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I “iii: J ‘ Bacteriology Wajor professor 1 , t'a_ ~ The Value of Certein Therapeutic Agents Against the Bacterial Infections Causing Conj nnetivitia and Rhinitis in Dogs Affected with Distemper. I." .J - J . Q39 I‘h’d "t" "lb .. .., ‘,-. I £1.49 ’ -. I «xii ' ." I i . ' I’: I I ,4 ‘II I. I , .' [. i. I r; r ~' ‘w -. s. P- . 'r ' I I 2 L’ I ' l I j i t l '1 q .‘ O . i t I .l’ r H' ‘ l . J .1 I {' ll“ THE VALUE OF CERTAIN THERAPEUTIC AGENTS AGAINSI‘ THE BACTERIAL INFECTIONS CAUSIM} CONJUNCI‘IVII'IS AND RHINITIS IN DOGS AFFECI‘ED WITH DISI‘El’IPER By flung, Gueh—dj en A THESIS 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 of DOCTOR 0F PHIIDSEHY Department of Bacteriology and Public Health 1951 ACKNOWLEDGEMENT The author wishes to express her grateful appreciation to Dr. H. J. Stafseth, head of the Department of Bacteriology and Public Health, Michigan State College, for his generous guidance during the progress of this work. She is also deeply indebted to Dr. F. E. Eads, assistant professor, Department of Surgery and Medicine, Michigan State College, for suggestions and materials. \ Hsiung, Gueh-djen candidate for the degree of Doctor of Philosophy Final examination, May 11, 1951, 10:00 A. M. , Bacteriology Building, Room 12 Dissertation: The Value of Certain Therapeutic Agents Against the Bacterial Infections Causing Conjunctivitis and Rhinitis in Dogs Affected with Distemper Outline of Studies Major subject: Bacteriology Minor subjects: Biochemistry, Animal Pathology Biographical Items Born, September 16, 1918, Hupeh, China Undergraduate Studies, Ginling College, 1938-42 Graduate Studies, Michigan State College, 1947-51 Experience: Technician in Bacteriology, North West Epizootic Prevention Bureau, Lanchow, China, 1942-46 Graduate Council Fellowship, Michigan State College , 1948-51 Member of Society of the Sigma Xi, Society of American Bacteriologists In memory of my brother Colonel L. C. Hsiung and his wife Mrs. Rosetta Ling Hsiung CONTENTS IMODMION O O O O O O O O O O O O O O O O C O O O O O C . HISTORICAL REVIEU I. Bacteriological Studies on Canine Distemper . . . . II.Therapellticlgentseeeeeeeeoeeeeeoeo III. Serological Studies on Identification of Organisms . MATERIALSANDMETHODS I. BacterialFlorasonyesandNose. . . . . . . . . . II. Bacteriological Studies on the Effects of Treatment . III. Serological Studies on Some Strains of the IBOla'ted Oz‘ga'nimns O 0 O O O O O O O O O O O O 0 O RESUIE'S I. Comparison of Bacterial Florae in Distemper Infected DogsandNoninfectedDogs. . . . . . . . . . . . II. Comparison of Bacteria Found in Distemper Infected Dogs During Different Seasons . . . . . . . . . . III. Bacteriological Studies on the Effects of Treatment. IV. Serological Studies on Neigsegg W “and MW masses var. slim Isolated From the Dogs Affected With Distemper . . . . . . . DISCU$ION O O O O O O O O O O O O O O O 0 0I O O O O O O O O SWY eeeeooeeoeeeooooeooooeeeo mmseoeooeeeeeeeeeeoeoeoooooe FIGIRES 21 23 32 38 57 67 :l 3 INTRODUCTION Canine distemper is a highly infectious disease of dogs. The cause of this disease was first discovered to be a filtrable virus by Carrd in 1905. Young dogs under one year of age are easily infected. During the course of the virus infection covering a period of five to twolve days, the dog's resistance becomes so low that it cannot fight off bacterial invasion. The germs which are present in the bacterial phase of the disease are secondary in- vaders and cause most of the symptoms. is a result of bacterial findings in canine distemper, Ferry (1910) came to the conclusion that the ocular, nasal, cutane- ous, and nervous symptoms are the result of secondary infection, and death in most cases results from these secondary invaders. If this is true, the distemper vaccine will have little or no direct effect. it the seventieth meeting of the American Veterinary Medical Association, Dr. E. A. Cahill (1933) stated during a dis- cussion “Regardless of whether the filtrable virus is the primary cause and m W a secondary invader of distemper or vice versa, there is increasing evidence throughout the country of a very great need on the part of the practitioner for something besides the regilar treatment for canine distemper.... There is increasing evidence that the antibronchiseptieus serum and the anti- filtrable virus serum are not always one hundred percent success- ful. . . ." The prevalence and methods of treatment of canine dis- temper have long been problems of economic importance to the veter- inarian and the dog owner. Many vaccines and ears have been used for the treatment of canine distemper, but published data on local application of therapeutic agents against secondary invaders of the eyes and nose are quite limited. The object of this study was to determine the efficacy of the various drugs that are applied locally against secondary infection in dog distemper. HIMICAL REVIEW I.Bte lo eonC D r Canine distemper is said to have been known in the time of Aristotle. One of the first to advance a definite conception of the nature of the disease was Jenner (1815). He recognized the contag- ious character of the disease, and noted that the causative agent re- tained its infectious prOperties for a long time after separation from sick dogs. Jenner was the first to differentiate between dis- temper and rabies. Early studies on the microbiology of canine distemper were directed at the discovery of the primary cause of the disease. A number of different organisms were found, and results could not be confirmed; therefore the question of etiolog was not definitely established until 1905 , when some dis covered the filtrable virus of distemper. The first bacteriological investigation of canine distmper was by Scanner (1875). He found a small and exceedingly slender ha.- cillus in the blood of diseased dogs a few hours after they died of distemper. Kraaewski (1881) found a micrococcus. Marcone and Meloni (1904) found cocci similar to staphylococci. Rabe (1883) and Mathis (1887) cultivated streptococci and staphylococci from the pustular contents, nasal exudate, the con- Junctival secretion, the blood and various organs. They claimd that these organisms were specifically related to distemper. Millais (18%) found a long bacillus which liquefied gela- tin, descending as a flaky mass in the almost clear fluid which be- came covered by a whitish scum. The worker also found a micrococcus which was thought to be the cause of lung lesions. Galli-Valerio (1896) found a bacillus on agar plates, from the mcus of the respiratory tract. It ranged in size from 0.3 x 1.2 to 0.3 x 2.5 microns. It was often dumbbell shaped. The ba- cillus was gram-positive and was motile. This organism was also isolated from the lungs, brain, spinal cord and pus from the frontal sinus. Taty and Jacquin (1898) found a diplococcus in the central nervous system which they regarded as the cause of the nervous form of disteaner. Jess (1899) isolated an ovoid bipolar-staining bacillus from the nasal discharge, blood, and conjunctival. secretion of dis- temper dogs. Copeman (1900) found a cocoa-bacillus in smears from broth, not infrequently in chains and sometimes of considerable length. It was gram-negative, but grew readily on agar at 36°C. Lignieres (1903) isolated and described an organism which was also studied by Phisalix (1903). They obtained from the blood of the heart and internal organs an organism in the form of a long bacillus, which, after passage through guinea pigs or on cultures, soon changed into a short coccobacillus. To this organism was given the name Pagteurella wig. Carre (1905) claimed that canine distemper was caused primarily by a filtrable virus, and that the disease as a whole consisted of a series of progressive secondary infections caused by a number of cultivable bacteria. Hewer (1906) stated that cocci which gave the reactions of the pyogenic staphylococci were obtained in pure culture from the nose and bronchi. W films was most frequently found, although SW m was often present. He iso- lated several bacteria from a few cases of distemper, but could not prove their importance. Ferry (1910) described a microorganism which, in 1911, he named BM W. This organism differed from the organisms described by Galli-Valerio (1896) , Copeman (1900) , Lignieres (1903), Phisalix (1903) and Hewer (1906). Ferry (19m) fetnld that when cultures were taken early in the disease, B. M ga__n_i_s was found in the respiratory tract in every case. If cultures were taken in the first stage of distemper, they were un- contaminated. Purulent discharges from the eyes and nose were due to secondary infections and were not true manifestations of dis- temper. He concluded that B. W was the primary and es- sential etiological factor in canine distemper. McGowan (1911), at the Royal College of Physicians, Edin- burgh, Scotland, reported the same organism as that described by Ferry. He isolated this bacillus without difficulty from the meo- purulent nasal discharge from the trachea or lungs, but not from the blood. This organism, when applied to nasal mucous membranes, produced the clinical symptoms of distemper. In one of the ex- perimental dogs, the nose was absolutely plugged with pus on the fifteenth day after inoculation, and cultures from the nose taken then gave staphylococci only. Staphylococci and also bacilli. were shown in large numbers in cultures from the nose on the six- teenth day after inoculation. Torrey and Belle (1913) obtained cultures from the eyes by streaking directly on agar plates. The cultures from nasal exudate were emllsified in sterile solution and plated. Their bac- terial findings were as follows: 1. During incubation period - trgptogocgs fegglis was found in the eyes. We M. We Wilma. W W. Altamira: Maudie. Eastman: saris. 13.81.9213: 9211, and 32:22. resell: Were found in the nose. 2. During first week of symptoms - Streptococci and 5139. £21.- imsis were found in the eyes. 13.. We, Also. epidgrmigig and m m were found in the nose. 3. During second week of symptoms - Gram-positive diplococci, Alba. W and a few streptococci were found in the eyes. Streptococci, albococci, and B. mm were found in the nose. 4. Chronic cases, three or more weeks - The same results were obtained as above. 5. Recovered cases - Very few organisms were found in the eyes and they were the same as above. 6. Fatal cases - Only one out of five cases yielded 513. W and M. M. Streptococci, albococci and a few colonies of B. mm were found on plates from the nasal exudate. In sumry, among the different organisms isolated, two percent of the cultures from the eyes and 56 percent of the cultures from the nose were B. bmnggepticug. Theyconcluded that B. W was the infective agent of the disease, but certain symptoms such as those of the eyes and nose might be due wholly or in part to second- ary infection. Streptococci, B. 991; and Bagging out were the most frequently encountered secondary invaders. Of these, the most important, as far as the clinical picture and the severity of the disease was concerned, were the streptococci. It was their ob- servation that the secondary invaders began to mlltiply in the tissue only after the animal had become exhausted by the toxin of the bacilli. Ferry (1912a, 1912b) reported that B. We may be the cause of a severe infection among laboratory animals other than dogs. He changed the name of the microorganism from B. W to B. W. Taking into consideration the combined re- sults of McGowan (1911) , Torrey and Rahe (1913) , and himself, he concluded that the condition known as distemper in the dog, and certain other animals, was an acute infectious disease due to B. W. This organism produced a catarrhal inflammation, primarily of the larynx and trachea, and possibly of the nasal cavity. The infection often extended to other mucous surfaces, resulting in general infection, followed by many complications and sequelae due to secondary infections. The mortality rate was from 60 to 90 percent. The investigator (1913, 1914) strongly suggest- ed that suspensions of both live and killed B. bronflgeptigg would protect dogs from natural distemper. Schoichi (1932) stated in his bacteriological studies of distemper that 50 percent of the dogs showed pure cultures of B. mm which in 35 percent of the animals was associated with streptococci, staphylococci and even B. gg_1_i,. Lockhart, Ray and Barbee (1925) published the results of their work which showed that a true virucidal serum could be pre- pared in dogs hyperimmnized against the filtrable virus of Carré. They concluded that this serum was a reliable immizing agent, producing an immunity of long duration after injection into dogs. They were the first to make a useful antidistemper serum. Dunkin and laidlaw (1926a, 1926b) reported a filtrable virus as the cause of canine distemper and concluded that B. Mi- m was only a secondary invader. Research on distemper virus vaccine was conducted by them. In the same year Pugh (1926) stated that distemper is due to a filter-passing virus and regarded B. bronchigeptiggg as a secondary invader. Lockhart (1927) stated that true distemper is a systemic disease, and that the things which are usually considered visible symptoms are secondary in character, being produced by organisms which are ordinarily of low virulence. These are capable of pro- ducing disturbances in devitalized tissue. The bacteria recover- able from distemper cases vary greatly, but generally W bignchigeptimg, Stafllococgg and Streptococggs are found. Schlingman (1931) advanced the possibility of a hemolytic streptococcus being associated with canine distemper. Good results in the treatment of distemper were obtained from the use of an anti.