SOME ASPECTS 6N CHICK. MORTALITY WITH PURE AND MIXED INFECTIONS OF THE PRGTOZOAN PARASITES, EIMERIA TENELLA AND EIMERIA NECATRIX AND IN VITRO STUDIES ON THE EFFECT OF AUREOMYCIN ON THE SPOROZOITE$ AND MEROZOITES OF EL TENELLA Thesis I’or II“ Degree OI pI’I. D. MICHIGAN STITE UNIVERSITY William Dunsmore WiIson E957 THESIS A (two This is to certify that the thesis entitled Some Aspects on Chick Mortality with Pure and Mixed Infections of the Protozoan Parasites, Eimeria tenella and Eimeria necatrix and In Vitro Studies on the Effect of Aureomycin on the Sporozoi-tes and Merozoites of E. tenella. presented by William Dunsmore Wilson has been accepted towards fulfillment of the requirements for _Bh..D.._ degree in chy 2/1 ZZI/lj}L// Auf/uui% Major professor 4 Date W— L! BRA R y Michigan Sta” University 0-169 SOME ASPECTS ON CHICh MORTALITY WITH PURE AND MIXED INFECTIONS OF THE PHOTOZOAN PARASITES, EIMERIA TENELLA AND EIMERIA NECATRIX AND ;§_VITR0 STUDIES ON THE EFFECT OF AUREOMXCIN ON TnE ssoaozoxTes AND MEROZOITES OF g. TENELLA By William Dunsmore Wilson A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1957 To ACKNOWLEDGMENTS Dr. William D. Lindquist, for the guidance and advice that made this work possible; 2r. Emanuel Waletzky, American Cyanamid Company, whose suggestions were most helpful and who generously supplied the Aureomycin; Lederle Laboratories Division, American.Cyanamid Company, Pearl River, New York, for making this work possible in the form of a Research Fellowship and by underwriting the cost of chicks and other expendable materials; and Dr. William D. Eaten, for his statistical assistance, The author wishes to express his sincere appreciation. TABLE OF CONTENTS INTRODUCTION 0 o o o o o o o o o o o o o o o 0 LITERATURE ' O O O O O O O O O O O O O I O O 0 MATERIALS AND METHODS . . . . . . . . . . . . . . . . ‘0 Pllre and l‘lixed Infections. o o o o o o o o e o 1. Source of chickens 2. Distribution of chickens 3. Care and management h. Source of oocysts . Single oocyst isolation 6. Preparation of culture 7. Oocyst culture 8. Administration of oocysts 9. Dosage of oocysts 10. Feed 11. Housing and utensils B. The Effect of Aureomycin on the Sporozoite and Merozoite Stages of E. tenella . . . . . . . . RBULTS C O O O O O O O O O O O O O O O O A. Single Oocyst Isolation, Pure and Mixed 1*nf00t10n8 0.000000000000000 B. The Effect of Aureomycin on the Sporozoites and Merozoite Stages of‘E. tenella . . . . . DISCUSSION 0 O O O O O O O O O O O O O O O O A. Pure and Mixed Infections . 1. Pure infection . . . . . 2. Mixed infections . . . . B. The Effect of Aureomycin on the Sporozoite and Merozoite Stages of E. tenella . . . . . . . SUM! O O O O O O O O O O O O O O O C O ‘ CONCLUSIONS 0 O O O O O O O O O O O O O O O O BIBLIOGRAPHY . . C O C O C O O 0 C O O O O O 0 APPENDIX 0 O O O O O O O O O O 0 O O O O 0 PAGE 63 67 68 71 82 LIST OF TABLES TABLE PAGE I. Percent mortality of chickens with pure infections of E. tenella and E. necatrix and with mixed infections where the number of oocysts per bird was varied for‘fi. tenella and constant for E. necatrix . . . . . . . . . . . . . . . . . . . . 50 II. Percent mortality or chickens with pure infections of E. tenella and E. necatrix and with mixed infections where the number of oocysts per bird was varied for ‘E. necatrix and constant for E. tenella . . . . . . . 51 III. Comparison of the average mortality caused by pure andmxedinreCtion eeeeeeoeeeeoeoooSB IV. Relationship between the total number of deaths and the days after infection on which these occurred . . Sh V. The effect of various levels of Aureomycin in the feed on the mortality of chicks infected with ‘2. tenella and E. necatrix . . . . . . . . . . . . . 56 VI. In vitro studies of the effect of Aureomycin on the —sporozoite and merozoite (Generations I and II) stages OfEimeria tenella o e e e e e e o e o o e e o 57 INTRODUCTION The magnitude of literature dealing with poultry coccidia would lead one to believe that these protozoan para- sites have been "tamed." Indeed, certain phases (e.g., life cycles) have been determined in great detail. However, in other phases (e.g., mortality with respeCt to inocula) there is much to be desired. In some studies where mortality is used as an indication of resistance, immunity and drug effect it has been reported that a few thousand oocysts were capable of causing 50 percent mortality while in other experiments, one hundred thousand oocysts were reported to cause little or no mortality. Recognizing these inconsistencies, cecal lesions have been graded and weight gains and losses have been utilized in conjunction with mortality. However, these have not proved entirely satisfactory since uniform grading of lesions is difficult and weight gains, to be significant, require many weeks of chicken care and management if very young chicks are used. Indagations of a correlation between age and mortality have been recorded, but although much work has been reported, this question remains unsettled. Associated with mortality results, one must be aware of the fact that the two most pathogenic coccidia of chickens (Eimeria tenella and g. necatrix) cannot be readily separated by size, sporulation time or location of oocysts. They can easily be distinguished from each other by pathological sequalae and by merozoite infections. Nevertheless, there are undoubt- edly many cases of mixed infections reported as a single in- fection. What effects mixed infections would exercise on mor- tality is not known. Mixed infections have been reported in drug studies, but the controls consisted of a like mixed in- fection, which gives no indication of the effect of individual species. Of the prodigious amount of literature on the prophy- laxis and'chemotherapy of poultry coccidiosis, the majority of conclusions have been based on mortality and weight gains which are difficult to confirm due to fluctuations of both criteria. Other methods (e.g., histOpathology) have been em- ployed as an aid in determining which, if any, phase of the life cycle is affected by a compound. Originally a program was set up to investigate the in- fluence of bacteria on coccidia and to determine what role Aureomycin HCI* might play in the overall picture. However, before one can investigate these questions others must first be answered. Therefore, the problems to be studied which are unre- solved are: 1. Can LDSO determinations be made for poultry coccidia? 2. If so, can these results be duplicated? *Aureomycin3 HCl 8 crystalline chlortetracycline H01. 3. 5. What influence, if any, does mixed infection play in mortality? Does the administration of Aureomycin to test animals affect mortality? If so, is this influence exerted by direct action on the parasite? LITERATURE Until Tyzzer's (1929) salient monograph, all coccidia of the fowl were known and studied as Eimeria avium, with few exceptions. Railliet and Lucet (1891) described Coccidium tenellum from the diseased ceca of chickens which Railliet (1913) later corrected to Eimeria tenella. Gerard (1913) described what he thought was a new species, Eimeria bracheti, which, from his description, was later considered to be E, tenella. Other workers of this period, Cole and Hadley (1908), Hadley (1909), Eckardt (1903), Fantham (1910, 1915), and Young (1929) indicated E,‘glggm as the etiological agent of white diarrhea, roup, blackhead in fowls, fowl paralysis, and liver involvement, as well as being transmissible from grouse to chicken and from chicken to turkeys, pigeons, geese, ducks and other fowl. All of these have later been refuted by the original author himself or by others, such as Johnson (1923) and Tyzzer (gg.‘g;§.). Isolation, deve1Opmental time, topographical distribu- tion, associated gross pathological changes and symptoms, in- testinal mucosa, morphology (including size and shape of oocysts), pathogenicity and the reaction of host-tissue were all taken into consideration by Tyzzer (o . git.) in describing E. tenella and three new species: Eimeria mitis, Eimeria acervulina and Eimeria maxima. Thus, for the first time morphological characteristics were established whereby new species of coccidia could be discovered and described. Due to the work of Tyzzer (2g. 313.), Johnson (1930), Tyzzer, Theiler and Jones (1932), and Levine (1938, l9h2a) we now recognize eight species of chicken coccidia, each of which pursues its own independent life cycle, as being responsible for coccidiosis of chickens. Of these eight, only two, E.) tenella and Eimoria necatrix, Johnson (92, git.) will be sub- sumed in this investigation. Eimeria tenella, Railliet and Lucet (1891) Life~cycle= Rivolta (1878) described white points the size of a poppy seed in the submucosa of the intestines of fowl and Schaudinn (1900), Fantham.