ABSTRACT STUDIES ON CLOSTRIDIUM PERFRINGENS TYPE C TOXIN INJECTED INTRAVENOUSLY IN DAIRY CALVES by John R. Welser Clostridium perfringens Type C (beta) toxin was obtained from the research division of a veterinary pharma— ceutical company* for experimental work on the bovine species. Intravenous injection into white mice (17-20 Gm.) was used to check the type and minimal lethal dose of the toxin. White mice were also used to determine the anti— toxin level in calves‘ sera, to determine how long the toxin remained in the blood stream of the calves, and to determine if the urine voided from the calves following toxin administration contained a lethal factor. In preliminary work, it was found that the toxin would not kill calves, but would kill goats and mice. The routes of administration tried in calves were intravenous, oral, and direct injection into the intestinal tract. How— ever, if the toxin was combined with trypsin, death could be produced. Six calves, whose sera was negative for antitoxin, were injected intravenously at the rate of 15,000 mouse MLD/lOO pounds body weight. The symptoms shown following John R. Welser administration of the toxin were: coughing, lacrimation, colic, urination, slight respiratory distress, temperature increase of one degree, doubled heart and respiratory rate, decrease in packed cell volume, increase in the total leuko- cyte count, and an inverse relationship between the numbers of neutrophils and lymphocytes. Sera samples taken from the jugular vein of six calves after intravenous injection of the toxin and injected into mice showed that the toxin was removed from the calves' blood stream within twenty-five minutes. A factor lethal to mice appearing in the calves' urine collected two to three hours after toxin injection suggests that the toxin may be excreted in a relatively unchanged form. *Haver-Lockhart Laboratories, Kansas City, Missouri: Dr. F. W. Binkley, research clinician. Toxin was originally sent to Dr. G. R. Moore, Director of large animal clinics, Michigan State University. STUDIES ON CLOSTRIDIUM PERFRINGENS TYPE C TOXIN INJECTED INTRAVENOUSLY IN DAIRY CALVES By John R. Welser A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Surgery and Medicine 1962 ACKNOWLEDGMENTS my sincere appreciation is extended to all those who so willingly contributed their time and interest in making this study possible. Special notes of thanks are offered to the follow- ing people: Dr. G. H. Conner, Professor, Department of Surgery and Medicine, for his physical help, guidance, counseling and especially, his encouragement; Dr. M. P. Rines, Assistant Professor, Department of Surgery and Med- icine, for his constructive criticism, disciplined think— ing, and advice; Dr. G. R. Moore, Professor and Director of Large Animal Clinics, Department of Surgery and Medicine, for providing the facilities, materials, ideas, and for his cooperation; Dr. D. J. Ellis, Assistant Professor and Director of Farm Services, Department of Surgery and Med- icine, who was a source of inspiration and evaluation; and Dr. C. C. Beck, Assistant Professor, Department of Surgery and Medicine, for his stimulating ideas and reference ma- terial. The author is also deeply indebted to Dr. W. 0. Brinker, Professor and Head, Department of Surgery and Medicine, for his helpful interest and guidance. Recogni- tion is due to Dr. Virginia Mallman and Mrs. Marian Bennett for their knowledge and advice concerning the bacteriolog- ical aspects of this study. Haver-Lockhart Laboratories (Kansas City, Missouri) and Dr. F. W. Binkley, Director of Research, provided the toxin and suggestions which were necessary to initiate my research. To them and to all others who contributed in any manner, my sincere thanks. PART I. II. III. IV. INTRODUCTION. TABLE OF CONTENTS REVIEW OF LITERATURE. . . . . . History . . Morphology. Classification and Conditions Type A. Type E. Type C. . Type D. . Type E. . Type F. . Type C. . . Epizootiology . . . . . . . Case history. . . . . . . . Pathogenesis. . . . . . . . Symptoms. Necropsy. Histopathology. . . . . . . Comparisons: Other species Comparisons: Other types Type A. Type E. Type D. Type E. Diagnosis Treatment MATERIALS AND METHODS . RESULTS AND DISCUSSION. . . . . Typing of toxin . . . . . . . Sera antitoxin test . . . . . Minimal lethal dose of toxin. Type C toxin. Preliminary work. . . . In goats. Trypsinated toxin Orally. . Intraduodenally . . . Intravenously . . . . . . . and trypsin . Temperature reaction. Heart and respiratory rate. Persistence of toxin in the Effect on blood counts. Urine tests . . . . V. SUMMARY AND BIBLIOGRAPHY. CONCLUSIONS blood 53 53 57 64 68 69 LIST OF TABLES TABLE Page I. Typing of toxin . . . . . . . . . . . . . . . . . 44 II. Test for serum antitoxin. . . . . . . . . . . . . 46 III. Calculation of LD50 for Clostridiumgperfringens type C toxin. . . . . . . . . . . . . . . . . . . 48 IV. Persistence of intravenously injected toxin in calves' Sera. O O O O O O O O O O O O O O O O O O 57 V. Effect of toxin on differential blood count . . . 61 VI. Effect of toxin on packed cell volume in calves . 64 VII. Urine study . . . . . . . . . . . . . . . . . . . 66 LIST OF CHARTS CHART Page I. Body Temperature Reaction to Toxin Administra- tion 0 O O O O O O O O O O O O O O O O 0 O O O O 54 II. Respiratory Rate Following Toxin Administration. 55 III. Heart Rate Following Toxin Administration. . . . 56 IV. Persistence of Intravenously Injected Toxin in Calves' Sera as Tested by the Death of Mice. . . 59 V. Total Mice Dead and Rate of Dying. . . . . . . . 60 VI. Leukocyte Counts Following Toxin Administration. 62 VII. Variation of Polymorphonuclear Cells and Lympho— cytes as a Result of Toxin Administration. . . . 63 VIII. Packed Cell Volume Variation due to Toxin Admin- istration. . . . . . . . . . . . . . . . . . . . 65 PART I INTRODUCTION The clostridial group of organisms are responsible for many of the more serious disease maladies of domestic animals. Included in a list of the common diseases caused by them would be blackleg, tetanus, malignant edema, entero- toxemia, botulism, red water, and infectious necrotic hepa- titis. In foreign countries, people refer to black disease, struck, braxy, lamb dysentery and pulpy kidney, each caused by a species of the clostridia (22). This study deals with just one of the clostridia--Clostridium4perfringens, type C and its toxin. According to the literature, the toxin produced by Clostridiumperfringens, type C, is responsible for entero- toxemia in cattle, a disease characterized by sudden death. The amount of toxin necessary for death is not known, nor are the symptoms in cases where cattle are subjected to sub-lethal doses of toxin. This project was originally designed to determine the minimal lethal dose and symptoms observed of type C toxin in calves. Later the design was modified to include the toxin's effect in blood and urine as well as the length of time necessary for the toxin to disappear from the blood of calves following its intravenous administration. PART II REVIEW OF LITERATURE History: In veterinary medical literature, the term entero- toxemia was apparently first used by Bennetts in 1952 to describe an acute disease of sheep. Later acute hemorrhagic enteritis was observed in young, suckling