I "eterinao" Parasitology, 41 ( 1992 ) 17-22 Elscvicr Science Publishers B.V., A m s t e r d a m
17
Distribution of oocysts, sporocysts and sporozoites of Eimeria tenella and Eimeria maxima in the digestive tract of chicken N. Shiotani a, E. Baba b, T. Fukata b, A. Arakawa b and T. Nakanishi a "Department of Food Science. Junior College of Osaka Jos&gakuen. Tennoji, Osaka 543. Japan bDepartment of l'etermary Science. College of Agriculture. University of Osaka t're~'cture. Sakai. Osaka 591. Japan (Accepted 10 July 1991 )
ABSTRACT Shiotani, N., Baba, E., Fukata, T., Arakawa, A. and Nakanishi, T., 1992. Distribution of ooc.vsls, sporocysts and sporozoites ofEimeria tenella and Eimeria maxima in the digestive tract of chicken. Vet. Parasitol., 41:17-22. The distribution ofoocysts, sporocysts and sporozoites of Eimeria tend& and Eimeria maxima in the digestive tract of chicken and in excreta was investigated. At 1 h after the oral inoculation of E. tcnella oocysts, the number of sporocysts in the cecum was 3.4 × 10 6 and decreased gradually thereafter, and the number of sporozoites in the cecum increased and remained at a high level until 12 h after the inoculation. Small numbers of sporocysts and sporozoites of E. tenella were found in other intestinal sites. A great number o f / ' . maxima sporozoites was found, especially in the jejunum. 2 h after the inoculation. The findings that the largest populations of sporozoitcs of E. tenella and E. maxima were found in the cecum and the jejunum, respectively, indicate that the site specificity of sporozoite invasion lbr each species is determined before the invasion takes place.
INTRODUCTION
Coccidia have been known to be characterized by strong host and site specificity. Eimeria tenella parasitizes only chicken ceca when inoculated orally. When it is inoculated parenterally, it also reaches the cecum and completes its life cycle (Davies and Joyner, 1962; Sharma, 1964; Leathem, 1969). On the other hand. coccidia o f the small intestine (Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria praecox and Eimeria necatrix) do not develop at o th er sites or organs such as the liver and spleen (Long and Millard, 1976, 1979; AI-Attar and Fernando, 1987; Fernando et ai., 1987). One of the factors involved in determining the site of sporozoite invasion of E. tenella may be the delayed excystation from sporoeysts. ~.3 1992 Elsevier Science Publishers B.V. All rights reserved 0 3 0 4 - 4 0 1 7 / 9 2 / $ 0 5 . 0 0
l8
N SIII~iI-~,NI E 1 AI..
The purpose of the present study was to compare the distribution of oocysts, sporocysts and sporozoites of E. tenella and E. maxima in the digestive tract after the oral inoculation of oocysts. MATERIALS AND METHODS
Chickens White Leghorn, Hy-Line ~, male chickens were purchased from a local commercial hatchery when less than 1 day old and were kept in coccidia-free electrically heated battery brooders with continuous artificial illumination. Feed and water were available ad libitum. The composition of the basal ration has previously been described (Arakawa and Ohe, 1975 ). Chickens used in all experiments were 4 weeks old.
Coccidia The E. tenella strain used in this study was originally supplied by the National Institute of Animal Health in Tsukuba. Oocysts were obtained from infected feces using a salt flotation technique (Long, 1971 ) and sporulated at room temperature in an aqueous solution of 2% potassium dichromate. The E. maxima strain used in this study was obtained from the University of Georgia in Athens, USA.
Distribution o f oocysts, sporocysts and sporozoites in the digestive tract Chickens were inoculated orally with 5 × 105 sporulated oocysts and four birds were killed at each time interval of 1,2, 3, 6, 12 and 24 h post-inoculation (p.i.). The digestive tract was immediately isolated and pieces approximately 5 cm in length were taken from five regions: stomach, duodenum, jejunum, ileum and cecum. Intestinal contents from each piece of the digestive tract and cecum were weighed and diluted with phosphate-buffered saline (Schmatz et al., 1984). The numbers of oocysts, sporocysts and sporozoites per gram of intestinal content were determined by hemocytometer under a microscope. Excreta were collected at 1,2, 3, 6, 12 and 24 h p.i. The numbers ofoocysts, sporocysts and sporozoites were examined in the same manner as the contents from the digestive tract. Excreta discharged in 24 h were pooled and weighed to extrapolate the numbers of oocysts, sporocysts and sporozoites discharged.
