EXPERIMENTAL LEPTOSPIROSIS: THE EARLY PATHOGENESIS OF LEPTOXSPIRA [OMONAA INFECTION 2N YOUNG swm: Thesis for the Degree of M. 5. MICHIGAN STATE UNIVERSITY Stuart Duane Sleight 1959 J z " " 3‘ "“U'i‘ehg ' .4 “EDI. r“: . LIBRARY Michigan State University EXPERIMENTAL LEPTOSPIROSIS: THE EARLY PATHOGENESIS OF LEFTOSPIRA POMONA INFECTION IN YOUNG SWINE by STUART DUANE SLEIGHT 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 Veterinary Pathology I959 Stuart Duane Sleight ABSTRACT An experiment was conducted on the pathogenesis of _ experimental Leptosgira pomona infection in young swine dur- ing the l4 day period following infection. Serological re- sponses were followed using the agglutination-lysis test with E. gomona antigen. A comparison was made between pre- infection and post-infection hematoldgical values. Only minimal changes were observed. Clinical manifestations were slight with a transient febrile response being the only recognizable symptom. Pathological alterations were observed in the kidneys, renal lymph nodes, adrenal glands and brains of the infected animals. Gross kidney lesions consisted of grayish white foci on the cortical surface. The microscopic renal lesions consisted primarily of an intertubular leucocytic infiltra- tion with lymphocytes predominating. The renal lymph nodes were edematous. Small areas of lymphocytic infiltration were observed in the adrenal glands. Meningoencephalitis, characterized by perivascular lymphocytic infiltration, typified the brain lesions. The chronology and severity of the lesions were determined. ACKNOWLEDGMENTS I would like to express my sincere appreciation to Dr. R. F. Langham and Dr. R. L. Morter for their assistance and guidance during the course of this work. I also wish to thank Dr. C. C. Morrill and Dr. L. C. Ferguson for their counsel and for providing the facilities of their respective departments. I am grateful_to Mrs. Athalie Lundberg and Mr. John Kramer for their technical assistance. E “4—"- IV '—‘<‘ —1.‘* ”129’- “..- ‘ —_. TABLE CF CONTENTS REVIEW OF THE LITERATURE. MATERIALS AND METHODS EXPERIMENTAL RESULTS. . . DISCUSSION. . . SUMNIARV O C . O O O O O O TABLES I through 7. . . . FIGURES I through l8. REFERENCES. . . . . . . . LIST OF TABLES Average daily temperature5. . . . . . . . . . . . Period of highest temperature response. . . . . . Summary of hematological data . . . . . . . . . . Differential leucocyte counts . . . . . . . . . . Antibody titers for L. Eomona in sera of infected pigs. 0 o o o o o o o o o a 0 Summary of guinea pig inoculations with homo- genized tissues from infected pigs. . . . . . . . Pathological changes in L. pomona infected swine . iv Page I\) \fo l\) C\ I‘D C O Kidney of pig l3 days after infection with £0 EOmOna. Xé. O O O O O O O D O O O O O O I 0 Kidney of normal control. Xifi. . . . . . . . . Section through cortex of kidney at day A to show early infiltration of lymphocytes betveen tUDUIeSo X600. 0 I O O O O O O O O O O I O O 0 Same kidney as Figure 3 showing a more diffuse infiltration with heter0phils, SOme plasma cells, and numerous lymphocytes. x600. . . . . Kidney at day 8 demonstrating an extensive intertubular infiltration of leukocytes. x98 . Kidney at day 9 with marked leukocytic accumu- lation in the medulla. x98 . . . . . . . . . . Kidney at day 9 showing peri-renal-corpuscular infiltration with lymphocytes. x72C. . .'. . . Kidney at day 9 to show pyknotic nuclei in tubular cells. XSCO. . . . . . . . . . . . . . Kidney at day 9. A. HydrOpic degeneration of proximal convoluted tubule. B. Hyaline casts in lumen of tubuleS. C. Lymphocytes in inter- tubular areas. x600. . . . . . . . . . . . . . Kidney at day IA to show collagenous fibers in area of leukocytic infiltration. x320. . . . . Adrenal gland at day l2 to show lymphocytic focus in zona fasciculata. x600. . . . . . . . Edema of renal lymph node at day ll. x75 . . . Lymphocytic infiltration of meninges and cortex of brain at day ll. xlSO . . . . . . . . . . . Higher power of Figure l3. x65C. . . . . . . . Perivascular infiitration of lymphOCytes around small arteriole of cerebrum at day 2. x900. . Same as Figure I5 only different pig at day l2. X900. 0 O O O O O O O O D O O O O O O 0 O O O O Page k»! U] 36 \fl (T) \N \O Figure 17. LIST OF FIGURES (contd.) Page Ferivascular hemorrhage and lymphocytic in- filtration around vein in :erebrum at day l l O X6CC‘. 0 I O O O O O O I O O O I O O 0 O 0 0 5C Small areas of encephalomalacia in cerebrum at day l2. (Same pig as Figure l5). x606. . . . . 5‘ vi INTRODUCTION Leptospirosis caused by Leptospira 223222 is a disease of considerable economic importance and public health signifi- cance (BI, 36, 46, A9). The role of swine is important not only because of adverse effects in infected animals (6, 7, 9, l4, 20, 37, 43, #4), but also because swine are believed to be the greatest reservoir of the disease for other livestock as well as man (7, 22, 4i). The pathogenesis of g. gomona infections in feeder- type hogs was investigated by Morse 5;;gl. (4i), and the pathological alterations were described by Langham ££.2L- (30). It was not in the scope of their investigations to study the early pathological manifestations. Considerable interest was shown by those working on the leptospirosis project at Michigan State University in the chronological development of the early lesions in swine. While renal lesions had been reported as early as 7 days after infection in one animal (42), a controlled experiment on the pathogenesis of the lesions had not been undertaken. The purpose of this experiment was to study this phase of experimental leptospirosis in swine and, in addition, to make further hematological, bacteriological, serological, and clinical observations during the two-week period follow- ing infection. REVIEW OF THE LITERATURE The initial isolation of g. gomona was made in l936 from the blood of a dairy farmer