range calves for a number of years in Colorado. After several erroneous diagnoses were made, the disease was specifically identi- fied and the term enterotoxemia applied to it (12). Various other names have been given to this condi— tion in calves, most of them based on the gross pathology. Among the more common are acute hemorrhagic enteritis, bo- vine hemorrhagic enterotoxemia, and hemorrhagic enterotox- emia (12, 26). Merchant defines the condition as follows: "Bovine hemorrhagic enterotoxemia, an acute disease of young calves, lambs, and sheep, caused by Clostridium perfriggens Type C (beta toxin), characterized by sudden onset, hemor- rhagic enteritis and early death (26)." Griner, who has done considerable work in this field, describes it as fol- lows: "Acute hemorrhagic enteritis, a newly recognized infectious disease resembling enterotoxemia of lambs, seen in young suckling range calves in Colorado, characterized by acute, sudden onset, hemorrhagic enteritis, severe hem- orrhage into the lumen of the small intestine and early death (17)." Clostridiumgperfringens has previously been desig— nated as Clostridium welchii, Clostridium ovitoxicus, ngilf lusgphlegmones—emphysematosae, Welch bacillus, and gas bacil- lus. It was originally isolated in 1892 from the foamy organs of a cadaver by Welch and Nuttall, and called Bagil- lus aerogenes capsulatus (21, 37). In 1898, Veillon and Zuber called it Bacillus perfringens; it was then referred to as Clostridium welchii in English speaking countries, and as Clostridium perfringens in France. In 1930, it was designated Clostridium perfringens by Bergey (21, 7). Morphology: Clostridium perfringens is a large, thick, straight- sided, encapsulated, non motile, gram-positive rod occur- ring either singly or in pairs, but seldom in chains. In- dividual cells are about 1 micron wide and from 4—8 microns long. The spores are oval, small, and cause little swell- ing of the rods. The rods vary in their ability to sporu- late, and spores will not form in a highly acid medium. Neither are they apt to be found in media that contain fer— mentable carbohydrates (7, 21, 37). The formed spores are not highly resistant, being destroyed at 100 C. in less than 5 minutes (21). The bacillus is a capsulated, strict anaerobe that grows readily in deep brain meat infusion broth, agar and gelatin media. 0n agar plates, round, entire, slightly raised, opaque center colonies are formed, varying from smooth convex discs with an unbroken edge to an umbonate rough col— ony with a crenated edge. The colonies are surrounded by a zone of hemolysis on blood agar plates. Clostridium_per- fringens will form small, biconvex colonies in deep agar, and if fermentable sugar is present, the media will frag— ment due to gas formation (7, 21, 37). Clostridium perfringens will produce acid and gas in glucose, maltose, lactose, levulose, galactose, mannose, and sucrose broth. Neither mannitol nor salicin is fermented and the fermentation of inulin and glycerol is variable. Gelatin is rapidly liquefied, but coagulated egg medium and Leoffler's blood serum are not liquefied by the bacil- lus. In broth cultures, excellent growth takes place as the media become cloudy. Good growth with gas formation is observed in cooked meat medium; however, the meat frag- ments are not digested. Litmus milk shows stormy fermenta- tion, coagulation, and acid with gas formation. Hydrogen sulfide is produced by Clostridium perfringens while indole is not (7, 21, 37). Stormy fermentation of milk, non—motility, and wide distribution in feces, sewage and soil are considered by Bergey's Manual to be the distinguishing characteristics of Clostridium perfringens (7). Classification and Conditions Caused: Clostridium perfringens, the most prolific toxin producer of the clostridia, is divided into 6 types, A through F. From these 6 types, 15 soluble antigenic toxic fractions have been identified and designated by letters of the Greek alphabet (17,38). Oakley and Warrack indicate that each of the 6 types produce one or more antigenic fractions. Generally, the one in greatest quantity is termed major and the others are termed minor (17, 42). Wilsdon (1931) first established the immunological relationship of the four main strains by comparing the antigenic properties of culture filtrates based on the presence of three lethal factors, which he called W, X, Z (17, 37, 42). In 1933, elaborating on Wilsdon's work, Clenny, Barr, Llewellyn, Jones, Dalling and Ross showed that Wilsdon's Z fraction was composed of at least 3 toxin components, and they proposed a Greek al- phabet terminology (37). Today the system is still termed the Wilsdon's clas- sification and the soluble antigenic toxic fractions are classified by their activity on lecithin, hyaluronidase, desoxyribonucleic acids, ability to cause lysis of erythro- cytes, necrosis following intracutaneous injection and death following intravenous inoculation (21). Identification is based on toxin neutralization and determination of enzymatic activity (21). for the methods used. See C. L. Oakley and G. L. Warrack (30) The following table gives the tabulation of the properties and distribution among the types of the known toxin soluble antigen of Clostridia perfringens (17, 30). Tabulation of the Toxin Antigen of Clostridium perfringens Names Alpha Beta Gamma Delta Ep- silon Eta Theta Iota Kappa Lambda Mu Nu Activity Biological Lethal, necrotic Hemolytic Lethal necrotic Lethal Lethal hemolytic Lethal necrotic Lethal Lethal hemolytic Lethal necrotic Lethal necrotic ? Spreading factor affects capacity of leukocytes to stain Filtrates Biochemical Lecithinase ? ? ? Collagenase Proteolytic enzyme Hyaluronidase Occurrence in fil— trates of Clostridium perfringens types A B C D E F +++ + + + + + +++ +++ + + + + ++ ++ +++ (+?) ++ + + + + ++ ++ + + + + + + ++ + Deoxyribonucle- + + + + + + ase i = produced by some strains + & ++ +++ = = minor toxin major toxin Some strains of Clostridium perfringens may lose their capacity to produce particular antigens and are called degraded strains (30). Dalling and Ross, 1938, stressed that there are Optimal cultural conditions for the produc- tion of each toxin, and unless these conditions are met, a parent strain may fail to yield a measurable quantity of one or the other toxin types (6). Likewise, laboratory toxicogenic types can be varied from poor to good by vary- ing the media (1). Many times, this accounts for incorrect typing. Toxins are believed to be products of bacterial metabolism. They consist of separate fractions having dual or separate specific action on certain cells or tissues (15). Oakley defines a toxin as a substance of high molecular weight, generally a protein, capable of damaging animal cells and possessing antigenicity which is capable, upon injection into a living animal, of exciting the production of substances called antitoxins; these are able to neutral- ize its toxic properties (28). The ability of the bacillus to produce hyaluronidase, a spreading factor, may be linked with pathogenicity and rapid spreading of the infection. Collagenase, which is present in culture filtrates of Clostridium perfringens, is believed to be partly responsible for the solvent action on supporting connective tissue as