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 L161 O-1096 
 
BULLETIN NO. 246 
 
 CLOSTRIDIUM BOTULINUM TYPE C 
 
 A PATHOGENIC ANAEROBE ASSOCIATED WITH A 
 LIMBERNECK-LIKE DISEASE IN CHICKENS AND DUCKS 
 
 BY ROBERT GRAHAM AND I. B. BOUGHTON 
 
 URBANA, ILLINOIS, OCTOBER, 1923 
 
CONTENTS OF BULLETIN No. 246 
 
 PAGE 
 
 INTRODUCTION 3 
 
 SPECIMENS 2770, 2771, AND 2772 . 5 
 
 History 5 
 
 Clinical Symptoms 5 
 
 Gross Pathology 5 
 
 Bacteriologic Findings , 6 
 
 SPECIMENS 3419 AND 3420 
 
 History 
 
 Gross Pathology 
 
 Bacteriologic Examination 
 Cultural Characters . 
 
 SPECIMENS 3421, 3422, AND 3423 16 
 
 History 16 
 
 Clinical Symptoms 16 
 
 Gross Pathology 16 
 
 Bacteriologic Examination 17 
 
 Pathogenesis 17 
 
 SPECIMENS 3466, 3467, AND 3468 23 
 
 BOTULINUS ANTITOXIN TYPE C 28 
 
 THE DEVELOPMENT OF TYPE C TOXIN IN SHELLED CORN 31 
 
 SUMMARY 32 
 
 NOTE. The presence of Clostridittm botulinum type C in the digestive tubes 
 of a horse and a steer following a fatal illness is noted in footnotes on pages 
 28 to 30. 
 
CLOSTRIDIUM BOTULINUM TYPE C 
 
 A PATHOGENIC ANAEROBE ASSOCIATED WITH A 
 LIMBERNECK-LIKE DISEASE IN CHICKENS AND DUCKS 
 
 BY ROBERT GRAHAM, CHIEF IN ANIMAL PATHOLOGY AND HYGIENE AND 
 I. B. BOUGHTON, ASSOCIATE IN ANIMAL PATHOLOGY 
 
 INTRODUCTION 
 
 The possible etiologic relation of botulinus toxin to a spontaneous 
 syndrome in chickens commonly referred to as limberneck was re- 
 ported by Dickson 1 of California in 1918. In a number of other 
 states, including Michigan, Washington, and Idaho, avian botulism 
 has also occurred in connection with human botulism as a result of 
 feeding poultry the unused portions of the contaminated food. In 
 Illinois the sporadic occurrence of avian botulism type A unassociated 
 with the disease in the human family has been suggested in the results 
 of bacteriologic findings and toxicogenic tests of the gastric contents 
 of clinically affected chickens. One type A outbreak following the 
 feeding of contaminated garbage, was reported from Urbana in 1921. 2 
 During the month of August, 1922, another outbreak which occurred 
 in Piatt county, Illinois, came to the attention of the authors through 
 Dr. T. J. Foster, of Monticello. Spoiled over-ripe tomatoes were sus- 
 pected as the cause. A similar clinical disease of chickens occurred 
 at the White Arrow Farms, Argenta, Piatt county, Illinois, in Sep- 
 tember, 1922. Clostridium botulinum type B was demonstrated in the 
 intestinal contents and liver of one of three chickens submitted for 
 examination from this latter outbreak. This strain, however, like other 
 cultures of type B, when fed in massive amounts to healthy chickens, 
 failed to induce illness. Definite evidence incriminating type B 
 Clostridium botulinum or its toxin as a primary factor in poultry 
 losses has not been obtained in cases coming to the attention of the 
 authors. 
 
 In the course of certain bacteriologic studies of limberneck-like 
 diseases of poultry resembling botulinus poisoning conducted during 
 the last two years, it became apparent that neither C. botulinum 
 
 1 Rockefeller Inst. Med. Research, Monograph No. 8, 1918. 
 'Journal of Infectious Diseases, 28, 4, 317-322. April, 1921. 
 
4 BULLETIN No. 246 [October, 
 
 type A nor the toxin elaborated by the type A organism was de- 
 monstrable in the tissues of certain affected fowls. The limberneck- 
 like syndrome in spontaneously affected chickens, however, was indis- 
 tinguishable from botulinus poisoning. Bacteriologic evidence sug- 
 gesting the character of the causative agent was not obtained in all 
 cases, but in five of these outbreaks definite and specific bacteriologic 
 evidence suggests that certain clinical symptoms of leg weakness and 
 limberneck occurring in poultry are associated with a pathogenic 
 anaerobe which in preliminary studies appears to be a distinct and 
 separate species resembling, if not identical with, C. botulinum type C 
 isolated from the larvae of Lucilia caesar by Bengtson 1 at the Hygienic 
 Laboratory, Washington, D. C., and also the toxicogenic anaerobe 
 isolated from bovine cases of bulbar paralysis by Seddon 2 of the Vet- 
 erinary Institute, University of Melbourne. From three of these five 
 affected flocks, C. botulinum type C or a closely related anaerobic, 
 toxin-producing, spore-forming rod has been isolated in what appears 
 to be pure culture. The cultural and immunologic characters are dis- 
 tinct from those of either type A or type B botulinus organisms but 
 are analogous to those of type C and the parabotulinus organism 
 of Seddon, cultures of which were supplied, respectively, by Dr. Gr. W. 
 McCoy, Director of the Hygienic Laboratory, Washington, D. C., and 
 Dr. J. B. Buxton, Professor of Animal Pathology, Cambridge Univer- 
 sity, England. In the other two affected flocks the toxic anaerobe was 
 not isolated in pure culture, but the presence of the specific type C 
 bacterial toxin in impure culture was demonstrated. 
 
 In the affected farm flocks 3 thus far observed, type C avian botul- 
 ism appears less fatal than type A. Weakness of the cervical muscles 
 (limberneck) has not been as frequently noted in outbreaks of type C 
 in chickens as in the two outbreaks of type A which have been 
 observed, tho this symptom occurs in the final stages of a highly fatal 
 type of both the spontaneous and the experimentally induced disease. 
 Healthy chickens under experimental conditions are less susceptible to 
 the filtered and the unfiltered cultures of type C than to those of 
 type A. In fact, some difficulty has been experienced in reproducing 
 fatal cases of the avian type C poisoning, which leads to the belief 
 that secondary predisposing factors, such as parasitism or other bac- 
 terial infections, may influence the susceptibility of different chickens 
 and account for certain irregularities in the development of the dis- 
 ease under farm conditions. The potency of the toxin in nature may 
 also play an important part. 
 
 *U. S. Pub. Health Serv., Pub. Health Epts., 37, 164. 1922. 
 2 Jour. Compar. Path, and Ther., 35, 147. 1922. 
 
 * The disease has been observed in one large poultry establishment in addi- 
 tion to the cases referred to in this bulletin. 
 
1923] CLOSTRIDIUM BOTULINUM TYPE C 5 
 
 SPECIMENS 2770, 2771, AND 2772 
 
 History. During the month of July, 1921, Drs. Brown and Hinkle 
 of Decatur, Macon county, Illinois, submitted three chickens to the 
 Laboratory of Animal Pathology and Hygiene for bacteriologic ex- 
 amination. A letter accompanying the specimens stated that these 
 fowls had been ill for a period of two weeks. Eight of the half -grown 
 chickens in one pen had died. The remaining fowls on the premises 
 (one thousand) remain- 
 ed healthy. The first no- 
 ticeable symptom of ill- 
 ness, according to the 
 owner, was lameness or 
 weakness of the right leg 
 followed by incoordina- 
 tion in walking. Later 
 both legs became weak 
 and the chickens were 
 unable to stand. The 
 eyes were partly closed 
 
 and the affected chickens FIG. i.Ascaridia perspicilli, found in chicken 2770 
 appeared dull and ex- 
 hausted. In the final stages of the disease the comb and wattles were 
 a bluish color. Mildly affected birds recovered. An inquiry into the 
 history of the flock over a period of years failed to reveal that this or 
 a similar disease had previously occurred on the premises, tho symp- 
 toms of roup had been recognized by the owner on different occasions. 
 No lesions of roup, however, were encountered in the birds submitted 
 for examination. 
 
 Clinical Symptoms. One of the three chickens (2770) died on the 
 way to the laboratory. The feathers of the posterior abdominal region 
 were smeared with soft, pasty feces. The comb and wattles were dark 
 in color. A slimy mucus filled the mouth and nasal cavities. Chickens 
 2771 and 2772 appeared weak and unable to stand, but were conscious 
 and observant of their surroundings when aroused. The eyes were 
 dull and the lids were partly closed. The comb and wattles were not 
 discolored. 
 
