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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., University of 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 thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f P o u l t r y S c i e n c e The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada i i ABSTRACT The present investigation was conducted to determine the ef f e c t of an i n f e c t i o n with Eimeria acervulina on vitamin A de f i c i e n t birds. One hundred day-old chicks were divided into 2 equal groups, and one group was raised on 440 I.U. vitamin A per kilogram of feed, while the second group received the normally recommended 4400 I.U. per kilogram. At 5 weeks of age, half the birds from each group were given an immunizing i n f e c t i o n with 3 m i l l i o n oocysts of E. acervulina. The c l i n i c a l symptoms were more acute, and the oocyst production, mortality, and weight losses s i g n i f i c a n t l y higher, i n the birds on the low vitamin A d i e t . When the birds were 9 weeks old, h a l f were subjected to a second i n f e c t i o n and the other h a l f held u n t i l they were 19 weeks old. The 9-week-old birds were given an oral inoculation of 10 m i l l i o n oocysts to evaluate the e f f e c t of a low vitamin A diet on: the strength of the immune response; a primary i n f e c t i o n with E. acervulina i n 9-week-old birds; and the early tissue stages of the parasite i n immune and non-immune birds. The r e s u l t s from t h i s experiment showed that the degree of immunity i s not affected by a low vitamin A r a t i o n , but primarily determined by the severity of the i n i t i a l i n f e c t i o n ; and that the primary i n f e c t i o n i n 9-week-old chicks i s c l i n i c a l l y very similar to that i n younger birds. The h i s t o l o g i c a l examinations demonstrated that the low vitamin A l e v e l used i n th i s study d id not a f fec t the i n t e g r i t y of the i n t e s t i n a l epi thel ium, nor the development of the endogenous forms of the p a r a s i t e . I t was a lso shown that the sporozoite invasion was the same i n immune and non-immune b i r d s , but the schizont development was severely i n h i b i t e d i n the immune birds regardless of the vitamin A l e v e l . The non-immune b irds on the low vitamin A r a t i o n d id not show as d i s t i n c t a he terophi l response to i n f e c t i o n as d id 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 respect ive d ie ts u n t i l 19 weeks of age, and during t h i s per iod there was no c l i n i c a l evidence of a vi tamin A def ic iency , and the.growth rate was not s i g n i f i c a n t l y affected by the l e v e l of v i tamin A i n the d i e t . When 19 weeks o l d , the b irds were inoculated with 25 m i l l i o n oocysts of E . acervul ina to assess the e f fect of an extremely heavy primary i n f e c t i o n i n older b i r d s , and to evaluate the degree of immunity s t i l l reta ined 14 weeks af ter an i n i t i a l i n f e c t i o n . The morta l i ty was very h igh , and the weight losses s i g n i f i c a n t l y greater , i n birds on the low vi tamin A d ie tary l e v e l . Immunity, although decreased from the e a r l i e r experiment, was s t i l l present i n b irds on both l eve l s of v i tamin A. i v I t was concluded from these experiments that a suboptimal l e v e l of v i tamin A i n the d i e t increased the pathogenicity of E . acervul ina i n chickens of various ages. The low vitamin A l e v e l d id not, however, s i g n i f i c a n t l y a f fec t the b i r d ' s immune response. V TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS ...... V LIST OF TABLES. . . v i i LIST OF FIGURES ix ACKNOWLEDGEMENT . X I. INTRODUCTION 1 I I . REVIEW OF LITERATURE 2 A. Vitamin A and Coccidiosis 2 B. Physiological Role of Vitamin A 5 C. Eimeria acervulina 8 1. L i f e cycle 9 2. Pathogenicity 16 3. Histopathology 18 D. Immunity to Coccidiosis 19 II I . EXPERIMENTAL METHODS AND DESIGN 24 A. Experimental Diets 24 B. Brooding 25 C. Experimental Design 27 D. Methods 29 1. Oocyst determinations-and techniques. . 29 2. H i s t o l o g i c a l techniques 37 v i PAGE E- Experiments: Object, Design, Inoculation, and Methods. 39 1. Experiment number 1 40 2. Experiment number 2 42 3. Experiment number 3 44 IV. 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 . 59 2. H i s t o l o g i c a l examination 63 C. Experiment No. 3 73 1. Primary i n f e c t i o n 73 2. Secondary i n f e c t i o n 76 3. Post mortem and h i s t o l o g i c a l examinations .83 V. DISCUSSION. 86 A. Oocyst production 86 B. Weight Gains 92 C. Mortality 94 D. Immunity 95 VI. SUMMARY AND CONCLUSIONS 98 BIBLIOGRAPHY 100 vii:.. LIST OF TABLES TABLE PAGE I. Average d a i l y oocyst production i n m i l l i o n s i n 5-week-old chicks, on 2 dietary levels of vitamin A, following i n f e c t i o n with 3 m i l l i o n oocysts of E. acervulina per b i r d 52 II. Mean weight i n grams of 5-week-old chicks, on 2 dietary levels of vitamin A, after i n f e c t i o n with 3 m i l l i o n oocysts of E. acervulina com-pared to non-infected controls 53 II I . Daily mean weight changes i n grams of 5-week-old chicks, on 2 dietary levels of vitamin A, following i n f e c t i o n with 3 m i l l i o n oocysts of E. acervulina 54 IV. Total mean weight i n grams, over a 4 month period, of 4 groups of chickens on 2 dietary lev e l s of vitamin A, and with one group on each l e v e l infected at 5 weeks of age with 3 m i l l i o n oocysts of E. acervulina 55 V. Average d a i l y oocyst production i n m i l l i o n s i n 9-week-old chicks on 2 dietary levels of vitamin A, and 2 levels of immunity, aft e r i n f e c t i o n with 10 m i l l i o n oocysts of E. acervulina per b i r d 66 VI. Average weight i n grams of immunized 9-week-old chicks, on, 2 dietary levels of vitamin A, following a second i n f e c t i o n with E. acervulina 67 VII. Average weight i n grams of 9-week-old chicks, on 2 dietary levels of vitamin A, following a primary i n f e c t i o n with E. acervulina . . . . 68 VIII. Daily mean weight changes i n grams of 9-week-old chicks, on 2 dietary levels of vitamin A, following a primary i n f e c t i o n with 10 m i l l i o n oocysts' of E. acervulina 69 v i i i TABLE Page IX. Two week t o t a l weight gain i n grams i n immune and non-immune 9-week-old chicks, on 2 dietary levels of vitamin A, following i n f e c t i o n with 10 m i l l i o n oocysts of E. acervulina 70 X. Performance index of 9-week-old chicks, on 2 dietary l e v e l s of vitamin A and 2 levels of immunity, following i n f e c t i o n with 10 m i l l i o n oocysts of E. acervulina per b i r d 71 XI. Average d a i l y oocyst production i n m i l l i o n s i n 19-week-old chickens on 2 dietary levels of vitamin A, and 2 level s of immunity, after i n f e c t i o n with 25 m i l l i o n oocysts of E. acervulina per b i r d 79 XII. Average weight i n grams of 19-week-old chickens, on 2 dietary levels of vitamin A, and 2 levels of immunity, following i n f e c t i o n with 25 m i l l i o n oocysts of E. acervulina 80 XIII. Daily mean weight changes i n grams i n immune, and non-immune 19-week-old chickens on 2 dietary l e v e l s of vitamin A, following i n f e c t i o n with 25 m i l l i o n oocysts of E. acervulina. . . .... 81 XIV. The reproductive index of E. acervulina following increasingly larger i n f e c t i v e doses as reported by various authors . 88 i x LIST OF FIGURES FIGURE PAGE 1. Arrangement of chicks i n batteries for Experi-ment No. 1. . . . . . . 29 2. The ef f e c t of dietary vitamin A l e v e l on the mean weight of 5-week-old chicks following i n f e c t i o n with E. acervulina 56 3. A comparison of the mean weights of infected and non-infected chicks, on 2 dietary levels of vitamin A, over a 4 month period. 57 4. The ef f e c t of dietary vitamin A l e v e l on the 2 week weight gain of 5-week-old chicks following i n f e c t i o n with E. acervulina. . . . . 58 5. The ef f e c t of dietary vitamin A l e v e l on the 2 week weight gain of 9-week-old chicks following i n f e c t i o n with E. acervulina 72 6. The ef f e c t of dietary vitamin A l e v e l on the mean weight of 19-week-old immune and non-immune birds 14 days after i n f e c t i o n with E. acervulina 82 7. A comparison of the d a i l y oocyst production i n chicks of Experiments 1, 2, and 3 91 X ACKNOWLEDGEMENTS The author would l i k e to express her deep grat i tude to Professor Jacob B i e l y , of the Department of Poul try Science, for a l l the help 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 incent ive to the completion of th i s work. Sincere thanks are a lso extended to Professor Beryl E . March, of the Department of Poul try Science, for prov id ing the f a c i l i t i e s used i n th i s i n v e s t i g a t i o n , and e spec ia l l y for her astute observations and valuable .suggestions i n the planning and preparat ion of th i s repor t . Thanks are a lso due to Mr. Ian Garnett for h i s able ass istance i n the compilat ion of s t a t i s t i c a l data. F i n a l l y , gra te fu l apprec iat ion i s given for the generous f i n a n c i a l support received from: Panco Poultry Ltd. -Panco Research Award; Univers i ty of B r i t i s h Columbia - Graduate. Fel lowship; and H.R. MacMillan Education Fund - Queen E l i zabe th Scholarship . 1 I . INTRODUCTION Experimental evidence has demonstrated that Eimeria  acervul ina i s not a very pathogenic species of c o c c i d i a . How-ever, i t has been noted that under f i e l d condit ions an i n f e c t i o n with th i s paras i te i s often associated with severe weight losses , morbidi ty , and some degree of m o r t a l i t y . Therefore, i t may be assumed that the pathogenicity of E . acervul ina i s increased by c e r t a i n external factors such as environmental condit ions and d i e t . As i t has been demonstrated that low leve l s of vi tamin A i n the r a t i o n w i l l increase the degree of paras i t i sm i n a l l animals, i t i s suggested that the pathogenicity of E . acervul ina may be increased by a def ic iency of vitamin A. This thes is was examined i n the fol lowing inves t iga t ion which compared the ef fects of E . acervul ina in fec t ions i n b irds ra i sed on 2 l eve l s of v i tamin A i n the d i e t . The pathogenicity was assessed by a comparison of oocyst product ion, weight gains, and m o r t a l i t y . H i s t o l o g i c a l examination was used to determine i f any pathology of the i n t e s t i n a l epithel ium resu l ted from the low vitamin A d i e t . The response of the immune host to a c o c c i d i a l i n f e c t i o n was a lso invest igated i n b irds on both d ie tary vi tamin A l e v e l s . 2 I I . REVIEW OF LITERATURE A. Vitamin A and Coccidiosis The r e l a t i o n s h i p between vitamin A and parasitism i n chickens was f i r s t investigated over 40 years ago by Ackert and coworkers (1927). Experimenting with the chicken round-worm, Ascardia l i n e a t a , Ackert found that t h i s parasite became better established i n birds on a low vitamin A diet than i n birds receiving a normal r a t i o n . Further work by t h i s author (1931) (1942) v e r i f i e d the e a r l i e r findings, and showed that birds on a vitamin A d e f i c i e n t diet had a much slower p e r i s t a l s i s than birds receiving normal levels of the vitamin. He suggested that t h i s fact might account for the establishment of more and larger worms i n the low vitamin A group. Murphy et a l (1938) reported a c o r r e l a t i o n between caecal c o c c i d i o s i s and d i e t . During the course of an experiment, to assess the effects of varying amounts of cod l i v e r o i l i n a complete diet, the birds contacted caecal c o c c i d i o s i s . It was noted that the birds on the lower levels of o i l showed higher mortality, and took longer to regain t h e i r weights, than the birds getting 1% o i l i n t h e i r r a t i o n . Taylor and Russell (1946) established, through a series of experiments, that the minimum requirements for vitamin A recommended at that time were not adequate under 3 s tress condi t ions . Although 1200 I . U . of provitamin A per pound of d ie t were adequate for normal growth, b irds subjected to caecal c o c c i d i o s i s were far more affected by the disease than b irds fed higher l e v e l s . They recommended, therefore , that a 66% safety margin be added to the standard vitamin A l e v e l , to give 2000 I . U . / l b . feed to provide protect ion i n time of s t res s . This increased need for vi tamin A i n a c o c c i d i a l i n f e c t i o n i s a l l i e d to a drop i n vitamin A l i v e r reserves . Davies (1952) found that the normal l i v e r reserve of vitamin A i n a chick was about 292 I .U . /gram, and that th i s dropped to an average of 15 I .U. /gram during a mixed i n f e c t i o n with Eimeria t e n e l l a and E . neca tr ix . In these experiments the b irds were r e c e i v i n g 2100 micrograms of carotene i n the feed, equivalent to 3500 I . U . vi tamin A per pound of feed. Davis a t t r ibuted the l i v e r loss to the lack of conversion of carotene to vitamin A, as th i s changeover occurs i n the epithel ium of the i n t e s t i n e . Erasmus, Levine , and Scott (1958) (1960) bel ieved that the reported drop i n l i v e r reserves of v i tamin A could be due to a lowered feed intake during a c o c c i d i o s i s i n f e c t i o n . Therefore, i n t h e i r inves t igat ions 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 and vitamin A, the controls were r e s t r i c t e d to the same feed intake as the infected b i r d s . Employing th i s refinement of technique, and using a mixed cu l ture of E . t e n e l l a and E . acervul ina as 4 inoculum, they conducted experiments with low and high levels of vitamin A. The following conclusions were reached: 1) Following a c o c c i d i o s i s i n f e c t i o n , the birds on the high levels of vitamin A showed a better appetite, and faster growth rate, than the birds on low levels of dietary vitamin A. 2) The chick's requirement for vitamin A i s increased by c o c c i d i o s i s . This was confirmed by greatly reduced l i v e r storage of vitamin A i n a l l infected groups regardless of the l e v e l i n the d i e t , and when controls were r e s t r i c t e d to the same feed intake. 