“EH ‘ \“w‘ ‘ HM ’ w ‘\ ‘ ll H E ‘ h ‘ ‘ ‘ ;‘ ‘1 an ‘ E 1 H‘ i: Mxh I H‘ H w ‘ y w E H ‘ H El \ 1“ ‘ ‘ \ ~\| m—a‘ mc>° hus‘ .THS_ A STUDY OF THE EFFECT OF BABILEAHY WHITE BIAHRHEA 0N MATURE FDWLS AND THEIR PRUGEHY THESIS FOR DEGREE OF M. s. HAROLD CANFIELD 1 9 2 5 TTTTT -. m f \J L( a ' VL‘L X; 1 ~ jib/1.0L (‘VL ' ' .L.» . .J T" * g9 SUPPLEMENTAR MATEFHAL IN BACK OF BOOK | ‘ ‘ \ . . ”1“!!! . _. : uE.‘o .. . {Etlr ‘ .\ I. x m A STUDY OF THE EFFECT OF BACILLARY wBITE DIARRHEA ON MATURE FOWLS AND THEIR PROGEHY. A STUDY OF THE EFFECT O? BACILLARY WHITE DIAER'E-FEA ON MATURE FOWLS AND TEETH PROGEI‘IY. THESIS Submitted to the Faculty of the Michigan State College in partial fulfillment of the requirements for the degree Of Master of Science. m by Harold‘ggnfield June, 1925. TABLE OF CONTENTS IEITRODUCTION HISTORICAL BEFUE- EXPEDIMENTAL WORK Object of Experiment Plan of Experiment Procedure Preparation of Antigen Collecting Blood Samples Making T est 3 Feeding and management of Hens Incubation of Eggs Feeding and Breeding of Chicks Experimental Data Discussion of Data CONCLUSIONS ACKNOWLEDGMEHTS BIBLIOGRAPHY f3}.- C‘X ? P 0 e INTRODUCTION Bacillary white diarrhea is prdbably the cause of greater loss and more discouragement to the poultry raiser and hatchery- man than any other poultry disease. A study of this disease is of particular interest because a very large proportion of the chicks reared are purchased from hatcheries. Some idea of the magnitude of the industry can be gained.when We consider the fact that there are sixty-six hatch- eries in Ottawa County, Michigan, with incubator capacity in ex- cess of twenty thousand eggs each. More than six million chicks are sold from these hatcheries each year. Since a very great number of chicks are produced by hatcherymen, it seems very reas- onable to suppose that the control or spread of this disease is dependent largely upon the effort made by hatcherymen to produce clean stock. . . It was hOped that it would.be possible to demonstrate the losses that may be expected.in a flock of poultry from which the reacting birds have been eliminated only once, compared with the losses sustained.in flocks where the infection is very heavy. HISTORICAL RESUME It is quite definitely understood that the disease, ba- cillary white diarrhea has existed for a long time. As early as 1899, Rettger (1) described the organism found in chicks suf- fering from white diarrhea. Later he named this organism Bac- terium pullorum. In 1908 Bettger (2) discovered that a white diarrhea, fatal to young chicks, was caused by an organism hav- ing some characteristics common to typhoid and colon bacilli. He also noted that this disease was very hard to eradicate and occurred year after year among the young chicks. As a preventa- tive measure, he recommended cleaning and disinfecting incubators, brooders, and brooder houses. The following year, 1909, Rettger (3) came to the conclusion that fatal septicemia and white diar- rhea of young chicks were the same disease and the cause of this disease was an organism, which he called Bacterium pullorum. Dur- ing the same year, Rettger and Stoneburn (4) published a bulletin eXplaining that Bacterium pullorum had been isolated from the yolks .of fresh eggs. The conclusions were: "That the mother hen is the original source of infection of the chick. That a few chicks from infected hens have the disease when hatched. That the mortality depends upon the number and virulence of the organisms, mode and time of infection, and the vitality of the chicks. That most of the infected chicks die under four weeks of age. That a for may survive the attack, but remain weak through life and are very sus- ceptible to other disorders.“ In 1911, Rettger (5) reported the finding of Bacterium pul- lorum.in the ovary of an eight months old pullet, a survivor of an attack of white diarrhea. This showed conclusively that the laying hen is the carrier of Bacterium pullorum. Bettger and Stoneburn (6) reported in 1911 that a varying percentage of eggs from infected hens contained Bacterium pullorum. "Very few or as many as 70 per cent of the eggs from infected hens contain Bacterium pullorum.” He also states that this disease may be spread through the medium of infected food and water; and.normal chicks may acquire the disease by picking up infected drOppings or food contamtnated thereby. "Infection from chick to chick cannot, apparently, take place after they are three or four days of age.‘ Female chicks, which survive this disease, often harbor the infection and may become carriers of the organism, infection in the breeding'pens being perpetuated.in this manner. He also observes that infected hens are apparently poor lay- ers, especially in their second and subsequent laying season. In 1912 Bettger (7) reported that the greatest danger from infection with bacillary white diarrhea lies within the first forty- eight hours after hatching, but that chicks may acquire the disease up to the time they are four days old. He also found that hens may become carriers after they have reached maturity. He believes that the infection is acquired.through the mouth. The feeding of sour milk to young chicks was recommended as a means of preventing or at least'holdhng in check outbreaks of bacillary white diarrhea. He concludes that the disease can be eliminated from the farm only be rejecting all birds that harbor the disease and.by obtaining eggs or stock from sources where white diarrhea has not been known to exist. ‘ JOnes (8) reported in 1913 that he had found the magronOp- ic agglutination test to be of great value in detecting fowls that harbor