well as the disintegra- tion of muscle (15, 2). According to Griner, localized necrotizing action of tissues is a feature of the toxin of Clostridium_per- fringens; gas production and odor which rise from the hy- drolytic oxidative and deaminative action on affected tis- sues also result. The lethal effect is due to the combined effect of toxin components on vital tissues and cells of the host (15, 2). Clostridium perfringens may be found either alone or mixed with other bacteria in diseases of animals. The different strains vary greatly in pathogenicity, but most will kill or produce disease in man and the domestic ani— mals. As for the laboratory animals, most of the strains of Clostridium perfringens will kill mice, guinea pigs, pigeons and rabbits (21, 26). Since Clostridium perfringens is widespread in the soil and found in the alimentary tract of nearly all species of warm blooded animals, it is fre- quently found as a post mortem invader from the alimentary tract into the tissues of bloating cadavers of man and ani- mals. For this reason, caution must be taken in drawing conclusions based on the presence of the organism in the tissues collected after death. It is found most often in the so-called gas gangrene infection of man (21). The following is a list of the 6 toxigenic groups A through F of Clostridium perfringens, the conditions re- ported, history, and the species in which they occur: 10 Type A: l936--Rose and Graham reported enterotoxemia jaun— dice of sheep and calves in Australia, re- sembling acute leptospirosis due to the hemo— lytic activity of A toxin (3, 12). --gas gangrene in man and animals (17). l943--enterotoxemia in young calves b Macrae, Murray and Grant in England (17). —-traumatic wound infection of animals (21). l958--acute enterotoxemia in a 6-month old feeder steer (3). --sapr0phytic—intestinal tract of all species (1). Type B: l923--Gaiger and Dalling recorded the isolation of lamb dysentery in England (37). l925--Dalling, Allen, Mason showed the lamb dys— entery bacillus to produce a lethal toxin (37). l932--Gill reported pulp kidney disease of lambs in New Zealand (37 . l937--Dysentery reported in foals by Montgomerie and Rowlands (23). l938--Enteritis in foals by Mason and Robinson (23). l952—-Hepple reported necrotic enterotoxemia in calves (23). l956--Frank of Wyoming reported enteritis in lambs and calves (9). Type B produces 2 major toxin components, beta and epsilon (42). It should be noted that by suitable cultural methods, type B organisms could be made to produce almost pure beta or epsilon toxin (42). The capacity to produce toxin is readily lost by many strains of type B (9). Some believe it has not been definitely established as a disease ll entity in the United States (9, 12). Type C: l930--Isolated by McEwen and Roberts as the cause of "struck" in adult sheep in England, largely restricted to the Romney Marsh (37). l933--Heller gave an account of acute hemorrhagic enteritis in young shed born lambs in Cal- ifornia (l7). l951--Study started at Colorado A and M (17) was observed and confirmed in numerous areas in Colorado after being found in 6 calves at autopsy (18). l952--Acute hemorrhagic enteritis described by Griner and Bracken in 5 calves at autopsy (18). l953--Griner and Johnson reported similar hemor— rhagic enteritis of new born lambs (17). l953--Excessive mortality of newborn lambs 12-72 hours after birth (20). l953--Griner and Bracken reported hemorrhagic en- teritis and reproduction of the disease by feeding a whole broth culture of Clostridium perfringens type C combined with cornmeal and milk (15). 1955-—Fie1d and Gibson reported enteritis in pig— lets in England (12). l956-—Barrons observed one case of enteritis in goats (19). Type C produces a lethal necrotizing toxin isolated first by McEwen. A member of the Welch group, it was first designated Bacillusgpaludis, and later became type C in the Wilsdon classification, 1931 (17). It most commonly affects adult sheep, according to Griner (20). Type D: l93l—-Schofield reported enterotoxemia in cattle 24 . 12 l932--In western Australia, Clostridium perfringens type D isolated from the small intestine of sheep as the cause of enterotoxemia (l). 1939—-Gordon found type D toxin in Scotland in horses with grass disease (21). l954--Keast and McBarron reported enterotoxemia in cattle in Australia (l2, l6). --enterotoxemia in sheep and goats throughout the world (l2, l7). Epsilon toxin is excreted as a protoxin. Upon stand- ing, the toxin is converted to an active, highly lethal necrotizing toxin by proteolytic enzymes (38). This also can be accomplished by incubating the toxin at 37° C. with trypsin for one hour (22). This process increases the lethal quality of the toxin by approximately 300 times (22). Type E: l921--Gaiger and Dalling reported bacillary dys- entery in lambs and reproduction by feeding the intestinal contents of a natural infected lamb (27). l943--Bosworth observed hemorrhagic enteritis in calves 1-4 weeks old (12). l954—-Griner reported hemorrhagic enteritis in calves due to Type E. Type F: --causes enterotoxemia of man called enteritis necroticans. —-characterized by the ability of spores to withstand boiling for 4 hours (3, 12, 17, 21). In a review of the literature, it is noted that all of the types of Clostridium perfringens A through F have been reported as causing a condition in calves (3, 12). It also may produce a fulminating mastitis, but no attempt 13 at typing has been made (15). From the above reports, it becomes obvious that little evidence of a species Specificity for any of the six types can be found, and a clear cut classification is impossible (3, 12). Type C: Epizootiology: Griner and Bracken reported that hemorrhagic enter- itis occurred most often in vigorous thrifty young animals (12). This is confirmed by stockmen who rarely report losing a small weak calf. The animal lost is generally large, well formed and apparently vigorous (39). It occurs in calves 2-10 days of age having high producing cows as their dams (3, 12). Most of the other reports on the condition agree that it occurs in calves under 3 weeks of age (1, 3, 26, 40). However, Barner (2), along with Stableforth and Galloway (27) limit the disease to 3-5 days of age. Accordingly, it is generally agreed to occur most commonly in the beef breeds with the highest incidence being in the Hereford breed (l5, 17, 26). Griner further states that few instances of the condition have occurred in the dairy breeds (17). However, Beck and Ellis (3) state that it does occur in dairy cattle with the highest incidence being the Holstein and Guernsey breeds. 