3) Carotene as a source of vitamin A i s not as e f f e c t i v e as the preformed vitamin. The authors d i d not obtain consistent r e s u l t s on the c o r r e l a t i o n between vitamin A levels and the severity of the disease. However, Garriets (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 vitamin A i n the diet and the severity of caecal c o c c i d i o s i s . In fact he considered the degree of protection so convincing, he recommended that high levels (5000 I.U./kilo.) of vitamin A could 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 natural i n f e c t i o n from con-taminated l i t t e r , so his r e s u l t s are open to question. In a more controlled experiment, Waldroup et al (1963) concluded 5 that mortality was not affected by the l e v e l of vitamin A i n the d i e t , and found there was no histopathological differences i n the caecal epithelium between birds on low and normal vitamin A d i e t s . However they did report a s i g n i f i c a n t difference i n the weight gains, the higher vitamin A groups regaining t h e i r l o s t weight much more rapid l y than the birds on lower vitamin A l e v e l s . Panda and Combs (1964) working on l i v e r vitamin A values, reported that when excessive amounts of vitamin A were fed (up to 24,000 I.U./kilo.) the proportionate amount of vitamin A retained i n the l i v e r at the end of a mixed i n t e s t i n a l c o c c i d i a l i n f e c t i o n was higher than found with normal dietary intakes. B. Physiological Role of Vitamin A The reason for t h i s c o r r e l a t i o n between vitamin A and the severity 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 vitamin A i n the body. It has long been established that vitamin A i s necessary for normal functioning of the epithelium. As early as 1925 Wolbach and Howe studied the tissue changes r e s u l t i n g from avitaminosis A i n r a t s . They reported that a squamous, s t r a t i f i e d , k e r a t i n i z i n g epithelium slowly replaced the normal columnar epithelium of the mucous membranes, although the goblet c e l l s themselves were not affected. They found that the respiratory mucosa was f i r s t affected, followed by the 6 epithelium of the s a l i v a r y glands, reproductive, and urogenital t r a c t . There appeared to be no change i n the epithelium of the stomach and i n t e s t i n e s . However at a l a t e r date, Richards (1935) reported that the e a r l i e s t microscopic i n d i c a t i o n of avitaminosis A i n the rat was k e r a t i n i z a t i o n i n the digestive 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 e i f r i e d (1930) studied avitaminosis A i n chickens and found the t y p i c a l k e r a t i n i z a t i o n of mucosa as reported for the r a t . He reported that the histopathology of avitaminosis A i n the chick was primarily i n the respiratory t r a c t , wherein the normal tissue was replaced by squamous k e r a t i n i z i n g epithelium. This resulted i n a loss of such sp e c i a l i z e d structures as c i l i a t e d c e l l s and goblet c e l l s , and consequently i n a loss of the protective properties of the l i n i n g epithelium. In the alimentary t r a c t the changes were confined to the mucous glands and ducts of the upper regions. There were no experiments conducted on the i n t e s t i n a l epithelium. Elvehjem and Neu (1932), i n studies on avitaminosis A i n chicks, found one of the most consistent findings was an elevated u r i c acid content i n the blood. This l e v e l was dependent on the degree of kidney damage from k e r a t i n i z -ation of the tubules, preventing the normal elimination of u r i c acid from the body. The authors also commented on the character-i s t i c ataxia associated with avitaminosis A and attributed i t to 7 nerve damage rather than to uremia. However, l a ter studies by Wollam and M i l l e n (1955) have shown that an increase i n cere-brosp ina l f l u i d i s the main cause of th i s d i sorder . Roels (1967), i n a review of vi tamin A funct ions , has suggested that the general r o l e of th i s vitamin i n metabolism i s that of a "membrane active" compound, concerned with the s t a b i l i t y of c e l l membranes. I t i s a l so impl icated i n prote in and l i p i d metabolism and the s t a b i l i t y of lysosomes. A very consistent f ind ing i n cases of v i tamin A def ic iency i s a depressed glycogenesis r e s u l t i n g from impaired adrenal gland funct ion . The inf luence of vitamin A on the adrenocortex of chicks was invest igated by G l i c k (1963). He reported that 5-week-old b irds on a low vitamin A d ie t showed reduced adrenal c o r t i c a l funct ion as r e f l e c t e d by a s i g n i f i c a n t l y lower he terophi l increase after ACTH i n j e c t i o n . The r e l a t i o n s h i p between avitaminosis A and immunity to disease has been invest igated by many workers. In 1960, Harmon et a l demonstrated the e f fect of a vitamin A def ic iency on the antibody-producing a b i l i t y of swine. They reported that pigs on a low vi tamin A d ie t produced lower antibody t i t r e s against Salmonella pullorum than pigs on a normal d i e t . Yaeger and M i l l e r (1963) experimented with the s u s c e p t i b i l i t y of rats to Trypanosoma c r u z i under condit ions of avitaminosis A. They found that rats on vitamin A de f i c i en t diets showed less 8 resistance to i n f e c t i o n with t h i s parasite, and also to mis-cellaneous b a c t e r i a l i n f e c t i o n s , than animals receiving adequate amounts of the vitamin. Panda and Combs (1963) working with S. pullorum i n f e c t i o n i n chicks, investigated the effects of suboptimal levels of vitamin A i n the r a t i o n . They fed diets containing 9960 I.U. vitamin A per kilogram of feed, and 975 I .U./kilogram, and wer.e able to demonstrate a s i g n i f i c a n t l y lower agglutination response to S. pullorum, and also a lower bursa weight, i n birds on the lower levels of vitamin A. This implication of the bursa of Fabricius i n antibody response con-firms e a r l i e r work by Glick, Chang, and Jaap (1956). They reported that i n t h e i r investigation, only 8/75 bursectomized birds produced antibodies against S. typhimurium, compared to 63/73 of the controls. Challey (1962), working with Eimeria  t e n e l l a infections i n normal and bursectomized birds, observed a much higher mortality i n the bursectomized chicks. However, these findings were not confirmed i n l a t e r investigations by Pierce and Long (1965), and Rose (1968). C. Eimeria acervulina Tyzzer 1929 E. acervulina i s the species of Eimeria most commonly reported from diagnostic laboratories, and also the least well controlled by coccidiostats. I t affects the upper h a l f of the small i n t e s t i n e , p a r t i c u l a r l y the duodenum, usually causing 9 whi t i sh transverse streaks on the mucosa. This species was -. f i r s t described by Tyzzer (1929), af ter i s o l a t i o n from i n t e s t i n a l l e s ions . Acervul ina i s a L a t i n term which i s d e s c r i p t i v e of the species' i n t e s t i n a l pathology: acerv = a heap and l i n a = l i n e . Infect ions with th i s species are character ized by chronic , rather than acute, symptoms which include anorexia, d iarrhea , and reduced weight gains. E . acervul ina in fec t ions may be d i f f erenc ia ted from i n f e c t i o n with other species of Eimeria by the prepatent per iod of 4 days; large numbers of oocysts passed; and the r e l a t i v e lack of pathogenic i ty . 1. L i f e Cycle The l i f e cyc le begins with the oocyst passed out i n the feces. Tyzzer (1929) reported the oocyst as egg-shaped and having an average measurement of 19.5 by 14.3 microns, with a range of 17.7-20.2 microns i n length, and 13.7-16.3 microns i n width. Becker (1956) disagreed with these measurements, f ind ing the oocyst to be smaller on the average, and reported the average s i ze as 16.4 by 13 microns. Lund and Farr (1965) agree with these measurements, but other authors have l i s t e d d i f f erent average s izes : 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 th i s inves t iga t ion the average s i ze of the oocysts were 17.3 by 15 microns. This great v a r i a t i o n i n 10 reported sizes demonstrates the d i f f i c u l t y i n making a species diagnosis from the oocyst size alone. As with a l l species.of Eimeria, the newly passed oocyst i s unsporulated and consists of a dense, tough, outer covering surrounding a sporont or zygote. Oxygen, moisture, and heat are required for sporulation which begins with the sporont undergoing reduction d i v i s i o n and throwing off a polar body. The sporont then divides into 4 sporoblasts, each of which develops into a sporocyst. Two sporozoites develop i n each sporocyst, giving a t o t a l of 8 sporozoites per oocyst. This arrangement of 2 sporozoites within 4 sporocysts i s character-i s t i c of the genus Eimeria, and separates i t from the genus Isospora, i n which the oocyst contains 2 sporocysts, each having 4 sporozoites. Wilson and Fairbairn (1961), studying the sporulation of E. acervulina when incubated at 30°C, demon-strated maximum sporoblast formation at 10. hours, iand maximum sporozoite development at 20 hours. Edgar (1955) reported sporulation as early as 17 hours at 29°, and Krasser (1963). was able to 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 birds with oocysts showing sporulation at 13 hours. However a l l authors agree that maximum sporulation occurs between 24 and 27 hours at 28°-o 30 C. The sur v i v a l of Eimeria oocysts outside the body depends primarily on moisture as demonstrated by Davies and Joyner 11 (1955). Farr and Wehr (1949) i n a study of oocyst s u r v i v a l i n the s o i l , concluded that humidity i s the l i m i t i n g fac tor , and for normal v i a b i l i t y of the oocyst i t should exceed 90%. They were able to demonstrate the s u r v i v a l of E . acervul ina oocysts for over 86 weeks under moist shady condi t ions . Under normal condit ions a b i r d must ingest the sporulated oocyst to become in fec ted , and i n f e c t i o n begins with the f r a c -ture of the oocyst w a l l , excystat ion, and release of sporozoites i n the i n t e s t i n e . Doran and Farr (1962; 1965), Doran (1966), and Farr and Doran (1962), have reported t h e i r invest igat ions in to the factors involved i n the i n i t i a l sporozoite re lease . According to these authors, the fo l lowing steps are involved: 1) The wal l of the oocyst i s fractured through mechanical act ion of the g izzard and the sporocysts are re leased. 2) Through the act ion of pancreat ic j u i c e , i n the presence of b i l e , there i s a c t i v a t i o n of the sporo-zoi te and a probable d iges t ion of the sporocyst plug which allows the sporozoites to escape. From i n v i t r o experiments i t would appear that the c r i t i c a l enzymes i n the pancreat ic ju i ce 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 to note that B r i t t o n et a l (1964) have found a marked decrease i n excystat ion of oocysts i n birds on a low prote in d ie t which produces low t r y p s i n values . 12 Nyberg and co-workers (1968) have suggested that carbon dioxide i s the i n i t i a l s t i m u l i for the excystation of E. t e n e l l a oocysts, and Lotze and Leek (1968) have demonstrated that some sporozoites, after a c t i v a t i o n with carbon dioxide and enzymes, can escape through the micropyle of the sporocyst and emerge from an undamaged oocyst through an unseen hole. It i s apparent that not a l l the factors necessary for excystation have been elucidated. Experiments by Landers (1960) and Davies and Joyner (1962) have shown that i n t e s t i n a l infections with Eimeria species can be i n i t i a t e d through parenteral injections of sporulated oocysts. Doran (1966 a, b, c) has demonstrated that E. acervulina sporozoites can excyst and penetrate the v i l l a r epithelium as early as 10 minutes after ingestion of sporulated oocysts. He found that the time of greatest penetration was 1-2 hours after dosing, and that the area of greatest invasion was at a point just before the curvature of the duodenal loop, to about 2.5 cm. before the entry of the pancreatic duct i n t o the ascending duodenum. The endogenous stages of E. acervulina are confined to the epithelium of the small 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 (1967). Working with E. necatrix, Van Doorninck and Becker (1957) f i r s t demonstrated that the sporozoites were engulfed by macrophages 13 as they entered the epithel ium of the v i l l i , and were t rans -ported down through the lamina propr ia to the e p i t h e l i a l c e l l s of crypts before i n i t i a t i n g schizogony. Chal ley and Burns (1959) reported the same migration of sporozoites i n E . t e n e l l a , in f ec t ions , and Doran (1966c) i n E . acervul ina i n f e c t i o n s . Doran found that sporozoites are not seen i n the glandular epithelium u n t i l 18 hours after ingest ion of sporulated oocysts, so th i s f ind ing would preclude the p o s s i b i l i t y of d i r e c t pene-t r a t i o n . He described the sporozoite as being pointed a n t e r i o r l y and averaging 8 microns i n length and 1.6 microns i n width when free i n the lumen, and about 4 microns i n length when i n the t i s sue . I t i s character ised by a eos inophi l i c r e f r a c t i l e g lobule . The schizogonous cycle consists of 3 schizont stages according to Warren and B a l l (1967) and Long (1967). However, V e t t e r l i n g and Doran (1966) who have done an exhaustive study in to the endogenous forms of E . acervu l ina , report the presence of a 4th schizont stage. They demonstrated that the schizogonous cycle s tarts with the appearance of the sporozoite i n the e p i t h e l i a l c e l l at the base of the crypt . Here the sporozoite rounds up, d i s t a l to the nucleus and just below the brush border of the c e l l , and begins the trophozoite or growth phase. When i t reaches a c e r t a i n s i ze nuclear d i v i s i o n occurs and the f i r s t 14 generation schizont i s formed. This schizont i s mature from 36-48 hours after ingest ion of the oocyst, measures 10 by 7 microns, and contains from 8-16 merozoites. However, Long (1967) reports the s ize as 5-8 microns. When the merozoites are released they invade c e l l s i n the neck of the crypt and within 8 hours develop in to second generation schizonts , measuring 5 by 4 microns, and containing 16 merozoites on the average. These merozoites, when re leased , penetrate the c e l l s at the base of the v i l l i . The t h i r d generation schizont that matures 16 hours l a t e