Nuclear Activity in Sporozoa

by.

K. Morgan, D.Sc. (Southamptou)

A thesis submitted

for the

Degree of Doctor of Philosophy

in the

Faculty of Science

University of London

Dept. of Zoology and Applied Entomology, Imperial College Field Station, Ashurst Lodge, Sunninghill, Ascot, Berkshire August, 1973 2.

Abstract

Studies were made on nuclear activity in Eimeria tenella an eirneriine coccidian parasite of the chicken. Results from these studies were compared with other Telosporidea, particularly the

malarial parasites.

With scanning microdensitometry measurements of the relative amounts of DNA in individual parasite nuclei were made. It was

shown that the only stage of the life cycle of E. tenella which

is diploid is the zygote. Other stages were shown to synthesise

DNA, until they reached the diploid value, in preparation for

mitotic division. No synthesis of DNA occurred in the early zygote but nuclear division followed fertilization restoring the

haploid state. The remaining two divisions in the oocyst were

mitotic.

From autoradiographic experiments it was shown that the 3 parasite did not incorporate H-thymidine or its metabolites into

its DNA but did utilize 311-adenosine or its metabolites for both

its DNA and RNA. Attempts to culture the parasite in tissue

.cultures, to enable closer regulation of the radioactive labelled

compounds supplied, were unsuccessful.

Attempts were made to overcome the difficulties caused by

the oocyst wall in fixation and embedding for electron microscopy. 3.

Although these were not entirely successful it was concluded that the general ideas on wall formation in the oocyst were correct.

No definite conclusions could be made regarding the possible persistance of the nuclear membrane during the meiotic division of the zygote or regarding the presence of centrioles or functional equivalents.

From PAS staining of the polysaccharide in sporozoites it was shown that the sporozoites in any one sporocyst were either strongly PAS positive or lightly PAS positive and that in any one sample of sporocysts there were approximately 5Z of each type present. The possible significance of these results in terms of sexuality of the parasite was discussed. 4.

Acknowledgements

I should like to express my thanks to Dr. E.U. Canning for

her supervision, guidance and encouragement throughout this work.

--I am indebted to Dr. L.P. Joyner of the Central Veterinary

Laboratory, Weybridge for the original samples of Eimeria tenella

and to Dr. L.P. Long of the Houghton Poultry Research Station for

his advice on embryonic and tissue culture methods for the

growth of E. tenella. I am grateful to Dr. R.E. Sinden for his instruction in the

techniques of electron microscopy and for his helpful discussion

and advice; to Mrs. A. Bishton for her endless patience in

obtaining references; to Mrs. B. Spain, Mr. P. Nicholas and

Mr. J. Smith for varied assistance and to my friends and colleagues

at Imperial College Field Station for their help in so many ways.

My thanks are due to the Medical Research Council for

financing this work and to Professor T.R.E. Southwood for the

laboratory facilities provided.

Finally, I should like to thank Mrs. C. Gower for so

accurately and patiently typing this thesis. 5.

Table of Contents

Page

Abstract 2

Acknowledgements 4

Introduction 7

Introductory Review 9 Zygotic Meiosis 9 Microdensitometry in the study of nuclear DNA content 13 DNA in Telosporidea 18 Nucleic Acid Synthesis 25 Nuclear Division in Eimeria 29 Embryonic culture of Eimeria species. 32 Tissue culture of Eimeria species 35 Sexual differentiation in Coccidia 38 Materials and Methods 48 Parasite 48 Chickens 48 Embryos 50 Scanning Microdensitometry 50 (1)Preparation of material 50 (2)Feulgen staining method 52 (3)Measurement of stained nuclei 54 (1+) Optimum wavelength for absorption measurements 55 (5) Optimum staining time for the Feulgen • technique. 55 (6)DNA reference slide 56 (7)Colchicine controls. 56 Autoradiography 57 (1)Preparation of slides 57 (2)Preparation of autoradiographs 58 (3)Development of autoradiographs 58 (4)Autoradiographic controls 59 (5)Chicken infections 59 (6)Embryo infections 60 (a)Preparation of sporozoites 60 (b)Inoculation of sporozoites 61 6.

Page

(7) Tissue cultures 62 (a) Cleaning and sterilising procedures 63 (b) Culture media 63 (1)CAM cultures 63 (2)Kidney cultures 64 (c) Setting up cultures 65 (1)CAM cultures 65 (2)Kidney cultures 66 -.electron Microscopy 68 (1)Preparation of material 68 (2)Fixation and embedding 68 (3)Sectioning and staining 70 PAS-staining (Periodic acid - Schiff) 71 (1)McManus technique 71 (2)Chayen's technique 72

Results 73 Scanning Microdensitometry 73 (1)Optimum wavelength for absorption measurements 73 (2)Optimum staining time for Feulgen technique 73 (3)Feulgen staining of parasite 76 (4)DNase controls 78 (5)Colchicine controls 79 (6)DNA reference standard 81 (7)DNA values for Feulgen-stained parasite nuclei 82 Autoradiography 89 Chicken infections 91 Embryo infections 98 Resul4 of autoradiography 101 (a)f:H-thymidine 101 (b)) H-adenosine 103 Tissue cultures 105 CAM cultures 105 Kidney cultures 105 Electron Microscopy 107 PAS (Periodic acid - Schiff) staining 122

Discussion 126

References 158

Appendix 176 7.

Introduction

Studies were conducted on Eimeria tenella, an eimeriine coccidian parasite of the chicken, to elucidate some aspects of its nuclear activity and to relate these to other Telosporidea.

Ideas on the ploidy of the nuclei of Telosporidea have been based on light microscope observations of stained structures interpreted as chromosomes but in many cases the nuclei are very small and the chromosomes difficult to distinguish. Therefore with E. tenella the DNA was stained by the Feulgen technique and the relative amounts of DNA in the nuclei of different stages of the life cycle were measured by microspectrophotometry allowing determination of ploidy and of the position of meiosis in the life cycle.

Autoradiographic studies with radioactive nucleic acid precursors were carried out to determine some of the requirements for nucleic acid synthesis. Little is known about the require- ments for nucleic acid precursors in the Telosporidea and most of the'information available is based on work with malarial parasites.

Electron microscope studies were conducted on the early oocyst of E. tenella to determine a suitable method of fixation 8.

for observations on the first zygotic nuclear division to see whether it differed from asexual nuclear divisions of this parasite and from nuclear divisions described in other Telo- sporidea.

It has been reported that the differing amount of poly- saccharide present in the merozoites which initiate gametogony in E. tenella is a reflection of a state of sex determination i.e. one type develops into a macrogamete and the other develops into a microgamete. Studies on the polysaccharide content of sporozoites were carried out using the PAS technique to determine whether they also showed this differentiated state and whether this idea of polysaccharide content being a reflection of sexuality is valid. 9.

INTRODUCTORY REVIEW

Zygotic Meiosis

The development of Telosporidea includes an asexual phase

followed by gametogony leading to the formation of gametes and

sporogony leading to the formation of snorozoites which are

usually enclosed in a cyst. •

The parasites must either be diploid with a reduction division

occurring during gametogenesis or haploid with a reduction

division immediately after formation of the zygote.

The first report of reduction division occurring in the

zygote nucleus was that by Dobell and Jameson (1915) who worked on

the coccidian AFg,recrata oberthi and the gregarine Dinlocystis

schneideri. A fuller account of the chromosome cycle of

A. eberthi was given by Dobell (1925) and Jameson (1920) rerorted

further on D. schneideri.

They concluded that the chromosome number in these two species was constant throughout the life cycle and that the

"reduction division" described during gametogenesis by earlier workers was not a true reduction division which halved the chromosome number. They pointed out that up to that time no chromosome counts had bean conducted on any coccidian species and that in the two cases of gregarines, in which chromosome 10.

counts had been made, they believed that Tr6gouboffd (1914) had misinterpreted his observations on Stenophora juli by claiming to have observed chromosome reduction from 4 to 2 by an. unequal nuclear division in the macrogametes before, during or after conjugation and that Nulsow (1911) had probably been dealing with two species of Monocystis, one with 4 chromosomes and one with 8 chromosomes, when he reported chromosome reduction immediately preceding gamete formation in Monocystis rostrata.

They considered A. eberthi and D. schneideri to be haploid parasites and that a nuclear reduction could only occur in the zygote, immediately after fertilization, in the 1st nuclear division. They thought that this condition would be found throughout the coccidian and gregarine parasites.

Further work on other genera has increased support for this idea. Reichenow (1921) recorded zygotic reduction division in Karyolysus lacertae, Grenier (1921) in Adelea ovata (although she was not sure whether it occurred in the 1st zygotic division or later), Naville (1927) in Klossia helicina, Patten (1935) in Merocystis kathae, Yarwood (1937) in Adelina crvotocerci,

Nabih (1938) in Klossia loosi, Hauschka (1943) in Adelina deronis,

Grell (1953) in Eucoccidium dinophili and Noble (1938) in the gregarine Zygosoma globosum.

Canning and Anwar (1967) reported that in ameria tenella and E. maxima the zygote nucleus contained ten, filamentous 11.

headed chromosomes. which condensed as short rods at the breakdown of the nuclear membrane and separated into two clusters of five after the first nuclear.division without division of the chromo- somes into chromatids and without chiasmata. This phenomenon was more fully reported by Canning and Anwar (1968). The haploid number agreed with Scholtyseck's (1963) report of five rod-like chromosomes in the nucleus of the microgamete of

E. maxima during the process of fertilization.

In the HaemosPoridia, Bano (1959) investigated nuclear division in the oocysts of seven species of Plasmodium and found that the reduction division was zygotic. She confirmed the observation of Wolcott (1954) that the blood phases of P. vivax were haploid with two chromosomes and found that the diploid condition in the oocyst after fertilization consisted of two rod- like chromosomes and two dot-like chromosomes.

Garnham et al (1957) using the Feulgen technique on the exoerythrocytic schizonts suggested that there were four chromo- somes in the haploid condition in P. knowlesi

Canning and Anwar (1966, 1968) looked at the chromosomes of the zygote nucleus of Plasmodium gallinaceum and reported the presence of two Y-shaped chromosomes and two dot-like chromosomes which agreed with Bano's findings on the same species except that the shape of the larger pair of chromosomes differed slightly. 12.

Bano reported them as being J-shaped. Canning and Anwar (1969) recorded the presence of two homologous pairs of chromosomes in the oocysts of P. cynomolgi, P. vivax and P. berghei. One

Pair of chromosomes was elongate, sometimes Y-shaped and the other pair was round and dot-like.

--Subsequently Canning and Sinden (1973) reported that from- ultrastructural observations of P. berghei oocysts they thought the haploid number of chromosomes to be not less than 5 and might be as high as 10. The structures, interpreted as paired chromosomes, seen in the blood stages of malaria Parasites by

Wolcott (1954, 1957) and in the oocysts by Bano (1959) and

Canning and Anwar (1969) they believed were iin reality fragments . of an enlarged digitate nucleus, an interpretation in accord with the reconstruction of nuclear events in cocyst development advanced by Howells and Davies(1971). Canning and Sinden concluded that the belief that the first Post-zygotic division represented meiosis might still be correct but that their ultrastructural studies had not revealed further clues to the position of the reduction.

Canning and Anwar's (1968) Paper introduced the use of

Colcemid (Ciba), an alkaloid from the meadow saffron Colchicum autumnale, in the study of nuclear division in Eimeria. This alkaloid is similar in action but less toxic than colchicine and has the power to•inhibit mitosis in metazoan cells, arresting

division at metaphase by inhibiting the division spindle. They

hoped that colcemid might interfere with the oocyst meiotic

division so that chromosomes in their most compact form could he

studied at metaphase. The colcemid, however, appeared to have

no effect on nuclear division which continued normally and they

showed that colcemid-treated oocysts Produced normal infections in chickens. Nevertheless, if oocysts were incubated in 0.2%

(w/v) colcemid within four hours of removal from the host then

they could subsequently be well fixed by acetic alcohol. They suggested that a possible explanation for this phenomenon was

that the colcemid delayed hardening of the oocyst wall rendering it penetrable to fixatives.

Previously studies on the zygote nucleus of the Eimeriina had been made only on the marine forms. A. eberthi and M. kathae with thin and permeable walls. Now, however, the strongly resistant oocysts of Eimeria which had been a barrier to chromo- some studies amongst this group of the Eimeriina had been breached.

Microdensitometry in the study of nuclear DNA content

The deoxyribonucleic.acid (DNA) content of a- normal cell nucleus is related to the size and number of chromosomes present ik,

in that nucleus.-,- From studies on the chemical nature of chromo- somes in eukaryotic cells it was discovered that DNA was found to be located almost exclusively in the nucleus with the exception of organelles such as plastids, mitochondria, and the kineto-. plast-blepharoplast complex of trypanosomes. Moreover the amount of DNA per diploid set of chromosomes was constant for a given organism and equal to twice the amount present in the haploid sperm cells. For example Mirsky and Nis (1949) and Vendrely and

Vendrely (1952) found a very constant ratio of 4 between the

DNA content of the sperm nucleus and that of the erythrocyte nucleus in a number of species of fish, chicken and toad. These data were obtained by gross chemical analysis performed upon nuclei isolated from one organ or another; an enumeration of the nuclei in the suspension allowing the amount of DNA per nucleus to be calculated. The mean value of DNA per nucleus would not correspond to reality if the suspension of nuclei studied contained some which were in the process of synthesising

DNA before division as in rapidly growing embryonic tissues or if some polyploid nuclei were Present.

The introduction of the technique of cytophotometry made possible the direct measurement of the amount of DNA in a single nucleus chosen from others under the miCroscope. Cytophoto- 15.

metry in visible light, which has become an efficient tool for

the study of nuclear DNA content, was developed from ultraviolet

microphotometry introduced by Caspersson (1940). The wide use

of this technique has yielded highly reproducible results which

have been checked by counts of chromosomes, biochemical data and

ultraviolet microspectrophotometry. Thus it can he considered

a reliable and precise means of investigation.

The method consists of staining the nuclei to be measured

by a qualitative and quantitative stain for DNA and measuring

the absorption of light by the dye-combined DNA of the whole

nucleus. The staining method introduced by Feulgen and Rossen-

beck (1924) is the most widely used. The validity of this

technique is conditioned by the specificity for DNA of the

Feulgen reaction and it is imnortant to be certain that the reaction specifically stains DNA producing a colour intensity

Proportional to the concentration of the DNA in the nucleus.

The Feulgen technique depends upon the treatment of fixed tissues by mild acid hydrolysis which Feulgen showed could release aldehyde grouts from the deoxyl-lentose sugar of the DNA. The exnosed aldehydes are then demonstrated by immersing the tissues in Schiff's reagent (Basic fuchsin decolourised by sulphur dioxide) which results in purple staining of nuclear chromatin. 16.

- Lessler (1953), discussing the specificity of- the reaction, reported that among the investigators who had studied the reaction there was general agreement that under aroterly controlled conditions the Feulgen nucleal reaction was specific for DNA and for the deoxypentose nucleoprotein found in the nuclei of cells.

Kurnick (1955) in his review of the histochemistry of nucleic acids stated "For the qualitative localization of DNA, irrespective of its state of polymerization (provided it meets the criterion of acid insolubility which is characteristic of nucleic acid as opposed to smaller nucleotide complexes), the

Feulgen nucleal reaction is probably the stain of choice. Of course, in very low concentrations, as in some oocyte nuclei,

DNA staining may be so faint as to be indiscernible. Localisation is now generally considered to he accurate, although the recent data of Chayen and Norris (1953) on root meristem cells may demand re-evaluation of this conclusion".

Pearse (1968) discussed the numerous arguments for and against the Feulgen technique but stated that "its specificity for DNA is. able to survive the conflict reported below, and it may be used with confidence for the purpose".

As to its ability to act as a quantitative dye Lessler

(1951) applied the Feulgen method under strictly controlled 17.

conditions to varying quantities of extracted DNA dissolved in

20% gelatin. Examining the preparations in a specially constructed microcell under a spectrophotometer, he showed that the intensity of colour developed was proportional to the temperature and duration of hydrolysis andi within limits ,to the concentration of

DNA:— He concluded that using carefully controlled methods with a large number of readings per slide relative DNA concentrations could be measured but he felt that extrarolation to absolute amounts of DNA per nucleus from densitometric measurements of the Feulr7en colouration went beyond the data.

Similarly Wheeler (1969) using DNA/gelatin preparations showed that within the limits of biological error the readings obtained increased proportionally with the amount of DNA present in each preparation. A similar linear increase in readings was shown with Preparations of liver sections cut at various thicknesses. He concluded that the Yeulgen technique could be used both as a qualitative specific stain for DNA and also for the ouantitative measurement of this substance.

Vendrely and Vendrely (1956) reviewed the .early work on cytoohotometry, discussed the validity of the technique, and the use of the Feulgen reaction in quantitative nuclear studies.

They also reached the conclusion that cytophotometry and the 18.

Feulgen reaction'elere valid for this purpose despite early

criticisms, concerning methods of measurement and reliability

of the Feulgen techniqu, so long as carefully controlled

conditions were used.

Since this early work improvement in the method of cyto-

photometry has occurred and now integrating scanning_micro- spectrophotometers are available. One of these, the Vickers

M85 scanning microdensitometer, was used in the Present work.

As pointed out by Wheeler (1969) the use of an integrating micro-

densitometer eliminates one of the most important sources of

mismeasurement in cytophotometry - the distributional error which

is due to the fact that the stained substance in the tissue is

not homogeneously distributed.

So the use of cytophotometry coupled with the Feulgen technique under suitably controlled conditions is a valid technique for investigating the nuantity of DNA in different nuclei. Thus these techniques can be used to measure the DA in different stages of a parasite's life cycle and the amount of DNA present will indicate whether a particular stage is haploid or diploid.

DNA in Telosporidea

The demonstration of DNA in the various genera of the

TelosporideL, is covered by a vast histological literature mainly 19.

concerned with the detection of. DNA by the Feulgen reaction sometimes accompanied by other tests such as methyl green staining and induced fluorescence using acridine orange.

Generally DNA has been found by one means or another within the nuclei of all species studied. Amongst the Eimeriina

Sassuchin (1935) obtained negative results, using the Feulgen technique, in the macrogametocytes and oocysts of Eimeria

oerforans and E. faurei but positive results in the microgameto-

eytes. Cheissin (19/40) with E. magna found the zygote and

merozoite nuclei to he Feulgen positive whilst the macroametocyte

nucleus was Feulgen negative. Lillie (1947) obtained a weak

nositive Feulgen reaction with the zygote nuclei of E. stiedae.

Pattillo and Becker (1955) reported DNA activity in all the asexual stages of E. Brunetti and E. acervulina. Microgameto-

cytes were Feulgen positive. In E. acervulina DNA was not

detected in the macrogametocytes but in young Feulgen treated

macrogametocytes of E. Brunetti a thin, faint coloured ring

was observed. However indications of DNA disappeared from the

rounded macrogametocytes even before they commenced to elongate.

Positive evidence of it was not seen again until the mature

oocysts were in the lumen of the intestine after leaving the

sites of their development in the mucosal epithelium. 20.

-Ray and Gill .(1955) considered that unsporulated oocysts of

E. tenella were devoid of detectable amounts of DNA and concluded that DNA contained in the sporozoites formed from similar oocysts

must have been formed de novo during the sporulation process.

In about 1W of the oocysts they found Feulgen positive granules in. the cytoplasm. Tsunoda and Itikawa (1955) found a positive

Feulgen reaction in most stages of E. tenella but found the nuclei of young schizonts were often Feulgen negative.

Horton-Smith and Long (1963) reported a weak Feulgen reaction of the macrogametocyte nucleus of E. maxima.

Chcissin (1957, 1959) again reported a Feulgen positive reaction in all stages except macrogametes of E. magna and also of E. intestinalis and E. media. He reported that the DNA was distributed on the periphery of the nuclei of schizonts and merozoites as well as of growing microgametocytes, and only in microgametes did the DNA appear to fill up the whole nucleus and look like a compact body. The nucleus of the macrogamete gave 'a reaction with methyl green which he said was an argument not for the disappearance of DNA from the nucleus but for its transition into such a state that it could not be detected by the Feulgen reaction. He did not find any indication of DNA in the cytoplasm of oecysts of these species as reported by Ray and Gill (1955) in E. tenella. 21.

Dasgupta (1959) working with E. stiedae found most early oocysts to be Feulgen negative but a few were faintly Feulgen positive. He also repbrted the presence of minute Feulgen positive granules in the cytoplasm of some of these early oocysts.

All other stages had Feulgen positive nuclei except for the macrogametocyte but this did have minute Feulgen positive granules in the cytoplasm.

Canning and Anwar (1967, 1968) reported that the early oocyst nucleus of E. tenella and E. maxima was without detectable

DNA using Azar A. but that at the onset of nuclear division ETA was detectable as filamentous, beaded chromosomes.

Similar results to those found in Eimeria species have been reported in Isospora species.

Bray (1954) working with two new species of Isospora from the mongoose reported that the macrogametocytes of Isospora garnhami and Isosnora hoarei were Feulgen negative whilst the microgametes were Feulgen positive.

Similarly, Anwar (1966) showed that all stages of Isosrora lacazei from the greenfinch (Chloris chloris) rave a positive

Feulgen reaction located at the periphery of the nucleus except in macrogametes which were Feulgen negative.

Generally all stages of the life cycles of eimeriine coccidians have Feulgen Positive nuclei e7:cept for the macro- 22.

gametocyte and macrogamete stages. There is little evidence to support the observations of Ray and Gill (1955) and Dasgupta

(1959) that Feulgen positive granules appear in the cytoplasm in oocysts.

The literature on the other Telospordea is more contradictory • regarding the outcome of the Feulgen reaction. For example

Pawan (1931) could not demonstrate DNA by the Feulgen reaction in Plasmodium falciparum or P. vivax whilst Lewert (1952) reported that P. gallinaceum, P. lonhurae and P. vivax had positive trophozoites and schizonts. Similarly, Bray (1957) reported a negative Feulgen reaction in the nuclei of early pre- erythrocytic schizonts in P. cynomolgi but Dasgupta (1959) reported them as giving a positive reaction.

From our knowledge of DNA being the hereditary material of an organism it is very unlikely that DNA does not exist in those stages where the Feulgen reaction has not demonstrated its

Presence. As already stated, Cheissin (1959) considered that the negative Feulgen reaction of the nucleus of macrogametes was probably due to the fact that the Feulf3en reaction was not sensitive enough. Pattillo and Becker (1955) were of the same opinion and quoted Alfert (1950) who, in a study of oogenesis and cleavage in the mouse, showed that in egg cells the Feulgen staining was diluted below the concentration at which it is visi ble. 23.

- • Pollister et al .(1951), also working on mice, showed very clearly that the amount of DNA per primary oocyte nucleus was constant and that this amount became progressively diluted as the nucleus increased in time. The decrease and disappearance of the Feulgen staining in oocytes was thus due to the progressive dilution of a constant amount of DNA in an increasing nuclear volume. Mulnard (1952) reached the same conclusion for oogenesis in the insect Acanthoscelides obtectus, Swift and

Kleinfeld (1953) obtained similar results in the grasshopper

Melanoplus differentialis and Alfert and Swift (1953) found the same p:attern in the annelid SabellariL.

With the Feulgen reaction the need for a carefully conducted, adequately controlled technique has been emphasised by many authors. Failure to follow such a course can lead to ambiguous results. The amount of DNA in the different stages of a parasite's life cycle will vary as with the state of the DNA i.e. whether it is being replicated or not. Both these factors could alter the ability to detect visually the presence or absence of a positive Feulgen reaction.

The amount of DNA present in the nucleus has been calculated for some species. For example Bahr and Mikel.(1972) reported that nuclei from mature schizonts of Plasmodium berghei contained 24.

0.49 x 10-13 gm of DNA per nucleus and Perrotto et al (1971) 13 that there was 1.0 0.2 x 10 gm of DNA per Toxonlasmo gondii

Parasite.

Chemically the nucleic acids of parasites correspond to those of free-living organisms. Whitfeld (1953a) showed that the absorption spectrum of the nucleic acids of P. berchei corresponded

exactly to the absorption spectrum of yeast nucleic acid. He also reported thatadenine, guanine, cytosine and thymine were the nitrogenous bases of the DNA. Gutteridge et al (1971) investigating malarial DNA reported that it was almost certainly double-stranded as both the density in caesium chloride and the absorbance at 260 nm increased on heating. Its failure to renature rapidly after heat denaturation suggested that it existed as a linear molecule rather than in a circular form.