(l910), Hadley (1911), and Gerard (1913) gave general accounts of life history, in some cases figures of various stages of development. It was not until Tyzzer (1929) and later Scholtzseck (1953) that the life cycle and morphological characteristics of E. tenella were described in detail. In fact it was not until 1929 when one could be sure that one, and only one, organism was involved in any of the aforementioned descriptions. A brief resume of the life cycle of this parasite is as follows: oocysts passed out of the chicken in the feces-~sporulation with the development of four sporocysts which are made up of a total of eight sporo- zoites--fully sporulated oocysts eaten by the chicken-- sporozoites liberated in the small intestine--migrate to coca-- penetrate epitheliumr-schizont (first generation) formed in which develops merozoites (first generation)--merozoites liber- ated (an estimated 900 from one sporozoite) from the enlarged cell--lumen of coca (2 1/2 to 3 days after oocysts eaten)-- merozoites invade epithelium of the fundi of the glands--develop into schizonts (second generation)--merozoites, 200-300 from one first generation merozoite (second generation), develop-- merozoites liberated five days after infection and penetrate glandular epithelium--a few may develop into third generation schizonts and.merozoites, but the majority develOp into micro- and macrogametocytes--micro- and macrogametes formed-emicro- gametes liberated--penetrate macrogametes--oocysts liberated-- pass out in the feces (total of seven days after ingestion of the sporulated oocysts). This prepatent period, time of oocyst ingestion to time of passage, may, under certain conditions, require only six days for this species and E, necatrix (Edgar, 1955). Pathology: Due to conflicting reports and misunderstanding of the etiological agents of chicken coccidiosis, it is small wonder that the true gross pathology and histOpathology picture was non-existant until the work of Tyzzer (1929). Since white diarrhea, roup, blackhead, fowl paralysis and other diseases of fowl were attributed to coccidiosis, it was almost impossible to separate the pathological picture of E, tenella from that caused by other species of coccidia and other etiological agents. Nonetheless, from descriptions of the parasite and the pathology involved, one is able to assume in some cases, e.g., Gerard (QB. git.), that one of the main etiological agents involved was g. tenella. However, the true pathological picture, in which the etiological agent of cecal coccidiosis was itself the instigator, was not des- cribed in detail until 1929 (Tyzzer), 1937 (Mayhew), and in 1953 (Greven). While histopathological events are taking place in the ceca of infected birds during the formation of the Generation I and II schizonts and merozoites, it is not until the Generation II merozoites are liberated that external symptoms of infected birds are observed. This usually occurs on the fifth day as large quantities (dependent upon the magnitude of the infection) of unclotted or partially clotted blood is passed in the feces. Presence of blood is due to the leakage and rupture of blood vessels as developmental stages reach maturity and are released into the lumen. Whereas gross external pathology cannot be observed until 5 days after infection, a microscopic study of the feces from 2 1/2 to 6 days after infection will reveal merozoites and on the seventh day, oocysts. Gross pathological studies on the ceca of infected birds reveal observable hemorrhages, either as small pinpoint spots or a profuse area, dependent upon the severity of infection, on the fourth day after infection. This hemorrhaging increases on the fifth day and then diminishes until approxbmately seven days after infection, and the ceca are mottled reddish or a milky white in color due to the loss of hemoglobin and the presence of a large number of oocysts. After this period, the ceca essentially return to normal. It should be noted, however, that the ceca are usually larger than they were pre- vious to the infection and that the efficiency of the ceca may be impaired due to connective tissue which replaces cells which have been destroyed. It is possible for unusual gross changes to occur if a large core (formed by mixtures of des- troyed tissue and clotting blood) is retained, or adhesions occur or if the ceca are ruptured. See Appendix, Plate I. Oocyst studies: Henry (1932) found that the cyst wall of various species of Eimeria (g. tenella and E, necatrgx excluded) contained up to three parts. Gill and Ray (l95hb) reported a hyaluronic acid type polysaccharide (HAP) in the protective covering of the oocyst of E, tenella which ensures safe pas- sage of oocysts through acidic medium of the proventriculus of birds. The latter authors also believe these mucopoly- saccharides are of great value to the organism by inhibiting intra-cytoplasmic clotting, helping in multiplication and gamete formation and by protecting merozoites against coagu- lation. The sporocyst wall and oocyst wall (g. brunetti and E9 acervulina contain some polysaccharide, either free and/or combined and some protein. However, the so-called plastic granules which later make up a large part of the oocyst wall are composed largely of muco-protein (Pattillo and Becker,' 1955). The actual structure of the oocyst wall is not known. I Warner (1933) found that birds did not become infected from soil which had been free of infected birds for 81-370 days, but that seeded soil gave positive results for 197 days. Intermittent freezing of oocysts in soil had no effect for at least 12 weeks and drying killed oocysts of E, tenella in four weeks. (Patterson, 1933). Dessication, putrefaction, lack of air and direct sunlight prevent sporulation and destroy oocysts rapidly (Benedetti, l9hh). E, tenella oocysts will sporulate at 29° C at a relative humidity as low as 60 percent, although 'the outer wall is broken and spores are released (Brotherston, 19h8). Warner (22. git.) and Ellis (1938) reported that chicken eggshmmersed in suspensions of sporulated oocysts no longer contained viable oocysts on the shell after normal incubation periods. Farr and Wehr (19h9) reported‘fi. tenella as having disappeared from the soil or .11 test plots in less than a year and that oocysts of g, acervulina were recovered from the plots after 86 weeks. Koutz (1950) found that‘§.ltgggllg oocysts were infective (both by feeding soil and under natural conditions) for 272 days exposure from September to June, but did not survive a severe winter and partial summer up to 322 days. In other tests the same author found that E, tenella 10 oocysts did not survive as long as did E, maxima, E, acervulina, and‘g, mitig, Delaplane and Stuart (1935) reported oocysts surviving in plot soil through the winter and spring for months after the removal of fowls from the plot. Soil of a wooded range showed viable oocysts at 15-18 months after removal of all fowl. In studies with deep litter, Boughton (1939) found that excessively wet litter inhibited sporulation while a limited amount of moisture enhanced sporulation. Koutz (1953) in his work with deep litter found that the oocysts of E. tenella and other Eimeria remained viable during the one year the experiments were carried out. Tomhave (19h9) stated that death losses were lower and chickens weighed more when raised on dry litter which contained oocysts, as opposed to chickens raised on wet litter containing oocysts. Baby chicks started out on re-used built up litter (compost litter) became heavily infected with coccidiosis and were not as heavy as those raised in batteries free from coccidia (Skoglund, 1952). Methyl bromide appeared to be immediately effective against g, tenella oocysts in the soil (Clapham, 1950). Boney (l9h8) found that this same gas, when used as a fumigant would kill sporulated oocysts when used at the rate of one pound per one thousand square feet. In l9u0, Horton-Smith, Taylor and Tuttle reported ammonia fumigant effective in destroying oocysts. 11 Fish (1931) reported that 5 percent colloidal iodine (Iodine Suspensoid, Merck) and 2-5 percent cresol produced 100 percent mortality of unsporulated oocysts in less than 2h hours and that ultraviolet light was also effective. How- ever it is important to remember that in laboratory tests the oocysts have been relatively freed from most excess organic matter. Therefore, chemical or physical methods may be effec- tive but under natural conditions may be quite ineffectual. One should also take cognizance of the fact that a compound must kill rapidly to be practical. Andrews (1933) found that oily mixtures of phenolics were effective against the oocysts of g, tenella. Using colloidal iodine, Chandler (1933) re- ported three gallons of suspension containing 0.2-0.h percent iodine content per 100 square feet of surface were sufficient to accomplish practical disinfection providing the broader floors were thoroughly cleaned. Chandler (92, git.) also reports that to exert lethal action on oocysts, the colloidal iodine must be in contact with them, while the iodine is in the free state, from one to two minutes. Anderson and Mall- mann (19h5) reported that, of the compounds tested, colloidal iodine was the only compound which possessed marked powers of penetrability. Uricchio (1953) reported that allylacetone and dibromobutene (pure and in emulsions composed of 50 per- cent sorbitol esters of mixed fatty acids) prevented sporula- tion and killed sporulated oocysts. 