r , or 56-72 hours after the i n i t i a l inocu la t ion of oocysts, i s s imi lar i n s i ze to the 2nd generation schizont but divides in to only 8 merozoites on the average. These 3rd generation merozoites invade the e p i t h e l i a l c e l l s of the v i l l i , mostly i n the middle reg ion , and develop in to the fourth generation schizonts which are larger than the previous 2 generations, measuring about 8 microns and containing 28-36 large merozoites. The gametogenous stages are i n i t i a t e d by the 3rd or 4th generation merozoites and are usua l ly 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 after oocyst i n o c u l a t i o n , measure about 7 microns i n diameter, and when mature contain about 90 micro-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 anter ior end. The macrogametes are 15 seen f i r s t at 88-90 hours, and when f e r t i l i z e d by the released microgametes develop into oocysts. The oocysts are voided from the body 96-97 hours af ter inocu la t ion of the i n f e c t i n g dose, and th i s short prepatent period 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 . acervu l ina . There are varying reports on the times, s i zes , and locat ions of the d i f f erent generations of schizonts . Tyzzer (1929) could not d i f f e r e n t i a t e between the schizogonous stages and made reference only to the fact that schizonts were found near the t ips of the v i l l i , which would probably re fer to the 3rd or 4th generation. The fact that there i s v a r i a t i o n i n the times of maturation for the i n d i v i d u a l schizonts makes i t d i f f i c u l t to accurately d i s t i n g u i s h the exact stage encountered. However, a l l the invest igators are i n agreement that there are stages i n the endogenous development progressing from the base of the crypt to the t i p of the accompanying v i l l i . Pout (1967) reported that the endogenous phases of the parasi tes seem to "ride" i n the migrat ing host c e l l s so that "trains" of paras i tes can be seen proceeding up the v i l l u s . He found th i s most marked at day 4 when maturing oocysts were seen at the t i p s of the v i l l i with la ter generations of schizonts 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) reported that a l l the stages from sporozoite to gamete occur wi th in the c e l l s 16 i n the gland invaded by the sporozoite and i n the v i l l i adjacent to i t . They speculate that th i s movement further toward the lumen with each successive generation 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 migrat ion. This con-centrat ion of the endogenous forms i n l imi t ed areas accounts for the white-grey ra i sed streaks which character ize a l i g h t i n f e c t i o n with E . acervu l ina . 2. Pathogenicity E . acervul ina has never been considered a very pathogenic species of Eimeria . Tyzzer (1929) (1932) reported that the pathogenicity of the organism depended on the durat ion , as we l l as the in tens i ty of i n f e c t i o n . He stated that extensive invo lve -ment of the epithel ium i n severe cases would ser ious ly i n t e r f e r e with d igest ion and absorpt ion. Dickinson and Scbf i e ld (1939) invest igated the c l i n i c a l manifestations of large doses of E . acervul ina , (35-50 m i l l i o n oocysts /bird) and confirmed Tyzzer's contention that mor ta l i t y was not a fac tor . They demonstrated a decrease i n food consumption and body weight from 4-10 days after i n o c u l a t i o n , and reported that the re turn to pre - inocu la t ion weight took 18-24 days. Dickinson (1941) invest igated the effects of b i r d age and oocysts dose oh patho-g e n i c i t y . He concluded that a d a i l y ingest ion 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 with e a r l i e r work that there was no m o r t a l i t y , the losses i n body weight were temporary, and that i f the i n f e c t i v e dose was large enough there were c l i n i c a l symptoms on day 4-9. These symptoms were anorexia, depression, loss of weight, and scanty, loose droppings. Morehouse and McGuire (1956) (1958) disagreed with the assumption that E . acervul ina could not cause m o r t a l i t y . They demonstrated that successive inoculat ions with large numbers of oocysts could r e s u l t i n 75% morta l i ty i n some groups. They a lso reported that weight losses could reach 20% with a s ing le heavy i n f e c t i o n . These authors a lso demonstrated that the t y p i c a l i n t e s t i n a l les ions of an E . acervul ina i n f e c t i o n j were present only i n very l i g h t in fec t ions of under 500,000 i oocysts. Heavier i n f e c t i v e doses of 5 m i l l i o n or more oocysts / b i r d resu l ted i n a marked inflammation and thickening of the i i n t e s t i n a l w a l l . Hein (1968a) recent ly invest igated the effects of v a r i a b l e doses of E . acervul ina oocysts on young ch icks . She found that the s ize of dose was d i r e c t l y corre la ted with;the sever i ty of the les ions and the degree of weight l o s s . Anorexia and morbidity were seen only at the higher l eve 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 . There was no morta l i ty i n any groups, but pathogenicity of the paras i te was higher i n the younger b i r d s . The large numbers of oocysts needed tio give c l i n i c a l evidence of disease i s r e la ted to the f indings of a l l 18 inves t igators that a high oocyst production i s t y p i c a l of i n f e c t i o n with E . acervu l ina . Morehouse and McGuire (1958) reported that one i n f e c t i v e dose of 5 m i l l i o n oocysts resu l ted i n a t o t a l e l iminat ion 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 sporulated oocysts. 3. Histopathology The patho log ica l f indings i n a b i r d infected with E . acervul ina depend on the number of sporulated oocysts ingested at one time. The previous ly mentioned white patches or streaks r e s u l t i n g from agglomerations of schizonts or oocysts, appear i n l i g h t in fec t ions and are found mainly i n the duodenal loop. As the i n f e c t i o n increases there i s a more extensive invo lve -ment of the same fundamental type accompanied by an edematous swel l ing of the e p i t h e l i a l layer and lamina propr ia causing a thickening of the i n t e s t i n a l w a l l . Hein (1968a) reported that , with heavy i n f e c t i o n s , the les ions extended further down the i n t e s t i n e . An extensive v a s o d i l a t i o n occurs i n heavy infec t ions r e s u l t i n g i n inflammation i n the invaded areas of the i n t e s t i n e . Sloughing of the i n t e s t i n a l epithelium i s present i n both l i g h t and heavy in fec t ions according to Morehouse and McGuire (1958). Pout (1967) has shown that on day 4 there i s a s i g n i f i c a n t decrease i n the height of the v i l l i to the t o t a l mucosal 19 thickness i n E. acervulina i n f e c t i o n s . D. Immunity to Coccidiosis Tyzzer (1929) investigated immunity i n c o c c i d i a l i n f e c -tions and concluded that the most pronounced protection occur-red i n species that penetrate deeply and tend to be retained i n host tissues - E. t e n e l l a for example. He reported poor immunity i n species such as E. acervulina that develop s u p e r f i c i a l l y i n the epithelium. Peterson (1949) reported that repeated exposures to E. acervulina are necessary for a good degree of immunity. He found that 3 l i g h t inoculations of oocysts (10-50 thousand) i n 2-week-old chicks gave complete c l i n i c a l protection when challenged 3 weeks l a t e r . However, although no lesions were present, a few oocysts were s t i l l passed i n d i c a t i n g that complete immunity was not achieved. This confirms e a r l i e r work by Dickinson (1941) who reported the presence of oocysts i n the droppings of c l i n i c a l l y immune birds. . However he did f i n d that a single large inoculum of oocysts was as e f f e c t i v e as several small doses i n confering t h i s immunity. He demonstrated that retention of protective immunity at 70 days, was i d e n t i c a l either following a single dose of 25 m i l l i o n oocysts/bird of E. acervulina or multiple small doses t o t a l l i n g t h i s amount. Rose and Long (1962) investigating complete immunity with Eimeria species, infected birds with 500,000, 5 m i l l i o n , and 10 m i l l i o n 2 0 oocysts of E. acervulina on 3 consecutive weeks. When challenged 18 days l a t e r with 20 m i l l i o n oocysts, the birds were completely immune to the parasite as v e r i f i e d by negative f e c a l oocyst counts. Hein (1968b) produced e f f e c t i v e protective immunity with 2 small immunizing doses 2 weeks apart. The inoculum con-s i s t e d of 80,000 and 160,000 oocysts of E. acervulina, and the birds were challenged with 5 m i l l i o n oocysts at 28 days after the second inoculation. Becker (1935) was one of the f i r s t to study the nature of c o c c i d i a l immunity. He experimented with E. m i y a i r i i (syn. E. nieschulzi) i n rats and concluded that there was no generalized host response. Unable to demonstrate protective humoral a n t i -bodies, he speculated that i n f e c t i o n resulted i n a l o c a l i z e d response, and the nature of t h i s was possibly a " s e n s i t i s a t i o n " of the epithelium which blocked the entrance of sporozoites. Morehouse (1938) agreed with t h i s hypothesis, and i n further studies with E. m i y a i r i i reported that s i g n i f i c a n t l y fewer sporozoites invaded the immune epithelium. However, various investigations into c o c c i d i a l immunity i n chickens have not supported these findings. Both Tyzzer (1932), i n studies on E. necatrix immunity, and Horton-Smith and co-workers (1963) examining the immune response to E. t e n e l l a , found no difference i n sporozoite invasion i n immune or susceptible birds. It was 21 found that the immune mechanism i n h i b i t e d the sporozoites from developing in to schizonts , as no evidence of f i r s t or second generation schizonts were observed. The presence of some type of protec t ive humoral a n t i -bodies was demonstrated by Burns and Challey (1959) and Horton-Smith, Beat t i e , and Long (1961). In separate studies these inves t igators both used the same technique of l i g a t i n g one caecum and then introducing an i n f e c t i o n of E . t e n e l l a into the other. Af ter an appropriate, period of time, the i so la t ed caecum was then exposed to i n f e c t i o n and was found to be r e s i s t a n t . P ierce and co-workers (1962) reported the presence of p r e c i p i t a t i n g antibodies i n b irds made immune to E . t e n e l l a . However the b irds degree of res i s tance to further i n f e c t i o n d id not co incide with the l e v e l of c i r c u l a t i n g ant ibodies . Rose (1963) examined the nature of th i s humoral antibody. Using E . s t iedae , and several chicken species of c o c c i d i a , she was able to st imulate the production of serum antibodies using antigens composed of in tac t oocysts; crushed oocysts; and t i s sue stages of the paras i t e s . The antibodies 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 able to corre la te the presence of antibodies with res is tance to i n f e c t i o n , although the antibodies do exhibi t des truct ive a b i l i t i e s as evidenced by t h e i r capacity to lyse 22 both sporozoites and merozoites i n v i t r o . Rose (1963) has demonstrated l y s i n g of sporozoites i n v ivo fo l lowing intravenous i n j e c t i o n i n b irds showing a high l e v e l of antibody. However, there i s no apparent destruct ion fo l lowing o r a l dosing. It has been conc lus ive ly demonstrated by many authors that there i s no "booster" ef fect on the l e v e l of c i r c u l a t i n g antibodies when the b i r d i s challenged with succeeding infec t ions of c o c c i d i o s i s . I t has a lso been reported that the sera of r e s i s tan t b irds does not always contain antibody. Horton-Smith and co-workers (1963) studied the effects of immunity on the endogenous stages of the paras i te and concluded that both schizogonous and gametogenous stages are i n h i b i t e d . Horton-Smith (1963) has reported that the second schizogony i s the stage of the l i f e cyc le which promotes the immune response under natura l in fec t ions . This f i nd in g was confirmed by Rose (1967b) working with E . t e n e l l a and E . neca tr ix . Rose (1963) has suggested that c i r c u l a t i n g antibodies may be important only when combined with a l o c a l factor such as a c e l l u l a r response. Various i n v e s t i -gators have studied the r o l e of c e l l u l a r immunity as the protect ive factor against c o c c i d i o s i s . Becker (1935) suggested that the l o c a l i z e d response may be due to "sensit ized macro-phages". Van Doorninck and Becker (1957), Doran (1966c), and Chal ley and Burns (1959) i n t h e i r studies on the transport of 23 sporozoites by the macrophages, have speculated on the r o l e these c e l l s play i n the immune response mechanism. Horton-Smith (1963) and Pierce et a l (1962), have demonstrated the presence of numerous pyron inophi l i c c e l l s - resembling plasma c e l l s - and also leukocytes i n the caeca of immunized birds after challenge with E . t e n e l l a . In d i scuss ing these plasma-l i k e c e l l s , Horton-Smith suggests that the macrophages may ingest not only sporozoites but also eosinophi ls which have come i n contact with t h i s antigen. Macrophages may then transform into the plasma c e l l s and produce antibody which destroys the sporozoite and i n h i b i t s further growth. However, to date, the exact mechanism of the immune response to c o c c i d i o s i s has not been e luc idated . . 