STUNES ON CAN‘NE Lfi9YOSP‘IROSlS: L WALL} AflON. GP LABGMTQRY DMGNOSTZC. PROCEBURQ. it. SERGLCQZCAL aawwmmofl 9‘? THE mcmmfi C}? LATER? Eflfiflfififl ifl THE LANSWG ME‘CWGAN REA 13mm €20: m 3.299309 65% M. 3. MkQfiGAN STATE COLLEGE Jahis Porter Newman 19:33 V- I"? '. '1 I I " a TIHESIB ' ' 9P1M1 :l'i“:9l, Ill" ‘ , if \- . f I“ l A: t (1?: \..§: . .21; It) ‘ - ','l’ "I k 'i ‘ t . ; L ' w .1; R . lic‘uf‘l', _‘ ; ‘ K _ '1 l3 s .. h" I ll‘ t, f h \ J. .' l " ' ‘ :5 l ,. a l, E w ’17 - / “2-“ '. a; , 1" I I fi' TV : ,1 t , -' I l r s _' 4;!" l ‘ f ; k Lr , q ‘ ‘ E L I X J . e .111 z Tlns 1s to certify that the "it. .1 thesis entitled 1. Evaluation of "7 Studies on canine leptoapiroaie: laboratory diagnostic procedures. II. Serological deternination of the incidence of latent infection 1 in the Lansing lichigan area tn} ., y W - presented by W ‘ J. P. Kellen . 1: fig? ' has been accepted towards fulfillment of the requirements for l I __12_ Se _degree inJMQJ-OU Date 4 .t.‘ 7-5. x’ =6 ‘ r .}§!“IX“I1* W/fl . t;il J jor professor 4' f"; . September 2, 191.8_ H 7 .erfr “u; L_.,v ‘ Y , 1 .h" / r (.1, t f r ‘A J I :1 g. a . r STUDIE 0N CANINE MEWS: I. EVALIWI‘ION 01' LABORATGZY DIAGNOSTIC PROCEDURES. II. SEROLOGIGAL DWW OF Tm IMIDENCE 0? mm MOTION IN mm mm; MICEIGJLN AREA by Jam! row was arms Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree or msmorscrm Department of Bacteriology and Public Health 1948 II'HESW The author is grateful to Dr» EL 3. Etafseth, Professor and.Head of the Department of BacteriolOgy and Public Health, Michigan State College, whose efforts and understanding have made this work possible, and to Dr. W. N. Mack, Assistant Professor of Bacteriology, for his generous help and guidance in the writing of this thesis. 21788.1. TABLE OF CONTENTS Introduction00000O...OOOOIOOOOOOOOOOOOOOOOO0.00......O... Historical R°v1e'oeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Disease......................................o 891301081031 tests............................. mthOdB and Materialauuun........................... Preparation of.materials for.miscroscopic, cultural and ”mlogical Studieseeeeeeeeeeeeee Isolation of the organism..................... Preparation of antigens....................... Positive and negative sera.................... Agglutination test............................ Interpretation of the agglutination reactions. Results of Experiment 1................................. Results of Experiment 2................................. Discussion.............................................. WCOOOOOOOOO0.000000000000000.000000000000000000000 mtomtm CitedOOOOOOO0.0000......OOOCOOOOOOOOOOOOOOOOO 11 12 13 15 16 18 21 25 29 IDERODTBTION Prior to July, 1945, canine leptospirosis had only been clinically diagnosed in the Lansing, Michigan, area. At this time a dog dexnon- strating icterus was admitted to the Veterinary Clinic at Michigan State College. The animal died 12 hours after presentation. An au- tOpsy was performed, and the lesions were highly suggestive of lep- tospirosis. A guinea pig was inoculated intraperitoneally with the blood of the dog's heart and developed icterus and died 9 days after inoculation. Leptospirae were danonstrated in the kidney tissues by darkfield examimtion and sections stained by a silver impregnation method. Cultures were also obtained and serologically the organian appeared to be Leptogpira canicola. Because of the similarity in clinical symptoms of canine lepto- spirosis, dog distemper, black tongue, and various gastro-intestinal disturbances, the clinician is often unable to make a differential di- agnosis. As a result, the immediate instigation of specific therapeu- tic measures is not possible. The literature revealed that man laboratory procedures are recom- mended as aids in the diagnosis of leptospirosis. Hanover, an evalua- tion of them was not available. Experiment I was planned to evaluate the relative merits of the several recomended laboratory procedures for a rapid, accurate diagnosis of leptospirosis. Canine leptospirosis appeared to be more prevalent in the ensuing two years than previously. The epizootological possibility that many dogs in this area were excreting the organisms in their urine, thus serving as sources of infection to dogs and man, was considered of im- pomcee Meyer and coworkers (1939a) reported that sera obtained from ten dogs in the Detroit, Michigan, area were serologically positive to I": icterohaemorrham the causative agent of Weil's disease in man. Since that time no further studies in Michigan have been reported. To add to Meyer's data relative to the incidence of the disease in Michigan and to determine the incidence of latent canine lepto- spirosis in the Lansing, Michigan, area, Experiment 11 was planned. HISTORICAL REVIEW Disease Twanty-eight years after Wail described the disease in nan, which bears his name, Inada and coworkers (1916), who experimentally trans- mitted the disease to guinea pigs in 1913, isolated and cultured the causative agent, which they named gpirochaeta icterohaanorrhagiae. Unacquainted with the work of lands, Euebener and Reiter (cited by Kaleb-Sorgdrager, 1939) and Uhlenhuth and Frame (1915) transmitted the disease to guinea pigs and named the organism gpirpchaeta 9.9.9.293 and Spirochaeta icterognes, respectively. Ncguchi (1917) isolated the same organism from wild rats in New York City and, because of its fine minute windings and dissimilarity to other spirochetes, prOposed the name, Leptcspira icterohaamorrhagiae. That Weil's disease (leptospirosis) may occur in the canine has been suspected for some time. Krmbein and Frieling (1916) reported an unconfined case of an association of two humus with a case of ca- nine jaundice, later developing typical Neil's disease. Coumont and Durand (1917) demonstrated puppy (canine) susceptibility to Leptogira icterohaanorrhagiae by subcutaneous and intraperitoneal inoculation or . ingestion of infectious liver material, although Nicolle and Lebailly I (1918) were unsuccessful in their attempts. Perhaps the first to suspect that there was another species of Leptogpira which my infect dogs were Lukas (1924) and Krivacek (1924) , who demnstrated a spirochete in the tissues of dogs, which had died of canine typhus or Stuttgart's disease, and called it §pirochete