METENIER, G. 1973. Localisation intracellulaire et characterisation de deux types de malate dehydrogenase chez Trichomonas vaginalis,Donne 1836. J. Protozool. 20 (2) : 320-327. 211

CHANCE, M.L. 1972. DNA base composition differences between species of Leishmania. Trans. R. Soc. trop.Med.Hyg. 67 : 24-25.

CHANCE, M.L., PETERS, W. and GRIFFITHS, M.W. 1973. A comparative study of DNA in the genus Leishmania. Trans. R.Soc.trop.Med.Hyg. 67 : 24-25

CHANCE, M.L., PETERS, W. and SHCHORY,L. 1974. Biochemical taxonomy of Leishmania. (1) Observations on DNA. Ann.trop.Med.Parasit. 68 (3) : 307-316

CHANCE, M.L. and WARHURST, D.C. 1973. DNA relationships in the subgenus Vinckeia. J. Protozool. 20,(4). Abs. 97 : 524.

CHUTE, H.L., ZARKOWER, A., O'MEARA, D.C. and WITTER, R.L. 1969. Acid and alkaline phosphatase levels in coccidiosis infected chickens. Avian Diseases. 5 : 107-116.

CLARK, G.W. and COLWELL, D.A. 1974. Eimeria dalli sp.n. (Protozoa: Eimeriidae) from Dall sheep Ovis dalli. J. Protozool. 21 (2) : 197-199.

CLARK, W.N. and HAMMOND, D.M. 1969. Development of Eimeria auburnensis in cell cultures. J. Protozool. 16 : 646-654.

CLERK, G. 1973. Staining procedures used by the Biological Stain Commission. 3rd ed. Williams and Wilkins Co. Baltimore.

CLEVELAND, L.R. 1949. Hormone induced sexual cycles of Flagellates.I. Gametogenesis, fertilisation and meiosis in Trichonympha. J. Morph. 85 : 197-296.

CORBETT, J.J. 1973. LDH isozyme variation during the populatior cycle in Tetrahymena pyriformis. J. Protozool. 20 (4).Ab.68 : 514.

CORDERO del CAMPILLO, M. 1959. Estudios sobre Eimeria falciformis (Eimer 1870) parasito del raton. An.Fac.Vet.Leon. 4 : 55-73.

COURTNEY, H., FORRESTER, D.J., ERNST, J.V. and NESBITT, J.A. 1975. Coccidia of Sandhill Cranes, Grus canadensis. J. Parasit. 61 : 695-699.

CUCKLER, A.C., MCMANUS, E.C. and CAMPBELL, W.C. 1969. Development of resistance in Coccidia. Acta Vet. (Brno). 38 : 87-99.

DAVIES, B.J. 1964. Disc electrophoresis - II. Method and application to human serum proteins. Ann. N.Y. Acad.Sci. 121 : 404-427.

DAVIES, J.F.M. and JOYNER, L.P. 1962. Infection of the fowl by the parenteral inoculation of oocysts of Eimeria.Nature. Lond. 194 : 996-997.

DAVIS, L.R., BOUGHTON, D.C. and BOWMAN, G. W. 1955. Biology and pathogenicity of Eimeria alabamensis (Christensen 1941), an intranuclear coccidkintof cattle. Amer.J.Vet.Res. 16 : 274-281.

DE VOS, A.J. 1970. Studies on the host range of Eimeria chinchillae, De Vos and Van der Westhuizen, 1968. Onderstepoort J. vet. Res. 37 : 29-36. DIEHL,E.J. and RISBY,E.L. 1974. Serum changes in rabbits experimentally infected with Trypanosoma gambiense. Am.J.Trop.Med.Hyg.23 :1019,1022. 212

BRYANT, E. 1974. On the adaptive significance of enzyme polymorphisms in relation to environmental variability. Amer. Natur. 108 : 1-19.

BURO, N.C. and WELER, D.L. 1974. Purification and characterisations of malic enzyme of Entamoeba invadens : evidence for isoenzymes. J. Protozool. 21(5) : 796-802.

BUXTON, A. 1954. Antibody production in avian embryos and young chicks. J. gen. Microbiol. 10 : 398-410.

CAMPBELL, W.C. and BARRY, T.A. 1970. A biochemical method for the detection of anthelminthic activity against liver fluke (Fasciola hepatica). J. Parasit. 56 : 325-331.

CANNING, E.U. 1962. Sexual differentiation of merozoites of Barrouxia schneideri (Butschli). Nature, Lond., 195 : 720-721.

CANNING, E.U. and ANWAR, M. 1968. Studies on meiotic division in coccidial and malarial parasites. J. Protozool. 15 : 290-298

CANNING, E.U. and MORGAN,K. 1975. DNA synthesis, reduction and elimination during life cycles of the Eimeriine Coccidian Eimeria tenella and the Haemogregarine, Hepatozoon domerguei Expl Parasit. 38 : 217-227

CARTER, R. 1970. Enzyme variation in Plasmodium berghei.Trans.R. Soc. trop. Med. Hyg. 64 : 401 -406.

CARTER, R. 1972. Electrophoretic forms of GPI in oocysts and blood parasites of Plasmodium berghei. Tran.R.Soc.trop Med. Hyg. 66 : 542

CARTER, R. 1973. Enzyme variation in Plasmodium berghei and Plasmodium vivax. Parasitology. 66 : 297-307.

CARTER, R. and MCGREGOR, I.A. 1973. Enzyme variation in Plasmodium falciparum in the Gambia. Trans.R. Soc. trop. Med. Hyg. 67 : 830-837.

CARTER, R. and VOLLER, A. 1973. Enzyme variants in primate malaria parasites. Trans. R. Soc. trop. Med.Hyg. 67 : 14-15

. CARTER, R. and VOLLER, A. 1975. The distribution of enzyme variation in populations of Plasmodium falciparum in Africa. Trans.R.Soc. trop.Med.Hyg. 69 : 371-376

CARTER, R. and WALLIKER, D. 1975. New observations on the malaria parasites of rodents of the Central Africa Republic: Plasmodium vinckei petteri subsp. nov. and Plasmodium chabaudi Landau, 1965. Ann.trop.Med. Parasit. 69 (2) : 187-196.

CATCHPOLE, J., NORTON, C.C. and JOYNER, L.P. 1975. The occurrence of Eimeria weybridgensis and other species of coccidia in lambs in England and Wales. Br.vet.J. 131 : 392-401.

CHALLEY, J.R. and BURNS, W.C. 1959. The invasion of the caecal mucosa by Eimeria tenella sporozoites and their transport by macrophages. J. Protozool. 6 : 238-241.

CHAMPION, L.R. 1954. The inheritance of resistance to caecal coccidiosis in the domestic fowl. Poult. Sci. 33 : 670-681. 213

DIKOVSKAYA, V.E. 1974. Intraspecific variability of Eimeria tenella. Parazitologiya 8 (b) : 548-552.

DOBELL, C. 1922. The discovery of the coccidia. Parasitology 14 : 342-347.

DOORIS, P.M. and MCGHEE, R.B. 1976. Immunologic and electrophoretic characteristics of two species of Crithidia. J. Protozool. 23 (3) : 433-437.

DORAN, D.J. 1953. Coccidiosis in the kangaroo rats of California. Univ. Calif. Pubis. Zool. 59 : 31 -60.

DORAN, D.J. 1970. Eimeria tenella: From sporozoites to oocysts in cell culture. Proc. helminth. Soc. Wash. 37 : 84-92.

DORAN, D.J. 1971a. Increasing the yield of Eimeria tenella oocysts in cell culture. J. Parasit. 57 : 891 -900.

DORAN, D.J. 1971b. Survival and development of five species of chicken coccidia in primary chicken kidney cell cultures. J. Parasit.: 1135-1137.

DORAN, D.J. 1971c. Comparative development of Eimeria tenella int primary cultures of kidney cells from the chicken, pheasant, partridge and turkey. J. Parasit. 57 : 1376-1377

DORAN, D.J. 1973. Cultivation of coccidia in avian ambryos and cell cultures. In D.M. Hammond with P.L. Long (eds.). The Coccidi, Univ. Park.Press. Baltimore : 45-80.

DORAN, D.J. 1974. Eimeria tenella : Merozoite production in cultured. cells and attempts to obtain development of culture-produced merozoites. Proc.Helm.Soc. Wash. 41 (2) : 169-173.