living near Pomona, South Queensland, Australia (l2). Serological studies of this strain, later referred to as g, pomona (l5), differentiated it from the then known serotypes of leptospirae (24, 25, 32). Mochtar (34), in l940, isolated g, gomona from swine in Java, and Johnson (24) in Australia made porcine isolations in l942. Gsell (50), in l944, proved that "swineherds disease", known as a dissase of man since l933, is caused by g. gomona. He isolated the organism from swine as well as man. The first cultural isolation of L, gomona from swine in the United States was made by Gochenour (2|) in l952, al- though Monlux gt‘gl. (35) had demonstrated the presence of leptospirae in swine kidneys as early as l948. In l952, Bohl and Ferguson (5) isolated the organism from porcine urine and Bryan gt'gl. (8) isolated L, Eomona from aborted swine fetuses. Results of serological surveys indicate an incidence of from 3 to 25 per cent among the swine population and a distribution in all geographic sections of the United States (5. 7. 9. 23. 38, 45. 47). The U. S. Department of Agricul- ture has estimated the loss from leptospirosis in farm ani- mals at greater than lOO million dollars per year (49). While the greatest share of this loss is due to bovine in- fection, a significant loss occurs in swine, primarily from abortion and the birth of weak or unthrifty pigs (36). The 3 economic loss in feeder-type hogs is difficult to assess, but probably does not exceed l to 5 per cent of the value of an infected drove (36). Swine are apparently the natural host and reservoir of g, pomona infection for other animals as well as man (7, 22, 4|). Morse (4i) gives several reasons for this. First, a prolonged period of leptospiremia occurs, during which con- siderable numbers of leptospirae are present in the blood of essentially asymptomatic swine. Second, leptospirae are very numerous in porcine urine 20 to 30 days after infection, and contamination of feed, bedding, and water makes transmission possible. Third, hogs excrete‘g. gomona in their urine longer than do other animals. The ability of swine to transmit L, gomona infection to other hogs, cattle, sheep, and goats has been shown experi- mentally (lo, 39, 43). Human cases of leptospirosis in the United States have been traced to contact with infected swine urine as well as infected tissues (28). The public health . aspects of leptospirosis in animals have been reviewed by Larson (3|) and Reinhard (46). Clinical reports on the symptoms noted in naturally occurring outbreaks of leptospirosis in feeder-type hogs vary greatly. Many infections are asymptomatic or so slight as to go unnoticed (7, 47). Bryan (9) reported the following symptoms noted by practitioners: fever, anorexia, hemo- globinuria, icterus, and anemia. Observations reported in Wisconsin included encephalitis, hyperirritability, incoordination, and pyrexia (37). Bennett (47) has described a case with prominent convulsive and encephalitic symptoms. The most important and often only symptom of g. pomona infection in sows is abortion or the birth of weak and un- thrifty pigs (6, I4, 20, 37, 44). The number of infected sows in which abortion occurs has been reported to range from 20 to loo per cent (43). Abortion appears to depend on the stage of gestation at time of exposure, occurring mainly during the last three weeks of the gestation period (44). Sows which abort once have not been observed to abort again due to g. pomona infection (42, 44). Bryan (9) reported abnormal milk and reduced milk flow as an occasional symptom in sows. Workers in this country have found that pigs inocu- lated experimentally with L, pomona usually fail to develoo clinically recognizable symptoms (IO, 4i, 47). Morse gt‘gl. (4i) in studies of the pathogenesis of ‘L. gomona infections in swine found a leptospiremia of 3 to 9 days duration occurring in some animals until the tenth day following inoculation. Leptospiral antibodies were de- tected as early as day 7 and by day lo there were strong re- actions of 50 per cent agglutination or more at dilutions of l0“2 or l0‘3. Leptospiremia was not found to be terminated absolutely with the appearance of specific antibody. Morse postulates that this may be due to antibody neutralization due t°.lfl.lL12 lysis of large numbers of leptospirae. Maximum serum antibody response of i0"7 to lO‘8 usually “I o occurred 3 to 4 weeks following inoculation and diminished to lO'2 to l0"5 by postexposure month 4 or 5. Titers were found to persist as long as one year. Urinary antibody levels increased gradually to a maximum of IO”3 at three to four months. Leptospirae were not found in urine with antibody levels higher than lO” or with a reaction lower than pH 6.3. The greatest number of leptospirae were excreted in the urine during days 20 to 30 and the maximum duration of the renal carrier state was found to be l22 days. ’ Burnstein and Baker (l0) infected swine subcutaneously, intranasally, and by contact exposure. They observed pyrexia of 24 to 36 hours' duration 7 to l3 days following inoculation. Leptospiruria was observed as early as day l2 and as long as until day l59. The specific gravity of the urine was normal (l.025 - l.O40) until 3 weeks following inoculation at which time it became lower, usually less than l.OlO and remained low for as long as 5 months. Albumin was found in the urine concurrently with the lowering of the Specific gravity. Hematological studies made during the first two weeks of infection have revealed normal values with few exceptions (40, 47). Morse gt‘gl. (40) observed anisocytosis and reticu- locytosis in 2 pigs and a decrease in erythrocytes and hemo- globlin in another. Langham 3; El- (36) found that the two most important macroscopic changes in g. gomona infected pigs were scattered to numerous grayish white foci in the kidneys, extending in 6 some cases through the cortex into