- serum and a mixed bacterin. He (1932) reported on the bacterio- logical studies of canine distemper in one hundred naturally infect- ed cases. From the different organs such as the lower trachea, lungs, liver, spleen and heart blood he isolated B. W from 81 percent of the animals, streptococci from nine percent, M. £31.]; from six percent and colon-typhoids from four percent. W was and §t_ap_h. M were seldom present. They should be considered as secondary invaders. Pyle (1934) reported a bacteriological examination of 146 spleens, taken from distemper infected puppies late in the filtrable virus stage of the disease. At this time there was a second rise in temperature, reaching a high level simultaneously with the appear- ance of the characteristic distemper conjunctivitis and rhinitis. W W was isolated from 20 spleens. An unknown 10 micrococcus and Staphylococcus 31M were isolated from one spleen each. The remaining 124 spleens were bacteriologically sterile. Two of the 13 spleens from distemper infected adult dogs showed §_al_. gntgritidig, whereas 21 spleens from distemper infected ferrets all proved to be sterile. In no instance was 51. broncgiseptigug iso- lated from any of 180 spleens examined. Regenos (1935) stated in his comprehensive studies on canine distemper: "In canine distemper, there is no question but that the filtrahle virus is the usual primary causative agent and that the following bacterial organisms should be considered as having etiological significance: E. W, streptococci, fig}. W B, Bel. entegitidis, and staphylococci. The occurrence and importance will vary with the seasons and localities. In general, the importance of the organisms is in the order given." Similar statements were made by Whitney (191.0) . Greene (1943) and Schlotthauer (1949) claimed that the cause of canine distemper is a specific filtrable virus. The dis- ease is always complicated by mmerous secondary invaders, e. g. Bg. m, Brucgua W, various staphylococci and streptococci. Hsiung, Eads and Stafseth (1950) found that W W var. 993a: and species of streptococci were the commonest organisms found in serous and mcopurulent ocular discharges from dogs affected with distemper. II. Therapeutic Agents Adler (1937) found that successful results were obtained with polyvalent antiserum in cases of distemper in dogs and cats, complicated with suppurative keratitis, caused by such microorgan- im as streptococci, staphylococci, pneumococci and other common pyogenic bacteria. This treatment hastened the local and general antibacterial responses , both humoral and cellular. Greene (1943) claimed that the secondary infections in canine distanper may be treated with one of the sulfonamides. Ophthalmic ointments and nasal solutions should be used as supple- mental treatment besides the antidistenmer serum and mined infect- ion serum. The control of canine distemper is largely dependent upon the use of biclogics. Homologous antidistemper serum (canine) has been used extensively for the last few decades. Schlotthauer (1949) reported that the efficiency of this antiserum for treatment varied with the state of virulence of the virus that was present in the animal 13063. In 1950, Eads stated: "The aim in treating distemper- affected dogs is to promote everything that tends to conserve the energ and vitality of the subjects. It is, therefore , essential that the patients should be kept as comfortable as possible.” Sulfa Drugs The use of sulfa drugs is effective in reducing the mor- tality due to distemper in dogs and has been reported by many workers. Marcus and Necheles (1938) demonstrated in their work that sulfanilamide and prontosil could be used successfully in the treat- ment of distemper in dogs, since in most of the fatal cases, strepto- cocci, staphylococci and B. mm were more fatal than the virus itself. Bryan (191.1) reported that sulfapyridine was apparently a specific therapeutic agent in the treatment of canine distemper in the early stages of the disease. . The combined use of sulfapyridine and homologous anticanine distemper serum was even more effective. Guyton (1941) reported that sulfanilamide in ointment form was a suitable preparation for local use. In eighteen cases of catarrhal conjunctivitis, due to ordinary infectious organisms, such as staphylococci and streptococci, five percent sulfanilamide oint- ment gave encouraging results. He suggested that sulfathiazole might prove to be better when used locally for certain types of in- faction. Richtner (1942) claimed that the bacteria disappeared rapidly from the nose upon local treatment with sulfathiazole in certain acute inflammatory conditions. Thygeson and Braley (191.3) found that the use of five per- cent sulfathiasole ointment was effective in the treatment of chronic conjunctivitis caused by staphylococci. They also found that in cases in which the Moral: Axenfeld diplobacillus was present, treat- ment with zinc sulfate was useless until the staphylococci were eliminated. Alvaro (1945) reported from Brazil that local sulfonamide therapy was very effective in a number of well-defined eye diseases. Since sulfonsmides have only bacteriostatic action, it is essential that the drug be applied frequently. Repeated instillations and applications of suitable ointment appeared to be the method of choice. He also stated that the sulfonamides are almost innocuous to the ocular tissues when applied locally, and he recomended the local use of sulfonamides because of easy penetration and tolerance. Robson and Scott (191.2) suggested that the local applica- tion of certain sulfonamides might be of value in the treatment of infective conditions of the eye. According to their emerimental results, 30 percent sodium sulfacetamide had produced no irritation or other ill effects. In 1943 these workers also claimed that a 30 percent solution of sodium sulfacetamide gave the same results as penicillin, by which B3931. m was eliminated from the flora of the conjunctival sac. Fifteen percent solution of solubilized sulfathiasole was less effective, and 2.5 percent sodium sulfaceta- mide was of little or no value. Cortes (191.7) reported that cases of acute and purulent conjunctivitis responded to sulfacetamide. The drug was adminis- tered locally and systemically. Kuhn (1947) , in Scotland, found that neither a solution nor an ointment of 30 percent sodium sulfacetamide was irritating and no allergic reactions occurred. He used a drop of the so— lution every four hours for three days after the removal of a foreign body. Benedict and Henderson (1947) demonstrated that a 30 per- cent sodium sulfacetamide gave the best results in average cases of acute catarrhal conjunctivitis and acute conjunctivitis associated with purulent or mlcOpurulent discharges. Leopold (1948) reviewed the merits of sulfonamide drugs for local use. He investigated the penetration into the anterior chamber of the eye by most available sulfonamide compounds. The concentration of the drug in the aqueous humor was determined in normal eyes and eyes in which the cornea had been damaged. The dmgs in various concentrations were applied as drOps with and with- out detergents and in various ointment bases. He concluded that the penetration of locally applied sulfonamides depends on the physi- cal form of the compound, its solubility, the vehicle, the presence of a detergent and the state of the cornea. Of the preparations available , sodium sulfacetamide , sulfadiazine, and sulfapyridine would appear to be the drugs of choice, in the order mentioned. Eads (1949) found that sulfamerasine was very useful in the treatment of a variety of conditions , commonly found in small. animal practice, namely, distemper and respiratory infections. He stated that the use of sulfamerazine for infections due to bacteria 15 associated with clinical distemper and respiratory infections such as bronchitis. rhinitis, laryngitis, etc. , was of definite value in his study. Bacitracin Johnson, Meleney and Auker (1945, 1947) have shown that, in general, bacitracin is effective against the same bacteria as penicillin, and, in addition, the organisms are often more suscept- ible to bacitracin than to penicillin, in a ratio of five to one. In one hundred cases of surgical infections treated locally with bacitracin, favorable response was evident in 88 percent of the patients. Bellows and Farmer (1948a, 1948b) reported that a baci- tracin-sensitive hemolytic M. gum infection can be prevented when treated with bacitracin within a definite time interval in em- perimental eye infections. Good results were obtained in acute infections in clinical cases of conjunctivitis that had been treated with bacitracin. Miller, Slatkin, and Johnson (1949) reported that 500 units per gram of bacitracin, effective against gram-positive organisms, were incorporated into several ointment bases. Superiority of baci- tracin over sulfonamides and penicillin rests in the low rate of sensitization of the patient. Thus far only the 0. 