ISOLATION AND CHARACTERIZATION OF

DENSE GRANULES OF EIIKEW TEWLLA SPOROZOITES

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

MUHAMMAD JAVAID TAKIR

In partial fulfilment of requirements

for the degree of

Master of Science

December, 1998

O Muhammad Javaid Tahir, 1998

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Abstract

Isolatioo and Characterization of Dense Granules of Eüneria tenella Sporozoites

Muhammad Javaid Tahir, DVM (PAK), M.Sc. University of Guelph, 1998

Advisor: Dr. J. R Barta

Subcellular hctionation using sucrose gradient centrifugation was established for

isolating dense granules and other organelles of Eimenà tenella sporozoites. Identity of

the organelles was confimed using transmission electron rnicroscopy. Spherical dense

granules measured 180I16.3 nm (n=25) in diameter. BALB/c mice were immunized with

dense granules or other organelles. Post-immune sera were tested for organelle-specific

antibodies against whole sporomites with indirect fluorescent antibody assay (IFA).

Splenocytes fiom immunized mice were fuseci with NS-I myeloma cells to give hybrids.

About half of the hybrid cultures showed growth and were tested using for antibody using

F A against sporozoite antigens. Twelve hybnds secreted antibodies recognizing

rhoptries or micronemes. One hybnd (designated DBO-5F4) gave discrete positive

labeling of dense granules and detected a 38 kDa band in immunoblots of whole

sporozoites. This was the first successful isolation of dense granules from sporozoites.

Specific antibodies generated can be used for M e r characterization of dense granule

proteins.

Acknowledgements

I am thankfid to my advisor Dr. J. R. Barta for providing me an opportunity to

work in his laboratory. Any element of accomplishment in this study could not have seen

the light of day without his generous help, advice and encouragement.

1 benefited a lot fiom the thoughtful ideas and words of wisdom of Dr. M. A.

Feniando and Dr. M. A. Hayes, both of whom were also on my advisory cornmittee.

Many thanks to my fellow graduate students D. Johnston, R Carreno, K.

Strickler, S. Kopko and S. Lee for good companionship and a iively work atmosphere in

the laboratory.

1 am indebted to my niends E. Simko, M. Farooq, A. Kidwai and S. Saini for

keeping my morale up whenever I was stressai.

Laboratory technicians J. Cobean and S. Slack were quite help hl as they

maintained a continuous supply of parasites needed for this work. The care given to my

experimental animais by Dave, Tony and Jackie in the OMAFRA isolation unit is

appreciated.

My research was supported by gants fkom the Nahiral Sciences and Enginee~g

Research Council of Canada and the Ontario Ministry of Agriculture, Food and Rural

Affâirs to Dr. I. R. Barta. My shidies were supported by a fellowship fiom Overseas

Projects Corporation of Victoria (OPCV) of Melbourne, Australia.

Finally, the help and support that 1 received fkom my brother Dr. M. Parvez while

pursuing this study is grate fully achowledged.

Table of Contents

PAGE ABSTRACT .........................................................................................................................

. . ................................................................................................. TABLE OF CONTENTS 21

LIST OF FIGURES .......................................................................................................... iv

............................................................................................................ LIST OF TABLES v

GENERAL MORPHOLOGICAL FEATURES OF APICOMPLEXAN PARASITES ......................... ,.. 1 ................................................................................................................ Micronemes 3

Rhoptries ..................................................................................................................... 4 ....*.................. ......................................*.................................... Dense granules ... 5

............................................................................................ COCCIDIA AND COCCIDIOSIS 7 ................................................................................................... The genus Eimeria 7 ..................................................................................................... Poultry Coccidiosis 9

INTRODUCTION

MATERIALS AND METHODS ...................................................................................... 14

PARASITE PROPAGATION ............................................................................................... 14 PARASITE EWREICATION ............................................................................................... 15

Oocyst flotation .......................................................................................... .. ............. 15 . . . Surface Stenlization of Oocysts ............................................................................... 16 Excystation .............................................................................................................. 16 Purification of sporozoites - Isopycnic Centrifugation ............................................. 17 Purification o f sporozoites - Glass Wool Column Filtration .................................... 18

ORGANELLAR ISOLA~ON FROM SPOROZOITES .............................................................. 18 Sonication ............................................................................................................... 18 French Press .............................................................................................................. 18 Discontinuous Sucrose Gradient ........................................................................... 19

E L E ~ O N MICROSCOPY ............................................................................................... 20 Fixation of Organelle specimens for Transmission Electron Microscopy (TEM) ... 21 Mouse hunization ................................................................................................ 22 Indirect Fluorescent Antibody Assay P A ) ............................................................. 23

.............................. IFA Of SPOROZOITES D W G PENETRATION OF ~ T U R E D CELU 25 Isolation o f Splenocytes ........................................................................................... 26

.................................................... Fusion of Splenocytes and NS-1 Myeloma Cells 26 ......... Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 28

WESTERN BLOT ....................................................................................................... 2 9

List of Figures

PAGE Figure 1. Electron micrograph of sporozoite of Eimeria renella. the apical complex is

usually considered to include the micronemes and hopaies as well as the conoid and apical rings. .......................................................................................................... 1

Figure 2- Typical life cycle of parasites of the genus Eimeria ( f?om Gardiner et al., 1 998). ......................... ,., ............................................................................................ 8

Figure 3. Illustration of the order and volumes of different parts of the discontinuous gradient used to isolate organelles fiom ENneria teneiIa sporozoites after French Press disniption. ......... ............................ ... ........................................... 20

Figure 4. Location of organelles within the discontinuous sucrose gradient after centrifugation at 125,000 x g for 90 minutes ........................................................ 34

Figure 5 - Transmission electron micrograph of organelles collected above the 1.4M/l S M interface in the step gradient (Magnification 60,000~). ........ ........ .. .... .. 3 5

Figure 6 - Transmission electron micrograph of organelles collected above the 1 SM/ 1.6' interface in the step gradient (Magnification 60,000~). .......... .... .. ..... .. . 3 5

Figure 7 - Transmission electron micrograph of arnylopectin granules found pelleted at the bottom of the step gradient (Magnification 60,000~). ........................................ 36

Figure 8 - Indirect fluorescent antibody assay of sera collectecl fkom mice imrnunized 14 days previously with isolated subcellular organelles (Scale bar measures 50 p). . 3 7

Figure 9 - Indirect fluorescent antibody w a y of sera collectecl nom mice boosted 4 days previously with isolated subcellular organelles (Scale bar measures 50 prn). . .. .. . .. . 3 8

Figure 1 0 - Indirect fluorescent antibody assay of hybridoma supernatants MR-2D2, MR-3C4, MR- 1 D 1 and MR-2H 1 0 (Scale bar measures 50 pm). ... .. .. . .. .. . . . . . . . . . .. . . . . 40

Figure 1 1. Indirect fluorescent assay of hybridoma supernatants MR-2F6, MR-LE6 , MR-1 G6 and MR- 1 C 1 0 (Scale bar measures 50 pm). ..... . .. . . . .. .... .... ... .. ... .. .. .. . .. .. .. .. . 4 1

Figure 12 Indirect Fluorescent Antibody assay of hybridoma supernatant (DB O-5F4) (Scale bar measures 50 pm). ......-........... .... .... . . . . . . . . . . . . . . . . . . .. . . . . . 42

Figure 13. Redistribution of antigen recognized by hybridoma antibody DBO-5F4 in sporozoites of Eimeria tenella following invasion of cultured chicken fibroblasts detected using F A (Scale bar measures 50 p). .. ...... .. ..... .. ..... .. ...... ... . .. .... . . . . . . . . . . 44

Figure 14. Electrophoretically separated proteins of Eimeria tenella sporozoites and isolated dense bodies ................... ,., ................................................................. 45

Figure 15. Electrophoretically separated proteins of Eimeria tenella sporozoites transferred to Immobilon-P membrane and probed with sera f?om mice irnmunized with dense bodies or with sera fkom mice immunized with micronemes, rhopûies and mitochondna .............................................................................................. 46

Figure 16. Reactivity of hybridoma MR- 1C IO amd MR-2H 1 O with electrophoresed antigens of sporozoites of Eirneria tenella. ............................................................. 47

List of Tables

PAGE Table 1. Infecthg dosages of sporulated oocysts given to chickens per os

at various ages. ......................................................................................................... 14 Table 2. Composition of layen used in a discontinuous sucrose gradient

to separate Iysed organelles obtained h m French Press disruption of Eimeria tenello sporozoit es. ............................................................................... 1 9

Table 3. Number of spleen cells and myeloma cells fûsed for hybrid ceil production ..... 27

With the advent of electron microscopy, ultrastructural studies of the parasitic protozoa

were undertaken. It was quickly recognized that one group of sporozoan parasites d

possessed a special collection of organelles known as the apical complex. The apical

complex is comprised of a conoid and associated apical rings, one or more rhoptries, and

(often numerous) smaller micronemes.

Micronemes &

Granules

FIGURE 1. ELECTRON MIçROGRAPH OF SPOROZOITE OF ~E!MERLQ TENELLA. THE APICAL COMPLEX IS USUALLY CONSLDERED TO NCLUDE THE MlCRONEMES AND RHOPTEUES AS WELL AS THE CONOD AND APICAL RMGS.

Some authon include extrusomes hown as dense granules in the apical complex

and othen consider them separate fiom the complex. This information was used for the

taxonomie revision of the protists and Ied to the establishment of the phylum

Apicomplexa by Levine in 1970. The structural conservation of the apical complex has

fiuthered our tmderstanding of the host cell-parasite phmornena in the protozoan phylum

Apicornplexa

Apicomplexan zoites (sporozoites, merozoites, tachyzoites, bradyzoites,

cystozoites) are motile, infective stages that invade cells of various tissues. In addition to

their motiiity, zoites are characterized by possessing ultmstmcturally a pellicle, conoid,

polar ring(s), rhoptries, micronemes and dense granules concentrateci in the apical part of

the parasites. Numerous investigations (see Dubremetz, 1998 for a review) have

suggested that the three distinct extmornes (rhoptries, micronemes and dense bodies)

present in ail apicomplexan zoites may be involved in zoite-host cell interactions.

Various approaches have led to the identiiication of their protein contents and to partial

understanding of the role of these organelles. These include: isolation and analysis of

organelles; identification of protective antigens or T-ce11 epitopes which were later found

to be Iocated in organelles; analysis of culture supernatants for "exoantigens"; or

screening for antibodies inhibithg invasion in vitro.

Although the organelles are conserved among different apicomplexan parasites

and despite seemingly conserved functions, very few homologies have been found

beyond the genus level. However our howledge is too hcomplete to detect homologies

which would very likely point to consewed properties and would help identiQ conserved

fünctions. Coccidian sporozoites and merozoites are characterized by their ability to

penetrate into intact host cells without damaging the host cell membrane. Within the

cytoplasm of infected cells, the parasites reside within a vacuole comprised of a modified

host cell membrane. The invasion process of coccidian zoites has been divided into: 1)

recognition of host cell, 2) internalization of zoite and 3) maturation of the vacuole into a

metabolic cornpartment suitable for parasite growth. Based largely on ultrastructural

obseryations, organellar involvernent in cell invasion has been suggested as follows: 1)

micronemes at recognition; 2) rhoptries during internakation and 3) dense granules

during parasitophorous vacuole maturation (see Hemphill et al. 1998). However more

detailed investigations are warranted to confirm these inferences.

Micronemes

These tiny organelles are abundant in apicomplexan zoites. Their abundance

allowed eariy studies by subceiluiar fractionatïon. Micronemes of Plasmodium spp.,

Sarcocystis spp. and ToxopZkrsma gondii exocytose binding proteins, as a fïrst step

towards specific interaction, leading to the formation of a moving junction (Dubremetz,

1998).

Micronemes proteins that show adhesive domains homologous to mammalian

proteins (e.g. thrombospondin) have been identifieci in Plnsmodium spp.

