EXPERIMENTAL LEFTOSPIROSIS: PATHOLOGY or Lmosmgé Pea-Mow: INFECTION m MALE CATTLE Thesis for the Degree .05 M. 5. MICHIGAN STATE UNWERSITY Osman Abd-Elaziz Atallah 19.6.3 LIBRARY Michigan State University ABSTRACT EXPERIMENTAL LEPTOSPIROSIS: PATHOLOGY OF LEPTOSPIRA POMONA INFECTION IN MALE CATTLE by Osman Abd-Elaziz Atallah An experiment using ten male cattle was conducted to study experimental Leptospira pomona infection. Inoculums in one or more animals consisted of leptospiremic guinea pig, hamster or cattle blood. In one animal bovine urine obtained during leptospiruria was used. The subcutaneous route of inoculation was used in all animals while two bulls were simultaneously inoculated intraperitoneally. In addition to gross and microscopic pathological studies; clinical, serological, hematological and bacteriological observations were recorded.. Clinically, fever was observed for periods ranging from two to seven days. Temporary anorexia was seen in only one animal during the thermal reaction. Leptospiremia in two animals was detected on the fourth, fifth and/or sixth day after inoculation. Leptospiruria in four ani- mals started between the fourteenth and twenty-third day Osman Abd-Elaziz Atallah after inoculation and continued until at least the thirty- eighth day after inoculation. Initial serological responses occurred between the fifth and the twelfth day after in- oculation. Maximum titers observed ranged from 102 to 108 using the microscopic agglutination-lysis test. The hemo- globin, packed cell volume, erythrocyte and leukocyte values were slightly decreased in all animals except one. The relative lymphocytic and monocytic counts were slightly raiSed. Grossly, the renal surface in three animals had,vari- able numbers of small white foci. These foci extended into the renal cortex and medulla. The renal lymph nodes of one animal were enlarged and edematous. Microscopic changes were demonstrated in particular locations of the genital tract (testes, rete testes, epididymides, seminal vesicles and prostate gland). These lesions were focal and diffuse interstitial cellular (mostly lymphocytes) infiltration of variable extent. Sim- ilar microscopic lesions were seen in the kidneys (partic- ularly in the cortex), liver and heart. These involved areas showed vacuolar degeneration in the renal and semini- ferous tubules, necrosis in the liver and an increased number of Anitschkow myocytes in the myocardium. EXPERIMENTAL LEPTOSPIROSIS: PATHOLOGY OF LEPTOSPIRA POMONA INFECTION IN MALE CATTLE BY OSMAN ABD-ELAZIZ ATALLAH A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Veterinary Pathology 1963 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation and gratitude to Dr. S. D. Sleight, Department of Veteri- nary Pathology, for his faithful guidance and assistance during the course of this research, without which, the work entailed in this thesis would have been more difficult. Appreciation is also due to Dr. C. C. Morrill, Chair- man of the Department of Veterinary Pathology, Dr. R. F. Langham and Dr. H. D. Hafs for their aid, suggestions, continued interest and encouragement. The author would like to thank Mrs. Athalie M. Lund- berg for her technical assistance and advice. Finally, appreciation is also extended to Michigan Artificial Breeders' Cooperative, Incorporated, for pro— viding, in part, financial support for this study. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . REVIm OF LITERATUm . O O C O O O I 0 MATERIALS AND METHODS . . . . . . . . EXPERIMENTAL RESULTS . . . . . . . . . Clinical Serological Hematoloqical and Bacteriological Pathological DISCUSSION 0 O O O O O O O O O O O O 0 SUMMARY . . . . . . . . . . . . . . . TABLES I through VI . . . . . . . . . FIGURES 1 through 28 . . . . . . . . . REFERENCES 0 O O O O O O O O O O O O 0 iii Page 13 17 17 18 18 19 25 37 39 50 64 LIST OF TABLES Table Page 1. Material and Methods of Inoculation and Day of Necropsy . . . . . . . . . . . . . . 39 2. Average Daily Temperature of Experimental Bulls O O O O O O O O O O O O O O O O O O O 41 3. Antibody Titers for L, pomona in Sera of Injected Bulls . . . . . . . . . . . . . . . 42 4. Mean (i) and Standard Deviation (8) Of the Hematological Values . . . . . . . . . . 43 5. Mean (x) and Standard Deviation (O) of the Differential Leukocyte Count . . . . . . . . 4S 6. Histopathological Findings in the Experi- mental Cattle . . . . . . . . . . . . . . . 47 iv Figure LIST OF FIGURES Page Section through the kidney to show destruc- tion of the renal tubules and lymphocytic infiltration. H. & E. X680 . . . . . . . . 50 Section through the kidney to show lymphocytic infiltration, syncytial giant cells and a thickened Bowman's capsule. H. & E. X680 . . . . . . . . . . . . . . . . 50 Section through the renal cortex to show vacuolar degeneration in the tubules, thickened Bowman's capsule and lymphocytic infiltration. H. & E. X411 . . . . . . . . 51 Section through kidney of the calf to show tubules distended with neutrophils and con- nective tissue proliferation around the tubules. H. & E. X160 . . . . . . . . . . . 51 Section through the kidney to show lympho- cytic infiltration in the renal medulla. H. & E. X411 0 O O O O O O O O O O O O O O O 52 Section through the calf kidney to show lymphocytes and neutrophils in interstitial tissue and a thickened Bowman's capsule. H. & E. X680 . . . . . . . . . . . . . . . . 52 Section through renal papillae and minor calyx to show lymphocytic infiltration. H. & E. X160 0 O O O O O O O O O 0 w. O O O O 53 Section through kidney of control bull to show lymphocytic infiltration around the arcuate artery. H. & E. X160 . . . . . . . 53 Section through the liver to show lympho- cytic infiltration in the area of the hepatic trinity. H. & E. X411 . . . . . . . 54 Figure 10. 11. 12. 13. 14. 15. 16.. 17. 18. 19. 20. Section through the liver to show lympho- cytic infiltration and slight edema in the interlobular areas. H. & E. X411 . . . . Section through the liver to show pyknotic nuclei of the hepatic cells. H. & E. X160 A higher power of Fig. 11 to show kanotic nuclei of hepatic cells. H. & E. X680 . . Section through the wall of the gall bladder to show lymphocytic infiltration in the lamina propria. H. & E. X160 . . . . . . Section through the testis of the calf to show scattered lymphocytes among the seminiferous tubules. H. & E. X1700 . . . Section through the seminiferous tubule to show syncytial giant cells in the lumen of the tubule and a thickened basement mem— brane. H. & E. X680 . . . . . . . . . . . Section.through‘the testis to show lympho- cytic infiltration among seminiferous tubules with vacuolar degeneration. H. & E. X160 . . . . . . . . . . . . . . . . . . . Section through the testis to show lympho- cytic infiltration among seminiferous tubules without vacuolar degeneration. H. & E. X160 . . . . . . . . . . . . . . . Section through testis to show lymphocytic infiltration among seminiferous tubules with vacuolar degeneration. H. & E. X411 . . . Section through epididymis to show lympho- cytic infiltration. H. & E. X411 . . . . Section through seminal vesicle to show lymphocytic infiltration of follicular type. H. & E. x56 0 O O O O O O O O O O 0 vi Page 54 55 55 56 56 57 57 58 58 59 59 Figure 21. 22. 23. 24. 25. 26. 27. 