AN ANTIBIOTIC APPROACH TO THE PROBLEM OP PULLORUM DISEASE By Abe Pital A THESIS Submitted, to tbe School of G-raduate Studies of Michig State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology and Public Health Year 1952 ACKNOWLEDGEMENTS The writer wishes to express his appreciation to Dr. E. H. Lucas for his help and guidance throughout the course of this study. The w r i t e r w o u ld also like to express his thanks to D r s . H. J. Stafseth, R. Czarnecki and A. C. Groschke for their valuable suggestions and constructive criticism. The w r i t e r is also grateful to those individuals who assisted h i m during various phases of this study* TABLE OP CONTENTS Page 1 INTRODUCTION 2 REVIEW OP LITERATURE ....................................... » MATERIALS AND M E T H O D S ....................................... 28 In Vitro Experiments .......... • ~K~, Sensitivity d e t e r m i n a t i o n s .................... Bo Antibiotic stability in tbe presence of feed constituents. . . . < > ....................... Co Effect of penicillin on S. p u l l o r u m . . . . o . D. The enhancement of penicillin action by cobalt against S.. pullorum . . • • • • . . • . 28 28 I n Vivo Experiments. . . . . . . . .................. 7T* P e e d . . . . . . B. General arrangement of each experimental s e r i e s ............ . . . . . . . .............. C. Preparation of the i n o c u l u m . .................. D. Meth od of antibiotic t h e r a p y ................ E. Preparation of an antigen-mash . . • • • • • • P. Met ho d of cobalt administration to chicks. • • G. Recovery of the infecting organism • • • • • . 37 37 RESULTS AND DISCUSSION 32 3!? 38 38 38 39 If0 iji if2 .................. The Sensitivity of £3. pullorum to Aureomycin, Chloromycetin and S t r e p to my ci n . Iflf The Sensitivity of S., pullorum to Penicillin . . . . If7 The Enhancement of Penicillin Action Against _S. pullorum by Proper Concentrations of Cobalt. . • 63 The Sensitivity of £>. pullorum to Garlic Extract . . 68 The Stability of Aureomycin, Chloromycetin, Streptomycin, Penicillin and Garlic in the Presence of Various Feed Mixtures, • • . • • • • a . . . . . 68 A n In Vivo Evaluation of l£ Experimental Series. • • A. General considerations ......................... B. Specific considerations.................... J6 76 79 TABLE OF CONTENTS CONTINUED Page The Percentage of Carrier Birds Produced by Treatment with. Aureomycin, Chloromycetin, Penicillin and Penicillin plus C o b l a t ............. 87 SUMMARY-........................................................90 BIBLIOGRAPHY ................. '..................... II4.8 INTRODUCTION Serious outbreaks of pullorum disease still occur among young chicks despite all protective measures* It is In such instances that a reliable antibiotic agent would be of immeasurable value. The use of antibiotics in the control of pullorum disease can be considered from several important aspects. First of all, the drug should lend itself to oral admin­ istration (In feed or water), since it is obvious that methods involving the injection of a therapeutic agent are inefficient when applied to practical situations. Secondly, the antibiotic must maintain its potency under a variety of conditions and should possess a demonstrated affinity for the infecting organism. Important, Finally, and most the antibiotic agent should eliminate the organism from the body of the host that survives infection. Drugs which merely suppress the symptoms without elimin­ ating the infection only serve to perpetuate the cycle of transmission. The propagation of the carrier state is ultimately far more serious than the losses that might be sustained, from non-treated flocks. The present problem was undertaken with the above considerations in mindo REVIEW OP LITERATURE I. General The causative agent of pullorum disease was first isolated by Rettger in 1900 (98)0 He described the con­ dition as a fatal septicemia in young chickens. Rettger, Kirkpatrick and Jones In 1911+ (100) established the cycli­ cal transmission of this disease and emphasized the importance of infected ovaries in producing the carrier state among recovered birds. In 1915 Rettger ejb al (99) announced the practical utilization of a macroscopic tube agglutination test for the detection of infected birds* Hinshaw, Upp and Moore (52) definitely proved that incubator transmission of pullorum disease was a serious control problem. Runnells, Coon, Parley and Thorp (109) in 1927 intro­ duced the rapid agglutination test as a diagnostic aid. This test was soon followed by a simplified modification devised by Bunyea, Hall and Dorset (16). In 1931 Schaffer, MacDonald, Hall and Bunyea (111) and Coburn and Stafseth (23) proposed a rapid test employing whole blood with a stained antigen,, Insko (5 7 ) in 19 i+l stressed the value of formaldehyde as a fumigant, but warned against the use of this method as a substitute for adequate sanitation. 3 I n 19lp- Youmie (131) reported the existence of sero­ logic variants in naturally infected flocks* The problem of antigenic variants was further investigated by Edwards and Bruner (36) who found that variants were stabilized with larger amounts of the X I I 2 factora The effect of diet upon the eventual course of pullorum infection has been sporadically investigated throughout the years, but has never yielded any conclusive results* Rettger (100) indicated that sour milk feeding had a beneficial influence on the growth of chicks and lessened mortality from all causes. However, he doubted the value of sour milk as a therapeutic measure for pullorum disease 0 Roberts, Card and Severans (103) maintained that environ­ mental factors influence resistance and susceptibility to infection in a variety of diseases* They demonstrated that one type of feed may cause a greater mortality than another, both among chicks inoculated with Salmonella pullorum and among non-inoculated birds. I n one experiment 80*5 percent of the inoculated chicks fed a chick m ash survived, while only 1 9 «7 percent survived among chicks fed a laying mash* I n experiments upon non-inoculated chicks, 98*1 percent of those fed a chick mash lived, while a survival rate of 71*8 percent was attained in those fed a laying mash. The two feeds differed in proportions of nutritive and of fibrous material. A recent and more intensive study on the effect of k r diet in pullorum infection has been conducted by M a n n (7 8 , 7 9 , 80). He maintained that pullorum disease could not be sufficiently controlled by blood testing, w h e n feeds of 20 percent protein content were fed to young chicks as the sole diet. Furthermore, M an n presented a new concept of the infective process as encountered in the usual cases of pullorum disease. Salmonella pullorum was regarded as a potential pathogen which must exist in a symbiotic relation­ ship with other organisms in order to produce a pathogenic complex. Organisms of the "welchii type 11 are considered as the principal symbionts and a chick diet for the initial suppression of such symbionts is proposed. During recent years the modification of intestinal flora by diet and antibiotics has occupied the attention of many investigators. The relationship of the intestinal flora to specific disease processes is still a problem of intensive research. Hull and Rettger (f?5>) in 1917 stated that lactose, milk and mixed grains are specific articles of the diet which exert an influence on intestinal bacteria. They found that feeding of lactose to white rats for a three-day period, brought about a complete transformation of the intestinal flora. They also found that the feeding of a high carbohydrate diet to typhoid patients tended to reduce the putrefying types of bacteria and encouraged the emergence of "acidophilic 11 forms. Johansson, Sarles and Shapiro (£9) studied the effect of various carbohydrates in a biotin-deficient ration upon intestinal bacteria of chickens. They found that dextrin stimulated the development of large numbers of coliform bacteria. Lactose-containing diets also encouraged a fecal coliform flora, but lactic acid bacteria proliferated more extensively in the intestines of birds fed such a dieto The effect of feeding sucrose as the principal carbohydrate produced a marked depression on fecal coliforms. Birds on a dextrin diet appeared to have the greatest numbers of microorganisms at all levels of the intestinal tract, Romoser, Shorb, Combs and Pelczar (107) reported on the effect of diet composition and antibiotics on cecal bacteria and growth of chicks. Aerobacter aerogenes appeared to increase in the ceca of chicks fed penicillin at a level of 15>0 ppm, and increased further when a diet containing both penicillin and lactose was given. The addition of lactose and procaine penicillin to the diet produced a greater response in chicks than when penicillin alone was used, Groschke and Evans (14-7) found that streptomycin and aureomycin exerted a definite stimulating effect on growth of young chicks. Streptomycin brought about a marked decrease in the coliform count of chick feces. It is suggested that the growth effect produced by antibiotics is mediated in some way through a changed intestinal micro­ flora. Anderson, Cunningham and Slinger (3) studied the effect of diets containing 17 , 2 0 , 2 3 , and 26 percent protein on the cecal bacterial flora of chicks. These diets were 6 employed with, and without antibiotics. An increase in protein content decreased the count of anaerobic and microaerophilic types, while aerobic forms remained constant. Penicillin lowered the pH, Both aerobic and anaerobic groups of organisms were influenced (increased count) by penicillin up to a protein content of 23 percent, by a definite decrease with 26 percent* predominated over proteolytic types. followed Aciduric forms Penicillin enhanced the coliform groups and depressed the enterococcal types. The use of aureomycin decreased the counts of both aciduric and proteolytic organisms with proteolytic forms being more inhibited, Coliform counts were also increased w ith this antibiotic, but the significance was reduced at a 23 percent protein level followed by a slight decrease with 26 percent. The research being conducted on antibiotic feed supplements is voluminous and at the present time there is no proved explanation for the interaction between anti­ biotics and the microflora of normal animals. Recently Juices (60) attempted to summarize the various viewpoints regarding the growth-promoting effects of antibiotic feed supplements. According to this summary two theories are currently popular. One theory assumes that antibiotics have a direct vitamin effect on the animal, while the other holds that antibiotics act indirectly through an effect on the microorganisms in the intestine. The second theory is more widely accepted at the present time and is based on several assumptions: (1 ) toxin-pro- 7 ducing bacteria are eliminated, are eliminated, (2 ) competing microorganisms (3 ) vitamin-producing microorganisms are favored and finally (I4.) beneficial changes occur in the chemistry of the microorganisms. The second theory is supported by the observation that growth-stimulating effects of antibiotics are most pronounced w hen the animals are kept under unsanitary conditions and are suffering from subacute intestinal diseases. The effect of antibiotics on protein requirements of chicks and other animals is still under investigation — in some cases antibiotics have lowered the protein require­ ments of the animal. Some investigators believe that the response to an antibiotic is partly dependent on the type of feed ration employed. The composition of the microflora present in the intestinal tract also constitutes one of the many variable' factors affecting the response to a particular antibiotic. There is found among the many breeds of domestic fowl, a variability in resistance and susceptibility to disease. There not only exists a b r e e d difference, but also a variation in genetic makeup within a particular breed, Stafseth and Weis ne r (121) reported a breed difference in susceptibility to a variety of diseases. During an eight-year period of observation they found that White Leghorns were more susceptible to a wide range of diseases, while such breeds as Columbian Rocks, Dominiques and Black Minorcas were more resistant. 8 In a study of selection for resistance to fowl typhoid, Lambert (6 7 , 6 8 , 6 9 ) found that the results from five generations of selection showed a marked increase in resistance among the selected birds. The observed mo rt a l ­ ities in the selected stock from the first to the fifth generation were as follows: 3 9 *8 , 2 9 «3 » 1 ^. 1+j l £®0 and 9»1+ percent. In the unselected birds (controls) the respective mortalities were 8 9 *6 , 9 3 *2 , 8 6 .2 , 8 6 .14. and 85>.0 percent® Lambert also demonstrated a breed difference in sus­ ceptibility to artificial infection with the organism producing fowl typhoid« The Rhode Island Red and White Wyandotte chicks showed the greatest rate of mortality as well as total mortality, while the white Plymouth Rock chicks exhibited the slowest rate and the least total mortality® The White Leghorn was found to be the most resistant parent breed® In experiments designed to ascertain breed suscepti­ bility to infection with S_. pullorum, Hutt and Scholes (£6 ) noted that White Leghorns were always more resistant to artificial infection with £3. pul lo ru m® Leghorn chicks not artificially infected, but exposed to the disease by contact were much more resistant than Rhode Island Reds and Barred Rocks exposed in the same manner. Hutt and Scholes concluded that resistance to £>. pullorum is a characteristic of the Leghorn breed. DeVolt, Quigley and Byerly (30) concluded from their work, that strains of relatively pullorum-resistant chickens 9 can be developed b y artificial selection and strains of relatively resistant chickens develop by natural selection in tbe presence of natural infection* Roberts and Card (102) in a ten-year genetic study of resistance to pullorum disease found tbat heredity Is an important factor* of the following: strains, Evidence for hereditary factors consisted (a) selection produced more resistant (b) selected stock was consistent in maintaining resistance through successive generations, is dominant to immunity, (c) resistance and (d) blood differences occur in resistant birds such as increase of erythrocytes and decrease In ne u t r o p h i l e s * I n a further study Severans, Roberts and Card (118) reported the following significant facts: in the number of lymphocytes (a) a decrease (by X-ray) produced a decrease in resistance to pullorum disease (b) the spleens of resistant chicks were larger than those of susceptible chicks, (c) a removal of the spleen from resistant chicks produced a decrease in the number of lymphocytes w ith a consequent reduction in resistance, level possessed by chicks, at the time the infecting organism reaches the blood stream, of resistance, (d) the lymphocyte determines the degree and (e) the temperatures of resistant chicks were slightly higher than those of susceptible chicks* Bell (5>) investigated the physiological factors associated w it h genetic resistance to fowl typhoid. findings reveal that the ability of polymorphonuclear His leucocytes to digest phagocytosed bacteria appears to be a major factor in genetic resistance to Salmonella gallinarum* Differences in body temperature also play an important supporting role* More recently Lerner and Taylor (70) obtained evidence for genetic differences in resistance to a respiratory disease among chickens, tentatively diagnosed as atypical infectious coryza* II* Sulfonamides The use of chemotherapautic agents in the control of poultry diseases has mainly been limited to the sulfonamides* Delaplane (29) in 19^4-1 successfully treated infectious coryza with sulfathiazole• He emphasized the fact that sulfathiazole was bacteriostatic rather than bactericidal* Prior to the work of Delaplane, toxicity studies were conducted on some of the sulfa compounds (8 l, 1 0 1 ). Richardson found that the daily administration of 0*5 to 2*0 g of sulfanil amide per kg to mixed breeds of chickens produced an intense cyanosis within four to five days* This was attributed to the conversion of hemoglobin into methemoglobin by sulfanilamide* Severans, Roberts and Card (117) tested sulfonamides with respect to their efficacy in reducing mortality from pullorum disease. Sulfadiazine and sulfamerazine were found to be the most effective, Sulfasuxidine, phthalyl- sulfathiazole and sulfanilamide were the least effective, 11 while sulfathiazole and sulfaquanidin© ware intermediate• Female birds (treated with; sulfadiazine and sulfamerazine at one day of age) reacted negatively to pullorum agglutin­ ation tests at nine months of age* Mullen (8 6 ) fed sulfamerazine to poults from pulloruminfected flocks at a concentration starting mash. of 0 .5> percent in He recorded beneficial results in mortality reduction, but w a r n e d against use of the drug for periods longer than five days. After five days the treated mash had an adverse effect. MacNamee (77) reported good results with sulfamerazine administered in the drinking water at a concentration of 0.5> percent. Medication after the fifth day offered no beneficial results. Sulfamerazine was also used prophy- lactically. Bottorff and Kiser (13) evaluated sulfadiazine, sulfamethazine and sulfamerazine against pullorum disease in day-old chicks. sulfadiazine, Experimental trials revealed that sulfamethazine and sulfamerazine were equally effective in curtailing mortality due to pullorum disease. Xn the treated groups the minimum reduction in mortality was 30 percent and the maximum £6 percent. There was no significant weight gain at twenty-one days in the treated groups and no evidence of toxic effects was noted. About 90 percent of the surviving chicks reacted to the rapid whole blood testa 12 Beneficial effects were obtained by H o l t ma n and Fisher (5 3 ) with, sulfonamides in the treatment of fowl typhoid® Failure of sulfamerazine therapy to control pullorum outbreaks in chicks has been reported b y Petersen (89)0 Considerable p r o t e c ti on was afforded b y a 0.2 percent concentration of sodium sulfamerazine to young chicks i n ­ fected orally and b y atomizing £>. pullorum in the brooders Roberts, Card and Alberts (2 ), (IQlj.) stated that adminis­ tration of sulfonamides in the drinking w a t er did not p r o ­ duce quite as good results as w h e n g iven in dry feed. They u r ge d treatment w i t h sulfonamides immediately after exposure. Adverse effects upon egg pr od uction following sulfa treatment has been noted b y Bankowski (I4.) , Nine sulfonamides were evaluated by Pomeroy, m ac he r and Roepke sulfapyrazine, (92), Sulfadiazine, Fenster- sulfamerazine, sulfaquinoxaline and sulfamethazine were the most effective against p u l lo ru m disease in chicks, Sulfasuxidine, sulfathalidine and combinations of both drugs were ineffective. Birds that survived the infection w i t h drugs continued to