DORAN, D.J. and AUGUSTINE, P.C. 1973. Comparative development of Eimeria tenella from sporozoites to oocysts in primary kidney cell cultures- from gallinaceous birds. J. Protozool. 20 (5) : 658-661

DORAN, D.J., VETTERLING, J.M. and AUGUSTINE, P.C. 1974. Eimeria tenella : an in vivo and in vitro comparison of the Winsconsin, Weybridge and Bellsville strains. Proc.helminth.Soc.Wash. 41 : 77-79.

DUBBS, C.A. 1966. Ultrasonic effects on isoenzymes. Clin.Chem. 12 : 181 -186.

DUBEY, J.P. 1976. Reshedding of Toxoplasma oocysts by chronically infected cats. Nature, Lond. 262 : 213-214.

DUSZYNSKI,D.W. 1971. Increase in size of Eimeria separata oocysts during patency. J. Parasit. 57 : 948-952.

DUSZYNSKI,D.W. and MARQUARDT, W.C. 1969. Eimeria (Protozoa : Eimeriidae) of the cotton tail rabbit Sylvilagus audobonii in north eastern Colorado with descriptions of three new species. J. Protozool. 16 : 128-137.

EBERT, F. 1973. Charakterisierung von L. donovani - Stamen mit der Disk-Elektrophorese. Tropenmed. Parasit. 24 : 517-524. 2114

EBERT, F. 1974a. Elektrophoretische Untersuchungen an Leishmania-tropica- Stgmmen. Tropenmed. Parasit. 25 : 49-53.

EBERT, F. 1974b. Vergleichende elektrophoretische Untersuchungen an Erreger- Stgmmen der cutanen Leishmaniase der Neuen Welt und ihre Beziehurigen zu Leishmania donovani und L.tropica. Tropenmed. Parasit. 25 : 259-266.

EDGAR, S.A. and SEIBOLD, C.T. 1964. A new coccidium of chickens, Eimeria mivati sp.n. (Protozoa Eimeriidae) with details of its life history. J. Parasit. 50 : 193-204.

EDWARDS, A.J., BURT, J.S. and OLGIVIE, B.M. 1971.' The effect of immunity upon some enzymes of the parasitic nematode, Nippostrongylus brasiliensis. Parasitology 62 : 339.

EIMER, T. 1870. Ueber die ei-oder kugelformigen sogenannten Psorospermium der Wirbeltiere. Wurzburg. 58.

EL-KASBY, A and SYKES, A.H. 1973. The role of chicken macrophages in the parenteral excystation of Eimeria acervulina. Parasitology 66 : 231- 239.

FAIR, D.S. and KRASSNER, S.M. 1971. Alanine aminotransferase and aspartate aminotransferase in Leishmania tarentolae. J. Protozool. 18 (3) : 441 -444.

FARR, M.M. 1953. Three new species of coccidia from the Canada goose, Branta canadensis (Linné 1758). J. Wash. Acad.Sci. 43 (10) : 336-340.

FAYER, R. and HAMMOND, D.M. 1967. Development of first-generation schizonts of Eimeria bovis in cultured bovine cells. J. Protozool. 14 : 764-772.

FERNANDO, M.A. and REMMLER, 0. 1973a. Four new species of Eimeria and one of Tyzzeria from the Ceylon jungle fowl, Gallus lafayetti. J. Proto- zool. 20 : 43-45.

FERNANDO, M.A. and REMMLER, 0, 1973b. Eimeria diminuta sp.n. from the Ceylon jungle fowl Gallus lafayetti. J. Protozool. 20 : 357

FISH, F.F. 1931. Quantitative and statistical analyses of infections with Eimeria tenella in chickens. Am.J.Hyg. 14 : 560-576.

FITZEGERALD, P.R. 1965. The results of parenteral injections of sporulated or unsporulated oocysts in calves. J. Protozool. 12 : 215-221

FITZEGERALD, P.R. 1970. Development of Eimeria stiedae in avian embryos. J. Parasit. 56 : 1252-1253.

FOWLER, J.F. and REEVES, E.L. 1974a. Detection of relationships among Microsporidian isolates by electrophoretic analysis : hydrophobic extracts. J. Invertebr. Pathol. 23 : 3-12.

FOWLER, J.F. and REEVES, E.L. 1974b. Detection of relationships among Microsporidian isolates by electrophoretic analysis : hyrophilic extracts. J. Invertebr. Pathol. 23 : 63-69

FOWLER, J.F. and REEVES, E.L. 1974c. Spore dimorphism in a Microsporidian isolate. J. Protozool. 21 (4) : 538-542. 215

FRANDSEN, J.C. 1968. Eimeria stiedae : Cytochemical identification of acid and alkaline phosphatases, carboxylic ester hydrolases, and succinate lactate, and glucose-6-phosphate dehydrogenases in endogenous stages from rabbit tissues. Expl. Parasit. 23 : 398-411.

FRANDSEN, J.C. and COOPER, J.A. 1972. Enzymes of coccidia. Purification and properties of L-lactate dehydrogenase from Eimeria stiedae. Expl. Parasit. 32 (3) : 390-402.

FRANDSEN, J.C. and ENNIS, T.H. 1974. Properties of glucose-6-phosphate dehydrogenase from Eimeria stiedae (Protozoa: Coccidia). J. Protozool. 21 (3) : 433.

FRENKEL, J.K. 1973.. Toxoplasmosis: parasite life _cycle, pathology and immunology. In D.M. Hammond with P.L. Long (eds.). The Coccidia. Univ. Park Press. Baltimore.

FRIEDMAN, H. and GABY, W.L. 1960. Immunological unresponsiveness to Shigella antigens in chickens. J. Immun. 84 : 441-448.

GARDENER, P.J. and HOWELLS, R.E. 1972. Isoenzyme variation in leishmanial parasites. J. Protozool. 19 (Suppl) : 47.

GARDENER, P.J., CHANCE, M.L. and PETERS, W. 1974. Biochemical taxonomy of Leishmania. II Electrophoretic variation of malate dehydrogenase. Ann. trop.Med.Parasit. 68 (3) : 313-325.

GARDINER, J.L. and MCLOUGHLIN, D.K. 1963. Drug resistance in Eimeria, tenella III Stability of resistance to Glycarbylamide. J.Parasit. 49 : 657-659.

GELDERMAN, A.H., KEISTER, D.B., BARTGIS, I.L. and DIAMOND, L.S. 1971a. Characterisation of the deoxyribonucleic acid of representative strains of Entamoeba histolytica, E. histolytica-like amoebae and E. moshkovskii. J. Parasit. 57 : 906-911.

GELDERMAN, A.H., BARTGIS, I.L., KEISTER, D.B. and DIAMOND, L.S. 1971b. A comparison of genome sizes and thermal-denaturation derived base composition of DNA's from several members of Entamoeba (histolytica group). J. Parasit. 57 (4) : 912-916.

GODFREY, D.G. and KILGOUR, V. 1972. The antigenicity of the alanine aminotransferase of Trypanosoma brucei brucei. Trans.R.Soc.trop. Med.Hyg. 66 :.351-352.

GODFREY, D.G. and KILGOUR, V. 1973. The relative activities of alanine and aspartate aminotransferases in bloodstream trypanosomes. Trans. R.Soc.trop.Med.Hyg. 67 : 260.

GOURLAY, R.N. BROCKELSBY, D.W. and SELWOOD, S.A. 1970. Disc electrophoresis as an aid to the identification of Piroplasms. Vet.Rec. : 629-630.

GRELL, K.G. 1953. Entwicklung and Geschlechtsbestimmung von Eucoccidium dinophili. Arch. Protistenk. : 156-186.

HABERKORN, A. 1970a. Die Entwicklung von Eimeria falciformis (Eimer,1870) in der weil3en Maus (Mus musculus). Z. Parasitenkde 34 : 49-67. 216

HABERKORN, A. 1970b. Zur EmpfUnglichkeit nicht spezifischer Wirte fUr Schizogonie-Stadien verschiedener Eimeria-Arten. Z. Parasitenkde. 35 : 156-161.

HABERKORN, A. 1971a. Zur Wirtsspezifit5t von Eimeria contorta n.sp, (Sporozoa : Eimeriidae). Z. Parasitenkde 37 : 303-314.