They found that the base compositions calculated from thermal denaturation temperatures were similar to those calculated from buoyant density determinations suggesting that it was unlikely that the DNA contained any bases other than adenine, cytosine, guanine and thymine. However the base comrosition of avian malarial parasite DNA was at least 5.; lower than my ever recorded previously. They concluded that the differences in base composi- tion of DNA of rodent and avian malarial parasites from nrimte 25.

malarial parasites could be a. reflection of markedly different genomes. Apart from. the differences in base composition, the physico-chemical properties of the main comnonents of DNA from primate and rodent malarial narasites were very similar to those of DNA from mammalian cells. DNA from avian malarial parasites appeared to have some unusual properties.

Nucleic Acid Synthesis

Some Telosnoridea can utilise nucleotides, nucleosides or simpler precursors for nucleic acid synthesis.

Clarke (1952a, b) and 'ihitfeld (1953a, b) showed that in malarial Parasites rapid incorporation of inorganic Phosphate into nucleic acids occurred in various species whilst Anfinsen et al (1946) found the addition of both °urines and pyrimidines was necessary to ensure maximal in vitro multinlication of

Plasmodium kno,,!lesi.

Using P. knowlesi in culture Gutteridge and Trigg (1970) showed that radioactive pyrimidines (thymine, thymidine, de- oxycytidine, cytidine, uracil and uridine) were not utilised for

nucleic acid synthesis though the pyrimidine nrecursor, orotic acid, was utilised to a limited extent. They found that absence of pyrimidines from the culture media did not affect the rro-dth

Of parasites so it seemed that P. knclesi could synthesise the 26.

nyrimidine ring. All radioactive purines tested (adenine, adenosine, deoxyadenosine, guanine, guanosine and hypoxanthine) were incorporated into nucleic acids but they could not detect 14 incorporation of C-formate. The parasites did not grow as well in culture media without added purine as did control cultures with-purine thus suggesting that P. knowlesi was unable to synthesise the purine ring and was dependent on an exogenous source of purine for DNA and RNA synthesis.

Other workers have shown that malarial parasites do not incorporate purine precursors into their DNA but utilise preformed

purines for DNA synthesis. There arc Bungener and Nielsen

(1968) working with P. herghei and P. Ifinckei, Polet and Barr

(1968) with P. knowlesi, Van Dyke et al (1970) with P. hercrhei, and Walsh and Sherman (1968) with P. lophurae.

Polet and Barr (1968) and Van Dyke et al (1970) have also shown that malarial parasites cannot incorporate thymidire and

depend on the utilization of 7yrimidine precursors to synthesise

DNA.

Trager (1971) followed the incorporation of labelled precursors

into lo,)hurae develoning extracellularly in vitro. He showed

that incorporation of adenine was reduced in the presence of

ATP or sur7,gesting competition at an uptae site. 27.

- - Tracy and Sherman (1972) showed that the same parasite had

a remarkable ability, both intracellularly and extracellularly,

to take up and utilize certain exogenous purines such as adenosine,

inosine andehypoxanthine. These studies indicated that this

species has a functional purine salvage Pathway by which these

precursors can be converted to both adenine and guanine nucleo-

tides.

Working with Toxoplasma pondii Perrotto et al (1971)

reported that DNA synthesis could occur independently of the host

cell and that incorporation of preformed pyrimidines (thymidine,

cytidine) as well as Pyrimidine Precursors (orotic acid) occurred.

However the pyrimidine precursors were preferentially utilised

over preformed pyrimidines. There seemed to be little apparent

utilization of purine precursors (formate, glycine) but utilization

of exogenous purines (adenine, guanine) readily occurred. The

DNA synthesis of the parasite differed from that of the host cell

in that there was no apparent feedback inhibition by high

concentrations of thymidine nor detectable de novo ourine bio-

synthesis. They thought that these differences in DNA bio-

synthesis might enable the parasite to replicate more efficiently

within host cells. They also showed that labelled inorganic

phosnhate.and glucose were incorporated into T. condii DNA.

'Thus this parasite differs from malarial parasites in that it

will utilize preformed nyrimidines. 28.

Roberts et al (1970) showed a lack of incorporation of

3H-thymidine into Eimeria callospermophili in cell cultures. 3 In one experiment free H-thymidine was available to the host

cell and developing schizonts of the parasite whilst in their

second experiment only - H-thymidine or its metabolites which had

been incorporated into the nucleoside or nucleotide pool of

the host cell before inoculation of sporozoites was available

for DNA synthesis during schizogony. They examined the cells under the electron microscope after autoradiography and found

in both experiments that 5H-thymidine was incorporated into the

host cell nuclei but that no silver grains were observed over

the parasite nuclei indicatinP: that no incorporation of H-

thymidine had occurred. In both experiments some silver grains

were seen over the cytoplasm of host cells, but these were more

numerous in the second experiment. These results suggest that

the parasite does not utilize thr:iidine directly from the media,

from degraded host cell nuclei, or from nucleoside or nucleotide

pools of the host cell. The presence of some silver grains in

the cytoplasm of the host cells indicated that thymidine metabolites

(produCed by desethylation or enzyme action) were utilised in

1RIA synthesis. It thus seems that E. callor nrobably synthesises thymidine de novo. There appeers to be no informa-

tion concerning uptake of purines or purine precursors by any snecies of Lameria. 29.

Nuclear Division in ameria

Nuclear division in rimer ia species is not well understood.

However recent observations by light and electron microscopy have

added some insight into this Phenomenon.

The nuclear membrane in 7, imeria anpears to remain intact

during nuclear divsion except in the oocyst.

The division of the nucleus of sporozoites of Eimeria callosnermophil.i has been observed in cell culture in living specimens (Speer and Hammond, 1970) and under the electron

microscope (Roberts and Hammond). Throug,,hout the division

Process a prominent nucleolus was present and this, with the nucleus itself, beca me elongate and finally dumbell shaped.

After the daughter nucleoli serarated the nuclear membrane

became infolded in the area between them finally forming two sep?rate nuclei.

Using the el etron microscope, 'Roberts and Hammond saw a pair of centrioles adjacent to the nucleus in. some specimens whilt in others a ccntriole was -seen at one Pole with micro- tubules prencnt at the 1a e_ margin of tha nucleolus. Hammond

(1971) suf7Tests .'..lis couid represent an arrangement of spindle fibres similar to that retorted for trypanosomes by Rudzinska and Vickerman (193). Roberts and Hamond furtlwr reported 30.

that clumps of granules were seen in the nucleus of E. callosrerno- nhili when division was almost complete. These granules appeared to have a randOm distribution in the nucleoplasm and were thought to possibly represent chromatin.

The nuclear divisions associated with the formation of mer6Zoites differed from those during the early development of - the schizont. In the former the spindle apparatus was peripheral whilst in the latter, when observed, it was centrally placed.

When centrioles were observed they occurred in pairs adjacent to each pole of the peripherally placed spindle apparatus.

Lee and Millard (1971) reported that in E. praecox schizonts centrioles were clearly associated with the dividing nuclei.

During the division the nuclear membrane remained intact and there was an intranuclear spindle apparatus. - Similarly,

Heller (1971) reported a typical intranuclear spindle apparatus during nuclear divisions in the small schizonts of E. stiedae.

Scholtyseck (1965) reported that the microgametocyte nuclei of L. nerforans stretched during. microgametogenesis to form the microgamete nuclei without further division. However

McLaren (1969) considered that in E. tenella the peripheral nuclei elongated with the nuclear chromatin aggregating in the peripheral half of each nucleus and that it was only these 31.

aggregates which gave rise to the gamete nuclei. She felt that the part of the,gametocyte nucleus which was devoid of chromatin probably gave rise to the perforatorium and the three flagella of the gamete with the residual material being left in the gameto- cyte cytoplasm as a gametogenic cyst.

Hammond et al (1969) with E. auburnensis and Snigirevskaya

(1969) with E. intestinalis and y magna observed nuclear divi- sions associated with microgamete formation that were similar to those associated with merozoite formation. Snigirevskaya reported the presence of a mitotic spindle with a peripheral location in the dividing nuclei of microgamatocytes with a centriole located adjacent to each of the Poles and Hammond et al observed pairs of centrioles in association with the roles of the peripherally located spindle apparatus. It therefore seems that a similar pattern of nuclear division is associated with both merozoite and microgamete formation in some species.

From light microscope observations Canning and Anwar (1968) reported that in E. tenella at the beginning of zygotic meiosis the nucleolus and nuclear membrane disapared and a spindle was formed. .The only ultrastructural studies on the oocynts have been on the occyst wall (Scholtyseck and '3eissenfels. 1956;

Nyberg and Knapp, 1970b) and on the srorocysts and sporozoites 32.

(Ryley 1969; Roberts et al, 1970). Nyberg and Knapp (1970a)

reported scanning electron microscope studies on E. tenella

oocysts. No information, at the ultrastructural level, on

nuclear divisions has been reported for Eimeria oocysts.

Embryonic culture of Eimeria species

The first report of an eimeriine parasite completing its

endogenous life cycle in an embryo was that of Long (1965)

. working with Eimeria tenella and the developing chick embryo.

He injected sporozoites into the allantoic cavities of chick

embryos anddemonstrated the different stages of the parasite's

development in the chorioallantoic membrane (CAM) of the developing

embryo. The oocysts produced from such an infection, when

sporulated, produced typical caeca' infections in one-week-old

chickens. The life cycle in the embryo appeared to be slightly

delved with large numbers of oocysts occurring after the ninth

day of infection and the phase of schizogony being extended.

'Subsequently Long (1966) showed that, besides E. tenella,

E. Brunetti and E. mivati would complete their endogenous life

cycle in the CAM of the chick embryo but that E. necatrix did

not develop beyond late schizogony. E. acervulina and E. maxima

did not develop.. Thus the four species which would experimentally 33.

complete their endogenous cycle in the chick caeca would grow in CAM tissue but the two which were restricted to an intestinal development were not able to grow in the CAM tissue. Attempts to induce gametogony and oocyst production by the introduction of second generation merozoites of E. necatrix into the allantois were unsuccessful although similar stages of E. tenella initiated infection culminating in oocyst Production 1-2 days after inocu- lation into the allantois. Again the development appeared to be delayed.

The incubation temperature of the embryos in the first two reports were 38°C and 39°C respectively which is a few degrees below the normal body temperature (21- 1= 42°C) of the normal host, the chicken. Long (1970a) found an incubation temperature of 41°C was more suitable for the development of the parasite.

He showed that mortality caused by E. tenclia in embryos was dose related, that the "W" (Weybrid.,e) and "H" (Houghton) strains

Produced different mortality levels with the "W" strain being more virulent, and that there were differences in susceptibility of the three strains of 'ghite Leghorn embryos to E. tenella 0 infections. The production of poor infections at 38 C were in conflict with Ryley's (1968) results using 9-day-old embryos but

Ryley did not get infections in embryos with. E. brunetti. 31+.

Long (1970b) introduced a new method for evaluating infections

in chick embryos. This method, chorioallantoic membrane foci

counting, was used to evaluate anticoccidial agents. The chorio-

allantoic membrane foci are macroscopic and are associated with

colonies of second generation schizonts.

--Fitzgerald (1970) reported that E. stiedae, a mammalian

eimeriine, would also develop in the CAM of chick embryos. He could not find any oocysts or schizonts but did find early

gametocytes in the epithelial cells lining the CAN of the chick embryo.

If E. tenella sporozoites are introduced intravenously into embryos (Long, 1971) then schizogony and gametogony occur in the liver of the chick embryo with gametogony occurring more freely in dexamethasone-treated embryos. Long (1971) also showed that schizonts developed in the CAN of a corticosteroid-treated goose embryo when sporozoites were inoculated via the allantoic cavity.

Heceated passage of E. tenella through chick embryos produces a strain which loses its ability to produce large second genera- tion schizonts characteristic of this species (Long 1972). He reported that by the 37th nassae no haemorrhn,7e or deaths occurred in embryos. Youiv: chickens inoculated with embryo- passaged oocysts. ft d not develon leisons characteristic of 35.

E. tenella and again large second generation schizonts were not seen. However two passages of the "embryo adapted" strain in chickens was sufficient.to restore pathogenicity and the re- appearance of the characteristic second generation schizonts

These results indicate that the form and structure of schizonts and -merozoites are greatly influenced. by host factors.

Differences in embryonic response have been shown by

Jeffers and Wagenbach (1969). They reported a significantly greater mortality among female embryos than among male- embryos during the acute haemorrhagic response of the embryos to E. tenella infection. This sex difference in resnonse to parasitism was apparently a. general phenomenon and was found amonz embryos from widely different genetic s"urces.

Lee and Millard (1972) showed that the ultrastructure of the different stages of D. tenella in CAM infections was similar to the ultrastructure of those stages in normal caccal infections.

Tissue culture of Eimeria s:lecies

The first renort of this tyre of worn was that of Patton

(1965) who demonstrated the development of first generation schizogony of Eimeria tene?-a in culturad bovine kidney cells and japanese luail fibroblasts. At the same time Strout et al 36.

(1965) reported the invasion of a number of different types of cell in tissue culture by sporozoites of E. acervulina and the development of trophozoites.

Subsequently Doran and Vetterling (1967a, b, 1968) cultured other Eimeria species from turkeys and chickens (E. meleaa7rimitis and- E. necatrix) in a variety of cells through at least one cycle of asexual development.

Fayer and Hammond (1967) showed that sporozoites of E. bovis invaded a variety of bovine cultured. cells. Hcwever the sroro- zoites only grew to the first generation schizogony stage in secondary cultures of spleen, kidney and thymus. Later Hammond and Fayer (1968) found most rapid growth of first generation schizonts of H. bovis in bovine tracheal cell line cultures.

!1atsuoka et al (1969) Produced first generation schizonts of E. tenella in cell line bovine embryonic trachea.

By this stage it wa.s clear that the sporozoites of Eimeria species were canable of invading a wide variety of cultured animal cells but the conditions for schizongony were not provided in all cell types and initiation of infection by sporozoites did not result in the continuation of the life cycle to the gameto-

onous stage.

Bedrnik (1967a), using an inoculum of second generation 37.

merozoites of B. tenella, obtained further schizogony in so- called chick embryo "fibroblast" cultures and HeLa cells.

Later Bedrnik (1967b) obtained a small number of gametocytes and oocysts in the same "fibroblast" cultures but said the sexual stages were not in fibroblasts but in epithelial-like cells.

Using spOrozoites to initiate infection Bedrnik (1969) obtained only the first asexual generation.

Strout and Ouellette (1969) first reported the occurrence of a small number of macro- and microp;ametocytes in primary chick embryo kidney cells inoculated with E. tenella sporozoites.

By using cultures set un from CAT4 cells from chick embryos infected 4-6 days previously, Long (1969) obtained the production of gametocytes and infective oocysts of B. tenella.

Strout and Ouellette (1970) and Doran (197a) both recorted completion of the life cycle of T]. tenella in embryonic chick kidney cells. Doran (loc. cit.) also obtained complete develop- ment in kidney cell cultures from 2-3-week-old chicks but found embryonic kidney cultures more favourable for oocyst production.

Doran (1971) also reported completion of the life cycle of

E. tenella in both chicken and pheasant kidney cell cultures but only to ,1;ametogony in partridge cells and to mature second generation schizonts in turkey cel ls. Oocysts were found on the sixth d.,y in the chick cells and on the ninth day in pheasant cells,- 38.

At this time,.E. tenella was the only species of Eimeria whose complete endogenous life cycle had been achieved after sporozoites had been inoculated into cell cultures and this had only occurred in embryonic chick kidney cells, chick kidney cells and pheasant kidney cells. When initiating the tissue cultures from CAMS, the CAM cells were already parasitised.

Subsequently there have been many reports of different species of Eimeria Partially developing in tissue cultures and they have been used for studies of penetration and ultrastructural morphology.

Speer and Hammond (1972) showed the development of gameto- cytes and oocysts of E. magna from rabbits in bovine kidney cells.

This was the first renort of-the development in cell culture of macro- and microgamonts of an eimeriine species coming from a mammal.

Sexual differentiation in Coccidia

In the subclass Coccidia anisogametes are produced; the

"females" being large macrogametes developing from the macro- gametocytes without change and the ":males" being motile micro- gametes differentiating from microgametocytes after nuclear and cytoplasmic divisions. 39.

The determinant for the production of either male or female stages could be,environmental or genetical.

Considering environmental influences, the.merozoites would be bisexual and would produce male or female gametocytes depending upon some aspect of the environment. The merozoites could firSt- produce one type of gametocyte and this production result in some substance either being taken up from or excreted into the environment. This substance could have suppressed develop- ment of the other type of gametocyte or could now stimulate its production.

According to Grell (1967), in Eucoccidium dinophili a parasite of the body cavity of the archiannelid, Dinochilus gyrociliatus, infections with different numbers of snores showed that after weak infections no microgamonts or microgametes act:ear within the body cavity; only macrogamonts arise from sporozoites.

These develop without fertilization and meiosis i.e. Partheno- genetically. The absence of meiosis is clearly recognisable in life. because the characteristic stage of the "fertilization // spindle" is missing. Using only one spore for infection he found that the dumber of macrogamonts Produced. was variable in most cases there being only one but occasionally un. to six. Each macrogamont produced about 250 sores and if these were produced rarthogenetically from a single spore infection and used for mss ko.

infection then the next generation produced macrogamonts, micro- gamonts and microgametes. Thus bisexual reproduction including fertilization'and meiosis were possible.

Grell drew two conclusions from these experiments:

(1) Sex determination could not be genotypic. If it were, one spore could not yield both sexes.

(2) Srorozoites were not sexually determined when emerging from the spores. A weak infection produced only macrogamonts but a heavy infection ello,:fed the development of some sporozoites into microgamonts.

He thou:ht that the macrogamonts might Produce a substance which had to exceed a certain threshold concentration in order to effect the determination of sporozoites to microgamonts. He also thought it possible that a substance inhibiting the determina- tion of microgamonts might be withdrawn by the macrogamonts from the body cavity.

Grell also suggested that in Coccidia t:hare gametogony was oreceedeci by schizogony sex determination could be environmental too, occurring either before, during or after schizogony. If a change of host occurred, as in Ac_weefata eberthi, where schizogony takes place in a crab and gametogony and sporogony in a cuttlefish it is probable that sex determination happens during the growth i)hse of the i-ametes. Evidence against environmental determination being a general rule is shown by the adeleine Coccidia in which association of gametocytes occurs at an early stage of development from merozoites.

Therefore it would seem unlikely that a differential host influence is responsible. Also the uresence of one "sex" could not influence the development of neighbouring merozoites into the other

"sex" as it is frequently possible to get two male gametocytes associating or less frequently two female as shown in Adelina

(Canning, 1963).

Klimes et al (1972) pointed out that, although in vivo it is quite uncommon to find a number of E. tenella gametocytes develoring in one cell, in chicken kidney cell cultures the most prevalent forms are nests of gametocytes developing inside one host cell. They found entirely male, entirely female and mixed nests of gametocytes. If sexual differentiation was deter:lined by environmental conditions then one would expect that all the gametocytes in a nest of them in one cell would be the same sex.

Phenotypic determination of gametes would thus seem to be ruled out in this case. This has not been evident before as in vivo only one gametocyte of E. tenella develops in any one intestinal cell.

Edgar and Seibold (1964) described 3-5 F;Fv2tocytes of E.

:nivati developing in one ocithelial cell but did not say. whether 1+2.

they were all microgametocytes, all macrogametocytes, or a mixture of both. However their illustration concerning this phenomenon shows them to be all macrogametocytes.

If sex determination is not environmental then it must be genetic. As first shown by Dobell and Jameson (1915) the reduction division occurs in the first division of the zygote and this now appears to be universally accepted for Coccidia. The oocyst is bisexual as shown by the ability of a single oocyst to produce a patent infection, Jones (1932) showed this first with a single sporulated oocyst of E. maxima. Becker (1934) established a patent infection in rats with a single sporulated oocyst of

miyairii and found. asexual stages, gametocytes and gametes of both sexes in the intestinal epithelium. Reyer (1937) obtained a patent infection using a single oocyst of Barrouxia schneideri but was unable to obtain a ratent infection using a sin7le srorocyst which in this case has only one srorozoite.

He concluded the snorozoites were unisexual and 'the sexes were separated at the reduction division.

Other attempts to prort/ce singleL77orocyst and s/;:orozoite infections have been made without success T)erhars due to technical difficlties. Long (1959) attemated to produce single sporulated snorocyst infections with F]. maxima but failed. 43.

However he did succeed with 16 and 50 free snorocysts.

Sexual differentiation must take Place at some point between the zygote and the recognisable male and female gametocytes.

If genetically determined then the male and female characteristics must be senarated at nuclear division during sporogony or schiZogony.

Canning (1962) pointed out the three results one would expect depending UDOfl when the separation occurred:

(1)if separation occurred at zygotic meiosis, sporocysts would. be unisexual (male or female) and a single sporocyst would produce all male or all female infections,

(2)if at nuclear division within the sporocyst, then there would be male and female snorozoites within a single srorocyst which would produce a patent infection,

(3)if at schazogony, sporozoites would be bisexual and a single snorozoite would be sufficient for a patent infection.

Also she said that if male or all female infections resulted from inoculation of a single sporozoite then phenotyPic sex determination could be ruled out. .5imilarly the existecce of two sexually different tyres of rerozoites and the inability of a single snorocyst to produce both sexes w ould . indicate z:,3-otic meiosis as the Point of sex sep7iration.

There haire been many rerorts alloginp7 differences in merozoites recognisable at schizogony.

Yarwood (1937) and Hauschka (1943) discussed the early reports on sexual dimorphism. In some cases, as reported by

Hauschka, alleged sexual differentiation in merozoites, when re-investigated by other workers, appeared to be due to infections of more than one parasite being confused.

Yarwood herself reported that the male and female charac- teristics of Adelina cryntocerci were senarated during bipolar division of schizonts into merozoites.

Hauschka was not convinced by Yarwood's evidence and pointed to some flaws in the sexual-polarit7 scheme of Joyet--Lavergn e

(1926), Naville (1927, 1931) and Yarwood (1937). These were:

(1)the failure to account for the numerical_ difference existing between microgametocvtes and macrogametocytes,

(2)the assumption that both sexes could arise from the haploid nucleus of one and the same merozoite in one and the same environment,

(3)the occasional occurrence of an infection showing only unmated fema?es. (Hauschka himself describes such a case in

Adelina deronis).

Hauschka believed that in Adelina deronis the gametocytes of the two sexes arose from different schizonts of the second 45.

generation, the sex difference not being apparent until these schizonts were mature. He believed that the formation of male and female gametocytes at opposite poles of the same schizont in

Klossia and Adelina would be understandable if the organisms were diploid and meiosis was pre-gametic but that as these coccidians were -haploid throunhout their life cycle except in the synkaryon stage the idea was not acceptable.

Pell5rdy (1965) reported a number of cases where different types of schizonts or merozoites have been described. These were R?..y and Das Gupta (1937) in ':Ienyonella hoarei, Putherford

(1943) in Mmeria irresidua, Fh manna, H. media and H. perfor7,ns,

Pell'rardv (1953) in E. niriformis and Kotr,n. and Pell (1937) in E. stiedae.

Canning (196i) recorded differences in the carbohydrate reserves and the nucleus of male and female gametocytes in

3arrouxia schneideri and that there was some indication that these differences were reflected in merozoites from different schizonts. Schellack (1912) also recop;nised the presence of more than one type of merozoite in various Coccidia including

Barrouia but concluded they did, not renresent sexually dimorphic forms. Canninr; felt her results indicated that the forms mirht well be male and female merozoites but that the problem of whether these in turn were derived from sexually differentiated 46.

sporozoites remained unsolved.

Edgar and Seibold (1964) mentioned the possibility of sexual dimorphism in Eimeria mivati where they recorded the presence of a number of colonies of small schizonts with smaller merozoites, similar to the third generation stages, but present when the larger fourth generation schizonts with larger merozoites were present. They suggested the smaller merozoites might give rise to one of the sexual stages whilst the larger merozoites might give rise to the other.

Klimes et al (1972) reported the presence of two distinct types of merozoites which initiated gametogony in Eimeria tenella grown in cultured chicken kidney cells. One tyre was strongly

PAS-positive whilst the other had only a small number of positive granules. The PAS-positive merozoites continued as positive trothozoites and later macrogametocytes. The relatively negative merozoites developed into negative trophosoites and microgametocytE:s which were PAS-negative. This difference in staining could also be seen in whole unruptured schizonts, which were either pos:rtive or negative, but only of the second or third generation. The residual bodies of second generation schizonts were also PAS-positive in female schizonts and PAS- negative in male schizonts. 47.