react positively to the rapid whole b lood agglutination test at five months of age, Roberts, Eisenstark, and Alberts (105) demonstrated that S, p u l l or um could be adapted to grow in the presence of sodium sulfamerazine. The adapted pu llorum strains were found to be more virulent for chicks than the non-adapted strains, Cole (2 I4.) and D ic kinson and Stoddard (31) could not 13 eliminate p u l l o r u m infection from yearling hens by p r o ­ longed feeding with, sulfa drugs* Swales (126) contended that the use of sulfonamides to reduce mo r ta li ty from p u ll o r u m disease cannot be justified in Canada* He stated that birds w i t h suppressed infection remain carriers* Chang and Stafseth (21) found that normal chicken serum w a s bacteriostatic for S* p ul lo r u m and g reatly enhanced the antibacterial activity of s ulfadiazi n e 0 Schweinburg and Rutenberg (111}.) justify the use of sulfonamide mixtures only w h e n in vitro sensitivity tests demonstrate that the components of the mixture either possess an additive or potentiating effect. They suggested that additive effects may fu nction in the m a jority of coccal infections, but not for infections due to gram-negative organ!sms• X n a review of therapeutics, Biester and Schwarte indicated that such compounds as chinosol, metaphen, furic acid, hydrochloric acid, mercuric chloride, p ot assium permanganate, (7) sul­ resorcin, sulfocarbolates and hypochlorite solutions have b ee n found to be without any beneficial effect in treating pullorum infection* The sulfa compounds were the first to exhibit promising results in reducing m o rt a li ty among young chicks* III * Au re omycin - Chloromycetin - Streptomycin A l t h ou gh the n ewer antibiotics have been used in the Illfield of veterinary medicine, their application in con­ trolling poultry diseases has been rather limited. The use of newer antibiotics in reducing pullorum infection has been limited almost exclusively to streptomycin. The number of experiments reported with this antibiotic is limited. Aureomycin was discovered b y Duggar (32) during a program of intensive investigation to detect antibioticproducing organisms. Collins, Paine, Wells and Finland (25) evaluated the use of aureomycin against Salmonella typhos_a, various severe salmonella infections and a colon bacillus bacter­ emia. The clinical and bacteriologic findings suggested that aureomycin h a d some beneficial effects, but the results in general were not strikingo Brainerd, Lennette, Meiklejohn, Bruyn and Clark (lip) stated that aureomycin appears to exert a greater or lesser suppressive effect on the infecting organism without p ro ­ ducing its complete destruction. Beneficial effects from aureomycin appeared to be limited or absent in typhoid, Salmonella, and Shigella infections. Aureomycin produced beneficial results in the rickettsial diseases and in brucella infections. The sensitivity of five enteric strains of gramnegative bacilli to aureomycin was determined by Alexander, Leidy and Redman (1). They found that all of the strains were completely Inhibited by less than 25 micrograms of i5 aureomycin per ml of* broth, medium,, In a series of* in vitro experiments, Jackson, Gocke, Collins and Finland (£8 ) showed that aureomycin inhibited 91 percent of 3 5 strains of Salmonella typhosa within a range of 1 2 .5 -25 micrograms of aureomycin per ml of medium. Fifty-five Salmonella strains (except £>. typhosa) including S. pullorum were sensitive to aureomycin within a range of 25-50 micrograms per ml of broth medium. Aureomycin was fifth in order of diminishing activity for S>. typhosa and other salmonellae among seven antibiotics tested. There did not seem to be any definite correlation between the sensitivity of any given strain to one antibiotic and its sensitivity to any of the others — S3, pullorum appeared to be an exception in that it was more sensitive than the other salmonellae to all of the antibiotics tested. Knight, Sanchez, Sanchez, Shultz and McDermott (6 6 ) reported that the results of aureomycin therapy in typhoid fever were not uniform and frequently negligible. The use of aureomycin in veterinary medicine is now receiving wider application. found that aureomycin produces Peterson and Hpias (90) beneficial results In the treatment of blue comb disease in chickens. Burkhart (17) in a review of aureomycin therapy in veterinary medicine stated that this antibiotic has been effective in treating a large number of animal diseases. In vitro tests upon a variety of animal pathogens has revealed that all were inhibited by one microgram per ml of aureo- 16 mycin with. the exception of Salmonella g a l l ln ar mn . All were sensitive to low concentrations of aureomycin with, the exception of Pseudomonas aeruginosa. Thus far there is little evidence of development of drug resistant strains with aureomycin. Xt was found to be effective in treating chicks infected w it h Pasteurella m u l t o c i d a . The survival rate of treated chicks w a s 50 to 8 0 percent higher than that of untreated controls* Chloromycetin was discovered by Burkholder (37) in avast screening program for newer antibiotics. Xt possesses a wide range of activity against bacteria, rickettsiae and certain large viruses. Scheidy (113) reported that this antibiotic is of value in veterinary medicine, in the treatment of urinary tract infections, especially diarrheas and systemic bacterial infections in dogs* Eastman, Schlingman, Manning and Eads (35) cited the value of Chloromycetin in the treatment of both large and small domestic animals. septicemia, Such diseases as hemorrhagic conjunctivitis, keratitis, infectious diarrheas and otitis externa have responded very well to Chloromycetin t h e r ap y. Pharmacological and pathological studies of Chloro­ mycetin in animals were conducted by Gruhzit, Pisken, Reutner and Martino (I4.8 ). They found that Chloromycetin was relatively non-toxic to animals and possessed no cum­ ulative toxic effect on oral dosage of 1 0 0 to 2 0 0 gm of Chloromycetin per kg per day over a four month period. Oral administration of Chloromycetin to dogs produced measurable 17 b l ood levels for* eight hours, The use of Chloromycetin In the treatment of poultry diseases has not b e e n reported to any appreciable extent* Chloromycetin has been widely used for the symptomatic treatment of typhoid fever (11, 66) • Rettger (100) in 191J+ called attention to the similarity between the chronic typhoid carrier and the chronic carrier of pullorum disease among infected hens* Xt has' therefore been of interest to follow the success of Chloromycetin therapy in typhoid fever with a view toward possible application in pullorum di sease• Knight, Sanchez, Sanchez, along w i t h Gabinus Shultz and McDermott (66), (lj-2) and Cook and M a r m l o n (28), noted dramatic responses to Chloromycetin therapy in typhoid fevero Stryker (12£) reported the failure of Chloromycetin to benefit a chronic typhoid carrier. Bailey (119) Smadel, Wo od wa rd and emphasized the fact that relapses occurred with Chloromycetin therapy and proposed an eight day period of treatment in acutely ill patients* M ore recently Boger, stated that, Schimmel and Matteucci (11) as the number of chloromycetin-treated cases of typhoid fever increases, it becomes apparent that, although patients respond dramatically to treatment, they are not always bacteriologically controlled* with moderate frequency* Relapses occur Bacteremia has persisted during Chloromycetin treatment in a number of cases and It is now 18 acknowledged that Chloromycetin is not effective in treating the carrier state. Chloromycetin is considered b a c t e r i o ­ static and tnis p r o b a b l y accounts for failure to eliminate the carrier condition. I n vitro studies w it h S. typhosa reveal that a concentration of 1 0 0 0 micrograms per ml of Chloromycetin fails to exert a bactericidal effect. Jackson, Gocke, Collins and Finland (£8 ) found that on a we ig h t basis Ch lo romycetin was third and streptomycin sixth in activity against S. typhosa and other salmonellae including ,S. p u l l o r u m (seven antibiotics were evaluated). Alexander, L e i d y and Redman (1) compared the in vitro action of streptomycin, Chloromycetin and aureomycin against eight gram-negative organisms, strains. including five enteric C hl oromycetin in a concentration of 10 micrograms per cubic mil l il it er of broth m e d i u m exerted a more rapid bactericidal action against Hemophilus pertussis and Hemophilus parapertussis than did aureomycin. A ur eomycin and Chloromycetin were equal in their speed of lethal action against Hemophilus influenzae and £J. t y p h o s a . Stasis of growth with Chloromycetin pe rsisted through twenty-four hours for all organisms tested except Psuedomonas a e r u g i n o s a . Of the newer antibiotics, streptomycin has received wid er application as a therapuetic agent in the control of p ul lo ru m disease. Streptomycin was discovered by Waksman (1 1 2 ) and is active against a large number of gram-positive and gram-negative organisms. activity against fungi, It possesses little to no viruses, yeasts, protozoa and 19 rickettsiae• Benson (6 ) reported that streptomycin was of value in checking pullorum disease in baby chicks, but did not eliminate the carrier condition,, G-watkin (£0) conducted a series of experiments to determine the effect of streptomycin on pullorum infection. He found that streptomycin in the drinking water protected baby chicks artificially infected at two days of age. Streptomycin was not effective in curtailing pullorum infection after symptoms had appeared following artificial infection. Chang and Stafseth (21) noted that streptomycin exhibited bactericidal action against £S. pullorum in a concentration of 31 micrograms of streptomycin per ml of tryptose broth medium. The antibacterial action of strepto­ mycin was not influenced by the serum of normal or infected chickens. They also found active blood levels in strepto­ mycin-treated birds at the end of three hours following intramuscular injection. Kirkpatrick, Moses and Baldini (6 £) noted beneficial results with streptomycin feed supplements in the treatment of infectious enteritis of quail. Hughes and Farmer recorded the order of descending resistance to streptomycin of a number of animal pathogens. The streptococci head the list with the Salmonella, Erysipelothrix rhusiopathiae, Erysipe1 othrix m o n ocytogenes, Pasteurella and Escherichia coli following in order. 20 Sev en strains of S_* typhosa and 5>7 strains of S a l ­ mon el l a covering fift e en species (all isolated from carriers) were tested for in vitro sensitivity to streptomycin* All strains w e r e in hibited b y concentrations ranging from O.OOI4. to 0 o061|_ micrograms of streptomycin per ml of b r o th med iu m (Ip-). Santivanez (110) stated that S. p u l l o r u m resisted the action of streptomycin (6 ,25>:» 1 2 *£, 2 £>, 5>0 , 1 0 0 units) at zero and four hours, and 2 0 0 but at twelve hours it was inhibited b y $ 0 units of streptomycin per ml of b r o th medium, ml* and at 20 hours by 1 2 * 5 units of streptomycin per M a r k e d turbidity appeared after 2l± hours of incubation* Salm onella strains representing sixty different types were tested for streptomycin sensitivity by S e l i gm an n and W a s s e r m a n n (116)• Most of the strains yie ld ed in a range of from four to eight units* Mouse experiments utilizing p e r os i n fection w it h Salmonella t y p h i- mu ri um , Salmonella interitidis et a l * coupled w i t h oral or oral and subcutaneous treatment resulted in the suppression of the normal fecal flora along w i t h the pathogens* A f t er termination of treatment the fecal flora and salmonellae reappeared* IV* Penicillin Pen ic il li n was discovered by Fleming (I4-O) , although in the past few years this has b e e n disputed b y Brunei (15>) . The dramatic action of p e ni cillin against diseases caused b y gram-positive pathogens has obscured its value as an 21 offactive agent in suppressing gram-negative organisms (with the possible exception of Neisseria gonorrhoeae ) 0 Bigger and Daly (9) in 19lj-6 reported unusual success in the treatment of typhoid carriers with penicillin and sulfathi azole, Comerford and K ay (27) encouraged employment of the method used by Bigger in the treatment of typhoid carriers. They were able to duplicate the successful results of Bigger with penicillin and sulfathiazole 0 McSweeney (82) modified the regimen of Bigger as far as dosage was concerned and also obtained good results in the treatment of .typhoid fever with penicillin<> Parsons (8 8 ) claimed that penicillin therapy in typhoid did not produce very striking results, but admitted that the regimen recommended b y McSweeney was not followed closelyo Bigger and Daly (8 ) again reported in 19l|-9 the successful treatment of chronic typhoid carriers with penicillin and sulfathiazole 0 Wit h the discovery of Chloromycetin and its pronounced effect against £3. typh os a, the work of Bigger and McSweeney was obscured until 195>1 when Boger, Schimmel and Matteucci (1 1 ) found that when penicillin was maintained at effective plasma levels, the symptoms of typhoid disappear dramatically and the carrier condition was eliminated. They stated that sulfathiazole has no appreciable effect on S^ typhosa and that penicillin by itself is very effective in suppressing 22 this organism. They blam ed the early failures of penicillin therapy on inadequate b lood levels, A min im um concentration of 1 0 units per ml of p l a s m a is recommended together w i th an agent such as Benemid to delay renal excretion and maintain proper blood levels of penicillin. There are m a n y reports in the literature concerning the in vitro action of p e n i c i l l i n on S_. typhosa and other s al mo ne ll ae • Evans (38) in 19U-& tested 66 different strains of £>, typhosa .against various concentrations of penicillin. He found that all strains we r e completely inhibited b y 2 £ units of pe ni c il li n p e r ml of broth medium. Sixty-three strains were completely i n hibited by 20 units of penici ll in per ml and 15> units of p e n i ci l li n per ml of b r o t h m e d i u m was sufficient to inhibit completely the growth of 5>0 strains, A concentration of 2«£ units of penicillin p e r ml retarded growth of 28 strains. Mi c e inoculated intraperitoneally with virulent typhoid appeared to be benefited by penicillin therapy, Pratt and D u f r en o y (96) stated that penici ll in affects aerobic gram-positive and gram-negative organisms through the same chemical systems. The concentrations of penicillin required to inhibit gram-negative organisms is, however, m uc h larger. They also r e v e a l e d that trace amounts of cobalt added to agar m a r k e d l y reduced the amount of penicillin needed to inhibit an organism. Trace amounts of cobalt are especially effective against gram-negative organisms that 23 are ordinarily resistant to penicillin. Cobalt also appeared to exert the same beneficial effect in vivo (9 3 » 9k., 12L\.) * ■Stewart (122) found that S. typhosa, Salmonella para­ typhi B and strains of Proteus vulgaris were inhibited by penicillin in concentrations of 8 - 2 0 units per ml. Thomas and Hayes (128) tested 18 strains of S. typhosa and found that the growth of 1 $ were inhibited by 5 units of penicillin per ml of broth medium or less. During a 2 k - hour period of incubation at 3 7 ° C there was a 3 £ percent loss of penicillin potency* Miller, Wilmer and Verwey (83) noted that w hen S. typhosa was used as the infecting organism in mice, there were marked differences in the amount of penicillin r e ­ quired for therapy depending upon the number and frequency of the doses used. Spicer and Blitz (120) pointed out the fact that a few viable organisms always remain in cultures containing penicillin, but their multiplication was inhibited, which suggested that individual members of the bacterial population react differently to antibiotic influence. Eagle, Fleischman and Musselraan (33) studied the bactericidal action of penicillin in vivo and the partici­ pation of the host during therapy* They found that sur­ viving organisms damaged by effective penicillin levels did not resume multiplication for a number of hours and were susceptible to the defense mechanisms of the host in the 214absence of penicillin* Eagle (3 I4-) in a further study of penicillin concluded that its bactericidal action was more rapid iri vivo than in vitro * The bactericidal action in the infected animal is the sum of the direct effect of penicillin itself, plus the bactericidal action of the host mechanisms on infecting organisms, w h ic h are acted upon b y the drug and thus made susceptible• George and Pandalai (!)