5 percent so- lution was found to produce sensitivity. 16 Streptomycin In 1944 streptonvcin was shown by Schatz et al. in i_n_ vivo and i_n_ vitro experiments to be bacteriostatic against certain gram- positive organisms as well as a wide variety of gram-negative forms. Robinson, Graessie and Smith (1945) found that streptom- cin was active in vitro against a variety of gram-negative and gram- positive bacteria. The former included W, W, menial-is, .smsslls. illshsialls. Emails and miss. The gram- pcsitive organisms were strains of §_tg_e_p. hemolflilggg, gm. m and animus 2.....aisaneumo . Owens (1946) reported one case of a severe corneal infect- ion caused by M. 991;, which responded satisfactorily to local application of streptomycin. Alberstadt and Price (1946) treated nine patients for corneal infections with streptomycin applied locally. In spite of the fact that adequate bacteriological data were unobtainable , they concluded that the addition of this antibiotic to the usual form of treatment definitely shortened the healing time. Leopold and Nichols (1946 , 1949) reported that the local use of streptonycin gave the same result as penicillin, though the spectrum of its activity was not the same as the latter. Bellows, Burkholder, and Farmer (1947) demonstrated that experimental corneal ulcers, produced by injection of W m- m, were prevented by applications of saline solution contain- ing 10,000 mcg. per m1. of streptomycin. In the same year, Bellows 17 and.Farmer (1947a) found that in acute and chronic conjunctivitis, where known organisms were present before treatment with strepto- mycin, the sac became sterile after a few days of instillation thera- py. Healing generally was prompt if complicating factors were ab- sent. Furthermore, these investigators (1947b) reported that strep— tomycin is safe and non-irritating to the surface of the eyeball in concentrations up to 10 ,000 S units per m1. local application of streptomycin decreases the amount of secondary infections accompanya ing vaccinia infections of the cornea. Kellberg (1947) found that streptomycin's main effective- ness has been in conquering those very persistent, lowgrade bacill- ary infections that commonly complicate distemper in dogs. Frenken (1948) reported that some surprising results were obtained in the local treatment of purulent rhinitis with streptom- cin. A solution of one gram of streptonwcin in 30 ml. of saline solution was recommended to be given on three consecutive days, three times daily, in doses of two and half to three ml. No relapses oc- curred during the period of four months ' observation. Grignolo (1948) reported that in 72 patients, streptomycin 'was administered dulseveral ways into the eye. Nb improvement was noted as result of treatment of corneal ulcerations due to pneumo- cocci, staphylococci or streptococci. Streptomycin appeared to be effective only against .12. 2911.. means W. 29.21.111.12 m and a few other organisms. Lepri (1950) found that 50,000 units of streptomycin in- 18 jected subconjunctivally in rabbits showed the same manner of dif- fusion as penicillin, but at a much slower rate. Eads (1951b) reported that streptomycin showed little, if any, value in the treatment of canine distemper even when administer- ed at the rate of 11,000 S units per pound of body weight, four times per day for ten days. Penicillin Penicillin in a valuable drug in the treatment of some in- fections. It is selective in its antibiotic action and it is not effective in all infections. Most of the organisms which respond favorably to penicillin therapy are gram-positive. Abraham, Chain, Fletcher, Gardner, Heatley, Jennings and Florey (1941) demonstrated in four cases that the local application of penicillin to the human eye resulted in rapid relief from pain and resolution of the inflammation. Swabs from the eye of one patient revealed M. mg. Robson and Scott (1943) found that penicillin gave a defi- nite beneficial reaction subsequent to local application in the eyes. m. w was the microbe eliminated from the conjuncti- val sac. When'treatment was begun. 24 hours after inoculation of the organisms, little or no benefit was produced by the application of penicillin. The importance of early treatment and repeated ap- plications in clinical use of this drug should be emphasized. Riser (1945) stated that penicillin is bacteriostatic rather - than bactericidal in its curative action. It has been used 19 in aqueous solutions for injections, solid tablets for oral adminis- trations, and ointment for local applications. Leopold and La Motto (1945) showed that the penetration of penicillin into the eye is greatly enhanced in the presence of in- fections or. abrasions of the cornea. The dramatic cures that have resulted from the use of penicillin have led to rather indiscrimin- ate use of the drug. Penicillin therapy aimed at the secondary invaders in canine distemper has been reported by Davidson (1945) . In a single complicated case of canine distemper, he obtained recovery in five days, using 10,000 Oxford units of penicillin intravenously and intra- muscularly. The purulent discharge from the eyes had decreased greatly in amount after 24 hours from the first injection. Only a scanty discharge appeared after the second injection and a small amount of catarrhal exudate after the third treatment. Costi and Alvarez (1947) reported good results by the local use of crystalline penicillin in acute, subacute and chronic con- junctivitis. Garcia (1947) worked on the concentration of penicillin in various ocular tissues following various methods of administration. He found that penicillin reaches the highest concentration in the tissue when it is given locally. Good results were obtained in the treatment of conjunctivitis. Penicillin was used locally in a; total of 153 cases by Bitran (1947). He concluded that the drug should be used locally in 20 ophthalmology and not systemically. Sorsby and Ungar (1946) and Minton (1946) reported that pure penicillin is well tolerated by the eye when applied locally in ointment containing up to 100,000 units per gram. Micuda and Holt (1947) reported on the use of penicillin in canine distemper meningitis. Dramatic results were obtained when intraspinal injection of penicillin in saline solution was used. Collins (1948) in his studies on penicillin in veterinary medicine, came to the conclusion that, although the canine distemper virus is not amenable to penicillin activity, the