Bacterium pullorum in their ovaries. Serum dilutions of 1-50, 1-100, and 1-200 were recommended as being the most prac- tical. He states that the ovaries of fowls, harboring Bacterium pullorum, are not always pathological, but that seventy-five per' cent of the carriers' ovaries are cystic. In 191% Rettger (9) stated that ovarian infection and ger- minal transmission of bacillary white diarrhea had.been conclus- ively demonstrated. The cycle of the disease was outlined as follows: "The disease, primarily, infects young chicks which fre- quently survive and become permanent bacillus carriers, the ovary being the important seat of infection. The eggs from such car- riers often harbor the organism of the disease in the yolk. Chicks that develop in infected eggs become in turn infected and have the disease at the time of hatching. The disease is transmitted to normal chicks through the infected drOppings.' He concludes that there is no evidence to indicate that germinal transmission through the male takes place. Horton (10) found that sulphocarbolates have very little, if any, value in the treatment of bacillary white diarrhea. Rettger, Kirkpatrick, and Jones (11) reported that sour milk has a very beneficial influence on the growth of young chicks. waever, they did.not regard the sour milk as an important agent in the prevention and suppression of white diarrhea. Gage and Hyland (12) found that the testing of eggs from infected hens was too slow and unreliable to be used as a prac- tical method for detecting fowls carrying Bacterium pullorum. The macroscOpic agglutination test was recommended as a practical method. I Kaup (13) advised the feeding of sour milk to young chicks as soon as they were taken from the incubator or nest. He be- lieved that it would.ward off attacks of white diarrhea and also produce a greater gain. Rettger, Kirkpatrick, and Jones (14) reported in 1916 that a single set of agglutination tests and the elimination of the reactors was not an absolute guaranty that the flock was rid of ovarian infection of white diarrhea but that the percentage of carriers was greatly reduced. Page (15) claimed that bacillary white diarrhea was com- pletely stamped out in the flock that was tested for two years, when the directions were carried out. Gage and Martin (16) described the histo-pathology of the intestines of young chicks infected.with Bacterium pullorum as follows: "From a study of sections made from the intestines there was revealed marked injury to the mucosa associated.w1th hyper- emia, hemorrhagic exudation, and leucocytic infiltration. In the individuals where the disease had run a longer course there were exhibited processes of regeneration. In many instances there was a thickening of the intestinal Wall. There was a marked.fi- brcblastic proliferation and wherever there was any of the co- 1umnar epithelium intact there was active secretion of mucous. Therefore, with these pathological conditions associated to- gether and repeated observations confirming them, it is evident that the important histo-pathological conditions in the intest- ines cf young chicks, dead of Bacterium pullorum infection, cor- respond.to either an acute or beginning chronic condition of catarrhal inflammation.” In 1917, Hadley (17) reports: “We can no longer regard Bacterium pullorum infections as limited to young stock. We have found that Bacterium pullorum was the causative agent in an epi- demic in adult fowls, indistinguishable in its clinical picture and pathological manifestations from fowl typhoid. The primary observations and the experimental features of the study lead to the conclusion that latent Bacterium pullorum infection was stim- ulated into active manifestations of fatal generalized infection or other pathological changes, following the feeding of a ration containing a large preportion of roughage in the form of oat hulls." Ward and Gallagher (18) reported that very satisfactory re- sults were obtained with an intradermal test for Bacterium pul- lorum. Their test fluid consisted of a killed and carbolized culture of Bacterium pullorum that had been grown for about a month. They reported that the results obtained.checked very closely with the agglutination test. Brown (19) reported the results of experiments conducted from 1912 to 1917 with a view to determining the influence of natural and artificial incubation upon white diarrhea in chicks. "En 1912, six hundred Barred Plymouth Rock eggs laid by a flock infeccted with white diarrhea were secured. One-half of these eggs were hatched artificially and the other half were hatched with hens. Enough of the more vigorous pullets of each flock were saved to produce eggs for the following year, and each year the eggs used were from the last generation. The two flocks were always penned together and the males mated with them each year were hen hatched." The percentage of loss each year from white diarrhea in the artificially hatched lot has shown no indication of diminish- ing, amounting to 23.75, 38.50, 26.75, 30.75, Mum, and 69.1% per cent respectively, thus showing a material increase in 1917." ”Each season since 1913 one hundred fifty eggs from the naturally hatched line have been incubated artificially with the result that, with the exception of the first and second genera- tions, no traces of white diarrhea have been found. The percent- ages of loss from white diarrhea, up to the age of twentybfive days, for the seasons 1913 to 1917 from eggs artificially hatched from the second, third, fourth, fifth, and sixth generations, respectively, of the naturally hatched line are as follows: 1913 chicks, 25 per cent; 191H chicks, H per cent; and none for 1915, 1916, and 1917 chicks. In 1919, Sherago and Benson (20) reported on a comparative study of the agglutination test for Bacterium pullorum infection and the intradermal test prcposed by Ward and Gallagher. ”Both tests were used on 134 fowls and the most typical reactors to either or both tests were used in retests to check the results obtained at first and!) observe the influence of previous injections with the intradermal test fluid on subsequent agglutination and intradermal tests.