I ) I S ] R I B t r r l ( ) N (.)F P-~RASITES IN DI(.;ESTIVE TR.~(q" OF CHICKEN
19
RESULTS
The results of hourly distribution of oocysts, sporocysts and sporozoites in the digestive tract after the oral inoculation ofoocysts are given in Figs. 1 and 2.
OocvsL~ Small numbers of E. tenella oocysts (less than 3.1 × 10 4 g - ' of contents) were recovered from all sites of the digestive tract. Eimeria maxima oocysts ( 1 . 3 × 10 5 ) were recovered only in the cecum at 12 h p.i. (Fig. 2). 4
~
3
~
2
]
h P[
4
6
h
PI
2 1
sS
~
0
•~
o
0
0 0
~
4
~
3
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2
I
2 h PI
12h
PI
U
o
1
0
ts
~
0
u o o ,,..,
4
~
0
3 h PI
3
4
24
h
Pl
3
C
~"
0 ,.,3 E Z
2
,,A k
2
1
1
o
o
Fig. 1. H o u r l y d i s t r i b u t i o n o f E. tenella oocysts, sporocysts and sporozoites in the digestive tract. O o c y s t s ( © . . . . . © ), s p o r o c y s t s ( r--] _ [ ] ) and sporozoites ( A - - - A ) were counted in the stomach ( S ) . d u o d e n u m ( D ) , j e j u n u m ( J ) , i l e u m ( 1 ) , c e c u m ( C ) and excreta ( E ) . V a l u e s are means o f f o u r replications.
N. %111()I.kNI l i t , \ I
]{)
4
"~
(, h P I
1 h P1
3
kO
c
2
x ;~
1
.J " ' "
S
v
" ° "
v
- - -
~
J
D
-
I
~
E
S
J
D
I
C
E
tl P I
Q C :I E
~.~SA " g x l 0 6
4
.4 h
',
I
/
ll
t
I I a ,,
\ t
\
g ~4
c Q
4
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3 ~
2
E z ....
0
S
J
I
E
S
~_ - - - - ~ O
J
......
~_ - - - - ~ l
C
E
Fig. 2. l l o u r l y d i s t r i b u t i o n o f 1-. m a x i m a o o c y s t s , s p o r o c y s t s and sporozoitcs in the digcstix c tract. (_)ocysts ( O . . . . . 0 ), s p o r o c ~ s t s ( U. ~ ) and sporozoites ( & - - - & ) w e r e c o u n t e d in the s t o m a c h ( S ), d u o d e n u m ( D ). j e j u n u m ( J ), i l e u m ( 1 ). c e c u m ( C ) a n d e x c r e t a ( E ). V a l u e s arc m e a n s o f [bur replications.
In both species, a total of 6 . 3 × 10 ~ oocysts, representing i.3% of oocysts inoculated, were discharged into the excreta in 24 h. ,~'po roL'y.~ l.~
At I h p.i. 3.4)< 10 6 sporocysts of E. tenella were found in the cecum. Alter 2 h p.i.E, tenella sporocysts in the cecum decreased gradually (Fig. 1 ). Very few E. maxima sporocysts were observed in all sites of the digestive tract (Fig. 2 ). Eimeria tenella sporocysts were shed into the excreta and the total number o f E . tenella sporocysts recovered from the excreta was 2 . 6 × 10 5. l-imeria maxima sporocysts were not recovered from the excreta.
