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Vitamin A deficiency and Eimeria acervulina infection in the chick Coles, Barbara Margaret 1969

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VITAMIN A DEFICIENCY AND EIMERIA ACERVULINA INFECTION IN THE CHICK  by  BARBARA MARGARET COLES B.S.A., U n i v e r s i t y o f B r i t i s h Columbia, 1949  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE m^rtWFemSimE  i n the Department of Poultry  Science  We accept t h i s t h e s i s as conforming r e q u i r e d standard  to the  THE UNIVERSITY OF BRITISH COLUMBIA September, 1969  In p r e s e n t i n g an the  this  thesis  advanced degree at Library  shall  fulfilment of  University  of  make i t f r e e l y  I f u r t h e r agree tha for  the  in p a r t i a l  permission  s c h o l a r l y p u r p o s e s may  by  his  of  this  written  representatives.  be  available  granted  gain  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  by  shall  permission.  Poultry  for  for extensive the  It i s understood  thesis for financial  Department o f  British  Science Columbia  requirements  Columbia,  Head o f my  be  I agree  r e f e r e n c e and copying of  that  not  the  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  ii  ABSTRACT  The present i n v e s t i g a t i o n was conducted  t o determine the  e f f e c t of an i n f e c t i o n w i t h E i m e r i a a c e r v u l i n a on v i t a m i n A deficient birds. equal groups,  One hundred day-old c h i c k s were d i v i d e d i n t o 2  and one group was r a i s e d on 440 I.U. v i t a m i n A per  k i l o g r a m o f feed, w h i l e the second recommended 4400 I.U. per k i l o g r a m .  group r e c e i v e d the normally At 5 weeks o f age, h a l f the  b i r d s from each group were given an immunizing i n f e c t i o n w i t h 3 m i l l i o n oocysts of E. a c e r v u l i n a .  The c l i n i c a l symptoms were  more acute, and the oocyst p r o d u c t i o n , m o r t a l i t y , and weight l o s s e s s i g n i f i c a n t l y h i g h e r , i n the b i r d s on the low v i t a m i n A diet. When the b i r d s were 9 weeks o l d , h a l f were s u b j e c t e d t o a second old.  i n f e c t i o n and the other h a l f h e l d u n t i l they were 19 weeks The 9-week-old b i r d s were g i v e n an o r a l i n o c u l a t i o n of 10  m i l l i o n oocysts t o e v a l u a t e the e f f e c t of a low v i t a m i n A d i e t on:  the s t r e n g t h o f the immune response;  a primary i n f e c t i o n with  E. a c e r v u l i n a i n 9-week-old b i r d s ; and the e a r l y t i s s u e of the p a r a s i t e i n immune and non-immune from t h i s experiment  birds.  stages  The r e s u l t s  showed t h a t the degree of immunity i s not  a f f e c t e d by a low v i t a m i n A r a t i o n , but p r i m a r i l y determined the s e v e r i t y of the i n i t i a l i n f e c t i o n ; and t h a t the primary i n f e c t i o n i n 9-week-old c h i c k s i s c l i n i c a l l y very s i m i l a r t o t h a t i n younger b i r d s .  The h i s t o l o g i c a l  examinations  by  demonstrated t h a t the low v i t a m i n A l e v e l used i n t h i s not a f f e c t development  the i n t e g r i t y of the i n t e s t i n a l  e p i t h e l i u m , nor the  of the endogenous forms of the p a r a s i t e .  a l s o shown t h a t the s p o r o z o i t e  study d i d  I t was  i n v a s i o n was the same i n immune  and non-immune b i r d s , but the s c h i z o n t development was i n h i b i t e d i n the immune b i r d s r e g a r d l e s s  severely  of the v i t a m i n A l e v e l .  The non-immune b i r d s on the low v i t a m i n A r a t i o n d i d not show as distinct  a h e t e r o p h i l response  to i n f e c t i o n as d i d the  other  groups. The remaining b i r d s , h a l f which were immunized,were maintained on t h e i r r e s p e c t i v e  d i e t s u n t i l 19 weeks of age,  d u r i n g t h i s p e r i o d there was no c l i n i c a l evidence deficiency,  of a v i t a m i n A  and the.growth r a t e was not s i g n i f i c a n t l y  by the l e v e l of v i t a m i n A i n the d i e t .  and  affected  When 19 weeks o l d ,  the  b i r d s were i n o c u l a t e d w i t h 25 m i l l i o n oocysts of E . a c e r v u l i n a t o assess the e f f e c t  of an extremely heavy primary i n f e c t i o n  o l d e r b i r d s , and t o evaluate r e t a i n e d 14 weeks a f t e r  the degree of immunity s t i l l  an i n i t i a l i n f e c t i o n .  v e r y h i g h , and the weight l o s s e s s i g n i f i c a n t l y on the low v i t a m i n A d i e t a r y l e v e l .  The m o r t a l i t y was greater,  Immunity, although  from the e a r l i e r experiment, was s t i l l present l e v e l s of v i t a m i n A .  in  i n birds decreased  i n b i r d s on both  iv I t was concluded from these experiments t h a t a suboptimal l e v e l of v i t a m i n A i n the d i e t i n c r e a s e d the p a t h o g e n i c i t y of E.  a c e r v u l i n a i n chickens of v a r i o u s ages.  l e v e l d i d n o t , however, response.  significantly affect  The low v i t a m i n A the b i r d ' s immune  V  TABLE OF CONTENTS  PAGE  ABSTRACT  i i  TABLE OF CONTENTS  ......  LIST OF TABLES. . . LIST OF FIGURES  I. II.  v i i ix  ACKNOWLEDGEMENT .  X  INTRODUCTION  1  REVIEW OF LITERATURE  2  A. V i t a m i n A and C o c c i d i o s i s  2  B. P h y s i o l o g i c a l Role of V i t a m i n A  5  C. E i m e r i a a c e r v u l i n a  8  1. L i f e c y c l e  III.  V  9  2. P a t h o g e n i c i t y  16  3. H i s t o p a t h o l o g y  18  D. Immunity t o C o c c i d i o s i s  19  EXPERIMENTAL METHODS AND DESIGN  24  A. Experimental D i e t s  24  B. Brooding  25  C. Experimental Design  27  D. Methods  29  1. Oocyst determinations-and t e c h n i q u e s . .  29  2. H i s t o l o g i c a l techniques  37  vi  PAGE  E- Experiments:  Object, Design, I n o c u l a t i o n ,  and Methods.  IV.  39  1. Experiment number 1  40  2. Experiment  number 2  42  3. Experiment number 3  44  RESULTS  47  A. Experiment No. 1  47  B. Experiment No. 2  59  1. C l i n i c a l r e s u l t s 2. H i s t o l o g i c a l  examination  C. Experiment No. 3  V.  VI.  .  59 63 73  1. Primary i n f e c t i o n  73  2. Secondary i n f e c t i o n 3. Post mortem and h i s t o l o g i c a l examinations  76  DISCUSSION.  .83 86  A. Oocyst p r o d u c t i o n  86  B. Weight Gains  92  C. M o r t a l i t y  94  D. Immunity  95  SUMMARY AND CONCLUSIONS BIBLIOGRAPHY  98 100  vii:..  LIST OF TABLES  TABLE  I.  II.  III.  IV.  V.  VI.  VII.  VIII.  PAGE  Average d a i l y oocyst p r o d u c t i o n i n m i l l i o n s i n 5-week-old c h i c k s , on 2 d i e t a r y l e v e l s o f v i t a m i n A, f o l l o w i n g i n f e c t i o n with 3 m i l l i o n oocysts o f E. a c e r v u l i n a per b i r d  52  Mean weight i n grams of 5-week-old c h i c k s , on 2 d i e t a r y l e v e l s of v i t a m i n A, a f t e r i n f e c t i o n w i t h 3 m i l l i o n oocysts of E. a c e r v u l i n a compared t o n o n - i n f e c t e d c o n t r o l s  53  D a i l y mean weight changes i n grams of 5-week-old c h i c k s , on 2 d i e t a r y l e v e l s of v i t a m i n A, f o l l o w i n g i n f e c t i o n w i t h 3 m i l l i o n oocysts o f E. a c e r v u l i n a  54  T o t a l mean weight i n grams, over a 4 month p e r i o d , of 4 groups o f chickens on 2 d i e t a r y l e v e l s o f v i t a m i n A, and w i t h one group on each l e v e l i n f e c t e d a t 5 weeks o f age w i t h 3 m i l l i o n oocysts of E. a c e r v u l i n a  55  Average d a i l y oocyst p r o d u c t i o n i n m i l l i o n s i n 9-week-old c h i c k s on 2 d i e t a r y l e v e l s of v i t a m i n A, and 2 l e v e l s of immunity, a f t e r i n f e c t i o n w i t h 10 m i l l i o n oocysts o f E. a c e r v u l i n a per bird  66  Average weight i n grams o f immunized 9-week-old c h i c k s , on, 2 d i e t a r y l e v e l s o f v i t a m i n A, f o l l o w i n g a second i n f e c t i o n with E. a c e r v u l i n a  67  Average weight i n grams of 9-week-old c h i c k s , on 2 d i e t a r y l e v e l s o f v i t a m i n A, f o l l o w i n g a primary i n f e c t i o n w i t h E. a c e r v u l i n a . . . .  68  D a i l y mean weight changes i n grams o f 9-week-old c h i c k s , on 2 d i e t a r y l e v e l s of v i t a m i n A, f o l l o w i n g a primary i n f e c t i o n w i t h 10 m i l l i o n oocysts' of E. a c e r v u l i n a  69  viii  TABLE  IX.  X.  XI.  XII.  XIII.  XIV.  Page  Two week t o t a l weight g a i n i n grams i n immune and non-immune 9-week-old c h i c k s , on 2 d i e t a r y l e v e l s o f v i t a m i n A, f o l l o w i n g i n f e c t i o n w i t h 10 m i l l i o n oocysts of E. a c e r v u l i n a  70  Performance index o f 9-week-old c h i c k s , on 2 d i e t a r y l e v e l s of v i t a m i n A and 2 l e v e l s o f immunity, f o l l o w i n g i n f e c t i o n w i t h 10 m i l l i o n oocysts o f E. a c e r v u l i n a per b i r d  71  Average d a i l y oocyst p r o d u c t i o n i n m i l l i o n s i n 19-week-old chickens on 2 d i e t a r y l e v e l s o f v i t a m i n A, and 2 l e v e l s of immunity, a f t e r i n f e c t i o n with 25 m i l l i o n oocysts of E. a c e r v u l i n a per b i r d  79  Average weight i n grams o f 19-week-old c h i c k e n s , on 2 d i e t a r y l e v e l s o f v i t a m i n A, and 2 l e v e l s of immunity, f o l l o w i n g i n f e c t i o n with 25 m i l l i o n oocysts o f E. a c e r v u l i n a  80  D a i l y mean weight changes i n grams i n immune, and non-immune 19-week-old chickens on 2 d i e t a r y l e v e l s of v i t a m i n A, f o l l o w i n g i n f e c t i o n with 25 m i l l i o n oocysts o f E. a c e r v u l i n a . . . . . . .  81  The r e p r o d u c t i v e index of E. a c e r v u l i n a f o l l o w i n g i n c r e a s i n g l y l a r g e r i n f e c t i v e doses as r e p o r t e d by v a r i o u s authors .  88  ix  LIST OF FIGURES  FIGURE  1.  2.  3.  4.  5.  6.  7.  PAGE  Arrangement o f c h i c k s i n b a t t e r i e s f o r E x p e r i ment No. 1. . . . . . .  29  The e f f e c t of d i e t a r y v i t a m i n A l e v e l on the mean weight of 5-week-old c h i c k s f o l l o w i n g i n f e c t i o n with E. a c e r v u l i n a  56  A comparison of the mean weights of i n f e c t e d and n o n - i n f e c t e d c h i c k s , on 2 d i e t a r y l e v e l s of v i t a m i n A, over a 4 month p e r i o d .  57  The e f f e c t of d i e t a r y v i t a m i n A l e v e l on t h e 2 week weight g a i n o f 5-week-old c h i c k s f o l l o w i n g i n f e c t i o n with E. a c e r v u l i n a . . . . .  58  The e f f e c t o f d i e t a r y v i t a m i n A l e v e l on the 2 week weight g a i n of 9-week-old c h i c k s f o l l o w i n g i n f e c t i o n with E. a c e r v u l i n a  72  The e f f e c t of d i e t a r y v i t a m i n A l e v e l on t h e mean weight o f 19-week-old immune and non-immune b i r d s 14 days a f t e r i n f e c t i o n with E. a c e r v u l i n a  82  A comparison of the d a i l y oocyst p r o d u c t i o n i n c h i c k s o f Experiments 1, 2, and 3  91  X  ACKNOWLEDGEMENTS  The  author would l i k e t o express her deep g r a t i t u d e t o  P r o f e s s o r Jacob B i e l y , of the Department of P o u l t r y for  Science,  a l l the h e l p and encouragement he has given throughout the  course of t h i s  study.  His u n f a i l i n g good humour and enthusiasm  have been a great i n c e n t i v e t o the completion of t h i s work. S i n c e r e thanks are a l s o extended t o P r o f e s s o r B e r y l E . March, of  the Department of P o u l t r y S c i e n c e ,  facilities  used i n t h i s  investigation,  f o r p r o v i d i n g the and e s p e c i a l l y  for her  a s t u t e o b s e r v a t i o n s and v a l u a b l e .suggestions i n the p l a n n i n g and p r e p a r a t i o n of t h i s r e p o r t .  Thanks are a l s o due to M r . Ian  Garnett f o r h i s a b l e a s s i s t a n c e data.  Finally,  i n the c o m p i l a t i o n of  grateful appreciation is  f i n a n c i a l support r e c e i v e d from:  given f o r the  statistical generous  Panco P o u l t r y L t d . - P a n c o  Research Award; U n i v e r s i t y of B r i t i s h Columbia - Graduate. F e l l o w s h i p ; and H . R . MacMillan Education Fund - Queen E l i z a b e t h Scholarship.  1  I.  INTRODUCTION  Experimental evidence has demonstrated t h a t  Eimeria  a c e r v u l i n a i s not a very pathogenic s p e c i e s of c o c c i d i a . ever,  How-  i t has been noted t h a t under f i e l d c o n d i t i o n s an i n f e c t i o n  with t h i s p a r a s i t e i s  o f t e n a s s o c i a t e d w i t h severe  l o s s e s , m o r b i d i t y , and some degree of m o r t a l i t y .  weight Therefore,  it  may be assumed t h a t the p a t h o g e n i c i t y of E . a c e r v u l i n a i s i n c r e a s e d by c e r t a i n e x t e r n a l f a c t o r s such as environmental c o n d i t i o n s and d i e t . of  v i t a m i n A i n the r a t i o n w i l l i n c r e a s e the degree of p a r a s i t i s m  i n a l l animals, i t E.  As i t has been demonstrated t h a t low l e v e l s  is  suggested t h a t the p a t h o g e n i c i t y of  a c e r v u l i n a may be i n c r e a s e d by a d e f i c i e n c y of v i t a m i n A .  T h i s t h e s i s was examined i n the f o l l o w i n g i n v e s t i g a t i o n which compared the e f f e c t s  of E . a c e r v u l i n a i n f e c t i o n s  on 2 l e v e l s of v i t a m i n A i n the d i e t .  in birds raised  The p a t h o g e n i c i t y was  assessed by a comparison of oocyst p r o d u c t i o n , weight and m o r t a l i t y . if  gains,  H i s t o l o g i c a l examination was used t o determine  any pathology of the i n t e s t i n a l e p i t h e l i u m r e s u l t e d from the  low v i t a m i n A d i e t .  The response of the immune host t o a  c o c c i d i a l i n f e c t i o n was a l s o i n v e s t i g a t e d i n b i r d s on both dietary vitamin A levels.  2  II.  A.  V i t a m i n A and C o c c i d i o s i s The  r e l a t i o n s h i p between v i t a m i n  chickens was f i r s t and  REVIEW OF LITERATURE  coworkers  worm, A s c a r d i a better  investigated  (1927).  over 40 years ago by Ackert  Experimenting w i t h the c h i c k e n round-  l i n e a t a , A c k e r t found t h a t t h i s p a r a s i t e  established  i n b i r d s on a low v i t a m i n  b i r d s r e c e i v i n g a normal r a t i o n . (1931)  A and p a r a s i t i s m i n  became  A d i e t than i n  F u r t h e r work by t h i s author  (1942) v e r i f i e d the e a r l i e r f i n d i n g s , and showed t h a t  b i r d s on a v i t a m i n  A d e f i c i e n t d i e t had a much slower  p e r i s t a l s i s than b i r d s r e c e i v i n g normal l e v e l s of the v i t a m i n . He suggested t h a t t h i s f a c t might account f o r t h e establishment of more and l a r g e r worms i n t h e low v i t a m i n et a l (1938) r e p o r t e d and  diet.  A group.  Murphy  a c o r r e l a t i o n between c a e c a l c o c c i d i o s i s  During the course o f an experiment, t o assess the  e f f e c t s of varying  amounts o f cod l i v e r o i l i n a complete d i e t ,  the b i r d s c o n t a c t e d c a e c a l c o c c i d i o s i s .  I t was noted t h a t the  b i r d s on the lower l e v e l s of o i l showed h i g h e r m o r t a l i t y , and took longer t o r e g a i n t h e i r weights, than the b i r d s g e t t i n g 1% o i l i n their ration.  Taylor  and R u s s e l l  (1946)  established,  through a s e r i e s of experiments, t h a t the minimum for vitamin  requirements  A recommended a t t h a t time were not adequate under  3  stress conditions.  Although 1200 I . U . of p r o v i t a m i n A per pound  of d i e t were adequate caecal coccidiosis  f o r normal growth, b i r d s s u b j e c t e d  were f a r more a f f e c t e d  b i r d s fed h i g h e r l e v e l s .  to  by the d i s e a s e than  They recommended, t h e r e f o r e ,  that a  66% s a f e t y margin be added to the standard v i t a m i n A l e v e l , g i v e 2000 I . U . / l b .  feed t o p r o v i d e p r o t e c t i o n i n time of  stress.  T h i s i n c r e a s e d need f o r v i t a m i n A i n a c o c c i d i a l i n f e c t i o n a l l i e d t o a drop i n v i t a m i n A l i v e r r e s e r v e s .  Davies  to  is  (1952)  found t h a t the normal l i v e r r e s e r v e of v i t a m i n A i n a c h i c k was about 292 I . U . / g r a m , and t h a t t h i s dropped t o an average  of  15 I . U . / g r a m d u r i n g a mixed i n f e c t i o n w i t h E i m e r i a t e n e l l a and E.  necatrix.  In these experiments  micrograms of carotene i n the f e e d , v i t a m i n A per pound of f e e d .  the b i r d s were r e c e i v i n g equivalent  t o 3500 I . U .  Davis a t t r i b u t e d the l i v e r  t o the l a c k of c o n v e r s i o n of carotene t o v i t a m i n A, as changeover occurs i n the e p i t h e l i u m of the i n t e s t i n e . Levine,  and S c o t t  (1958)  loss  this Erasmus,  (1960) b e l i e v e d t h a t the r e p o r t e d drop  i n l i v e r r e s e r v e s of v i t a m i n A c o u l d be due t o a lowered intake during a c o c c i d i o s i s investigations  infection.  feed  Therefore, i n t h e i r  on the i n t e r r e l a t i o n s h i p between c o c c i d i o s i s  v i t a m i n A, the c o n t r o l s were r e s t r i c t e d t o the same feed as the i n f e c t e d b i r d s .  2100  Employing t h i s refinement  and u s i n g a mixed c u l t u r e of E . t e n e l l a  of  and  intake  technique,  and E . a c e r v u l i n a as  4  inoculum, they conducted experiments with low and h i g h of v i t a m i n A.  The  levels  f o l l o w i n g c o n c l u s i o n s were reached:  1) F o l l o w i n g a c o c c i d i o s i s i n f e c t i o n , the b i r d s on  the  h i g h l e v e l s of v i t a m i n A showed a b e t t e r a p p e t i t e , and  f a s t e r growth r a t e , than the b i r d s on low  of d i e t a r y v i t a m i n 2) The  levels  A.  c h i c k ' s requirement f o r v i t a m i n A i s i n c r e a s e d  coccidiosis.  T h i s was  confirmed  by  by g r e a t l y reduced  l i v e r storage of v i t a m i n A i n a l l i n f e c t e d groups r e g a r d l e s s of the l e v e l i n the d i e t , and when c o n t r o l s were r e s t r i c t e d t o the same feed i n t a k e . 3) Carotene as a source  o f v i t a m i n A i s not as  effective  as the preformed v i t a m i n . The  authors  d i d not o b t a i n c o n s i s t e n t r e s u l t s on  the  c o r r e l a t i o n between v i t a m i n A l e v e l s and the s e v e r i t y of the disease.  However, G a r r i e t s (1961) found a d i r e c t r e l a t i o n s h i p  between the l e v e l of v i t a m i n A i n the d i e t and the s e v e r i t y of caecal c o c c i d i o s i s .  In f a c t he considered  the degree of  p r o t e c t i o n so c o n v i n c i n g , he recommended t h a t h i g h  levels  (5000 I . U . / k i l o . ) of v i t a m i n A c o u l d be used p r o p h y l a c t i c a l l y . However, t h i s author r e l i e d on n a t u r a l i n f e c t i o n from contaminated l i t t e r ,  so h i s r e s u l t s are open t o q u e s t i o n .  more c o n t r o l l e d experiment, Waldroup et al  (1963)  In a  concluded  5  t h a t m o r t a l i t y was the d i e t , and  not a f f e c t e d by the l e v e l of v i t a m i n A i n  found t h e r e was  no h i s t o p a t h o l o g i c a l d i f f e r e n c e s  i n the c a e c a l e p i t h e l i u m between b i r d s on low and normal v i t a m i n A diets.  However they d i d r e p o r t a s i g n i f i c a n t d i f f e r e n c e i n  the weight g a i n s , the h i g h e r v i t a m i n A groups r e g a i n i n g t h e i r l o s t weight much more r a p i d l y than the b i r d s on lower v i t a m i n A levels.  Panda and Combs (1964) working on l i v e r v i t a m i n A  v a l u e s , r e p o r t e d t h a t when e x c e s s i v e amounts o f v i t a m i n A were fed  (up t o 24,000 I . U . / k i l o . ) the p r o p o r t i o n a t e amount of  v i t a m i n A r e t a i n e d i n the l i v e r at the end of a mixed c o c c i d i a l i n f e c t i o n was  intestinal  h i g h e r than found with normal d i e t a r y  intakes.  B.  