HABERKORN, A. 1971b. The problem of host specificity and variability in the pathogenic behaviour of coccidia. Vet. Med.Rev.1971 (2/3) : 341-348.

HADLEY, P.B. 1911. Eimeria avium. A morphological study. Archiv. f. Protistenk. 23 : 7.

HAMMOND, D.M. 1973. Life cycles and development of coccidia. In D.M. Hammond with P.L. Long (eds.) The Coccidia. Univ.Park Press, Baltimore : 45-80.

HANSON, A.W. and CANNEFAX, G.R. 1967. Development of immune tolerance in the chick embryo to Borrelia hispanica. J. Bact. 94 : 1359-1365.

HEIN, H. 1971. The effects of cross infections with Eimeria brunetti in chickens immunised with multiple doses of Eimeria maxima. Exp. Parasit. 22 : 12-18.

HENRY, D.P. 1932. Coccidiosis of the guinea pig. Univ. Calif.Publ. Zool. 37 : 211 -268.

HORTON-SMITH, C. and LONG, P.L. 1965. The development of Eimeria necatrix, Johnson, 1930, and Eimeria brunetti, Levine, 191 2 in the caeca of the fowl (Gallus domestica). Parasitology. 55 : 401 -405.

HORTON-SMITH, C. and LONG, P.L. 1966. The fate of sporozoites of Eimeria acervulina, E. maxima and E. mivati in the caeca of fowl. Parasitology 56 : 569-574.

HOWELLS, R. E. and MAXWELL, L. 1973a. Further studies on the mitochon- drial changes during the life cycle of P. berghei : electrophoretic studies on isocitrate dehyrdogenase. Ann.trop.Med.Parasit. 67 (3) : 279-283.

HOWELLS, R.E. and MAXWELL, L. 1973b. Citric acid cycle activity and chloroquine resistance in rodent malaria parasites: the role of the reticulocyte. Ann.trop.Med.Parasit. 67 (3) : 285-300.

HUNTER, R.L. and MARKERT, C.L. 1957. Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels. Science 125 : 1294-1295.

ITAGAKI, K., TSUBAKURA, M. and TAIRA Y. 1972. Basic biological studies on avian coccidium development of Eimeria tenella, E. brunetti, and E. acervulina in chick embryos. Jap.J.vet.Sci. 34 : 143-149.

JARUMILINTA, R. and MAEGRAITH, B.G. 1961. The patterns of some proteo- lytic enzymes of Entamoeba histolytica and Acanthamoeba sp. II. The action of E. histolytica and Acanthamoeba sp on various syn- thetic substrates. Ann.trop. Med. Parasitol. 55 : 518-528. 217

JEFFERS, T. K. 1974. Eimeria tenella : Incidence, distribution and anticoccidial drug resistance of isolates in major broiler-producing areas. Avian Dis. 18 : 74-84.

JEFFERS, T. K. 1975. Attenuation of Eimeria tenella through selection for precociousness. J. Parasit. 61 : 1083-1090.

JEFFERS, T. K. and WAGENBACH, G. E. 1970. Embryonic response.to Eimeria tenella infection. J. Parasit. 56 : 656-662.

JOHNSON, W. T. 1923. Avian coccidiosis. Poult. Sci. 2 (5) : 146.

JOHNSON, W. T. 1924. Eimeria avium and the diagnosis of avian coccidiosis. Poult. Sci. 3 (2) : 41.

JOHNSON, W. T. 1930. Coccidiosis of the chicken with special reference to species. Stn.Bull.Ore.agric.Exp.Stn. 358 : 3-33.

JONES, E. E. 1932. Size as a species characteristic in coccidia. Variat- ions under diverse conditions of infections. Arch.Profistenke. 76 : 130-170.

JOSEPH, T. 1969. Eimerians occurring in or infective to both the fox squirrel Sciurus niger rufiventer and the grey squirrel S.carol- inensis. J. Protozool. 20 : 509.

JOYNER, L. P. 1969. Immunological variation between two strains of Eimeria acervulina. Parasitology. 59 : 725-732.

JOYNER, L. P. 1970. Coccidiosis : problems arising from the development of anticoccidial drug resistance. Expl.Parasit. 28 : 122-128.

JOYNER, L. P. and LONG, P. L. 1974. The specific characters of the Eimeria, with special reference to the coccidia of the fowl. Avian Path. 3 : 145-147.

JOYNER, L. P. and NORTON, C.C. 1969. A comparison of two laboratory strains of Eimeria tenella. Parasitology. 59 : 907-913.

JOYNER, L. P. and NORTON, C.C. 1972. The development of Eimeria acervulina in the caeca of young fowls. Parasitology 64 : 479-483.

JOYNER, L. P. and NORTON, C.C. 1975. Transferred drug-resistance in Eimeria maxima. Parasitology 71 : 385 - 392.

KARN, R. C. and HUDOCK, G. A. 1973. A photorepressible isozyme of malic enzyme in Euglena gracilis. Strain 2. J. Protozool. 20 : 316-320.

KATES, J. B. and GOLDSTEIN, L. 1964. A comparison of the protein composition of three species of Amoebae. J. Protozool. 11 (1) : 30-35.

KHEYSIN, Y. M. 1947a. Mutability of the oocysts of Eimeria magna. ' Zool.Zhurn. 26 : 17-29.

KHEYSIN, Y. M. 1947b. A new species of intestinal coccidia of the rabbit - E. coecicola. Dokl. ANSSR 55 : 181-183.

KHEYSIN, Y. M. 1957. Variability of the oocysts of Eimeria intestinalis - the parasite of the domestic rabbit. Vestn.LGU.ser.Biol. 2 : 43-52

KHEYSIN, Y. M. 1972. Life cycles of coccidia of domestic animals. Univer- sity Park Press ed. K.S. Todd. 218 KILGOUR, V., GARDENER, P.J., GODFREY, D.G. and PETERS, W. 1974. Demon- stration of electrophoretic variation of two aminotransferases in Leishmania. Ann.trop.Med.Parasit. 68 (2) : 245-246.

KILGOUR, V. and GODFREY, D.G. 1973. Species-characteristic isoenzymes of 2 aminotransferases in trypanosomes. Nature.Lond. 244 : 69-70.

KILGOUR, V., GODFREY, D.G. and BELLO, N.K. 1975. Isoenzymes of two aminotransferases among Trypanosoma vivax in Nigerian cattle. Ann.trop. Med. Parasit. 69 (3) : 329-335.

KILLICK-KENDRICK, R. 1974. Parasitic protozoa of the blood of rodents. A revision of Plasmodium berghei. Parasitology. 69 : 225-237.

KIMURA, M. 1969a. The rate of molecular evolution considered from the standpoint of population genetics. Proc.Nat.Acad.Sci. 63 : 1181-1188.

KIMURA, M. 1969b. The number of heterozygous nucleotide sites maintained in the finite population due to a steady flux of mutations. Genetics 61 : 893-903.

KING, J.L. and JUKES, T.H. 1969. Non-Darwinian evolution. Science 164 : 788-798.

KIRBY, K.S. 1965. Isolation and characterisation of ribosomal ribonucleic acid. Biochem. J. 96 : 266-269.

KLIMES, B., ROOTES, D.G. and TANIELIAN, Z 1972. Sexual differentiation of merozoites of Eimeria tenella. Parasitology, 65 : 131-136

KOGAN, Z.M. 1962. Variability of shape in oocysts of chicken coccidia and its biological significance. Zool.Zhurn 41 : 1317-1326.

KOGAN, Z.M. 1965. Variability of the oocysts of chicken coccidia Eimeria necatrix and factors which determine it. Zool. Zhurn 44 : 986-996.

KRASSNER, S.M. 1968. Isoenzymes in the culture forms of Leishmania tarentolae. J. Protozool. 15 : 523-528.

KUCERA, M. and WEISER, J. 1975. Lactate dehydrogenase isoenzymes in the larvae of Barathra brassicae and Galleria mellonella during Micro- sporidian infection. J.Invertebr.Pathol. 25 : 109-114.

LATNER, A.L. and SKILLEN, A.W. 1968. Isoenzymes in Biology and Medicine. Academic Press. London.

LAURENT, M. and STEINERT, M. 1970. Electron microscopy of kinetoplast in DNA from Trypanosoma mega. Proc.natn.Acad.Sci.U.S.A. 66 : 419-424.