28. A higher power for Fig. 20 to show lympho- cytic infiltration around the crypts of the seminal vesicle. H. & E. X160 . . . . . . . Section through adrenal gland to show lymphocytic and eosinophilic infiltration in zona glomerulosa. H. & E. X160 . . . . . A higher power of Fig. 22 to show lympho— cytes and eosinophils. H. & E. X640 . . . . Section through adrenal gland to show lymphocytic infiltration in zona fascicu- lata. H. & E. X411 . . . . . . . . . . . . A higher power of Fig. 24 to show lympho- cytes and eosinophils in zona fasciculata. H. & E. X680 0 O O O 0 O O O O O O O O O O 0 Section through a lymph node to show edema of the capsule with eosinophilic infiltra- tion. H. & E. X160 0 O O O O I O O O C O 0 Section through lymph node to show edema and plasma cells. H. & E. X680 . . . . . . . . Section through lymph node to show a megakaryocyte. H. & E. X1700 . . . . . . . vii Page 60 6O 61 61 62 62 63 63 INTRODUCTION Leptospira pomona infection has been shown to be a major cause of abortion in pregnant female cattle (2, 6, 10, 16, 17, 21). Due to the gradual replacement of nat- ural breeding with artificial insemination in the past several years and the great importance of the bull in this respect, leptospirosis in male cattle was selected for experimental study. The purpose of this present study was to investigate the clinical, hematological, serological and pathological effects of L, pomona infection in bulls with particular emphasis on the pathological aspect. REVIEW OF THE LITERATURE Pathogenic leptospires were first seen in New Orleans by Stimson (55) in 1907 in sections of kidney from a hu- man patient who was believed to have died of yellow fever. Michin and Aéinov (42) in 1935 in North Caucasus observed spirochetal forms in eight cultures made from the heart blood, liver and spleen of a calf which had died of ictero- hemoglobinemia. They injected this culture into guinea pigs and white mice. These animals died on the seventh or eighth day after injection. Clayton and Derrick (12) in 1936 isolated leptospires from a dairy farmer near Pomona in Australia. Jungherr (30) in 1944 described the patho- logic changes in three fatal bovine cases in which he dem- onstrated leptospires in Levaditi—stained kidney sections. This was the first microscopic demonstration of the lepto- spiral organism in cattle in the United States. Marsh (35) in 1945 reported 25 fatal cases in calves six to ten weeks old. Leptospires were demonstrated in Levaditi-stained renal and hepatic sections. Baker and Little (2) in 1948 were the first to isolate the causative agent of lepto- spirosis in cattle in the United States. They injected abnormal milk into seven-day-old chick embryos, guinea pigs and cultural media and Obtained positive results. Leptospirosis has been reported in most areas of the United States (2, 5, 8, ll, 25, 29, 35, 48, 52, 55, 59). Many investigators reported that leptospiral infection may be transmitted through urine (2, 4, 20, 21, 28, 45, 47, 61), semen (53;, blood (2, 3, 36, 45) and abnormal milk (2) from carrier animals. Contaminated water (22) and food (28) were believed to play a role in the transmission. Some arthropods (Ornithodoros turicala) may (2, 9) or may not (57) transmit the disease. Many natural reservoirs for leptospires have been found, including the jackal (64), rat (28), opossum (49), brown rat (43), cottontail rabbit, marsh rabbit, red fox, gray fox, dog, raccoon, striped skunk, spotted skunk, otter, wildcat and house cat (38). The route of infection was suggested to be the mucous mem- branes and/cn:the abraded skin (20, 62, 47). Experimental subcutaneous injection was more effective than intranasal in- oculation while feeding did not transmit the disease (2, 3, 36, 45). A wide variety of clinical symptoms was observed in lepto- spiral infected cattle with calves showing more severe symp- toms than adults. Calves showed a rapid course in fatal cases, a high mortality rate and evidences of hemolytic anemia such as icterohemoglobinemia, jaundice and hemoglobinuria (2, 3, 15, 28, 35, 42, 51, 63, 64). The thermal reaction was subnormal, normal or above normal (2, 10, 63). Sev- eral other nonspecific symptoms such as weakness, anorexia, stunted growth, dullness, bumping pulsation of the heart, labored respiration, depression, weight loss and difficulty in standing were reported in calves (6, 15, 28, 59, 64). Leptospiruria started during the second or third week after inoculation (19, 26) and continued for a few months (30). The clinical symptoms in adult cattle were generally mild but some showed signs of hemolytic anemia such as hemoglobinuria (2, 34, 37, 50) and icterus (37). Abortion in the last trimester of pregnancy and lowered milk pro- duction caused major economic losses (2, 6, 10, 17, 16, 21, 26, 27, 37, 60, 41, 44, 50, 52). Leptospiremia on the fourth and fifth day after inoculation (10, l8, l9) and leptospiral antibodies in the blood serum on the sixth day after inoculation have been demonstrated (10, 42, 61, 64). Other symptoms reported included pale color of the teats (37), fever (10, 16, 19, 29, 34, 37, 41, 44, 61), normal (52) or subnormal (29) temperature, anorexia (10, 16, 46), evidence of pulmonary congestion (29), depression, tachy- cardia (44), convulsions and rarely death (19, 37). Some workers reported breeding difficulties (50) while others found no statistically significant change in the reproductive efficiency of cattle (33). In one experimental group of heifers, hemoglobinuria, albuminuria or leptospiruria could not be demonstrated. The heifers showed retained placenta, photophobia, con— junctival hyperemia and mucopurulent discharge from the eyes(l8). Naturally infected steers showed fever (34), hemo- globinuria (34), enlarged superficial lymph nodes, diar- rhea, anorexia, rough hair coat, high morbidity and low mortality (46). One steer was unable to stand and died (37). Ramirez _E_§l, (46) showed that 88 percent of one group of steers had a positive serum titer against L, hardjo while only 7 percent reacted with L, pomona antigen. No leptospires were isolated or demonstrated from these steers. Work with leptospirosis in bulls has not been reported frequently in the literature. Some experimental investi- gators in this field observed fever or subnormal temperature, leptospiremia, weak rapid pulse, rapid respiration, hemo- globinuria and rarely death (19). In experimentally in- fected six-months to one-year-old bulls, symptoms included a slight fever on the day of inoculation, fever of three days duration after inoculation, leptospiremia on the first and fourth day and leptospiruria 21 days after in- oculation. A maximum antibody titer of 1:6,400 was re- corded on the 26th day after' inoculation in.a two—month- old calf (26). Pathological findings associated with this disease in the bovine are of wide variation. Generally the lesions reported in the literature were mostly attributed to damages of the capillaries, small veins and parenchymatous tissues especially of the kidney and liver (15). Some of these findings were hydrothorax, hydroperitoneum, hydropericardium, subserous and subcutaneous hemorrhages, reddish tinted fibrinéon the diaphragm, patches of congestion in the abomasum and duodenum and a thickened and congested wall of the gall bladder (17, 30, 50). There was evidence of jaundice in the aorta, tendons and connective tissue (1, 14, 15, 28). A marked yellow gelatinous edema was reported in the mediastinum just dorsal to the sternum (28). The renal lesions have been considered to be highly significant in the pathology of leptospirosis. Grossly, the kidney was enlarged (15), turgid with indistinct lobulation (61), dark in color (14, l) or pale with red- dish-brown spots (26) or white spots (2, 15, 28) on the cortical surface. These spots extended into the cortex and occasionally reached the medulla (24, 29, 44, 46, 52, 61). Te Punga (61) described the renal cut surface as showing considerable exudate. The renal pelvis was either empty or contained yellowish sandy material and several brownish calculi in some of the renal calyces (24). Microscopically, the kidneys were reported to have edema (15) and cellular infiltration (mostly lymphocytes and monocytes mixed with fewer neutrophils and eosinophils) in the interstitial tissue of the cortex, corticomedullary junction and medulla and in the lumens of the affected tubules (2, 3, 14, 15, 24, 52). The renal tubules were destroyed in the area of the cellular