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BIOCHEMICAL AND BIOLOGICAL CHARACTERS OF EIMERIA

SPECIES

by 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 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 charac-

E. acervulina.

ters.

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, Pharmaceuticals 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.

7

CONTENTS

Page Abstract .. .. .. .. .. .. ..

..

.. 3

Acknowledgements .. .. .. .. ..

..

.. 5

Introduction .. .. .. .. .. ..

..

.. 14

.. .. .. Introductory Review ....

..

.. 16

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

.. 16

.. .. 2. Species Characters of Eimeria (A) Morphological Characters .. .. i) The Oocyst .. .. .. .. ii) Endogenous stages .. • • • •

Oe

.. 16 .. 17

.. 19

.. .. .

.. 21 .. 21 .. 23

(B) Biological Characters .. • • • • i) Pathogenicity • • ' ' .. • .. Period ii) Prepatent iii) Patent Period .. .. .. iv) Reproductive potential .. v) Sporulation time .. • • vi) Development in culture • • vii) Drug resistance • . • • • viii) Host and site specificity

• • • • • • .. . • • • • • •

.. 24 .. 24 .. 24 .. 26 .. 26 .. 27 .. 28 .. 28 .. 29

(C) Immunological Specificity.

.

.. 29

• •

.. 32

.

(D) Concluding Remarks

Materials and Methods

••



••

34



34

1.

Parasites

2.

Experimental Birds

3.

• • .. 37 •• Bird Infections • • (A) Isolation Procedures .. ... 37 (B) Standardisation of Dose and Inoculation procedures 38 .. • • .. 38 (C) Single Oocyst • • .. • . .. 38 •• (D) Abnormal Infection Routes

4.

Cortisone Treatment • •

5.

Avian Embryos • • • •

.. 39 '' ' . • • . .. 39

6.

Avian Embryo Infections • •

••





••

••

34

• • .. 39

• ▪



8

and

7. Isolation

Harvesting

Page Oocysts •

of

• 41

• •

41

Collection

of

oocysts

from

faeces OS 00 00

(B)

Collection

of

oocysts

from

caeca

of

oocysts

from

embryos 06 00 00 42

(C)

Collection

(D)

Sporulation

(E)

Cleaning

and

..

..

sterilising

..

sporozoites

9. Preparation

of

merozoites

..

..

..

• •



• •





• •

• • ..

-

(A)

Preparation

of

merozoites

from

chicken

(B)

Preparation

of

merozoites

from

embryo

in vitro

.. 66 06 41

• ..

oocysts

of

• .. 42 4

..

..

42

• .. 43

caeca

.•

43

and

. 43

• culture ..

• .. •

• •

.. • Electrophoresis ..

• • ..

• •

• .. 44

.. .. • samples

• .. 44

(A)

Preparation

of

enzyme

(B)

Polyacrylamide

(C)

Disc

(D)

Thin-Layer

(E)

Enzyme

gradient

• gels ..

(B)

Analytical

• solutions

acid

Caesium

Culture

(A)

Sterilisation

(B)

Monolayer

(C)

Suspension

(D)

Organ

.. •

.. •

Staining

14.

Cytochemistry

15.

.. • Measurements

16.

Serum

samples

• . .

. 47

• ..

• .. .

. 62

density

• •

• • .. .. ''

• .

. 63

• .. .

. 63

..

• •

• •

• . . .

• .

. .. • •

• •

• .. 64

• .. •

• .. 64

• •

• •

• •

• .. •

• •

• -.



Characterisation

criteria •

General

• •

of

Protozoa

• •

• .. •

• .

• . .

Considerations •

Sarcodina



by

• •

• •

• •

• •



• .. •



• •

.

Mastigophora .. .. .. .. 0 a

Ciliophora

..

..

Sporozoa

.

5)

Microsporidia

.. .. .. .. . .

in

relation

to

• •

parasitic

69

69 .

. 74

.

4)

(B) Isoenzymes

65

72 • ..

2)

• • •

.

biochemical

• •

..

. 65

69 ' •

3)

..

64

Eimeria species

• 1)

.

• . . 65

Introduction The

. 63

• •

PART I : Biochemical characters of

(A)

• 62

• .. •

• • ..

• •

gradient

• .. •

cultures

Techniques

• .. .. 46

• •

• .. •

• cultures. ..

13.

.. • 45

• •

• .. .. .. 45

• • ..

• procedures ..

slices ..

• .

• •

Isolation

Chloride

centrifugation •

Tissue

• .. gels

• .. •

• gels

Starch

assay

11. (A) Deoxyribonucleic

12.