14 Cattlemen agree that the most common predisposing factors listed include overeating and dams producing an abundance of milk (1, 3, 12, 15, 26). According to Griner, unfavorable cold, wet and windy weather at calving increases the incidence (17, 15). Baldwin reports an increase in incidence in the west when an increase in the number of spring storms is noted (1). However, Merchant reports there is no relationship to season, climate or weather (26). Griner reports mortality in herds is 1—10% with unconfirmed reports up to 30%. Most stockmen are of the Opinion that morbidity of the disease is greater than mor- tality. They believe that animals develop subacute infec- tions from which they frequently recover but remain un- thrifty (15). This is somewhat supported by Griner and Baldwin along with serological studies, which showed that 14% of the cows tested had normal antitoxin, and 24% of the calves had antitoxin (l7). Baldwin reports that western cattlemen who have vaccination programs observed a drop in the incidence of debilitating diarrhea in calves (1). Case History: Due to the sudden onset and short course of entero- toxemia, the history is usually the finding of calves pros- trate or dead (12). Beck and Ellis report the common his- tory to be an animal seen normal one night or morning and upon the next observation by the owner, to be found dead (3). 15 Baldwin, however, reports a period of listlessness and in- appetence 12-24 hours prior to onset (1). There is usually a history of an excellent diet or a good milking dam (39). However, there is no agreement on the theory of an increase in incidence in calves from mature cows versus calves from heifers (2, 39). Reports appear in the literature of individual cows that have lost their calves for 2-3 consecutive years (12, 39). Pathogenesis: Enterotoxemia as a term was first used by Bennetts in 1932 to describe an acute disease of sheep caused by epsilon toxin of Clostridium perfringens type D. Since then, it has been applied to toxemia occurring in other species caused by Clostridium perfringens (l2). The pathogenesis of this disease is unknown (15). In the laboratory, anaerobic conditions, 37 C. temperature, and a media rich in protein and carbohydrate, gave maximum growth in four hours (1). Clostridium perfringens is a saprophyte, normal in the lower intestine of all domestic animals. The fact that it is a spore former coupled with its saprophytic ex- istence, gives a large pOpulation wherever livestock exist, thus making enterotoxemia an ever present possibility (1). We know it is a pathogen, according to Beck and Ellis (3), from post mortem findings and animal tests with l6 toxin produced from cultures obtained from the small intes- tine of actual cases. Support is given to this by other examples of organisms that are residents under normal con- ditions and become pathogens; examples are streptococci, coliforms, and staphylococci (31). Griner states that enterotoxemia is related to pri- mary indigestion in an animal on a high carbohydrate ration or an increase in feed, or a sudden change in type of feed, which creates a favorable intestinal medium for the produc- tion of large quantities of toxin (l2, 15). This is sup- ported by Merchant who states: "Spores of the organisms are ingested from the environment; inflammation and engorge- ment of the bowel causes anaerobic conditions for the growth of the organism and the development of toxin. The intestinal stasis is conducive to absorption of toxin and its distribu— tion by the blood stream. The toxin is produced in the intestine due to enteritis and probably overfeeding which causes the stasis and resulting absorption of toxin (26)." Baldwin (1), Burns Co. Symposium (21), Bullen, and Batty (8), and Smith and Jones (35) all subscribe to the idea of overeating as the trigger mechanism. Baldwin claims, however, that the static condition of the bowel prevents flushing, and the rich feed provides the media (1). 0n the other hand, Burns Co. Symposium (21) along with Bullen and Batty, maintain it is due to an overflow of unfermented starch from the rumen into the small intestine. Roberts 17 claims the organism is markedly susceptible to acidity, and that overloading the rumen with high protein feed swamps the stomach acid allowing the organism to grow rapidly (37). Beck and Ellis proposed that the trigger mechanism in the older cattle is stress as related to liver function (3). Their proposal is based on liver function and the role of bile in controlling Clostridium perfringens. Since bile is the alkaline reserve for proper pH in the body, and under conditions of stress the gall bladder does not empty properly, a more acid medium is created. The lack of bile gives decreased fat absorption; hence, food material in the gut becomes coated with fat, which results in protein putrifaction. Further support is gained from the fact that bile is used in bacteriology to inhibit gram positive or- ganisms and a lack of it would tend to let them grow more profusely (3). Bullen and Batty (8), in experimental work, found that when concentrated diphtheria antitoxin was dripped into the duodenum of normal sheep, small but constant amounts are absorbed into the blood, showing that the intestine is very slightly permeable to this protein. The rate of ab- sorption of the antitoxin is not significantly affected by the sheep's overeating or by acid conditions in the rumen. However, in experimental enteritis, the rate of absorption is greater than normal, which shows that the permeability of the intestine is increased (8). 18 Likewise, massive quantities of the toxins of any of the enterotoxemia organisms can be fed to susceptible animals without evoking any signs of ill health. Therefore, it must be concluded that some unknown mechanism initiates the state of permeability, or more specifically, necrosis of the mucosa (37). Toxin found in the peritoneal fluid but absent in the thoracic fluid, suggests a direct diffusion, but the missing link is an explanation of the permeability of the intestinal wall to the toxin (37). Enterotoxemia has been reproduced in experimental sheep by the following ways: (1) previously injuring the lambs' alimentary tract; (2) functionally impairing it with opium and belladonna; (3) distending it with excessive amounts of milk and irritating it with a heavy feeding of cornmeal; (4) ligation of the jejunum and feeding culture (35). It has also been reproduced in sheep by feeding them whole broth cultures as well as intestinal contents of sheep that have died from the condition (27). Not much success has been reported in reproducing enterotoxemia in calves. Griner reports one success out of four tries using whole broth culture of Clostridium per— fringens (18). One other report in the literature of re- producing the condition, included the feeding of cornmeal or some other irritant food to the calves prior to feeding the culture of Clostridium (35). 