P h y s i o l o g i c a l Role of Vitamin The reason  A  f o r t h i s c o r r e l a t i o n between v i t a m i n A  and  the s e v e r i t y of c o c c i d i o s i s i n f e c t i o n must depend on the r o l e of v i t a m i n A i n the body. v i t a m i n A i s necessary As e a r l y as 1925  I t has  long been e s t a b l i s h e d t h a t  f o r normal f u n c t i o n i n g of the  epithelium.  Wolbach and Howe s t u d i e d the t i s s u e changes  r e s u l t i n g from a v i t a m i n o s i s A i n r a t s .  They r e p o r t e d t h a t a  squamous, s t r a t i f i e d , k e r a t i n i z i n g e p i t h e l i u m slowly r e p l a c e d the normal columnar e p i t h e l i u m of the mucous membranes, the g o b l e t c e l l s themselves were not a f f e c t e d . the r e s p i r a t o r y mucosa was  although  They found t h a t  f i r s t a f f e c t e d , f o l l o w e d by  the  6  e p i t h e l i u m of the s a l i v a r y glands, tract.  r e p r o d u c t i v e , and u r o g e n i t a l  There appeared t o be no change i n the e p i t h e l i u m of the  stomach and  intestines.  However at a l a t e r date, Richards  r e p o r t e d t h a t the e a r l i e s t m i c r o s c o p i c A i n the r a t was  (1935)  i n d i c a t i o n of avitaminosis  k e r a t i n i z a t i o n i n the d i g e s t i v e t r a c t , which  showed up c l i n i c a l l y as a g a s t r o - e n t e r i t i s . s t u d i e d a v i t a m i n o s i s A i n chickens  and  Seifried  found the  k e r a t i n i z a t i o n of mucosa as r e p o r t e d f o r the r a t . t h a t the h i s t o p a t h o l o g y  (1930)  typical He  reported  of a v i t a m i n o s i s A i n the c h i c k  was  p r i m a r i l y i n the r e s p i r a t o r y t r a c t , wherein the normal t i s s u e was  r e p l a c e d by squamous k e r a t i n i z i n g e p i t h e l i u m .  This r e s u l t e d  i n a l o s s of such s p e c i a l i z e d s t r u c t u r e s as c i l i a t e d c e l l s g o b l e t c e l l s , and consequently  i n a l o s s of the p r o t e c t i v e  p r o p e r t i e s of the l i n i n g e p i t h e l i u m .  In the alimentary  the changes were c o n f i n e d t o the mucous glands upper r e g i o n s .  Elvehjem and Neu  tract  and ducts of the  There were no experiments conducted on  i n t e s t i n a l epithelium.  and  the  (1932), i n s t u d i e s on  a v i t a m i n o s i s A i n c h i c k s , found one of the most c o n s i s t e n t f i n d i n g s was l e v e l was  an e l e v a t e d u r i c a c i d content  dependent on the degree of kidney  i n the blood.  This  damage from k e r a t i n i z -  a t i o n of the t u b u l e s , p r e v e n t i n g the normal e l i m i n a t i o n of u r i c a c i d from the body.  The  authors  a l s o commented on the  character-  i s t i c a t a x i a a s s o c i a t e d with a v i t a m i n o s i s A and a t t r i b u t e d i t t o  7  nerve damage r a t h e r than t o uremia. Wollam and M i l l e n brospinal f l u i d is  However, l a t e r s t u d i e s by  (1955) have shown t h a t an i n c r e a s e i n c e r e the main cause of t h i s  disorder.  Roels  (1967), i n a review of v i t a m i n A f u n c t i o n s , has suggested t h a t the g e n e r a l r o l e of t h i s v i t a m i n i n metabolism i s t h a t of a "membrane a c t i v e " compound, concerned w i t h the s t a b i l i t y of membranes.  cell  I t i s a l s o i m p l i c a t e d i n p r o t e i n and l i p i d metabolism  and the s t a b i l i t y of lysosomes.  A very c o n s i s t e n t  cases of v i t a m i n A d e f i c i e n c y i s  a depressed  finding in  glycogenesis  r e s u l t i n g from i m p a i r e d a d r e n a l gland f u n c t i o n .  The i n f l u e n c e  of v i t a m i n A on the adrenocortex of c h i c k s was i n v e s t i g a t e d by Glick  (1963).  He r e p o r t e d t h a t 5-week-old b i r d s on a low v i t a m i n  A d i e t showed reduced a d r e n a l c o r t i c a l f u n c t i o n as r e f l e c t e d by a significantly  lower h e t e r o p h i l i n c r e a s e a f t e r ACTH i n j e c t i o n .  The r e l a t i o n s h i p between a v i t a m i n o s i s A and immunity to d i s e a s e has been i n v e s t i g a t e d  by many workers.  In 1960,  Harmon  et a l demonstrated the e f f e c t of a v i t a m i n A d e f i c i e n c y a n t i b o d y - p r o d u c i n g a b i l i t y of swine.  They r e p o r t e d t h a t  on a low v i t a m i n A d i e t produced lower antibody t i t r e s Salmonella pullorum than pigs on a normal d i e t . Miller  pigs  against  Yaeger and  (1963) experimented w i t h the s u s c e p t i b i l i t y  of r a t s  Trypanosoma c r u z i under c o n d i t i o n s of a v i t a m i n o s i s A . found t h a t r a t s on v i t a m i n A d e f i c i e n t  on the  d i e t s showed  They  less  to  8  r e s i s t a n c e t o i n f e c t i o n with t h i s p a r a s i t e , and  a l s o t o mis-  c e l l a n e o u s b a c t e r i a l i n f e c t i o n s , than animals r e c e i v i n g adequate amounts of the v i t a m i n .  Panda and  S. pullorum i n f e c t i o n i n c h i c k s ,  Combs (1963) working w i t h  i n v e s t i g a t e d the e f f e c t s of  suboptimal l e v e l s of v i t a m i n  A i n the r a t i o n .  containing  A per k i l o g r a m of feed,  9960 I.U.  vitamin  They fed d i e t s and  975  I .U./kilogram, and wer.e able t o demonstrate a s i g n i f i c a n t l y lower a g g l u t i n a t i o n response t o S. pullorum, and bursa weight, i n b i r d s on the  a l s o a lower  lower l e v e l s of v i t a m i n  A.  This  i m p l i c a t i o n of the bursa of F a b r i c i u s i n antibody response confirms e a r l i e r work by G l i c k , Chang, and reported  Jaap (1956).  t h a t i n t h e i r i n v e s t i g a t i o n , only  b i r d s produced a n t i b o d i e s 63/73 of the c o n t r o l s .  against  Challey  8/75  They  bursectomized  S. typhimurium, compared t o (1962), working with E i m e r i a  t e n e l l a i n f e c t i o n s i n normal and  bursectomized b i r d s , observed  a much h i g h e r m o r t a l i t y  i n the bursectomized c h i c k s .  these f i n d i n g s were not  confirmed i n l a t e r i n v e s t i g a t i o n s  P i e r c e and  C.  Long (1965), and  Eimeria acervulina E. a c e r v u l i n a  reported  Tyzzer  from d i a g n o s t i c  by  (1968).  1929  i s the s p e c i e s  of E i m e r i a most commonly  l a b o r a t o r i e s , and  c o n t r o l l e d by c o c c i d i o s t a t s . small  Rose  However,  a l s o the  least well  I t a f f e c t s the upper h a l f of  i n t e s t i n e , p a r t i c u l a r l y the duodenum, u s u a l l y  causing  the  9  w h i t i s h t r a n s v e r s e s t r e a k s on the mucosa. first  d e s c r i b e d by Tyzzer  lesions.  Acervulina is  the s p e c i e s ' line.  (1929), a f t e r i s o l a t i o n from i n t e s t i n a l  a L a t i n term which i s d e s c r i p t i v e of  i n t e s t i n a l pathology:  Infections  with t h i s  r a t h e r than a c u t e ,  acerv = a heap and l i n a =  s p e c i e s are c h a r a c t e r i z e d by c h r o n i c ,  symptoms which i n c l u d e a n o r e x i a , d i a r r h e a ,  and reduced weight g a i n s . differenciated  This s p e c i e s was -.  E . a c e r v u l i n a i n f e c t i o n s may be  from i n f e c t i o n w i t h other s p e c i e s of E i m e r i a by  the prepatent p e r i o d of 4 days; l a r g e numbers of oocysts passed; and the r e l a t i v e lack of p a t h o g e n i c i t y .  1. L i f e The l i f e feces.  Tyzzer  Cycle c y c l e begins w i t h the oocyst passed out i n the (1929) r e p o r t e d the oocyst as egg-shaped and having  an average measurement of 19.5 by 14.3 m i c r o n s , w i t h a range of 17.7-20.2 microns i n l e n g t h , Becker  and 13.7-16.3 microns i n w i d t h .  (1956) d i s a g r e e d w i t h these measurements,  oocyst t o be s m a l l e r on the average, s i z e as 16.4 by 13 m i c r o n s . these measurements, average s i z e s :  f i n d i n g the  and r e p o r t e d the  Lund and F a r r  average  (1965) agree w i t h  but other authors have l i s t e d  different  17 by 14 microns (Morehouse and McGuire,  1958);  18.3 by 14.6 microns (Edgar, 1955); and 17.4 by 13 microns (Long, 1967).  For t h i s  i n v e s t i g a t i o n the average s i z e of the  oocysts were 17.3 by 15 m i c r o n s .  This great v a r i a t i o n i n  10  r e p o r t e d s i z e s demonstrates the d i f f i c u l t y i n making a s p e c i e s d i a g n o s i s from t h e oocyst s i z e alone. As w i t h a l l s p e c i e s . o f E i m e r i a , t h e newly passed i s u n s p o r u l a t e d and c o n s i s t s of a dense, tough, surrounding a sporont or zygote.  oocyst  outer c o v e r i n g  Oxygen, moisture, and heat  are r e q u i r e d f o r s p o r u l a t i o n which begins w i t h t h e sporont undergoing  r e d u c t i o n d i v i s i o n and throwing o f f a p o l a r body.  The sporont then d i v i d e s i n t o 4 s p o r o b l a s t s , each o f which develops  i n t o a sporocyst.  Two s p o r o z o i t e s develop i n each  s p o r o c y s t , g i v i n g a t o t a l o f 8 s p o r o z o i t e s per o o c y s t .  This  arrangement of 2 s p o r o z o i t e s w i t h i n 4 s p o r o c y s t s i s c h a r a c t e r istic  of t h e genus E i m e r i a , and separates i t from t h e genus  Isospora, i n which the oocyst c o n t a i n s 2 s p o r o c y s t s , each having 4 sporozoites.  Wilson and F a i r b a i r n  (1961), s t u d y i n g t h e  s p o r u l a t i o n o f E. a c e r v u l i n a when incubated a t 30°C, demons t r a t e d maximum s p o r o b l a s t formation a t 10. hours, iand maximum s p o r o z o i t e development a t 20 hours.  Edgar  (1955) r e p o r t e d  s p o r u l a t i o n as e a r l y as 17 hours a t 29°, and Krasser was  (1963).  a b l e t o i n i t i a t e a s l i g h t i n f e c t i o n i n b i r d s with oocysts  showing s p o r u l a t i o n at 13 hours.  However a l l authors  agree  t h a t maximum s p o r u l a t i o n occurs between 24 and 27 hours at 28°o 30 C.  The s u r v i v a l of E i m e r i a oocysts o u t s i d e the body depends  p r i m a r i l y on moisture  as demonstrated by Davies and Joyner  11  (1955).  F a r r and Wehr (1949) i n a study of oocyst s u r v i v a l  in  the s o i l , concluded t h a t humidity i s the l i m i t i n g f a c t o r , and for  normal v i a b i l i t y of the oocyst i t  should exceed 90%.  They  were able t o demonstrate the s u r v i v a l of E . a c e r v u l i n a oocysts for  over 86 weeks under moist shady c o n d i t i o n s . Under normal c o n d i t i o n s a b i r d must i n g e s t the s p o r u l a t e d  oocyst t o become i n f e c t e d , t u r e of the oocyst w a l l , i n the i n t e s t i n e .  and i n f e c t i o n begins w i t h the  excystation,  Doran and F a r r  and r e l e a s e  of  sporozoites  (1962; 1965), Doran  and F a r r and Doran (1962), have r e p o r t e d t h e i r i n t o the f a c t o r s i n v o l v e d i n the i n i t i a l  frac-  (1966),  investigations  sporozoite  release.  A c c o r d i n g t o these a u t h o r s , the f o l l o w i n g steps are i n v o l v e d : 1) The w a l l of the oocyst i s  f r a c t u r e d through mechanical  a c t i o n of the g i z z a r d and the s p o r o c y s t s are r e l e a s e d . 2) Through the a c t i o n of p a n c r e a t i c j u i c e , i n the presence of b i l e , t h e r e i s  a c t i v a t i o n of the s p o r o -  z o i t e and a probable d i g e s t i o n of the sporocyst p l u g which allows the s p o r o z o i t e s t o  escape.  From i n v i t r o experiments i t would appear t h a t critical  the  enzymes i n the p a n c r e a t i c j u i c e are t r y p s i n and  chymotrypsin, and i t i s  i n t e r e s t i n g t o note t h a t B r i t t o n et  al  (1964) have found a marked decrease i n e x c y s t a t i o n of oocysts b i r d s on a low p r o t e i n d i e t which produces low t r y p s i n v a l u e s .  in  12  Nyberg and  co-workers  (1968) have suggested t h a t carbon  d i o x i d e i s the i n i t i a l s t i m u l i f o r the e x c y s t a t i o n of E. oocysts, and Lotze and Leek  tenella  (1968) have demonstrated t h a t some  s p o r o z o i t e s , a f t e r a c t i v a t i o n with carbon d i o x i d e and  enzymes,  can escape through the m i c r o p y l e  emerge  of the sporocyst  from an undamaged oocyst through an unseen h o l e .  and  I t i s apparent  t h a t not a l l the f a c t o r s necessary f o r e x c y s t a t i o n have been elucidated. Joyner  Experiments by Landers  (1960) and  Davies  and  (1962) have shown t h a t i n t e s t i n a l i n f e c t i o n s w i t h  Eimeria  s p e c i e s can be i n i t i a t e d through p a r e n t e r a l i n j e c t i o n s of sporulated oocysts.  Doran (1966  a, b, c) has  E. a c e r v u l i n a s p o r o z o i t e s can excyst and  demonstrated t h a t  penetrate  the  e p i t h e l i u m as e a r l y as 10 minutes a f t e r i n g e s t i o n of oocysts.  He  sporulated  found t h a t the time of g r e a t e s t p e n e t r a t i o n  1-2  hours a f t e r dosing,  was  at a p o i n t j u s t b e f o r e the c u r v a t u r e  t o about 2.5  villar  cm.  and t h a t the area of g r e a t e s t  invasion  of the duodenal  before the entry of the p a n c r e a t i c duct  the ascending duodenum.  The  endogenous stages  was  loop, into  of E. a c e r v u l i n a  are c o n f i n e d t o the e p i t h e l i u m of the s m a l l i n t e s t i n e as described  f i r s t by Tyzzer  (1929) and  confirmed by Doran (1966,  a, b, c ) , V e t t e r l i n g and Doran (1966), and Pout Working with  E. n e c a t r i x , Van  Doorninck and  (1967).  Becker  (1957)  first  demonstrated t h a t the s p o r o z o i t e s were engulfed by macrophages  13  as they entered the e p i t h e l i u m of the v i l l i ,  and were t r a n s -  ported down through the lamina p r o p r i a t o the e p i t h e l i a l of c r y p t s before i n i t i a t i n g schizogony.  C h a l l e y and Burns  r e p o r t e d the same m i g r a t i o n of s p o r o z o i t e s infections,  and Doran (1966c)  Doran found t h a t s p o r o z o i t e s  (1959)  in E. tenella,  in E . acervulina infections. are not seen i n the g l a n d u l a r  e p i t h e l i u m u n t i l 18 hours a f t e r i n g e s t i o n of s p o r u l a t e d so t h i s  cells  oocysts,  f i n d i n g would p r e c l u d e the p o s s i b i l i t y of d i r e c t pene-  tration.  He d e s c r i b e d the s p o r o z o i t e as b e i n g p o i n t e d a n t e r i o r l y  and averaging 8 microns i n l e n g t h and 1.6 microns i n width when f r e e i n the lumen, and about 4 microns i n l e n g t h when i n the tissue.  I t i s c h a r a c t e r i s e d by a e o s i n o p h i l i c r e f r a c t i l e  globule. The schizogonous  cycle consists  a c c o r d i n g t o Warren and B a l l  of 3 s c h i z o n t  stages  (1967) and Long (1967).  However,  V e t t e r l i n g and Doran (1966) who have done an exhaustive i n t o the endogenous  forms of E . a c e r v u l i n a , r e p o r t the  of a 4th s c h i z o n t s t a g e .  They demonstrated t h a t the  study presence  schizogonous  c y c l e s t a r t s w i t h the appearance of the s p o r o z o i t e i n the e p i t h e l i a l c e l l at the base of the c r y p t .  Here the  sporozoite  rounds up, d i s t a l t o the nucleus and j u s t below the brush border of the c e l l , and begins the t r o p h o z o i t e or growth phase. it  reaches a c e r t a i n s i z e n u c l e a r d i v i s i o n occurs and the  When first  14  generation schizont i s  formed.  T h i s s c h i z o n t i s mature from  36-48 hours a f t e r i n g e s t i o n of the o o c y s t , measures 10 by 7 m i c r o n s , and c o n t a i n s from 8-16 m e r o z o i t e s . (1967) r e p o r t s the s i z e as 5-8 m i c r o n s . are r e l e a s e d they invade c e l l s  However, Long  When the merozoites  i n the neck of the c r y p t and  w i t h i n 8 hours develop i n t o second g e n e r a t i o n s c h i z o n t s , measuring 5 by 4 m i c r o n s , and c o n t a i n i n g 16 merozoites on the average.  These m e r o z o i t e s , when r e l e a s e d , p e n e t r a t e the  at the base of the v i l l i .  The t h i r d g e n e r a t i o n s c h i z o n t t h a t  matures 16 hours l a t e r , or 56-72 hours a f t e r the i n o c u l a t i o n of o o c y s t s ,  is  cells  initial  s i m i l a r i n s i z e t o the 2nd g e n e r a t i o n  s c h i z o n t but d i v i d e s i n t o o n l y 8 merozoites on the average. These 3rd g e n e r a t i o n merozoites invade the e p i t h e l i a l c e l l s the v i l l i ,  of  mostly i n the middle r e g i o n , and develop i n t o the  f o u r t h g e n e r a t i o n s c h i z o n t s which are l a r g e r than the p r e v i o u s 2 g e n e r a t i o n s , measuring about 8 microns and c o n t a i n i n g 28-36 large merozoites.  The gametogenous  stages are i n i t i a t e d by the  3rd or 4th g e n e r a t i o n merozoites and are u s u a l l y found i n the t i p s of the v i l l i .  The microgametocytes which appear f i r s t  at  around 84 hours a f t e r oocyst i n o c u l a t i o n , measure about 7 microns i n diameter, and when mature c o n t a i n about 90 m i c r o gametes.  These average 3 by 0.5 microns and have 2 long  f l a g e l l a a r i s i n g from the a n t e r i o r end.  The macrogametes are  15  seen f i r s t at 88-90 h o u r s , and when f e r t i l i z e d by the microgametes develop i n t o o o c y s t s .  The oocysts  released  are v o i d e d from  the body 96-97 hours a f t e r i n o c u l a t i o n of the i n f e c t i n g dose, and t h i s short prepatent p e r i o d i s c h a r a c t e r i s t i c of i n f e c t i o n with E . a c e r v u l i n a . There are v a r y i n g r e p o r t s on the t i m e s ,  sizes,  l o c a t i o n s of the d i f f e r e n t generations of s c h i z o n t s .  and Tyzzer  (1929) c o u l d not d i f f e r e n t i a t e between the schizogonous  stages  and made r e f e r e n c e o n l y t o the f a c t t h a t s c h i z o n t s were found near the t i p s of the v i l l i , 3rd or 4th g e n e r a t i o n .  which would probably r e f e r t o  The f a c t t h a t there i s v a r i a t i o n i n the  times of m a t u r a t i o n f o r the i n d i v i d u a l s c h i z o n t s makes difficult  the  it  t o a c c u r a t e l y d i s t i n g u i s h the exact stage encountered.  However, a l l the i n v e s t i g a t o r s are i n agreement t h a t t h e r e are stages i n the endogenous development p r o g r e s s i n g from the base of the c r y p t t o the t i p of the accompanying v i l l i .  Pout (1967)  r e p o r t e d t h a t the endogenous phases of the p a r a s i t e s seem t o "ride" i n the m i g r a t i n g host c e l l s  so t h a t " t r a i n s " of  can be seen proceeding up the v i l l u s .  parasites  He found t h i s most marked  at day 4 when maturing oocysts were seen at the t i p s of the  villi  with l a t e r generations of s c h i z o n t s t r a v e l l i n g behind them lower down i n the v i l l i .  V e t t e r l i n g and Doran (1966) r e p o r t e d t h a t  a l l the stages from s p o r o z o i t e t o gamete occur w i t h i n the  cells  16  i n the gland invaded by the s p o r o z o i t e to i t .  They s p e c u l a t e  and i n the v i l l i  adjacent  t h a t t h i s movement f u r t h e r toward the  lumen w i t h each s u c c e s s i v e g e n e r a t i o n i s more l i k e l y due to the flow of mucous than to e p i t h e l i a l c e l l m i g r a t i o n . c e n t r a t i o n of the endogenous forms i n l i m i t e d areas  This con-  accounts  for the white-grey r a i s e d streaks which c h a r a c t e r i z e a l i g h t i n f e c t i o n with E . a c e r v u l i n a .  2.  Pathogenicity  E . a c e r v u l i n a has never been c o n s i d e r e d a very s p e c i e s of E i m e r i a . pathogenicity  Tyzzer (1929)  pathogenic  (1932) r e p o r t e d t h a t  the  of the organism depended on the d u r a t i o n , as  as the i n t e n s i t y  of i n f e c t i o n .  He s t a t e d t h a t e x t e n s i v e  well  involve-  ment of the e p i t h e l i u m i n severe cases would s e r i o u s l y  interfere  with d i g e s t i o n  (1939)  investigated E.  and a b s o r p t i o n .  D i c k i n s o n and S c b f i e l d  the c l i n i c a l m a n i f e s t a t i o n s  acervulina,  of l a r g e doses of  (35-50 m i l l i o n o o c y s t s / b i r d )  and confirmed  T y z z e r ' s c o n t e n t i o n t h a t m o r t a l i t y was not a f a c t o r .  