 Gross Pathology. Post-mortem changes in the dead chicken 
 (2770) masked any gross lesions which might have been present before 
 death. In the other two chickens (2771 and 2772) petechial hemor- 
 rhages and areas of congestion and catarrhal inflammation of the 
 mucosa of the small intestine were observed. A mass of Ascaridia 
 perspicilli that partially closed the lumen was found in chicken 2770 
 (see Fig. 1). 
 
(3 
 
 BULLETIN No. 246 
 
 [October, 
 
 Bacteriologic Findings. The heart's blood- of specimens 2771 
 and 2772 was seeded on tubes of slant agar and found negative to 
 Pasteurella avium. Rabbit inoculation likewise failed to yield any 
 evidence of Pasteurella infection. A composite sample of the in- 
 testinal contents of the three specimens, and also separate pieces of 
 liver tissue from each, were sown in glucose pork broth and brain 
 media. After attenuating in a water bath at 80 C. for fifteen min- 
 utes in order to destroy vegetative organisms, the tubes were incubated 
 seven to ten days at room temperature in a sealed jar, the oxygen 
 pressure of which had been reduced by vacuum. The lethal charac- 
 ter of both the intestinal and liver cultures (impure) was observed 
 by feeding .5 to Ice. to guinea pigs. Death was also induced in other 
 guinea pigs by feeding similar amounts of the sterile filtrate. The 
 symptoms observed preceding death resembled those of botulinus 
 poisoning. 
 
 Neither A nor B botulinus antitoxins injected subcutaneously in 
 guinea pigs at the time they were fed toxic cultures 2770, 2771, and 
 2772, afforded any protection (see Tables 1, 2, and 3). These anti- 
 toxins also failed to protect guinea pigs when injected simultaneously 
 twenty-four hours previous to their being fed culture filtrates 2770, 
 2771, and 2772, and at the time of feeding, altho a non-specific 
 
 TABLE 1. NON-PROTECTIVE CHARACTER OF BOTULINUS ANTITOXINS A AND B 
 AGAINST CULTURE FILTRATE 2770 
 
 Identification 
 
 Treatment, 
 
 September 15, 1921 
 
 Results 
 
 Guinea 
 pig 
 
 No. 
 
 Weight 
 
 963 
 
 300 gms. 
 
 . 5 cc. culture 
 mouth 
 
 filtrate 
 
 2770 
 
 by 
 
 tOO units type A botulinus anti- 
 toxin subcutaneously 
 
 Died 9/17/21 
 
 489 
 
 300 gms. 
 
 . 5 cc. culture 
 mouth 
 
 filtrate 
 
 2770 
 
 by 
 
 100 units type B botulinus anti- 
 toxin subcutaneously 
 
 Died 9/17/21 
 
 544 
 
 300 gms- 
 
 . 5 cc. culture 
 mouth 
 
 filtrate 
 
 2770 
 
 by 
 
 Control 
 
 Died 9/16/21 
 
 TABLE 2. NON-PROTECTIVE CHARACTER OF BOTULINUS ANTITOXINS A AND B 
 AGAINST CULTURE FILTRATE 2771 
 
 Identification 
 
 Treatment, 
 
 September 9, 1921 
 
 Results 
 
 Guinea 
 pig 
 
 No. 
 
 Weight 
 
 987 
 
 300 gms. 
 
 .5 cc. culture 
 mouth 
 
 filtrate 
 
 2771 
 
 by 
 
 100 units type A botulinus anti- 
 toxin subcutaneously 
 
 Died 9/10/21 
 
 979 
 
 300 gms. 
 
 .5 cc. culture 
 mouth 
 
 nitrate 
 
 2771 
 
 by 
 
 100 units type B botulinus anti- 
 toxin subcutaneously 
 
 Died 9/10/21 
 
 485 
 
 300 gms. 
 
 .5 cc. culture 
 mouth 
 
 filtrate 
 
 2771 
 
 by 
 
 Control 
 
 Died 9/10/21 
 
1923} 
 
 CLOSTRIDIUM BOTULINUM TYPE C 
 
 TABLE 3. NON-PROTECTIVE CHARACTER OF BOTULINUS ANTITOXINS A AND B 
 
 AGAIMST CULTURE FILTRATE 2772 
 
 Identification 
 
 Treatment, September 15, 1921 
 
 Results 
 
 Guinea 
 pig 
 No. 
 
 Weight 
 
 984 
 
 300 gms. 
 
 .5 cc. culture 
 mouth 
 
 filtrate 
 
 2772 by 
 
 100 units type A botulinus anti- 
 toxin subcutaneously 
 
 Died 9/19/21 
 
 543 
 
 300 gms. 
 
 .5 co. cultuie 
 mcutn 
 
 filtrate 
 
 2772 by 
 
 100 units type B botulinus anti- 
 toxin subcutaneously 
 
 Died 9/16/21 
 
 072 
 
 300 gms. 
 
 .5 cc. culture 
 mouth 
 
 filtrate 
 
 2772 by 
 
 Control 
 
 Died 9/16/21 
 
 and incomplete protection was occasionally observed. These findings 
 were carefully checked by demonstrating the protective character of 
 the antitoxins against known A and B filtered cultures of the botulinus 
 organism. The preliminary results of typing tests were conflicting 
 only when less than lethal amounts of the culture or culture filtrate 
 were used. 
 
 Stained smears of seven- to ten-day-old cultures from the liver tis- 
 sues of specimens 2770, 2771, and 2772 showed at least two types of 
 anaerobes a facultative anaerobic, Gram-positive diplococcus and a 
 few terminal spore-bearing, Gram-negative rods. The diplococcus 
 proved to be non-pathogenic. An effort to purify and cultivate the 
 spore-bearing anaerobe by heating and seeding in glucose agar failed. 
 While the cultural characters of this organism were not determined, 
 preliminary immunologic tests suggested the presence of a toxin- 
 producing anaerobe which was capable of inducing the botulism 
 syndrome but which was apparently neither A nor B type of (7. 
 botulinum. The methods used and the media in which subcultures 
 were seeded, while satisfactory for the isolation of types A and B, 
 did not seem to meet the requirements of the toxin-producing rod 
 encountered. 
 
 Bacteriologic examination of soil samples obtained from the 
 chicken yards from which specimens 2770, 2771, and 2772 had been 
 received failed to show the presence of a pathogenic anaerobe. In 
 view of the positive results obtained later from other soils, however, 
 the authors are inclined to think that these negative results may be 
 attributed to the media employed, which have since appeared to be 
 unsatisfactory for the isolation of this organism. 
 
 During the year following the discontinuing of the above bacterio- 
 logic study, the intestinal contents and internal organs of several 
 suspected chickens submitted from different flocks for diagnosis were 
 cultured for the presence of toxic anaerobes, but always with negative 
 results. 
 
8 BULLETIN No. 246 [October, 
 
 SPECIMENS 3419 AND 3420 
 
 History. One hen (3419) and one rooster (3420) were received 
 from a sick flock belonging to Mr. John Thomen, GTreenup, Cumber- 
 land county, Illinois, on July 1, 1922. The hen appeared dull and 
 droopy, while the rooster was moribund and suffered from a severe 
 diarrhea. Neither showed symptoms of limberneck. A complete 
 history of the disease in the flock was not obtained, tho a letter ad- 
 vised that the flock had been fed the carcasses of chickens that had 
 died of limberneck. It was estimated by the owner several months 
 later that sixty birds had died of the disease. 
 
 Gross Pathology. Intestinal parasitism was encountered in speci- 
 men 3420 at autopsy, about twenty tapeworms and numerous 
 Heterakis papillosae being found in the ceca. Diffuse areas of 
 catarrhal enteritis were found in the duodenum. Specimen 3419 
 was held for observation. It appeared normal and active after the 
 second day. 
 
 Bacteriologic Examination. Aerobic cultures from the heart's 
 blood of specimen 3420 were negative, but the anaerobic cultures in 
 meat-mash medium from the intestinal contents and liver proved lethal 
 to guinea pigs. The symptoms induced resembled botulism, but the 
 course of the disease was in no way modified by the subcutaneous in- 
 jection of botulinus antitoxins of either type A or type B. Botulinus 
 antitoxin type C, however, apparently provided a specific protection 
 (see Table 4). Colonies in deep glucose-agar cultures as well as 
 
 TABLE 4. PROTECTIVE CHARACTER OF BOTULINUS ANTITOXINS TYPE C 
 AGAINST CULTURE FILTRATE 3420 
 
 Identification 
 
 
 Treatment, 
 
 July 25, 1922 
 
 Results 
 
 Guinea 
 pig 
 
 No. 
 
 Weight 
 
 659 
 
 300 gms. 
 
 .5 cc. culture 
 mouth 
 
 filtrate 3420 
 
 by 
 
 1 cc. type C botulinus antitoxin 
 296 1 subcutaneously 
 
 Remained healthy 
 Released 8/25/22 
 
 799 
 
 300 gms. 
 