melanoggnes canis. Okell, Dalling and Pugh (1925) proved that infectious jaundice, occurring naturally in dogs, was caused by a spirochete. They were able to isolate 3 strains of the organian, which corresponded in every way to _L_. icterohaemorrhagigg obtained from rate. It was suggested that rats transmitted the disease to dogs by contaminating the food and bedding with their body excretions and that the organian may be found in the urine of the dog as well as of area. The first to remain and describe the symptcns and pathology of canine leptospirosis, as caused by a new species, were narenbeek and Schuffner in 1931 (1933). The species is now known as _I_._. canicola. The strain isolated was agglutinated by specific serum to a titer of 1:10,000 and with serum specific for l... icterohaemorrhagigg to a titer of 1:25. The epidaniological importance of this discovery was empha- sized by Klarenbeek (1934) , who danonstrated that L. canicola may also cause infection in man. . ‘ IReports of the occurrence of canine leptospirosis in the United States have been comparatively recent. Jungherr (1957) appears to be the first to report the canine disease in the United States. He re- ported an' outbreak in a pointer kennel in which 41 of 42 dogs, less than 1 year of age died of the disease., Mloying Levaditi's silver stain, spirochetes were danonstrated in the liver, lung and kidney tissue, and the Lieberkuhun glands of the colon. Although guinea pig inoculations and subcultures were initially successful, the strain was eventually lost. The epizootological data indicated that the outbreak was caused by .1: icterohaemorrhagiae. However, the organim's virulence for the guinea pig was characteristic of Ir canicola. Meyer and coworkers (1938) were the first to isolate a. canicola from dogs in this country. Two cases of human canicola infection were also reported by these authors. Their darkfield examination of wild rat kidneys indicated that 33 per cent harbored the organim. Meyer and coworkers (1939a) published an excellent review of their studies on all aspects of the canine disease in 1939. The incidence of latent canine leptospirosis in Pennsylvania was reported by Raven (1941), whose results indicate that 28-38 per cent of the animals emined had apparently been infected at one time. Greene (1941) employed the macroscopic agglutination test and reported the re- sults of his survey conducted in southern California. Twenty-nine per cent of the dogs he examined were serologically positive to l... canicola and none to Er icterohawnorrhagiae. One of a hundred feline and 2 of 426 human specimens dunonstrated a positive reaction to _I_._. icterohae- morrlngiae; all were negative to 13: canicola. Elander and Perry (1940) and Bloan (1941) presented a study on 6 and 9 cases of canine leptospirosis, respectively. Bloom emphasized that only 5 of the 9 cases he observed danonstrated jaundice, and dis- cussed in detail his pathological findings. The first isolation of Ir icterohaanorrhagiae fran a canine in the United States was reported by Randall (1944). He anployed the golden hamster, following the recamnendations made by Morton (1942) that the golden hamster (Cricetus auratus) was the animal of choice for the iso- lation of am Serological tests The adhesion, complacent—fixation, and agglutination-lye“ tests have been deployed in the serological identification of Leptospira and diagnosis of infection. The application of these tests has greatly en- hanced the studies conducted in the past and provided a firm foundation for future observations. Laveran and Meanil (1901) demonstrated the fact that particles; such as, blood platelets or bacteria, becane adherent to trypanosomes or spirochetes in the presence of their own imune scrun. Brown and Davis (1927) described the uses of the thrombocytobrin or Rieckenberg reaction and proposed the name, ”adhesion test." The authors concluded from their experiments that _I_... icterohaanorrhpflg and .1: icteroids were identical and in addition they serologically examined 100 London wild rats. Brown (1935) demonstrated that the test could be employed in the serological diagnosis of Weil's disease in man. Noguchi (1920) was the first to carry out the complement-fixation test with Leptospira antigen and obtained positive results. He un- ployed the test in his comparative investigations between 3': icteroides and Ir. icterohaemorrhagiae strains. It was proven that a specific complement fixation in Ieil's disease was possible, although fixations were only achieved with 0.1 m1 imune sermn. Bessunans and Nelis (1928) tried the complwnent fixation test with Leptcspira antigen, which had been concentrated by centrifugation and suspended in physiological salt solution. Deploying this as antigen, they obtained positive fixation with 3 acre fran hman patients and 1 some from a dog with Weil's disease. They also dunonstrated that positive Wasserman-reacting sera gave negative complement-fixation reactions with Leptospira antigens. Pot and Dornick (1936) examined 100 human sera, employing the com- planent fixation and agglutination-hale test. Although the aggluti- nation lysis test consistently gave higher titers, the complement- fixntion test was recounnended as a reliable diagnostic procedure for Neil's disease. The effect of imune serum (convalescent serum from patients with Neil‘s disease) on cultured spirochetes was initially discussed by Ito and.latsu2ki (1916). They state that the sera had a "slight bacteri- cidal action." mrtin, Pettit, and Vaudremer (1917), who first em- ployed Leptogpira cultures in serological diagnosis, reported that serum.from.a patient with Weil's disease agglutinated gpirochetae icterohemorrhagiae in 1:500 dilution. Narmal and syphilitic sera were negative. Schuffner'and.Mbchtar (1927) gave a very thorough account of the agglutination-lysis reaction.and suggested the important point that lye sis of leptospirae takes place without the influence of complacent. They observed that agglutination takes place only in the low serum.di- lutions with lysis occurring in the higher dilutions. It was also ob- served that there will always be sane leptospirae which escape the lytic process in those dilutions where this takes place. The use of formalin-killed cultures in the agglutination