LEATHEM, W.D. 1969. Tissue and organ specificity of Eimeria tenella (Railliet and Lucet, 1891) Fantham, 1909 in cecectomised chickens. J. Protozool. 16 : 223-226.

LEE, D.L. and LONG, P.L. 1972. An electron microscopal study of Eimeria tenella grown in the liver of the chick embryo. Int.J.Parasit. 2 : 55-58.

LEVINE, N. D. 1953. A review of the coccidia from the avian orders. Galliformes, Anseriformes and Charadriiformes, with descriptions of three new species. Am.Midl.Nat.Monogr. 49 : 696-719. 219

LEVINE, N.D. 1962. Protozoology today. J. Protozool. 9 : 1-16.

LEVINE, N.D. 1963. Coccidiosis. A Rev.Microbiol. 17 : 179-198.

LEVINE, N.D. 1973a. Introduction, history and taxonomy. In D.M. Hammond with P.L. Long eds., The Coccidia. Univ. Park Press, Baltimore : 1-22.

LEVINE, N.D. 1973b. Historical aspects of research on coccidiosis. In Proc.Symp. on Coccidia and related organisms. Guelph, Ontario, Canada : 1-10.

LEVINE, N.D. and IVENS, V. 1965. The coccidian parasites (Protozoa, Sporozoa) of rodents. Illinois biol.Monogr. No.33.

LEVINE, N.D. and IVENS, V. 1970. The coccidian parasites (Protozoa, Sporozoa) of ruminants. Illinois biol.Monogr. No.44.

LEVINE, P.P. 1938. Eimeria hagani n.sp. (Protozoa: Eimeriidae) a new coccidium of the chicken. Cornell Vet. 28 : 263-266.

LEVINE, P.P. 1942. A new coccidium pathogenic for chickens Eimeria brunetti n.sp. (Protozoa: Eimeriidae). Cornell.Vet. 32 : 430-439.

LEWONTIN, R.C. 1974. The Genetic basis of evolutionary change. Columbia Univ.Press.

LONG, P.L. 1965. Development of Eimeria tenella in avian embryos. Nature Lond. 208 : 509-510.

LONG, P.L. 1966. The growth of some species of Eimeria in avian embryos Parasitology 56 : 578-581.

LONG, P.L. 1968. The effect of breed of chickens on resistance to Eimeria infections. Brit. Poult.Sci. 9 : 71-78.

LONG, P.L. 1969. Observations on the growth of Eimeria tenella in cultured cells from the parasitised chorioallantoic membranes of the developing chick embryo. Parasitology 59 : 757-765.

LONG, P.L. 1970a. Some factors affecting the severity of infection with Eimeria tenella in chick embryos, Parasitology 60 : 435-447.

LONG, P.L. 1970b. Development (Schizogony) of Eimeria tenella in the liver of chickens treated with corticosteroid. Nature, Lond. 225 : 290-291.

LONG, P.L. 1971. Schizogony and gametogony of Eimeria tenella in the liver of chick embryos. J. Protozool. 18 (1) : 20-23.

LONG, P.L. 1972a. Eimeria tenella : Reproduction, pathogenicity and immunogenicity of a strain maintained in chick embryos by serial passage . J. comp.Path.Ther.82 : 429-437.

LONG, P.L. 1972b. Observations on the oocyst production and viability of E.mivati and E. tenella in the chorioallantois of chicken embryos incubated at different temperatures. Z.Parasitenkde 39 : 27-37.

LONG, P.L. 1972c. Eimeria mivati in chick embryos: Reproduction, pathogenicity and immunogenicity of a strain maintained in embryos by serial passage. J.comp.Path.Ther. 82 : 439-445. 220

LONG, P.L. 1973a. Studies on the relationship between Eimeria acervulina and Eimeria mivati. Parasitology 67: 143-155.

LONG, P.L. 1973b. Pathology and pathogenicity of coccidial infections. In D.M. Hammond with P.L. Long (eds.), The Coccidia. Univ.Park Press, Baltimore : 253-294.

LONG, P.L. 1973c. The growth of Eimeria in cultured cells and in chicken embryos: A review. In Procs.Symp. on Coccidia and Related Organisms. Guelph, Ontario, Canada : 57-80.

LONG, P.L. 1974a. Experimental infection of chickens with two species of Eimeria isolated from the Malaysian jungle fowl. Parasitology 69 : 337-3147.

LONG, P.L. 1974b. Further studies on the pathogenicity and immunogenicity of an embryo-adapted strain of Eimeria tenella. Avian Path. 3 (4) : 255-268.

LONG, P.L., FERNANDO, M.A. and REMMLER, O. 1974. Experimental infections of the domestic fowl with a variant of Eimeria praecox from the Ceylon jungle fowl. Parasitology, 69 : 1-9.

LONG, P.L. and MILLARD, B.J. 1968. Eimeria : Effect of metichlorpindol and methyl benzoquate on endogenous stages in the chicken. Expl. Parasit. 23 : 331 -338.

LONG, P.L. and MILLARD, B.J. 1973. Eimeria infection of chick embryos: the effect of known anticoccidial drugs against E.tenella and E. mivati. Avian Path. 2 : 111-125.

LONG, P.L. and MILLARD, B.J. 1975. Pathogenicity, immunogenicity and site specificity of an attenuated strain of Eimeria tenella. J. Protozool. 22 (53A) :. 154.

LONG, P.L. and MILLARD, B.J. 1976a. The detection of occult coccidial infections by inoculating chickens with corticosteroid drugs. Z.Parasitenkde. 48 : 287-290.

LONG, P.L. and MILLARD, B.J. 1976b. Studies on site finding and site specificity of Eimeria praecox, Eimeria maxima and Eimeria acervulina in chickens. Parasitology 73 (3) : 327-336.

LONG, P.L. and ROSE, M.E. 1970. Extended schizogony of Eimeria mivati in betamethasone treated chickens. Parasitology 60 : 147-155.

LONG, P.L. and ROWELL, J.G. 1958. Counting oocysts of chicken coccidia. Lab.Pract. 7 : 515-518.

LOPEZ, T. and MELTON, C.G. Jr. 1975. Cell volume and DNA content of Trypanosoma lewisi and Trypanosoma mega. J. Parasit. 61 : 209-212.

MANDEL, M. 1967. Nucleic acids of protozoa.in Florkin M and Scheer B.T. eds. Chemical Zoology. Academic Press, Inc. New York : 541-572.

MANDEL, M. and HONIGBERG, B.M. 1964. Isolation and characterisation of deoxyribonucleic acid of two species of Trichomonas Donné. J. Proto- zool. 11 : 114. 221

MARINCEK, M. 1965. Eimeria subepithelialis chez la carpe. In Progress in Protozoology. 2nd Int.Conf.Protozool. London. Abs : 160-1.62.

MARQUARDT, W.C. 1966. Attempted transmission of the rat coccidium Eimeria nieschulzi to mice. J. Parasit. 52 : 691-694.

MARQUARDT, W.C. 1975. Host and site specificity in the coccidia. In Hammond, D.M. with Long, P.L. eds. The Coccidia. Univ.Park Press Baltimore : 23-44.

MARQUARDT, W.C. 1976. Some problems of host and parasite interactions in the coccidia. J. Protozool. 23 (2) : 287-290.

MAYBERRY, L.F., and MARQUARDT, W.C. 1973. Transmission of Eimeria seperata from the normal host, Rattus, to the mouse Mus musculus. J. Parasit. 59 : 198-199.

MAYBERRY, L.F., PLAN, B., WASH, D.J., MARQUARDT, W.C. 1975. Genetic dependence of coccidial transmission from rat to mouse. J. Protozool. 22 : 28A.

MCDOUGALD, L.R. and JEFFERS, T.K. 1976. Eimeria tenella (Sporozoa, Cocci- dia) : Gametogony following a single asexual generation. Science 192 : 258-259.

MCLAREN, D.J. 1969. Observations on the fine structural changes associated with schizogony and gametogony in Eimeria tenella. Parasit- ology 59 : 563-574.

MCLOUGHLIN, D.K. 1969. The influence of dexamethasone on attempts to transmit Eimeria meleagrimitis to chickens and E. tenella to turkeys. J. Protozool. 16 : 145-148.

MCLOUGHLIN, O.K. 1970. Coccidiosis: experimental analysis of drug resistance. Expl. Parasit. 28 : 129-136.