relative sensitivi- ty of the usual secomiary invaders associated with the virus-caused disease, indicated the use of the substance for the treatment of the secondary complications. Holstege (1950) claimed that penicillin salve was a valu- able agent in the treatment of external diseases of the eye, and when it was combined with a sulfonamide, its range of indication extended to almost all infectious external diseases of the eye. Lugossy (1950) enumerated various infectious diseases of the eyelid, orbit and globe, in which penicillin should be given either by local application, or by the subconjunctival or intra- mscular route. He stated that penicillin should be given for two additional days after a clinical cure had been noted, in order to prevent a remission. Hsiung, Beds and Stafseth (1950) found that calcium peni- cillin ointment was highly bacteriostatic to organisms found in either serous or mlcopurulent discharges from the conjunctiva. There 21 was a marked decrease in the number of bacterial colonies present four hours following the application of the penicillin ointment. Eads (1951b) reported that amorphous penicillin at the rate of 1,000 to 1,500 units per pound of body weight, given from four to six times daily, was an effective agent in controlling the secondary invaders of distemper. He (1951a) also stated that he liked the use of penicillin ointment for local application. III. Serolo cal udie 0 Id ntifi ation 0r Van de Velde (1898) first demonstrated specific aggluti- nation betwoen a univalent serum and its homologous streptococcus. Later Kinsella and Swift (1917, 1918a, 1918b) reported that the classification of hemolytic and non-hemolytic streptococci was de- termined by the complement fixation reactions between the organisms and their antisera. Dochez, Avery and Lancefield (1919) studied the bioloy of streptococci. Antigenic relationships among strains of Strepto- 99.95315 M were demonstrated by them. Four biological types of streptococci were identified by means of the agglutination re- actions and protection. Avery and Heidelberger (1923, 1925) found that pneumococci can be distinguished readily one from another serologically due to the antigenic composition of the capsules. The antigenic complexity of streptococci was studied ex- tensively by Lancefield (1925, 1928, 1933). In 1933 she claimed 22 that hemolytic streptococci can be differentiated serologically. Sic classified 106 strains of streptococci, isolated from man, ani- mals, milk and cheese, into five groups by means of precipitation reactions. The antisera for the precipitation tests were prepared by injection of heat killed cells intravenously into rabbits. The application of the serological method in the differen- tiation of strains of organisms was demonstrated by Hucker (1932). He attempted to utilize the agglutination reaction in the separation of the genera Leuggnogtgg and Strepjggocgg. He concluded that these species showed evidence of a large amount of strain specificity. Stockinger and Carpenter (1944) studied the differences in cross reactivity among the species of Neisseria and indicated that there was an iunnunological relationship between certain strains. The application of serology in the differentiation of strains of W W was reported by flvaro and Mc- Cleskey (191.7) . In their studies , both precipitation and agglutina- tion methods were employed. Of these two methods, the agglutination test was the most useful in showing type relationship. 23 MATERIALS AND METHODS The investigations leading to the present report were begun in June 1943 with the purpose of determining the effective- ness of various therapeutic agents on the growth of the bacteria in the conjunctival sac and nasal cavity in dogs affected with distemper. LW For the purpose of checking the variety of organisms present in the eyes and nose of dogs without distmper, 22 animals were used. Fourteen dogs infected with distemper were chosen for comparison. Swabs were taken along the conjunctivae and nostrils of different dogs and sent to the laboratory imediately with a record as follows: Sample No. Case No. Date Owner Address Breed Age Sex Clinical diagnosis Medication Material submitted These swabs were streaked directly on blood agar plates, and then placed in tryptose broth or smisolid brain-heart infusion. After W incubation at 37°C discrete colonies with different charac- 24 teristics were transferred to tryptose agar slants in order to ob- tain pure cultures for identification. Blood agar slants were used for those organisms which failed to grow on the tryptose agar slants. One loopful of the 24—hour broth culture or the semisolid brain-heart infusion was streaked on another blood agar plate to detect any organism that was absent on the previous allture. The morphology of the organisms from the slants was stud- ied i‘nGram stain preparations. Then the organisms were placed into three groups, namely, gram-positive cocci, gram-negative cocci, and gram-negative rods. Identification and clas sification were based on biochemical reactions according to Bergey's Manual of Determinative . Bacteriology (191.8). Basic media used for the preliminary studies included fermentation broths , nitrate peptone broth, litlls milk and gelatin, which were prepared as follows : Limantaiismm Tristan ------------------ - 1 p. NaCI -------- — ......... - 0,5! madO'liMicator--e--—------- :- 1 ml. Distilledwater -------- ------ .. 100 ' Andrade's indicatorwas preparedbyadding 12.17 111. of 1 11mm into 100 al. of 0.2% squeousacidfuchsin. aw Pam!” -------fl---- ----- an--- 1 a. BBB ----- ----- ..... ---.... 0.2!. Dextrose----- ------- - ...... — 5 ' NSC]. ----- '- -------- ---—-- 5 u Distilledwater ---------—------ 1000 m1. W Bglgtiogl Sulphanilicacid---------------- 8 @- 25 B o: naphthylamine .............. 5 Glacial acetic acid - - - - .......... 250 Diflmfi Hater ---------- - u u - - 7% £5? Equal amounts of solution A and B were added to the allture. A red color indicated a positive reaction. 1M Lit-1s -------- ........... 2 acnmflk .................. 1000 This medium was sterilized at 10 lbs. for 15 minutes. 4. WM (Dirac dehydrated) BQCtO-Mdm-In- ------ ----- 3 a. 5 Bacto—peptons------ ----- ------ " Bacto-gelatin ---------------- 120 " Distilled water ---------------- 1000 m1 Special media were used for further studies according to the different groups. A. Gram-positive cocci with heavy growth on the tryptose agar slants were grouped as W and the following media were inoculated: Nitrate peptone broth , litms milk , gelatin, nannitol fermentation broth, one percent, and monium phosphate agar. The color of the colony of the organism was alum recorded for the sake of identifi- cation. 1 0 1 00 nasal. ------------------ 0.0 Distilledwater ------------- 100 One ml. of 1.6 percent alcoholic brom—cresol plrplswasaddedtolOOOml. cftheabovenedium for the indicator. When the organisms utilised ammonium phosphate as sole source of nitrogen, themedium medilmwanged from plrple to yellow. 