the medulla, and edema of the renal lymph nodes. Burnstein and Baker (lo) described similar kidney lesions which appeared no earlier than three weeks after pyrexia and progressively increased in number thereafter. As the duration of the infection progressed the kidneys appeared shrunken, with small bands of fibrous tissue extending into the parenchyma. In pigs killed during pyrexia 7 to l3 days following exposure, Burnstein and Baker (l0) observed microscopically an occasional focal area of "mononuclear" infiltration of the interstitial tissue of the kidney and areas of degenera- tion in the liver. Upon microsc0pic examination, the grayish white foci in the kidneys have been found to consist of an intertubular and perivascular infiltration of numerous lymphocytes, plasma cells and some macrophages (lo, 30). Langham £3.2L- (30) observed varying degrees of degeneration and necrosis in the proximal and distal convoluted tubules in the inflammatory areas. An intertubular proliferation of fibroblasts and increase of collagenous fibers, although not extensive, was also noted. Minor changes were occasionally observed in the renal corpuscles. The pigs in Langham's experiment were killed 35 to 2l4 days after exposure. Morter, Morse, and Langham (42) have found that the active inflammatory processes in the porcine kidney persist for as long as l0 to l4 months after initial infection and for 8 to lo months after leptospirae can be demonstrated in the urine. They also observed grossly a few grayish white foci and, upon microscopic examination, small areas of lymphocytic infiltration in kidneys of two swine killed 7 and l3 days after inoculation. MATERIALS AND METHODS Thirty, apparently healthy, crossbred pigs, 3 months of age, were used as experimental animals. By random number assignment, 24 of the pigs were divided into three groups of 8, and 6 were selected to serve as controls, each group be- ing assigned separate pens. The killing date for each ani- mal was determined by random numbers before inoculation. Two infected pigs were killed daily starting on post-infection day three. A control pig was killed on alternate days. All serological studies were performed by the modified microscopic agglutination-lysis test (l8, 40, 4|) using 5. ,Eomona (strain Johnson) live antigen. Two pre-infection serum samples per pig were obtained at 2| day intervals. No pre-infection antibody reactions were demonstrated. The animals were fed free choice a standard starter ration without antibiotics. Water was provided 39 libitum. No significant level of intestinal parasites was found on examination of representative fecal samples. To establish normal hemoglobin, packed cell volume, erythrocyte, leucocyte, differential leucocyte (l3), blood non-protein nitrogen (I3, 26, 27, 48), and blood creatinine levels (l3, i9, 20, 48), at least two blood samples were taken from each pig before inoculation. Two pre-inoculation rectal temperatures were taken on each pig. Post-inoculation temperatures were recorded daily. The 24 pigs to be infected were inoculated with 2 ml. of‘g. gomona (strain Ohio) infected cavian blood (40). Titra- tion using ten-fold serial dilutions and guinea pig inocu- lation established the presence of at least |O4 organisms per ml. (40). The strain oflg. pomona used was isolated from porcine urine by workers at the Ohio Agricultural Experi- ment Station, Wooster, Ohio, approximately two years ago and has been maintained in our laboratory by continual hamster or guinea-pig passage.’ The six control animals were inocu- lated with 2 ml. of non-infected cavian blood each. All animals were given sodium pentobarbital intra- venously to effect before necropsies were performed. Five consecutive daily blood samples for hematological studies were obtained from each infected pig killed after and including post-infection day 6, the fifth bleeding being made on the day of necropsy. As bleedings were started on post-infection day 2, those pigs killed on post-infection days 3, 4, and 5 were bled 2, 3, and 4 times reSpectively. Control animals were bled terminally. Modified microscopic agglutination-lysis tests for g. pomona antibodies were made on the terminal blood samples of all pigs killed up to and including post-infection day 6. On days 7 and 8, the ten samples taken for hematological studies were tested, and on day 9 and each day thereafter all remaining pigs were tested daily. Ten-fold serial dilu- tions were used to determine the end point of the reaction. On post-infection days 4 through 7, approximately lO O.l ml. of b|ood from each pig was inoculated into each of 3 tubes of modified Chang's medium (ll, 37). The cultures were incubated at 30°C. and examined for the presence of leptOSpirae by dark field microscopy at l4 and 28 days. Terminal urine samples were obtained from I? pigs and were clinically tested for albumin, occult blood, pH (4), specific gravity, and urine sediment (l3). Tissues were saved from the kidneys, liver, spleen, renal lymph nodes, heart, adrenals, skeletal muscle, brain, spinal cord, and pituitary gland of each pig. Immediately after necropsy all tissues were placed in one or more of the following fixatives: Zenker's fluid, l0 per cent neutral formal-saline solution, and Carnoy's fluid (32). The following staining procedures were used: hema- toxylin and eosin, Sudan IV for fat, Best's Carmine stain for glycogen, and Steiner and Steiner's method for spirochetes (33). Samples of both kidneys, liver, spleen and brain were obtained for guinea pig inoculation from each pig killed through post-infection day 8. Tissues were homogenized in 0.85 per cent sterile sodium chloride solution to give approximately a ten per cent tissue suspension (40). Three weanling guinea pigs were inoculated with 2 ml. kidney sus- pension, two with 2 ml. liver and spleen suspension, and two with 2 ml. of brain suspension for each pig killed. On