MCLOUGHLIN, D.K. 1973. Pathogenesis and virulence-variation among avian coccidia. In. Procs.Symp on Coccidia and Related Organisms, Guelph, Ontario : 83-91.

MICHAEL, E. and HODGES, R.D. 1973. Enzyme cytochemical observations on the tissue stages of the life cycle of Eimeria acervulina and Eimeria necatrix. Int.J. Parasitol. 3 : 681-690.

MITCHELL, J.M., DARON, N.H. and FRANDSEN, J.C. 1976. Comparative properties of Eimeria stiedai and rabbit muscle aldolases. Fed.Proc.35 : 7.

MOLNAR, K. and HANECK, G. 1974. Seven new Eimeria spp. (Protozoa, Coccidia) from freshwater fishes of Canada. J. Protozool. 21 (4) : 489-493.

MOMEN, H. (1975). Enzyme variation in rodent piroplasms. Trans.R.Soc. trop.Med.Hyg. 69 : 478.

MOMEN, H., ATKINSON, E.M. and HOMEWOOD, C.M. 1975. An electrophoretic investigation of the malate dehydrogenase of mouse erythrocytes infected with Plasmodium berghei. Int.J. Biochem. 6 : 533-535.

MOMEN, H. and CHANCE, M.L. 1976. DNA buoyant density of rodent piroplasms. Trans.R.Soc.trop.Med.Hyg. 70 (1) : 13-14. 222 MONTVALO, F. and REEVES, R.E. 1968. Electrophoretic characterisation of ameba] glucose phosphate isomerases. Expl.Parasit. 22 : 129-136.

MOORE, E.N. and BROWN, J.A. 1951. A new coccidium pathogenic for turkeys, Eimeria adenoides n.sp. (Protozoa: Eimeriidae). Cornell.Vet. 41 : 124-125.

MOORE, E.N. and BROWN, J.A. 1952. A new coccidium of turkeys, Eimeria innocua n.sp. (Protozoa: Eimeriidae). Cornell.Vet. 42 : 395-402.

MOORE, E.N., BROWN, J.A. and CARTER, R.D. 1954. A new coccidium of turkeys : Eimeria subrotunda n.sp. (Protozoa: Eimeriidae). Poult. Sci.`33 : 925-929.

MORGAN, K. 1973. Nuclear activity in Sporozoa. PhD thesis. University of London.

MOVSESIJAN, M., SOKOLIC, A., TANIELIAN, Z., and ALI, N.A. 1975. Circula- ting and local antibodies in Eimeria tenella infection. Acta Vet (Belgr) 25 (2) : 59-64.

NEWTON, B. and BURNETT, J.K. 1972. DNA of k'inetoplastidae: A comparative study. In Comparative Biochemistry of Parasites ed. Van den Bossche, London, Academic Press : 185-188.

NIELSEN, P.J. and ANDRONIS, P.T., 1975. Further electrophoretic characterisation of strains of Tetrahymena pyriformis. J.Protozool. 22 (2) : 185-187.

NORTON, C.C. 1967. Eimeria colchici sp.nov. (Protozoa: Eimeriidae), the cause of caecal coccidiosis in English covert pheasants. J. Protozool. 14 : 772-781.

NORTON, C.C. and JOYNER, L.P. 1975. The development of drug-resistant strains of Eimeria maxima in the laboratory. Parasitology. 71 : 153-165.

NORTON, C.C., JOYNER, L.P. and CATCHPOLE, J. 1974. Eimeria weybridgensis sp.nov and Eimeria ovina from the domestic sheep. Parasitology. 69 : 87-95

NORTON, C.C. and PEIRCE,.M.A. 1971. The life cycle of Eimeria bateri (Protozoa, Eimeriidae) in the Japanese quail Coturnix coturnix japonicum. J. Protozool. 18 : 57-62.

NYBERG, P.A. and KNAPP, J.E. 1970. Effect of sodium hypochlorite on the oocyst wall of Eimeria tenella as shown by electron microscopy. Proc. helminth.Soc.Wash. 37 : 32-36.

OELSHEGEL, F.J., SANDER, B.J. and BREWER, G.J. 1975. Pyruvate kinase in malaria host-parasite interaction. Nature. Lond.255 : 345-347.

ORNSTEIN, L. 1964. Disc electrophoresis. I. Background and theory. Ann. N.Y. Acad.Sci. 121 : 321-344.

OXBROW, A.I. 1973. Strain specific immunity to Plasmodium berghei: a new genetic marker. Parasitology 67 : 17-27.

PARR, C.W. and GODFREY, D.G. 1973. The measurement of enzyme ratios as a means of differentiating trypanosomes. Trans.R.Soc.trop.Med.Hyg. 67 : 260. 223

PARR, C.W. and TAYLOR, A.E.R. 1974. Phosphoglucomutase variation in trypanosomes. Proc. 3rd Int.Cong.Parasit.Munich. 3 : 1470.

PATTILLO,W.H. 1959. Invasion of the cecal mucosa of the chicken by sporozoites of Eimeria tenella. J. Parasit. 45 : 253-258.

PEARSE, A.G.E. 1972. Histochemistry, Theoretical and Applied. 3rd ed. Churchill Livingstone, London.

PELLERDY, L. 1964. An attempt to subdivide the unwieldy genus Eimeria. Proc. 1st Int.Cong.Parasit.Rome.ed. A.Corradetti, Pergamon Press, New York. 1 : 284-286.

PELLERDY, L. 1965. Coccidia and coccidiosis. Akad.Kiado. Budapest.

PELLERDY, L. 1969a. Parenteral infection experiments with Eimeria stiedai. Acta vet.Acad.Sci.hung. 19 : 171-182.

PELLERDY, L. 1969b. Attempts to alter the host specificity of Eimeriae by parenteral infection experiments. Acta.Vet. (Brno) 38 : 31-35.

RAILLIET, A. 1913. Quel nom dolt-on donner a la coccidie intestinale de la poule. Arch.Paras. 16 : 147.

RAILLIET, A. and LUCET, A. 1891. Notes sur quelques espaces de coccidies encore peu etudiees. Bull.Soc.zool.Fr.16 : 246.

RAUSCHER, F.J., REYNIERS, J.A. and SACHSTEDER, M.R. 1962. Japanese quail egg embryo as a host for viruses. J. Bact. 84 : 1134-1139.

RAY, H.N. 1945. On a new coccidium Wenyonella gallinae n.sp., from the gut of the domestic fowl, Gallus gallus domesticus. Linn.Curr.Sci. 14 : 275.

REEVES, R.E. and BISCOFF, J.M. 1968. Classification of Entamoeba species by means of electrophoretic properties of amoebal enzymes. J. Parasit. 54 : 594-600.

REEVES, R.E., LUSHBAUGH, T.S. and MONTVALO, F.E. 1971. Characterisation of deoxyribonucleic acid of Entamoeba histolytica by caesium chloride gradient centrifugation. J. Parasit. 57 : 939-944.

REEVES, R.E., MONTVALO, F., and SILLERO, A. 1967. Glucokinase from Entamoeba histolytica and related organisms. Biochemistry. 6 : 1752-1760.

REICHENOW, E. 1921. From Prowazek's Handbuch der pathogenen Protozoen, 3 : 1253.

REID, W.M. 1973. A diagnostic chart for nine species of fowl coccidia. University of Georgia, College of Agriculture Experimental Station Research Report 163 : 18.

REID, W.M. 1975. Progress in the control of coccidiosis with anticoccidials and planned immunization. Am.J.vet.Res. 36 (4) : 593-596.

REID, W.M., SHARMA, N.W. and KEENER, J. 1961. Intestinal species of coccidia in chickens from Georgia. J. Parasit. 47 (suppl) : 45. 224

REID, W.M., TAYLOR, E.M. and JOHNSON, J. 1969. A technique for demonstration of coccidiostatic activity of anticoccidial agents. Trans. Am.Microsc.Soc. 88 :148-159.

RIVOLTA, S. 1878. Della gregarinosi dei polti e dell' ordinamento delle gregarine e dei psorospermi degli animale domestica. Giorn. Anat. Fisiol e PatolAnimale, Pisa 10 (4) : 220-234.