• •

(A)

8. Preparation

10.

• •

• .

.. . g

diseases

85 .

86

Page Results

•• 88



species 88

Eimeria

1)

Identification of enzymes in oocysts of

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 .. • • • • • • (a) Oocysts .. .. .. .. (b) Sporozoites .. .. .. .. (c) Merozoites .. .. .. .. •



• •





• •





• •





• •

100 100 101 101

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 ii) Differences within species (b) Characterisation of

Eimeria

106 113 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 ..

•• • •

(d) Identification of an isolate from quail 8) The use of (A) The i) ii) iii)

enzyme markers in genetic studies E.acervulina complex : • • • • Oocyst sizes • • • • • • Enzyme types • • • • • • Ability to grow in embryos

••



125 126 128 128 128 128 128

(a)E.acervulinavar.mivati (Houghton) • • 128 (b)E.acervulina (Weybridge) •• • • • • 130 (c) E.acervulina ('M' and Houghton strains) 130 iv) v)

(B) The

Investigations into the viability of 'contaminating oocysts' enclosed in incubating eggs • • 131

E.acervuZina (Weybridge) and E.acervulimavar.mivati (Houghton) ..• • 131

The cross between

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

11)

Isoenzymes in relation to coccidiosis (a) E.tenelia infections (b) E. stiedai infections

145

•• • • 146 146 •• • • 146

10

Page 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 Eimeria • • .. • • • • • • • • i) The value of electrophoretic data .. .. ii) Assessment of relationships within the genus iii) Speciation .. .. •• " " " iv) Adaptive significance of enzyme variations ..

158 158 160 161 162

(E) Population structure with particular reference to laboratory cultures .. .. .. .. • • • . • • 163 ' ' (F) Clones of coccidia .. .. .. .. .. .. 166 .. .. .. 169 (G) Sexual differentiation • • . (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 3. Specificity of culture

Eimeria

spp 181

Eimeria spp of birds in vitro and embryo 183 • • • • ' ' • • " "

Results .. .. .. .. .. .. .. .. . 186 1. Development of Eimeriaspp in chicken embryos .. 2. Development of

3.

••

186

E.tenella (Houghton) in avian embryos 186

Development of Eimeriaspp in vitro (a) Cells cultures (b) Cell suspensions • • (c) Organ slices •





• •







• •





189 189 193 193

E.tenella (Houghton) in Coturnix coturnix japonica .. • • .. 193 5. Site specificity of E.tenella in chicks . . 197 4.

Attempts to obtain development of

6. Cytochemistry . • • • • • • • 199

Discussion References

204

• •

00 00

Subsidiary matter

0

. 0

0

. .

00 041

0 •

00

08

209

23o

11

LIST OF TABLES

Page

TABLE

Eimeria cultures used in this study

One:

Details of

Two:

Buffer systems used in thin-layer starch gel

35

48

electrophoresis .. Three:

List of Enzymes and buffer systems

Four:

Enzyme assay solutions

Five: .

Incubating media

Six:

Enzymes identified in water soluble extracts of

••

••

Eimeria tenella oocysts Seven:





.



• •





• •



50

••

52



••

.

66



89

• •

Enzymes identified in water soluble extracts of oocysts of

90

Eimeria spp

Eight:

Measurements of oocysts .of

Nine:

Spectrophotometric analysis of AP levels in the serum

E. acervulina

••

••

of two—week-old Ranger cockerels inoculated with 4 1 x 10 oocysts of E. teneZla (Houghton) ..

Eimeria spp from the chicken

Ten:

Biochemical key to certain

Eleven:

Electrophoretic migration of ten enzymes in with reference to

129

147 153

Eimeria spp

E. tenella (Houghton) ..

154

12

LIST OF FIGURES

FIGURE

One:

Page Inoculation of sporozoites into a 10—day7old chicken

•.

embryo

Two:

• •

• •



.

• •

.•

Variation in the migration of G6PDH in oocysts of

E. acervulina

strains, attributable to differences 95

in electrophoretic conditions

Three:

Glucose phosphate isomerase in oocysts of different ages

Four:

Leucine aminopeptidases in sporulated and unsporulated oocysts of

Five:

E. tenella • • • • • •

Purified second generation merozoites of E.

Total' proteins of

Eimeria

spp demonstrated on poly104

Eimeria

Electrophoretic mobility of enzymes in

spp 108

from the chicken

Eight:

Glucose phosphate isomerase activity in mixed populations of

Nine:

E. acervulina

(Weybridge) and

E. tenella

(Houghton)

114

E. tenella

Intraspecific variation of glucose phosphate isomerase in

E. acervuZina Eleven:

116

Electrophoretic mobility of enzymes in

Eimeria

spp from

the rabbit

Twelve:

119

Electrophoretic mobility of enzymes in

Eimeria

spp from

the sheep ..