-Related to this finding, Scholtyseck et al (1969) examining the merozoites of E. tenella under the electron microscope found that the presence or absence of carbohydrate inclusions was apparent. They considered that there were two possibilities for this. Firstly they thought there might be a difference in carbohydrate content between different merozoite generations or secondly that the carbohydrate represented a reserve of material used to provide energy for movement and was used during the motile phase producing the observed difference in carbohydrate levels. This second possibility was supported by light micro- scope histochemical studies of Gill and Ray (1954) and-Rootes and

Long (1965).

However Klimes et al (1972) pointed out that as the difference in PAS-staining, which reflects the difference in carbohydrate levels, could be seen in unbroken mature schizonts it did not seem to be influenced b:r depletion of the carbohydrate store durin27 movement as su_ested above. 48.

MATERIALS AND METHODS

Parasite

Experiments were carried out on Eimeria tenella obtained by passage, through two week-old cockerels, of original samples of the Weybridge strain received from Dr. L.P. Joyner, Central

Veterinary - Laboratory, Weybridge, Surrey.

The cockerels were infected per os with'sborulated oocysts suspended in 1.0 ml of distilled water or of 2.5;5 (w/v) aqueous potassium dichromate solution.

Oocysts were harvested after seven days from the cockerels' faeces or by squeezing the oocysts from the dissected caeca.

To obtain infected caeca. with the parasite in different stages of its life cycle doses were given as set out in Table 1

Chickens

The chickens used to maintain a collection of Eimeria tenella oocysts were obtained as day-old coccidia-free Ranger cockerels from Sterling Poultry Products Limited. They were kept at o -^- 3'1- , rfo twot- , :seeks at which stare they were transferred to a different building and infected with the parasite. Here they were kept at an ambient temperature of 25°C in metal ca ses, 49,

Table 1 Dosage of sporulated oocysts for Eimeria tenella

infections.

Dose of Time from Approximate Stage of Life Cycle Required sporulated infection to oocysts removal of caeca (Days)

Tronhozoites 1,000,000 1-1a

Early first generation schizonts 1,000,000 2-2

Late first generation schizonts 1,000,000 3-3

Early second generation schizonts 50,000 4-43 Late second generation schizonts 50,000 5-5-;,. Early - late gametogony 5,000-10,000 6-64 Late gametogony - oocysts 1,000-5,000 7-71-

Oocysts 1,000 8-83 50•

given water ad libitum and fed on chick starter mash No. 508

(which is coccidiostat free) obtained from British Oil and Cake

Mills Limited (B.O.C.M.)

Embryos

-The- embryos used for cultivation of Eimeria tenella were obtained as white, fertilised, Apollo eggs from Sterling Poultry

Products Limited. They were incubated at 39°C in a humid atmosphere for eleven days before introduction of T.]imeria sporozoites into the allantoic cavity. The eggs were turned between one and three times a day before infection but. after infection were left undisturbed until the infected membranes of the egg were harvested.

Scanning Microdensitometry

(1) Preuaration of material

Smears made from infected chicken caeca at different stages in the parasite's life cycle were wet fixed in Carnoy's fluid

(Absolute alcohol : chloroform : glacial acetic acid, 6 : 3 : 1) or in acetic alcohol (1 : 3) for two hours.

For secti_ons, portions of infected caeca were flushed with distilled water to remove the contents before fiy..ation in

Carnoy's fluid for two hours. After dehydration and embeddin 51.

in wax, sections were cut at 1+-5tUM.

To prepare oocysts chickens were killed on the seventh day of infection, the caeca.were removed, and placed in distilled o, water at 4 The caecal contents were flushed out in a stream of distilled water and discarded. The oocysts were removed from the- host cells by scueezing the caeca between the fingers.

These oocysts were washed repeatedly in cold distilled water and finally concentrated in a small volume by centrifugation.

Sporulation was carried out in distilled water at 26°C with air bubbled through the medium, After one hour the oocysts were concentrated by centrifugation and placed in 0.25 (w/v) aqueous colchicine (B.D.H. Chemicals Ltd.) solution at 26°C for one and a half hours. After this time the oocysts were washed free of colchicine with distilled water and sporulation was continued under the original conditions. The colchicine was used to make fixation easier,possibly by delaying the hardening of the oocyst wall. Samples or oocysts were removed at various time intervals after the colchicine treatment and fixed in acetic alcohol or

Carnoy's fluid for two hours.

After fixation the oocysts were washed in absolute alcohol and then prepared for sectioning. Originally the oocysts were concentrated in 70 alcohol and transferred to a cavity in a small cube of mouse brain tissue also in 70 alcohol. The cavity 52.

had been lined with glycerol albumen and on immersion of the whole in 90% alcohol the albumen coagulated fixing the oocysts in situ for subsequent easy handling in dehydration, embedding and sectioning. However, it was found that it was easier to put the oocysts in 1% agar solution in a TAAB embedding capsule at the

70% alcohol stage and centrifuge the capsule until the agar hardened. The oocysts were thus concentrated in the tip of the capsule. The agar tip was cut off for dehydration, embedding and sectioning.

Blood smears, for the use of erythrocyte nuclei as standards for DNA readings, were made from healthy chickens and were wet fixed in Carnoy's fluid or in acetic alcohol for two hours.

(2) Feulgen staining method

To stain the deoxyribonucleic acid (DNA) of the experimental material, the qualitative and quantitative staining technique using basic fuchsin was used (Feulgen and Rossenbeck, 1924).

Sections, after removal of wax, and smears were taken throuFJ1 descending grades of alcohol to distilled water and were then hydrolysed in 111 HCl at 6000 for eii7ht minutes if fixed in Carnoy's fluid or for ten minutes if fixed in acetic alcohol. Optimum hydrolysis times for this technisne were worked out by F:aver (1932).

After hydrolysis and rinsing in distilled water the nrerarations were placed in Schiff's reagent for ninety minutes at room 53.

temperature. They were then rinsed in three changes, each of two minutes duration, of a freshly prepared solution of equal parts of 1% sodium metabisulphite solution and 0.1N HC1. A further rinse in distilled:eater was followed by dehydration in alcohol, clearing in xylol, and mounting in Canada balsam. All slides and coverslips used had been cleaned in acid alcohol and were of the same quality and thickness.

Accompanying each test slide through the above procedure was a chicken blood smear as a DNA reference slide of nucleated chicken erythrocytes.

Deoxyribonuclease (DNase) control slides were used to show that the Feulgen method stained only the DNA. These controls were treated with 0.1% DNaoe in veronal-HC1 buffer at pH7 to which had been added an equal volume of 0.05M cysteine hydrochloride in

0.05M sodium acetate solution at Pf47. Magnesium chloride was also added to give a concentration of 1%. The slides were treated with this solution for 6 hours at 37°C. Similar preoarations were treated in the same way except that 0.1M zinc sulphate was added to inhibit the prase. The cysteine hydrochloride inhibited any proteolytie activity present in the "crystalline" DNnse whilst the magnesium ions were included to activate the DNase.

All controls were fixed in the same solution as the e:('eeri- menta] slides they accomeanied. 54.

(3)Measurement of stained nuclei

The Feulgen-stained nuclei were measured on the Vickers 1185 microdensitometer which'is an automatic scanning microspectro- photometer for the measurement of the absorption properties of microscope specimens in transmitted light.

--All specimens measured were mounted in Canada balsam on microscope slides of the same quality and thickness.

Measurements of the different specimens which were to he compared were made at the same wavelength and measuring spot size

()N, 60 (573 nm) and spot size 2) under the same power objective

(x100 oil immersion lens with ALP 1(ND = 1.524) non-drying immersion oil).

The integrated readings of the densitometer were directly proportional to the amount of stain present and thus the amount of DNA present. Therefore-the integrated readings obtained from different specimens could be directly compared after any necessary adjustments, as indicated by their DNA reference standard, had been mde.

The Power of objective used was chosen according to the three criteria:-

(1)the specimen image had to be as large as possible whilst remain:1hp, within the total :neasuring area,

(2)sufficient scnning had to be transmitted to the 55.

measuring photomultiplier within the limitations imposed by high electronic gain.noise, monochromotor band width, spot size and selected wavelength,

(3)the objective depth of focus had to be sufficient to accommodate the whole depth of the specimen.

(4)OPtimum wavelenth for absoLation measurements.

For the most accurate results the absorption of the DNA- stain complex must be measured at its optimum wavelength.

A smear of chicken blood cells was stained by the above

Feulgen method and the absorption of one erythrocyte nucleus was measured at various wavelengths on the Vickers n85 scanning micro- densitometer. The wavelength which gave the highest reading for absorption was selected as the optimum wavelength.

(5)OPtium staining time for the FT:II:Ten technique.

To find the optinlum staining time for the Feulgen method, a number of smears of chicken blood were prepared and fixed in

Carnoy's fluid. After hydrolysis the slides were placed in

Schiff's reagent and one removed at the following time intervals: , , flour, acurs, 2-?1:- hours and 20 hours. i,fter completing the staining procedure the erythrocyte nuclei were measured on the

1185 densitometer at A 60 (573 nm). Four readings of tw.nty- five nuclei per slide were measured and the ootimun stainin tifne 56.

selected.

(6)DNA reference slide.

Some variation in depth of staining was bound to occur between slides stained at different times due especially to the quality of the stain. Some reference point cf stable PNA content had to be used as a standard for comparison with each batch of coccidial slides.

For this purpose ram's sperm and chicken erythrocytes were considered. These were both stained by the Feulgen method and measured on the 1,25 densitometer.

From the noint of view of staining and measurement both candidates seemed good. However, the ram's sperm could only be supplied a few times a year and it is probable that the amount of DNA in ram's sperm nuclei may decrease under storage conditions

(Dr, H.M. Lott, A.R.C. Animal Research Station, Cambridge : nersonal communiction). As nucleated chicken erythrocytes were readily available in a fresh condition they were chosen as the

DNA reference standard,

(7)Colchicine controls.

To ensure that the colchicine was not affecting the nuclear divisions during snorulation some samples of oocysts were treated as above except that thf.., colchicine treatment was omitted and the 57.

oocysts ground up in a tissue homogeniser to break the oocyst wall

before fixation.• This procedure was not used routinely as many

of the oocysts became toe, damaged for the nuclei to be found

after staining. The stained nuclei were measured on the N85

densitometer.

Also a group of colchicine-treated oocysts were allowed to

complete sporulation and these sporulated oocysts were fed to

three chickens. The faeces from each chicken were examined for

oocysts from the sixth day after infection.

Autoradiography

(1) Preparation of slides.

Microscore slides were soaked overnight in an acid cleaning

solution consisting of 100 gm. of potassium dichromate in 850 ml

of water with 100 ml of concentrated sulphuric acid added. The

. slides were then washed for several hours in cold running tap-

water and rinsed in two changes of distilled. water for thirty

minutes each. The slides were then dipped into a freshly

prepared, filtered, aqueous solution cf 0.5 7,e1 atin/0.05

chrome alum ard allowed to drain and cry in a dust-free at;Nosphere.

This coating of "subbing" solution on the slide provided mood

adhesion both for the sections and the emulsion. of the auto-

radiopTa-ohic film. 58.

(2) Prenarat,:ion of autoradiographs

Subbed slides with 3 kuhlwax sections of the material to be autoradiographed were dewaxed in xylol, hydrated, and immersed in distilled water.

The subsequent techniques were conducted in a cool, slightly humid darkroom lit with 15 watt bulbs covered by Wratten No. 2 filters.

Strips of Kodak AR-10 fine grain autoradiographic film were cut from their glass Plates and floated for 3 minutes on water in a water bath at 25°C to allow for expansion of the film.

The slide, with the sections to be autoradiogranhed, was placed under the floating film and-raised out of the water so that the film wrapped round the slide over the sections and overlapped onto the back of the slide. After drying in warm dust-free air the slides were placed in a light-tight plastic box with a small quantity of silica gel. The boxes were stored in a refrigerator at 40C until ready for development.

(3)Develoianent of autoradiogranhs.

Autoradiogranhs were removed from their boxes at time intervals from 2 days to 3 months of exposure and developed in

Kodak D-19 developer for 4 minutes at 20°C. They were then washed briefly in distilled water and fixed for 10 minutes. 59.

After thorough washing to remove any traces of photographic agents the slides were allowed to dry for 24 hours. They were then stained with methyl green-nyronin, Giemsa or Harris' haematoXylin.

(4)Autoradioeranhic controls.

Controls were needed with each batch of autoradiographs to exclude the presence of spurious counts and the possible loss of counts through some process reducing the efficiency of the recording medium. To control against false positives produced by chemozrephy, heat, light and oressore, en identical but non- radioactive specimen was included witn each batch of slides.

To control against false negatives due to loss of silver grains from areas in which they should be found, one experimental slide from each hatch of autoradiographs was exposed to light and replaced in the container holding the rest of the slides so that exposure and development could proceed under identical conditions.

(5)Chicken infections

All infections for autoradiogranhic experiments were produced by giving two-week-old cockerels oer os 5,000 - 10,000 sporulr.!.ted oocysts of . tenella in 1.0 ml of aqueous 2 .5h potassium dicrom7Yte solutjon. 60.

(6) Embryo infections

(a) Preparation of sp2rozoites

Sporulated oocysts'were collected from chickens' faeces

7 days after infection. The faeces were suspended in 2.5% aqueous potassium dichromate solution and filtered through a

40 mm-mesh sieve. The filtrate was concentrated by centri- fugation and treated with sodium hypochlorite solution at

4°C for 10 minutes (after Wa=:enbachi et al. 1966). This treatment breaks up the host cells and faeces whilst strilizing the oocysts. The resultant suspension was concentrated by centri- fugation in sterile centrifuge tubes and the oocysts resuspended in sterile 20% (w/v) aqueous sodium chloride solution. The floating oocysts were harvested and placed in sterile rhosphate buffered 0.9% (w/v) saline at pH 7.6. They were repeatedly washed in buffered saline until the smell of hypochlorite solution had disappeared.

After concentration the oocysts were ffround up in a sterile homogeniser to release the scorocysts. These were incllb,!ted ir

0.5 (W/V) bile salt (Difco)/0.25% (w/v) trypsin (1:250 powder,

)ifco) in rhosnhate buffered saline at on 7.6 and 39°C for 2 hours. This solution caused excystation of the onorozoites which were then concentrated and w3ched free of excyst g medium with sterile phosohate buffered c'slino at nH 7.0. The sr:orozoites 61.

were separated. from emnty oocyst shells and srorocysts by differential centrifugation and concentrated in sterile Phosphate buffered saline at PH 7;0.

The glassware and buffered saline were sterilised by auto- claving at 15 lbs/sa,. in. Pressure for 20 minutes.

(b) Inoculation. of soorozoites.

11-day-old developing chick eggs were candled to determine the position of the major blood vessels on the chorioallantoic membrane. A mark was made on the shell to one side of a large blood vessel to indicate the site for injection of the parasites.

The concentrated snorozoites were counted in a haemocytomcter and diluted with buffered saline until there were apProximately

5000 in 0.05 ml of solution.

The egs were swabbed with 70, alcohol and a hole made in the shell at the injection site with a sterile needle. The snorozoites, in 0.05 ml of medium, and 0.05 ml of antibiotics

(penicillin 2000 i.u./strentomycin 2.000 .::ere injected into the allantoic cavity using sterile syringes (see Fig. 1). The hole in the shell was sealed by paintini:: collodion solution over it. The eggs were re:Jlaced in the incubator and maintained at

400C Instead of 390e. 62.

Fig. 1 The embryonic membranes of the chick on the eleventh

day of incubation. (After Payne, 1972)

amniotic co.iry fusion of chocio-ollacook Ayiekdrem% olloetois and amnion ..""" / oVniection.

shell

shell oesbcose

1air sac

allostoic comq

albumen extroesebrioeic 'cc bah cavil/

yolk sac

(7) Tisslu,- cultures

Two methods were tried for groyinp; t(.rne-2_la in tissue cultures. These ,,:ere based on Losi;'s (1969) chorioallantoic tisr-_-;e culture r:ethori nud Dornn's (197a)) chicen kidney cell c ulture method.

For both -:.2thods P;-3u7 (1965) w,i,s used as a i:uidc for the rstancThrd nrecdures of tissue culture. 63.

(a)Cleaning and sterilising procedures.

All glassware, filter holders and instruments were cleaned in 2% Decon 75 solution'(Decon Laboratories Ltd.). Coverslins were cleaned in two changes of absolute alcohol and one of ether.

Glassware and instruments were wranped in Alcan foil and autoclaved at 15 lbs/so. in. pressure for 20 minutes after which they were placed in sterile cabinets and irradiated with ultra- violet light. Phosphate buffered saline was placed in clean glass bottles and mitoclaved as above.

Both the CO /air mixture and tryptose phosphate broth were 2 sterilised by filtration through 0.22 ',um millenore filters and

pre-filters in Swinnex 25 filter holders.

Before use the sterile cabinets were swabbed out with 70 alcohol and irradiated with ultra-violet light for two days.

The room in which the tissue cultures were preared was swabbed down with 70% alcohol, particularly all bench surfaces, before work began. The room was srrayed with alcohol the night before setting up tissue cultures.

(b)Culture mathn

(1) CAM cultures

The growth medium used was that of Long (Personal communication) which consisted of 64.

84 ml Medium_199

10 ml Tryptose Phosphate broth

5 ml Foetal calf serum

1 ml - Antibiotics

Each ml of tissue culture medium finally contained

100 i.u. penicillin and 100 iwg streptomycin

The tryptose phosPhate broth contained:

10.00 ml 1C10 dextrose solution

10.00 gm Tryptose

2.50 gm Sodium chloride

125 gm Di-sodium hydrogen orthophosphate

The volume was made an to 500 ml with de-ionised distilled

water.

(2) Kidney cultures

The medium used was "Doran's growth medium":

5 ml Foetal calf serum

10 ml 0.5 lactalbLrlin hydrolysate

-82.5 ml Hank's balanced salt solution

1.5 ml T.C. sodium bicarbonte

1.0 ml Antibiotics.

Each nil of growth medium finally contained 50 i.u.

penicillin and 50 pg streptomycin.

Lactroumin hydrolysate was made by dissolving 50 gm in

1 litre of de-ionised ..a 1e filtered througli a `:;eit-2., clarjf-in 65.

filter and autoclaved. It was stored at 4°C.

The above tissue culture materials were supplied by Difco

Ltd.

(c) Settingun cultures

(1) CAM cultures

The general methods employed were those of Long (1969).

Infections of E. tenella in chick embryos were set up as described on p.60-61. After 3 to 4 days the infected CAlls were removed from four eggs under sterile conditions, washed two or three times in sterile Hanks solution and transferred with a little Hanks solution' to a glass netri dish where they were chopped into small nieces and macerated with scapels. The pieces of tissue were washed, in a water bath, in Hanks solution for =} hour at 39°C then placed in 5 (w/) trypsin (Difeo

1:2'50) in Hanks solution for -;;- hour at 39°C to break up the tissue into small ar7f7rep:ates of cells. The suspension of cell a=71rep;ates was filtered through a 1.50pormesh sieve 2nd centrifuged for 10 minutes at 1000 r.n.m. The sediment was washed two or three times in growth medium and firslly reuspended in a small volume of it. The number of cell ag:7reFates (anpro7-:imatf:ly

8 cells rear a;TreL;nte) were counted usin a hamocytometer.

Alicuots of 5 ml containing up to 120,000 areF;ates were than n.(36E:a to strile ulestic naLri dishos (Sterilin Ltd.) in which 66.

were placed sterile cover-glasses. The dishes were placed in plastic sandwich boxes, gassed with a sterile 95% air/5% CO2 mixture and placed in an incubator at 41°C. Each sandwich box also contained an open dish of sterile, distilled water to which had been added a few drops of "Marinol" - an antibacterial agent - in order to maintain humidity. The growth medium in the dishes

was changed when necessary.

After two attempts to grow CAM cultures, both of which resulted in a fungal infection that prevented the CAM from growing and which could not be eliminated in snite of the sterile

precautions undertaken, Amphotericin B (Flow Labs., Ltd.,

Edinburgh) was added to the medium at a concentration of 5preml,

(2) Kidney cultures.

The method was based on Doran (1970b) and the details were supplied Long (Personal communication).

Three or four 10 day-old chickens were killed, the kidneys removed under sterile conditions, and placed in sterile phosphate

buffered O.97, saline (DH 7.4) at 41°C in a petri dish. After

the connective tissue had been removed the kidneys were chopped un very Finely, placed in a flask with mhos*" to buffered saline at 41°C and washed with more of this solution until the blood and fatty tissue was removed. Trypsin was added to a concentra-

tion of 5. (w/v) and the solution incubated at 41°C, whilst beirn): 67.

stirred with a magnetic stirrer, for 15 minutes. The supernatant

was poured off and stored at 410C with 1 ml of foetal calf serum

which inhibited the trypsin. The tissue was again incubated as above for 15 minutes, the supernatant was decanted off and 1 ml

of foetal calf serum added to it. The remaining tissue was

incubated for two three-minute oeriods, the supernatant being removed each time and the trypsin inhibited.

The four supernatants were added together and filtered

through stainless steel mesh (500/2/n.and 250/"..m.). The filtrate

Was centrifuged for 10 minutes at 1000 r.n.m. The supernataut was removed, the sediment resuspended in p.osihr,te buffered saline at 41°0 centrifuged as alove end then the sedi-ert "'S resus-

pended in Doran's growth medium. The cell aggregates were counted using a haerocytomet`er. 5 mi aliquots, containing up to 25,000

ggregates, were nipetted into sterile netri dishes and cover- slips added. These dishes were placed in sandwich boxes and

treated in te same -ranner as the i cell cultures.

Sterile srorozoites, nreoared as on u. 60, were suspended

jr porantS medium and '::ere 7Apetted into the dishes after 2 (yr-

when the kidney' cultures bed. estat)lishe,i

Coverslis were stained ir tjright's stain or Giemsa. 63.

Electron microscopy

(1)Preparation of material

Oocysts were obtained from infected chicken caeca as described on p. 51 . In some cases they were cleaned from cellular debris with 3% sodium hyPochlorlte solution at 0°C for 10 minutes and then floated on a saturated salt solution. After washing free from the hynochlorite and salt solutions the oocysts were snoru- leted in distilled water at 2.6°C with air bubbled through the medium. In other cases the hypochiorite treatment was omitted.

Oocysts were removed from the snorulation medium for fixation at various time intervals.

(2)Fixation and embedding.

Initially the following fixation schedule was followed:

Oocysts were fixed overnight in T, AB glutaraidebyde in 0.1M phosnhate buffer oh 7.4 at 4°C. After washing. for 3-12 hours in 3 chanmes of 0.15M nhosphate buffer at pH 7.4 t;_ -y were nest-osmicated ill 1; osmiifl in 0.1M buffer at 11 °C for 1 hours.

They .were then transferred to 70 alcohol at room temperature for

15 rinutes, dehydrated 71.1 three ch-nr-es of dry absolute alcohol

(15 minutes each) and transferred to an absolute alcohol/arsldite

(W50) mixture ,4hich was rotated overnight. They were trans- ferred to araldite for 1 day at room temperature and then djspensed into fr-sb aral-iite in embeddin er)psq?eri „hi,-11 were centri- 69.

fuged to concentrate the oocysts at the bottom of the capsule.

The araldite was Polymerised at 60°C for 2 days.

The oocyst wall of. the parasite, which hardens after release

from the host cell and becomes impermeable to most substances,

caused difficulty in the fixation and embedding procedures

therefore the following modifications were tried:

(a)The oocysts were treated with 0.2 (w/v) colehicine

solution for 2 hours after collection, washed free from colchicine,

and fixed as above.

(b)The oocysts were gently ground up in a homogeniser

nrior to fixation as above.

(c)Fixation was carried out as above but at room temperature

and under reduced pressure.

(d)Fixation in glutaraidehyde was carried, out at 39°G and

in osmium at 4°C.

(e)The oocysts were gently ground un in a hooe,:iser

prior to fixation at 39°C in elutaraldeyde and 4°C for osmium,

(f)Oocysts wcre treated with 0.2a, (w/v) trypsiP/0.% (w/v)

bile :.0.1.ution at l'?9(1(; for 1 hour, 1; hours, 2 hour, 4 hnurs,

6 'nouns, and 2/4- hours befo5-e 17xation as in the org3nel

(g)Oocysts were treated with 7,DTA/lvso7.7me solution

•(110 m:,-/m1 lysozym7! flnd ])17A in 0.I 17)hosh:to buffer)

at 370° for h minutes, 15 minute3, milnAtes. 1 hnur, hodrs, 70.