DIS'I-RIB[ VIION ()F P~RASITFS IN I)I(.iES'I-IVE T R A C T OF CHICKEN
2 1
Sporozoites Few E. tenella sporozoites were recovered from the stomach and duodenum. At 1 and 2 h p.i.E, tenella sporozoites were found widely distributed in the jejunum, ileum and cecum. After 3 h p.i. the numbers of E. tenella sporozoites in the jejunum and ileum decreased, while in the cecum a valuc higher than 1.1 × 106 was maintained until 12 h p.i. (Fig. 1 ). A great number of E. maxima sporozoites (approximately 8.9× 10c') were found, especially in the jejunum at 2 h p.i. (Fig. 2). Sporozoites ofE. tenella (1.4× 105 ) and of E. m a x i m a ( 6.3 × 104 ) were discharged into the excreta. DISCUSSION
In the present experiment, at 1 h p.i., the largest number ofE. tenella sporocysts was recovered from the cecum, but hours later, the number of sporocysts decreased gradually, and conversely sporozoites increased. It was found that most orE. tenella sporozoites excyst in the cecum after arrival. The number orE. tenella sporozoites was largest in the cecum compared with the numbers at other sites of the digestive tract, from 2 to 12 h after oral inoculation ofoocysts. The distribution of E. m a x i m a sporozoites was different from that ofE. tenella sporozoites. A large number of E. maxima sporozoites was found only in the jejunum at 2 h p.i. This finding indicates that the distribution of sporozoites in the digestive tract is directly related to the site specificity of invasion. The observations by Doran et al. ( 1962 ) that E. tenella sporozoites excyst in vitro more slowly than E. acervulina suggest that the slow excystation is a characteristic property of E. tenella. Slow excystation may account for the long passage through the small intestine before reaching the cecum. Numerous E. maxima sporozoites were already observed in the jejunum at 2 h p.i. Similar to E. acervulina (Doran et al., 1962), E. maxima sporozoites excyst more rapidly than E. tenella, indicating that rapid excystation is a characteristic property of small intestinal Eimeria. It is well known that bile is indispensable for the excystation of Eimeria oocysts in vivo. Speer et al. (1970a,b) observed that, in kidney cell culture. bile is associated with the stimulation of the motility of sporozoites and their further development. They also showed that the bile requirement of Eimeria for excystation is dependent upon species of Eimeria. Rose and Hcsketh ( 1983 ) demonstrated that E. acervulina was more susceptible to bile than E. tenella, using chickens in which the bile supply to the intestine was interrupted by ligation of bile ducts or diversion of the bile out of the intestine. It seems that E. maxima may also be susceptible to bile, as is E. acervulina, because of its rapid excystation. The low susceptibility of E. tenella to bile,
22
N SIIIOT-'~NI H
\1
resulting in delayed excystation, might be an important factor for the cecal parasite.
REFERENCES AI-Attar, M.A. and Fernando, M.A., 1987. Transport of Fimeria necalrix sporozoites in the chicken: effects of irritants injected intraperitoneally. J. Parasitol., 73: 494-502. -~rakawa, A. and Ohe, O., 1975. Reduction of Clostridium petJ~'ingens by feed additive antibioties in the ceca of chickens infected with l:'imeria wnella. Poult. Sci., 54: 1000-1007. Davies. S.F.M. and Joyner, L.P., 1962. Infection of the fowl by the parenteral inoculation of oocysts ofl,tmerm. Nature, 194: 996-997. Doran, D.J., Lahn, T.L. and Rinaldi, R.. 1962. Exc,vstation and locomotion of l-imerta aucrvultna sporozoites. J. Parasitol., 48: 32-33. Fcrnando, M.A., Rose, M.E. and Millard, B.J., 1987. l'Smerta spp. of domestic fowl: lhe migration ofsporozoiles intra- and extra-enterically. J. Parasitol., 73: 561-567. Leathern. W.D., 1969. Organ specificity of Eimeria tenella in cecectomized chickens. J. Prmozool., 16: 223. Long. P.L., 1971. Maintenance of intestinal protozoa in vivo with particular reference to l-'t. meria and ltistomonas. In: A.E.R. Taylor and R. Muller ( Editor), Proceedings of the 9th ,Symposium of the British Society for Parasitology. Blackwells, Oxford. pp. 65-75. Long. P.L. and Millard, B.J.. 1976. Studies on site finding and site specificity ot" Eimeria praccox. Eimeria m a x i m a and l-imeria acervulina in chickens. Parasitology, 73: 327-336. Long, P.L. and Millard, B.J., 1979. Rejection of Eimeria by foreign hosls. Parasitology. 78: 239247. Rose. M.E. and Hesketh, P., 1983. Infections with I-imeria species: the role of bile. J. Parasiml., 69: 439-440. Schmalz, D.M., Crane, M.S.J. and Murray, ILK., 1984. Purification of Eimeria sporozoites b,~ DE-52 anion exchange chromatography. J. Protozool., 31 : 181-183. Sharma, N.N., 1964. Response of the fowl ( Galllls dome.~ticus ) to parenteral administrmion of seven coccidial species. J. l'arasitol., 50:509-517. Speer, C.A., Hammond, D.M. and .Anderson, L.C., 1970a. Development of Eimeria callospermophili and E. hilamellata from the Uinta ground-squirrel, Slwrm~q~hilus armatus, in cultured cells. J. Protozool., 17: 274-284. Spcer. C.A., Hammond, D.M. and Kelley. G.L.. 1970b. Stimulation of motility in merozoitcs of five l-imeria species by bile salts. J. Parasitol., 56: 927-929.