24 I I I . EXPERIMENTAL METHODS AND DESIGN A. Experimental Diets 1. Basal Diet No. 1: 21% protein. Ingredients Percent i n ra t i o n Ground wheat 74.5 Soybean meal 12.0 Herring 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 kilogram Manganese sulf a t e 12.5 grams Riboflavin 0.33 grams Vitamin D3 440 I.G.U. 2. Experimental Diet No. 2: Basal Diet No. 1 plus 440 I.U. vitamin A* per k i l o -gram of feed. * Vitamin A palmitate - Rovemix A-325. Hoffman-La Roche Ltd. 25 3. Experimental Diet No. 3: Basal Diet No. 1 plus 4400 I.U. vitamin A* per k i l o -gram of feed. B. Brooding One hundred day-old White Leghorn cockerels were placed i n e l e c t r i c a l l y heated battery brooders with wire screen f l o o r s . For the f i r s t 4 days the chicks were a l l fed Basal Diet No. 1 containing no supplementary vitamin A. At 4 days of age the chicks were wing banded, d i s t r i b u t e d randomly into 4 sections of the brooder, and fed t h e i r experimental d i e t s . The 2 top sections of the brooder were used for chicks on Diet No. 2, low vitamin A; and the 2 lower sections were used for chicks on the normal vitamin A d i e t , No. 3. This arrangement prevented the chicks on the low vitamin A diet accidently getting a higher intake of vitamin A from s p i l l e d feed. During the f i r s t 2 weeks i n the brooder any weak or deformed chicks were k i l l e d . Both feed and water were available at a l l times. The chicks were weighed at 18 days of age, and the average for each group was: Diet No. 2: 171 grams Diet No. 3: 170 grams * Vitamin A palmitate - Rovemix A-325. Hoffman-La Roche Ltd. At th i s time the growing chicks were r e d i s t r i b u t e d in to 6 sections of the brooder to give 15-16 chicks to each sect ion instead of 25. A l l the b irds on the low vitamin A d ie t were al igned on one s ide of the battery brooder, and the b irds on the normal vi tamin A on the other. This arrangement el iminated any chance of feed contamination from one group to the other, and yet standardized brooder p o s i t i o n s . At 4 weeks of age the chicks were again weighed and had the fo l lowing averages per group: Diet No. 2: 294 grams Diet No. 3: 296 grams The b irds were then transferred to the experimental room and placed i n non-heated wire b a t t e r i e s . '.' I Brooder Quarantine Methods During the time the chicks were i n the brooder room, s t r i c t quarantine methods were prac t i sed to ensure that they remained c o c c i d i a - f r e e . These included: 1. Before the chicks were introduced, the brooder and troughs were cleaned, s t e r i l i z e d , and re-painted with aluminum paint to e l iminate any previous contamination. 2. The f loor of the room, tab les , and sink were scrubbed with undi luted bleach (sodium hypochlorite) before chick in t roduc t ion , and twice a week thereafter to 27 destroy any oocysts which might have been c a r r i e d i n . 3. A l l c leaning equipment such as brooms, scrapers , sponges, plus containers used i n the brooder room were bought new and never removed from the room. 4. Every e f for t was made to keep the room f l y - f r e e . An i n s e c t i c i d e ba i t was present to k i l l any f l i e s that d id enter. 5. Only one person, the author, tended the chicks during the course of the ent i re experiment. To ensure that these precautions were s a t i s f a c t o r y , f eca l 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 negative f indings that these procedures were success fu l . C. Experimental Design When the b irds were transferred to the experimental room at 4 weeks of age, they were div ided in to 3 weight categories: l i g h t , average, or heavy, based on the average weight of 295 grams. Each of the 2 d ie tary groups was subdivided in to 4, each subgroup containing 10 b i r d s : 3 l i g h t , 4 average, and 3 heavy. These groups were designated as fol lows: Low vitamin A Diet No. 2: Groups A, B, C, D. Normal vi tamin A Diet No. 3: Groups E , F , G, H. 28 Extra b irds were held i n case they were needed for 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 irds were caged i n 3 b a t t e r i e s , each containing 9 sec t ions . The b irds were kept 3 and 4 to a sect ion for most of the experimental work. .As the experiments progressed, and the number of b irds diminished, i t was poss ib le to allow the older b irds more space, p lac ing 2 to a sec t ion . The arrangement of the b irds i n the bat ter ies i s shown i n Figure 1. To ensure that the contro l groups were kept completely free from i n f e c t i o n u n t i l chal lenge, the fo l lowing precautions were undertaken: 1. The non-infected c o n t r o l groups C, D, and G were kept i n a separate battery (No. 3) during the course of the f i r s t experiment. Contro l group H was placed i n the top row of battery No. 2, and was separated from the infected group by a buffer layer of extra b i r d s . Modif icat ions of t h i s plan were made for the second experiment. 2. The bags of feed for each d ie t were div ided in to 2 l o t s . One was used for the infected group and the other for the c o n t r o l s . 3. The c o n t r o l b irds were always fed, watered, and cleaned f i r s t . A l l u tens i l s used for the c o n t r o l groups were Group A Group B Gr oup C Group H E x t r a B i r d s 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. 3 Fig u r e 1 Arrangement of c h i c k s i n b a t t e r i e s f o r Experiment No. 1. separated from those used for the infected groups. A separate coat was worn for tending each group. 4. No personnel , other than the author, were permitted i n the experimental room during the ent i re course of the experiments. Per iod ic checks of the feces from the non-infected groups revealed no evidence of oocysts. D. Methods 1. Oocyst determinations and techniques The cu l ture of E . acervul ina used i n these experiments was received from the Norwich Pharmacal Co. i n August, 1968 and was termed American Cyanamid s t r a i n . O r i g i n a l l y i t was intended to use t h i s cu l ture only to in fec t b irds for oocyst, product ion, but numerous t r i a l s on young b irds d id not produce enough oocysts for a large scale i n f e c t i o n , and so most of the o r i g i n a l cu l ture was used for Experiment 1. The oocysts passed by the birds i n t h i s experiment were c o l l e c t e d and used for Experiment 2, and the oocysts from Experiment 2 were harvested and used for Exper-iment 3. a) C a l i b r a t i o n of oocyst numbers for inocu la t ion (1) T o t a l number of oocysts required for the exper-iment i s determined. 11 (2) The number of oocysts present i n 1 ml . of the stock cu l ture i s ca lcu la ted by d i l u t i n g th i s amount i n 500 ml . water, for example; counting the number i n 1 cu . mm. by use of a hemo-cytometer; m u l t i p l y i n g th i s f igure by 1000 to give the number of oocyst i n 1 m l . ; and m u l t i -p l y i n g t h i s amount by 500 to give the number i n 1 ml . of o r i g i n a l c u l t u r e . (3) The amount of cu l ture required for the experiment i s then removed, centri fuged and washed several times, and resuspended i n p h y s i o l o g i c a l sa l ine (0.9%) to 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 sporulat ion of oocysts As previous ly s tated , oocysts were harvested from Exper-iments 1 and 2 to be used for inoculum i n the succeeding exper-iments. The procedures for c o l l e c t i o n and sporulat ion were sub-jected to much experimentation to determine the best method for the preparat ion of the large numbers of oocysts required for these experiments. The usual 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 for the c o l l e c t i o n of oocysts from the feces. However, i t was found that none of the methods 32 t r i e d y i e lded a high enough percentage of oocysts from the f e c a l mass, and so the fo l lowing modi f icat ion was devised to recover the maximum number of oocysts. (1) The infected feces were c o l l e c t e d and mixed with enough 2% potassium dichrornate, i n a shallow pan, to give a slushy mixture. (2) This mixture was covered with a damp c lo th and l e f t at room temperature (26°c) for 48 hours to allow sporulat ion of the oocysts. There was a great deal of d i f f i c u l t y encountered at th i s stage. The degree of sporulat ion was extremely low, averaging about 20% i n the ear ly e f f o r t s . Incubation at higher temperatures d id not prove e f f e c t i v e , nor d id constant a g i t a t i o n , bubbling i n oxygen, or other modi f icat ions . Storage of the feces i n the r e f r i g e r a t o r was found to be the p r i n c i p a l factor l i m i t i n g sporu la t ion , and i t was found that by processing feces immed-i a t e l y a f ter c o l l e c t i o n a much higher rate of sporulat ion could be acquired. However, the highest sporulat ion effected never extended beyond 50-60%. 33! (3) There was no evidence that add i t i ona l spor-u l a t i o n occurred af ter 48 hours, so at th i s time the mixture was passed i n sequence through 2 s ieves , 30 and 100 meshes per l i n e a r inch re spec t ive ly , using a je t of tap water to force i t through. (4) The co l l ec ted washings were put into 1000 ml . graduates, and water added, i f required , to completely f i l l the container . The suspension was allowed to stand overnight, and then the supernatent was decanted and the sediment mixed with 1000 m l . tap water and again l e f t to s e t t l e out. This operation was repeated 2 or 3 times to remove as much extraneous debris as poss ib le from the oocysts. (5) Fol lowing the l a s t decanting, the sediment was mixed with 2% potassium dichromate and stored ' i n the r e f r i g e r a t o r u n t i l required . This method gave the maximum number of oocysts but d id r e s u l t i n a cu l ture heav i ly contaminated with f e c a l m a t e r i a l . However, no i l l e f fects were observed which could be a t t r ibuted to th i s con-tamination, and as the cu l ture was used short ly after c o l l e c t i o n , the oocysts d id not degenerate from the b a c t e r i a l contamination. c) C a l c u l a t i o n of the d a i l y oocyst production per b i r d The number of oocysts passed d a i l y by an infected b i r d i s one c r i t e r i o n of the b i r d ' s response to the disease. In these experiments, the t o t a l f e c a l output from each inoculated group was c o l l e c t e d d a i l y , s t a r t i n g on the 4th day af ter inocu la t ion and continuing for 7 days. After t h i s time the c o l l e c t i o n s were made every second day for the next 4 to 8 days depending on condi t ions . The t o t a l number of oocysts per b i r d per day was ca lcu la ted by the fo l lowing methods: (1) The t o t a l d a i l y feces from each group was put in to a container and weighed. I f the samples were not processed immediately, they were mixed with enough 2% potassium dichromate to keep them moist and then placed i n the r e f r i g e r a t o r u n t i l processed. (2) The t o t a l sample was then thoroughly mixed i n a basin and a 10 gram sample removed. I f the feces had been mixed previous ly with potassium dichromate a sample ca lcu la ted to be equivalent to 10 grams of untreated feces was used. (3) This 10 gram a l iquot was then mixed with 500 cc of tap water i n a Waring blender and agitated at f u l l speed for lh minutes. 35 (4) Immediately fo l lowing homogenization, 6 separate 1 ml . samples of the suspension were removed and placed i n a tes t tube. This sample was mixed 10 times with a Pasteur p ipet te and one drop was then removed and placed i n one chamber of the hemocytometer. This procedure was repeated to f i l l the second chamber. (5) The t o t a l number of oocysts present i n the 5 large squares of each chamber were counted. I f the 2 chambers showed more than a 10% d i s -crepancy i n the counts, a further 2 samples were counted and an average taken. (6) The t o t a l number of oocysts per sample was c a l -culated as fol lows: Average hemocytometer count = Y (number i n 1 cu . mm) Number i n 1 ml . = 1000Y Number i n 500 ml . = 500 X 1000Y The number of oocysts i n 500 ml . i s equivalent to the number i n 10 grams of feces, and from th i s f igure the t o t a l number of oocysts per group could be determined. This amount was d iv ided by the number of b irds i n the group to give the oocys t s /b ird /day . Using th i s method of c a l i b r a t i o n , each oocyst counted i n the hemocytometer was equivalent to 500,000 oocysts, or 50,000 per gram of feces. Therefore, i f the numbers were below th i s f igure per gram, the sample would appear to be negative. Due to the large amount of extraneous matter present i n the f e c a l samples, a smaller d i l u t i o n factor was not poss ib le when using the hemocytometer. Therefore, to avoid the fa l se assumption that a sample having below 50,000 oocysts per gram was negative, any sample showing one or no oocysts was subjected to the McMaster Counting Technique as follows: (1) The t o t a l d a i l y f eca l mass per group was we l l mixed and a 10 gram a l iquot removed and mixed i n a beaker with 95 ml . of a saturated so lu t ion of sodium c h l o r i d e . (2) Using a Pasteur p ipe t t e , the 0.15 ml . chamber of a McMaster s l i d e was f i l l e d with the coarse suspension. The oocysts f loated free of the debris and pressed against the bottom of the scored cover s l i p where they could be counted. (3) The average of 2 counts was taken to give the t o t a l number of oocysts i n 0.15 ml . The t o t a l number of oocysts i n 10 grams was ca lcu la ted as fol lows: T o t a l oocysts i n 0.15 ml . = Y T o t a l i n 95cc (10 grams) = Y X 100 X 7 Using th i s technique, each oocyst counted was equivalent to 700 oocysts, or 70 per gram of feces. d) C a l c u l a t i o n of the reproductive index The reproductive index or reproductive p o t e n t i a l i s a measure of immunity or res i s tance offered by the b i r d against a c o c c i d i a l i n f e c t i o n . As such i t i s a useful t o o l i n the assess-ment of the host 's reac t ion and was ca lcu la ted for each b i r d at the conclusion of each experiment. I t i s ca l cu la ted as follows: R I = T o t a l number of oocysts passed T o t a l number of oocysts inoculated 2. H i s t o l o g i c a l techniques a) F i x a t i o n Immediately after a b i r d was k i l l e d , the duodenal loop was removed and placed i n Bouin's f i x a t i v e . The formula for th i s so lu t ion i s : Saturated p i c r i c ac id 75cc Formalin 25cc Ace t i c ac id 5cc b) Dehydration and embedding (1) A 3-5 mm sect ion of the duodenum, d i s t a l to the turn of the loop, was removed and placed i n a mesh p l a s t i c capsule (Tissue Tek) for p r o -cess ing . 