infiltration (l, 2, 14). There were hemorrhages around the arcuate and inter- lobular blood vessels and blood pigments in the superficial tubules (15). The renal epithelium of the proximal con- voluted tubules appeared as solid masses with intracel- lular hyaline droplets (15) or swollen with finely vacuolated cytoplasm which contained hemosiderin and no clear nucleus (24). In other instances, the renal epithelium showed hypertrophy (52), vacuolation (19, 29), atrophy (52, 51) or necrotic changes (19, 24, 29, 52). Tubular basement membranes were thickened (24, 52) or absent (19). A few renal epithelial cells were replaced by masses similar to giant cells of the Langhan's type (24, 61). There were degenerative changes (24, 46, 52) or discrete fibrosis (57). The tubular lumens contained protein precipitate (2), granular debris (14), strongly eosinophilic hyaline, hematin or cellular casts (4, 7, 15, 19, 24, 29, 61), leptospire-like structures (1, 14, 15), inflammatory cells and desquamated necrotic epithelium (24, 52). The tubular lumens were dilated and lined with a thin layer of epithe- lium (24, 61). The renal medulla was either normal (2, 3) or had alternate pale and dark striations (61), swollen tubular epithelium and/or calcium in the renal papillae and interstitial tissue (24). Glomerular changes, though not extreme, were reported. These included thrombi in the glomerular capillaries (15) and increased cellularity (24, 61) and congestion (29) of the capillary tufts to completely fill the corpuscular cavities (15). The glomerular tufts showed various stages of degeneration (19, 29). Bowman's capsules were thickened (24, 29, 46, 50, 61). There was thickening and metaplasia of the corpuscular parietal cells (29, 61). Eosinophilic granular material was reported within Bowman's capsule (19,29). Hemoglo- binuria was reported in some cases (1, 14, 15). Macroscopically, the liver was enlarged (l, 14, 15, 61) with distinct lobulation (61), friable (1, l4) and red- dish—brown, yellowish-tan or orange in color (14). The liver had necrotic areas especially in the caudate lobe (61). Microscopically, the hepatic cells were distorted (15, 28), showing finely granular (24) or fragmented and lysed (15) cytoplasm which contained brown pigments (l4) and leptospires (1, 14, 15). There was centrolobular necrosis (l4, 19, 29) and sinusoids distended with blood in the necrotic areas (15, 24). The interlobular connective tis- sue and the portal tracts contained cellular infiltrates including lymphocytes (15, 28), histiocytes, plasma cells, a few neutrophils (14, 15, 24, 28) and excess fibrous tis- sue (29, 61). Hadlow §§_§1, (25) reported a small thrombus in one interlobular vein, hydropic degeneration of the hepatic cells and a mild fatty change but no leptospires were seen in silver-impregnated sections. Bile stasis 10 was reported in the canaliculi and interlobular ducts (14). The gall bladder was distended (l, 61) with bile that was usually thick, granular and of yellow colOr (14). It had a thickened congested wall (61). The spleen showed engorged red pulp (14, 24, 32) and some hemorrhages (24, 32), scattered neutrophils (14, 24, 32), megakaryocytes (around the follicles) 24, 32), plasma cells, eosinophils (24, 29), and macrophages engulfing erythrocytes (l4) and iron pigments (24, 29). The splenic follicles were inactive (14, 24, 29). The lymph nodes were edematous (l4), hyperemic (l4), juicy and of pinkish or red color (1,14). Their sinusoids contained erythrocytes, neutrophils (l4) and leptospire- like structures especially in the mesenteric lymph nodes (1, 29). Jungherr (26) observed lymphoid necrobiosis. The lungs have been described as partially collapsed (14), with atelectasis (24), subpleural hemorrhage (1), and pulmonary adhesions (29) on the apical lobes. Micro- scopically, there were alveloar emphysema (l4) and edema (l, 14,) thickened alveolar walls (24), some fibrin (14, 29) and a few neutrophils (l4), histiocytes and lympho- cytes (24). Leptospires were found (1, 14) in the pulmonary tissues. 11 Lesions reported in the heart included a pale, friable and flabby myocardium (1), and petechial hemorrhages in the epicardium (l4). Microscopically, the cardiac muscle showed degenerative changes (1, 61), diffuse areas of fatty infiltration (61), and leptospires in silver-stained sec- tions (1). Hadlow g§_§l, (24) observed some macrophages loaded with hemosiderin in the wall of the uterus. The fetal membranes from abortion cases were light brown (17, 16, 61) and translucent (16, 61) with uniformly edematous (17, 16, 40, 61) intercotyledonary areas. The cotyledons were leathery (61), harsh to touch, dry (61), or moist, atonic (l7) and light fawn in color (60). Morter §t_§l,.(44) reported the pathological changes in the placenta after various number of days following ex- perimental L, pomona inoculation of pregnant heifers. Ten days after inoculation, there were hemorrhagic areas and hemolysis in the cotyledons. There were vascular and necrotic changes in the chorionic maternal junction 25-28 days after inoculation. By 47 days after inoculation, the fetal villi were absent and the normal relationship between the fetal and maternal tissue was absent. 12 t al, (61) in 1953 reported a dry and leath- Te Punga ery vaginal wall with concentric and longitudinal stria- tions mostly at the cervical end. They also found an edematous wall of the urinary bladder with congested small blood vessels in the mucosal surface. Awrorow §t_§1, (l) and Baker §t_§l, (2) found red-colored urine in the uri- nary bladder. In a thorough review of the literature only Awrorow (1) observed necrotic areas in the muzzle, gums, tongue and skin around the eye. He also reported catarrhal gastritis and edema in the subcutaneous and subserous tissues. The pathological findings in the aborted fetuses were nearly the same as the general lesions mentioned above ex- cept for the fact that the fetuses were usually aborted 24 hours after death (16, 61). This fact altered the pathological picture due to advanced postmortem changes. The adrenal glands of the aborted fetuses were congested, enlarged and abnormally colored (61). No lesions have been reported in the genital system of male cattle. MATERIALS AND METHODS (Nine healthy bulls were inoculated with Leptospira pomona (strain Ohio) and one bull was used as a control. Three of the animals were not inoculated by the author, but histological sections and experimental data from these three animals were evaluated and included in the results of this work. The microscopic agglutination-lysis test (43) showed that all animals were serologically negative for leptospiral antibodies prior to inoculation. The pro- cedures for inoculation and the necropsy schedule are out- lined in Table 1. Four methods were used for determining leptospiremia and leptospiruria in the experimental animals: 1. Guinea pig inoculation: Weanling guinea pigs were inoculated intraperitoneally with two ml. of diluted urine (one volume centrifuged urine added to one volume buffered saline) at three—day intervals starting from the second week after inoculation until the time of necropsy. At necropsy of each bull, pieces (about one cc.) of kidney, spleen and liver were pooled and homogenized in buffered ‘normal saline to make a 10 percent final suspension. Two m1. of this suspension were inoculated intraperitoneally into each of four weanling guinea pigs. All guinea pigs 13 14 were checked twice (at two and three weeks after inocula- tion) by the microscopic agglutination-lysis test for anti- body production against L, pomona. The bovine blood, urine or tissue suspension was considered to have contained leptospires if the serum from the corresponding guinea pigs contained antibody for L, pomona at a dilution of 102 or higher at two to three weeks after inoculation. 