. a .n 3‘ o F; .0 3. duo "- .Jr. 2 Arm .1 H___:__:_:_.:_,: a ’. f \ n - 3 {fir-1t .Li‘.“ . ’s .. \:Ig ., . V. ; n ‘ - g 7“ '. ‘~ I '9 a, Km «.7 4.3 WWW”WWW”!!!WWW 1293 00676 8539 PLACE IN RETURN BOX to ram ova this checkout from your record. OID FINES return on or before date due. DATE DUE DATE DUE DATE DUE “Hg. (1 3 1999’ “' 1 .1994 A? I! MSU Is An Affirmative Action/Equal Opportunity Institution EXPLRIMENTAL Leprosgggg PonoNA INFECTIONS OF LELR by James Alton Pay Leptospirosis is a major disease problem in domestic animals and is of importance as a disease of man. mild ani- mals have also been established as hosts for the disease and have many times been incriminated as the source of infection for both domestic animals and man. Serum agglutinins against Leptosnira nomona have been demonstrated in the sera of deer in several states in the United States. In order to learn more about L. oomona infection in deer, this study was undertaken. Six anparently normal female deer (Ddocoileus virgini- anug) were infected with L. Qomona strain Ohio by subcu- taneous inoculation with infected guinea pig blood. The 3 animals were studied to determine the clinical signs, tne antibody response both in the serum and urine, the duration of leptosniruria, the existence of leptospirae in body tis- sues and fluids, and the gross and microscooic pathology of the disease. The only clinical sign observed was a slight depression during the acute phase of the disease. The infection did not cause abortions in these animals. James Alton Pay Maximum serum antibody titers of 107 through 1010 were found. These titers gradually declined until postinocula- tion day 319, when they ranged from 102 through 104 in the surviving animals. Urine antibody levels were considerably lower than those of the serum. The maximum titer demon— strated was 104, which was found in one animal on postinocu- lation day 56. The duration of leptospiruria varied considerably from animal to animal, ranging from 27 to 56 days following inocu— lation. Leptospirae were demonstrated in the blood during the acute nhase of the disease and from the kidneys of the one animal sacrificed during the urinary shedding nhase of the disease. Pathologic changes were limited to the kidneys and were less extensive than those found in other species in- / fected with L. oomona. The microscopic alterations were occasional areas of leukocytic infiltration, primarily lymphocytes. These were located both periglomerularly and in the interstitial tissue surrounding the tubules. Vary- ing degrees of tubular degeneration were also found, mainly in the proximal convoluted tubules. EKPERIMhNTAL LEPTOSPIBA POMONA INFECTIONS IN LLER by James Alton Bay A THESIS Submitted to The College of Veterinary Medicine Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1961 hm” IS—ICIV) ["2 15-7 '5'" f I ACKNOWLEDGMENTS I wish to express my thanks to Dr. L. C. Ferguson and Dr. R. L. Mortar for their assistance and guidance during this study. I acknowledge and thank the Michigan Department of Con- servation which provided the animals and their maintenance. I would like to thank Dr. L. D. Fay, Mr. F. G. Youatt, and the biologists of the Roselake Vildlife Experiment Station for their parts in the maintenance, restraint and .post-mortem studies of the deer. Also I want to express my thanks to Dr. R. F. Langham for his suggestions and assistance with the histOpathologic aSpects of this problem. My sincere thanks goes to Dr. R. C. Balding for the time and suggestions he contributed. I am very grateful to Mrs. Athalie Lundberg for her technical assistance. Many thanks, too, to my wife, Nancy, for the hours spent typing, and most of all for her encouragement. ii. TABLE OF CONTLNTS INTRODUCTION LITERATURE REVIEW MATERIALS AND METHODS Experimental animals Inoculum Collection of specimens Clinical studies Bacteriologic studies Antibody detection Pathologic studies RESULTS Clinical findings Bacteriologic findings Antibodies detected Pathologic findings DISCUSSION SUMMARY REFERENCES 111. 