 .5 cc. culture 
 mouth 
 
 filtrate 3420 
 
 bar 
 
 Control 
 
 Died 7/26/22 
 
 Furnished by Dr. G. W. McCoy, Director, Hygienic Laboratory, Washington, D. C. 
 
 liver-agar anaerobic plates yielded a pathogenic spore-bearing 
 anaerobe in pure culture. 
 
 Cultural Characters. The following description of the cultural 
 characters of pathogenic anaerobe 3420 is based upon a study of 
 single-colony subcultures in glucose agar. Non-toxic subcolony cul- 
 tures from single colonies have been encountered, but a study of the 
 morphology of the organism together with the growth characteristics 
 
1923} CLOSTRIDIUM BOTULINUM TYPE C 9 
 
 leads to the belief that a pure culture may contain not only non-toxic 
 cells, as mentioned by Bengtson, 1 but also colonies which on trans- 
 plantation fail to elaborate toxin. The reaction of media or conditions 
 of growth, such as temperature and oxygen requirements, may possibly 
 influence the number of toxic cells in a culture, since serum agar or 
 glucose agar in inverted petri-dish plate cultures incubated at 37 C. 
 yield a relatively high number of non-toxic colonies. The appear- 
 ance of the colonies on subculturing in glucose-agar tubes, the char- 
 acter of growth of subcultures in meat media as well as the morphology 
 and staining characters do not serve to distinguish toxic from non- 
 toxic cultures obtained from single colonies. 
 
 Conditions of Growth. Clostridium botulinum type C is a strict 
 anaerobe. It grows well in media containing meat (beef muscle, pork 
 muscle, liver) in unsealed tubes. Scant growth on solid media may 
 be obtained in an atmosphere of hydrogen, providing a heavy inoculum 
 is used. Satisfactory anaerobiosis may be secured in glucose-agar 
 plate cultures by sealing the inverted petri dish in the lid. More 
 consistent growth has been obtained in subcultures from glucose agar 
 when heavily inoculated. Growth occurs at 22 C. but more rapidly 
 at 37 C. 
 
 Morphology. The organism is a clostridium with rounded ends 
 about .9 micron wide and 5 to 8 microns long (see Figs. 2 and 3). 
 In old meat-mash cultures longer and more slender forms have been 
 observed. Cultures in meat mash forty-eight hours old incubated 
 at 37 C. frequently show terminal spores. Often two or more 
 organisms are observed in short chains, while in old cultures a few 
 long chains may extend across the field. Intracellular or extracellular 
 spores are present depending on the age of the culture. The latter 
 are numerous in old meat-mash cultures. The spores are oval in 
 shape, bulging the cell wall, and occur singly at the end of the vege- 
 tative cell. They are not heat resistant. An exposure to 80 C. for 
 five minutes is generally sufficient to render them inert. 
 
 Motility. In examining cultures twenty-four to forty-eight hours 
 old in cover-glass films, no motility has been observed. 
 
 Staining. The Clostridium stains readily with ordinary aniline 
 dyes. In young cultures the organism is Gram-positive. This char- 
 acter, however, is not constant as Gram-negative cells appear after 
 three or four days. Many Gram-negative staining organisms may 
 contain Gram-positive granules in cultures five days old. 
 
 Plate Cultures. In petri-dish cultures made anaerobic according 
 to Krumwiede and Pratt 's method, scant growth may be obtained in 
 glucose and in liver agar (see Fig. 4). In the former medium, gas in 
 small amounts is produced. The growth is translucent, whitish in 
 color, and made up of cottony, loosely constructed colonies. In older 
 
 'U. S. Pub. Health Serv., Pub. Health Rpts., 37, 2252. 1922. 
 
10 
 
 BULLETIN No. 246 
 
 [October, 
 
 FIG. 2. SPOKES OF C. botulinum TYPE C (3420). 
 AN OLD CENTEIFUGALIZED MEAT-MASH CULTURE, x 1200 
 
 FIG. 3. C. botulirmm TYPE C (3420). A CENTRIFUGALIZED 
 MEAT-MASH CULTURE FORTY-EIGHT HOURS OLD. x 1200 
 
1923} CLOSTRIDIUM BOTULINUM TYPE \\ 
 
 plate cultures the growth becomes more compact and opaque with a 
 faint resemblance to frosted glass. In liver-agar plates the character 
 of growth is similar, but disrupted and broken by gas production. The 
 distinctness of the growth disappears in old plate cultures, and sub- 
 cultures from plates are obtained with difficulty. Serum-agar plate 
 cultures incubated in hydrogen atmosphere (Brown jar) show a faint 
 growth at points heavily inoculated. The edges of the colonies are 
 irregular and have many short, fine, spiney projections extending out 
 irregularly. Stained smears of plate colonies fail to show spores. A 
 larger percentage of non-toxic subcultures have been obtained from 
 colonies in plates than from colonies in glucose-agar shake cultures. 
 
 Meat Medium. In meat-mash medium of Bengtson (one part 
 finely chopped lean meat to two parts water) the most favorable 
 growth has been obtained. Gas production occurs in twenty-four to 
 forty-eight hours at 37 C. The meat remains undigested, and the 
 liquor relatively clear. The meat is not discolored during growth. 
 Gas production is generally very marked. The bubbles are large and 
 do not collect on the surface of the liquid but may be seen in the 
 meat along the sides of the tubes (see Fig. 5, A). After six to eight 
 days, gas production generally subsides, and the inoculated tube 
 appears clear. Spore formation occurs in meat mash in thirty-six 
 hours at 37 C. A slight odor is noticeable in actively growing cul- 
 tures, but it does not resemble the butyric acid odor of either the 
 A or the B botulinus organism. 
 
 Liver Medium. In liver medium made in the same proportion as 
 the meat medium employed by Bengtson, the growth is similar to 
 that in meat-mash cultures and resembles the growth of B. novyii and 
 certain non-pathogenic, sporulating anaerobes. 
 
 Glucose Broth. In glucose broth a suspended flocculent growth 
 occurs at 37 C. The flocculi settle to the bottom of the tube in a 
 white, cottony mass or collect as a fine sediment along the side of the 
 tube, leaving the broth clear. Acid is produced but no gas. 
 
 Glucose Agar. In 2-percent glucose-agar deep tubes at 37 C. 
 growth occurs two or more centimeters below the surface. The colonies 
 are translucent, white, and irregular in shape, with fine, cottony, 
 fiber-like projections. The maximum growth is generally attained on 
 the fourth or fifth day, after which the distinctness of the growth 
 decreases. Similar types of colonies occur in liver agar with mild 
 gas production (see Fig. 5, B). 
 
 One-tenth Percent Agar. In .1 percent agar, well defined sepa- 
 rated colonies have not been observed at 37 C. Two or more centi- 
 
12 
 
 BULLETIN No. 246 
 
 [October, 
 
 meters below the surface the inoculated tubes show a uniform 
 cloudiness. 
 
 MUk. In milk, acid is produced. 
 
 The cultural characters of value in differentiating Clostridium 
 botulinum types A and B from type C or the parabotulinus organism 
 of Seddon are summarized in Table 5. 
 
 TABLE 5. CULTURAL CHARACTERS DIFFERENTIATING CLOSTRIDIUM BOTDLINUM 
 TYPES A AND B FROM TYPE C AND PARABOTULINUS OF SEDDON 
 
 
 Clostridium botulinum 
 types A and B 
 
 Clostridium botulinum type C or 
 parabotulinus 'Seddonl 
 
 Glucose agar 
 
 Gas 
 
 No gas 
 
 Glucose agar 
 
 Disc colonies 
 
 Branching colonies 
 
 Glucose broth 
 
 Even cloudiness 
 
 Flocculent growth 
 
 Glucose broth 
 
 Acid and gas 
 
 Acid 
 
 
 
 
 
 
 
 Milk 
 
 No change 
 
 Acid 
 
 Motility 
 
 Motile under cover glass 
 
 Non-motile under cover glass 
 
 
 
 
 
 Resistant to heat 
 
 Non-resistant to heat 
 
 
 
 
 Pafho genesis. The non-lethal character of small amounts of the 
 type C nitrate 3420 given by the mouth is indicated in the results 
 obtained in chickens, horses, pigs, calves, and rabbits, which are 
 reported in Table 6. Larger amounts, however, when fed to these 
 animals in some cases produced illness. In chickens the symptoms 
 experimentally induced resemble clinically those of the disease 
 occurring under farm conditions. 
 