2 hourS, 4 hours, 6 hours and 24 hours before fixation as in the original schedule. Oocysts prepared by methods (f) and (g) were embedded in wax, sectioned at 10[4,611, stained in Giemsa and examined under the light microscope.

(h) Oocysts were gently ground up in a ho:aogeniser prior to o fixation at 39 ,u in glutaraldehyde and at 4 C in osmium but were embedded in Spurr's resin instead of Araldite epoxy resin. For this the following schedule was followed:

After fixation the oocysts were washed in 70 alcohol for

15 minutes and three changes of absolute alcohol for 15 minutes each. The oocysts were concentrated in 2 ml of the final alcohol wash and an equal volume of resin added. to the alcohol. The solution was rotated overnight at room temperature and then an eclual volume of resin was added again with further rotation overnight. The oocysts were spun down, excess resin -poured off and fresh resin added, again rotating the mixture overnight.

Finally after concentration and after the excess resin had been poured off, the oocysts were embedded in a TAB embedding capsule in fresh resin and this was ce,itrifured to sediment the oocysts in the tin of the capsule. The resin was polymerised for 12 hours at 70°C.

(3) Sectioning and staninr

Sections for electron mioroscony :ore cut on F',11 LX3-3 ultraicrotolne with prloss and fio:,te6 on 111"; acetone 71.

solution. They were picked up on TAAB 100 micron conner grids which were covered with formvar and carbon-coated.

The sections were stained with 2% (w/v) aqueous uranyl acetate solution for 10 minutes, rinsed in distilled water and dried. They were then stained in lead citrate solution for

10 minutes, washed in 0.021 sodium hydroxide, rinsed in distilled water and dried. The sections were examined on a Phillips

EM300 electron microscope.

Some sections were cut at 1pArn, stained with toluidine blue and examined by light microscopy.

PAS-staininf' (Periodic acid-Schiff)

Scorocysts were released from fully sporulated oocysts by grinding in a tissue homogeniser. They were then smeared onto slides and wet fixed in 0arhoy's fluid for 1 hour or Schaudinn's fluid for 50 minutes. With Sc!haudinn fixed material any free mercuric chloride was removed by 70?; alcohol to which a few droos of iodine alcohol had been added.

After fixation the material was hydrted in :iesending, fTrades-of alcohol and f!ln-Tlly plar:ed in distilled water. Two technilues were employed for staff

1) Nnus technilue (quoted in Pe71rse, 19:68).

The hydrated :.;irir.s were oxidir;ed for 10 minutes in aqueous Periodic wached r1.1in;.: watar for !7iinutefs. 72.

They were then treated with Schiff's reagent for 20 minutes, washed in running water for .5 minutes, dehydrated, cleared in xylene and mounted in DePeX.

2) Chayen's technique (1969)

This employed 2 modifications Of 14cNanus' method by using a reducing solution between periodic acid treatment and Schiff's solution and by washing after Schiff's solution with S02 - water instead of tap-water. Three controls were incorporated. The first control slide was treated with M/20 alcoholic solution of hydrochloric acid at room temperature, the second had no prior treatment, and the third was treated with an acetylation solution

(acetic anhydride 16 ml, dry pyridine 24 ml) for 13 hours at o00 C before treatment with Schiff's reaf7ent.

For the PAS stained slides sporocysts were examined and the number of heavily-stained and lihtly-stained sporocyoLs wee counted. 73.

Results

ScanningMi ri

(1)Optimum wavelength for absorption measurements

For accurate quantitative measurement of a DNA-Feulgen complex with the Vickers M85 scanning microdensitometer it is essential to

measure the absorption of monochromatic light at the optimum wavelength i.e. the wavelength which gives the highest absorption reading.

The absorption measurements at different wavelengths of one

Feulgen-stained erythrocyte nucleus are set out in Table 2 and

Figure 2. The wavelength which gave the maximum absorption (the highest integrated reading in absorption units) wash 60 on the wavelength dial. This value is equivalent to a wavelength of

573 nm and corresponds to the setting found by Gutteridge and

Trigg (personal communication) for Feulgen-stained malarial DNA.

This optimum wavelength also gave the smallest standard error over 10 consecutive readings (Table 2) and was used for all future measurements of Feulgen-stained nuclei.

(2)Optimum staining time for the Feulgen technique

Absorption measurements were made on Feulgen stained chick erythrocytes which had been stained for varying lengths of time.

Table 3 shows the range of readings obtained and the mean absorption value per nucleus after varying staining times. For individual X -4 4o 45 5o 55 56 57 58 59 6o 61 62 63 64 65 7o 1 36 67 112 130 141 139 142 140 150 138 142 140 133 124 24 Readings 2 33 65 110 134 137 140 140 140 150 138 142 140 136 4 122 22 in 3 35 68 118 137 137 137 138 139 149 141 144 139 135 125 24 densito- 4 35 68 114 135 138 140 138 142 152 137 141 140 137 125 26 meter 5 37 69 118 135 137 141 137 142 150 138 143 137 130 123 22 absorption 6 33 68 114 135 136 142 141 143 152 134 142 137 130 124 24 units 7 35 65 115 133 137 142 140 138 15o 138 141 136 135 124 24 8 33 69 115 135 136 143 139 140 150 135 140 140 132 123 22 9 29 68 117 135 137 143 136 134 152 141 142 140 131 123 24 10 32 71 118 134 1 6 141 136 139 152 137 140 142 131 120 24 arithmetric 33.8 67.8 115.1 134.3 137.2 140.8 138.7 139.7 150.7 137.7 141.7 139.1 133.0 123.3 23.6 mean standard 0.73 0.41 0.60 0.82 0.47 0.41 error (-0 o.56 0.85 0.56 0.47 0.60 0.63 0.79 0.37 0.69

Table 2 Variation of absorption with wavelength ()\ ) of Feulgen stained chicken erythrocyte nucleus (X40 objective; spot size 2)

Fig. 2 Variation of absorption values obtained at different wavelengths when measuring Feulgen stained DNA with the Vickers M85 scanning micro- densitometer. (Means of 10 readings given). X60 (573 nm) is the optimum wavelength.

Chicken red blood coil nucleus—Feulgen stained Variation of absorption with wavelength x 40 objective, spot size 2.

r--

40 45 50 55 56 57 56 59 60 61 62 63 64 65 70

A Reading 76.

readings see Appendix - Table 1 and Fig. 3 for a summary histogram.

Table 3 Variation of absorption values of Feulgen-stained chicken erythrocytes with staining time. (Measured under X100

oil immersion objective with spot size 2; 25 nuclei per

sample - four readings per nucleus).

Staining time Absorption Units

hours Ran e Mean Standard error I 241-289 264.86 +1.12 11. 436-511 465.56 +1.89 2 417-511 448.10 +2.32 20 352-426 379.35 ±1.98

MINIIMEIN=1■1•1111•01;1•02•••••■•••••

From the mean values obtained 1i hours staining gives the maximum absorption reading for the Feulgen-stained chick erythrocyte nucleus (465.56 + 1.89 units). This time was used for all future

Feulgen staining.. (3) Feulgen staining of the parasite Feulgen positive nuclei were found in all stages of the life cycle except in the macrogametocytes and macrogametes. These Fig. 3 Variation of absorption values obtained for different staining times with the Feulgen technique. (Mean of 25 nuclei given).

Chicken R.B.C.s - Feuigen stained Variation of absorption with staining time x100 oil obj., spot 2.

C e300

0 .0• 4

100

0 12 112 212 20 STAINING TIME -Hrs. 78.

findings are in general agreement with those of previous workers

with eimeriine coccidians.

Early schizont nuclei were often pale stained. Older schizont nuclei, merozoite nuclei, microgametocyte nuclei and

microgamete nuclei were distinctly Feulgen positive.

- Nuclei of early sporulating oocysts were Feulgen-positive.

The Feulgen positive material appeared as fine clumps mainly near to one edge of the zygote at the 22 hour stage. Pairing of chromosomes in two rows of five parallel with the elongate meiotic spindle (as described by Canning and Anwar, 1968) was not seen but the stretching of the Feulgen-stained DNA in an arch across the oocyst was seen. After the first zygotic division the resultant nuclei were seen as condensed clumps of chromatin at opposite ends of the region across which the chromatin had stretched. Later nuclei in the sporulation were also seen as condensed clumps. No indication of "centriolar granules"

(Canning and Anwar, 1968) nor of Feulgen-positive granules in the cytoplasm in oocysts as described by Ray and Gill (1955) and

Dasgupta (1959) was seen with the Feulgen stained material.

(4) DNase controls

DNase treated smears of nucleated chick erythrocytes and of sections containing E. tenella failed to stain with the Feulgen technique whilst those treated with tilt DNase incubation medium 79.

but with the DNase inactivated by 0.1M zinc sulphate showed the usual magenta colour of the Feulgen reaction. Thus the Feulgen stain was shown to be specific for DNA.

(5) Colchicine controls

Oocysts at the stage after the first zygotic division were stained without previous colchicine treatment. Absorption values for these nuclei (Fig. 4 and Table 4; Appendix Table 2 for readings) all fell within the range of those that had been treated with colchicine (Fig. 7; Appendix Table 6).

Table 4 Range of DNA values for nuclei after the first zygotic

division with and without colchicine treatment.

Nucleus Range of DNA values in standard units

.111M=MMENIMI. • rm.. SI

Post 1st Division

(a)Colchicine 14.20 - 32.00

(b)No colchicine 14.30 - 30.80

Two nonparametric statistical tests were applied to the two sets of data. The Mann-Whitney U test was applied to see if there was any difference in location between the groups i.e. Fig. 4 DNA values obtained for E. tenella zygote nuclei which had no colchicine treatment;

15

Zygote: 12 Post first division nuclei No colchicine 9 x100 oil obJ., spot 2.

6 •I

3

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 ______)Amount of DNA (standard units) 81.

whether one group was by and large greater or lesser than the other. .The Wald-Wolfowitz runs test was applied to see if the two groups of data differed in any way e.g. in central tendency, in variability, in skewness or whatever (see Siegel, 1956 for the tests used). Using 2 tailed tests in both cases values of p obtained were 0.3030 and 0.3320 respectively. Thus the two groups of data are not significantly different.

Further colchine-treated oocysts which were allowed to complete sporulation and which were subsequently fed to three chickens produced normal infections in these chickens and oocysts were passed on the seventh day as in a normal infection. It was concluded that the colchicine did not affect the DNA of the parasite and so readings of DNA from colchicine treated oucysts were considered as normal values.

Colchicine was used in these studies as Colcemid was no longer available in this country at the time. Similar results to those found by Canning and Anwar (1968) using Colcemid were obtained. Thus colchicine, too, does not interfere with nuclear division of the oocyst and delays hardening of the oocyst wall. (6) DNA reference standard Because of unavoidable differences in quantitative staining by the Feulgen reaction due to the effects of variation in batches of stain and the length and temperature of hydrolysis, 82.

chick erythrocyte9ontrol slides were made with each experimental slide. The average value from 25 nuclei per standard slide was calculated from the absorption measurements. This value was used to convert the parasite DNA absorption units to standard comparable units. The erythrocyte nuclei contained a fixed amount of DNA as they were non-dividing cells. Therefore variation in their absorption values reflected variation due to the staining procedure.

Converting the mean value for the control slide to 1000 units produced a conversion factor by which the absorption readings of the test slide had to be multiplied to convert them to standard comparable units. For example if the mean value of the erythrocyte nuclei on a test slide was 500 units then conversion to 1000 units gave a factor of 2. This factor was then used to convert the absorption values of parasite nuclei on the accompanying experi- mental slide to standard units.

(7) DNA values for Feulgen-stained parasite nuclei.

The Feulgen-stained nuclei from different stages of the life cycle.of Eimeria tenella were measured on the Vickers M85 micro- densitometer.

The arithmetric mean of 4 readings on a particular nucleus was multiplied by the appropriate conversion factor derived as above. The resultant value in standard units could then be used to compare the amount of DNA in different nuclei. 83.

Table 5 summarises the corrected values of DNA for E. tenella nuclei in different stages of the parasite's life cycle. Figures

5-8 are histograms of these different stages. The individual readings are given in the Appendix - Table 3-7. Measurements for microgamete nuclei were made on free micro- gametes. These nuclei had values from 13.25 - 15.11 units.

The arithmetric mean of the corrected values was 13.95 4. 0.11 units. As the gametes must be haploid, the value of 13.95 units was taken to represent the haploid amount of DNA. Schizont nuclei had values of 14.64 - 31.85 units. These readings were taken from immature schizonts in which the nuclei were still dividing and from mature schizonts with newly-formed merozoites. The full range from the haploid amount of DNA up to twice that value indicated the presence of nuclei actively synthesising DNA in preparation for division. Newly-formed oocyst nuclei i.e. ones within 2i hours of the start of sporulation had values of 27.20 - 31.60 units with an arithmetric mean of 29.75 + 0.17 units. This mean value, approximately twice that of the microgamete nuclei, was taken to represent the diploid amount of DNA. Nuclei after the first division in the oocyst i.e. the products of the first zygotic division had values from 14.20 -

32.00 units. 84.

The products of the second nuclear division of the oocyst had values from 14.40 - 29.14 units.

Table 5. Range of DNA values (standard absorption units) of

E. tenella nuclei throughout the life cycle.

Nucleus Range of DNA Arithmetric mean

values of values

/8.111/IIPINIOI■ey

Schizont 14.64-31.85 WI& Micro garnet e 13.25-15.11 13.95+0.11 Zygote:

Pre first division 27.20-31.60 29.75+0.17

Post first division 14.20-32.00

Post second division 14.40-29.14

Conclusions from these values are: (1)The microgametes are haploid

(2)Non-dividing schizont nuclei are also haploid but as DNA is synthesised in preparation for the next division the DNA value increases from the haploid amount to twice that value and mitotic division restores the haploid state. Fig. 5 DNA values obtained for E. tenella microgamete nuclei and for newly formed zygote nuclei.

15

Zygote 12 gametgametee Pre nuclei first division 9 nuclei

Both x100 oil obi., spot 2.

s. 8

T

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Amount of DNA (standard units)

Fig. 6 DNA values obtained for E. tenella schizont nuclei

15

Schizont nuclei 12 x100 oil obi., spot 2.

9

•

w 3

0 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Amount of ON A ($tammo units) Fig. 7 DNA values obtained for E. tenella post-first division nuclei of the zygote.

15

Zygote: 12 Post first division nuclei x100 oil obl.,opot 2. 9

0 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 _____4Amount of DNA Ostanderd units co

Fig. 8 DNA values obtained for E. tenella post-second division nuclei of the zygote.

15

Zygote: 12 Post second division nuclei x100 oil obi,spot 2.

0 L_ 12 13 14 15 16 17 16 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 _____44mount of DNA (stanclordunits) 89.

(3)The newly formed oocyst nucleus is diploid, as a result of the fusion of the haploid microgamete and haploid macrogamete.

(4)After the first zygotic division the resultant nuclei are haploid. These nuclei synthesise DNA until they have the diploid amount of DNA in preparation for further division.

(5)After the second zygotic division the resultant nuclei are haploid and synthesise DNA until they contain the diploid amount at which stage they will divide to produce haploid sporo- zoites.

Thus the only stage of the life cycle of E. tenella which is diploid is the zygote. Other stages are haploid and synthesise

DNA until they produce an amount equal to the diploid amount, in preparation for mitotic division. No synthesis of DNA occurs in the early zygote but nuclear division follows fertilization and the haploid state is restored immediately. Therefore the first zygotic division is meiotic and,is a single step reduction division.

The remaining two divisions within the oocyst are mitotic.

Autor'adiography To determine whether Eimeria tenella would incorporate a

pyrimidine or a purine into its DNA during growth, radioactive

DNA precursors were made available to the parasite and their

possible presence in the parasite was investigated by the use of

Kodak-AR 10 stripping film in the technique of autoradiography. 90.

All radioactive materials used (Table 6) were obtained from The Radiochemical Centre, Amersham„ Buckinghamshire as aqueous, sterilized solutions with a tritium label.

Table 6 Radioactive DNA precursors used

Substance Specific Activity (millicuries per millimole)

Thymidine ((methyl)-T) 2,000 Adenosine - H3(G) > 5,000

Guanosine, -8-H3 > 2,000 Orotic acid -5-H3 >15,000 (a pyrimidine precursor)

These tritium labelled compounds emit / -particles from the radioactive tag. These /3 -particles strike the photographic emulsion of the autoradiographic film over the specimen being

autoradiographed and activate the silver halide grains, forming a latent image which, upon development of the emulsion, will depict the distribution of radioactive material within the specimen. Thus one can determine whether the labelled precursor offered to

the parasite has been utilised by it. 91.

Chicken infections

Infections in chickens were initiated with 5,000-10,000 sporulated oocysts of E. tenella adminstered 292-...aa in 1.0 ml of aqueous 2.5% potassium dichromate solution.

Experiment I

-For this experiment four groups of chickens were set up:

(a)Chickens injected intra-peritoneally with 100iLidc of tritiated thymidine 144 hours after initiation of infection

(b)Uninfected birds injected intra-peritoneally with 100 pe of tritiated thymidine at the same time as Group (a)

(c)Infected birds not treated with tritiated thymidine

(d)Chickens treated with neither E. tenella nor tritiated thymidine.

One bird from each group was killed at 168 hours and one at

192 hours after initiation of the infection. The caeca were removed, fixed in Carnoy's fluid for 2 hours, dehydrated and embedded in 60°C M.P. paraffin wax. Sections were cut at 3p.ent and autoradiographs set up. After exposure and staining, the autoradiographs were examined under the light microscope.

Autoradiographs of up to 2 months exposure were searched for the presence of silver grains. Neither the host cells nor the

parasites from chickens injected with tritiated thymidine produced silver grains in the stripping film over it nor did the non- 92.

radioactive controls. Light exposed controls developed normally showing no loss of silver grains therefore there was no latent image-fading. Thus both host material and parasite were negative. The possible explanations for these results are:

(1)that the 3H-thymidine was not circulated to the caeca from the injection site, (2)that the host tissue did not take up the radioactive label and therefore this label was unavailable to the parasite,

. (3) that the 3H-thymidine was taken up by the host and/or parasite but at such a low level that the technique was not sensitive enough to reveal it. A range of larger doses of 3H-thymidine was used in Experiment

2. Ex eriment 2 For this experiment 3H-thymidine was injected intra-peritoneally into chickens 144 hours after ipitiation of an infection of

E. tenella. The different dose levels used were 1000 pc, 800 p-c,

400 /Pc, 200 pc, 100 /Pc and a non-radioactive control. 24 hours after injection of the 3H-thymidine the chickens were killed and the caeca fixed in Carnoy's fluid for 2 hours. Wax sections of the tissue were prepared and autoradiographs set up. The autoradiographs were exposed for increasing time intervals, stained and examined. - 93.

Table 7 summarises the results of this experiment. After

2 weeks exposure autoradiographs, made from chickens given 1000 /Ac

of 3H-thymidine, had large numbers of silver grains in the

emulsion over caecal tissue- nuclei but there was only an occasional

grain over a parasite (Fig. 9). The number of grains over a

group of parasites was not above the general background level

found in a similar area of emulsion away from the tissue. After

1 month's exposure grains were also found in the emulsion above

tissues exposed to 800 /Lit: of 3H-thymidine but again there was

no significant labelling over parasites.

Tissues from the remaining dose ranges showed no incorporation

of radioactive material after 1 month's exposure. The non- radioactive control had no more grains than the very low background level. The light-exposed control developed normally showing no loss of silver grains thus there was no latent image fading.

DNase treated slides had no more grains than the low back-

ground level. Therefore the presence of grains over tissues exposed to 1000 it.c and 800 tt,c of 3H-thymidine indicated the

presence of radioactively-labelled thymidine in the DNA of the caecal tissue.

As no silver grains were present over the parasites it was concluded that E. tenella did not utilize the 3H-thymidine during DNA synthesis. As the label was supplied 144 hours Table 7 Presence or absence of silver grains, after different exposure times, in autoradiographs from chickens given varying doses of 3H-thymidine.

7 Dose of Exposure Times 'H-thymidine 2 days 1 week 2 weeks 1 month

cuc) Caecal Parasite Caecal Parasite Caecal Parasite Caecal Parasite tissue tissue tissue tissue loco IMP OE 00 Soo LOO 200 loo

Non-radioactive control 00 00 Light exposed control

+ = presence of silver grains = absence of silver grains 950

Fig. 9 Autoradiograph of E. ten4la-infected caecal tissue treated with 1000 A's of H-thyrnidine. Stained with methyl gr ee pyronin. X 270 o = oocvst of E. tenella SG = silver grains over host cell nuclei) 96.

after initiation of the infection when gamete formation was prevalent, it was possible that the parasite had taken up the labelled precursor but in amounts too small to be detected by the procedure used. In Experiment 3 1000 of 3H-thymidine was injected into chickens in which the parasite was at the stage of schizogony.

Experiment 3

For this experiment 1000 ic,c of 3H-thymidine was injected intraperitoneally into a chicken 96 hours after• initiation of an infection of E. tenella. A non-radioactive control was also set up.

Again silver grains were found over caecal tissue nuclei after 2 weeks exposure of the autoradiographs. However there was no incorporation of radioactive label into the developing schizonts of the parasite. The light-exposed and non-radioactive controls responded normally.

It was concluded that E. tenella did not incorporate the radio.active label of the 3H-thymidine during its growth and nuclear division as investigated during schizogony and gametogony.

Experiment 4

Infected chickens were given doses of 3H-adenosine, 3H- guanosine or 3H-orotic acid as set out in Table 8. 97.

Table 8 Dosage of 3H-labelled nucleic acid precursors

Substance Dose Time injected after (pc) initiation of infection (hours)

3H-adenosine 1000 144 3H-guanosine 1000 144

3H-orotic acid 1000 96

3H-orotic acid 1000 144

24 hours after injection of the labelled compound the chickens were killed, thecaeca fixed and prepared for autoradio- graphy. Unfortunately during the course of this experiment the chickens were accidently fed on B.O.C.M. starter mash No. 510, which contained the coccidiostat "pancoxin". The parasites were prevented from developing and were not present for the investiga- tion of the uptake of the purines and the pyrimidine precursor. After 2 weeks exposure the autoradiographs indicated that the caecal tissue had taken up both 3H-adenosine and 3H-guanosine. No indication of the uptake of 3H-orotic acid was found until 47 days of exposure when silver grains were found over the caecal tissue. 98.

With all three precursors the background levels of grains on developed slides, was high.

Embryo infections

Because of the difficulty of ensuring that there was enough radioactive substance available to the parasite whilst it was growing in caecal tissue it was decided to set up infections of

E. tenella in developing chick embryos and to introduce the radio- active substance into the allantoic cavity of the egg at different stages of the parasite's development. The techniques for setting up the embryo infections have been described (pp 60-62)

24 hours after setting up the infected embryos either 1000 /c.v of 3H-thymidine or of 3H-adenosine were injected into the allantoic cavity of the expe?"imental eggs through the same perforation that had been used for inoculation of the parasites. This hole was resealed with collodion solution. The eggs were maintained in the incubator at 40°C and removed 2 or 5 days later. Non- radioactive controls were also taken at these times.

The infected chorioallantoic membranes were removed from the eggs, washed several times in warm distilled water, to remove any free radioactive material and blood, and infected pieces were fixed in Carnoy's fluid for 2 hours. After dehydration and embedding in wax, sections were cut at 3/mmand autoradiographs prepared. 99.

Some sections were treated with DNase as described on p 53

some with RNase, some with both and some with 5% (w/v) trichloro-

acetic acid before being autoradiographed.

For RNase-treated controls the sections were incubated in

•0.3 mg per ml. crystalline RNase in distilled water pre-adjusted

to pH 6.8 for 3 hours at 60°C. As a control for the specificity of RNase similar preparations were incubated in the medium to

which had been added 0.1M zinc sulphate to inactivate the enzyme.

For controls treated with 5% trichloroacetic acid the sections

were incubated with this solution at 90°C for 30 minutes.

Some infected embryos, to which labelled precursors had not

been added, were removed at daily intervals and infected pieces

of chorioallantois were fixed in Carnoy's fluid for 2 hours.