(circumsporozoite protein, TRAP; Robson et al. 1988). Micronemes were k t isolated in

Sarcocystis muris by Dubremetz and Dissous (1 980) and immunological cross reactivities

arnong several Sarcocystis species were reported (Pohl et al. 1989). Cloning and

sequencing of a major 16 kDa protein suggested the presence of a binding domain (Klein

et al. 1992). Pre-invasion tramfer of microneme proteins to the host cell surface hints at

their possible role in hoa ce11 recognition (Entzeroth et al. 1992) or perhaps motility.

Several proteins known to be involved in PIasmodium merozoite-erythrocyte interactions

have evenhially been localized to micronemes by immunoelectron microscopy. These

include erythrocyte binding antigen of P. fakiparurn and the Du£@ binding antigen of P.

vivax and P. howlesi (see Camus and Hadley 1985, Adams et al. 1992). The

circumsporozoite protein (CSP), involved in host ceiI recognition, has been detected in

micronemes (Fine et ai. 1984). One of the ligands was encoded by a sequence also fomd

in tlmmbospondin that binds to Matides and cholesterol-3-sulfate (Cerami et al. 1992).

In Eimeria tenella, microneme proteins Etmic-1 (10OkDa) and Etmic-2 (SOkDa) were

cloned, sequenced and shown to possess thmmbospondin-like domains suggesting a

binding ability (Tomley et al. 1991,1996).

In Toxop~arma gondii three microneme proteins (MIC1; 60 kDa, MIC2; 120 kDa,

MIC3: 90 kDa) were studied by Achbarou et al. 199 1. MIC3 has demonstrateci

membrane-binding ability.

In Cryptospondiurn parvum, severai microneme proteins have been described

(Bonin et al. 199 1; 1993). They are al1 giycosylated and one of them shares antigenic

epitopes with rnacrogamete granules. Knocking out the TRAP gene in Plarmodium sp.

sporozoites using homologous recombination has suggested a role for this microneme

protein in the gliding motility of the zoites (Sultan et al., 1998; Spaccapelo et al 1997).

A large number of rhoptry proteins have been described in Plasmodium spp. ( e.g.

RHOP 1 .2,3; RAP 1,2 by Perkins. 1992). Genes for three have been cloned and sequenced

but this has not resulted in knowledge of their hction. Most of the rhoptry proteins are

located in the posterior bulge of the organelle whereas only two have been localized to

the duct which elongates towards the apex of the zoite (Roger et al., 1988, Crewther et al,

1990). In Toxoplma gondii, several rhoptry proteins are known and for one, ROP-1

(previously described as Penetration Enhancing Factor, PEF), the gene has been entirely

sequenced. It is subdivided into one acidic and one basic domain that may pemit a broad

spectnun of binding capabilities (Schwartzman, 1986; Ossorio et ai, 1992). A family of

proteins (ROP 2,3,4) in the 55-60 kDa range has also been identifid These proteins are

synthesized as pre-proteins that undergo post-translation modification (Sadak et al, 1988).

ROP2 has been partly sequenced and shown to elicit a strong T cell response. ROP2 has a

putative trammembrane domain close to its C terminus (Saavedra et al, 1992). Several

other rhoptry proteins (42,55,57,59 60 and 60.5 kDa) have also been descnbed (Leriche

and Dubremetz 199 1). In Eimeria tenelh, rhoptq organelles fiorn sporomites contain at

least 60 independent polypeptides. In Eimeriu spp. and Piumodium falcipamm, there is

poor conservation of rhoptry epitopes among various stages of the life cycle (Doury et al,

1994; Tomley 1991). During host ceil invasion, the rhoptry proteins are exocytosed and

found within the parasitophorous vacuole (PV) and on the PV membrane (Rick et al.

1998).

Dense granules

In contrast to micronemes and rhoptries, the dense granules were initially ignored

as to their role in host-parasite interactions. Recentiy they have been looked upon as a

major source of components that rnodify the PV some fime afler invasion. Of the apical

organelles the dense granules are exocytosed last of dl, about 20 minutes d e r

completion of invasion in the case of Tgondii (Cesbron-Delauw, 1994). Sarcocystis spp.

were the fint coccidia in which dense granules were characterized (Dubremetz and

Dissous, 1980) and later these were shown to exocytose into the parasitophorous vacuole

(Entzeroth, 1984). Out of the three proteins (20,26 and 32 kDa) described nom dense

granules, protease activity has been reported in a 26 kDa hction (Strobel et al. 1992).

The dense granules have been extensively studied in Toxoplma gondii where nine

separate dense granule proteins (GRA 1-7, NTPl and NTP2) have been characterized. Of

these, a function is asmied only for the closely related NTPase isoenzymes 1 and J I with

a probable role in purine salvage (Asai et al 1995; Cmther and Sibley 1997). GRA 1

(22,23,27 kDa) is a soluble protein with two calcium binding domains (Cesbron-Delauw

et al. 1989). GRA2 (28,28.5 kDa) has two amphipathic a-helixes that may bind to

membranes (Mercier et al. 1993). G W (30 kDa) becomes associated with the

parasitophorous vacuole membrane (PVM) and extensions of the PVM which protmde

into the cytoplasrn (Bermudes et al. 1994). GRA 4 (40 kUa ) and GRA 5 (21 kDa) have

transmembrane domains (Mevlec et al. 1992; Lecordier et al. 1993). The 32 D a GRA6

has two hydrophobic regions with characteristics of transmembrane domains. The C-

terminal hydrophilic region is comprised of 24% glycine residues, suggesting a structurai

role for GRA6 in the vesicular network found in the PV (Lecordier et al. 1995). GRA7

has a molecular mass of 25 kDa and a membrane-spanning domain (Jacobs et al. 1998).

Ail of these proteins are components of the dense granule matrk and al1 granules contain

these nine proteins in tachyzoites as well as bradyzoites. Differentiai M c k i n g of dense

granule proteins to various locations in the PV suggests unique functional and structural

attributes for each of these molecules (Achbarou et al. 1991).

In Toxoplarma gondii, excreted/secreted antigens (ESAs) have been investigated

as vaccine candidates. Dense granules proteins appear to be a major contributor to the

ESAs. These antigens have been used to irnmunize ndnu rats against placental

transmission of T. gondii. In addition, partial protection has been achieved by

immunization of mice with purified GRA2 and GRAS protein (Cesbron-Delauw, 1994).

In Plasmodium spp. two dense grande antigens (i.e. Pfl WRESA and RIMA) have been

cloned and sequenced (Aikawa et ai 1990; Trager et al 1992). A dense granule serine

protease @47) with a possible role in erythn,cyte invasion has been descnbed (Blackrnan

et al. 1998). In the closely related parasite, Neospora caninum, a gene (NCDG1)

encoding a 32 kDa dense grande protein has been cloned and sequenced. The proposed

amino acid sequence has three hydrophobie stretches, one being long enough to span the

PV membrane (Laiiy et al. 1997).

Coccidia and Coccidiosis

The coccidia are a large ubiquitous group of protozoa classified under the phylum

Apicomplexa on the basis of certain anatomical features that were revealed by electron

microscopie studies, collectively known as the apical complex (Levine 1980). n i e most

important coccidial parasites in poultry and cattle management are species of the genus

Eimeria. These parasites are characterized by oocysts that contain four sporocysts, each

containhg two infective sporozoites.

The genus Eimeria

The life cycle shown in Figure 2 is characteristic of most monoxenous coccidia of

the genus Eimeria. Both the asexual and sexual stages of reproduction are completed in

one host. The cycle commences when the unsporulated oocysts are passed in the faeces

of infected hosts. The oocysts lie dormant until environmental conditions (optimal

moisture, oxygen temperature) favor spodation (Current et al., 1990). Oocyst

spodation begins with the h t nuclear division, which is considered to be meiotic

(Canning and Morgan, 1975). Cytokinesis leads to the fonnation of sporoblasts, which

fiirther develop into four spomcysts. It is beiieved that a finai mitotic division occurs

pnor to the formation of the haploid sporozoites (Canning and Anwar, 1968).

Eimeria n a -

FIGURE 2. TYPICAL LEE CYCLE OF PARASITES OF THE GENUS ELMERIA (FROM GARDINER ET AL., 1998).

The life cycle proceeds when the sporulated oocyst is ingested by a suitable host.

Once ingested, the oocyst wall is disrupted and four sporocysts are released. In the

chicken, the grinding action of the gizzard facilitates the release of the sporocysts. The

next step, which is mediated by chemical rather than physical forces, involves the release

of the two sporozoites fkom each sporocyst. Sporozoite release is accomplished through

the actions of trypsin and chymotrypsin, which degrade the sporocystic plug or Stieda

body (Speer and Duszynski, 1975) and bile salts, which are believed to stimulate

sporozoite motility (Current et al. 1990). The sporozoites then enter host epithelial cells,

particularly on the intestinal v i a , where they undergo asexual reproduction (merogony)

and round up into ht-generation schizonts (Levine, 1982). Each meront contains many

nucleated, sickie-shaped cells h o w n as kt-generation merozoites. Thae merozoites

subsequently break out h u g h the epithelial cell membrane, enter the lumen and infect

other epithelial celis on the intestinal villi. AsexuaI reproduction (maogony) continues

for several rounds before proceeding to the sexual stage of reproduction. Most of the

pathologicd changes to the intestine associated with coccidiosis occur h m these

repeated rounds of merogony. M e r entering epithelid ceils, the last-generation

merozoites initiate sexual reproduction (gamogony) through the formation of gamonts.

Some of these will fom microgametes (male) and divide asexualiy by multiple fission

(schizogony) to produce a large number of flagellated microgametes while others form

macrogamonts (fernale) producing one macrogamete without M e r multiplication.

Microgametes then break out of an infected ce11 to penetrate and fertilize the intracellular

macrogamete. At this time syngarny takes place to form a zygote, which now contains a

diploid amount of DNA, This is the only stage in the life cycle in which the organism is

diploid (Canning and Morgan, 1975). The -te has eosinophilic granules or wall

forming bodies around its periphery, which flatten out and fuse to form a protective

three-layered wall. The resultaot unsponilated oocyst is released into the intestinal lumen

where it is passed out in the faeces to begin the cycle agai..

PouItry Coccidiosis

Coccidiosis in chickens is a variable form of gastroenteritis (enteropathy) caused

by intracellular apicomplexan protozoa of the genus Eimeriiz. Following bacterial

diseases, it is the second largest problem worldwide in the poultry industry. Coccidiosis

is prevalent wherever the poultry are reared in crowded conditions. The short life cycle

of this monoxenous parasite coupled with a very high reproductive capacity renders it of

great significance.

Damage to the digestive tract is caused by substantial replication within epithelial

cells of the intestine of up to seven Eimenia species. As discussed above, the life cycles

of Eimmia species are completed withh a single host (5-7 days) and comprise discrete,

expansive phases of asexual reproduction (two or more generations of merogony)

followed by a s e d phase (gamogony) that culminates in the production and excretion

into the faeces of large numbers of tough, resistant cysts (oocysts). Most important types

of intensively managed poulîry are reared directly on the floor throughout their life and

these management conditions facilitate the faecal-oral route of infection. The

introduction of highly productive chicken breeds in the 1940's and the resulting intensive

farming practices provided an ideal situation for the propagation of coccidia and for

producing clinical and sub-c linical coccidiosis. Chemotherap y was the main weapon

agauist coccidiosis d e r better management conditions. New dmgs were added to the

existing pool of chemoprophylactic and chemotherapeutic agents, as the parasite became

refiactory to early anticoccidials after repeated exposure (Chapman 1978). As a rneasure

of the scale of this problem, around 30x10~ bkds are bred annually worldwide for their

meat and around 200x 1 o6 breeding birds are reared for their eggs (Kaufmann 1 996;

Shirley and Bedniik 1997).

Large annual expenditures on anticoccidials (over US $300 million per mm),

continued emergence of dnig resistant strains, dong with the observations regarding the

development of immunity in exposed birds, focused the attention of researchers towards

immunological control. Efforts aimed at developing anticoccidial vaccines have not yet

been successful, partly because of the lack of interspecies cross-protection. The

development of effective anticoccidial vaccines has M e r been complicated by the

evidence of significant antigenic differences among various strains of the same coccidiai

species (Martin et al. 1997). This implies that there exist gaps in our understanding of the

host parasite relationships at the sub-cellular and orgaaismal levels.