12 Fish (23. git.) found that the thermal death time of unsporulated oocysts is inversely proportional to the degree of heat used (2h hours at h5° C - 5 seconds at 80° C) and that moist heat at 55° C killed both sporulated and unsporu- lated oocysts. Perard (1925) reported the Eimeria pgrforang (host: rabbit) oocysts were unable to sporulate if kept in an aqueous medium for one day at h0° C and that 80 percent of the oocysts were killed within 20 minutes in water at 55° 0, within 10 seconds at 80° C, and within 5 seconds in boiling water. The same author also presented evidence that dessica- tion destroys oocysts completely, putrefaction was detrimen- tal and that no sporulation occurred at 0° C and 2° C. In experiments with_§. perforans and E. Eggg§_(host: rabbit) Becker and Crouch (1931) found that g. m oocysts completed sporulation as follows: at 25° 0 - 50 percent sporulation within 8h hours, at 33° C - 80 percent sporulation within 72 hours: and that similar tests with g. perforans resulted as follows: at 25° C - 50 percent sporulation within k8 hours, and at 33° C - 100 percent within h8 hours. In 195h, Edgar found that E, tenella oocysts' maximwm sporulation appeared to take place at 29° C for 22-2u hours and at 28° C for 27-30 hours. However, E, tenella and g, necatrix oocysts became infective in 18 hours (Edgar, 1955). Up until this report by Edgar, the sporulation time (time involved from oocysts passage until it became infective) was considered to be approximately 2h-h8 hours. 13 Pratt (1937) reported that he was unable to initiate excystation of oocysts by chemical means, however, Smetana (1933) and Goodrich (l9hh) were successful with the use of a trypsin solution. Immunity: Beach and Corl (1925) observed that chickens with previous infection with'g. gzigm_(§§. tenella) deveIOped some resistance to reinfection. The first experimental evidence showing that immunity to coccidiosis may be acquired through previous infection was by Johnson (1927). While working with mixed species of Eimeria he likewise observed that a high degree of susceptibility was maintained in both developing and mature cagenreared fowl. Concerning this last observa- tion, workers are not in agreement. There are those who maintain that the younger the birds the more susceptible they are, while others feel that very young birds are comparatively immune (Becker, 1952). Tyzzer, Theiler and Jones (1932) using.§, necatrix observed that younger birds fed massive doses of oocysts were less severely affected than older birds. Brackett and Bliznick (1952), using the same pathogen, con- cluded that younger birds are more severely affected than older birds. Gordeuk gt 31. (1951) found that chicks as young as one day were susceptible to cecal coccidiosis and developed some immunity. In 1936, Herrick, Ott and Holmes reported chickens up to and including 15 months of age were susceptible to E, tenella infection, and that those three months or older were more resistant than those younger than two months. Horton-Smith (19h?) demonstrated that if chickens are kept free of infection, six month old animals are just as susceptible as younger birds. Gardiner (1955) using E. tenella found that chicks one to two weeks old were more resistant than older chicks and that the greatest susceptibility during the first six weeks of their life occurred at the age of four weeks. While the question of age and susceptibility still remains un- resolved,other workers, Mayhew (193A), Herrick (193h), Rosen- berg, Alicata and Palafox (195M) reported that hereditary re- sistance and susceptibility to cecal coccidiosis exists in chickens. Both Champion (l95h) and Rosenberg, gt’gl. (l95h) reported that selective breeding was effective in establishing lines of chickens resistant and susceptible to cecal coccidi- osis, that factors for resistance or susceptibility do not exhibit a strong order of dominance, that sex-linked, maternal effect or cytoplasmic inheritance do not play a significant role in resistance or susceptibility. Along this same line Rosenberg (l9h1) reported that the Barred Plymouth Rock and Jersey White Giant had higher mortality rates than did the White Leghorn, New Hampshire and Rhode Island Reds. Johnson's (1927) was not only the first reported study on immunity but was the first to use graduated doses of sporu- lated oocysts to accomplish this end. Tyzzer (1929), Farr (19h3), Jankiewicz (19h2) and Babcock and Dickinson (195h) have all reported immunity studies with varying dosage levels. 15 The latter authors reported that the time for immunity to develop (chickens receiving a total dosage of 1,050) is h days longer than for those whose graduated dose totaled 2,125. These authors further indicated that the minimum time required for immunity (600 oocysts the first day and 1,000 the second day) was six days with a culture more than 300 days old and three days with a culture less than 150 days of age. Jankiewicz (19h2) reports that immunity increases with increase of oocysts given from 50-3,000 and that from 6,000-100,000 the immunity is the same. Gordeuk gt 3;, (1951) indicated that in addition to age and degree of exposure other unknown factors are in- cluded in the immunity process. Using compost litter to rear chickens for the purpose of deve10ping immunity is at best unreliable (Houtz, 1955). That reinfection may occur was pointed out by Tyzzer (1929), Tyzzer, Theiler and Jones (1932), Farr (l9h3) and Waletsky and Hughes (l9h9). Herrick (1935) showed, upon sub- sequent reinfection, no observable effect on chickens one year after immunity developed. Waletsky and Hughes (22. 213.) point out that in labora- tory studies on immunity it would be advisable to use more than a two week interval between primary and challenge inoculum due to duration and incidence of pathological sequalae which may give erroneous results in acquired immunity studies. Many authors, among which are Allen and Farr (19h3), Horton-Smith and Taylor (19h5). Waletsky and Hughes (l9h6). 16 Seeger (19h6), Swales (l9h6), Koutz (l9h8), Thorp, 23 3;. (l9h7), Goldsby and Eveleth (1950), and Cuckler and Malanga (1955) have reported a substantial degree of acquired immunity following infection and using various medicated diets simul- taneously. Using shmilar procedures, a practical vaccine was announced by Edgar (1956). Some attempts have been made using attenuated oocysts in producing immunity in chickens. Jankiewicz and Scofield (l93h) used heated oocysts. Waxler (l9hla) used x-ray treated oocysts and Uricchio (1953) used oocysts altered by ultrasonics, radium, freezing and heat. The latter author found that the most effective degree of immunity produced was by feeding fif- teen day old birds 100,000 oocysts exposed to ~5° C for five days while the former two reported some success with heat and x-ray treated oocysts. A point raised by Babcock and Dickin- son (195h) that, in the works of Jankiewicz and Scofield (22, ‘g$£.) and Waxler (gp.‘g;§.), it was doubtful whether or not attenuation occurred since the results obtained might have been due to the death of some of the oocysts, thus reducing the number of viable oocysts in the dose. This might also be raised in the case of Uricchio (2p,‘g$§.). Attempts by Tyzzer (1929), Tyzzer, Theiler and Jones (1932) and Goldsby and Eveleth (1950) to demonstrate humoral antibodies yielded inconclusive results. Tyzzer in attempting to immunize chickens against E, tenella was unsuccessful using serum from immune birds, oocysts and merozoites injected in 17 order as above, subcutaneous and intraperitoneal, parenteral, and intravenous. Precipitin tests using dried and ground oocysts as the antigen were unsuccessful as reported by Tyzzer g; 3;. Goldsby and Eveleth (23. 