Thirteen:

122

Intraspecific variation of glucose phosphate isomerase in

E. bateri.

Oocysts of

127

E. bateri

Fourteen: E. acervulina /E. acervulina var.mivati Fifteen:

114

Intraspecific variation of glucose phosphate isomerase and hexokinase in

Ten:

98 102

acrylamide gradient gels

Seven:

98

tenella

(Houghton) isolated from chicken caeca .

Six:

14 0

cross

132

Gludose phosphate isomerase in populations in the 1314 E. acervuZina/E. acervuZina var.mivati cross

13

LIST OF FIGURES (cont.)

Page

FIGURE

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)

Twenty: Twentyone: Twenty7 two:

167

Transfer of characters during meiosis

172

E. tenella

(Houghton) infection in chorioallantoic

Twentythree:

E. acervulina var.mivati

Twentyfour:

E. tenella

Twentyfive:

Stages of

Twentyseven: Twenty.eight: Twentynine: Thirty:

147

Inheritance of characters within an oocyst

membrane of chicken embryo ..

Twenty six:









.

infection in chorioallantoic

membrane of chicken embryo ..





. •

• •

E.tenella

.188

(Houghton) in chorioallantoic

.

membrane of duck embryo

E. tenella E. tenella

• •





E. tenella

Schizont of

• •

• •

••





••

in monolayer chicken kidney cells

E. tenella

(Houghton) in quail kidney cell

E. tenella

Sporozoites of

••

• E. tenella

Schizonts of

Thirtytwo:

Trophozoites of

Thirtythree:

Stages of

Thirtyfour:

SporozoiteS of

E. tenella

194 195

195

198

(Houghton) in chicken kidney

200

cells stained for LDH (Houghton) in chicken kidney cells

201

stained for G6PDH

G6PDH

192

(Houghton) in the liver of a

19-day-old 'tolerant' chicken

E. tenella

191

(Houghton) in the caecum of a

quail

Thirtyone:

190

(Houghton) in chorioallantoic

membrane of pheasant embryo .. Stages of

• •

(Houghton) in chorioallantoic

membrane of quail embryo Stages of

188

(embryo-adapted) infection in chorioallantoic

membrane of chicken embryo .

Stages of

187

E.tenella

(Houghton) stained for LDH and

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 Coccidia are are found in the genera Eimeria and Isospora. 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 ubiquity 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 abnormalities 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 reluctance 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. Understandably, 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 The concept of the fixed individual species of Eimeria. species is held no more and there is an increasing awareness 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 locality, 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 residual 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 dosage 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 selecting 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,substantially 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. acervuline (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. acerna var.diminuta (Long 1974a)showed that its ability to develop in chicken embryos was similar to that of E. acervuZina 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. (C) i) Immunological Specificity 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. Partial 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. Similarly, 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 careful 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% potassium dichromate (K2Cr207) until required.

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

Species

Eimeria cultures used in this study:

Line

Strain

Number on arrival

Supplied by



E.tenella

Houghton

E.necatrix

HPRS

Houghton Embryo-adapted

E.acervulina

HPRS

Weybridge

21

WL

Weybridge

227

CVLW

Weybridge

48

CVLW

9,11

WL

Weybridge

53

CVLW

" (w4o)*

7

" (W44)*

E.brunetti

67,70,127

WL

Houghton

HPRS

Houghton

HPRS

var.mivati E.acervulina

6

Ongar

MB

Ongar 17,20

Weybridge

WL

Weybridge

63

CVLW

imi

16

CVLW

CVLW

F.maxima var. indentata '.maxima

WL

statyl-resistant

5

CVLW

4

WL

Weybridge (W74) Weybridge

Weybridge

statyl-resistant

85,86,87,88,89

CVLW

90,91,99

CVLW

16

CVLW

20

CVLW

27

CVLW

32

CVLW

17

CVLW

9,10

CVLW

robenidine-resistant clopidol-resistant sulphaquinoxaline-resistant robenidine-dependent Houghton Norwich

6

CVLW

36 TABLE ONE (continued)

Species

Strain

Line

Number on arrival

E.ovina

Supplied by

CVLW

E.weybridgensis

18

CVLW

6

CVLW

E.ninakohlyakimovae E.stiedai

ICI

E.magna

ICI •

Weybridge

W15

E.coecicola

CVLW CVLW'

E.intestinaZis

CVLW

E.bateri

?