post-infection day 9 and thereafter, kidney suspensions only were used and three guinea pigs were inoculated per pig. Two guinea pigs were inoculated with kidney suspension from each control animal. The original inoculum was con- sidered to have contained leptospirae if the serum of the guinea pigs contained antibody for g. pomona at a dilution of IO”2 or higher three to four weeks after inoculation (40, 4|). l2 EXPERIMENTAL RESULTS The only significant clinical symptom attributable to‘g. pomona infection was a transient febrile response. The average daily temperature recordings are summarized in Table l. Individuals showing febrile reSponse and the post-infection day on which the response occurred are given in Table 2. Sixteen infected animals had temperatures of lO4.0°F. or higher (Table 2) during the observation period with the highest reading being l05.6°F. No blood or blood pigments were visible in the urine. However, 8 of l7 urine specimens examined were positive to the occult blood test. Clinical examinations for urine albumin, pH, and sediment gave essentially normal results. Hematological data obtained during the course of the experiment is summarized in Table 3. Average normal values for the 6 control animals and pre-infection deter- minations for the 24 infected pigs are given. Daily post- infection averages for the pigs bled each day together with a comparison of normal values for these animals are also listed in Table 3. The average erythrocyte, packed cell volume, and blood non-protein nitrogen values did not significantly change after the animals were infected. Average hemoglobin determinations did not vary more than 0.6 gms. per lOO ml. except on day lO when there was a fail ofl.l5 gms. per IOO mi. from normal values. Leucocyte counts were subject to considerable indi- vidual variation. However, the average values indicated an initial slight leucocytosis of approximately 3,000 leucocytes per cu. mm. until day 4. Daily averages fell progressively until day 7 when a slight leucopenia of about 2400 leucocytes per cu. mm. below normal values was evident. The leucopenia was followed by a second rise until a slight leucocytosis again occurred by day 9 and lasted until day l4. One animal (3—7) showed a consistent marked leucocytosis on all 5 days that values were determined. As this animal was the only one showing this extremely elevated leucocyte count, leucocyte averages excluding 3-7 are given in Table 3. Blood creatinine levels did not vary markedly after infection except for 2 animals on day 7 which had levels of 2.5 mg. per lOO ml. Results of differential leucocyte counts are given in Table 4. In 5 animals a post-infection decrease in heterophils with a corresponding increase in lymphocytes occurred (Table 4). Significant serum titers appeared first on day 8. The maximal agglutination-lysis reactions that occurred were present in serum dilutions ranging from l0"'3 to l0"8 with‘g. pomona antigen. Table 5 summarizes the post-infec- tion agglutination-lysis test results. Terminal blood sam- ples from the 6 control animals were negative. Leptospira were demonstrated in tissues of the in- fected swine by inoculation of guinea pigs with homogenized i4 tissues (Table 6). These findings indicate thatlg. gomona was present in the kidneys of the infected swine from day 4 until the experiment was concluded, in the liver and/or spleen at least from day 4 until day 8, and in the brain tissue from day 6 at least until day 8. Serum from guinea pigs inoculated with homogenized kidney tissue from the control animals was negative indicating that g, pomona was not present in the non-infected swine. Macroscopic lesions were observed in the kidneys of all infected pigs killed on day 7 and thereafter. The kid- ney lesions consisted of grayish white areas or foci located principally in the cortex and occasionally in the medulla. The size of the lesions varied from the limit of visibility to approximately 5 mm. in diameter (Fig. l). An indication of the individuai variation in the severity and extent of the gross lesions is given in Table 7. The first kidney lesions were slight, with the edges of the affected areas being poorly defined and the lesion itself being slightly raised and swollen. However, by day 9, marked gross kidney lesions were observed in pig 4-l which had more than 50 per cent of the cortical surface affected. Moderate to marked lesions occurred in 8 of l2 infected pigs killed during the last 6 days of the experiment (Table 7). Edematous and enlarged renal lymph nodes were a con- sistent finding on day 9 and thereafter (Fig. i). There were no other gross lesions observed which could be attributed to infection with E. pomona, nor were any 15 significant gross lesions observed in the control animals (Fig. 2). In Table 7, an indication of the chronological appear- ance and extent of the microscopic lesions is given. The first renal lesions were observed in the cortex of pig 3-4 killed on day 4 (Figs. 3 and 4). The majority of these early lesions were rather discrete with infiltrations of small numbers of lymphocytes between the tubules (Fig. 3). An occasional lesion was rather diffuse with intertubular in- filtrations of heterophils, some plasma cells, and numerous lymphocytes (Fig. 4). Similar lesions to those described above with marked individual variations were observed in kidney sections from all animals killed on day 5 to day l4. Figure 5 shows an extensive intertubular infiltration of leukocytes in the kidney of animal 4-2 killed on day 8. Marked leucocytic accumulations were found in the medullary portion of the kidney of pig 4-l killed on day 9(Fig. 6). Occasionally, the infiltrations would surround the renal corpuscle (Fig. 7). A few degenerative changes were observed. An occasional tubule had pyknotic nuclei (Fig. 8). Hyaline casts were found in a few tubules (Fig. 9) and hydrooic degeneration