RIVOLTA, S. and SILVESTRINI 1873. Psorospermosi epizootica nei gall- inacei. Giorn. Anat.Fisiol.e.Patol Animale, Pisa. 5 : 42

ROBERTS, W.L., HAMMOND, D.M., ANDERSON, L.C. and SPEER, C.A. 1970. Ultrastructural study of schizogony in Eimeria callospermophili. J.Protozool. 17 (4) : 584-592.

ROMMEL, M. 1969. Untersuchungen Uber Infektionsverlauf sowie Ausbildung und Natur derImmunitat an experimentell mit Eimeria scabra (Henry, 1931) und E.polita (Pellerdy 1949) infizierten Schweinen. Habil- itationsschrift der Veterinarmedizinischen Fakult5t der Freien Uni- versitat, Berlin. 1969. •

ROMMEL, M. 1970. Studies on the nature of the crowding effect and of the immunity to coccidiosis. Proc.2nd Int. Cong.Parasit.J.Parasit. 56 (4) : Sec. 2 : 468.

ROSARIO, V.E. 1976. Genetics of chloroquine resistance in malaria parasites. Nature Lond. 261 : 585-586.

M.E. 1967a. Immunity to Eimeria tenella and Eimeria necatrix infections in the fowl. Influence of the site of infection and stage of parasite. II. Cross protection. Parasitology. 57 : 567-583.

ROSE, M.E. 1967b. Immunity to E.brunetti and E.maxima infections in the fowl. Parasitology. 57 : 363-370.

ROSE, M.E. 1973. Immunity. In Hammond D.M. with P.L. Long eds. The Coccidia. Univ.Park Press Baltimore : 295-342. •

ROSE, M.E. 1975. Infections with Eimeria maxima and Eimeria acervulina in the fowl : effect of previous infections with the heterologous organism on oocyst production. Parasitology 70 (2) *: 263-275.

ROSE, M.E. and LONG, P.L. 1962. Immunity to four species of Eimeria in fowls. Immunology. 5 : 79-92.

ROSENBERG, M.A. 1941. A study of the *inheritance of resistance to Eimeria tenella in the domestic fowl. Poult.Sci. 20 : 472.

ROWE,E., GIBSON, I., and CAVILL, A. 1971. The effects of growth conditions • on the esterases of Paramecium aurelia. Biochem.Genet.5 : 151 -159.

RUBIN, H.L., FANISHIER, L., CORNELIUS, A. and HUGHES, W.F. 1962. Toler- ance and immunity in chickens after congenital and contact infection with an avian leukosis virus. Virology 17 : 143-156.

RUFF, M.D., WERNER, J.K., and DAVIS, G.M., 1971. Effect of extraction procedures on disc electrophoretic patterns of S.japonicum proteins. Jap.J.Parasit. 20 (5) • 341-358. 225

RYLEY, J.F. 1973. Cytochemistry, physiology and biochemistry. In D. M. Hammond with P.L. Long eds. 'The Coccidia'. Univ.Park Press, Baltimore.

RYLEY, J.F. and BETTS, M.J. 1973. Chemotherapy of chicken coccidiosis. Adv.Pharmacol.Chemother. II : 221-293.

RYLEY, J.F. and WILSON, R.G. 1972. The development of Eimeria brunetti in tissue culture. J. Parasit. 58 : 660-663.

SCHILDKRAUT, C.L., MARMUR, J. and DOTY,P. 1962. Determination of the base composition of deoxyribonucleic acid from its buoyant density in CSCI. J. molec.Biol. 4 : 430-443.

SCHNEIDER, A. 1875. Note sur la psorospermie oviforme du poule. Arch. Zool.Exp.Gen. 4 : xl-xliv.

SCHNUR, L.F. and CHANCE, M.L. 1976. The characterisation of African leishmanial strains : a comparison of differentiating methods. Trans.R.Soc.trop.Med.Hyg. 70 (1) : 14.

SCHOLTYSECK, E. 1954. Untersuchungen fiber die bei einheimischen Vagelarten vorkommenden Coccidien der Gattung Isospora. Arch.Prvti•.tenk.100 : 91-112.

SCHOLTYSECK, E. 1963. Untersuchungen fiber die KernverhSltnisse and das Wachsturn bei Coccidiumorphen unter besonderer Berticksichtigung von Eimeria maxima. Z. Parasitenkde. 22 : 428-474.

SCHOLTYSECK, E. 1973. Ultrastructure. In D.M. Hammond with P.L. Long eds. The Coccidia. Univ.Park Press. Baltimore : 81-144.

SHAH H.L. and JOHNSON, C.A. 1971. Eimeria bateri, Bhatai, Pandey and Pande, 1965, from the Hungarian Quail Coturnix c. coturnix in the United States and its attempted transmission to the chicken. J. Protozool. 18 (2) : 219-220.

SHARMA, N.W. 1964. Response of the fowl (Gallus domesticus) to parenteral administration of seven coccidial species. J. Parasit. 50 : 509-517.

SHAW, C.R. 1970. How many genes evolve? Biochem. Genet. 4 : 275-283.

SHAW, C.R. and PRASAD, R. 1970. Starch gel electrophoresis of enzymes - a compilation of recipes. Biochem. Genet. 4 : 297-320.

SHERMAN, I.W. 1961. Molecular heterogeneity of lactic dehydrogenase in avian malaria (Plasmodium lophurae). J.exp.Med.114 : 1049-1062.

SHERMAN, I.W. 1962. Heterogeneity of lactic dehydrogenase in infra- erythrocytic parasites. Ann.N.Y.Acad.Sci. 24 : 944-953.

SHERMAN, I.W. 1964. Heterogeneity of lactic dehydrogenase in avian malaria demonstrated by the use of coenzyme analogs. Proc.lst Int.Congr. Parasit.Rome. ed. Corradetti A. Pergamon Press 1,A2 : 73.

SHERMAN, I.W. 1966. Malic dehydrogenase heterogeneity in malaria. (Plasmodium lophurae and P. berghei). J. Protozool. 13 : 344-349. 226

SHERMAN, I.W., PETERSON, I., TANIGASHI, L. and TING, I.P. 1971. The Glutamate dehydrogenase of Plasmodium lophurae. Expl. Parasit. 29 : 433-439.

SHIBALOVA, T.A. 1970. Cultivation of the endogenous stages of chicken coccidia in embryos and tissue cultures. J. Parasitol.56 (4) : 315-316.

SHIRLEY, M.W. 1975. Enzyme variation in Eimeria species of the chicken. Parasitology 71 : 369-376.

SHIRLEY, M.W. and MILLARD, B.J. 1976. Some observations on the sexual differentiation of Eimeria tenella using single sporozoite infections in chicken embryos. Parasitology 73 (3) : 337-341.

SKANDAR, M.V.F. 1973. Frequencia de coccidiosis en ganada bovino y sa identification en Mexico. Veterinaria 4 (11) : 131-136.

SMITH, B.F. and MCSHAW, W.H. 1949. The effect of the protozoan parasite, Eimeria stiedae, on the succinic dehydrogenase activity of liver tissue of rabbits. Ann.N.Y.Acad.Sci. 52 : 496-500.

SMITH, I. 1968. Chromotographic and electrophoretic techniques. Volume IT, Zone Electrophoresis. Heinemann, London.

SOLOMON, J.B. 1968. Immunity to Salmonella gallinarum during ontogeny in the chicken. II Induction of tolerance or priming by single doses of live or killed bacteria. Immunology. 15 : 207-218.

STAMLER, S.J. 1974. Multiple forms of enzymes exhibited during macrostome formation in Tetrahymena vorax. J. Protozool. 21 (3) : 433.

STEIGER, R., KRASSNER, S.M. and JENNI, L. 1974. Comparison of specific and relative alanine and aspartate aminotransferases of Trypanosoma brucei sub-group trypanosomes. Acta trop. 31 (3) : 202-218.

STEPHENS, W.P. 1974. Insects. In Biochemical and Immunological taxonomy of animals.C.A. Wright ed. Academic Press : 303-349.

STOTISH, R.C. and WANG, C.C. 1975. Preparation and purification of merozoites of Eimeria tenella. J. Parasit. 61 : 700-703.

TAIT, A. 1970. Enzyme variation between syngens in Paramecium aurelia. Biochem. Genet. 4 : 461-470.