19 Symptoms: There is general agreement that the symptoms for Clostridium_perfringens type C enterotoxemia vary depending on the severity of intoxication (l, 15, 39, 40) and range from a subacute form as recognized by Griner (12) to sudden death. The acute form is often preceded by a period of 12-24 hours of listlessness, weakness and inappetance (l, 39, 26). Following this, acute colicy pains set in along with uneasiness, straining to defecate and kicking at the abdomen (1, 3, 15, 25, 39, 40). Hemorrhagic diarrhea may or may not occur depending upon the duration of the condi- tion (1, 3, 26, 39, 40). Baldwin states: "If the animal lives over 6 hours, a hemorrhagic diarrhea is observed with the feces containing large quantities of fresh undigested blood (1)." When and if the bloody scours do appear, it generally indicates that the disease is in an advanced stage (15). Just prior to death, the animal becomes prostrate, develops opisthotonus, tetanic spasms and toxic symptoms. The entire course of enterotoxemia is usually from 2-24 hours (1, 25, 26, 39, 40). The temperature remains normal throughout the course of the condition, becoming subnormal as the animal approaches death (15, 39). The subacute enterotoxemia, as recognized by Griner, is characterized by diarrhea, listlessness and anorexia, 2O followed by acute colic, straining, and kicking at the ab- domen (40). It somewhat resembles calf scours in that most animals recover but remain unthrifty (39). This Opinion is supported by serological studies where the dams showed no antitoxin titer, although their calves had titers up to 16 units (12, 39). The two field case reports in the literature, one by Beck and Ellis (3) and the other by Gregory (10), support the above symptoms. Necropsy: In enterotoxemia, caused by type C of Clostridium perfringens, one can always observe an acute hemorrhagic enteritis Of the jejunum and ileum, involving as much as from 20 consecutive feet to the entire small intestine (3, 15, 18, 19, 26, 39). Griner reports that in some cases, the enteritis is necrotic with desquamation Of the mucosa (18). The lumen of the intestine has much free blood and necrotic tissue debris in it (3, 15, 39). Merchant states that a light fibrinous exudate covers the serosa of the intestine, but Griner describes it as a mild fibrinous per- itonitis covering inflamed intestines (l5). Extensive hem- orrhage into the lumen and wall of the intestines was ob- served by Griner (15), and in another case, he reported subserosal hemorrhage along the entire digestive tract (18). This is confirmed by all other authors who observed petechial or ecchymotic hemorrhages throughout the remainder of the 21 digestive tract (3, 26, 39). Stableforth and Galloway (37) state that hemorrhages occur in the mesentery also. Petechial or ecchymotic hemorrhages occur on the epicardium, thymus, diaphragm, abomassum and inconsistently on the parietal pleura (3, l2, 17, 26, 39). In addition, Griner reports subepicardial and endocardial hemorrhages (12). The mesenteric lymph nodes show serohemorrhagic lymph— adinitis (3, 15, 26). Beck and Ellis (3) report that nearly all lymph nodes are swollen and hemorrhagic. The peritoneal cavity contains a small quantity of serosanguinous fluid and an excess of fluid with some clotting is noted in the pericardial sac (15). Moderate pulmonary congestion was noted by Merchant (26). The ab— omassum is frequently distended with milk. Its mucous mem- brane is hyperemic and covered with a thick mucus (l2, 15). In the field cases, Beck and Ellis (3) Observed a severe toxemia. A central nervous system involvement was substantiated by finding petechial hemorrhages on the brain and cord. The kidneys showed hemorrhages and the large intestine was distended with gas (3). Gregory (10) Observed the following post mortem lesions: cecum distended with gas, the small intestines empty with a mucoid cast, and the walls of both the large and small intestines show- ing severe edema. The kidneys were highly congested with a soft friable parenchyma (10). 22 Histopathology: Very little work has been done on the histopathology of type C enterotoxemia. Griner reports that most of the changes occur in the small intestine (15). Extensive ne- crosis and hemorrhage are the principal lesions occurring in the mucosa and submucosa of the small intestine with the necrotic process extending to the muscularis mucosae. Vary- ing degrees Of hemorrhage, vascular congestion, edema and distention Of the lymphatics in the muscularis and subser- osal occurs (17). Frequently, the villi are completely destroyed, presumably by the necrotic action of the toxin (l7). Toxin degeneration of the liver and kidney parenchyma is consistently observed (15). The kidneys, myocardium, thymus and mesenteric lymph nodes show areas of hyperemia, hemorrhage and toxic degeneration (18). Hemorrhages in the perivascular spaces of the brain stem, as well as sub— epicardial and focal myocardial hemorrhages, may be Observed (15, 18). A smear of the lumen contents of the small intes- tine reveals large quantities of intact and hemolytic eryth- rocytes, fragments Of necrosed villi, polymorphonuclear leukocytes and many gram positive rod shaped bacilli occur- ring singly or in short chains (l5, 17, 18). Comparisons: other Species A review of the literature concerning the symptoms, 23 pathology and histopathology of type C enterotoxemia in other species reveals a marked similarity to the condition occurring in calves. In sheep, the condition is character- ized by sudden onset, early death, and post mortem lesions of a severe hemorrhagic enteritis (1, 17, 20). Shivering, bleating and other signs of chilling are shown as initial symptoms (17, 20). The rest of the symptoms and pathology are identical, except that there is general agreement that the condition is less severe in lambs (l, 20, 17). More work has been done on the histopathology in lambs, and the lesions shown in liver and kidney sections are as follows: A. Liver--central congestion and cloudy swelling of hepatic cords, swollen parenchymal tissue and pigmentation Of cytoplasm. B. Kidneys--hyperemia and hemorrhage in the parenchy- mal and stromal tissues, swollen epi— thelium of the convoluted tubules and granulated cytoplasm (17). According to Stableforth and Galloway (37), the post mortem findings and pathologic changes seen in C enterotoxemia occurring in the English Romney Marsh area, differs from that described by Griner and Johnson in the United States (20). Piglets showing hemorrhagic enteritis similar to calves have been Observed in England and the United States. 24 The autopsy reports in both cases showed an acute necrotic hemorrhagic enteritis of the ileum and jejunum (1, l2). Comparisons: other types In the case of other types of Clostridium perfringens, A, B, D, E, causing enterotoxemia in calves, a striking similarity is noted. A short review of the lesions Observed and the main difference is presented. Type A. The symptoms and pathology Observed in type A en- terotoxemia are very similar to type C. Differ- ences include: the presence of diarrhea, a temper- ature increase (103-106), more gas formation caus— ing more gas to be found in the digestive tract, and a closer resemblance to acute leptospirosis due to the hemolytic activity of type A toxin (12, 25, 32, 34). Much more work has been done on the histopathology of type A, and the following les- ions are noted: a. heart-—cloudy swelling of the fibers, occas- ional hyaline degeneration, petechial hem- orrhages beneath the covering of serous mem- branes, occasional degeneration and necrosis with slight calcareous infiltration in the Purkinji cells, and hemorrhages in the epi- cardium, coronary furrow and subendocardium. 