 The fact that small amounts of the culture or culture nitrate may 
 be consumed by fowls as well as by other domestic animals without 
 producing illness suggests that either unusually large amounts are 
 necessary to produce the disease or the invasive character or potency 
 of the toxin in nature is greater than that of the toxin produced in 
 artificial cultures. When subcutaneously injected, both the unfiltered 
 and the filtered culture are more quickly fatal to horses, cattle, and 
 sheep than when given in the feed, tho some chickens have withstood 
 single subcutaneous injections of 3 cc. and 4.5 cc., which represented 
 .05 cc. and .01 cc., respectively, per gram live weight. 
 
 Baeteriologic examination of soil samples obtained during the 
 month of January, 1923, from the chicken yard from which specimen 
 3420 was received, yielded positive evidence of C. botulinum type C. 
 The relation of the soil contamination to the disease occurring in the 
 fowls, however, is not known. 
 
B 
 
 FIG. 4. C. botulinum TYPE C (3420) : INVERTED PETRI-DISH CULTURES 
 (A) IN GLUCOSE AGAR; (B) IN LIVER AGAR 
 
FIG. 5. C. botulinum TYPE C (3420) : TUBE CULTURES (A) IN 
 MKAT MASH; (B) ix LIVER AGAB 
 
1923] 
 
 CLOSTRTDIUM: BOTULINUM TYPE C 
 
 15 
 
 TABLE 6. EFFECT OF SMALL AMOUNTS OF TTPE C CULTURE FILTRATE 3420 
 
 GIVEN BY MOUTH 
 
 Identification 
 
 Treatment, July 26, 1922 
 Filtrate 3420 by mouth 
 
 Results 
 
 No. 
 
 Species 
 
 Weight 
 
 3518 
 866 
 
 Chicken 
 
 1050 gms. 
 
 1 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3519 
 862 
 
 Chicken 
 
 1130 gms. 
 
 2 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3520 
 852 
 
 Chicken 
 
 1310 gms. 
 
 4 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3521 
 
 872 
 
 Chicken 
 
 1300 gms. 
 
 5 cc.' 
 
 Remained healthy 
 Released 8/11/22 
 
 3522 
 768 
 
 Chicken 
 
 610 gms. 
 
 8 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3523 
 856 
 
 Chicken 
 
 1170 gms. 
 
 10 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3525 
 18B 
 
 Horse 
 
 1550 Ibs. 
 
 10 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3526 
 21A 
 
 Horse 
 
 1500 Ibs. 
 
 20 cc. 
 
 Remained healthy 
 Released 8/1 1/22 
 
 3527 
 13DJ 
 
 Pig 
 
 60 Ibs. 
 
 1 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3528 
 30H 
 
 Pig 
 
 50 Ibs. 
 
 2 cc. 
 
 Remained healthy 
 Released 8/1 1/22 
 
 3529 
 10H 
 
 Pig 
 
 40 Ibs. 
 
 3 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3530 
 2DJ 
 
 Pig 
 
 251be. 
 
 6 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3531 
 99DJ 
 
 Pig 
 
 25 Ibs. 
 
 12 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3532 
 5H 
 
 Pig 
 
 30 Ibs. 
 
 24 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3533 
 184 
 
 Calf 
 
 400 Ibs. 
 
 5 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3534 
 187 
 
 Calf 
 
 400 Ibs. 
 
 10 cc. 
 
 Remained healthy 
 Released 8/11/22 
 
 3535 
 188 
 
 Calf 
 
 400 Ibs. 
 
 20 cc. 
 
 Remained healthy 
 Released 8/1 1/22 
 
 3536 
 820 
 
 Guinea pig 
 
 280 gms. 
 
 .5 cc. 
 
 Died 7/28/22, 7 a.m. 
 
 3537 
 684 
 
 Guinea pig 
 
 330 gms. 
 
 1 cc. 
 
 Died 7/28/22, 7 a.m. 
 
 3538 
 721 
 
 Guinea pig 
 
 370 gms. 
 
 1.5 cc. 
 
 Died 7/28/22, 7 a.m. 
 
 3540 
 622 
 
 Rabbit 
 
 2830 gms. 
 
 2 cc. 
 
 Transitory illness; re- 
 covered 
 Released 8/11/22 
 
 3541 
 613 
 
 Rabbit 
 
 2900 gms. 
 
 
 4 cc. 
 
 Remained healthy 
 Released 8/1 1/22 
 
 3.542 
 612 
 
 Rabbit 
 
 2600 gms. 
 
 8cc. 
 
 Died 7/28/22, 10 a.m. 
 
16 
 
 BULLETIN No. 246 
 
 [October, 
 
 SPECIMENS 3421, 3422, AND 3423 
 
 History. Two chickens and one duck from a farm flock of two 
 hundred fowls were submitted by Mr. A. E. Davidson of Mt. Sterling, 
 Brown county, Illinois, on July 12, 1922, for bacteriologic examina- 
 tion. From the history of the outbreak, given by the owner of the 
 flock, it was learned that the disease had appeared simultaneously 
 with the feeding of a certain lot of wheat. Twelve mature hens, four- 
 teen half-grown pullets, and one duck had died, and several birds 
 
 were dull and droopy. 
 Some chickens of the flock 
 were normal in appearance 
 but were unable to walk. 
 The ground of the chicken 
 lot was strewn with feath- 
 ers that had fallen from the 
 affected fowls. A similar 
 disease, according to the 
 observation of the owner, 
 had occurred on these prem- 
 ises the preceding summer. 
 A part of the chicken range 
 on this farm was bottomland and overflowed each spring. 
 
 Clinical Symptoms. Chicken 3421 died en route to the laboratory. 
 Chicken 3422 showed symptoms of leg weakness, remaining in a nor- 
 mal sitting posture (see Fig. 6). Duck 3423 was moribund (see 
 Fig. 7). Apparently it could not raise its head, but frequently 
 moved it from side to side on the ground. The eyes were closed. 
 When aroused the duck responded only by quacking faintly. 
 Respirations were slow, deep, and regular. Diarrhea was a promi- 
 nent symptom. The feathers came out when the duck was handled. 
 
 Fig. 6. Chicken 3422, apparently healthy but 
 unable to stand 
 
 FIG. 7. DUCK 3423 IN A MORIBUND CONDITION 
 
 Gross Pathology. In the crop of chicken 3421 fully fifty fly larvae 
 were found. A few Heterakis perspicilli were noted in the small 
 
1923] CLOSTRIDIUM BOTULINUM TYPE C 17 
 
 intestine. Aside from small areas of hyperemia in the intestinal mu- 
 cosa, no lesions were found. The intestinal contents of the three speci- 
 mens were extracted for thirty minutes in saline solution and 1 cc. 
 of the solution was then fed to guinea pigs. Symptoms of botulism 
 and death followed in twenty-four hours or less. 
 
 Bacteriologic Examination. Aerobic cultures of the internal 
 organs failed to reveal evidence of Pasteurella avium. Anae- 
 robic cultures from the intestinal contents and liver of the three 
 specimens proved lethal when fed to guinea pigs. A faintly Gram- 
 positive, spore-forming rod, which culturally resembled anaerobe 
 3420, was isolated from the liver of chicken 3421 by the deep-tube 
 method. The feeding of culture 3421, as well as the culture nitrate, 
 to guinea pigs produced a fatal botulinus syndrome. No protection 
 was obtained by injections of either A or B botulinus antitoxins. 
 Specific protection, however, was apparently afforded by botulinus 
 antitoxin type C, received from the Hygienic Laboratory, Washington, 
 D. C. (see Table 7). 
 
 TABLE 7. PROTECTIVE CHARACTER OF BOTULINUS ANTITOXIN TYPE C 
 AGAINST CULTURE FILTRATE 3421 
 
 Identification 
 
 Treatment, July 25, 1922 
 
 Results 
 
 Guinea 
 pig 
 
 No. 
 
 Weight 
 
 671 
 
 300 gms. 
 
 .5 cc. culture filtn 
 mouth 
 
 ite 3421 by 
 
 1 cc. type C botulinus antitoxin 
 296 l subcutaneously 
 
 Remained healthy 
 Released 8/25/22 
 
 654 
 
 300 gms. 
 
 .5 cc. culture filtrate 3421 by 
 mouth 
 
 Control 
 
 
 Died 7/27/22 
 
 "Furnished by Dr. G. W. McCoy, Director, Hygienic Laboratory, Washington, D. C. 
 
 Pathogenesis. The subcutaneous injection of .01 cc. to .001 cc. of 
 culture and culture nitrate 3421 proved fatal to guinea pigs and 
 rabbits, as did also the feeding of .5 cc. to guinea pigs. The feeding 
 of 10 cc. of culture and culture nitrate to horses, calves, and pigs for 
 ten consecutive days was without noticeable effect (see Table 8). A 
 chicken (3667) fed 3 cc. of the culture daily for ten consecutive 
 days succumbed, but another chicken (3668) fed the culture nitrate 
 in similar amounts each day showed no ill effect. Two dogs, one 
 consuming 5 cc. of the culture and the other 5 cc. of the culture 
 nitrate, for ten consecutive days, remained healthy. The toxic prop- 
 erty of the culture nitrate was determined at the beginning and the 
 termination of t,he feeding period by feeding .5 cc. to guinea pigs. 
 
