EXPEREMEN'FM INFECTEQN CF PREGNANY EWES WITH LEPTOSFERA POMGNA; AND §NTESTENAL Aaségfiafiwa? mfifim. ANTEBQDIES EN LAMBS Thesis ‘or flu Degree 05 M. S. MICE :17"ng ‘{ “if"; :21. wt. 5‘1233123; flame Juliam Linciqvis‘: 1957 ICHIGA um» Mm mm M A]MmM\ilikifli\‘Iiil‘fli’a‘iir‘ifill l HI 31293 006121432 ‘ EXPERIMENTAL INFECTION OF PREGNANT EWES WITH LEPTOSPIRA POMONA, AND INTESTINAL ABSORPTION OF LEPTOSPIRAL ANTIBODIES IN LAMBS by KAARE JULIAN LINDQVIST A THESIS Submitted to the College of Veterinary Medicine Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1957 I. II. III. IV. VI. VII. VIII. IX. TABLE OF CONTENTS INTRODUCTION . . . . . . . REVIEW OF THE LITERATURE . MATERIAL AND METHODS . . . EXPERIMENTAL RESULTS . . . 1. Group I . . . . . . . 20 GroupII....... 3. Absorption of Leptospiral DISCUSSION AND CONCLUSIONS I. The Disease in Sheep 2. Necropsiea e e e e e e 3. Lambing and Lambs . . . Antibodies in O O h. Leptospirae in Milk . . . . S. Antibody Transfer to the Newborn SMARY C O O O O O O O O 0 FIGURE 0 O O O O O O O O O TABLES 2 through 23 . . . REFERENCES . . . . . . . Page 21 27 27 32 39 1.2 1+2 so 52 53 51+ 57 so 61 82 LIST OF TABLES TABLE Page 1. Colostral transmission of antibody substances . . . 20 2. Normal hematological data (December, 1956) . . . . 61 3. History of the ewes . . . . . . . . . .0. . . . . 62 h. Duration of leptospiremia in Group 1, determined by blood cultures . . . . . . ... . . . . . . . 63 5. Serum antibody titers of the ewes in Group I (L... Romanatiters) eeeeeeeeeee.e-eee 6h. 6. Serum antibody titers of the ewes in Group I (L, icterohaemorrhagiae, AB titers) . . . . . . . 6h 7. Temperatures, hemoglobin values, erythrocyte and total leucocyte counts of Group I . . . . . . . 6S 8. Differential leucocyte counts of Group I . . . . . 67 9. Duration of urinary excretion of leptospirae in Group I, determined by animal inoculations . . . 68 10. Antibody titers for L. pomona in the urine of GroupI eeeeeeeeeeeeeeeeeeee 68 ll. Antibody titers for L. pomona in colostrum (or mdlk) and sera of ewes, and sera of lambs in Group]: eeeeeeeeeeeeeeeeeeee 69 12. Hematological observations of the lambs in GroupIatbirth................ 7O 13. The course of'L. pomona infection in ewe 79h . 71 1h. Temperatures and hematological data obtained in Group II after inoculation of blood from Ewe 79k. 72 15. Temperatures and hematological data of Group II following inoculation with 3 x 108 leptospirae . 7h 16. Duration of leptosphxnda in Group II, determined .bybIOOdCUIturea oeeeeeeeeeeeeee 76 17. Serum antibody titers in Group II . . . . . . . . . 76 TABLE 18. 19. 20. 21. 22. 23. Duration of urinary excretion of leptospirae in Group II, determined by animal inoculation . . . . Antibody titers in the urine of Group II . . . . . . Hematological data of lambs in Group II at birth . . Antibody titers for L, pomona in colostrum and milk whey, as well as sera o ewes and lambs in GroupII eeeeeeeeeeeeeeeeeeee The course of L. pomona infection in a lactating ewe (758) exposed 30 days post partum . . . . . . . . Development of serum titers in lambs to which antiserum was administered per g§_at various ages . Page 77 77 78 79 80 81 ACKNOWLEDGMENTS The author wishes to express his sincere thanks to the W. K. Kellogg Foundation whose generosity made his stay in this country possible. Thanks are also due to Dr. E. V. Morse both for pro- viding facilities for, and for his continued interest in this study. The valuable assistance rendered by Mrs. Athalie Lundberg and Mr. Leonard Eames in the course of this work is greatly appreciated. INTRODUCTION This investigation is one aspect of a general research program concerning leptospirosis presently being conducted at Michigan State University. Numerous reports indicate that leptospirosis caused by Leptospira pomona is one of the most prevalent diseases of domestic animals in the United States (70, 71, 95). There- fore, the disease is of considerable economic importance to the livestock industry (M6, 56). The public health signifi- cance is indicated by the fact that the disease is trans- missible from swine and cattle to man and that farm animals constitute the most important reservoir of the etiological agent (28, h2, A3, 85). Leptospira pomona is frequently incriminated as the cause of abortion in cattle and swine (9, 58, 95). The course and effect of Leptospira pomona infection in pregnant sheep have not been subjected to extensive investigations. The purpose of this work has been to study the bacteriological, hematological and clinical aspects of the disease in preg- nant ewes, and to ascertain the effect of infection upon the fetuses at various stages of gestation. The antibody content of milk from.the ewes, as well as the development of antibody titers in the sera of lambs ingesting colostrum, was deter- mined e Upon completion of this work, some experiments were carried out on the intestinal absorption of leptospiral anti- bodies in normal lambs of different ages by feeding high titer serum. REVIEW OF THE LITERATURE l. The Recognition of Leptospiragpomona In 1936 a leptospira was isolated from the blood of a dairy farmer living near Pomona in south Queensland, Aus- tralia, who had suffered from a fever of eight days duration. This strain was referred to as the Pomona strain and was later shown to differ serologically from other known sero- types of leptospirae (15, 19, 20, ho, h7, 91, 92). Subse- quent to the establishment of the specificity of the organism, the name Leptospira pomona was proposed by Derrick in l9h2 (20). In 1938 an epidemic of leptospirosis in swine care- takers caused by a strain which was designated Honakow (type II) occurred in Russia (88). Sera from convalescents agglu- tinated this strain in high titers as did those from the pigs with which they had been associated. In the same year a strain designated Mezzano was isolated from a case of "rice field fever” in a man in Italy. Both the Russian and Italian strains have been shown to be identical with Leptospira pomona (2, 3, 27, 7h). As early as in 19h0, Mochtar (53) isolated Leptospira pomona from.swine in Java. Leptospira pomona has been incriminated in Switzerland and Germany to be the causa- tive agent of certain cases of serous meningitis in man affecting those persons who have been in close contact with pigs. Since swine appeared to be the reservoir of the infectious agent, the disease became known as "swineherds disease" (27, 29, 7S). 2. Bovine Leptospirosis The first etiologically defined case of bovine lepto- spirosis in the United States was reported by Jungherr (#1) in 19hh. He was able to demonstrate the presence of leptoe spirae in kidney sections from diseased animals. The agent was not isolated; therefore, the serotype is not known. It has been assumed that it was'g. pomona. Previously, clinical syndromes of icterus, hemoglobin- uria and abortions had been reported in cattle in the United States which in retrospect may very well have been lepto- spirosis (#8, 70, 8h). Mathews (R9) in 19h6 was able to transmit a febrile disease of cattle to calves and guinea pigs, by the inocula- tion of blood from affected animals. Leptospirae could be demonstrated in tissue sections from some cases. Baker and Little (u') in 19h6 isolated an agent from cattle which had acute mastitis.‘ At that time the agent was considered to be a virus. Later, however, the condition was shown to be due to a leptospira (h"). The microorganism.was found to be serologically indistinguishable fromnggptggpigg pomona (26). The course of Lgptggpigg pgmgng infection in cattle may be inapparent or may be characterized by severe symptoms of anemia, icterus and hemoglobinuria, especially in calves. Abortions are not infrequent and usually occur in the latter half of pregnancy (3, 2k, 30, 33, 52, 70, 81, 87). It is not known whether the abortion is a physiological reaction or whether it is directly mediated by the lepto- spirae (23, 57). Preliminary evidence indicates a hemolytic endotoxin or exotoxin may be involved (23). Only one report has been found describing the isola- tion of leptospirae from aborted bovine fetuses (6h). The almost consistent failure to demonstrate leptospirae in aborted bovine fetuses is in great contrast to what has been described in porcine leptospirosis. Leptospirae can be found rather easily in aborted pigs or piglets born dead or weak (23). Ferguson gt £1. (23) stated that there was an apparent lack of erythrocytes in the heart, blood vessels and other tissues of aborted bovine fetuses. This phenomenon has also been observed in naturally-occurring leptospiral abortion in cattle. Infected cattle may shed leptospirae in the urine for three months after the infection (86). This probably repre- sents the extreme rather than the usual duration of the carrier state in cattle (55). 3. Porcine Leptospirosis Monlux,‘g£_§;. (5h) in l9u8 found leptospirae in sections of kidneys from.a diseased hog and this is thought to be the first case of porcine leptospirosis noted in the United States. In 1950, Bohl and Ferguson (22) discovered that anti- bodies against Lgptospira pomona were present in the sera of Ohio swine. The first isolations of this organism from in- fected swine in the United States were performed in 1952 by Gouchenour, gt 5;.(25). Bohl and Ferguson (8) cultured lepto- spirae from an "apparently healthy,” but serologically positive, pig. Recently a number of isolations have been made from aborted fetuses (12, 13, 73) and from.stillborn and weak pigs (9). The serotype involved in these instances was‘g. pomona. Although the organisms may be found in ”apparently healthy" pigs, the disease in pregnant sows mabee extremely severe. Abortions frequently occur as well as the birth of dead or weak piglets. These may be the only manifestations commonly found (A, ll, 73, 80). Icterus and hemoglobinuria commonly associated with the disease in cattle, sheep and horses have not been reported in swine in the United States. Infected pigs may harbor leptospirae in their kidneys for long periods of time. The microorganisms may be eliminated in the urine in large numbers (h). Thus, swine represent important carriers of these organisms and a potent source of infection for other animals and man. The duration of the carrier state in swine has been investigated by Baker (R) who found it to last longer than five months. Schmid and Giovanella (78) reported that the elimination of leptospirae in the urine of experimentally- infected pigs commenced approximately three weeks after the infection and that the organisms were still present in large numbers for six months. The number of organisms decreased gradually and one year after the infection only very few, if any, leptospirae were observed in the urine. Horse (55) found that in an experimentally—infected group of twenty pigs only one pig was shedding leptOSpirae on the 122nd day after inoculation. This was determined by intraperitoneal inoculation of urine into groups of hamsters. Two different strains, one of bovine origin and one of porcine origin, served to infect the hogs. Extensive and detailed information concerning human, porcine, bovine and equine leptospiroses is given in the re- views by Halch-Sorgdrager (91), Broom (10), Reinhard (70), Bohl and Ferguson (8), and the monographs by Gsell (27), Van Thiel (90) and Rimpau (7M. h. Ovine Leptospirosis Only a few reports have been published concerning leptospirosis in sheep. Therefore, it was deemed necessary to review these findings in more detail. The Annual Report (1950-1951) of the New Zealand Department of Agriculture reportaifbr the first time the oc- currence of leptospirosis in sheep (31). Hartley (31) in 1952 described the findings on which this first diagnosis was based and also presented data concerning a more recent outbreak. In the first outbreak in 1950 the diagnosis of leptospirosis was made on the clinical finding of "redwater" and pathological changes similar to those observed in bovine leptospirosis. Moreover, leptospirae were observed in sec- tions of liver and kidney stained according to the method of Levaditi. In the second outbreak, occurring in 1951, the clinical observations and pathological changes