“ From the results obtained, the authors conclude that the intradermal test is so inconsistent as to be worthless as a di- agnostic agent for Bacterium pullorum infection in adult fowls. As possible reasons for the inconsistency of the test, it was suggested that edema is likely to develop and persist for some time after injection, even with sterile water. Any protein is likely to give the same results. The experiments also indicated that a previous injection of the intradermal fluid caused at least 85 per cent of the birds retested to react to the agglutination test regardless of their reaction in the original test. In 1919 Rettger, Kirkpatrick, and Card (21) reported that, due to the results obtained by experimentation, they had con- cluded that transmission of infection from hen to hen, through infected litter and by ordinary association, is rare. No definite conclusions were drawn regarding the part played by the male in the transmission of the organism, but it was thought that this method of transmission could.not be doubted. It was recommended that the males should.not be allowed to run with the females except during the breeding season. Gage (22) made the statement in 1919 that he firmly believed that Rettger and Stoneburn were justified in their conclusion that white diarrhea, as Poultrymen understand it, is a bacillary disease caused.by Bacterium pullorum. he 1922 Gage and.Flint (23) report on the control of bacillary white diarrhea: “The number of cases of bacillary white diarrhea is being reduced each year.“ "From review of the data obtained during the past year, there are indications of improvement among the breeding flocks in the State as a result of the testing work.” 'Of the 110 flocks tested during 1921-1922, twenty-seven were found to be free from bacillary white diarrhea. It is not easy to rid flocks of this disease, especially if the original infection is great. t can be seen from the following table that the infection may be eliminated by testing the original birds, the pregeny, and the prOgeny of the progeny; which means that elimination is possible only after a series of tests. 10 Reduction of Percentage of Infection as Determined.by Testing Work on Eighteen Different Flocks 1919-1921 Flock Infection in Infection Infection in No. original flock in prOgeny prOgeny of progeny per cent per cent per cent 1 2O 11 6 2 2 0 O 3 O O O h 16 7 l 5 5 0 '- 6 it 3 o 7 12 H u 8 h - 2 9 32 21 6.1; 10 0 - 0 11 3 - O 12 31 125 - 13 12+ 25 is - 15 O O - 16 1+.9 o. 3 - 17 7. 6 5 u 18 6.3 1.7 - 11 In 1922 Gage (24) made a general report concerning the diagnosis of Bacterium pullorum infection in the domestic fowl: "Eighty-three specimens showing leg weakness or paralysis, were examined for Bacterium pullorum. The organism was isolated from only five of the eighty-three. This evidence does not in- dicate that 1eg*weakness is due to Bacterium pullorum." "In 1917 and 1918 several sets of experiments were carried out under the best known conditions for poultry husbandry. Eggs from sixty hens, known to have reacted positively to the aggluti- nation test, were set in an electrobator. When tested at the end of the first seven days of incubation, thirty were found to be in- fertile and two were found dead in the shell. Of the twenty-eight left, ten were hatched. Three died at the end of the first day and Bacterium pullorum was isolated from the unabsorbed yolk. All eggs containing fully developed chicks were examined especial- ly for Bacterium pullorum.with the following results. The egg number in each case represents the number of the hen laying the egg.” 12 Table Showing Results of Tests for Bacterium pullorum in Dead Chicks from Eggs Produced by Positively Reacting Birds. gEEber 3:312:33? gggber giiigiégm 8001 + 7925 - 838“ «0- 7993 - e388 - su3o o- 8002 - 8430 - 8002 - 85 65 +- su3o + 8388 4- 7925 - 7995 t 8565 - 814-30 - 8001 + 85814- - "From this table it will be seen that with the methods used it was not possible to detect Bacterium pullorum in all the dead chicks, although adult hens were all positively react- ing to the agglutination test. From eight, Bacterium pullorum was isolated without difficulty and the cultures were negative from the other ten. After three months, following out three sets of incubation, the author was able to obtain from the three sets of eggs set, sixty in each lot, all from positively reacting hens, seven liva- ble chicks in the first set, nine in the second set, and nine in the third set." These chicks were all given the number of the 13 parent stock from which they came: 7811, 7895, 7925, 7997, 7998, 8001, 8002, 8020, 8082, 808”, 8094, 8139, 8171, 8180, 8202, 8204, each, 8294, 838%, 8388, 8389, su3o, 8431, s5u4, 8565, 8810. "These twenty-five birds, all reared from positively ag- glutinating hens, were yarded together and blood taken at vari- ous intervals to determine whether their blood would show any agglutinative power.” Nov. 7, 1917 serum Dilution of Aug. 26, 1917 Dilution of serum * serum Aug. 3, 1917. Dilution of July 21, 1917 serum Dilution of July 17, 1917 8 erum Number Dilution of Records of Agglutination Tests on Chicks Hatched from Eggs Laid by Positively Reacting Hens. Chicks 1M. o o- o 000000000000 coca-r 0 ° 0,, O OOOI-I o 00 o- 0 0000000 0 o cog-1: o oo «- o Oooooooooooo 008-1: 0 °° o- o 000000000000 001'“! ° 00 0- 0 000000000000 00036: 090° 0- 00000000000000 .OOOI-I 00-00 9" 00000000000000 009-1: 00-00 °" 0‘000000000000 COB-I 090° 6' 00000000000000 OOI-I Oo—oo o- 00000000000000 0 000-000-- OCOZ-I 0000 0 00°00 0 ° 0 0001-1 0000 0 0000000 00 P000- OOQ-I 0000 0 0000000 0009.00.» 