2. Dark-field examination: Daily examination of the urine and blood serum was done. 3. Medium inoculation: Four tubes of Stuart’s (Difco) media enriched with 10 percent rabbit serum were each in- oculated daily with three drops of blood from the experi- mental cattle starting with the first day of inoculation and continuing for 14 days. These tubes were incubated at 300 C. and checked weekly for leptospires by dark-field microscopy. They were not discarded until the sixth week. 4. Serological tests: The microscopic agglutination- lysis test was run on the bovine serum collected daily starting with the first day of inoculation. The antigen used was living L, pomona(strain Johnson) incubated at 300 C. for five to seven days in liquid Stuart's medium enriched with 10 percent rabbit serum. 15 Blood was collected daily from the jugular vein for at least a week before inoculation to establish the normal erythrocyte, leucocyte and differential leucocyte, hemo- globin and packed cell volume values (13). The nonprotein nitrogen values were determined once a week (31, 32, 63). The forementioned blood study was continued after inocula- tion until the necropsy of the cattle. Urine was collected weekly to check for albumin, blood, sugar, bile pigment, specific gravity, pH., ketone bodies and sediment. Rectal temperatures were recorded daily. The male cattle received 10-20 ml. chlorpromazine hyrochloride (25 mg. per cc. with benzyl alcohol 2 percent preservative), followed by 100-200 ml. pentobarbital- sodium (one grain per cc. in 10 percent propylene glycol and water) prior to necropsy. At the time of necropsy, tissues were saved from the kidneys, liver, spleen, eyes, adrenal glands, renal lymph nodes, testes, epididymides, prostate gland, urethra, urinary bladder, cardiac muscle, skeletal muscle, brain, spinal cord, thyroid gland and pituitary gland from each bull. These collected tissues were immediately placed in one or more of the following fixatives: Zenker's fluid, 16 10 percent neutral formalin solution, Carnoy's fluid and Bouin's fluid (41). The following staining methods were used: hematoxylin and eosin for general characteristics, Sudan IV and Oil- Red-O for fat, Best's carmine stain for glycogen and Warthin-Starry method for leptospires (41). This experiment was also designed to study semen from male cattle infected with L, pomona. A Plectron electric ejaculator was used to collect semen from bulls number I and 298. Semen tests were run for volume, color, sperm concentration, motility and morphology, live-dead count and the presence ofLL, pomona. However, animals were very sensitive to the stimulation and often went down in spite of reasonable means of restraint. Many of the semen samples were highly contaminated with urine and there was considerable struggle to obtain even these contaminated samples. For these reasons, this phase of the investiga- tion was discontinued. EXPERIMENTAL RESULTS The clinical, hematological, serological and patho- logical findings in the animals experimentally inoculated with L, pomona are reported with major emphasis on the pathological aspect. Clinical: All the animals showed good appetite and condition during the course of the experiment. The average daily temperatures are recorded in Table II. The tempera- ture range for the control animal was 100.10 to 102.0°F. Five animals had fevers of variable duration and with peaks ranging from 103.00 F. to 106.40 F. Two animals had normal temperatures (2647, I) while one had a subnormal tempera- ture of 96.00 F. on intermittent days (3247) (Table II). The fever was generally observed between the fourth and ninth days after inoculation. The urine analyses for albumin, blood, pH, sugar, specific gravity, bile pigment, ketone bodies and sediment showed no deviation from the normal values. Leptospires could be demonstrated by dark-field microscoPy in four ani- mals (3108, 3514, 3249, 3425) but were not seen in the others. Leptospiruria started between the 14th and the 23rd day after inoculation and continued until at least the 38th 17 18 day after inoculation in those animals in which it was ob- served. Urine from animals (3425, 3514) was tested for the presence of antibody against L, pomona and was found negative for 60 days after inoculation. Guinea pigs inocu- lated with the saline tissue suspension from the bovine kidney, spleen and liver (298,2647, 3247, 3268, 3249) failed to show any antibody response against L, pomona. Serological: Serum—antibody response against L, pomona was recognized (in the infected animals) on the 5th to the 12th day after inoculation. Maximum titers against L, pomona ranged from 102 to 108 (Table III). Two animals' (I,II) sera were negative for leptospiral antibodies through- out the test period. Hematological: Table IV shows the average (i) and the standard deviation (8) of hemoglobin, packed-cell volume, erythrocytes, leukocytes and blood nonprotein nitrogen (NPN) values before and after inoculation. The postinocula- tion values for the hemoglobin, packed cell volume, erythro— cytes and leucocytes showed a slight persistent fall in all the animals except bull 3247 which had a slight rise in the values for hemoglobin and packed cell volume while the erythrocyte values remained the same before and after in- oculation. The blood nonprotein nitrogen showed no devia- tion from normality. 19 Table V represents the arithmetic mean (i) and the standard deviation (5) of the differential leukocyte counts before and after inoculation in the bulls. The post-in- oculation absolute lymphocyte counts were raised in four animals (2647, I, 3514, 3248). The lymphocyte count went down in three animals (298, 3247, 3108). The values for the segmented neutrophils were slightly decreased in five bulls (I, II, 3425, 3514, 3248) and slightly elevated in four. The post-inoculation monocyte counts were Slightly raised in six animals while three animals had a slight fall (298, II, 3514). The post-inoculation eosinophil counts fell in seven animals and were slightly raised in the other two (2647, I). Leptospiremia was demonstrated in two animals (298, 3108). It started on the fourth, fifth and/or sixth day after inoculation. Its duration ranged up to at least two days. No leptospires could be isolated from the blood of the other animals. Pathological: The renal surface of the calf had in- numerable white small foci. These foci extended through the renal cortex and into the medulla. Two bulls (3425, 3514) had numerous whitish lesions similar to those found in the calf. The renal lymph nodes of the calf were 20 enlarged and edematous. The myocardium appeared flabby in one animal (3249). No other significant gross lesions were observed. Table VI summarizes the histopathological findings. Generally, the kidneys had interstitial cellular infiltra- tion mostly with lymphocytes, some plasma cells and a few neutrophils, macrophages and fibroblasts (Figs. 1, 2, 3, 4, 5, 6, and 7). Neutrophils were also seen inside the lumen of some renal tubules in the calf (Fig. 4). The tubules were distended with neutrophils. A few segments of the renal tubules in the area of the cellular infiltra- tion lost their normal architecture and resembled syncytial giant cells (Figs. 1 and 2). Some of the Bowman's capsules around and in the area of cellular infiltration were thick- ened (Fig. 3). The subcapsular spaces of the renal cor- puscles contained several pinkish rounded bodies varying in diameter from 10-30 u. Several renal tubules contained similar granular pinkish material. The tunica adventitia of the arcuate artery contained degenerated collagen which appeared as homogeneous dark-pinkish foci. The renal papillae of the calf had several dark—bluish foci resem- bling areas of calcification. A kidney section of the control bull had a focus of lymphocytes around the arcuate artery (Fig. 8). 