were essentially similar to those noted in the first outbreak. Darkfield examination <1“ urine from a living male lamb revealed numerous viable leptospirae and the organisms were found to infect guinea pigs. Sera from recovered animals agglutinated a strain of Leptospira pomona to a titer of 1:2000. Hebster (31) in a personal communication to Hartley, states that he had also been able to demonstrate live leptospirae in the urine from infected sheep. Ovine leptospirosis, as described by Hartley (31) occurred mainly in lambs although a few ewes contracted leptospirosis and died. Icterus and hemoglobinuria were the only prominent signs observed. In a personal communication to R. L. Mortar in 1956, Hartley stated that leptospirosis is quite prevalent as a clinical entity in sheep in certain areas in New Zealand. \0 Furthermore, he indicated that ovine leptospirosis usually occurred as eXplosive outbreaks among lambs on lush pastures which were heavily stocked. Affected animals showed a high temperature, the urine was invariably red in color and the animals often died within two or three days. He noted that survivors and animals which developed subclinical infections remained renal carriers for many months. Usually evidences of interstitial nephritis were lacking. Experiments were performed on several occasions to infect lambs, "hoggets" and pregnant ewes with known virulent calf leptospiral strains and with strains isolated from sheep, but no clinical signs except a mild rise in temperature and inconsistently lepto- spiruria were produced. The strains of leptospirae isolated from cattle and sheep in New Zealand appeared to be serologi- cally identical and indistinguishable from Leptospira pomona (32). Since Hartley's first descriptions of leptospirosis in sheep, further outbreaks have been recorded in New Zealand. Salisbury in 1953 investigated an outbreak of the disease in a small flock in which eight lambs died out of a total of seventy. After the last death occurred, urine samples were collected from the lh5 animals which remained, and of these 13 were shown ”to be positive" (76). Seddon (79) in his paper on diseases of domestic animals in Australia, mentioned the occurrence of lepto- spirosis in three pet lambs on a farm where five weeks 10 previously there had been a severe outbreak of the disease among calves. All three lambs excreted leptospirae in their urine and one lamb was still doing so eight weeks after the onset of symptoms. Salisbury and McDonald (77) reported that death is the first indication of leptospiral infection in sheep. Losses are greatest among young lambs and yearlings. Icterus and hemoglobinuria are usually present. Webster and Reynolds (93) in New Zealand had the oppor- tunity to study a severe outbreak of ovine leptospirosis in a large flock of ADC ewes and their lambs. Morbidity approached 100 percent, and there was a mortality of 18 percent. The symptoms were milder in the older sheep; most of which gave no clinical evidence of infection. A slight transient hemo- globinuria was observed in some of the older ewes. A strain isolated from that outbreak was identified serologically as ‘E. pomona. The same authors also described another outbreak of ovine leptospirosis in a flock of 162 unweaned lambs. Thirty of the lambs died and there was clinical evidence of leptospirosis in 38 of the survivors, as follows: 19 had elevated temperatures up to 106° F; 5 had hemoglobinuria; while others were icteric and anemic. A strain of L. pomona was obtained by inoculation of blood from febrile lambs into guinea pigs. None of the ewes gave evidence of clinical infection, although some had a leptospiruria. The authors stress the fact that in both of these natural outbreaks the ll lambs were unthrifty owing to prolonged wet weather producing rank unpalatable pastures and waterlogged conditions. The lambs in the first flock were also heavily burdened with in- testinal parasites. Webster and Reynolds (93) further reported vaccination experiments in sheep with heat-killed whole cul- ture. Twenty lambs were vaccinated in one trial and resisted challenge (three weeks after vaccination and again 11 months later) with 10 ml. of a 7-day old culture of L. pomona (96.7 x 106 organisms per m1.). Twenty control sheep became infected by the same challenge dose; 16 subsequently developed leptospiruria which persisted for up to 3 months. The shedding of leptospirae was followed by microscopic examination of sedi- ments of urine samples stained by Fontana's method. Only one of a total of 28 control sheep develOped the acute Clinical syndrome which was considered as being as typical of lepto- spirosis in the young. The authors attributed this to the fact that the experimental animals had reached adolescence and had passed their most susceptible age. They also ob- served in the natural outbreaks that the majority of naturally infected sheep ceased to excrete leptospirae in the urine after 2 or 3 months. In some, however, leptospiruria persisted mmch.longer; one sheep was shedding leptospirae consistently in the urine for 9 months. In the vaccination trials all vaccinated and control animals were thriving and fattening well when challenged. {fine one lamb which did manifest typical acute symptoms was thee smallest and least thrifty. Hemoglobinuria was not 12 detected in any of the experimentally infected control lambs. The pathogenicity of the strain used was not questioned, since guinea pigs were readily infected, and severe illness developed in both authors due to accidental infection which occurred at the time the sheep were challenged. Ovine leptospiral abortion was first observed in the United States by Beamer (6) in 1952. Many of the ewes on an Illinois farm aborted and several showed a positive agglutination-lysis test. The agent involved was not reported. The first etiologically defined cases of ovine lepto- spirosis in the United States were described by Beamer gt 3}. (7) in 1953. These authors reported an outbreak of the disease in a flock of 108 ewes shipped from New Mexico to a farm.in Illinois. Five months later, six or seven deaths occurred, and shortly afterward, 19 of the remaining ewes aborted. Sub- sequently, depression and loss of condition were evident. The placental membranes were usually expelled within a few hours after the abortion. Of these 19 ewes, 15 died. At necropsy some ewes revealed icterus, swollen kidneys, mild fatty changes in the liver and marked hematuria. Although no leptospirae could be demonstrated histologically in the kidneys, darkfield examination of the stomach contents of an aborted fetus revealed motile organisms morphologically resembling leptospirae. A guinea pig inoculated with the stomach contents of this fetus died three days later. Histo- logical examination of three other aborted fetuses revealed 13 forms which were suggestive of leptospirae. Unfortunately, the microorganisms could not be identified with certainty since cultures were not made. Serum samples from some of the ewes were examined for "leptospiral antibodies" by the agglutination-lysis test and some titers were found to be 1:1000. Although leptospirae were not demonstrated by cul- tural methods, the authors believe that the clinical and pathological findings, the results of the agglutination-lysis test and the demonstration of leptospirae from an aborted fetus warrant a diagnosis of leptospirosis. The investigat- tors did not indicate which serotype might be incriminated. A disease in calves which were on the same farm.had been ten- tatively diagnosed as leptospirosis a few months previous to the outbreak in the sheep. A serum sample from one calf showed a positive agglutination-lysis titer of 1:1000. Again, it was not stated which species of Leptogpira was involved. Andres (l) reporting an outbreak of leptospirosis in cattle in l95h, mentioned that some yearling ewes on a farm aborted at about the time that the disease also appeared in the cattle. Evidence of Leptogpira pomona infection was sub- stantiated by positive serum titers. Morter and Morse (59) in 1956, using experimentally infected calves as carriers, reported that four 3-h year old ewes placed in contact with the infected calves on the seventh day following inoculation, did not show any evidence of infec- tion. Heifers, pigs and one goat which were also in contact with these calves became infected. In a more recent publication Morse 23 31. (61) stated that sheep are quite susceptible to experimental Lepto- gpira pomona infection. Eight u-S months old lambs were used. Two strains of Leptospira ppmona which had been maintained in continual hamster or guinea pig passage since their recovery from.field cases were employed. One was obtained from an in- fected dairy cow and the other from the urine of an infected hog. Both strains proved to be pathogenic. The clinical manifestations of leptospirosis in sheep were characterized by pyrexia, anorexia, depression and polypnea. In some cases hemoglobinuria and icterus were observed, and in two lambs lameness was also noted. Five to eight days following inocu- lation, the lambs were found to pass through a leptospiremic phase of one to three days duration, during which the animals showed a high temperature. Sometimes leptospirae were de- tected in the blood on the day prior to the rise in tempera- ture. Leptospiruria was demonstrable as early as the seventh day following exposure in one lamb, but generally leptospirae were not shed in the urine prior to the twelfth or fourteenth day. Leptospiruria was maximal and most consistent after 20 to 30 days. The authors further stated that darkfield microscopy consistently failed to reveal the presence of leptospirae in urine samples which were later proved to be positive by animal inoculation. The maximal duration of the renal carrier state for sheep was found to be 62 days. The majority of the sheep, however, did not excrete organisms in 15 the urine for longer than an days. Transmission from infected sheep to normal sheep, swine and goats did not occur. A marked decrease in the number of erythrocytes and low hemo- globin levels were noted. The differential counts remained essentially unaltered, although a mild leucocytosis could be observed in some of the sheep. Due to the close relationship between the ovine and caprine species, the report by Van Der Hoeden (89) on Lepto- gpira grippotyphosa infection in goats and the experimental caprine Leptospira pomona infections by Morse and Langham (60) are worth noting. Van Der Hoeden in 1953 (89) described an outbreak of caprine leptospirosis in Israel. In this enzootic, which was found to be caused by Leptospira grippotyphosa, mainly goats were involved, although serological evidence of infection was found in several other species including sheep. Considerable losses were sustained only in goats. The main clinical find- ings were icterus, hemoglobinuria and abortion in the majority of the pregnant goats. The disease in sheep showed an atypical and benign course which was of short duration. The patho- genicity of a strain of Lgptospira grippotyphosa isolated from a dead goat was tested in several species of animals. Two Arab sheep used in this experiment remained overtly healthy, but developed high agglutinin titers for the infecting organ- ism. One sheep slaughtered forty-six days after the inoculation 16 showed interstitial nephritis but no leptospirae were ob- served microscopically, and cultures from this animal remained sterile. Morse and Langham (60) observed that in experimentally- infected goats the course of Leptospira pomona infection ap- peared to be occult; the characteristic clinical manifesta- tions of depression, anorexia, polypnea, hemoglobinuria and icterus were absent. Transmission of the disease from in- fected hogs to one goat was observed. Leptospiral antibodies were present in high titers in the sera of all animals and leptospirae were found in urine samples and in the kidneys of three goats at the time of necropsy. The pathological changes due to the disease in goats were also described. 