TODD, K.S. and HAMMOND, D.M. 1968a. The life cycle and host specificity' of Eimeria callospermophili Henry, 1932 from the Uinta Ground Squirrel Spermophilus armatus. J. Protozool. 15 : 1-8.

TODD, K.S. and HAMMOND, D.M. 1968b. Life cycle and host specificity of Eimeria larimerensis Vetterling, 1964, from the Uinta ground squirrel Spermophilus armatus. J. Protozool. 15 : 268-275.

TODD, K.S., HAMMOND, D.M. and ANDERSON, L.C. 1968. Observations on the life cycle of Eimeria bilamellata Henry, 1932 in the Uinta ground squirrel Spermophilus armatus. J. Protozool. 15 : 732-740.

TODD, K.S. and LEPP, D.L. 1972. Completion of the life cycle of Eimeria vermiformis. Ernst, Chobotar and Hammond 1971 from the mouse, Mus musculus, in dexamethasone-treated rats Rattus norvegicus.. J. Parasit. 58 : 400-401. 227

TODD, K.S., LEPP, D.L. and TRAYSER, C.W. 1971. Development of the asexual cycle of Eimeria vermiformis, Ernst, Chobotar and Hammond 1971, from the mouse Mus musculus in dexamethasone-treated rats, Rattus norvegicus. J. Parasit. 57 : 1137-1138.

TOYE, P.J. 1974a. Isoenzymic differences between culture forms of Trypanosoma rangeli, T.cruzi and T. lewisi. Trans.R.Soc.trop. Med. Hyg. 68 : 266.

TOYE, P.J. 1974b. Isoenzyme variation in isolates of Trypanosoma cruzi. Trans.R.Soc.trop.Med.Hyg. 68 : 147.

TSUKAMOTO, M. 1974. Differential detection of soluble enzymes specific to a rodent malaria parasite, Plasmodium berghei, by electrophoresis on polyacrylamide gels. Tropical Medicine. 16 (2) : 55-69.

TSUKAMOTO, M., YAMAGUCHI, K., TSUENDA, K. and NAKABAYASHI, T. 1975. Field trials of biochemical blood examination in relation to chemotherapy of malaria patients in Palawan Island, the Philippines. Tropical Medicine. 17 (1) : 13-26.

TYZZER, E.E. 1929. Coccidiosis in gallinaceous birds. Am.J.Hyg. 10 : 269-383.

TYZZER, E.E., THEILER, H. and JONES, E.E. 1932. Coccidiosis of gallinaceous birds II. A comparative study of species of Eimeria of the chicken. Am.J. Hyg. 15 : 319-393.

VAN DOORNINCK, W.M. and BECKER, E.R. 1957 Transport of sporozoites of •i Eimeria necatrix in macrophages. J. Parasit. 43 : 40-44.

VERWEY, J. 1926. Infectieproven met vogelcoccidien.Zool.Ber. 9 : 497.

VETTERLING, J.M. 1976. Eimeria tenella : Host specificity in gallinaceous birds. J. Protozool. 23 (1) : 155-158.

VETTERLING, J.M. and DORAN, D.J. 1969. Storage polysaccharide in coccidial sporozoites after excystation and penetration of cells. J. Protozool. 16 : 772-775.

VETTERLING, J.M., DORAN, D.J. and AUGUSTINE, P.C. 1973. Eimeria tenella: Pathogenicity versus development in cell culture of three strains. 48th Annual Meeting of the American Society of Parasitologists.

VETTERLING, J.M. and WIDMER, E.A. 1968. Eimeria cascabeli sp.n. (Eim- eriidae, Sporozoa) from rattlesnakes, with a review of the species of Eimeria from snakes. J. Parasit. 54 : 569-576.

VINOGRAD, J. and HEARST, J.E. 1962. Equilibrium sedimentation of macro- molecules and viru ses in a density gradient. Fortschr. Chem.org. NatStoffe. 20 : 372-422.

VISVESVERA, G.S. and BALAMUTH, W. 1975. Comparative studies on related free-living and pathogenic amebae with special reference to Acanth- amoeba. J. Protozool. 22 (2) : 245-256.

VON BRAND, T. 1973. Biochemistry of parasites. 2nd Edition. Academic Press.

WACHA, R.S. and CHRISTIANSEN, J.L. 1974. Systematics of the eimerian parasites from North American snakes of the family Colubridae and their prevalence in the colubrids of Iowa. J. Protozool. 21 (4) : 483-489. 228

WACHA, R.S. and CHRISTIANSEN, J.L. 1976. Coccidian parasites from Iowa turtles:systematics and prevalence. J. Protozool. 23 (1) : 57-63.

WAGENBACH, G.E. 1969. Purification of Eimeria tenella sporozoites with glass bead columns. J. Parasit. 55 : 833-838.

WALLIKER, D. 1976. Genetic factors in malaria parasites and their effect on host-parasite relationships. In Symposia of the British Society for Parasitology. Vol. 14. edited by A.E.R. Taylor and R. Muller.

WALLIKER, D., CARTER, R. and MORGAN, S. 1971. Genetic recombination in malaria parasites. Nature.Lond. 232 : 561-562.

WALLIKER, D., CARTER, R. and MORGAN, S. 1973. Genetic recombination in Plasmodium berghei. Parasitology 66 : 309-320.

WALLIKER, D., CARTER, R. and SANDERSON, A. 1975. Genetic studies on Plasmodium chabaudi : recombination between enzyme markers. Parasitology. 70 : 19-24.

WALLIKER, D., SANDERSON, A., YOELLI, M. and HARGREAVES, B.J. 1976. A genetic investigation of virulence in a rodent malaria parasite. Parasitology. 72 : 183-194.

WANG, C.C. 1974. Sporulation of E. tenella. Proc. 3rd.Int.Cong.l-arasit. Munich. 3 : 64.

WANG, C.C. and STOTISH, R.L. 1975. Changes of nucleic acids and proteins in the oocysts of Eimeria tenella during sporulation. J. Protozool. 22 (3) : 438-443.

WANG, C.C., STOTISH, R.L. and POE, M. 1975a. Dihydrofolate reductase from Eimeria tenella: rationalization of chemotherapeutic efficacy of pyrimethamine. J. Protozool. 22 (4) : 564-568.

WANG, C.C., WEPPELMAN, R.M. and LOPEZ-RAMOS, B. 1975b. Isolation of amylopectin granules and identification of amylopectin phosphorylase in the oocysts of E. tenella. J. Protozool. 22 (4) : 560-564.

WENYON, C.M. 1926. Protozoology. William Wood and Company. New York.

WILLIAMS, R.B. 1973. Effects of different infection rates on the oocyst production of Eimeria acervulina or Eimeria tenella in the chicken. Parasitology. 67 : 279-288.

WRAXALL, B.G.D. and CULLIFORD, B.J. 1968. A thin-layer starch gel method for enzyme typing of bloodstains. J. Foren.Sci.Soc.8 : 81-82.

WRIGHT, C.A. 1974. Biochemical and immunological taxonomy of animals. Academic Press.

YAKIMOFF, W.L. and RASTEGAIEFF, E.F. 1931. Zur HUhnerkokzidiose in Russland. 261.Bakt.,I.Abt.Orig. 123 : 1-14.

ZEE, D.S. and ZINKAM, M.D. 1975. Demonstration of parasitism in tissues by electrophoretic definition of parasite enzymes in host tissues. J. Pediat. 86 (3) : 408-409. 229

ZMERZLAYA, Y.I. 1965. Coccidiosis enteritis in carp. Avtoref. Diss L. : 1-16.

ZUCKERKANDL, E. and PAULING, L. 1962. In Horizons in Biochemistry. M. Kasha and B. Pullman eds. Academic Press : 189-225. 230

SUBSIDIARY MATTER

The following publications are submitted as subsidiary matter:

1 Electrophoretic variation of enzymes in chicken coccidia. Trans. R. Soc. trop. Med. Hyg. 69 : 436-437, 1975. 2 Development of Eimeria terzeZ/a in quail embryos. Trans. R. Soc. trop. Med. Hyg. 70 : 21, 1976. 3. Electrophoretic variation of enzymes in mammalian coccidia. Trans. R. Soc. trop. Med. Hyg. 70 : 21, 1976. 4. Further electrophoretic studies on species of Eimeria. Parasitology, 73 (2), 1976. Proc. Brit. Soc. Parasit. : IV. 231

Electrophoretic Variation of Enzymes in Chicken Coccidia

Species of eimeriine coccidians parasitizing fowls show variation in their morphological and biological characters and, in general, they can be considered to be immunologically distinct. However, differences are not always easy to detect and as the number of laboratory isolates increases, an additional method of characterization would be advantageous. The use of biochemical criteria for the recognition of particular protozoal populations is becoming increasingly important, as in studies of malaria parasites (CARTER, 1970), trypanosomes (KILGOUR and GODFREY, 1973) and leishmaniae (GARDENER et al. 1974). Results were presented of similar methods used to provide definable biochemical characters for chicken Eimeria spp.