38 (2) Dehydration started with 3 changes of 70% ethyl alcohol, within a 48 hour period, to remove as much p i c r i c acid as possible from the t i s s u e . This was followed by 2 hours or more i n 95% alcohol; 2 changes i n 99% alcohol of one hour each; and clearing i n 2 changes of x y l o l , one hour each. (3) The dehydrated tissue was then transferred to 3 changes of wax i n a 60° oven, and embedded i n "Tissue Tek" embedding rings. c) Sectioning The sections were cut at 5 microns using a L e i t z micro-meter. The ribbons were floated on water, and the sections af f i x e d , 3-4 per s l i d e , using egg albumen or g e l a t i n . d) Staining A great deal of experimentation was undertaken to fi 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 sporozoites i n the t i s s u e . P a t t i l l o (1959) recommended PAS Feulgen for t h i s purpose; Clarkson (1958) and Shortt and Cooper (1948), a Giemsa modification; Horton-Smith and co-workers (1963) recommended McFarlane's (1944) Picro-Mallory stain; and Challey and Burns (1959) used Groat's hematoxylin followed by Shorr's s t a i n . A l l these recommendations were t r i e d i n addition to Groat's 39 Tetrachrome S t a i n . None of these methods proved very s a t i s -factory and eventual ly 2 methods were used exc lus ive ly . These were a standard hematoxylin and eosin technique and a modified Groat's Tetrachrome S t a i n . The standard method followed was: (1) The sections were dewaxed through 2 changes of x y l o l and hydrated through 99%, 95% and 70% a lcoho l s . (2) Stained i n H a r r i s ' Hematoxylin for 5-10 minutes, then decolourized i n ac id a lcohol and blued i n ammonia water. (3) Counterstained i n e i ther aqueous eosin (standard H and E ) , or i n Biebr ich Scarlet-Orange II s t a i n (Groat's Tetrachrome). (4) Washed, dehydrated, and c leared i n x y l o l . (5) The .sections were mounted with "Pro-Tex" mounting medium and covered with 22mm by 50mm c o v e r s l i p . With both these s ta ins the r e f r a c t i l e vacuole i n the sporozoite was stained red , making i t easy to i d e n t i f y . How-ever, although the Groat's modi f icat ion gave a better d i f f e r -en t ia t i on of blood c e l l s , i t tended to overs ta in , and therefore the standard H and E was the recommended method. E . Experiments The 8 groups of b irds were used i n 3 d i f f e r e n t exper-iments. The f i r s t experiment u t i l i z e d 5-week-old b i r d s , the 40 second 9-week-old birds, and the t h i r d 19-week-old birds. 1. Experiment Number 1 a) Object a) To assess the response of 5-week-old chicks, on 2 levels of vitamin A, to a primary i n f e c t i o n with E. acervulina. b) To immunize half the chicks of each dietary group against E. acervulina. b) Design The groups of chicks were arranged according to the pre-viously discussed experimental-design. Four groups, each con-s i s t i n g of 10 birds, were infected. The groups were as follows: Low vitamin A diet: Groups A and B infected Groups C and D controls Normal Vitamin A diet : Groups E and F infected Groups G and H controls. c) Inoculation The culture used for t h i s f i r s t experiment was the o r i g i n a l culture of sporulated E. acervulina oocysts received from Norwich Laboratories. An inoculum was c a l i b r a t e d to con-t a i n 3 m i l l i o n oocysts per ml. of physiological s a l i n e , and each b i r d from groups A, B, E, and F was inoculated o r a l l y with 41 1.0 ml . - 3 m i l l i o n oocysts. This approximated 8000 oocysts per gram of body weight. The b irds were starved 24 hours before i n o c u l a t i o n . d) Methods (1) S t r i c t hygienic and quarantine techniques were employed to ensure that the contro l groups remained free 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 infected b irds were cleaned every second day, from day 4 to the end of the experiment, to ensure there was no r e -i n f e c t i o n of the b i r d s . (3) Fecal 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 c a l -cu la ted . (4) The infected b irds were weighed at the time of i n o c u l a t i o n , on day 4, and on every second day fo l lowing th i s for about 20 days. (5) The non-infected b irds were weighed weekly. (6) In th i s experiment, and i n the 2 that fol lowed, the weights between groups were sub-jected to an analys is of variance to determine 42 i f the differences were s t a t i s t i c a l l y s i g n i f i c a n t . 2• Experiment Number 2 a) Object To compare, i n 9-week-old birds raised on 2 levels of vitamin A: (1) The degree of acquired immunity retained 4 weeks after a primary i n f e c t i o n with E. acervulina. (2) The early tissue stages of E. acervulina i n immune and non-immune chicks. (3) The response bf susceptible 9-week-old chicks to a primary i n f e c t i o n with E. acervulina. b) Design Before t h i s experiment was conducted, the groups of birds were re-arranged i n numbers, keeping the weights as uniform as possible. The groups were adapted for t h i s experiment as follows: Low vitamin A diet: Group A - 10 birds - To be given a second i n f e c t i o n . Group B - 8 birds - Non-infected immune controls. Group C - 10 birds - To be given a primary i n f e c t i o n . Group D - 9 birds - Non-infected non-immune controls. 43 Normal vitamin A diet: Group E - 11 birds - To be given a second i n f e c t i o n . Group F - 9 birds - Non-infected immune controls. Group G - 11 birds - To be given a primary i n f e c t i o n . Groupi.H;- 9 birds - Non-infected non-immune controls. The i n f e c t i o n of groups A and E would determine the degree of immunization retained 4 weeks after a primary infection; and the i n f e c t i o n of groups C and G would examine the response of these 9-week-old birds to a primary i n f e c t i o n . c) Inoculation The culture consisted of sporulated oocysts of E_. acervulina harvested 4 weeks e a r l i e r from Experiment No. 1. The inoculum was calib r a t e d to give 5 m i l l i o n oocysts per ml. of saline and the birds were infected per os with 2 ml., giving 10 m i l l i o n oocysts per b i r d . This averaged out to 11,000 oocysts per gram of body weight. d) Methods (1) Three birds from each group were k i l l e d 3 hours after inoculation, and another 3 birds were k i l l e d at 48 hours. The duodenum was removed from each b i r d and fixed i n Bouin's solution. (2) Pooled f e c a l samples from each group were taken d a i l y and processed to determine the t o t a l number of oocysts passed per b i r d per day. 44 (3) Standard hygienic practises were observed to prevent i n f e c t i o n of the control groups. (4) The groups B, D, F, and H, were held for 10 weeks following Experiment No. 2, and during t h i s period they were weighed once a week. (5) The remaining birds i n Groups A, E, C, and G were eliminated 3 weeks after inoculation when Experiment No. 2 was terminated. 3. Experiment Number 3 a) Object To compare the response of 19-week-old birds, raised on 2 leve l s of dietary vitamin A, to an i n f e c t i o n with E. acervulina i n order to determine: (1) The degree of immunity retained by birds 14 weeks after a primary i n f e c t i o n . (2) The response of older birds to a heavy primary i n f e c t i o n . b) Design A l l the remaining groups were infected i n t h i s 3rd exper-iment. They were: Low vitamin A diet: Group B - 7 birds - primary i n f e c t i o n 14 weeks e a r l i e r . 45 Group D - 8 birds - No previous i n f e c t i o n . Normal vitamin A diet: Group F - 8 birds - primary i n f e c t i o n 14 weeks e a r l i e r . Group H - 8 birds - No previous i n f e c t i o n . c) Inoculation The oocysts used for t h i s experiment were harvested from Experiment No. 2. They were stored i n the usual manner and re-suspended i n physiological saline to give 2.5 m i l l i o n oocysts ml. A l l the birds were inoculated o r a l l y with 25 m i l l i o n oocysts, a dose of 10 ml. per b i r d . This was roughly equivalent to 14,000 oocysts per gram of body weight. d) Methods (1) As there were no non-infected controls i n t h i s experiment, the hygienic techniques were not as s t r i c t as i n the previous experiments. How-, ever, precautions were maintained to omit extraneous contamination and to prevent re-i n f e c t i o n from the contaminated feces. (2) Two birds from each group were k i l l e d on the 4th day after inoculation. These birds were autopsied and c l i n i c a l evidence of the disease was noted." The duodenal loop, and any other 46 areas of the in te s t ine showing gross l e s ions , were removed and f ixed i n Bouin's so lut ion for sect ioning and h i s t o l o g i c a l examination. Da i ly pooled fecal, samples from each group were taken and the number of oocysts determined per b i r d . A record of weights was maintained fo l lowing the rout ine of the previous two experiments. 47 IV. RESULTS A. Experiment No. 1 This experiment examined the response of 5-week-old chicks, on low and normal levels of vitamin A, to a primary-i n f e c t i o n with E. acervulina. C l i n i c a l l y , the birds on low vitamin A rations (AB) were more affected by the disease than the birds on normal vitamin A rations (EF). Both groups of birds began to show symptoms of the disease at day 3, when a s l i g h t drop i n feed consumption was observed. A watery diarrhea was apparent by day 4, with scanty mucoid droppings c h a r a c t e r i s t i c a l l y stained green. Anorexia and adipsia were pronounced i n both groups on day 4 and 5, and the anorexia continued i n the low vitamin A group u n t i l about day 14. The normal vitamin A group (EF) regained t h e i r appetites by day 7-8. The droppings of the low vitamin A group (AB) were scantier and more watery than group EF throughout the acute stage of the disease (days 4-6), and did not return to normal consistency u n t i l day 12, compared to day 7 for group EF. There was no mortality i n the normal vitamin A group, but 2 birds died in group AB. The f i r s t died on day 10 and showed a 27 percent loss i n body weight at that time, and the second died on day 12 after losing 22 percent body weight. Neither bird revealed 48 severe i n t e s t i n a l pathology, but both were very dehydrated and atonic. The low vitamin 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, giving a reproductive index of 205. The birds on normal dietary levels of vitamin A had a t o t a l average production of 360 m i l l i o n oocysts and a R.I. of 120 for the same period. As shown i n Table 1 both groups had a peak oocyst; production on day 5, but group AB maintained a higher d a i l y l e v e l throughout the course of the experiment. Periodic f e c a l examinations for the presence of oocysts were made during the 3rd week, and i t was found that group EF was e s s e n t i a l l y negative by day 18, but the birds of group AB were s t i l l passing several m i l l i o n oocysts/bird/day. However, a l l the birds were negative by the time the 2nd exper-iment was instigated at day 28. Table II shows the t o t a l weights of the 4 experimental groups during the course of Experiment No. 1. The groups a l l had similar weights at the s t a r t of the experiment but by day 4, the two infected groups of birds were s i g n i f i c a n t l y lower i n weight (P <£ .01) than the controls. The infected low vitamin A group of birds (AB) were an average of 28 grams l i g h t e r i n weight than group EF on day 6 and th i s difference increased to 80 grams by day 14. There was no s i g n i f i c a n t difference i n 49 weight between the 2 control groups throughout the experiment. The low vitamin A control group was about 4 percent less i n average t o t a l weight than the other 3 groups at the s t a r t of the experiment, and maintained th i s difference with the other control group u n t i l the end. However there was a marked difference between the 2 infected 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 diminishing, was maintained u n t i l day 21 (P <^ ".05) . By day 14 of the experiment the low vitamin A group had l o s t 25 percent of i t s body weight compared to i t s control, while the normal vitamin A group had l o s t only 16 percent as i l l u s t r a t e d i n Figure 2. It took several weeks before the weights of the infected groups were s t a t i s t i c a l l y equal to those of the control groups as shown i n Table IV. Figure 3 graphically compares the weights of infected groups B and F, and t h e i r non-infected controls, D and H respectively, from November 1st to February 23rd. During t h i s 4 month period the non-infected controls did not show a s i g n i f i c a n t difference i n weight. Groups B and F were infected at 5 weeks, and at 6 weeks showed a s i g n i f i c a n t (P <^.01) d i f f -erence i n weight from t h e i r control groups. This difference was maintained i n the normal vitamin A groups, F and H, u n t i l 4 weeks 50 after the date of inoculation. The low vitamin A groups B and D showed a s i g n i f i c a n t difference i n the t o t a l weights of the birds u n t i l 6 weeks after inoculation. However, there was a s i g n i f i c a n t difference i n weight CP <Cbetween groups B and H u n t i l 9 weeks after inoculation at which time, the birds were 14 weeks old. The weight changes which occurred during the 2 weeks following i n f e c t i o n are shown i n Table I I I . This table gives the weight gained or l o s t i n 2 day interv a l s from day 4 to 14 of i n f e c t i o n , plus the infected groups weight gains compared to the controls. During the acute stage of i n f e c t i o n , between days 4 and 6, the low vitamin A group l o s t an average of 25 grams per bi r d , while the normal vitamin 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 loss i n weight while group EF had started to gain and by day 14 both groups were showing si m i l a r weight increases. However, the low vitamin A group showed an average gain of only 64 grams per bi r d during the 14 days, compared to 145 grams for the normal vitamin A group. This was s i g n i f i c a n t l y d i f f e r e n t at the .01 pr o b a b i l i t y l e v e l . The controls showed no s i g n i f i c a n t difference i n the t o t a l average weight gained over t h i s same period. Figure 4 i l l u s t r a t e s t h i s difference i n weight gains, and gives a graphic picture of the deleterious effects of a low vitamin A 51 d i e t . The chicks on the low vitamin A r a t i o n had a weight gain 63 percent lower than that of t h e i r contro l group, while the infected b irds on normal vitamin A rat ions 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 erent react ions to an i n f e c t i o n with E . acervul ina i n 5-week-old birds on normal and low vitamin A r a t i o n s , shows that the sever i ty of the disease i s almost doubled i n the b irds on low d ie tary vitamin A. The performance index has been used by Stock? Stevenson; and Hymas (1967), and Stevenson (1969), to evaluate the t o t a l effects of c o c c i d i o s i s i n d i f f eren t groups of b i r d s . It encompasses the weight gain , mor ta l i t y and oocyst product ion, and although an a r t i f i c i a l method of comparison, i t does give an i n d i c a t i o n of t o t a l e f f ec t . I t was modified i n th i s inves t iga t ion to give some .standard of comparison between experiments, and the formula devised was: average gain + percentage s u r v i v a l - oocyst production i n m i l l i o n s . Using th i s formula the P . I . of the low 10 vi tamin A group (AB) was 64 + 90 - 61 = 93, and that of the normal vitamin A group (EF) was 145 + 100 - 36 = 209. Thus i t can be seen that by taking morta l i ty into account plus the sever i ty of the disease i n the surv ivors , the low vitamin A group was over twice as severely affected by the i n f e c t i o n with E . acervul ina as was the normal vitamin 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 vitamin A d ie t (AB) Normal vitamin A d ie t (EF) 4 5 6 7 8 9 10 12 14 T o t a l oocysts per b i r d Reproductive Index 55 125 95 85 75 70 55 (19) 35 (18) 20 (18) 615 205 20 125 60 50 30 30 25 15 5 360 120 * Surviv ing b irds out of 20. 