5. Passive Antibody_Transfer in the Newborn The early reports concerning antibody transfer in the newborn appear to be quite contradictory, since results obtained from experiments with one species were thought to be valid for other species. Frequently, it was not definitely ascertained whether antibody in the newborn was derived from placental transfer or obtained through ingestion of colostrum containing antibodies. Furthermore, due consideration was not always given to the time factor involved in the intes- tinal absorption of colostral antibodies. No attempt will be made to cover in full the numerous publications that have {i 17 appeared on this subject. Attention will be mainly focused on some major facts and differences among various species of animals and some of the more recent observations in lambs. Bauer (5) pOinted out that colostrum plays an impor- tant role in the develOpment of immunity in the newborn. Famulener (21) systematically evaluated the function of earlymammary secretions in the transmission of antibodies from the mother to offspring. Working with goats, he found that colostrum plays a greater role in the transmission of hemolysin-type antibody to the offspring than that which oc- curs due to placental transfer. Howe (37) showed that globulins were lacking in the serum of the newborn calf. After the ingestion of colostrum the serum globulin content rose rapidly as compared to the sera of calves which did not receive colostrum. Little and Orcutt (AS) reported that calves born to "Brucella abortus immune cows" possessed no agglutinins to this organism in their sera at birth, but soon after inges- tion of colostrum the agglutinins appeared. The same con- clusions may be drawn from the work of Huddleson (39) and Connaway (18). Smith and Little (82) investigated the significance of colostrum to the newborn calf. They showed that 75 to 80 percent of tne calves that failed to get colostrum died of septicemia which they thought was due to Escherichia coli. Calves which received colostrum, only for a very short time, l8 survived. In a subsequent paper (83) the same authors con- firmed their previous findings on the importance of colostrum to the newborn calf. They fed newborn calves, which were deprived of colostrum, a cow's serum.containing high agglutinin titers for Brucella abortus. The serum was given during the first hours of life and the agglutinin content of sera from the calves was shown to contain high titers when tested a few hours later. Serum titers had not been evident at birth. The feeding of high antibody content serum to a calf slightly older than three days of age failed to raise the existing titer above that obtained by a previous feeding. The authors stated that the bovine placenta is impermeable to brucella agglutinins and that colostrum seemed to be the most efficient transporting agent for antibodies. Rather gt 51., in a series of publications (65, 66, 67, 68, 69), reviewed the literature on the subject and pre- sented additional evidence for the importance of colostrum in antibody transport for a number of species. The authors also considered the mechanism of transplacental passage of antibodies which occurred. Transplacental passage of anti- bodies may well be due to anatomical differences of the placentae of different species of animals. These differences offer a possible explanation for the occurrence of ig_gtg£g transfer in man, guinea pigs and rabbits, and for the ab- sence of such transfer in the mouse and ruminants. They further concluded that in cattle, goats and sheep, colostrum is essential for the transmission of antibodies to the young. 19 Several reports (35, 36, 62, 63, 69) substantiate the importance of colostrum in producing antibody titers and probably engendering passive immunity in newborn pigs. It is generally agreed that the porcine placenta is impermeable to any appreciable amount of protective substances and that the function of antibody transfer is accomplished by ingestion of the colostrum. It is important to consider that this passive transfer of antibodies may interfere with active im- munization in early days of life (36, 38). Although there seems to be a general agreement that antibodies are absorbed gig the gastrointestinal tract during the first hours of life, at least in certain species, com- paratively little work has been published on the duration of this absorption phenomenon in the newborn. Hill and Hardy (3h) stated that the epithelial cells of the villi of kids and lambs up to 36 hours old contained large numbers of eosinophilic globules which they believed to be colostral proteins. This work was based upon histo- logical and histochemical studies of intestinal epithelium of lambs and kids which were killed after receiving colostrum. Mason 33 El: (50), working with lamb dysentery, i.e., Q._perfringens, type B, found that the antitoxin in the colostrum of ewes was absorbed by newly-born lambs. Adsorp- tion was shown to take place for as long as eighteen hours, but did not occur in a four day old lamb. 20 Comline t a1. (17) demonstrated histologically droplets which were considered to be normal colostral globulins in the intestinal mucosa of 9 to 36 hour old calves. very few droplets were colostrum was given to The same authors (16), tinins showed that the Only a observed within the cells when the calves 63 to 65 hours after birth. working with Brucella abortus agglu- absorption took place in the small intestine of calves. transmission of antibody substances This author presented the following \ A review is given by McGirr (51), concerning the from mother to offspring. table: TABLE 1 COLOSTRAL TRANSMISSION-OF ANTIBODY SUBSTANCES Antibody Transmission Period from Birth 82:2:83 Placental Type Placental Colostral Egggggtwgicgflfiggitinal bodies Occur Man haemochorial +++ ‘1 first few days Mouse haemochorial + +++ probably at least 20 da. Rat haemochorial + +++ " " " " " 631298 haemochorial +++ - " " " 10 " Rabbit fiiifiié’fifiiifiinh ”I ’ " " " " " Dog endotheliochorial .1 +++ probably 10-12 days Sheep syndesmochorial - +++ less than k days Goat syndesmochorial - +++ " " " " Cow syndesmochorial - +++ 2h hours Pig epitheliochorial - +++ no data availaole Horse epitheliochorial - +++ " " " 21 MATERIAL AND METHODS Thirteen yearling western range ewes were purchased from the University flock in September, 1956. These animals were selected from a group of twenty-one ewes, the sera of which showed no antibody titer when tested with L. pomona and‘g. icterohaemorrhagiae AB antigens using a modified micro- scopic agglutination-lysis test (61). The ewes were kept on pasture and were fed hay and oats until the experiments were started. While still on pasture, the animals received two treatments with phenothiazine (25 grams per sheep per treat- ment) at one-month intervals to remove intestinal parasites. In addition, they were fed 39 gig. commercial phenothiazine salt for one week after the last treatment. Two purebred Hampshire rams were allowed to stay for one month each with hthe ewes on pasture. To detect when the ewes had been bred, the rams carried a harness to which a piece of colored chalk was secured. The ewes were examined daily for colored spots on their loins, indicating that they had been bred. The ewes were again bled in December for agglutination-lysis tests. Normal values of hemoglobin, erythrocytes and leucocytes as 'well as differential leucocyte counts and nonprotein nitrogen CNPN) values were obtained. The agglutination-lysis tests “Kith‘L. pomona antigen were all negative even at the lowest diJlution (1:10). Standard clinical hematological techniques 22 were employed to ascertain levels for various blood cells and blood hemoglobin values (uh). However, in the case of leuco- cyte counts of the newborn lambs, it was found necessary to increase the concentration of acetic acid in the diluting fluid to h percent to obtain complete hemolysis of red cells. The hematological findings before commencement of the experiments and a history of the ewes, are presented in Tables 2 and 3. The ewes were divided at random into two groups, one of which consisted of numbers 737, 786, 832, 836, and number 79h as control. This group was infected at about midpregnancy (72-87 days of pregnancy). Each animal was inoculated sub- cutaneously with 5 cc. of heparinized blood obtained from in- fected guinea pigs. This blood was obtained when the maximal pyretic response (10h.5-106.0) was manifested. Ewe 79h re- ceived 5 cc. of normal guinea pig blood. Prior to inocula- tion, each animal received 1 cc. of adrenalin solution (0.1 percent) subcutaneously. The other group consisted of numbers 608, 761, 763, 76k, 770, 777, 781 and number 758 as control. The latter group was infected in late pregnancy (131-136 days of preg- nancy). After serving as control in the first group, Ewe 79h 'was infected and was killed for autopsy at the height of the temperature response. Blood from Ewe 791;. was used as inoculum IYJr the second group, but failed to infect the animals, which 23 were then inoculated subcutaneously with 10 cc. of a 9-day-old blood culture from sheep in the leptospiremic phase. Each sheep, except Ewe 770 and Ewe 608, received approximately 3 x 108 leptospirae. The number of organisms was determined by counting in a Petroff-Hauser counting chamber. Ewes 770 and 608, which only received blood from Ewe 79h, showed no signs of infection either clinically or serologically. Ewe 770 was kept with the group to determine if transmission would take place. A portion of the culture used as inoculum in the second group of ewes was Seitz-filtered and 10 cc. of the filtrate was inoculated subcutaneously into Ewe 608. The filtrate had been frozen for 2h hours and thawed before inoculation. When tested for sterility in Chang's medium, however, it was shown to con— tain living leptospirae although none was observed on darkfield examination of the filtrate. Ewe 758 was exposed 20 days after lambing with 10 cc. of heparinized blood from infected guinea pigs at the height of pyretic response. Samples of milk were inoculated in groups of hamsters (2-3 hamsters) from the first to the 13th day after inoculation. During the experimental period, each group of ewes was kept in a room 10 x 18 feet. The controls were kept in a separate room approximately 5 x 10 feet. As soon as possible after lambing, blood and colostrum :flunples from the ewes, as well as fetal membranes were obtained. The blood and colostrum from the ewes were examined for the presence of L. pomona antibody titers. Samples of fetal membranes were homogenized in a tissue grinder and injected intraperitoneally into guinea pigs as described elsewhere. The blood from the lambs was cultured immediately in Chang's fluid medium, and hematological examinations as well as agglutination-lysis tests were performed. The strain of Q. pomona selected for exposure was originally isolated by horse (57) from bovine urine obtained during active infection in a Wisconsin dairy herd. It has been maintained since isolation by continual passage through guinea pigs and has been designated strain Wickard. The agglutination-lysis test, using living Q. omona, strain Johnson, cells as antigen, was conducted with sera at tenfold dilutions starting at 1:10. One-tenth ml. of the serum dilution was mixed with 0.1 ml. of the antigen culture. The tubes were either incubated in a thermostatically-controlled waterbath at 37° C for two hours or overnight at room tempera- ture. The tubes were then examined microscopically at 100x using a microscope fitted with an Abbe condenser into which 'was fitted a star diaphragm. This produced a modified dark- field type illumination which was found to be satisfactory for reading the tests. Detection of agglutinins in the urine was conducted 111 a similar manner, except that the urine was diluted 1:5, 1:10, 1:100 and 1:1000. Urine samples were obtained by tem- porary occlusion of the sheep's nostrils. Milk whey was produced for the agglutination-lysis test by adding two drops of concentrated rennin solution to approximately five ml. of milk. The mixture was allowed to stand at room temperature for a few hours or overnight at h° C. The whey was then separated by centrifugation. Ten- fold dilutions were employed in the agglutination-lysis tests. All agglutination-lysis titers were based on the 50 percent endpoint unless otherwise indicated. Animal inoculation techniques were employed to deter- mine the presence of leptospirae in urine, milk and tissues. Two to four guinea pigs (250 g. average) or hamsters (four to five weeks old) were inoculated intraperitoneally. Approxi- mately IUD percent tissue emulsion, suspended in 0.85 percent sterile sodium chloride solution, served as the inoculum. In the case of contaminated fetal membranes, thorough wasning and rinsing in sterile saline, previous to homogenization, was performed. Hamsters received 0.5 to 1.0 cc. of urine; guinea pigs received 1 to 3 cc. of urine or 2 to 3 cc. of the tissue suspension. After 21 to 30 days the animals were sacrificed. If their sera contained 2. pomona agglutinins, the inocula were considered to have contained L. pomona. The strain, E. omona, Wickard did not kill guinea pigs or hamsters. 