215.) reported that using cecal lining and contents of birds in the acute state of in- fection as the vaccine failed to produce immunity. In l95h, McDermott and Stauber demonstrated agglutinins for the first time in the sera of experimental birds. These agglutinins persisted for at least thirty days after infection and had maximum serum titers reaching 1-320 between the tenth and fifteenth days post infection. The antigen used by these authors was a suspension of merozoites. Becker (l93h) indicated that immunity may not be merely a depletion of available epithelial cells and also that if it is purely a local defense reaction, the immune principle is capable of spreading from cell to cell. Andrews (193h) reported that chickens developing non-fatal coccidial infections either (1) develop an immunity, or (2) the infec- tion becomes patently chronic with oocysts discharging indefi- nitely and birds susceptible to superimposed reinfection. Physiology, metabolism and digestion: In 193ha, Mayhew found that normal birds, with ceca removed, attained normal weights in a short time as compared with those recovering from coccidiosis and that layers laid a normal number of eggs. Various authors agree that the ceca of chickens are not essential structures and are concerned 18 with absorptive and cellulose digestion processes (Dukes, 1955). Collier and Swales (19u8) found that two-thirds of the metabolic activity of cecal tissue resides in the mucosa and that cecal tissue parasitized with E, tenella showed no increase of respiration from the normal but rather a slight decrease in active parasite stages. Smith and Herrick (1944) presented evidence that parasitized (E, tenella) tissue gave a marked increase in 02 consumption of the epithelial cells. In l9h9, Ripson gg‘gi. studied three areas of the ceca (neck, middle and tip) and found that in uninfected birds there was a significant difference in the Q02 of these three areas. They also found that after infection these 002 areas were more uniform and that upon reinfection, the coccidia were more evenly distributed in these three areas. That coccidia causes some effect on the temperature regulation mechanism was shown by Herrick (1950). He found that uninfected birds,when subjected to cold, increased their metabolic rate and maintained their body temperature within one degree of normal and that with infected birds, of like age and size, the metabolic rate decreased and the body temp perature of some drOpped as much as twenty degrees centigrade. In 195h, Levine and Herrick reported that chickens in- fected with E, tenella were so weakened that they could only do one-half as much work (over a three-minute test period) as could uninfected birds. These same authors (1955) found that this loss of ability to do muscular work was more l9 pronounced in infected Single-Comb White Leghorns than Barred Plymouth Rocks, and that while the leg muscles of these birds contained the same amount of actomyosin per gram,’the ratio of actin to myosin was 1:2h for the Leghorns and 1:8 for the B. P. Rocks. Glycogen studies of E; tenella, Edgar 93.3}. (19hh) and Gill and Ray (l95ha), have resulted in showing the presence of this polysaccharide in developing macro- and microgametocytes, macro- and microgametes, freshly passed oocysts, aged oocysts that failed to sporulate, and excysted sporozoites. G111 and Ray (22. gig.) indicate that the parasite metabolizes its gly- cogen from the host's blood sugar, possibly through the activity of akaline phosphatase. Ray and Gill (195h) also reported that the heavily infected zone of the ceca was devoid of akaline phosphate activity but that activity of this enzyme was demon- strated in various stages of the parasite. In 1950, Daugherty and in 1952, Daugherty and Herrick reported that material taken from the infected cecum.inhibited the phosphorylation process and that the possible effects of cecal coccidiosis may be mediated through carbohydrate metabolism interfer- ences. The distribution of acid phosphatases and 5- nucleo- tidase and their probable role in protein and polysaccharide metabolism was reported by Gill and Ray (195hc). Pratt (19h0) reported that severe coccidiosis (E, tenella) caused an in- crease in blood sugar which was probably due to cecal hemor- rhages since a similar increase could be initiated by 20 artificial bleeding. In l9hl, this same author reported changes in liver and muscle glycogen levels during infection and advanced the hypothesis that by some mechanism, the addi- tional blood sugar had come from material (probably lactic acid) from the muscle when body fluids were taken in to re- place that lost from cecal pouches during the acute stage of infection. Herrick (1950) reported liver and muscle glycogen were lowered on the fourth or fifth day and that feeding sugar solution increased the blood sugar but not the stored glycogen as is seen with uninfected birds. Feeding concen- trated saline to chickens during the hemorrhagic phase will permit the rise in the blood sugar to be maintained at a lower level (Waxler, l9hlb). Pattillo and Becker (1955) in their study with Eimeria brunetti and Eimeria acervulina of the chicken report the distribution of glycogen, free aldehydes, protein-carbohydrate complex, lipids, desoxyribonucleic (DNA), and ribonucleic (RNA) acid in the life cycle stages of these parasites. The distribution of DNA and RNA in various endogenous stages of E, tenella have also been reported (Ray and Gill, 1955). In 1933, Herrick reported that the total nitrogen, non-protein nitrogen and hemoglobin of the blood was reduced during‘E. tenella infection. Evidence is presented by Todd and Hansen (19h8) that induced mild hyperthyrosis (thyroprotein thyroxine in diet) resulted in an increase of the mean oocyst count per gram 21 of feces. Gill (1955) presented evidence that in infected birds (300 oocysts per bird) induced hyperthyroidism stepped up the rate of production four-fold whereas induced hypothy- roidism slightly lowered oocyst production as compared with the controls. Similar results were reported by Todd gg_§l. (19119). Wheeler 23 _a_1. (19118) reported thiouracil did not alter mortality or loss of body weight as compared with in- fected controls, but that thyroprotein showed more benefits. Natt and Herrick (1955, 1956) report the use of the hematocrit as a quick and easy method for determining the severity of hemorrhage in coccidial infection and that chickens who lost 29 and h8 percent of their blood volume in ten suc- eessive bleedings required the same amount of time for the eryth- rocyte count to return to normal as those with cecal coccidiosis. They further report decreases in body weight, percent corpus- cular volume and blood volume, but no change in plasma volume during the hemorrhagic phase of cecal coccidiosis. Schildt and Herrick (1955) found that in most infected birds the feed was retained in the crop during the fifth day post infection and if not retained food passage was delayed while no change was noted with respect to food passage in the gizzard and small intestine. They also reported crop activity decreased on the third day after infection and that this activity ceased on the fifth day in most cases and that two weeks were required before the cr0p activity returned to normal. Jones (193h) reported that chickens maintained on high protein diets maintained more normal weights, developed R) N immunity slower, and produced more oocysts than did birds on normal diet, although Allen (1932) found that this same diet caused infected birds to be slower in gaining back weights apparently due to chronic coccidiosis. Vitamin A at various levels and in dietary substances failed to produce any notice- able effect of resistance to coccidiosis (Wickware, 19h9, and Jones, 2p.‘g;£.). Vitamin K afforded some control of hemor- rhage in cecal coccidiosis (Baldwin 23 g;., l9hl), but Hawkins (19h5) reported the exact opposite. Kennard and Chamberlin (19h9) reported that chicks on old built-up litter made the best rate of growth with lowest mortality with the complete or incomplete (all plant) diet. Becker and Waters (1938) found that combinations of large amounts of dried skim.milk or dried buttermilk and wheat middlings in the ration pre- disposed chickens unfavorably to cecal coccidiosis attacks. While E. R. Becker did much work in this area with coccidia of the rat, Eimeria neischultzi, the exiguity of literature dealing with diet in E, tenella and E. necatrix infections is most pronounced. Delaplane (1953) stated there is no clearly recognized relationship between poultry nutrition and cocci- diosis. With respect to another possible predisposing factor in cecal coccidiosis, Riedel (1950) reported that weight and morbidity records indicate ascarid infected birds were less resistant to coccidiosis than controls. 23 Eimeria necatrix, Johnson (1930) General: Johnson (1930) and Tyzzer 25‘2E. (1932) independently discovered Eimeria necatrix and since the former's work was the first published, to him goes the credit for describing this species. However, it is worth mentioning that both of the above used Tyzzer's (1929) criteria for species differen- tiation and were in agreement in their descriptions, but Tyzzer.2§42l. (22. cit.) contained more detail. Life cycle: The stages in this parasite's life cycle are the same as those present in‘E, tenella, the difference being in the type of cells parasitized. Sporozoites liberated from in- gested sporulated oocysts penetrate the epithelial cells of the small intestine and develop first generation schizonts and merozoites. These merozoites invade the adjacent gland epithelium.and develop into the second generation schizonts and merozoites, the latter when liberated proceed to the ceca where they invade the surface epithelium and develop into the sexual stages. As withuE. tenella, the oocysts are passed in the faces in seven days. E, necatrix infections can be initiated by introducing its Generation I merozoites into the crop or the intestine of chickens. 