26 B. Gran-positive cocci with pin-point colonies and fine youth on tryptose agar or blood agar slants were streaked on blood agar plates. According to their henolytic characteristics they were sub— divided into hemolytic, viridans and nonhemolytic streptococci. The huolytic streptococci were inoculated into the following media: tryptose broth, 6.5 percent sodium chloride, sodinn hippurate broth, lit-1s milk and fermentation broths (lactose , nannitol , glycerol, sorbitel, and trehalose, all one percent). W Tryptose --------- - -------- 2 p, Dextrose - - - - - -------------- 1 " N801 ------- ...... .n..... 0.5 fl Distilled water ---------------- 100 m1, This nediun was used for the determination ofothe growth when the cultures were incubated at 45 G and 10°C. W Tryptosebroth ----- ----------- 100 nl. Sodium hippurate, - - ------------- 1 p. This medium was tubed in four n1. Sodiun hippirate broth cultures were incubated at 37°C for four to five days. The clear broth above the growth was decanted into a second tube. This broth was acidified by adding one drop of concentrated sul- furic acid. Then the acid broth was extracted with twotofivenl. ofether, anddecanted intoathird tube. Two ml. of Zwicker's solution was added to the ether extract. , The needle crystals at the interface meant benzoic acid, therefore hippurate was split by the organisms. O Guwl. 510 percent) ------ - ------- - 40 n1. Pyridm ------------ - I- - I- I- - - 10 II Distilled water ............... 50 I 27 0. Gram-negative bacilli were inoculated into the following media: Lactose motility medium, Kligler iron agar slant , fermentation broth (dextrose, lactose, maltose, mannitol, and sucrose, all one percent), indol medium, citrate agar slants and methyl red Voges Proskauer medium. Gelatin stab cultures and litms milk were used specially for the identification of Bruceyé Mam ngtgge motilitz medium Motility test medium (Difco) - - - - - - - - 1.8 gs. Beef extract (Difco) - - ------- - - - 0.2 " KZHPOA ------------------- 1.0 " Lactose ------------------ 1.0 " Andrade's indicator ------------ 1.0 ml. Distilled water - - - - - - - - ------ 100 " Indgl medium Tryptone ----------- - - - - - - 1.0 " Na01 ------------------- 0.5 " Distilled water ------ - - - - - - - 100 m1. e e e t Paradimethylaminobenzaldehyde ------- 5.0 gm. Amyl alcohol -------- - ------ 75 m1. H01 (concentrate c.p.) ------ - - - - 25 " A red color indicated the positive reaction. V an r te rea e t figlgtign L naphthol -------------- 5.0 gn. Alcohol (95 percent) ----------- 100 ml. Soluti B KOH ------ - - - - - -------- 1.0 gm. Distilled water ------------- 100 ml. 1 1.2 m1. of solution A w 3 added to twa m1. culture which was incubated at 3 G for 21. hours. Then 0.1. ml. of solution B was added to the same tube. A pink or red layer indicated the development of acetyl- methylcarbinol after 10 to 20 minutes. For the methyl red test the culture was incubated for 48 hours at 37°C. Three drops 01' .02 percent methyl red were added to five m1. of culture. A red color indicated the presence of acid. 28 D. Gram-negative cocci were streaked on blood plates for the oxidase test. A one percent para-aminodimethyl-aniline monohydrocbloride solution was poured on the incubated plates and poured off again in- nediately. Colonies of bacteria forming indophenol oxidase turned pink,changing to maroon and finally black. The following mdia were inoculated for the identification of m: Fermentation broths (dextrose, lactose, maltose, mannitol and sucrose, all one percent), nitrate peptone broth, litmus milk, gelatin stab, and indol medium. 11. MW Effectg of Treatment Dogs with a clinical diagnosis of canine distemper in the veterinary hospital at Michigan State College and showing evidence of conJunctivitis or rhinitis with either a serous or mcopurulent discharges were selected for this study. These patients ranged in age from five months to three years. The animals were maintained in the comfortable kennels at the hospital during the period of treat- ment. Daily clinical observations were recorded. Prior to the application of the various treatments , swabs were taken of the conjunctivae and nostrils of the dogs. These swabs were handled as previously described (Part I). Immediately thereafter one of the therapeutic agents was applied and distributed over the corneas,conjunctivae and nasal cavity. At specified intervals after the drug had been adminis- tered such as 4, 21., 1.8, 72, 96, and 1“ hours, swabs were taken 29 and cultured as indicated above. In some cases swabs were taken after one week of treatment to determine the degree of bacterio- static activity of the compound. Eight different kinds of therapeutic agents were used and were supplied through the courtesy of the following compan- ies: ' 1. W (mixture of antiviral and antibacterial sera): Pitman Moore Company, Indianapolis, Indiana. 2. fillfathiazole ointment (five percent): J en—Sal Laboratory, Kansas City, Missouri. 3. figdium flacetamide solution (30 percent): Schering Corporation, Bloomfield, New Jersey. 1.. B ent ointmen (500 units of bacitracin per gram) : Upjohn Company, Kalamazoo, Michigan. 5. W (500 units per gram): Upjohn Company, Kalamazoo, Michigan. 6. Wish (100,000 8 units per m1.): Merck and Compamr, Rahway, New Jersey. 7. P t eni n at (28,600 units per gram): Upjohn Company, Kalamazoo, Michigan. 8. Calcium penicillin ointment (14,300 units per gram): Parke, Davis and Company, Detroit, Michigan. III. Serologigal Studies on Some Strains WM Application of serology in the differentiation of thme strains offleiaamWO-Ddtwo “niacin-1222922211 m var. m was made in this work. Agglutination tests were carried out for this purpose. One strain of 15. W was isolated from dog No. 37, and one strain of M. m var. gm; was isolated from dog No. 7740. H. W (lumen strain) No. 1010005. mm. m (lmman strain) N0. 202were ob- tained from the diagnostic laboratory, Michigan Department of Health, Lansing, Michigan. .11. W Abbott No.5 was obtained from Miss Lisa Neu, Department of Bacterioloy and Public Health, Michigan State College. All these human strains were newly isolated from the threats of different individuals. Was: The antigenswerepreparedfromtheabove culmresgrown on tryptose agu' slants at 37°C for 21. hours, and washed off with 0.5% phenolated saline. The heavy suspension was filtered thread: a cotton filter into bottles with glass beads, shaken and diluted to turbidity No. l of Molarland' s nerheloneter. This turbidity equalled approximately 300,000,000 organises per I1. WW: Each strain of the above organisms was indected into chickens and rabbits for the production of antibodies. These animals were injected intravenously with y'adually increased amounts of 2!.» 31 hour living cultures (0.25 m1., 0.5 101., 0.75 m1., and 1 n1.) as shown in tables 16 and 20. Killed cultures were also used for the production of antiserum of M. W var. we as shown in table 21. These cultures were heated at 65° to 70°C for one hour in the water bath. Injections were made twice a week for a period of three to fourweeks. The animalswere bled sixto tendays afterthe last injection. W8 For the cross agglutination reaction between the differ- ent strains of E. W, antisera from chickens produced by injection with each organim were used. Pour dilutions 1.25, 1-50, 1.100 , and 1-200 , of the antisera were introduced into separate tubes. To the fifth tube negative serum from a normal chicken was added. There was no serum in the sixth tube. The last two tubes were used as controls. One ml. of the antigen was added to each of these tubes. After mixing the contents thoroughly, these tubes were incubated at 37°C. Remlts were read after 24 and 1.3 hours of incubation. Agglutination tests were also set up in the same manner with antiserum No. 37 from rabbits, using antigen of the three strains of H. W. Normal rabbits serum was used as con- trol. In the same manner cross agglutination tests on u. m- m var. m from dogs and man were made. lntisera from rabbits produced by injection of living and killed cultures were used. Normal rabbit serum was employed as control. 