test was reported by'walcheSorgdrager (1939). He observed that only agglutina- tion occurred when.the killed culture was employed. The titer obtained by this method was equal to that obtained uploying the living organ- ism.in the lysis test. The first macroscopic agglutination test was described and employed by Pot (1936), who examined sera fran 26 patients positive to Schuffner's agglutination test. Twentybfive of the 26 gave corresponding results; one was negative. finith and Tulloch (1937) reported on a macroscopic plate aggluti- nation test and grew the organism.in.8chuffner's media, using it in the live state. Having previously demonstrated that 55 C for 5-30 minutes and .1-5 per cent formalin altered the morphology of the or- ganism, the authors believed that this would be alleviated if the orb ganism.was first sensitized with its specific antibody. This proved to be true and, upon incubation of the serumrantigen dilutions at 3V and 55 C, floccules were easily observed with a hand lens and no dif- ficulty was experienced in interpreting the results. .All human and guinea pig sera examined gave almost identical reactions with the m- croscopic plate agglutination test as those observed with.8bhuffner's agglutination-lysis test. Brown (1939) employed the rocking slide macroscopic agglutination test and recommended it for use as a rapid presumptive serological test for Neil's disease. The rocking slide method and the macroscopic meth- od of Pot gave consistently identical results. Schuffner's technique, ‘which.was employed in conjunction with these two, gave titers three times higher. Starbuck and ward (1942) made a comparison of the macroscopic plate agglutination test and the standard microscopic agglutination test, employing 356 human sera. The L. ictemMmrngiae antigen for the macroscOpic test was supplied by Joyner and consisted of a cone centrated suspension of leptospirae, tO‘lhIOh gentian violet had been added. They reported that thezmicroscopic test was more accurate but believed the macroscopic plate test sufficiently specific and sensi- tive to be of value in the diagnosis of leptospiral Jaundice in hmnans. WOMAND MAME gemmtion of materials for microscopic, cultural, and serological studies Blood and urine, obtained from normal and Wig-infected dogs, were employed as the original materials on which microscopic, cultural, and serological examinations were conducted. The equipment employed in the collection of the above materials was sterilized by autoclaving at 15 lbs. pressure for 45 minutes or by dry heat at 180 C for 4 hours. Microscopic examinations conducted on stained sediment and dark- field preparations were made, employing the 101 ocular and 97X objec- tive. Observing aseptic precautions, 8-10 ml of blood was drawn from the radial or jugular vein of each dog with a sterile syringe and needle. The blood was then expelled into a sterile rubber-stoppered, 30 ml vaccine vial containing 8-12 glass beads and canpletely defibri- nated by shaking. The vials, containing the defibrinated blood, were then centrifu- galized at 1,600 rpm for 10 minutes to remove the blood cells. The serum was then removed aseptically with a sterile 5 ml pipette and ex- pelled into a second sterile vial and recentrifugalized at 2,800 rpm for 1 hour. The second centrifugation, the purpose of which is to throw down the leptospirae, was conducted only on the samples intended for microscOpic and cultural studies. The serum, unployed in the sero- logical studies, was placed in a third sterile vial, and 0.01 ml of the sediment was placed on each of 3 glass slides. One slide was 10 stained with Giemsa's stain, one with Pontana's, and the third used for darkfield examination. The urine specimens were collected by catheterization as aseptically as possible. The external genitaliae were cleansed with Roccal and care exercised in introducing the catheter to avoid contamination prior to insertion into the urethral opening. Wherever possible 20-30 ml were collected in sterile 30 m1 vaccine vials. Two centrifugation procedures were conducted on the urine speci- mens and 3 slides prepared for examination as was done with the blood sediments. The Giemsa staining procedure, as outlined by Stavitsky (1945), was conducted on one blood and one urine sediment slide from each dog. The stain was prepared by adding, to 50 ml of boiled distilled water, 2 drops of 0.5 per cent sodium bicarbonate, 1.5 ml methyl alcohol, and 1.25 ml of Giemsa stainl (stock solution). All nears were allowed to dry overnight, fixed in methyl alcohol for 3 minutes, and then stained with the prepared Giemsa stain for 12-16 hours. Positive slides were employed as controls in conjunction with each group of slides stained. In the Giansa preparations the organians stained reddi sh-viclet and the background, a lighter pinkish-blue. The urine and blood sediment slides, that were stained by Pontana's method, were also allowed to dry on the slide overnight. Fixation was accomplished by placing the slides in Ruge's acetic acid-formalin-water solution for 2 minutes. The staining procedure, employing Pontana's tannic acid mordant and silver nitrate solutions, was then completed and the preparations emined by microscopy. Positive slides were @- 1Nationel Analine and Chemical Co., Inc., New York, New York ployed as control. By this method the organises stain black against a light-brown background. Prepared slides of the centrifugalized blood and urine sediments were also examined by darkfield illumination. One-tenth milliliter of each sediment was placed on a clean glass slide and imediately covered with a No. 1, 22m square cover glass. Care was exercised to prevent the occurrence of air bubbles in the preparation, for they reflected the light, making the examination for Leptospira difficult. The edges were then sealed with paraffin to prevent evaporation, and a careful microscopic examination was nmde for 15 minutes, employing the 10X ocular and 971 objective. A second preparation for darkfield examina- tion was nede of all specimens in which suspicious leptospirae were observed. The organisms in these preparations appeared as bright sil- ver threads against a black background. Lerolation of the cm Schuffner's modification of Verwoort's median (cited by Meyer, 1939) was used exclusively in the cultural studies conducted on blood and urine sediments and the production of antigen. Nine milliliter amounts were dispensed in sense an culture tubes to which 1 ml of sterile, in- activated, rabbit serum was added prior to use. Rabbit serum known not to contain antileptospiral substances was used. The remaining blood and urine sediments were suspended in 2 m1 of Schuffner's medium (minus rabbit sermn). One milliliter amounts of this suspension were used to inoculate 2 tubes of Schuffner's medim. The proportions of sediment to media were 1:10-1:20 and yielded the best results by trial experiments. 