21 The livers of all the experimental cattle, including the control, had a few lymphocytes and immature fibro- blasts in the interlobular connective tissue and in the areas of the portal triads (Figs. 9 and 10). The livers of two animals (3247, 3425) had a few small degenerative and necrotic foci (Figs. 11 and 12) and in some regions a hypertrophy of Kupffer cells. Collagen breakdown was evident in the interlobular connective tissue of the liver (2647, 3247, 3425). The gall bladder of one animal (3247) had follicles of lymphocytes (Fig. l3)in the lamina propria around the crypts. The testicular interstitial tissue in four animals (3247, 3425, 3514, 3108) had several foci of predominantly lymphocytic infiltration (Figs. l4, 16, 17, 18 and 19). In these areas tubular cells were in some instances under- going degeneration (Figs. 15 and 16). The rete testes in two animals (3514, 3108) had a few lymphocytes in the interstitial tissue. The intertubular connective tissue of the epididymides in two animals (3514, 3108) had lymphocytic infiltration (Fig. 19). This lesion in the calf (3108) contained both lymphocytes and neutrophils. 22 The seminal vesicles in two animals (3247, 3425) had extensive lymphocytic follicles in the lamina propria (Figs. 20 and 21). The connective tissue between the acini of the pros- tate gland of the calf had lymphocytes, eosinophils and collagen breakdown. Another animal (298) had only lym- phocytic infiltration in the interstitial tissue of the prostate gland. Microscopically the hearts of two animals (2647, 3249) had epicardial cellular infiltrations of mostly eosinophils and a few lymphocytes. Animals 3425 and 3249 had a few lymphocytes among the myocardial fibers. One animal (3247) had increased numbers of Anitschkow myocytes in the myocardium. The spleen was congested in all the animals including the control. The splenic red pulp contained numerous macrophages (3247, 3249), plasma cells (3249), eosinophils (3248, 3247) a few megakaryocytes and excess brownish pig- ments (298, 3249) both free and within the macrophages. There was also some edema (3425). The cerebrum in one animal (298) showed focal areas of malacia in the cortex. 23 The adrenal glands in two animals (2647, 3514) had a few lymphocytes and eosinophils in the zona glomerulosa and fasciculata (Figs. 22, 23, 24 and 25). There was early connective tissue proliferation in the capsule of the ad- renal gland in one animal (3249). The lymph nodes of five animals (3248, 2647, 3247, 3514, 3249) had eosinophilic infiltration both in the capsule and the pulp (Fig. 26). Edema and plasma cells were demonstrated in the lymph nodes of the calf (3108) (Fig. 27). The lymph nodes of only one animal (3247) had megakaryocytes (Fig. 28) and erythropoietic foci. Dark bluish foci resembling areas of calcification were in the lymph nodes of two animals (2647, 3108). Brownish pigments were seen in the lymph nodes of three animals (2647, 3247, 3249). Collagen breakdown, fibrin and neutrophils were observed in the lymph nodes of animal 2647. g No microscopic lesions were demonstrated in the vas deferens, bulbourethral glands, urethra, penis, urinary bladder, aorta, eyes, thalamus, brain stem, medulla ob- longata, spinal cord, thyroid gland or pituitary gland. Several incidental pathological findings were observed during this experiment. These findings included ciliated 24 protozoa in the pulmonary alveoli and bronchioles of two animals (3248, 3249) and plant material in the alveoli and bronchioles of one bull (3249) accompanied by exces- sive eosinophilic infiltration in the pulmonary tissue particularly around the small arterioles. The cardiac muscle of two animals (2647, 3249) had several sarcocysts as did the cremaster muscle in one (3247). DISCUSSION In a thorough review of the literature, no references to pathological changes in the genital system of bulls could be found. Tammemagi §L_§L, (60) could not find lesions in the testes of boars experimentally infected with L, pomona. Stoenner _L_§L, (58) reported orchitis during the convalescent period in humans naturally-infected with L, ballum. The present work demonstrated that the testes, rete testes, epididymides, seminal vesicles and prostate gland had microscopic lesions due to L, pomona infection. The testes of the calf had only a few lymphocytes in- filtrating between the seminiferous tubules (Fig. 14). The testes of three adult animals (3247, 3425, 3514) had mild to extensive intertubular lymphocytic infiltrations (Fig. 17). Several seminiferous tubules, in the areas of lymphocytic infiltrations, were undergoing vacuolization (Fig. 16). In analyzing these lesions, it seemed that the testicular lesions of the adult animals were more exten- sive than those seen in the calf. No clear-cut conclusion could be established because of the number of the animals used in the experiment. However, the factors which dif- ferentiate the adult animal from the immature one could 25 26 have some relation to the extent of the pathological le- sions seen in the testes. No leptospires could be demon- strated in silver-stained testicular sections but lepto- spires were isolated from the semen of two animals (3514, 3425). Leptospires were isolated from the semen beginning on the 15th to the 18th day after inoculation and contin- uing at least until the 38th day after inoculation. Leptospires isolated from the semen are most likely con- taminants from the urine but the occurrence of testicular lesions suggested the possibility of the presence of lepto- spires without urine contamination. That the leptospires in the semen of shedding animals are virulent enough to produce the disease in cows served by these animals was shown by Sleight gL_§L, (53). They demonstrated the pos- sibility of spread of L, pomona among cattle by shedder bulls through either natural coitus or artificial insem- ination. The significance of the previously mentioned testicular lesions needs more study. The importance of the epididymis in the bovine gen- ital tract is well established. In two of the animals (3514, 3108), there were a few scattered foci of lympho- cytes in the interstitial tissue as shown in Fig. 19. 27 It is interesting to note that the two animals which had this particular lesion were also the youngest (one year and eight weeks respectively) of all the experimental animals. The scope of this work did not cover the effect of these epididymal lesions on the semen- The seminal vesicles of two animals (3247, 3425) had lymphocytes in an arrangement resembling a follicle in the lamina propria (Figs. 21 and 22). This lymphocytic tissue was not seen inside the crypts. Whether this 'lesion was due to infection with L, pomona or whether this was an aberrant lymph follicle could not be determined. The effect of this tissue on the physiology of the sem- inal vesicle is not known. The prostate gland of two animals (298, 3514) had a few lymphocytes, eosinophils and collagen breakdown. These lesions in both the bovine male genital tract and accessory sex organs point up the need for more work in this area. No leptospires could be demonstrated in silver-impreg- nated sections from the genital system or in sections from the other tissues. This failure to demonstrate leptospires could be due to technical difficulties in the staining procedure. 