26 Leptospirae were cultured from blood in Chang's medium (1h), containing 10 percent sterile filtered rabbit serum and 0.01 percent hemoglobin (Difco). The same amount of blood was drawn from the jugular vein of each sheep. Each of five screw—cap tubes, containing ten m1. of medium, was inoculated with 0.1 to 1.0 cc. of the blood sample. In a few instances, dilutions were made of the blood sample before inoo inoculation to ascertain the maximal number of organisms at different stages of infection. Cultures were incubated at 30° C. for approximately 31 days. Darkfield microscopy exami- nation (600x) were made at 1h to 16 days and again at 28 to 31 days to detect leptospirae. Normal lambs for studies on intestinal absorption of leptospiral antibodies were obtained from the University flock. The sera and colostrum from the mother ewes were tested for the presence of leptospiral antibodies and were found to be negative. The lambs were allowed to stay with the ewes during the experiments. Serum containing high antibody titers (1:10"6 or 1:10'9) against E. omona, was obtained from the infected sheep. The serum was Seitz— filtered and was administered to the lambs of different ages through a stomach tube. The sera of the lambs were tested at various intervals for the presence of leptospiral anti- bOdieS o 27 EXPERIMENTAL RESULTS 1. Group I This group consisted of ewes numbered 737, 786, 832, 836 and 79h which served as the control. The group was infected at about midpregnancy. The exact stages of pregnancy are given in Table 3. Blood cul- tures were made daily from the third to the twelfth day after exposure. The time and duration of leptospiremia are pre- sented in Table A. Agglutination-lysis tests were performed with serum samples obtained during the experimental period. The titers obtained are tabulated in Tables 5 and 6. The hematological data are presented in Tables 7 and 8. No reticulocytes were observed in blood smears with one exception, namely, Ewe 786. A count of 0.8 percent reticulocytes was obtained on the 11th day after inoculation in that animal. The duration of urinary excretion of leptospirae was investigated from the 12th to the 73rd day after inoculation. At the same time the urine was examined for the presence of antibodies against E. pomona. The results obtained from these investigations are shown in Tables 9 and 10. The urine (samples were all negative for hemoglobin when examined with the benzidine test (123). Hemoglobinuria or icterus were not observed. Darkfield microscopy (600x) consistently 28 failed to reveal the presence of leptospirae in urine samples which were later proved positive by animal inoculation. All ewes except 836 and 794, which were killed for necropsy before lambing, lambed normally. The gestation periods are presented in Table 2. As soon as possible after lambing, samples of colostrum and sera were obtained from the ewes, as well as placental membranes. Colostrum and placental membranes did not contain viable leptospirae as determined by negative agglutination-lysis tests of sera from guinea pigs inoculated with these materials. Colostral whey and sera were examined for the presence of antibodies against E. pomona by the agglutination-lysis test. Serum samples were obtained from the lambs soon after birth and at different intervals until the end of the experimental period. The titers of sera from the ewes and the lambs as well as the titers of colostral or milk whey at various times after lambing, are shown in Table 11. Since the ewes in this group all lambed during the night, samples could only be obtained several hours after birth. The lambs had in all cases obtained colostrum by sucking the ewes. This accounted for the high serum titers obtained in the lambs at the time _ of the first bleeding. Blood cultures were made from the newborn lambs. No leptospirae were observed in these cultures. Hemoglobin, red and white cell determinations as well as differential counts and reticulocyte counts were made of the blood 29 samples obtained from the lambs at the time of the first bleeding. The hematological results are found in Table 12. The characteristic course'of infection in Group I is shown in Figure l. Ewe 836 was killed for necropsy 23 days after infec- tion, at 110 days of pregnancy. The serum titer of the ewe 7 was 10- with ;. pomona antigen. The ewe was killed by intra— venous injection of "Halatol" and subsequent exsanguination. At necropsy the macroscopic lesions were as follows: There were a few scattered grayish-white foci measuring about 1 mm. in diameter in the cortex of the kidneys. The right kidney showed a scar measuring about 2 cm. in diameter at the end of this structure. There were a few calcified areas in the lungs probably due to parasites. The uterus contained one live fetus. Cultures were made in Chang's medium from amnionic fluid, fetal stomach contents and fetal blood. The cultures remained sterile. Agglutination-lysis tests performed with Q. pomona antigen on amnionic fluid, fetal serum and stomach contents were all negative. Emulsions of fetal organs (kidney, liver, spleen, lung) stomach contents and amnionic fluid were injected intraperitoneally into groups of guinea pigs. When these animals were sacrificed one month later, their sera were all negative in the agglutination-lysis test with‘g. pomona antigen. 3O Emulsions of kidneys from Ewe 836 were also injected into 5 hamsters and h guinea pigs. Two of the guinea pigs and h of the hamsters showed positive titers when sacrificed. An emulsion of placental membranes from Ewe 836 was injected into 3 hamsters and 3 guinea pigs. These animals failed to develop titers against E. pomona. Ewe 79h served as control in Group I. The ewe re- ceived 5 cc. of normal guinea pig blood subcutaneously at the same time as the rest of the group received blood containing leptospirae. The ewe was bled according to the schedule for the group. The hematological observations are presented in Tables 7 and 8. Ewe 79h was originally intended to become the source of inoculum for the next group of sheep. She was therefore inoculated subcutaneously with 33-day-old culture of E. pomona obtained from sheep in the leptospiremic phase. The inoculum contained 1.2 x 108 organisms, determined by counting in the Petroff-Hauser counting chamber. The ewe was then 123 days pregnant. Blood cultures were made 5 hours after inoculation. Two cc. of blood were cultured in each of 5 tubes containing 10 ml. medium. In addition, 5 tubes were inoculated with 0.2 cc. blood. After 13 days of incuba- tion, u out of 5 tubes with 2 cc. inocula were positive, whereas only one was positive of the others. Blood cultures were then made daily in 10 tubes, 5 of which received 1 cc. inoculum and 5 received 0.2 cc., each. The blood cultures with 1 co. inocula contained innumerable leptospirae at 31 darkfield examination 7 days later. The tubes with 0.2 cc. inocula only contained 8—12 organisms per field. On the third day following infection, tenfold dilutions of blood were inoculated into series of 5 tubes containing 10 ml. medium. 0 After 22 days of incubation at 30° 0., growth of leptOSpirae l .. dilution was observed in all 5 tubes inoculated with 10- q . q o a -2 10 o of blooa, in u out of S tuoes receiVinb the 10 dilution, and in 2 out of q receivinb the 10-3 dilution. Only one out I 1 of 10 tubes inoculated on the fourth day after infection with 1 cc. blood, was positive, and all tubes inoculated on the fifth day, were negative for leptospirae. Blood was drawn on the fourth and fifth day frou this animal and inoculated in 10 cc. amounts subcutaneously into ewes in Group II. Since the inocula obviously contained very few organisms, if any, the sheep in Group II did not show any evidence of infections during the 6 days which were allowed to pass before reinocu- lation with leptospira culture. he course of the infection in Ewe 79h is shown in Table 13. Ewe 79h was killed for necropsy by intravenous "Hala- tol" injection and exsanguination 5 days following infection. The macroscopic lesions consisted of edematous lymphnodes and a few small greyish-white lesions in the kidneys. The uterus contained one live, male fetus, 38.5 cm long. Edema was ob; served in the neck region and scrotum of the fetus. The cotyledons were very congested. Cultures were made for leptospirae from fetal blood, stomach content and amnionic 32 fluid. These cultures remained sterile. No leptospiral antibodies were detected in fetal serum, stomach content or amnionic fluid. Samples of liver, spleen, lung, kidneys and stomach content from the fetus, as well as amnionic fluid, were pooled and injected into S guinea pigs. The sera of these guinea pigs did not contain antibodies against E. pomona when the animals were sacrificed one month later. On the day of necropsy, the urine from Ewe 79h con- tained hemoglobin as shown by a positive benzidine test and lack of red cells upon microscopic examination of sediment. Cultures of urine.remained sterile. Samples of cotyledons were injected into 3 guinea pigs. They all showed a positive serum reaction when killed one month later. Two guinea pigs were inoculated with emulsion of brain from Ewe 79h. One died after h days. The other was heartbled for blood cultures when sick 9 days following inocu- lation. A positive culture was obtained from this guinea pig and one month later its serum showed a positive.§. pomona titer. An emulsion of kidneys from Ewe 79h was inoculated into h guinea pigs, which upon sacrifice showed titers against ‘E, pomona. The serum antibody titers of Ewe 79h on the fourth and fifth day of infection were 10'1 and 10‘3, respectively. 2. Group II Group II consisted of ewes numbered 761, 763, 76h, 770, 777, 781, 608 and 758 as control. This group was infected 33 during late pregnancy as shown in Table 3. Hematological observations made 3 months before infection are presented in Table 2. As previously mentioned, Group II was inoculated with blood obtained from Ewe 79h during the febrile response h and 5 days after exposure. As serum samples obtained from Ewe 79h at the same time showed positive leptospiral agglutinin titers, it was felt that the chances of infecting group II with this material were minimal. Clinical and hema- tological observations were made daily for 6 days after the inoculation of blood from Ewe 79h. These data are presented in Table 1h. Blood cultures obtained daily during this period remained sterile and no serum antibody titers developed. The data obtained during this period will thus represent "normal" hematological findings in this group. ho reticulo- cytes were observed. A 9-day-old culture, obtained from the blood of an infected sheep was then used as inoculum. Ten ml. of culture, containing 3 x 107 organisms per ml. were injected subcu- taneously into 761, 763, 76h, 777 and 781. Ewe 770, which had only received blood from 79h, was not inoculated and was allowed to stay with the infected ewes. Ewe 770 remained uninfected throughout the entire experimental period as determined by the absence of serum antibody titers. Urine from this animal did not contain antibodies or live leptospirae. 