-3) reported that penicillin- resistant organisms became penicillin sensitive w hen grown in the presence of other organisms or their metabolic products o Thomas and Levine (127) found that S* pullorum was inhibited by 10 units of penicillin per ml in beef extract broth. Jackson, Gocke, Collins and Finland (£8 ) found that S . p u l l o r u m •was inhibited by 3 o8 micrograms of penicillin per ml of broth medium* The oral use of penicillin has ma n y decided advantages, but the dosage must be increased above that employed in parenteral administration. Keefer (6 3 ) contended that oral doses, 3 "to £ times the parenteral dose, give results comparable to those given parenterally. Ross, Burke and McLendon (108) noted that adequate blood levels of penicillin could be attained by oral administration when the drug was protected against in­ activation b y gastric acidity* r 25 Robinson, Hirsh, and Dowling (106) also reported that oral administration of pe ni ci ll in in dosages five times the customary intramuscular dosage p roduced results comparable to those obtained w i t h parenteral penicillino Collins, Seeler and F i n l an d (26) found that Caronamide in sufficient dosage enhanced and p r o l o n ge d pe ni cillin blood levels (penicillin was administered o r a l l y ) • During recent years m a n y substances have b e e n u sed to m a i n t a i n and prolong effective b l oo d levels of penicillino A few of the b e t te r k n o w n enhancing substances are Caronamide, V. vitamin K and B o r r e l i d i n (i-j-9> 91* 95* 1224-, 130) • Antibiotic Substances from Highe r Plants Antibiotic substances from hi gh e r plants have h ad limited application in the treatment of hum an and animal diseases. K a h n (6 l) in 1933 noted that b a na na powd er fed to infants pro du ce d a conversion of the intestinal flora from an almost completely gram-negative to an almost completely gram-positive state. Bogert (12) in a review of the dietary uses of the banana indicated that bananas have b e e n of benefit in alleviating m any intestinal disturbances including diarrheas of typhoid fever* Osborn (8 7 ) in 1914-3 reported on the antibacterial properties of hig he r p l a n t s 0 Lucas and Lewis (7 6 ) in 19^4- found that there was a 26 similarity of antibacterial actions throughout a given plant genus. They noted wide differences in the potency of active principles with genera and species. Little and G-rubaugh (73) in 19^4-6 noted that some crude plant juices were m uch more active against animal pathogens than against those causing diseases of plants. Inhibition also appeared to be more pronounced against the gramnegative test strains than against gram-positive organisms. The juices of beans, corn, cabbage, tomatoes and cauli­ flower were tested. I n I 9 J4J4-S Cavallito, Buck^ Suter (19) and Gavallito and Bailey (20) announced the isolation of an antibacterial substance from garlic cloves. The substance was found to be active against both gram-positive and gram-negative bacteria and was given the name of Allicin. Rao, Rao, Natarajan and Venkataraman (97) reported in 19i+6 that Mycobacterium tuberculosis was inhibited by garlic extract. I n 19J-I-7 Stoll and Seebeck (123) isolated and defined the properties of alliin which is characteristic of certain kinds of garlic. They found that when alliin was acted upon by the enzyme alliinase, the antibacterial substance allicin was produced. I n 19^4-1 Block and Tarnowski (10) reported favorable results from feeding bananas to dysentery patients. More recently Scott, McKay, Schaffer and Fontaine found several antibiotic substances in the banana with therapeutic possibilities. (ll5) 27 The use of rutin, a flavonol glucoside, ment of capillary fragility in the t reat­ (I4-6 ) has led some workers to investigate w i der application of this drug in a variety of diseases • L e v i t a n (71, 72) claimed that rutin contributed to the stability of the tissue ground substance and that spread of infection and malignant growth is par tl y deter­ m i n e d b y the degree of permeability of ground substance, Clark and M a c K a y (22) stated that judging by their experimental results it is unlikely that rutin is a v i t a mi n­ like compound. They further claimed that rutin does not exert any specific or therapeutic effect, but may elicit a non-specific stress or " alarm reaction" w h i c h might explain some of the observed, non-specific physiological phenomena, Gottshall, Jennings, Weller, Redemann, Lucas and Sell (L|J?) reported the presence of antibacterial substances in seed plants w h i c h are effective against M y c o b a ct er iu m tuberculosis o There are a number of higher plants w h i ch yield extracts exhibiting antibacterial properties, Hydrastis canadensis produced extracts effective in vitro against Micrococcus pyogenes var, culosis (I4I4., 61, 8 7 ) • aureus and M yc ob ac te ri u m tu ber­ Trigonella Foenum-graecum possesses m i l d antibacterial activity, Hy pericum perforatum is h i g h l y active against gram-positive organisms, M yc o b a c t e r i u m tuberculosis (7k-» 75?) • including MATERIALS AND METHODS I n Vitro Experiments A. Sensitivity determinations The tube dilution method was used in ascertaining the sensitivity of £>. pullorum # 8 9 8 1 7 to aureomycin hydrochloride*15-, Chloromycetin'', plex) streptomycin (calcium chloride com­ and penicillin G-'* (oral, buffered, potassium salt). Veterinary preparations of streptomycin and Chloromycetin were used for in vivo experiments. The usual method of two-fold dilutions was modified to some extent and a stepwise increment of five was employed in the higher concentrations and a two-fold dilution in the lower ranges. Each series of concentrations extended from 35 micrograms to 0,625 micrograms per ml of broth medium. The units of penicillin per ml ranged from 100 to 5 units* Stock solutions of aureomycin and Chloromycetin*5"5'" were prepared by dissolving appropriate amounts in distilled w ater In a concentration of 250 m g per 100 ml of solution, ‘''"The above antibiotics were kindly supplied through the courtesy of the following agencies: Aureomycin - Lederle Laboratories, Pearl River, N.Y.; Chloromycetin - Parke-Davis Detroit, Mich.; Penicillin - The Upjohn Co., Kalamazoo, M ich ^“^Sensitivity determinations with Chloromycetin were later repeated, when a purified batch became available for this study. 29 A solution of streptomycin was made b y adding an appro­ priate quantity of distilled water to the dry powder con­ tained in a sterile vial. This was further diluted with distilled water to give a solution containing 2 f?0 m g of streptomycin base p e r 100 ml. A penicillin stock solution was prepared by dissolving an oral, buffered tablet in a given quantity of distilled water. All the stock solutions were Seitz filtered to insure sterility and immediately used for the various tests. I n order to prepare desired concentrations of a p a r ­ ticular antibiotic, varying amounts were taken from each stock solution and further diluted w it h sterile, water. distilled This was so calculated that one ml of the diluted stock solution when added to 9 ml of seeded broth gave the indicated concentrations. Penassay broth (DIfco) was employed for the deter­ min at i on of sensitivity to aureomycin, penicillin and Chloromycetin. M y c in broth was used for streptomycin. A flask containing 200 ml of broth was Inoculated with one ml of a 2 Lj.-hour broth culture of S_. pullorum #89817. Nine ml of this seeded broth was then dispensed to each tube. The addition of one ml of antibiotic solution to each tube brought the total volume to 10 ml. Each test series as well as each concentration of antibiotic was repeated at least in triplicate. Controls consisted of placing one ml of each concentration into 9 ml of unseeded broth. A second control consisted of only one 30 tub© c o n t ai ni ng 10 ml of seeded b r o t h without the addition of any antibiotic m at erial* The tubes w e r e i n c u b a t e d for 21{. h o u rs at 37° C. and e xamined for visible turbidity. The lowest concentration of antibiotic p r e v e n t i n g g r ow th i n 2ij. h o u r s was ta ken as the endpoint reading* The s e n s i t i v i t y of j3. p u l l o r u m #89 81 7 to garlic extract could not be d e t e r m i n e d b;/-. the t u b e - d il ut i on method, since garlic extract imparts a c lo udiness to b r o t h media. An agar p l at e m e t h o d w a s d e vi se d for tes ti n g sensitivity* A stock so lu ti on of garlic extract was p r e p a r e d by b l e n d i n g 20 g of d e h y dr at ed garlic w i t h I4.OO ml of distilled water. This p r o d u c e d a stock solution c o n t ai ni ng 200 garlic u nits p e r ml. (The garlic unit is d e f i n e d as the amount of active pri n ci pl e c o n t a in ed i n one ml of a solution p r e ­ p a r e d b y b l e n d i n g 0.1 g of deh yd ra te d garlic w i t h I4.OO ml of d i s t i l l e d water.) A f t e r blending, the m i x t u r e w a s c e n t r i f u g e d and the clear supernatant l i q u i d Seitz f i l t er e d to insure sterility. Various amounts of sterile garlic s o l u ti on and P e n a s s a y seed agar w e r e u n i f o r m l y m i x e d to give a range of garlic units f r om one to fifty p e r m l of g a r l i c- ag ar mixture. # 8 9 8 1 7 w a s u s e d for A 2l^-hour culture of S_. p u l l o r u m streaking each plate. The plates were i n c u b a t e d at 37° C • and re a d w i t h i n I4.8 hours. The lowest c o n c e n t r a t i o n of garlic units completely in hi bi ti ng growth of the test or ga n i s m was r e g a r d e d as the endpoint reading. Control plates co ntained seeded agar wit ho ut g a r l i c 0 31 A second metho d for determining 'sensitivity was developed and differs somewhat from the above procedure. Two ml of a 2Lj.-hour culture of S_. pullorum # 8 9 8 1 7 was added to 200 ml of penassay seed agar. Various concentrations of garlic standard were placed in a series of Petri dishes and seeded agar added in amounts necessary for the various desired concentrations. The lowest concentration of garlic completely inhibiting the growth of the test organism for 7 2 hours was regarded as the endpoint. The following table illustrates the metho d of obtaining the range of concentrations used in this study* TABLE 1 PREPARATION OP GARLIC DILUTIONS Garlic units Ml of seeded agar Concentration per ml of mixture in units H• O 20 19.9 1 0. 2 ko 19.8 2 0.3 60 19.7 3 0 *14- 80 19.6 o.£ 100 19«5> 5 H • O Ml of standard garlic solution 200 19.0 10 2.0 lj.00 18.0 20 3.0 600 17.0 30 I4-00 800 16.0 b-o S.o 1000 15.0 5o 32 B. Antibiotic stability in tbe presence of feed constituents Six feed samples of varying composition were utilized for stability tests. Aureomycin was incorporated at a level of 1 mg per g of feed. Chloromycetin was added to prepared feed samples in concentrations of 1 mg per g of feed, but two forms of this antibiotic were employed. One form consisted of Chloromycetin plus binder (1 m g containing 0.5 m g of pure antibiotic). The other form, which was obtained at a later date, was a purified preparation (1 m g = 1 m g of pure n a t i b i o t i c ) . The latter preparation was evaluated in a commercial feed sample. base Streptomycin was incorporated at a concentration of 0 .25> mg per g of feed, while penicillin levels consisted of 1000 units per g of feed. sample. G-arlic constituted l£ percent of each feed Feed samples containing varying amounts of i n ­ gredients were prepared as shown in Table 2 0 Aureomycin and streptomycin were also combined with regular commercial feed samples. A veterinary streptomycin mixture was used here instead of the calcium chloride complex. This mixture was an oral preparation w h i c h con­ tained 0 o 36 S of streptomycin base per g of mixture (this mixture was later evaluated qualitatively). The sample feed mixtures containing antibiotic material were stored for a one-month period and then tested for antibiotic activity. The method used was in general the same for all samples. Five g of reea-mixture were suspended in a dilution 33 TABLE 2 PREPARATION OP SAMPLE FEED MIXTURES* Ingredient Peed mixture number 3 5 h 2 1 6 280 92 0 0 0 0 0 0 0 0 222 72 92 280 150 300 150 300 0 0 2.2. 2. 72 0 0 20 20 20 20 20 20 Limestone k k k k- k- k Bone meal 2 2 2 2 2 2 Salt 2 2 2 2 2 2 15 30 15 30 15 30 Corn Dextrin Soybean meal Sucrose Alfalfa meal Percent protein ■“'Total weight of each sample = J4-OO 8 blank containing 50 ml of distilled water (for aureomycin 9 streptomycin and Chloromycetin samples) Ten g of feed- garlic mixture were suspended in 50 ml of distilled water 9 while penicillin--feed s ample s were added at the rate of one gram in 100 ml. The penicillin solution produced after settling of feed particles was further diluted to give a theoretical concentration of one unit per ml of solution, AID- dilution blanks were vigorously shaken in order to p ro ­ duce a complete solution of the particular antibiotic. shaking, After the suspensions were allowed to settle and the clear liquid portions decanted into a Petri dish. Standard 314paper discs (7 I4-O-E, 12,7 m m diameter) were immersed in the solution and placed on seeded agar plates which, were p r e ­ pared in the following manner: 18 ml of penassay base agar was poured into Petri plates and allowed to solidify* A flask containing 200 ml of penassay seed agar was then inoculated with 2 ml of a 2 l4.-h.our broth culture of S_. pullorum # 7 1 1 and vigorously rotated to insure a uniform distribution of the organism. Three ml of the seeded agar was then overlayered onto the solidified base and the Petri dish gently rotated back and forth to distribute the seed agar uniformly over the entire surface. Micrococcus pyogenes var. aureus # 9 1 l4i4- was used as the test organism in penicillin potency determinations. The zones of inhibition observed were then evaluated for actual antibiotic concentration by referring to standard reference curves (Figs. 2 - 10). Theoretically the anti­ biotic concentration per ml of feed-water suspension was k nown from the amount of feed sampled and the level of antibiotic concentration originally added. However, any inactivation by feed constituents or any strong adsorption of the antibiotic to feed, w o u l d result in the formation of inhibition zones that were smaller than those produced by theoretical concentrations. Controls consisted of feed samples without antibiotics* Standard reference curves were prepared b y utilizing a m odified version of the paper disc plate method for streptomycin assay (129). Essentially the plates were 35 prepared like those described for stability testing. Originally M. pyogenes var. aureus #91^-4- was employed as the test organism for preparing penicillin reference curves and stability tests. S.* This organism was later replaced by #89817 wh ich revealed moderate to high sensi­ tivity in the presence of appropriate amounts of penicillin. The reference curves employed in this study consisted u sually of four to five points — each point being the average of triplicate or quadruplicate trials. Sterile discs were immersed in different concentrations of anti­ biotic solution and placed on seeded plates. The zones of inhibition were- measured after 18 hours of incubation at 37° C. S.. pullorum consistently showed poor growth on mycin agar w h i c h was replaced by penassay seed agar (in strepto­ m y ci n determinations). I n reading streptomycin plates it was found that this antibiotic produced elliptical zones of inhibition with a consistent long axis. The long axis was therefore used in measuring zones of inhibition. Antibiotic stability tests were conducted at intervals of one month and twelve months (penicillin tested at one and six mon th intervals). G. Effect of penicillin on S_. pullorum The following numbered strains of S_. pullorum were obtained from the Michigan State College Poultry Pathology Laboratory: 89817, 130l|.-BS, 1273-1, 1300-B1, 711 and 5. 36 Each, strain was tested by the previously described seededplate method. Concentrations of £0 and 100 units per ml of penicillin were used. The action of penicillin on j3. pullorum was determined by subculturing into plain penassay broth from various tubes containing different concentrations of penicillin. These transfers were carried out at 2Lj-, I4.Q and 72 hours. Some surviving organisms were identified by the usual sugar reactions in order to ascertain any damage that m a y have occurred through penicillin contact. Agar plates were streaked in addition to subculturing in broth. D* The enhancement of penicillin action by cobalt against S . pullorum A series of seeded plates were prepared with E>. pullorum #89817 as the test organism. A n aqueous solution of cobalt was prepared by dissolving cobalt chloride in a concentration of 0.2 m g per ml of solution. Three ml of this solution was carefully added to each seeded plate. then incubated for 30 minutes at 37° C. The plates were After incubation the cobalt solution was carefully decanted from each plate. Sterile paper discs were immersed in a stock solution of penicillin (containing 100 units per ml) placed on each plate. and immediately Controls consisted of seeded plates without addition of cobalt. All plates were then incubated for 2 I4. hours at 37° CJ and read. I n order to measure the degree of enhancement by cobalt, 37 a standard reference curve was made with, seeded plates containing £>. p u l l o r u m * Six plates were used for each concentration of penicillin* The range of concentrations extended from 5>0 units per ml of solution to 1^00 units* Toxicity tests on cobalt were performed by the paper disc method. The discs were immersed in a solution of cobalt and placed on plates seeded with £>. pullorum. The formation of a zone of inhibition would indicate an in­ hibiting effect by the selected concentration of cobalt* In Vivo Experiments A • Peed Regular commercial starter feed was used in all experiments with the exception of the first and third series. Feeds that did not contain any antibiotic supplements were chosen. In the first series, birds were given feeds containing 15 and 30 percent protein. In the same series another group was supplied with a diet of oats for 72 hours prior to feeding w ith 15 and 30 percent protein. In the third series of experiments one of the feeds contained sucrose as the principal carbohydrate. These special feeds were composed of the following ingredients: 38 Percent protein 30 15 Ingredient 0 Sucrose Corn Soybean meal Alfalfa Bone meal 73.k 17.0 5.0 3.0 Sucrose15$ prote 0 35.4 55.0 5.0 3.0 60.3 0 30.0 5.0 3.0 Calcium carbonate Salt BY feed Viadex APF 0.5 0 .5 0.3 0.1 0.1 0.5 0.5 0.3 0.1 0.1 Magnesium sulfate Choline chloride Niacin 0.05 0 0.05 0.05 0 0.05 0.05 0.1 0.05 0.5 0.5 0.3 0.1 0.1 All above figures given in percent, B, General arrangement of each, experimental series Each series consisted of six experimental groups and. two control groupso One control was composed of birds which were artificially infected, b u t did not receive aa y therapeutic treatment. The second control consisted of birds that were not infected and did not receive any anti­ biotic therapy. A total of 25 birds were included in each experimental group. Pour different breeds of chicks were used in the course of this study. C. Preparation of the inoculum Large nutrient agar slants (20 m m tubes) were inoculated with a 2 l4.-h.our culture of S. pullorum # 8 9 8 1 7 and incubated for 2 I4. hours. To each slant 5 ^ml of sterile physiological 39 saline was added and the organisms gently removed with, a wire loop. The bacterial suspensions were pooled in sterile flasks. F or the first 5 experimental series the suspension was adjusted to tube number J4. of the McFarland nephelometer (1,200,900,000 organisms per ml). Beginning with the sixth series the number of organisms per ml of suspension was determined by plating methods. The average number of organisms was found to be approximately 1,350,000,00C per ml of saline suspension. The inoculum was administered orally to each chick in a volume of 0 . 5 ml* D. M e t h o d of antibiotic therapy I n general most of the antibiotic agents were admin­ istered as a prophylactic measure. The period of pr o p h y ­ lactic feeding started w h en the birds were one day of age and continued for 72 hours in the first two series of experiments. This period was reduced to Lj.8 hours in succeeding series and finally down to 2 I4. hours# W h e n the time of prophylactic feeding was terminated, the birds were inoculated w it h the infecting organism and continued on a therapeutic diet for the duration of the experi m en t• All the antibiotic agents were incorporated into feed at the various indicated levels. first series, I n several groups of the dietary constituents were evaluated for their ability to check the course of infection. 14.0 E. P reparation of an antigen-mash The following _S. pull or um strains were u s e d i n p r e ­ p a ri ng a m i x e d antigen: 1300-B1, and 8 9 8 1 7 * 130l|.-BS, 1273-1* 5* 711 All strains were obtained from the M i c h i g a n State College Poultry Pathology Laboratory. A n t i g e n preparations were added to the feed and removed after the birds were inoculated itfith the infecting organism. Large concentrations of the various p u llorum strains were p r e p a r e d b y adding 2 ml of a 2ij.