28 The pathological changes seen in the other organs were, for the most part, as reported by previous workers. Macroscopically, lesions similar to the small white foci on the renal surface have been described by several investigators (2, 15, 28). Microscopically, intertubular and periglomerular lym- phocytic infiltration was present in the renal cortex and sometimes in the medulla. This finding is in agreement with the results of many other workers (2, 3, 14, 15, 24, 52). Only the calf had neutrophils distending the renal tubules. Many neutrophils were mixed with lymphocytes and a few eosinophils between the renal tubules. This type of cellular response was earlier reported by several authors (2, 3, 14, 15, 24, 52). The neutrophilic response of the calf could be a manifestation of a more severe inflammatory response in younger animals. Renal tubules in the area of the extensive cellular infiltration had degenerative changes and atrophy similar to that reported by earlier workers (1, 2, 14). In the middle of the cellular infil- tration, there were several syncytial giant cells (Figs. 1, 2) like those described by Hadlow §L_§L, (24) and Te t QL, (61). These giant cells could be the remnants Punga of destroyed renal tubules which coalesced to resemble 29 giant cells. Early fibrosis around the areas of cellular t EL- (46) . infiltration is in agreement with Ramirez Several renal tubules and Bowman's capsules contained granular pinkish material. This could be unabsorbed plasma protein in the glomerular filtrate (23). Similar t El: (2) inside granular material was reported by Baker the lumen of the renal tubules and Bowman's capsule. Leptospira-like structures in the renal tubules (l, 14, 15) could not be demonstrated in this present work. A few Bowman's capsules in the areas of cellular infiltra— tion had hyalinized thickened walls (Fig. 3). This is in agreement with several reports (24, 30, 50, 51, 56). Several Bowman's capsules were indistinct while the glomeruli showed increased cellularity similar to the t aL, (24) and Te Punga (61). description by Hadlow These changes made the glomeruli appear as collections of dark nuclei among the renal tubules. The renal papillae of the calf had a few dark-bluish focal areas. These are probably similar to areas of calcification reported by Dodd in leptospiral-infected calves (15). The kidney of the control animal had a focus of lymphocytic infil- tration beside the arcuate artery (Fig. 8). Whether this was normal lymphocytic tissue or whether an incidental 3O infection may have caused this lesion could not be determined. Macroscopically the liver was unchanged. Several in- vestigators reported enlargement (1, 14, 15, 60), fria- bility (1, l4), reddish—brown to distinct yellowish color (14) and necrotic areas particularly in the caudate lobes (59) related to acute leptospirosis. Microscopically, the liver of all animals had a few lymphocytes and early connective tissue proliferation in the areas of the hepatic trinity and interlobular connec- tive tissue (Figs. 9 and 10). Similar lesions were ear- lier reported in several publications (15, 28). The finding of this lesion in all bulls, including the control, tends to minimize the relationship of this lesion to the experimental infection. There were small foci of degen-' eration and necrosis in the hepatic parenchyma (Figs. 11 and 12). The liver in the present work did not show dis- torted, lysed or fragmented hepatic cells, leptospires inside the cells or sinusoids distended with blood in the necrotic areas as were reported by several investigators (15, 24, 28) who were dealing with acute cases of the disease. 31 Microscopically the gall bladder in one bull had lymphocytes in a follicular type arrangement in the lamina propria particularly around the crypts (Fig. 13). This finding could be normal according to Bailey (1958), who reported lymph nodules in the wall of the human gall bladder. The spleen was enlarged in all the animals. This was most likely due to the anesthesia used at the time of necropsy (54) and less likely due to leptospirosis. Sev— eral reports on acute bovine leptospirosis described en- gorged red pulp of the spleen (14, 24, 29). Microscopically, the spleen contained excessive num- bers of macrophages, plasma cells and eosinophils plus a few megakaryocytes. Considerable brownish pigment was both free and within the macrophages. Similar lesions were observed by earlier workers (14, 24, 29). The lymph nodes did not show definite gross lesions. Some previous reports indicated edema, hyperemia and pinkish or red color:hlcases of acute bovine leptospirosis (1, l4). Microscopically, some of the lymph nodes had various numbers of eosinophils, plasma cells, macrophages, mega- karyocytes and neutrophils. These microscopic findings 32 concur with the results in an earlier report (14). Sev- eral workers (11, 26, 29) observed leptospire-like struc- tures and lymphoid necrobiosis in the lymph nodes that could not be seen in the present experiment. These lymph nodes were edematous and had bluish foci similar to areas of calcification. These bluish areas may have been arti- facts since special stains for calcium were not run. Brownish pigments and a few erythropoietic centers were very likely related to hemolytic changes. The lungs did not show any gross pathological altera- tions. A few earlier reports included partial collapse of the lung (14, 24), subpleural hemorrhage (l) and pul- monary adhesions on the apical lobe. No specific microscopic lesions were present in lung sections examined. Other reports described alveolar emphysema (l4), edema (l, 14), thickened alveolar walls (25), little fibrin (14, 29), some neutrophils (l4), histiocytes, lymphocytes (24) and leptospires (1, 14) in the pulmonary tissue. Grossly, the heart of one animal was flabby with no other macroscopic lesions in the other bulls. Previous work by other investigators demonstrated friable, pale and flabby myocardium (l) and petechial hemorrhages in 33 the epicardium (l4). Microscopically the hearts of three animals (2647, 3514, 3249) had a few eosinophils and lym— phocytes particularly in the epicardium. Other previous reports indicated myocardial degeneration, diffuse areas of fatty infiltratiOn and leptospires in silver-stained sections of the myocardium (l, 60). Two animals (I, II) did not become infected as indi- cated by the negative agglutination-lysis tests (Table III). Leptospires were isolated from the inoculum used in the attempt to transmit the disease to the bulls. The reason why these animals did not become infected could be due to one or more of the following factors: 1. As reported by Hamdy §L_§l, (26) the bulls may have obtained immunity from their dams which were pre- viously infected. 2. The bulls may have had a high natural resistance to the infection. 3. Unknown nutritional factors may have inhibited infection. 4. Some virulence factors of the organism for the host may have been absent at the time of inoculation. 34 These two bulls were not necropsied because they were uninfected. Clinically, four infected animals (298, 3108, 3514, 3425) had fevers ranging from 102.5° F. to 106.40 F. and varying in duration from one day to four days. The start of this fever reaction varied from the third to the seventh day after inoculation. One animal (2047) did not show fever and animals 3247 had a subnormal temperature (960 F.) as shown in Table II. These variations in the degree of pyrexia, its day of start, duration and presence or ab- sence may be related to the virulence of the infective organism and the resistance of the host. As reported previously (2, 3, 15, 62), immature animals seem to be more susceptible to infection. The calf (3108) had the highest temperature (106.6o F.) with a long duration (four days). Of interest was the apparent relationship of route of inoculation to response. Bulls 3514 and 3425 were inoculated by both the intraperitoneal and subcut- aneous routes. These animals had higher fevers (103.0 and 105.00 F.) than bulls 2647 and 3247 which were inoculated subcutaneously only. The appetite and general condition of the animals were generally good during the course of the experiment. One 35 animal (3108) had depressed appetite during pyrexia. These findings are in concurrence with the experimental work done by Hamdy and Ferguson (26). Leptospiruria was recognized in four animals (3108, 3514, 3425, 3249). Leptospires were seen in the urine of the calf starting from the 23rd day after inoculation. This leptospiruria