Samples of oocysts were obtained from Wellcome Research Laboratories, Berkhamsted, Central Veterinary Laboratory, Weybridge, and Houghton Poultry Research Station. Sporulated oocysts in distilled water were mechanically homogenated in an ice bath using glass beads. The resulting suspension was centrifuged at 30,000 g. for 45 min. at 4°C. The clear supernatant was either used immediately for electrophoresis on thin layer starch gel plates (WRAXALL and CULLIFORD, 1968) or stored temporarily at -20°C.

The following species have been examined: E.tenella, E.necatrix, E.brunetti, E.maxima and E.acervulina. The enzymes which have been identified and studied include lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PD), ph phOglucose isomerase (PGI) and leucine aminopeptidase (LAP)

Simple observation of electrophoretic migration revealed one enzyme band for LDH (FRANDSEN and COOPER, 1971) and for G6PD. Five sub-bands have been detected for PGI using a phosphate buffer (DELORENZO and RUDDLE, 1969). As found by WANG (1974), there was variation between isoenzymes of LAP of sporulated and unsporulated oocysts. Two bands were produced for LAP of sporulated oocysts.

The variation in the mobility of the enzymes allowed all the species to be distinguished. It was interesting to find that only slight differences in LDH and G6PD occurred between E.teneZla and E.necatrix, species which have certain biological characters in common.

It has not been possible using the same enzymes to recognize differences between Houghton samples of E.mivati and E.acervulina. This provides further support for the view of LONG (1973) that E.mivati is not sufficiently different from E. acervuZina to warrant a separate specific status. Pre- liminary experiments indicate variation between the Weybridge and Houghton strains of E.tenella. References: CARTER, R. (1970). Trans.R.Soc.trop.Med.Hyg., 64,401. DELORENZO, R.J. and RUDDLE,F.H. (1969). Biochem.Genet., 3,151. FRANDSEN,J.C. and COOPER,J.A. (1971).The ASB Bull.,18,2,35. GARDENER,P.J.,CHANCE,M.L. and PETERS,W. (1974).Ann.trop.Med.Parasit.,68,317. KILGOUR,V. and GODFREY,D.G. (1973).Nature New Biology,244,69. LONG,P.L. (1973).Parasitology,67,145. WANG,C.C. (1974). Proc.Third.Internat.Congress Parasit.Munich,3,1464. WRAXALL,B.G.D. and CULLIFORD,B.J. (1968).J.Forensic.Sci.Soc., 8,81. 232

Development of Eimeria tenella in quail embryos

Avian Eimeria spp exhibit a very strong host specificity: it is rare for a parasite to complete its development in more than one host genus. The chicken was found by VETTERLING (1973), to be the only suitable host for E. tenella out of eight genera of gallinaceous birds, including quail. Previous attempts to infect quail embryos with normal pathogenic strains of E. tenella have been unsuccessful (LONG,. 1965; LONG and MILLARD, 1975), although LONG and MILLARD (1975) reported that a strain which had adapted to development in the chick chorioallantoic membrane was capable of infecting quail and duck embryos. A corticosteroid-treated goose embryo supported development of schizonts (LONG, 1971).

Coturnix coturnix japonica eggs were obtained from birds reared on a coccidiostat-free diet. On the seventh day of incubRtion at 39-41°C, the developing embryos were inoculated with 2x10 - sporozoites of the Houghton strain of E. tenella, via the allantoic cavity. Stages of the parasite were later detected in the chorioallantoic membrane. Large second generation schizonts,characteristic of pathogenic strains, were found at 116 hours. Smaller schizonts confined to epitheli al cells were also present. Small numbers of oocysts, capable of sporulation, were recovered at 160 hours.

References:

LONG, P.L. (1965), Development of Eimeria tenella in avian embryos, Nature, 208, 509-510 LONG, P.L. (1971), Schizogony and gametogony of Eimeria tenella in the liver of chick embryos. Journal of Protozoology, 18, 17-20. LONG, P.L. & MILLARD, B.J. (1975). Pathogenicity, immunogenicity and site specificity of an attenuated strain of Eimeria tenella. Journal of Protozoology, 22, 28th Annual Meeting of the Society,of Protozoologists, Abstract I54,53A. VETTERLING, J.M. (1973). Host specificity studies in eight species of gallinaceous birds. Journal of Protozoology, 20, 510 233

Electrophoretic variation of enzymes in mammalian coccidia

Variations in the mobility of enzymes, as shown by electrophoresis, have proved useful in characterizing many groups of Protozoa including the eimeriine coccidians of chickens (ROLLINSON, 1975; SHIRLEY, 1975). The present report concerns the use of electrophoretic techniques to distinguish certain Eimeria spp of mammals.

Samples of oocysts were provided by the Central Veterinary Labor- atory, Weybridge and ICI (Pharmaceuticals) Ltd., Macclesfield. Preparation of the water soluble extracts and electrophoresis were carried out as described by ROLLINSON (1975), except that enzyme stabilizers as used by KILGOUR and GODFREY (1973) were added.

The following species have been examined: E. coecicola, E. intestinalis, E. magna and E. stiedae from the rabbit and E. ninakohlyakimovae and E. weybridgensis of sheep. Enzymes which have been identified and studied include: lactate dehydrogenase, glucose 6- phosphate dehydrogenase, phosphoglucose isomerase, leucine aminopeptidase and aspartate aminotransferase.

When the isoenzyme patterns, obtained by visualization of enzymatic activity in the gels, were compared, extensive variation was found between the species.

Similar methods may prove useful in clarifying the relationships of other groups of coccidia.

References:

KILGOUR, V., & GODFREY, D.G. (1973). Species-characteristic isoenzymes of two aminotransferases in trypanosomes. Nature New Biology, 244, 69-70. ROLLINSON, D. (1975). Electrophoretic variation of enzymes in chicken coccidia. Transactions of the Royal Society of Trop- ical Medicine and Hygiene, 69, 436. SHIRLEY, M.W. (1975). Isoenzymes in the coccidia, Journal of Protozoology, 22, 28th Annual Meeting of the Society of Protozoologists, Abstract 162, 55A. 234

Further Electrophoretic Studies on Species of Eimeria

Thirteen enzymes (lactate dehydrogenase,ck-glycerophosphate dehydrogenase, glucose 6-phosphate dehydrogenase, hexose 6-dehydrogenase, 6-phosphogluconate dehydrogenase, leucine aminopeptidase, aspartate aminotransferase, phosphoglucomutase, alkaline phosphatase, hexokinase, adenylate kinase, fructose 1,6-diphosphatase and glucose phosphate- isomerase) in the Eimeria spp of the chickens have been examined by thin-layer starch-gel electrophoresis. A high frequency of inter- specific variation occurred except between E.mivati (Houghton) and E.acervulina (Houghton, 'M', Ongar and Weybridge), which could only be distinguished by variations found in the mobilities of glucose 6-phosphate dehydrogenase and glucose phosphate isomerase.

Evidence that these strains are closely related has also been obtained from crosses between E.mivati (Houghton) and E.acervulina (Weybridge). The parent strains differ in their ability to passage in eggs and in their electrophoretic form of glucose phosphate isomerase (GPI). E.acervulina (Weybridge) contains GPI-2 andliwill not passage in eggs when small concentrations of sporozoites, 1 x 10 , are inoculated into eggs; E.mivati (Houghton) will passage at such concentrations and contains GPI-1. Oocysts have been observed from the cross between these two strains which are characterized by their ability to develop in eggs using only small sporozoite inoculations and by containing GPI-2.

These results uphold the view of LONG (1973), Parasitology 67, 143-55), based on biological and immunological evidence, that E.mivati, at least the Houghton isolate, can be considered to belong to the species E.acervulina.