They  demonstrated a decrease i n food consumption and body weight from 4-10  days a f t e r  i n o c u l a t i o n , and r e p o r t e d that the r e t u r n  to p r e - i n o c u l a t i o n weight took 18-24 investigated genicity.  the e f f e c t s  days.  D i c k i n s o n (1941)  of b i r d age and oocysts dose oh patho-  He concluded t h a t a d a i l y i n g e s t i o n  of 5000 oocysts  was the margin between c l i n i c a l and s u b c l i n i c a l i n f e c t i o n s .  He  17  concurred w i t h e a r l i e r work t h a t t h e r e was no m o r t a l i t y , l o s s e s i n body weight were temporary, and t h a t i f the  the  infective  dose was l a r g e enough t h e r e were c l i n i c a l symptoms on day These symptoms were a n o r e x i a , d e p r e s s i o n , scanty,  loose droppings.  4-9.  l o s s of weight,  Morehouse and McGuire  (1956)  and  (1958)  d i s a g r e e d w i t h the assumption t h a t E . a c e r v u l i n a c o u l d not cause mortality.  They demonstrated t h a t s u c c e s s i v e i n o c u l a t i o n s  l a r g e numbers of oocysts c o u l d r e s u l t groups.  with  i n 75% m o r t a l i t y i n some  They a l s o r e p o r t e d t h a t weight l o s s e s c o u l d reach 20%  w i t h a s i n g l e heavy i n f e c t i o n . t h a t the t y p i c a l i n t e s t i n a l  These authors a l s o demonstrated  l e s i o n s of an E . a c e r v u l i n a i n f e c t i o n j  were present oocysts.  only i n very l i g h t i n f e c t i o n s  Heavier i n f e c t i v e  of under 500,000 i  doses of 5 m i l l i o n or more o o c y s t s /  b i r d r e s u l t e d i n a marked inflammation and t h i c k e n i n g of intestinal wall.  Hein  (1968a) r e c e n t l y i n v e s t i g a t e d  the  i  the effects  of v a r i a b l e doses of E . a c e r v u l i n a oocysts on young c h i c k s . found t h a t the s i z e of dose was d i r e c t l y c o r r e l a t e d severity  She  with;the  of the l e s i o n s and the degree of weight l o s s .  Anorexia  and m o r b i d i t y were seen only at the higher l e v e l s of i n f e c t i o n 5 m i l l i o n and 20 m i l l i o n o o c y s t s / b i r d . i n any groups, but p a t h o g e n i c i t y the younger b i r d s . c l i n i c a l evidence  There was no m o r t a l i t y  of the p a r a s i t e was higher i n  The l a r g e numbers of oocysts needed tio g i v e of d i s e a s e i s r e l a t e d to the f i n d i n g s  of  all  -  18  investigators  t h a t a h i g h oocyst p r o d u c t i o n i s  i n f e c t i o n with E . a c e r v u l i n a . r e p o r t e d t h a t one i n f e c t i v e  t y p i c a l of  Morehouse and McGuire  (1958)  dose of 5 m i l l i o n oocysts  resulted  i n a t o t a l e l i m i n a t i o n of 684 m i l l i o n oocysts per b i r d ,  and Long  (1967) has demonstrated 2000 m i l l i o n per b i r d from a dose of 100,000 s p o r u l a t e d o o c y s t s .  3.  Histopathology  The p a t h o l o g i c a l f i n d i n g s  i n a b i r d infected with E .  a c e r v u l i n a depend on the number of s p o r u l a t e d oocysts at one t i m e .  ingested  The p r e v i o u s l y mentioned white patches or  r e s u l t i n g from agglomerations in light infections  of s c h i z o n t s  or o o c y s t s ,  streaks appear  and are found mainly i n the duodenal l o o p .  As the i n f e c t i o n i n c r e a s e s  there i s  a more e x t e n s i v e  involve-  ment of the same fundamental type accompanied by an edematous s w e l l i n g of the e p i t h e l i a l  l a y e r and lamina p r o p r i a c a u s i n g a  t h i c k e n i n g of the i n t e s t i n a l w a l l . w i t h heavy i n f e c t i o n s , intestine.  Hein  (1968a) r e p o r t e d t h a t ,  the l e s i o n s extended  f u r t h e r down the  An e x t e n s i v e v a s o d i l a t i o n occurs i n heavy  infections  r e s u l t i n g i n inflammation i n the invaded areas of the  intestine.  Sloughing of the i n t e s t i n a l and heavy i n f e c t i o n s Pout  e p i t h e l i u m i s present  i n both  a c c o r d i n g t o Morehouse and McGuire  (1967) has shown t h a t on day 4 t h e r e i s  decrease i n the h e i g h t  of the v i l l i  a  (1958).  significant  t o the t o t a l mucosal  light  19  thickness  D.  i n E. a c e r v u l i n a i n f e c t i o n s .  Immunity t o C o c c i d i o s i s Tyzzer  t i o n s and  (1929) i n v e s t i g a t e d immunity i n c o c c i d i a l i n f e c -  concluded t h a t the most pronounced p r o t e c t i o n occur-  red i n species host  t h a t penetrate  deeply and  t i s s u e s - E. t e n e l l a f o r example.  i n species  tend t o be r e t a i n e d i n He r e p o r t e d  poor immunity  such as E. a c e r v u l i n a t h a t develop s u p e r f i c i a l l y i n  the e p i t h e l i u m .  Peterson  (1949) r e p o r t e d  t h a t repeated  exposures t o E. a c e r v u l i n a are necessary f o r a good degree of immunity.  He  found t h a t 3 l i g h t i n o c u l a t i o n s of oocysts  thousand) i n 2-week-old c h i c k s gave complete c l i n i c a l when c h a l l e n g e d were present,  3 weeks l a t e r .  a few  oocysts  complete immunity was by D i c k i n s o n  However, although no  droppings of c l i n i c a l l y  protection lesions  were s t i l l passed i n d i c a t i n g t h a t  not a c h i e v e d .  (1941) who  (10-50  reported  T h i s confirms e a r l i e r work  the presence of oocysts  i n the  immune b i r d s . . However he d i d f i n d  a s i n g l e l a r g e inoculum of oocysts  was  small doses i n c o n f e r i n g t h i s immunity.  as e f f e c t i v e as He  that  several  demonstrated t h a t  r e t e n t i o n of p r o t e c t i v e immunity at 70 days, was  identical  e i t h e r f o l l o w i n g a s i n g l e dose of 25 m i l l i o n o o c y s t s / b i r d  of  E. a c e r v u l i n a or m u l t i p l e s m a l l doses t o t a l l i n g t h i s amount. Rose and Long  (1962) i n v e s t i g a t i n g complete immunity w i t h  s p e c i e s , i n f e c t e d b i r d s w i t h 500,000, 5 m i l l i o n , and  10  Eimeria  million  20  oocysts of E. a c e r v u l i n a on 3 c o n s e c u t i v e weeks.  When c h a l l e n g e d  18 days l a t e r w i t h 20 m i l l i o n o o c y s t s , t h e b i r d s were completely immune t o the p a r a s i t e as v e r i f i e d by negative f e c a l counts.  Hein  oocyst  (1968b) produced e f f e c t i v e p r o t e c t i v e immunity  w i t h 2 s m a l l immunizing doses 2 weeks a p a r t .  The inoculum  con-  s i s t e d of 80,000 and 160,000 oocysts o f E. a c e r v u l i n a , and the b i r d s were c h a l l e n g e d w i t h 5 m i l l i o n oocysts a t 28 days a f t e r the second i n o c u l a t i o n . Becker  (1935) was one o f the f i r s t t o study the nature of  c o c c i d i a l immunity.  He experimented w i t h E. m i y a i r i i  n i e s c h u l z i ) i n r a t s and concluded host response.  (syn. E.  t h a t t h e r e was no g e n e r a l i z e d  Unable t o demonstrate p r o t e c t i v e humoral a n t i -  bodies, he s p e c u l a t e d t h a t i n f e c t i o n r e s u l t e d i n a l o c a l i z e d response,  and t h e nature of t h i s was p o s s i b l y a " s e n s i t i s a t i o n "  of t h e e p i t h e l i u m which blocked the entrance of s p o r o z o i t e s . Morehouse (1938) agreed w i t h t h i s h y p o t h e s i s , and i n f u r t h e r s t u d i e s w i t h E. m i y a i r i i r e p o r t e d t h a t s i g n i f i c a n t l y s p o r o z o i t e s invaded t h e immune e p i t h e l i u m .  fewer  However, v a r i o u s  i n v e s t i g a t i o n s i n t o c o c c i d i a l immunity i n chickens have not supported  these f i n d i n g s .  Both Tyzzer  (1932),  i n s t u d i e s on  E. n e c a t r i x immunity, and Horton-Smith and co-workers (1963) examining t h e immune response  t o E. t e n e l l a , found no d i f f e r e n c e  i n s p o r o z o i t e i n v a s i o n i n immune or s u s c e p t i b l e b i r d s .  I t was  21  found t h a t the immune mechanism i n h i b i t e d the s p o r o z o i t e s developing i n t o schizonts,  from  as no evidence of f i r s t or second  g e n e r a t i o n s c h i z o n t s were observed. The presence of some type of p r o t e c t i v e humoral a n t i bodies was demonstrated by Burns and C h a l l e y Smith,  Beattie,  investigators  and Long (1961).  (1959) and H o r t o n -  In separate s t u d i e s these  both used the same technique of l i g a t i n g one  caecum and then i n t r o d u c i n g an i n f e c t i o n of E . t e n e l l a i n t o other.  A f t e r an a p p r o p r i a t e , p e r i o d of t i m e , the  the  isolated  caecum was then exposed t o i n f e c t i o n and was found t o be resistant.  P i e r c e and co-workers  (1962) r e p o r t e d the  presence  of p r e c i p i t a t i n g a n t i b o d i e s i n b i r d s made immune t o E . t e n e l l a . However the b i r d s degree of r e s i s t a n c e  to further i n f e c t i o n d i d  not c o i n c i d e w i t h the l e v e l of c i r c u l a t i n g a n t i b o d i e s . (1963) examined the nature of t h i s humoral a n t i b o d y . stiedae,  Rose Using E .  and s e v e r a l c h i c k e n s p e c i e s of c o c c i d i a , she was able  t o s t i m u l a t e the p r o d u c t i o n of serum a n t i b o d i e s u s i n g composed of i n t a c t oocysts; of the p a r a s i t e s .  crushed oocysts;  and t i s s u e stages  The a n t i b o d i e s were demonstrated by agar  p r e c i p i t a t i o n and complement f i x a t i o n t e s t s .  Rose and Long  (1962) were not a b l e t o c o r r e l a t e the presence of with resistance  antigens  to i n f e c t i o n ,  destructive a b i l i t i e s  antibodies  although the a n t i b o d i e s do e x h i b i t  as evidenced by t h e i r c a p a c i t y t o  lyse  22  both s p o r o z o i t e s  and merozoites  in vitro.  demonstrated l y s i n g of s p o r o z o i t e s  Rose  (1963)  i n vivo following  i n j e c t i o n i n b i r d s showing a h i g h l e v e l of a n t i b o d y .  has  intravenous However,  t h e r e i s no apparent d e s t r u c t i o n f o l l o w i n g o r a l d o s i n g .  It  has  been c o n c l u s i v e l y demonstrated by many authors t h a t t h e r e i s "booster" e f f e c t  on the l e v e l of c i r c u l a t i n g a n t i b o d i e s when the  b i r d i s c h a l l e n g e d w i t h succeeding i n f e c t i o n s  of  coccidiosis.  I t has a l s o been r e p o r t e d t h a t the s e r a of r e s i s t a n t does not always c o n t a i n a n t i b o d y . (1963) s t u d i e d the e f f e c t s  birds  Horton-Smith and co-workers  of immunity on the endogenous  of the p a r a s i t e and concluded t h a t both schizogonous gametogenous  no  stages are i n h i b i t e d .  stages  and  Horton-Smith (1963) has  r e p o r t e d t h a t the second schizogony i s the stage of the  life  c y c l e which promotes the immune response under n a t u r a l i n f e c t i o n s . T h i s f i n d i n g was confirmed by Rose t e n e l l a and E . n e c a t r i x .  (1967b) working w i t h E .  Rose (1963) has suggested t h a t  c i r c u l a t i n g a n t i b o d i e s may be important only when combined w i t h a l o c a l f a c t o r such as a c e l l u l a r response.  Various  gators have s t u d i e d the r o l e of c e l l u l a r immunity as protective  factor against c o c c i d i o s i s .  Becker (1935)  investithe suggested  t h a t the l o c a l i z e d response may be due to " s e n s i t i z e d macrophages".  Van Doorninck and Becker (1957), Doran (1966c), and  C h a l l e y and Burns (1959) i n t h e i r s t u d i e s on the t r a n s p o r t of  23  s p o r o z o i t e s by the macrophages, have s p e c u l a t e d on the r o l e these c e l l s Smith  p l a y i n the immune response mechanism.  (1963) and P i e r c e et a l  (1962), have demonstrated the  presence of numerous p y r o n i n o p h i l i c c e l l s cells  Horton-  - r e s e m b l i n g plasma  - and a l s o leukocytes i n the caeca of immunized b i r d s  after challenge with E . t e n e l l a . like cells,  In d i s c u s s i n g these plasma-  Horton-Smith suggests t h a t the macrophages may  i n g e s t not only s p o r o z o i t e s but a l s o e o s i n o p h i l s which have come i n contact with t h i s antigen. i n t o the plasma c e l l s  Macrophages may then transform  and produce antibody which destroys  s p o r o z o i t e and i n h i b i t s f u r t h e r growth.  However, t o d a t e ,  the the  exact mechanism of the immune response t o c o c c i d i o s i s has not been e l u c i d a t e d .  .  24  III.  A.  Experimental  EXPERIMENTAL METHODS AND DESIGN  Diets  1. B a s a l D i e t No. 1:  21% p r o t e i n .  Ingredients  Percent  i n ration  Ground wheat  74.5  Soybean meal  12.0  H e r r i n g meal  8.0  Dried d i s t i l l e r s solubles  2.0  Bonemeal  2.0  Limestone  1.0  Iodized s a l t  0.5 Amount per k i l o g r a m  Manganese s u l f a t e  12.5 grams  Riboflavin  0.33  V i t a m i n D3  2. Experimental  grams  440 I.G.U.  D i e t No. 2:  B a s a l D i e t No. 1 plus 440 I.U. v i t a m i n A* per k i l o gram o f f e e d .  * V i t a m i n A p a l m i t a t e - Rovemix A-325. Roche L t d .  Hoffman-La  25  3. Experimental  D i e t No. 3:  Basal D i e t No. 1 plus 4400 I.U. v i t a m i n A* per k i l o gram of feed.  B.  Brooding One hundred day-old White Leghorn c o c k e r e l s were p l a c e d  i n e l e c t r i c a l l y heated b a t t e r y brooders with wire screen For the f i r s t  floors.  4 days the c h i c k s were a l l f e d Basal D i e t No. 1  c o n t a i n i n g no supplementary v i t a m i n A.  At 4 days o f age the  c h i c k s were wing banded, d i s t r i b u t e d randomly i n t o 4 s e c t i o n s of the brooder, and f e d t h e i r experimental  diets.  The 2 top  s e c t i o n s of the brooder were used f o r c h i c k s on D i e t No. 2, low v i t a m i n A; and the 2 lower s e c t i o n s were used f o r c h i c k s on the normal v i t a m i n A d i e t , No. 3.  T h i s arrangement prevented the  c h i c k s on the low v i t a m i n A d i e t a c c i d e n t l y g e t t i n g a h i g h e r i n t a k e of v i t a m i n A from s p i l l e d  feed.  During the f i r s t  i n the brooder any weak or deformed c h i c k s were k i l l e d .  2 weeks Both  feed and water were a v a i l a b l e a t a l l times. The for  c h i c k s were weighed a t 18 days of age, and the average  each group was: D i e t No. 2:  171 grams  D i e t No. 3:  170 grams  * V i t a m i n A p a l m i t a t e - Rovemix A-325. Roche L t d .  Hoffman-La  At t h i s  time the growing c h i c k s were r e d i s t r i b u t e d i n t o 6  s e c t i o n s of the brooder t o g i v e 15-16 i n s t e a d of 25.  c h i c k s t o each  section  A l l the b i r d s on the low v i t a m i n A d i e t were  a l i g n e d on one s i d e of the b a t t e r y brooder, and the b i r d s on the normal v i t a m i n A on the o t h e r .  This arrangement e l i m i n a t e d any  chance of feed contamination from one group t o the o t h e r , yet  s t a n d a r d i z e d brooder  and  positions.  At 4 weeks of age the c h i c k s were again weighed and had the f o l l o w i n g averages per group: D i e t No. 2:  294 grams  D i e t No. 3:  296 grams  The b i r d s were then t r a n s f e r r e d to the experimental room and p l a c e d i n non-heated wire  '.'  batteries.  I Brooder Quarantine Methods During the time the c h i c k s were i n the brooder room,  s t r i c t q u a r a n t i n e methods were p r a c t i s e d t o ensure t h a t remained c o c c i d i a - f r e e . 1.  These i n c l u d e d :  Before the c h i c k s were i n t r o d u c e d , the brooder and troughs were c l e a n e d ,  sterilized,  and r e - p a i n t e d w i t h  aluminum p a i n t t o e l i m i n a t e any p r e v i o u s 2.  they  The f l o o r of the room, t a b l e s , with undiluted bleach  contamination.  and s i n k were scrubbed  (sodium h y p o c h l o r i t e )  before  c h i c k i n t r o d u c t i o n , and twice a week t h e r e a f t e r  to  27  d e s t r o y any oocysts which might have been c a r r i e d i n . 3. A l l c l e a n i n g equipment such as brooms, s c r a p e r s , sponges, p l u s c o n t a i n e r s used i n the brooder room were bought new and never removed from the room. 4.  Every e f f o r t was made t o keep the room f l y - f r e e . i n s e c t i c i d e b a i t was present t o k i l l did  any f l i e s  An  that  enter.  5. Only one p e r s o n , the a u t h o r , tended the c h i c k s d u r i n g the course of the e n t i r e  experiment.  To ensure t h a t these p r e c a u t i o n s were s a t i s f a c t o r y ,  fecal  samples were taken p e r i o d i c a l l y and examined for the presence of oocysts.  I t would appear from the n e g a t i v e  procedures were  C.  f i n d i n g s t h a t these  successful.  Experimental Design When the b i r d s were t r a n s f e r r e d t o the experimental room  at 4 weeks of age, light, grams.  average,  they were d i v i d e d i n t o 3 weight  or heavy, based on the average weight of  295  Each of the 2 d i e t a r y groups was s u b d i v i d e d i n t o 4,  each subgroup c o n t a i n i n g 10 b i r d s : heavy.  categories:  3 light,  These groups were designated as Low v i t a m i n A D i e t No. 2:  4 average,  follows:  Groups A , B, C , D.  Normal v i t a m i n A D i e t No. 3:  Groups E , F , G, H .  and 3  28  E x t r a b i r d s were h e l d i n case they were needed f o r replacement, and were maintained on t h e i r s t a r t i n g d i e t s . The b i r d s were caged i n 3 b a t t e r i e s , sections.  each c o n t a i n i n g 9  The b i r d s were kept 3 and 4 t o a s e c t i o n f o r most of  the experimental work.  .As the experiments p r o g r e s s e d , and the  number of b i r d s d i m i n i s h e d , i t was p o s s i b l e t o allow the o l d e r b i r d s more space,  p l a c i n g 2 to a section.  the b i r d s i n the b a t t e r i e s  is  The arrangement of  shown i n F i g u r e  1.  To ensure t h a t the c o n t r o l groups were kept completely f r e e from i n f e c t i o n u n t i l c h a l l e n g e ,  the f o l l o w i n g p r e c a u t i o n s  were undertaken: 1. The n o n - i n f e c t e d c o n t r o l groups C , D, and G were kept i n a separate b a t t e r y  (No. 3)  the f i r s t experiment.  d u r i n g the course of  C o n t r o l group H was p l a c e d i n  the top row of b a t t e r y No. 2, and was separated from the i n f e c t e d group by a b u f f e r l a y e r of e x t r a b i r d s . M o d i f i c a t i o n s of t h i s p l a n were made f o r the  second  experiment. 2.  The bags of feed f o r each d i e t were d i v i d e d i n t o 2 lots.  One was used f o r the i n f e c t e d group and the  other for the c o n t r o l s . 3. The c o n t r o l b i r d s were always f e d , watered, and cleaned first.  A l l utensils  used f o r the c o n t r o l groups were  Group A  Group H  Group B  Extra Birds  Gr oup C  Group F  B a t t e r y No'.  1  B a t t e r y No.  2  Group C  Group D  Group G  B a t t e r y No.  Figure  1  3  Arrangement o f c h i c k s E x p e r i m e n t No. 1.  i n batteries for  separated from those used f o r the i n f e c t e d groups. separate  A  coat was worn f o r t e n d i n g each group.  4. No p e r s o n n e l ,  other than the a u t h o r , were p e r m i t t e d  i n the experimental room d u r i n g the e n t i r e course of the  experiments.  P e r i o d i c checks  of the feces from the n o n - i n f e c t e d  r e v e a l e d no evidence  D.  of  groups  oocysts.  Methods  1. Oocyst determinations  and techniques  The c u l t u r e of E . a c e r v u l i n a used i n these experiments  was  r e c e i v e d from the Norwich Pharmacal Co. i n August, 1968 and was termed American Cyanamid s t r a i n . use t h i s  c u l t u r e only to i n f e c t  but numerous t r i a l s  b i r d s f o r oocyst,  to  production,  on young b i r d s d i d not produce enough oocysts  for a l a r g e s c a l e i n f e c t i o n , was used f o r Experiment 1. this  O r i g i n a l l y i t was intended  and so most of the o r i g i n a l c u l t u r e The oocysts passed by the b i r d s i n  experiment were c o l l e c t e d  and used f o r Experiment 2, and  the oocysts from Experiment 2 were h a r v e s t e d and used f o r Experiment  3. a) C a l i b r a t i o n of oocyst numbers f o r (1)  inoculation  T o t a l number of oocysts r e q u i r e d f o r the iment i s  determined.  