Sections were cut at 5pokand stained in Giemsa. Examination of

these sections showed that the parasite had developed in the

chorioallantoic membrane as first described by Long (1965).

Development in different eggs proceeded at different rates probably

due to variation in temperature within the incubator. Long

(1965, 1970a) has shown that small variations in temperature

affect development of E. tenella grown in chick embryos. At an

incubation temperature of 38°C mature second generation schizonts

were seen between 6 and 9 days after inoculation of sporozoites -

whilst at 39°C and 41°C the first mature second generation

schizonts were seen at 3i days.

▪ ▪ 100.

Despite this difference in developmental rates it was possible

to produce schizonts between 3 and 6 days after inoculation of

sporozoites. From observations on serial sections it was apparent

that these schizonts showed greater variation in size than occurs

in similar schizonts in the caeca of the chicken. The schizonts

grew in the CAM tissue either singly or in clusters of up to about

12 in number (Fig. 10).

4

• • ... 4111, 5(.411zor4T 4,444 I NUCL t-24'

O qfp. 11 • t JP... 4• • • • it • 1, 1

6.-r "-Ut_. r- ' 41" 7•4 •-• • -• • ▪ * I ON? • **,.. • AL,* • 4". ". a 4 • . •ib• • z • •

Fig. 10 Group of schizonts growing in CAM tissue; four day

infection, Giemsa stained. X270. 101.

After 6 days infection the CAM tissue tended to be in a state of necrosis with blood clots in the allantoic fluid.

Many of the embryos were dead at this stage due to haemorrhage caused by the developing schizonts. At no time were gametocytes, gametes or oocysts seen. Death of embryos due to haemorrhage could have been reduced by administering smaller doses of sporozoites

but this led to difficulty in finding infected pieces of CAM tissue which would be required for autoradiography.

Results of autoradIagmtta

•As 1000 of 3H-thymidine . or adenosine had been administered

1 day after initiation of infection of E. tenella in the embryos, tissue taken 3 days and 6 days after initiation of the infection had been in the presence of the radioactive label for 2 and 5 days respectively.

(a) 311-thymiaine,

For tissue treated with 3H-thymidine silver grains were seen over the nuclei of CAM tissue after 5 days exposure but 2 weeks exposUre gave a better picture. In proliferating areas between

50% and 100% of CAM cells were labelled. None of the developing parasite schizonts had silver grains over them. (See Fig. 11)..

Pretreatment of the tissues with DNase and trichloroacetic acid resulted in autoradiographs with no grains above background level over the CAM tissue. Pretreatment with RNase did not 102.

Fig. 11 Autoradiograh of 3-day CAM infection treated with 1000 p-c of 'H-thymidine. Stained with Harris' haematoxylin. X 504 A = Schizont of E. tenella B = Silver grains over host cell nucleus

•

f •

• 103.

affect the production of silver grains. Therefore it was concluded that the CAM tissue had utilised the 311-thymidine in synthesis of its DNA but the parasite had not. CAM cells infected with

E. tenella appeared to be more heavily labelled than non-infected cells. It is possible that the presence of the parasite had caused enhanced DNA synthesis in the host cell.

(b) 311-adenosine

For tissue treated with 3H-adenosine there was uptake of the labelled material in both 3 and 6 day infected material as shown by the presence of silver grains after 5 days exposure. With 6 day infections the labelling was a lot heavier than in 3 day infections. This was a reflection of the longer time that the precursor had been available.

Uptake of the labelled material by the developing parasites was clearly demonstrated by the schizonts in both 3 and 6 day infections (Fig. 12).

Pretreatment with RNase reduced the labelling of both CAM cells and schizonts as did pretreatment with DNase. Pretreatment with trichloroacetic acid and with DNase and RNase gave auto- radiographs in which the number of silver grains over CAM tissue and parasite was not above background level.

Light-exposed controls developed normally and non-radioactive controls produced no grains. 101+.

Fig. 12 Autoradiogra h of 3-day CAM infection treated with 1000 fcc of H-adenosine. Stained with Harris' haematoxylin. X 1560.

A = Schizont of E. tenella B = Host cell nucleus

• !ft 401, • sift. • r •

•

r#* 4, A/. 105.

Thus both the CAM cells and parasites incorporated the radioactive material into their DNA and RNA.

Tissue cultures

In order to use smaller amounts of radioactive material end to regulate when the material was available to the parasite tissue cultures of infected CAM cells and of kidney cells were prepared.

CAM cultures

With the first attempts to set up CAM cultures as described on p 65 fungal infections were produced in the dishes preventing the CAM cells from growing. This fungal infection could not be eliminated in spite of the sterile precautions undertaken and it was suspected that the fungal spores may have been introduced when the sporozoites were inoculated into the eggs. Therefore

Amphotericin B (Flow Labs Ltd., Edinburgh) was added to the culture medium at a concentration of 5p/gAn1 and this procedure eliminated the fungal growth except for a few isolated cases.

However the only growth obtained on the coverslips were a few isolated CAM cells which were unparasitised. This may have been caused by excess trypsinization as Long (1969) reported that the majority of stages grown were situated in "islands" of epithelial-like cells.

Kidney cultures

Monolayers of cells from trypsinised chick kidneys were produced as described on p 66 . With this technique it was 106.

possible to produce coverslips with large groups of cells growing although they were in isolated clumps.

Addition of 100,000 living sterile sporozoites suspended in Doran's medium 2 to 3 days after initiation of the cultures did not produce infected cells on the coverslips. In many cases the kidney cells died off but even in those in which the kidney cells remained alive infections were not produced.

Reasons for lack of infection may be that not enough sporozoites were initially added to the cultures, that the inoculum contained

too much debris (oocyst and sporocyst shells, oocysts and sporo- cysts) which disrupted the cell layer and caused death of cells containing parasites, or that the cell layer was not confluent

enough initially. Fayer and Hammond (1967) found that the fluid obtained after mechanical release of sporocysts from E. bovis oocysts killed cells within hours when inoculated in cell cultures.

Doran (1970a) reported that it was likely that the residue oocyst and sporocyst shells, oocysts and sporocysts caused lack of

proportionality and greater loss of parasites in cultures of

bovine embryonic kidney cell cultures inoculated with 500,000 and 1 million sporozoites from uncleared suspensions of E. adenoeides sporozoites.

As no consistent cultures of growing parasites could be

produced with either CAM or kidney cell cultures no experiments 107.

using radioactive-labelled compounds could be conducted.

From the autoradiographic experiments undertaken it can be concluded that Eimeria tenella does not incorporate 3H-thymidine or its metabolites into its.DNA either directly from the allantoic fluid or from the host cell in which it grows. However it does utilize 3H-adenosine or its metabolites,into both its DNA and

RNA from the host cell in which it grows and perhaps directly from the allantoic fluid before penetration.

Electron Microscopy

Difficulties with fixation of oocysts and penetration of embedding media were experienced with E. tenella oocysts.

With fixation in glutaraldehyde at 4°C and osmium at 4°C and with embedding in Araidite some of the oocyst material was fixed but structural details could not be adequately observed and many oocysts had collapSed.

Since colchicine treatment enabled oocysts to be fixed for light microscopy as in the densitometry work it was hoped that this procedure would produce better fixation and embedding of the material but this was not the case.

Attempts to alter the permeability of the oocyst wall by fixation under reduced pressure and by enzyme treatment were also unsuccessful. Again some fixation of oocysts occurred but 108

structural components could not be easily discerned.

Increase in the temperature at which fixation was carried out gave some improvement as did gently grinding up the oocysts in a homogeniser before fixation. However the latter procedure produced only a small number of oocysts which were not too damaged for their structure to be seen. A combination of these last two treatments proved the best method for preparation.

The use of Spurr's resin, a less viscous embedding medium than Araldite, did not improve the embedding of oocysts to any appreciable extent. Nyberg and Knapp (1970b) reported that after treatment with sodium hypochlorite solution the oocyst wall of E. tenella consisted of a single layer, the outer primary layer having been removed during the treatment, but the other structures in the

treated oocysts appeared similar to those in untreated oocysts.

However, in this work the outer,primary layer was not removed by

sodium hypochlorite treatment although partial separation of the inner and outer layers did occur. The treatment did not give improved subsequent fixation of oocysts. Therefore the treatment

was omitted as this left some debris with the oocysts giving

better cutting properties to the embedding media and allowing

some observations on merozoites and on early oocysts during wall

formation. 109.

An early zygote of E. tenella in the process of wall formation is shown in Fig. 13. This process is probably initiated by fertilization of the macrogamete by the microgamete. On the upper margin of the zygote the dark wall-forming bodies I (Schol- tyseck et al, 1971) can be seen. These bodies fuse together to form the continuous dark layer seen at the lower margin of the zygote and are identical to the plastinoid granules described by

Cheissin (1957). The more loose, sponge-like wall forming bodies

II have already started to lay down the inner wail of the zygote.

Fig. 14, which is of material from a macrogamete, shows the structure of these wall forming bodies more clearly.

The bulk of the early zygote contains electron-pale ovoid bodies (Fig. 13, -A) which contain a polysaccharide (amylopectin) probably in association with protein (Riley et al, 1968).

The nucleus of the zygote can be seen lying in a central position (Fig. 13, -N) and clumps of granules, possibly representing chromatin, appear to have a random distribution in the nucleoplasm.

However a distinct nuclear envelope cannot be seen at this stage probably because of the compaction of the amylopectin granules.

It seems unlikely that a nuclear envelope would not be present at this stage although McLaren (1969) reported the absence of a nuclear membrane in the mature macrogamete of E. tenella. However this was not confirmed by Scholtyseck et al, (1971). 110.

Abbreviations used for electron micrographs of E. tenella - Figs. 13-21

A = Amylopectin

CH- = Chromatin

M = Membrane

N' = Nucleus

NE = Nuclear envelope

NU = Nucleolus

R= Ribosomes

S = Substiedal body

WFB I = Wall forming body type I

WFB II = Wall forming body type II

1 = Thin outer lamella of oocyst wall

2 = Layer of oocyst wall formed by wall-forming bodies type I 3 = Layer of oocyst wall formed by wall-forming bodies type II

Fig. 13 Faly 2-,y()te of E. 17. :211a in the process

of wall formation.

X 5,586,

-• 14 forinr!'. bodics I and II from a

Xl• 13629 11 2

Fig-g , 13 113.

The nucleus from an older zygote, in which completion of the primary layer formed from the wall-forming bodies I has occurred, shows the presence of a nuclear envelope (Fig. 15, -NE). A typical nuclear envelope is a complex, double membraned structure which consists mainly of an inner and outer nuclear membrane between which is a perinuclear space. However in this nucleus only the outer membrane is distinct due to the ribosomes present on it. The inner membrane does not have ribosomes on it and so is difficult to see. The perinuclear space is obscured probably due to shrinkage and compression during fixation embedding and cutting. The nucleus is =12: 4.9 by 3.`+/,c-min size.

The significance of the arrowed structures, which appear to be extranuclear, is unknown. The presence of numerous free and membrane-bound ribosomes can be seen between the amylopectin granules. Fig. 16 shows a zygote after completion of the oocyst wall.

The trilaminate nature of the oocyst wall can be distinctly seen.

At one end the thin outer membrane has lifted off during preparation of the material. The nucleus lies eccentrically and is compact.

In Fig. 17, an enlargement of this nucleus, the outer nuclear membrane can be seen on the upper margin. Again the clumps of granules, possibly representing chromatin, appear to have a random distribution in the nucleoplasm. Fig. 15 High power of nuclear region from a zygote after

completion of the wall formed from wall-forming

bodies I.

X. 18,150 115. Fic. 16 Zyota after conpletion of oocyst wall 4,956

Fig. 1? H$_7;11 :o'er of nuclear region frog zyzote after conoletioL of oocyst wall 16,355 Fag® 17 W

ee,

3, . • 117.

Fig. 18 shows a zygote nucleus at the beginning of the first

nuclear division. Along the upper margin of the nuclear material

there appears to be an accumulation of ribosomes. Only at the

left part of the nucleus can a membrane (M) be seen adjacent to

the nucleoplasm. The nature of the dense blocks of material (Y)

at the left end of the nucleus is not known. The dense

irregularly spherical particles are —J1-- 53 nm in diameter. Material X in Fig. 15, where the particles are 35 nm in diameter,

may be the same material at-an earlier stage. The association

of ribosomes with this material Y suggests that it may consist of

protein.

Whether material Y is nuclear or extranuclear is uncertain

although a clump of similar material can be seen in the cytoplasm

(Fig. 19). If it is extranuclear this would suggest that the

nuclear envelope is absent, as the only membrane present (M in

- Fig. 18) appears to be continuous with this material on its

periphery and does not occur in the region Z. The absence of a

nuclear envelope could be the result of poor preservation but as

other membranes are present in the cytoplasm of the same oocyst

(Fig. 19) this argues against such a possibility and indicates that the nuclear envelope may disappear at the beginning of the

first nuclear division in the oocyst.

However the difficulty in clearly visualising the nuclear

envelope is emphasised by Figs. 20 and 21. Fig. 20 allows a 118.

Fig. 18 Nuclear region of zygote at the beginning of the

first nuclear division.

X 44,118 119. Fis, 19 Zygote at the, beginning of the first nuclear division

showins dense bodies in the cytoplasm.

X 18,810

Fig, 20 Nuclear region of sporocyst.

X 28,842

121,

Fig. 21 Nuclear region of merozoite. X 23,100. 122.

nucleus from a sporocyst before complete differentiation of sporozoites. The condensation of material, which is probably chromatin, at the edge of the nucleus is clearly visible and in this region the presence of a nuclear envelope can be seen but its presence at other margins of the nucleus is more difficult to visualise. The structure S at the left end of the sporocyst is the substiedal body. The Stieda body which is normally anterior to the substiedal body has been broken off during preparation.

Similarly in Fig. 21, which shows part of a merozoite, the nuclear envelope can be seen at the periphery of most of the nucleus but not in all regions.

Therefore definite confirmation of Canning and Anwar's

(1968) report that the nuclear envelope disappears at the beginning of the first zygotic division must await further improvement in fixation and embedding of the zygote although there is some indication that their idea may be correct.

Similarly the presence of centrioles or "centriolar granules" was not seen in this work perhaps due to poor fixation.

PAS (Periodic acid - Schiff) staining

The periodic acid - Schiff reaction is a diagnostic histo- chemical test for carbohydrate material. The test conditions are such that they are conductive to oxidation (to the aldehyde) 123.

of the -CHOH-CHOH- groups of carbohydrates but not allow over- oxidation. The fuchsin of the Schiff reagent becomes recoloured and attached to the polysaccharide thus indicating its presence.

Using a reducing rinse- between the periodate solution and the

Schiff's reagent is necessary (Chayen et al, 1969) to remove any adsorbed traces of periodate which would recolour the Schiff's reagent and thus give a bogus result. Using a S02-water rinse after treatment with the Schiff's reagent is necessary (Chayen et al, 1969), as it is with the Feulgen reaction, because if unbound Schiff's reagent is not washed out of the material it will oxidise into the coloured pararosaniline form which is not a

Schiff complex and is spurious. Rinsing with water is likely to produce this coloured form from any adsorbed leuco-basic fuchsin.

The control treatments ensured that any resultant positive reaction was due to the -CHOH-CHOH- groups. Material which is positive to the simple PAS reaction but negative to PAS after the benzoylation treatment contains the -CHOH-CHOH- grouping and almost certainly contains carbohydrate. The treatments with

Schiff's reagent without prior oxidation and with M/20 alcoholic solution of HC1 for 5 minutes guarded against a positive reaction being due to pre-existing aldehydes or ketones. 124.

Oocysts were ground in a homogeniser to release the sporocysts which were smeared on to slides, wet fixed in Carnoy's fluid or

Schaudinn's fluid and subjected to the PAS reaction. The numbers of sporocysts strongly and weakly positive to the reaction were counted. Using the McManus technique the results set out in Table 9 were obtained.

Table 9 Response of E. tenella sporocysts to the McManus technique of PAS staining

PAS+ Slightly PAS+ Unclassified

Carnoy fixed 2110(45.8„;) 256(48.5) 28(5.3%) Schaudinn fixed 371(45.,0::;) 430(52.4:0 22(2.6%) .1■12a.al AVIMI

The sporocysts gave two reactions to the PAS technique.

One group of sporocysts contained sporozoites which gave a strong

PAS positive reaction whilst the second group contained sporozoites which gave a slight PAS positive reaction. A small number of sporocysts were not classifiable into either group as they appeared to have an intensity of stain somewhere between the other two types. The two sporozoites in any one sporocyst both gave the came reaction, 125.

Using Chayen's technique the results set out in Table 10

were obtained. , The same overall pattern occurred as with the

McManus technique. Early oocysts and sporoblasts gave a positive

PAS reaction. All three types of control gave a negative reaction showing that the technique used was staining the polysaccharide of

the sporozoites.

Table 10 Response of E. tenella sporocysts to the Chayen technique

of PAS staining

PAS+ Slightly PAS+ Unclassified

Carnoy fixed 102(43.4%) 122(51.9%) 11(4.7%) Schaudinn fixed 146(44.8%) 165(50.6%) 15(4.6%)

From these results it was concluded that E. tenella has two

types of sporocyst. One type has sporozoites which give a strong

PAS reaction and therefore contains more polysaccharide than the second type which has sporozoites giving a relatively weak PAS reaction. Secondly it was concluded that in any sample of sporocysts there are --A-504,6 of each type of sporocyst. 126.

Discussion

If an organism has a stage of sexual reproduction in its life cycle with the formation of a zygote by fusion of a male and female gamete the next generation would have double the number of chromosomes normally present unless reduction of chromosome number occurs at gametogenesis or after the zygote has been formed.

Normally this doubling of chromosome number does not occur and the chromosome number remains constant in any species from one generation to the next indicating that doubling of chromosome number is of little or no advantage to an organism. In fact this procedure would rapidly lead to large unbalanced cells with immense nuclei.

However, cells containing more than two sets of chromosomes may arise giving triploid, tetraploid or polyploid forms.

Occasionally whole organisms are tetraploid but polyploid cells are usually abnormal. Also in certain differentiated cells the chromosomes are multistranded (polytene) as for example in the salivary gland nuclei of Drosophila.

In many eukaryotic organisms the reduction process normally occurs at gametogenesis with the somatic cells being diploid and the gametes haploid. In this reduction process, called meiosis, each chromosome usually has a homologue so that a diploid.consti- 127.

tution of 4 chromosomes would consist of two pairs, one of each pair being present in the haploid gamete. At fertilization a zygote would be formed with the 2 homologous pairs which in many higher organisms•would now divide mitotically to produce a diploid adult. In this way each parent contributes one of a homologous pair of chromosomes to its offspring.

The advantage of homologous pairing derives from differences in biological function between chromosomes. If each pair affect different characteristics then to have every pair present is of advantage to an organism. Meiosis ensures the presence of one chromosome of each pair in a gamete by separating the 2 chromosomes of each homologous pair into separate gametes. If no pairing occurred the chromosomes could undergo a random reduction producing gametes with varying numbers. of chromosomes or gametes with the haploid number but with the wrong assortment of chromosomes allowing the formation of zygotes lacking at least one essential chromosome with a resultant loss in viability.

In this type of meiosis the diploid amount of DNA is doubled, producing the 2 chromatids per chromosome which are visible after pairing of homologous chromosomes and crossing-over of genetic material between chromosomes can take place. The homologous centromeres move to opposite poles of the division spindle and the first meiotic division occurs reducing the nuclei to the diploid condition (2N). The second meiotic division follows 128.

when the centromeres divide and the homologous chromatids move to opposite poles of the division spindle. Haploid gametes are thus

produced.

The genetically important features of meiosis i.e., the

pairing of homologous chromosomes, and crossing-over of genetic

material, makes recombination of heritable characters possible and this recombination is the basis of much of the variation which sexual reproduction provides.

However meiosis in the Telosporidea appears to differ from this type of eukaryotic meiosis. Many workers now consider that the first zygotic division is the place where meiosis occurs and not during gametogenesis. Thus the parasites are haploid through- out their life cycle except immediately after fusion of the gametes when a diploid zygote is formed. This idea is based on numerous cases of chromosome counting as reported in the Introductory

Review.

With Eimeria tenella and Eimeria maxima Canning and Anwar

(1967) reported that the meiotic division was zygotic reducing the diploid zygote nucleus with 10 chromosomes to 2 haploid nuclei each with 5 chromosomes in a single step.

All reports of Telosporidea being haploid with a zygotic reduction have been based on light microscopical observations of the number of chromosomes present at different divisions during 129.

the life cycle. yowever, as shown by Canning and Sinden (1973), the interpretation of these observations may be incorrect. From their electron microscopic observations on the oocyst nucleus of plaanodinahei they believed previous reports of structures interpreted as chromosomes (Wolcott 1954, 1957; Bano, 1959;

Canning and Anwar, 1968) were in fact fragments of an enlarged digitate nucleus. This report, in agreement with the hypothesis of Howells and Davies (1971) regarding nuclear events in oocyst development of P. berFhei, must cast doubt on other reports of chromosome numbers in Telosporidea where the nuclei are small.

The number of chromosomes present in any stage of a parasite's life cycle will be directly related to the quantity of DNA present in the nucleus of that stage. Therefore measurements of the quantity of DNA present in individual parasite nuclei of.the different stages in the life cycle will enable the determination of which stages are haploid, which are diploid and at which stage meiosis occurs. For this reason the DNA of Eimeria tenella was stained by the Feulgen reaction and the intensity of the

DNA-Schiff complex in individual nuclei measured on a Vickers M85 scanning microdensitometer.

From reports in the literature of Feulgen staining of various

Telosporidea it might seem that the technique was too variable to be of any use. However if the Feulgen reaction is conducted 130.

under prepoerly controlled conditions it can be used with confidence as a qualitative and quantitative stain within certain limits.

Failure to take account of the following points explains why variations in the Feulgen reaction is met with in the literature:

(1)Loss of sensitivity of the Schiff reagent due to a change in pH,

(2)Variation in the intensity of Feulgen colouration with duration and temperature of hydrolysis e.g. loss of colour intensity of Feulgen colouration with overhydrolysis,

(3)Variation in the intensity of Feulgen colouration dependent upon the fixative,

(4)Inadequate rinsing of fixative from the material,

(5)Inadequate rinsing of stained material in the bisulphite rinse which removes unbound stain. As pointed out by Chayen et al (1969) inadequate rinsing will give spurious results due to oxidation i.e. the leuco-form of the dye will be converted back to the pararosaniline form which is not a Schiff-complex.

"For this reason it is non-sensical to wash the sections with tap-water or with water to which an oxidant has been added because these will. produce the coloured pararosaniline form (of the dye) from any adsorbed leuco-basic fuchsin. Yet some workers actually recommend that it is done".

(6)Dilution of the DNA-Schiff complex in a large nucleus 131,

below the level at which it is visible.

(7) Possible reduction in Feulgen staining due to the

physical state of the deoxyribonucleoprotein (i.e. diffuse or compact) which is a reflection of its metabolic activity.

For quantitative work it is almost impossible to achieve

exactly the same conditions for separate batches of slides

therefore in this work nucleated chicken erythrocytes were used as a standard DNA reference with each experimental slide and the amount of DNA-Schiff complex in the parasite nuclei converted to comparable units. From the microdensitometry readings it seems

that Eimeria tenella is haploid throughout its life cycle except after fusion of the gametes when a diploid zygote is formed.

Division of this diploid nucleus occurs without synthesis of

DNA and thus produces two haploid nuclei in one step. The remaining two zygotic divisions are mitotic producing haploid sporozoites. The schizonts appear to be produced by mitosis with

each nucleus synthesising DNA until the diploid amount is produced and then dividing to form 2 haploid nuclei. This process of schizogony differs from that reported by Gutteridge and Trigg

(1972) for Plasmodium knowlesi in which they studied the incor-

poration of 3H-adenosine whilst the parasites grew in culture.

The cultures started off as late trophozoite stage parasites.

Incorporation of 3H-adenosine occurred until nuclear division 132,,

began, ceased during this period and began again as soon as parasites had invaded new red cells. They concluded that the

S-phase occurred during the ring and trophozoite stages of the intra-erythrocytic cycle with the G1 and G2 phases, if they occurred at all, being of very short duration and occurring at the young ring and late trophozoite stages respectively.

Schizogony they thought consisted of a "multiple mitosis" which occurred without further DNA synthesis and yielded a 16-nuclear schizont. With Eimeria tenella the number of nuclei per schizont is many times that found in the erythrocytic schizonts of

Plaslorljnm species therefore an enormous quantity of DNA would

have to be produced in a small volume in young Eimeria schizonts if all the DNA were produced before any nuclear division occurred.