003": 0000 0 0000000 0000-000» 001.1: 0000 0 0000000 coco-.006» 00°C POGO O 00000~00P00 0002-1 ooooooo 0001-1; o-ooo o 000009.00 9000000000 009-1: 0-000 o Ooooo‘ho COB-I P000 0 00300 00000000 001'“! 000° 0 OOOQOo-OOOOOOOOO ”0-00 0 Oo-POOPOOO 00000—0 8288:; go-oo o oo-ceoowooo owoo-o 009-1: 9-0-00 0 owe-owe—ooo— Geno-6'0 003-1 ““00 0 OFF'OPFOOc—o Opp—«.0 ~OOI‘I ““00 O Oofi'oo-e—ooo- Owe-eche- Hmmwwflmogaimdomdi%mo$o 8$R$$8°No 895388$m39m3 0 - no agglutination ? - doubtful agglutination c - complete agglutination “These experiments indicate that in chicks, hatched from eggs laid by positively reacting hens, at least six months' time should elapse before the normal agglutinative power of such sera would be sufficiently definite to furnish indication of past or present infection. The birds reared from hens 8001, 8139, and 8810 never showed an agglutinative power in their blood core. The length of time a serum maintains its aggluti- native power has not yet been determined.“ In 1923, Gage and Flint (25) made further report on the prOgress of the work on cOntrol of bacillary white diarrhea. “The hatchability of the eggs has greatly improved. Previ- ous to testing and before control measures were followed.by lo- cating and eliminating disease carriers, great losses were sus- tained in the twenty-nine poultry plants now free from disease.“ “After a series of tests had indicated that there were no reactors in these twenty-nine flocks, this department sought to find.where eggs had been sold for hatching, so that a record could be made of the value of the test as a means of producing disease free flocks, from which clean stock could be produced. Data have been obtained by personal visits and.by correspondence. The reports thus far received have been most striking. On one‘ of the large farms where the breeding'birds were infected.with Bacterium pullorum, it became almost impossible to rear even a small percentage of the chicks hatched. Testing was begun in 16 1919 and the agglutination test revealed 27 per cent of the birds in the breeding pen infected.” ”In 1920 another test was run and the percentage of in- fection had drOpped to 20 per cent. The progeny of this flock was tested in 1921 and only 6.5 per cent were found infected. During the present season, 1922-1923, the breeding flock was found to be free from birds which could be classed as carriers. This flock being established as a disease-free flock, records were obtained from all persons receiving eggs for hatching from this flock.” ‘ "The results were gratifying. From 1110 tested breeding birds 11,600 eggs were incubated and 8,700 chicks were hatched, 92.9 per cent of which were reared. This is considerably better than in 1919 when less than 15 per cent matured, the deaths in the first few days after hatching being due to infection with Bacterium pullorum.” "The eXperienoe of the last five years has indicated that poultrymen must test their breeding birds consecutively until no reactors are found in the breeding flock." ”When close coOperation does not exist between the poultry- man and the scientific worker, the disease condition is not im- proved and may be even worse than when work was started." In 1923 Beaudette, Bushnell, and Payne (26) reported on work which they had done on the relation of Bacterium.pullorum to the hatohability of eggs. 17 ”During four years (1919-1922 inclusive) 1,462 fertile eggs were obtained from the infected hens and 553 or 37.8 per cent hatched. Twenty-five eggs were examined which contained dead embryos. Twelve or 483per cent yielded a culture of Bao- terium pullorum." A Comparison of the Hatchability of Eggs from In- fected and Non—infected Hens. (1922) Number Eggs Per cent Per cent of Hens Incubated Fertile Hatched Infected #1 861 69.57 53.59 Non-infected 218 6,387 77.10 65.10 Total 259 7,2ua 76.21 63.86 Summary of Data on Hatching Record of Hens Known to be Infected with Bacterium Pullorum. Number Eggs Fertile Per cent of Eggs Bacterium of Hens Incubated Eggs Fertile Eggs Examined pullorum Hatched Found 3n 2,073 1,u62 37.8 25 12 18 Comparison of Hatchability of Fertile Eggs from Infected Hens with the Flock Average. Year Infected Hens Per cent Hatched by Number Fertile Per cent Entire Flock of Eggs Eggs Hatched ___ 1919 3 123 18.9 30.3 1920 13 569 23.3 32.0 1921 15 278 us.n 53-9 1922 28 492 54.u I 63.9 "The percentage of fertile eggs hatched from the entire flock for the four years was ”3.3 per cent as compared with 37.8 per cent hatchability of fertile eggs from the infected hens during this period.‘I ”There was considerable variation in the hatchability of eggs from the different hens. N0 definite eXplanation based on known facts can be given for this variation. In those cases in which the hatchability of fertile eggs from infected hens was below the average hatched, it might be assumed that the organism was present in a large percentage of the eggs used for hatching purposes. Yet this does not seem to be an ade- quate eXplanation, because it is known positively that the organism may be present, the egg may hatch, and the chick suf-l for from white diarrhea after hatching." 