12 12 13 14 14 15 16 18 18 18 21 TABLE 1. TABLE 2. TABLE 3. TABLE 4. The duration of leptospiremia in deer artificially infected with Leptosnira nomona as determined by inoculation of guinea pigs and artificial medium. The duration of leptospiruria in deer artificially infected with Leptosnira pomona as determined by inoculation of guinea pigs. The presence of leptospirae in various tissues and body fluids of deer arti— ficially infected with Lentospirg pgmgpa as determined by the inoculation of guinea pigs. Antibodies detected in serum and urine samples from deer artificially infected with Lentospira pomona as determined by the agglutination and7or lysis of living LeotOSpira pgmppg. iV. Page 19 20 F») U) aiid LIST OF FIGURES FIGURE 1. Kidney with periglomerular leukocytic accumulations. FIGURE 2. Kidney with leukocytic accumulations in the intertubular areas, degenerate epithelium of the proximal convoluted tubules, and hyaline casts in the lumen of the tubules. FIGURE 3. Kidney with iron deposits in the epithe— lium of the proximal convoluted tubules. 2a 29 INTRODUCTION Because of its importance in veterinary medicine and public health, leptospirosis has been studied extensively during the last few years. Morse (1955) estimated the losses in the livestock industry due to this disease at more than $112,000,000 annually. Other reports establish its veterinary significance and high incidence in animals (York, 1951; Reinhard, 1953; Galton £3 31., 1958). Leptospirosis is also a frequent disease of man through- out the world (Buckland and Stuart, 1945; Kirschner, 1954; Dvoskin and Hook, 1956; Seiler gt g1., 1956; Murphy and Alex— ander, 1957; Jellison e3 a1., 1958; Hale and Cathey, 1958). Since it is a zoonosis (Gsell, 1952; Steele, 1960) and trans- mission from man to man occurs infrequently if ever, the oc- currence of this disease in the livestock population is also significant from the public health standpoint. Galton (1959) reported a study on the source of 146 cases of human lepto- spirosis. Her findings were as follows: 1) 56 (38 per cent) had contact with infected cattle or swine. 2) 39 (26 per cent) had contact with presumably infected water. 3) 21 (14 per cent) had contact with dogs. 4) 19 (13 per cent) had contact with rats. 5) 6 (4 per cent) had contact with wild animals. 6) 5 (3 per cent) had contact with other animals or possibly contaminated environment. Epidemiologically, rats, dogs and swine have for years been considered the primary animal carriers of leptospirosis. l 2 Recent studies have shown various species of wildlife fre- quently to be infected. In many cases they have been direct- ly incriminated as the source of human and domestic animal infections. Deer have been suspected of contributing to the spread of leptospirosis. Various workers have found serologic evi- dence of leptospirosis in deer in the United States. More specifically, Fay and Youatt (1958) demonstrated antibody titers of 1:100 or higher for Leptospira pomona in 20 per cent of randomly selected deer sera. Since leptospirosis often causes abortions in domestic Species, it was postulated that the disease could adversely affect reproduction in deer herds. In order to obtain information regarding the effect of lep- tospirosis in deer and the potential epidemiologic importance of the infection in this species, a cooperative study between the Michigan Department of Conservation and Michigan State University was undertaken. In this study six presumably pregnant does were artificially infected with L. nomona (Ohio). Studies were made of clinical signs, antibody content of both the urine and serum, duration of leptospiruria, existence of leptospirae in the body tissues and fluids and gross and mi- croscopic pathology of the disease. LITERATURE nrvxaw The first recorded observation of pathogenic lepto- spirae was that of Stimson (1907) working in New Orleans. He observed the organisms in human kidney sections which were stained by Levadidi's silver impregnation method. His descriptions of the organisms closely fit those of lepto- spirae. Stimson named the organisms Spirgchaeta interrogans and thought they were the cause of yellow fever since yellow fever had been diagnosed as the cause of the patient's death. When his sections were reviewed by