-hour b r ot h culture of each strain to a flask containing 500 ml of nutrient broth. The number of organisms per ml of b r ot h was determined by plating methods. Several procedures were u se d for attenuating the organisms. Afte r a 2l|.-hour peri o d of incubation at 37° C in either nutrient or tryptose broth, the organisms were / killed by adding three volumes of absolute alcohol and the mixture allowed to stand for one hour. The ethanol- broth mixture was then po ur e d over a g iven quantity of feed and m i x ed thoroughly. The alcohol was evaporated by a stream of air at room temperature. Af ter evaporation the feed on w h i c h the organisms were adsorbed was thoroughly stirred and added to the bulk quantity of feed. Other methods for antigen pr o duction involved heat attenuation and adsorbing of the organism on c h a r c o a l . The procedures used for charcoal adsorption and heat attenuation were as follows: kl 1. Heat attenu at io n - 512 mi of antigen broth was h e at ed to a temperature of 90— 95° C and h eld at this tem pe r­ ature range for 30 minutes. The b r o th w a s then cooled w ith frequent stirring and incorporated into 10 kg of feed by thorough mixingo 2. Charcoal adsorption - 512 ml of antigen broth was heated and cooled in the same m a n n e r as described above. Two h u n d r e d g of activated carbon (Norit A) was gradually added to the cooled b r o t h w i t h continuous stirring. The resulting suspension was thoroughly m i x e d w i t h two kg of feed and dried. The dry product was further m i x e d wi t h the remainder of feed to give a total of 10 kg. P. M e t h o d of cobalt administration to chicks Chicks were allowed to drink water w h i c h contained cobalt chloride at a concentration of 0.2 m g per ml of solution. Cobalt-water was given for 2i+ hours prior to the time of artificial infection. During this period the chicks were kept on p l a i n starter feed with ou t antibiotic. Thirty minutes after infection each chick was again given 0*5 ml of cobalt w a t e r (0.2 m g per ml) orally. Cobalt wa ter and p l ai n feed w ere then removed from the troughs and feed containing p e n i c i l l i n at a level of 2290 units per g and p lain w a t e r were substituted* The pe n i c i l l i n used in the last series of experiments was an oral p r e p a ra t io n w h i c h assayed 1560 ■units per mg. Calcium carbonate h a d to be added as a buffer for this par ti c ul ar preparation. U2. G. R e c o v e r y of the i n f e ct in g orga ni s m The f o l l o w i n g m e t h o d was u se d for isolating £>. p ullorum from dead chicks: Portions of the heart, lung, liver and intestine w er e p l a c e d i n tetrathionate enrichment broth and incubated for 18 h ours at 37° C. P r o m h ere mate ri al was streaked on b i s mu th sulfite agar and incubated from 2 I4. to J4.8 hours. Typical colonies w e r e t h e n transfe rr ed to Kligler*s iron agar slants. I f a characteristic r eaction for S* p u l l o r u m appeared, transfers were made to Sims medium, sucrose and m a n n i t o l broth. The use of b i s mu th sulfite agar as a choice m e d i u m for the i s o l a ti on of _S. p u l l or um has b e e n c o n fi rm e d b y other Investigators (1 8 )• A large number of birds from these experiments was kept for carrier studies. Most of the birds w ere sa cri­ ficed at twelve w eeks of age at 9 m o n th s of age) of £>. p u l l o r u m . (aureomycin birds sacrificed and carefully examined for the presence Portions of the heart, liver, spleen, lung, testes or ovaries and intestinal b i f r i ca ti on from each bird were p o o l e d in tetrathionate broth. A f t e r enrichment, transfers were m a d e to b i s mu th sulfite agar, Kligler's medium, etc. I n ex amining a large num be r of birds it was imperative not to carry over inf ec ti ng organisms fr o m one b i r d to another by w a y of instruments and hands. Therefore, after each d i s s e c ti on all Instruments were immersed in a germicidal detergent for three to five minutes In detergent). (hands were also washed A f t e r this period of time, the Instruments were removed and the excess fluid allowed to drain. This procedure did not appear to interfere with the isolation of S. p u l l o r u m . Previously, all instruments were dried with sterile gauze pads to remove residual detergent solution. However, the usual number of isolations was obtained w i t h either method* X n order to ascertain the efficacy of this m e t h o d of instrument and h an d sterili­ zation, swabs w e r e taken from the instruments and hands and p l a c e d in tetrathionate enrichment. Prom here, material was t r an sferred to the various differential media. The virulence of the infecting organism was at first mai nt ai ne d by chick passage, but it was later found that virulence could be m a i n t a i n e d i n stock cultures (nutrient agar slants) b y transferring the organism every two weeks. jS. p u l l o r u m # 8 9 8 1 7 is a standard strain, produce gas at 37° C 0 but does not RESULTS A N D DISCUSSION The Sensiti vi t y of S. pull or um to Aureomycin, Chloro m yc et in and Streptomycin There are m a n y factors w h i c h affect the response of a given bacterial p o p u l a t i o n to a selected concentration of an antibiotic. This is true for both in vitro and in vivo studies® In p e r f o r m i n g the tube-dilution assay, certain con­ ditions m ust be standardized so that results can be d up ­ licated w i t h i n allowable limits. A number of factors in this procedure can be regulated, while others are almost impossible to control. Reasonable accuracy can be attained by this met ho d in replicate trials, mind: if the following factors are kept in (1 ) the test organism must be sensitive to the antibiotic, (2 ) the organism must be of the same strain and age in e v e ry assay, (3 ) incubation temperatures should be the same in every trial. Generally, the duration of incubation is shorter w he n the temperature is increased. Endpoint readings are sharper at certain incubation temper­ atures and tend to shift either into a higher or lower concentration w i t h a change in the time of incubation. Variable endpoint readings m a y be due to the development of k$ bacterial resistance or the gradual destruction of the antibiotic, (l±) for certain antibiotics the number of organisms initially present must be constant in every test and finally (5 ) the composition of the test medium should not be varied. However, a given sample of antibiotic may be assayed under identical conditions and still exhibit a variable potency in successive tests. A partial explanation for this phenomenon m a y be due to the fact that any given bacterial population is not perfectly homogeneous in regard to the minimal concentration of antibiotic necessary to inhibit or kill each individual member. used as an example, If penicillin is (other antibiotics could be included) only those cells that are in an active metabolic phase would be susceptible to minimal inhibiting concentrations of the antibiotic. As the more sensitive members of the bacterial population are killed, there is a release of cellular constituents which may serve as essential nutrients for those members that are in a phase of decreasing m e t a ­ bolic activity. These inactive cells become metabolically active and are subject to minimal inhibiting concentrations of the antibiotic. Thus there may appear waves of growth within a definite period of incubation time and a consequent shift In the endpoint reading. The in vivo activity of antibiotic agents may not necessarily parallel the in vitro activity. Certain con­ stituents of* normal body fluids may inactivate all or a portion of the antibiotic or else enhance its antibacterial properties. Eagle, Fleischman and Musselman (33) found that organisms damaged by penicillin contact in vivo, did not recover as rapidly as those contacted in vitro. They also noted that penicillin-damaged organisms were suscepti­ ble to the defense mechanisms of the host in the aosence of any demonstrable penicillin levels for a certain length of time• Variations in sensitivity tests have been reported by Jaclcson, Gocke, Collins and Finland (58)* They used a number of salmonella species including £>. typhosa and S_„ pullorum. mycin. They reported a four-fold variation with strepto­ A two-fold difference was found in tests with Chloromycetin at different times. Results w it h aureomycin varied as much as eight-fold from one test to another. The authors conclude that the results are valid only for the conditions under which the tests were performed. They obtained the following endpoint readings for S^. pullorum; streptomycin, 2 £ micrograms per m l .5 aureomycin, 12.5 micrograms; and Chloromycetin, 1.6 micrograms per ml (all readings in i{.8 hours) • X n general most investigators have reported that many species of Salmonella are susceptible to low concentrations of streptomycin as determined by in vitro methods (ip., 1 , 1 1 6 )• The in vivo activity of aureomycin against various Salmonella species has not been very pronounced. This is hi especially true for infections due to S. typhosa (ll|., 25>, 66). The in vivo activity of Chloromycetin against £>. typhosa is much superior to that of aureomycin (66). The in vitro sensitivity of _S. pullorum # 8 9 8 1 7 to Chloromycetin (impure and purified forms), aureomycin and streptomycin has been recorded in Tables 3 - 6 . It will be noted that there occurs a shift in the endpoint readings (more marked with streptomycin), but this recorded difference is within allowable limi ts 0 The Sensitivity of S. pullorum to Penicillin As was mentioned previously, the unusual success of treating infections caused by gram-positive organisms with penicillin, h a d obscured the possible use of this anti­ biotic against gram-negative organisms. Previously, organisms which required concentrations above one or t wo units per ml for inhibition were thought of as being rather resistant to penicillin (in view of the fact that a large number of streptococci were inhibited by concentrations as low as 0.06 units per ml). The term units, by which penicillin concentrations are designated, can be rather deceiving when sensitivity concentrations are considered. If a certain batch of penicillin has been assayed at 1^60 units per mg, it would mean that an organism inhibited by 10 units per ml Is actually subjected to approximately 6 micrograms per ml on a weight basis. Vihen units are evaluated in micrograms, the term assumes nev*r meaning as far as sensitivity deter- TABLE 3 THE SENSITIVITY OF SALMONELLA PULLORUM TO AUREOMYCIN Micrograms of aureomycin per ml of broth Positive control •Negative control Seeded broth plus aureomycin 1 2 3 Experiment 1 15 + 44* 44- — — — — — — - 10 4- — — - _ - 35 30 25 20 — — _ d 2.5 4- - 4- 1.25 0 „625 4 4 * — 4 4 4 4 4 4 - — - — — — — — — 444 _ — — — — — 4 4* 44 44- 15 10 4 4 5 + — — — — — — — 4— — 2.5 1.25 4 4 — - — - — - 0.625 + 4- 4- - - - _ - Experiment 2 15 10 5 - 2.5 1.25 0.625 - - - Experiment 3 - - - — 4■ positive control - seeded broth without aureomycin Negative control - un3 eeded broth plus aureomycin + = presence of growth; - = complete absence of growth i+9 TABLE ^ THE S E N S I T I V I T Y OF SALMONELLA PULLORUM TO CHLOROMYCETIN (IMPURE") Micrograms of Chloromycetin per ml of broth Positive control Negative control Seeded b r o t h plus chloromyc etin 1 2 3 Experiment 1 32 30 22 20 12 + + + + 10 2 2.2 1.22 + + + + 0.622 + _ — — _ — — — — — - — _ — — ~ — - — + + + + + + + + — _ — — — am — — Experiment 2 12 10 2 2.2 1.22 0.622 + — — — — + + ■4* + + + + + _ — — + + — — — — — — — — — + + - + + + + + + + + + Experiment 3 12 10 2 2.2 1.22 0.622 + + *4" + + + Positive control - seeded broth, without Chloromycetin Negative control - unseeded broth plus Chloromycetin + = presence of growth; - = complete absence of growth 50 TABLE 5 THE S E N S I T I V I T Y OP SALMONELLA PULLORUM TO CHLOROMYCETIN (PURSl Micrograms of Chloromycetin p er ml of br oth Positive control Negative control Se eded b r o th plus Chloromycetin 1 2 3 Experiment 1 25 12.5 6.25 3«125 l .56 0.78 0.39 0.195 0.0975 o.Oi+875 + + + + -i- — — — - _ — — — — _ — — — - _ — — + + + + + — — + + -i+ + + + + + + + + + + + + + + -t+ _ — — — — — — — — — — — — — — — + + + + 4- — — “ + + + + + + + + + + + + + - Experiment 2 25 12.5 6.25 3.125 1.56 0.78 0.39 0.195 0.0975 0.0^875 + Positive control - seeded broth without Chloromycetin Negative control - unseeded b r o t h plus Chloromycetin + =- presence of growth; - — complete absence of growth 5i TABLE 6 THE SENSITIVITY OF SALMONELLA PULLORUM TO STREPTOMYCIN Micrograms of* streptomycin p er ml of b roth Positive control Negative control Se eded broth plus streptomycin 1 2 3 Experiment 1 — — — - — — — - + 4+ + + + + 4- _ + 444* — — — — — — _ — — - — — - + + + 44* 4- 444- 35 30 25 20 15 4* + 4+ 4- — — — 10 5 2.5 + + + -H 4- — — — — — 5 444- 2.5 1.25 0.625 44+ 1.25 0.625 — — — - _ _ Experiment 2 15 10 - Experiment 3 15 5 + 44- 2.5 1.25 0.625 444- 10 im — + — + — -t. — + 4* 4* 444- 444- — Positive control - seeded broth, without streptomycin Negative control - unseeded broth plus streptomycin 4- = presence of growth; — = complete absence of* growth 52 ruinations are concerned. Pratt and Du frenoy (96) stated that penici l li n affects aerobic gram-positive and gram-negative organisms by blocking ttie catabolism of mononucleotides. However, the minimal concentration of pe ni ci ll in required to produce this effect is far greater for gram-negative organisms than for grampositive . Reports in the literature concerning the use of penicillin against gram-negative organisms are rather numerous (8 , 9? 11, 27, 38, 83, 127). Stewart that "When applied to gram-negative bacteria, (122) stated the term sensitivitry to pe ni ci ll in must be interpreted w i t h rese r­ vations." I n his study of p en icillin action on gram-negative organisms, the range of p e n i c il li n sensitivity extended from 8 - 1 2 8 units of pe ni c il li n per ml of broth medium. A shifting endpoint was obtained w h e n S>. pullorum #89817 be came subjected to varying concentrations of penicillin (tube dilutions). However, the endpoint reading (in 2 J4. hours) was constant for a given experiment, but tended to shift in repeated d eterminations. This was observed despite every effort to duplicate each step in successive trial assays. This m ay be explainable on the basis that bacterial populations exhibit post lytic growth responses w hen in contact with penicillin. It is reas on ­ able to assume that various members of the bacterial p o p ­ u lation po ssessed different sensitivities to inhibiting concentrations. The phenomenon of bacterial lysis and •53 release of essential nutrients has already been discussed. It will be noted that in high, concentrations of p e n ­ icillin (9 0 - 1 0 0 units per ml) the endpoint is constant. The fact should be mentioned that endpoint readings were made in 2.\\ hours and that the true endpoint at the end of 72 hours was an entirely different value (determined by subculture technique rather than visible turbidity). This determination will be discussed shortly. The sensitivity of S_. pullorum to penicillin has been reported sporadically in the literature, but rarely as a specific eva lu at io n against S. p u l l o r u m . This organism has served as one of the m any species in a bacterial spectrum. W i t h the reported success of penicillin in the t reat­ ment of typhoid infections (9> 1 1 } 2 7 ) , it was decided to evaluate penicillin against the test strain of jS. pullorum used in this study as well as a number of other pullorum strains. The results presented in Table 7 were obtained on plates seeded wi t h different pullorum strains (obtained from the Mic hi ga n State College Poultry Pathology L a b o r a t o r y ) • The results obtained in successive- tube dilution tests are given in Tables 8 and 9« The minimal inhibitory concentration of penicillin varied in some of these sensitivities tests, but once the endpoint reading was attained (in 21). hours), constant thereafter. However, it remained it was also noted that tubes showing a definite presence of growth did not increase in visible turbidity after 2 14. hours, and as a matter of fact 54 TABLE 7 THE PLATE SENSITIVITY OP SIX SALMONELLA PULLORUM STRAINS TO PENICILLIN Strain number Plates minus peni cillin ___________ Units per ml 3.125 6.25 12.5 25 + 1273-1 + 1300-B1 + -t- 20.0 25-5 + + + H -0 . O + 24 - 3 + + + + 17.8 23.0 711 + + -t- + 17.3 22.8 5 + + + + + H -'J • O 1304-BS 100 50 23.3 + 4- + + + 19.8 2^.0 89817 + + = complete growth, of test organism zone diameter m easured in m m in 48 hours all tubes exhibited a decreasing amount of turbidity. At 72 hours and 96 hours all the tubes became clear and a slight sediment was visible at the bottom of each tube. This was observed in every test. phenomenon was not noted with aureomycin, streptomycin. A comparable Chloromycetin or W i t h these antibiotics the endpoint reading (in a given experiment) w o ul d shift after 2 4 hours to the next highest dilution. A similar action was seen on plates seeded wi t h £>. p u l l o r u m . Penicillin produced a zone of inhibition that was very sharp and distinct. The borders of such zones retained their sharpness for weeks without any encroachment of the test organism. aureomycin, In plate assays with Chloromycetin and streptomycin, the zones of inhibition w o u ld begin to disappear after 48 hours and 55 TABLE 8 THE PENICILLIN SENSITIVITY OP A STOCK CULTURE OF SALMONELLA PULLORUM # 89817 N o 0 of trials Positive control Negative control Units per ml Experiment 1 1 2 3 k 100 + + -t- - — - - + "• ... Experiment 2 1 2 + + 3 + b- + 60 — + + — + — - + 55 + + + + + +• + + 50 4-5 4-0 35 - - - - - mm mm - - + 50 - + + - + + + + - 4-5 4-0 35 •w — •— _ _ - - + Experiment 5 k + + + 6.25 — Experiment Jfc 1 2 3 + 1 2 .5 55 60 65 70 75 8 o 85 90 95 1 0 0 2 3 *4- k + - 1 2 3 25 mm Experiment 3 1 50 - 30 25 - - - - 20 - 20 15 10 5 ■f* + -H + + + + + + + *4“ + + + + + Positive control - seeded broth, without penicillin Negative control - unseedod broth plus penicillin + = presence of growth; - = complete absence of growth 56 TABLE 9 THE PENICILLIN SENSITIVITY OP A RECENT ISOLATE OP SALMONELLA PULLORUM # 8 9 8 1 7 Units per ml Positive control Negative control 1 Trials 2 3 Experiment 1 100 95 90 85 80 + 4+ + + 75 70 65 60 55 + + — — — — __ 4- — — — - 50 45 40 35 30 + 4444- _ — 25 20 15 10 5 + + + 4- _ — - 444- - 444- - — + 4“ + + 4- 4- 444- 4- + + + + + 44* 44- 4 + 44- + 4- 44* + 4- + + 44- + 44* +■ + 4 4* + + + + + 4* 4- 4- 444- + + 44- 4* 44- + Experiment 2 100 95 90 Positive control ~ seeded broth, without penicillin Negative control - unseeded broth plus penicillin 4- = presence of growth; - = complete absence of growth 44- 57 w i t h i n a few days only a slight haze of inh i bi ti on was vi s i b l e • In the p en ic i l l i n - t u b e assay w h i c h em pl oy ed a tenday isolate of S^. p u l l o r u m , the gradual turbidity occurred. clearing of visible At t h e end of 96 hours all tubes were clear and it was dec id ed to subculture each tube into p l a i n brotho The results p r o d u c e d b y subculturing are re corded in Table 10« The results of subcul tu ri ng ind ic at ed that p e n i c i l l i n exerted a b a c t e ri ci da l effect w i t h i n 96 hours. However, a further d e t ai le d study w a s n e e d e d for d e t e r m in in g more accurately the l e n g t h of time r e q u i r e d for a bactericidal effect to become evident. A tube d i l u t i o n series was p r e p a r e d w h i c h exte nd ed f r o m 5 units of p e n i c i l l i n p e r ml of b r ot h m e d i u m to 35 units. p e n a ss ay broth. Subcult ur es w e re made in The results f r o m this study are recorded in Table 11. It is evident that the use of a b r o t h m e d i u m is superior to that of a solid m e d i u m for subculturing techniques. Some organisms w h i c h survived p e n i c i l l i n contact continued to p roliferate w h e n s u bc ul tu re d in broth, b u t did not grow w hen streaked on agar plates. The b a c t e r ic id al act io n of p e n i c i l l i n against S_. p u l l or um could have a partial ex pl an at i on in the theory of post-l yt ic growth responses. As d e s cr ib ed previously, members of any ba ct er i al p o p u l a t i o n will vary in response to a min im al in hi b it in g concen tr a ti on of penicillin. 