19 "The correlation between infection and low hatchability as indicated.by the records for 1922 seemed to Justify the ap- plication of the tests the following year. In 1923, one hundred eighty-three fowls were tested and.nineteen or 10.3 per cent of the flock were found to be infected. The percentage of fertile eggs that were hatched from infected and non-infected hens is given in the table below, which shows that 18.2 per cent more of the fertile eggs from the non-infected hens hatched.than from the infected hens. It will also be noted that the fertility of eggs from non-infected hens was 33.4 per cent more than from in- fected hens. The difference in hatchability of fertile eggs from infected and non-infected hens was greater in 1923 than in l922, yet the removal of infected birds from.this flock would only have increased the hatchability by 0.8 per cent.“ Hatchability of Fertile Eggs from Infected and Non-infected Hens. (1923) Number Eggs Per cent Per cent Per cent of Hens Incubated Fertile Fertile Total In- Hatched cubated Hatched Infected 19 387 57.0 45.2 25.8 Non-infected 164 5,066 90.4 63.4 57.4 Total 183 5.453 58~0 52-5 55-1 'That low hatchability due to infection by this organism exists in flocks other than the one reported, was shown by the large number of inquiries received regarding losses in the shell.” 20 ”For illustration, we mention the hatching record of a flock of twenty-six fowls of which eleven or 43.3 per cent were found to be infected in 1922. The infected fowls were eliminated and the flock replenished.with new hens, and prior to 1923 hatch- ing season the flock was again tested and found to be free from the infection. A comparison of the hatching record shows that the percentage of fertile eggs hatched was increased from 35.18 per cent to 97.14 per cent or a difference of 61.96 per cent. This difference cannot be attributed.to better management, be- cause both hatches received the same attention. It might be added that hens were always used for hatching in this flock. Two dead embryos were produced during this year, due to chilling. Of the sixty-eight chicks produced none died of white diarrhea." 'From our study of Bacterium pullorum tn relation to poor hatchability it appears that this organism is, at least, one factor to be taken into consideration. While the eggs from all infected hens do not show a uniformly low hatchability, the av- erage hatchability of eggs from infected hens is below the av-. erage for non-infected hens.” "The fertility of eggs from infected hens seems to be con- siderably lower than the fertility of eggs from non-infected hens. From this it might be supposed that the organism.may exert its influence at different stages of the incubation period. The egg may be rendered infertile or death of the embryo may take place at any stage of the incubation period. Usually the largest mor- 21 tality takes place about the nineteenth day of incubation. Bacterium pullorum infection of chicks from the eggs has long been known, but, in view of our eXperiments, t appears that we must recOgnize another loss due to this organism.” In 1923, Hitchner (27) reported that blood sera from hens in heavy laying condition gives very unsatisfactory results in the macr0s00pic agglutination test for bacillary white diarrhea. He claimed that sera has a high fat content that causes cloudy reactions. He recommends starving the birds for at least thirty- six hours before bleeding in order to get clear sera. It is generally understood that this cloudiness of reaction is due to a protein in the sera and.not to fat. Gage and Flint (28) made a third report on the control of bacillary white diarrhea in 1924. They stated that the livabil- ity of chicks at nearly all plants which buy from bacillary white diarrhea free flocks, had.been excellent and that the hatchabil- ity of eggs had been normal. Further claim was made that the presence bacillary white diarrhea in the breeding birds seriously influenced the hatchability. On the other hand, if bacillary white diarrhea is not present, it is no assurance that the hatch will be above normal. Low hatchability of eggs isnot altogether a disease problem. 'However, when chicks are once procured free from Salmonella pullora infection the chances of their maturing are enhanced tre- mendously. White diarrhea control has advanced to day in Massa- chusetts to a point where the original source of infection is being eliminated and chicks hatched have a fair show to live." ”The lowest percentages of livability, reported by customers, of day old chicks from bacillary white diarrhea free poultry plants were 71.80 per cent, 71.87 per cent, and 76.# per cent. These low livability percentages can readily be eXplained. In the first in- stance, the loss was due to poor breeding methods; in the second, the chicks were taken care of by an ineXperienced poultryman who was not fully trained in brooder management; and in the third, the low percentage of livability was due to lack of attention to the details of brooder management." "or the 20,665 day old chicks sold from representative ba- cillary white diarrhea free breeding flocks, aside from the three mentioned above, the livability averaged.nearly 90 per cent. Many individual plants have reported that nearly 100 per cent of all chicks received from bacillary white diarrhea free flocks were living and in good health.” 