Sellards (1940), the photomicrographs showed organisms which are undoubtedly lep- tOSpirae. The first isolation of leptospirae from a human in the United States was reported by Wadsworth et a1. (1922). This infection was due to the accidental inoculation of a laboratory worker. Inada and Ido (1915) working in Japan, Hubener Reiter (1915) and Uhlenhuth and Fromme (1915) working independently in Germany are credited with the first isolations of patho- genic leptospirae from cases of Weil's disease. Noguchi (1917) described the morphology and studied the immunologic and serologic properties of strains of leptospirae isolated in Belgium, America, and Japan. He concluded that the strains were identical and named the organisms Leptospira icteroides. Noguchi (1919) isolated L. icteroides from a man suffering from "yellow fever" and also concluded that it was the cause of this disease. During the next 20 years, serotypes of leptospirae were 3 4 isolated in various areas of the world. Most significant to the study presented here was the isolation by Clayton gt 31. (1937) of a leptOSpiral species from the blood of a man suffering with an acute febrile condition. Serologic comparison of this strain to others isolated throughout the world proved that it was antigenically distinct (Lumley, 1937). Derrick (1942) suggested the name L. pomona for this organism, since it was initially recovered from a man in Pomona town- ship, Queensland, Australia. Until 1944 only L, igperohaemorrhagiae and L. canicola were thought to be present in North America.. The only ani- mals known to be infected on this continent were man, rat and dog (Reinhard, 1953). Junherr (1944), using silver im- pregnation techniques, observed leptospirae in sections of kidney, liver, and mesenteric lymph nodes of an ox. Lepto- spirae had previously been reported only once in the bovine. This report was made by the Russian workers, Muchin and Azinov (1935). I Baker and Little (1946) studied an atypical mastitis in cattle. After considerable study, including guinea pig in— fections and neutralization studies with convalescent sera, they decided that the condition was caused by a virus. Later (Baker and Little, 1948), when studying the same condi- tion in other cattle, they found a leptospiral Species to be the etiologic agent. Gochenour et a1. (1950) found this strain to be serologically indistinguishable from L. pgmgga. They thereby became the first to identify L. oomona as an 5 infecting serotype in the United States. Since 1950, naturally occurring L, pomona infections have been reported in the United States in the porcine (Gouchenour §§_gL., 1952; Bryan gt aL., 1953; Baker, 1954; Bohl gt aL., 1954; Ferguson gt gL., 1955), ovine (Beamer gt gL., 1953), equine (Roberts, 1958a) and canine (Murphy et al., 1958; Morter 2£.§l-: 1959), L, pomona is considered to be the major infecting leptospiral serotype of the bovine, porcine, ovine and equine in the United States. Until 1953, rats, dogs (Steel, 1960) and pigs (Gsell, 1952; Morse and Langham 1958) were considered the primary animal carriers of leptospirosis. Yager gt,§L. (1953), while investigating an enzootic area of bovine leptospirosis due to L. omona, isolated L. ballum from one of two Opossums and nine of 27 house mice (Mus Musculus) trapped in Virginia. These findings stimulated a renewed interest in the role of wild animal species other than rats as carriers of pathogenic leptospirae. Van der Hoeden (1957) reported a vole (Microtus arvalis) .to be the main natural reservoir for L, grippotyphosa in Israel. Previously cattle were incriminated in the role. He pointed out that the human case incidence rose when the vole population increased. He also isolated L. ballum from voles and reported the Jackal to be a natural host for L. canicola in Israel. In studies of leptospirosis in Denmark, Borg-Peterson and Fennestad (1956) isolated L. ppmona from 3 of 14 field 6 mice (Adonemus agararius). This rodent is found only on Lol- land Island and Falster Island. L. pomona agglutinins were found in 13 of 200 cattle sera and 5 of 138 swine sera from the animals of Lolland-Falster Islands, while none were found