3h The ewe had one lamb, the serum titer of which remained negative for leptospiral antibodies. At lambing, neither serum or colostral whey from the ewe contained detectable anti- bodies. Thus, no transmission took place from the infected sheep to Ewe 770 or its lamb. Ewe 608 received 10 ml. of a frozen and thawed fil- trate of the leptospira culture. The culture was filtered through a Seitz filter. When tested for sterility in Chang's medium, the filtrate was, however, shown to contain viable leptoSpirae. Ewe 608 subsequently developed antibody titers both in the blood serum in the milk and in the urine. Blood cultures obtained from Ewe 608 from the first day following inoculation until the occurrence of antibodies in the serum on the seventh day after infection revealed no growth of lep- tospirae. The temperature also remained normal during this period. No leptospirae could be demonstrated by animal inoculation of urine from Ewe 608, as shown in Table 18. Temperatures and hematological data obtained after inoculation of Group II with L. pomona culture are found in Table 15. The duration of leptospiremia in this group is presented in Table 16. The serum antibody titers obtained at various intervals are recorded in Table 17. The duration of urinary excretion of live leptospirae is presented in Table 18, and the urine antibody titers are found in Table 19. Darkfield microscopy did not reveal the presence of leptospirae in Group II urine samples which were later proved to be positive by animal 35 inoculation. Only Ewe 777 showed hemoglobinuria. This occurred on the sixth day following infection and was determined by the benzidine test. hicroscopic examination of the urine sediment did not reveal the presence of erythrocytes. Ewe 763 was killed by intravenous injection of "Halatol" 4 days after infection, i.e., at 131 days of preg- nancy. Except for pseudotuberculosis-like lesions in the submandibular lymph-nodes, no macrosc0pic pathological changes were observed. Blood cultures obtained about 6 hours before necrOpsy showed one positive tube out of 5. Blood cultures taken at the time of necropsy were all negative. Samples of colostrum, brain, kidneys and placental membranes from the ewe were injected into groups of guinea pigs. Only the kid- neys were shown to contain viable leptospirae. The uterus contained one live fetus. No macroscopic lesions were ob- served in the fetus or placental membranes. Cultures from fetal blood, stomach contents and amnionic fluid did not reveal leptospirae. Agglutination-lysis tests with E. pomona antigen or fetal serum, stomach content and amnionic fluid were negative. Emulsions of fetal liver, spleen, kidneys and stomach contents as well as fetal blood and amnionic fluid did not contain leptospirae. The ewes numbered 758, 761, 76h, 770, 777, 781, and 608 lambed after normal gestation periods as shown in Table 2. Samples of serum, colostrum, amnionic fluid and fetal mem- branes were obtained immediately after birth from all of these 36 ewes. At the s(me time blood cultures were made from the newborn lambs. Sera obtained from the newborn lambs showed no antibody titers. All blood cultures were negative for leptospirae. Colostrum and samples from fetal membranes did not contain live leptospirae, with one exception. Two guinea pigs were inoculated with an emulsion of fetal mem- branes from Ewe 781. One of these guinea pigs died h days afterwards. The remaining one had a positive serum titer for E! pomona. Ewe 781 did not allow its two lambs to nurse. One lamb died 3 days after birth. Emulsion of liver, kidney, spleen and brain did not contain leptospirae. The serum of this lamb had a 10'2 antibody titer, which was due to the fact that the lamb had managed to obtain some milk containing antibodies from other ewes. The other lamb of Ewe 781 was killed ig_extremis when 32 days old. This lamb had managed to survive by obtaining milk from other ewes. The lamb had for the past two weeks suffered from pneumonia with inter- mittent fever with temperatures of 106.3° F. Blood cultures obtained during the febrile periods were all negative for leptospirae. Although the serum from this lamb had shown a 10'2 titer on the fourth day after birth, it remained nega- tive from the seventh day of age till death. At necropsy large areas of pneumonia were observed, which involved the apical and cardiac lobes as well as the anterior portions of the diaphragmatic lobes. Some caseous areas were observed 37 within the pneumonic consolidations. The liver had a very bright yellow color. Emulsions of kidney, liver and spleen, brain and lungs were injected into groups of guinea pigs. None of the guinea pigs showed any macroscopic lesions when sacrificed one month later. Only one out of 3 guinea pigs injected with lung emulsion showed positive leptospiral anti- body titers, the sera of the other guinea pigs were negative. Cultures on blood agar plates from lungs, liver, spleen and brain revealed a gram negative rod in pure culture. The organism is 'ifficult to classify, but has most characteristics in common with Brucella bronchiseptica. It does, however, liquefy gelatin and is urease negative, which may indicate that it may belong to the genus Alkaligenes. Ewe 781 was killed for necropsy 3h days after lambing, i.e., hh days after infection. The macroscopic pathological observations consisted of numerous greyish-white lesions extending through the depth of the cortex of the kidneys. The mediastinal and bronchial lymph nodes were greatly enlarged and showed caseous areas. A culture made from the bronchial lymph nodes revealed a pure culture of (kmwnebacterium pgeudotuberculosis. Several large caseous areas were found in the diaphragmatic and cardiac lobes of the lungs. The kidneys, liver and uterine mucosa were shown not to contain vialble leptospirae by guinea pig inoculations. Serum samples were obtained immediately after birth from the lambs in Group II. At the same time samples of 38 colostrum were obtained. Samples of lagbs' sera and milk from the ewes were taken at various intervals till about 3 weeks after birth. The results of agglutination-lysis tests performed on these samples with.£. pomona antigen are pre- sented in Table 21. Ewe 738, which had served as control in Group II and had been kept in a separate room, had two lambs. Serum samples from this ewe and its lambs, as well as milk whey, did not contain antibodies at the time of lambing. As a pre- liminary experiment concerning the absorption of antibodies through the intestinal tract, the lambs received milk or serum containing leptospiral antibodies through a stomach tube. At the age of 52 hours one lamb received 20 ml. sheep serum which had a titer of lO-u; the other lamb received 75 ml. of milk collected from the infected ewes, and which showed a titer of 10-3. The lambs were bled lh hours later. The lambs did not show any reaction even in the 10..1 dilution in agglutination-lysis tests. At the age of 75 hours the lambs received additional amounts of serum (27 ml.) and milk (60 ml.) respectively. They were again bled after 18, 2h, and uh hours. No antibody titers were found in these samples. Ewe 758 was inoculated subcutaneously with 10 cc. of blood obtained from infected guinea pigs during the pyretic response. This was done to follow the shedding of leptospirae in the milk, and to ascertain if the nursing lambs would contract the disease. The ewe was infected 30 days after lambiné. BlOUd cultures were taken from the ewe from the day of inoculation until the ninth day. hilk samples were obtained from the first day through the thirteenth day following inoculation. Samples of milk were inoculated into groups of hamsters and examined for the presence of lepto- spiral antibodies. The temperatures of the ewe and lambs H were recorded da 1y. No rise in temperature was observed in the lambs and no antibody titers were found in their sera When examined one month after the inoculation of the ewe. The results are sumharized in Table 22. The urinary excretion of leptospirae in Ewe 753 was followed by animal inoculation. One out of 3 hamsters inoculated with urine obtained 25 days after infection showed antibody titer. Hamsters inoculated with urine obtained 17, 27 and he days following infection were negative. The antibody titers of the urine were negative on the 17th day and were 10'1 and 10"3 on tae 27th and hoth day, respectively. 3. Absorption of Leptospiral Antibodies in Lambs On the basis of the experiments carried out with the lambs of Ewe 758 it seemed likely that, either the lambs were too old (52 hours) and absorption did not take place, or the amount of antibody given was too small to be absorbed in detectable serum concentrations. An arbitrarily-chosen dose of 50 cc. of serum with a titer of 10"6 was given to a MO lO-hour old lamb weighing 6 pounds. The lamb was bled after 6, 12, 2h, 30 and 36 hours, and showed the following titers, respectively: 10-3, 10’3, 104+ and lO-u. The lamb was bled again h8 hours after the administration of serum. he serum titer of the lamb had dropped to a 1 reaction, i.e., 25 2 and 10-3 percent agglutination-lysis, in the 10-1, 10' dilutions. At the same time, i.e., AB hours after the first serum administration and 53 hours after birth, this lamb again received about 50 cc. immune serum. Subsequent bleed- ings and agglutination-lysis tests failed to reveal an in- crease in the serum titer. Another lamb, 2h hours old and weighinb 8 pounds, re- ceived through stomach tube, 75 cc. of a serum with a titer of 10-6. Subsequent bleedinss and abglutination-lysis tests did not reveal any titers in the serum of this lamb. These preliminary experiments indicated, then, that serum antibodies, when administered 22£.2§: were absorbed from the alimentary tract of a lO-hour old lamb, and that no detectable antibodies were demonstrated in the serum of a lamb receiving serum at the ape of 2h hours. Furthermore, the absorption seemed to occur quite rapidly as a titer of 10-3 was found 6 hours after the administration of serum. The amount of immune serum used, namely about 8.5 cc. per pound body weight, appeared to be sufficient to enable de- tection of serum antibody titer in the lamb. The lambs suf- fered no ill effects from the administration of serum per gs. Ln Several workers (3h, 50, 17, 51) had indicated that absorption of globulins or antibodies from colostrum might occur up to the age of 36 hours or even h days. To obtain additional data a group of 9 lambs was given serum with an antibody titer of 10'9. The ages of the lambs, their weights, amounts of serum administered as well as the results obtained, are presented in Table 23. It may be deducted from this table that absorption of antibodies took place from the alimentary tract of lambs which were approximately 15 hours old. The antibodies were not absorbed in detectable amounts in lambs older than about 25 hours. Since these experiments were conducted late in the lambing season, few lambs were available, therefore it was not possible to study lambs 15 to 2h hours old. #2 DISCUSSION AND CCNCLUSIONS l. The Disease in Sheep Ovine leptospirosis does not seem to be prevalent in the United States. This may be due to the fact that the disease may run an inapparent course with a low mortality rate. The disease will then only be detectable by serological means. The author is not aware of any reported serolo¢ical surveys pertaining to ovine leptospirosis in the United States. Ovine leptospirosis may, however, appear as a severe disease with symptoms of icterus and hemoglobinuria as well as a high mortality rate, eSpecially in lambs. The disease as such is a problem to farmers in New Zealand where it causes considerable losses (31, 76, 77). The factors respon- sible for the difference in prevalence may be many and varied. American breeds of sheep have been found to be sus- ceptible to experimental Q. pomona infection. Differences in resistance of breeds of sheep, then, apparently do not play any part. The virulence of E. pomona may differ from one strain to another. Information pertaining to the com- parative virulence of E. pomona strains is not known to the author. The difference in virulence, then, may account for the reported prevalence of ovine leptospirosis