Introduction

The interactions between apicomplexan zoites and their host cell have been

summarized as the identification and penetration of a host ceiî foiîowed by the maturation

of the parasitophorous vacuole (PV). During the maturation of PV into a metaboiicaily

active compartment, apical organelles such as micronemes, rhoptries and dense granules

are exocytosed fiom the zoite into the developing PV. In the late 19707s, a number of

researchers began to identie and characterize the apical organelies to better understand

the biology of the parasite and thereafter devise effective nonchernical means of control.

To accomplish this, parasites were hctionated to isolate sub-cellular organelles and the

latter were studied in t m s of their size and polypeptide contents. To accornplish this,

mono-specific probes (generally polyclonal and monoclonal antibodies) against the

organelles were generated and used to screen peptides produced by genes in stage-

specific cDNA libraries. The first parasites tu be examined contained plentifid organelles

or were of medical a d o r veterinary importance such as Sarcocystis species, T. gondii or

Plasmodium spp. In a search for possible fhctions of these apical organelles,

researchers started looking for the existence of conserved features at an intra- and inter-

species level. To facilitate this, a wider range of parasites must be examined.

The discharge of the protein contents of micronemes, rhoptries, and dense

granules during invasion of host ce11 that has been studied in various apicomplexan

parasites has led to the suggestion that these organelles are involved in the process of

recognition, host ce11 invasion and parasite adaptation (Bannister et al. 1975; Entzeroth et

al. 1986; Sibley & Krahenbuhl,l988; Cesbron-Delauw et al. 1988; Ton et al. 1989;

Aikawa et al. 1990; Charif et al. 1990; Leriche & Dubremetz 1990; Dubremetz and

Dissous 1980; Dubremetz 1993; Rick et al 1998;). The precisely controlled chronology

of the exocytosis of apical organelles is quite intnguing and forms the basis of the

ongoing search aimed at describing thae events in fûnctional and stnictural tams

(Carnithers and Sibley, 1997).

Eimeria tenella is one of the most pathogenic and costly eimerian parasites of the

domestic chicken. Its economic significance makes it a subject of ongoing research in

order to Save the poultry industry fiom losses brought about in the fonn of diminished

growth, morbidity and mortality. Studies on the apicai organelles of E. tenella have been

limited (e.g. Tomley et al. 199 1, 1996). Unfortunately, these authors were unable to

purify sufEcient dense granules fiom this parasite to permit characterization. Part of the

difficulty in studying dense granule organeiles in E h m a tenella may be the lirnited

number of these organelles relative to the other extrusornes such as micronemes and

rhoptries (Figure 1). Yet study of the dense granules in E. tenella may help us understand

the biology of parasite at the individual and group level and rnay later contribute to an

integrated strategy to control the parasite. This is essential before we can look for

conserved molecules at a broader level and select possible vaccine candidates fiom the

characterized components of the extnisornes.

The principal objective of the present work was to devise a means of isolating

dense granules in sdficient numbers and purity to permit the generation of dense body-

specific antibodies. These antibodies could then be usai to partially characterize the

contents of the dense granules and investigate their fate during ce11 penetration.

Materials and Methods

Parasite Propagation

White Leghom chickens (University of Guelph strain) raised under coccidia-fke

conditions in the OMAFRA isolation facility were used for propagation and production

of Eimmia tenella USDA#80 oocysts for use in the experiments. The chickens were

infected with parasites through oral inoculation; the dosage varied with bird age. The

dosage regimen for the E. tenellu oocysts for inf'ting the birds is shown in Table 1.

TABLE 1. ~ F E C T I N G DOSAGES OF SPORULATED OOCYSTS GNEN TO CHICKENS PER OS AT VARIOUS AGES.

The infection was established by adrninistering oocysts (previously counted using a

hemocytometer) suspended in 1 ml of l x Phosphate BufTered Saline (PBS, pH 7.4) using

a blunt nosed feeding tube. The feeding tube, attached to a 10 ml syringe, was passed

down the mouth into the crop where the inoculum was dispensed. Forty to fifty birds of

the same age were used for each propagation, with 4-5 birds placed in a cardboard box

measuring 20"x 16"x 18". The boxes were raised off the floor to rninimize contamination.

The rooms for housing the chickens at the isolation facility were sterilized with s t e m and

ammonia prior to use. The chickens were offered unmedicated water and fed chick

starter ration with 20% protein content (Shur-Gain, FIoradaIe, Ontario) ad libitum.

Identical housing conditions were maintained for each propagation. The birds were

kilîed eight days post infection and theïr ceca were removed and placed in a beaker kept

on ice. The ceca were cut open lengthwise and the contents and mucosa were smped

into sterile 2.5% potassium dichromate solution. These cecal scrapings were

homogenized in a blender to fkee the oocysts and passed through a 1 mm mesh sieve into

1000 ml Erhlenmeyer flasks. Flasks were fiIled with only about 400 ml of homogenate to

allow enough space for ventilation. The flasks were incubated at 26' C for 5 - 6 days

with constant shaking to permit the oocysts to sponûate.

Parasite Purification

The sporulated oocysts in 2.5% potassium dichromate were centrifbged at 1000xg

for 10 minutes and the sediment saved The pellet was resuspended in 1 x PBS and

centrifuged a second time to replace the potassium dichromate with lx PBS.

For the separation of oocysts fkom fecdtissue debris saturated stenle sodium chlonde

floatation was used according to the procedure described by Rose et al. (1984). The

washed pellet of the oocysts was mixed with saturated sodium chloride solution. The

mixture was blended briefly and then transferred into 50 ml centrifuge tubes. Two ml of

distilled water were layered carefùlly on the top of each tube. The tubes were centrifuged

at lOOOxg for 15 minutes. The oocysts fomed a whitish layer on the top of the salt

solution and were collected by aspiration. The oocysts were washed 4 times with

distilled water using centrifugation to collect the oocysts between two washings (1000xg

for 10 minutes). The pelleted oocysts were stored in lx PBS at 4 O C and were used in

experimentation for up to 4 months after flotation.

Surface Sterilization of Oocysts

The oocysts were surface steriiized by exposure to commercial bleach. The 1 x

PBS suspension of oocysts was cenûifbged at lOOOxg for 10 minutes and the supernatant

discarded. The peliet was resuspended in ~avex@ bleach (5% available chlorine - sodium

hypochlorite) and left on ice for 10 min with occasional shaking. The bleach stenlization

was completed by adding at les t five volumes of sterile l x PBS or distilled water and

centrifiiging the mixture at l OOOxg for 10 minutes. Pelleted, sterilized oocysts were

washed repeatedly in lx PBS (5 or 6 times or untii all trace of bleach was removed).

Surface-sterilized oocysts were stored in lx PBS at 4" C.

The sporozoites were excysted through mechanicd and enzymatic digestion of the

oocyçts and sporocysts respectively. The oocysts in 1 x PBS were spun at 1 OOOxg for 10

minutes in a tapered 15 ml centrifuge tube. The supernatant was discarded and 0.9 ml of

the thick white slurry of oocysts was added to 3.3 gm of glass beads (Fem micro beads

class 4A, Cataphote Division, Jackson, Mass.) contained in a g l a s vial. The stoppered

via1 was held in a vibrating Mickle shaker for 10-12 seconds. The released sporocysts

were separated fkom the g l w beads by washing with 1 x PBS and the resulting washhgs

containhg sporocysts, oocyst walls and a few intact oocyst were Eltered through a

Nitex@ mesh sieve with 10 p pores (Advance Process Supply Company, Toronto, ON).

The filtrate was collected in a beaker on ice and then centrifiiged at lOOOxg for 10

minutes to collect the spomcysts.

The pelleted sporocysts were suspended in excystation fluid (0.25% trypsïn

(Sigma, St. Louis, Mo), 5% chicken bile (v/v) in l x PBS, adjusteci to pH 7.4 with 1.0 M

NaOH) and placed in shaking water bath at 41" C in flasks, each containing 40-50 ml of

sporocyst suspension, for 90 to 120 minutes. When the major@ of the sporozoites had

exited the sporocysts as detennined by microscopie examination of excysting parasites,

the excystation was halted by 3-fold dilution with l x PBS. Four centrifugations at

1 OOOxg for 10 minutes were used to wash the excystation fluid with 1 x PBS. The pellet

containing sporozoites, sporocyst walls and the unexcysted sporocysts was suspended in

Ix PBS.

Purijication of Sporozoites - Percdl Gradient Cenhr~gatioon

The mixture of sporozoites, sporocyst walls and unexcysted sporocysts was

Eactionated using a ~ e r c o l f (Pharmacia Uppsala, Sweden) gradient. Ninety- percent

isotonic Percoll was prepared by adding 10x PBS to Percoll. The gradient was created by

rnixing 3 parts of 90% isotonic Percoll with 2 parts of sporozoite suspension in l x PBS.

The resulting mixture was centrifûged at l8OOOxg for 20 minutes. The sporozoites were

recovered fkom near the bottom of the resulting continuous gradient and washed 4 times

with l x PBS to remove the Percoii. The centrifugations between the washings were done

at lOOOxg for 10 minutes each time. The purïfied sporozoites were stored ovemight in

lx PBS at 4" C.

Purifcation of sporozoites - Glus Wool Coiumn Filnation

A two centimeter long glas column tightly packed in a glass Pasteur pipette was

employed as an altemate to the Percoll gradient for the purification of excysted

sporozoites. The sporozoite suspension was added drop-wise on to the column and the

f i h t e was collected in a glass beaker kept on ice.

Organeiiar Isolation fkom sporozoites

Sonication

Different concentrations of the sporozoites ranging h m 50-300~ 106 per ml (as

detennined by hemocytometer counts) in homogenization medium were subjected to

sonication using a Branson Sonifier 250@ (Branson Sonic Power Company Danbury, CT

O68 10) at 40% duty cycles @ setting 3. Sporozoites were exposed for a total of 2

minutes in 10-1 5 second bursts and the sarnples were kept on ice during the entire

disruption process. The degree of dimqtion was followed microscopically.

French Press

Sporozoites were counted using a hemocytometer and then transferred from 1 x

PBS and diluted to 0.25 M sucrose homogenization medium (HM) to a h a i

concentration of 1 x 108 sporozoites/ml. The sporozoite disruption was carried out using

French Press Ce11 (American Instrument Company, Silver Spring, MD) with a constant

pressure of 600 pounds per square inch (psi). The pressure valve was released to allow

the lysate to trickle through slowly in order to ensure uniform dimption. The pre- and

post-disruption samples were kept on ice. The lysate was centrifiged at 400xg for 15

minutes to remove imbroken sporozoites/sporocysts and then homogenized using a

Dounce cell homogenizer with five gentle up/down stmkes of the pestle.

DLscontinuous Sucrose Gradient Ce~trrigafion

Sucrose solutions of ciiffixent molanties (025,1.4,1.5 or 1.6M) were prepared in

5 mM tri-ethanolamine (TEA SMM) and 1 mM ethylene diamine tetraacetate (EDTA 1

mM). These solutions were used to construct the gradient used for separatiun of

organelles from the ce11 lysate (Table 2 and Figure 3).

TABLE 2. COMPOSITION OF LAYERS USED iN A DISCONTINUOUS SUCROSE GRADIENT TO SEPARATE LYSED ORGANELLES OBTAiNED FROM FRENCH PRESS DISRUPTlON OF EIÀUERIA TENELLA SPOROZOITES.

1 Sucrose 1 0.25 M 1 1.5 M

TEA 1 5 m ~

Sucrose solutions were carefully layered upon one another using bent tip g la s

pipettes, to avoid mwig. Twelve-miliiliter transparent Beckman centrifuge tubes were

used to contain the gradient. The tubes were placed in the buckets of a SW41 Ti rotor.