53 TABLE II 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 Infected birds Non-infected birds Low vitamin Normal vitamin Low A Normal A Day A diet (AB) A diet (EF) diet diet 0 396 397 380 398 2 434 436 4 409 416 465 486 6 383 412 8 378 ** 433 10 397 (19)a ** 455 12 425 (18) ** 499 14 460 (18) ** 542 601 632 16 497 (18) ** 570 18 538 (18) ** 621 20 581 (18) * 662 737 786 a. birds surviving out of 20. * s i g n i f i c a n t difference i n weight (P <T.05) between AB and EF. ** highly s i g n i f i c a n t difference i n weight (P <^.01) between groups AB and EF. 54 TABLE III 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 vitamin A rat i o n Infected (AB) Control (CD) Normal vitamin A rat i o n Infected Control (EF) (GH) 0-4 4-6 6-8 8-10 +15 -25 - 3 +19 +85 +19 - 4 + 21 +22 +88 10-12 12-14 +27 +36 +45 +43 Total weight gain i n 2 weeks* +64c +221a +145b +234a Grams per day of gain 4.5 +16 10.5 +17 * means showing the same l e t t e r did not d i f f e r s i g n i f i c a n t l y (P <T.01) . 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 vitamin A Infected Control B D Normal vitamin A Infected control F H 180 5 - infected 377 6 8 10 12 14 16 18 377 564 820 1066 1348 1520 1695 178 369 470 741 997 1221 1431 1579 1695 175 387 420 674 984 1188 1472 1660 1818 171 392 500 802 1076 1293 1567 1690 1848 * no s i g n i f i c a n t difference (P <^.05) between groups B, D, F, H. 56 3)AV 0 DAY 14-Figure 2. The e f f e c t of dietary on the mean weight of following i n f e c t i o n wi vitamin A l e v e l 5-week-old chicks th E. acervulina 57 Figure 3. A comparison of the mean weights of infectedu and non-infected chicks, on 2 dietary levels of vitamin A, over a 4 month period 58 Low VITAMIN A NoftrtflL VITAMIN A r Z W 5 I S O loo -• So •• CONTROL I N ^ C T E J ) C O N T R O L Tupecreo) Figure 4. The eff e c t of dietary vitamin A l e v e l on the 2 week weight gain of 5-week-old chicks following i n f e c t i o n with E. acervulina 59 B. Experiment No. 2 The primary purpose of th i s experiment was to study the ear ly endogenous forms of E . acervul ina i n immune and non-immune 9-week-old chicks rece iv ing 2 d i f f eren t l eve l s of vitamin A i n the r a t i o n . Six b irds from each group were k i l l e d for h i s t o l o g i c a l examination of the i n t e s t i n e s , leaving only 4-5 b irds per group, and t h i s small experimental number may have resu l ted i n some of the c l i n i c a l r e s u l t s appearing less s i g n i f i c a n t than they a c t u a l l y were. 1. C l i n i c a l Results C l i n i c a l l y , the immune groups A" and E , showed l i t t l e response to t h e i r second i n f e c t i o n . A s l i g h t anorexia was noted on days 4-6 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 feces. The non-immune groups, C and G, rece iv ing t h e i r primary i n f e c t i o n with E . acervu l ina , mani-fested the t y p i c a l symptoms of the disease on days 4-8, but these symptoms were not as severe as i n the younger b irds of Experiment 1. There was no morta l i ty i n e i ther of these groups, but both groups had a marked anorexia and watery, scant droppings on days 4-6. Diarrhea was most severe on day 5, e s p e c i a l l y i n the low vitamin A group (C). The b irds on normal l eve l s of v i tamin A (G) regained t h e i r appetites more quick ly 60 than those i n group C, and showed less evidence of diarrhea after day 5. A comparison of the oocyst numbers passed i n the course of the disease i s shown i n Table V. The protective effects of immunity from a primary i n f e c t i o n can be seen i n groups A and E. There was no evidence of the disease on day 4, and day 5 showed a low peak for both groups. The low vitamin A group (A) appeared to have a more s o l i d immunity as the birds passed an average of only 6 m i l l i o n oocysts per b i r d i n the 14 days following inoculation, compared to 70 m i l l i o n for group E. The more rapid drop i n d a i l y oocyst production i n group A, compared to group E, i s also i n d i c a t i v e of a better immune response. Groups C and G, receiving t h e i r primary i n f e c t i o n at 9 weeks, produced high numbers of oocysts, but unlike the 4-week-old birds of Experiment 1, the peak production was on day 4 as well as 5. The d a i l y course of oocyst production duplicated the findings of Experiment 1. The low vitamin A group maintained a high d a i l y l e v e l , while the normal vitamin A group peaked on day 4 and dropped abruptly to lower and more rapidly decreasing l e v e l s . The r e l a t i v e t o t a l s of oocysts between the two groups on a primary i n f e c t i o n also followed the re s u l t s of Experiment 1. The low dietary vitamin A group birds had twice the number of oocysts, and twice the reproductive 61 index, of the birds from the normal vitamin A group. Table VI compares the t o t a l average weight, over a period of 2 weeks, of the birds immunized i n Experiment 1 when ha l f the group i s re-infected with a large number of oocysts. It can be seen that the second i n f e c t i o n did not aff e c t the average weight i n either the low or normal dietary vitamin A groups. In fact, as shown by the t o t a l weight gains over t h i s same period i n Table IX, the i n f e c t i o n even appeared to accelerate weight gains i n the low vitamin A group (A). The 2 groups of birds, C and G, subjected to a primary i n f e c t i o n of E. acervulina at 9 weeks, did not suffer the weight losses of t h e i r 4-week-old counterparts i n Experiment 1. As shown i n Table VII, the low vitamin A group (C) l o s t an average of 19 percent body weight, i n the 14 days following i n f e c t i o n , compared to the non-infected controls. The normal vitamin A group (G) showed only a 4 percent loss i n body weight. Table VIII shows the weight gains and losses during these 14 days. It can be seen that the maximum weight loss was on day 6 i n both groups, although the gain between day 0 and 4 was far below that of the two control groups. The low vitamin A group (C) showed a greater weight loss during the acute stages of the disease and did not match the recovery rate of group G. Figure 5 i l l u s t r a t e s the difference i n weight gain between these 2 groups, and demonstrates that group C had a weight gain equal to 62 only 26 percent of that made by i t s control group D, and only 30 percent of the gain made by group G. The normal vitamin A group (G) had a weight gain equal to 80 percent of i t s control group H. The degree of protection afforded by immunization with a single i n f e c t i o n of E. acervulina i s shown i n Table IX. Even with the small numbers of birds used i n t h i s experiment i t can be seen that with low dietary levels of vitamin A, the pro-te c t i v e properties of the immunization are well defined. The immune group A had a weight gain following i n f e c t i o n which p a r a l l e l e d that of the uninfected control group D. But the non-protected group C had a weight gain 74 percent below that of the control group. With normal levels of vitamin A i n the ra t i o n , the degree of protection obtained by immunization i s not so dramatic. Group E had a weight gain equal to only 88 percent of the uninfected control group H, but equalled that of i t s immune control F. However group G, with a primary i n f e c t i o n , had a weight gain equal to 80 percent of the uninfected control group H,so the r e l a t i v e degree of protection offered by immunization i s not as large as seen i n the low vitamin A groups. The performance indices for the 4 infected groups i n Experiment 2 are shown i n Table X, and these confirm the advantage of immunization i n the low vitamin A groups. The 63 di f ference i n the P . I - for the 2 groups rece iv ing a primary i n f e c t i o n with E . acervul ina (C and G) , i s p a r t i c u l a r l y s t r i k i n g . The normal vi tamin A (G) group has a P . I . more than 3 times that of the low vitamin A group (C). 2. H i s t o l o g i c a l Examination .3 hours post inocu la t ion: A l l the sections revealed eos inophi l i c r e f r a c t i v e bodies i n the epithel ium of the crypts and v i l l i epithel ium which were construed to be sporozoi tes . This presence of sporozoites i n the crypts 3 hours after inocu la t ion does not conform with most reports which state that sporozoites take 12-18 hours to migrate to the crypt s . I t would suggest that there i s a d i r e c t invasion of the crypts with a heavy inocu la t ion of oocysts. A very obvious f ind ing was an accumulation of heterophi l s i n the lamina p r o p r i a . There was a very heavy concentration 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 irds showed sections s i m i l a r to the other groups. The sections from the other 2 b irds of group C revealed only a few he teroph i l s . There d id not appear to be any di f ference i n e p i t h e l i a l i n t e g r i t y between the groups, and there was no evidence of squamous epithel ium and k e r a t i n i z a t i o n i n the groups on low vitamin A l e v e l s . 64 48 hours post inoculation: In the 2 immune groups, A and E, examination of the sections showed very few trophozoites and almost no schizonts. One b i r d i n the low vitamin A group (A) appeared to have an area of i n f e c t i o n retained from the immunizing dose given i t 4 weeks e a r l i e r . There was evidence of gametocytes and oocysts present i n the tissue of one crypt and accompanying v i l l u s showing that t h i s b i r d was harbouring a s l i g h t latent i n f e c t i o n . There were sporozoites present i n the crypts and v i l l i of almost a l l the immune birds but very few had developed into trophozoites. The 2 groups receiving t h e i r primary i n f e c t i o n , C and G, showed large numbers of trophozoites and schizonts. The schizonts measured about 5 microns i n diameter and were probably 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 areas encompassing a crypt and neighbouring v i l l i . Most of the forms were near the base of the v i l l i , but i n many areas the whole of the e p i t h e l i a of the v i l l i was par a s i t i z e d with endogenous forms i n varying degrees of development. There were also many sporozoites s t i l l unchanged i n the tissue, and i n fact one b i r d from the normal vitamin A diet-had only sporozoites present and showed no evidence of trophozoites or schizonts. There was no d e f i n i t e evidence that the low dietary vitamin A groups had a greater incidence of invasion or schizont development. However, 65 there was an ind i c a t i o n that a more extensive area of the int e s t i n e was invaded i n the suboptimal groups, and a larger sample number may reveal 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 Low vitamin A rat i o n Normal vitamin A rat i o n Immune (A) Non-immune (C) Immune (E) Non-immune (G) 0 200 0 230 4.5 10 12 14 ,02 .01 .005 210 60 50 130 110 65 25 10 18 17 25 8 .6 .1 .006 .005 110 20 15 15 20 5 10 Total oocysts per b i r d 6 Reproductive Index 0.6 860 86 70 426 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 Low vitamin A Normal vitamin A Day Infected Non-infected Infected Non-infected (A) (B) (E) (F) 0 782 735* 828 * 860 4 883 805* 918 * 945 6 938 835 951 985 8 968 880* 989 1030* 10 1013 * 920 1024 1065* 12 . 1032 961 1046 1108 14 1076 * 990 1096 * 1130 * 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 Infected (C) v i tamin A Non-infected (D) Normal Infected (G) v i tamin A Non-infected (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 Per-centage loss i n body weight com-pared to con-t r o l s 19% 4% * extrapolated weights 69 TABLE VIII 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 Low vitamin A levels Normal vitamin A levels Days Infected non-infected Infected non-infected (C) (D) (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 * extrapolated 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 vitamin A le v e l s : Grams Group A: Immune, infected: 294 B: Immune, non-infected 255 C: Non-immune, infected 74 D: Non-immune, non-infected 285 Normal vitamin A lev e l s : Group E: Immune, infected 268 F: Immune, non-infected 270 G: Non-immune, infected 246 H: Non-immune, non-infected 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 Index A - Low vitamin A rati o n - secondary i n f e c t i o n 393 C - Low vitamin A r a t i o n - primary i n f e c t i o n 88 E - Normal vitamin A r a t i o n - secondary i n f e c t i o n 361 G - Normal vitamin A r a t i o n - primary i n f e c t i o n 303 * 2 week average weight gain + percentage su r v i v a l -oocyst production 10 72 Low V I T A M I N A NORMAL VITAMIN A ISO--z er 2 H 15*0--X Ui 3 loo--5 o - . CONTROL TnfGCT£S Figure 5. The ef fect of d ie tary vitamin A l e v e l on the 2 week weight gain of 9-week-old chicks fo l lowing i n f e c t i o n with E . acervul ina 73 C. Experiment No. 3 In t h i s experiment the birds were subjected to a massive i n f e c t i o n of 25 m i l l i o n oocysts E. acervulina to assess the response of immune and non-immune older birds raised on 2 dietary l e v e l s of vitamin A. The term "immune" i s used to denote a primary i n f e c t i o n at an e a r l i e r date, and does not indicate any p a r t i c u l a r degree of immunity. 