25 kidney--extensive degeneration and necrosis of convoluted tubules, precipitation in the lumen of the tubules, subcapsular hemorrhages, congestion and hemorrhage in the medulla, and a friable parenchyma. spleen--petechial hemorrhages throughout and pulpy in consistency. lung--slight to marked congestion, serous exudate in the alveoli. liver--cloudy swelling and karyolysis of some small groups of cells. lymph nodes-—edema and peripheral congestion with slight hemorrhage (32). No agreement can be found among authors as to the age at which type A occurs. Macrae (25) reports that it occurs in the first week Of life, but Schofield (32) states it occurs between 6-10 weeks, and Shirley (34) claims that it is seen only in older cows. Type B. According to Hepple (23), in calves a severe diar- rhea fatal in 1-4 days is caused by type B. Small yellowish diphtheritic patches distributed in necrotic areas along with congestion of the mucous membrane Of the caecum and colon (23) are the major differences noted. The liver, spleen and kidneys are intensely congested with blood. The lungs are reported to be normal (23). Type D. Type E. 26 Histopathology of the mesenteric lymph nodes shows an active vascular reaction with hyperemia of venulae and cellular infiltration. The kidneys show subcapsular hemorrhage, swelling of the con- voluted tubules, areas of congestion in the medulla and some nuclear fragmentation (23). Enterotoxemia caused by type D is reported in young calves by Griner, Aichelman, and Brown (12), and Schofield (31), and in cows by Keast and McBarron (24). It is characterized by central nervous symp- toms, convulsions, incoordination, blindness, opis- thotonus and head pushing (12). A severe pulmonary edema and diffuse red specking of the trachae with much froth in the air passages is seen (6, 24). Considerable gas was also reported to be present in the caecum and colon by Keast and McBarron (24). Griner and associates (12), along with Keast and McBarron (24), agree that the condition closely resembles acute D enterotoxemia of sheep. The diarrhea present in type E enterotoxemia ranges from yellow to orange mucus. All other symptoms reported by Griner were similar to type C (12). To quote Griner, "The symptoms of enterotoxemia caused by the 6 types of Clostridium perfringens in various 27 species are in general similar to those caused by type D in lambs (12)." Recent findings by Griner (13) in acute and subacute forms of Clostridium perfringens type D enterotoxemia in lambs are of valueto this study. The histOpathology of the brains Of dying lambs is very similar in location to that of animals with the subacute form. MicrOSOOpic foci Of softening or liquefaction necrosis are formed in the basal ganglia, thalamus, internal capsule, substantia nigra, subcortical white matter, and cerebellum of affected lambs. The lesions are characterized by vascular congestion, de- generation of endothelium and walls of the vessels, pronounced perivascular edema and varying degrees of intercellular edema. Pathologic changes in the neurons and neuroglia appear to be related to the increase in plasma transudate (13). The use Of radioactive 1131 revealed a sharp increase in the distribution of the isotOpe in the brains Of lambs intoxicated with Clostridium_perfringens type D toxin, thus indicating a marked increase in vascular permeability (19). Also, the chronologic pathogenesis of encephalic lesions in type D intoxicated mice appeared to be related to an initial increase in vascular permeability followed by edema, softening, liquefaction necrosis, and healing by glial scar- ring (13). It therefore appears that the primary action Of Clostridium perfringens type D toxin is on the vascular system, causing an increase in permeability (19). These 28 findings are in agreement with the findings of Bullen and Batty (8), who reported that oral administrations Of type D toxin increased the permeability of the mouse intestine. NO gross lesions were reported to occur by either author. Diagnosis: Due to the rapid course Of enterotoxemia, symptoms are frequently missed (12). A presumptive diagnosis of hemorrhagic enterotoxemia can be made when young suckling calves are found dead, and autopsy shows extensive hemor- rhagic enteritis (l). The classic method of identifying individual species of bacteria by the morphological, cul- tural, physiological and pathogenic features is helpful and should be a standard procedure in the laboratory; how- ever, it doesn't definitely type the toxin (28). Field diagnosis can be accomplished by the use of specific type antitoxin or vaccine. By injecting part of the subjects with type A, type B, type C, or type D, respec- tively, and leaving part to serve as a control, one is able to determine the causative organism. However, this method is very impractical (l2). Confirmation of a diagnosis can be Obtained by send- ing 25-50 cc. of intestinal contents to a diagnostic lab- oratory (15). Here, the presence of gram positive, nonmotile, medium sized bacilli morphologically resembling Clostridium perfringens seen in a direct smear of intestinal contents 29 is considered helpful (17, 26). Upon anaerobic incubation on blood agar, a hemolytic zone around each colony should be produced. A stormy fermentation is shown in milk (17). Typing of the toxin is carried out in the labora- tory utilizing various tests. Perhaps the most practical is by toxin neutralization with specific antitoxin or sera. Sera has antitoxin in it for neutralizing toxins produced by various species in the intestine or the media of the laboratory. Each serum is type specific and may have 1 to 6 recognized antitoxins in it. All of the 6 known types Of Clostridium perfringens can be identified by recognizing the toxin or combination of toxins produced (41). The anti- toxin sera are prepared in hyperimmune horses and interna— tional standards have been established for each serum (37). Routine laboratory diagnosis is performed on the supernatant fluid of the intestine (9), or more preferably on the bacteria-free filtrates of the intestinal contents (15). The intestinal contents are diluted with equal parts of distilled water and filtered through a seitz filter. The filtrate is then inoculated into mice, 0.3 cc. (41) intravenously into the lateral tail vein. If toxin is present, death will occur in 30 minutes to 3 hours (17). If the filtrate is lethal, prepare mixtures Of 0.9 cc. Of test fluid plus 0.3 cc. of the different types Of sera A, B, C, and D. Incubate the mixtures for 1/2 hour at room temperature and inject 0.4 cc. (41) or 0.3 cc. (1?) Of each 30 mixture intravenously into mice along with 0.3 cc. of the test fluid as a control. The results are read up to 3 days (41). A simple typing chart adapted from Frank (9) is as follows: Typing of Clostridium perfringens. Filtrate Filtrate Interpretation Neutralized by Not neutralized of results Serum type by Serum type Toxin Type A -- A A B A, C, D B, E B C A, D B C D A, C E D Bosworth A, B, C, D Lambda E Type D toxin is neutralized by its own antitoxin and type B antitoxin, but not A or C antitoxin (37). Type C antitoxin will neutralize the filtrate of a young culture of type B, because at this time the beta, gamma, and delta toxins are formed while epsilon protoxin is in its nontoxic state. After time, however, the epsilon protoxin becomes activated, and type C antitoxin will no longer neutralize type B toxin. Intravenously, the beta toxin causes an increase in respiratory rate, followed closely by chronic nervous spasms, hind limb extension and death. Therefore, it should be kept in mind that the beta toxin will usually kill mice within minutes after injection (37). 