12 All cultures were incubated at 30 C with periodic macroscopic and darkfield examinations performed to determine the presence or absence of growth. Three transfers were made at 7-day intervals to new cul- ture medium, regardless of the results of these examinations. All cul- tures were held for 4 weeks before being discarded as negative. Preparation of antijggng The antigens were made from cultures of 3': canicola and .]_'.._. 3.939.127 hasmorrhagiae, which were obtained through the courtesy of Dr. K. E. Meyer of the George Williams Hooper Foundation. To make it possible to employ freshly-prepared antigen, antigen production was continued throughout the experiment. It is necessary that a medium supporting heavy growth of lepto- spine, in an otherwise clear medium, free from cltsnps and particulate matter, be employed for antigen production. Trials were made with the various media as recommended by Noguchi and Battistini (1926), Korthof (1932), Starbuck and Ward (1942), and Chang (1947); however Schuffner's modification of Verwoort's medium was found to be far superior to the rest. The following slight modifications were made in Schuffner's medium, adapting it to our use: d 1. It was found that filtration. of the supernatant fluid through a No. 6 Seitz filter, employing a type ST discl, gave the same results as a Berld’eld filter of carifying porosity. 2. The growth of the Leptospira is dependent to a great de- IHercules Filter Corporation, Paterson, New Jersey 13 gree upon the pH of the medium. The original recommen- dations that the pH fall in the 6.8-7.2 range allows too wide a pH variation. A pH of 7.15-7.25 was found to be more conducive to growth of both species of Leptogira. 3. The medium was tubed and autoclaved inmediately. 4. Inactivated rabbit serun was added to the mediun prior to use. For antigen production 0.5 ml of each stock culture was pipetted into a number of culture tubes containing 10 m1 of Schuffner's median. Inoculated tubes were incubated at 30 C for 4-5 days. At this time the organisms were still young and vigorously growing. Macroscopic and microscopic examinations were made of all antigen tubes to check growth, morphology, and the presence of bacterial contamination. All tubes, in which atypical morphology, poor growth, or in which bacterial I contamimtion was observed, were discarded. The antigens unployed in the agglutination test were prepared by addim 0.15 per cent neutral formalin to each tube. The tubes were then centrifugalized for 10 minutes at 1,600 rm to remove amr clumps of organisms which might interfere with the interpretation of the ag- glutination reaction. The sensitivity of the antigen was checked by employing known neg- ative and positive sera of varying titers. Those antigen suspensions yielding satisfactory results were then stored in the refrigerator. Min and pggative sera Positive and negative inactivated rabbit sera were used as controls in the agglutination test. Negative sera were also added to Bchuffner's median, 10 per cent by volumeJHto enhance growth. 14 The negative control sera were obtained from normal rabbits and examined by the agglutination test for the presence of agglutinins. The negative sera to be added to Schuffner's median were examined by cultural methods in addition to the agglutination test. The cultural examination consisted of adding 1 ml of each serum to be tested to each of 4 tubes of culture media. Two tubes were in- oculated with .1: canicola and 2 with $9 icterohaanorrhggigg, as out- lined in the procedure for antigen pro‘duction. Microscopic darkfield counts were made on the first, third, and fifth days of incubation and all tubes examined macroscopically and microscopically at the end of the incubation period. Any negative serum demonstrating inhibition of growth or variability in morphology was discarded. Positive control sera were produced according to the following procedure. Vaccines were prepared from 6-dayoold cultures of each spe- cies, to which 0.15 per cent neutral fonnalin was added to fill the or- ganians. Three z-ml. injections of the killed preparations were given intravenously into the marginal ear vein of a rabbit every 5th day. A final injection, consisting of 2 ml of the live culture, was administered on the 15th day. Antibody production in the rabbits took place with almost autanatic regularity. 0n the 5—6th day after the first injection the titer was approximately 1:400; on the 12th day the titer was 1:3,000; on the 18th day the titer was 1:6,000-12,000; on the 24th-30th day the titer was l:24,000-30,000. The titer will fall gradually (if additional injections are not given), and in the case of the animals examined, the titer after 5 months was down to 1:300-400. Although individual rabbits will vary somewhat to antigenic response, 7 of the 8 rabbits employed for positive l5 sera production gave the typical reaction as outlined above. "Booster shots” were administered periodically, usually consisting of a 2-ml injection of the fonnalin-killed culture and a 2-ml injection of the living culture at weekly intervals. It might be of interest to point out that the first sign of an- tibody in the serum does not appear until the 4th day after the first 2 ml of vaccine is administered. 