 Massive amounts of the culture have been fed to chickens, horses, 
 cattle, and pigs. Some chickens have displayed a marked resistance 
 to the culture and culture filtrate in the feed, consuming as high 
 as one gram per thirty grams live weight, or approximately 3 percent 
 
18 
 
 BULLETIN No. 246 
 
 [October, 
 
 
 Results 
 
 
 Remained healthy 
 Released 8/22/22 
 
 Remained healthy 
 Released 8/22/22 
 
 Remained healthy 
 Released 8/22/22 
 
 Remained healthy 
 Released 8/22/22 
 
 Remained healthy 
 Released 8/22/22 
 
 Remained healthy 
 Released 8/22/22 
 
 Marked leg weak- 
 ness 8/16 
 Died 8/19/22 
 
 Remained healthy 
 Released 8/22/22 
 
 Remained healthy 
 Released 8/22/22 
 
 Remained healthy 
 Released 8/22/22 
 
 Died 8/8/22 
 
 Died 8/9/22 
 
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 Identification 
 
 
 Animal 
 
 Horse A, No. 21 
 
 Horse B, No. 18 
 
 2 
 
 o 
 
 fe 
 
 J5 
 
 3 
 
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 4 
 
 o 
 
 3 
 a 
 
 
 
 Pig; black and 
 white male, No. 
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 1 
 
 67 
 
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 Chicken; white leg- 
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 iL 
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 11 
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 Dog; brindle 
 female 
 
 Dog; black and 
 white male 
 
 Guinea pig, No. 
 606 
 
 Guinea pig, No. 
 602 
 
 * 
 
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CLOSTRIDIUM BOTULINUM: TYPE C 
 
 19 
 
 TABLE 9. RESISTANCE OP CHICKENS TO TYPE C CULTURE FILTRATE 3421 
 
 GIVEN BY MOUTH 
 
 Identification 
 
 Treatment, October 31, 1922 
 
 Results 
 
 Chicken No. 
 
 Weight 
 
 455 
 
 1860 gms. 
 
 7.7 cc. 1 culture filtrate 3421 by mouth 
 
 Remained healthy 
 Released 11/14/22 
 
 456 
 
 2530 gms. 
 
 21 cc. 2 culture filtrate 3421 by mouth 
 
 Remained healthy 
 Released 11/14/22 
 
 457 
 
 2110 gms. 
 
 35 cc.* culture filtrate 3421 by mouth 
 
 Remained healthy 
 Released 11/14/22 
 
 458 
 
 2180 gms. 
 
 36.6 cc. culture filtrate 3421 by mouth 
 
 Remained healthy 
 Released 11/14/22 
 
 459 
 
 2000 gms. 
 
 66 cc. 4 culture filtrate 3421 by mouth 
 
 Remained healthy 
 Released 11/14/22 
 
 460 
 
 2370 gms. 
 
 79 cc. 4 culture filtrate 3421 by mouth 
 
 Remained healthy 
 Released 11/14/22 
 
 'Approximately 1 cc. of toxin to 240 grams live weight. 
 Approximately 1 cc. of toxin to 120 grams live weight. 
 "Approximately 1 cc. of toxin to 60 grams live weight. 
 Approximately 1 en. of toxin to 30 grams live weight;. 
 
 of their body weight. Occasionally other chickens have succumbed fol- 
 lowing the ingestion of smaller amounts of the culture. The resist- 
 ance which some healthy chickens possess is shown by the data in 
 Table 9. The tolerance of healthy chickens to both the culture and the 
 culture nitrate under experimental conditions, together with the fact 
 that the course of the disease is more rapidly fatal in experimental 
 chickens affected with contagious epithelioma, suggests the possible 
 part played by secondary or predisposing causes. 
 
 In feeding C. botulinum type C (3421) to horses, it was found 
 that 50 grams of the culture per 1000 pounds live weight was sufficient 
 to induce a transitory illness (see Table 10). One horse given 100 
 grams per 1000 pounds live weight, developed symptoms of weak- 
 ness, paralysis, obstinate constipation, and incoordination of move- 
 ment, followed by decumbency on the fourth day. In the decumbent 
 position the feet were spasmodically moved in a running motion, 
 while the animal remained nervously alert and conscious of the sur- 
 roundings. Death followed on the eighth day. 
 
 Three out of four cattle, weighing from 240 to 985 pounds each, 
 which were fed from 100 to 400 grams, per 1000 pounds live weight, 
 of C. botulinum type C, culture 3421, showed marked symptoms of 
 incoordination, dulness, and stiffness (see Table 10). The posterior 
 limbs were unsteady, the animals falling on their knees if urged to 
 walk rapidly. After exertion the animals appeared nervous and 
 excitable, but following rest they resumed a more normal attitude. 
 The symptoms gradually subsided in a few days. The daily tempera- 
 
20 
 
 BULLETIN No. 246 
 
 [October, 
 
 
 
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 BULLETIN No. 246 
 
 [October, 
 
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1923] CLOSTEIDIUM BOTULINUM TYPE C 23 
 
 ture of the animals did not show any appreciable elevation during the 
 course of the illness. One cow (497) given 200 grams of C. botulinum 
 type C, culture 3421, per 1000 pounds live weight, remained unaffected. 
 
 The results of several experiments indicate a resistance of sows 
 and pigs to C. botulinum type C. Nursing pigs twenty-four days 
 old were fed .1 cc., .2 cc., .4 cc., .8 cc., and 1 cc., per pound live weight, 
 of culture 3421 with no apparent effect other than a slight elevation 
 of the rectal temperatures between the .fourth and sixth days (see 
 Table 11). Nursing pigs thirty to thirty-four days old which were 
 fed 1 cc., 1.5 cc., and 2 cc., per pound live weight, of C. botulinum 
 type C, culture 3421, also showed no illness (see Table 12). Massive 
 amounts of C. botulinum type C culture were fed to sows at the time 
 young pigs were being suckled, but without noticeable effect in either 
 the sow or the pigs (see Table 13). 
 
 Bacteriologic examination of soil samples obtained several months 
 later from the chicken yard from which specimens 3421, 3422, and 
 3423 were received, proved negative to C. botulinum type C. 
 
 SPECIMENS 3466, 3467, AND 3468 
 
 Three chickens, Nos. 3466, 3467, and 3468, were received for ex- 
 amination on July 20, 1922, from Mr. I. L. Durbin, of Taylorville, 
 Christian county, Illinois. Fifteen half-grown chickens had died in 
 
 FIG. 8. CHICKENS 3466 AND 3467. TYPICAL SPONTANEOUS 
 CASES OF TYPE C BOTULINUS POISONING 
 
 this flock. The clinical symptoms displayed by the three chickens on 
 their arrival consisted of weakness and diarrhea. The eyes were 
 closed, and the chickens seemed to be asleep (see Fig. 8). One of 
 them (3468) appeared during the examination to be greatly im- 
 proved over its previous condition as described by the owner and 
 was returned to the flock. Chickens 3466 and 3467 were killed for 
 autopsy. No gross lesions were encountered. 
 
24 
 
 BULLETIN No. 246 
 
 [October, 
 
 Anaerobic cultures of the intestinal content and spleen tissue of 
 chicken 3466 yielded toxic cultures. The purification of this strain 
 
 FIG. 9. CHICKEN 554, SHOWING CLINICAL ASPECTS OF BOTULINUS POISONING, 
 TYPE C, EXPERIMENTALLY INDUCED BY FEEDING (See Table 15) 
 
 by plate and deep-tube cultures was not accomplished. Guinea pigs, 
 however, which were fed .5 cc. of the toxin elaborated in impure 
 
 TABLE 12. RESISTANCE OF PIGS TO MASSIVE AMOUNTS OF TYPE C CULTURE 
 3421 GIVEN BY MOUTH 
 
 Identification 
 
 Treatment, November 13, 1922 
 
 Results 
 
 Pig No. 
 
 Sex 
 
 Age 
 
 Weight 
 
 534 
 
 2 
 
 M 
 
 30 days 
 
 81bs. 
 
 8 cc. culture 3421 by mouth 
 
 Remained healthy 
 Released 12/1/22 
 
 535 
 3 
 
 M 
 
 30 days 
 
 91bs. 
 
 18 cc. culture 3421 by mouth 
 
 Remained healthy 
 Released 12/1/22 
 
 536 
 4 
 
 F 
 
 30 days 
 
 10 Ibs. 
 
 Control 
 
 Remained healthy 
 Released 12/1/22 
 
 537 
 5 
 
 F 
 
 30 days 
 
 9^ Ibs. 
 