31 Stableforth and Galloway (37) consider intracutane- Ous tests in guinea pigs more sensitive than mouse intra- venous tests. The beta toxin gives a purplish congested area which eventually becomes necrosed. The lesion is ir- regular in shape and spreads due to the presence of hyaluron- idase (37). Rabbits and mice may also be used for the in- tracutaneous tests (41). It may be assumed that when beta toxin is demonstrated, the causative organism is either type B or 0. Likewise, if epsilon toxin is observed, B or D could be the causative organism (9). Beta toxin is in highest concentration in early cultures and is readily inactivated by trypsin (28). Therefore, using the methods of Bosworth and Glover (1934), epsilon toxin of Clostridium perfringens can be demonstrated by activating the filtrate with trypsin and retyping again in mice to differentiate between types B and C (23). In the case of culture filtrates that are typed B, they should be rechecked in 72 hours for the presence of epsilon toxin (25). Montgomerie clearly states in his work that he was never able to demonstrate the presence of both beta and epsilon toxin in a single sample of intestinal contents (27). This was confirmed by Frank (9). However, Frank states he has found flocks of sheep with both types C and D occurring in them (9). Toxin neutralization tests can also be used to measure antitoxin unitage of animal titer (l7). 32 Some laboratories prefer to grow the organism in 1% glucose broth, modified Brewer's media or similar medium. It is filtered through Seitz sterilizing pads before being examined for toxins (30, 17). The filtered toxins are then tested by toxin neutralization tests and for haemolysin, lecithinase and collagenase (30, 37, 41). See Table I, page 4, for results and Oakley and Warrack (30) for pro- cedures. Barner states that the diseases to be considered in making a differential diagnosis are the following: (1) hemorrhagic septicemia; (2) coccidiosis; (3) listeriosis; (4) plant or chemical poisoning; (5) botulism; (6) white muscle disease (2). Griner reports that a positive diagnosis of type C enterotoxemia can be made if the following three conditions are met: 1. Smears of intestinal contents show organisms resembling Clostridium perfringens. 2. Types B and C antitoxin neutralize the toxin. 3. Type C antisera prevents the condition (20). Treatment: Few diseases or conditions of domestic animals demon- strate the value of preventative medicine as does entero- toxemia (12). In many cases, enterotoxemia is observed in animals too young to be actively immunized by vaccine, so 33 control must take on the form of passive immunity by: (l) administration of hyperimmune antitoxin serum as soon after birth as possible; (2) ingestion Of colostrum milk from a dam previously immunized with toxoid (17, 26, 39). Older animals can be protected either actively or passively by using antitoxin sera, a vaccination with highly antigenic toxoids, or bacterins (12, 26). Antitoxin is derived from horses which have been hyperimmunized against the type Of toxin desired (39). PrOphylactic dosage is 10 cc./calf subcutaneously. The antitoxin titer lasts approximately 3 weeks in the calf (l, 17, 39, 40). Due to the similarity in symptoms of en- terotoxemia, which is caused by the various types of Gigg- tridium perfringens, it is recommended that in actual cases the practitioner use the combination antitoxin B.C.D. rather than risking greater losses (3, 22, 42). It should be noted that Clostridium perfringens type B.C.D. antitoxin is actu- ally a combination of type C and D antitoxin and does not contain 3 major fractions or antitoxins as the name implies (l). Therapeutically, antitoxin can be Of some help in the early stages of the condition in dosages Of 25-30 cc. intravenously or subcutaneously (l, 39, 40). The above dosages are based on the products having a minimum of 1500 units of a specific antitoxin/cc. (1, 39). It must be kept in mind that when using hyperimmune serum, one is injecting 34 a foreign protein; therefore, precautions should be taken to handle any foreign protein or anaphylactic reaction (1, 3). To provide passive immunity to the offspring, the dam is usually vaccinated 2-4 months prior to calving with a booster injection given 3 weeks after the initial injec- tion. Griner states, however, that ranchers Object to the 2 injections being given within a 3-week period (12). A single booster dose is recommended for the second year and years following. The recommended dosage is 5 cc. in cows with 30 toxoid units/cc. being the minimum standard for the vaccine (ll, 17, 22, 26, 39, 40). Initial injections will give a titer in 3 weeks (12), and the second injection provides a higher response in 7 days (39). Toxoids give much better results than bacterins (39). They can be used in younger animals and produce a higher longer lasting titer. Lambs as young as 15 days have produced immunity (12, 22). Bacterins cannot be used in animals under 8 weeks of age. If used in older animals, the resulting immunity is lower and short lived (22). The toxoid product is activated with trypsin and then detoxified to produce the vaccine. Toxoid immunity comes from a sensitization to the antigen. The second dose of toxoid has a booster effect by sensitizing the animal to the specific toxin. Upon contact with the specific toxin, the animal immediately responds with a rapid production of antitoxin (22, 39, 42). A small percentage of animals fails 35 to respond and never does develop an antitoxin titer (42). The Object of vaccinating close to the end of preg- nancy is to Obtain as high an antitoxin titer as possible at the time of calving. The higher the blood antitoxin of the dam, the higher the colostrum antitoxin and the greater the protection to the calf (l). The effectiveness Of active immunizing agents against the organism is debated and difficult to determine because of the unpredictability Of natural occurring cases (35). Griner and Baldwin found in their work that anti- toxin may be cumulative in calves, since, in many cases, the antitoxin titer Of the calf exceeded that of the dam (1, l7). Griner also found that 14% of the cows and 24% of the calves he tested had natural occurring titers (l7). PrOphylactic chemotherapy, in the form Of feed ad- ditives, has been used and is currently being evaluated. McGowan reported significant results with chlortetracycline, as a feed additive, in the control Of enterotoxemia in lambs (l2). Griner reports in another article that antibiotics are of value when used prOphylactically, but their value as therapeutic agents is doubtful, as they are not capable of neutralizing the lethal toxin (15). In Hagan and Bruner, it states that the addition of sulfur to the ration to re- strict the intake of feed, has been successful in reducing enterotoxemia (21). In the Annual Review of Microbiology (43), Williams 36 showed that oral and cloacal administration of Clostridium perfringens had no effect on the growth of the chickens. He further stated that his findings failed to support the hypothesis that Clostridia in the feces elaborate toxins which inhibit growth, and that the growth-stimulating ef- fect of antibiotics in the diet, is due to the suppression of these Clostridia (43). PART III MATERIALS AND METHODS The Clostridium perfringens type C (beta) toxin used for this experimental work was obtained from the re- search division Of a veterinary pharmaceutical company.