of some proxi- mal convoluted tubules was evident (Fig. 9). A slight in- crease in collagenous fibers was observed in a few areas in the kidney of pig 4-6 killed on day l4 (Fig. l0). Areas of lymphocytic infiltration, not as extensive as the renal lesions, were observed in sections from the adrenal glands of 6 animals (Fig. ll). IR V Microscopic examination of the renal lymph nodes verified the grossly observed edematous condition (Fig. I2). Brain lesions, located principally in the cerebrum, consisted of meningoencephalitis (Figs. l3 & l4) character- ized by perivascular infiltration and hemorrhage (Figs. l5, l6 and l7) and microencephalomalacia (Fig. l8). Lymphocytes were the predominant inflammatory cell present. The brain lesions, though not extensive, were present in all animals killed after day II (Table 7). No lesions were observed in brain sections of any of the non-infected control animals. Upon examination of kidney sections taken from the control animals, an accumulation of lymphocytes in the medullary Portion, in close proximity to the hilus, was ob- served in 5 of 6 animals. These infiltrations were not seen in the cortical portion and the lymphocytes were less concentrated than similar lesions in the region of the hiluses of the infected animals. No leptOSpirae could be demonstrated in the Steiner and Steiner stained sections. Microscopic lesions were not observed in the liver, spleen, heart, sketetal muscle, or spinal cord of either the infected or control animals. ‘7 DISCUSSION Experimental L. pomona infection in this group of young swine was a very mild clinical infection with a slight febrile reaction being the only recognizable symptom. This concurs with previous experimental cases of L. pomona in- fection in swine reported in the United States (l0, 4|, 47). While naturally occurring infections in swine apparently may produce more severe symptoms such as marked anorexia, hemoglobinuria, icterus, anemia, incoordination and convul- sions, such outbreaks are typically asymptomatic (7, 9, 36, 46). The diversity in reported symptoms may be due to vari- tions in the virulence of the infecting organism or its serotype, host susceptibility, and the type of management practices. Factors which could be eXpected to increase host susceptibility were noticeably absent in this experimental group of animals. The animals were fed a balanced ration, water 39 llbitum, and housed in a heated building. No apparent parasitism or concurrent bacterial or viral infec- tions were present. While the animals were readily infected, godd husbandry practices may have been important in the mild- ness of the clinical infection. The febrile period in experimental L. gomona infec- tions in swine has been observed to occur from approximately the 4th to the 9th day after infection (l6, 4|). While the temperatures of the infected pigs in this experiment were not markedly elevated, the increases that were recorded gener- ally did occurduring this period (Table 2). l ,0 \J The slight leucopenia may be related to the concurrent pyrexia and leptospiremia. However, Ferguson and Powers (l6) observed a mild leucocytosis during pyrexia in a group of experimentally infected gilts. Morse EE.EL° (4|) observed no significant alterations in the leucocyte counts. While variations from normal values may occur during the febrile period, these changes are so subject to group and individual variation that they are of little diagnostic value. Terminal samples from 5 of the last l4 pigs killed showed an absolute and relative decrease in heterophils and an increase in lymphocytes. There did not appear to be any significant alterations in the differential leucocyte counts in those animals killed during the period considered to be the grexic phase with but two exceptions (4-l and 4-2). This contrasts with the findings of Ferguson and Powers (I6) in that changes they observed were mainly an absolute increase in heterophils during pyrexia. It would seem, in view of present knowledge, that unlike many infectious diseases, L. pomona infection in swine produces no predictable altera- tion in the differential leucocyte determinations. The absence of marked alterations in the erythrocyte, the packed cell volume, and the hemoglobin values may be due to the relative resistance of porcine erythrocytes to the hemolytic effects of‘L. pomona (l, 2, 3). Experimental work with hemolysins, which have been concentrated from the supernates of broth cultures of L. pomona, indicate that the ovine and bovine erythrocytes are highly susceptible to I“. la the hemolysins while the pig erythrocytes are relatively resistant (2). Hemoglobinuria, icterus, and anemia are common findings in experimental and natural ovine and bovine L. pomona infections (2, l7. 39). Such manifestations are rarely observed in swine. The infrequent occurrence and mild nature of the hemolytic features undoubtedly are among the factors which contribute to the usual asymptomatic course of L. pomona infection in immature swine. There was some evidence that a slight degree of hemo- lysls was taking place in the infected pigs. Urine samples were positive for occult blood in 8 instances, and on day l0 hemoglobin values were depressed l.l5 gms. per lOO mls. from normal determinations. It is possible that at the per- iod of the marked increase in antibody titers, which occurred from days 8 to lO, destruction of the leptospiral organisms liberated sufficient hemolysins to slightly alter the hemo- globin values (l7). The antibody responses of the infected pigs were similar to those in the experiment of Morse‘gL‘gL. Titers rose rapidly after day 7 to levels of l0"8 in some cases by day ii. The period of leptospiremia was not established in this experiment due to the contamination of media when making blood inoculations. It cannot be definitely stated that leptospiremia terminated with the appearance of