58 TABLE 10 THE SURVIVAL OP SALMONELLA PULLORUM AFTER 96 HOURS OF PENICILLIN CONTACT AS DETERMINED BY SUBCULTURE TECHNIQUE Units per ml of subcultured tube + + + + + 100 95 90 85 80 + + + + + 75 70 65 60 55 5o i+5 k.0 35 30 25 20 15 10 5 Positive control + + + + + + + + Negative control Trials 1 _ _ "— T ....... 3 — - - - — - - — — - — — — — - - mm - — — — - — - - - - - — — — — — — — - _ — — _ - - - - - - - - - - - — — — — — — — _ — - - - - - - - — - - + +• + + mm Positive control - seeded broth without penicillin Negative control - unseeded broth plus penicillin + - presence of growth; - — complete absence of growth TABLE 11 THE GROWTH OF SALMONELLA PULLORUM IN THE PRESENCE OF PENICILLIN FOR SUBCULTURE STUDIES 2 i|. hours of incubation Trials Positive control 1 2 4 4- 3 + Units per ml Negative control - Units 1 per ml 20 25 15 10 5 4* 4- 444- 444- 4 4 4 4 4 4 +■ 2 lt-hour reading 1 35 30 2 3 444- l4.8 -h.our re ading 1 2 3 4 4 4- 72- hour re a ding 1 2 3 Subcultures in broth after 2 I4. hours of Incubation 4 444 4 4 35 4 4 4 4 4 4 4 4 30 4 4 4 4 4 4 4 4 25 - 20 10 4 4 4 5 + 15 4 4 44- 4 ■I* + 4 4 4 4 4 4444- 4444- 4444 4 4 4 4 - 4 4 4 4 4 4 Subcultures in broth after I4.8 hours of incubation 35 30 25 20 15 10 5 44- 4 444- - 4 •f 4 4 44- 4* + 44- *4* — _ — + 44- 4444- 4444- — 444- _ — — 4 — 4 4 4 4 + 4 4 Subcultures In broth, after 72 hours of incubation 6o T A BL E 11 CONTINUED S u b c u lt u re s on solid m e d i a U n it s p e r ml 35 30 25 2 ^--hour incub. 2 1 3 mm + + 20 15 10 5 4 .8 --hour incub. i 2 3 - + + + + + + + 7 2 -hour incub. 1 2 3 mm mm mm mm mm 4* 4- — — - - — - — — — — — — — _ _ _ _ _ _ 444* + + - + + - - - — + 4- + - Pos it i ve control - seeded b r o t h w i t h o ut p e n i c i l l i n N e g a t i v e control - u n s e e d e d b r o t h plus p e n i c i l l i n + = p r e s e n c e of growth; - ^ comp l et e absence of g r ow th 6l Those members that are k i l l e d first, release essential nutrients u p o n lysis of the cells. P e n i c i ll in is k n o w n to exert its lethal action against those cells active m e t a b o l i c state. that are in an Those m e m b er s of the bacterial p op ul at io n that are less active are stimulated to greater metabolic activity w h e n essential nutrients are made available t h ro ug h lysis of more increased me t ab ol ic susceptible members* The activity of for m er ly inactive cells, renders these cells susceptible to a minimal inhibiting c on centration of penicillin. Thus the k i l l i n g action of p en i c i l l i n is exe rt ed t h r o u g h a series of cycles,- in w h i c h non-susceptible cells are continu al l y made until a point is rea ch e d w h e r e killed. susceptible all cells are eventually This p h e n o m e n o n m a y not onl;/ explain shifting endpoint readings, b u t also the rather slow bactericidal action obse rv ed in this study. A be g i n n i n g bactericidal action was no ted a fter I4-8 hours of pe n i c i l l i n contact minimal in h i b i t i n g concentrations of p e n i c i l l i n ) • (for Apparen t ly some organisms were able to survive 72 hours of pen ic il li n contact. I n 25 units per ml of p e n i c i l l i n and in 20 units, one out of three tubes still showed growth. These o r g a n ­ isms either d ev eloped resistance or the antibiotic un der­ went deterioration. Thomas and H a yes (128) found that during a 2l 4.-h.our per i o d of i n c u ba t io n at 37° C there was a 35 percent loss of p e n i c i l l i n potency. This is Interesting w h e n minimal inhibiting concentrations of pe n i c i l l i n are considered* 62 In the pres en t study 20 units of p e n i c i l l i n per ml of broth, m e d i u m m a y be c o n s id e re d as the bacter ic id al point for S_, p u l l o r u m #89 81 7 at the end of 72 hours, but this c o n c e nt r at io n m a y have a lower value p ot en cy at 37° G) • end­ (decrease in W h e n viewe d in this respect the b a ctericidal c o n c e n tr at io n of p e n i c i l l i n is p r o b a b l y lower than 20 unit s p e r ml of broth m e d i u m 0 It was of i nterest to determine whet he r _S. p u l l o r u m h a d b een dam ag ed by p e n i c i l l i n contact, so as to Influence the biochemical r e a c t i o n s characteristic for this organism. The organisms were r e m ov ed f r om several different c o n c e n ­ trations of p e n i c i l l i n in w h i c h they still ex h ib it ie d a growth response and p l a c e d in the f o l l o w i n g differential media: K l i g l e r ’s i ron agar, and Sims medium. sucrose and mannitol b r oth The f o l l o wi ng reactions we r e obtained: TABLE 12 THE B I O C H E MI CA L C H A R A C T ER IZ AT IO N OP S AL MO NE LL A P ULLORUM A F T E R CONTACT WITH- SEVERAL V A R Y I N G CONCEN T RA TI ON S OP PENICILLIN U nitage deriva “ K l i g l e r ’s Individual sugars Sims tion of select " Butt Slant M annitol Sucrose Gas H 2 S Mo tility ed transfer I{.8-hour pos, control A AK A AK — 5 units 20 units 30 units A A A AK AK AK A A A AK AK AK — 7 2 -hour 10 units 20 units 25> units A A A AK AK AK A A A AK AK AK — A — acid reaction; AK — _ - — — alkaline r ea ct i o n ■ 4- ■* + + + - + + + - 63 Apparently, p e n i c il li n contact does not alter the biochemical activity of* S. p u l l o r u m . on differential m e d i a are typical this study. The r e c o rd ed reactions for the strain used in M ic ro sc op ic examination of these organisms did not reveal any gross morphological changes# Occasionally, the organism appeared more elongated than usual. The Enhanc em en t of Penicil l in A c t i o n Against S. p u l l o r u m by Proper Concentrations of Cobalt I n 19i4-7 Pratt, D ufrenoy and Strait (93? 9i+, 121+) reported that trace amounts of cobalt enhanced the e f fective­ ness of penicillin. They found that trace amounts of cobalt added to agar plates increased the effectiveness of dilute penicillin solutions in producing inhibition zones. test organism us e d was Mi cr o co cc us pyogenes var. The aureus. A four-fold to eight-fold enhancement of penici ll in action was recorded. The degree of enhancement depended on the test organism u s e d and the minimal inhibiting concentration of p en icillin that could be detected by their m e t h o d of testing. A cobalt concentration of one m g pe r liter of nutrient agar was found to be the m ost optimal. They also observed that the time required to produce discernible zones of inhibition was m u c h shorter than on plates containing no cobalt. They po s tulated that trace amounts of cobalt lowered the required minimal inhibiting concentration of penicillin. These investigators found further evidence for the 61*. enhancement of penicillin action by cobalt. Test plates seeded with Escherichia coli and incubated for 16 hours at 37° G produced readable zones of inhibition with 10 units of penicillin per ml of aqueous solution. 12 m m in diameter The zones measured without the addition of cobalt. However, when the test agar contained cobalt chloride in a concen­ tration of one m g per liter of nutrient agar, a zone of inhibition measuring l£ m m in diameter was produced by only one unit of penicillin per ml of aqueous solution. This was not the result of an additive effect of two antibiotic agents. A solution containing only cobalt chloride did not have any inhibitory effect on the test organism. The cobalt effect on penicillin was also noted in serial dilution experiments. Tubes containing cobalt required only half the usual minimal inhibiting concen­ tration of penicillin. The cobalt effect on penicillin was also noticed with such test organisms as Proteus vulgaris and Bacillus subtilis. It was also found that a short period of incubation with cobalt solution prior to penicillin contact produced the most striking results. This period of incubation varied with the test organism employed. The in vivo effect of cobalt on penicillin was then determined. given Mice were inoculated w it h £>. typhosa and then micrograms of cobalt chloride. vivo incubation period, After a short in 2000 units of crystalline sodium benzyl penicillin was injected. This combination exerted a protective effect equal to 1+000 units of penicillin. Con­ centrations as low as 61+ micrograms of cobalt chloride produced some enhancement of penicillin effectiveness. Cobalt in these concentrations wa^s non-toxic and conferred no protection on the animals. The results of cobalt action on penicillin reported by Pratt and Dufrenoy (121^) were extremely interesting and the possibility of extending their findings to such an organism as S_. pullorum seemed plausible. The first task was to find a suitable concentration of cobalt that would be effective against S. p u l l o r u m . The recommended concentration of one m g per liter of solution was not effective. A much higher concentration of cobalt was found to give very striking results. The concentration used in this study was 0.2 mg of cobalt chloride per ml of distilled water. This concentration was not toxic to S. p ullorum, although a concentration of 0.J+ m g per ml appeared to have some slight toxic effect. Plates seeded with pullorum and incubated w i t h cobalt solution for 30 minutes produced zones of inhibition that were three to ten mm. larger than control plates containing no cobalt. This is illustrated in Figure 1. The cobalt enhancement of penicillin action against S>. pullorum was then evaluated by means of a penicillin reference curve for this particular test organism (Figure 11). It was found that cobalt produced approximately a four-fold degree of enhancement. I n other words, 100 units per ml of 66 Figure 1. The enhancement of penicillin action against Salmonella pullorum by cobalt Both plates were seeded with Salmonella pullorum #89817 and subjected to equal concentrations of penicillin (100 u/ml). The zone of inhibition on plate A measured 22 mm, while that of plate B measured 32 mm. Organisms on plate B were incubated for 30 minutes in the presence of cobalt (0.2 mg/ml) before penicillin contact. Organisms on plate A received no cobalt treatment* 67 penicillin plus cobalt was equivalent to i+OO units per ml of penicillin without cobalt* The concentration of cobalt used in this study may not be the optimal, since there was not enough time to investigate a series of concentrations below 0.2 mg. The information obtained here was later used for in vivo experiments which will be discussed shortly. Pratt, Dufrenoy and Strait (93) proposed some theories regarding the me chanism of cobalt action. They stated that "the effect of cations m a y ultimately be associated w ith the formation of complexes with "*SH containing groups or with some other essential component of an energy-providing oxidation-reduction system. The degree of inhibition effects exhibited by the various cations may be related to the degree of binding of the cations in the complex. Thus cadmium and silver, wh ich form stable complexes, highly toxic, whereas cobalt, are which forms loose complexes with "*SH groups is m uch less toxic." The authors also postulated a general theory regarding the role of cobalt. They stated that penicillin exerts its greatest antibiotic effect on susceptible organisms when they are in a phase of logarithmic increase. P en i ­ cillin exerts its optimal effect on bacteria which are thriving in an enviornment favorable for t h eir growth. Conditions wh ich favor the multiplication of bacteria also increase the rate of penicillin action. Suitable concen­ trations of cobalt stimulate the metabolism or growth of 68 the organisms and u l t i m a t e l y render t he m more susceptible to p e n i c i l l i n action, Tlie results of cobalt action on ;S. p u l l o r u m are recorded in Table 13* M a r k e d in vitro enhancing effects can be duplicated if three ml of cobalt solution is u s e d in a concentration of 0,2 m g p e r ml* The Sensiti v it y of S. p u l l o r u m to Garlic Extract Alicin, the active p ri nciple of garlic cloves, has b e en found to exhibit a rather wide antibacterial spectrnm. Its action in vitro against S_* p u l l o r u m # 8 9 8 1 7 was very pronounced. The zones of i n h i b i t i o n p r o d u c e d on seeded plates were very sharp and clear* There was a m a r k e d similarity b e t w e e n these zones and those p r o d u c e d by p e n i ­ cillin. A streak-piate technique and a seeded agar m e th od were u s e d in testing S_* p u l l o r u m se ns it i vi ty (Tablesll^. and l £ ) • It appears that b o t h m e t ho d s can be u s e d for the d e te r mi na ti on of garlic sensitivity* The St ability of Aureomycin, Streptomycin, Chloromycetin, P en ic il li n and Garlic in the Presence of Various F e e d M i x t ur es (Tables 16 and 17) The results obtained from stability determinations Indicate that all the antibiotics tes t ed were relatively 69 TABLE 13 THE IN VITRO ENHANCEMENT OF PENICILLIN ACTION BY APPROPRIATE CONCENTRATIONS OF COBALT Trials 1 2 3 Toxicity control 2 ml cobalt (o ,2 m g / m l ) minus penicillin Penicillin 2 ml cobalt (0,2 mg/ml) 30 min, incub. 100 u/ml minus cobalt k 4+ 44* 26 26 21}.«5 21}- 23 22.5 22 21.5 5 6 7 8 4444- 27.5 29 26 28 23 o5 21,5 23 22.5 44* 444- 28 26,5 25 26 25 o5 22 21 21 21.5 22+ 26.3 22,2 9 10 11 12 13 Average diameter _________________ Penicillin 100 u/ml_________________ 1 ml cobalt 2 ml cobalt (0,2 mg/ml) minus cobalt (0,2 mg/ml) 30 mln. 1 2 0 min. 30 mln, incub, incub, incub. 1 2 3 k 5 6 7 8 9 10 Average diameter 21}..5 28.5 26,5 26 27 30 30 32 29 29 2i+ 25 26.5 25 27 23 22.5 23 o 5 22 21.5 26.5 2k. 5 26 27-5 27.5 28.5 28 29 25.5 25.5 >4. 22.5 20.5 20.5 22 22,5 29 2 5 *5? -- 26,2 —— — 22 70 TABLE 13 Trials cobalt 2 ml 1 2 3 25 26 2i+ 25 k 5 6 7 8 Average diameter 1 2 3 1-!* tt It tt 1! 26 2 3 o5 21+ 21+ 28.5 27.5 30 31.5 —_ 22+.7 29.1 29.4 — — 22.5 21 22 22 23 23 21.5 2k 22.1+ minus cobalt 23 23 21+.5 23 —— 22+ 26.6 23.14- 23 06 /V Toxicity control 3 ml cobalt (0.2 mg/ml) minus penicillin 30 min. in cub 0 + + + + 5 6 7 8 Average diameter minus cobalt 26 26.5 27 27 tt — Average diameter 1 2 3 2+ Penicillin 100 u/ml 30 min. incub 0 (0.2 mg/ml) cobalt (0.U- mg/ml) 2 ml 3 ml 3 ml zone 31 29 RPen 28 28.5 H 28.5 29«5 It 27 31 Penicillin 100 u/ml 30 min. incub 0 1 ml cobalt 0.2 mg/ml 0.002 mg/ml Tri als Trials CONTINUED + + + + Peni cillin 3 ml cobalt (0.2 mg/ml) 30 min. incub. 2k 23.5 22.5 22+ 100 u/ml minus cobalt 31.5 29 27 29.5 23.5 23 22.5 23 30 29.5 30 26.5 22.5 23 22.5 22.5 22.8 29.1 + = complete growth, of organi sm zone diameter measured in mm •ttReplicates not carried out beyond last reading 71 TABLE ljj. A STREAK-PLATE METH OD FOR DETERMINING THE GARLIC SENSITIVITY OF SALMONELLA PULLORUM Units per ml of mixture 1 2 3 k 5 10 20 30 k.0 Trials 1 (ij-8-hour _2_---r e ad .) Agar without garlic + + + + + + + + + + + + + + + + — — — _ + + + + + — — — 50 + - presence of growth; - = complete absence of growth TABLE 15 A SEEDED-AGAR METHOD FOR DETERMINING GARLIC SENSITIVITY OF SALMONELLA PULLORUM THE Units per ml of mixture Trials 1 (72-hour r e a d . ) 2 Agar without garlic 1 2 3 1+ + + + 4- + + + + + + + + 5 10 20 30 -f* + + + + + + + + + + = presence of growth.; - = complete absence of growth 72 TABLE 16 THE STABILITY OP SELECTED ANTIBIOTICS IN PEED SAMPLES A FTER A ONE-MONTH PERIOD OP STORAGE Antibiotic Aureom y ci n C h lo ro ­ mycetin Strepto­ m y ci n F e e d mixture Trials 3 A v e . zone diameter 1 2 l5?£ p rotein 23 22 21 22 22 2>0% protein 23-5 22 22 22 22.Ij. Sucrose + 1^>% prot e in 23 22 22 22 22.3 Sucrose + 30% protein 22. £ 22 20 20.5 21.3 D extrin + 1.5% p r o te in 23-5 23 05 23 05 23 23.14- Dextrin + 3 0 % protein 2k 2k 23 23 23-5 Commercial 20 20.5 21 21 20 06 1 21 20 19 19 19.6 30% protein 18.3 18 l8o.5 1 7 «.5 18.1 Sucrose + p rotein 23 21 20.5 20o5 21.2 Sucrose + 3 0 /o pro te i n 20 20 20o5 13.5 19-8 Dextrin + 1 5 ^ pro te in 20 •5 20.5 2 1 .5 .2 1 . 5 21 Dex tr i n + 3 0 ?& protein 20 18 17 l8o5 1 8 oil- Commercial 25 2k 25 25-5 21).09 15>% protein 15 15 15 15-5 15-1 3 0 % protein 15.5 15 1 5 . 5 16 15-5 Sucrose + protein 15 15 16 16 15-5 Sucrose + 3 0 % protein 16 16 16 16 16 prot ei n k 73 TABLE 16 Peed mixture Strepto­ mycin Penicillin G-arl i c k Ave. zone diameter Dextrin + 1^% protein 16 15 15 15 15.2 Dextrin + protein 16 15 15 15.5 15.1+ ±5% protein 22 22 21 22 ro H • 1 Tri als 2 3 30% protein 26 27 25 o5 2k 25.5 Sucrose + 1$% protein 25 26 25 2lj-.5 25.1 Sucrose + 30% protein 23.5 2k 2k 25 2i|.ol Dextrin + l5$> protein 23 2k 25 2k 2k Dextrin + 30% protein 25 21J..5 2L|.o5 25.5 2ko9 13% protein 21.5 22.5 22 20.5 21.6 30%> protein 23 23 23.5 22.5 23 Sucrose + 15$ protein 22 20.5 22.5 21 21.5 Sucrose + 30%> protein 22 22 23.5 23 22.6 Dextrin + 1%% protein 2Lj_•5 26 26 27 25 09 Dextrin + 30% protein 2k 2k 2k 23 23.8 Zone diameter measured in mm Control (feed minus antibiotic) - no antibiotic detected in all tested feed samples The zones of inhibition produced by theoretical of antibiotic in tested feed samples (from reference Aureomycin - 2J?.0 m m Penicillin — 2^.3 Chloromycetin - sample mixture 20.3 m m ; commercial sample 25*7 1,1111 Streptomycin - l5«3 111111 Garlic - 22.0 mm CD Antibiotic CONTINUED activity levels curve data) m 7k TABLE 17 SUMMARY OP ANTIBIOTIC STABILITY AFTER A ONE-YEAR PERIOD . 