exper-  11 (2)  The number of oocysts present i n 1 m l . of stock c u l t u r e i s  the  c a l c u l a t e d by d i l u t i n g t h i s  amount i n 500 m l . water,  f o r example;  counting  the number i n 1 c u . mm. by use of a hemocytometer; m u l t i p l y i n g t h i s g i v e the number of oocyst plying this  f i g u r e by 1000  to  i n 1 m l . ; and m u l t i -  amount by 500 t o g i v e the number i n  1 m l . of o r i g i n a l c u l t u r e . (3)  The amount of c u l t u r e r e q u i r e d f o r the  experiment  i s then removed, c e n t r i f u g e d and washed  several  times,  saline  and resuspended i n p h y s i o l o g i c a l  (0.9%) t o give a f i n a l d i l u t i o n of about 1 m i l l i o n oocyst/ml.  The washing i s necessary  to  remove the potassium dichromate i n which the oocysts are suspended. b) C o l l e c t i o n and s p o r u l a t i o n of As p r e v i o u s l y s t a t e d , iments iments.  oocysts  oocysts were h a r v e s t e d from Exper-  1 and 2 t o be used f o r inoculum i n the succeeding  exper-  The procedures f o r c o l l e c t i o n and s p o r u l a t i o n were sub-  j e c t e d to much experimentation to determine the best method f o r the p r e p a r a t i o n of the l a r g e numbers of oocysts r e q u i r e d f o r these experiments.  The u s u a l methods recommended i n l i t e r a t u r e  u t i l i z e d some type of f l o t a t i o n f o r the c o l l e c t i o n of oocysts from the f e c e s .  However, i t was found t h a t none of the methods  32  t r i e d y i e l d e d a h i g h enough percentage mass,  of oocysts from the  and so the f o l l o w i n g m o d i f i c a t i o n was d e v i s e d t o  the maximum number of (1)  fecal  recover  oocysts.  The i n f e c t e d  feces were c o l l e c t e d  and mixed  w i t h enough 2% potassium dichrornate, i n a shallow pan, t o g i v e a s l u s h y m i x t u r e . (2)  T h i s mixture was covered w i t h a damp c l o t h and left  at room temperature  (26°c)  f o r 48 hours  t o allow s p o r u l a t i o n of the o o c y s t s .  There was  a great  deal of d i f f i c u l t y  stage.  The degree of s p o r u l a t i o n was  low,  encountered at  a v e r a g i n g about 20% i n the e a r l y  I n c u b a t i o n at h i g h e r temperatures effective,  nor d i d constant  i n oxygen,  or other m o d i f i c a t i o n s .  this  extremely efforts.  d i d not prove  agitation,  bubbling  Storage of  the feces i n the r e f r i g e r a t o r was found t o be the p r i n c i p a l f a c t o r  l i m i t i n g s p o r u l a t i o n , and  i t was found t h a t by p r o c e s s i n g feces immediately  after  collection  a much h i g h e r r a t e  s p o r u l a t i o n c o u l d be a c q u i r e d .  However,  h i g h e s t s p o r u l a t i o n e f f e c t e d never beyond 50-60%.  of  the  extended  33!  (3) There was no evidence t h a t a d d i t i o n a l s p o r u l a t i o n occurred after  48 h o u r s , so at  this  time the mixture was passed i n sequence through 2 sieves,  30 and 100 meshes per l i n e a r i n c h  respectively,  u s i n g a j e t of tap water to  force  i t through. (4)  The c o l l e c t e d washings were put i n t o 1000 m l . graduates,  and water added, i f r e q u i r e d ,  completely  fill  the c o n t a i n e r .  to  The suspension  was allowed to stand o v e r n i g h t ,  and then  the  supernatent was decanted and the sediment mixed w i t h 1000 m l . tap water and again l e f t t o out.  T h i s o p e r a t i o n was repeated 2 or 3 times  t o remove as much extraneous from the (5)  settle  d e b r i s as  possible  oocysts.  F o l l o w i n g the l a s t d e c a n t i n g ,  the sediment  was  mixed w i t h 2% potassium dichromate and s t o r e d '  i n the r e f r i g e r a t o r u n t i l r e q u i r e d .  T h i s method gave the maximum number of oocysts but d i d r e s u l t a c u l t u r e h e a v i l y contaminated w i t h f e c a l m a t e r i a l . ill  However, no  e f f e c t s were observed which c o u l d be a t t r i b u t e d to t h i s  tamination,  and as the c u l t u r e was used s h o r t l y a f t e r  in  con-  collection,  the oocysts d i d not degenerate from the b a c t e r i a l c o n t a m i n a t i o n .  c)  C a l c u l a t i o n of the d a i l y oocyst p r o d u c t i o n per b i r d  The number of oocysts passed d a i l y by an i n f e c t e d b i r d one c r i t e r i o n of the b i r d ' s response experiments, was c o l l e c t e d  the t o t a l  to the d i s e a s e .  is  In these  f e c a l output from each i n o c u l a t e d group  d a i l y , s t a r t i n g on the 4th day a f t e r  and c o n t i n u i n g f o r 7 d a y s .  inoculation  A f t e r t h i s time the c o l l e c t i o n s  were  made every second day f o r the next 4 to 8 days depending on conditions.  The t o t a l number of oocysts per b i r d per day was  c a l c u l a t e d by the f o l l o w i n g methods: (1)  The t o t a l d a i l y feces from each group was put i n t o a c o n t a i n e r and weighed. were not processed immediately,  I f the  samples  they were mixed  w i t h enough 2% potassium dichromate to keep them moist and then p l a c e d i n the r e f r i g e r a t o r until (2)  processed.  The t o t a l sample was then thoroughly mixed i n a b a s i n and a 10 gram sample removed.  If  the  feces had been mixed p r e v i o u s l y w i t h potassium dichromate a sample c a l c u l a t e d t o be  equivalent  t o 10 grams of untreated feces was used. (3)  T h i s 10 gram a l i q u o t was then mixed w i t h 500 cc of tap water i n a Waring blender and a g i t a t e d at f u l l  speed f o r lh minutes.  35  (4)  Immediately f o l l o w i n g homogenization, 1 m l . samples  6  separate  of the suspension were removed  and p l a c e d i n a t e s t tube.  T h i s sample was  mixed 10 times w i t h a Pasteur p i p e t t e  and one  drop was then removed and p l a c e d i n one chamber of the hemocytometer. repeated t o f i l l (5)  This procedure was  the second chamber.  The t o t a l number of oocysts present  i n the 5  l a r g e squares of each chamber were counted.  If  the 2 chambers showed more than a 10% d i s crepancy i n the c o u n t s ,  a f u r t h e r 2 samples  were counted and an average (6)  taken.  The t o t a l number of oocysts per sample was c u l a t e d as Average  follows: hemocytometer count  = Y (number i n 1 c u . mm)  Number i n 1 m l .  = 1000Y  Number i n 500 m l .  = 500 X 1000Y  The number of oocysts i n 500 m l . i s number i n 10 grams of f e c e s ,  and from t h i s  equivalent to f i g u r e the  number of oocysts per group c o u l d be determined.  the  total  T h i s amount  was d i v i d e d by the number of b i r d s i n the group t o g i v e the oocysts/bird/day.  cal-  Using t h i s method of c a l i b r a t i o n , each oocyst counted i n the hemocytometer was e q u i v a l e n t t o 500,000 o o c y s t s , per gram of f e c e s .  or 50,000  T h e r e f o r e , i f the numbers were below  this  f i g u r e per gram, the sample would appear t o be n e g a t i v e . to the l a r g e amount of extraneous matter present samples,  i n the  Due fecal  a s m a l l e r d i l u t i o n f a c t o r was not p o s s i b l e when u s i n g  the hemocytometer.  T h e r e f o r e , t o a v o i d the f a l s e  assumption  t h a t a sample h a v i n g below 50,000 oocysts per gram was negative,  any sample showing one or no oocysts was  t o the McMaster Counting Technique as (1)  subjected  follows:  The t o t a l d a i l y f e c a l mass per group was w e l l mixed and a 10 gram a l i q u o t removed and mixed i n a beaker w i t h 95 m l . of a s a t u r a t e d s o l u t i o n of sodium chloride.  (2)  Using a Pasteur p i p e t t e ,  the 0.15 m l . chamber of a  McMaster s l i d e was f i l l e d w i t h the coarse  suspension.  The oocysts f l o a t e d f r e e of the d e b r i s and pressed against  the bottom of the scored cover s l i p where  they c o u l d be counted. (3)  The average of 2 counts was taken t o g i v e the number of oocysts i n 0.15 m l .  The t o t a l number of  oocysts i n 10 grams was c a l c u l a t e d as  follows:  T o t a l oocysts i n 0.15 m l .  =  Y  T o t a l i n 95cc  =  Y X 100 X 7  (10 grams)  total  Using t h i s technique, oocysts,  each oocyst counted was e q u i v a l e n t t o  or 70 per gram of  700  feces.  d) C a l c u l a t i o n of the r e p r o d u c t i v e index The r e p r o d u c t i v e index or r e p r o d u c t i v e p o t e n t i a l i s measure of immunity or r e s i s t a n c e coccidial infection.  As such i t  a  o f f e r e d by the b i r d a g a i n s t a is  a u s e f u l t o o l i n the  assess-  ment of the h o s t ' s r e a c t i o n and was c a l c u l a t e d f o r each b i r d at the c o n c l u s i o n of each experiment. R I  =  I t i s c a l c u l a t e d as  follows:  T o t a l number of oocysts passed T o t a l number of oocysts i n o c u l a t e d  2. H i s t o l o g i c a l  techniques  a) F i x a t i o n Immediately a f t e r  a b i r d was k i l l e d ,  removed and p l a c e d i n Bouin's f i x a t i v e . solution  the duodenal loop was  The formula f o r  this  is: Saturated p i c r i c a c i d  75cc  Formalin  25cc  Acetic acid  5cc  b) Dehydration and embedding (1)  A 3-5 mm s e c t i o n of the duodenum, d i s t a l t o  the  t u r n of the l o o p , was removed and p l a c e d i n a mesh p l a s t i c capsule cessing.  (Tissue Tek) f o r p r o -  38  (2) Dehydration s t a r t e d w i t h 3 changes  of 70% e t h y l  a l c o h o l , w i t h i n a 48 hour p e r i o d , t o remove as much p i c r i c a c i d as p o s s i b l e from the t i s s u e . T h i s was  f o l l o w e d by 2 hours or more i n 95%  alcohol;  2 changes  i n 99% a l c o h o l of one hour  each; and c l e a r i n g i n 2 changes hour  of x y l o l ,  one  each.  (3) The dehydrated t i s s u e was changes  of wax  then t r a n s f e r r e d t o 3  i n a 60° oven, and embedded i n  "Tissue Tek" embedding r i n g s . c) S e c t i o n i n g The s e c t i o n s were cut at 5 microns u s i n g a L e i t z m i c r o meter.  The r i b b o n s were f l o a t e d on water, and the s e c t i o n s  a f f i x e d , 3-4 per s l i d e , u s i n g egg albumen or g e l a t i n . d) S t a i n i n g A great d e a l of e x p e r i m e n t a t i o n was  undertaken t o f i n d a  d i f f e r e n t i a l s t a i n which would c l e a r l y show the s p o r o z o i t e s i n the t i s s u e .  Pattillo  purpose; C l a r k s o n  (1959) recommended PAS  (1958) and S h o r t t and Cooper  m o d i f i c a t i o n ; Horton-Smith and co-workers McFarlane's  Feulgen f o r t h i s (1948), a Giemsa  (1963) recommended  (1944) P i c r o - M a l l o r y s t a i n ; and C h a l l e y and  Burns  (1959) used Groat's hematoxylin f o l l o w e d by Shorr's s t a i n . these recommendations were t r i e d i n a d d i t i o n t o Groat's  All  39  Tetrachrome S t a i n .  None of these methods proved very  f a c t o r y and e v e n t u a l l y  2 methods were used e x c l u s i v e l y .  were a standard hematoxylin and e o s i n technique G r o a t ' s Tetrachrome S t a i n . (1)  satisThese  and a m o d i f i e d  The standard method followed  was:  The s e c t i o n s were dewaxed through 2 changes of x y l o l and hydrated through 99%, 95% and 70% a l c o h o l s .  (2)  Stained i n H a r r i s '  Hematoxylin f o r 5-10 minutes,  then  d e c o l o u r i z e d i n a c i d a l c o h o l and blued i n ammonia water. (3)  Counterstained i n either E),  aqueous e o s i n  (standard H and  or i n B i e b r i c h S c a r l e t - O r a n g e I I s t a i n  (Groat's  Tetrachrome). (4)  Washed, dehydrated, and c l e a r e d i n x y l o l .  (5)  The .sections were mounted w i t h "Pro-Tex" mounting medium and covered w i t h 22mm by 50mm c o v e r s l i p .  With both these s t a i n s the r e f r a c t i l e vacuole i n sporozoite ever,  was s t a i n e d r e d , making i t  easy t o i d e n t i f y .  although the G r o a t ' s m o d i f i c a t i o n gave a b e t t e r  entiation  of blood c e l l s ,  i t tended t o o v e r s t a i n ,  and  the How-  differtherefore  the standard H and E was the recommended method.  E.  Experiments The 8 groups of b i r d s were used i n 3 d i f f e r e n t  iments.  The f i r s t  exper-  experiment u t i l i z e d 5-week-old b i r d s ,  the  40  second 9-week-old b i r d s , and  the t h i r d 19-week-old b i r d s .  1. Experiment Number 1 a)  Object a) To assess the response of 5-week-old c h i c k s , 2 l e v e l s of v i t a m i n w i t h E. b)  The  A, t o a primary i n f e c t i o n  acervulina.  To immunize h a l f the c h i c k s group a g a i n s t  b)  E.  acervulina.  Design  v i o u s l y discussed  experimental-design.  s i s t i n g of 10 b i r d s , were i n f e c t e d . Low  vitamin  A diet:  to the  pre-  Four groups, each con-  The  groups were as  follows:  Groups A and  B infected  Groups C and  D controls  Normal V i t a m i n A d i e t :  The  of each d i e t a r y  groups of c h i c k s were arranged a c c o r d i n g  c)  on  Groups E and  F infected  Groups G and  H controls.  Inoculation  c u l t u r e used f o r t h i s f i r s t experiment was  o r i g i n a l c u l t u r e of s p o r u l a t e d  E. a c e r v u l i n a oocysts  from Norwich L a b o r a t o r i e s .  inoculum was  An  t a i n 3 m i l l i o n oocysts per ml. b i r d from groups A,  B, E, and  the received  c a l i b r a t e d t o con-  of p h y s i o l o g i c a l s a l i n e , and F was  inoculated  o r a l l y with  each  41  1.0 m l . - 3 m i l l i o n o o c y s t s . gram of body weight.  This approximated 8000 oocysts per  The b i r d s were s t a r v e d 24 hours before  inoculation. d) Methods (1)  S t r i c t h y g i e n i c and q u a r a n t i n e techniques were employed t o ensure t h a t the c o n t r o l groups remained f r e e of i n f e c t i o n .  These were d i s -  cussed under Experimental Methods and Design Section C. (2)  The cages of the i n f e c t e d b i r d s were cleaned every second day, from day 4 t o the end of the experiment, t o ensure t h e r e was no r e i n f e c t i o n of the  (3)  birds.  F e c a l samples were c o l l e c t e d d a i l y and the number of oocysts per b i r d per day was  cal-  culated. (4)  The i n f e c t e d b i r d s were weighed at the time of i n o c u l a t i o n , on day 4, day f o l l o w i n g t h i s  and on every second  f o r about 20 days.  (5)  The n o n - i n f e c t e d b i r d s were weighed weekly.  (6)  In t h i s  experiment, and i n the 2 t h a t  followed,  the weights between groups were sub-  j e c t e d t o an a n a l y s i s of v a r i a n c e t o determine  42  i f the d i f f e r e n c e s were  statistically  significant. 2• Experiment a)  Number 2  Object  To compare, i n 9-week-old b i r d s r a i s e d on 2 l e v e l s of v i t a m i n A: (1) The degree of a c q u i r e d immunity r e t a i n e d  4  weeks a f t e r a primary i n f e c t i o n w i t h E. acervulina. (2) The e a r l y t i s s u e stages of E. a c e r v u l i n a i n immune and non-immune c h i c k s . (3) The response  bf s u s c e p t i b l e 9-week-old c h i c k s  t o a primary i n f e c t i o n with E. b)  acervulina.  Design  Before t h i s experiment  was  conducted,  the groups of b i r d s  were re-arranged i n numbers, keeping the weights possible.  The groups were adapted  as uniform  f o r t h i s experiment  as  as  follows: Low  vitamin A diet: Group A - 10 b i r d s - To be given a second i n f e c t i o n . Group B -  8 b i r d s - Non-infected immune c o n t r o l s .  Group C - 10 b i r d s - To be given a primary i n f e c t i o n . Group D -  9 b i r d s - Non-infected non-immune c o n t r o l s .  43  Normal v i t a m i n A d i e t : Group E - 11 b i r d s - To be g i v e n a second i n f e c t i o n . Group F -  9 b i r d s - N o n - i n f e c t e d immune c o n t r o l s .  Group G - 11 b i r d s - To be given a primary i n f e c t i o n . Groupi.H ;  9 b i r d s - N o n - i n f e c t e d non-immune c o n t r o l s .  The i n f e c t i o n of groups A and E would determine the degree of immunization r e t a i n e d 4 weeks a f t e r a primary i n f e c t i o n ; the  and  i n f e c t i o n o f groups C and G would examine the response of  these 9-week-old b i r d s t o a primary i n f e c t i o n . c) I n o c u l a t i o n The c u l t u r e c o n s i s t e d of s p o r u l a t e d oocysts of E_. a c e r v u l i n a h a r v e s t e d 4 weeks e a r l i e r from Experiment No. 1. inoculum was  c a l i b r a t e d t o g i v e 5 m i l l i o n oocysts per ml. of  s a l i n e and the b i r d s were i n f e c t e d per os w i t h 2 ml., g i v i n g m i l l i o n oocysts per b i r d . per  The  10  T h i s averaged out t o 11,000 oocysts  gram of body weight. d) Methods (1) Three b i r d s from each group were k i l l e d  3 hours  a f t e r i n o c u l a t i o n , and another 3 b i r d s were killed  at 48 hours.  The duodenum was  removed  from each b i r d and f i x e d i n Bouin's s o l u t i o n . (2) Pooled f e c a l samples from each group were taken d a i l y and processed t o determine the t o t a l number of oocysts passed per b i r d per day.  44  (3) Standard h y g i e n i c  p r a c t i s e s were observed t o  prevent i n f e c t i o n of the c o n t r o l groups. (4) The groups B, D, F, and H, were h e l d f o r 10 weeks f o l l o w i n g Experiment No.  2, and d u r i n g  this  p e r i o d they were weighed once a week. (5) The remaining b i r d s i n Groups A, E, C, and G were e l i m i n a t e d Experiment No.  3 weeks a f t e r i n o c u l a t i o n when 2 was  terminated.  3. Experiment Number 3 a) Object To compare the response of 19-week-old b i r d s , r a i s e d on 2 l e v e l s of d i e t a r y v i t a m i n  A, t o an i n f e c t i o n with E. a c e r v u l i n a  i n order t o determine: (1) The degree of immunity r e t a i n e d by b i r d s 14 weeks a f t e r a primary i n f e c t i o n . (2) The response of o l d e r b i r d s t o a heavy primary infection. b) Design A l l the remaining groups were i n f e c t e d i n t h i s 3rd experiment.  They were: Low v i t a m i n  A diet:  Group B - 7 b i r d s - primary i n f e c t i o n 14 weeks earlier.  45  Group D - 8 b i r d s - No previous  infection.  Normal v i t a m i n A d i e t : Group F - 8 b i r d s - primary  infection  14 weeks  earlier. Group H - 8 b i r d s - No p r e v i o u s  infection.  c) I n o c u l a t i o n The oocysts used f o r t h i s experiment were h a r v e s t e d from Experiment No. 2.  They were s t o r e d i n the u s u a l manner and  re-suspended i n p h y s i o l o g i c a l s a l i n e t o g i v e 2.5 m i l l i o n ml.  oocysts  A l l the b i r d s were i n o c u l a t e d o r a l l y w i t h 25 m i l l i o n  oocysts, a dose of 10 ml. per b i r d .  This was roughly e q u i v a l e n t  to 14,000 oocysts per gram of body weight. d) Methods (1) As t h e r e were no n o n - i n f e c t e d c o n t r o l s i n t h i s experiment, the h y g i e n i c techniques were not as strict  as i n the p r e v i o u s experiments.  ever, p r e c a u t i o n s were maintained extraneous infection  contamination  t o omit  and t o prevent r e -  from the contaminated  feces.  (2) Two b i r d s from each group were k i l l e d 4th day a f t e r i n o c u l a t i o n . a u t o p s i e d and c l i n i c a l was noted."  How-,  on the  These b i r d s were  evidence  of the d i s e a s e  The duodenal loop, and any other  46  areas of the i n t e s t i n e showing gross  lesions,  were removed and f i x e d i n B o u i n ' s s o l u t i o n sectioning  and h i s t o l o g i c a l  D a i l y pooled fecal, samples  for  examination. from each group were  taken and the number of oocysts determined per bird. A r e c o r d of weights was maintained f o l l o w i n g r o u t i n e of the previous two  experiments.  the  47  IV.  A.  RESULTS  Experiment No. 1 This experiment examined the response of 5-week-old  c h i c k s , on low and normal l e v e l s o f v i t a m i n A, t o a primaryi n f e c t i o n w i t h E. a c e r v u l i n a . C l i n i c a l l y , the b i r d s on low v i t a m i n A r a t i o n s  (AB) were  more a f f e c t e d by the d i s e a s e than the b i r d s on normal v i t a m i n A rations  (EF).  Both groups of b i r d s began t o show symptoms of the  d i s e a s e at day 3, when a s l i g h t drop i n feed consumption observed.  