in sera from other parts of Denmark. They concluded that this incriminated this mouse as the principle carrier of L, pomona in Denmark. In a two-year study of leptospirosis in rodents com- pleted in 1955, Brown and Gorman (1960) reported L. ballum to frequently infect the house mouse (Mus muculus) in south- western Georgia. In the same area this serotype was isolated occasionally from the roof rat (Rattus rattgg), cotton rat (Sigmodon hispidis), and the field mouse (Peromyscus polio- pgtis). No other serotype of leptospira was found. Galton gt aL. (1956) isolated L, grippotyphosa from ra- coons. These animals were trapped within 50 miles of a Flori- da farm on which the cattle had serum agglutinins for L. ggip- pgtyphosa. From New Zealand, Webster (1957) reported the natural infection of the hedgehog (Erinaceug europaeus) with L, pomona. These animals were trapped on farms where leptospirosis had been diagnosed in cattle. He experimentally infected hedge- ‘hogs with the L, pomona strain isolated from this species and produced classical signs of leptospirosis. Death was ob- served in the young and half-grown animals, while pregnant animals aborted. Infected animals developed high serum ti- ters, and leptospiruria was demonstrated on the 16th to 18th day of infection. Van der Hoeden (1957) reported natural infections in hedgehogs in Israel. Serologic evidence of leptospirosis was found in 21 (33.3 per cent) of 63 animals tested. L. canicola was isolated nine times from ten animals sero- logically positive for this serotype. He also isolated L. ggippptynhosa once from five L. grippotyphosa serologic- ally positive animals. He reported the isolation of L, papir ggla and L, gripootyphosa from goats. He found a significant serum titer for L. sejroe in 1 of 59 bats (Hgssetus aegytiacus). From a study of various wild animal species, Galton 21 file (1957) reported the isolation of two leptospiral sero- types new to the United States. They isolated leptospirae of the L. mitis-hyos serogroup from four Opossums, and L. agg- tralis from two raccoons. Similarly, Roth and Knieriem (1958) reported finding natural L, pomona infection of one opossum. This animal was trapped in Louisiana. Leptospiral isolations from wild animal species in the United States were again reported by McKeever nggL. (1958). In the six month study ending March, 1956, kidneys from 846 wild animals (14 species) trapped in southwestern Georgia were studied bacteriologically. The incidence of infection was 5.4 per cent. L, pomona was isolated once from raccoons (Procyon lgtor), ten times from striped skunks (Menhitis mephitis), and two times from wildcats (Helis rafa). The incidence of leptospiral infection and the serotypes of the strains isolated from the various wild animal species stud- ied were as follows: 8 l) Opossum 6.1 per cent; L, ballum, L, mgpis-ngg group 2) Gray Fox 3.9 per cent; L, hallum* 3) Raccoon 3.8 per cent; L, ballpm? L, pgmgna, L. anagralis A, L. grippgtyohosaf L. Lewdomadi§* 4) Striped skunk 13.6 per cent; L, pomonaf L. Lallum* 5) Wildcat 7.5 per cent; L, ppmgnaf L. ballpm* *New host records. Tissues from the cottontail rabbit, marsh rabbit, fox squir- rel, red squirrel, feral dog, spotted skunk, otter and feral house cat were negative when cultured. In a survey of leptospirosis, the Ohio Department of Agriculture (1958) found serum agglutinins for L, pomona in Ohio wildlife. The agglutinins were found in the sera of 43 (19 per cent) of 224 deer, 16 (22 per cent) of 70 raccoons, 14 (25 per cent) of 55 foxes, and 6 (54 per cent) of 11 skunks. In contrast to these studies, Manges and Galton (1958) reported only six serologic indications of leptospiral in- fection and no isolations from 131 wild animals studied in Oregon. Significant titers for L, sejroe were found in 12 per cent of 173 cattle studied in this area. Baduieri (1958) reported the findings of studies of wild birds in northern Italy. Leptospirae were observed in the tissues of 12 of "hundreds" of wading birds obtained from Italian rice fields. He was able to isolate L, Lafigzigg from these tissues five times. These birds migrate from northern Italy to central