0n the other hand, merozoites of E, tenella are ablerto cause infection only when injected into the intestine 2k or cloaca, Levine (19h0). Tyzzer (22. cit.) was unable to initiate E. tenella infection withczloacal injection of mero- zoites. Pathology: In acute infections, hemorrhages of the small intestine occur on about the fifth day post infection as a result of the growth and liberation of the second generation merozoites. As with E, tenella, merozoites of this species may be seen in the feces before the blood is observed. Gress pathological studies of the small intestine in the earlier stages, toward the end of the fourth day, will show whitish opacities (schizonts), especially in the middle one third of small intestine, through the serous and muscular coats, but not visible through the mucous surface. On the fifth day these opacities are larger (approximately one milli- meter in diameter) and the small intestine shows a pronounced swelling, various degrees of distension, a dull reddish hue and puncturte hemorrhages. 0n the seventh day, the intestine appears pale and the white opacities are not so readily dis- tinguished. In cases recovering from.an acute attack, there may be badly damaged intestine which will show scar tissue having re- placed destroyed glandular tissue, the result of which may impair the efficiency of the digestive processes of the small intestine. See Appendix, Plate 11. 25 WhilelE, necatrix infection is readily distinguished from E, tenella on the basis of gross pathology features, its sporozoites, merozoites and oocysts have morphological charac- ters which separate each from the other, Tyzzer.2£.2l, (1932). Oocysts: Oocysts sizes range from 22.7 x 18.3/u.maximum to 13.2 x 11.3,): minimum with 16.7 x 1L1.2/u mean, Tyzzer 21; fl.- (gp, _o_;_t_); 2114, x 17.2/1 mean Edgar (1955): and a 19.7 _+_ 1.82/u.x 16.7 1,1.2/0 mean with a length range of 12.1 -28.9/u x and a width range of 10.8 - 23/u Becker 23 2;. (1956). Immunity, physiology, metabolism and digestion: Chickens recovering from a severe infection oféE. necatrix.are clinically protected against reinfection, however, a very light infection results in slight protection, Tyzzer 23‘2E. (22,,222.). As with‘E, tenella authors are not in agreement concerning the relationship between age and sus- ceptibility of the chicken. In one instance young birds (8 days old) were found to be less susceptible than older (35 days old) birds, Tyzzer‘gg'EE. (22. 223.) and vis—a-vis evidence was presented that show younger birds (9 days old) to be more susceptible than birds 10 to 12 weeks of age, Brackett and Bliznick (1952). While there is a prodigious amount of literature avail- able with respect to E, tenella, the paucity of that concerning ‘E, necatrix is conspicuous. 26 MATERIALS AND METHODS A. Pure and Mixed Infection Studies 1. Source of Chickens Chickens (White Rock - Vantress Cross - straight run) were obtained from Hess's St. Louis Hatchery, St. Louis, Michigan. The majority of these birds were sent to Michigan State University via parcel post and arrived at the East Lansing postoffice, where they were promptly picked up the same day they were hatched. Occasionally, although handled in a similar manner, chicks were sent through the University mail and did not reach this area until they were one day old. 2. Distribution of Chickens Upon their arrival, the chicks were immediately taken to the animal disease barn and placed in a brooder, which was in an isolated room.' When the birds attained the desired age for the experi- ments, (ranging in age from 13-18 days, the majority being 1k and 15 days old), they were removed from the brooder and placed in large paper bags (thirty chicks per bag). They were taken to their respective isolated rooms and distributed in groups of ten, one group for each of the three levels in each brooder. 27 3. Care and Management During the first part of the experiment, Room 2 was used to maintain stock birds and controls. Later this room was used for experiments with Aureomycin and the stock birds were raised in Room 1. Feed (in 50 lb. bags) was always maintained in Room 1 and removed to the other rooms as needed. Upon entering the entrance room, street clothes were removed. At the door leading into the hallway, street shoes were removed and knee boots, maintained in the hallway, were put on. These boots were worn only in the hallway. The first room.entered, on all occasions, was that containing the stock birds. These chickens were fed and watered before proceeding to the other rooms. The procedure for entering all rooms was as follows: hallway boots were removed at the doorway into the wash room, hands were washed and since no boots were main- tained in these rooms this area was traversed in stockinged feet. Upon opening the door into the experimental room, boots, which were maintained in and never removed from this room, were put on. Clean coveralls were taken into roomm which were used for experiments on the same day as the birds and inoculating material. These coveralls were put on when entering this room and worn until leaving the room. Coveralls were removed from the room.with the fecal can when the "run" was terminated. Chicks, when received, were taken to the stock room (1). Here they were placed in the five level brooder (never 28 more than 70 birds per one level). The temperature of the brooder levels used for rearing younger birds was maintained at approximately 33° C. The temperature of the lowest level, used for older control birds, was approximately 25° C. Feed and water was suppliedlgg libitum, except for a three-hour period prior to infection. When the birds reached the desired age they were taken off feed and water for three hours. At the end of this period they were placed in large paper bags (thirty birds per bag) and removed to the desired rooms along with coveralls and inocula vials. (Birds were removed and inoculated one by one and placed in the brooder. After inoculation feed and water trays were filled and chicks fed and watered 22 libitum until they were sacrificed. These brooders were maintained at approximately 25° C and their rooms were maintained at a temperature which varied from 22° C to 28° C. Exceptions will be noted later. A11 birds were fed and watered every day. When birds died, a necropsy was performed to ascertain whether or not coccidia could have caused their death. At the end of the "run," birds were sacrificed and the oocysts collected as mentioned under collection of oocysts. Dead birds were placed in the fecal cans as was the fecal material from the fecal trays and the remaining feed. The brooder, except for the frame, was disassembled, thoroughly cleaned, washed and re- assembled. The floor and walls were likewise cleaned. The fecal can and coveralls were removed and taken out of the 29 building via the door opposite the entrance to the hall. The coveralls were taken to a container and later cleaned. The fecal cans were taken to the incinerator and the contents burned. Using a hood, which contained one water and one steam jet, the cans were cleaned and taken to a deep sink, immersed in boiling water for thirty minutes, dried, and re- turned to their respective rooms. Upon the completion of all the "runs" (E, tenella, E. necatrix, mixed infections and drug test) the floors of the hallway and wash rooms, as well as the entrance room, were washed with a 2 percent colloidal iodine solution. This solu- tion was then permitted to dry on the floor. To determine if accidental coccidial infection had oc- curred, the following procedures were followed. When the birds became seven days old and every other day thereafter, until the sixth day post-infection, composite fecal samples were collected and examined for oocysts. The sugar flotation concentration method (Morgan and Hawkins, 1953) was used as follows: the fecal material was mixed with water, allowed to stand for one-half hour, mixed, strained through two layers of cheesecloth, and poured into two 100 ml. round bottom centrifuge tubes (approximately one-quarter full). Saturated sugar solution was added until the tubes were three-quarters full. The suspension was thoroughly mixed and more sugar solution was added until the tubes were filled. The tubes were then centrifuged for one minute at approximately 1000 rpm. 30 Using a headed glass rod, the material from the surface film of the sugar solution was transferred to a glass slide and covered with a cover slip. An examination of this material was made, using a compound microscope (100X), for the presence of oocysts. h. Source of Oocysts Oocysts of both species studied (E, tenella and E. necatrix) were obtained from the Stamford Laboratories of the American Cyanamid Company. Their origin and data are as follows: ‘E. tenella originating from Beltsville, Maryland; 103 previous passages; last harvest, prior to arrival (April 23, 1956) at Michigan State University, was on March 28, 1956. ‘E. necatrix originating from California (some doubt exists as to this origin); 28 previous passages, last harvest, prior to arrival (as above), was on March 3, 1956. The preservation medium, in both cases, was a two percent potassium dichromate solution. 