A watery d i a r r h e a was  was  apparent by day 4, w i t h scanty  mucoid droppings c h a r a c t e r i s t i c a l l y s t a i n e d green.  A n o r e x i a and  a d i p s i a were pronounced i n both groups on day 4 and 5, and the a n o r e x i a continued i n the low v i t a m i n A group u n t i l about day 14.  The normal v i t a m i n A group  day 7-8.  (EF) r e g a i n e d t h e i r a p p e t i t e s by  The droppings of the low v i t a m i n A group  (AB) were  s c a n t i e r and more watery than group EF throughout the acute stage of the d i s e a s e  (days 4-6), and d i d not r e t u r n t o normal  c o n s i s t e n c y u n t i l day 12, compared t o day 7 f o r group EF.  There  was no m o r t a l i t y i n the normal v i t a m i n A group, but 2 b i r d s d i e d i n group AB.  The f i r s t  d i e d on day 10 and showed a 27 percent  l o s s i n body weight at t h a t time, and the second d i e d on day 12 a f t e r l o s i n g 22 percent body weight.  Neither b i r d revealed  48  severe i n t e s t i n a l pathology, but both were v e r y dehydrated and atonic. The low v i t a m i n A group passed a t o t a l average of 615 m i l l i o n oocysts per b i r d between days 4 and 14, g i v i n g a r e p r o d u c t i v e index of 205.  The b i r d s on normal d i e t a r y  levels  of v i t a m i n A had a t o t a l average p r o d u c t i o n of 360 m i l l i o n oocysts and a R.I. of 120 f o r the same p e r i o d .  As shown i n  Table 1 both groups had a peak oocyst; p r o d u c t i o n on day 5, but group AB maintained a h i g h e r d a i l y l e v e l throughout the course of the experiment.  P e r i o d i c f e c a l examinations f o r the presence  of oocysts were made d u r i n g the 3rd week, and i t was found t h a t group EF was e s s e n t i a l l y n e g a t i v e by day 18, but the b i r d s of group AB were s t i l l p a s s i n g s e v e r a l m i l l i o n o o c y s t s / b i r d / d a y . However, a l l the b i r d s were n e g a t i v e by the time the 2nd experiment was i n s t i g a t e d a t day 28. Table I I shows the t o t a l weights of the 4 experimental groups d u r i n g the course of Experiment No. 1.  The groups a l l had  s i m i l a r weights a t the s t a r t of the experiment but by day 4, the two i n f e c t e d groups of b i r d s were s i g n i f i c a n t l y lower i n weight  (P <£ .01) than the c o n t r o l s .  group of b i r d s  The i n f e c t e d low v i t a m i n A  (AB) were an average of 28 grams l i g h t e r i n  weight than group EF on day 6 and t h i s d i f f e r e n c e i n c r e a s e d t o 80 grams by day 14.  There was no s i g n i f i c a n t d i f f e r e n c e i n  49  weight between t h e 2 c o n t r o l groups throughout the experiment. The low v i t a m i n A c o n t r o l group was about 4 percent l e s s i n average t o t a l weight than the other 3 groups a t the s t a r t of t h e experiment, and maintained t h i s d i f f e r e n c e w i t h t h e other c o n t r o l group u n t i l t h e end.  However t h e r e was a marked d i f f e r e n c e  between t h e 2 i n f e c t e d groups.  There was a s i g n i f i c a n t  difference  i n mean weight between day 8 and 18 (P <f .01) which although d i m i n i s h i n g , was maintained u n t i l day 21 (P  <^".05) .  By day 14  of t h e experiment the low v i t a m i n A group had l o s t 25 percent o f i t s body weight compared t o i t s c o n t r o l , w h i l e the normal v i t a m i n A group had l o s t only 16 percent as i l l u s t r a t e d i n F i g u r e 2. I t took s e v e r a l weeks b e f o r e t h e weights o f the i n f e c t e d groups were s t a t i s t i c a l l y equal t o those of the c o n t r o l as shown i n Table IV.  groups  F i g u r e 3 g r a p h i c a l l y compares t h e weights  of i n f e c t e d groups B and F, and t h e i r n o n - i n f e c t e d c o n t r o l s , D and H r e s p e c t i v e l y , from November 1st t o February 23rd.  During  t h i s 4 month p e r i o d t h e n o n - i n f e c t e d c o n t r o l s d i d not show a s i g n i f i c a n t d i f f e r e n c e i n weight.  Groups B and F were i n f e c t e d  at 5 weeks, and a t 6 weeks showed a s i g n i f i c a n t erence i n weight from t h e i r c o n t r o l groups.  (P <^.01) d i f f -  This d i f f e r e n c e was  maintained i n t h e normal v i t a m i n A groups, F and H, u n t i l 4 weeks  50  a f t e r the date of i n o c u l a t i o n .  The low v i t a m i n A groups B and  D showed a s i g n i f i c a n t d i f f e r e n c e i n the t o t a l weights of the b i r d s u n t i l 6 weeks a f t e r i n o c u l a t i o n . s i g n i f i c a n t d i f f e r e n c e i n weight  However, t h e r e was a  CP <Cbetween  groups B  and H u n t i l 9 weeks a f t e r i n o c u l a t i o n a t which time, the b i r d s were 14 weeks o l d . The weight changes which occurred d u r i n g the 2 weeks f o l l o w i n g i n f e c t i o n are shown i n Table I I I . T h i s t a b l e g i v e s the weight gained  or l o s t i n 2 day i n t e r v a l s from day 4 t o 14  of i n f e c t i o n , p l u s the i n f e c t e d groups weight gains compared t o the c o n t r o l s .  During the acute stage of i n f e c t i o n , between days  4 and 6, the low v i t a m i n A group l o s t an average of 25 grams per b i r d , w h i l e the normal v i t a m i n A group l o s t only 4 grams. Between days 6 and 8 group AB s t i l l  showed a s l i g h t l o s s i n  weight w h i l e group EF had s t a r t e d t o g a i n and by day 14 both groups were showing s i m i l a r weight i n c r e a s e s .  However, the low  v i t a m i n A group showed an average g a i n of only 64 grams per b i r d d u r i n g the 14 days, compared t o 145 grams f o r the normal v i t a m i n A group. probability level.  This was s i g n i f i c a n t l y d i f f e r e n t a t the .01 The c o n t r o l s showed no s i g n i f i c a n t d i f f e r e n c e  i n the t o t a l average weight gained  over t h i s same p e r i o d .  Figure  4 i l l u s t r a t e s t h i s d i f f e r e n c e i n weight g a i n s , and gives a graphic p i c t u r e of the d e l e t e r i o u s e f f e c t s of a low v i t a m i n A  51  diet.  The c h i c k s on the low v i t a m i n A r a t i o n had a weight g a i n  63 percent lower than t h a t of t h e i r c o n t r o l group, w h i l e  the  i n f e c t e d b i r d s on normal v i t a m i n A r a t i o n s were only 30 percent lower i n weight gains  from t h e i r c o n t r o l .  A comparison of the d i f f e r e n t w i t h E . a c e r v u l i n a i n 5-week-old A rations,  reactions  t o an i n f e c t i o n  b i r d s on normal and low v i t a m i n  shows t h a t the s e v e r i t y  of the d i s e a s e i s  doubled i n the b i r d s on low d i e t a r y v i t a m i n A . index has been used by Stock? Stevenson; Stevenson  (1969), t o evaluate  in different  groups of b i r d s .  The performance  and Hymas (1967), and  the t o t a l e f f e c t s It  almost  of  coccidiosis  encompasses the weight g a i n ,  m o r t a l i t y and oocyst p r o d u c t i o n , and although an a r t i f i c i a l method of comparison, i t effect.  does g i v e an i n d i c a t i o n of  I t was m o d i f i e d i n t h i s  investigation  .standard of comparison between experiments, devised was:  average g a i n + percentage  production i n m i l l i o n s . 10  Using t h i s  total  t o g i v e some  and the formula  survival -  oocyst  formula the P . I .  v i t a m i n A group (AB) was 64 + 90 - 61 = 93,  of the  and t h a t of  low  the  normal v i t a m i n A group (EF) was 145 + 100 - 36 = 209.  Thus i t  can be seen t h a t by t a k i n g m o r t a l i t y i n t o account p l u s  the  severity  of the d i s e a s e i n the s u r v i v o r s , the low v i t a m i n A  group was over twice as s e v e r e l y E.  affected  by the i n f e c t i o n with  a c e r v u l i n a as was the normal v i t a m i n A group.  52  TABLE I  AVERAGE DAILY OOCYST PRODUCTION IN MILLIONS IN 5-WEEK-OLD CHICKS, ON 2 DIETARY LEVELS OF VITAMIN A , FOLLOWING INFECTION WITH 3 MILLION OOCYSTS OF E . ACERVULINA PER BIRD  Day  Low v i t a m i n A d i e t (AB)  Normal v i t a m i n A d i e t (EF)  4  55  20  5  125  125  6  95  60  7  85  50  8  75  30  9  70  30  10  55  (19)  25  12  35  (18)  15  14  20  (18)  5  T o t a l oocysts per b i r d  615  360  Reproductive Index  205  120  * S u r v i v i n g b i r d s out of  20.  53  TABLE I I  MEAN WEIGHT IN GRAMS OF 5-WEEK-OLD CHICKS, ON 2 DIETARY LEVELS OF VITAMIN A, AFTER INFECTION WITH 3 MILLION OOCYSTS OF E. ACERVULINA COMPARED TO NON-INFECTED CONTROLS  Day  Infected birds Low v i t a m i n Normal v i t a m i n A d i e t (AB) A d i e t (EF)  0  396  397  2  434  436  4  409  416  6  383  412  8  378  ** 433  10  397 (19)a ** 455  12  425 (18)  14  460 (18) **  16  497  18  538 (18) ** 621  20  581 (18)  Non-infected birds Low A Normal A diet diet 380  398  465  486  601  632  737  786  ** 499 542  (18) ** 570  *  662  a. b i r d s s u r v i v i n g out o f 20. * s i g n i f i c a n t d i f f e r e n c e i n weight  (P <T.05) between AB and EF.  ** h i g h l y s i g n i f i c a n t d i f f e r e n c e i n weight groups AB and EF.  (P <^.01) between  54  TABLE I I I  DAILY MEAN WEIGHT CHANGES IN GRAMS OF 5-WEEK-OLD CHICKS, ON 2 DIETARY LEVELS OF VITAMIN A, FOLLOWING INFECTION WITH 3, MILLION OOCYSTS OF E. ACERVULINA  Between Days  Low v i t a m i n A ration I n f e c t e d (AB) C o n t r o l (CD)  Normal v i t a m i n A ration Infected Control (EF) (GH)  0-4  +15  4-6  -25  - 4  6-8  - 3  + 21  8-10  +19  +22  10-12  +27  +45  12-14  +36  +43  T o t a l weight g a i n i n 2 weeks* Grams per day of gain  +64c  4.5  +85  +19  +88  +221a  +145b  +234a  +16  10.5  +17  * means showing the same l e t t e r d i d not d i f f e r (P <T.01) .  significantly  55.  TABLE IV  TOTAL MEAN WEIGHT IN GRAMS, OVER A 4 MONTH PERIOD, OF 4 GROUPS OF CHICKENS ON 2 DIETARY LEVELS OF VITAMIN A, AND WITH ONE GROUP ON EACH LEVEL INFECTED AT 5 WEEKS OF AGE WITH 3 MILLION OOCYSTS OF E. ACERVULINA  Age weeks  Low v i t a m i n A Infected Control B D  Normal v i t a m i n A Infected control F H  180  178  175  171  5 - infected  377  369  387  392  6  377  470  420  500  8  564  741  674  802  10  820  997  984  1076  12  1066  1221  1188  1293  14  1348  1431  1472  1567  16  1520  1579  1660  1690  18  1695  1695  1818  1848  * no s i g n i f i c a n t d i f f e r e n c e  (P <^.05) between groups B, D, F, H.  56  3)AV 0 F i g u r e 2.  DAY 14-  The e f f e c t of d i e t a r y v i t a m i n A l e v e l on the mean weight of 5-week-old c h i c k s f o l l o w i n g i n f e c t i o n wi th E. a c e r v u l i n a  57  F i g u r e 3.  A comparison of the mean weights of i n f e c t e d u and n o n - i n f e c t e d c h i c k s , on 2 d i e t a r y l e v e l s of v i t a m i n A, over a 4 month p e r i o d  58  Low  VITAMIN A  NoftrtflL  VITAMIN A  r  ISO Z  W  loo -•  5  So ••  CONTROL  F i g u r e 4.  IN^CTEJ)  CONTROL  Tupecreo)  The e f f e c t of d i e t a r y v i t a m i n A l e v e l on the 2 week weight g a i n of 5-week-old c h i c k s f o l l o w i n g i n f e c t i o n with E. acervulina  59  B.  Experiment No. 2 The primary purpose of t h i s experiment was t o study  the  e a r l y endogenous forms of E . a c e r v u l i n a i n immune and nonimmune 9-week-old  chicks receiving 2 d i f f e r e n t  v i t a m i n A i n the r a t i o n . for h i s t o l o g i c a l  S i x b i r d s from each group were k i l l e d  examination of the i n t e s t i n e s ,  4-5 b i r d s per group, and t h i s  1.  appearing l e s s  than they a c t u a l l y were.  Clinical  Clinically, response  l e a v i n g only  s m a l l experimental number may  have r e s u l t e d i n some of the c l i n i c a l r e s u l t s significant  l e v e l s of  Results the immune groups A" and E , showed  t o t h e i r second i n f e c t i o n .  noted on days 4-6  A slight  little  a n o r e x i a was  i n both groups, and although the droppings  of group E were s l i g h t l y watery on day 5, there was no other evidence  of abnormal f e c e s .  The non-immune groups, C and G,  r e c e i v i n g t h e i r primary i n f e c t i o n with E . a c e r v u l i n a , manif e s t e d the t y p i c a l symptoms of the d i s e a s e on days 4-8,  but  these symptoms were not as severe as i n the younger b i r d s of Experiment 1.  There was no m o r t a l i t y i n e i t h e r  of these groups,  but both groups had a marked a n o r e x i a and watery, droppings on days 4-6. especially  scant  D i a r r h e a was most severe on day 5,  i n the low v i t a m i n A group ( C ) .  The b i r d s on normal  l e v e l s of v i t a m i n A (G) r e g a i n e d t h e i r a p p e t i t e s more q u i c k l y  60  than those i n group C, and showed l e s s evidence a f t e r day  of d i a r r h e a  5.  A comparison of the oocyst numbers passed i n the of the d i s e a s e i s shown i n Table V. immunity from a primary E.  There was  course  The p r o t e c t i v e e f f e c t s of  i n f e c t i o n can be seen i n groups A  no evidence  of the d i s e a s e on day  showed a low peak f o r both groups.  The  and  4, and day  5  low v i t a m i n A group  (A) appeared t o have a more s o l i d immunity as the b i r d s passed an average of only 6 m i l l i o n oocysts per b i r d i n the 14 days f o l l o w i n g i n o c u l a t i o n , compared to 70 m i l l i o n f o r group E. The more r a p i d drop i n d a i l y oocyst p r o d u c t i o n i n group A, compared t o group E, i s a l s o i n d i c a t i v e of a b e t t e r immune response.  Groups C and G, r e c e i v i n g t h e i r primary  i n f e c t i o n at  9 weeks, produced high numbers of o o c y s t s , but u n l i k e the week-old b i r d s of Experiment 1, the peak p r o d u c t i o n was 4 as w e l l as 5.  The  d a i l y course of oocyst  d u p l i c a t e d the f i n d i n g s of Experiment 1. group maintained  day  production  The  low v i t a m i n A  4 and dropped a b r u p t l y t o lower and more  r a p i d l y decreasing l e v e l s .  The r e l a t i v e t o t a l s of  groups on a primary  r e s u l t s of Experiment 1. had  on  a h i g h d a i l y l e v e l , w h i l e the normal v i t a m i n A  group peaked on day  between the two  4-  The  oocysts  i n f e c t i o n a l s o f o l l o w e d the  low d i e t a r y v i t a m i n A group b i r d s  twice the number of oocysts, and twice the r e p r o d u c t i v e  61  index, of the b i r d s from the normal v i t a m i n A group. Table VI compares the t o t a l average weight, over a p e r i o d of 2 weeks, of the b i r d s immunized  i n Experiment 1 when h a l f the  group i s r e - i n f e c t e d w i t h a l a r g e number o f o o c y s t s .  I t can be  seen t h a t the second i n f e c t i o n d i d not a f f e c t the average weight i n e i t h e r the low or normal d i e t a r y v i t a m i n A groups.  In  f a c t , as shown by the t o t a l weight gains over t h i s same p e r i o d i n Table IX, the i n f e c t i o n even appeared t o a c c e l e r a t e weight gains i n the low v i t a m i n A group (A). The 2 groups of b i r d s , C and G, s u b j e c t e d t o a primary i n f e c t i o n of E. a c e r v u l i n a at 9 weeks, d i d not s u f f e r the weight l o s s e s of t h e i r 4-week-old  c o u n t e r p a r t s i n Experiment 1.  shown i n Table V I I , the low v i t a m i n A group  (C) l o s t an average  of 19 percent body weight, i n the 14 days f o l l o w i n g compared t o the n o n - i n f e c t e d c o n t r o l s . group  As  infection,  The normal v i t a m i n A  (G) showed only a 4 percent l o s s i n body weight.  Table  V I I I shows the weight gains and l o s s e s d u r i n g these 14 days. I t can be seen t h a t the maximum weight l o s s was  on day 6 i n  both groups, although the g a i n between day 0 and 4 was t h a t of the two c o n t r o l groups.  f a r below  The low v i t a m i n A group  (C)  showed a g r e a t e r weight l o s s d u r i n g the acute stages of the d i s e a s e and d i d not match the r e c o v e r y r a t e of group G.  Figure  5 i l l u s t r a t e s the d i f f e r e n c e i n weight g a i n between these 2 groups, and demonstrates that group C had a weight g a i n equal t o  62  only 26 percent o f t h a t made by i t s c o n t r o l group D, and only 30 percent o f the g a i n made by group G. group  The normal v i t a m i n A  (G) had a weight g a i n equal t o 80 percent of i t s c o n t r o l  group H. The degree o f p r o t e c t i o n a f f o r d e d by immunization w i t h a s i n g l e i n f e c t i o n o f E. a c e r v u l i n a i s shown i n Table IX.  Even  w i t h the s m a l l numbers of b i r d s used i n t h i s experiment i t can be seen t h a t w i t h low d i e t a r y l e v e l s of v i t a m i n A, t h e p r o t e c t i v e p r o p e r t i e s o f t h e immunization a r e w e l l d e f i n e d .  The  immune group A had a weight g a i n f o l l o w i n g i n f e c t i o n which p a r a l l e l e d t h a t o f t h e u n i n f e c t e d c o n t r o l group D.  But t h e non-  p r o t e c t e d group C had a weight g a i n 74 percent below t h a t of the c o n t r o l group.  With normal l e v e l s of v i t a m i n A i n t h e r a t i o n ,  the degree of p r o t e c t i o n obtained by immunization i s not so dramatic.  Group E had a weight g a i n equal t o only 88 percent  of t h e u n i n f e c t e d c o n t r o l group H, but e q u a l l e d t h a t o f i t s immune c o n t r o l F.  However group G, w i t h a primary i n f e c t i o n ,  had a weight g a i n equal t o 80 percent of t h e u n i n f e c t e d  control  group H,so t h e r e l a t i v e degree of p r o t e c t i o n o f f e r e d by immunization i s not as l a r g e as seen i n the low v i t a m i n A groups. The performance i n d i c e s f o r the 4 i n f e c t e d groups i n Experiment 2 a r e shown i n Table X, and these c o n f i r m t h e advantage o f immunization i n t h e low v i t a m i n A groups.  The  63  difference  i n the P . I - f o r the 2 groups r e c e i v i n g a primary  i n f e c t i o n w i t h E . a c e r v u l i n a (C and G ) , i s p a r t i c u l a r l y striking.  The normal v i t a m i n A (G) group has a P . I . more than  3 times t h a t of the low v i t a m i n A group (C).  2.  H i s t o l o g i c a l Examination .3 hours post i n o c u l a t i o n : A l l the s e c t i o n s r e v e a l e d e o s i n o p h i l i c  i n the e p i t h e l i u m of the c r y p t s and v i l l i construed to be s p o r o z o i t e s . the c r y p t s 3 hours a f t e r  bodies  e p i t h e l i u m which were  T h i s presence of s p o r o z o i t e s  in  i n o c u l a t i o n does not conform w i t h most  r e p o r t s which s t a t e t h a t s p o r o z o i t e s migrate to the c r y p t s .  refractive  take 12-18 hours  to  I t would suggest t h a t there i s  a direct  i n v a s i o n of the c r y p t s w i t h a heavy i n o c u l a t i o n of o o c y s t s . very obvious  f i n d i n g was an accumulation of h e t e r o p h i l s  lamina p r o p r i a .  A  i n the  There was a very heavy c o n c e n t r a t i o n of these  c e l l s i n groups A , E , . and G, but i n group C only one of the 3 b i r d s showed s e c t i o n s s i m i l a r t o the other groups.  The  s e c t i o n s from the other 2 b i r d s of group C r e v e a l e d only a few heterophils.  There d i d not appear t o be any d i f f e r e n c e  in  e p i t h e l i a l i n t e g r i t y between the groups, and t h e r e was no evidence  of squamous e p i t h e l i u m and k e r a t i n i z a t i o n i n the  on low v i t a m i n A l e v e l s .  groups  64  48 hours post i n o c u l a t i o n : In the 2 immune groups, A and E, examination o f the s e c t i o n s showed very few t r o p h o z o i t e s and almost no s c h i z o n t s . One  b i r d i n t h e low v i t a m i n A group (A) appeared t o have an area  of i n f e c t i o n r e t a i n e d from t h e immunizing dose given i t 4 weeks earlier. in  There was evidence  o f gametocytes and oocysts  present  t h e t i s s u e of one c r y p t and accompanying v i l l u s showing t h a t  t h i s b i r d was h a r b o u r i n g s p o r o z o i t e s present  a slight latent infection.  i n the c r y p t s and v i l l i  There were  o f almost a l l t h e  immune b i r d s but very few had developed i n t o t r o p h o z o i t e s . The  2 groups r e c e i v i n g t h e i r primary i n f e c t i o n , C and G,  showed l a r g e numbers o f t r o p h o z o i t e s and s c h i z o n t s .  