antibody. However, the organisms were still present in liver, Spleen, and brain tissue of pigs killed on day 8 which indicates that viable organisms were present in these organs after the 20 appearance of circulating antibody. Unfortunately, due to a lack of guinea pigs, it could not be determined at what point the organisms were no longer present in liver, spleen, and brain and only present in the kidney. The characteristic grayish white foci in the kidney, which were Observed as early as day 7, have been described by many workers (l0, 30, 4|, 47). The significant features of the gross renal lesions were the early appearance and relative severity. Burnstein and Baker (l0) observed no gross lesions until approximately 3 weeks after pyrexia. Langham and Morter (42) observed small lesions in each of two pigs killed 7 and l3 days after infection. All of the infected animals in this experiment showed gross renal lesions on day 7 through day l4 and 8 pigs showed moderate to marked lesions. The early appearance of renal lesions in contrast to the report of Burnstein and Baker, may have been due to a strain difference in the infective organism. The lack of early marked lesions in Langham's and Morter's work was probably due to relatively few animals being observed prior to day l4. Even though the gross renal lesions were extensive, kidney function was evidently normal. Total blood non- protein nitrogen values and blood creatinine levels were in the normal range and albuminuria was not evident. The microscopic appearance of the kidney sections helped explain this lack of kidney malfunction. There were very few de- generative changes that could be observed. Occasional 2| pyknosis and a slight degree of hydropic degeneration were the only degenerative changes seen, even in areas of exten- sive infiltration. The termination of this experiment at l4 days did not enable the period of initial kidney malfunction to be deter- mined, but Burnstein and Baker (l0) previously observed that disturbances in renal function apparently begin to take place about 20 days after infection. The edematous renal lymph nodes, previously described by Langham gL‘gL. (30), appeared nearly concurrently with the gross renal lesions. This edema apparently may persist for long periods of time as Langham observed extensive edema in the lymph node of one pig killed I46 days after exposure. The edema in the renal lymph node is likely a response to the inflammatory reaction in the kidney and may aid in the localization of the organism. The appearance of microscopic lesions in the kidney on day 4 indicates that lesions develop very early,during the leptospiremic and pyrexic phase of the disease. While a small portion of the kidney was affected in the earliest lesions, by days 8 and 9 marked infiltrations of leucocytes, predominantly lymphocytes, were present (Figs. 5 & 6). This lymphocytic reaction during the acute phase of the disease and the persistence of this reaction during the chronic stage of leptospirosis (30, 42) may be due to a distinctive antigen-antibody relationship, the precise characteristics of which have not yet been determined. It is hoped that 22 fluorescent antibody techniques will help clarify this correla- tion between leptospira infection and lymphocytic infiltration. The lesions observed in the adrenal glands were very discrete and probably would cause very little functional dis- turbance in this organ. The brain lesions consisting of meningoencephalitis and characterized by perivascular infiltration and hemorrhage were similar to the lesions described by Morse 2; 3L. (39) in ovine leptospirosis and by Koppisch and Bond (29) in human cases. These porcine lesions were observed primarily in the cerebrum while the human and ovine lesions were seen throughout the brain. Nervous symptoms have been described in cases of leptospirosis in swine and this fact, together with the posi- tive animal inoculations from brain tissue, made logical the finding of these brain lesions. While the lesions in this group of pigs were not extensive, it is not difficult to visualize that with a multiplicity of the lesions observed nervous manifestations could appear. Lesions have been described in the liver of L. pomona infected swine by Burnstein and Baker (l0). The lesions consisted of "mononuclear" infiltration with areas of degenera- tion. Similar lesions were not seen in this group of pigs. It is probably that, although the liver may be slightly affected in some cases, the damage is usually not severe in L. pomona infection in swine. Carditis, splenitis. and myositis have been described 23 in human cases of leptospirosis (29) but lesions in these areas were not observed in this experiment. The inability to observe leptospirae in specially stained tissue sections emphasizes the need for improved techniques in this field. While the use of serological methods using laboratory animals is relatively satisfactory in the verification of infection, much more information could be obtained about the host-parasite relationship if improved methods for observation of the leptospirae in tissues could be developed. Fluorescent antibody techniques offer con- siderable promise and future research is being formulated along this line. 24 SUMMARY A controlled experiment using 30 pigs was conducted to study the pathogenesis of L. gomona infection in young swine during the l4 day period following inoculation with live organisms. Demonstrable microsc0pic renal lesions were present by day 4. These early lesions consisted primarily of an intertubular leucocytic infiltration with lymphocytes the predominant inflammatory cell. Only minor degenerative changes were observed, and kidney function was apparently normal. Gross kidney lesions, consisting of grayish white foci on the cortical surface, appeared on day 7 and were extensive by day 9. Microscopic brain lesions were present by day ii. The meningoencephalitis was characterized by perivascular lymphocytic infiltrations in the cerebrum. Hematological values were only slightly altered from normal determinations. Serological and clinical findings substantiated previous experimental evidence. Swine appear clinically to adapt quite readily to L. pomona infection and this further enhances their ability to serve as carrier animals. r 9) TABLE AVERAGE DAILY TEMPERATURES* f Controls Infected pigs Pre-infection l02.7 l02.8 Day l lO3.3 l03.2 Day 2 lO3.l -lO3.2 Day 3 lO3.3 IC3.2 Day 4 |O3.2 lO3.6 Day 5 lO3.0 lC3.5 Day 6 lO3.0 lO3.6 Day 7 lO2.S lC3.4 Day 8 lO3.2 lO3.6 Day 9 lO3.5 lO3.9 Day l0 lO3.l lO2.9 Day II lO2.8 lO3.2 *Temperatures are expressed ____- 4- in degrees Fahrenheit. K) C‘\ TABLE 2 PERIOD OF HIGHEST TEMPERATURE RESPONSE Pig Number Highest Temperature* .