 Control 
 
 Remained healthy 
 Released 12/1/22 
 
 538 
 6 
 
 M 
 
 30 days 
 
 9^ Ibs. 
 
 Control 
 
 Remained healthy 
 Released 12/1/22 
 
 539 
 7 
 
 F 
 
 30 days 
 
 11 Ibs. 
 
 11 cc. culture 3421 by mouth 
 
 Remained healthy 
 Released 12/1/22 
 
 540 
 8 
 
 F 
 
 30 days 
 
 6 Ibs. 
 
 12 cc. culture 3421 by mouth 
 
 Remained healthy 
 Released 12/1/22 
 
 541 
 1 
 
 M 
 
 34 days 
 
 15 Ibs. 
 
 15 oc. culture 3421 by mouth 
 
 Remained healthy 
 Released 12/1/22 
 
 542 
 2 
 
 F 
 
 34 days 
 
 14 Ibs. 
 
 21 cc. culture 3421 by mouth 
 
 Remained healthy 
 Released 12/1/22 
 
 543 
 3 
 
 F 
 
 34 days 
 
 14 Ibs. 
 
 Control 
 
 Remained healthy 
 Released 12/1/22 
 
 544 
 4 
 
 M 
 
 34 days 
 
 18 Ibs. 
 
 Control 
 
 Remained healthy 
 Released 12/1/22 
 
 545 
 6 
 
 M 
 
 34 days 
 
 6 Ibs. 
 
 6 cc. culture 3421 by mouth 
 
 Remained healthy 
 Released 12/1/22 
 
 546 
 7 
 
 M 
 
 34 days 
 
 12 Ibs. 
 
 18 cc. culture 3421 by mouth 
 
 Remained healthy 
 Released 12/1/22 
 
1923] 
 
 CLOSTRIDIUM BOTULINUM TYPE C 
 
 25 
 
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26 
 
 BULLETIN No. 246 
 
 [October, 
 
 TABLE 13. RESISTANCE OF Sows AND NURSING PIGS TO TYPE C CULTURE 3421 
 
 FED TO Sow 
 
 Identification 
 
 Treatment, November 8, 1922 
 
 Results 
 
 No. 
 
 Animal 
 
 Sex 
 
 Age 
 
 Weight 
 
 506 
 OPC 
 
 Sow 
 
 F 
 
 2 yrs. 
 
 450 Ibs. 
 
 150 cc. culture 3421 by mouth 
 
 Remained healthy 
 Released 11 /2S/22 
 
 681 
 1 
 
 Sucking 
 pigs 
 of 
 Sow 
 OPC 
 
 M 
 
 12 days 
 
 91bs. 
 
 None 
 
 Remained healthy 
 Released 11/23/22 
 
 682 
 2 
 
 M 
 
 12 days 
 
 gibs. 
 
 None 
 
 Remained healthy 
 Released 11/23/22 
 
 683 
 3 
 
 F 
 
 12 days 
 
 91bs. 
 
 None 
 
 Remained healthy 
 Released 11/23/22 
 
 684 
 4 
 
 F 
 
 12 days 
 
 9H Ibs. 
 
 None 
 
 Remained healthy 
 Released 11/23/22 
 
 685 
 5 
 
 F 
 
 12 days 
 
 10 Ibs. 
 
 None 
 
 Remained healthy 
 Released 11/23/22 
 
 686 
 6 
 
 F 
 
 12 days 
 
 10 Ibs. 
 
 None 
 
 Remained healthy 
 Released 11/23/22 
 
 687 
 7 
 
 F 
 
 12 days 
 
 9 Ibs. 
 
 None 
 
 Remained healthy 
 Released 11/23/22 
 
 TABLE 14. PROTECTIVE CHARACTER OF BOTULINUS ANTITOXIN TYPE C 1 TO 
 CULTURE FILTRATE 3466 
 
 Identification 
 
 Treatment, August 5, 1922 
 
 Results 
 
 Guinea 
 & 
 
 Weight 
 
 662 
 
 350 gms. 
 
 .5 cc. culture filtrate 3466 by 
 mouth 
 
 .5 cc. botulinus antitoxin type C, 
 296 subcutaneously 
 
 Died 8/6/22, 
 8 a.m. 
 
 687 
 
 350 gms. 
 
 .5 cc. culture filtrate 3466 by 
 mouth 
 
 1 cc. botulinus antitoxin type C, 
 296, subcutaneously 
 
 Remained healthy 
 Released 9/1/22 
 
 753 
 
 400 gms. 
 
 . 5 cc. culture filtrate 3466 by 
 mouth 
 
 1.5 cc. botulinus antitoxin type 
 C, 296, subcutaneously 
 
 Remained healthy 
 Released 9/1/22 
 
 253 
 
 400 gms. 
 
 . 5 cc. culture filtrate 3466 by 
 mouth 
 
 Control 
 
 Died 8/6/22, 
 8 a.m. 
 
 'Furnished by Dr. G. W. McCoy, Director of Hygienic Laboratory, Washington, D. C. 
 
1923] 
 
 CLOSTRIDIUM BOTULINUM TYPE C 
 
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28 
 
 BULLETIN No. 246 
 
 [October, 
 
 TABLE 15. RESISTANCE OF CHICKENS FED MASSIVE AMOUNTS OF TYPE C 
 
 CULTURE 3466 
 
 Identification 
 
 Treatment, November 14, 1922 
 
 Results 
 
 Chicken 
 No. 
 
 Weight 
 
 549 
 
 2460 gms. 
 
 10 cc 
 
 2 meat-mash culture 3466 by mouth 
 
 Remained healthy 
 Released 12/12/22 
 
 550 
 
 1280 gms. 
 
 10 cc 
 
 * meat-mash culture 3466 by mouth 
 
 Remained healthy 
 Released 12/12/22 
 
 551 
 
 2320 gms. 
 
 38 cc 
 
 4 meat-mash culture 3466 by mouth 
 
 Remained healthy 
 Released 12/12/22 
 
 552 
 
 1810 gms. 
 
 30 cc 
 
 4 meat-mash culture 3466 by mouth 
 
 Leg weakness and prostra- 
 tion 11/18 Died 11/24/22 
 
 553 
 
 2130 gms. 
 
 71 cc 
 
 6 meat-mash culture 3466 by mouth 
 
 Remained healthy 
 Released 12/12/22 
 
 554 
 
 2130 gms. 
 
 71 cc. 
 
 5 meat-mash culture 3466 by mouth 
 
 Leg weakness 11/15 
 Died 11/16/22 
 
 Culture was impure, containing a spore-forming rod resembling B. sporogenes. 
 'Approximately 1 gram of m.m. culture to 240 grams live weight. 
 'Approximately 1 gram of m.m. culture to 120 grams live weight 
 Approximately 1 gram of m.m. culture to 60 grams live weight. 
 'Approximately 1 gram of m.m. culture to 30 grams live weight. 
 This chicken showed slight symptoms of ocular roup at the time of treatment. 
 
 cultures were protected by injections of 1 and 1.5 cc. of type C anti- 
 toxin (see Table 14). The toxin elaborated in impure cultures of 
 anaerobe 3466 was also fed in varying amounts to six chickens. Two 
 of these developed typical symptoms of botulinus poisoning and died. 
 (See Table 15 and Fig. 9). 
 
 Cultures of soil samples obtained several months later from the 
 chicken yard of the premises where chicken 3466 had been kept, 
 proved positive to C. botulinum type C as determined by toxin- 
 antitoxin tests on guinea pigs, using unpurified cultures and culture 
 filtrates. 
 
 BOTULINUS ANTITOXIN TYPE C 
 
 An antitoxin has been prepared from goats and calves by giving re- 
 peated injections subcutaneously of non-lethal amounts of the culture 
 filtrate of type C strains isolated from chickens. The specific pro- 
 tective character of this antitoxin has been consistently demonstrated 
 in guinea pigs. The antitoxin of the homologous type C strain pro- 
 tects against the toxin of the two other strains isolated from sporadic 
 cases of leg weakness and limberneck in poultry described in this bulle- 
 tin, as well as against the toxin of a strain 1 isolated from the stomach 
 
 1 Since this report was written, Dr. H. H. Seely of White Hall, Illinois, has 
 submitted to the Laboratory of Animal Pathology and Hygiene for bacteriologic 
 examination a sample of stomach contents (526) from a horse, following death. 
 An anaerobic toxin-producing, spore-forming rod was isolated from this specimen 
 by seeding dilutions of the original sample in glucose agar tubes and incubating 
 
CLOSTRIDIUM BOTULINUM TYPE C 
 
 29 
 
 contents of a horse and one 1 from the intestinal contents of a steer, 
 the type C strain of Bengtson, and the parabotulinus organism of 
 Seddon (see Table 16). The antitoxin prepared against the fly-larvae 
 strain of Bengtson protects against the toxin elaborated by the three 
 chicken strains, the type C strains from a horse and a steer, as well as 
 against the parabotulinus toxin of Seddon. The type C antitoxin 
 prepared against the toxin of chicken strains has also been used to 
 determine the presence of type C strains in cultures from two samples 
 of soil from poultry yards, as well as in certain fly larvae received from 
 Dr. E. W. Saunders, of St. Louis, Missouri. The prophylactic value 
 of the botulinus antitoxin type C in healthy animals is suggested in 
 experimental results, but there is as yet but limited field evidence 
 
 at 37 C. Single isolated colonies proved to be similar, culturally and morpho- 
 logically, to C. botulinum type C. The identity of the anaerobe was finally deter- 
 mined by positive immunologic tests, reported in the following table. 
 