The haploid value of the microgametes is slightly low when compared with the diploid value of the newly formed zygote.

The microgametes are in a resting state of development awaiting

fusion with a macrogatnete before they continue development.

Therefore the DNA in the nucleus will be in a repressed state.

Because of this state the stainability of the DNA by the Feulgen reaction may have altered slightly. There have been a number of reports showing a changed stainability of DNA due to this repressed

state. Yataganas et al (1970) showed that late polychromatic

erythroblasts gave a 10% lower DNA-Feulgen value compared with 133,.

pro-erythroblasts, basophilic erythroblasts and early poly- chromatic erythroblasts which contained the same amount of DNA.

Similar 1C% lower Feulgen-DNA values innon-dividing cells have been shown in mature lymphocytes and neutrophils (Killander and

Rigler, 1965, 1969) in hen erythrocytes (Bolund et al, 1969) and in spermatocytes (Gledhill et al, 1966).

The small variation between the haploid values for schizont nuclei, for nuclei resulting from the first zygotic division and for nuclei resulting from the second zygotic divisions is due to variation in the degree of separation from neighbouring nuclei and in the clarity of background around the nuclei which will affect the accuracy of the densitometric measurements.

Failure to detect DNA by the Feulgen technique in macro- gametes is probably due to the repressed state of the DNA and to its dilution in a large volume as suggested by Cheissin (1959).

The life cycle of E. tenella as regards the ploidy of the nuclei is summarised in Fig. 22.

The zygotic meiosis occurs without doubling of DNA and produces two haploid nuclei thus it appears to be a simple one- step reduction division without chiasmata and centromeres being formed as reported by Canning and Anwar (1968). Thus exchange of genetic material in crossing-over cannot occur. The only variation in the genome which could occur will be as a result of mutations or perhaps by distribution of the chl-omoso!ileb- to the Fig. 22. Life cycle of Eimeria tenella showing ploidy of nuclei

Sporulation OUTSIDE Newly formed zygote---.. meiosis---9. 2 haploid -> 4 haploid 9. 8 haploid HOST (Diploid - 2N) nuclei nuclei nuclei •-■

•■••••••••••••••.• ■ ••••■•••■•

IN HOST ••••••••■■■•• •••••■••■■••■••.11 -

Fertilization sporozoite (N) Schizogony

microg-mete (N) uacrogamete (N) merozoites (N) Schizogony

Gametogony • merozoites (N) 135,

product nuclei. The amount of this variation is likely to be very small and will depend upon which essential genes are present on which chromosomes. If all the haploid chromosomes contain some essential unique genes not present on the others of the haploid set then all these chromosomes will be essential for a viable product. Thus at meiosis the separation of the chromosomes must be well ordered to ensure viable offspring. The apparent pairing of chromosomes on the spindle of the dividing oocyst described by Canning and Anwar (1968) would thus be the point at which this ordering takes place. However this process may still allow some variation as normally at meiosis the different pairs of homologous chromosomes align themselves on theequator of the division spindle in an independent fashion. Thus the haploid nuclei produced as a result of meiotic division in E. tenella oocysts may contain a mixture of the original microgamete and macrogamete complement of chromosomes.

Pairing of chromosomes at meiosis without chiasmata has been reported for a number of animal species particularly in insects

(e.g. Hughes-Schrader (1943) working on meiosis of the mantid

Callimantis antillarum Sassure). There appears to be no information on E. tenella regarding the proportion of the oocysts produced which are viable therefore the accuracy of this pairing process and the segregation of chromosomes cannot be estimated. Brackett and Bliznick (1952) recorded that for each oocyst of E. tenella 136,

inoculated in light infections approximately 400,000 oocysts were produced. Therefore some oocysts defective due to wrong chromosome numbers or types may well be present. These should not affect the reinfection of new hosts to any great extent despite the possible additional loss of oocysts due to lack of sporulation caused by unfavourable environmental conditions, failure to gain entry to a new host, failure to excyst and failure to develop due to host responses. In fact some of these factors such as lack of sporulation and of development may be caused by the presence of the wrong chromosome constitution.

One-division meiosis has been reported in the polnnastigote flagellates (Oxymonas doroaxostylus and Saccinobaculus ambloaxo- stylus) by Cleveland (1950a, b). In mitosis both centromere and chromosome duplication occurs. In meiosis, of both one- division and two-division type, suppression of centromere and

.chromosome duplication occurs. As Cleveland says "In ordinary or two-division meiosis, the centromeres are not duplicated during the first division but the chromosomes are; and in the second division the centromeres are duplicated, while the chromosomes are not. . In one-division meiosis, neither the chromosomes nor the centromeres are duplicated in the first nuclear division.

Thus reduction in the number of chromosomes and centromeres occurs immediately and no second division is necessary. It is as 137.

simple as that. In other words, meiosis as we know it in higher organisms requires two nuclear divisions because the suppression of chromosomal and centromere duplications does not occur at the same time, during the same nuclear division".

This explanation seems to be a reasonable interpretation of

the observed phenomena in both these flagellates and in Eimeria

tenella. Two-division meiosis has a great evolutionary advantage over one-division meiosis in that tetrad formation and crossing over occurs in the former allowing greater chance for variation and change in the organism. In one-division meiosis it is impossible because the prophase and metaphase chromosomes are not each composed of two chromatids, as in the first division of two division meiosis, so that crossing-over, if it occurred, would have to take place between chromosomes.

The extent to which Eimeria tenella is typical of other

Telosporidea is uncertain. From densitometry readings on Feulgen stained nuclei of Hepatozoon domerguei there is some indication that the undivided zygote nucleus may contain up to 4 times the haploid amount suggesting that DNA is replicated by the parental chromosomes before reduction in 2 stages to the haploid amount

(Canning, personal communication). From electron microscope studies on Plasmodium berFhei oocysts (Canning and Linden, 1973;

Howells and Davies, 1971) the nucleus appears to become polyploid 138.

without division until just before sporozoite formation when nuclear fragmentation occurs producing spheroidal nuclei which undergo a final simple division prior to entering the sporozoites.

Further densitometry work on Feulgen stained DNA of parasite

nuclei should be conducted on different Telosporidea to determine if different genera have similar or different patterns of ploidy in their life cycles.

During the endogenous stages of a coccidial life cycle nuclear and cytoplasmic growth and division occur. For this,

nucleic acid synthesis is a prerequisite. The parasite has two

potential sources of nucleic acid precursors. The parasite may utilize the purines and pyrimidines of the host cell, either by

degrading host nucleic acids or by incorporating unpolymerised

purines and pyrimidines. The parasite may also be capable of

purine and pyrimidine synthesis from simpler compounds. From ultrastructural studies of Eimeria species in vitro it has been

shown that sporozoites and immature schizonts ingest host cell

cytoplasm (Sampson and Hammond, 1971; Scholtyseck and Strout,

1968) but there is no evidence that the parasite utilizes host

cell nuclear DNA.

From the autoradiographic experiments it was found that

Eimeria tenella did not utilize 3H-thymidino which had been incorporated into host cells nor 3H-thymidine direct from the 139.

allantoic fluid but did utilize either 3H-adenosine and/or its

metabolites which had been incorporated into host cells or

3H-adenosine direct from the allantoic fluid. These results

suggest that E. tenella probably synthesises thymidine, a pyri-

midine nucleoside de novo but probably utilizes preformed

adenosine, a purine nucleoside. Whether it is capable of

synthesising the pyrimidine and purine rings can not be determined

without information on whether it incorporates their precursors.

The result with 3H-thymidine agrees with the findings of

Roberts et al (1970) who found no incorporation of 3H-thymidine

by E. callos-cermo hili during its asexual development in vitro.

He concluded that the parasite did not utilize thymidine directly from the media, from degraded host cell nuclei, or from nucleoside or nucleotide pools of the host cell.

Similarly, Ouellette et al (1973), working with the asexual stages of E. tenella cultured in embryonic chick kidney cells found that developing parasites did not incorporate 3H-thymidine either when host cells were labelled prior to infection or when the cultures were labelled for 30 minutes, 48-72 hours after infection. However continuous exposure of infected cultures to

3H-thymidine for up to 18 hours resulted in light labelling of cell cytoplasm and schizonts. This labelling in schizonts was always associated with labelling of host cell cytoplasm and since 11+0.

it was RNase resistant they thought it possible that the label

was associated with mitochondrial DNA. They found that 3H-

cytidine and 3H-uridine, both pyrimidine nucleosides, were incorporated into parasites developing in cultures that were labelled before infection and in cultures labelled for 30 minutes,

48-72 hours post infection. They believed that much of the

3H-cytidine label in parasites came via the host cell cytoplasm from label originally bound to RNA in the cell nuclei. With 3H-uridine the label was accumulated more readily by the parasites

than by the cells and they thought that the RNA was being more rapidly synthesised in the parasites because they observed that

the transfer of nuclear label to the cytoplasm appeared to

proceed more rapidly in the parasites than in the cells.

There have been no reports on the uptake of purine nucleosides, such as adenosine, by any species of Eimeria. Clearly further work on incorporation of purine.and pyrimidine nucleosides and their precursors into the parasite is essential to elucidate the sources of precursors for the nucleic acids and their routes of synthesis in Eimeria parasites. However from evidence so far available it seems probable that the parasite can salvage compounds from host cell RNA.

Eimeria tenella seems to be related to Toxoplasma gondii in its ability to utilize exogenous pyrimidine nucleosides. Kishida 141.

and Kato (1965) reported little detectable uptake of exogenous

thymidine in T. gondii whilst Perrotto et al (1971) reported

incorporation of preformed pyridimidines (including thymidine

provided at concentrations higher than 10 71.1) but that pyrimidine

precursors were preferentially utilized over preformed pyrimidines.

With malarial parasites e.g. P. knowlesi it has been shown

that radioactive pyrimidines were not utilized for nucleic acid

synthesis though the pyrimidine precursor, orotic acid, was

utilized to a limited extent (Gutteridge and Trigg, 1970).

Considering the use of exogenous purine nucleosides T. gondii

seems to make little apparent utilization of purine precursors

but readily utilized exogenous purines (Perrotto et al, 1971).

Similarly P. knowlesi incorporated a range of purines but not a

purine precursor (Gutteridge and Trigg, 1970). E. tenella utilized

the exogenous purine adenosine but there is no information on the

- utilization of purine precursors.

The oocyst wall in Eimeria'species of Coccidia has presented

a great barrier to studies on the nuclear divisions of the zygote.

Monne and Haig (1954), using the light microscope, reported

that the oocyst wall was composed of 2 layers, the outer one

consisting of a quinone-tanned protein and the inner one consisting

of a protein lamella firmly associated with a lipid on its inner

surface. With the electron microscope Scholtyseck and Weissenfels

(1956) confirmed that the flooyst wall consisted of 2 primary

layers plus a thin-lamella noverinr the external surface of the 142.

outer primary layer. Scholtyseck and Voigt (1964) similarly reported that with E. perforans there was this additional outer membrane around the whole oocyst.

McLaren (1969) reported that in E. tenella the oocyst wall consisted of three layers: the outermost layer being the original limiting membrane of the macrogamete and the other two layers corresponding to the primary layers described by Monn7e and Hoenig

(1954). She thought that the outer primary layer was formed from "dense bodies" seen in the macrogamete and the inner primary layer was formed from "wall-forming bodies" seen in the macrogamete.

Nyberg and Knapp (1970b) observed the presence of the two primary layers of the E. tenella oocyst wall but did not observe the thin lamella covering the outer primary layer when using the electron microscope. Similarly Roberts et al (1970) did not see a limiting membrane around E. larimerensis oocysts but did with

E. callospermophili oocysts.

Scholtyseck et al (1971) reported that the two primary layers of the walls of Eimeria oocysts were formed from wall forming bodies I and wall forming bodies II. Wall forming bodies I, which correspond to the "dense bodies" of McLaren, formed the outer primary layer and wall forming bodies II, which correspond to the "wall forming bodies" of McLaren, formed the inner layer.

From Scholtyseck's diagrammatic representation of wall formation 143.

in the oocysts of Eimeria species the outer limiting membrane of the oocyst appeared to be the original macrogamete limiting membrane as described by McLaren.

From the electron microscope observations on E. tenella in this work the results found by Scholtyseck et al (1971) seem to be the proper interpretation of oocyst wall formation in

Eimeria species. Also from this work the ease with which the outer membrane lifts off from the remniming layers was noted and this may account for the failures to observe this layer as reported above.

There have been no reports on nuclear divisions of the oocyst as studied by electron microscopy in Eimeria species and no definite conclusions could be reached in this work as to whether the nuclear membrane remained intact during the meiotic division of E. tenella oocysts or as to whether centrioles or

"centriolar bodies" are involved in this division. Even reports by Porchet-Hennere and Richard (1971) on the adeleine coccidian ApgreRata eberthi which has a much thinner, more permeable oocyst wall has little information on the actual nuclear divisions during sporogony as observed by electron microscopy due to poor fixation. However they report on the presence of 2 centrioles and a zone of microtubules between a peripheral nucleus and the neighbouring oocyst wall, corresponding to an extranuclear spindle, shortly after fertilization.

Vivier et al (1972) reported some observations on Hepatozoon domerguei an adeleine coccidian. Again very incomplete observations were reported and concerned only the division stages which follow the initial meiosis. However they observed the following features:

(1)the persistance of the nuclear envelope,

(2)spindle fibres, originating in a depression of this envelope on the external nuclear surface, which extended into the cytoplasm and to the interior of the nucleus,

(3)the possible presence of a centriole or functionally equivalent structures,

(4)division of the nucleus by lateral stretching.

Because of the few effectual observations they could not draw any general conclusions on the mode of nuclear division but restricted themselves to saying there was a certain resemblance between nuclear division of Heptazoon and Eimeria relating to the invagination of the nuclear envelope as Dubremetz (1971 and personal communication) described an invagination of the nuclear envelope, opposite centrioles, with the presence of intranuclear spindle fibres in schizogony of E. necatrix. They also pointed out the similarity with nuclear division in the Nicrosporidia 14-5.

where spindle microtubules arise in a depression of the nucleus

but in the absence of centrioles (Vivier, 1965; Vavra, 1965).

Of the number of electron microscope studies carried out on

malarial oocysts (Canning and Sinden, 1973; Duncan et al, 1960;

Garnham et al, 1969; Howells and Davies, 1971; Terzakis et al

1966,_1967; Terzakis, 1968, 1971; Vanderberg et al, 1967;

Vanderberg and Rhodin, 1967) only Canning and Sinden (1973) and

Howells and Davies (1971) present a comprehensive account of the nuclear changes involved.

Howells and Davies (1971), working on the post-meiotic

nuclear divisions of Plasmodium berghei oocysts, reported that

nuclear division proceeded without the disappearance of the

nuclear membrane, a result in accord with the previous studies

on nuclear division in the erythrocytic stages of plasmodia

(Aikawa and Beaudoin, 1968; Ladda, 1969). They observed the

presence of more than one spindle in a single nuclear section

within the immature oocyst which they thought indicated multiple

and presumably repeated division of the chromosomal material

within a single nucleus. They also observed structures, probably representing centrioles in longitudinal sections, which did not

appear to be attached to the terminal plaques of the spindles.

Canning and Sinden (1973) reported on the first nuclear

division of P. berghei oocysts and found that the nuclear membrane 146.

remained intact throughout the period of nuclear activity.

Spindle microtubules were found extending across the small

•nuclei to centriolar plaques in the nuclear envelope. The

presence of kinetochores in association with the spindles was also

reported.

-All the reports on nuclear divisions in the non-oocyst

stages of Eimeria species describe the nuclear membrane as

remaining intact during nuclear division. Roberts et al (1970)

reporting their studies of schizogony in Eimeria callospermophili

considered the retention of an intact nuclear membrane during

division to be a common phenomenon in the sporozoan species

discussed in their paper including Toxoplasma, Eimeria and

Plasmodium.

No firm evidence to contradict this idea has been elucidated

from this work and from the evidence of nuclear divisions in

endogenous stages of Eimeria species and from the nuclear divisions

in the oocysts of adeleine coccidia and plasmodia it seems

likely that nuclear divisions in the eimeriine oocyst may follow

a similar pattern. However it should be pointed out that the

only evidence concerning the first nuclear division of an oocyst

is from Plasmodium berghei (Canning and Sinden, 1973). The

nuclear material in this species appears to undergo repeated

divisions within the same nuclear membrane before finally 11+7.

dividing to form individual nuclei which undergo a final simple division prior to entering the sporozoites, a situation which is radically different from Eimeria in which the nucleus divides to form two daughter nuclei at the initial meiosis.

Definite evidence concerning the nuclear membrane in eimeriine oocysts during nuclear division must await improvements in fixation and embedding. Perhaps the use of a water-soluble embedding resin, such as Durcupan, which circumvents the ordinary dehydra- tion procedures would minimize denaturation and allow further study of this nroblem.

Similarly the presence or nature of the "centriolar granules" described in the oocysts of Eimeria tenella from light microscope observations by Canning and Anwar (1968) could not be determined.

The presence of centrioles has been reported by many workers in studies of. nuclear division of endogenous stages of Eimeria.

Roberts et al (1970) reported that the structures referred to as centrioles in E. calloseermonhili differed from basal bodies in having 9 single tubules around the periphery and 1 central tubule (basal bodies are short cylinders with 9 double or triple fibres around the periphery and lack central tubules. They thought that the centrioles probably divided before nuclear division but the intranueleas spindle apparatus did not appear to be attached to them. 148,

From work on many animal cells centrioles appear to play a decisive role in determination of the locations of the poles of the spindle during nuclear division but it is not known whether the centriole directs the synthesis of spindle macromolecules, is involved in their assembly to form larger structural components of the spindle, determines the orientation of spindle components, or whether it plays only an indirect role in these processes.

However the presence of typical centrioles is not necessary for spindle formation for it is lacking in higher plants and only appears in lower plants when a motile microgamete is being formed.

The typical nine-triplet-tubule structure of the centriole is related to the formation of cilia and flagella and is not essential for spindle formation (Luyk, 1970).

The absence of centrioles has been reported in the first and second meiotic divisions of the mouse oocyte (Szollosi et al,

1972). They observed that the spindle microtubules terminated in a number of electron dense filamentous structures referred to as microtubule foci.

In malarial parasites centriolar plaques have been described and symmetrical and cylindrical structures of these plaques indicate that they are centriolar equivalents (Robinow and Marak,

1966; Aikawa and Beaudoin, 1968). These plaques show a close relationship with the nuclear microtubules and are located on the 149.

nuclear membrane. It is possible that these centriolar plaques may be the places where synthesis and organisation of the nuclear microtubules occurs.

It is possible that the large arrowed structure in the invagination of the nuclear membrane in Fig. 15 is in some way related to the centriolar plaques of malarial parasites or the

centriolar granules" described by Canning and Anwar (1968).

That centrioles may not be present in the oocyst nuclear divisions but are in nuclear.divisions of endogenous stages should not be surprising as Luykx (1970) describes cases in which morphology of spindle poles alters in successive divisions of the same cell line and cases in which cells with centrioles arise by division from cells without recognixable centrioles. It is possible that all eukaryotic cells might use the same few basic devices for establishing the poles of a spindle. Variations in pole morphology would then be only a consequence of differing relative contribu- tions of these devices to spindle formation or might be the result of other differences in cell organisation - such as differences in number and kinds of inclusions, in size and shape of the cells, or in the stability of the nuclear envelope.

The dense, irregularly spherical particles seen adjacent to the nucleoplasm in Fig. 18 and in the cytoplasm in Fig. 19 resemble the Type I crystalloid particles described by Trefiak 150.

and Desser (1973) in the ookinetes of Leucocytozoon simondi,

L. ziemanni, Plasmodium gallinaceum, Parahaemoproteus fringillae and Parahaemonroteus velans. They describe them as being lipid-

protein in nature with particles 25-40 nm in diameter which are formed after fertilization. Porchet-Henner‘ and Richard (1969, 1971) and _Porchet-Hennere and Vivier (1971) have described such inclusions in sporoblasts of several coccidian species and they suggest the crystalloid arises from the Golgi apparatus, is

probably protein in nature and serves as a source of energy.

It is possible that the particles seen in E. tenella are also part of such a crystalloid and serve as a source of energy during sporulation.

From the PAS staining of the E. tenella sporocysts carried out in this work it seems that there are 2 types of sporocyst distinguishable by the amount of polysaccharide (amylopectin) in their sporozoites. In one type the sporozoites stain heavily with the PAS technique whilst in the other the sporozoites stain lightly. From counts of samples of mechanically released sporocysts there are approximately 50% of each type.

Klimes et al (1972) reported that with E. tenella grown in tissue culture 2 distinct types of schizont and merozoite could

be distinguished by their relative staining with the PAS technique.

They found that the less PAS reactive schizonts and merozoites 151..

gave rise to microgametocytes whilst the strong PAS reactive ones gave rise to macrogametocytes. The difference of PAS staining of sporozoites may also be a reflection of a state of sex differentiation. It may be that the relatively PAS negative sporozoites eventually give rise to male gametes and the PAS positive ones will give rise to female gametes. Thus each sporozoite would be unisexual.

If this is the case then one would expect the point of differentiation to be the 1st zygotic division or the nuclear division within the sporocyst. If the former occurred the sporocysts would be unisexual (male or female) and if the latter the sporocysts would be bisexual with both a male and a female sporozoite. If the difference in PAS reaction is a reflection of sexuality then the results suggest that the 1st nuclear division is the point of sex determination as at no time was a sporocyst seen with different kinds of PAS stained sporozoites in it. From studies of meiosis in insects it has been shown that the chromosomes which associate in pairs at prophase are of paternal and maternal origin respectively and that each chromosome maintains.its morphological individuality throughout successive cell divisions (Sutton, 1902). Therefore, if sex differentiation in Eimeria was determined by a specific pair of homologous chromo- somes - one chromosome being in the male gamete and its homologous chromosome being present in the female gamete - then pairing of 152.

chromosomes at meiosis (Canning and Anwar, 1968) would ensure that these chromosomes were segregated at division with one chromosome going to one pole of the spindle and the homologous one going to the opposite pole. As a result of the two following mitotic divisions two sporocysts containing male sporozoites and two sporocysts containing female sporozoites would be produced.

The remaining chromosomes from the male and female gametes would pair as homologous chromosomes at meiosis but would align them- selves on the aluator of the spindle in an independent fashion.

If the gene (or genes) which determine the amount of poly- saccharide produced in sporozoites and merozoites were present on the sex chromosomes, the female chromosome containing the gene determining the presence of a large amount of polysaccharide and the male chromosome containing its allele determining the presence of a small amount of polysaccharide, then at meiosis the gene and its allele would be segregated. As a result half the sporozoites produced would contain the gene producing a large amount of polysaccharide and half would contain the gene producing a small amount of polysaccharide. Thus one oocyst would produce two sporocysts containing sporozoites with a large amount of polysaccharide and two sporocysts containing sporozoites with a relatively small amount of polysaccharide. This would explain why any sample of sporocysts looked at in this work contained 153.

approximately 50% which contained sporozoites giving a strong PAS reaction and approximately 50% which contained sporozoites giving a weak PAS reaction.

The presence of amylopectin in many species of coccidial sporozoites has been reported by numerous workers using the PAS technique or electron microscopy but in no case has the presence of two distinct groups of sporozoites containing different amounts of amylopectin been described. One reason could simply be that they were not looking for them so the presence of two distinct groups could be missed especially under the electron microscope.

Another reason could be that the difference was no longer apparent at the time they looked at them. In the present work the sporocysts looked at were from freshly released and sporulated oocysts but in many of the above cases the oocysts looked at had been stored for some time. Vetterling and Doran (1969) found that in E. acervulina the polysaccharide content of sporozoites fell from 33.3 g glucose/106 oocysts at 3 months to 7.8 lag at 2 years and 1.5 114g at 6 years. This loss of polysaccharide on storage might hide any differences in amylopectin levels due to sexuality.

Differences'in polysaccharide content in merozoites have also been reported by Canning (1962) in Barrouxia schneideri and

Scholtyseck et al (1969) in. Eimeria tenella. Scholtyseck's 154.

explanation was that either they were looking at merozoites from different generations or that the carbohydrate represented a reserve of material used to provide energy for movement and was used during the motile phase producing the observed differences.

This second possibility was supported by Gill and Ray (1954) and

Rootes and Long (1965).