in some areas, e.g., New Zealand, and the scant reports of the disease in other areas, e.g., the United States. ’43 The experimentally-produced disease in pregnant ewes does not exhibit the serious symptoms and high mortality rate sometimes reported in natural outbreaks. On the other hand, a disease is very often not reported unless it is the cause of serious loss of animals or decrease in yield. In certain areas, then, a higher incidence of the disease may be found, due to adequate diagnostic facilities and reporting services. The mild symptoms of disease commonly observed in experimental L. pomona infection of sheep, can only be at- tributed to leptospiral infection on the basis of bacterio- logical and serological investigations. This is in a5reement with the statements made b: Hartley (32) in New Zealand: d J-r~ We have, on several occasions, tried to infect lambs, hog;ets and pregnant ewes with both kxow- virulent calf strains and with sheep strains but have produced no clinical signs (except a mild rise in temperature and sometimes leptospiruria). An important factor which will account for some of the differences observed between the naturally-occurriné isease and the experimentally—induced disease, is the con- .02 ditions under which the experiment is performed. Under natural husbandry sheep may be subjected to various factors predisposing for disease. These may be more or less heavy infestation with various parasites, e.g., lungworms or gastro-intestinal parasites. Sheep are sometimes kept on submarginal pastures which are incapable of supplying the animals with the necessary nutrients or essential minerals. Under such circumstances the animals may be rendered more susceptible to infection. Animals used for experimental purposes are, on the other hand, commonly kept under good hygienic conditions, fed an adequate diet, and in general not subjected to undue stress. They are often routinely treated for parasites, as the case was in this experiment. Differences in climatic conditions and animal husbandry practices may be considered important factors influencing the prevalence and severity of ovine leptospirosis. Moderate temperatures and heavy rainfall will favor survival of L. pomona outside the host. Crowded pastures where several Species may be feeding will enhance the possibilities of spread of the disease. Several outbreaks of ovine leptospirosis (6, 7, 79) have been described following close contact with diseased cattle. Deer may serve as the source of infection since serological evidence indicates that leptospirosis has oc- curred in this game animal (94). Although the inocula used in this experiment contained a much higher number of organisms than natural conditions would provide, the disease was very mild. The sheep passed through a pyretic stage during which symptoms of anorexia and polypnea were present. Hemoglobinuria occurred only in Ewes 79h and 777. In group I there was a marked and uniform decrease in hemoglobin levels and erythrocyte counts. But this drop was just as pronounced in the control sheep. The most marked drop in hemoglobin and red cell levels was observed #5 during the course of the disease in Ewe 79h (Table 13). At the time of necropsy, 5 days after inoculation, the urine contained hemoglobin. In group II marked depression in these levels was not detected. Hemoglobinuria may have been present in the experimental ewes for such short periods of time as to escape detection. On the other hand, an intravascular des- truction of red cells with resulting hemoglobinemia may have occurred without hemoglobinuria. Hemoglobinuria only occurs when the amount of free hemoglobin in the circulating blood plasma exceeds the threshold of the kidneys for hemoglobin. An accurate means of following red cell destruction would be to record free hemoglobin in the blood plasma, or possibly erythrocyte fragility. This, however, has not been done in these experiments. Degrees of hemolysis judged by the color of the serum are subject to too many inaccuracies to be of much value. Leptospiremia is readily demonstrated in experimental ovine leptospirosis. Leptospirae have been cultured from the blood as early as the first day after inoculation and as late as the seventh day. Tables 4 and 16 present the days after infection when positive blood cultures were obtained from the ewes. All h ewes in group I showed positive blood cultures on the fifth and sixth day after infection at the beginning of the febrile period. Two ewes (786 and 832) showed posi- tive blood cultures as early as the third day after infection; the first blood cultures were made on the third day after us inoculation in this group. In group II (Table 16) 3 ewes showed positive blood cultures on the first day after inocu- lation. On the second, third and fourth day, all inoculated ewes (except Ewe 608) had positive cultures. The inoculum used in this group contained a very large number of leptospirae (3 x 107 per m1.). Ewe 79h which received 1.2 x 108 organisns yielded positive blood cultures 5 hours after subcutaneous inoculation. Blood cultures from Ewe 79h were positive for h days after inoculation. When the presence of leptospiremia (Tables h and 16) is correlated with the temperature of the animals (Tables 7 and 15), it is observed that positive blood cultures are more likely to be obtained during, or even be- fore, the rise in temperature. In the case of Ewe 79h, it was observed that the temperature was normal during the first two days after infection. Blood cultures obtained on these days were positive for leptospirae. The temperature rose to th.3-105.u° F. on the third day after inoculation. Positive cultures were obtained on that day from blood diluted 10'3. Two of S inoculated tubes were positive. On the fourth day after inoculation the temperature was lOS.S° F. Only one out of IO tubes inoculated with 1 cc. of blood was positive. Five tubes inoculated with 0.2 cc. blood were negative (Table 13). Positive blood cultures, then, were obtained from.ewes 786, 832 and ewe 79h, and from all infected ewes in group II (except Ewe 608) before any rise in temperature was observed. Leptospiremia thus occurs rapidly after A7 experimental infection of sheep with massive inocula of E. pomona. The term septicemia may well be used for the con- dition observed during the febrile period with symptoms of anorexia, polypnea and positive blood cultures. It is some- times asserted that in septicemia organisms are multiplying in the blood, whereas in bacteremia they are derived from infected tissues. Such a distinction clearly cannot be drawn from the results of blood cultures. There are, how- ever, indications that bacteria may multiply in the blood stream. The septicemic phase of any bacterial infection is probably the manifestation of a rapid and continuous in- vasion of the blood stream from foci which have been established earlier in the disease. The appearance of antibodies in the serum.ends the septicemia as can be shown by comparing Tables h and 5 and Tables 16 and 17. In this connection the patho- genesis of relapsing fever (Borrelia recurrentis infection) is borne in mind. The antibody titers increase very rapidly from negative at the time of leptospiremia to 10"9 in a matter of 3 or u days (Tables 5 and 17). The titers may re- main rather high (lO'u to 10'6) for at least 3 months. During, or soon after the rise in temperature a de- crease in the number of leucocytes was observed in all in- fected ewes in group I (Table 7), in Ewes 761, 763 (Table 15) and in Ewe 79h (Table 13). A rise, however, in the number of leucocytes in Ewe 76h was observed. This may be accounted for on the basis of a concurrent suppurative AB pododermatitis. Ewe 777 showed a slight decrease in the number of leucocytes at the rise in temperature, and then a rapid increase which occurred at the time of hemoglobinuria. Leukopenia, although of moderate degree, has been recorded in the course of leptospira infection in these sheep. This phenomenon is known to occur in certain infectious diseases. It has been observed in experimental leptospirosis in calves (70). Some alterations were observed in the differential counts of the infected ewes (Tables 8 and 15). The percentage of eosinophils decreased during the infection in most of the animals. A slight relative increase in percentage of seg- mented neutrophils and a relative decrease in percentage of lymphocytes were also observed. Acute infections with neutrophilia are commonly associated with lymphOpenia. Antibodies were present in the urine of most of the infected animals (Tables 10 and 19). They did not appear in the urine until about 3 weeks after the infection. The possible sources of the urinary antibodies are either local antibody production in the kidneys or leakage of serum anti- bodies due to damage of renal tissue when the organisms became localized in the kidneys. Tests for protein (Heller's test, uh) performed on urine samples have all been negative, although antibody titers have been positive in 10"3 and lO-u dilution of urine. M9 In the case of damage to renal tissue with subsequent leakage of serum into the urine, one would expect a positive reaction for albumen. The amount of serum protein leaking into the urine may, on the other hand, be very minute and still give a positive antibody titer. The test for albumen may then be too insensitive to detect traces of albumen. The presence of antibodies in the urine may interfere with the detection of leptospirae in the urine samples since the organisms are lysed. Inoculation of urine into animals should be performed as soon as possible after sampling to avoid in- activation of the leptospirae. The maximal duration of leptospiruria has been found to be 35 days (Tables 9 and 18). Ewe 781, shedding lepto- spirae on the 35th day, was killed for necropsy on the hhth day after inoculation. Leptospirae were not harbored in the kidneys at the time of necropsy. The number of organisms ex- creted in sheep urine seems to be rather low. Organisms have not been observed in darkfield micrOSCOpical examination of fresh ovine urine. Only one or a few of a group of hamsters or guinea pigs inoculated with l-3 cc. of the same urine sample may show serological evidence of infection. It thus appears that sheep experimentally infected with'g. pomona are not very important as reservoirs and shedders of the organisms. This is in agreement with the results obtained by Morse gt £1. (61). So 2. Necrgpsies Ewes 763 and 79h were killed for necropsy h and 5 days after infection, respectively. Blood cultures obtained from Ewe 763 at the time of the necropsy were all negative. One out of 10 tubes inoculated with blood from Ewe 79h on the day before necropsy, was positive. All cultures obtained on the day of necropsy were negative. The serum antibody titer at the time of necropsy was 10'3 in Ewe 79h and negative in Ewe 763. Both ewes were pregnant (Table 3). Both fetuses were alive at necropsy. Cultures and animal inoculations failed to detect leptOSpirae in amnionic fluid, fetal blood and stomach contents and fetal organs. No antibodies were detected in amnionic fluid, stomach contents or fetal serum. In Ewe 763 only the kidneys were shown to contain viable leptospirae. The brain, samples of cotyledons and kidneys of Ewe 79h were shown to contain leptospirae. In ewes 763 and 79h leptospiral invasion of the fetuses or amnionic fluid had not taken place. Detectable transplacental anti- body transfer to the fetuses had not occurred. These two ewes, 763 and 79h, were killed at a time when the leptospirae were disappearing from.the blood stream. In Ewe 79h the microorganisms obviously were still present in other tissues in addition to the kidneys, while in Ewe 763 only the kidneys harbored live leptospirae. Ewe 79h also exhibited the more marked gross pathological renal changes and hemoglobinuria. Ewe 836 was necropsied 23 days after infection. 