5. Single Oocyst Isolation A piece of soft glass tubing (inside and outside diameters, three and five millimeters, respectively) was heated and drawn out into a fine capillary. By breaking this capillary at the approximate point of smallest diameter, two capillary pipettes were obtained. To ascertain if a capillary was open, rubber tubing with a mouth piece (type used with a Thoma red blood cell pipette) was attached and the mouth piece 31 placed in the mouth. The capillary pipette tip was immersed in a drop of water on a glass slide, which had been placed on the stage of a stereoscopic microscope. Air was then blown through the pipette. Whether or not air was passing through was determined by looking for air bubbles through the lenses of the microscOpe (magnification I 20x). The oocyst culture was diluted until a drop, when placed on a glass slide and observed with a compound micro- scope (100x) revealed one or no oocyst per micros00pe field. The mouthpiece of the rubber tubing was placed in position, the capillary pipette tip was placed in tap water and allowed to fill by capillary action. After removal from this water, the tip was immersed in the drop of diluted culture on the slide and moved into the observable field of the microscope. A very small amount of water was blown out of the capillary and held out by applying the tip of the tongue to the mouth piece. By gentle manipulation, the tip of the pipette was brought up to an oocyst. When the tongue was released, capillary action drew the oocyst into the capillary. The pipette was then removed from the water and the water and oocyst in the capillary blown out onto a clean slide and checked under a compound microscope (as above) to determine if only one oocyst was present. This oocyst was repipetted by a similar pipette, which was neither placed in the drop containing the oocysts nor into the water used to fill the former pipette. 32 The material from the latter pipette was blown into a number five gelatin capsule. This capsule was placed in the esophageal-pharyngeal region of a bird, two weeks old, and blown into the crop using a short piece of flamed glass tubing whose inside diameter was slightly less than that of the cap- sule. ' Chicks two weeks old were brought into the laboratory and infected as above. Immediately after the single oocyst was given to each bird, they were taken to their respective isolated room in the animal disease barn. Here they were placed in the brooder and remained there for six days. On the sixth day, the birds were brought into the laboratory to prevent oocysts from more than one bird contaminating the brooder and room, and each bird individually isolated. 0n the seventh day post-infection, the birds were sacrificed (one at a time) and the ceca were removed with forceps, weaned in running tap water and placed in sterile petri dishes, which were numbered in the order of ceca removal. In order to avoid contamination from one bird to another, a sterile pair of scissors and forceps were used for each pair of ceca re- tnoved. Birds carcasses were destroyed. The material from the ceca in each petri dish was examined (in the same order as their removal) by direct smear under the compound micro- scope (lOOx). The material from the first cecum examined which contained oocysts, was placed in tap water and treated as described under preparation of cultures. These oocysts 33 constituted the so-called "pure line strain" which was used throughout this experiment. Both species were treated in a like manner. 6. Preparation of Culture On the eighth or ninth day post infection, enough re- maining birds were sacrificed (in their respective rooms) to obtain a quantity of oocysts necessary for the next ‘run." Usually for E. tenella this required only five to ten birds for more than an adequate supply. For'E. necatrix fifteen to twenty blrds were needed to collect slightly over the amount of oocysts needed. In collecting‘E. tenella oocysts, the ceca were removed from the sacrificed birds and cut lengthwise. The contents were removed and placed into a screw cap jar (100 ml. capacity) which was one-half filled with tap water. The cecal epithelium was not scraped and it was hoped that the number of nonfertile oocysts were thus kept at a minimum. With E. necatrix infected and sacrificed birds, the ceca were never cut open for oocyst collecting purposes. Since no cores ever formed and the ceca walls were rarely enlarged, the material inside the ceca was forced through a cut in the distal end of the blind sack. By holding the ceca against the rim of the jar with the fingers of one hand and grasping the distal or constricted end of the ceca in the other, the ceca would be pulled through this constriction 3h thus forcing the cecal fluid into the water inside the screw cap jar. The oocyst collection of both species was never made on the same day. This material was brought back to the laboratory and placed in a metal "Waring Blender" jar and subjected to the blender's agitation for one to three minutes. The ceca cores from birds infected with‘E. tenella required more time than the material collected from the ceca of E, necatrix infected birds. The material from the blender jar was poured into two 100 ml. round bottom centrifuge tubes and centrifuged at approximately 1000 rpm for one to one and one-half minutes. The supernatant was poured off and the tubes refilled with tap water. This procedure was repeated until the supernatant was clear (usually four or five washings). The supernatant was then removed and a two percent potassium dichromate solu- tion (15-25 m1.) added to one tube. This tube was thoroughly shaken and the suspension poured into the other tube, which was shaken in a similar manner. The combined material from these tubes was poured onto a series of sieves (sieve sizes National Bureau of Standards Series of 100, 200, and 325). The material, whose diameter exceeded approximately hh/p was retained and the smaller material, including the oocysts, passed through and collected in a 250 ml. Erlenmeyer flask beneath the funnel holding the screens. Fifty to one hundred ml. of two percent potassium dichromate solution was poured 35 onto the screens to wash through as many oocysts as possible. The material collected in the flask was poured into sterile petri dishes (each filled one-quarter to one-third from the bottom) and the oocysts were allowed to sporulate (three to four days) at room temperature. Once each day during sporulation, the material in each petri dish was agi- tated with a clean applicator stick. All of the equipment used in this procedure, except the culture and petri dishes, was autoclaved for at least ten minutes at 2h8° F. and sixteen lbs. of steam pressure. All laboratory equipment used in subsequent procedures with these pathogens was likewise autoclaved before being used again. On the third or fourth day after collection, the cul- ture was pipetted from the petri dishes into centrifuge tubes and centrifuged (as above) until all potassium dichromate solution was removed. Tap water was added to one tube which was then shaken and poured into the other tube. This tube was shaken and the culture poured into a 100 m1. prescription bottle and the culture diluted with tap water to the h0-60 ml. level. This bottle, containing the culture ready to use, was placed into the walk-in refrigerator (2° C) until needed for the next "run," which in most cases was three to five days after being placed in refrigerator storage. 36 7. Oocyst Counts After removal from the walk-in refrigerator, the cul- ture was taken to the laboratory. The bottle was shaken vigorously for approximately one to two minutes in an up and down, as well as a rotating manner, in order to get the oocysts evenly distributed throughout the medium. Immediately after this a W.B.C. (white blood cell) pipette (with rubber tubing and mouth piece in position) was inserted into the middle of the culture suspension. Material was withdrawn up to the bottom of the W.B.C. pipette bulb and transferred immediately under a hemocytometer cover slip (both sides) which was in position on the hemocytometer counting chamber (Spencer Bright Line). No dilution factors were involved, the W.B. C. pipette used merely as a means of transfer. Using the areas of the counting chamber, which are nor- mally used for W.B.C. counts, the number of sporulated oocysts were counted. All eight of these areas were utilized in each count, thus making a total area of eight square millimeters (mm2). To find the number of sporulated oocysts per one mm2, the total number counted was divided by eight (e.g., if 80 sporulated oocysts were counted in eight mm2, then there were 80/8 or 10 aporulated oocysts per mmz). To get volume (cubic 2 was multi- millimeter or mm3), the number of oocysts per mm plied by ten since the distance between the cover slip and counting chamber is 0.1 mm. To get the number of sporulated oocysts in one nfilliliters (ml.), the number of oocysts *0ne(nn4)equals approximately one cubic centimeter (cc.). 