0n the 4th day the agglutination re- action is negative, on the 5th day agglutination can be detected in dilutions of 1:100-500. All positive and negative control sera were filtered through ger- micidal discsl, using a No. :5 Beitz filter, inactivated at 55 c for so minutes prior to storage in the refrigerator. It was noted that the titer of the positive sera dropped during storage; therefore fresh bleedings were made every month. wimtion test The agglutination test, as outlined by Schuffner and Mochtar (1927) , was conducted on all sera examined. The macroscopic plate test, as re- commended by Brown (1939), was used in trial experiments and found not to be as sensitive as the microscOpic test originally devised by Schuf- fner. Serum dilutions of 1:5, 1:50, 1:500, and 1:5,000 were made in 121 75 mm tubes according to the following table: Tubefi 1 2 3 4 Buffer" 1.2 ml 0.9 ml 0.9 ml 0.9 ml 861'“ 0e3 1111*" Del m1** 0e]. m1** Del 1111 Dilution 1:5 1:50 1:500 1:5,000 118 rabbit serum *The buffer consisted of Schuffner's medium min 2 to 3, and 3 to 4. M0.1 m1 is transferred fran tube 1 to 2, lCentral scientific Company, Chicago, Illinois 16 Serum-antigen dilutions were also made in 12x75 mm tubes in du- plicate sets, employing .11' canicola as the antigen in one set and _1_... icterohaanorrhagiae in the other according to the following tables 3° germ Ant iggn Buffer Dilution 1 0.15 ml of 1:5 0.15 ml 0 1:10 2 0.15 ml of 1:5 0.15 ml 0.10 ml 1:30 3 0.15 ml of 1:50 0.15 ml 0 1:100 4 0.15 ml of 1:50 0.15 ml 0.10 ml 1:500 5 0.15 ml of 1:500 0.15 ml 0 1:1,000 0 0.15 ml of 1:500 0.15 ml 0.10 ml 1:3,000 '7 0.15 ml of 1:5,000 0.15 ml 0 1:10.000 8 0.15 ml of 1:5,000 : 0.15 ml 0.10 ml 1:30.000 9 None 0.15 ml 0.15 ml Control“ *One set of each positive and negative control sera was set up with each series of tests conducted. After the serum-antigen dilution tubes were shaken for 5 minutes and incubated in a water! bath for 3 hours at 3'7 0, l loopful of each dilution was placed on a clean glass slide and covered with a No. 1, 22mm square, cover glass. All slides were examined by darkfield il- lmnination, employing the 10X ocular and the 10X, 43X, and if the re- actions appeared doubtful, the 9'IX objective. Integretation of the agglutination reactions: The varying degrees of agglutination observed were arbritarily given plus values to standardize the readings as follows: 1 plus, mall clumps of 3—10 leptospirae and many free ones; 2 plus, numerous small clumps of leptospirae and many free ones; 3 plus, nunerous large clumps of leptospirae and few free ones; 4 plus, amorous large clumps of leptospirae and only an occassional free one. sera , danonstrating a partial reaction in the lower sew-antigen dilutions, were retested. 1'7 Difficulty was encountered in properly evaluating the agglutina- tion reactions which occurred in the lower serum-antigen dilutions. As yet, the frequency and possible limits of nonspecific reactions have not been sufficiently investigated. It is possible that the low titer reactions may indicate a recent or previous leptospiral infec- tion or may be entirely nonspecific in nature. 18 'RESULTBOFEXPERDENT]. In an attempt to evaluate some of the laboratory diagnostic pro- cedures,:micorscopic, cultural, and serological examinations were cone ducted on the blood and.microscopic and cultural examination on the urine of 30 naturallybinfected dogs. The results of this evaluation are sunrnarized in Table l. Nflcroscopic blood and urine sediments, stained by Giemsa's and Fontana's methods, were conducted. Table 1 shows that the number of speciments, in which leptospirae were detected by these methods, were very small. Rene of the Giemsa blood preparations and only 1 (3.33%) of the urine preparations demonstrated Leptospiras. Three (10%) of the Fontana blood preparations and l (3.33%) of the urine preparations de- monstrated,Leptospira. In addition to the above two microscopic procedures, darkfield examinations were made. In '7 (23.33%) of the blood preparations and 1 (3.33%) of the urine preparations, Leptospira appeared to be present. The urine sediment fram the same dog was positive in all 3 of the microscopic examinations. The results of the cultural examinations of blood.compare favor- ably with those obtained by darkfield examination. Isolations were obtained fram 6 (20%) of the samples cultured. .All the organisms cul- tured appear to be‘gp canicola as evidenced by serological examination. The results, obtained by attempting to culture Leptospira frum urine sediment, were all negative. unfortunately, it was impossible to culture urine sediment without bacterial contamination; as a result this diagnostic procedure was considered unreliable. 19 The results of the 3 serological tests conducted at 6-8 day inter- vals on the 30 infected dogs are sumnarized in Table 2. The results re- corded for the first test designate the highest dilution in which ag- glutination was observed. For the second and third tests, only the di- lutions, in which a 4-plus agglutination reaction in the 1:100 serum dilution, or higher, was considered of diagnostic significance. Sixteen (53.33%) of the animals tested were males. The ages for. all the animals tested ranged from 6 months to 7 years. On the initial test, 14 (46.66%) danonstrated an agglutination re- action to one or both antigens. However, all of the 30 samples demon- strated some degree of agglutination on the second and third tests. 0f the 14 sera which demonstrated some degree of agglutination in the initial test, only one yielded a 4-p1us reaction in the 1:100 sermn dilution. The results of the second test indicate that 16 of the sera yielded 4-plus reactions in the 1:100 serum dilution, whereas on the third test all gave 4-p1us reactions in the 1:100 serum dilution or higher. . The degree of agglutination observed in the first, second and third tests varies considerably. They range from negative to 4 plus in 1:100, 4 plus in 1:10—10,000, and 4 plus in 1:100-30,000, respectively. As was expected, the titers rose as the disease progressed, the strongest reactions in the higher dilutions occurring in the third test. It should not be assmned that an end titer of 1:30,000 (Table 2) is the highest titer that may be expected. Other examinations, not re- corded here, reveal that the titer may reach at least 1: 300,000. Arbritarily, the infected dogs were divided into 3 age groups, as follows: 1 month to 1 year, 1 year through 3 years, and 4 years through '7 years. Nine (30%) of the dogs were in the 1st group, 14 (46.66%) in the second, and 7 (23.34%) in the third group, respectively; Rene of the animals in this study were over 7 years of age; in fact, the author is yet to observe a clinically active case