4., Antibiotic feed mixture Ave. zone diameter (mm) Zone produced byoriginal concentration (from ref. curve data) Aureo-l5% protein Aureo-30^ protein A u r e o - 1 ^ protein-sucrose Aureo-30^ protein-sucrose Aureo-l5% protein-dextrin Aureo-30% protein-dextrin Aureo-commercial sample 21.8 21.2 18.8 CM-15# protein CM- 3 0 ^ protein CM-l5/£ protein-sucrose CM-30?o protein-sucrose CM-15/2 protein-dextrin CM- 3 0 ^ protein-dextrin CM-commercial sample 20.5 18.7 19 o2 20.5 21.0 18.7 19.5 23.6 25.3 SM-l55> SM-30^ SM-l5% SM- 3 0 ^ SM-1^ SM-30/& protein protein protein-sucrose protein-sucrose protein-dextrin protein-dextrin Garlic-l5% Garlic-30?o G-arlic-l5?e Garlic- 3 0 ^ Garlic-1^ Garlic-30^ protein protein protein-sucrose protein-sucrose protein-dextrin protein-dextrin Aureo — aureomycin CM = Chloromycetin SM = streptomycin 19.0 20.8 21.7 20.6 15.3 15.2 15.5 no zones 15.3 no zones 20.5 20.0 20.8 19.3 21 o 5 22.0 21+.5 tt Tt II It tt u 11 II ft It II 15.3 11 tl IV It It 22.0 11 It tt tt tt 75> stable over a one-year period. Aureomycin appeared to undergo a slight to moderate decrease in potency at the end of four weeks, but this was probably due to an initial adsorption to feed constituents rather than outright deterioration. This conclusion is based on the fact that similar zones of inhibition were produced at the end of twelve months (identical amounts of feed-mixture were tested at one and twelve mont hs )• The slight variations in zone diameter are negligible when the errors inherent in plate-assay procedures are considered. A number of in vivo experiments covered a period of two to three weeks and it was necessary to know whether a particular antibiotic would remain stable for that length of time. It was also of interest to know whether these antibiotics would retain their potency over a protracted period of time under practical storage conditions. At the end of one year, streptomycin exhibited evidence of deterioration in two of the sample mixtures. Penicillin mixtures produced zones of inhibition that were almost identical at one and six month intervals. This is probably due to the fact that oral penicillin preparations are usually well buffered to withstand gastric acidity. Qualitative determinations were made on veterinary preparations of Chloromycetin and streptomycin. These antibiotic mixtures produced ill-defined zones of inhibition that could not be evaluated with accuracy. 76 An Xn Vivo Evaluation of 15 Experimental Series (Tables 18 - lp6) A» General considerations A total of four series failed to produce valid results (Series 2, 5* 10 and lip) • These failures were evidenced by very low or non-existent mortality in all experimental groups. for successful, In this study, the only criterion artificial Infection was a demonstrable difference in mortality among non-treated, and treated infected groups. infected controls However, this method of evaluation may be somewhat severe, in view of the fact that chicks infected with S . pullorum may exhibit symptoms of the disease without succumbing to the infection (these birds are dangerous because they may be carriers)• The infected controls of the present experimental series (invalid series) pasted vents, exhibited such pullorum symptoms as drowsiness, ruffled feathers, and respiratory difficulties. extreme thirst However, within a few days all symptoms disappeared and to all outiirard appearances the chicks appeared healthy. There Is a good possibility that £3. pullorum could have been recovered, swabs had been taken at this time. if cloacal Several factors may have contributed to low or no mortality among the non­ treated, infected controls. One factor may be that a critical number of Infecting organisms are required to produce drastic results (in certain 77 cases) • In series ll}., the usual method, of preparing an inoculum, resulted in a bacterial count of 900,000,000 organisms per ml of saline suspension (the usual average being 1,350,000,000 organisms per ml of suspension). On the other hand, in series 10, the bacterial count was 1 ,600,000,000 organisms per ml of saline suspension. Another factor to consider is the present day practice of careful genetic selection for resistant birds. Genetic variations among different breeds and within a particular breed only serve to complicate the results obtained In studies of this nature. It is known, for instance, that the White Leghorn breed is rather resistant to pullorum infection (30, 56). The virulence of the infecting organism is also of prime importance. This factor cannot be predicted with certainty from one experiment to the next. Certain pro­ cedures such as chick passage are of benefit, but even here the method can be open to question. It was found in this study that the virulence of the infecting organism could be maintained for a long period of time in stock cultures, if transfers were made every two weeks. The degree of pullorum infection is also dependent upon the age of the chick at the time of exposure. In this particular experiment a 72-hour period of prophylactic feeding was used in the first two experimental series. This was later changed to lj.8 hours and then to 2 hours. It Is a known fact that chicks rapidly develop resistance 78 to pullorum infection after* the first day of life. A rti­ ficial exposure to infection beyond the first day will not produce as many fatalities as infection at one day of a g e« This may have been a factor in causing low mortality rates among some non-treated, infected controls. Chicks which died of pullorum infection during the experimental periods were necropsied and examined for any gross lesions. Usually consolidation of the lungs and nodulation of the heart appeared in 12 to lip days after infection. Chicks dying of overwhelming infection during the first two or three days generally did not exhibit any unusual lesions. An attempt was made to isolate the in­ fecting organism from each chick that died, but this was not always possible in every experimental group and there­ fore only a representative number were sampled in such instances. It should be stress,ed that in vivo experiments are usually subject to many variables. Some of these variables are known, while others remain undetected. A large number of experiments are therefore necessary In order to determine the validity of all results obtained. In this particular study, a number of experiments have been repeated in successive trials. However, this Is not sufficient evidence for making definite conclusions about the efficacy of any particular antibiotic therapy (or other treatment). The experiments in this study have merely provided a basis for further work. The results indicate a trend or direction 79 that should bo followed either with additions or modifi­ cations of present procedures. B. Specific considerations 1. The value of feed modifications with respect to protein and carbohydrate content. Mann (78, 79* 80) con­ siders S_. pullorum as only a potential pathogen and that its existence is dependent on a symbiotic relationship with certain gram-positive organisms. He therefore claims that a diet which suppresses gram-positive organisms will check the eventual course of pullorum infection. proposes that chicks be fed an oat diet for 72 Mann hours in order to establish an acid condition in the intestinal tract. This will provide an environment unfavorable to gram-positive organisms of the "welchii type” , which are regarded as the principal symbionts for £3. pullorum. After 72 hours, the oat diet was supplanted by feed con­ taining lip percent protein. Mann holds that feeds con­ taining 20.percent protein favor the growth of grampositive symbiontso The work of Mann is interesting in view of our present knowledge concerning the effect of antibiotics on the normal intestinal microflora. Hoxvever, the findings reported by Mann are vague in many places and the proof of symbiotic relationships are inconclusive. The inclusion of oats in the diet would appear to be r ather unnecessary, since the intestinal tract of young chicks is normally in 80 an acid condition, Nevertheless it was of interest to test the hypothesis of M a n n in this study. His proposals w e r e modified so that feeds containing two extremes of protein content were used, A 30 percent protein feed was employed in order to exaggerate the harmful effects of high protein diets on the course of pullorum infection (as believed by Mann), The results of the first series of experiments indicate that oat feeding and a lowered protein content of feed do not reduce mortality. diets, When aureomycin was added to these there was a m a r ke d decrease in mortality. Perhaps more experiments w o ul d be needed to confirm or refute the findings by Mann, but at present the in d i ­ cations do not seem to support the original hypothesis. Johansson, Sarles and Shapiro (59) found that sucrose as the principal carbohydrate in chick feed, produced a m a rk ed depression on coliform organisms. other hand, Dextrin, on the stimulated the development of organisms at all levels of the intestinal tract. It was of interest to determine the effect of these carbohydrates on the eventual course of pu llorum infection. There was no opportunity for evaluating dextrin, but sucrose was included in one experi­ mental group. The results were not striking and the usual mortality rates from infection per os were obtained. aureomycin group of this particular series The (Series 3) had a 1 3 « 3 percent mortality, while for sucrose it was 2 i-j_ percent. However, a combination of sucrose plus aureomycin resulted 81 in no mortality. There may be a synergistic effect between sucrose and aureomycin, but this cannot be stated with certainty from one group of experiments. 2. Aureomycin. This antibiotic appears to be effective in reducing mortality rates due to pullorum infection. The synergistic effect of this drug in combination w i th other antibiotics remains to be determined. Of special interest w ould be the use of aureomycin along with appropriate levels of penicillin. 3. Chloromycetin. This drug also seems to be of value in the treatment of pullorum infection. Its effectiveness in synergistic combinations remains to be ascertained. Chloromycetin was combined with aureomycin and also with penicillin, but the results were not conclusive as far as a synergistic phenomenon is concerned. I4-. Penicillin. The use of oral penicillin in feed mixtures involves some important considerations. The amount of penicillin given by oral administration must be at least three to five times greater than a parenteral dose. is due to several factors. First, This absorption of oral peni­ cillin in the intestinal tract is erratic. Secondly, organisms present in the intestinal tract may inactivate a large proportion of the original concentration by elabora­ tion of the enzyme penicillinase. Thirdly, the gastric juices may also destroy penicillin to a certain extent, even 82 tta.ou.gta. oral preparations are highly buffered.-® Therefore taigta concentrations of penicillin must be used to offset ttaese various factors, Ttae effect of penicillin on ttae course of pullorum infection taas been beneficial in a number of experimental groups (Series 3, 7, 9, 12, 15) • Ttae erratic nature of penicillin absorption at inadequate concentration levels is best illustrated by ttae results obtained in several experimental groups. In Series 3* 7 and 9, low mortality figures at 6 6 0 and 3 3 0 units per g of feed were recorded. Poor results were obtained in Series 13* which included penicillin groups having concentrations of 2 0 0 0 and 2 2 9 0 units per g of feed. Series 12 and 15, Favorable results w ere observed In at levels of 2936 and 5500 units of p e n i ­ cillin per g of feed. It is therefore extremely important to maintain an adequate dosage level. This level is probably well above 5 0 0 0 units of penicillin per g of feed. From in vitro results, ttae Indications are that p e n i ­ cillin blood levels of 1 5 to 20 units per ml of plasma may be necessary at all times during ttae acute fection. However, stage of i n ­ a study should be made on ttae actual blood levels of chicks being fed a penicillin masta0 Perhaps the actual concentration of penicillin will have to be based on a series of blood level determinations plus a number of in vitro studies on various pullorum strains. The in vitro studies indicated that appropriate con­ centrations of cobalt markedly reduced ttae minimal inhibiting 83 concentration of penicillin. Cobalt produced a four-fold enhancement of penicillin action. also observed in vivo. This phenomenon was Penicillin, when incorporated at a level of 2 2 9 0 units per g of feed, did not reduce m o r ­ tality figures to any appreciable extent, but with cobalt treatment there was a striking reduction in mortality. Series 15 the non-treated, In infected control had a mortality figure of 32 percent, while three cobalt groups had m o r ­ talities of 12 and 0 percent respectively. The proper use of cobalt may greatly reduce the required amount of penicillin. Studies should also be made on the optimal concentration of cobalt necessary for maximum enhancement (in v i v o ) of penicillin action. Sodium benzoate \tfill prolong penicillin blood levels after oral A b f i in i s t P a t * oH This is due bo the fact that sodium benzoate inactivates the enzyme penicillinase which is produced by such intestinal organisms as Escherichia c o l i . Sodium benzoate was used in Series 7> 8 and 9. However, no conclusive results were obtained since low concentrations of penicillin were employed in these groups. In vitro studies should also be conducted with various concentrations of sodium benzoate. Cobalt along w i t h sodium benzoate may prove to be valuable agents in reducing the minimal Inhibiting concentration of penicillin. There are indications that a marked increase in oxygen uptake occurs when certain bacterial species are placed In contact with appropriate concentrations of cobalt. It would 8JU. be extremely interesting to p erform a series of manometric studies on the oxygen uptake of S>. pullorum in the presence of cobalt. It might also be m en tioned that penicillin at higher concentrations did not appear to exert any toxic effect on chicks. Cobalt without the addition of penicillin may enhance the virulence of S . pullorum. This m a y be true in view of the current belief that cobalt accelerates metabolic activity among certain microorganisms. 5>. Streptomycin. The results obtained by using a veterinary streptomycin mixture were not very pronounced. Prophylactic feeding of this mixture did not seem to reduce the symptoms of pullorum infection. mycin, Birds given aureo­ Chloromycetin and penicillin (in adequate dosages) exhibited slight transient symptoms. The use of other streptomycin preparations m a y produce b e t t e r results. However, it is a well k n o w n fact that a number of organisms will quickly develop resistance to rather high concen­ trations of streptomycin. Therefore, it w o u l d be Important to conduct a series of in vitro tests on the development of resistance to streptomycin by S. p u l l o r u m . Prom in vitro determinations it wo uld appear that streptomycin is mainly bacteriostatic in action against £>. p u l l o r u m . These factors m a y preclude the further use of streptomycin in the control of pu llorum infection. 85 6 . Antibiotic substances from higher plants. Anti­ biotic substances from higher plants do not appear to be beneficial in the control of pullorum disease. Such sub­ stances as Trigonella Foenum-graecum actually exaggerated the symptoms of this disease. Birds that were given this preparation developed extreme pasting of the vent and p r o ­ nounced respiratory symptoms. this group, 60 percent. Mortality was very high in The use of banana meal may have possibilities, but there is not sufficient evidence for any definite conclusions. recorded for non-treated, series of experiments. A mortality of 3i{-«5 percent was infected controls in the third A mortality of 16 percent occurred among birds given banana meal diet. (Maqueno variety) in the For many years the beneficial results of banana feeding were thought to be due to the exceptional nutritional qualities of this plant. The recent discovery of several promising antibiotic fractions in the banana by Scott, McKay, Schaffer and Fontaine (115) has put new emphasis on the use of the ban^ia for therapeutic puproses* Garlic is perhaps the most promising of all the higher plants. The in vitro actioh of garlic against S. pullorum is very similar to that of penicillin. Zones of inhibition on seeded plates were very sharp and remained clearly de­ fined for many weeks without any encroachment by the test organism. The effect of garlic appears to be bactericidal for S . p u ll o r u m . The use of garlic in several in vivo experiments did not produce very favorable results. However, 86 this m ay have been due to the fact that the concentrations employed were too h ig h and actually exerted a toxic effect on the chicks. Experiments should be tried in which very low levels are used over a longer period of time. I n the present study garlic constituted 15? and 6 percent of feed mixtures respectively, Xt would be of interest to use a concentration of one or two m g of garlic powder per g of f eedo 7 o The use of an antigen-mash. In this experiment oral antigen preparations were Incorporated In the feed. The antigen was either adsorbed on pl a in feed or on a charcoal preparation. The results obtained in these experimental groups were poor, and it appeared that char­ coal h a d a slight toxic effect on young chicks w h e n given for a prolonged period of time* Ingestion of a p repared antigen would ■undoubtedly have a decided advantage over conventional methods of immunization. A n antigen-mash m a y have value in protecting young chicks from later infection* What appeared to be a successful method of immunization against pullorum disease was reported b y Morcos, Zaki and Zaki in 191+6 (85) • Their m e t h o d of immunization consisted of testing mature chickens for the presence of pullorum infection by the agglutination technique* proved to be absolutely negative, If these chickens they were inoculated intramuscularly with a heat-killed antigen (1 0 ^ organisms per ml). Each bird received 0.5 ml, one ml, and one ml at 8? w e ek ly intervals. Two weeks after the last injection the birds were tested by the agglut in at io n method. Each bird p o s s e s s e d a h i g h titer as compared to non-va c ci na te d controls. The eggs of vaccinated birds were collected and kept for hatching. controls. The same was done w i t h n o n - v a c ci na te d E ig h t y chicks f rom vaccinated parents survived, while n i n e t e e n survived from the n o n- va cc i na te d controls (these chicks were not exposed to JS. p u l l o r u m ) . W h e n the chicks were two months old they w e r e tested b y the a g g l u ­ tination method. The chicks f rom vaccinated birds a titer of b e t we en 1 :80 and 1:320, while gave the n on -v accinated chicks did not exceed a ti ter of 1:£. The authors conclude that "agglutinins were t r a n s ­ m i t t e d f rom the va c ci na te d m o t h e r to the chick through the egg” o The Percentage of C a rr ie r Birds P roduced b y Treatment w i t h Aureomycin, Chloromycetin, P en ic il li n plus Cobalt P e n i c il li n and (Table JU-7) A total of 278 birds were retained for carrier studies. Most of the bi rds were killed at three months of age (aureomycin group nine months) the p resence of S.. p u l l o r u m . and carefully examined for The aureomycin group c o n ­ tained the smalUe st numb er of birds (thirteen). The p er centage of carriers recorded among the several different groups w e r e a3 follows: C hl oromycetin — first group, aureomycin, 0 percent, 0 percent; second group, 28 88 percent, and