17
Distribution of oocysts, sporocysts and sporozoites of Eimeria tenella and Eimeria maxima in the digestive tract of chicken N. Shiotani a, E. Baba b, T. Fukata b, A. Arakawa b and T. Nakanishi a "Department of Food Science. Junior College of Osaka Jos&gakuen. Tennoji, Osaka 543. Japan bDepartment of l'etermary Science. College of Agriculture. University of Osaka t're~'cture. Sakai. Osaka 591. Japan (Accepted 10 July 1991 )
ABSTRACT Shiotani, N., Baba, E., Fukata, T., Arakawa, A. and Nakanishi, T., 1992. Distribution of ooc.vsls, sporocysts and sporozoites ofEimeria tenella and Eimeria maxima in the digestive tract of chicken. Vet. Parasitol., 41:17-22. The distribution ofoocysts, sporocysts and sporozoites of Eimeria tend& and Eimeria maxima in the digestive tract of chicken and in excreta was investigated. At 1 h after the oral inoculation of E. tcnella oocysts, the number of sporocysts in the cecum was 3.4 × 10 6 and decreased gradually thereafter, and the number of sporozoites in the cecum increased and remained at a high level until 12 h after the inoculation. Small numbers of sporocysts and sporozoites of E. tenella were found in other intestinal sites. A great number o f / ' . maxima sporozoites was found, especially in the jejunum. 2 h after the inoculation. The findings that the largest populations of sporozoitcs of E. tenella and E. maxima were found in the cecum and the jejunum, respectively, indicate that the site specificity of sporozoite invasion lbr each species is determined before the invasion takes place.
INTRODUCTION
Coccidia have been known to be characterized by strong host and site specificity. Eimeria tenella parasitizes only chicken ceca when inoculated orally. When it is inoculated parenterally, it also reaches the cecum and completes its life cycle (Davies and Joyner, 1962; Sharma, 1964; Leathem, 1969). On the other hand. coccidia o f the small intestine (Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria praecox and Eimeria necatrix) do not develop at o th er sites or organs such as the liver and spleen (Long and Millard, 1976, 1979; AI-Attar and Fernando, 1987; Fernando et ai., 1987). One of the factors involved in determining the site of sporozoite invasion of E. tenella may be the delayed excystation from sporoeysts. ~.3 1992 Elsevier Science Publishers B.V. All rights reserved 0 3 0 4 - 4 0 1 7 / 9 2 / $ 0 5 . 0 0
l8
N SIII~iI-~,NI E 1 AI..
The purpose of the present study was to compare the distribution of oocysts, sporocysts and sporozoites of E. tenella and E. maxima in the digestive tract after the oral inoculation of oocysts. MATERIALS AND METHODS
Chickens White Leghorn, Hy-Line ~, male chickens were purchased from a local commercial hatchery when less than 1 day old and were kept in coccidia-free electrically heated battery brooders with continuous artificial illumination. Feed and water were available ad libitum. The composition of the basal ration has previously been described (Arakawa and Ohe, 1975 ). Chickens used in all experiments were 4 weeks old.
Coccidia The E. tenella strain used in this study was originally supplied by the National Institute of Animal Health in Tsukuba. Oocysts were obtained from infected feces using a salt flotation technique (Long, 1971 ) and sporulated at room temperature in an aqueous solution of 2% potassium dichromate. The E. maxima strain used in this study was obtained from the University of Georgia in Athens, USA.
Distribution o f oocysts, sporocysts and sporozoites in the digestive tract Chickens were inoculated orally with 5 × 105 sporulated oocysts and four birds were killed at each time interval of 1,2, 3, 6, 12 and 24 h post-inoculation (p.i.). The digestive tract was immediately isolated and pieces approximately 5 cm in length were taken from five regions: stomach, duodenum, jejunum, ileum and cecum. Intestinal contents from each piece of the digestive tract and cecum were weighed and diluted with phosphate-buffered saline (Schmatz et al., 1984). The numbers of oocysts, sporocysts and sporozoites per gram of intestinal content were determined by hemocytometer under a microscope. Excreta were collected at 1,2, 3, 6, 12 and 24 h p.i. The numbers ofoocysts, sporocysts and sporozoites were examined in the same manner as the contents from the digestive tract. Excreta discharged in 24 h were pooled and weighed to extrapolate the numbers of oocysts, sporocysts and sporozoites discharged.