23 EXPERIMENTAL WORK. Object of the EXperiment. Past experience has shown that bacillary white diarrhea is very common in the poultry flocks of Michigan. [here this infection exists in the flock, the mortality of chicks seems to increase each year unless some control measure is practiced. In many cases the mortality of young chicks increases to such an extent that it is impossible to keep more than twenty to thirty per cent of the chicks alive for six weeks. However, most of the infected chicks die between four and fourteen days of age. At present the most practical control of the disease is accomplished by locating the infected.birds in the breeding flock with the macroscOpic agglutination test and eliminating these infected individuals sometime during the winter before eggs are saved for hatching purposes. It is quite generally known that a single agglutination test does not eliminate all infected fowls. This experiment was planned to make a comparative study of the egg production of both the hens giving a positive reac- tion and those giving a negative reaction to the macroscOpic agglutination test for Bacterium pullorum, using two tubes of gntigen with serum dilutions of 1-40 and l-lOO. It was also planned to compare the fertility and the hatchability of eggs from non-reacting birds. 2» Lastly, it was planned to compare the quality and liva- bility of chicks from reacting hens with the quality and liva- bility of chicks from negatively reacting hens. From this study it was hOped to determine the efficiency of a single agglutination test in eliminating the reacting fowls from a poultry flock and the extent of the losses sustained.by infection with this disease. Plan pf the Experiment. The plan of this experiment was: '(a) To apply the macroscOpic agglutination test for Bac- terium pullorum to all the hens in a given flock of poultry. (b) To place in a pen, separate from the main flock, all birds giving a positive reaction to the test. (0) To give the same care, feed, and attention to infected and.non-infected birds. { (d) To keep trap nest records for at least twelve weeks of all birds in the entire flock. (a) To incubate eggs from infected and non-infected hens and keep records of the fertility and hatchability of these eggs. (f) To feed and care for the chicks hatched for at least fourteen days and keep records of the deaths during this period and also note, if possible, the causes of death. Procedure. Preparation of Antigen. About 30 c.c. of liver infusion agar (2 per cent agar) was placed in each of twenty sterile Kolle flasks. These flasks, 25 containing the agar, were sterilized.in the autoclave with fifteen pounds steam pressure for fifteen minutes. as soon as the agar was cool the flasks were then inoculated with one c.c. of Bacterium pullorum (strain No. 51) and then incubated for forty-eight hours at 37°C. The resulting growth was washed from the surface of the agar with phenolated physiological salt solution, care being taken to keep the volume of the suspension as small as possible. About 50 c.c. of a fairly heavy suspension of organisms was collected and filtered through cotton. Trial dilutions were made to find out what prcportion of phenolated physiolOgical salt solution should be added to the suspension to make an antigen having the same turbidity as tube Ho. 1 of McFarland's Nephelometer. Three blood samples were used in testing the new antigen. In a previous test, one sample had given a strong positive reac- tion, another had given a.weak positive reaction and the third had given a negative reaction. The antigen was found to be satisfactory and stored in the ice box for future use. Collecting the Blood Samples. About two c.c. blood were collected in small sterile glass vials from each hen, the blood being taken from the wing. As soon as the sample was drawn the vial was laid in slanting posi- 26 tion on a shelf until the blood coagulated, forming a blood slant. The blood samples were left at room temperature for about six hours in order to allow the serum to separate from the clot and then stored in the ice box until about two or three hundred samples had been collected. Making the Tests. When a sufficiently large number of blood samples had been collected, the macroscopic agglutination test was applied to each sample for Bacterium pullorum. For making this test clean Wasserman tubes were placed.in racks holding thirty-two tubes and.numbered in pairs, one to sixteen inclusive. One c.c. of Bacterium pullorum antigen, diluted so that the turbidity was the same as No. 1 tube of Mo- Farland's nephelometer was measured.into each tube. Two tubes were used for each sample. In the first tube was placed 0.025 c.c. of blood serum and 0.01 c.c. in the eecOnd tube, giving di- lution of l-MO and 1-100 respectively. The serum and antigen were thoroughly mixed.by shaking. After incubating at 37°C. for twenty-four hours the tubes were examined and results recorded. Feeding and Management of the Hens. Of the five hundred ninety-three hens tested, fifty-eight reacted to the agglutination test. The fifty-eight hens react- ing to the test were placed in pen No. l} with seven male birds, two of which were infected. These birds were given the same feed and care as other birds at the poultry plant. The ration consisted of dry mash (20 parts bran, 20 parts ground corn, 20 parts ground cats, 20 parts middlings, and 10 parts dried beef scraps), scratch grain (equal parts of corn and wheat), sprouted oats, and semi-eclid buttermilk. The hens were given free access to the dry mash at all times and the scratch grain was fed twice daily, the caretaker using his own Judgment as to the quantity to be fed. As much semi- solid buttermilk was smeared on the wall as the hens would clean up in fifteen minutes. Each morning about ten o'clock, sprouted oats were fed, allowing about one cubic inch of sprouted oats for each hen. Oyster shell, charcoal, grit, and