third group, i^OO units pe r g feed, 16 percent; penicillin -- 31-9 percent, ££00 units per g, 1 0.5> percent; p en i cillin (2290 units per g) plus cobalt, 6.6 percent and non-treated, infected controls, 16 percent. The listed results cannot be taken at face value. Some important factors must be considered before any definite analysis can be made. First of all, a m uc h larger group of birds from repeated experiments would have to be sampled. Secondly, birds given different antibiotics (or different dosage levels of the same antibiotic) be isolated with in their respective groups. should This must be done in order to evaluate the efficacy of a particular antibiotic (or dosage level) organism from the host. in removing the infecting Recovered birds w h i c h no longer harbor the infecting organism (due to an effective anti­ biotic) may again become infected with the same organism if placed in contact w ith other birds that are still infected (due to an ineffective antibiotic). Unfortunately, the facilities for extensive carrier studies were rather limited in this experiment and various groups had to be kept in the same housing area. Finally, the optimal concentration of antibiotic necessary to eliminate carriers, m ay not have b e e n attained in a number of experimental true of penicillin. groups. This is probably very It is believed that the effective con­ centration of penicillin required to eliminate carriers is well beyond the highest levels employed in this study (a limited supply of p e n i ci l li n p r e v en te d the use of higher concentration levels). The number of infecting organisms u s ed for artificial i n f ec ti on m a y be far in excess of that likely to be encount er ed in natural infections. This possibility m ight decrease the required minimal, inhibiting concentration of antibiotic for natural infections. (as indicated in this study) However, artificial infections can neve r duplicate the conditions operating in nature and therefore all determinations m ust be b a s ed on laboratory situations* SUMMARY Aureomycin, Chloromycetin and penicillin were effective in reducing fatalities among young chicks arti­ ficially infected with S. p u l l o r u m . The protective action of a veterinary preparation of streptomycin and that of garlic was not very pronounced® In vitro studies indicate that iS. pullorum #89817 is sensitive to low concentrations of aureomycin (2 micrograms of aureomycin per ml of broth medium) and Chloro­ mycetin (1o5>6 micrograms per ml of broth medium). S_. pullorum # 8 9 8 1 7 also possesses a moderate sensitivity to penicillin (15 > units per ml), ml) garlic (20 to 30 units per and streptomycin (6.6 micrograms per ml). pullorum strains, Five in addition to # 8 9 8 1 7 * were also found to be sensitive to penicillin action. Antibiotic substances from higher plants did not seem to exert any beneficial effect on the course of pullorum infection. I n vitro determinations indicate that garlic may have therapeutic possibilities. The concentrations employed in this study were too toxic for young chicks. Appropriate concentrations of cobalt chloride markedly enhanced the action of penicillin against S_. pullorum. tests. This was true for both in vivo and in vitro Cobalt produced approximately a four-fold enhance­ ment of penicillin action. 91 A n attempt to immunize young chicks against pullorum infection by use of an antigen-feed did not produce any significant results. However, the possibility of immuni­ zation by other methods still existso Aureomycin, Chloromycetin, streptom;/cin and garlic were found to be relatively stable in various feed mixtures over a one-year period. Penicillin was also found to be stable when tested at the end of a six-month period. Carrier birds were found among those groups treated with Chloromycetin, pe ni cillin and penicillin plus cobalt* No carriers were found among birds treated w i t h aureomycin. The small number of birds in the aureomycin group, precludes any definite statements regarding the value of this anti­ biotic in the elimination of p ullorum carriers* A combination of cobalt plus penicillin produced the lowest carrier percentage among the larger groups of birds tested (6.6 percent). TABLE 18 EVALUATION OP THE EFFECT OP FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 1) Experimental group protein (infected control) Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days 0 2k 72 9 37.5 30$ protein 0 25 72 6 2ij.o0 Oats - followed by 15 % protein 0 25 72 7 28,0 Oats - followed by 30$ orotein 0 25 72 7 28.0 1% protein + aureomycin 1 29 72 1 3«k 30$ protein + aureomycin 1 29 72 0 0 Oats - followed by 1% protein + aureomycin 1 29 72 3 10.3 Oats - followed by 30% protein + aureomycin 1 29 72 2 6o9 Breed of chicks: White Leghorn TABLE 19 THE RECOVERY OP SALMONELLA PULLORUM PROM DEAD CHICKS Experimental group Growth on bismuth sulfite agar Kliglers Butt Slant (SERIES l) Individual sugars Mannitol Sucrose Sims h 2s Motility Oats-30% Oats-30/6 Oats-30^ Oats-30^ protein protein protein protein + + + + A A A A AK AK A A A A AK A AK AK AK NR 4* + N N 0ats-15$ 0ats-l5$ Oats-15$ 0ats-15$ protein protein protein protein + + + + A A A A AK AK AK AK A A A A AK AK AK AK + + + + 30$ protein + + + + A A A A AK AK AK AK A A A A AK AK AK AK + + + + 15$ protein l$ fo protein 1$fo protein 1S i protein 15% protein + + + + + A A A A A AK AK AK AK AK A A A A A AK AK AK AK AK + + + + + mm N N N N N + A AK A AK + 30$ protein 30$ protein 30$ protein protein + aureomycin Gas mm- - - N N N N am - - - - am am am - - - - - - ma - - - mm - - mm - N N \% 0ats-l5$ protein + aureomycin 16 positive isolates A - acid reaction NR - no reaction AK - alkaline reaction F - reaction not carried further TABLE 20 EVALUATION OP THE EFFECT OP FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 2) Experimental group Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days Non-infected control 0 30 0 0 0 Infected control 0 23 0 1 4*3 32 72 1 3.1 Penicillin 550 units Sucrose 0 30 72 0 0 Sucrose + aureomycin 1 25 72 0 0 Aureomycin 1 25 72 1 4 150 25 72 16 25 72 2 Garlic Banana 150 Breed of chicks: White Leghorn Feed: Kellogg starter 64*0 8 TABLE 21 THE RECOVERY OF SALMONELLA PULLORUM FROM DEAD CHICKS Experimental group Growth on bismuth sulfite agar Aureomycin Banana Penicillin Garlic + *!* + + Garlic Garlic Garlic Garlic Garlic Garlic Garlic Garlic Garlic 12 positive isolates Kliglers Butt Slant A A A AK . a?: AK AK + + + + A A A A + + + + + A A A A A h (SERIES 2) Individual sugars Mannitol Sucrose A Sims h 2s Motility A A AK AK AK AK + + AK AK AK AK A A A A AK AK AK AK + + + + AK AK AK AK AK A A A A A AK A AK AK AK + N + + + ; -v + Gas « • - - f - - - «■» MB mm - ~ - •* N N - - - — mm A - acid reaction All - alkaline reaction N - reaction not carried further vO vn TABLE 22 EVALUATION OF THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 3) Experimental group Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds 0 0 Percent mortality in 21 days Non-infected control 0 2k Infected control 0 29 0 10 3k»S 25 2k 1 Jj-oO 25oO Penicillin 660 units ’ 0 Sucrose 0 2k 2k 6 Sucrose + aureomycin 1 25 2k 0 Aureomycin 1 30 2k k 13.3 ' 60 27 2k 6 22,2 25 2k k 16,0 Garlic Banana 150 Breed of chicks: White Leghorn Feed: Kellogg starter 0 TABLE 23 THE RECOVERY OF SALMONELLA PULLORUM FROM DEAD CHICKS Experimental group Infected Infected Infected Infected Growth on bismuth sulfite agar Kligl ers Butt Slant (SERIES 3) Individual sugars Mannitol Sucrose h 2s control control control control + + + + A A A A AK AK AK AK A A A A AK AK AK AK + + Infected control Infected control Infected control + + + A A AK AK AK AK A A A AK AK AK + + N Aureomycin Aureomycin Aureomycin Aureomycin + + + + AK A A A AK All AK AK AK A A A AK AK AK AK Banana Banana Banana Penicillin + + + + A A A A AK AK AK AK A A A A Garlic Garlic + + A A AK AK Sucrose Sucrose Sucrose Sucrose Sucrose + + + + + A A A A A AK AK AK AK AK 20 positive isolates Sims Motility Gas + - mm - - n, - - N N N + + + N N mm - - - - - AK AK AK AK + + + + _ - — - - A A AK AK + + - - A A A A A AK AK AK AK AK + + + + + A - acid reaction AK - alkaline reaction N - reaction not carried further - - _ - — — mt TABLE 2lj. EVALUATION OF THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED VJITH SALMONELLA PPLLORUM (SERIES Ij.) Experimental group Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days Non-infected control 0 25 0 1 Infected control 0 2b 0 7 29.2 Penicillin - 1 660 units 29 4-8 lb - ltf.3 Penicillin - 2 990 units 25 i+8 11 bb*o Aureomycin - 1 1 2b ^8 b- 16.7 Aureomycin - 2 1 25 US 1 ij-.o Antigen mash (ethanol attenuated) 0 25 kB k 16.0 Antigen mash (heat attenuated) 0 25 lj.8 6 21)..0 Breed of chicks: Rhode Island Red Feed: Kellogg starter TABLE 25 THE RECOVERY OF SALMONELLA PULLORPM FROM DEAD CHICKS Experimental group Infected Infected Infected Infected Antigen Antigen Antigen Antigen Antigen Growth on bismuth sulfite agar control control control control mash mash mash mash mash (ethanol) (ethanol) (heat) (heat) (heat) Aureomycin - 1 Aureomycin - 2 Kliglers Butt Slant (SERIES if) Individual sugars Mannitol Sucrose Sims h 2s Motility + + + + A A A HS AK AK AK HS A A A N AK AK AK N + + N + + + + + A A A A A AK AK AK AK AK A A A A A Alt AK AK AK AK + + + + + + + HS A HS AK N A N AK N + 4* Penicillin Penicillin Penicillin penicillin Penicillin - 1 1 1 1 1 + + + + + A A A A A AK AK AK AK AK A A A A A AK AK. AK A AK + + + N + penicillin penicillin Penicillin Penicillin Penicillin - 1 1 1 1 2 + + + + + A A A A A AK AK AK AK AK A A A A A AK AK AK AK AK + + + + + 2 2 2 2 2 + + + + + A A A A A AK AK AK AK AK A A A A A AK AK AK AK AK + + + + + Penicillin Penicillin penicillin penicillin Penicillin - - 23 positive isolates Gas mm - - - N N _ - - MS - ~ - N - N - — - — - N - N tm — «v - - — - — — - - — - — — — A - acid reaction HS - Excess H 2S AK - alkaline reaction N - reaction not carried further TABLE 26 EVALUATION OF THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 5) Experimental group Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days Non-infected control 0 18 0 1 5.6 Infected control 0 30 0 3 10.0 Penicillin - 1 660 units 2k w 8 33.3 Penicillin - 2 990 units 25 lj.8 3 12,0 1 25 14-8 3 12.0 Aureomycin - 2 2 25 148 1 Ij-.O Antigen mash - 1 (ethanol attenuated) 0 2k CO 5 20,8 Antigen mash - 2 (ethanol attenuated) 0 25 l ^.0 100 Breed of chicks: Rhode Island Red Feed: Kellogg starter CD Aureomycin - 1 TABLE 27 THE RECOVERY OF SALMONELLA PTJLLORUM FROM LEAD CHICKS Experimental group Growth on bismuth sulfite agar Kliglers Butt Slant (SERIES 5) Individual sugars Mannitol Sucrose Sims Gas h 2s Motility . Non-infected control Infected control Infected control Infected control + + + + A A A A AK AK AK AK A A A A AK AK AK AK + + + + Antigen Antigen Antigen Antigen NR + + N A A A N AK AK AK N A A A N AK AK AK NR N NR N N N N N + A AK NR N + mash mash mash mash - 1 1 1 2 Aureomycin - 1 Aureomycin - 1 Aureomycin - 1 Aureomycin - 2 Penicillin Penicillin Penicillin Penicillin - 1 1 1 1 Penicillin Penicillin Penicillin Penicillin - 1 1 1 1 16 positive isolates - — N + + + N N - - •a - - - N N N N N N N N A All + - - N N N N N N AK N N A AK N N N + N _ K - NR NR A N N N N + A AK + + A A AK AK + A A AK AK NR N N + A A AK + + AK A - acid reaction Ak - alkaline reaction N A N N AK + - - A A A AK AK AK + + + A AK + - _ - N A A N AK N N M N — AK + N - reaction not carried further NR - no reaction •m 101 Penicillin - 2 Penicillin - 2 Penicillin - 2 - TABLE 28 EVALUATION OP THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 6) Experimental group Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days Non-infected control 0 25 0 1 1+.0 Infected control 0 25 0 7 28 «0 9 37.5 CO 8 32.0 3.7 Penicillin - 1 660 units 21+ Penicillin - 2 990 units 25 Aureomycin - 1 1 27 CO 1 Aureomycin - 2 2 27 l)fl 1 3.7 Antigen mash - 1 (heat attenuated) 0 25 kS 6 21+.0 Antigen mash - 2 (heat attenuated) 0 25 kd 8 32.0 102 Breed of chicks: New Hampshire Feed: Kellogg starter TABLE 29 THE RECOVERY OF SALMONELLA PULLORUM FROM DEAD CHICKS Experimental group Infected Infected Infected Infected Infected Infected Antigen Antigen Antigen Antigen Antigen Antigen control control control control control control mash mash mash mash mash mash Growth on bismuth sulfite agar Kliglers Butt Slant (SERIES 6) Indi vidual sugars Mannitol Sucrose Sims Motility «v N - + + + + + + A A A A A A AK AK A AK AK AK A A A A A A AK AK A AK AK AK + + N + + + - 1 1 1 1 1 1 + + + + + + A A A A A A AK AK AK All AK AK A A A A A A AK AK AK AK AK AK + + + + + + Antigen mash Antigen mash Antigen mash Antigen mash Antigen mash Antigen mash - 2 2 2 2 2 2 j. + + + + + A A A A A AK All All AK AK A A A A A AK AK AK AK AK AK AK + + + + + + + + A A AK AK AK AK + + Aureomycin - 1 Aureomycin - 2 A A A A Ga; H23 mm ~ •* mm - . N — - mm mm- _ - _ - - TABLE 29 Growth on bismuth sulfite agar Kliglers Butt Slant Individual sugars Mannitol Sucrose Sims h 2s Motility Penicillin Penicillin Penicillin Penicillin Penicillin - 1 1 1 1 1 + + + + + A A A A A All AK AK AK AK A A A A A AK AK AK AK AK + + + + + Penicillin Penicillin Penicillin Penicillin Penicillin - 1 1 1 2 2 + + + + + A A A A A AK AK AK AK AK A A A A A AK AK AK AK AK + + + + + Penicillin Penicillin Penicillin Penicillin Penicillin - 2 2 2 2 2 + + + + A A A A A AK AK AK AK AK A A A A A AK AK AK AK AK + + + + + 3i| positive isolates Gas . - - - - - - - - - ■M - — — — - •m _ - _ — mm - A - acid reaction AK - Alkaline reaction N - reaction not carried further •*701 Experimental group CONTINUED TABLE 30 EVALUATION OF THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 7) Experimental group Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days Non-infected control 0 30 0 2 6.7 Infected control 0 23 0 7 304 Rutin - 1 1 25 kQ 3 12.0 Rutin - 2 2 2k ks 5 20.8 Antigen mash - 1 (heat attenuated) 0 25 I f i. 6 2lp.O Antigen mash - 2 (heat attenuated) 0 2k lj.8 7 29*2 Sodium benzo'ate control 1 22 w 5 22,7 25 lj.8 2 8.0 Penicillin + sodium benzoate 660 units 105 Breed of chicks: New Hampshire Feed: Kellogg starter TABLE 31 THE RECOVERY OP SALMONELLA PULLORUM PROM DEAD CHICKS Experimental group Growth on bismuth sulfite agar Kliglers Butt Slant (SERIES 7) Individual sugars Mannitol Sucrose Sims h 2s Motility Non-infected control Non-infected control Infected control Infected control Infected control + + + + + AK AK A A A AK AK AK A AK AK Alt A A A AK AK AK AK AK N N + N + Infected Infected Infected Infected + + + + A A A A AK AK AK AK A A A A AK AK AK AK + + + + + + + + + AK A AK AK AK AK AK Alt A AK AK AK AK AK N + + + + AK N N AK AK AK + N N + + + Sodium Sodium Sodium Sodium Sodium control control control control benzoate benzoate benzoate benzoate benzoate Antigen Antigen Antigen Antigen Antigen Antigen mash mash mash mash mash mash - 1 1 1 1 1 1 control control control control control + + NR + + + A A A A HS N A A A AK HS N AK AK Alt A A A A N N A A A Ga; N N N - N N ■■ N - - - - - N N _ — - wm mm - mm N N + N - - - - - mm 106 TABLE 31 Experimental group Antigen Antigen Antigen Antigen Antigen Antigen mash mash mash mash mash mash - Growth on bismuth sulfite agar 2 2 2 2 2 2 CONTINUED Kliglers Butt Slant Individual sugars Mannitol Sucrose Sims h 2s Motility + + + + + + A A A A A A AK AK AK AK AK AK A A A A A A AK AK AK AK AK AK + + + + + + Penicillin - 1 Rutin - 1 Rutin - 1 + + + A A A AK AK AK A A A AK AK AK + + + Rutin Rutin Rutin Rutin Rutin + + + + + A A AK AK AK AK AK A A AK AK AK AK AK + + + N + - 2 2 2 2 2 27 positive isolates A AK A A - acid reaction A AK A Gas «■» - - - - ** _ M M - • N mm N •* HS - Excess H£S AK - alkaline reaction HR - noreaction N - reaction not carried further 107 TABLE 32 EVALUATION OF THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 8 ) Concentration of antibiotic agent mg/g feed Number of birds ■J Hours of prophylactic feeding Number of dead birds Percent mortal ity in 21 days Non-infected control 0 32 0 0 Infected control 0 25 0 6 2lj.,0 Rutin - 1 1 25 1*8 1 ij-.O Rutin - 2 2 25 llfl 5 20,0 Antigen mash - 1 (heat attenuated) charcoal adsorbed 0 25 1*8 0 0 Antigen mash - 2 (heat attenuated) 0 2k CO 6 25*0 Sodium benzoate control 1 25 4CD =- Experimental group 2 8.0 6 25.0 Penicillin + sodium benzoate 660 units 2k 108 Breed of chicks: Barred Rock x White Rock Feed: Kellogg starter 0 TABLE 33 Experimental group Infected Infected Infected Infected Infected Infected Growth on bismuth sulfite agar + + control control control control control control Antigen Antigen Antigen Antigen Antigen Antigen mash mash mash mash mash mash - Penicillin + sodium benzoate Penicillin + sod, penicillin + sod, Penicillin +■ sod. Penicillin + sod. Penicillin + sod. Rutin Rutin Rutin Rutin Rutin Rutin - + AK AK AK AK AK AK + + A A AK AK A A AK AIi + + + + A A A A A A A .AK AK AK AK AK N H A A A A A AK AK AK AK AK A A A A A A A A AK AK AK AK N N + + + + + N A A A A N AK AK AK AK N N + + + + + + + + + + A A A A A A AK AK AK AK AK .AK A A A A A A AK AK AK AK AK AK + + + + + + + 1 2 2 2 2 2 23 positive isolates Gas **• - + A A A A A A + + + benz, benz. benz. benz. benz. Sims H2S Motility AK AK AK AK AK AK + 2 2 2 2 2 2 Indi vi dual sugars Mannitol Sucrose A A A A A A + + Sodium benzoate control Sodium benzoate control Kligl ers Butt Slant (SERIES 8) + + + + - - - + - - N + N - N + + + + + - N — — - N M a* N N — - - — - — . — A - acid reaction N - reaction not carried further AK - alkaline reaction — 601 THE RECOVERY OP SALMONELLA PPLLOKPM PROM DEAD CHICKS TABLE 3k EVALUATION OF THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 9) Experimental group Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days 0 30 0 0 0 Infected control 0 25 0 5 20.0 Rutin - 1 0.25 A 2 8.3 Rutin - 2 0.5 A 7 29.2 Antigen mash (heat attenuated) 0 25 i>8 7 28.0 25 lj.8 0 0 330 units CO Penicillin + sodium benzoate CO Non-infected control Chloromycetin 1 25 M3 0 0 Streptomycin 1 A it.8 2 8.3 Breed of chicks: Barred Rock x White Rock Feed: Kellogg starter TABLE 35 THE RECOVER! OP SALMONELLA PULLORUM PROM DEAD CHICKS Experimental group Growth on bismuth sulfite agar Infected control Infected control Antigen mash (heat) Antigen mash (heat) Antigen mash (heat) Antigen Antigen Antigen Antigen mash mash mash mash (heat) (heat) (heat) (heat) Rutin - 1 Rutin - 1 Rutin - 1 + + + + + + 4+ + + 44- Kliglers Butt. Slant A A N A A AK AK N AK AK 4+ H M A A A A AK AK AK AK A A A A AK AK AK AK + + + 4* M M A A A AK AK AK A A A AK AK AK + + 4- AK AK AK AK AK AK A A A A A A AK AK AK AK AK AK + + + + + + AK AK A A AK AK + + 44- Streptomycin Streptomycin 4* 4- A A - - - 19 positive isolates + + Sims h 2s Motility AK AK HS AK AK A A A A A A + Individual sugars Mannitol Sucrose A A HS A A Rutin - 2 Rutin - 2 Rutin - 2 Rutin 2 Rutin 2 Rutin 2 4- (SERIES 9) + + N - mm N - a* - - — - - - — - — mm “ — HS - Excess H2S N - reaction not carried further 112 A - acid reaction AK - alkaline reaction Gas TABLE 36 EVALUATION OF THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 10) Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days Non-infected control 0 30 0 0 0 Infected control 0 25 0 0 0 Chloromycetin 1 25 1(8 0 0 Streptomycin 