The paired buckets were balanced using an electronic weighing scale up to the third

decimal point (0.001 g) before loading on to the rotor. The gradient was spun in a

Bechan LS-55 Ultracentrifuge (Beckman Instruments Inc., Palo Alto, CA 94304) at

27000 rpm (125000xg) for 90 minutes. The segments of the lysate containhg different

organelles moved to different gradient zones according to the specific gravity of the

EDTA

Lysate

5 m M

1 mM

Yes

5 m M 5 mM

1 mM

No

1mM

No

1 mM

No

respective organelles. The fkactionated organelles were coiiected h m each isopycnic

zone, labeled with the zone of origin, and then diluted to 12 ml with 0.25M HM and spun

at 27000 rpm (125000xg) in SW 41 Ti rotor for 10 minutes. Each resulting pellet was

harvested and divided into two portions. One third was us& for transmission electron

microscopie identification of the organelle(s) present and the remainder was stored at -20'

C for use in the immunization of mice.

EIecbon Microscopy

l FIGURE 3. ILLUSTRATION OF THE ORDER AND VOLUMES OF DIFFERENT PARTS OF THE DISCONïïNüOUS GRADIENT USED TO ISOLATE

l ORGANELLES FROM EIMERIA T E N E U SPOROZOITES AFTER FRENCH PRESS DISRUPTION.

The organelle specimens obtained fiom the discontinuous sucrose gradient were

transferred to 1.5 mi microcentrifuge tubes. Specimen processing was done according to

the procedure descnbed by Barta et al. (1987) [see Appendix 2 for reagents].

Fixation of Organelle spechens for Trausmisssi4n Electron Microsco~ W M )

The samples were fixeci in primary fixative for one hour at room temperature with

continuous, gentle agitation in a tissue rotator and then washed in buffer (3x 10 minutes).

The specimens were post-fked with osmium tetroxide for one hour in dark at room

temperature. Osmium tetroxide was added to the buffér shortly before use to prevent

degradation of this reactive compound. Post-fixation, the specimens were washed 1 x5

minutes in b a e r followed by 3x5 miautes washings with double distillecl water.

Pnor to ernbedding in epoxy resin, the water in the fixed specimens was gradually

replaced with ethanol. For dehydration an qua1 volume of 50% ethanol2x5 minutes

was mixed with the specirnen followed by 2x5 minutes b a h in an ascending ethanol

series (70% ethanol90% ethanol95% ethanol 100% ethanol) and an additional 5 minutes

soak in 100% ethanol. A 50/50 mixture of ethanol concentrations was used between each

change of concentration (Le. 50/50 mixture of 70%/90% ethanol between the 70% and

90% ethanol dehydration steps).

For infiltration, Epon 8 12 (Appendix 2) was used as the ernbedding medium for

the fked organelle specirnens. Since Epon 812 is not soluble in ethanol, the specimens

were transferred fkom ethanol to propylene oxide, an intermediate solvent. Once in

propylene oxide, the addition of resin was accomplished as follows: 3: 1 mixture of

propylene oxide:Epon 8 12 for 20 minutes; 1 : 1 propylene oxide:Epon 8 12 for 20 minutes;

1 :3 propylene oxide:Epon 8 12 for 20 minutes; and 100% Epon 8 12 for 60 minutes.

Finally, the tissue in 100% Epon 8 12 was left at 60° C overnight for polymerization of the

resin.

Ultrathin sections (about 75 nm) of the embedded organelle fiactions were cut using an

Ultracut E microtome (C. Reichert AG. Hemalser Hauptstrasse 219 A- 1 170 Wien

Austria) and loaded ont0 carbon-formvar coated copper EM grids.

Staining of Sections for TEM

EM grids loaded with sections were floated on drops of stains (Appendix 2) as

follows: 1) sanirateci uranyl acetate in 70% ethanol (prepared fiesh) for 10 minutes; 2)

washed through 70% ethano1 and double distiiied water; 3) stained on modified

Reynold's Lead Stain for 2 minutes; 4) washed thoroughly with double distilled water,

and 5) blotted dry. Stained specimens were observed using JOEL 200 transmission

electron microscope operated at an accelerating voltage of 80 kV.

Mouse lmmunization

Once the identity of the organelles was confimed using transmission electron

microscopy, aliquots of the material stored at -20' C were used as antigen to immunize

mice for the generation of polyclonal antisera and plasma cells for hybridorna production.

Six to seven-week-old BALB/c mice (M/s Charles River, Quebec) h o w d in standard

cages (water and food ad lib) at the OMAFRA animai isolation facility were used.

Isolated organelle samples were thawed and each pellet was brought up to 0.4 ml by the

addition of l x PBS. To obtain a 40:60 mixture of antigen: adjuvant, 0.6 ml of ~itermax"

Gold adjuvant (Cyfrex Corporation, Norcross, GA) was homogenized with 0.4 ml antigen

suspension using a Becton Dickinson Multifit homogenizer (Randoti g las technology

Inc. CA. 9 10 16). Each mouse was injectai with 0.4 ml antigedadjuvant homogenate,

haif intramuscularly and half subcutaneously at the base of the neck.

Mer two weeks, the antiiody response was detenniaed by indirect fluorescent antibody

assay (IFA). Anaesthetized mice were bled by gently pressing microhematocrit capillary

tubes in the rctroorbital space. The sera were separated and stored at -20' C.

After confirming that the primary immunization was successfbl in that antibodies

directed against the apical organeiles and dense bodies had been elicited, the mice were

administered a second injection of 4O:6O mix of antigen:adjuvant three weeks after the

initial immunization. For this injection, each mouse received a 0.2 ml dose

subcutaneously in the neck region. Four days after injection, the mice were anaesthetized

and bled by cardiac puncture for the collection of sera These post-boost sera were

separated and stored at -20" C. The mice were kiiled and the spleens were removed

aseptically and processed (below) for the generation of hybridoma antibodies.

Indireci Flrrorescerat Antibudy Assay (IFA)

The IFA was used to hvestigate antibody response in the irnmunized mice after

both primary and secondary immunizations. Its main components were Eimeria tenella

USDA #8û sporozoites fixed onto Shandon ~ u l t i s p o t ~ slider (Shandon Inc. Pittsburgh,

PA), primary antibody (post-immunization sera) and fluorescein isothiocyanate (F1TC)-

conjugated goat anti-mouse IgG (H+L) second antibody. One thousand Eimeria tenella

#80 sporozoites suspended in 5 pl lxPBS were transfemed to each spot of twelve spot

Shandon slides. The Shandon slides were air-dried and stored at -20" C. For IFA, the

slides stored at -20" C were thawed at room temperature and fixed in cold acetone for 10

minutes. After rehydration, the slides were placed in a hydrated box in order to avoid

drying during subsequent processing.

The slides were washed in filtered PBS-Tween (0.1% Tween 20 in l x PBS) for

10 minutes prior to incubation with 10% fetal caifsenim (FCS) in PBS-Tween for 45

minutes at room temperature. Specimem were rinsed twice with PBS-Tween and then

washed in PBS-Tween for an additional 10 minutes. Slides were then incubated for 45

minutes with 150 or 1 : 100 dilutions of the fkst antibody by adcihg 20-30 pl to each spot.

M e r incubation, the spots were rinsed twice with PBS-Tween, washed in PBS-Tween

for 10 minutes and again rinsed twice with PBS-Tween. The following incubations were

done in the dark. Slides were incubated with 20-30 pl of 1 : 100 dilution of FITC

conjugated anti IgG (H+L) second antibody (Cedarlane Laboratones, Hornby, Ont.) for

45 minutes. After incubation with this antibody, the spots were rinsed twice with PBS-

Tween, then washed in PBS-Tween for 15 minutes and rinsed twice with PBS-Tween.

PBS-Tween was withdrawn and the slides were mounted with buffered glycerol(9 parts

glycerol and 1 part 10x PBS ). Positive and negative controls, using MAb 1209(agauist

rehctile bodies of sporozoite) and control mouse semm respectively, were nin each tirne

the F A was conducted. Prepared slides were stored in the dark at 4" C. Al1 F A

preparations, whether of whole sporozoites or cultured cells (see below) were viewed

ushg a Provis microscope (Olympus Opticai Co. Ltd., Japan). Images of these

specimens were recorded using a CCD camera and associated image capture software

(SpotTH Camera and Software per. 1.2.11, Diagnostic Instruments Inc., Ont.). Digital

images were modified using only whole-image, photographie manipulations (image

brightness and image contrast).

IFA of Sporozoites daring Penetirtion of Cnltured Cells

Monolayers of chicken fïbroblasts were grown on multi-weii microscope culture

siides (Lab-Tek Chamber Slides for Tissue Culture, Nunc Inc. Naperville, IL) in a 10%

CO2 atmosphere at 37S°C. Sporozoites, isolated as aseptically as possible as outlined

above, were uioculated onto the monolayers of nbroblasts at a concentration of 3x 1 O'

sporozoites per each 64 mm2 well. Mer incubation for 15 minutes, unattached

sporozoites and sporocyst wall debris were removed fiom al1 wells by vigorous washing

with 20% Fetal Calf Senuzl-Dulbecco's Modified Eagle's Medium (FCS-DMEM). Wells

were fixed at 15 minutes, 45 minutes, 75 minutes and 120 minutes aAer initial inoculation

of sporozoites. Half of the wells at each tirne were fixeci in absolute methanol for 30

seconds and then covered with PBS. The other halfof the weils were k e d with neutral

bufTered forrnalin for 2 minutes before replacing the fixative with PBS. To detect dense

body antigens during ceil penetration, candidate hybridoma antibodies were used in an

IFA procedure (outlined above) to detect epitopes in the parasites and within infected

cells. The only modification necessary for the use of the IFA procedure with fixed cells

was to include 0.1% saponin (Sigma, St. Louis, MO) in d l reagent solutions to permit

entry of the antibodies and FITC conjugate into the cells and to the parasites within.

Uninfected cells were fixed sirnilady to act as control.

Ceii fusion for the production of monoclonal antibodies

Cell fusion for hybridoma and monoclonal antibody production was canied out

by fusion of NS- 1 cells (cell line derived fiom mineral oil induced plasmacytorna of

mice) with splenocytes of BALB/c mice immiinized with the dense grande or

mi~~~nemedrhoptry/mitochondria kt ions of Eirnea tenellu USDA #80. The fusion

was perfomed according to the method of G a l h et ai (1977) with minor variations.

NS-I cells were harvested b r n a 2 &y (flask) culture and their viability was

deterrnined by counting unstained cells in a hemocytometer 5 minutes after mbchg a 0.5

ml aliquot of cells with 0.4% trypan Mue.

Isolation of Splenocytes

The spleen was removed aseptically nom the i r n m k e d mice after they were

killed. A single cell suspension was prepared by mincing the tissue with scissors and

then forcing the pieces through a 60 mesh rnetallic sieve. The cells and debris that passed

through the sieve were suspended in 10 ml DMEM-20% FCS and allowed to settle at l xg

for 10 minutes. The supernatant was transfmed to another tube and the sediment

discarded. The supernatant was centrifuged at 400xg for 10 minutes. The pellet was

retained and mixed with 5 ml cold m C I (0.17M) to lyse erythrocytes. Five ml cold

DMEM-20% FCS was added to the ce11 suspension d e r lysis, the suspension was gently

mixed and then centrifuged at 400xg for 10 min at 4 ' ~ . The supernatant was discarded

and the pellet resuspended in 5 ml DMEM (without FCS) at room temperature. A viable

ce11 count was made by mixing an equal volume of cells with 0.4% trypan blue. The

cells were allowed to settle in a hemocytometer for 3 minutes before counting.

Fusion of Splenocytes and N ' I Myelonta Celis

NS- 1 myeloma cells (10x 106) were transferred to a 50 ml centrihige tube. Viable

spleen cells were added to the same tube (Table 3) and mixed. The cells were pelleted by

centrifugation at 400xg for 10 minutes at room temperature, then washed 1 x with 10 ml

DMEM- without FCS at 42O C and transferred to a spherical bottom g las test tube pnor

to fhion.

TABLE 3. NUMBER OF SPLEEN CELLS AND MYELOMA CELLS FUSED FOR MONOCLONAL ANTLBODY PRODlfcI?ON.