The polysaccharide provides a readily available source of energy to the parasite and Gill and Ray (1954) considered that the amount of reserve ran proportionally to the amount of work expected by the particular stage of the parasite. However

Klimes et al (1972) pointed out that in E. tenella the difference in PAS staining could be seen in unbroken mature schizonts so it did not seem to be influenced by depletion of the carbohydrate reserve during movement.

The newly formed oocyst is full of amylopectin as can be seen by PAS staining and by electron microscopy. During sporula- tion this polysaccharide is utilized and by 10 hours sporulation in E. acervulina when 4 sporoblasts have been formed there has been 50% utilization of this polysaccharide (Wilson and Fairbairn,

1961). The metabolism then appears to be at the expense of lipid reserves and there is some resynthesis of polysaccharide. It could be at this stage that the differences in PAS levels between sporozoites is determined. 155.

Vetterling and Doran (1969) have shown with E. acervulina,

E. necatrix, and E. meleagrimitis that sporozoites use about irds of their stored polysaccharide during excystation. As these reserves decline on storage the accompanying decline in infectivity may be due to an inability of the sporozoites to actively excyst and nenetrate a host cell. They also found that some individuals had a greater ability to survive than others (some surviving at least 2 years). However they did not know whether these individuals had greater polysaccharide reserves, metabolised at a slower rate or could survive on less polysaccharide.

Differences between "male" and "female" sporozoites in polysaccharide content may affect the relative ability of the sporozoites to excyst and penetrate a new host cell, the female with the bigger reserves standing more chance of success. Again if there are 2 types of merozoites then the one with the more reserve is most likely to be successful. Thus "female" sporo- zoites and merozoites would stand more chance of development than

"male" sporozoites and merozoites.

However a microgametocyte gives rise to many microgametes whilst a macrogametocyte gives rise to only one macrogamete.

As only one microgamete is needed to fertilise a macrogamete the production of vastly excessive numbers of microgametes would be 156.

wasteful as the developing microgametocytes would occupy host

cells which could be utilised by macrogametes. Some microgametes

are probably lost in the caeca or intestine and some never find a

macrogamete so a balance must be maintained between producing too

many or too few microgametes. The differences in polysaccharide

levels in male and female sporozoites and merozoites which will

alter their chances of successful development may be part of a

mechanism achieving this balance.

However all this must remain speculation until single sporo- zoite and single sporocyst infections can be produced. Only by finding out whether they are capable of producing patent infections

or only capable of producing one type of gamete can one be sure

whether the sporocysts and sporozoites are unisexual or bisexual.

With the advent of successful culturing of a number of species of

Eimeria parasites in tissue cultured cells it may be possible to use such tissue cultures to produce the required infections.

This technique would eliminate one of the main difficulties in

the past., i.e., finding the infection if it developed.

As to the future, elucidation of the anabolic and catabolic

pathways involved in nucleic acid synthesis in the Telosporidea would enable a better understanding of the action of chemothera- peutic drugs used against these parasites and may in turn lead to increased knowledge concerning the developmental processes involved in their growth and reproduction. 157.

Radioactive labelling of the parasites' DNA would enable its presence and distribution to be detected at the electron microscope level which would increase one's knowledge on its activity during nuclear division. Also if the DNA of one type of gamete, in parasites which have anisogametes, could be radio- actively labelled then the fate of this DNA after fusion of the gametes to form a zygote could be traced enabling a better understanding of the mechanisms involved during zygotic divisions.

Generalisations about nuclear activity in the Telosporidea should not be made as evidenced by the differences apparent between meiotic division in Hepatozoon domerguei and Eimeria tenella and between zygotic nuclear divisions in Plasmodium berghei and adeleine coccidia. Further comparative work concerning nuclear division, nucleic acid metabolism and the determination of sexuality should be conducted on different genera of the Telosporidea to ascertain any patterns of simil- arities and differences in these processes between different groups. 158.

References

AIKAWA, M. and BEAUDOIN, R. (1968). Studies on nuclear division of a malarial parasite under pyrimethamine treatment. J. Cell. Biol., 22 : 749-754. ALFERT, M. (1950). A cytochemical study of oogenesis and cleavage in the mouse. 25 : 381-409. ALFERT, M. and SWIFT, H. (1953). Nuclear DNA constancy: A critical evaluation of some exceptions reported by Lison and Pasteels. Eu12211222" 2 : 455-46o. ANFINSEN, C.B., GEIMAN, q.m., McKEE, R.W., ORMSBEE, R.A. and BALL, E.G. (1946). Studies on malarial parasites. VIII Factors affecting the growth of Plasmodium knowlesi in vitro. 84 : 607-77- ANWAR, M. (1966). Cytochemical studies on infections with Isos ora lacazei. Trans. R. Soc. trop. Med. Hy., 6o : 428-429. BAHR, G.F. and MIKEL, U, (1972). The arrangement of DNA in the nucleus of rodent malarial parasites. Proc. helminth. Soc. Wash. m : 361-372. (Special Issue). BANG, L. (1959). A cytological study of the early occysts of seven species of Plasmodium and the occurrence of post- zygotic meiosis. Parasitoloryo 42 : 559-585. BAUER, H. (1932). Die Feulgensche Nuklealfgrbung in ihrer Anwendung auf cytologische Untersuchungen. Z.Zellforsch. .mikrosk. Anat., 15 : 225-247,

BECKER, E.R. (1934). Infection with a single coccidian oocyst and its significance. Am. Nat., 68 : 571-574. BEDRNIK, P. (1967a). Further development of the second generation of Eimeria tenella merozoites in tissue cultures. Folia Parasit., 14 : 361-363. 159.

BEDRNIK, P. (1967b). Development of sexual stages and oocysts from the 2nd generation of Eimeria tenella merozoites in tissue cultures. Folia Parasit., 14 : 364.

BEDRNIK, P. (1969). Some results and problems of cultivation of Eimeria tenella in tissue cultures. Acta Veterinaria (Brno), 28 : 31-35. BOLUND, L., RINGERTZ, N.R. and HARRIS, H. (1969). Changes in the cytochemical properties of erythrocyte nuclei reactivated by cell fusion. J. Cell Sci., 4 : 71-87.

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. (1954). On the coccidia of the mongoose. Ann. trop. Med. Parasit., 48 : 405-415. BRAY, R.S. (1957). Studies on the exo-erythrocytic cycle in the genus Plasmodium. __LOnd•SCI..._1z1/getr...... _* el Mem., 12. H.K. Lewis and Co., Ltd., London.

BUNGENER, W. and NIVI,SEN, G. (1968). Nukleinsaurenstoffwechsel bei experimenteller Malaria. 2. Einbau von Adenosin und Hypoxanthin in die Nukleinsauren von Malariaparasiten (Plasmodium berghei und Plasmodium vinckei). Z. Tro-)enmed. Parasit., 19 : 155:197.

•CANNING, E.U. (1962). Sexual differentiation of merozoites of Barrouxia schneideri (Butschli). Nature, Lond., 222 : 720-721. CANNING, E.U. (1963). The use of histochemistry in the study of sexuality in the Coccidia with particular reference to the Adeleidae. Progress in ProtozooloDN, Proc. 1. Int. Congr. Protozool. Prague 1961 : 275g:q42.

CANNING, E.U. and ANWAR, M. (1966). Nuclear staining of Plasmodium oocysts. Trans. R. Soc. troatiell lia., 60 : 424. • •IN •• ■•• CANNING, E.U. and ANWAR, M. (1967). Nuclear division in Eimeria oocysts. Trans. R. Soc. trap. Med. Hyg., 61 : 451. 160.

CANNING, E.U. and ANWAR, M. (1968). Studies on meiotic division in coccidial and malarial parasites. J. Protozool., : 290-298. CANNING, E,U. and ANWAR, M. (1969). Meiotic division in oocysts of malaria parasites of mammals. Trans. R. Soc. trop. : 4-5.

CANNING, E.U. and SINDEN, R.E. (1973). The organisation of the ookinete and observations on nuclear division in oocysts of Plasmodium berghei. Parasitology, (In Press). CASPERSSON, T. (1940). Methods for the determination of the absorption spectra of cell structures. 31 R. microsc. Soc., 60 : 8-25. CHAYEN, J., BITENSKY, L., BUTCHER, R.G. and PDULTER, L.W. (1969). "A Guide to Practical Nistoehemistry". Oliver and Boyd, Edinburgh. CHAYEN, J. and NORRIS, K.P. (1953). Cytoplasmic localization of nucleic acids in plant cells. Nature, Lond., 171 : 472-473.

CHEISSIN, E.M. (1940). Rabbit coccidiosis. III. Eimeria manna and its development cycle. (Russian paper, English summary). The Herzen State Pedagouical_Instityte, Scientific Memoirs Division of Zoology, 30 5-920

CHEISSIN, E.M. (1957). Cytologische Untersuchungen verschiedener Stadien des Lebenszyklus der Kaninchen-coccidien I : Eimeria intestinalis E. Cheissin 1948. Arch. Protistenk., 102 : 265-290. CHEISSIN, E.M. (1959). Cytochemical investigations of different 'stages of the life cycle of coccidia of the rabbit. Int. ConoT. Zoo'. 15, London 1958, pp 713-716.

CLARKE, D.H. (1952a). The use of phosphorus 32 in studies on Plasmodium aallinaceum I. The development of a method for the quantitative determination of parasite growth and development in vitro. J. exp. Med., 96 : 439-450. 161.

CLARKS, D.H. (1952b). The use of phosphorus 32 in studies on Plasmodium gallinaceum II. Studies on conditions affecting parasite growth in intact cells and in lysates. J. exp. Med., 22 : 451-463.

CLEVELAND, L.R. (1950a). Hormone-induced sexual cycles of flagellates II. Gametogenesis, fertilization and one- division meiosis in Oxymonas. J. Morph., 86 : 185-214,

CLEVELAND, L.P. (1950b). Hormone-induced sexual cycles in flagellates III. Gametogenesis, fertilization and one- division meiosis in Saccinobaculus. J. Morph., 86 : 215-223.

DASGUPTA, B. (1959). The Feulgen reaction in the different stages of the life-cycles of certain Sporozoa. microsc. Sci., 100. 241-255.

DOBELL, C. (1925). The life history and chromosome cycle of Agftt2Lata eberthi (Protozoa : Sporozoa : Coccidia) farpzIpsiozz, 17 : 1-136.

DOBELL, C. and JAMESON, A.P. (1915). The chromosome cycle in coccidia and gregarines. Proc. R. Soc., Series B, : 83-94.

LORAN, D.J. (1970a). Survival and development of Eimeria adenoeides in cell cultures inoculated with sporozotes from cleaned and uncleaned suspensions. Proc. helminth. Soc. Wash., 37 : 45-48.

DORAN, D.J. (1970b). Eimeria tenella : From sporozoites to oocysts in cell culture. Proc. helminth. Soc. Wash., 37 : 84-92.

DORAN, D.J. (1971). Comparative development of Eimria tenella in primary cultures of kidney cells from the chicken, pheasant, partridge and turkey. J. Perasit., 52 1376-1377.

DORAN, D.J. and VL'TTERLING, J.M. (1967a). Comparative cultivation of poultry coccidia in mammalian kidney cell cultures. J. Protozool., 14 : 657-662. 162.

DORAN, D.J. and VETTERLING, J.M. (1967b). Cultivation of the turkey coccidium, Eimeria meleag.rimitis Tyzzer 1929, in mammalian kidney cell cultures. Proc. helminth. Soc. Wash., j : 59-65. DORAN D.J. and VETTERLING, J.M. (1968). Survival and development of Eimeria meleagrimitis Tyzzer 1929 in bovine kidney and turkey intestine cell cultures. J. Protozool., : 796-802.

DUBREMETZ, J.F. (1971). L'ultrastructure du centriole et du centroc8ne chez la Coccidie Eimeria necatrix; 'etude au cours de la schizogonie. J. Microscopic, 12 : 453-458. DUNCAN, D., EADES, J.,- JULIAN, S.R. and NICKS, D. (1960). Electron microscope observations on malarial oocysts (Plasmodium cathemerium). J. Protozool., : 18-26.

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. FAYER, R. and HAMMOND., D.M. (1967). Development of first- generation schizonts of Eimeria bovis in cultured bovine cells. J. Protozool., 14 : 764-772 FEULGEN, R. and ROSSEUBECX, H. (1924). Mikroskopisch-chemischer Nachweis einer Nucleinsaure vom Typus der Thymonucleinsaure and die darauf beruhende elektive Farbung von Zellkernen in mikroskopischen Praparaten. Hoppe-Seyler's Z. physiol. Chem., 135 : 203-248.

FITZGERALD, P.R. (1970). Development of E. stiedae in avian embryos. J. Parasit., 56 : 1252-1253. GARNHMI, P.C.C., BIRD, R.G., BAKER, J.R., DRESSER, S.S. and EL-NAHAL, H.M.S. (1969). Electron microscope studies on motile stages of malaria parasites. VI. The ookinete of Plasmodium berghei yoelii and its trans- formation into the early oocyst. Trans. R. Soc. troll. Med. Hvg., 6 : 137-194. 163.

GARNHAM, P.C.C., LAINSON, R. and COOPER, W. (1957). The tissue stages and sporogony of Plasmodium knowlesi.. Trans. R. Soc. trop. Med. HyR., -L175747

GILL, B.S. and RAY, H.N. (1954). Glycogen and its probable significance in Eimeria tenella Railliet and Lucet, 1891. Indian J. vet. Sci., 24 : 223-228. GLEDHILL, B.L., GLEDHILL, M.P., RIGLER, P. and RINGERTZ, H.R. (1966). Changes in deoxyribonucleoprotein during spermiogenesis in the bull. Expl Cell Res., 41 : 652-665. -. GRELL, K.G. (1953). Entwicklung und Geschlechtsbestimmung von Eucoccidium dinophili. Arch. Protistenk., 99 : 156-186.

GRELL, K.G. (1967). Sexual reproduction in Protozoa. In "Research in Protozoology, Volume 2". (Tze-Tuan Chen, editor) pp. 147-213. Pergamon Press. GRENIER, J. (1921). Cytologische Untersuchung der Gametenbildung und Befructung von Adelea ovata (A. Schneider). Zool. Jb., 42 : 327-362.

GUTTERIDGE, W.E. and TRIGG, P.I. (1970). Incorporation of radioactive precursors into DNA and RNA of Plasmodium knowlesi in vitro. J. Protozoal., 17 : 89-96. GUTTERIDGE, W.E. and TRIGG, P.I. (1972). Some studies on the DNA of Plasmodium knowlesi. In "Comparative Biochemistry of Parasites". (H. Van den Bossche, editor). pp. 199-218. Academic Press. GUTTERIDGE, W.E., TRIGG, P.I. and WILLIAMSON, D.H. (1971). Properties of DNA from some malarial parasites. Parasitology, 62 : 209-219. HAMOND, D.M. (1971). The development and ecology of Coccidia and related intracellular parasites. In "Ecology and Phsiolo'Parasites".yyof. (A.M. Fallis, editor5. pp. 3-20. University of Toronto Press, Canada. 164.

HAMMOND, D.M. and FAYER, R. (1968). Cultivation of Eimeria bovis in thred established cell lines and bovine tracheal cell line cultures. J. Parasit., : 559-568. HAMMOND, D.M., SCHOLTYSECK, E. and CHOBOTAR, B. (1969). Fine structural study of the microgametogenesis of Eimeria auburnensis. Z. ParasitKde., : 65-84. •HAUSCHKA, T.S. (1943). Life history and chromosome cycle of the coccidian Adelina deronis. J. Morph., 22 : 529-581. HELLER, G. (1971). Elektronenmikroskopische Untersuchungen zur Schizogonie in den sog. kleinen Schizonten von Eimeria stiedae. (Sporozoa, Coccidia). ProtistologicE777761-469.

HORTON-SMITH, C. and LONG, P.L. (1963). Coccidia and coccidiosis in the domestic fowl and turkey. Advances in Parasitoloqv, 1 : 67-107. HOWELLS, R.E. and DAVIES, E.E. (1971). Nuclear division in the oocyst of Masmodium berghei. Ann. trod. Med. Parasit., : 451-459. HUGHES-SCHRADER, S. (1943). Meiosis without chiasmata in diploid and tetraploid spermatocytes of the mantid Cal.limantis antillarum Saussure. J. Morph., Z3 : 111-141.

JAMESON, A.P. (1920). The chromosome cycle of gregarines, with special referene to Dinlocvstis schneideri Kunstler. quart. Jl microsc. Sci., 4 : 207-266. JEFFERS, T.K. and WAGENBACH, G.E. (1969). Sex differences in embryonic response to Eimeria tenella infection. J. Parasit., : 949-951. JONES, E.E. (1952). Size as a species characteristic in _coccidia: Variation under diverse conditions of infection. Arch. Protistenk., 76 : 130-170. JOYET-LAVERGNE, P. (1926). Recherches sur le cytoplasrne des Sporozoaires. Archs Anat. microsc., 22 : 1-128. 165,,

KILLANDER, D. and RIGLER, R. (1965). Initial changes of deoxyribonucleoprotein and synthesis of nucleic acid in phytohemaggl.utinine-stimulated human leucocytes in vitro. aplaLLLE., : 701-704. KILLANDER, D. and RIGLER, R. (1969). Activation of deoxyribo- - nucleoprotein in human leucocytes stimulated by phyto- hemagglutinin. Exel Cell Res., 54 : 163-170. KISHIDA, T. and KATO, S. (1965). Autoradiographic studies on intracytoplasmic multiplication of Toxoelasma gondli in FL cells. Biken J., 8 : 107-113. KLIMES, B., ROOTES, D.G. and TANIELIAN, Z. (1972). Sexual differentiation of merozoites of Eimeria tenella. Parasitology, 624 131-136. • KOTLAN, S. and PELLgRDY, L. (1937). Klserleti vizsgalatok a haziny,S1 dajcoccidiosisar61. II. (Experimental studies on hepatic coccidiosis in domestic rabbits II). K8zlem&nyek az OsszehasoulTAS 6let-es KOrtan KOreb81, 28 : 105-120.

KURNICK, N.B. (1955). Histochcmistry of nucleic acids. Int. Rev. Cytol., 4 : 221-268. LADDA, R.L. (1969). New insights into the fine structure of rodent malarial parasites. Iiilit. Med., 134 (Suppl) 825-864.

LEE, D.L. and MILLARD, B.J. (1971). Fine structure of the schizonts of Eimeria praecox. Int. J. Parasitol., 1 : 37-41. LEE, D.L. and MILLARD, B.J. (1972). Fine structural changes in Eimeria tenella, from infections in chick embryos and chickens, after exposure to the anticoccidial drug robenidene. Parasitolog, : 309-316. LESSLER, M.A. (1951). The Nature and Specificity of the Feulgen Reaction. Archs Biochem. Bionhys., 32 : 42-54. LESSLER, M.A. (1953). The nature and specificity of the Feulgen nucleal reaction. Int. Rev. Cytol., 2 : 231-247. 166.

LEWERT, R.M. (1952). Nucleic acids in plasmodia and the phosphorus partition of cells infected with Plasmodium za111m. J. infect. Dis., : 125-144. LILLIE, R.D. (1947). Reactions of various parasitic organisms in tissues to the Bauer, Feulgen, Gram and Gram-Weigert methods. J. Lab. clin. Med., 32 : 76-88. •LONG, P.L. (1959). A study of Eimeria maxima Tyzzer, 1929, a coccidium of the fowl (221122aallaa). Ann. trop. Med. Parasit., : 325-333. LONG, P.L. (1965). Development of Eimeria tenella in avian embryos. Naturet_ionl., : 509-510. LONG, P.L. (1966). The growth of some species of Eimeria in avian embryos. Parasitology, : 575-581. LONG, P.L. (1969). Observations on the growth of Eimeria tenella in cultured cells from the parasitized chorioallantoie me m branes of the developing chick embryo. Parasitolon,, : 757-765. LONG, P.L. (1970a). Some factors affecting the severity of infection with Eimeria tenella in chicken embryos. Parasitology, 60 : 435-447. LONG, P.L. (1970b). Eimeria tenella: chemotherapeutic studies in chick embryos with a description of a new method (chorioallantoic membrane foci counts) for evaluating. infections. L ParasitKa., : 329-338. LONG, P.L. (1971). Schizogony and gametogony of Eimeria tenella in the liver of chick embryos. J. Protozool., 18 : 17-20. LONG, P.L. (1972). Endogenous stages of an embryo-adapted strain of E. tenella. J. Protozool., 19 (Suppl.) : 12.

LUYKX, P. (1970). "Cellular Mechanisms of Chromosome Distribu- tion". International Review of C,1-.olory Suoniement 2. Academic Press, New York. MATSUOKA, T., CALLENDER, M.E. and SHUMARD, R.F. (1969). Embryonic bovine tracheal cell line for in vitro cultivation of Eimeria tenella. Am. J. vet. Res., 30 : 1119-1122. 167.

McLAREN, D.J. (1969). Observations on the fine structural changes associated with schizogony and gametogony in Eimeria tenella. Parasitology, 59 : 563-574.

MIRSKY, A.E. and RIS, H. (1949). Variable and constant components of chromosomes. Nature. Lond., 152 : 666-667. MONNE, L. and HONIG, G. (1954). On the properties of the shells of the coccidian oocysts. Ark. Zool., 7 : 251-256.

MULNARD, J. (1952). Etude quantitative de l'acide d4soxyribonucleic au cours de l'oogenese chez "Acanthoscelides obtectus sav" (Cor6optere). C.r. Ass. Anat7ET775:7457---7----"- MULSOW, K. (1911). Uber Fortpflanzungserscheinungen bei Mono- - cystis rostrata n, sp. Arch. Protistenk., 22 : 20-55.

NABIH, A. (1938). Studien uber die Gattung Klossia and Beschreibung des Lebenszyklus von Klossia loosii777,77p.). Arch. Protistenk., 91 : 474-515. NAVILLE, A. (1927). Recherches sur le cycle evolutif et chromo- somique de Klossia helicina (A. Schneider). Arch. Protistenk.

NAVILLE, A. (1931). Les Sporozoaires (cycles chromosomiques et sexualit6). 3•ierfL.Soc.P .92thf, 41 : 1-223

NOBLE, E.R. (1938). The life cycle of Lylosoma globosum sp. nov., a gregarine parasite of Urechis cauloo. Univ. Calif. Pubis Zool., : 41-66. NYBERG, P.A. and KNAPP, S.E. (1970a). Scanning electron micro- scopy of Eimeria tenella oocysts. Proc. helminth. Soc. Wash., 22 : 29-32. NYBERG, P.A. and KNAPP, S.E. (1970b). Effect of sodium hypo- chlorite on the cocyst wall of Eimeria tenella as _shown by electron microscopy. Proc. helmiath. Soc. Wash., 37 : 32-36. OUELLETTE, C.A., STROUT, R.G. and McDOUGALD, L.R. (1973). Incorporation of radioactive pyrirnidine nucleosides into DNA and RNA of Eimeria tenella (Coccidia) cultured in vitro. J. Protw,00l., 20 : 150-153. 168.

PATTON, R. (1935)- The life-history of Merocvstis kathae in the whelk Buccinum undulatum. Parasitology, 27 : 399-430. PATTILLO, W.H. and BECKER, E.R. (1955). Cytochemistry of Eimeria brunetti and E. acervulina of the chicken. J. Morph., 96 : 61-96. PATTON, W.H. (1965). Eimeria tenella: cultivation of the asexual stages in cultured animal cells. Science, N.Y., 122 : 767-769.

PAUL, J. (1965). "Cell and tissue culture". 3rd Edition.E. and S. Livingstone Ltd. PAWAN, J.L. (1931). Feulgen nucleal reaction and the malarial nucleus. Ann. tro). Med. Parasit., 25 : 185-187, PEARSE, A.G.E. (1968). "Histochemistry, Theoretical and Applied". Volume I. 3rd Edition. J. and A. Churchill Ltd., London.

PELLERDY, L.P. (1953). Beitrgge zur Kenntnis der Darmkokzidiose des Kaninchens, Die endogene Entwicklung von Eimeria piriformis. Acta vet. hung., 3 : 365-377. PELLERDY, L.P. (1965). "Coccidia and Coccidiosis". Akadgmiai KiadO, Budapest.