1. Primary Infection The 2 groups receiving a primary i n f e c t i o n with J). acervulina were D, low vitamin A diet, and H, normal vitamin A d i e t . Both groups of birds showed a severe reaction to the i n f e c t i o n . A l l birds had diarrhea by day 3, and the feces were almost completely l i q u i d with evidence of sloughed epithelium and b i l e present. By day 4 the birds of group D showed complete anorexia and adipsia but the birds of group H were not so severely affected. In both groups the birds were very depressed with congested or cyanotic combs and wattles, and pale, dry shanks. By day 6 the birds on normal vitamin A levels began to recover; the anorexia lessened and the feces, although scant and mucoid, were less l i q u i d . By day 8 the birds of th i s group looked normal, and by day 9 t h e i r appetites had completely returned and the feces were back to normal. The birds on the low dietary vitamin A l e v e l s , group D, had a much stronger reaction to the i n f e c t i o n . 74 The depression and diarrhea were more marked, and 4 out of the 6 birds died from the disease. The f i r s t death was on day 9, and t h i s b i r d had l o s t 25 percent of i t s body weight compared to i t s inoculation weight. Bird 2 died on day 11 having:lost 35 percent body weight; b i r d 3 on day 13 with a 40 percent loss i n body weight; and b i r d 4 on day 17 with a 48 percent loss i n weight. Post mortem examination of the dead birds revealed extreme emaciation and dehydration i n a l l of them, plus a nephritis characterised by pale, mottled kidneys containing heavy urate deposits. A l l birds showed some evidence of i n t e s t i n a l damage, mostly involving the duodenum. Bird 1 had the most severe pathology with areas of hemorrhage extending to the duodenal-jejunum junction. The other 3 birds did not show such severe damage but a l l revealed evidence of e p i t h e l i a l sloughing and the walls of the in t e s t i n e were thinner than usual. The birds of group D were moribund during most of the experimental period, and no evidence of normal feces was seen u n t i l day 12 i n the surviving birds. Anorexia was complete, i n a l l birds that died, from day 3 u n t i l t h e i r time of death. Table XI shows the oocyst production of the 4 groups involved i n t h i s 3rd experiment. The group D t o t a l i s probably lower than could be expected i f there had been a higher rate of s u r v i v a l . This low vitamin A group had i t s peak production on 75 day 5, while the normal vitamin A group, H, had a peak oocyst production on day 4 followed by an abrupt drop on day 5. It i s i n t e r e s t i n g to note that i n the primary i n f e c t i o n of a l l normal dietary vitamin A groups examined, the peak production i s followed the next day by a drop of almost exactly 50 percent. Table XII gives the t o t a l average weights of a l l the groups throughout the experiment. It can be seen that both the low vitamin A groups, D and B, were below the weights of groups F and H at the s t a r t of the experiment, but the differences 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 to the high standard deviation present i n a l l groups. However, by day 8, group D was s i g n i f i c a n t l y lower (P «=c.01) i n weight than group H, and by day 14 the survivors of group D retained t h i s difference at the 5 percent p r o b a b i l i t y l e v e l . By day 14 the surviving birds of group D had l o s t about 20 percent of th e i r s t a r t i n g body weights while the birds on higher levels of vitamin A (G) had l o s t only 2^ percent of t h e i r weight. The weight changes which took place during the course of the experiment are examined i n Table XIII. Both groups receiving a primary in f e c -t i o n showed a continual weight loss up to day 8. Coinciding with th e i r regained appetites, the birds of group H showed a dramatic recovery of th e i r l o s t weight between days 8 and 10, when they gained an average of 118 grams i n the 2 days. The 76 survivors of the dessimated D group actually began to show a weight gain between days 10-12, but the continuing losses of the moribund birds maintained the group losses shown on Table XIII. 2. Secondary Infection The low dietary vitamin A group (B) and the normal vitamin A group (F) were both given a primary i n f e c t i o n at 5 weeks old i n Experiment 1. This secondary i n f e c t i o n was given 14 weeks la t e r to test the degree of immunity retained i n both groups. Unlike the 2 groups receiving t h e i r primary i n f e c t i o n , these two immunized groups did not exhibit a marked c l i n i c a l response to the i n f e c t i o n . There was a s l i g h t scouring present on days 4.-6, and some evidence of anorexia. The low vitamin A group (B) showed less appetite during t h i s acute period than the normal vitamin A group (F), and the feces were more scanty. Also group B birds did not return to t h e i r pre-infection feed consumption by the end of the experimental period. One b i r d i n group B began to show ataxia and leg paralysis at day 14, and was k i l l e d at day 16. No evidence of co c c i d i o s i s was found i n the i n t e s t i n a l t r a c t , but microscopic examination revealed an overwhelming mold population present. The oocyst production for these 2 "immune" groups i s shown in Table XI, and unlike the findings i n Experiment 2, the low vitamin A birds had almost twice the oocyst production of 77 the normal group. This conformed more to the findings i n a primary i n f e c t i o n . However, i t can be seen that some immunity was retained i n both groups as the numbers of oocysts passed was far below that of groups D and H. An examination of the t o t a l weights throughout the exper-iment, as shown i n Table XII, demonstrates that group F was the only group to show an increase i n weight during the f i r s t 14 days after inoculation. Group B was 47 grams l i g h t e r than i t s inoculation weight at day 14, a 2 percent los 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 analysed i n Table XIII, i t can be seen that - both the immune groups l o s t weight during the f i r s t 6 days of the i n f e c t i o n , and group B l o s t more than twice that of group F. The reason for the weight loss i n group B between day 10-14 i s p a r t i a l l y explained by the f a i l i n g weight of the b i r d which was subsequently k i l l e d . It was demonstrated i n Experiment 2 that a primary i n f e c t i o n with E. acervulina gave marked protection against a secondary i n f e c t i o n 4 weeks l a t e r . From t h i s present experiment i t can be seen that t h i s protective immunity i s retained, i n a lowered degree, 14 weeks after the primary i n f e c t i o n . Unlike the re s u l t s of Experiment 2, the low dietary vitamin A group did not show a higher degree of immunity than the birds on a 78 normal vitamin A d i e t . The average weight losses, and the oocyst counts, were both higher i n group B thdn i n Group F. However, th i s experiment confirmed the findings of Experiment 2 that the degree of protection afforded by the immunity i s much more obvious i n the low vitamin A group. Although the normal vitamin A group (H) exhibited a severe weight loss i n the acute stages of the disease, t h i s loss was regained so ra p i d l y that i n 14 days after inoculation the group H weight was equal to the immunized low vitamin A group (B), and not s i g n i f i c a n t l y lower than the immune normal vitamin A group (F). On the other hand, a com-parison of groups B and D during t h i s period shows a s i g n i f i c a n t difference i n weights (P < .01) plus a high mortality rate i n group D. As i l l u s t r a t e d i n Figure 6, the normal vitamin A groups show only a 2 percent difference i n weight between the immune and non-immune groups, but thi s difference r i s e s to 18 percent i n the low vitamin 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 vitamin A ra t i o n Normal vitamin A ration Immune (B) Non-immune (D) 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 , 6 40 9 20 25 (5)* 6 20 10 10 20 (5) 7 10 12 0.1 2 (4) 3 4 14 0.03 2 (4) 0.08 1 Total oocysts per b i r d 145 474 90 330 Reproduc-t i v e Index 6 19 4 13 * number of surviving birds out of 6. 80 TABLE XII 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 vitamin A Normal vitamin A Immune (B) Non-immune (D) Immune (F) Non-immune(H) 0 4 6 8 10 12 14 21 1785 1737 1697 1726 1754 1752 1738 1889 1763 1668 1541 1394 1294(5) 1280(4) 1417(3) 1620(2) 1848 1823 1808 1866 1902 1912 1911 1945 1911 1826 1728 1707 1825 1848 1866 1922 * number of surviving birds out of 6. 81 TABLE XIII DAILY MEAN WEIGHT CHANGES IN GRAMS IN IMMUNE, AND NON-IMMUNE 19-WEEK-OLD CHICKENS ON 2 DIETARY LEVELS OF VITAMIN A, FOLLOWING INFECTION WITH 25 MILLION OOCYSTS OF E. ACERVULINA Between Low vitamin A Normal vitamin A days Immune(B) Non-immune(D) 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 of surviving birds out of 6. 82 IMMUNE 1800 -• 1500 --z H o S00 -• 6 0 O - . 3oo -• L O W V t T R M t M A N o R M f U . V l W I I N l A Figure 6. The effect of d ie tary vitamin A l e v e l on the mean weight of 19-week-old immune and non-immune birds 14 days after i n f e c t i o n with E . acervul ina 83 3. Post Mortem and H i s t o l o g i c a l Examinations Two birds from each group were k i l l e d on day 4 following inoculation, and the intestines checked for pathological lesions. Sections from each of the birds were examined to determine the extent of the p a r a s i t i c invasion and development. Post mortem findings: Group B - Low vitamin A diet - immune One of the birds showed a 3.5 percent weight loss at 4 days and examination of the inte s t i n e showed some sloughing of the epithelium i n the duodenum, and areas of e n t e r i t i s at the beginning 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 vitamin A diet - primary i n f e c t i o n Both birds k i l l e d showed c l i n i c a l signs of the disease and both had a 7 percent weight loss at day 4. Bird No. 1 demonstrated only some sloughing of the i n t e s t i n a l epithelium on post mortem examination, but b i r d No. 2, which had a more severe diarrhea, showed pronounced lesions i n the i n t e s t i n e . The entire i n t e s t i n e anterior to the yolk stalk revealed a spotty e n t e r i t i s . Group F - Normal vitamin A - immune Bird No. 1 showed no c l i n i c a l or post mortem signs of c o c c i d i o s i s . The second b i r d had a weight loss of less than 84 1 percent but exhibited severe diarrhea. However, there was no evidence of e n t e r i t i s on post mortem examination, although the duodenum and jejunum both showed severe sloughing of the epithelium. Group H - Normal vitamin A - primary i n f e c t i o n Both the birds had severe diarrhea and depression, and showed a 5 percent loss i n weight since inoculation. Post mortem examination showed that b i r d No. 1 had severe sloughing of the entire anterior portion of the i n t e s t i n a l mucosa, and the jejunum posterior to the duodenum was inflammed. The second b i r d showed less pathology, but severe e p i t h e l i a l sloughing was evident. H i s t o l o g i c a l examinations: Almost a l l the sections revealed evidence of severe sloughing of the v i l l i epithelium so that i n many cases the lamina propria was completely unprotected. There was also a great deal of edema evident i n the v i l l i of a l l groups. The difference between s l i g h t :and severe pathology i n the i n t e s t i n e appeared to be the degree of involvement. In the severe cases, a far greater number of c e l l s were invaded with few areas i n the sections being free of the parasites. The crypts and neighbouring v i l l i were packed with trophozoites and schizonts of d i f f e r e n t -sizes and stages of maturity. The gametocytes and oocysts present were not confined to the t i p s 85 of the v i l l i as reported from various sources, but were found also i n the c r y p t s . In the in tes t ines showing only sloughing of the epi thel ium, there were far fewer areas invaded, and the affected regions d id not show the same number of c e l l s af fected . Both the low vitamin A groups revealed a more extensive invo lve -ment than the groups on normal vitamin A d i e t s . The non-immune group D showed the most extensive invasion of the i n t e s t i n a l c e l l s and also proport ionate ly 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 investigation demonstrate that a diet containing only 10 percent of the recommended l e v e l of vitamin A does not necessarily a f f e c t the weight gains or c l i n i c a l health of the birds receiving i t . However, i f these same birds are subjected to c o c c i d i o s i s , the effects of t h i s suboptimal vitamin A become immediately apparent. In these experiments, i n f e c t i o n with E. acervulina was more severe i n a l l groups on the low vitamin A d i e t , as determined by a comparison of the oocyst production, weight gains, and mortality between birds on low dietary levels of vitamin A and normal l e v e l s . A. Oocyst Production Brackett and Bli z n i c k (1952b) have reported that the number of oocysts produced by a c o c c i d i a l i n f e c t i o n are affected by at least 6 factors. 1) The inherent pot e n t i a l of the parasite i n a non-immune host. 2) The immunity or resistance developed by the host. 3) The "crowding e f f e c t " . 4) Competition with other c o c c i d i a l species or i n f e c t i v e agents. 5) N u t r i t i o n of the host. 87 6) Strain differences of the host. The reproductive index, or p o t e n t i a l , of a coccidium i s the maximum number of oocysts produced per oocyst inoculated. It depends on the number of schizogonous generations and the numbers of merozoites produced In each generation. With E_. 8 x 20 x 16 x 10 x 36 acervulina, the t h e o r e t i c a l p o t e n t i a l i s = 455,000 per oocyst injected, i f the endogenous stages reported by V e t t e r l i n g and'Doran (1966) are correct. Hein (1968a) demonstrated that the reproductive potential dropped with increasing inoculum, which confirms findings by other authors and the re s u l t s from these experiments. Warren and B a l l (1967) reported the highest R.I. (100,000) of any inve s t i g a t i o n . This resulted from an inoculum of 500 oocysts i n 3-week-old chicks. Table XIV gives a l i s t of reproductive indexes obtained by various authors and demonstrates the decrease i n R.I. with increasing inoculum. The re s u l t s of t h i s investigation are also included. As f i r s t demonstrated by Brackett and Bli z n i c k (1952b) E. acervulina i s characterized by an extremely high oocyst production. Although i t s reproductive potential i s lower than that of E. brunetti and E. t e n e l l a , i t s oocyst pot e n t i a l i s far higher than any other avian species of c o c c i d i a . Tyzzer et al_ (1932), and Brackett and Bli z n i c k (1952b), have demonstrated TABLE XIV THE REPRODUCTIVE INDEX OF E. ACERVULINA FOLLOWING INCREASINGLY LARGER INFECTIVE DOSES AS REPORTED BY VARIOUS AUTHORS Inoculation - number of oocysts per b i r d R.I Age of birds Author 50 500 900 1250 320,000 500,000 1 m i l l i o n 3 m i l l i o n -vitamin 3 m i l l i o n -vitamin 5 m i l l i o n 10 m i l l i o n -vitamin 10 m i l l i o n -vitamin 20 m i l l i o n 25 m i l l i o n -vitamin 25 m i l l i o n -vitamin • normal A group • low A group • normal A group • low A group • normal A group • low A group 72,000 100,000 38,000 43,000 1,800 1,026 . 