DB-O (Dense bodies)

DB-1R (Dense bodies

The medium was decanted from the g las test tube and a stede cotton swab was used to

remove all traces of medium nom inside the tube. Two hundred pl of pre-warmed 30%

polyethylene glycol (PEG, MW = 1000) was added to the ce11 pellet and the cells were

resuspended by gentle tapping. Celk were imrnediately centrifûged at 500xg for 3

minutes at room temperature. The pelleted cells were incubated for an additional two

minutes. To avoid osmotic shock five ml of DMEM-without FCS was drop-wise added

without resuspending the ce11 pellet. The cells were rested for a M e r two minutes and

then the ce11 pellet was gently resuspended. M e r centrifugation at 400xg for 5 minutes,

the supernatant was removed and the cells were resuspended in 20%FCS in DMEM-with

hypoxanthine, aminopterine and thymidine (HAT) to yield a final concentration of NS- 1

cells of 3 . 3 ~ lo5/ml (30 mi for 10' NS-1 cells).

50x 10' spleen cells

105x 106 spleen ceils 1 10x 1 o6 NS- 1 celis

M R (MicronemesRhoptries)

Using a multi-channe1 pipette, the fused cells in suspension were transferred to

stede 96-well flat-bottom plates (50 pl per well). The plates were placed in a humidified

incubator at 37O C and 10% CO2 supply. 20% FCS in DMEM with HAT (50 pl per well)

was added to each well on day four and seven post-fusion. The screening for hybnd cells

10x 106 NS-1 cells

r

100x 10' spleen ceils 1 10x 106 NS- 1 cells 1 .

that secreted antiiodies was carrkd out using the IFA procedure descnied above h m

day 14 post-fusion onward. Culture supematants h m any weiis demonstrating growth

under the selection of the HAT hybridoma medium were screened.

Sodiuni Dodecyl Sulf~e-Poly~~erylamide Gel Electrophoresis (SDSPAGE)

Separation of antigens found in isolated organelles or whole parasites prior to

Western immuno-blotting was accompüshed using SDS-PAGE incorporating a 7.5, 10

and 15% resolving gel overlaid by a 5% stacking gel (Appendix 3). The resolving gel

was h t poured between the washed glass plates, topped with a thin layer of water-

sahirated butanol and allowed to set for 45 minutes at room temperature. Before pouring

the stacking gel, the butanol layer was removed completely. The well-forming comb was

inserted immediately after the stacking gel was poured.

The sarnpies for electrophoresis were prepared by mkhg Iml of 1 xPBS

containing 100x 1 o6 E i e a tenella #80 sporozoites with 1 ml of 2x reducing buffer

(Appendix 3). A low molecular weight broad range standard (range 6.5- 175 kDa, Bio-

Labs, Hercules, CA) was also nui simultaneously for calibration. Eimetfa tenella

USDA#80 sporozoites at a concentration of 50x 1 o6 /ml in 1 xPBS were mixed with an

equal volume of 2 x reducing b a e r and h z e n at -20" C. The samples and the low

molecular weight standard were boiled for 5-minutes before loading into wells. The

samples were was alectrophoresed with 1 x ninning buffer (6.0 g Tris, 28.8 g Glycine,

20.0 ml 10% SDS adjust volume to 2 liters with double distilled water and adjust pH= 8.3

with 10 N NaOH) in the inner and outer tanks. The electrophoresis was nui for 45

minutes at 10 mA after which the current was increased to 20 mA until the completion

(the samples and molecular weight markers reached within few mülimeters of bottom end

of the gel).

Western Blot

At the conclusion of electrophoresis the gel was removed and placed in transfer

buffer (39 mM Glycine, 48 mM Tris, 0.05% SDS, 20% Methanol)for 3x20 minutes. The

separated antigens were m f e r r e d nom the gel to Immobilon-P membrane (MiIlipore)

using the 2 1 17-250 Novablot Electrophoretic Transfer Kit-, Bromma, Sweden) with

a mode1 1000/500 power supply (Bio-Rad 3000 Xi, Japan). Rior to transfer Immobilon-

P membrane was treated with methano1 for 30-seconds and then placed in transfer buffer.

The membrane and gel were sandwiched on top and bottom between 6 sheets of filter

paper (Munktell grade IF), soaked in transfer buffer. The electrophoretic transfer was

carried out for 60 minutes at a constant current of 0.8 rnA/cm2 of gel.

Chemiluminescence detectioa of polyclonai or monoclonal antibodies

Chemiluminescence was used to detect the specificity of antibodies agaùist

various antigens of E. tenella sporozoites electrophoresed in the gel and transferred onto

the Immobilon-P membrane. Following the transfer the identity of the upper face of the

Immobilon-P membrane was preserved and the membrane was placed in blocking buffer

(0.5% 1-Block Reagent (casein - PE Biosystems, NJ) in 0.1% Tween-20 prepared in

IxPBS and heated to 70' C) for 30 minutes on a shaker. The membrane was washed in

wash bufTer (0.1% Tween-20 in IxPBS) 2x5 minutes. The Immobilon-P membrane was

sliced into 8 mm wide strips while retaining the molecular weight markers with two of

the strips. The identity of upper face of each strip was again preserved with a mark or

minute corner cut.

Each strip was placed in an antisenmi (1 : 100 dilution in wash buffer) or hybrid

cell supernatant (undiluted) for 30 minutes on a shaker. M e r rinsing in wash buffer 4x5

minutes, an alkaline phosphatase conjugated goat-autimouse anti IgG (H+L) second

antibody (1 5000 dilution in wash buffer) was added and aiiowed to react for 30 minutes.

The membrane was washed 4x5 minutes in wash buffer. The membrane stnps were

transferred to assay b a e r (0.96% diethanolamine, 0.02% MgClz p H 10) 2x5 minutes

and then incubated with nitroblock@ (Polybenzy ldimethy lvinylbenzy 1 ammonium

chloride -~ro~ix@) diluted 1:20 in wash buffer. Excess nitroblock was removed by sssay

b d e r washes (2x5 minutes). The alkaline phosphatse substmte CSPD (19-dioxetane

cherniluminescent enzyme substrate - ~ r o ~ i x @ ) diluted 1 : 100 in assay b d e r was

incubated with the strips for 5 minutes.

To detect the cherniluminescence, resulting nom the bond of protein, primary

antibody and AP- tagged second antibody, the Immobilon-P stnps were sandwiched

between acetate sheets and transferred to radiograph film cassette for exposure of XAR 5

imaging film (Kodak).

Parasite Propagation

The infection was established by atlministering oocysts suspended in Iml of I x

Phosphate BufFered Saline (PBS) containin2 the appropriate number of oocysts, using a

blunt nosed feeding tube. Approximately 2 - 4 x 10' oocysts were collectecl £iom a single

propagation fkom faeces of 40-50 chickens. Over 90% of the collectai oocysts

successfully sporulated and were available for purification and excystation.

Parasite Purification

Routinely, 1 . 5 ~ 1 O' purified sporozoites were recovered h m about 1 x 1 O'

sporuiated oocysts of Eimeriu tenella.

h i a t o n of ooeysts and sporocysts

The methods outlined in the Materials and Methods provided highly enriched

sporocysts for excystation that were essentially clear of oocyst wall debris. Recovenes

after breakage and filtering were approximately 2 sporocysts for every oocyst in the

initial oocyst preparation calculated fkom initial numbers of sporulated oocysts (4

sporocysts per oocyst) and the final yield of sporocysts.

Punfcarion of sporozoites

Salt notation and surface stenlization with sodium hypochlorite followed by in

vie0 excystation and isolation on a continuous isotonic Percoll gradient provided

sporozoites that were essentially devoid of any cellular, bacteriai and/or oocyst/sporocyst

wall contamination. AU sporozoites were dimpted using a French Press Ce11 within 24

hours of excystation and purification. Initially, sporozoites were isolated on Percoll

isotonic continuous gradient centrifbgation. This d t e d in highiy p d ï e d sporozoites

but with low recovery. As few as 1 sporozoite per cocyst (or iess) was routinely obtained

f ier purification. In an attempt to increase the nuniber of sporozoites isolated, giass

wool columns were used as an alternative to Percoll gradient centrifugation. The yield of

isolated sporozoites was increased at least two-fold but the purity of the preparation was

reduced with sporocyst walls, residual bodies and a few oocyst walls contaminating the

sporozoite preparation pnor to disruption in the French Press Cell(see below). It was

subsequently determineci that the purity of the sporozoites (fkee of any contaminating

sporocyst or oocyst walls) was cntical for the repeatable and efficient disruption of the

sporozoites and the success of the gradient centrifugation hctionation steps.

Organelle Isolation from sporozoites

Sonication

Sonication was used in initial attempts at disrupting parasites to collect the

çubcellular organelles. 50- 100x 1 o6 sporozoites per ml of homogenization medium (HM -

Appendix 1) resulted in good disruption but it was dificuit to standardize and most of the

disruprions aiso broke open the membrane bound organelles thus making the procedure

less usefiil.

French Press

After sporozoites were counted using a hemacytometer and transferred to KM, the

parasites were disrupted using the French Press. M e r many triais, it was determineci that

optimal disruption was accomplished at about 600 psi constant pressure and a sporozoite

concentration of 100x 106/ml in 0.25M sucrose homogenization solution. The

concentration of sporozoites was critical. Likewise, the sporozoites used in the press

needed to be fke of ail debris. Any large pieces of debns were prone to clog the needle

valve in the press and produced poor homogenate samples for separation on the sucrose

discontinuous gradient. Once optimized, greater than 95% of al1 sporozoites in a sample

could be reliably disrupted with many/most of the organelies remaining intact in the

homogenate.

Dkcontinuous Sucrose Gradient

Iiiitially, the SW-27 rotor was employed for centrifugation with the Bechan L8-

55 ultracentrifuge. The relatively wider and cloudy tubes needed larger volumes of

gradient solutions and the organelie hgments were not easily visible after centrifugation.

As an alternative the SW 41 Ti rotor with smaller buckets and 12 ml transparent

centrifuge tubes was used to contain the gradient during the centrifugation. Initially the

protocol for the discontinuous sucrose gradient described by Dubremetz and Dissous

(1980) for Sarcocystis tenella zoites was used. Observation s kom repeated sessions of

electron microscopie examination after lysis and hctionation guided the modification of

the gradient. Consequently, the discontinuous gradient was changed fiom 2.2M:4ml,

1.8M: 1 OmI, 1.6M: Sm1 (sample), 1.4M: 4 mi, 1 M: 10 ml, 0.25 M: 2nd with a total

volume of 35 ml to 1 -6M: 3mI, 1 .SM:2mi, 1.4M:2ml and 0.25M: Sm1 (sample) having a

total volume of 12 ml. The centrifugation time and rotational centrifûgd force were

increased fiom 1 hour at 113,000xg to 1.5 hours at 125,000xg. At the end of

centrifugation various organelles had formed discrete bands in different zones of the

gradient depending upon their respective densities.

<= Membranes

<= Micronemes/fUioptneslMitochondria

<= Dense Bodies

FIGURE 4. LOCATION OF ORGANELLES WITHïN THE DISCONTINUOUS SUCROSE GRADENT AFTER CENTRIFUGATION AT 125,000 x G FOR 90 MINUTES.

Electron Microscopy

Transmission electron microscopy was the nrst tool used to confinn the morphologie

identity of organelles d e r their isolation fiom the discontinuous sucrose gradient.

Samples were processed nom ail four fiactions used in the modified gradient (0.25 M,

1.4 M, 1 -5 M, and 1.6 M sucrose). Pelleted material £kom each fiaction was examined

nom at least two separate hctionation experiments.

The 0.25 M fraction contained prhcipally membranes and membrane fragments.

Occasional organelles (usually micronemes) were observed but the majority of the

material in this fkaction was composed of membrane fragments with the occasionai

ribosome attached (presumably of nuclear, c y t o p l d c andor plasmalemma ongin).

The 1.4 M fiaction (Figure 5) contained a mixture of organelles. Micronemes were

numerous in this ûaction. Rhoptries were present as well but were much less numerous

than the micronemes. in addition to the two extnisomes, mitochondria with their

vesicular cristae were commonly observed in this fiaction.