37 per mm3 was multiplied by 1000, since 1 mm3 equals approxi- mately 0.001 ml. Thus in the examples used above we have: 80 oocystfister 8 mm2 8 x 10 to cube x 1000 conversion factor ' 100,000 00 cysts /ml. 01‘ Ember of oocystsjer 8 m2 2 till d x 10,000 -= number of oocysts/ml. m u ze 0n the basis of the above count the necessary dilution was calculated in order to get the desired number of oocysts per ml. (e.g, if there were 100,000 sporulated oocysts per ml. in the culture and a suspension of 20,000 per ml. was desired, by dividing 100,000 by 20,000 a factor 5 is obtained. This number 5 means that there are 5 groups of 20,000 oocyst in 1 ml. containing 100,000. By diluting 1 ml. containing 10C’9000 sp0rulated oocysts with Li ml. of tap water we obtain 5 ml, each of which contains 20,000 sporulated oocysts. If 30 m1. of suspension containing 20,000 sporulated oocysts per ml. is desired, the following calculation is made: 30 o 5 I 6 1:Lix6=6:2)4.-30 In these experiments three different dosages were used in ea ch series of "runs," so that three checks were made on the c>I'zi.ginal culture count. If any one of these varied more than 5,000 in either direction (e.g., 20,000 3 5,000) re- checks were made on the dilutions and original count until all 38 dilutions, when calculated from the original, fell within the desired range when counted as above. In all cases the oocyst suspension was prepared in screw cap vials of a size which best suited the total number of m1. desired for each run (e.g., if 10 ml. were needed at least 11 ml. were prepared in a 25 ml. capacity vial, if 30 ml. were desired, a 115 ml. capacity vial was used and at least 31 ml. were prepared. 8. Administration of Oocysts The vials containing the proper number of oocysts per ml. were taken to the animal housing area. These vials were taken to their respective rooms. Each bird was given 1.0 m1. of the desired inoculum and placed in the brooder as follows: lowest number of oocysts per bird group on the top level, and Proceeding downward with the other groups so that the group 01‘ birds receiving the greatest number of oocysts per bird was on the bottom level. The inoculum was administered with an Exax serological Pipette (1 ml. capacity). This pipette was inserted into the deSiI‘ed inoculum, filled and exhausted 8 times, filled to I”We the 0.0 mark and the culture was held at that position in the pipette by holding the index finger of the right hand over the pipette bore. The material was carefully allowed t0 run down to the 0.0 mark. The pipette was removed and placed into the crop by holding the bird in the left hand, grasp ing the beak with the thumb and index finger, the mouth 39 was forced open, the pipette inserted into the esophagus and at the same time the neck was stretched out by placing the butt of the left hand on the dorsal thorax area and extending the thumb and index finger holding the beak straight up and the pipette pushed into the crop. When properly inserted, the index finger holding the inoculum in the pipette was released and the inoculum drained into the crop. As the pipette was being withdrawn, the remaining culture was blown out with air from the mouth. In order to avoid the possibility of carrying over a large number of oocysts from a vial containing a larger number of oocysts to one with a lower number, the bird group receiving the lowest number of oocysts per inoculum were the first to be infected, followed by the group which received the next highest number, and so on until all were infected. In order to help keep accidental contamination of either- of the two species with each other occurring, their inoculation occurred on separate days, e.g., birds were in- feCted with E. tenella on Monday and with E. necatrix on Tuesday. Mixed infections were carried out using the same vials (different pipettes) as those used in pure infections and usually this group of chicks received its inoculation 0n the same day as the second "pure" group. In the example above. this would occur on Tuesday, I A separate vial Or I! pure" inoculum was prepared at the same time as the other Vials for studies with Aureomycin. The birds used in these LLO experiments were inoculated after the birds which were used to maintain pure oocysts. Vials and pipettes were autoclaved after being used. 9. Dosage of Oocysts g. tenella: The range of inocula given varied from 20, 000 sporulated oocysts to lh0,000 per bird. However, of the eighteen runs made, ten were as follows: Group I-— 20,000, Group II-—h0,000, Group IIl--80,000 sporulated oocysts per- bird. E. necatrix: Range of inoculum given varied from 20,000 to 360,000 sporulated oocysts per bird. As with g. M: of 18 runs made, 10 were made at the levels of 20, 1+0 o and 80 thousand per bird. Mixed infection: In preliminary runs the number of oocysts given per bird was as follows: E. tenella, 20,000 to lli0,000; E. necatrix, 50,000 to 280,000. Five runs were made where the three groups of ten birds received 14.0,000 E. necatrix oocysts and Group I received 20,000; Group II § received 1,0000; and Group III received 80,000 g. tenella oocysts. Five runs were made where the total numbers involved Were the same as above except the oocyst number of Q. necatrix was varied and that of E. tenella was constant at LL0,000. lo ‘ Feed The feed used throughout the entire experiment was n on‘antibiotic, non-medicated chick starter mash. It was hl supplied by the Valley City Milling Company, Portland, Michi- gan. Lbs . per mix 1 520 Auoo 2 .280 20 £200 Lioo 3000 60 ‘80 0 S 10 0.5 ”1 13h Aureomycin in the following manner. Its composition was as follows: Ingredients Ground yellow corn Pulverized oats Delsterol u percent soybean oil meal Red fish meal Meat scraps Middlings 17 percent alfalfa meal Poultry mineral A Ground fermentatknisolubles Condensed fish solubles Vitamin A Premix Salt Manganese Sulphate 812 supplement Choline chloride Total lbs. ASS 100 0.5 220 S 50 100 25 15 20 10 1.25 2.5 2 oz. 2/3 lbs. 1/2 lb. loos 13/21-L For the antibiotic study, this same feed was supplemented Three separate weights (2 -5, 5 and 10 grams) of Aureomycin were weighed on an analyti- cal balance and placed in small separate screw cap vials. Tk11?aca was pinched shut with the thumb and index finger and he 1d shut for about one or one and one-half minutes to enhance the retention of this fluid. Fluids thus injected go almost irmnediately to the ceca (Dukes, 1955). Another bird was simi- lar- ly given the remaining ml. and both were properly banded fol" future observations. This injection method was used throughout each time interval and for each test material. Th3— 8 method is similar to that described by Levine (19140) for cloacal merozoite infection. Sporozoite and merozoite collections, as well as thevELted and untreated collection material and the inoculation or the birds, were carried out in a walk-in incubator (37° C). so Chicks were removed from the incubator immediately after inoculation and maintained in the same carton in which they were brought into the laboratory. No food or water was supplied to the birds receiving sporozoites until the fol- lowing day in order to avoid contamination. Merozoite- infected birds were immediately removed to cages after inocu- la and given feed and water. Other control chicks were treated as follows: 1 ml. of buffered saline was given by cloaca to two birds for each time interval, and two chicks received no inocula but were maintained with the other chicks at all times. Two chicks were given 1 ml of sporulated oocyst by cloaca while two others were given 1 ml. of a mixture of oocysts and spores. The latter mixture was obtained by subjecting a sporulated oocyst suspension to the action of an all-glass tissue homogenizer for a very short time. Attempts were made to avoid releasing sporozoites which had occurred due to rapid and prolonged action. Although no sporozoites were observed under a microscope, the possibility does exist that a few were released. Merozoites stages (Generation I and II) were collected from the ceca on the third and fifth days post infection, respectively, and treated in a similar manner as the sporo- zoites previously described. 0n the seventh day post infection, since this type of :Lnfection does not alter the duration of the life cycle 1+7 (Levine, l9h0), all birds were sacrificed and examined for oocysts as described under single oocyst infection collection. In the case of negative results (no oocysts) the contents of the ceca of these birds were checked by the sugar flotation method described under pure and mixed infections. In the Second experiment, where one level of Aureomycin was substituted with a higher level, the centrifugation (as before) of Generation I merozoites was increased to 2000 rpm. Since no infections occurred with this stage in the first experiment, the possibility existed that these merozoites were not in the sediment when subjected to 1000 and 900 rpm due to their small size (2-h,u x l-l.