1 25 -CPD" Experimental group 0 0 23 1(8 0 0 Penicillin 660 units Antigen mash (heat attenuated) charcoal adsorbed 0 20 kS if 20.0 Streptomycin + aureomycin 1 19 1(8 2 10.5 Breed of chicks: Barred Rock x White Rock Feed: Kellogg starter 113 TABLE 37 EVALUATION OF THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 11) Experimental group Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days Non-infected control 0 32 0 1 3.1 Infected control 0 25 0 8 32.0 Chloromycetin 1 22 2k 3 13.7 Streptomycin 1 25 2k 11 [}4.0 Aureomycin + streptomycin 1 2k 2k 2 8.3 Aureomycin + Chloromycetin 1 25 2k 3 12,0 Antigen mash (heat attenuated) charcoal adsorbed 0 25 2k 12 i|.8.0 60 25 2k 15 60.0 Trigonella 111}. Breed of chicks: Barred Rock x White Rock Feed: Kellogg starter TABLE 38 THE RECOVERY OP SALMONELLA PULLORUM FROM DEAD CHICKS Growth on bismuth sulfite agar Kliglers Butt Slant Individual sugars Mannitol Sucrose Sims h 2s Motility Infected control + A AK A AK + Antigen mash. Antigen mash + + A A AK AK A A AK AK + + .Aureomycin + streptomycin + A AK A AK Chloromycetin + A AK A Streptomycin Streptomycin + + A A AK AK Trigonella + A AK Q positive isolates Gas - mt - am + - - AK j- - - A A AK AK + + - mm A AK + - - am A - acid reaction AK - alkaline reaction SIT Experimental group (SERIES 11) TABLE 39 EVALUATION OP THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 12) Experimental group Concentration of aitibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortal, ity in 21 days 1 Non-infected control dU-3 g)“ 0 43 0 0 0 Infected control (1XU- g) 0 25 0 6 24.0 Penicillin - 1 (1^-3 g) 2933 units 23 0 1 4.3 Penicillin - 2 (137 g) 2936 units 23 0 2 8.7 2.75 25 2k 8 32.0 Streptomycin (143 g) Hydrastis (123 g) 10 14 24 2 14.3 Hypericum (130 g) 10 25 24 7 28.0 25 24 0 0 Chloromycetin (ISO g) 1.5 116 v-Average weight per chick Breed of chicks: White Leghorn Feed: Kellogg starter TABLE i|.0 THE RECOVERY OF SALMONELLA PULLORUM-FROM DEAD CHICKS Experimental group Infected Infected Infected Infected Infected Growth on bismuth sulfite agar control control control control control Kliglers Butt Slant (SERIES 12) Individual sugars Mannitol Su> rose Sims h 2s Motility + + + + + A A A A A AK AK AK AK AK A A A A A AK AK AK A K AK + + + + + Hydrastis Hydrastis Penicillin + + + A NR A AK NR AK A N A AK N AK + N + Hypericum Hypericum Hypericum Hypericum Hypericum + + + + + A A A A A AK AK AK AK AK A A A A A AK AK AK AK AK + + + + + A A A A AK AK AK AK A A A A AK AK AK A1C A A A A AK AK AK AK A A A A AK AK AK AK Penicillin + sod. benz. Penicillin + sod. benz. Streptomycin Streptomycin Streptomycin Streptomycin Streptomycin Streptomycin + + + + + + + A - acid, reaction AK - alkaline reaction wmm - - - - - - a* - _ m N - N _ - - - - - - - - - — mm + + + + - - - + - Ml + - - + — N - reaction not carried further NR - no reaction 117 20 positive isolates + Gas TABLE i|l EVALUATION OP THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 13) Experimental group Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days 0 35 0 1 2o8 Infected control 0 25' 0 10 4-0.0 7 28.0 Penicillin - 1 2290 units •UA C\J Non-infected control 24- penicillin - 2 2290 units 25 24- 6 21+.0 Penicillin - 3 3500 units 25 2k 5 20,0 Penicillin - 4- 2000 units 214. 2k 7 28.0 1.5 25 21+ 3 12.0 1.5 + 114-5 units 25 21+ 3 12.0 Chloromycetin Chloromycetin + penicillin 118 Breed of chicks: White Leghorn Feed; Kellogg starter TAGLE i-2 THE RECOVERY OF SALMONELLA PULLORUM FROM DEAD CRICKS Experimental group Infected Infected Infected Infected control control control control Infected Infected Infected Infected control control control control Chloromycetin Chloromycetin Chloromycetin Penicillin - 1 Penicillin - 1 Penicillin Penicillin Penicillin Penicillin Penicillin + + A A + ak: AK AK AK + A .Alt A A N A + A A A A Alt AK Alt AK A A A A AK AK AK AK A A A A A AK AK AK Alt Alt A A A A A AK AK Ait AK A It A A A A A .AK AK AK Alt Alt A A A A A AK Alt AK AK AK A AK AK AK A A A A A AK AK AK AK AK + + + + + + + + - 1 - 1 + + - 1 - 1 - 1 + + + - 2 - 2 - 2 - 2 - 2 Kliglers Individual sugars Butt Slant Mannitol Sucrose + + + + A A A A AK AK AK Alt N AK Sims E^S Motility Gas N - N - + + - — - + + + _ - - -r + - - - — — - - + + 7 i> iT + + + + + + + + - + + + + + — _ _ — — _ _ 119 Penicillin Penicillin Penicillin Penicillin Penicillin Growth on bismuth sulfite agar (SERIES 13) TA3LE l±2 Experimental group Penicillin - 3 Penicillin - 3 Penicillin - 3 Penicillin - 3 Penicillin Penicillin Penicillin penicillin penicillin Penicillin Penicillin C-rowth on bismuth sulfite agar + Kliglers Butt Slant NR N N AK A AK -r - A A A A AK AK AK AK A A A A AK AK AK AK + + + _ — - ~ - - + - - + + + A A A AK AK AK A A A AK AK AK + + + - - - - + A AK A AK + - - + A AK A AK + - - + A AK A AK + ** *• ij. ij. ij. Penicillin + Chloromycetin Penicillin + Chloromycetin Penicillin + Chloromycetin 35 positive isolates Gas Motility + + N - i| - ij. - ij. - B^S AK AK + + + + - Sims A A + + + - Individual sugars Mannitol Sucrose AK AK A A NR A - CONTINUED A - acid reaction AK - alkaline reaction • mm - - N N - _ NR - no reaction N - reaction not carried further TABLE k-3 EVALUATION OF THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH SALMONELLA PULLORUM (SERIES 1 k) Experimental group Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days Non-infected control 0 30 0 1 3*3 Infected control 0 25 0 1 I4-0O Penicillin - 1 14-500 units 24 24 1 ko2 penicillin - 2 l^OO units 25 2k 1 If..0 Penicillin + cobalt (in feed) 2290 units 25 24 3 12.0 Chloromycetin - 1 2.2 25 2k 0 0 Chloromycetin - 2 2,2 25 2k 2 8.0 Cobalt control (2.5 mg/15 lb feed) 0 25 2k 7 28,0 Breed of chicks: White Leghorn Feed: Kellogg starter TABLii IjijTHE RECOVERY OP SALMONELLA PULLORUM PROM DEAD CRICKS Experimental group Growth on bismuth sulfite agar Kligl ers Butt Slant (SERIES I4 ) Individual sugars Mannitol Sucrose Sims h 2s Motility + + A A AK AK A A Alt AK + + + + + + A A A A A AK AK AK AK AK A A A A A AK AK AK AK A It + + + + + Chloromycetin - 2 Chloromycetin - 2 Penicillin - 1 + + + A A A AK AK AK A A A AK AK AK + + + mt Penicillin Penicillin Penicillin Penicillin + + + + AK A A A AK AK Alt AK N nA A A ?.T AK AK AK R + + + N — Non-infected control Infected control Cobalt Cobalt Cobalt Cobalt Cobalt control control control control control + + + + cobalt cobalt cobalt cobalt 13 positive isolates - Gas - _ *■ - mm - — N - — A - acid reaction AK - alkaline reaction N - reaction not carried further 122 TABLE i|5 EVALUATION OF THE EFFECT OF FEEDING THERAPEUTIC AGENTS TO CHICKS INFECTED WITH S ALMONELLA PULLORUM (SERIES 15) Experimental group Concentration of antibiotic agent mg/g feed Number of birds Hours of prophylactic feeding Number of dead birds Percent mortality in 21 days Non-infected control d k 7 g )* 0 35 0 1 2.8 Infected control (121). g) 0 25 0 8 32.0 5500 units 25 2k 2 8.0 Penicillin - 2 (126 g) 5500 units 25 2k k 16.0 Penicillin + cobalt (0.2 mg/ml) - 1 (139 g) 2290 units 25 2k 1 4.0 Penicillin + cobalt (0.2 mg/ml) - 2 (135 g) 2290 units 25 2k 3 12.0 Penicillin + cobalt (0.2 mg/ml) - 3 (137 g) 2290 units 25 2k 0 0 25 2k 1 lf.0 Chloromycetin (iii-3 g) 1 ''Average weight per chick Breed of chicks:1'White Leghorn Feed: Kellogg starter 123 Penicillin - 1 (125 g) TABLE 1+6 THE RECOVERY OP SALMONELLA PULL0RUM FROM DEAD CHICKS Experimental group Growth on bismuth sulfite agar Kliglers Butt Slant Individual sugars Mannitol Sucrose control control control control control + + + + + A A NR A A AK AK NR AK AK A A Infected control Infected control Infected control Chloromycetin + + + + A A A NR AK AK AK NR A A A N Penicillin Penicillin Penicillin Penicillin Penicillin Penicillin - 1 2 2 2 2 2 + + + + + + NR A A A NR A NR AK AK AK NR AK N A A A N A AK AK AK N AK Penicillin Penicillin Penicillin Penicillin + + + + cobalt - 1 cobalt - 2 cobalt - 2 cobalt - 2 + AK AK AK AK A A A A AK AK AK AK Infected Infected Infected Infected Infected 15 positive isolates 4- A A + A + A (SERIES 15) A A N A - acid, reaction AK - alkaline reaction Sims H2S Motility Gas + + N + + N - AK AK AK + + - N N N N N N + 4* N - N - 4- - - N N N 4" - - - «•* — — AK AK N AK AK N _ + + + + - NR - no reaction N - reaction not carried further 121+ TABLE 47 THE ISOLATION OF SALMONELLA PULLORTJM FROM SUSPECTED CARRIER BIRDS Non-treated Infected Controls Bird number 301 302 303 304 305 Tetrathionate enrichment of pooled organs + + + + _L Growth on bismuth sulfite agar Transfers to differential media — — — 309 310 + + + + + — + — — + " 311 312 313 314 315 + + + + + — + + — + + — “ 316 317 -t+ + + + i■— “ + — — — — — - 323 324 325 + + + + + 326 327 328 329 330 + + + + + + + + — + 331 332 333 334 335 + + + -t* + — — + — - 306 307 308 318 319 320 321 322 — r- — + TABLE 14-7 Bird number Tetrathionate enrichment of pooled organs 336 337 338 339 314-0 + + + + + 3IP314-2 31+3 3l4l4- + + + + + 3k5 Growth on bismuth sulfite agar 314-7 314-8 314-9 350 3£L Transfers to differential medi — + + - + _ + + + + + + 314.6 CONTINUED - — - — - — — - _ _ + + — — - — - — - - Non-treated Infected Controls Further Characterization (Biochemical) of Suspected Carriers Bird number 309 312 313 316 327 329 33k . 338 339 314-7 Kligl ers Butt Slant A A A A A A Excess h 2s A A A AK A AK AK AK AK Exce S 3 h 2s AK AK AK Individual sugars Sucrose Mannitol h 2s A N A A A AK N AK AK AK + N + + + A N Alt N + N A A A Alt AK AK 4+ + 8 positive isolations A - acid, reaction N - reaction no? carried further Sims Motility Gas mm N N - - - - - — _ _ N N - - - - - - AK - alkaline reaction NR - no reaction 127 TABLE J 4.7 Aureomycin 3ird number Tetrathionate enrichment of pooled, organs CONTINUED (1 mg/g feed) Growth on b i smuth sulfi te agar 3503 35>Ol43505 3509 + + + + _ 3510 3511 3512 3513 35l5 35l6 3517 3521 3537 Transfers to differential media — - - - - - + + + + _ _ - - - — - - + + + + + _ — - - - TABLE 1+7 CONTINUED Chloromycetin - Group I Bird number 3802 Growth on bismuth sulfite agar 4 + Tetrathionate enrichment of pooled organs + + (1,5 mg/g feed) Individual sugars Kligl ers Butt Slant Mannitol Sucrose A HS A HS A AK HS AK HS AK A N A N AK AK N AK N AK N 4 - - N N N N N N N N N Mi N N N N N N N N N N N N N N N N N N N A N N N N AK N N N N N N N N N N 4 - - N N N N N N N N N N N N N N N N N N + + 3911 38lli 3315 3917 3819 4 4 4 4 4 4 4 4 4 4 HS HS NR A A HS HS NR AK A N N N AK N 3822 3823 3827 4 4 4 4 X HS NR A A HS HS NR A AK HS 4 4 4 HS A NR HS A NR 3831 4 4 4 3933 383+ 3836 + + 3828 4 3 positive isolates j. 4 N 4 4 4 + Ga* N 3803 3'905 3809 3810 4 Sims k 2s Motility A - aoid reaction HS - Excess H2SAK - alkaline reaction NR - no reaction N - reaction not carried further 128 TABLE kl CONTINUED Chloromycetin - Group II Bird number 3020 Growth on bismuth sulfite agar Tetrathionate enrichment of pooled organs Eli gl ers Butt Slant (2,2 mg/g feed) Individual sugars Mannitol Sucrose h 2s Sims Motility Gas 3023 302k 3051 3053 + + j. + + + + + + + A HS A HS NR A HS A HS NR N N N N N N N N N N N N N N N N N TJ N N I'T N N N N 305143057 305S 3059 3066 + + + + + + + + + + HS A A A A HS AK A AK AK N A N A A N AK N AK AK N + N + + N N - N — N + - 3063 3070 308C 30314. + + + + + + + + + + NR A A NR A NR AK AK NR AK N AK A N A N AK AK N AK N N + N + N N N - N N — N - ^827 + h.829 1+830 I4J86I+ 1+365 j. + + + + + HS A HS A A HS A HS AK AK N N N A A N N N AK AK N N N + + N N N N N N + + + A NR A A HS AK NR AK A HS A N A A N AK N AK A N + N + N N 3098 I+867 i+870 I4S73 A+8?5 + + + + + 7 positive isolates + " acid reaction HS - Excess H 2S AK - alkaline reaction NR - no reaction N - reaction not carried further - - - N + N — — N N N N 129 1-1-871 + + + TABLE I4.7 CONTINUED Chloromycetin - Group III Bird number Growth on bismuth sulfite agar Tetrathionate enrichment of pooled organs Kli glers Butt Slant (1.5 mg/g feed) Individual sugars Mannitol Sucrose + + + + + + + + + -f NR HS A NR NR NR HS AK .NR NR 333? 3333 3334 3337 3362 + + f+ + + + + + + NR NR HS NR A NR NR HS NR AK N N N N AK 3369 3373 3391 3853 3863 + + + + + + + + + + A HS NR NR HS AK HS NR NR HS 3881 3887 3896 5826 + + + + + + + + HS A NR HS it-837 f8ip. + + + + + + + + NR NR NR NR 1^8 No positive isolations N N N N N N N N N N N N N N N N N N N AK N N N N N N N •N N N N N N N N AK N N N N AK NN' N N N N N N N N N N N N N N N N N HS AK NR HS N AK N N ' N AK N N N N N N N N N N NR NR NR NR N N N N N N N N N N N N N N N N N N AK N N . N ' N AK N■ N Gas A " acid reaction HS - Excess H2S AK - alkaline reaction NR - no reaction N - reaction not carried further'- • N N N N N N N N 130 3305 3306 3334 331s 332^ Sims. h 2s Motility TABLE i|7 penicillin Bird lumber 818 819 820 821 822 Growth on bismuth sulfite agar Tetrathionate enrichment of pooled organs + + + + + + + + + + + + + 828 829 830 831 832 + 833 83^1835 836 837 838 A A A A A AK AK AK AK AK A A A A A AK AK AK AK AK + A A A A A AK AK AK AK A A A A A AK AK AK AK AK + + + + + + + ■f + + + + + + + + + + + + + + + + + + + + + + AK Sims H2S Motility Gai N N N + + + + - - - - - - mm - + + + mm _ - mm mm mm + - — + - - + _ + - - + - - + - - + - - A NR NR NR NR NR AK A AK + NR NR NR NR NR N N N N N N N N N N N HS HS HS HS A HS HS HS HS A N N N N N N N N N N T'T N N N N N N N N N N N N N N N N N N N N N N N N N N N N 131 887 888 890 891 892 A AK AK AK AK + 823 824 825 826 827 . A A A A A A A A A + Individual sugars Mannitol Sucrose A AK AK AK AK + + + (1|500 u/g feed) Kligl ers Butt Slant A + CONTINUED TABLE 1+7 Penicillin Bird number Growth on bismuth sulfite agar 866 867 868 869 870 + + + + + 871 872 873 874 875 876 877 878 379 880 881 882 883 88k 88^ 856 Tetrathionate enrichment of pooled organs CONTINUED (l+$0Q u/g feed) Continued Kliglers Butt Slant + + + + + NR A NR A NR NR A NR A NR + + + + + + + + + + NR NR NR NR HS NR NR NR NR HS + + + + + + + A HS NR HS NR A HS NR H3 NR NR HS HS HS HS NR NR HS HS HS HS NR ' j. + + + + 0- + + + + 4* + + '+ N N N N AT X. N N 7“ N Sims h 2s Motility N N N N TvT N N N N N N N N Iff M N N N N N N N N N N N N N N N N N N N N N N N N N N T\T N N N N N N N N N A T A* N X'i N N N N N N N AT IN TVT xi N N N N N N N N N N N N AT ^7■ N AT 11 AT N AT Xi N N Gas N AT A - acid reaction HS - Excess H2S AK - alkaline reaction NR •- noreaction N - reaction not carried further N N N N N N N N N IT 132 15 positive isolates Individual sugars Mannitol Sucrose TABLE ij.7 CONTINUED Penicillin Bird number 3326 3329 3330 3333 3339 33Nt 3355 3358 3359 3377 3378 3379 3383 3386 3387 3338 3389 3393 3399 3400 Tetrathionate enrichment of pooled organs + - + - + + + 4" Kligl ers Butt Slant N N N A N • Individual sugars Mannitol Sucrose Sims H2S Motility Ga; N N N AK N N N N AK N N N N AK N N N N N N N N N N N N H N N N N N N N N J. + + + + + HS A N N HS HS AK N N HS N AK N N N N AK N N N N N N N N + + A A A N N A AK AK N N AK A AK N N AK AK AK N N N + I N IT N HS AK N AK N N A N AK N K AK IT 11 11 N N N IT N N N N + - - AK N AE HS AK A N A N AK AK j. mm N N N + IT N — — + - •f* + + mm + - + - + + + T l« 11 N N N N IT N 133 3530 357(|3582 3598 3599 Growth on bismuth sulfite agar (5500 u/g feed) TABLE 47 Bird number Growth on bismuth sulfite agar 3600 384.7 3856 3861 a. 3864 + 3872 mm «* 4833 4835 4836 4838 + 4840 — 4«44 4849 + pm ■* 4 positive isolates CONTINUED Penicillin (5500 u/g feed) Tetrathionate enrichment of pooled organs Kligl ers Butt Slant Continued Individual sugars Mannitol Sucrose Sims H2S Motility - Gas + + + + + N N N HS HS F H F HS HS F H F N N N N F N N F H N F F F F F F N F F N F N + + N N N N HS H F N N HS F F N F F F F F N F F F F F F N F F F F F F N TiyVT A A A F F N H F N F F JL 1. + + + + + AT N F N F F A F F F A " acid reaction . HS ■- Excess h 2s AK - alkaline reaction FR - noreaction F - reaction not carried further N TABLE ii7 CONTINUED Penicillin {2290 u/g feed) - Cobalt (0.2 mg/ml water) Eir numb 3302 3303 3307 Growth on ilsmuth sulfite agar + - + 3308 mm 3309 — 3312 3313 3317 3320 3322 3323 3325 3329 ,3331 3338 331+1 3342 + + - - — - + + + + Tetrathionate enrichment of pooled organs Kli gler3 Eutt Slant + + + T + NR N HS N IT NR N HS N H N N N 17 IT N 11 N H II + + + + + HS HS N N 11 HS HS H N II N N N II II + + + + + N HS HS A HS 11 HS HS A HS II I\T 17 A TvT + 17 HS A N 17 HS HS AK N HS 3352 3353 + + •• + + _L N HS A N l\uT 3365 3372 3379 3380 3381 + + + + + + + + + HS HS A IT HS 3351 - Individual sugars Mannitol Sucrose a . Sims h 2s Motility 11 7\T Ga; N 11 11 :I N N II II 1J N N N N II N II 11 N N 11 II 11 II N 11 N 11 11 II II II IT IT II 11 11 A II 11 11 N II M II N N 11 11 IT N N II 11 A II 17 17 11 AK N 11 11 11 N N 11 II II II N II IT 11 ■ II II N A N 11 V 11 AK N II M 11 + N N 11 II IT 11 - - A'* N N N IT N N IT II H VjJ vn. CONTINUED Penicillin (2290 u/g feed) - Cobalt (0.2 mg/ml water) Bird number 3382 3331j. 3388 3392 3394 3398 38.00 3832 3830 3882 38U9 3882 3888 3387 3888 Growth on bismuth sulfite agar mm mm mm + + + + + Tetrathionate enrichment of pooled organs T,,. , .l^iglerg— Butt Slant Continued _ Mannitol s.li^a.r>.g.----------Sucrose H2S Motility - TABLE I1.7 + + + + + N N N il HS N IT N N HS R N N IT N N N N N R IT N N N N R N N N N R N R IT R + + IT A HS 'A A N AK HS AK AK IT A N A A N AK N AK AK N + N IT R - - IT IT + - - AK N HS N N AK N N N N AK N R N H N R N N N T\T IT R IT IT R -f + + + + mm 1 T «« + + + + + + + A HS HS A N AK HS HS AK N AK N N AK II AK N N AK N N N IT N N R N R N R IT R H R + + IT N AK N AK N • N N N AK N IT N N K IT R N N R R N N R IT R R R - 3889 3862 3868 3866 3968 + + + 3869 3073 3876 3878 3879 _ J. - + - + + + 4. A N HS HS ' N HS HS N N N N "M 136 + A N HS N N mm Qas TABLE 1+7 CONTINUED Penicillin (2290 u/g feed) - Cobalt (0.2 mg/ml water) Bird number Growth on bismuth sulfite agar 3882 3883 3884 3886 + 3888 3891 3895 4342 + 4343 4846 4847 — - - - I4. positive isolates Tetrathionate enrichment of pooled organs Kliglers Butt Slant Continued Individual sugars Mannitol Sucrose Sims H2S Motility Gas + + + + HS N N N HS N N N N N N N N N N N N N N N N N N N N N N N 1 + + + A N N N AK N N N AK N N N AK N N N N N N N N N N N N N N N + + + N N N N N N N N N N N N N N N N N N N N N A - acid reaction HS - Excess H2S AK - alkaline reaction NR - noreaction N - reaction not carried further 137 LOG CONCENTRAT ION-MICROGRAMS PER ML ZONE s /NH/BITION, M M ^ 0 * ^ i \ o 0 u F h h n * r-'-h H VjO CD Ei LOG CONCENTRATION MICRO GRAMS PER ML 1.8 N N “ T" T T FT L OG CONCENTRA TION M/CROGRAMS PER M L « T 5 § S | i | ^ H § •T o LOG CONCENTRA TION-MICROGRAMS PER ML ZO/VE 3 $ /NH/B/T/OA/, M M R 'f LOG CONCENTRA TION MICROGRAMS PER ML H -P" ro LOG CONCENTRATION MICRO GRAM 3 PER ML bo N & N * Ni * T CoO & $ H -PU) (y/V/TS PER LOG CONCENTRATION i K * , K Ni i i tr'O- , S >i ML Cl ^ Ki 'Ik r 1-- 1 ---- C^v VJ 8 ^ 8 1 o ^ • n S Co frs , S LOG C O N C E N T R A T I O N - U N / r s PER M L ZOA/£ INHIBITION O X 0 H “F” VA 114-6 r / G . 10 G A R L I C - R E F E R E N CE C U RVE F O R 5 A L M O N E L L A P U L L O R U M (lcthl 12 mo M s J 2 5 20 ZONE INHIBITION, MM Jo 1 5 10 zoo o U N IT S zso PER 200 ML OY LO G CONCENTRATIONNi Sv Pj t-S °o K UNITS PER ML Ni !\i “I Ni i J\i Oi 1--- 1--- r N ' " f e p * | £ H -F7 ->i BIBLIOGRAPHY 1* Alexander, H. 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