__ Day Recorded 2-2+ l-l 3-6 l-6 3-8 I no ear 2-9 3-3 4-4 l-7 4-5 l03.9 lO3.5 lO4.2 lO3.9 IO4.3 lO3.7 lO4.4 lO3.7 l05.6 l05.5 lO4.4 lO4.i lO4.6 l04.8 l04.6 l04.7 l04.3 lO4.4 lO4.4 l05.C NJ U‘l # 1? C\ U1 KN h (I) \O \O \I l\) 0\ U1 V & 8 *Temperatures are expressed **Highest temperature recorded. in degrees Fahrenheit. ll 27 KEY hemoglobin in grams per lOOml. blood packed cell volume exprESsed as per cent. erythrocytes in millions per cmm. leucocytes per cmm. non-protein nitrogen in mg. per l00 ml. blood creatinine in mg. per IOO ml. blood TABLE 3 SUMMARY OF HEMATOLOGICAL DATA AVERAGE VALUES Hgb Hct Rbc Wbc an Creat. Pre. Inoc. ll.78 39.25 6.57 20200 42.3 l.39 Controls ~ Post Inoc. l2.23 39.58 7.l2 620066 39.25 l.44 Infected Pre. Pigs égfggtéon ll.20 37.l9 6.57 l7944 40.86 l.36 Pre. Inf. ll.27 37.l0 6.49 l8l05 38.45 l.40 Day 2 Post Inf. ll.0 8.20 6.72 2l00 40.60 l.42 Pre. Inf. ll.27 37.l0 6.49 l8l05 38.45 l.40 Day 3 Boat Inf. ll.6O 6.20 6.86 2| 7. 0 Pre. Inf. IT.25 37. . l 235 39 5 . 3 Day 4 f . Lfiost Inf. l0.92 27.20 6.42 2|060 28.50 l.05 Pre. Inf. ll.l5 37.75 . O *l 250 37,.3 l.37 Day 5 Post Inf. ll.8O 26.60 6.64 *lTBEO 22.20 l.25 Pre. Inf. ll.l6 37.90 .5 *l 2 5 l. 7 l.35 Day 6 Post Inf. l0.46 56.20 6.52 *IZI40 42.40 l.46 Pre. Inf. ll.OO 37. 5 . *l7595 2.77 l.35 Day 7 _, Post. Inf. ll.O7 “37.l0 6.4 *l l 0 4 . 6 l.61_ Pre. Inf. l|.00 37.40 6.48 *l79l5 42.37 l.34 Day 8 Post Inf. l|g34 28.00 None *lZl80 42.22 l.25 Pre. Inf. IO.96 37.35 . 3 *l7 5 l.70 l.37 Day 9 figgt Inf. lO.9O 26.50 6.24 *l8580 2Z.28 |.58 Pre. Inf. ll.l6 3 .90 .7 I729O 2.00 l.33 Day l0 Post Inf. l0.0| 436.00 6.82 l8220 28.56 |.52 Pre. Inf. ll.l2 37.75 . 3 l7! 0 2.l0 l.3 Day ll “ Post Inf. lO.Zl 25.85 6.40 2l42O 436.86 l.25 Pre. Inf. ll.30 37.90 .79 l 300 4l.90 l.32 Day l2 . Vfiost Inf.. ||.7l .08 6. 208 0 44. 0 l.42 Pre. Inf. ll.67 38.25 6.87 l7933 33.25 l.29 Days T38: l4 Post. Inf. *Indicates that values for pig 3-7 were excluded. 29 m n we Tm m .Lcwnumui n lam m. m . o. mum 6 mm m. m _ .cc_usca I c .N w. w llw 0. Lee 0: _ m K Elm m hi CF. Tech I n m A. N: e m Aim .6 bell» low __ n CELL.“ m. om N w TN v _l¢# m. _m mm m n .Ccaumca 6 Ida, mm a m u \l. um Tom, em 6 o .cc_-mca o n mm mm m o w mu m am on m m .Cc_iocd as e as _m n n . .N m-m e .6 rmml m m .Lmoisca 3. MN w_ A N N DIM a mm .m m m .Cc_lmca . n mm mm m _ w on. m Km mm a _ .Lc_lmca e no om o. _ e mum m mm m. A. _ .cc_lmcu s mm mm A o In .l. Tm lem .lom m . c_ioL s .3 mml 2 mm m in e m mp, _ET L .wcmumca . s m .o _m 0. O ***o Ni . mm QMII IMI m a*.mc_loga o S 4 m a w pcaou mo moo LmnEsc m_m WHZDOQ mH>DOUDw4 4-I>\N\N +-+4-+-++-+-++ +-++ ++-+-++ +-++ +-+I I I \l + indicates that the serum of the guinea pigs contained anti- body for L. gomona at a dilution of IO"2 or higher three to four weeks 3 er Inoculation with the respective tissues of the infected pigs. indicates serum of guinea pig was negative. ommcceoEoc Lm_:omm>_cou n Id o>_mcmpxm n +++ co.zmuw..mc. Lm_:omm>_ood n no upmeoooz n ++I m m: cm. H . n + oz Sofia mo> oz oz mo> +++ + ¢. qu oz 2 mo> mm> oz 02 +++ ++ a. mI_ mm> 2 mo> mo> 02 oz +++ +++ n. mIm oo> zwmd mm> oz oz oz + + m. mI_ mo> Ho oo> oz oz oz ++ +++ m. mIq mwz Edna mo> oz oz oz + +++ w. NI. oz Effii 2; oz oz oz + + I I: mo> Ewan mm> oz oz oz ++ +++ __ MIn mm> oz mm> oz mm> oz ++ ++ o. mlm 02 oz mm> 02 02 oz ++ +++ 0. Law 0: . oz oz mo> oz mo> wo> +++ + m wIm oz 02 mm> oz wm> mm> +++ +++ m .Id 02 oz mm> 02 oz 02 +++ + m mi. oz oz oz oz oz oz + + w NI: oz oz oo> oz oz oz ++ + m nIm 02 oz 02 02 Oz 02 ++ + N wlm OZ 02 OZ 02 02 OZ IT I 0 0|— oz oz oz oz oz oz + I o oIn 02 OZ 02 OZ 02 02 + I m T..— oz oz oz oz oz oz + I m qu oz 02 oz 02 oz 02 + I a JIM oz oz oz oz oz oz I I o on oz oz oz oz oz oz I I m mIm oz oz oz oz oz oz - I I n «I. moooz zoco_x o__oo.om_ cos»; P: mefltu Izaak mo zoco_x c_ mco_mo4 co_woowc_ mcofifl mooBoI. .mcmm so msocmmmzoo 2.332 c_ 968 cozocuZIEH moon; Louis .382 £95 «Ema oomoococH flaccid $2.53» ozeaooosoz 30.5 930 .02 m: wzmzm omeummzH <2020u .4 z” muoz FIGURE l8. Small areas of encephalomalacia in cerebrum at day l2. (Same pig as Figure l5). x600. U'l l\.) REFERENCES Alexander, A. D., Smith, 0. H., Hiatt, C. W., and Gleiser, C. A.: Presence of hemolysin in cultures of pathogenic leptospires. Proc. Soc. Exptl. Biol. and Med., 2;, (l956): 205-2ll. Bauer, D. C.: Personal communication. Bauer, D. C., and Morse, E. v.: Variation and hemolysis production in relation to virulence of L tos ira pomona. Proc. Soc. Exptl. Biol. and Med., 98, (I95 : SOS-SOo. Berrier, H. H.: Diagnostic Aids in the Practice of Veterinary Medicine. St. Louis: C. w. Alban & Co., (I958). Bohl, E. H., and Ferguson, L. C.: Leptospirosis in domes- tic animals. J.A.V.M.A., l2l, (l952): 42l-428. Bohl, E. H., Powers, T. E., and Ferguson, L. C.: Abortion in swine associated with leptospirosis. J.A.V.M.A., I24, (l954): 262-264. ’ Bohl, E. H., Powers, T. C., and Ferguson, L. C.: Lepto- spirosis in swine. Proceedings, Fifty-Eighth Annual Meeting of the U. S. Livestock Sanitary Association, (1954): 365-367. Bryan, H. 8., Rhoades, H. E., and Willigan, D. A.: Studies on leptospirosis in domestic animals: II.Isolation of Le tos ira omona from aborted swine fetuses. Vet. Med., fig il9535: 438-439. Bryan, H. 8.: Studies on leptOSpirosis in domestic ani- mals. III. Incidence of leptOSpirosis in cattle and swine in Illinois. J.A.V.M.A., l24 (l954): 423-426. Burnstein, T., and Baker, J. A.: Leptospirosis in swine caused by Le tos ira gomona. J. Infect. Dis., 23 (l954): 53- .. Chang, S. L.: Studies on Le tos ira icterohemorrhagiae. J. Infect. Dis., QL, (l9z75: 2S-34. Clayton, G. E. E., Derrick, E. H., and Cilento, R. W.: The presence of leptospirosis in a mild type (seven- day fever) in Queensland. Med. Jl. Austr., L, No. l8, (I937): 647-654. Coffin, D. L.: Manual of Veterinary Clinical Pathology. Ithaca, New York: Comstock Publishing Co. Inc., l953. 