Ascot

I

Ascot

CVLW

Weybridge

CVLW

Macster Field Isolate

MSD

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. Control 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. (C) Single Oocysts 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 microscopic 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 candled 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-dayold chicken embryo

14 1

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 Infections) 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 homogenised 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. Unsporulated oocysts were obtained by carrying out all procedures at 5°C.

42

(C) Collection of Ooycsts from Embryos 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. Merozoites 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 Whirlimixer. For the demonstration of malate dehydrogenase and malic enzyme the homogenate was further disintegrated 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. (C) Disc Gels 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 individually. 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 methanol-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

Buffer Number

Buffer

Electrode Components (per litre)

pH

Gel Components (per litre)

pH

1

0.214M Phosphate 0.027M Citrate

29.1g.K2HPO4 5.7g Citric Acid

7.0

1.06g K2HPO4 0.254g Citric Acid

7.0

2

0.3M Borate

18.55g Boric Acid 2.0g NaOH

8.0

1.86g BoricAcid 0.48g NaOH

8.5

3

0.2M Phosphate

5.8

50 ml of electrode buffer

5.8

4

0.378M Tris 0.165M Citrate

460 ml 0.2M NaH2PO42H2O 40 ml 0.2M Na2HPO4 45.8g Tris 34.2g Citric Acid

6.0

33.3m1 of electrode buffer

6.0

5

0.15M TrisCitric Acid

9.0

1.81g Tris 9.0

6

0.14M Tris 16.35g Tris 0.043M Citric Acid 9.04g Citric Acid

7.0

66.7m1 of electrode buffer

18.16g Tris

7.0

TABLE TWO (cont.) Buffer Systems used in Thin Layer Starch Gel Electrophoresis

Buffer Number 7.

8

9

Buffer

Electrode Components (per litre)

p

Gel Components (per litre)

pH

0.004M Na2EDTA 0.10M Borate 0.18M Tris

1.44g EDTA 6.18g Boric Acid 21.80g Tris

8.6

0.0546M Tris 0.245M Boric Acid

6.61g Tris 15.17g Boric Acid

7. 5

0.12g Tris 1.79g Boric Acid

7.5

0.2M Phosphate

255m1 0.2M NaH2PO4H20 245m1 0.2M Na2HP04'7H20

6.8

65m1 of electrode buffer

7.0

200m1 of electrode buffer

, 8.6

50

TABLE THREE

List of Enzymes and Buffer Systems

Abbreviation 1) 2)

AcP

Enzyme

Buffer System

Esterase ('non specific')

2,8

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

-35)

LAP

phosphate lyase

Leucine Aminopeptidase

2,5,7

L-Leucyl-peptide hydrolase 6) H6DH

Hexose 6- dehydrogenase

4,6

7)

Glucose 6- phosphate dehydrogenase

2,5,6,8

G6PDH

D-Glucose 8)