of the infection in dogs over 7 years old. The occurrence of specific and paraspecific reactions are summarized in Table 3. These results are based on the reactions observed for each antigen in the third agglutination test. It was noted that only 10 (33.33%) of the sera failed to demonstrate any paraspecific reactions with _I.._. icterohaanorrhagLag antigen. Further- :more, as the specific titer increased, there was also an increase in de- gree and frequency of the paraspecific reactions. It has been reported by Meyer and coworkers (1939), Haven (1941), Walch-Sorgdrager (1939) , that infections due to l:- canicola yield higher and stronger reactions than those due to _I:._. icterohawnorrhggigg. The results of the third test indicate that 22 (73.33%) of the sera demonp strated 4-p1us reactions in dilutions of 1:1,000 or higher. TABLE 1 ngpgrative:microscopic and cultural findings in 9_p9sitive dogs Dog Giemsa Fontana Darkfield Culture N°° Bld. f U. Bld. U. Bld. U. 1310. U. LP 1 - - - - + - + -* LP 3 - + - + - 4 - - LP 5 ' - + - + - + «- 1P 8 - - - - + - - - LP 10 - - - - + - - - LP 15 - - + - + - - - IP 19 - - - - - - + - 1.? 21 - - + - + - + - LP 25 - - - - + - + - I“All urine cultures became overgrown.with bacteria. TABLEZ Serological results of 3 tests made at 6-8 deLintervals on 30 Leptospira-infected dogs No. lst Test 2nd Test 3rd Test Sex Age w LP 1 0 2+, 1:10 K0, 1:300 M 8mos. LP 2 31:133. 1:11:33. 3:; 1:13:33. 1 3m- LP 3 2321:330 3.1113300311113300.) M 41010. LP 4 g 2:, 1:30 E, 1:300 M In” I? 5 3 2+. 1:30 A 1:100 F 5““ LP 6 8 fl 2:11:10 21. 1:1,000 l' lyr. :2 v 1”'1‘1° 311113 1111333 . .m... LP a 1:: 1:13 1:: 1:133 3:1:11‘333. . 2m- 1. 9 1:11:13 2:1:333 31133333. I 2.... 131° 3 f1).«.,1::'.0 f1.11:100 M 11”“ LP 11 3:: 1:30 It: 1:300 Z: 1:203:30 M 7 5’". 1112 3:11:33 211E133 3321:3330. M 3m: ... 1:112 2:112 2:11.322. . m- “’14 3:: 1:13 2:1:fow0::1:10?380 1’ 2m- 0 0 0 I? 15 0 4+, 1:100 4+, 1:3,000 M 53"" I"First line: titer of sero-reaction for _];._. icterohaanorrhagiae antigen; second line for _I_._. canicola. TABLE 2 (continued) No. 1st Test 2nd Test 3rd Test Sex Age LP 16 3 2+ 1:10 2+. 1:300 11 8 “1°" 12 1" 1:; 1:13 :1: 1:130 41:; 133000 F 4 ”'3' LP 18 13, 1:30 2+: 1:50 2+. 1:1:000 M 1 ’1" LP 19 3 :1: 1:33 211111333 1 2 m- I: 2° 11:1:33 3; 1:133 3:113:33: r 4 m: LP 21 3 2:54:10 2+. 1:100 3' 11 m" 0 4+. 1:300 4+. 1:3.000 1" 22 0 4+: 1:300 4+: 1:10.000 M 2 “'5' LP 23 g 3; 1:13 1:: 1:13:00 F 6 yr" . 2: 2 :1; 1:12. 2:11:21. . .. m... LP 25 g 2:. 1:100 2+. 1:100 1' 7 m" L. .. 1:; 1:13. 1:11:13: 1111:1133: M 1 ... . 27 1+. 11:2 21:2. 1 m I? 28 g a; 3138 2:;133000 M 9 mos. LP 29 8 2:11:18 fl;1:1jgoo M 3 ”3' 19 3° 8 2: 1113300 1:: 1133300 L“ 1 71" TABEE 3 Titer of 31:3ch and gregpecific reactions in 30 dogs (Active infection with P: canicola) 5 Paraspecific Specific z 3 ’ _1_._. icterohaanorrhggiae titer . canicola a '3 v. titer 8 £2. 0., Reg. 10 30 100 500 1,000 3,000 10,000 100 4 4 0 0 0 0 0 0 0 300 4 3 0 0 1 0 0 0 0 1,000 6 2 0 1 2 1 0 0 0 3.000 6 1 0 0 1 2 2 0 0 10,000 8 0 0 1 0 1 3 3 0 30,000 2 0 0 0 o 0 0 1 1 Total 30 10 0 2 4 4 5 4 1 — -..- v-— -—. O ‘r'IT-‘f' ._. -1 ._.- to 1m. canicola. 7-day culture from Semifner's modu- ium. lbntm It's-E. X 1'50. Isolated. from canine. Jilly. 19‘s. 21 RESULTS OF ECPERMNI' 2 In an attempt to ascertain the magnitude of reservoir leptospiral infection in the Lansing, Michigan, area, 5 ml of blood was drawn asep- tically fran the radial vein of 500 dogs. These animals were hospital- ized in the Veterinary Clinic at Michigan State College or present in nearby kennels. At the time of bleeding, all dogs appeared normal, none showing any clinical evidence of leptospirosis. History relative to leptospirosis was unobtainable in most instances. Reteste were not conducted on any of the dogs. For all specimens that were found un- satisfactory for any reason, second samples of blood were obtained. All sen were inactivated in a water bath at 56 c for 30 minutes. This sera was stored in the refrigerator at 4 0 until tested. The ag- glutination test was conducted on all samples within 3 days after col- lection. Formalin-killed 3:. canicola and _I_... ic:terohaanorrhagiae antigens were employed in the agglutination test. A l-plus reaction, or higher, in the 1:10 serum dilution or greater, was considered indicative of a latent infection. A majority of the positive sera demonstrated a stronger reaction to one antigen than to the other. In these instances the antigen which gave the stronger reaction in the highest dilution was considered spe- cific. The antigen giving the weaker reaction with the same serum was considered paraspecific. Of the 500 canine sera examined. 148 (29. 6%) gave a positive re- action to one or both antigens. The remaining 352 (80.4%)gave no re- action to either antigen. One hundred and thirtyoone (26.2%) dmon- 22 strated a stronger reaction with g. canicola antigen and 13 (2.6%) a stronger reaction with _]:.._. icterohaanorrhagiae antigen. Four of the samples (.8%) reacted with an equal intensity to both the I? canicola and p. icterohaemorrhagiae antigens. The details are given in Table 4. Table 5 summarizes the incidence of leptospirosis among the vari- our age groups. Arbritarily, age divisions were made dividing the group of dogs tested into four age groups. The groups were: 1 month to 1 year; 1 year through 3 years; 4 years through 7 years; and 8 years through 12 years. The incidence of infection in the various age groups was 26.90%, 17.40%, 42.60%, and 57.40% respectively. As is substanti- ated in Table 5, the greater the age the animal achieves, the greater the Opportunity for exposure. Attention is called to the 1 year through 3 year group (Table 5), which does not conform to this expected pattern. TIO hundred and twanty- one of the 500 degs, comprising nearly one-half of the animals tested, were in this group. Seventeen and forty-hundreths per cent of this group demonstrated Leptoepira imune bodies. Attention is also called to the fact that 25.70% of the positive agglutination reactions occurred in this group. The reason for this could possibly be explained by the fact that kennel dogs were included in this experiment. The majority of their ages ranged frcm 1 year through 3 years. These young dogs were kept in kennels and runs with oncrete floors. Rat infestation was not evident. They were not allowed to run free and roam as the pet dog. These kennel dogs had less chance of exposure to the infection than did the majority of other dogs tested. It is possible that, if kennel dogs had not been included in this study, the number of positive animals would have been greater in this age group. 