Africa where L, bataviag infections have 9 Valso been found. He experimentally infected these wild birds with.L. pomgna, L. igterobaemorrhagiae, and L. bataviag and was able to reisolate the organisms for ten days from the blood and after 26 days from the feces. He was able to re- isolate leptospirae from the feces of artificially infected domestic ducks for 38 days. The serum antibody titers pro- duced by these birds were low and were present for a short period of time, sometimes shorter than the period of organism shedding. Chalquest (1937) induced serum antibody production in chickens, Hungarian partridges, Pekin ducks, and ringneck pheasants with L, pomona by contaminating their drinking water with urine from a cow shedding leptospirae. He was un- able to isolate the organism from the tissues of these birds or their excreta by injecting emulsions of tissue or feces intraperitoneally into tao week old chicks. Gillespie gg,§L. (1957) were able to detect serum anti- bodies for L. pomona in chickens. These chickens were ob- tained from a ranch where L. nomona had been isolated from cattle. L, Lygg was isolated by Ferris gt QL. (1959) from a deer mouse. Agglutinins for L, ggippotyposa were found in the sera of cattle from the same area. Reports of leptospirosis in deer have been few in num- ber and limited to serologic studies. This is probably due to the difficulties encountered in obtaining fresh urine or kidney tissue from these animals. Experimental infections 10 are also difficult to study due to the wild nature of this species; the animals often injure themselves during restraint and confinement. Wedman and Driver (1957) reported finding serum aggluti- nins for L, pomona in deer in Minnesota. They found an inci- dence of 16 per cent in 187 samples tested. In a study of Illinois deer, Ferris g3 al. (1958) re- ported finding serum agglutinins for L. pomona in 10.2 per cent of 343 samples tested. They also found titers for L. gripnotyphosa in 9.8 per cent of the samples. As indicated previously the Ohio Department of Agricul- ture (1958) reported finding serum antibodies for leptospirae in deer. Using an L, pomona antigen, they demonstrated anti- bodies in 43 (19 per cent) of 224 serum samples tested. The incidence of L, pomona antibodies found in the sera of Ohio deer is similar to that found in Michigan deer by Fay and Youatt (1958). In a survey of deer killed in 1957 and 1958 they found that 20 per cent (558) of the sera showed agglutination-lysis reactions at dilutions of 1:100 or higher. In contrast to these reports, Richardson (1958) stated that Delaware workers had failed to demonstrate evidence of leptospirosis in the deer of that state. In a survey of southwestern deer, Shotts §L_aL, (1958) found significant titers in only 1.73 per cent of 403 sera processed. Reynolds and Smith (1958) failed to demonstrate L, pomona antibodies in deer from Massachusetts. It should be noted that Shotts §L_§L. and Reynolds and Smith used the Stoener plate test 11 and the Stoener capillary tube test respectively. These tests are considered less sensitive than the agglutination- 1ysis test. MATERIALS AND METHODS Experimental animals. Eight apparently normal, adult, deer (Odgcoileus zirgipianps) served as the experimental ani- mals for this study. Six of the animals, females bearing Game Division ear tag numbers 129, 132, 147, 160, 853 and 864, were obtained from the deer herd at the Houghton Lake Wildlife Experiment Station. These animals had been natur- ally bred and were assumed to be pregnant. The seventh ani- mal, number 870, was a nonpregnant doe that had been held at the Rose Lake Wildlife Experiment Station for two years. The eighth animal, a male number 37101, served as a negative con— trol. All animals were negative by serologic tests for L. pomona (table 4).. During this study, the animals were main- tained by the Department of Conservation at the Rose Lake Wildlife Experiment Station in pens constructed for the con— finement of wild animals. These pens were made of eight-foot wire fence and were approximately 20 feet wide and 40 feet long. All animals were kept in individual pens excepting animals number 147 and 870, which shared a pen 40 feet by 40 feet in size. Each pen had a small shed which provided shel- ter. The animals were fed a ration of commercial deer pel- lets and shelled corn supplemented with hay. Inoculum. Leptospira pomona, strain Ohio, was used to infect does number 129, 132, 147, 160, 853 and 864 while ani- mal 870 was not infected. The leptospiral strain used was isolated from hog urine at the Ohio Agricultural Experiment Station in 1956. Following isolation, it was maintained by 12 .. 