§,n) as compared with the size (lo/u x 2/u) (Tyzzer, 1929) of the Generation I1 merozoites. As with pure and mixed infection studies, utmost care was exercised to prevent accidental infection with any oocysts. Since g, tenella merozoites (Generation II) failed to cause infection when introduced via the crop (Levine, 1940), passed .feces presented no problem with this stage of infection. The other two stages have not previously been reported upon when treated in a similar manner. Since with sporozoite infection the possibility exists for both oocysts and spores to be Present with the sporozoites as collected, no oral inocula were made. However, oral inocula with Generation I merozoites were attempted. Birds were supplied feed and water 3g libitum after .hm>culation, exceptions noted previously. AB RESULTS A. Single Oocyst Isolation, Pure and Mixed Infections Of a total of ten two-week old chicks, each of which received one‘g. tenella Sporulated oocyst, only one, the ninth bird examined, had become infected (oocysts were demon- strated by direct smear). Only one of the two ceca contained oocysts. A similar group of ten chicks, each of which re- ceived one g, necatrix sporulated oocyst, yielded, on the fourth bird examined, oocysts in both ceca of this same bird. Preliminary investigations with sporulated oocysts of E. tenella at an inocuhmilevel varying from 20 to lhO thousand per bird, indicated that no thousand (1.5 thousand) would cause approximately 50 percent mortality. Infections with sporulated oocysts of E. necatrix in passages 3 through 7 gave results which indicated a very erratic type of infec- tion. The oocysts collected in passage number 7 had to be used in three different "runs" due to insufficient oocyst collection. However, average results of mortality (0-100 percent) indicated that 50 to 70 thousand sporulated oocysts would produce approximately 1i} percent mortality. Thus, on file basis of these results, 80 thousand sporulated oocysts amould produce approximately the same mortality as E. tenella at the no thousand level. For further "runs" with pure and L9 mixed infections, the inocula levels of 20, so and 80 thousand sporulated oocysts per bird were used. Since mixed infection studies require controls which could alSo be used to determine if the do and 80 thousand inocula of g, tenella and g. necatrix would produce an LDSO' both experiments were run as one unit. Table I shows the results of five "runs" where, in the mixed infections studies, the inocula of g. tenella oocysts were varied, while the inocula of E, necatrix oocysts were constant. Table II shows the results of five "runs" where (with respect to dosages in mixed infections) the converse is true. The average of these ten "runs" with respect to each other (I-V compared with VI-X) is given in Graph I. A comparison of the relationship between the average percent mortality caused by mixed infection and the average percent mortality caused by the total of both species of pure infection is given in Table III. In an attempt to determine if mixed infection caused any change in mortality with respect to the number of days after infection when compared with that of either of the two Pure strains, mortality dates were recorded and are listed in Table IV. Eimeria necatrix Eimeria tenella Mixed Infection TABLE I 50 PERCENT MORTALITY OF CHICKENs* wITH PURE INFECTIONS OF E. TENELLA AND E. NECATRIX AND WITH MIXED INFECTIONS WHERE THE NUMBER OF OOCYSTS PER BIRD WAS VARIED FOR 3. TENELLA AND CONSTANT FOR g, NECATRIx** Rune II III IV Passage Number Age of birds in days Thousands of oocysts per bird Percent mortality Passage Number Age of birds in days Thousands of oocysts per bird Percent mortality Passage Number Age of birds in days Thousands of 000 etc biyd er §.tene11a Thousands of oocysts or bird, -. necatrix Percent mortality 9 9 9 1n 1n 1h 20 h0 80 30 to 10 7 7 7 15 15 20 no 15 15 20 no 80 to to to no 70 10 10 10 17 17 17 20 to 80 30 30 60 18 18 18 20 no 80 o no 100 As above 18 18 18 20 no 80 to ho no 11 11 11 15 15 15 20 ho 80 10 30 10 16 16 16 20 ho 80 O 10 50 16 16 16 20 to 80 no no so 30 no 80 100 90 100 90 12 12 12 1h 1h 1h 20 so 80 10 no to 10 10 10 15 15 15 20 to 80 0 10 10 15 15 15 20 no 80 to ho L0 to no 50 13 13 1h 1h 20 no 0 no 11 11 15 15 20 no 15 IS 20 a0 80 no to no 30 80 90 *UPen birds in each group. hxad no deaths nor were oocysts passed. en control birds, for each run, given 1 cc. of tap water, 51 TABLE II PERCENT MORTALITY OF CHICKENS* WITH PURE INFECTIONS OF E, TENELLA AND E. NRCATRIX AND WITH MIXED INFECTIONS WHERE THE NUMBER OF OOCYSTS PER BIRD WAS VARIED FOR E. NECATRIX AND CONSTANT FOR E. TENELLA** Runs VI VII VIII IX X Passage Number 111 1h 1h 15 15 15 16 16 16 17 17 17 18 18 18 ‘13 giygirds 13 13 13 1h 11 lb, 15 15 15 15 15 15 15 15 15 Thousands of oocysts 20 no 80 20 ho 80 20 no 80 20 no 80 20 NO 80 per bird Percent 20 to 60 30 70 60 10 30 no 20 60 30 10 10 20 mortality Eimeria tenella N3322§° 12 12 12 13 13 13 1h 11 1h 15 15 15 16 16 16 ‘figiyfiirds mmm mmm mmm 111111 1111111 Thousands of oocysts 20 ho 80 20 to 80 20 no 80 20 to 80 20 no 80 per bird apercent 0 to 60 0 30 100 0 o 10 0 20 30 10 0 10 mortality is necatgix mes. Passage Number As above Ag f bi d 1; gays ” 8 1h 1A In 15 15 15 15 15 15 15 15 15 15 15 15 Thgusandst oer:g§g§h 8 to no no to NO to ho ho h0 hO #0 no no to no one Thousands °§r°§§¥§ts 20 no 80 20 no 80 20 no 80 20 no 80 20 so 80 E. necatrix Mixed Infections Per cent 1 lmbrtality 80 6O 9O 50 80 90 10 30 80 50 SO 60 ho 50 3o fl"Pen birds in each group. ”Ten controlbirds, for each run, given 1 cc. of tap water, had no mortality nor were oocysts passed. 52 Gmumzx COMPARISON OF AVERAGE MORTALITY RATES WITH PURE AND MIXED INFECTIONS OF RUNS I - V WITH RUNS VI - X 5p——9M1xed infection (g. tenella inocula varied at 20, no and 30 thousand) (E, necatrix inocula constant at h0,000) x———%Control (E. tenella pure infection) Hontrol (E. necatrix pure infection) Dotted line denotes the second experiment where the ‘5. necatrix oocysts inocula was varied -- g. tenella constant at N0,000 oocysts per bird per group. ~80 ’I I I I I / I I ’I ,’ ~60 I L50 ..................... 1 [)6 ,‘ o q I I I/ I ” // El I I I I I I f ’ ’ I I I I I I I I I I I I I ’ ’ "’ ’/ 20 I’ ’I __ I I ’I [I ’I .4 I I P10 I ’I I I I” 'I l q. T 21 no 80 ‘Number of oocysts per bird per group (in thousands) Percent mortality 53 TABLE III COMPARISON OF THE AVERAGE MORTALITY CAUSED BY PURE AND MIXED INFECTION Inocula Level Average Mortality in Percent E. tenella E. necatrix Total Mixed - __......._ " __........___. Infection Runs I - V 20,000 17 29 u& h0,000 39 12* 51 7h 80,000 01 53 72 Runs VI - X 20,000 2 an A6 h0,000 h2** 18 60 Sh 80,000 h2 8h 70 *In runs I-V, h0,000 oocysts of E. necatrix were given to chicks along with the three levels or E, tenella oocysts. In the totals the average percent mortality caused by this inocula level is included with those caused by the three levels of E, tenella. **The converse is true for runs VI-X. 5h TABLE IV RELATIONSHIP BETWEEN THE TOTAL NUMBER OF DEATHS AND THE DAYS AFTER INFECTION ON WHICH THESE 0000mm)"c Days After Total Infection 3 h S 6 7 8 9 Deaths Deaths due to .E- tenella O 0 6h 92 18 0 0 17h Percent - - 36.5 53-5 10 - - Deaths due to E. necatrix O 2 62 Sh 1h 0 0 132 Percent - 1-5 #7 h1 10-5 - - Deaths due to 0 0 hi 15h 29 7 0 231 mixed infection Percent - - 18 67 12 3 - *Preliminary data included with that in Tables I and II. 55 B. The Effect of Aureomyoin on the Sporozoites and Merozoite Stages of E. tenella The experiments to determine what effects various levels of Aureomycin in the feed might have on chick mor- tality are tabulated in Table V. The controls in these ex- periments correspond to those used in mixed infection studies and are so designated by the passage numbers which are the same as the number of serial passages after single oocyst isolation. The results of _i_r_1_ vitro experiments of Aureomycin, at the same levels as per the mortality studies, on the sporo- ZO 1te and merozoite stages of E. tenella are recorded in Table VI. 56 TABLE V THE EFFECT OF VARIOUS LEVELS OF AUREOMYCIN IN THE FEED ON THE MORTALITY OF CHICKS INFECTED WITH E, TENELLA AND E. NECATRIX Grams of R"1‘11 Passage Age of 1 Burn be :r Number (zhick? ggrggfigsnogegeed Controls da 3 _._ y 5*“ 10 20 ‘JL.. Pure E. tenella infections with medicated feed given to chicks (10 per group) the day before infection. (u0,000 sporulated oocysts per bird) IE 11 18 50 0 0 30 Percent XII 12 18 no 0 0 no Mortality 1LIII-N 13 16 no 10 0 no .— § 2. Pure E. necatrix infections with medicated feed given to chicks (10 per group) the day before infection. 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