23 Two hundred and seventy-nine (55.80%) of the dogs tested were males, of which 104 (70.40%) gave a positive serological test to one or both antigens. Ninety-one (89.50%) of the male reactors were po- sitive to 31° canicola antigen, 10 (9.62%) to 3': icterohaanorrhagiae, and 3 (2.88%) danonstrated an equal titer for both. Two hundred and twenty-one (44.20%) of the dogs tested were females, of which 44 (29.60%) gave a positive agglutination reaction to one or both anti- gens. Forty (90%) gave a positive reaction with .1_’.._. canicola antigen, 3 (6.82%) to 1:9 icterohaemorrhagiae, and 1 (2.28%) reacted with an equal titer to both. The percentage of male to female reactors was slightly more than 2:1. The results (see Table 6), relative to the sex incidence of the disease, is in accord with the findings of Meyer (1939) and Raven (1941). Their examinations also dealt predominantly with _I_.._. canicola infections. Klarenbeek (1934) reports that leptospirosis may occur up to 10 times as often in the male as in the fauale. The results indicate that _1[.._. ictm rrhagiae infections are predominate. Infection due to .I_._. canicola appears to produce a higher titer than _l_.-. icterohaanorrhagiae. This high titer was observed in the ser- ological results in the 500 nomal dogs tested. It was further sub- stantiated in the results of the 30 active infections of the disease. In Experiment 2, only 2 (15.4%) of the sera demonstrated a 1:1,000 or greater specific titer to l... icterohumrrhggigg. Whereas, 35 (25.7%) of the sera gave a 1:1,000 or greater specific titer with 1.... canicola. Non or paraspecific reactions, especially in the higher dilutions, occurred with sera giving a specific titer to both antigens. As is summarized in Table 7 and 8, 75 (57.25%) of the positiveIE? canicola samples and 5 (38.40%) of the‘ép icterohaemorrhagiae positive samples demonstrated paraspecific titers. The E: canicola-positive sera demon- strate a higher percentage of paraspecific reactions. Regardless of specific antigen, paraspecific reactions occurred more commonly in sera demonstrating a high specific titer. TABIE 4 The reactions of 148 serologically positive canine sera samples Number Per cent Per cent Specific Positive Titers of of of Reactors Total L. icterohaemorrhagiae l3 8. 8 2. 6 l... canicola 131 88. 5 26 . 2 ']_Z._. icterohaemorrhagiae and P: canicola equal Total 148 29. 6 TABLE 5 Age incidence of latent canine leptospirosis of the 500 dogs tested NUmber number Per cent Per cent °f Age Group Tested. Positive Positive Age Group Infected 1 mo. to 1 yr. 97 26 17.50 26.90 1 yr. through 3 yrs. 221 38 25.70 17.40 4 ”Se through 7 yrae 134 57 38e55 42e60 8 yrs. through 12 yrs. 47 27 18.25 57.40 TABLE 6 Sex incidence of latent canine leptospirosis in the Lansing, 94411 THREE 311140993 :93 yo iueo 195 2.88 2.28 seats rumba Suticseu xes JO °on 3 eatfisqxxomeeqomeiot "I" flunoaeu xas yo iueo Isa 9.62 6.82 eetSsqazcmesqoxeiot '1.3311°F°H res :0 'on 10 3 atootueo “T Sutiosea xsg JO iueo meg 87.50 Nflchigan, area atootuso “E 311140993 xes JO °on 91 ssoqoeeu dues sea szoaoaeu 'ou tsiom JO rues meg 99499.1. °ou 188 Nhles F79 55.80 104 70.40 Femle%21 44.20 44‘ 29.60 40 90.00 TABLE 7 Titer of specific and paraspecific reactions Infection with L. canicola SP°°1fi° g _I_._. ictefigfigfiia. titer . canicola‘ E titer z Neg. 10 30 100 500 1,000 10 s e 0 0 0 o 0 50 14 1o 4 0 o 0 0 100 59 19 a 12 0 0 0 500 55 15 5 7 e 0 0 1,000 21 5 2 5 5 4 0 5,000 8 1 0 1 5 4 0 10,000 2 0 o 0 o 2 0 30,000 4 0 0 o 1 2 1 Total 151* 56 19 25 17 12 1 I"4 sera, titers equal for both strains, not included TABLE 8 Titer of specific and paraspecific reactions Infection with L. icterohaamorrhagiae Paraspecific SP601f1° 3 L. canicola titer - 1219.22- '° 7 ““— emn‘hafii“ g Neg. 10 50 100 500 1,000 10 0 0 O 0 0 0 0 30 1 l 0 0 0 0 0 100 6 4 2 o 0 o 0 300 4 3 0 0 l 0 0 1,000 2 0 o 0 1 1 0 3,000 0 O 0 O O O 0 10,000 0 0 0 0 0 0 0 50,000 0 0 0 0 o 0 0 ___, ____ ____ ___m .__. .._. .__. Total 13 8 2 0 2 2 0 *4 sera, titers equal for both strains, not included 25 DISCU$ION Canine leptospirosis may be caused by either L. canicola or L. icterohaanorrhjgiae. The two species are culturally and morphologi- cally identical (Morton, 1943), although they may be differentiated serologically. The incubation period for the disease varies from 1-2 weeks with a mortality rate of 0-25 per cent. The organimns are pre- sent in the blood from the let to the 6-8th day after the appearance of symptoms or during the febrile period. After this time, they dis- appear from the blood and localize in the kidneys, resulting in lepto- spiruria. 0n the 9-10th day the antibody titer begins to rise, reach- ing a maximum within a few weeks, and is maintained for a period of several months. The dr0p to lower titer levels is gradual, and some titer may persist for as long as 2-3 years. Clinical observations of the 30 proven cases of canine leptospi- rosis due to L. canicola were similar to those observed by Jungherr (1937), Meyer (1939a), and Blender and Perry (1940). Taco distinct clinical types were observed, the icteric and the hemorrhagic. Icter- us occurred in 6 of the 30 cases. Depression, high tanperature, vomit- ing, posterior weakness, refusal to eat, and congested conjunctivae occurred in the majority of the cases. Dogs may become infected with _1_._. icterohaemorrhagigg through con- tact with infected rat urine. Rats from various parts of the world have been examined and as high as 45 per cent have demonstrated the or- ganisms in the kidney tubules (Ida and associates, 1917; Noguchi, 1917; Iobling and Eggstein, 1917; Langworth and Moore, 1927; Walch-Sorgdrager, 1939; Meyer and coworkers, 1939, and Monlux, 1948). 