water were be- fore the hens at all times. Trap nest records of the hens at the plant are kept through- out the year and during the hatching season each egg is marked with the number of the hen when it is taken from the trap nest. Incubation of the Eggs. The first lot of eggs from the infected hens was incubated in a Buckeye incubator and a Prairie State incubator was used for the second lot. The third lot of eggs from the infected.hens was incubated for eighteen days in a Petersime electric incubator and removed to a Newtown Mammoth incubator to finish hatching. The three lots of eggs from the hens showing a negative reaction were incubated in the Newtown Mammoth incubator. Feeding and Brooding the Chicks. All the chicks hatched were placed in brooder houses ten feet square, heated by hard coal burning brooder stoves. The temperature was kept about 95°F. under the canOpy. When forty- eight hours old the chicks were given a small amount of Domino Chick Starter on a newspaper, some fine grit, and.warm water. For the first week the chicks were fed five times daily as much Chick Starter as would be cleaned up in fifteen minutes. After the chicks were a week old the Chick Starter was fed in small feed dishes and the chicks were given free access to it at all times. A small amount of chick scratch feed and cat sprouts were added to the ration at this time. Tables No. I and II.* Table III. Showing the Livability of Chicks from Hens not Reacting to the Agglutination Test. First Lot Second Lot Third Lot Total of Hatched Hatched etched Three Lots March 2 March 17 March 26 ggnber of 31 “7 - 39 117 ’ icks Number of u l 2 7 Chicks Died Per cent 87.09 97.8 92.3 94.02 Livability ‘ These tables will be found in pocket on the cover. "89 Table IV. Showing the Livability of Chicks from Hens Reacting to the Agglutination Test. First Lot Second Lot Third Lot Total of Hatched Hatched Hatched Two Lots March 31 April 14 May 22 Number of 49 76 52 125 Chicks Number of #6 51 - 97 Chicks Died in 14 days Per cent 6.12 32.89 - 22.4 Livability Table V. Showing the Death Rate of Chicks from Hens Reacting to the Agglutination Test. Age of chicks l 2 3 h 5 6 7 8 9 10 ll l2 13 14 Chicks surviv- _itn days ing 1m day No. of chicks deadinLotflOClZLlBlljj 4JOOO j _ Appearance 0 ver - - x x x x x x r: rx 2: Ufiabsorbed “ 101k - - + f.) + + + + f + No. of chicks dead in Lot #2 4 2 O 2 29 11622 3 5 ,2 l 2 25 Appearance “ ofliver xx-xxpmrxrxrrnfr Uhabsorbed 101k f f - 4 + + r + + j + + 4} it Note: x - ochre color r - red lines rx - ochre color with red lines nf - necrotic foci xp - ochre colored patches - - unabsorbed yolk present .30. During the feeding period, all chicks that died were ex- amined for lesions. After fourteen days all the chicks still alive in the infected lots were killed and examined for lesions. The chicks from eggs laid by hens giving a negative reaction were not killed as they appeared very strong and sturdy, showing no symptoms of disease. Discussion of Data. It has been quite generally understood that birds infected with bacillary white diarrhea will produce practically as many. eggs during the first year as uninfected hens, but during sub- sequent years they are very poor producers. The data obtained in this eXperiment do not show much evidence that the one and two year old infected hens produce less than could be expected of them normally. The numbers in Table No. I preceded by the letter 0 represent pullets, numbers preceded by b represent year old hens, and.numbers preceded by a represent two year old hens. However, the average egg production of the positively re- acting hens was about three-fourths as great as the egg produc- tion of the hens in the check lot. The average production of the reacting hens was 37.37 per cent, while production of the hens in the check lot was ”5.56 per cent. The positively reacting hens apparently produced a higher percentage of infertile eggs and eggs having weak germs, than we. produced by the hens giving a negative reaction. The re- 31 actors produced an average of 20.8 per cent of infertiles and 17.5 per cent dead germs, while birds in the check lot produced an average of only 8.# per cent infertiles and 6.6 per cent dead germs. An average of Ml.9 per cent of the eggs laid by the hens in the check lot-produced chicks that died in the shell, while only 25.9 per cent of the reactor's eggs, which were incubated, produced fully formed chicks that failed to hatch. Chicks were hatched from 35.9 per cent of the reactors' eggs and 43.1 per cent of the check birds' eggs that were incubated. Apparently the only explanation for the small percentage of fully formed chicks failing to hatch from eggs laid by infected hens is that many of the embryos die in the early stages of in- cubation. The difference between the hatchability of eggs from in- fected hens and the hatchability of eggs from non-infected hens was far less marked than the difference in livability of chicks from the two lots. Of the chicks hatched from eggs laid by hens in the check lot, an average of 9M.O2 per cent were living and apparently vigorous on the fourteenth day, while an average of only 22.4 per cent of the chicks from reacting hens were living at that time. Of all of the infected chicks living at the end of the fourteenth day only four showed any indications of recovery. The rest of the chicks appeared stunted and diseased. All of the infected chicks, remaining alive on the fourteenth day, 32 were killed and examined. In all cases the livers showed marked indications of the presence of Bacterium pullorum. Cultures were made from three chicks hatched from eggs laid by hens in the check lot and eight chicks hatched from eggs laid by the reactors. Bacterium pullorum was found in each case. This shows that bacillary white diarrhea was not completely eradicated by one elimination of reactors. The tables showing the death rate of chicks from infected hens indicate that the greatest mortality occurs during the sixth and seventh days after hatching. The diicks from the infected hens appeared as healthy and strong as normal chicks when first hatched and did not show a particularly unhealthy appearance until they were five or six days old. 33 Cenclusions. From the data obtained the following conclusions could be drawn. 