The  s c h i z o n t s measured about 5 microns i n diameter and were f i r s t generation.  These were found l o c a l i z e d i n s p e c i f i c  encompassing a c r y p t and neighbouring were near t h e base of t h e v i l l i , the e p i t h e l i a o f the v i l l i  villi.  areas  Most of t h e forms  but i n many areas t h e whole o f  was p a r a s i t i z e d with  forms i n v a r y i n g degrees of development. sporozoites s t i l l  probably  endogenous  There were a l s o many  unchanged i n the t i s s u e , and i n f a c t one b i r d  from the normal v i t a m i n A d i e t - h a d only s p o r o z o i t e s present and showed no evidence d e f i n i t e evidence  of t r o p h o z o i t e s or s c h i z o n t s .  There was no  t h a t t h e low d i e t a r y v i t a m i n A groups had a  g r e a t e r i n c i d e n c e o f i n v a s i o n or s c h i z o n t development.  However,  65  t h e r e was an i n d i c a t i o n that  a more e x t e n s i v e area of the  i n t e s t i n e was invaded i n the suboptimal groups, and a l a r g e r sample number may r e v e a l  something more s i g n i f i c a n t .  66  TABLE V  AVERAGE DAILY OOCYST PRODUCTION IN MILLIONS IN 9-WEEK-OLD CHICKS ON 2 DIETARY LEVELS OF VITAMIN A, AND 2 LEVELS OF IMMUNITY, AFTER INFECTION WITH 10 MILLION OOCYSTS OF E. ACERVULINA PER BIRD  Day  Normal v i t a m i n A ration  Low v i t a m i n A ration Immune (A)  Non-immune (C)  Immune (E)  Nonimmune (G)  0  200  0  230  4.5  210  18  110  60  17  20  ,02  50  25  15  .01  130  8  15  .005  110  .6  20  10  65  .1  5  12  25  .006  14  10  .005  T o t a l oocysts per b i r d  6  Reproductive Index  0.6  860  86  70  10  426  43  67  TABLE VI  AVERAGE WEIGHT IN GRAMS OF IMMUNIZED 9-WEEK-OLD CHICKS, ON 2 DIETARY LEVELS OF VITAMIN A, FOLLOWING A SECOND INFECTION WITH E. ACERVULINA  Day  Low v i t a m i n A Infected Non-infected (B) (A)  Normal v i t a m i n A Non-infected Infected (F) (E)  *  0  782  735*  828  860  4  883  805*  918  945  6  938  835  951  985  8  968  880*  989  1030*  10  1013  1024  1065*  1046  1108  1096  1130  920  12  . 1032  961  14  1076  990  * extrapolated  figures  *  *  *  *  68  TABLE VII  AVERAGE WEIGHT IN GRAMS OF 9-WEEK-OLD CHICKS, ON DIETARY LEVELS OF VITAMIN A , FOLLOWING A PRIMARY INFECTION WITH E . ACERVULINA  Day  Low v i t a m i n A Infected Non-infected (D) (C) *  Normal v i t a m i n A Infected Non-infected (G) (H) *  0  924  885  924  940  4  952  955*  986  1025*  6  885  993  964  1056  8  874  1035*  1006  1105  10  909  1090*  1063  1165  12  928  1141  1105  1223  14  998  1170*  1170  1245*  Average P e r centage l o s s i n body weight compared t o c o n trols 19%  * extrapolated  weights  4%  * *  69  TABLE V I I I  DAILY MEAN WEIGHT CHANGES IN GRAMS OF 9-WEEK-OLD CHICKS, ON 2 DIETARY LEVELS OF VITAMIN A, FOLLOWING A PRIMARY INFECTION WITH 10 MILLION OOCYSTS OF E. ACERVULINA  Between Days  Low v i t a m i n A l e v e l s Infected non-infected (C) (D)  Normal v i t a m i n A l e v e l s Infected non-infected (G) (H)  0-4  +28  +70  +62  +85  4-6  -67  +38  -22  +31  6-8  -11  +42  +42  +49  8-10  +35  +55  +57  +60  10-12  +21  +51  +42  +58  12-14  +70  +29  +65  +29  * e x t r a p o l a t e d weights  70  TABLE IX  TWO WEEK TOTAL WEIGHT GAIN IN GRAMS IN IMMUNE AND NON-IMMUNE 9-WEEK-OLD CHICKS, ON 2 DIETARY LEVELS OF VITAMIN A, FOLLOWING INFECTION WITH 10 MILLION OOCYSTS OF E. ACERVULINA  Low v i t a m i n A l e v e l s : Group A:  Grams  Immune, i n f e c t e d :  294  B:  Immune, n o n - i n f e c t e d  255  C:  Non-immune, i n f e c t e d  74  D:  Non-immune, n o n - i n f e c t e d  285  Normal v i t a m i n A l e v e l s : Group E:  Immune, i n f e c t e d  268  F:  Immune, n o n - i n f e c t e d  270  G:  Non-immune, i n f e c t e d  246  H:  Non-immune, n o n - i n f e c t e d  305  71  TABLE X  PERFORMANCE INDEX OF 9-WEEK-OLD CHICKS ON, 2 DIETARY LEVELS OF VITAMIN A AND 2 LEVELS OF IMMUNITY, FOLLOWING INFECTION WITH 10 MILLION OOCYSTS OF E. ACERVULINA PER BIRD  Group  Performance  A - Low v i t a m i n A r a t i o n  - secondary infection  C - Low v i t a m i n A r a t i o n  393  - primary i n f e c t i o n  88  E - Normal v i t a m i n A r a t i o n  - secondary infection G - Normal v i t a m i n A r a t i o n - primary infection  * 2 week average weight g a i n + percentage s u r v i v a l oocyst  production 10  361 303  -  Index  72  Low  VITAMIN  A  NORMAL VITAMIN A  ISO-z  er  2 H  15*0--  loo-X Ui  3  5o-.  CONTROL  F i g u r e 5.  TnfGCT£S  The e f f e c t of d i e t a r y v i t a m i n A l e v e l on the 2 week weight g a i n of 9-week-old c h i c k s f o l l o w i n g i n f e c t i o n with E . acervulina  73  C.  Experiment No. 3 In t h i s experiment the b i r d s were subjected  t o a massive  i n f e c t i o n of 25 m i l l i o n oocysts E. a c e r v u l i n a t o assess the response of immune and non-immune o l d e r b i r d s r a i s e d on 2 d i e t a r y l e v e l s of v i t a m i n  A.  The term "immune" i s used t o denote  a primary i n f e c t i o n a t an e a r l i e r date, and does not i n d i c a t e any  p a r t i c u l a r degree of immunity.  1. Primary The  Infection  2 groups r e c e i v i n g a primary i n f e c t i o n with J).  a c e r v u l i n a were D, low v i t a m i n diet.  A d i e t , and H, normal v i t a m i n  Both groups of b i r d s showed a severe r e a c t i o n t o the  infection.  A l l b i r d s had d i a r r h e a  by day 3, and the feces were  almost completely l i q u i d with evidence of sloughed and  A  b i l e present.  epithelium  By day 4 the b i r d s of group D showed complete  a n o r e x i a and a d i p s i a but the b i r d s of group H were not so severely affected.  In both groups the b i r d s were very depressed w i t h  congested or c y a n o t i c  combs and w a t t l e s ,  and p a l e ,  dry shanks.  By day 6 the b i r d s on normal v i t a m i n  A l e v e l s began t o recover;  the a n o r e x i a l e s s e n e d and the f e c e s ,  although scant and mucoid,  were l e s s l i q u i d . and  By day 8 the b i r d s of t h i s group looked normal,  by day 9 t h e i r a p p e t i t e s  feces were back t o normal.  had completely r e t u r n e d and the The b i r d s on the low d i e t a r y  vitamin  A l e v e l s , group D, had a much s t r o n g e r r e a c t i o n t o the i n f e c t i o n .  74  The d e p r e s s i o n and d i a r r h e a were more marked, and 4 out of the 6 b i r d s d i e d from the d i s e a s e . and t h i s b i r d had  percent body weight;  weight.  f i r s t death was  on day  9,  l o s t 25 percent of i t s body weight compared t o  i t s i n o c u l a t i o n weight.  body weight;  The  B i r d 2 d i e d on day 11 h a v i n g : l o s t 35  b i r d 3 on day 13 w i t h a 40 percent l o s s i n  and b i r d 4 on day 17 w i t h a 48 percent l o s s i n  Post mortem examination  of the dead b i r d s r e v e a l e d  extreme emaciation and d e h y d r a t i o n i n a l l of them, p l u s a n e p h r i t i s c h a r a c t e r i s e d by p a l e , m o t t l e d kidneys c o n t a i n i n g heavy urate deposits.  A l l b i r d s showed some evidence of i n t e s t i n a l  damage, mostly i n v o l v i n g the duodenum.  B i r d 1 had the most  severe pathology with areas of hemorrhage extending t o the duodenal-jejunum j u n c t i o n .  The  other 3 b i r d s d i d not show such  severe damage but a l l r e v e a l e d evidence of e p i t h e l i a l s l o u g h i n g and the w a l l s of the i n t e s t i n e were t h i n n e r than u s u a l . b i r d s of group D were moribund d u r i n g most of the p e r i o d , and no evidence of normal feces was the s u r v i v i n g b i r d s .  A n o r e x i a was  complete,  The  experimental  seen u n t i l day 12 i n i n a l l birds that  d i e d , from day 3 u n t i l t h e i r time of death. Table XI shows the oocyst p r o d u c t i o n of the 4 groups i n v o l v e d i n t h i s 3rd experiment. lower than c o u l d be expected survival.  The group D t o t a l i s probably  i f t h e r e had been a h i g h e r r a t e of  T h i s low v i t a m i n A group had i t s peak p r o d u c t i o n on  75  day 5, w h i l e the normal v i t a m i n A group, H, had a peak oocyst p r o d u c t i o n on day 4 f o l l o w e d by an abrupt drop on day 5.  It is  i n t e r e s t i n g t o note that i n the primary i n f e c t i o n of a l l normal d i e t a r y v i t a m i n A groups examined,  the peak p r o d u c t i o n i s  f o l l o w e d the next day by a drop of almost e x a c t l y 50 p e r c e n t . Table XII gives the t o t a l average weights of a l l the groups throughout the experiment.  I t can be seen t h a t both the  low v i t a m i n A groups, D and B, were below the weights of groups F and H at the s t a r t o f the experiment, but the d i f f e r e n c e s were not s t a t i s t i c a l l y s i g n i f i c a n t due t o the h i g h standard d e v i a t i o n present i n a l l groups. was  s i g n i f i c a n t l y lower  However, by day 8, group D  (P «=c.01) i n weight than group H, and  by day 14 the s u r v i v o r s of group D r e t a i n e d t h i s d i f f e r e n c e at the 5 percent p r o b a b i l i t y l e v e l .  By day 14 the s u r v i v i n g  b i r d s of group D had l o s t about 20 percent of t h e i r  starting  body weights w h i l e the b i r d s on h i g h e r l e v e l s of v i t a m i n A had l o s t only 2^ percent of t h e i r weight.  The weight  (G)  changes  which took p l a c e d u r i n g the course of the experiment are examined  i n Table X I I I .  Both groups r e c e i v i n g a primary i n f e c -  t i o n showed a c o n t i n u a l weight l o s s up t o day 8.  Coinciding  with t h e i r r e g a i n e d a p p e t i t e s , the b i r d s of group H showed a dramatic r e c o v e r y of t h e i r l o s t weight between days 8 and when they gained an average of 118 grams i n the 2 days.  10, The  76  s u r v i v o r s of the dessimated D group a c t u a l l y began t o show a weight g a i n between days 10-12, but the c o n t i n u i n g l o s s e s of the moribund b i r d s maintained  the group l o s s e s shown on Table X I I I .  2. Secondary I n f e c t i o n The  low d i e t a r y v i t a m i n A group (B) and  the normal v i t a m i n  A group (F) were both given a primary i n f e c t i o n at 5 weeks o l d i n Experiment 1. This secondary i n f e c t i o n was  given 14 weeks  l a t e r t o t e s t the degree of immunity r e t a i n e d i n both groups. U n l i k e the 2 groups r e c e i v i n g t h e i r primary these two  immunized groups d i d not e x h i b i t a marked  response t o the i n f e c t i o n . on days 4.-6, group  infection,  and  There was  some evidence  clinical  a s l i g h t scouring  of a n o r e x i a .  The  present  low v i t a m i n A  (B) showed l e s s a p p e t i t e d u r i n g t h i s acute p e r i o d than  the normal v i t a m i n A group ( F ) , and the feces were more scanty. A l s o group B b i r d s d i d not r e t u r n t o t h e i r p r e - i n f e c t i o n feed consumption by the end  of the experimental  group B began t o show a t a x i a and was  killed  at day  16.  No  of c o c c i d i o s i s was  the i n t e s t i n a l t r a c t , but m i c r o s c o p i c  The  oocyst p r o d u c t i o n  One  l e g p a r a l y s i s at day  evidence  overwhelming mold p o p u l a t i o n  period.  bird in  14,  and  found i n  examination r e v e a l e d  present. f o r these  2 "immune" groups i s  shown i n Table XI, and u n l i k e the f i n d i n g s i n Experiment 2, low v i t a m i n A b i r d s had  an  almost twice the oocyst p r o d u c t i o n  the of  77  the normal group. primary i n f e c t i o n .  This conformed more t o the f i n d i n g s i n a However, i t can be seen t h a t some immunity  was  r e t a i n e d i n both groups as the numbers of oocysts passed  was  f a r below t h a t of groups D and H. An examination of the t o t a l weights throughout the exper-  iment, as shown i n Table X I I , demonstrates t h a t group F was the only group t o show an i n c r e a s e i n weight d u r i n g the 14 days a f t e r i n o c u l a t i o n .  Group B was  first  47 grams l i g h t e r than  i t s i n o c u l a t i o n weight a t day 14, a 2 percent l o s s .  However, by  day 21 the 2 groups had the same r e l a t i v e weights as they had at day 0.  When the weight changes are a n a l y s e d i n Table X I I I , i t  can be seen t h a t - both the immune groups l o s t weight d u r i n g the first  6 days of the i n f e c t i o n , and group B l o s t more than twice  t h a t of group F.  The reason f o r the weight l o s s i n group B  between day 10-14  i s p a r t i a l l y e x p l a i n e d by the f a i l i n g weight  of the b i r d which was I t was  subsequently k i l l e d .  demonstrated i n Experiment 2 t h a t a primary  i n f e c t i o n w i t h E. a c e r v u l i n a gave marked p r o t e c t i o n a g a i n s t a secondary i n f e c t i o n 4 weeks l a t e r . it  From t h i s p r e s e n t experiment  can be seen t h a t t h i s p r o t e c t i v e immunity  i s retained, i n a  lowered degree, 14 weeks a f t e r the primary i n f e c t i o n .  Unlike  the r e s u l t s of Experiment 2, the low d i e t a r y v i t a m i n A group d i d not show a h i g h e r degree of immunity  than the b i r d s on a  78  normal v i t a m i n A d i e t .  The average weight l o s s e s , and the oocyst  counts, were both h i g h e r i n group B thdn i n Group F.  However,  t h i s experiment confirmed the f i n d i n g s of Experiment 2 t h a t the degree o f p r o t e c t i o n a f f o r d e d by t h e immunity obvious i n t h e low v i t a m i n A group. A group  i s much more  Although the normal v i t a m i n  (H) e x h i b i t e d a severe weight l o s s i n t h e acute stages  of the d i s e a s e , t h i s l o s s was r e g a i n e d so r a p i d l y t h a t i n 14 days a f t e r i n o c u l a t i o n t h e group H weight was equal t o t h e immunized low v i t a m i n A group  (B), and not s i g n i f i c a n t l y lower than t h e  immune normal v i t a m i n A group  (F).  On the other hand, a com-  p a r i s o n of groups B and D d u r i n g t h i s p e r i o d shows a s i g n i f i c a n t d i f f e r e n c e i n weights group D.  (P <  .01) p l u s a h i g h m o r t a l i t y r a t e i n  As i l l u s t r a t e d i n F i g u r e 6, the normal v i t a m i n A  groups show only a 2 percent d i f f e r e n c e i n weight between t h e immune and non-immune groups, but t h i s d i f f e r e n c e r i s e s t o 18 percent i n the low v i t a m i n A group.  79  TABLE XI  AVERAGE DAILY OOCYST PRODUCTION IN MILLIONS IN 19-WEEK-OLD CHICKENS ON 2 DIETARY LEVELS OF VITAMIN A, AND 2 LEVELS OF IMMUNITY, AFTER INFECTION WITH 25 MILLION OOCYSTS OF E. ACERVULINA PER BIRD  Day  Low v i t a m i n A r a t i o n Immune (B) Non-immune  (D)  Normal v i t a m i n A r a t i o n Immune (F) Non-immune (H)  4  3  40  8  135  5  40  220  30  60  6  40  60  10  30  7  25  50  20  30  8  6  55  9  20  25 ( 5 ) *  6  20  10  10  20 (5)  7  10  ,6  40  12  0.1  2 (4)  3  4  14  0.03  2 (4)  0.08  1  T o t a l oocysts per b i r d 145 Reproductive Index  6  474  90  330  19  4  13  * number of s u r v i v i n g b i r d s  out of 6.  80  TABLE X I I  AVERAGE WEIGHT IN GRAMS OF 19-WEEK-OLD CHICKENS, ON 2 DIETARY LEVELS OF VITAMIN A, AND 2 LEVELS OF IMMUNITY, FOLLOWING INFECTION WITH 25 MILLION OOCYSTS OF E. ACERVULINA  Day-  Low v i t a m i n A Immune (B) Non-immune (D)  Normal v i t a m i n A Immune (F) Non-immune(H)  0  1785  1763  1848  1911  4  1737  1668  1823  1826  6  1697  1541  1808  1728  8  1726  1394  1866  1707  10  1754  1294(5)  1902  1825  12  1752  1280(4)  1912  1848  14  1738  1417(3)  1911  1866  21  1889  1620(2)  1945  1922  * number of s u r v i v i n g b i r d s out of 6.  81  TABLE X I I I  DAILY MEAN WEIGHT CHANGES IN GRAMS IN IMMUNE, AND NONIMMUNE 19-WEEK-OLD CHICKENS ON 2 DIETARY LEVELS OF VITAMIN A, FOLLOWING INFECTION WITH 25 MILLION OOCYSTS OF E. ACERVULINA  Between days  Low v i t a m i n A Immune(B) Non-immune(D)  Normal v i t a m i n A Immune (E) Non-immune(H)  0-4  -48  -95  -25  -85  4-6  -40  -127  -15  -98  6-8  +29  -147  +58  -21  9-10  +28  -100(5)*  +36  +118  10-12  - 2  -2(4)  +10  +24  12-14  -14  +137(3)  -1  +17  * number o f s u r v i v i n g b i r d s out o f 6.  82  IMMUNE  1800 -•  1500  --  z H  S00 -• o 60O-.  3oo -•  LOW  F i g u r e 6.  VtTRMtM  A  NoRMfU.  VlWIINl  The e f f e c t of d i e t a r y v i t a m i n A l e v e l on the mean weight of 19week-old immune and non-immune b i r d s 14 days a f t e r i n f e c t i o n with E . a c e r v u l i n a  A  83  3. Post Mortem and H i s t o l o g i c a l Examinations Two b i r d s inoculation,  from each group were k i l l e d  and the i n t e s t i n e s  on day 4  following  checked f o r p a t h o l o g i c a l  lesions.  S e c t i o n s from each of the b i r d s were examined t o determine the extent of the p a r a s i t i c i n v a s i o n Post mortem  and development.  findings:  Group B - Low v i t a m i n A d i e t - immune One of the b i r d s  showed a 3.5 percent weight l o s s a t 4  days and examination of the i n t e s t i n e showed some s l o u g h i n g o f the  e p i t h e l i u m i n the duodenum, and areas of e n t e r i t i s a t the  b e g i n n i n g of the jejunum.  The second b i r d showed no c l i n i c a l or  post mortem evidence of i n f e c t i o n , and had not l o s t weight. Group D - Low v i t a m i n A d i e t - primary i n f e c t i o n Both b i r d s k i l l e d  showed c l i n i c a l  signs of the d i s e a s e  and both had a 7 percent weight l o s s a t day 4.  B i r d No. 1  demonstrated only some s l o u g h i n g of the i n t e s t i n a l e p i t h e l i u m on post mortem examination, but b i r d No. 2, which had a more severe d i a r r h e a , showed pronounced l e s i o n s The e n t i r e i n t e s t i n e a n t e r i o r  i n the i n t e s t i n e .  t o the y o l k s t a l k r e v e a l e d a  spotty e n t e r i t i s . Group F - Normal v i t a m i n A - immune B i r d No. 1 showed no c l i n i c a l coccidiosis.  or post mortem signs o f  The second b i r d had a weight l o s s o f l e s s than  84  1 percent but e x h i b i t e d severe d i a r r h e a .  However, t h e r e was no  evidence o f e n t e r i t i s on post mortem examination,  although the  duodenum and jejunum both showed severe s l o u g h i n g of the epithelium. Group H - Normal v i t a m i n A - primary  infection  Both the b i r d s had severe d i a r r h e a and d e p r e s s i o n , and showed a 5 percent l o s s i n weight examination  since inoculation.  Post mortem  showed t h a t b i r d No. 1 had severe s l o u g h i n g of the  e n t i r e a n t e r i o r p o r t i o n of the i n t e s t i n a l mucosa, and the jejunum p o s t e r i o r t o the duodenum was inflammed.  The second b i r d showed  l e s s pathology, but severe e p i t h e l i a l s l o u g h i n g was e v i d e n t . Histological Almost  examinations:  a l l the s e c t i o n s r e v e a l e d evidence of severe  s l o u g h i n g of the v i l l i  e p i t h e l i u m so t h a t i n many cases the  lamina p r o p r i a was completely unprotected. great d e a l of edema evident i n the v i l l i  There was a l s o a  of a l l groups.  The d i f f e r e n c e between s l i g h t :and severe pathology i n the i n t e s t i n e appeared  t o be the degree  of involvement.  In  the severe cases, a f a r g r e a t e r number of c e l l s were invaded w i t h few areas i n the s e c t i o n s being f r e e of the p a r a s i t e s . The c r y p t s and neighbouring v i l l i  were packed with t r o p h o z o i t e s  and s c h i z o n t s of d i f f e r e n t -sizes and stages of m a t u r i t y .  The  gametocytes and oocysts present were not c o n f i n e d t o the t i p s  85  of the v i l l i  as r e p o r t e d from v a r i o u s s o u r c e s ,  a l s o i n the c r y p t s .  but were found  In the i n t e s t i n e s showing only  sloughing  of the e p i t h e l i u m , t h e r e were f a r fewer areas i n v a d e d , and the affected  regions  d i d not show the same number of c e l l s  Both the low v i t a m i n A groups r e v e a l e d a more e x t e n s i v e ment than the groups on normal v i t a m i n A d i e t s .  