26 Canine _L_. canicola infections are maintained by simple contact from dog to dog. Natural infections have not been reported in other animals except man. The elimination of the organisms in the urine is responsible for the spread of the disease. The organims are capable of living outside of the body for varying periods of time (Davidson and coworkers, 1934). Meyer and coworkers (1938) and Walch-Sorgdrager (1939) have not encountered _l_._. canicola in the rat population. It is believed Justified to conclude that the rat is not the natural host for L. canicola and need not be considered in the epizootology of the disease. The results of Experiment 1 indicate that none of the laboratory diagnostic procedures are sufficiently reliable at all times. It is impossible for a veterinarian to diagnose canine leptospirosis, except clinically, when the animal is first present. Only 10 per cent and 23.33 per cent of the stained and darkfield blood sediment preparations, respectively, demonstrated Leptogira. Their efficiency as diagnostic procedures is apparent. False positives in stained anears are practi- cally nonexistent, although caution should be exercised in the inter- pretation of darkfield preparation. Schultz (1923) and Hall (1925) observed motile filamentous elements in the blood, highly suggestive of living organisms. The ”pseudospirochetes" have been observed in the blood of normal and diseased individuals. The novice may be mis- led by their presence. If one thoroughly acquaints himself with the morphology and motility of these "pseudospirochetes' and Lepjospira, fewer false positives will be recorded. The cultural examinations of blood yielded isolations in 6 (20%) 0f the animals examined. Had cultural examinations of blood been made 27 on or before the 4-5th day of illness, more satisfactory results would have been obtained. This confirms the results of all workers in their isolation attempts from humans infected with Leptospira (Davidson, et a1., 1934; Snith and Tulloch, 1937; Packchanian, 1941; Bertucci, 1945). Table 2 indicates that the accuracy of the agglutination test ins creases as the disease progresses. The results reported here (Table 2) indicate that at the end of 12—15 days and 21-30 days of illness it is approximately 50 and 100 per cent accurate, respectively. .All workers (Bertucci, 1945) agree that strong false positive reactions are non! existent. If serial agglutination tests are conducted, the exact status of the titer*may be determined. .A 4-p1us agglutination reac- tion in the 1:100 serum dilution was employed as the diagnostic cri- terion. Experiment 2 indicates that at least 29 per cent of the ani- mals in this area appear to demonstrate some titer to one or both an- tigens. (Although the agglutination test is a rapid, accurate, laboratory diagnostic procedure, it must be emphasized that its usefulness is lim- ited. At the time an accurate diagnosis is obtained by this method, the animal is either dead or on its way to recovery. Hence, the imme- diate instigation of specific therapeutic measures is not possible. It would appear, however, that it is fully reliable in retrospective diagnosis as the results of Experiment 1 and 2 indicate. The incidence of latent canine leptospirosis has been summarized from comparable reports in Table 9. The world incidence is 0-45 per cent, the highest incidence occurring in.Holland, where perhaps the 4most extensive studies on both human.and canine leptospirosis have been conducted (references previously cited). In the United States the 28 range is 0-33. 33 per cent, the highest occurring in Pennsylvania and none reported from Alabama. The results reported here correlate closely with other reports from this country, although the incidence of L. icterohaanorrhagiae canine infection is slightly lower. There appears to be an explanation for the difference between the results obtained by Meyer (1939) and the results reported here. The 10 samples examined by Meyer were obtained from the Detroit area, from which Molnar and Kasper (1938), Molnar and Meyer (1940), Molnar, Meyer, and Raskin (1948) reported 75 human cases of Neil's disease. In addition, Meyer and associates (1939) reported that 16 per cent of the rats in that area demonstrated renal leptospiral infections. Thus, because of enviromnental conditions which exist in the Detroit area, the incidence of L. icterohaamorrhagiae infections is higher than in the Lansing area. It should be mentioned, however, that the canine sera samples submitted to the author for examination from the Detroit area, indicate that L. canicola infections are also present. Evidence supported by the findings in Experiment 2 (Table 4), in- dicate that many of the atypical canine illnesses may be subclinical or latent leptospirosis. Leptospiruria is known to occur in those dogs infected and their role as spreaders of the disease to other dogs and man should be emphasized. me Incidence of latent canine leptospirosis Po-mve 3233?... State L. canicola Mann. Nunber or ‘ Dogs Holland3 10 20.0 4 6.0 50 Gemanys 6 12.0 5 6.0 48 oemav?’ 5 1.5 1 0.5 200 Delmarks 1 1.9 18 54.0 55 Alabams 0 0.0 0 0.0 21 California3 20 26.0 o 0.0 75 New York3 10 9.0 5 2.7 111 Pennsylvania1 11 22.0 5 6.0 50 Pennsylvannz 26 24.8 9 6.5 105* Michigan3 0 0.0 10 76.0 15 Michigan‘ 151 26.8 15 2.6 500" 1Dogs frun the' urban area - reported by Raven (1941) 2Dogs from the rural area - reported by Raven (1941) sported by K, 1'. Meyer (1939a) ported by author *rive sera danonstrated an equal titer for both antigens. I""‘l’our sera demonstrated an equal titer for both antigens. 1. 2. 3. 4. 5. 29 SUMMARY Staining, cultural, and serological procedures were conducted on blood and urine sediments of 30 naturally-infected dogs to deter- mine their value as laboratory diagnostic procedures of infection. The staining and cultural procedures are considered unreliable as diagnostic procedures, although the agglutination test was approxi- mately 50 per cent and 100 per cent accurate after the 2nd and 3- 4th week of illness, respectively. A The sera from 500 normal dogs were examined by the agglutination test in retrospective diagnosis. Twenty-nine and six-tenths per cent of the dogs demonstrated some titer to either L. canicola or L. icterohaanorrhagiae antigens, or both. I'venty-six and two- tenths per cent of these were positive to L. canicola, two and six-tenths per cent were positive to L. icterohaemorrhagiae and eight-tenths per cent demonstrated an equal positive titer to both. 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