'Tne ewe was then 110 days pregnant. The serum titer at the time of necropsy was 10-7. No antibodies were detected in amnionic fluid, fetal serum or stomach contents. The fetus was alive and did not harbor leptospirae, as determined on the basis of appropriate cultures or animal inoculations. Only the kidneys of Ewe 836 were shown to contain leptospirae. The absence of antibody titers in the fetus in the presence of high antibody titers in the ewe clearly indicates that trans- placental transfer of leptospiral antibody does not occur; a fact which is further substantiated by the absence of lepto- spiral antibodies at the time of birth in lambs born to ewes with high serum antibody titers (Table 21). Ewe 781 was killed for necropsy 3h days after lambing. Animal inoculation failed to reveal leptospirae in the tissues, including kidneys. One lamb belonging to Ewe 76h died the day following birth. Another lamb which belonged to Ewe 781 died three days after lambing. None of these lambs showed any evidence of‘g. pomona infection proved by animal inoculations of tis- sue samples. The cause of death in these lambs remains un- determined. The second lamb of Ewe 731 was killed at the age of 32 days. The lamb had been ill for aoout 8 days. Blood cultures obtained during febrile attacks were negative for leptospirae. At necropsy extensive pneumonia was found. An organism resembling grucella bronchiseptica was isolated 52 from the lungs, liver, spleen and brain. Guinea pigs in- jected with such material remained healthy and no lesions were observed when the animals were sacrificed one month after inoculation. At that time one out of three guinea pigs, inoculated with lung emulsion, had a titer against L. pomona. This phenomenon, for which there is no obvious ex- planation, may be attributed to a laboratory error. 3. Lambing and Lambs The ewes lambed normally. The gestation periods are shown in Table 3. The gestation periods for the ewes lie all within the range of normal variation, which is lhO to 160 days. In group I samples of fetal membranes were obtained probably several hours after birth. In group II samples of fetal mem- branes were obtained immediately after birth. The fetal membranes, except those from.Ewe 781, and all colostrum samples did not contain viable leptospirae. Injection of fetal mem- branes from Ewe 781 into 2 guinea pigs resulted in death in h days in one and an agglutination-lysis titer in the other guinea pig. A urine sample obtained h days after lambing was proved to contain leptospirae by animal inoculation. Whether the leptospirae were derived from the placental mem- branes themselves or from contamination with urine cannot be determined with certainty. Considering the presence of high serum antibody titer in the ewe and the absence of lepto- Spirae in the blood of her lambs at birth, it would seem likely 53 that con amination with infective urine was the source of the leptospirae infecting the guinea pig. Colostrum and milk whey from infected ewes were shown to contain antibodies against E. pomona (Tables 11 and 21). On some occasions the titers in the whey were higher than in the blood serum of the ewe. The sera of the lambs did not contain leptospiral antibodies at birth. A rapid appearance of titers occurred after the lambs had obtained colostrum. In most lambs the antibody titers of the sera disappeared approximately 3 weeks after birth. Blood samples obtained from Ewe 770, which was not infected, and her lamb, remained negative. No antibody titer was found in colostral or milk when from that ewe. Transmission of the disease, then, from the ewes to their lambs did not take place. The presence of antibodies in the sera of these lambs during the most active shedding period, would appear to offer an explanation. Ewe 770 and her lamb, however, did not have serum antibody titers and should have been quite susceptible. Transmission did not occur even to these animals. h. Leptospirae in Milk_ At the time of lambing, i.e., from 7-81 days after infection (Table 3), none of the colostrum samples contained leptOSpirae. Ewe 758 was infected to determine the extent of excretion of leptospirae in the milk. Leptospirae were 51+ shed in the milk from the fifth to the ninth day after inocu- lation (Table 22). Blood cultures were positive only on the sixth and seventh day following inoculation. The two lambs of Ewe 758, which nursed the ewe for one month during this eXperiment, did not show any fever and did not develOp anti- body titers. Transmission of the disease, then, did not take place, although the milk contained leptospirae for five days. At the time of inoculation of the ewe, the lambs were 30 days old. One lamb received high titer serum while the other re— ceived high titer milk at 52 and 75 hours of age. Antibodies were not demonstrated in the lamb's sera. The number of organisms shed in the milk was probably too low to cause in— fection through ingestion. No symptoms of mastitis occurred in Ewe 758. It is borne in mind at this point that Baker and Little (h') isolated E. pomona from acute mastitis in cattle. S. Antibody Transfer to the Newborn It has previously been pointed out that no detectable antibodies were found in the sera of ovine fetuses, although the serum titers of the ewes were very high. This has also been found to be true for lambs at the time of birth. Shortly after the lambs had nursed infected ewes, antibodies appeared in their sera. The serum titers of the lambs in- creased rapidly during the first two days after birth and often reached higher values than those of the sera or milk 55 wheys of their mothers (Tables 11 and 21). Transplacental transfer of leptospiral antibodies did not occur in detectable amounts in the lambs. The presence of antibodies in the sera of young lambs born to infected ewes is due wholly to intes- tinal absorption from high titer colostrum. The antibody titers of the whey decreased rapidly after the first few days of nursing. The titers of the lambs decreased slowly to nondetectable levels approximately 3-h weeks after birth. The sudden occurrence of serum antibodies in the lambs after the first nursings and the subsequent disappear- ance of titers in spite of continuous ingestion of milk con- taining antibodies stimulated the following studies on the influence of the age upon antibody absorption. A Eh-hour-old lamb did not snow antibody titers after receiving 75 ml. of serum with a titer of lO'é. Ab- sorption was then shown to take place in a lO-hour-old lamb which received through stomach tube 50 ml. serum with a titer of 10-6. The results of further experiments with lambs of various ages are shown in Table 23.. On the basis of these experiments it may be concluded that the age factor plays an important role in the intestinal absorption of colostral antibodies in the newborn. Absorption of anti- bodies occurs in newborn lambs up to the age of 15-16 hours. At the age of 2h hours or older, detectable amounts of leptospiral antibodies have not been found in lambs. Due to \fl 0" a lack of lambs for further studies on this phenomenon, the age period from lS-2h hours was not investigated. 57 SUMMARY A study was made on experimental infection of pregnant ewes with Lgptospira pomona. The ewes were inoculated subcu- taneously either with blood from infected guinea pigs in the leptospiremic phase or with a culture of leptospirae. The ewes became ill after an incubation period of 3-6 days. They showed elevated temperatures and had a leptospiremia which lasted from 2-5 days. In some ewes, leptospirae were present in their blood before the rise in temperature. Hemoglobinuria was observed in only 2 of 12 infected ewes. The ewes subse- quently developed serum antibody titers, and shed leptospirae in the urine. The maximal duration of leptospiruria was found to be 35 days. Leptospiral antibody titers were first found in the urine approximately the third week after inocu- lation. A non-infected ewe and its lamb, which were allowed to stay with the infected sheep, did not contract the disease. The fetuses of 2 ewes, killed at the end of the lepto- spiremic phase (h-S days after inoculation) and the fetus of an ewe killed 23 days after inoculation, were alive at the time of necropsy. The fetal tissues, blood or amnionic fluids did not contain leptospirae or leptospiral antibodies. The rest of the infected ewes and the control ewes lambed normally. Leptospirae were not detected in their 58 colostrum at the time of lambing. Placental membranes did not contain leptospirae, with one exception. It is question- able whether the microorganisms in the latter case were de- rived from the placental membranes themselves, or were due to contamination with infected urine. High leptospiral antibody titers were found in colos- tral whey at birth. No titers were found in newborn lambs. A few hours after sucking, the sera of the lambs showed antibody titers and the whey titers of the ewes drOpped rapidly. One ewe was inoculated 30 days after lambing. Lepto- spirae were shed in the milk from the fifth to the ninth day after inoculation. The duration of leptospiremia in that ewe was from the sixth through the seventh day following inoculation. The nursing lambs did not contract leptospirosis, although the ewe was shedding leptospirae in the milk. Hematological observations were made during the course of the infection in the ewes. A slight decrease was noted in hemoglobin levels and erythrocyte counts in some of the ewes. The significance of this is doubtful, as a de- crease in these figures was also noted in the control ewes. During the pyretic response, there was a decrease in the total number of leukocytes and a relative neutrOphilia and lymphopenia. There was also a decrease in percentage of eosinophils during pyrexia. A study was also made on the influence of the age of the lambs upon intestinal absorption of leptospiral anti- bodies. hilk or serum containing antibodies, was administered through a stomach tube to normal lambs of different ages. Absorption occurred in lambs younger than 15 l/2 hours old, but not in lambs older than 2h hours. Unfortunately, lambs were not available for studies on antibody absorption in the period from 15 l/2 to 2h hours of age. 60 mHmmH|COHpesHpsHmww pdooaed Om mm eemmoadwm* OOOHQ .HE OOH nod madam CH EHQOHonoL .580 Add OH K manhooanpmae .880 new MOH x mephooodeH .OOH + .m mmoamoo SH endpwaedeop umpmcfiuao COHuomHCH nepww whee "wmmHomp¢ a assassisasmmw A: n im fish in #7...— Lm 1.0 \\ Lib \. \. ... ..o \\\\ \\\..\.\‘ mophooodog :OH \\ mopmoohnuhhm :HH .rNH GHQOHonmm A.MH f: rmH n a u u n + + + + + measuHSo COOHm 0-0a o-oa o-oa muoa mnoa - - - - - . wa 03m an oopdmmoadma .H 35090 no“ oodepno GOHpomhdH mo amazoo OHQmHampomamso H .OHm #maopHp Edaew TABLES 2 through 23 W TABLE 2 NORMAL HEMATOLOGICAL DATA (DECEMBER, 1956) 61 3:? giggin Erythro Leucocytes Eosin Biidf::::t::;p::u;::o Retic NPN 608 13.7 11.70 8800 6 1 25 68 0 0 A9.0 737 12.0 11.15 12u50 2 0 38 63 1 0 81.5 758 13.8 10.16 9300 3 2 16 78 1 0 83.0 761 13.3 11.50 12850 3 0 18 78 1 0 50.0 763 18.1 9.18 7100 5 5 27 61 2 0 83.0 76h 13.7 10.6u 7250 h 2 36 58 0 0 ul.S 770 13.9 10.99 9850 0 3 22 72 3 0 83.0 777 13.3 12.28 7900 7 2 25 65 1 0 A3.7 781 11.3 9.22 9800 a 1 32 63 0 0 u3.0 786 16.2 13.73 8300 6 0 19 7h 1 0 h3.0 79h 12.7 13.00 7550 1 1 14 Ba 0 0 53.0 832 13.2 11.39 11550 6 0 27 66 1 0 59.0 836 12.7 10.30 8600 5 1 23 70 1 0 58.0 Hemoglobin in grams per 100 ml. blood Erythro = erythrocytes in millions Eosin = eosinophils Band = juvenile neutrOphils Segm = polymorphnucleated neutrophils Lympho = lymphocytes Mono = monocytes Retic = reticulocytes NPN = mg percent nonprotein nitrogen in blood serum 62 mGHQEwH am anodes emaawa 6m\ma\oa one am mma sm\mm\m 6m\mH\OH was deQEwH mma enemas eoaaws 6m\am\0H doe ms sea sm\mm\m 6m\~\aa + oa\oa own and Had sm\sa\m em\em\0H awe 62 min RES} 633): E concedes soc HmH sm\om\m 6m\om\oa can :2 m1: REA} 63834. is denEmH Hma 666906 eoadas 6m\jm\oa mes mma mea sm\ma\m 6m\om\oa Hes mCHnEwH aopmw empomnnw .Howpcoo Hma sm\oa\m 6m\ma\6H ems we so 01H so 0W\O\HH so me mad am\m\s 6m Hm\oa ems and sea sm\ma\m em\om\oa woe Amhmov Ammmbv GOHumH300dH ehommb QOHpmpmeO deQEwA wGHooosm sonfidz coapmumow Ho COprAdQ no COprASQ Mo open Ho mama COprOHHHpGoOH mg Mme mo MmOBmHm m mqm4a TABLE 1; DURATION OF LEPTOSPIREMIA IN GROUP I, DETERMINED BY BLOOD CULTURES 63 Ewe Days after Inoculation N°‘ 3 u 5 6 7 8 9 10 11 12 737 - - + + - - - - - - 786 + - + + - - .. - .. - 832 + + + + + - - - - - 836 9k TABLES 5 and 6 SERUM ANTIBODY TITERS OF THE EWES IN GROUP 1* TABLE 5 ‘Q. pomona titers Ewe Days after Ex;osure NO- 3 u 5 6 7 8 9 10 11 12 23 25 59 69 78 82 97 737 - — - - 1 ‘1 3 u 7 6 u a 3 u 786 - - - - l 3 h 6 7 7 5 5 5 6 832 - - - - - 1, 3 5 9 9 7 6 836 - ~ - - - .1 3 5 IO 9 7 7 6 TABLE 6 ;. icterohaemorrhagiae, AB titers Ewe Days after Exposure N . ° 3 u 5 6 7 8 9 10 11 12 23 25 59 737 - - - - - - j, 3 3 3 2 786 - - - - - - 1 3 1 3 3 3 832 - - - - - - '1 2 2 3 836 - - - - - - ‘1 2 3 2 2 *The titers are expressed highest serum dilutions lysis. dilution of serum. as negative exponents of the showing 50 percent agglutination- : : 25 percent agglutination—lysis in the 10"1 .Efio pod mopmoooneH N .00504 .550 pod mQOHHHHe CH manhooanuhae u .ongpmam UOOHQ .HE OOH nod msdam CH CHQOHonoQ u .pwm .mo CH maanAedEop u .dEoB ommw mm.oa o.HH ommo ms.HH H.3H ooos :m.ma o.ma mm omoo ms.o m.o m.HoH omos mm.HH H.WH o.HoH ooms ms.aa m.:~ m.aoa NH omom m6.o m.o o.aoa ooas ma.oa m.HH m.oo~ omoo mm.ma o.mH m.aoa Ha ooms oo.o o.o m.HoH omso mm.oa o.HH m.HoH omss so.ma m.ma o.ooH oH omoo oo.o m.oH e.moa ooom oa.oa o.HH o.HoH oooo NH.HH m.ma m.HoH o oomm oo.o a.oa m.soa omm: os.aa o.HH m.moa oooo 4H.HH s.mH m.moa m oooo NH.HH m.HH 3.6oa ooom oo.oH o.aa e.mofl oamo ss.o H.:H :.moa s oosm ma.oH H.HH o.ooH omso oa.ma m.ma o.6oH oooo mm.HH s.ma o.ooH o oooo mm.oa m.HH m.moa oomm om.oa m.ma a..oa omoo o:.ma m.ma o.moa m oooo s:.aa m.ma o.moH omsm oo.aa m.:a o.:oa ooom :m.ma H.3H o.moH : omso o:.HH a.ma o.moa ooos oe.ma s.ma o.moa oooo mm.ma o.ma :.ooH m .donoA .ohflphnm .bwm .aSoB .oosoH .omnpmam .Qmm .dEeB .oodoA .oasumam .nmm .dEoB COprHMMMWW ‘MHm 63m was was ans men when Hmdomd.mo MBZDOU MEMOOODMA Adn u m gouge when mm ammoaem: p<* mHHQdoGHmoe u m o oo om o o H mo :m o 6* o HN Hm o o o HN Hm o o o NN NH o 6 mm H mo mH o m H mN Hm o o o no 6H o o H oN mm o H o Ho NH o m NH o oo Nm o m m MN :m o H m mN mm o H H mN mm o H o we on o m HH o mN em o a H o6 Hm o m m NN om o o H oN om o H H om NH o m oH o oN oH o m m mo mm o o o mN am o H o oN om o m H mm mH o a o o NN om o m H me mm H o o N: mm o o o no on o H o MN :m o m m Hmoommo ommmemmH onNmHmH ommomfiH HoosmmH N H oN 6H H e o on Ho o o o om N: m H o mN om m o o NN mm o o o m sN mH : m H mo om o o H MN om o o o mm mmmH.H H om on H m m m mN 6H o N m oo Nu m o m oN :m o 2 H Nm mm o o H HN mm o o a H no em o N m mo mm o o m oN mm o o o Ho on o o m me on o a m 2 H m m m z a m m a z a m m a s g m m m s H m m m eOprHmMmmw HHospeoov :oN saw one can mmo 63a omN 62m NHN can when H moose mo mszooo mosoooemq HeHazmmmaaHa @ mam: 68 TABLE 9 DURATION OF URINARY EXCRETION OF LEPTOSPIRAE IN GROUP I, DETERMINED BY ANIMAL INCCULATIONS 3:? Days after Inoculation 12 18 22 21 29 31 33 no 59 73 737 + - - - - - - ‘ 786 - — + - + + - - - 832 - — + + - - - - 83 69:- + - - *Ewe 836 killed for necropsy on the 23rd day after inoculation. TABLE 10 AKTIBODY TITERS roa ;. POMONA IN THE URINE OF GROUP 1* Ewe Days after Inoculation N°‘ 12 18 22 _ 2a 29 33 no 59 73 737 - - - - - - - - 786 - — — - - - 2 2 832 - - — - 2 3 2 2 836 - - - r . fThe titers are eipressed as negative exponents of the highest urine dilutions showing SO percent agglutination-lysis with 'E. pomona antigen. 69 .doHudHHO HIOH dH mHmmHaCOprdHHSHm.m mm unfl .mHmmHIGOHpandemmm accused Om wdeosm meHpSHHb Essen pmengn one He mphmdmeo e>Hpmwmd one we Oommeadxo one mumqu mfla* - a m o m m m s o m o m N N N o o o N Nmo psaH N N m N N N N N N m m m m m a e 6 Now sHHs O N mmw Edamm - a o N 6 o o N N m o N N o N ooN pamH N m a m a a N m m a o N owN sHHs 0 0 own. Fdhom - H. m a m s m m NmN pseH N N m a N NHN sHHs : : NHN asses om Nm m: 6N 3N oH oH mH :H HH NH HH oH o o N o m a m N H Hve woowwq 1| mpAHm Loewe mmmm 90 nonewz 0| '1‘ li :l": '7"!!! | *H mDomO 2H mmfldq mo €Mflm 924 . amaza ho Hpmmec on» we Ummmoadwo one mbepHp hcoanc4+ mmeoodoE H a manhoosdsmH u A .320 new mopmooosoq mHHnmoapsod oobooHozczdaoShHOQ u m .EEo mom moumoOAQHHam mHHhiompso: eHHdo>sn u m .eopo .HE OOH so; madam CH CHQOHmoaes H .Qmm mHHhmochoo u m .mo CH oaspsnedsop n .QEoB .mOHpNHSOQQH sebum when m mmdoaooc new eoHHHM * msoH o 6: N6 o m oon ooo.omo.m 6.o . N.moH = m H-oH o om o6 o H omam ooo.ooN.N m.6 + N.moH = 6 H Nm Nm o oH oooN ooo.omm.o m.o + n.6oH = m + woNOH : N omN6 ooo.omm.o m.HH + N.NoH N66 H u H 66 on o m om66 ooo.omo.HH N.NH + 6.NoH mason m armsopHa ; H 6 do n IHm u! m m endes coprinoocH . MWwoM mandoo HprcohmMMHQ mophooodoq mephcoanumam .pmm OOOHM .ufioe sebum oSHB esoN Ham 2H onNowaHH «Hosom .H so Hmmeoo was MH aflmda 72 TABLE 1A TEMPERATURES ALD HEAATOLOGIOAL DATA OBTAINED IN”GROUP II AFTER IAOOULATIOB OF BLOOD EROM BWE 79Aw Days after v, _ Differential Counts Inoculation Temp. dgb. Erythro. Leuco. E B S L 1 ‘1' 1‘1 .Ewe 758 (control) 1 101.2 12.7 10.03 8650 A 0 37 58 1 2 101.3 12.3 9.33 7150 9 0 AA 61 1 3 102.0 12.3 8.96 9600 6 0 A9 AA 1 A 101.0 11.3 8.38 8350 11 O 33 56 0 5 102.0 11.3 8.53 9900 7 O 39 5A 0 6 102.9 10.A 9.57 7550 11 0 A8 A1 0 Ewe 761 1 101.8 11.3 9.91 9500 A 0 A2 51 3 2 101.7 11.7 9.0 11250 2 0 3A 6A 0+ 3 101.8 10.8 10.9 11350 3 2 39 SA 2 A 101.3 10.7 9.A8 11850 6 0 28 65 1 5 101.6 10.A 9.00 11150 7 O 32 61 0 6 102.1 10.7 10.19 10000 7 0 33 58 2 Ewe 763 1 101.9 13.2 10.51 10850 9 O 7 53 1 2 101.8 12.3 9.79 9200 9 0 33 58 0 3 102.A 11.7 9.AA 7800 7 0 38 5A 1T A 102.5 12.0 9.52 9100 10 0 30 59 1 5 102.3 12.5 9.81 7700 9 0 27 6A 0 6 102.5 11.2 9.23 8650 7 0 37 56 O Ewe 76A 1 103.1 11.7 10.61 9650 2 1 58 39 O 2 102.9 11.7 9.70 9700 2 0 AA 5A 0 3 102.3 11.2 10.10 10A50 2 O 55 A3 0 A 101.2 11.5 9.98 10500 2 0 60 38 O 5 102.7 11.0 10.37 11550 0 0 53 A7 0 6 102.A 9.6 8.87 9650 5 0 50 A2 3 Ewe 770 1 101.5 13.7 11.02 8250 A O 31 65 0 2 101.6 13.0 9.83 10100 2 0 A1 57 O 3 102.A 12.2 11.23 9350 6 O 26 68 0 A 101.1 12.0 8.83 8050 7 O 21 72 0 5 102.3 12.9 9.70 10950 7 0 3% 59 0 6 102.0 11.3 9.0A 7950 3 0 3 58 3 TABLE 1A, continued 73 Days after Temp. Hgb. Erythro. Leuco. Differential Counts Inoculation E B S L M Ewe 777 1 102.5 13.7 11.90 12100 9 O 56 35 0 2 102.3 13.3 11.19 9850 15 0 5O 35 0 3 102.1 13.0 11.21 10650 8 O 56 36 O A 102.5 13.0 12.28 12650 11 0 A6 A2 1 5 102.2 13.0 10.01 12150 8 0 A5 '5 2 6 101.8 11.0 9.22 10000 A O 59 37 O Ewe 781 l 102.A 11.0 8.8A 900 5 O 51 Ah 0 2 101.7 11.2 8.75 8000 7 O 33 60 O 3 102.6 10.A 8.96 8300 6 0 A0 SA 0 A 102.0 10.7 7.90 9150 10 O 32 5O 0 5 102.3 10.8 9.00 8150 9 O 31 6O 0 6 102.A 9.; 8.0A 9600 8 O 33 S7 2 Ewe 608 1 101.9 12.0 9. 88 11650 9 O 31 6O 0 2 101.5 12.0 8.50 9500 9 O 39 52 0 3 101.6 11.3 9.32 8A00 6 0 3A 59 1 A 102.0 11.0 8.98 7000 9 0 3A 56 1 5 102.A 11.3 9.80 7850 6 0 A3 A9 2 6 102.A 11 3 9.27 9600 5 0 3A 50 1 Temp. = temperature in °F. Hgb. = hemoglobin in grzi. s per lCC ml. blood Erythro.= erythrocytes in millions per 0mm. Leuco.= leucocytes per cmh. E. = eosinophils B = juvenile neutrophils S = polymorphnucleated neutrophils L = lymphocytes M = monocytes *Blood contained too few leptospirae to infect ewes. Data serve as control information. 'tBasOphils 78 TABLE 15 TEMPERATURES AND HEMATOLOGICAL DAT OF GROUP II FOLLOWING ROCULATIOK WITH 3 x 10 LEPTOSPIBAE Days after ..- t C Inoculation Temp. Hgb. Erythro. Leuco. Diiieren 131 ounts E B s L 1 Ewe 758 (control) 2 102.2 3 102.5 A 102.5 5 102.0 10.7 9.01 6A00 9 0 A9 A2 0 6 102.3 11.1 8.85 7A5O 10 0 38 52 0 7 102.6 10.3 .28 7000 15 0 AS 39 1 8 102.2 Ewe 761 2 102.A 10.3 8.88 10A50 7 0 36 56 1 3 103.0 10.5 8.82 7250 9 0 AA A7 0 A 10A.O 11.3 9.23 6950 6 O 30 63 1 5 103.0 10.A 9.08 6200 3 0 28 69 0 6 101.8 10.A 8.25 7750 3 0 22 75 O 7 101.8 10.5 8.33 9300 7 0 30 63 0 8 102.6 10.1 8.8 7650 2 0 3o 67 1 Ewe 763 2 102.6 10.7 8.63 9750 7 0 35 S7 1 3 10A.5 11.0 8.83 7A50 7 0 A3 5A 1 A 10A.7 10.7 8.83 5700 6 O 38 55 1 Ewe 7611 2 103.8 11.3 9.89 8500 0 0 51 At 1 3 105.9 9.0 8.03 10000 0 0 78 22 0 A 105.6 10.7 9.A2 11600 0 0 78 22 O 5 10A.3 11.3 9.80 11950 O 0 6A 35 1 7 101.8 11.3 9.8A 11700 2 0 72 25 1 8 103.8 11.0 9.32 12500 3 O 56 A0 1 Ewe 770* 2 102.7 10.6 8.90 9100 A 0 36 60 0 3 102.2 11.7 9.60 9700 A 0 29 67 0 A 102.2 10.A 8.91 9A50 3 0 3A 63 0 5 102.2 11.0 8.87 9350 1 0 18 81 0 6 102.1 11.0 8.87 9950 1 0 38 60 1 7 101.6 10.7 9.69 11600 A 0 2A 70 2 8 102.8 11.5 9.53 9500 .1 0 29 70 0 TABLE 15, continued 75 Differential Counts *Not infected Days after .. e .12 >u Inoculation Temp. Hgb. Erythro. Le 00. E B S L A Ewe 777 2 102.7 11.7 9.57 1095 0 8 0 5A 38 0 3 10A.3 12.7 10.81 12 0‘0 5 O 8 37 O A 103.6 11.7 10.AO 8750 O O ~5 3A 1 5 lOA.1 12.0 9.36 9100 O 0 77 23 O 6 102.5 11.3 9.25 12050 O O 73 27 O 7 lOC.8 10.7 8.95 15000 0 O 1 25 A 8 102.3 9.3 8.78 10900 3 0 A5 51 1 Ewe 781 2 102.8 9.6 8 .3 7650 2 0 A5 52 1 3 105.5 8.8 8. 35 9A00 3 O 62 35 0 A 103.5 9.6.10 7550 O 0 56 A3 1 5 103.8 9.3 8.35 “A50 0 O 51 A9 0 6 103.8 9.8 8. 61 7800 l O 51 A8 0 7 100.8 9.0 8.00 3500 7 O 38 52 3 8 102.5 8.8 7.60 8750 2 O 2 67 2 Ewe 608 2 102.010.5 8.59 9000 8 0 A3 A7 2 3 102.0 10. 3 8.77 8000 7 0 A0 53 O A 102.0 10.1 8.7A 7150 6 0 9 55 0 5 102.2 9. 8 7.50 9250 8 0 32 60 O 6 101.8 10.A 9.07 8500 7 0 A1 52 0 7 102.’ 10.1 8.77 9800 10 0 A7 A0 3 8 102.3 10.1 8.30 7150 5 0 13 82 0 Temp. = temperature in °F. Hgb. = hemoglobin in grams per 100 ml. blood Erythro.= erythrocytes in millions per 0mm. Leuco. = leucocytes per 0mm. E = eosinophils B = juvenile neutrophils S = polymorphnucleated neutrOphils L = lymphocytes M = monocytes 76 TABLE 16 DURATION OF LEPTOSPIRELIA IN GROUP II, DETEREINED BY BLOOD CULTURES 3:? Days after Inoculation l 2 3 A 5 6 7 761 - + + + - - - 763 - + + + - - - 76A + + + + - - - 777 + + + + - .. - 781 + + + + - - - 608 - - - - - - - TABLE 17 SERUM ANTIBODY TITERS IN GROUP 11* 3:? Days after Infection 21 5 6 7 8 10 1A 17 31 37 67 761 - 2 A 6 8 7 5 A 768 - - 2 5 8 9 7 5 5 777 - - A 7 7 9 9 7 7 781 - - 3 7 6 9 8 9 608 - - - 1 2 A 5 2 2 *Titers are expressed as the negative exponents of the highest serum dilutions showing 50 percent agglutination- 1ysis with L. pomona antigen. 77 TABLE 18 DURATION OF URINARY EXCRETION OF LEFTOSPIRAE IK GROUP II, DETERMINED BY ANILAL INOCULATION 5:? Days after Inoculation 1h 23 35 S2 65 81 761 + - - - - ' 76h - - - ’ ‘ ' 777 ' + + ’ ' 7 781* + + + 608 - ' ’ ' ' * . Ewe 781 killed for necropsy Mu days after infection, kidneys negative. TABLE 19 ANTIBODY TITERS IN TEE URIHE OF GROUP 11% fig? Days after Inoculation 16 22 35 52 65 81 761 - - - 1 2 2 76h - - 3 3 2 3 777 - - 2 2 1 3 781 - 2 2 608 - - - 1/2 - - wThe titers are expressed as the negative exponents of the highest urine dilutions showinb 50 percent agglutination-lysis with L. pomona antigen. 70 TABLE 20 HEEATOLOGICAL DATA OR LAMBS IN GROLP II AT BIRTH Lamb of Ewe W b. Erythro.. Leuco. Differential C0 t3 R E B S L M 1 758 15.5 11.t5 5750 0 0 12 58 o 0 2 758 18.1 10.06 3800 O 0 22 78 0 0 1 761 13.3 9.37 5350 2 761 13.7 9.26 6850 1 766 15.0 11.59 1050 0 o 20 00 0 0 2 76h 14.7 11.17 L500 0 O 19 81 0 0 1 770 1A.5 11.15 2050 0 0 AZ 58 0 O 1 777 11.1 10.70 A250 C 0 10 9O 0 0 2 777 11.: 10.95 1750 c c 30 7c 0 0 1 781 7.1 11.;A 365C 0 0 19 81 0 0 2 781 15.7 10.27 3320 O 0 18 82 O 0 Hgb. hemoglobin in grams per 100 ml. blood. Erythro. erythrocytes in millions per cum. Leuco. leucocytes per 03m” eosinophils juvenile neutrophils polymcrjhnuCICated neutrophils lymphocytes monocytes reticulocytes per thousand red cells II U H II II I! H II II :HZt‘UQUHfi 79 TABLE 21 ANTIBODY TITERS FOR L. POMONA IN COLOSTRUM AND MILK WHEY, AS WELL AS SERA OF EWES AND LAMBS IN GROUP II Lamb or Days after Birth Ewe No. sample 1 2 3 u 5 6 7 9 12 17 18 19 20 21 23 I??? 1 serum - 6 5 h 3 .1 L32; 3 serum - h 5 A 3 3 ‘: Ewe 761 whey h S 5 h 3 serum 7 5 L;2E 2 serum - 2 2 :_ - - Ewe 7 61+ whe y 2 S U. 3 2 2 serum 9 L??8 serum - - whe v - - Ewe 770 serAm - - L??? 1 serum - 3 2 2 ’ L??? 2 serum - 3 3 l - - whey 3 7 A 2 3 Ewe 777 serum 7 9 9 L??? 1 serum - 3 2 - - L%§E 2 serum - 3 2 ‘ whey 7 A 7 5 Ewe 781 serum 9 L318 serum - 3 2 3 - . whey 3 L t l Ewe 608 serum 5 *The titers are expressed as the negative exponents of the highest serum or whey dilutions showing 50 percent igglutination-lysis. 1’= 25 percent agglutination-lysis in the 10' serum dilution. 80 .mhopméwn m we mmsomm one“ uuofipwasoonw Hmmcouapommapca m9 womafipmpomk a. 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