affected. involve-  The non-immune  group D showed the most e x t e n s i v e i n v a s i o n of the  intestinal  c e l l s and a l s o p r o p o r t i o n a t e l y more oocysts i n i t s  sections  than any other group.  86  V.  DISCUSSION  The r e s u l t s of t h i s i n v e s t i g a t i o n demonstrate t h a t a d i e t c o n t a i n i n g only 10 percent of the recommended l e v e l of v i t a m i n A does not n e c e s s a r i l y a f f e c t the weight gains or c l i n i c a l h e a l t h of the b i r d s r e c e i v i n g i t .  However, i f these  same b i r d s are s u b j e c t e d t o c o c c i d i o s i s , the e f f e c t s of t h i s suboptimal v i t a m i n A become immediately experiments,  apparent.  i n f e c t i o n w i t h E. a c e r v u l i n a was  In these  more severe i n a l l  groups on the low v i t a m i n A d i e t , as determined  by a comparison  of the oocyst p r o d u c t i o n , weight g a i n s , and m o r t a l i t y between b i r d s on low d i e t a r y l e v e l s of v i t a m i n A and normal l e v e l s .  A.  Oocyst  Production  B r a c k e t t and B l i z n i c k number of oocysts produced  (1952b) have r e p o r t e d t h a t the  by a c o c c i d i a l i n f e c t i o n are  a f f e c t e d by at l e a s t 6 f a c t o r s . 1) The i n h e r e n t p o t e n t i a l of the p a r a s i t e i n a nonimmune h o s t . 2) The  immunity or r e s i s t a n c e developed  3) The  "crowding  by the h o s t .  effect".  4) Competition with other c o c c i d i a l s p e c i e s or i n f e c t i v e agents. 5) N u t r i t i o n of the h o s t .  87  6) S t r a i n d i f f e r e n c e s of the h o s t . The r e p r o d u c t i v e index, or p o t e n t i a l , of a coccidium i s the maximum number of oocysts produced  per oocyst i n o c u l a t e d .  I t depends on the number of schizogonous numbers of merozoites  produced  generations and  In each g e n e r a t i o n .  a c e r v u l i n a , the t h e o r e t i c a l p o t e n t i a l i s  the  With E_.  8 x 20 x 16 x 10 x 36  = 455,000 per oocyst i n j e c t e d , i f the endogenous stages r e p o r t e d by V e t t e r l i n g and'Doran (1966) are c o r r e c t .  Hein  (1968a)  demonstrated t h a t the r e p r o d u c t i v e p o t e n t i a l dropped w i t h i n c r e a s i n g inoculum,  which confirms f i n d i n g s by other  and the r e s u l t s from these experiments.  authors  Warren and B a l l  r e p o r t e d the h i g h e s t R.I.  (100,000) of any i n v e s t i g a t i o n .  r e s u l t e d from an inoculum  of 500  Table XIV g i v e s a l i s t  (1967) This  oocysts i n 3-week-old c h i c k s .  of r e p r o d u c t i v e indexes obtained by  v a r i o u s authors and demonstrates the decrease i n R.I. w i t h i n c r e a s i n g inoculum.  The r e s u l t s of t h i s i n v e s t i g a t i o n are  also included. As f i r s t demonstrated by B r a c k e t t and B l i z n i c k  (1952b)  E. a c e r v u l i n a i s c h a r a c t e r i z e d by an extremely h i g h oocyst production.  Although  i t s r e p r o d u c t i v e p o t e n t i a l i s lower  than  t h a t of E. b r u n e t t i and E. t e n e l l a , i t s oocyst p o t e n t i a l i s f a r higher than any other a v i a n s p e c i e s of c o c c i d i a . (1932), and B r a c k e t t and B l i z n i c k  Tyzzer e t al_  (1952b), have demonstrated  TABLE XIV THE REPRODUCTIVE INDEX OF E. ACERVULINA FOLLOWING INCREASINGLY LARGER INFECTIVE DOSES AS REPORTED BY VARIOUS AUTHORS  I n o c u l a t i o n - number of oocysts per b i r d  R.I  Age o f b i r d s  50  72,000  unknown  500  100,000  3 weeks  900 1250 320,000 500,000  38,000 43,000 1,800 1,026  1 million 3 m i l l i o n -• normal v i t a m i n A group 3 m i l l i o n -• low v i t a m i n A group 5 million 10 m i l l i o n - • normal v i t a m i n A group 10 m i l l i o n - • low v i t a m i n A group 20 m i l l i o n 25 m i l l i o n - • normal v i t a m i n A group 25 m i l l i o n - • low v i t a m i n A group  Author  . 302  6 weeks  B r a c k e t t and B l i z n i c k (1952b) Warren and B a l l (1967) Long (1967) Hein (1968a) Hein (1968a) Rose and Long (1962) Hein (1968a)  120  5 weeks  Coles  205 44  5 weeks 6 weeks  Coles  43  9 weeks  86 8  9 weeks 6 weeks  13  19 weeks  Hein  19  19 weeks  Coles  7 6 6 3  weeks weeks weeks weeks  Hein  (1968a)  Coles Coles  Coles  (1968a) oo oo  89  t h a t the h i g h e s t y i e l d s of oocysts are obtained w i t h  light  i n f e c t i o n s i n the case of E. t e n e l l a , E. b r u n e t t i , E. maxima and E. n e c a t r i x , and w i t h h i g h inoculums the oocyst p r o d u c t i o n decreased.  However, these authors  found t h a t E. a c e r v u l i n a d i d  not show t h i s c o r r e l a t i o n , and i n s t e a d r e v e a l e d an almost l i m i t e d oocyst p r o d u c t i o n c a p a c i t y . f i n d i n g i s r e l a t e d t o a lower  un-  I t would appear t h a t t h i s  immune response  to this species,  and i f t h i s i s the case the higher oocyst p r o d u c t i o n shown by the groups on a r a t i o n low i n v i t a m i n A may s u p p r e s s i o n of the immunological  be due t o a  mechanisms i n the  deficient  host. I t has been r e p o r t e d by s e v e r a l authors t h a t age i s a f a c t o r i n oocyst p r o d u c t i o n ; the o l d e r the b i r d s , the g r e a t e r the p o t e n t i a l p r o d u c t i o n .  Hein  (1968a) demonstrated t h a t an  i n f e c t i v e dose of 5 m i l l i o n oocysts of E. a c e r v u l i n a i n 2week-old c h i c k s gave a t o t a l p r o d u c t i o n of 37 m i l l i o n ,  while  the same inoculum  225  million.  Krassner  i n 6-week-old c h i c k s gave a t o t a l of (1963) r e p o r t e d t h a t the mean oocyst  p r o d u c t i o n i n c r e a s e s p r o p o r t i o n a t e l y w i t h host age,  and  a t t r i b u t e d t h i s f i n d i n g t o the f a c t t h a t more i n t e s t i n a l are a v a i l a b l e f o r p a r a s i t e p e n e t r a t i o n , i n o l d e r b i r d s .  cells In t h i s  present i n v e s t i g a t i o n the maximum oocyst p r o d u c t i o n occurred i n 9-week-old b i r d s f o l l o w i n g an i n f e c t i v e dose of 10 m i l l i o n  90  oocysts.  The low v i t a m i n A group had a t o t a l oocyst p r o d u c t i o n  of 860 m i l l i o n , twice the p r o d u c t i o n of the b i r d s on normal l e v e l s of v i t a m i n A . conditions  T h i s d o u b l i n g of oocyst p r o d u c t i o n under  of low d i e t a r y v i t a m i n A was seen a l s o i n the 5-week-  o l d b i r d s , but i n the 19-week-old  b i r d s the m o r t a l i t y was  h i g h t h a t an a c c u r a t e assessment was d i f f i c u l t t o make.  Figure  7 compares the oocyst p r o d u c t i o n i n the 3 experiments. et a l  Ackert  (1931) suggested t h a t the g r e a t e r p a r a s i t i s m w i t h nematodes  i n b i r d s on low v i t a m i n A d i e t s was due t o reduced If this this  so  is correct,  peristalsis.  i n c r e a s e d oocyst p r o d u c t i o n may r e s u l t from  same phenomena, as a slower p e r i s t a l s i s  g r e a t e r number of s p o r o z o i t e s  would allow a  more time t o invade the  cells.  The n u t r i t i o n a l requirements of the coccidium can be i n v o l v e d i n t o t a l oocyst p r o d u c t i o n . investigated  Warren  (1968)  has  the v i t a m i n requirements of E . a c e r v u l i n a and has  found t h a t t h i a m i n e , r i b o f l a v i n , b i o t i n and n i c o t i n i c a c i d are essential ever,  f o r optimum oocyst development  he r e p o r t s t h a t a d i e t d e f i c i e n t  and p r o d u c t i o n .  How-  i n vitamin A resulted  i n an oocyst p r o d u c t i o n twice t h a t of the normal c o n t r o l s . Britton,  Hill  and Barber  (1964) have demonstrated t h a t  low  p r o t e i n l e v e l s i n the d i e t cause reduced oocyst p r o d u c t i o n because a low t r y p s i n s e c r e t i o n r e s u l t s excystation.  i n a decreased  91  LOW ISO I  VlTf»MlN A  NOKMAL Viraciw A  o IOO +  > o O  50 4  EXPERIMENT No I.  F i g u r e 7.  a)ftY V  r  6  r  a  'lO  f  ll  ^  A comparison of the d a i l y oocyst p r o d u c t i o n i n c h i c k s on Experiments 1, 2, and 3  92  Long (1968a) r e p o r t e d t h a t the breed  and  s t r a i n o f the  b i r d plays a s i g n i f i c a n t part i n i t s s u s c e p t i b i l i t y to a c o c c i d i a l i n f e c t i o n as demonstrated by the oocyst  counts.  He  found t h a t White Leghorns are more s u s c e p t i b l e than Rhode I s l a n d Reds.  B.  Weight Gains In the 3 experiments comprising  main f i n d i n g s was  t h i s study  one  of the  a much reduced weight g a i n f o l l o w i n g i n f e c t i o n  i n the b i r d s on low  l e v e l s of v i t a m i n A.  In Experiment 1,  using  5-week-old c h i c k s and an inoculum of 3 m i l l i o n o o c y s t s , the v i t a m i n A group had percent  a 2 week weight g a i n amounting t o only  of t h a t shown by i t s n o n - i n f e c t e d c o n t r o l , w h i l e  normal v i t a m i n A i n f e c t e d group had of i t s c o n t r o l . and 80 percent  In Experiment 2, these  respectively.  of Erasmus, S c o t t , and Levine The weight l o s s was the acute  a gain equal t o 70  37  the  percent  f i g u r e s were 26  percent  These f i n d i n g s concur with  those  (1960) and Waldroup et a l (1963).  due mainly t o the anorexia c h a r a c t e r i z i n g  stage of the i n f e c t i o n .  r e p o r t e d t h a t from day 4-6  Reed and P i t o i s  (1965)  f o l l o w i n g J a n i n f e c t i o n with  E.  a c e r v u l i n a , feed and water i n t a k e s are reduced at l e a s t 50 cent.  low  Preston-Mafham and  Sykes  per-  (1967b), u s i n g e q u a l i z e d  feedings between i n f e c t e d and c o n t r o l b i r d s , found t h a t with a E. n e c a t r i x i n f e c t i o n 70 percent  of the weight l o s s was  due  to  93  a r e d u c t i o n i n feed i n t a k e , i n the i n t e s t i n e . niques,  and 30 percent to impaired a b s o r p t i o n  Erasmus et a l  reported s i m i l a r findings  (1960), u s i n g the same t e c h i n b i r d s on 2 l e v e l s of  v i t a m i n A s u b j e c t e d t o a mixed i n f e c t i o n of E . a c e r v u l i n a and E. tenella.  An a n a l y s i s  of t h e i r r e s u l t s  show t h a t the  v i t a m i n A group was 9 percent lower i n weight than i t s  low non-  i n f e c t e d c o n t r o l , w h i l e the h i g h v i t a m i n A group was only 4 p e r cent lower than i t s  control.  This difference  i s probably due to  a decreased a b s o r p t i o n i n the lower v i t a m i n A group. (1968), and Preston-Mafham and Sykes  Long  (1967b) r e p o r t e d t h a t an  i n f e c t i o n w i t h E . a c e r v u l i n a r e s u l t e d i n a l o s s i n serum proteins  i n t o the gut lumen, as demonstrated by the Pontamine  sky blue dye t e s t . infection, and i s  This l o s s i s  at a maximum 3-5 days  c o i n c i d i n g w i t h the p e r i o d of depressed  absorption,  i n d i c a t i v e of a change i n gut p e r m e a b i l i t y .  r e p o r t e d changes i n p e r m e a b i l i t y 3^-7 hours a f t e r which he a t t r i b u t e d to the s p o r o z o i t e  after  Long a l s o inoculation  entry i n t o  epithelial  investigation,  i t was  cells. In the experiments  of t h i s  t h a t the feed and water i n t a k e of the b i r d s d u r i n g the stage was p r a c t i c a l l y n i l .  d i d not e q u a l i z e  acute  The a n o r e x i a was more pronounced i n  the low v i t a m i n A groups i n a l l the 3 experiments. experiments  noted  feed i n t a k e between the  As these infected  94  and  non-infected  c o n t r o l s , and  groups showed a g r e a t e r  as the low v i t a m i n A i n f e c t e d  degree of anorexia,  i t would f o l l o w t h a t  the main weight d i f f e r e n c e s between the groups were due greater  C.  l o s s of  to this  appetite.  Mortality M o r t a l i t y i s not a c h a r a c t e r i s t i c f i n d i n g i n an i n f e c t i o n  w i t h E. a c e r v u l i n a .  T h i s i s e s p e c i a l l y t r u e when the b i r d s  subjected  t o only one  Dickinson  and  McGuire  i n f e c t i v e dose as demonstrated by  S c o f i e l d (1939) and Hein  (1956) r e p o r t e d  s i n g l e 5 m i l l i o n oocyst c o u l d be due  6 percent  (1968a).  Morehouse  and  m o r t a l i t y i n chicks a f t e r a  i n o c u l a t i o n , but t h i s i s unusual  t o a p a r t i c u l a r l y v i r u l e n t s t r a i n of the  In the 3 experiments r e p o r t e d  i n t h i s paper, the  and  organism.  highest  m o r t a l i t y appeared i n 19-week-old, non-immune b i r d s on the l e v e l of d i e t a r y v i t a m i n A. d i e d , and percent  death was  are  low  Four of the 6 b i r d s i n t h i s group  a s s o c i a t e d with a weight l o s s of over  of the t o t a l body weight.  p r i m a r i l y t o the severe anorexia  T h i s weight l o s s was and  due  a d i p s i a d i s p l a y e d by  i n f e c t e d b i r d s on the low v i t a m i n A d i e t .  Age  may  30  the  have been a  f a c t o r which aggravated the e f f e c t s of the i n f e c t i o n i n e x p e r i ment No.  3..  Tyzzer  et a l (1932) r e p o r t e d  t h a t o l d e r b i r d s were  more s u s c e p t i b l e t o i n f e c t i o n w i t h E. n e c a t r i x and be t r u e w i t h E. a c e r v u l i n a .  Brackett  and  t h i s same  may  B l i z n i c k (1952a), a l s o  95  working with  E. n e c a t r i x , demonstrated t h a t with equal  oocyst  i n g e s t i o n the younger b i r d s were more s u s c e p t i b l e , but when the dose was  c a l c u l a t e d to g i v e equal numbers of oocysts  per gram of  body weight, the o l d e r b i r d s were the more s e v e r e l y a f f e c t e d .  D.  Immunity As demonstrated by the higher  reproductive t h e r e was  oocyst  production  and  index i n b i r d s on the low v i t a m i n l e v e l i n the  diet,  a delay i n the appearance of the immune response.  reason f o r t h i s delay may  be a s s o c i a t e d with a reduced  The  adrenal  c o r t i c a l f u n c t i o n under low v i t a m i n A c o n d i t i o n s as r e p o r t e d Glick  (1963).  I t was  e a r l i e r demonstrated by C h a l l e y  that i n birds a f f e c t e d with the s e c r e t i o n of adrenal  by  (1962)  E. t e n e l l a there i s an i n c r e a s e i n  c o r t i c o s t e r o n e s , and he a t t r i b u t e d  t h i s i n c r e a s e t o the s t r e s s of the d i s e a s e and the s y t h e s i s o c c u r r i n g i n the body as a r e s u l t .  increased  G l i c k (1963) found  t h a t the r e d u c t i o n of adrenal c o r t i c a l hormones c o u l d be  deter-  mined by a lower h e t e r o p h i l count i n the c i r c u l a t i o n f o l l o w i n g an i n j e c t i o n of ACTH.  Newcomer (1957) has  suggested t h a t  i n c r e a s e d h e t e r o p h i l s are a measure of the response of b i r d s t o acute s t r e s s .  The  appearance of numerous h e t e r o p h i l s i n the  lamina p r o p r i a of b i r d s 3 hours a f t e r an i n o c u l a t i o n with a c e r v u l i n a oocysts, would agree with t h i s suggestion.  The  E. fact  96  t h a t 2 of the 3 b i r d s on low v i t a m i n A l e v e l s d i d not show the l a r g e number of h e t e r o p h i l s observed i n the other groups, c o r r e l a t e with the r e p o r t of reduced a d r e n a l c o r t i c a l  may  secretions  under low v i t a m i n A c o n d i t i o n s . The  s t r e n g t h of the immunity, once e s t a b l i s h e d , was  not  r e l a t e d t o the l e v e l of v i t a m i n A i n the d i e t , but r a t h e r t o the s e v e r i t y of the i n i t i a l i n f e c t i o n . r e p o r t s by Tyzzer  (1929) and  This f i n d i n g confirms  l a t e r f i n d i n g s by Hein  earlier  (1968b) t h a t  the degree of immunity t o c o c c i d i o s i s i s d i r e c t l y r e l a t e d t o the s e v e r i t y of the i n f e c t i o n . been w i d e l y  i n v e s t i g a t e d , and  The nature of t h i s immunity i t i s generally considered  that a  l o c a l f a c t o r i s p r i m a r i l y r e s p o n s i b l e f o r the p r o t e c t i o n . (1963) suggests t h a t a s e n s i t i v i t y of the i n t e s t i n a l  has  Rose  epithelium  r e s u l t s from the presence of c i r c u l a t i n g a n t i b o d i e s , and  this  s e n s i t i v i t y i s r e s p o n s i b l e f o r the i n h i b i t i o n of s c h i z o n t development.  The  e f f e c t s of immunity are demonstrated i n the  s e c t i o n s taken 48 hours a f t e r the s t a r t of i n f e c t i o n .  The  immune b i r d s , r e g a r d l e s s of the l e v e l s of v i t a m i n A f e d , showed an i n h i b i t i o n of p a r a s i t e development. on endogenous forms confirms Pierce Burns  the f i n d i n g s of Long, Rose,  (1963), Horton-Smith and (1967).  This e f f e c t of immunity and  Long (1963) and Leathern and  A l l these i n v e s t i g a t o r s found t h a t there was  i n v a s i o n of the v i l l i  by the s p o r o z o i t e , and  a migration  an  t o the  97  crypt.  However, the  sporozoites  or e l s e a t r o p h o z o i t e  e i t h e r f a i l e d t o develop f u r t h e r ,  developed which d i d not mature i n t o a  schizont. The  b i r d s on the  lower d i e t a r y l e v e l of v i t a m i n  A did  not r e t a i n t h e i r immunity as w e l l as the b i r d s on normal d i e t s , but  the  d i f f e r e n c e was  t h e r e was 100  considerable  not  significant.  protection  still  However, the  f a c t that  e f f e c t e d by both groups  days a f t e r a primary i n f e c t i o n , i s very i n t e r e s t i n g as most  investigators report E. a c e r v u l i n a . oocysts has acervulina effective  a shorter  period  of p r o t e c t i v e  immunity w i t h  However an immunization dose as h i g h as 3 m i l l i o n  not been r e p o r t e d ,  and  i t would appear t h a t w i t h  an inoculum of t h i s s i z e must be used t o e s t a b l i s h immunity.  E.  98  VI. 1.  SUMMARY AND CONCLUSIONS  Experiments were conducted to i n v e s t i g a t e  the  p a t h o l o g i c a l e f f e c t s of an i n f e c t i o n w i t h E . a c e r v u l i n a i n b i r d s fed a d i e t  c o n t a i n i n g 440 I . U . v i t a m i n A per k i l o g r a m , compared  to b i r d s fed a s i m i l a r d i e t c o n t a i n i n g 4400 I . U . v i t a m i n A per kilogram.  2. No symptoms of d e f i c i e n c y were observed i n the v i t a m i n A groups, and no s i g n i f i c a n t  weight d i f f e r e n c e s  low were  recorded between the n o n - i n f e c t e d normal v i t a m i n A and low v i t a m i n A groups.  3. The e f f e c t  of the low l e v e l  of v i t a m i n A i n the  diet  was not manifested u n t i l the b i r d s were i n f e c t e d w i t h E . acervulina.  4.  F o l l o w i n g an i n f e c t i o n w i t h E . a c e r v u l i n a , the b i r d s  on the low l e v e l gains,  of v i t a m i n A showed s i g n i f i c a n t l y  lower weight  and h i g h e r m o r t a l i t y and oocyst p r o d u c t i o n , than d i d the  b i r d s r e c e i v i n g the h i g h e r l e v e l  5.  B i r d s fed the low l e v e l  slower development infection.  of v i t a m i n A .  of v i t a m i n A demonstrated a  of the immune response  to E . a c e r v u l i n a  99  6. to  d e g r e e o f immunity  the s e v e r i t y  7. did  The  The  not r e s u l t  of the i n i t i a l  low  t o E. a c e r v u l i n a  levels  sporozoite similar  l e v e l of v i t a m i n A used i n these experiments  i n a loss  of e p i t h e l i a l i n t e g r i t y i n the  birds  effects  intestine.  o f low v i t a m i n  on t h e endogenous s t a g e s o f t h e p a r a s i t e .  invasion  of the i n t e s t i n a l e p i t h e l i u m  i n a l l groups.  development  related  infection.  8. T h e r e were no d e t e c t a b l e dietary  was  The  of the f i r s t  on b o t h d i e t a r y  inhibitory  effect  levels  of vitamin  A.  The  a p p e a r e d t o be  o f immunity  g e n e r a t i o n s c h i z o n t was  A  also  on t h e  similar i n  100  BIBLIOGRAPHY  A c k e r t , J . E . 1942. N a t u r a l r e s i s t a n c e t o h e l m i n t h i c J . P a r a s i t o l . 28:1.  disease.  Ackert, J.E.; M c l l v a i n e , M.F.; and Zimmermann, N.B. 1927. R e s i s t a n c e t o p a r a s i t e s a f f e c t e d by f a t s o l u a b l e v i t a m i n A. J . P a r a s i t o l . 