FIGURE 5 - TRANSMISSION ELECTRON MKROGRAPH OF ORGANELLES COLLECTED ABOVE THE 1.4M/1 .SM MTERFACE IN THE STEP GRADIENT. NUMEROUS MICRONEMES AM) SOME RHOPTRIES ARE VISBLE. THE FRACTION ALSO CONTAMED SOME MIToCHOrnRiA

(MAGNIFICATION 60,000~)

The 1.5 M ûaction was comprised almost uniformly of spherical, electron dense bodies

m e a s h g l8Wl6.3 nm in diameter (n = 1 5) that corresponded morphologically to dense

bodies of sporozoites (Figure 6).

FIGURE 6 - TRANSMISSION ELECTRON MICROGRAPH OF ORGANELLES COLLECTED ABOVE THE 1 .5M/1.6M INTERFACE iN THE STEP GRADIENT. THE FRACTION CONTAMED ONLY HIGHLY PURIFIED DENSE BODIES (~~GNIFICATION 60,000~).

The 1.6 M fraction and pellet contained electron-lucent oval structures (Figure 7) that

were morphologically indistinguishable from the aumerous amylopectin granules

typically found in apicomplexan zoites.

FIGURE 7 - TRANSMISSION ELECTRON MICROGRAPH OF AMYLOPECTM GRANULES FOUND PELLETED AT THE BOTTOM OF THE STEP GRADIENT. THESE POLYSACCHARIDE STORAGE GRANULES REMAMED UNSTAMED

Mouse Immunization and Ce11 fusion for Monoclonal Antibody Production

AAer confimihg the morphologie identity of the organelles by transmission electron

microscopy, each of the rhoptry plus microneme and dense body fraftions was used to

immunize the BALB/c mice. After two weeks, the immune response was investigated by

IFA assay and, fùiding the IFA results positive, each mouse was re-immunized with the

same antigenic preparation. Four days later, sera were collected from al1 mice and a

second IFA was carried out to assess the secondary response. In al1 cases a stronger

response was recorded (Figures 8 and 9). Two mice imrnunized with purified dense

bodies and a third mouse irnmunized with the microneme/rhoptry/mitochondrion bction

were then exsanguinated under anesthesia and killed. The sera were separated and

preserved at -20' C. The spleens were processed for ce11 fusion. Fusion of splenocytes

with NS-1 cells was carried out according to Galfie et al. (1 977). The fused cells were

FIGURE 9 - ~NDIRECT FLUORESCENT ANTIBODY ASSAY OF SERA COLLECTED FROM MICE BOOSTED 4 DAYS AFTER SECOND MMUNiZATION WlTH A MIXTURE OF MICRONEMES, RHOPTRIES AND MITOCHONDRIA COLLECTED FROM THE 1.4 M FRACïTON OF THE GRADENT (A, B) OR DENSE BODIES ISOLATED FROM THE 1.5 M FRACTION OF THE GRADIENT (C, D). IN BOTH CASES, THE M G E N WAS EMULSiFIED M T I T E W GOLD (SCALE BAR

MEASURES 50 j.lM).

Indirect Fluorescent Antibody @'A) Screening of Prirnary Hybrid CeU Cultures

In addition to the use of IFA as primary saeening test to verify the specificity of

anti'bodies nom polyclond antisera, IFA was also used to detect secreting hybridomas

after fision. Alrnost one-half (about 700 wells) of the micro-wells cultured with celis

following fusion showed ceU growth and were screened for anti'body production.

Undiluted supematant fiom ail welis that showed growth was used in an IFA with whole

air-dried sporozoites as the target antigen. During this s d g îwelve weUs derived

fiom the single microneme/rhoptry imrnunized mouse were observed to be F A positive.

From the pair of mice immunized with dense bodies, almost 500 wells demonstrated

growth. Despite this, only a single well contained hybrid cells that produced antibodies

directed against the dense bodies.

The clones positive for antibodies to micn>nemes/rhoptries gave fluorescence in

the apical region of the sporozoites where these organelles are situated (Figure 10) or

gave a generalized surface fluorescence (Figure 1 1). The single micro-well containhg

hybrids secreting antibodies against the dense granules labeled discrete dense bodies

distrîbuted Ui the middle of the sporozoites (Figure 12). FA-positive clones were

subsequently transferred to 24-well plates to allow the population to expand and later to

vented culture flasks. The supematant fiom culture flasks was used as a source of

primary antibody for IFA and western blot analysis. Ce11 lhes expressing antibodies that

recognized sporozoite antigen were cryoprotected with dimethyl sulfoxide (DMSO) and

stored in Liquid nitrogen to await cloning and M e r study.

FIGURE f 0 - DIRECT FLUORESCENT ANTlBODY ASSAY OF HYBRID CELL SUPERNATANTS MR-2D2 (PANEL A), MR-3C4 (PANEL B), MR-1 DI (PANEL C) AND MR-2H 1 O (PANEL D). STRONG FLUORESCENCE IN THE APICAL REGION RESULTED FROM RECOGNlTION OF THE TIGHTLY PACKED MICRONEMES AND RHOPTRIES FOUND IN THAT AREA OF THE SPOROZOITE (SEE FIGURE 1 - SCALE BAR MEASURES 50 w).

FIGURE 1 1. IND~RECT FLUORESCENT ASSAY OF HYBRID CELL SUPERNATANTS MR-2F6 (PANEL A), MR-1 E6 (PANEL B), MR- lG6 (PANEL C) AND MR- 1 C 1 O (PANEL D). NOTE THAT PANELS A AND B DEMONSTRATE MORE LiMITED APICAL STAiNiNG THAT MAY HIGHLIGHT THE RHOPTRIES. MR- I G6 DEMONSTRATED A PWCTATE STAMMG PATTERN THAT WAS MOST INTENSE AROUND THE PERlPHERY AT THE CELL MEMBRANE. HYBRIDOMA MR- I C 1 O DEMONSTRATED GENERAL SURFACE~NT'ERNAL STAlNCNG (SCALE BAR MEASURES

50 Ph

FIGURE 12 INDIRECT FLUORESCENT ANT[BoDY ASSAY OF iiYûRID CELL SUPERNATANT @BO-5F4). ALTHOUGH THE MTMSITY OF THE LABEL VARLES BECAUSE OF DIFFERMT ANTIBODY CONCENTRATIONS, THE LOCALiZATION OF ORGANELLES REMAMS SAME. THE DISCRETE GRANULAR PATTERN OF FLUORESCENCE CONFORMS TO THE KNOWN DISTRIBUTION OF DENSE GRANULES iN THE PERINUCLEAR REGION BETWEEN THE ANTEMOR AND POSTERIOR REFRACTILE BODIES (SEE FIGURE 1 FOR COMPARISON - SCALE BAR

MEASURES 50 w).

IFA of Sporozoita daring Penetration of Cuïtured CeUs

Sporozoites penetrated the avian fibroblasts rapidly and were observed withh

cells within 15 minutes of being placed on the rnonolayer of cells. Between 15 and 75

minutes after inoculation onto the monolayer the sporozoites were observed to be within

the fibroblasts and could be seen to be within a parasitophorous vacuole. No fluorescence

was observed in any of the specimens k e d with methanol prior to I F k In contrast,

antriodies f?om hybrid cells designated DBO-5F4 recognized antigen within the

sporozoites @oth intracellula. and extracellular) that had been fixed in buffered formalin

(Figure 1 3). The inclusion of O. 1 % saponin was required for labeling of either

intracellular or extracellular sporozoites. Even formalin- fixed sporozoites were not

recognized by DBO-5F4 if saponin was not included in the F A procedure.

The dense bodies remaineci within intracellular sporozoites for the nIst 75

minutes (Panel A, Figure 13). At 2 hours post-inoculation, the intracellular sporozoites

demonstrated an dtered distribution of antigen recognized by DBO-5F4. The subcellular

organelles identified by this hybridoma using IFA were observed to be released into the

parasitophorous vacuole of infected cells (Panels B-D, Figure 1 3). Some parasites

appeared to be in the process of discharging these granules into the developing

parasitophorous vacuole. Distinct granules a d o r more diffuse staining were observed

directly adjacent to the sporozoites within parasitophorous vacuoles. This movement of

the antigen to an extra-sporozoite location was never observed with extracellular

sporozoites. These observations suggest that the antigenic material released in response

to the penetration of the host celi by the parasite.

SDSPolyacrykmide Gel Eleeh-ophoresis for protein analysis

Eimeria tenefla #$O sporozoites (50x 1 o6 /ml and 2 5 0 ~ 1 06/ml) were

electrophoresed ushg 15% polyacrylamide resolving gel under both reducing and non-

reducing conditions. Broad range low molecuiar weight molecular weight markers were

used to make a comparative assessrnent of the relative rate of migration (M,) of various

resolved bands. After electrophoresis gels were : 1) stained with Coomassie blue and the

images of the bands were scanned and electronically stored, 2) electrophoretically

transferred to nitrocellulose membrane (~mmobilon-PT. The Immobilon-P was sliced

and each strip was used to perform the western blot using the supernatant nom an IFA

positive hybrïd cells. The western blot images on the ~rnmobilon-P@ were recorded on

radiographie films that were scanned and electronically stored.

FIGURE 14. ELECTROPHORETICALLY SEPARATED PROTEMS OF EIMERL~ TENELLA SPOROZOlTES AND ISOLATED DENSE BODIES. LANE 1 - SPOROZO~TES ( 5 0 ~ 1 @?ML); LANE 2- BIORAD BROAD RANGE SDS-PAGE STANDARDS (FROM THE BO?TOM, THESE STANDARDS HAVE THE FOLLOWING

APPROXIMATE M ~ s [KDA]: 6.5, 16.5,25, 32.5,47.5,62,83, AND 175); LANE 3 - S m ~ o z o i ~ ~ s (NUMBERS 2 5 0 ~ 1 o~/ML); LANE 4 - PUMFIED DENSE BODIES.

On electrophoresis whole sporozoites resolved into about 32 major bands of

varying thickness with a relative rate of migration (M,) of 6.5-1 75 kDa (Fig. 14, lane 1

and 3). An aliquot of dense granule isolate with reasonable purity (see Figure 6) showed

45

12 bands within 35-85 kDa range (Fig. 14, lane 4). Many of the major antigens fiom the

whole sporozoites (such as the strong bands found at about 23,45 and 100 kDa) were

removed during the purification process. The 23 kDa band probably corresponds to the

highiy immunogenic rehctile body antigen (Hertzenberg et ai 1995). Post immunization

sera (Figure 8) were used to label eIectrophoreticaI1y separated antigen of sporozoites of

Eimeriu teneDa in a Western blot procedure (Figure 15). Bound antibody was detected

using goat anti-mouse, alkaline phosphatase-conjugated anti IgG (H+L) second antibody.

The bound secondary antibodies were detected using cherniluminescence. Sera fkom the

two mice immunized with only purified dense bodies (mouse DBO -Lane 2; Lane 3

mouse DB 1 R) recognized relatively few (but mostly the same) antigens. In contrast, the

mouse imrnunized with the 1 -4 M fraction (micronemes/rhoptries/mitochonciria)

recognized a large number of bands, some quite strongly (Lane 4). Pooled pre-

immunization sera from these same mice did not recognize any antigens nom the

sporozoites (lane 1).