I ) I S ] R I B t r r l ( ) N (.)F P-~RASITES IN DI(.;ESTIVE TR.~(q" OF CHICKEN
19
RESULTS
The results of hourly distribution of oocysts, sporocysts and sporozoites in the digestive tract after the oral inoculation ofoocysts are given in Figs. 1 and 2.
OocvsL~ Small numbers of E. tenella oocysts (less than 3.1 × 10 4 g - ' of contents) were recovered from all sites of the digestive tract. Eimeria maxima oocysts ( 1 . 3 × 10 5 ) were recovered only in the cecum at 12 h p.i. (Fig. 2). 4
~
3
~
2
]
h P[
4
6
h
PI
2 1
sS
~
0
•~
o
0
0 0
~
4
~
3
"~
2
I
2 h PI
12h
PI
U
o
1
0
ts
~
0
u o o ,,..,
4
~
0
3 h PI
3
4
24
h
Pl
3
C
~"
0 ,.,3 E Z
2
,,A k
2
1
1
o
o
Fig. 1. H o u r l y d i s t r i b u t i o n o f E. tenella oocysts, sporocysts and sporozoites in the digestive tract. O o c y s t s ( © . . . . . © ), s p o r o c y s t s ( r--] _ [ ] ) and sporozoites ( A - - - A ) were counted in the stomach ( S ) . d u o d e n u m ( D ) , j e j u n u m ( J ) , i l e u m ( 1 ) , c e c u m ( C ) and excreta ( E ) . V a l u e s are means o f f o u r replications.
N. %111()I.kNI l i t , \ I
]{)
4
"~
(, h P I
1 h P1
3
kO
c
2
x ;~
1
.J " ' "
S
v
" ° "
v
- - -
~
J
D
-
I
~
E
S
J
D
I
C
E
tl P I
Q C :I E
~.~SA " g x l 0 6
4
.4 h
',
I
/
ll
t
I I a ,,
\ t
\
g ~4
c Q
4
PI
3 ~
2
E z ....
0
S
J
I
E
S
~_ - - - - ~ O
J
......
~_ - - - - ~ l
C
E
Fig. 2. l l o u r l y d i s t r i b u t i o n o f 1-. m a x i m a o o c y s t s , s p o r o c y s t s and sporozoitcs in the digcstix c tract. (_)ocysts ( O . . . . . 0 ), s p o r o c ~ s t s ( U. ~ ) and sporozoites ( & - - - & ) w e r e c o u n t e d in the s t o m a c h ( S ), d u o d e n u m ( D ). j e j u n u m ( J ), i l e u m ( 1 ). c e c u m ( C ) a n d e x c r e t a ( E ). V a l u e s arc m e a n s o f [bur replications.
In both species, a total of 6 . 3 × 10 ~ oocysts, representing i.3% of oocysts inoculated, were discharged into the excreta in 24 h. ,~'po roL'y.~ l.~
At I h p.i. 3.4)< 10 6 sporocysts of E. tenella were found in the cecum. Alter 2 h p.i.E, tenella sporocysts in the cecum decreased gradually (Fig. 1 ). Very few E. maxima sporocysts were observed in all sites of the digestive tract (Fig. 2 ). Eimeria tenella sporocysts were shed into the excreta and the total number o f E . tenella sporocysts recovered from the excreta was 2 . 6 × 10 5. l-imeria maxima sporocysts were not recovered from the excreta.