 TABLE A. PROTECTIVE CHARACTER OF TYPE C ANTITOXIN 3421 AGAINST TOXIN 526 
 
 Identification 
 
 Treatment, December 11, 1922 
 
 Results 
 
 Guinea pig 
 
 No. 
 
 Weight 
 
 1467 
 
 250-300 gms. 
 
 100 units Botulinus antitoxin type A 
 5 cc. culture filtrate 526 by mouth 
 
 Died 12/12/22, 3 
 
 p.m. 
 
 1465 
 
 250-300 gms. 
 
 100 units Botulinus antitoxin type B 
 5 cc. culture filtrate 526 by mouth 
 
 Died 12/12/22, 10 a.m. 
 
 1681 
 
 250-300 gms. 
 
 1 cc. BotuTnus antitoxin type C (3421) 
 .5 cc. culture filtrate 526 by mouth 
 
 Remained healthv 
 Released 1/30/23 
 
 Symptoms. The symptoms observed in the spontaneously affected horses con- 
 sisted of a period of stupor of ten to twelve hours, followed by decumbency. Evi- 
 dence of paralysis or weakness of the posterior quarters was noticeable when the 
 affected animals attempted to rise. Irritability and hypersensitiveness, together 
 with accelerated respiration, preceded death. After lying down, animals survived 
 from twelve hours to several days. The temperature ranged fom 98.8 F. to 
 101.8 F. ; pulse, weak, 54 to 65 ; respiration 16 to 20. Four other horses in this 
 herd died during a period of twelve days. 
 
 Feed. The horses had been fed over-ripe clover hay, timothy hay, and corn. 
 They had also had access to pasture. Samples of the feeds submitted were tested 
 for the presence of toxin, with negative results. No evidence of the presence of 
 toxin-producing anaerobes in the feed was obtained by cultural methods. 
 
 Soil. Samples of soil from the yard where the horses died yielded cultures 
 which proved toxic for guinea pigs when given by the mouth. The intoxication 
 was offset only by injecting type C antitoxin. The soil culture was not obtained 
 free from contamination either by seeding in glucose-agar tubes or by plating. 
 
 The distribution of C. ~botulinum in nature forbids a diagnosis of botulism on 
 the basis of bacteriologic findings alone. Symptoms in spontaneously affected 
 horses, together with evidence of the presence of C. botulinum in the digestive 
 tube of fatally afflicted animals and its presence in the soil of the premises on 
 which the horses died, are worthy of consideration in investigational studies look- 
 ing to the establishment of the causative agent of similar diseases of horses, of 
 unknown etiology. 
 
 1 On January 2, 1923, Dr. H. E. Rinehart of Bushville, Illinois, submitted kid- 
 neys (780), lung (781), heart (782), trachea (783), and intestinal content (784) 
 of a three-year-old steer that had died after an illness of twenty-four hours. Two 
 other steers in a herd of 113 in the same feed lot had died from an acute disease. 
 Petechial and ecchymotic hemorrhages were very noticeable on the heart, lung, 
 and serous covering of the intestines received for examination. 
 
30 
 
 BULLETIN No. 246 
 
 [October, 
 
 TABLE 16. PROTECTIVE CHARACTER OF TYPE C ANTITOXIN 3421 AGAINST 
 HOMOLOGOUS AND HETEROLOGOUS TOXIN 
 
 Guinea 
 pig 
 
 No. 
 
 Treatment 
 
 Results 
 
 Date 
 
 Toxin, by mouth 
 
 Type C antitoxin 
 3421, 
 subcutaneously 
 
 659 
 
 9/25/22 
 
 .5 cc. avian strain culture filtrate 3421 
 
 2 cc. goat antitoxin 
 
 Remained healthy 
 
 654 
 
 P ,'25/22 
 
 .5 cc. avian strain culture filtrate 3421 
 
 None 
 
 Died 9/26/22 
 
 671 
 
 9/25/22 
 
 .5 cc. avian strain culture filtrate 3420 
 
 2 cc. goat antitoxin 
 
 Remained healthy 
 
 799 
 
 9/25/22 
 
 . 5 cc. avian strain culture filtrate 3420 
 
 None 
 
 Died 9/26/22 
 
 264 
 
 9/25/22 
 
 . 5 cc. fly larvae strain culture filtrate 2070, 
 Bengtson 
 
 2 cc. goat antitoxin 
 
 Remained healthy 
 
 726 
 
 9/25/22 
 
 . 5 cc. fly larvae strain culture filtrate 2070, 
 Bengtson 
 
 None 
 
 Died 9/26/22 
 
 1308 
 
 1/19/23 
 
 . 5 cc. avian strain culture filtrate 3466 
 
 2 cc. goat antitoxin 
 
 Remained healthy 
 
 1865 
 
 1/19/23 
 
 . 5 cc. avian strain culture filtrate 3466 
 
 None 
 
 Died 1/21/23 
 
 1259 
 
 11/26/22 
 
 . 5 cc. avian strain culture filtrate 487 
 
 2 cc. goat antitoxin 
 
 Remained healthy 
 
 1274 
 
 11/26/22 
 
 . 5 cc. avian strain culture filtrate 487 
 
 None 
 
 Died 11/27/22 
 
 1681 
 
 12/11/22 
 
 . 5 cc. equine strain culture filtrate 526 
 
 2 cc. calf antitoxin 
 
 Remained healthy 
 
 1465 
 
 12/11/22 
 
 .5 cc. equine strain culture filtrate 526 
 
 None 
 
 Died 12/12/22 
 
 1853 
 
 1/23/23 
 
 . 5 cc. bovine strain culture filtrate 784 
 
 2 cc. calf antitoxin 
 
 Remained healthy 
 
 1502 
 
 1/23/23 
 
 . 5 cc. bovine strain culture filtrate 784 
 
 None 
 
 Died 1/24/23 
 
 947 
 
 7/6/23 
 
 .5 cc. parabotulinus strain culture filtrate, 
 Seddon 
 
 2 cc. caif antitoxin 
 
 Remained healthy 
 
 935 
 
 7/6/23 
 
 .5 cc. parabotulinus strain culture filtrate, 
 Seddon 
 
 None 
 
 Died 7/8/23 
 
 A composite sample of kidney, lung, and heart was injected subcutaneously 
 into a healthy rabbit. Six days later the rabbit was found dead. Direct cultures 
 from the heart's blood of the rabbit yielded a small, Gram-negative rod indis- 
 tinguishable from Pasteurella bovisepticum. Cultures of the intestinal content 
 (784') of the steer in meat -mash media proved fatal to guinea pigs when given 
 by the mouth. The symptoms in guinea pigs were similar to the symptoms of 
 botulism. Single colonies in glucose agar were subcultured and purified. The 
 results, which are shown in the following table, suggest its close relation to 
 C. botulinum type C. 
 
 TABLE B. PROTECTIVE CHARACTER OF TTPE C ANFITOXIN 3421 AGAI.VST TOXIN 784 
 
 Identification 
 
 Treatment, January 19, 1923 
 
 Results 
 
 Guinea pig 
 No. 
 
 Weight 
 
 1651 
 
 250-300 gms. 
 
 100 units Botulinus antitoxin type A 
 .5 cc. culture filtrate 784 by mouth 
 
 Died 1/22/23 
 
 1159 
 
 250-300 gms. 
 
 100 units Botulinus antitoxin type B 
 .5 cc. culture filtrate 784 by mouth 
 
 Died 1/21/23 
 
 1652 
 
 250-300 gms. 
 
 1 cc. Botulinus antitoxin type C (3421) 
 5 cc. culture filtrate 784 by mouth 
 
 Remained healthy 
 Released 1/30/23 
 
 The widespread distribution of C. botulinum type C in nature may be the 
 explanation of the positive findings in the intestinal tube of the steer. In view 
 of the susceptibility of cattle to type C poisoning under experimental conditions, 
 the possible relation of this organism to acute paralytic diseases of cattle may 
 be worthy of further study. 
 