GDH

-6-

phosphate: NADP oxireductase

Glutamate dehydrogenase

3,6

L-Glutamate: NAD(P) oxireductase (deaminating) 9) 0.(.-GPDH

0(-Glycerophosphate dehydrogenase

off-Glycerol

-3-

1,6

phosphate: (acceptor) oxi-

reductase 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

6- Phospho -D- gluconate: NADP oxireductase 15) SDH

Succinate dehydrogenase Succinate: (acceptor) oxireductase

5,6,7,9

51 TABLE THREE (cont.) List of Enzymes and Buffer Systems

Abbrevi ation

Buffer System

Lnzyme

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

* Buffer Nos. underlined indicate most commonly used buffer

7

TABLE FCUR

Enzyme Assay Sclutions

Enzyme

1) Esterase

Substrate

1 ml , rock, 0 naphthyl

5 mg, Fast Blue RR

Buffer

4 m1,0.5M Tris-HCI,pH 7.0

4 ml, H2O

acetate

2) AcP

Coenzyme + other additives

10 mg Nack- naphthyl

5 mg, Black K Salt

8 m1,0.05M Acetate,pH 5.0

10 mg Fast Blue RR

5 m1,0.5M Tris-HC1,pH 8.5

phosphate

3) AP

6 mg Na B - naphthyl phosphate

10 mg MgSO4 7H20 5 ml H2O

4) FDP

6 mg Fructose

1,6-

phosphate

6 mg NADP 1 mg PMS 5 mg NBT • 40 ut PGI 10 ut G6PDH

5 mg MgC1 2 5 ml H2O

2 m1,0.5M Tris-HC1,pH

7.5

TABLE FOUR (zont.) Enzyme Assay S;lutions

Enzyme

5) LAP

Substrate

4 mg L - leucyl 0naphthylamide ,

6) H6DH

1 ml 1M Galactose phosphate

Coenzyme t other additives

5 mg Black K Salt 5 ml H2O

Buffer

5 ml 0.02 M Tri-maleate pH 6.0

5 mg MgC1 2

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

9) ot.GPDH

8 mg Na o(-glycerophosphate

Coenzyme + other additives

5 mg NAD

Buffer

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, pH 7.0

5 mg NAD 6 mg NBT 1 mg PMS

8 ml 0.25M Tris-HC1,pH 8.0

TABLE i

Enzyme

12)

ME

FOUR (cont.)

Enzyme Assay Solutions

Substrate 1 ml 1M Na L-malate

Coenzyme + other additives

5 mg NADP

Buffer. 8 ml 0.25M Tris-HCI,pH 8.0

6 mg NBT

pH 7.0

1 mg PMS 13)

LDH

1 ml 1M Na DL-lactate

2 ml 0.5M Tris-HC1,pH 7.0

5 mg NAD 3 mg NBT

pH 7.0

1 mg PMS 6 mi H2O

14)

6PGDH

10 mg Na

3

6-phosphogluconate



5 mg NADP

2 ml 0.5M Tris-HC1,pH 7.0

6 mg NBT 1 mg PMS 6 ml H2O



TABLE FOUR (cont.) Enzyme Assay Solutions

Enzyme 15)

SDH

Substrate

Coenzyme + other additives

5 ml 0.05

5 mg NAD

1 ml 0.1M Sodium succinate

Buffer K2HPO4 ,pH 7.0

2 mg ATP

4 mg NBT 1 mg PMS 1 ml EDTA

16)

AK

2 ml 0.5M Tris-HC1,pH 7.0

5 mg NADP

10 mg Glucose

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

17) HK

Substrate

10 mg Glucose

Coenzyme + other additives

5 mg NADP

Buffer

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 pyruvate

5 mg ADP . 5 mg NADH ca. 20 ut LDH

4 mg MgC1 2 After 1 hr incubation 1 mg PMS 4 mg NBT

7 ml 0.433M Glycine, pH 9.0

TABLE FOUR (.::ont.) Enzyme Assay Solutions

Enzyme 19) GPI

Substrate 6 mg Fructose 6-phosphate

Coenzyme + other additives 5 mg NADP

Buffer 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 100 ut MDH 0.5 ml O. 1Mck-ketoglutarate After 1 hr incubation 2 mg MTT 1 mg PMS

5 ml 0.1M phosphate, pH

7.4

TABLE

FOUR (cont.)

Enzyme Assay Solutions

Enzyme

21) ALAT

Substrate

10 mg L-alanine

Coenzyme + other additives

It mg NADH 50 ut LDH

Buffer

5 ml 0.1M phosphate, 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 2 mg Pyridoxal phosphate 6 mg NBT 1 mg PMS 10 ut GDH

10 ml 0.5M Tris-HCl, pH 8.0



TABLE FOUR (c.,ont.) Enzyme Assay Solutions

Substrate

Enzyme

23) PGM

10 'mg Glucosel-phosphate

Coenzyme + other additives

6 mg NADP 20 ut G6PDH

Buffer

.2 ml 0.5M Tris-HCl 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 4 mg MgCl2

5 ml 0.1M phosphate pH 7.4

After 1 hr incubation 2 mg MTT 1 mg PMS

25) TO

None

6 mg NBT 1 mg PMS

5 ml 0.5M Tris-HCl 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 homogenised 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 predigested 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 Centrifugation 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.). Particularly dirty glassware was treated with chromic acid. Coverslips 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 prefilters 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. Penicillin 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%. (C) Suspension Cultures 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 paraffin wax or paraplast. Sections were stained with Ehrlich's haematoxylin as described by Clerk (1973) or by the Giemsacollophonium 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, counterstained in Orange G and mounted in glycerine jelly. Observations 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 magnifications 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

LDH

Substrate

13.2 ml 1M Na 1M DL-Lactate (pH 7.0)

Incubating Solution

NAD 2 mg 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

2

Succinate

0.5 ml 2 0.1M NaCN 0.1 ml

0.05M MgC1

PBSA•(pH 7.4) 1.5 ml NBT 1 mg LAP

4 mg L-leucyl -B naphthylamide

Fast Blue B salt 2 mg 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

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