concurred with previous reports (19, 26) although, Fennestad t El: (18) could not demonstrate leptospires in the urine of 21 experimentally infected heifers. The inability to demonstrate leptospires in the urine of the infected bulls (298, 2647, 3247) could be due to the early appearance of leptospiral antibody in the urine but is more likely due to the failure of the organism to establish residence in the kidneys. Leptospiremia was detected in two animals (298, 3108). In one animal (298) leptospires were isolated from the blood cOllected on the morning of the fourth day after inocu- lation. No leptospires could be isolated from a blood sample collected in the evening of the same day or there- after. This was the first day of fever (103.2o F.) for this animal. Leptospires were isolated from the calf blood on the fifth and sixth days afixm'inoculation. Sev- eral other workers observed leptospiremia on the fourth 36 to eighth day after inoculation (l9,j19). Sera from seven animals (Table III) had positive micro- scopic agglutination-lysis test results with titers ranging from 101 to 108. The antibody responses started between the 5th and 12th days after inoculation. Similar data on leptospiral antibody response have been reported previously (10, 41, 50). The blood picture in general showed a slight decline in the hemoglobin concentration, packed-cell volume, eryth- rocytes, leukocytes (neutrophils and eosinophils) while both the lymphocytic and monocytic values were raised as shown in Table V. This rise in the mononuclears in the blood picture went hand in hand with the mononuclear infil- tration in various organs. The decline in the numbers of erythrocytes could be associated with the excessive brownish pigments in the spleen and lymph nodes whiCh indicated hemolysis. This hemolytic effect was demonstrated in lambs by Bauer _L_§L, (1961). SUMMARY AND CONCLUSIONS Experimental work was done on six male cattle and histopathological sections were analyzed from four addi- tional bulls to investigate the pathogenesis of L, pomona infection in these animals. The following were considered the most important findings: 1. The testes, rete testes, epididymides, seminal vesicles and prostate gland had areas of interstitial lymphocytic infiltration. This had not previously been reported. 2. The kidneys of three animals had variable numbers of small white foci on the cortical surface. 3. Microscopically, the renal lesions were similar to those previously described including particularly in- tertubularemd periglomerular lymphocytic infiltration (in addition to a few plasma cells, neutrophils and macro- phages) primarily in the cortex. 4. There were a few areascfifvacuolar degeneration in the renal tubules and glomeruli particularly in areas of cellular infiltration. 37 38 5. Clinical observations of the urine and nonprotein nitrogen indicated normal renal physiology. 6. Hematological studies indicated a slight drop in the hemoglobin, erythrocytes, total leukocyte count and packed cell volume with a rise in the relative values of lymphocytes and monocytes. 7. Leptospiremia (in two animals) was demonstrated on the fourth and fifth and/or sixth days after inoculation and leptospiruria was observed in four animals starting between the 14th and 23rd days after inoculation. 8. Serological and clinical findings were much as previously reported. 9. Generally the disease did not produce any definite clinical symptoms in the adult male cattle except temporary fever. Depressed appetite was observed in one animal (3108) during pyrexia. 39 Table I Material and Methods of Inoculation and Day of Necropsy Case No. Breed Age Inoculation Route Of Necropsy (Yrs.) Inoc. Day G 298 HF 3 13 ml. GP S/C PI 17 blood (1) G 2647 HF 2 12 m1. calf blood (1) S/C PI 24 G 3247 H 1.5 15 m1. calf urine (2) S/C PI 27 G 3248 1.0 3 ml. normal ham- S/C PI 90 ster blood. 12 HF Control ml. normal bo- vine blood (62 days later) I HF 2.0 8 m1. GP blood S/C not done (1) (AL was neg- ative up until PI day 25) II HF 1.5 3.0 m1 hamster S/C not done blood (AL was negative up to PI day 20) G 3108* HF 0.15 1 ml. hamster S/C PI 53 blood (1) G 3249* HF 1.5 5 ml. GP blood (1) S/C PI 400 F 3514* J 1.0 10 ml. GP blood s/c PI 57 (1) . 15 m1. GP blood (1) IP F 3425* HF 3.0 3 m1. GP blood IP PI 75 (l). '7 ml. ' GP blood (1) S/C Key for PI S/c IP GP 40 Table I (continued) Table I: = postinoculation subcutaneously = intraperitoneally = microscopic agglutination-lysis test = animals not infected by author, but tissues and data evaluated as part of this thesis = taken during leptospiremia = taken during leptospiruria = Hereford = Holstein-Friesian = Jersey = guinea pig 41 Table II Average Daily Temperature of the Experimental Bulls Case Numbers and Temperature Days 5:2; G 298 G2647 G3247 I II G3108 F3514 F3425 3248 8' .5 101.4 101.0 100.6 100.3 101.6 101.0 --- --- -—- Q) a: 1 101.4 101.5 100.6 100.2 101.6 100.8 --— --- —-- 2 100.6 102.1 100.6 100.4 101.7 101.0 102.0 —-— --— 3 101.6 102.1 101.4 100.0 101.4 103.0 --- --- -—— 4 101.8 103.2 100.6 98.0 101.0 101.2 --- —-— ——- 5 101.4 102.5 101.4 100.0 101.7 102.8 104.1 100.0 —-- 6 101.2 103.0 100.4 96.0 101.5 102.8 106.2 102.0 --- c 7 101.0 102.7 100.7 101.0 101.8 101.4 106.4 103.0 103.0 :3 8 102.0 102.5 100.4 101.0 101.6 101.4 103.2 103.0 104.0 .g 9 100.8 103.6 100.0 100.2 102.0 101.4 102.0 102.0 105.0 8 10 101.2 102.6 101.0 100.3 101.4 100.0 —-— 101.5 100.0 :3 11 101.8 102.0 101.2 101.0 101.6 101.0 --- ___ ___ § 12 100.8 102.1 100.0 100.0 101.8 101.0 -—- ___ ___ 13 101.8 102.0 100.4 96.0 101.6 101.6 --— --- ——- 14 102.0 102.0 100.4 100.0 101.6 100.0 --- --- --— 15 100.6 101.8 100.6 100.0 101.7 -—_ _-- -_- ___ 16 101.4 102.8 100.4 100.4 101.2 _-_ ___ ___ ___ 17 100.4 102.0 100.4 100.3 101.0 ___ _-_ -_- _-_ 18 100.8 102.0 100.5 100.0 101.3 ___ -__ _-- --- 19 100.1 --- 100.5 100.1 101.6 ___ ___ ___ --_ 42 Table III Antibody Titers* for L, pomona in Sera of Injected Bulls Case Number and Titer* Days After m l\ h m fl m Inoc. é'gg g g g H 3 iii 3 Ohm N N m H H m m m UULD (D (D (D (D In In 1 _ _ _ _ _ _ _ 2 _ _ _ _ _ - _ 3 _ _ _ _ _ _ 4 - _ _ _ _ - _ 5 — - 4 - - - - 6 - - 6 - - - - 7 - - 5 - - - - 8 - - 6 - - - - 9 - — 4 - - - - 5 10 - 3 5 1 - - 2 11 - 5 4 2 - - - 12 - 6 5 l - - 3 13 - 8 6 - — 6 14 - 7 8 - - 3 15 - 8 8 - - 16 - 8 7 - — 6 17 - 7 - - 7 7 23 - — - 7 * The titers are expressed as the exponents of the highest lO-fold serial serum dilution showing 50 percent agglutination-lysiS. 43 Table IV Mean (i) and Standard Deviation (8) of Hematological Values case Hb Hct. RBC WBC NPN No. G3248 AI § 13.85 § 43.14 E 10.07 Q = 8.15 E 29.85 (con- 6 12.19 6 t1.264 6 t1.272 6 = t1.004 6 t4.012 trol) PI § 13.18 § 39.73 i 7.46 i = 5.99 £ 29.07 6 t1.072 6 t2.429 6 12.91 6 = t1.24l 6 t4.449 G298 AI E 14.70 § 43.50 i 9.5 i = 6.90 i 32.00 6 to.lo 6 to.7 6 to.27 6 = to.15 6 t PI § 12.09 E 35.22 i 6.55 i = 6.54 § 37.00 6 12.88 6 t3.728 t1.236 6 = 1:1.326 6 t11.1 62647 AI 5 12.7 § 35.00 i 10.0 i = 10.0 E 26.00 6 to 6 to 6 to 6 = to 6 to PI i 11.61 § 32.92 i 6.97 Q = 9.28 § 28.50 t1.28 i2.837 6 11.241 6 = i1.288 6 $1.118 G3247 AI i 9.9 § 30.00 i 5.3 i = 10.05 i 32.0 6 to.35 6 to 6 to.82 6 = 10.813 6 to PI i 10.6 E 30.79 i 5.3 i = 9.0 § 32.0 6 10.906 6 £1,296 6 t0.85 = $0.643 6 to I AI E 16.92 § 47.25 i 9.82 Q = 8.07 i 34.8 6 10.92 6 £2,628 6 i0.64 6 = $1.445 6 i0 PI E 15.42 § 40.00 i 8.09 i = 6.71 E 28.35 6 t1.378 6 t3.8oo 6 to.745 6 = to.686 6 t5.949 44 Table IV (continued) case HB Hct. RBC WBC NPN No. II AI i = 12.98 i = 39.16 i = 7.8 i = 10.4 = 24.16 6 = £2.010 6 = £1,435 6 = £0.350 6 = £0.430 6 = £1.944 PI 2 = 11.93 i = 36.23 i = 6.9 i = 7.7 = 30.61 6 = £1,067 6 = £1.609 6 = £0.460 6 = £0.770 6 = £2.469 F3425 AI 2 = 13.62 x = 40.75 i = 9.76 i = 13.26 6 = £0.52 6 = £1.66 6 = £0.48 6 = £2.42 PI 2 = 13.11 i = 38.39 i = 9.09 2 = 8.05 6 = £1.039 6 = £2.55 6 = £0.63 6 = £1.51 F3514 AI R = 12.4 i = 28.0 i = .73 i = 9.05 