1923] 
 
 CLOSTEIDIUM BOTULINUM TYPE C 
 
 31 
 
 obtained under conditions properly controlled to justify the use of 
 the antitoxin in the prevention of type C poisoning in animals. 
 
 It has been observed that the subcutaneous injection of type C 
 toxin (filtered cultures) is more fatal to goats and sheep than is the 
 toxin of type A or B, and that these animals, like yearling calves, 
 may succumb even after they have withstood several injections of 
 toxin. Sheep appear to be even less resistant to subcutaneous injec- 
 tions of the filtered toxin than goats and calves. 
 
 The sterile filtrate of cultures used in hyper-immunizing animals 
 in the preparation of antitoxin has proven labile, the toxicity of the 
 filtrate decreasing rapidly upon exposure at room temperature. 
 
 THE DEVELOPMENT OF TYPE C TOXIN IN SHELLED CORN 
 
 The tolerance exhibited by chickens, horses, and cattle following 
 the ingestion of toxin developed in meat-mash medium led to experi- 
 ments designed to determine whether toxin may not develop in grains 
 and hays, and if so whether such toxin is as potent as that elaborated 
 
 TABLE 17. DEVELOPMENT OF TOXIN IN SHELLED CORN INOCULATED WITH 
 
 TYPE C TOXIN-FREE SPORES AND ITS FAILURE TO DEVELOP IN 
 
 SAMPLES OF OTHER FEEDS AND SOIL 
 
 Identification 
 
 Inoculum, 
 3/14/23 
 TypeC 
 spores, 3421 
 
 Treatment of guinea pigs 
 
 Sample 
 No. 
 
 - Sterile 
 
 Non-sterile 
 
 Culture given by 
 mouth 6/28/23 
 
 Results 
 
 1658 
 
 Soil 
 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1659 
 
 
 Soil 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1660 
 
 Shelled corn 
 
 
 10,000,000 
 
 .5cc. 
 
 Typical fatal symp- 
 toms of botulism 
 
 1661 
 
 
 Shelled corn 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 - 1662 
 
 Corn meal 
 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1663 
 
 
 Corn meal 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1664 
 
 Oats 
 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1665 
 
 
 Data 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1666 
 
 Ground alfalfa 
 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1667 
 
 
 Ground alfalfa 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1668 
 
 Ground timothy 
 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1669 
 
 
 Ground timothy 
 
 10,000,000 
 
 .Sec. 
 
 Negative 
 
 1670 
 
 Tankage 
 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1671 
 
 
 Tankage 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1672 
 
 Silage 
 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
 1673 
 
 
 Silage 
 
 10,000,000 
 
 .5cc. 
 
 Negative 
 
32 BULLETIN No. 246 [October, 
 
 in meat medium. The possibility of feeds becoming contaminated was 
 further suggested by the distribution of the spores in nature. 
 
 Duplicate series of samples of soil, shelled yellow corn, corn meal, 
 oats, ground alfalfa, ground timothy, tankage, and silage placed in 
 eight-ounce clear glass bottles, were inoculated with 10 million type C 
 spores (3421) each (see Table 17). Previous to inoculation one of 
 each of the duplicate samples was sterilized by fractional sterilization 
 and cultured to determine sterility. 
 
 The spores were washed ten times and were heated at 70 C. for 
 five minutes. The non-toxic character of the spore suspension was 
 established in guinea pigs, while subcultures showed that the vitality 
 of the spores was noticeably reduced. After allowing all the inocu- 
 lated samples to incubate at room temperature in a dark place for one 
 hundred and thirty-six days, 50 cc. of sterile saline was added to each 
 sample and mechanically shaken for five minutes. The liquor from 
 each bottle was then fed in amounts of .5 cc. to guinea pigs. That 
 from shelled corn (1660) was the only one of the series which proved 
 toxic (see Table 17). When injected subcutaneously, .0001 cc. of the 
 liquor proved fatal to guinea pigs. The intoxication induced by 
 subcutaneous injection and also that induced by feeding was offset 
 by type C antitoxin. 
 
 In other series of samples of grains and hays which have been 
 inoculated with toxin-free spores of type C, consistent growth has not 
 been obtained, but the results suggest that under favorable conditions 
 type C toxin may develop in shelled corn and possibly in other feeds. 
 
 SUMMARY 
 
 1. A disease of chickens and ducks characterized by the sudden 
 development of lameness or leg weakness, diarrhea, ptosis, prostra- 
 tion, and limberneck, analogous to avian botulism type A and fol- 
 lowed in some cases by recovery but in many others by death from 
 respiratory paralysis, has been encountered in five flocks in Illinois. 
 
 2. The results of toxicogenic tests and bacteriologic findings of 
 the intestinal contents and internal organs of affected chickens sug- 
 gest that one type of the avian botulism syndrome is closely associated 
 with the ingcstion of a bacterial toxin related to, if not identical with, 
 the toxin of the anaerobe isolated from the larvae of Lucilia caesar 
 by Bengtson of the Hygienic Laboratory, who designated it Bacillus 
 botulinus type C, or Clostridium botulinum type C. 
 
 3. The toxic anaerobe isolated from affected chickens is also closely 
 related to the parabotulinus organism isolated by Seddon of the Vet- 
 erinary Institute, Melbourne University, from cases of bulbar paraly- 
 sis of cattle. The antitoxin prepared from the avian type C strains 
 

 1923} CLOSTRIDIUM BOTULINUM TYPE C 33 
 
 protects guinea pigs against lethal doses of the parabotulinus toxin, 
 as well as against similar toxic anaerobes isolated from the stomach 
 contents of a spontaneously affected horse and the intestinal contents 
 of an affected steer in two outbreaks in Illinois. 
 
 4. The symptoms of type C botulinus poisoning in guinea pigs are 
 similar to the symptoms of type A and type B botulinus poisoning, 
 yet neither type A nor type B botulinus antitoxin affords protection 
 against the type C poisoning. In horses, also, type C poisoning re- 
 sembles types A and B poisoning. Since chickens are susceptible to 
 type A toxin as well as to type C, a diagnosis of the causative agent of 
 limberneck-like diseases may be expedited in the laboratory by im- 
 munologic tests. 
 
 5. C. botulinum type C grows less vigorously than type A or B 
 in media employed in the isolation of those strains. Single colony 
 subcultures of type C may sometimes fail to grow or for some reason 
 prove non-lethal. The spores are less resistant to heat than are the 
 spores of types A and B. 
 
 6. The resistance of certain chickens to type C toxin has been 
 suggested by the low mortality in some affected flocks as well as by 
 the tolerance of healthy chickens to the culture and culture filtrate 
 
 .under experimental conditions. Chickens of low vitality, suffering 
 from contagious epithelioma, have been shown to be more susceptible 
 to type C toxin than are healthy fowls. The exact significance of sec- 
 ondary factors such as parasitism and contagious epithelioma, which 
 might lower the vitality of chickens, independent of the important 
 factor of the quantity of toxin ingested, probably merits further 
 investigation. 
 
 7. It has been shown that horses, cattle, and sheep are susceptible 
 to the subcutaneous injection of C. botulinum type C. Small amounts 
 in the feed of horses and cattle daily for a period of ten consecutive 
 days failed to induce symptoms. Single massive amounts given by 
 the mouth, however, induced illness in cattle and death in horses. 
 
 8. Sows, nursing pigs, shotes, and dogs have been shown to be 
 resistant to the culture and culture nitrate of C. botulinum type C 
 when given in the feed. 
 
 9. An antitoxin has been prepared from goats and calves by re- 
 peated injection of the culture filtrate of C. botulinum type C. The 
 antitoxin is of prophylactic value in guinea pigs; it is also useful 
 in identifying the toxin in immunologic tests where the type C botuli- 
 nus organism is suspected. Sufficient evidence has not as yet been 
 obtained to show that the antitoxin is of value in the treatment or 
 prevention of type C poisoning occurring in poultry or other animals 
 under farm conditions. 
 
34 BULLETIN No. 246 
 
 10. The presence of C. botuUnum type C in the soil of two poultry 
 yards several months after manifest symptoms of the disease in 
 chickens occurred on the premises, as well as in the soil of a barn- 
 yard where horses had died of a paralytic disease, may be significant 
 of the distribution of this organism in nature. 
 
 11. The toxin-free spores of C. botuUnum type C are capable of 
 elaborating a highly lethal toxin in shelled corn sealed in glass bottles 
 and kept at room temperature. The development of the toxin in all 
 corn samples inoculated was not consistent under laboratory conditions. 
 
UNIVERSITY OF ILLINOIS-URBANA