DIS'I-RIB[ VIION ()F P~RASITFS IN I)I(.iES'I-IVE T R A C T OF CHICKEN
2 1
Sporozoites Few E. tenella sporozoites were recovered from the stomach and duodenum. At 1 and 2 h p.i.E, tenella sporozoites were found widely distributed in the jejunum, ileum and cecum. After 3 h p.i. the numbers of E. tenella sporozoites in the jejunum and ileum decreased, while in the cecum a valuc higher than 1.1 × 106 was maintained until 12 h p.i. (Fig. 1 ). A great number of E. maxima sporozoites (approximately 8.9× 10c') were found, especially in the jejunum at 2 h p.i. (Fig. 2). Sporozoites ofE. tenella (1.4× 105 ) and of E. m a x i m a ( 6.3 × 104 ) were discharged into the excreta. DISCUSSION
In the present experiment, at 1 h p.i., the largest number ofE. tenella sporocysts was recovered from the cecum, but hours later, the number of sporocysts decreased gradually, and conversely sporozoites increased. It was found that most orE. tenella sporozoites excyst in the cecum after arrival. The number orE. tenella sporozoites was largest in the cecum compared with the numbers at other sites of the digestive tract, from 2 to 12 h after oral inoculation ofoocysts. The distribution of E. m a x i m a sporozoites was different from that ofE. tenella sporozoites. A large number of E. maxima sporozoites was found only in the jejunum at 2 h p.i. This finding indicates that the distribution of sporozoites in the digestive tract is directly related to the site specificity of invasion. The observations by Doran et al. ( 1962 ) that E. tenella sporozoites excyst in vitro more slowly than E. acervulina suggest that the slow excystation is a characteristic property of E. tenella. Slow excystation may account for the long passage through the small intestine before reaching the cecum. Numerous E. maxima sporozoites were already observed in the jejunum at 2 h p.i. Similar to E. acervulina (Doran et al., 1962), E. maxima sporozoites excyst more rapidly than E. tenella, indicating that rapid excystation is a characteristic property of small intestinal Eimeria. It is well known that bile is indispensable for the excystation of Eimeria oocysts in vivo. Speer et al. (1970a,b) observed that, in kidney cell culture. bile is associated with the stimulation of the motility of sporozoites and their further development. They also showed that the bile requirement of Eimeria for excystation is dependent upon species of Eimeria. Rose and Hcsketh ( 1983 ) demonstrated that E. acervulina was more susceptible to bile than E. tenella, using chickens in which the bile supply to the intestine was interrupted by ligation of bile ducts or diversion of the bile out of the intestine. It seems that E. maxima may also be susceptible to bile, as is E. acervulina, because of its rapid excystation. The low susceptibility of E. tenella to bile,
22
N SIIIOT-'~NI H
\1
resulting in delayed excystation, might be an important factor for the cecal parasite.
REFERENCES AI-Attar, M.A. and Fernando, M.A., 1987. Transport of Fimeria necalrix sporozoites in the chicken: effects of irritants injected intraperitoneally. J. Parasitol., 73: 494-502. -~rakawa, A. and Ohe, O., 1975. Reduction of Clostridium petJ~'ingens by feed additive antibioties in the ceca of chickens infected with l:'imeria wnella. Poult. Sci., 54: 1000-1007. Davies. S.F.M. and Joyner, L.P., 1962. Infection of the fowl by the parenteral inoculation of oocysts ofl,tmerm. Nature, 194: 996-997. Doran, D.J., Lahn, T.L. and Rinaldi, R.. 1962. Exc,vstation and locomotion of l-imerta aucrvultna sporozoites. J. Parasitol., 48: 32-33. Fcrnando, M.A., Rose, M.E. and Millard, B.J., 1987. l'Smerta spp. of domestic fowl: lhe migration ofsporozoiles intra- and extra-enterically. J. Parasitol., 73: 561-567. Leathern. W.D., 1969. Organ specificity of Eimeria tenella in cecectomized chickens. J. Prmozool., 16: 223. Long. P.L., 1971. Maintenance of intestinal protozoa in vivo with particular reference to l-'t. meria and ltistomonas. In: A.E.R. Taylor and R. Muller ( Editor), Proceedings of the 9th ,Symposium of the British Society for Parasitology. Blackwells, Oxford. pp. 65-75. Long. P.L. and Millard, B.J.. 1976. Studies on site finding and site specificity ot" Eimeria praccox. Eimeria m a x i m a and l-imeria acervulina in chickens. Parasitology, 73: 327-336. Long, P.L. and Millard, B.J., 1979. Rejection of Eimeria by foreign hosls. Parasitology. 78: 239247. Rose. M.E. and Hesketh, P., 1983. Infections with I-imeria species: the role of bile. J. Parasiml., 69: 439-440. Schmalz, D.M., Crane, M.S.J. and Murray, ILK., 1984. Purification of Eimeria sporozoites b,~ DE-52 anion exchange chromatography. J. Protozool., 31 : 181-183. Sharma, N.N., 1964. Response of the fowl ( Galllls dome.~ticus ) to parenteral administrmion of seven coccidial species. J. l'arasitol., 50:509-517. Speer, C.A., Hammond, D.M. and .Anderson, L.C., 1970a. Development of Eimeria callospermophili and E. hilamellata from the Uinta ground-squirrel, Slwrm~q~hilus armatus, in cultured cells. J. Protozool., 17: 274-284. Spcer. C.A., Hammond, D.M. and Kelley. G.L.. 1970b. Stimulation of motility in merozoitcs of five l-imeria species by bile salts. J. Parasitol., 56: 927-929.