13:219. A c k e r t , J.E.; M c l l v a i n e , M.F.; and Crawford, N.Z. 1931. R e s i s t a n c e of chickens t o p a r a s i t i s m a f f e c t e d by v i t a m i n A. Am. J . Hyg. 13:320. A f i f i , A.K., and Acra, A.N. 1955. Concurrent demonstration o f DNA and 1,2, - g l y c o l s . 2. P e r i o d a t e o x i d a t i o n . S t a i n Technol. 30:119. A i t k e n , R.N.C. 1958. A h i s t o c h e m i c a l study of the stomach and i n t e s t i n e o f the c h i c k e n . J . Anat. 92:453. A y d e l o t t e , M. 1963. J . Nutr. 17:205.  Vitamin A d e f i c i e n c y i n chickens.  Brit.  A y l o t t , M.V.; V e s t a l , O.H.; Stephens, J.F.; and Turk, D.E. 1968. E f f e c t o f c o c c i d i a l i n f e c t i o n upon passage r a t e s of d i g e s t i v e t r a c t contents o f c h i c k s . P o u l t r y S c i . 47:900. Becker, E.R. 1935. The mechanism o f immunity i n murine coccidiosis. Am. J . Hyg. 21:389. Becker, E.R. 1956. Appropos oocyst measurements. P a r a s i t o l . 42:Suppl. 24.  J.  Benson, J.A. and Rampone, A . J . 1966. G a s t r o i n t e s t i n a l absorption. Ann. Rev. P h y s i o l . 28:201. B r a c k e t t , S., and B l i z n i c k , A. 1952a. The r e l a t i v e susc e p t i b i l i t y of chickens o f d i f f e r e n t ages t o c o c c i d i o s i s caused by E i m e r i a n e c a t r i x . P o u l t r y S c i . 31:146. B r a c k e t t , S. and B l i z n i c k , A. 1952b. The r e p r o d u c t i v e p o t e n t i a l of 5 s p e c i e s of c o c c i d i a of the c h i c k e n as demonstrated by oocyst p r o d u c t i o n . J . P a r a s i t o l . 38:133.  101  Bradley, O.C. and Grahame, T.D. 1960. The S t r u c t u r e of the Fowl. 4th ed. Edinburgh and London: O l i v e r and Boyd, p. 143. B r i t t o n , W.M.; H i l l , C.H.; and Barber, C.W. 1964. A mechanism of i n t e r a c t i o n between d i e t a r y p r o t e i n l e v e l s and c o c c i d i o s i s i n c h i c k s . J . Nutr. 82:306. Burns, W.G. and C h a l l e y , J.R. 1959. R e s i s t a n c e of b i r d s t o c h a l l e n g e with E i m e r i a t e n e l l a . Exp. P a r a s i t o l . 8:515. C h a l l e y , J.R. 1962. The r o l e of t h e bursa of F a b r i c i u s i n adrenal response and m o r t a l i t y due t o E i m e r i a t e n e l l a i n f e c t i o n s i n the c h i c k e n . J . P a r a s i t o l . 48:352. C h a l l e y , J.R. 1966. Changes i n adrenal c o n s t i t u e n t s and t h e i r r e l a t i o n s h i p t o c o r t i c o s t e r o n e s e c r e t i o n i n chickens s e l e c t e d f o r g e n e t i c r e s i s t a n c e and s u s c e p t i b i l i t y t o c a e c a l coccidiosis. J . P a r a s i t o l . 52:967. C h a l l e y , J.R and Burns, W.C. 1959. The i n v a s i o n of the c a e c a l mucosa by E i m e r i a t e n e l l a s p o r o z o i t e s and t h e i r t r a n s p o r t by macrophages. J . P r o t o z o o l . 6:238. Chodnik, K.S. 1947. A c y t o l o g i c a l study of t h e a l i m e n t a r y t r a c t of the domestic fowl (Gallus domesticus). Quart. J . M i c r o s c o p . S c i . 88:419. C l a r k s o n , M.J. 1958. L i f e h i s t o r y and p a t h o g e n i c i t y of E i m e r i a adenoeides Moore and Brown 1951, on t h e turkey p o u l t . P a r a s i t o l o g y 48:70. C l a r k s o n , M.J. 1959. The l i f e h i s t o r y and p a t h o g e n i c i t y o f E i m e r i a m e l e a g r i m i t i s Tyzzer, 1929, i n the turkey p o u l t . P a r a s i t o l o g y 49:70. Davies, A.W. 1952. Lowered l i v e r v i t a m i n A r e s e r v e s coccidiosis. Nature 170:849.  i n avian  Davies, S.F.M. and Joyner, L.P. 1955. Observations on the p a r a s i t o l o g y o f deep l i t t e r i n p o u l t r y houses. V e t . Record. 67:193. Davies, S.F.M. and Joyner, L.P. 1962. I n f e c t i o n of t h e fowl by p a r e n t e r a l i n o c u l a t i o n o f oocysts of E i m e r i a . Nature 194:996.  102  D a v i e s , S . F . M . ; J o y n e r , L . P . ; and K e n d a l l , S . B . 1963. .Coccidiosis. London: O l i v e r and Boyd, p . 216. D i c k i n s o n , E . M . 1941. The e f f e c t s of v a r i a b l e dosages of s p o r u l a t e d E i m e r i a a c e r v u l i n a oocysts on c h i c k e n s . Poultry S c i . 20:413. D i c k i n s o n , E . M . and S c o f i e l d , R . H . 1939. The e f f e c t of sulphur a g a i n s t a r t i f i c i a l i n f e c t i o n with E i m e r i a a c e r v u l i n a and Eimeria t e n e l l a . P o u l t r y S c i . 18:419. Doran, D . J . 1966a. P a n c r e a t i c enzymes i n i t i a t i n g e x c y s t a t i o n of E i m e r i a a c e r v u l i n a . P r o c . H e l m i n t h o l . Soc. Wash. 33:42. Doran, D . J . 1966b. L o c a t i o n and time of p e n e t r a t i o n of duodenal e p i t h e l i a l c e l l s by E i m e r i a a c e r v u l i n a s p o r o z o i t e s . P r o c . H e l m i n t h o l . Soc. Wash. 33:43. Doran, D . J . 1966c. The m i g r a t i o n of E i m e r i a a c e r v u l i n a s p o r o z o i t e s t o the duodenal glands of L i e b e r k u h n . J . P r o t o z o o l . 13:27. Doran, D . J . and F a r r , M.M. 1962. coccidium, Eimeria a c e r v u l i n a .  E x c y s t a t i o n of the p o u l t r y J . P r o t o z o o l . 9:154.  Doran, D . J . and F a r r , M.M. 1965. S u s c e p t i b i l i t y of 1-and 3d a y - o l d c h i c k s t o i n f e c t i o n w i t h the c o c c i d i u m , E i m e r i a acervulina. J . P r o t o z o o l . 12:160. Edgar, S . A . 1955. S p o r u l a t i o n of oocysts at s p e c i f i c temperatures, and notes on the prepatent p e r i o d of s p e c i e s of a v i a n c o c c i d i a . J . P a r a s i t o l . 41:214.  several  Elvehjem, C A . and Neu, V . F . 1932. Studies i n v i t a m i n A . a v i t a m i n o s i s i n the c h i c k . J . B i o l . Chem. 97:71. Erasmus, J . ; L e v i n e , P . P . ; and S c o t t , M . L . 1958. An i n t e r r e l a t i o n s h i p between c o c c i d i o s i s and v i t a m i n A n u t r i t i o n of c h i c k e n s . P o u l t r y S c i . 38:1202. Erasmus, J . ; S c o t t , M . L . , ; and L e v i n e , P . P . 1960. s h i p of c o c c i d i o s i s and v i t a m i n A n u t r i t i o n i n P o u l t r y S c i . 39:565.  A relationchickens.  103  F a r r , M.M. and Doran, D.J. 1962. Comparative e x c y s t a t i o n of 4 s p e c i e s of p o u l t r y c o c c i d i a . J . P r o t o z o o l . 9:403. F a r r , M.M. and Wehr, E.E. 1949. S u r v i v a l of E i m e r i a a c e r v u l i n a , E. t e n e l l a , and E. maxima oocysts on s o i l under v a r i o u s f i e l d conditions. Ann. N.Y. Acad. S c i . 52:468. G a r r i e t s , E. 1961. The p r o p h y l a c t i c a c t i o n of v i t a m i n A i n c a e c a l c o c c i d i o s i s by p r o t e c t i o n of e p i t h e l i u m . B r i t . Vet. J . 117:507. G l i c k , B. 1957. Experimental m o d i f i c a t i o n of the bursa of Fabricius. P o u l t r y S c i . 36:18.. G l i c k , B. 1963. I n d i r e c t evidence of the i n f l u e n c e of v i t a m i n A on the a d r e n a l c o r t e x of the c h i c k . P o u l t r y S c i . 42:1022. G l i c k , B.; Chang, T.; and Jaap, R. 1956. The bursa of F a b r i c i u s and antibody p r o d u c t i o n . P o u l t r y S c i . 35:224. Harmon, B.G.; M i l l e r , E.R.; U l l r e y , D.E.; and Hoefer, J.A. 1960. E f f e c t of v i t a m i n A d e f i c i e n c y on the antibody producing a b i l i t y of swine. J . Anim. S c i . 19:1265. Hein, H. 1968a. The pathogenic e f f e c t s of E i m e r i a i n young c h i c k s . Exp. P a r a s i t o l . 22:1.  acervulina  Hein, H. 1968b. R e s i s t a n c e i n young c h i c k s t o r e i n f e c t i o n by immunization w i t h two doses of oocysts of E i m e r i a a c e r v u l i n a . Exp. P a r a s i t o l . 22:12. H i l l e r m a n , J.P.; K r a t z e r , F.H.; and W i l s o n , W.O. 1953. Food passage through chickens and turkeys and some r e g u l a t i n g f a c t o r s . P o u l t r y S c i . 32:332. Horton-Smith, C. 1963. Vet. J . 119:99.  Immunity t o a v i a n c o c c i d i o s i s .  Brit.  Horton-Smith, C and Long, P.L. 1959. The e f f e c t s of d i f f e r e n t a n t i - c o c c i d i a l agents on the i n t e s t i n a l c o c c i d i o s i s o f the fowl. J . Comp. P a t h o l . Therap. 69:192. Horton-Smith, C. and Long, P.L. 1963. Behavior of..invasive stages of E i m e r i a t e n e l l a i n the immune fowl (Gallus domesticus). Exp. P a r a s i t o l . 14:66.  104  H o r t o n - S m i t h , C ; B e a t t i e , J . ; and Long, P . L . 1961. Resistance to E i m e r i a t e n e l l a and i t s t r a n s f e r e n c e from one caecum to the other i n i n d i v i d u a l fowls. Immunology 4:111. Howell, J . McC. and Thompson, J . N . 1967. Lesions a s s o c i a t e d w i t h the development of a t a x i a i n v i t a m i n A d e f i c i e n t c h i c k s . B r i t . J . N u t r . 21:741. Huber, W.G.  1962.  Current v i t a m i n A problems.  V e t . Med. 57:311.  Imondi, A . R . and B i r d , F . A . 1966. The turnover of e p i t h e l i u m i n the c h i c k . P o u l t r y S c i . 45:142. Jackson, A . R . B . sporozoites. Joyner, L . P . chickens.  intestinal  1964. The i s o l a t i o n of v i a b l e c o c c i d i a l P a r a s i t o l o g y 54:87.  1958. Experimental E i m e r i a m i t i s P a r a s i t o l o g y 48:101.  infection  in  Jungherr, E . 1945. A h y p o v i t a m i n o s i s i n commercial p o u l t r y f l o c k s on b a s i s of n a s a l h i s t o p a t h o l o g y . P o u l t r y S c i . 24: 112. Koutz, F . R . 1952. The e f f e c t of b u i l t - u p l i t t e r on the p a r a s i t i c ova and oocysts of p o u l t r y . P o u l t r y S c i . 32:313. K r a s s n e r , S . M . 1963. F a c t o r s i n host s p e c i f i c i t y i n f e c t i v i t y i n Eimeria acervulina i n f e c t i o n s . P r o t o z o o l . 10:327. Landers, E . J . 1960. Studies on e x c y s t a t i o n oocysts. J . P a r a s i t o l . 46:195.  and oocyst J.  of c o c c i d i a l  Leathern, W.D. and Burns, W.C. 1967. E f f e c t s of the immune c h i c k e n on the endogenous stages of E i m e r i a t e n e l l a . J. P a r a s i t o l . 53:180. Long, P . L . 1962. Observations on the d u r a t i o n of the a c q u i r e d immunity of chickens t o E i m e r i a maxima T y z z e r , 1929. P a r a s i t o l o g y 52:89. Long, P . L . 1967. Studies on E i m e r i a m i v a t i i n chickens and a comparison w i t h E i m e r i a a c e r v u l i n a . J . Comp. P a t h . Therap. 77:315.  105  Long, P.L. 1968a. The e f f e c t of breed of chickens on r e s i s t a n c e to Eimeria i n f e c t i o n s . B r i t . P o u l t r y S c i . 9:7.1. Long, P.L. 1968b. The pathogenic e f f e c t s of E i m e r i a praecox and E. a c e r v u l i n a i n the c h i c k e n . P a r a s i t o l o g y 58:691. Long, P.L. and M i l l a r d , B.J. 1968. Eimeria: e f f e c t of m e t i c l o r p i n d o l and methyl benzoquate on endogenous stages i n the c h i c k e n . Exp. P a r a s i t o l . 23:331. Long, P.L. and Rowell, J.G. 1958. coccidia. Lab P r a c t . 7:515.  Counting  oocysts of chicken  Long, P.L.; Rose, M.E.; and P i e r c e , A.E. 1963. E f f e c t s of fowl s e r a on some stages i n the l i f e c y c l e of E i m e r i a t e n e l l a . Exp. P a r a s i t o l . 14:210. L o t z e , J.C. and Leek, R.G. 1968. E x c y s t a t i o n of the s p o r o z o i t e s of E i m e r i a t e n e l l a i n a p p a r e n t l y unbroken oocysts i n the chicken. J . P r o t o z o o l . 15:693. Lund, E. and F a r r , M.M. 1965. Protozoa, i n : Diseases of P o u l t r y , ed. by B i e s t e r , H.E. and Schwarte, L.H. 5th ed. Ames, Iowa, Iowa S t a t e U n i v e r s i t y Press, p. 1056. McFarlane, D. 1944. P i c r o - M a l l o r y : an e a s i l y c o n t r o l l e d r e g r e s s i v e t r i c h r o m i c s t a i n i n g method. S t a i n Technol. 19:29. Moore, T. 1967. E f f e c t s of v i t a m i n A d e f i c i e n c y i n animals. In: The V i t a m i n s , ed. by S e b r e l l , W.H. and H a r r i s , R.S., New York, Academic P r e s s , p. 245. Morehouse, N.F. 1938. The r e a c t i o n of the immune i n t e s t i n a l e p i t h e l i u m of r a t s t o r e - i n f e c t i o n with E i m e r i a n i e s c h u l z i . J . P a r a s i t o l . 24:311. Morehouse, N.F. and McGuire, W.C. 1956. M o r b i d i t y and m o r t a l i t y among chickens i n f e c t e d with l a r g e numbers of the i n t e s t i n a l coccidium E i m e r i a a c e r v u l i n a Tyzzer 1929. J . P a r a s i t o l . .42: Suppl. 24. Morehouse, N.F. and McGuire, W.C. 1958. The p a t h o g e n i c i t y of E i m e r i a a c e r v u l i n a . P o u l t r y S c i . 37:665.  106  Murphy, R.R.; Hunter, J.E.; and Knandel, H.C. 1938. The e f f e c t s of r a t i o n s c o n t a i n i n g g r a d i e n t amounts of cod l i v e r o i l on the subsequent performance o f l a y i n g p u l l e t s f o l l o w i n g a n a t u r a l i n f e c t i o n of c o c c i d i o s i s . P o u l t r y S c i . 17:377. Newcomer, W.S. 1957. Blood c e l l changes f o l l o w i n g ACTH i n j e c t i o n i n the c h i c k . Proc. Soc. Expt. B i o l . Med. 96:613. Newcomer, W.S. and Connally, J.D. 1960. The bursa of F a b r i c i u s as an i n d i c a t o r o f s t r e s s i n immature c h i c k e n s . Endrocinology 67:264. Norwich Animal Industry Inc. Coccidiosis. 41 pp.  1968.  Technical B u l l e t i n .  Nyberg, P.A.; Baurer, D.N.; and Knapp, S.E. 1968. Carbon d i o x i d e as the i n i t i a l stimulus f o r e x c y s t a t i o n o f E i m e r i a t e n e l l a oocysts. J . P r o t o z o o l . 15:144. Panda, B. and Combs, G.F. 1963. Impaired antibody p r o d u c t i o n i n c h i c k s on low l e v e l s o f v i t a m i n A, pantothenic a c i d , or riboflavin. Proc. Soc. Expt. B i o l . Med. 113:530. Panda, B. and Combs, G.F. 1964. Studies on c o c c i d i o s i s and v i t a m i n A n u t r i t i o n of b r o i l e r s . P o u l t r y S c i . 43:154. Pande, P.G. and Krishnamurty, D. 1959. I n t e r - r e l a t i o n s h i p between hypovitaminosis A and A s c a r i d i a g a l l i i n f e s t a t i o n i n poultry. P o u l t r y S c i . 38:13. / i  P a t i l l o , W.H. 1959. Invasion of c e c a l mucosa of the c h i c k e n by sporozoites of Eimeria t e n e l l a . J . P a r a s i t o l . 45:253. Perek, M. and E c k s t e i n , B. 1959. The a d r e n a l a s c o r b i c a c i d content o f m o l t i n g hens and the e f f e c t o f ACTH on the adrenal a s c o r b i c a c i d content of l a y i n g hens. P o u l t r y S c i . 38:996. Peterson, E.H. 1949. C o c c i d i o s i s i n l a y i n g hens due presumably to E i m e r i a a c e r v u l i n a . Ann. N.Y. Acad. S c i . 52:464. P i e r c e , A.E. and Long, P.L. 1965. Studies on a c q u i r e d immunity to c o c c i d i o s i s i n b u r s a l e s s and thymectomised fowls. Immunology 9:427. P i e r c e , A.E.; Long, P.L.; and Horton-Smith, C. 1962. Immunity to E i m e r i a t e n e l l a i n young fowls (Gallus domesticus) Immunology 5:129.  107  Pout, D.D. 1967. V i l l o u s atrophy 213:306.  and c o c c i d i o s i s .  Nature  Preston-Mafham, R.A. and Sykes, A.H. 1967a. Changes i n p e r m e a b i l i t y of the mucosa d u r i n g i n t e s t i n a l c o c c i d i o s i s i n f e c t i o n i n t h e fowl. E x p e r i e n t a 23:972. Preston-Mafham, R.A. and Sykes, A.H. 1967b. F a c t o r s cont r i b u t i n g t o the weight l o s s d u r i n g i n t e s t i n a l c o c c i d i o s i s i n f e c t i o n s i n t h e fowl. Proc. Nutr. Soc. 26:27. Reid, W.M. and P i t o i s , M. 1965. The i n f l u e n c e of c o c c i d i o s i s on feed and water i n t a k e of c h i c k e n s . Avian D i s e a s e s , 9:343. Reid, W.M.; Womack, H.E.; and Johnson, J . 1968. C o c c i d i o s i s s u s c e p t i b i l i t y i n l a y e r f l o c k replacement programs. P o u l t r y S c i . 47:892. Richards, M.B. 1935. The r o l e of v i t a m i n A i n n u t r i t i o n . B r i t . Med. J . (19351) 1:99. R i t t e r , H.B. and Oleson, J . J . 1950. Combined h i s t o c h e m i c a l s t a i n i n g o f a c i d p o l y s a c c h a r i d s and 1,2 - g l y c o l groupings on p a r a f f i n s e c t i o n s of r a t t i s s u e . Am. J . Path. 26:639. Roels, O.A. 1967. Vitamin A. In: The V i t a m i n s , ed. by S e b r e i l , W.H. and H a r r i s , R.S., New York, Academic Press, v o l . 1:167. Rose, M.E. 1963. Some aspects o f immunity t o E i m e r i a infections. Ann. N.Y. Acad. S c i . 113:383. Rose, M.E. 1967a. Immunity t o E i m e r i a b r u n e t t i and E i m e r i a maxima i n f e c t i o n s i n the f o w l . P a r a s i t o l o g y 57:363. Rose, M.E. 1967b. Immunity t o E i m e r i a t e n e l l a and E i m e r i a n e c a t r i x i n f e c t i o n s i n the f o w l . I . I n f l u e n c e o f the s i t e of i n f e c t i o n and t h e stage o f t h e p a r a s i t e . I I . Cross p r o t e c t i o n . P a r a s i t o l o g y 57:567. Rose, M.E. 1968. The e f f e c t of splenectomy upon i n f e c t i o n with Eimeria t e n e l l a . P a r a s i t o l o g y 58:481. Rose, M.E. and Long, P.L. 1962. Immunity t o 4 s p e c i e s o f E i m e r i a i n t h e fowl. Immunology 5:79.  108  S h o r t t , H.E. and Cooper, W. 1948. S t a i n i n g of m i c r o s c o p i c s e c t i o n s c o n t a i n i n g p r o t o z o a l p a r a s i t e s by m o d i f i c a t i o n of McNamara's method. Trans. Roy. Soc. Trop. Med. Hyg. 41:427. S e i f r i e d , O. 1930. Studies J . Exp. Med. 52:519.  on A - a v i t a m i n o s i s  i n chickens.  Sharma, N.N. 1964. Response o f the fowl (Gallus domesticus) t o p a r e n t e r a l a d m i n i s t r a t i o n of seven c o c c i d i a l s p e c i e s . J . P a r a s i t o l . 50:509. Sharma, N.N. and Reid, W.M. 1962. S u c c e s s f u l i n f e c t i o n of chickens a f t e r p a r e n t e r a l i n o c u l a t i o n of E i m e r i a spp. J . P a r a s i t o l . 48:33. Smith, D.A. 1955. P a r a s i t i c i n f e c t i o n and n u t r i t i o n . Vitamins and Hormones. 13:239. Stevenson, G.T. 1969. T e c h n i c a l b u l l e t i n . Dow Chemical Co. Coyden 25 c o c c i d i o s t a t premix. The P r a c t i c i n g N u t r i t i o n i s t 3:2. Stock, B.L.; Stevensort, G.T.; and Hymas, T.A. 1967.. Coyden c o c c i d i o s t a t f o r c o n t r o l o f c o c c i d i o s i s i n chickens. P o u l t r y S c i . 46:485. T a y l o r , M.W. and R u s s e l l , W.C. 1946. The p r o v i t a m i n A requirement of growing c h i c k s . P o u l t r y S c i . 26:234. Turk, D.E. and Stephens, J.F. 1967. C o c c i d i o s i s and n u t r i e n t absorption. Proc. Maryland Nutr. Confer, p. 12. Tyzzer, Am.  E.E. 1929. C o c c i d i o s i s i n g a l l i n a c e o u s J . Hyg. 10:269.  birds.  Tyzzer, E.E.; T h e i l e r , H.; and Jones, E.E. 1932. C o c c i d i o s i s i n g a l l i n a c e o u s b i r d s . I I - A comparative study of s p e c i e s of E i m e r i a of the c h i c k e n . Am. J . Hyg. 15:319. Van Doorninck, W.M. and Becker, E.R. 1957. Transport of s p o r o z o i t e s of E i m e r i a n e c a t r i x i n macrophages. J . P a r a s i t o l . 43:40.  109  V e t t e r l i n g , J.M. and Doran, D.J. 1966. Schizogony and gametogony i n the l i f e c y c l e o f t h e p o u l t r y coccidium, E i m e r i a a c e r v u l i n a Tyzzer, 1929. J . P a r a s i t o l . 52:1150. Waldroup, P.W.; Simpson, C.F.; Cox, D.D.; and Harms, R.H. The e f f e c t s of f e e d i n g v a r i o u s l e v e l s o f v i t a m i n A on c h i c k s with c a e c a l c o c c i d i o s i s . P o u l t r y S c i . 42:274.  1963.  Warren, E.W. 1968. Vitamin requirements of the C o c c i d i a of the c h i c k e n . P a r a s i t o l o g y 58:137. Warren, E.W. and B a l l , S.J. 1967. Schizogonous stages of E i m e r i a a c e r v u l i n a Tyzzer, 1929. Nature, 214:829. Wilson, P.A. and F a i r b a i r n , D. 1961. Biochemistry of s p o r u l a t i o n i n oocysts of Eimeria a c e r v u l i n a . J . P r o t o z o o l . 8:410. Wolbach, S.B. and Howe, P.R. 1925. T i s s u e changes f o l l o w i n g deprivation of f a t soluable A vitamin. J . Exp. Med. 42:753. Woollam, D.H. and M i l l e n , J.W. 1955. E f f e c t of v i t a m i n A d e f i c i e n c y on the c e r e b r o s p i n a l f l u i d pressure on the c h i c k . Nature, 175:41. Yaeger, R.G. and M i l l e r , O.N. 1963. E f f e c t of m a l n u t r i t i o n on s u s c e p t i b i l i t y o f r a t s t o Trypanosoma c r u z i . V. Vitamin A d e f i c i e n c y . Exp. P a r a s i t o l . 14:9.  

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