302 120 205 44 43 86 8 13 19 unknown 3 weeks 7 weeks 6 weeks 6 weeks 3 weeks 6 weeks 5 weeks 5 weeks 6 weeks 9 weeks 9 weeks 6 weeks 19 weeks 19 weeks Brackett and Blizn i c k (1952b) Warren and B a l l (1967) Long (1967) Hein (1968a) Hein (1968a) Rose and Long (1962) Hein (1968a) Coles Coles Hein (1968a) Coles Coles Hein (1968a) Coles Coles oo oo 89 that the highest y i e l d s of oocysts are obtained with l i g h t i nfections i n the case of E. t e n e l l a , E. brunetti, E. maxima and E. necatrix, and with high inoculums the oocyst production decreased. However, these authors found that E. acervulina did not show t h i s c o r r e l a t i o n , and instead revealed an almost un-lim i t e d oocyst production capacity. It would appear that t h i s f i nding i s related to a lower immune response to t h i s species, and i f t h i s i s the case the higher oocyst production shown by the groups on a r a t i o n low i n vitamin A may be due to a suppression of the immunological mechanisms i n the d e f i c i e n t host. It has been reported by several authors that age i s a factor i n oocyst production; the older the birds, the greater the p o t e n t i a l production. Hein (1968a) demonstrated that an i n f e c t i v e dose of 5 m i l l i o n oocysts of E. acervulina i n 2-week-old chicks gave a t o t a l production of 37 m i l l i o n , while the same inoculum i n 6-week-old chicks gave a t o t a l of 225 m i l l i o n . Krassner (1963) reported that the mean oocyst production increases proportionately with host age, and attributed t h i s f inding to the fact that more i n t e s t i n a l c e l l s are available for parasite penetration,in older birds. In this present investigation the maximum oocyst production occurred i n 9-week-old birds following an i n f e c t i v e dose of 10 m i l l i o n 90 oocysts . The low vitamin A group had a t o t a l oocyst production of 860 m i l l i o n , twice the production of the b irds on normal l eve l s of vitamin A. This doubling of oocyst production under condit ions of low d ie tary vi tamin A was seen a lso i n the 5-week-old b i r d s , but i n the 19-week-old b irds the morta l i ty was so high that an accurate assessment was d i f f i c u l t to make. Figure 7 compares the oocyst production i n the 3 experiments. Ackert et a l (1931) suggested that the greater paras i t i sm with nematodes i n b irds on low vitamin A diets was due to reduced p e r i s t a l s i s . I f th i s i s c o r r e c t , increased oocyst production may r e s u l t from th i s same phenomena, as a slower p e r i s t a l s i s would allow a greater number of sporozoites more time to invade the c e l l s . The n u t r i t i o n a l requirements of the coccidium can be involved i n t o t a l oocyst product ion. Warren (1968) has invest igated the vitamin requirements of E . acervul ina and has found that thiamine, 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 ac id are e s sen t ia l for optimum oocyst development and product ion. How-ever, he reports that a d ie t d e f i c i e n t i n vitamin A resu l ted i n an oocyst production twice that of the normal c o n t r o l s . B r i t t o n , H i l l and Barber (1964) have demonstrated that low prote in l eve l s i n the d ie t cause reduced oocyst production because a low t r y p s i n secret ion re su l t s i n a decreased excystat ion. 91 o > o O ISO I IOO + 50 4 EXPERIMENT No I. LOW VlTf»MlN A NOKMAL Viraciw A a)ftY V r6 r a 'lO f l l ^ Figure 7. A comparison of the d a i l y oocyst production i n chicks on Experiments 1, 2, and 3 92 Long (1968a) reported that the breed and s t r a i n of 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 that White Leghorns are more susceptible than Rhode Island Reds. B. Weight Gains In the 3 experiments comprising t h i s study one of the main findings was a much reduced weight gain following i n f e c t i o n i n the birds on low levels of vitamin A. In Experiment 1, using 5-week-old chicks and an inoculum of 3 m i l l i o n oocysts, the low vitamin A group had a 2 week weight gain amounting to only 37 percent of that shown by i t s non-infected control, while the normal vitamin A infected group had a gain equal to 70 percent of i t s c o n t r o l . In Experiment 2, these figures were 26 percent and 80 percent respectively. These findings concur with those of Erasmus, Scott, and Levine (1960) and Waldroup et a l (1963). The weight loss was due mainly to the anorexia characterizing the acute stage of the i n f e c t i o n . Reed and P i t o i s (1965) reported that from day 4-6 followingJan i n f e c t i o n with E. acervulina, feed and water intakes are reduced at least 50 per-cent. Preston-Mafham and Sykes (1967b), using equalized feedings between infected and control birds, found that with a E. necatrix i n f e c t i o n 70 percent of the weight loss was due to 93 a reduct ion i n feed intake , and 30 percent to impaired absorption i n the i n t e s t i n e . Erasmus et a l (1960), using the same tech-niques, reported s im i lar f indings i n b irds on 2 leve ls of vitamin A subjected to a mixed i n f e c t i o n of E . acervul ina and E . t e n e l l a . An analys is of t h e i r re su l t s show that the low vitamin A group was 9 percent lower i n weight than i t s non-infected c o n t r o l , while the high vitamin A group was only 4 per-cent lower than i t s c o n t r o l . This d i f ference i s probably due to a decreased absorption i n the lower vitamin A group. Long (1968), and Preston-Mafham and Sykes (1967b) reported that an i n f e c t i o n with E . acervul ina resu l ted i n a loss i n serum proteins in to the gut lumen, as demonstrated by the Pontamine sky blue dye t e s t . This loss i s at a maximum 3-5 days after i n f e c t i o n , c o i n c i d i n g with the per iod of depressed absorpt ion, and i s i n d i c a t i v e of a change i n gut permeabi l i ty . Long a lso reported changes i n permeabi l i ty 3^-7 hours after inocu la t ion which he a t t r i b u t e d to the sporozoite entry in to e p i t h e l i a l c e l l s . In the experiments of th i s i n v e s t i g a t i o n , i t was noted that the feed and water intake of the b irds during the acute stage was p r a c t i c a l l y n i l . The anorexia was more pronounced i n the low vitamin A groups i n a l l the 3 experiments. As these experiments d id not equalize feed intake between the infected 94 and non-infected controls, and as the low vitamin A infected groups showed a greater degree of anorexia, i t would follow that the main weight differences between the groups were due to t h i s greater loss of appetite. C. Mortality Mortality i s not a c h a r a c t e r i s t i c finding i n an i n f e c t i o n with E. acervulina. This i s especially true when the birds are subjected to only one i n f e c t i v e dose as demonstrated by Dickinson and S c o f i e l d (1939) and Hein (1968a). Morehouse and McGuire (1956) reported 6 percent mortality i n chicks after a single 5 m i l l i o n oocyst inoculation, but t h i s i s unusual and could be due to 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 organism. In the 3 experiments reported i n t h i s paper, the highest mortality appeared i n 19-week-old, non-immune birds on the low l e v e l of dietary vitamin A. Four of the 6 birds i n this group died, and death was associated with a weight loss of over 30 percent of the t o t a l body weight. This weight loss was due primarily to the severe anorexia and adipsia displayed by the infected birds on the low vitamin A d i e t . Age may have been a factor which aggravated the effects of the i n f e c t i o n i n experi-ment No. 3.. Tyzzer et a l (1932) reported that older birds were more susceptible to i n f e c t i o n with E. necatrix and t h i s same may be true with E. acervulina. Brackett and B l i z n i c k (1952a), also 95 working with E. necatrix, demonstrated that with equal oocyst ingestion the younger birds were more susceptible, but when the dose was calculated to give equal numbers of oocysts per gram of body weight, the older birds were the more severely affected. D. Immunity As demonstrated by the higher oocyst production and reproductive index i n birds on the low vitamin l e v e l i n the diet, there was a delay i n the appearance of the immune response. The reason for t h i s delay may be associated with a reduced adrenal c o r t i c a l function under low vitamin A conditions as reported by Glick (1963). It was e a r l i e r demonstrated by Challey (1962) that i n birds affected with E. t e n e l l a there i s an increase i n the secretion of adrenal corticosterones, and he attributed t h i s increase to the stress of the disease and the increased sythesis occurring i n the body as a r e s u l t . Glick (1963) found that the reduction of adrenal c o r t i c a l hormones could be deter-mined by a lower heterophil count i n the c i r c u l a t i o n following an i n j e c t i o n of ACTH. Newcomer (1957) has suggested that increased heterophils are a measure of the response of birds to acute stress. The appearance of numerous heterophils i n the lamina propria of birds 3 hours after an inoculation with E. acervulina oocysts, would agree with t h i s suggestion. The fact 96 that 2 of the 3 birds on low vitamin A levels did not show the large number of heterophils observed i n the other groups, may correlate with the report of reduced adrenal c o r t i c a l secretions under low vitamin A conditions. The strength of the immunity, once established, was not related to the l e v e l of vitamin A i n the diet, but rather to the severity of the i n i t i a l i n f e c t i o n . This finding confirms e a r l i e r reports by Tyzzer (1929) and l a t e r findings by Hein (1968b) that the degree of immunity to c o c c i d i o s i s i s d i r e c t l y r elated to the severity of the i n f e c t i o n . The nature of t h i s immunity has been widely investigated, and i t i s generally considered that a l o c a l factor i s primarily responsible for the protection. Rose (1963) suggests that a s e n s i t i v i t y of the i n t e s t i n a l epithelium re s u l t s from the presence of c i r c u l a t i n g antibodies, and t h i s s e n s i t i v i t y i s responsible for the i n h i b i t i o n of schizont development. The effects of immunity are demonstrated i n the sections taken 48 hours after the s t a r t of i n f e c t i o n . The immune birds, regardless of the levels of vitamin A fed, showed an i n h i b i t i o n of parasite development. This e f f e c t of immunity on endogenous forms confirms the findings of Long, Rose, and Pierce (1963), Horton-Smith and Long (1963) and Leathern and Burns (1967). A l l these investigators found that there was an invasion of the v i l l i by the sporozoite, and a migration to the 97 crypt. However, the sporozoites either f a i l e d to develop further, or else a trophozoite developed which did not mature into a schizont. The birds on the lower dietary l e v e l of vitamin A did not r e t a i n t h e i r immunity as well as the birds on normal di e t s , but the difference was not s i g n i f i c a n t . However, the fact that there was considerable protection s t i l l effected by both groups 100 days after 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 a shorter period of protective immunity with E. acervulina. However an immunization dose as high as 3 m i l l i o n oocysts has not been reported, and i t would appear that with E. acervulina an inoculum of t h i s size must be used to establish e f f e c t i v e immunity. 98 V I . SUMMARY AND CONCLUSIONS 1. Experiments were conducted to invest igate the patho log ica l ef fects of an i n f e c t i o n with E . acervul ina i n b irds fed a d ie t containing 440 I . U . vitamin A per ki logram, compared to b irds fed a s i m i l a r d ie t containing 4400 I . U . vitamin A per k i logram. 2. No symptoms of def ic iency were observed i n the low vitamin A groups, and no s i g n i f i c a n t weight dif ferences were recorded between the non-infected normal vi tamin A and low vitamin A groups. 3. The e f fect of the low l e v e l of vitamin A i n the d ie t was not manifested u n t i l the b irds were infected with E . acervu l ina . 4. Fol lowing an i n f e c t i o n with E . acervu l ina , the b irds on the low l e v e l of v i tamin A showed s i g n i f i c a n t l y lower weight gains, and higher mor ta l i t y and oocyst product ion, than d id the b irds r e c e i v i n g the higher l e v e l of vitamin A. 5. Birds fed the low l e v e l of vitamin A demonstrated a slower development of the immune response to E . acervul ina i n f e c t i o n . 99 6. The degree of immunity to E. a c e r v u l i n a was r e l a t e d 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 . 7. The low l e v e l of v i t a m i n A used i n these experiments d i d not r e s u l t i n a l o s s of e p i t h e l i a l i n t e g r i t y i n the i n t e s t i n e . 8. There were no d e t e c t a b l e e f f e c t s of low v i t a m i n A d i e t a r y l e v e l s on the endogenous stages of the p a r a s i t e . The s p o r o z o i t e i n v a s i o n of the i n t e s t i n a l e p i t h e l i u m appeared t o be s i m i l a r i n a l l groups. The i n h i b i t o r y e f f e c t of immunity on the development of the f i r s t g e n e r a t i o n s c h i z o n t was a l s o s i m i l a r i n b i r d s on both d i e t a r y l e v e l s of v i t a m i n A. 100 BIBLIOGRAPHY Ackert, J.E. 1942. Natural resistance to helminthic disease. J. P a r a s i t o l . 28:1. Ackert, J.E.; Mcllvaine, M.F.; and Zimmermann, N.B. 1927. Resistance to parasites affected by fat soluable vitamin A. J. P a r a s i t o l . 13:219. Ackert, J.E.; Mcllvaine, M.F.; and Crawford, N.Z. 1931. 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