13 I , transfer every six months in Chang's semi-solid medium (Chang, 1947) containing 15 per cent sterile, normal rabbit serum. Before the deer inoculations were made, the Strain was passed four times in guinea~pigs by the intraperitoneal injection of guinea pig blood obtained at the height of febrile resnonse (105-106F). ‘Each doe inoculated received 4 ml of heparinized infect- ed guinea pig blood subcutaneously. Each ml of the inoculum contained approximately 5 x 101+ guinea pig infective doses (I.D.50) as determined by the intraperitoneal inoculation of guinea pigs with 10 fold dilutions of the blood in saline. Guinea pigs were considered infected if they developed anti- body titers of 103 or higher to L, pomona, strain Johnson, within 18 to 21 days following inoculation. Calculations were by the method of Reed and Muench as presented by Cun- ningham (1956). Collection of specimens. Blood samples were taken from the external jugular vein, using a California bleeding needle with an attached vial. Urine samples were obtained by cathe- terization with sterile, stainless steel female canine cathe- ters lubricated with sterile vaseline. A human vaginal speculum was used to retract the vaginal wall and expose the external urethral orifice. animal restraint was difficult and required the assis- tance of five biologists of the Rose Lake Station. Each ani- mal was caught with a large net and manually restrained in lateral recumbancy whenever injections were made or labora- tory specimens taken. 14 To establish the existence of leptospiremia, blood was collected from infected animals on postinoculation days four, six, eight and nine for bacteriologic studies. Serum for serologic studies was also collected on the same days. Serum and urine samples were collected from surviving, inoculated animals for the determination of antibody titers and lepto- spiruria on days 15, 21, 27, 32, 39, 49, 56, 67, 81 and 95 following inoculation. Serum samples for serologic studies were collected from animal 870 (uninfected penmate) at the same time. No samples were taken between day 95 and day 230 because of possible heat exhaustion of the deer due to re- straint in the summer months. Since leptospirae were not present in the urine samples obtained at day 95, only serum was collected at day 230. ‘Serum samples were obtained from all experimental animals at death. Samples for bacteriologic studies were obtained from animals number 129, 160, 853 and 864 when they were sacrificed. Sections for histopathologic examination were saved from animals number 129, 132, 160, 853, 864, 870 and 37101 during necropsy. QLinical studies. The infected animals were observed for clinical signs of illness such as anorexia, depression, icterus, fever, or lameness. Rectal temperatures were re- corded when specimens were obtained until 56 days following inoculation. Lacteriologic studies. Intraperitoneal inoculation of guinea pigs was used to determine the presence of leptospirae in blood, urine and tissue emulsions. Within five minutes of 15 the time the samples were obtained, two or three 150-250 gram guinea pigs were inoculated with 0.5- 3 ml urine or blood. To determine the presence of leptospirae in tissues 3 ml of a 10 per cent emulsion of the tissues in sterile saline was injected intraperitoneally into guinea pigs. Lep- tospirae were considered to be present if the guinea pig ser- um agglutinated and/or lysed L. pomona (Johnson) at dilutions of 1:1000 or higher 18-21 days following inoculation. Isolation of leptospirae from blood of each animal was also attempted by inoculating five tubes each containing 10 ml of Stuarts medium (Difco) with 10 per cent normal rabbit ser- um added. Each tube was inoculated with 3-5 drops of hepari- nized blood. The tubes of medium were incubated at 30C for 30 days or until growth was detected by the observation of leptospirae at 540x using dark field microscopy. Antibodygdetection. A modified agglutination-lysis test was used throughout this study to detect serum and urine, antibodies. Living 7414_day cultures of L. nomona, strain ' Johnson, groan in Stuarts medium with 10 per cent sterile, normal rabbit serum added, were used as antigen. Starting with serum or urine dilution of 1:5, ten fold serial dilutions were made with sterile saline in Kahn tubes. Two-tenths ml of each serum or urine dilution was then mixed with 0.2 ml of antigen resulting in final ten fold dilutions starting with 1:10. The mixtures were then incubated in a thermostatically controlled water bath for two hours at 37C. Agglutination and/or lysis of the leptospirae was determined microscopic- 16 cally at 100x magnification using a microscope equipped with an Abbe condenser which was fitted with a dark field stop. The end point was the highest dilution showing agglutination and/or lysis of 50 per cent or more of the leptOSpirae. Ti- ters are expressed as the number of reacting units per ml of serum or urine. Pathologic studies. A necropsy was conducted on each experimental animal. Animal number 870 and number 37101 were uninfected and served as controls. Immediately prior to sacrifice, the animals were anes— thetized with pentobarbital sodium injected intravenously. They were exsanguinated by cutting the external Jugular vein. Animals number 129, 160, 864 and 870 were sacrificed at postinoculation day 319. Animals number 132 and 853 were sacrificed at postinoculation days 117 and 22 respectively because of injuries sustained while being caught prior to re- straint. Animal 147 died on day 313 of unknown causes. Tis- sues were not saved from this animal for further studies be- cause of post-mortem decomposition. Sections of liver, kidneys, adrenals, spleen, brain and cervical spinal cord from animals 129, 160 and 864 were saved for histopathologic examination. In addition, renal lymph nodes were saved from animals 160 and 864. Brain, kidney, liver, spleen and cotyledons were saved from animal 853 and liver, kidney and spleen were saved from her fetus. Only kid— neys and cotyledons were saved from animal 132. Specimens for histopathologic examination were preserved in 10 per cent neu- 17 tral formalin-saline solution, Carnoy's fluid, or Zenker's fluid. Tissues for pathologic examination were embedded in paraffin and cut at a thickness of 6-8 microns. Stains util- ized were hemotoxylin and eosin for general histologic struc- ture or Prussian blue for iron pigments. RESULTS Clinical findings. Clinical manifestations of the di- sease were not pronounced even during its acute (leptospi- remic) phase. No anorexia, icterus or lameness was noticed. A slight depression was observed when samples were obtained during leptospiremia. ' The study of rectal temperatures obtained was inconclu- sive. Due to the activities of restraint, temperatures as high as 108.8 F were recorded. For this reason the body tem- peratures are not presented. No abortions were observed, however, only three of the animals were pregnant. Does number 129 and 147 gave birth to normal fawns with number 129 fawning twins. Number 853 was pregnant with a living, apparently normal fetus when she was sacrificed at day 22. Bacteriologic findingg. Leptospirae were demonstrated bacteriologically in the blood of all infected animals during the acute phase of the disease. The results presented in table 1 indicate that leptospiremia occurred in all animals on postinoculation days four, six and eight. On day nine" leptospirae were present in the blood of all animals except animal number 132. The data concerning leptospiruria are presented in table 2. It should be noted that the duration of leptospi- ruria varied considerably from animal to animal. Leptospirae were demonstrated in the urine of animals 129 and 147 until day 56. 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