14. '70 l8. I9. 20. 2'. 53 De Lay, P. D., Daley, R., Callahan, C.: LeptOSpirosis in relation to porcine abortion. Bull. Dept. of Agr" Calif-p E! (.955): '35. Derrick, E. H.: Le tos ira pomona. Med. Jl. Austr., I, No. I5, (l935%?'—%3TT Ferguson, L. C., and Powers, T. E.: EXperimental leptospirosis in pregnant swine. Am. J. Vet. Res., L1. (I956): 47l-477. Ferguson, L. C., Ramge, J. C., and Sanger, V. L.,: Experimental bovine leptospirosis. Am. J. Vet. Res., L2. (I957): 43-49. Folin: J. Biol. Chem. i1, (l9l4): A75. Folin and Wu: J. Biol. Chem. 2g, (l9|9): 8|. Gambrel, P. T., Gambrel, E. T., and Gambrel, L. E.: An outbreak of porcine leptospirosis. No. Am. Vet., 21! ('956): l9-2i- Gochenour, N. 8., Jr., Johnston, R. V., Yager, R. H., and Gochenour, W. S.. Porcine lethSpirOSIs. Am. J. Vet. Res., l2, (l952): IEC-lGC. ‘L‘ Gsell, O. R.: Epidemiology of the leptOSpiroses. Sym- posium on the leptCSpiroses. Army M. Serv. Graduate School, M. Sci. Pub. No. I, (l952): 34656. rosis in Hale, m. w.: Laboratory report on ospi 958): '95-'97. lept Georgia - l957. J.A.V.M.A., I22, (l Johnson, 0. fi.: The discovery of a fifth Australian type of leptospirosis. Med. Jl. Austr., (l942): 43l. Johnson, D. W., and Brown, H. E.: Mild leptOSpirosis in South Queensland. A classification of the infecting leptOSpira and a report of eight further cases of the disease. Med. Jl. Austr., i, No. l9, (l938): SOS-SI}. J. Am. Chem. Soc. 3g, (I924): 2066. J. Lab. & Clin. Med. l , (I929): I55. Klatskin, 3.: Leptospirosis. Yale J. of Bicl. and Med., g1, (I955): 243-266. KOppisch, E. and Bond, w. M.: The morbid anatomy of human leptospirosis: A report of thirteen fatal cases. Symposium on the leptospiroses. Army M. Serv. Graduate School, M Sci. PJb. No. I, (l952): SB-ICS. 3i. 40. 4|. Langham, R. F., Morse, E. V., and Morter, R.,L.: EXperi- mental leptospirosis. V. Pathology of Leptospira omona infection in swine. Am. J. Vet. Res., L9, I'9SO): 388-394. Larson, C. L.: The public health significance of the leptospiroses. Symposium on the leptospiroses. Army M. Serv. Grad. School, M. Sci. Publ., No. i (I952): I4-240 Lumley, G. F.: A serological investigation leading to the discovery of distinct serological groups of lepto- spirae causing leptospirosis as it occurs in Northern Queensland. Med. Jl. Austr., L, No. l8, (I937): 654-664. Methods for Medical Laboratory Technicians: U. 8. Govern- ment Printing Office, Washington, (l95I): 677-695 Mochtar, A. 0.: Over het voorkommen van Leptospirae bij varkens te Batavia. Geneesk. Tijdschr. v. Nederl. Indie., g, (I940): 2334-2339. Cited by Ferguson, L. C. Leptospirosis in swine. Proceedings, Fifty- Ninth Annual Meeting of the U. S. Livestock Sanitary Association, (l955): 332-336. Monlux, A. W., Siebold, H. R., Shalkop, W. T., and Davies, C. L.: Leptospirosis in hogs. No. Am. Vet., 22, (I952): 467-469. Morse, E. v.: The economic significance of leptOSpirosis in domestic animals. Proceedings, Ninety-Second Annual Meeting of the American Veterinary Medical Association, Minneapolis, August l5-l8, (I955): I62-l67. Morse, E. V., Allen, V., Krohn, A. F., and Hall, R.: LeptOSpirosis in Wisconsin. I. Epizootiology and clinical features. J.A.V.M.A., i 2, (I955): 4l7-42l. Morse, E. V., Allen, V., Krohn, A. F., and Hall, R.: Leptospirosis in Wisconsin. II. Serological studies. J.A.V.M.A., I 1, (I955): A24-426. Morse, E. V., Morter, R. L., Langham, R. F., Lundberg, A., and Ullrey, D. E.: Experimental ovine leptospirosis, Leptospira pomona infection. J. of Inf. Dis., lOl, I957 : :29-l3 o A Morse, E. V., and Langham, R. F.: Experimental lepto- spirosis. III. Caprine Legtosgira pomona infection. Am. J. Of VQto Res., 19, 3' : 3 - e Morse, E. V., Bauer, 0. C., Langham, R. F., Lang, R. w., and Ullrey, D. E.: Experimental leptospirosis. Iv. Pathogenesis of porcine Legtospira Q mona infections. Am. J. VEt. RESI, i2. ('95 : 3 - 9 o 47. 48. 50. VI Morter, R. L., Morse, E. V., and Langham, R. F.: Un- published data. Morter, R. L., and Morse, E. V.: Experimental lepto- Spirosis. II. The role of calves in the transmission of Leptospira pomona among cattle, swine, sheep, and goats. J.A.V.M.A.,ng§, (I956): 408-4l3. Powers, T. E., Bohl, E. H., and Ferguson, L. C.: Clinical studies on leptospirosis as a cause of abortion in swine. J.A.V.M.A., l29, (I956): 568-572. Reinhard, K. R.: Present knowledge and concepts of leptospirosis in farm animals. J.A.V.M.A., I22, ('953): 487-493. Reinhard, K. R.: Public health aspects of animal lepto- spiroses. Proceedings, Ninety-First Annual Meeting of the American Veterinary Medical Association, Seattle, Aug. 23-26. (I954): 455-460. Sippel, w. L.: Porcine leptospirosis. Iowa Vet., 25, Todd, J. C., and Sanford, A. H.: I94], Diagnosis by Laboratory Methods, Philadelphia, W. B. Saunders Co., pp. I99-292. U. S. Department of Agricultune: Losses in agriculture. A preliminary appraisal for review. A.R.S., U.S.D.A. (l954): I27-l59. Van Thiel, P Leiden, I . H.: The Leptospiroses. Univ. Pers. 948. Thesis “.3. Sleight, Stuart D. Axperimental lepto- spirosis: early patho- genesis...1n young swine. i IIHI' 69 4759 l I I'll ll 03 III I l 312 I , ,I I! III” l I III