1 2 3 4 FIGURE 15. ELECTROPHORETICALLY SEPARATED PROTEMS OF EIMERL~ TENEUA SPOROZOITES TRANSFERRED TO ~MMOBILON-P MEMBRANE AND PROBED WlTH SERA FROM DENSE BODY IMMUNIZED MICE (DBO - LANE 2; DB 1 R - LANE 3) OR MICE IMMLMIZED WITH MICRONEMES, RHOPTRIES AND MITOCHONDRIA CANE 4). PRE-CMMUNE SERUM WAS USED TO PROBE LANE 1. BOUND ANT'IBODY WAS DETECTED USMG A

GOAT-ANTIMOUSE IMMUNOGLOBULM CONJUGATED TO ALKALINE PHOSPHATASE. THIS WAS DETECTED USING CHEMILUMINESCENCE. LOCATIONS OF BIORAD BROAD RANGE SDS-PAGE STANDARDS ARE MARKED TO LEFT M KILODALTONS

Relatively few of the hybrïd celi a n t i i i e s produced detectable binding to

electrophoretically separated sporozoite antigens (Figure 16). Although most of the

FA-positive hybrid cells (mostly reacting to antigens f o n d in the 1.4 M fraction) were

tested against sporozoite antigens, only two MR- 1 C 1 O and MR-2H 1 O, demonstrated

reactivity in the western immunoblot. Antibodies secreted by hybridoma MR- 1 C 1 O

bound to a pair of bands with M,'s of 3 1 kDa and more weakly to bands at about 46 kDa

and 83 kDa Antibodies secreted by MR-2HIO bound to a diffhe antigenic band with a

M, of about 85 D a .

FIGURE 16. R E A ~ OF HYBRID CELL SUPERNATANT MR- 1C 10 (RIGHT LANE) WITH ELECTROPHORESED ANTIGENS OF SPOROZOITES OF ~~ TENELLA. A STRONG DOUBLET BAND WAS lDENTlFlED AT ABOUT 3 1 KDA AND A WEAKER BAND WAS RECOGNIZED AT ABOUT 46 KDA (THE LOCATION OF PART OF THE BIORAD BROAD RANGE STANDARD IS MARKED TO THE NGKT). IN A SEPARATE WESTERN IMMUNOBLOT, HYBRZD CELL SUPERNATANT MR-2H 10 (LEFT LANE) WAS SHOWN TO REACT WlTH A SINGLE BAND WITH A MR OF APPROXIMATELY 85 KDA (-0 w).

The hybrid cells secreting antibodies that putatively bound to dense granules

(DBO-5F4) produced antibodies that reacted with a number of bands with a M, of about

38 kDa (data not shown). Antibodies f?om these hybrid cells did not demonstrate binding

to any antigens that had been boiled in loading buffer pnor to electrophoresis. Weak

labeling of an antigenic band was detected only with sporozoites that were disrupted by

gentle dissociation in loading buffer at room temperature.

Discussion

The present work was initiateù p~c ipa l l y to generate mono-specific probes

against organeiles. The invasive stages of apicomplexan parasites possess a set of

srnetory organelles that facilitate host celi invasion, PV formation and maintenance of

this vacuole as an environment for survival and maintenance of the parasite. Rhoptries

and micronemes have been shown to discharge components that perturb the host ceil

membrane or act as parasite ligands for host cell surface receptors thus facilitating

intemalization of the parasite. It is generally believed that dense grandes are a main

source of exocytosed components that rnodiQ the PV soon after invasion (Dubremetz and

McKerrow; 1 995).

Considerable information has been generated on the role and the molecular

composition of dense grandes in Sarcocystk, Pla~modium. Babesia and Toxoplosma

gondii (Dubremetz and McKerrow, 1995; Cesbron-Delauw, 1994; Galinski and Bamwell,

1996; and Sam-Yellowe, 1996). For these species, electron microscopic observations

have demonstrated that the dense granule contents are exocytosed into the

parasitophorous vacuole shortly after invasion (Entzeroth, 1986; Entzeroth et al. 1986;

Jantzen and Entzeroth 1987; Leriche and Dubremetz, 1990).

In view of the economic importance of poultry parasite Eimeria tenella, the goal

of this study was to isolate dense granules fiom these parasites and then use these isolated

organelles to generate monospecific probes. The published protocol described by

Dubremetz and Dissous (1 980) for Sarcocystrs teneliu was used as a starting point for the

isolation of subcellular organelles. During the course of experimentation, numerous

modifications were necessitated. While the published sporozoite concentration of

100~10~/ml of homogenization medium tumed out to be workable, significantly altered

operating pressures had to be used within the French Press to get optimal disruption.

Although a constant pressure of 400 psi described by Leriche and Dubremetz (1 991) for

Toxopiusma gondii tachyzoites was too gentle for Eimeria tenella sporozoites, the 700

psi used for Surcocystls spp. (see Dubremetz and Dissous 1980; Strobel et al 1992), not

only successfully disrupted the sporozoites but also dismpted the subcellular huer

organelles. Using microscopie examination of the resulting lysate and the final isolated

organelles fiom each dimption as a guide, an empirically derived constant pressure of

600 psi was found to dismpt about 95% sporozoites. At this dismption pressure, the

majority of the subcellular organelles released into the homogenization medium remained

intact. Thus, the constant pressure of 600 psi was adopted as the standard disruption

pressure for Eheria tenelln sporozoites. The alternative method of disruption using

sonication occasionally gave reasonable disruptions with the advantage of greater speed,

but this rnethod was found to be impossible to standardize fiom one disruption to the

next The ability to standardize the disruption method was central to the success of

isolating intact organelles and thus sonication was abandoned in favor of the French Press

Cell.

For organelle separation firom the lysate resulting fkom disrupted sporozoites,

several modifications were required in the discontinuous sucrose gradient to reflect the

sedimentation behavior of the organelles of Eimeria tenella sporozoites. The recovery of

organelle fiactions from the gradient at the end of centrifugation showed that these have

unique buoyant densities. Unlike Sarcocystis tenella and Sarcocystis muris where the

dense granules move to the 1.8-2.2 M sucrose interface (Dubremetz and Dissous, 1980),

the same organelles were recovered fiom 1.5-1.6 M zone of the step gradient in the

present study. This, plus the relative scarcity of dense bodies within sporozoites, may

explain the inability of Tomley (1997) to isolate these organelles. Tomley (1 997)

observed micronema (primarily) at the 1 A-1.5 M interface using the same parasite. The

dense bodies and rhoptnes were fomd Iower in her linear sucrose gradient procedure. In

the present study , the much demer, non-proteinaceous amylopectin grandes were found

at the bottom of the gradient as previously observeci by Dubremetz and Dissous (1980)

and Tomley (1 997).

One crucial step in the isolation of organelles that had not been previously

discussed in the literature was the requirement for highly purifid sporozoites during

disruption. Apparently? the remaining sporocyst w d s and intact sporocysts disturbed the

smooth migration of organelles in the step gradient. Invariably, good disruptions of glass

wool column-purified sporozoites (as assessed by electron microscopie examination of

the lysate) never produced acceptable separation of the organelles after centrifugation.

Tomley (1997) suggests the use of DE-52 anion-exchange resin in conjunction with glas

wool for the purification of sporozoites. This procedure was not tested for efncacy in the

present study but would be expected to yield sporozoites of increased purity over those

obtained fiom glass wool alone. The preferred protocol in this laboratory was to use

isopycnic centrifugation with an isotonic Percoll gradient that yielded high purity

sporozoites. In the present study, the use of an optimued combination of Percoll

isopycnic isolation of sporozoites d e r excystation and a standardized dimption using a

French Press Ce11 was required for reproducible and acceptable parasite lysates. The

lysates containing intact organelles and other cellular components could then be used

successfiùiy in sucrose step gradients for isolation of the subcellular organelles.

Immunization and Adjuvant

The scarcity of the antigen obtained necessitated an amendment in the initial

protocol of this study in which rabbits were to be used in the production of polyclonal

antibodies. Instead, BALBIc mice were used to raise both polyclonal antisera and then

subsequently used as a source of splenocytes for organelie-specific hybrid ceii antibody

generation. In view of the limited amount of isolated antigen available for study, a strong

humoral adjuvant was used to maximize the antibody production in irnmunized mice.

For this reason, two synthetic block copolyrner adjuvants, ~ i t e r ~ a x " Classic and

~ i t e r ~ a x @ Gold were tried. Both of these adjuvants are significantly safer than Freund's

Complete Adjuvant (FCA). Using the antigenic preparation of organelles prepared in the

present study, TiterMax Gold dernonstrateci excellent immunogenicity when used for

primary and secondary hunizations. A ratio of adjuvant to antigen (isolated

organelles) of 6O:4O worked well. Standard use of two d l plastic syringes connected by a

three way stopcock was convenient and provided sufficiently homogenized materiai for

injection. Indirect fluorescent antibody assay using air-dried sporozoites and

fluorescence conjugated goat anti-mouse immunoglobulin was employed to monitor

immune response and the adjuvant performance (Figures 8 and 9). A satisfactory

immune response was seen at day 18 and this response was augmented significantly 4

days after repeat immunization at day 23.

Hybrid CeH Production

Cell funon was undertaken twice using immunized mouse splenocytes and NS- 1

myeloma cells (Ga& et al. 1977). On both occasions the fusion went weli and more

than one third of the seeded wells showed celi growth, indicating successful fusion of

myeloma ceUs with the splenocytes. The results of the first fusion were disappointing in

that no hybrid cells were found to be secreting parasite-specfic antibody. It was only

after the second fusion that twelve clones were found to secrete parasite-specific

antibodies against micronemes and rhoptries and one clone produced antibodies against

dense granules. To ensure that F A procedures were operating properly, monoclonal

antibody 1209 (Danforth and Augustine, 1983) was used as a positive control d u ~ g al1

IFAs. This antibody recognized ubiquitous coccidiai antigen (Hertzenberg et al. 1995)

and provided a clearly discemible label to both refiactile bodies within the sporozoites.

Identification of organelle-specific hybridoma antibodies is relatively uncommon when

only whole parasites are used as the Mmunogen @anforth, 1983; Danforth, personal

communication). The identification of numerous hybnd clones with patterns of

fluorescence suggestive of organelle-specific labeling supports the use of isolated

organelles for kunization. The pattern of fluorescence in case of microneme rhoptry

clones varied fiom relatively specific labeling of these organelles in the apex to

generalized fluorescence of the anterior hdf of sporozoite and in some cases intemal and

extemal label of the pellicle was recorded. The F A observations on the distribution of

microneme-specific antibody labeled antigeas are in general agreement with the known

pattern of microneme distribution within coccidian zoites (see also Figure 1). These

organelles are largely confïned to the apical end of eimenan zoites, generally anterior to

the anterior r e k t i l e body that is labeled by monoclonal antiibody 1209 (Dubremetz and

Dissous, 1980; Todey et al 1991, 1996). It is generaUy believed by these workers that

the micronemes discharge their contents onto the surface of coccidian zoites through the

apical end of the parasite. However, Barta (personal communication) has observed

networks of micronemes discharging their contents laterally to the surface of zoites in

regions 0 t h than through the apically located conoid. The fiinction of the micronemes

was once believed to be solely the recognition of a suitable host ceil. Micronemes of

many apicomplexan mites exocytose binding proteins, many displaying adhesive

domains homologous to mammalian proteins (e.g. thrombospondin), as a fht step

towards specific interaction that leads to the formation of a moving junction (Dubremetz,

1998). Pre-invasion tramfer of microneme proteins to the host ce11 surface suggested this

role in host ce11 recognition (Entzeroth et al. 1992). As in other parasites, Eimericl tenella

microneme proteins Elmic-1 and Elmic-2 were shown to possess thrombospondin-like

domains, again suggesting binding ability of these proteins which are localized to the

micronemes of apicomplexa. parasites (Tornley et al. 199 1, 1996). Interestingly,

removing the hinctional TRAP gene in Plasmodium sp. sporozoites (one of the well-

characterized thrombospondin-like proteins found within micronemes) using homologous

recornbination has suggested a role for this microneme protein in the gliding rnotility of

the zoites (Spaccapelo et al 1997; Sultan et al., 1998). Perhaps the microneme proteins

with binding domains have a more general hc t ion than mere involvement in ceIl

penetration. They may be essential components of the gliding motihty of apicomplexan

zoites. Some sort of binding molecule would be expected to be required to permit

sporozoites and other motile apicomplexan zoites to glide across solid substrates. The

same motility may be at least partly responsible for the active penetration of these

parasites into thw host ceb .