32 Culture from the eyes and nose of 22 dogs with diag- noses other than distemper were used for comparison with those of ll. dogs infected with distemper during the same season. The organ- isnsfoundineachcasearerecordedintableslandZ, andthe comparison are shown in table 3. TABLE 1 01101101348 FOUND IN OCULAR AND MEAL DISCHIRGES 0F 22 DOGS IVDT AFFECTED WITH DISTMEB Amount Case Clinical of No. Age Sex diagnosis g‘owth Organisms found 5686 * M Foreign body I M. Diogenes var. allns intestine M. candidus M. epidermidis 7037 ll. mo. F lie-nary neo- x M. citreus plasm 7131 2 yr, M Castration x M. pyogenes var. albus . M. candidus Henolytic strep. group C 7193 15 no. M Radial x Heulytic strep. paralysis group C * Owner unable to give correct age Case N0. 33 um 1 (Contimed) Clinical diagnosis Amount of growth Organisms found 71.21 7454 7455 7469 7524 7565 7579 7640 10 wk. 10 wk. 531‘. Fractured humerus Ear trim Eartrim Helminthiasis Fractured tnmems Eartrim Helminthiasie Chorea (or- with discharge) * Owner unable to give correct age M. pyogenes var. albus M. pyogenes var. aureus M. varians M. epidermidis H molytic strep. group D M. pyogenes var. albus Hemolytic strep. group C M. pyogenes var. aureus M. sp. Hemlytic strep. group C N. catarrhalis M. pyogenes var. albus M. epidermidis M. flavus Hemolytic strep. group C group D M. epidermidis M. candidus Hemolytic strep. N. catarrhalis M. pyogenes var. albus Nonhemolytic strep. Ps. aeruginosa M. pyogenes var. albus M. pyogenes var. albus M. candidns M. surantiacus Hemolytic strep. Nonhemolytie strep. Shigella 8p. 31+ TABIE l (Contimed) Amount Case Clinical of No. Age Sex diagnosis growth Organisms found 7645 3 yr. Ovariectom x M. pyogenes var. albus 7685 3 yr. Eye with xx M. flavus serous dis- Nonhemolytic strep. charge A. aerogenes N. catarrhalis 7688 2 yr. Helminthiasis x M. pyogenes var. albus I. epidermidis 7703 4 yr. Eczema x M. yogenes var. albue 7708 * Fractured x M. aurantiaous tibia Hemolytic strep. group C N. catmhalis 7726 1 yr. Castration x M. epidermidis M. flavus Hemolytic strep. group C 7734 8 yr. Dermatitis xx M. epidermidis M. pyogenes var. albus namely-tic strep. group C Viridans strep. 771.4 9 yr. Dermatitis xx M. epidermidis Bemlytic strep. group C N. catarrhalis l 1 yr. Normal dog 1 M. candidus la 2 mo. Ear trim x M. pyogenes var. albus M. anrantiacus Note: haunt of growth in the area of inoculation on blood agar plates indicated by: non: Numerous colonies xxx Less than 500 colonies * Owner unable to give correct age xx Less than 100 colonies x 140 colonies ‘ 35 TABIEZ ORGANISiS FOUND IN OCULLR AND NASAL DISCHARGES OF 14 UNTRENI‘ED DOGS AFFECTED WITH DISTD’IPER Case Clinical of N0. 186 Se: diagnosis growth Organisms found 771.0 1 yr. F Distanper Bemolytie strep. with con- group C vulsion N. catarrhalis 769) 7 yr. F Distemper and M. pyogenes var. albus pneumonia M. aurantiacus with labored Br. bronchiseptica breathing 7618 4 yr. F Distemper with M. epidermidis severe con- vulsion 1 Snell F Distemper M. pyogenes var. albus Br. bronchiseptica 2 Small F Distemper M. pyogenee var. albus Nonhemolytic strep. Br. bronchiseptica N. catarrhalis 3 * P Distemper M. pyogenes var. albus Hemlytic strep. group C N. catarrhalis I. * M Distemper Hemolytic strep. group C 5 * M Distemper M. pyegenes var. albus Hemolytic strep. group D E. coli N. catarrhalis * Age unknown These dogs were obtained frc The Humane Society 36 TABIE 2 (Continued) Amount Case Clinical of No. Age Sen diagnosis growth Organisms found 6 1" M Distemper xxx M. pyogenes var. albus M. sp. E. coli A. aerogenes 7 3 yr. F Distemper xx M. pyogenes var. albus 8 * F Distemper xx M. pyogenes var. albus M. pyogenes var. aureus Nonhemolytic strap. 9 * F Distemper mo: M. epidermidis Hemlytic strep. group C l. aeroganea Pr. mirabilis Br. bronchiseptica 10 * M Distemper x Viridans strep. 24 * M Distemper xxx M. pyogenes var. albus Henclytic strep. group C Pr. mirabilis Note: Amount of growth in the area of inoculation on blood agar plates indicated by: xxx: Numerous colonies xxx Less than 500 colonies xx Less than 100 colonies x 1.20 colonies *Ageunknown These dogs were obtained from The Humane Society 37 TABLE 3 COMPARIEJN 0F BACTERIAL FIDRAE OF DISTEMPE , INFECTED AND LDNINFECTED DOGS Name of organism Percentage of dogs found with the organisms 11. infected dogs 22 noninfected dogs A. W M. pyogenes var. albus 64.2 59.0 M. pyogenes var. aureus 7.1 9.1 M. aurantiacus 7.1 13.1 M. candidus - 22.7 M. citreus - 4.5 M. epidermidis 14. 3 36.3 M. flavus - 13.6 M. verians - 4.5 M. 8p. 7.1 9.1 B. W Hemolytic strep. group A - - group B - ' - group G 35.5 40.8 group D 7.1 13.6 Viridans strep. 7.1 27.2 Nonhemolytic strep. 14.3 13.6 C. Willi Br. bronchiseptica 28.5 - E. coli and A. aerogenes 14.3 4.5 Pa. aeruginosa - 4.5 Pr. mirabilis 14.3 - Saigella sp. - 4.5 D. W N. catarrhalis 28.5 22.7 Note: Identification of the organisms was based on biochemical reactions acco to Bergey' s Manual of Determinative Bacteriology (192.8 38 II. Wicca of Bacterial Flores Found in Distm Infected Dogs During Different Seasons According to the clinical records of the veterinary hospital at Michigan State College, 713 dogs were found to have canine distemper during the period of one year from June 1948 to Mu 1949. The umber of cases reported as canine distemper in each month is indicated in table 1.. The seasonal incidence and enzootic period of distemper during the winter season, from Nov- ember to April, is shown in figure 1. The various organism found in terms of percentage of cases (hiring this period are shown in table 5. TABLE 1.. NEEBER OF GAES 0F CANINE DISIEMPER IN VARIOUS WEEKS ' umber of cases Number of cases Month Year per month per day June 1948 46 1. 53 July 1948 24 .77 August 1943 24 .77 September 191.8 16 .53 October 191.8 25 .81 November 1948 64 2.13 December 1948 34 2.71 Jamar: 1949 1.14 3.68 rebruary 1949 81 2.89 March 1949 99 3.19 April 1949 81 2.70 HI? 1949 55 1.77 39 . TABLE 5 VARIOUS ORGANISMS FOUND IN DOGS INFECTED WITH DISI‘EMPEB DURIM} DIFFERENT SEASONS Percentage of dogs harboring the various 022m Sum Fall Winter Spring (June—Aug.) (Sop-3.3”) (Jan-Mar. ) (Main-May) Nana of organisms 1948 48 1949 1949 1. W M. pyogenes var. albus 73.3 44.4 100.0 62.5 M. pyogenes var. aureus 3.3 11.1 - - M. anrantiacus 10.0 22.2 54.5 12.5 M. candidus 10.0 11.1 36.3 12.5 M. conglomeratus - - - 12.5 M. caseolyticus 3.3 66.6 9.1 12.5 M. epidermidis 30.2 22.2 27.2 12.5 M. flma 6.6 2202 9.1 ‘- M. freudenreichii - 11.1 - - M. luteus 3.3 11.1 - 12.5 M. varians 3.3 33.3 18.1 - B. W Hmlytic strep. group A - - .. .. group B 3, 3 .. .. .. mp c 3606 22.2 54.6 - group D 36.6 22.2 91.0 .- Viridans strep. 13.3 55.5 18.1 62.5 Nonhemolytic strep. 30.0 66.6 27.2 25.0 C. G Br. bronchiseptica 16.6 33.3 54.5 50.0 E. coli and 1. aerogenee 20.0 33.3 36.3 12.5 P8. aeruginosa 3.3 - - - Pr. mirabilis 6.6 11.1 - 12.5 flagella. 