PERROTTO, J. KEISTER, D.B. and GELDERMAN, A.H. (1971). Incorpora- tion of precursors into Toxoplasma DNA J. Protozool., 18 : 470-473. PpLET, H. and BARR, C. (1968). DNA, RNA and protein synthesis in erythrocytic forms of Plasmodium knowlesi. Am. J. trop. Med. Hyg., 17 : 672-679. POLLISTER, A.W:, SWIFT, H. and ALFERT, M. (1951). Studies on the desoxypentose nucleic acid content of animal nuclei. J. cell. comp. Physiol., 3 : 101-119. PORCHET-HENNE4- E. and RICHARD, A. (1969). Structure fine du sporoblaste immature uninuclee d'Aggregata eberthi Lang (Sporozoaire, Coccidiomorphe). C.R. Acad. Sci. Paris, 269 : 1681-1683. 169.

PORCHET-HENNERE, E. and RICHARD, A. (1971). La Sporogene'se chez la Coccidie Agregata eberthi. Etude en micro- scopieelectronique. J. Protozool. 18 : 614-628. PORCHET-HENNERE, E. and VIVIER, E. (1971). Observations sur le "crystalloids" des Coccides. J. Protozool., 18 (Supol): 51

RAY, H.N. and DAS GUPTA, M. (1937). A new coccidian, Wenyonella hoarei n. sp. from an Indian squirrel. Parasitology, 29 : 117-119.

RAY, H.N. and GILL, B.S. (1955). Observations on the nucleic acids of Eimeria tenella Railliet and Lucet, 1891. Indian J. vet. Sci., 22. : 17-23.

REICHENOW, E. (1921). Die. HElemococcidien.der Eidechsen. Arch. Protistenk., 42 : 179-291.

REYER, (1937). Infektionsveruche mit Barrouxia schneidcri an Lithobius forficatus insbesondere zur Frage der Sexualitat der Coccidiensporozoiten. Z. ParasitKde., 9 : 478-522. ROBERTS, W.L., ELSNER, Y.Y., SHIGEMATSU, A. azad HAMMOND, D.M. (1970). Lack of incorporation of -)H-thymidine into Eimeria calloaml2ohili in cell cultures. J. Parasit., : 833-834. ROBERTS, W.L. and HAMMOND, D.M. (in preparation). Cited by Hammond, D.M. (1971).

- ROBERTS, W.L., HAMMND, D.M., ANDERSON, L.C. and SPEER, C.A. (1970). Ultrastructural study of schizogony in Eimeria callosrermo- ohili. J. Protozool., 17 : 584-592.

ROBERTS, W.L., SPEER, C.A. and HAMMOND, D.M. (1970). Electron and light microscope studies of the oocyst walls, sporocysts and excysting sporozoites of Eimeria calla- soermophili and Eimaria larimerensis. J. Parasit., : 918-926. ROBINOW, C.F. and MARAK, J. (1966). A fiber apparatus in the nucleus of the yeast cell. J. Cell Biol., 29 : 129-151. 170.

ROOTES, D.G. and LONG, P.L. (1965). Studies on the synthesis of glycOgen by Eimeria necatrix in the domestic fowl. Prop-ress in Protozooloui_Troc. 2. Int. Congr.Protozool. Lonclan12L. Abstract 173.

-RUDZINSKA, M.A. and VICKERMAN, K. (1968). The Fine Structure. In "Infectious Blood Diseases of Than and Animals". (D. Weinman and M. Ristic, eds.) Vol. I, pp 217-.-306. Academic Press, New York.

RUTHERFORD, R. (1943). The life-cycle of four intestinal coccidia of the domestic rabbit. J. Parasit., 29 : 10-32. RYLEY, J.F. (1968). Chick embryo infections for the evaluation of anticoccidial drugs. 22EaLL2LITL, : 215-220.

RYLEY, J.F. (1969). Ultrastructural studies on the sporozoite of Eimeria tenella. Parasitoloy, 59 : 67-72.

RYLEY, J.F., MANNERS, D.J. and STARK, J.R. (1968). Amylopectin, the storage polysaccharide of Eimeria tenella. J. Protozool., (Supple) : 31.

SAMPSON, J.R. and HAMMOND, D.M. (1971). Ingestion of host cell cytoplasm by micropores in Eimeria alabamensis. T s Parasit., L7 : 1133-1135. SASSUCHIN, D. (1935). Zum Studium der Protisten- und Bakterien- kerne. I. Uber die Nuclealreaktion und ihre Anwendung bei protozoologischen und bakteriologischen Untersuchungen. Arch. Protistenk., 84 : 186-198.

SC LACK, C. (1912). Untersuchungen uber die Coccidien aus Lithobius und Scolopendra (Barrouxia, Adelea, Eimeria). Verh. dt. Zool. Ges., 22 : 163-179a SCHOLTYSECK, E. (1963). Untersuchungen uber de Kernverhaltnisse und das Wachstum bei Coccidiomorphen ureter besonderer Berucksichtigung von Eimeria maxima. Z. ParasitKde. 22 : 428-474. 171.

SCHOLTYSECK, E. (1965). Die Mikrogametenentwicklung von - Eirner'forans.j.apu Z. Zellforsch. mikrosk. Anat., 66 : 625-642. SCHOLTYSECK, E. , MEHLHORN, H. and HAMMOND, D.M. (1971). Fine structure of macrogametes and oocysts of Coccidia and related organisms. Z. ParasitKde., 117 : 1-43. SCHOLTYSECK, E. and STROUT, R.G. (1968). Feinstrukturunter- suchungen uber die Nahrungsaufnahme bei Coccidien in Gewebekulturen (Eimeria tenella). Z. ParasitKde., : 291-300. SCHOLTYSECK, E., STROUT, R.G. and HABERKORN, A. (1969). Schizonten and Merozoiten von Eimeria tenella in Nakrophagen. Z. ParasitKde., 77717727;. SCHOLTYSECK, E. and VOIGT, W.H. (1964). Die Bildung der Oocystenhulle bei Eimeria Derforans (Sporozoa). Z. Zellforsch. mikrosk. Anat., 62 : 279-292. SCHOLTYSECK, E. and WEISSENFELS, N. (1956). Elektronenmikro- skopische Untersuchungen von Sporozoen. I. Die Oocystenmembran des Huhnercoccids, Eimeria tenella. Arch. Protistenk., 101 : 215-222.

SIEGEL, S. (1956). "Nonparametric Statistics for the behavioural sciences". McGraw-Hill Book Company, Inc. SNIGIREVSKAYA, E.S. (1969). Changes in some u1trastructures during microgametogenesis in the rabbit coccidia. Eimeria intestinalis and E. ma.zaa. (In Russian). Tsitologiya, 11 : 582-385.

SPEER, C.A. and HAMMOND, D.M. (1970). Nuclear divisions and refractile-body changes in sporozoites and schizonts of Eimeria callos.ermonhili in cultured cells. J. Parasit., : 4 1-4o7.

SPEER, C.A. and HAMMOND, D.M. (1972). Development of gametocytes and oocysts of Eimeria magna from rabbits in cell culture. Proc. helminth. Soc. Wash., 32 : 114-118. 172.

STROUT, R.G. and OUELLETTE, C.A. (1969). Gametogony of Eimeria tenella (Coccidia) in cell cultures. Science, N.Y., .1.62 : 695-696. STROUT, R.G. and OUELLETTE, C.A. (1970). Schizogony and gametogony of Eimeria tenella in cell cultures. Am. J. vet. Res., : 911-918. STROUT, R.G., SOLIS, J., SMITH, S.C. and DUNLOP, W.R. (1965). In vitro cultivation of Eimeria acervulina (Coccidia). 111.722.LIrsL., 17 : 241:2+6. SUTTON, W.S. (1902). On the morphology of the chromosome group in Bitsla:191: magna. Biol. Bull. mar. biol. Lab., Woods Hole, 4 : 24-39.

SWIFT, H. and KLEINFELD, R. (1953). DNA in grasshopper sperma- togenesis, oogenesis and cleavage. Phvsiol. Zool., 26 : 301-311. SZOLLOSI, D., CALARCO, P. and DONAHUE, R.?. (1972), Absence of centrioles in the 1st and 2nd meiotic spindles of mouse oocytes. J. Cell Sci., 11 : 521-542. TERZAKIS, J.A. (1968). Uranyl acetate, a stain and a fixative. J. Ultrastruc. Res., 22 : 168-184.

TERZAKIS, J.A. (1971). Transformation of the Plasmodium cynomolGi oocyst. J. Protozool., 18 : 62-73. TERZAKIS, J.A., SPRINZ, H. and WARD, R.A. (1966). Sporoblast and sporozoite formation in Plasmodium gallinaceum infection of Aedes aejypti. Milit. Med., 131 (Suppl)-- : 984-992. TERZAKIS, J.A., SPRINZ, H. and WARD, R.A. (1967). The transformation of the Plasmodium gallinaceum oocyst in Aedes aegvDti -mosquitoes. J. Cell Biol., 34 : 311-326. TRACY,.S.M, and SHERMAN, I.W. (1972). Purine uptake and utili- zation by the avian malaria parasite Plasmodium lor)hurae. J. Protozool., 19 : 541-549.

TRAGER, W. (1971). Malaria parasites (Plasmodium lophurae) developing extracellularly in vitro : incorporation of labelled precursors. J. Protozool., 13 : 392-399. 173.

TREFIAK, W.D. and DESSER, S.S. (1973). Crystalloid inclusions in species of Leucocytozoon, Parahaemoproteus and Plasmodium, J. Protozool., 20 : 73-80.

TREGOUBOFF, G. (1914). Sur l'evolution sexuelle de Stenophora juli A. Schneider (Frantzius) et la position systema- matique de la famine des Stenophorides Leger et Duboscq. Archs Zool. exp. gen (Notes et Revue), 2Lk : 19-30.

TSUNODA, K. and ITIKM/A, O. (1955). Histochemical studies of chicken coccidia (Eimeria tenella) I. On the nucleic acids, polysaccharides and phosphomonoesterases in their several developmental stages. Report No. 29 Government Ex erimental Station for AniniallaimLzluan (L,12) pp 73-82.

VANDERBERG, J. and RHODIN J. (1967). Differentiation of nuclear and cytoplasmic fine structure during sporogonic develop- ment ofertPla,iberhela. J. Cell Biol., 2? : C7-10.

VANDERBE2G, J., RHODIN, J. and YOELI, M. (1967). Electron microscope and histochemical studies of sporozoite formation in Plasmodium berghei. J. Protozool., 14 : 82-103.

VAN DYKE, K. , TREMBLAY, G.C., LANTZ, C.H. and LZUSTKIEWICZ, C. (1970). The source of purines and pyrimidines in Plasmodium berghei. Am. J. trop. :ed. Hyg., 19 : 202-208.

VAVRA, J. (1965). Etude au microscope electronique de la morphologie et du developpement de quelques micro- sporidies. C.R. Acad. Sci. Paris,261 : 3467-3470.

VENDRELY, R. and VENDRELY, C. (1952). Sur la teneur individuelle en arginine des sbermatozoides, compare EZ'la teneur individuelle en arginine des noyaux d' erythrocytes chez quelques esprThes de Poissons. C.r. he d. Seanc. Acad. Sci., Paris, 235 : 444-446.

VENDRELY, R. and VENDRELY, C. (1956). The results of cytophoto- nietry in the study of the deoxyribonucleic acid (DNA) content of the nucleus. Int. Revt Ltol., : 171-197. 174.

VETTERLING, J.M. and DORAN, D.J. (1969). Storage polysaccharide in cocc3.dial sporozoites after excystation and penetration of cells. J. Protozool., 16 : 772-775. VIVIER, E. (1955). Presnce de microtubules intranucleaires chez Motchnikowella hovassei Vivier. J. Microsconie, 4 : 559-502. VIVIER, E., PETITPREZ, A. and LANDAU, I. (1972). Observations ultrastructurales sur la sporoblastogeHe'se de 1' Hemogr,Sgarine, Hepatozoon domerruei (Coccidie Adeleidae). Protistolovica, 8 : 315-334.

WAGENBACH, G.E., CHALLEY, J.R. and BURNS, W.C. (1966). A method for purifying coccidian oocysts employing Clorox and sulphuric acid - dichromate solution. J. Parasit., : 1222.

WALSH, C.J. and SHERMAN, I.W. (1968). Purine and pyrimidine synthesis by the avian malaria parasite, Plasmodium 122hyrae. J. Protozool., 15 : 763-770.

WHEELER, E.E. (1969). An evaluation of cytochemical methods for the quantitative estimation of deoxyribonucleic acid (DNA). J. med. Lab. Technol., 26 : 314-339.

WHITFELD, P.R. (1953a). Studies on the nucleic acid of the malaria parasite, Plasmodium berghei (Vincke and Lips). Aust. J. biol. Sci., 67754-243.

WHITFELD, P.R. (1953b). The uptake of radioactive active phos- phorus by erythrocytic stages of Plasmodium berghei (Vincke and Lips). Aust. J. biol. Sci., 6 : 591-596. WILSON, P.A.G. and FAIRBAIRN, D. (1961). Biochemistry of sporulation in oocysts of Eimeria acervulina. J. Protozool., 8 : 410-416. WOLCOTT, G.B. (1954). Nuclear structure and division in the malaria parasite, Plasmodium vivax. J. Morph., 94 : 353-365. 175.

WOLCOTT, C.B. (1957). Chromosome studies in the genus Plasmodium. J. Protozool. 4 : 48-51. YARWOOD, E.A. (1937). The life cycle of Adelina crutocerci sp. nov. a coccidian parasite of the roach Cryptocercus punctulatus. Parasitolo7x, 29 : 370-390.

YATAGANAS, X., GAHRTON, G. and THORELL, B. (1970). DNA, RNA and hemoglobin during erythroblast maturation. A cyto- photometric study. Expl Cell Res., 62 : 254-261.

Addendum

PAYNE, L.N. (1972). The Chick Embryo. In "The UFAW Handbook on the Care and Management of Laboratory Animals". TUTTTTdir;;). 4th Edition. pp. 440-460. Churchill Livingstone, Edinburgh and London. 176.

.112221111.2c.

Table 1 Absorption units of Feulgen stained chick erythrocyte nuclei. Variation of absorption obtained for different staining times..

hour. 282 268 267 277 276 276 289 272 261 282 265 281 269 253 256 259 276 261 261 274 280 279 280. 265 265 275 260 276 272 253 255 257 280 267 269 270 280 270 283 267 272 276 260 284 272 249 257 259 274 261 262 276 277 275 232 265 264 282 256 279 271 248 255 260

269 274 261 263 247 255 254 250 245 271 268 264 263 241 258 257 255 245 271 274 259 261 241 256 256 255 247 267 271 265 256249 252 253 246 243

1i hours 448 491 485 450 446 446 46o 494 498 473 476 473 480 445 441 464 444 486 487 444 447 446 461 495 499 466 471 472 471 1443 440 46o 442 482 486 446 444 439 46o 501 501 468 476 470 471 447 436 461 445 479 484 448 453 436 458 496 505 471 474 473 474 439 438 463 46o 457 464 449 461 476 461 511 457 462 454 466 451 462 48o 457 510 459 460 454 463 452 46o 430 459 5o8 462 454 453 468 445 458 481 464 508 462 177.

Table 1 contd.

2-1 hours 429 425 437 436 439 445 466 433 41+3 491 438 424 452 511 454 424 429 423 429 434 434 1+38 463 43o 437 492 429 426 444 503 450 42o 427 422 436 430 437 437 458 435 439 491 430 423 448 507 451 417 426 422 432 430 1+35 440 463 437 444 496 426 421 451 505 452 422

485 431 438 1+53 438 447 484 472 446 483 435 444 452 434 445 1+91 1+76 444 480 438 443 452 434 446 1492 474 447 481 432 439 456 434 440 493 467 446

20 hours 421 409 372 366 396 368 362 365 372 366 367 363 387 364 356 359 420 412 373 361 389 369 363 366 373 364 368 361 384 371 356 352 426 411 363 358 392 373 36o 366 373 365 367 365 378 364 356 353 419 410 371 354 389 367 361 365 367 366 368 367 376 365 356 356

403 401 383 411 365 360 397 1+00 409 1+00 398 377 411 363 359 395 395 /402 405 399 374 414 364 364 393 397 /410 396 392 375 410 361 359 395 396 400 178.

Table 2 Nicrodensitometry readings of Feulgen-stained oocyst nuclei after the 1st zygotic division (no colchicine treatment) >■60, X100 oil objective, spot size 2

Absorption value DNA value Absorption value DNA value of nucleus (mean in standard of nucleus (mean in standard of 4 readings) units of 4 readings) units

6.50 14.30 9.25 20.35 7.00 15.40 11.00 24.20 12.50 27.50 12.50 27.50 12.00 26.40 7.25 15.95 11.50 25.30 13.25 29.15 12.25 26.95 13.0o 28.60 13.50 29.70 8.00 17.60 12.00 26.40 6.75 14.85 14.00 30.80 6.75 14.85 9.00 19.80 9.00 19.80 11.75 25.85 8.5o 18.70 11.50 25.30 1000 22.00 9.00 19.80 11.00 24.20 8.25 18.15 7.50 16.50 11.00 24.20 8.00 17.60

Chicken erythrocyte control (DNA standard) Range of absorption values 434-482 Mean absorption value 452.5 Standard error + 1.6 Conversion factor 2.2 179.

Table 3 Microdensitometry readings of Feulgen-stained microgamete

nuclei. X 60, X100 oil objective, spot size 2.

Absorption value DNA value Absorption value DNA value of nucleus (mean in standard of nucleus (mean in standard of 4 readings) units • (of 4 readings units

15.25 14.13 14.75 13.71 14.50 13.48 15.25 14.18 16.25 15.11 15.50 14.41 15.50 14.41 14.50 13.48 14.50 13.48 15.50 14.41 15.00 13.95 15.25 14.18 14.75 13.71 14.75 13.711 14,50 13.48 16.00 14.50 13.48 14.50 13.43 14.25 13.25 15,00 13.95 14.50 13.48 15.75 14.64

Chicken erythrocyte control (DNA standard):

Range of absorption values 1011-1170

Mean absorption value 1078.2

Standard error +7.6 Conversion factor 0.93 Table 4 Microdensitcmetry readings of Feulgen-stained E,chizont nuclei A 60, X100 oil objective, spot size 2

Absorption value DNA value Absorption value DNA value Absorption value DNA value of nucleus (mean in standard of nucleus (mean in standard of nucleus (mean in standard of 4 readings) units of 4 readings) units of 4 readings) units

23.00 21.39 16.50 15.34 16.75 15.57 20.00 18.60 19.00 17.67 16025 15.11 16.00 14.88 25.75 23.94 25.00 23.25 17.75 16.50 19.50 18.13 25.75 23.94 20.75 19.29 22.50 20.92 29.50 27.43 19.00 17.67 17.00 15.81 19.25 17.90 21.00 19.55 16.25 15.11 16.25 15.11 23.00 21.39 27.00 25.11 29.50 27.43 22.50 20.92 20.75 19.29 32.50 30.22 16.25 15.11 17.00 15.81 34.25 31.85 19.25 17.90 15.75 14.64 19.50 18.13 17.00 15.81 17.00 15.81 23.75 22.08 21.50 19.99 16.75 15.57 21.50 19.99 18.00 16.74 16.50 15.34 22.00 20.46 17.00 15.81 18.00 16.74 16.75 15.57 20.50 19.06 19.50 13.13 17.50 16.27 30.00 27.90 20.25 18,83 20.75 19.29 18.50 17.20

Chicken erythrocyte control (DNA standard): Range of absorption values 1011-1170 Mean absorption value 1078.2; Standard error +7.6; Conversion factor 0.93 181.

Table 5 Microdensitometry readings of Feulgen-stained undivided zygote nuclei in the oocyst. A 60, X100 oil objective spot size 2

Absorption value DNA value Absorption value DNA value of nucleus (mean in standard of nucleus (mean in stan- of 4 readings) units of 4 readings) dard units

18.00 28.8 18.25 29.2 18.25 29.2 18.25 29.2 18.00 28.8 18.25 29.2 18.00 28.8 17.75 28.4 17.25 27.6 18.00 28.8 17.50 28.0 13.25 29.2 18.00 28.8 18.00 28.8 19.00 30.4 18.00 28.8 18.00 28.8 18.50 29.6 18.75 30.0 18.00 28.8 19.75 31.6 17.75 28.4 18.25 29,2 19.00 30.4 17.00 27.2 • 18.00 28.8

Chicken erythrocyte control (DNA standard): Range of absorption values 581-640 units

Mean absorption value 607.3 units Standard error -15.2 units

Conversion factor 1.6 182,,

Table 6 Microdensitometry readings of Feulgen stained oocyst nuclei after the fist division of the zygote. A60 X100 oil objective, spot size 2

Absorption value Conversion DNA value of nucleus (mean factor in standard of 4 readings) units AVIC-A01.1.0...... 310.•••••••. 10.50 1.4 14.70 12.00 1.4 16.80 11.00 1.9 20.90 11.25 1.9 21.30 11.25 1.9 21.30 9.75 1.9 18.50 8.75 1.9 16.60 9.0o 1.9 17.10 7.50 1.9 14.20 7.5o 1.9 14.20 14.25 1.6 22.80 14.50 1.6 23.20 15.25 1.6 24.40 13.75 1.6 22.00 14.00 1.6 22.40 18.50 1.6 29.60 19.00 1.6 -30.40 20.00 1.6 32.00

Chicken erythrocyte controls (DNA standards)

1 2 3 Range of absorption values 659-795 470-536 566-624 Mean absorption value 710 515.1 591.3 Standard error +7.0 ±5.2 15.7 Conversion factor 1.4 1.9 1.6 183.

Table 6 Contd., 1

Absorption value Conversion DNA value of nucleus (mean factor in standard of 4 readings) units

--10.00 1.6 16.00 9.50 1.6 15.20 10.00 1.6 16.00 10.00 1.6 16.00 10.75 1.6 17.20 16.25 1.6 26.00 16.25 1.6 26.00 15.00 1.6 24.00 10.25 1.6 16.40 10.25 1.6 16.40 10.00 1.6 16.00 12.00 1.6 19.20 11.25 1.6 18.00 11.00 1.6 17.60 12.75 1.6 20.40 12.00 1.6 19.20 13.25 1.6 21.20

Chicken erythrocyte controls (DNA standards)

Range of absorption values 566-624 Mean absorption value 591.3

. Standard error 15;7 Conversion factor 1.6 184.

Table 6 Contd., 2

Absorption value Conversion DNA value of nucleus (mean factor in standard of 4 readings) units

•••••■•••■•••••••••■••1.0

12.50 1.6 20.00 13.25 1.6 21.20 14.00 1.6 22.40 14.25 1.6 22.80 14.75 1.6 23.60 15.25 1.6 24./10 15.50 1.6 24.80 17.25 1.6 27.60 19.75 1.6 31.60 15.00 1.6 24.00 15.00 1.6 24.00 15.50 1.6 24.80 15.75 1.6 25.20 12.00 1.6 19.20 11.25 1.6 18.00 10.00 1.6 16.00 9.25 1.6 14.80

Chicken erythrocyte controls (DNA standards) Range of absorption values 566-624 Mean absorption value 591.3

Standard error 5-7 Conversion factor 1.6 Table 7 Microdensitometry readings of Feulgen stained oocyst nuclei after the second division of-the zygote. . A 60, X100 oil objective spot size 2

Absorption value Conversion DNA value Absorption value Conversion DNA value of nucleus (mean factor in stand- of nucleus (mean factor in stand- of 4 readings ard units of 4 readings ard units

31.00 0.94 29.14 18.25 0.94 17.15 25.25 0.94 23.73 28.50 0.94 26.79 21.75 0.94 20.44 23.5o 0.95 22.32 27.00 0.94 25.38 17.00 0.95 16.15 27.00 0.94 25.38 29.00 0.95 27.55 26.00 0.94 24,44 30.25 0.95 28.73 15.75 0.94 14.8o 28.00 0.95 26.60 14.75 0.94 13486 20.25 0.96 19.44 15.75 0.94 14.80 16.50 0.96 15.84 16.75 0.94 15.74 17.75 0.96 17.04 15.50 0.94 14.57 20.50 0.96 19.68 18.75 0.94 17,62 15.00 0.96 14.40 16.25 0.94 15.27 15.50 0.96 14.88

Chicken erythrocyte controls (DNA standards) 1 2 3 4 RanE:e of absorption values 990-1122 1033-1105 1003-1053 1038-1034 Mear.. absorption value 1065.8 1055.8 1037.6 1053.9 StE.ndard error +10.0. _.+4.0 ±5.4 +2.0 Conversion factor 0.94 0.95 0.96 0.94