The identification of a specinc probe (hybndoma antibody DBO-5F4) is an

important contribution to the comparative study of the orgmellar biology of

apicomplexan zoites. Until the identification of this antiiody, there were no probes

available that specincaily labeled these organelles despite repeated attempts to produce

them (Tomley et al., 199 1; 1996). Antiiodies fmm hybnd cells designated DBO-5F4

produced granular fluorescence in the middie zone of the sporozoite. The quality of the

staining in this case was quite good as it was hast devoid of any non-specific labels.

The number of dense granules in sporozoites enumerated using F A varied nom 9-30.

Like most of the hctional amiautes of dense granules in other parasites like Sarcocystis,

ToxopIasrna~ Plasmodium and Neospora, the impact of the number of dense bodies on the

physiology of sporozoites or theV host is also unknown. The refinernent of this

monospecific probe by limiting dilution of DBO-5F4 (generation of a monoclonal

antibody) will certainy add to the strength and specificity of the present F A results. It

would be interesthg to know if antibodies DBO-5F4 clone can detect cross-reactive

epitopes in other members of the genus Eimeria or other related coccidia.

SDS-PAGE and Western Blotong

SDS- PAGE and western blotting were used for the separation of various proteins

of whole sporozoites and dense body hctions and detection of specificity of antibody

probes against these proteins. Only three of the IFA positive clones i.e. MR- 1C10,

MR2-H 1 O and DBO-5F4 reacted positively on western blots. MR- 1 C 10 supematant

highlighted a 3 1 kDa doublet. A weaker band was recognized at about 46 kDa In

another blot, supematant h m MR2-Hl0 reacted with a single band of approximately 85

kDa Only two microneme proteins, Et mic-1 (100 kDa) and Et mic-2 (50 ma) , have

been descnbed previously firom Eimenà tenellu by Tomley et ai. (199 1; 1996). The

present observations expand the number of putative microneme antigens for which

specific probes are now available.

DBO-5F4 supematant reacted with a 38 kDa band under both reducing and non-

reducing conditions. An aliquot of dense granule isolate was also electrophoresed

through a 10% polyacrylamide gel and it showed about twelve bands of vaxying thichess

in the 35-85 kDa range. AU known dense granule proteins studied in Toxoplasma,

Sarcocystis and Neospora have molecular weights ranging fiom 20-40 kDa

Electrophoresing the whole sporozoites resulted into about 32 bands between 6.5-175

kDa range.

Morphological and immunoelectron-rnicroscopic investigations have showed that

dense granules are exocytosed into parasitophorous vacuole by Plasmodiwn howlesi

(Bannister et al. 1975; Torii et al. 1989) by Sarcocystis muris (Entzeroth, 1985; Entzeroth

et al. 1986) and by Toxoplma gondii (Leriche and Dubremetz 1990). In Toxoplasrna

gondii penetration into the host ce11 is completed in 10 seconds while the dense granules

are released into PV about 20 minutes post-invasion, being the last of al1 apical

organelles to leave the parasite (Carruthers and Sibley 1997). Dense body discharge into

the parasitophorous vacuole was studied by Ui£êcting chicken fibroblast monof ayers with

fieshly excysted Eimeria tenella sporozoites. The cells were fixed at different times and

F A was conducteci. Out of the two batives use& i.e. neutrai buffered fonnalin and

methanol, the former gave far better resuits. The epitope recognized by supematant

DBû-5F4 was apparently alcohol-sensitive. Using this antiibody partial exocytosis was

observed by IFA at 75 minutes after inoculation of monolayen and there was reduced

label within the sporozoites. At 120 min, many of the dense granules had been released

into the PV(Figure 13). Although this suggests that the DBO-5F4 antibody is most likely

reactive with dense grandes, this pattern of exocytosis is seemingly delayed in

comparison to the one described for T. gondii. Further in vitro experimentation is

required to dehe more precisely the period of exocytosis fier celi penetration and these

observations will need to be corroborated using parasites derived fmm in vivo

experirnent ation.

This study was part of current efforts to elucidate the molecular biology of an

economicdy important parasite of poultry, Eimeria tenefla. In an attempt to isolate and

characterize dense body organelles of this parasite, the protocols described for

Sarcocystis spp. were adopted. Mer many unsuccessfid attempts and numemus

modifications to the initial procedures, dense bodies were isolated in highly enriched

form. The purifïed dense granule antigens were used to immunize BALB/c mice.

Polyclonal antisera obtained h m immunized mice were used for indirect fluorescent

antibody assays @A) to locate the dense granules in air-dried sporozoites. Splenocytes

from these immunized mice were fused with NS-I myeloma cells to produce hybrid ce11

cultures. One clone (designated DBO-5F4) was found to secrete antibodies against dense

granules by IFA. Supernatant fiom the same clone detected a 38 kDa band when

immunoblotting was perfonned on electrophoresed proteins of whole sporozoites. Using

IFA the same clone positively labeled dense granules in chicken fibroblasts infected with

fieshly excysted sporozoites of Eheria $enella, M e r confirming the specificity of the

antibody. The labeled dense bodies were observed to be exocytosed into the

parasitophorous vacuole starthg at about 75 minutes after initial contact with the cells in

vit?-o.

Possible avenues for fûture research include the cloning and sequencing of genes

encoding the dense granule proteins to investigate homologies to the same organelles of

other related parasites. The antibody identified in the present work @BO-5F4) will make

screening expression libraries possible. Later, parasite transformation (knocichg out

dense body genes using homologous recombination) may shed light on the d e of this

organelle in the biology of the parasite. Some potential questions awaiting answers are:

1) What is the role of parasite surface molecules in invasion? 2) What signaiing

mechanisms are involved in invasion? 3) Which host cell d a c e molecules are involved

in host-parasite interactions? and 4) What mechanisms trigger the exocytosis of apical

organelles?

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Appendix 1 - Common Solutions

Phosphate Buffer Saline(PBS)

Dissolve each salt completely before adding next one. Bring volume up to 1000 ml with double distilled water and sterilize by autoclaving. Store at room temperature.

Homogenizafion medium

Homogenization medium (HM) was used to protect the sporozoites during the

French Press process and later while loading the resulting lysate onto the step gradient.

Other fiactions of the gradient differed h m HM only in tems of sucrose content i.e.

they contained 1400 mM, 1500 mM, 1600 m M or 2000 mM sucrose

Appendix 2 - Fixation, Embedment and Staining for Transmission Electron Microscopy

TEM Fl'xrrlion Rotocd

Precautions: Use tightly stoppered vials for al1 processing steps and Wear @oves

thughout. Avoid breathing any fixatives; use a fume hood if possible or otherwise

arrange for adequate ventilation. Skin contact with the various resins is to be avoided in

particular.

Notes: Al1 chemicals used in kations and embeddhg are toxic and must be disposed of

properly. Uranyl acetate should be considerd radioactive waste and hmdied accordingly.

During the final steps of ethanol dehydration, absolute (200 proof) ethanol must be free

of moisture to ensure proper polymerization. Srnail (250 ml.) bottles of absolute ethanol

are the best source for such dcohol because they will be used relatively quickly and can

be bought fksh whenever M e r tissue is to be processed. Plastic Pasteur pipettes with

attached bulbs are best because 1) they do not drop small hgments of g l a s which cm

contaminate the specimen and cause severe damage to g las and diamond hives and 2)

the entire unit can be discarded after use to avoid additional contact with these chemicals.

The protocol is standard:

1) Collect organelles by cenhifugation at 16000xg in a microtentrifuge tube using

Eppendorf microfige. Decant the supernatant. Organelle specimens are centrifbged at the

end of each processing stage prior to decanting the reagent.

2) Primary Fixation - Fix pelieted organelles in primary fixative for one hour (RT).

3) Pass tissue through 3 x 10 minutes of b a r n wash.

4) Post-Fixation - Post-fix tissue (with Os0,-based haîive) for one hour in the dark at

RT. Add OsO, to the buffer shortly before use and store in the dark.

5) Wash tissue in 1 x 5 minutes of buffér wash followed by 3 x 5 minutes of ddH20.

When filling the vials the last time with distilled water, fill only one-half of the vial.

6) Dehydration - FiU the remaining halfwith 50% ethaaol. Then, 2 x 5 minutes with fiesh

50% ethanol. Continue likewise (a 50/50 mix between solutions) through the following

senes:

70% ethanol - 2 x 5 minutes

70%/90% mix - 5 minutes

90% ethanol - 2 x 5 minutes

90W95% mix - 5 minutes

95% ethanol - 2 x 5 minutes

95%/100% mix - 5 minutes

100% ethanol - 3 x 5 minutes .

. .-

Buffer Washes

Post-Fixative

0.2M Sorenson's Bmer 1 2.0 mi 1 2.0 ml 1 3.25 ml

0.2M Sorenson's Bmer with sucrose /IO ml

0.2M Sorenson's Buffer 1 2.0 ml 1 2.0 ml 1 2.0 ml Sucrose 0.14 g 0.2 g 0.27 g

Epon 812 Embedding Medium - Standard Hardness

Resin 812 min 51.13 g 50 g

Hardeners DDSA 27.02 g 25 g

Ratio of the DDSA and NMA detennines the overall hardness of the polymerized resin.

The more NMA, the harder the &al plastic; the more DDSA, the softer the final plastic.

The ber recipe is better for sectioning with a diamond hife, especially if extremeIy

thin sections are needd

Stainihg of Secfions

Float Sections on drops of solutions as follows:

Sahirated UranyI Acetate in 70% ethanol (make fkesh) for 10 minutes

Wash through 70% ethanol and then ddH20.

Stain on modined Reynold's Lead Stain for 2 minutes.

Wash thoroughly with ddH20.

Blot dry.

Observe using TEM.

Lead Citrate 1.33 g.

Sodium Citrate 1.76 g

1 NaOH 1 3 pellets (approx. 0.4 g) 1 - -- -

DdH20 - --

Bring total volume up to 50 ml.

Note: Stain unstable - diseard after 1 day.

Appendix 3 - Sodium Dodecyi Snlfatô.Poiyacry1amide Gel Electrophoresis

SDS-PAGE was performed accordhg to Laemrnli, 1970. A 15%, 10% or 7.5% resolving

gel and 5% stacking gel were prepared by mixing the following reagents.

Bis-Acryiamide (30%)

Tris-HCI 1SM (pH8.8)

Tris-HCIOSM @H 6-8)

SDS 10% (aqn.)

double distilled H20

TEMED*

*After mudng the other components of the gel, the solution was degassed for 10 minutes and the indicated catalyst reagents were added just prior to pouring.

0.825 ml

0.625 ml

0.010 ml

10% Amm. Persalfate*

4.50 ml

6.25 pl

10.00 ml

5-00 ml

0.20 mi

NasDTA 1M 1 0.010 ml

50 pl

Tris-HC1O.SM (pHo.8)

Glycerol

SDS 10% (aqueous)

0.04 ml 0.04 ml

4.00 ml

16 pl

8.5 ml

2-0 ml

4.0 ml

-- --- -

Pyronin Y (2 mg/ml aqueous)

dd Hz0

0.04 ml

6.65 mI

5.00 ml

0.20 ml

100 pl

- -

40 pl

560 pl

5.00 ml

5.00 ml

0.20 mt

7.35 ml

16 pl

9.00 ml

16 pl

100 pl IO0 pl

Appendix 4 - Coomassie Blue R-UO Polyacrylamide Gel Staining

Reagents

1 Coomassie Blue R-250 1 025g 1

1 Double distillecl water 1 39.75m.l 1 Stain

1 Acetic acid 1 7.51111 1

Methanol

Acetic Acid

Procedure

50ml

loml

Destain

1) Immerse gel after ninning in stain solution for 15 to 60 minutes with gentle

agitation.

2) Rernove from staining solution and immerse in destain wiîh gentle agitation.

Drain the destaining solution after 20 minutes and replace with fiesh destaining

solution.

Methanol

Water

3) Repeat d l the desired quality of stalliing is attained.

25ml

67Sml

IMAGE NALUATION TEST TARGET (QA-3)

APPLlED J IMAGE, lnc - = 1653 East Main Street - -. - Rochester. NY 14609 USA -- -- - - Phone: i l 6/482-O3W -- = Fa~r 71 61288-5989