1. The egg production of hens harboring bacillary white diarrhea infection is usually lower than egg production of hens free from this disease. .3. The fertility and hatchability of eggs is materially lowered by the presence of bacillary white diarrhea infection in the flock. 3. The hatchability of eggs and the livability of chicks is usually improved by even one elimination of hens reacting to the macrOSCOpic agglutination test. 4. 8 single elimination of hens reacting to the aggluti- nation test does not remove all birds harboring Bacterium pul- lorum. Acknowledgments. The writer takes great pleasure in expressing his grati- tude to Hr. C. G. Card, Assistant Professor of Poultry Hus- bandry. Hr. C. h. Ferguson, Assistant Professor of Poultry Husbandry, Dr. H. J. Stafseth, Research Associate and Associate Professor of Bacteriology, Er. W. L. Mallman, Research Assist- ant and Assistant Professor in Bacteriology, and Zr. C. F. Huffman, Research Assistant in Dairying for valuable suggestions and criticisms which have made this work possible. It is also a pleasure to thank the Departments of Poultry Husbandry and Bacteriology for the use of stock, equipment, and materials necessary in carrying on the experimental work. 1. Bibliography Bettger. Fatal Septicemia in Young Chickens. New York Medical Journal. Vol. 71. Page 803. Rettger. Fatal Septicemia in Young Chickens. Journal Medical Research. Vol. 18. Page 277. Bettger. Further Studies of Fatal Septicemia in Young Chicks or White Diarrhea. Journal Medical Research. Vol. 21. Pages 115-123. Bettger and Stoneburn. Bacillary White Diarrhea in Young Chicks. Connecticut Expt. Station Bulletin 60, 1909. Pages 33-57. Bettger. Bacillary White Diarrhea of Young Chicks. Abstract in Science. No. 8%}. Pages 547-543. Bettger and Stoneburn. Bacillary White Diarrhea of Young Chicks. Connecticut EXpt. Station Bulletin 65. Pages 279-301. 10. ll. 12. b4 0\, Bettger, Kirkpatrick and Stoneburn. Bacillary White Diarrhea of Young Chicks. Connecticut Expt. Station Bulletin 7h. Pages 153-185. Jones. The Value of the macroscOpic Agglutination Test in De- tecting Fowls that Harbor Bacterium Pullorum. Journal of Medical Research. Vol. 27, No. 4. Pages 471-9. Rettger. Ovarian Infection in the Domestic Fool and Direct Trans- mission of Disease to the Offspring. Journal Experimental Medicine. Vol. 19. Pages 552-561. Horton. Sulphocarbolates in the Treatment of White Diarrhea (bacillary form) of Young Chicks. American Veterinarian Review. Vol. us, No. 3. Pages 321-2. Rettger, Kirkpatrick and Jones. Bacillary White Diarrhea of Young Chicks. Connecticut EXpt. Station Bulletin 77. Pages 236-309. Gage and Hyland. On the Diagnosis of Infection With Bacterium pullorum in the Domestic Fowl. Massachusetts EXpt. Station Bulletin luS. Page 20. 13. 14. 15. l6.' 17. 18. 37' Kaupp. Some Further Studies of Chick Hortality. North Carolina Expt. Station Bulletin 235. Pages 11-15. Rettger, Kirkpatrick and Jones. BacillarY White Diarrhea of Young Chicks. Second Pregress Report on the Elimination of Infected Breeding Stock. Page. Report of the Department of Veterinary Science. Massachusetts Expt. Station Report. 1916. Pages 89-92. Gage and Martin. Notes on the Histo-PathOIOgy of the Intestines of Young Chicks Infected With Bacterium Pullorum. Journal Medical Research. Vol. 3%. Page leg. Hadley o Infections Caused.by Bacterium pullorum in Adult Fowls. Rhode Island Expt. Station Bulletin 172. 1917. Ward and Gallagher. An Intradermal Test for Bacterium pullorum Infection in Adult Foals. U. S. D. A. Bulletin 517. 19. 20. 21. 22. 23. 3s Bronn. Artificial vs. Natural Incubation. Influence on Baeillary White Diarrhea of Chicks. Minnesota Station Report, Crookstcn Substation. 1917-ls. Pages 54-86. Scherago and Benson. Experiments on the Intradermal Test for Bacterium.pullorum. Cornell Veterinarian. Vol. 9, l9l9. Page 111. Rettger, Kirkpatrick and Card. Bacillary White Diarrhea of Young Chicks. Ovarian Infec- tion of the Adult Fonl and Transmission of the Disease through the Oviduct. Connecticut EXpt. Station Bulletin 101. Pages 73-78. Gage. Notes on Ovarian Infection With Bacterium pullorum in the Domestic Foal. Journal Medical Research. Vol. 2%, No. 3. Page 491. Gage. Control of Bacillary White Diarrhea. Massachusetts Expt. Station Control Series Bulletin 22. 2h. 25. 26. 27. 28. 39 Gage. Concerning the Diagnosis of Bacterium pullorum In- fection in the Domestic Fcnl. Massachusetts Expt. Station Technical Bulletin 5. Gage. Control of Bacillary White Diarrhea. Massachusetts Expt. Station Control Series Bulletin 23. Beaudette, Bushnell and Payne. Relation of Bacterium pullorum to Hatchability of Eggs. Journal of Infectious Disease. Vol. 33. Page 331. Hitchner. The MacroscOpic Agglutination Test as Influenced.by the Fatty Content of the Blood Sera of Foals. Journal American Veterinary Medical Association. Vol. 63, No. 6. Pages 759-763. Gage and Flint. Control of Bacillary White Diarrhea. massachueetts Expt. Station Control Series 27. nail