6 = £1.13 6 = £0.0 6 = £0.48 6 = £0.64 pl i = 11.93 i = 35.37 i = 8.48 i = 8.75 6 = £3.07 6 = £2.22 6 = £1.85 6 = £2.21 G3108 AI R = 9.35 i = 28.25 i = 9.30 i = 11.47 6 = £0.20 6 = £0.35 6 = £0.43 6 = £0.81 pl i = 8.62 i = 26.07 i = 8.76 i = 11.36 6 = £1.61 6 = £4.23 6 = £1.68 6 = £3.31 Key for Table IV: Hb. Hct RBC WBC NPN AI P I hemoglobin in grams per 100 ml. blood. packed cell volume expressed as percent. erythrocytes in millions per cmm. total leukocytes in thousands per cmm. nonprotein nitrogen in mg. per 100 ml. ante-inoculation. postinoculation. blood. (n) f\) G) 45 Table V Mean (i) and Standard Deviation (6) of the Absolute Differential Leukocyte Count Case Neutrophils No. L E M s N G3248 AI § 77.57 i = 16.0 i = 0.0 i 5.71 § = 0.28 (Con- 6 18.00 6 = 16.928 6 = 10.00 6 12.289 6 = 10.00 tr°1) pl § 81.46 Q = 14.46 i = 0.69 i 3.00 i = 0.53 6 111.34 6 = 19.433 6 = 11.315 6 12.915 6 = 10.871 0298 AI § 58.0 i = 25.0 Q = 0.0 § 9.0 i = 7.00 6 10.0 6 = 10.0 6 = 10.0 6 10.0 6 = 10.0 pl i 51.5 i = 45.37 i = 0.0 i 1.75 i = 0.0 6 13.714 6 = 112.55 6 = 10.0 6 11.769 6 = 11.38 G2647 AI E 59.00 § = 30.50 i = 0.0 i 1.50 i = 0.0 6 15.056 6 = 12.906 6 = 10.0 6 12.012 6 = 10.0 pl § 56.46 i = 34.75 i = 1.70 § 11.55 i = 1.65 6 19.083 6 = 112.00 6 = 10.55 6 16.300 6 = 11.264 03747 AI § 36.66 i = 15.00 E = 0.66 E 46.66 i = 1.00 6 16.557 6 = 11.0 6 = 11.153 6 13.834 6 = 11.00 PI § 59.83 i = 22.11 i = 3.00 § 26.11 i = 2.11 6 17.733 6 = 17.238 6 = 12.061 6 110.50 0 = 11.378 l AI E 37.5 i = 45.5 i = 0.0 E 11.25 i = 2.75 6 18.584 6 = 116.55 6 = 10.0 6 17.931 6 = 112.60 pl E 18.37 i = 29.12 i = 0.0 § 11.37 i = 8.37 6 18.426 6 = 17.669 6 = 10.0 6 _15.282 6 = 13.376 46 Table V (continued) Case Neutrophils E M No. N II AI 2 = 90.0 i = 7.28 i = 0.70 i = 1.42 i = 0.85 6 = £2,449 6 = £2,868 6 = £1,253 6 = £0.9 6 = £0.686 PI i = 91.38 i = 7.23 i = 0.76 i = 0.76 i = 0.07 6 = £5,357 6 - £3,898 6 = £1.476 6 = £1,005 6 = £0.00 F3425 AI 1 = 44.7 i = 18.17 i = 1.0 i - 31.7 i = 1.41 6 = £14.2 6 = £7.83 6 = £1.11 6 = £18.5 6 = £1.00 pl i = 80.12 i = 11.37 i = 11.2 i = 5.0 i = 2.5 6 = £10.00 6 = £5.06 6 = £1.08 6 = £3.11 6 = £1.74 F3514 AI i = 72.0 i = 13.5 i = 1.0 i = 11.0 i = 2.50 6 = £17.0 6 = £77.3 6 = £0.0 6 = £6.29 6 = £0.71 pl i = 81.78 ' = 7.57 i = 1.0 i = 8.14 i = 1.42 6 = £9.62 = £5.17 6 = £0.78 6 = £4.15 6 = £1.99 03108 AI i= 74.0 i = 24.0 i = 0.0 i = 2.0 i = 0.0 6 = £00.0 6 = £0.0 6 = £0.o 6 = £0.0 6 = £0.0 pl i = 66.97 i = 78.84 i = 1.23 i = 1.92 i = 76 6 = £11.6 6 = £11.99 6 = £1.24 = £2.059 6 = £2.68 Key for Table V: AI P KIHEZUDF'H = ante-inoculation with leptospires. postinoculation with leptospires. lymphocytes (number of cells per cmm. segmental neutrophils per' cmm. non segmented neutrophils per- cmm. eosinophils per' cmm. = monocytes per' cmm. 47 Table VI Histopathological Findings in the Experimental Cattle Case Number and Finding No. Tissue and Lesion G3248 G298 G2647 G3247 F3425 F3514 G3108 G3249 Kidney: a. H-UHQ Him 0.016 Neutrophils Lymphocytes Plasma cells Macrophages Thick. Bow. Caps. Collag. breakdown Degeneration Early Conn. tissue Calcification Liver: a. b. c. d. e. f. Lymphocytes Collag. breakdown Degeneration Enlarged Kupffer cells Early conn. tissue Erythropoiesis Gall Bladder: a. Lymphocytes Seminal Vesicles: a. Lymphocytes Epididymis: a. b. Lymphocytes Neutrophils Testis: a. b. c. d. Lymphocytes Deg. tubules Sync. giant cells Thick. basement membrane + l++l| I + + + + + n + + + + l fi-+ + + + + + + + + + l + + + + + + + + + I + ++l+l| Table VI (continued) 48 NO. Tissue and Lesion Case Number and Finding G3248 G298 G2647 G3247 F3425 F3514 63108 G3249 10. ll. 12. 13. Prostate gland: a. Lymphocytes b. Eosinophils c. Collag. breakdown Cremaster muscle: Sarcosporidiosis Rete testis: a. Lymphocytes Cardiac muscle: a. Lymphocytes b. Eosinophils c. Anitch.myocytes d. Sarcosporidiosis Lung: a. Excess lymphocytes b. Eosinophils c. Ciliated protozoa d. Plant material e. Giant cells and macrophages Spleen: a. Macrophages b. Congestion c. Plasma cells d. Eosinophils e. Edema f. Excess pigment Cerebrum: a. Focal malacia l+l + + u + + + l + + + + + n + + + + + + + + + + + + 49 Table VI (continued) Case Number and Finding No. Tissue and Lesion G3248 G298 G26470 G3247 F3425 F3514 G3108 G3249 13. 15. Adrenal Gland: a. b. c. Lymphocytes Eosinophils Early conn. tissue in the capsule Lymph Node: a. b. c. d. e. f. g. h. i. j. k. Edema Eosinophils Plasma cells Macrophages MegakaryOcytes Neutrophils Fibrin Collag. breakdown Calcification Erythropoiesis Pigments + + + + l + l + I + + I + + + I Fig. 1. Section through the kidney. A. Destruction of the renal tubules. B. Area of lymphocytic infiltration. H. and E. Fig. 2 Section through the kidney. A. Lymphocytic infiltra- tion. B. Syncytial giant cells. C. Thickened Bowman's cap- sule. H. and E. X680 C O 1 .0. x Fig. 3. Section through the renal cortex. A. Vacuolar de- generation in tubules. B. Thickened Bowman's capsule. C. Lymphocytic infiltration. H. and E. X411 \ I ‘ .~‘s§¥ , 3 x , . - , N. Fig. 4. Section through kidney of calf. C. Distended tu- bules with neutrophils. D. Connective tissue proliferation. H. and E. X160 ' y“ ‘ i: ‘ 7" y ' D- . h ‘ - ’1 ~ 3 . A"' _ (4 l - A- -_ - ‘ Fig. 5. Section through the kidney to show lymphocytic infil- tration in medulla. H. and E. X411 ‘E‘..' ,J i \O I ‘. I",‘ - I Fig. 6. Section through calf kidney. E. Lymphocytes and neutrophils in interstitial tissue. F. Thickened Bowman's capsule. H. and E. X680. S. Minor calyx. T. Focus of lymphocytic infiltration. H. and E. x160 ~5"* 2' ' , i i. ‘I ;r. ".,.1 i .1: i i“ I“ _ «_ 1- ._ ~~ A “68!fi" 6211.1;1_.;_ '11 Fig. 8. Section through kidney of control bull. 0. Focus of lymphocytic infiltration. P. Arcuate artery. H. and E. X160 '. I 'i‘{ ‘ 3 ". 5’ gfl)‘ , I 4‘ . 4.....- o . Flg. . Section t roug t e lver. ‘. Lymphocytic infiltra- tion in hepatic trinity. B. Bile duct. H. and E. X411 '2.- - _ 3 .231, , ' w ‘j‘- Fig. 10. Section through liver. S. Lymphocytic infiltra- tion. T. Slight edema. U. Hepatic lobules. H. and E. X411 1g. ec 1on roug e lver. . ’y no 1c nuclei of hepatic cells. H. and E. X160 '- ‘ ‘ . 3f )5". pr 6‘}:- 1' tin" Fig. 12. Higher power of Fig. 11. K. Pyknotic nuclei of hepatic cells. H. and E. X680 56 Lymphocytic infiltration in lamina propria. T. Crypts. H. and E. X160 r. Fig. 14. Section through the testis of the calf. 2G. Scattered lymphocytes among seminiferous tubules. H. and E. X1700 57 Fig. 15. Section through seminiferous tubule. T. Syncytial giant cells in lumen of tubule. U. Thickened tubular base- ment membrane. H. and E. X680 ‘ I ' ‘ :0 u. I J. - c ._1 1;_ . l H;h__ .EZHQLi __v , ' Fig. 16. Section through the testis. E. Lymphocytic infiltra- tion among seminiferous tubules. F. Tubules with vacuolar de- generation. H. and E. X160 1 58 Lymphocytic infil- tration among seminiferous tubules not showing vacuolar de- generation. Section through the testis. D. 17. Fig. and E. X160 Ho Seminiferous tubules Lymphocytic infiltration. Section through testis. H. 18. Fig. showing vacuolar degeneration. I. and E. X411 H. 59 \5”. i C Section through epididymis. L. Lymphocytic infil- .\ Fig. 19. tration. H. and E. X411 Fig. 20. Section through seminal vesicle. D. Follicular type lymphocytic infiltration. E. Crypt. H. and E. X56 Fig. 21. High power *for LFig. 20 H. Crypt of seminal vesicle. I. Lymphocytic infiltration. H. and E. X160 Fig. 22. Section through adrenal gland. S. Lymphocytic and eosinophilic infiltration. T. Zona glomerulosa. H. and E. X160 \ Fig. 23. Higher power for Fig. 22. Q. Lymphocytes and eosinophils. H. and E. X680 Fig. 24. Section through“adrenal gland. T. Small focus of lymphocytic infiltration in zona fasciculata. H. and E. X411 Fig. 25. Higher power for Fig. 24. V. Lymphocytes and eosinophils. W. Zona fasciculata. H. and E. X680 {d Fig. 26. Section through a lymph node. E. 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