81). 3.3 11.1 - - D. G t i N. catarrhalis 33.3 - 9.1 - Note: Identification of the organisms was based on biochemical reactions accord to Bergey's Manual of Deteminative Bacteriology (191.8 III. WW Effectg Q: Tregtment A study of the effects of local treatment with various therapeutic agents in canine distemper was made on 46 cases. The amount of growth on blood agar plates made before and after treat- ment, is indicated by a number of Is on an arbitrary scale. In all, swabs were examined as follows: 1. W Distemper serum was drapped into the eyes and nose of four dogs infected with distemper. The organisms found before and after the treatment are shown in table 6 and the ineffectiveness of local serum therapy is indicated in figure 10. 2. wgthiggglg ointment (five percent) This drug was used on seven dogs which showed some degree of clinical improvement. The results obtained from this agent ap- plied locally is shown in table 7 and figure 10. 3. W (30 percent) This solution was used as eye and nose drew on four dogs affected with distemper. Not very such evidence of improvement was shown clinically. The results are indicated in table 8 and figure 10. 4. WM (500 units of bacitracin per gram) The drug was applied to the conjunctivae once daily for three days. No change was apparent during the first 1.8 hours but there was a sudden drop in gowth after the third application in case No. 5708. The results are shown in table 9 and figure 10. 5. W (500 units per gram) Six dogs were treated with this ointment. There was some evidence of gradually decreasing powth. The activity of this drug is shown in table 10 and figure 10. 6. W (loqooo S units per m1.) This solution was dropped into the nose and eyes of four dogs infected with distmper. Clinical observations showod very slight improvement after treatment and this was not constant. The antibacterial activity of this agent is indicated in table 11 and figure 10. (28,600 units per gram) This compound was used on five dogs. The results are shown in table 12 and figure 10. Not very much effectiveness can be seen in these five cases. 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SEROLOGICAL SI'UDIES 0N NEISSERIA CATARRHALIS AND MICROCOCCUS PYOGENES VAR, ALBUS ISOLATED FROM THE DOGS AEEECTED WH‘H DISIEME’ER 1. W The biochemical reactions of the two human strains (cul- tures No. 101 and Abbott No. 5) and one strain from dog No. 37 of y. W are shown in table 15. The period of immization of the animals for the production of antisera and the time of bleeding are shown in table 16. Tables 17 and 18 indicate the results of the cross agglutination reactions or antisera from chickens and rabbits respectively. Although there is definite cross agglutination be- tween strains No. 101 and Abbott No. 5, there is no evidence that the two human strains of E. W: and the strain isolated from dog No. 37 are antigenically related. Table 17 indicates that the antisera from the chickens give stronger positive results than the antisera from rabbits (table 18) used in these agglutination tests. 2. W: m m. aims in. biochemical reactions of the hmnan strain (calms No. 202) and strain No. 771.0, isolated from a dog, are shown in table 19. Only rabbits were used for the production of the antisera for these two organisms. Table 20 and 21 show the period of immunization with living and killed organisms and time of bleeding of the animals. The results of agglutination are shown in table 22. Here again it is indicated that there is not nuch serological relationship between these two strains of u. m var. m. 58 TABLE 15 BIOCHEMICAL REACTIONS OF THE THIEE SIRAINS 0F NEISSEIRIA CKI‘ARRHALIS Culture number Media 101 Abbott 5 37 (human origin) (human origin) (canine origin) Oxidase test + + + Dextrose — .. .. Lactose - - .. Maltose — .. - Mannitol - .. - Indol - .. - Litms - — - Nitrate + - I + Note: + Positive reaction - Negative reaction These three strains are all gran-negative diplococci. They are nonhanolytic with big colonies on blood agar plates. On first isolation from dogs ,thqshowed abundant growth on tryptose agar slants. The human strain showed only scanty growth on primary isolation. 59 _ Fl H H H H H H H mb. 2.. mm. I-‘lI-lr-IHu-I nip. mm. Ht-ir-IHI-l mp. um. HHHHHHHH mb. mm. HHHHH mp. mm. r-ll-ll-lr-IH mp. mm. HHHHHI—‘IHH HHHHH mb. mm. Ht-lr-lc-lt-Iu-l't-lc-I mp. on. 83 8:34 cannula 8:24 Rona RIMIH mTomnNH 3:84: SAXAH 313:3 matmumfl Sue-ma 3,-8.3 mimufi A8453 hm Agatha gEv 3.3534940 amamez E: a ho BQSHE 0H as h H sounds A35 m “530.4 m m .. 4 notes Ass-=5 8H .3 .932: no 33 don: Hid: oohnoo mama»: “9on HHHHHH mb. “0 mm. HHHHI-lr-l HHHH HHHHH EHm H H H H H lelm omlmum RIHIm omlbmlm von , RIMNIN H RIbHIN H RIMHIN H RnoHnm H omuonm H leum nfipgm 38a 8 .83“ 83 8.84 m .Ha 9“ n3 $83 no 325 n 4 noxuflno con: 35 m #vopnd. And-55 3.38 «833:4 HOH 38388 «H Em: R 53% 8395 sec «888m 28: m as o ,.o 2.833 .m 335 83.23 5h @888“ 98: m 3a m .m 8833 3H 35. 5638 8h cooaxfi 8:. n 3. 4 93:3 downs?" 0350mm: I Hag—8n « Inno- oz 0 3303.» 3.3.."qu paw—"Hm + 558 330% .3332 a 33039 0533A woman: .1. 3503 .Ho 8303.." 05363 9893 .11 .302 II I + ++ .11. I... I I I I II II I I man—.3935 II + ... ++ .71. I I I I I I II II I I cgndvg I I + ++ ++ +++ I I I I I I I I I I I I o 55003.5 I I I I I I I I I I I + I I H + + + h gnaw: I I I I I I I I I + ++ .71. I I + + ++ +++ H “Panda—.5 I I 2? .Ho .Ho .H9 I I .1 .1 .Ho .Ho I I + ++ .1 ..H0 m Iguana»: I I I I I I I I I + ++ .11 I I I + ++ +++ d 5503qu A62 Hausdv .oz 550m com 03 can can con 8H Hogan—6 550m o a IH IH RIH «NIH o a IH IH omIH mNIH o n IH IH RIH mmIH hm. m 3.39.4 HOH a EH8 NE madman—Ho flan—”E ho gum gm. a fig 2384a HOHHHHHDHGS mug 5H an. 62 .3398... 33.502 H3339 H H.333." 05$qu .333 + gen 02 o 303.." cinnamon woman: .1 I980 p.340." Haoz a H3903." 03338" 93.56 .I.+ u 302 II+ + + + III I I I II I I I I 0.33.3 pm 35333 III I .1. I III I I I II I I I I magnum hm 3.98de III I H + III I I I II I I I I dfifipun pm 5.5335 A62 35.un .02 550m com 09” com 8H com 09” nfipsg gm on In IHRIHmNIH oz IHIHOmIHmNIH oz IH IHomIHmNIH Agv Agv Aggy cannon mama»: hm m 3.35 . HQH mama 893 km Egg EH: 3840 flag ho mg an. E9 ho mag HOHHHZHSAGS 3” H.849 63 TABLE 19 BIOCHEMICAL REACTIONS OF THE TWO STRAINS OF MICROOOCCUS PIDGENES VAR. ALBUS Gulmrcnumber Media 202 771.0 (human origin) (canine origin) Anoninn phosphate - .. agar Nitrato broth 4- + Gelatin liquefication + (24 hours) + (1.8 hours) Lima nil]: acid and curd acid and curd Indol — - Dextrose + + hotel. 4- + Melton + + Hannitol + (one weak) 4- (one weak) 3163080 4- + Glycerol + + Baffin” + + Note: + Positive reaction - Negativa reaction Both organism are gran-poaitiva cocci , hoavy whitish growth on tryptose agar slants, and alpha hemolytic colonies on blood agar plates. 64 mb. 3. ma. nu. mm. ma. mb. mm. mm. 83 8.34 H 8.3.“. a. and: H 8.3 E. 8.3 m. 8.1 «a. 84.3 mm. 033 33.5 8F HH .1. 5 8333 «a 38 Ans—.35 conga domfihd ABBHHB Ed 39: 51> mmzmoowm goo8omo§ FD”: manna B ZOHSHEEZH 839 65 1‘ OOHm HMIbHIM H H H H Hmlmlm H H H H H33 H H H H Hnlmmlm H H H H Hnloalm H H H H HmIoHrN H H H H H33 H H H H Hmualm H H H H Hmlwlm m m . m . m H34 .H3 «H :oHfio a: no 8.8 H: HHP Hp > Honda 153 H333 . H855 833 83.3 SE «8 Ammmanbo QHHHHMV mama! .mu> mmzuwomm mDoooooflUHzanH3.maHmm