* The toxin was sent air express in three shipments, Febru- ary 9, June 17, and July 20, 1961. It was packed in dry ice and arrived in a semi-frozen state. Upon arrival, it was allowed to liquefy at room temperature, and was then transferred to 1 cc. and 5 cc. vials, refrozen immediately, and stored at -5 F. until used. The toxin was kept frozen to avoid a loss in potency and when needed, it was lique- fied at room temperature and used promptly. Accompanying the toxin was the following descrip- tion on how it was Obtained:** "In my previous work in screening organisms to ob- tain this beta toxin, found one of the 'American type culture collection' #3626 Clostridiumgperfrin— gggg Type agni to give the best results in my hands. Media: A. Take 5 pounds fresh ground beef liver, cover *Haver-Lockhart Laboratories, Kansas City, Missouri, Dr. F. W. Binkley, research Clinician. Toxin was originally sent to Dr. G. R. Moore, Director of Large Animal Clinics, Michigan State University. **Quoted directly from Dr. F. W. Binkley's descrip- tion. 37 38 with distilled water; boil for thirty minutes and press. B. Heat broth and add 1% peptone (50 Gm.), 0.1% dibasic potassium phosphate (5 Gm.), 0.02% crystine (1.25 Gm.). C. Adjust to pH 8.2. D. Boil for 3 minutes and filter (coarse). E. Make up to 5,000 cc. with distilled water and fill into flasks (300 00.). F. Fill deep anaerobic tubes containing small quantity of pressed liver (use these for seed tubes). G. Sterilize for 45 minutes at 15 pounds pressure. The seed is carried in (F) above and three quick transfers are made--Monday, 8:00 A.M.; Tuesday, 8:00 A.M.; Tuesday, 4:00 P.M.--then inoculate flask (E) with one tube of (F) Wednesday 8:00 A.M. Harvest 2:00 P.M. (Wednesday) or 6 hours growth. The next step is to centrifuge to pull out cells and lower particulate, then filter through ST -3 Hercules filter pad to remove cells and leave pure filtrate of toxin. In looking up the habits Of this organism, you will find that the Beta toxin comes Off first, between 4 and 8 hours; between 8 and 12 hours, the Theta toxin comes off and later, the Epsilon toxin. The A toxin comes off in unknown quantity any- time. However, your toxin has proven to be al- most completely pure Beta. Beta toxin here should titer out to be between 800 and 1000 MLD/cc. for mice. Previous experience indicates that 1 cc. of this i.v. to a ewe will kill in 15 minutes if she had no protective antibodies." Prior to experimentation, the toxin was typed, using sera Obtained from Wellcome Research Laboratories, London, England. The sera, which are type specific, contain the antitoxins for neutralizing the toxins produced by the various species of Clostridium perfringens organisms. The 39 method Of typing was in accordance with directions accom- panying the toxin as follows: Three white mice (17-20 Gm.) were injected intravenously with 0.3 cc. of test fluid (toxin) and, if this proved lethal, mixtures were prepared using 0.9 cc. of test fluid and 0.3 cc. of the different type sera A, B, C, D, Bosworth. The mixtures were allowed to stand at room temperature for half an hour; then 0.3 cc. Of each mixture A, B, C, D, Bosworth and, as a control, 0.3 cc. of test fluid, was injected into white 17-20 Gm. mice. Four mice were used for each mixture. The results were read up to three days and the interpretation was based on a chart similar to the table on page 30. The minimal lethal dose (MLD) of each shipment of toxin was determined by intravenous inoculation into white 17-20 Gm. mice. After thawing at room temperature, the toxin was agitated and 0.1 cc. was pipetted into a 10 cc. volumetric flask which was then filled to volume with .85% sodium chloride solution (saline) to make a 1-100 dilution. Serial dilutions were made from the 1—100 dilution by taking 1 cc. and diluting with the proper amount of saline to make the desired dilution. A preliminary screening of the toxin was done at dilutions Of l-lOO, 1-200, 1-300, 1-400 on three white mice per dilution. After estimating the MLD, five dilutions were prepared-—one at, two above, and two below the estimated endpoint--each differing by a dilution factor Of 25. The diluted toxin was then injected intravenously 40 into 10 mice for each dilution. The results were read be- tween 5 minutes and 4 hours and the MLD 50 was figured for mice. Trypsinated toxin was prepared by mixing 0.1 cc. Of 0.25% trypsin solution (pH of 8 or higher) to 1 cc. of toxin. The mixture was incubated for one hour at 37 C. and injected. White mice* weighing between 17 and 20 grams were used for titrating the toxin, checking the sera and other procedures conducted in this research project. Before in- jection, the mice were placed in a pail and warmed with a 100 watt light bulb to cause engorgement in the coccygeal veins. Care was taken not to overheat the mice. Intravenous injections were made into the lateral coccygeal vein using a 0.5 cc. tuberculin syringe equipped with a 1 inch, 27 gauge needle. For restraint, they were placed in a holder made from a large centrifuge tube. Before injecting intra- venously, the tails were wiped with 50% alcohol. After injection, mice were placed in a clean, dry cage until the results were recorded. In all cases, results were taken between 5 minutes and 4 hours except in the case of typing, where the time lapse was up to 3 days. For this study, 15 clinically normal steer calves *Obtained from Rawley Farms, Plymouth, Michigan, and Spartan Research Animals, Okemos, Michigan. Seven hun- dred twenty-five mice were used in this problem. 41 were obtained from the Michigan State University dairy farm. Seven Of the calves were Holsteins, three Brown Swiss, three Jerseys, and two Guernseys. They varied in age from 1 week to 11 months and in weight from 60 to 345 pounds. All the calves were housed in the basement of the large animal clinic. Serum from each calf was checked for Clostridium perfringens antitoxin prior to use in this experimental work. Ten cc. samples of jugular vein blood were centri- fuged at 1350 rpm. for two minutes and the serum removed. Then 1 cc. of the toxin was diluted to contain 12 MLD/cc. One cc. of the diluted toxin (l2 MLD/ca) was incubated with 1 cc. of the serum at 37 C. for 1 hour. Following incuba- tion, 0.3 cc. of this mixture was injected intravenously into each of three mice. As a control, 1 cc. of 12 MLD/cc. toxin was incubated with 1 cc. of normal saline solution and 0.3 cc. of the resulting mixture was also injected into each of three mice. Death of the mice indicated that the calf did not have any protective antibodies at this level. If the mice lived, the calf was not used, since it presum- ably had antitoxin against the Clostridiumgperfringens toxin. After determining that the sera of the calves did not contain antitoxin and much preliminary work, six calves were given intravenous doses of toxin at the rate of 15,000 mouse MLD's per 100 pounds of body weight. Prior to injecting the Clostridium perfringens toxin into the calves, rectal temperatures, heart rates, respiratory 42 rates, blood and urine samples were taken for control data. Control urine samples were collected by stimulating the steer to urinate by massaging the prepuce with warm water. A plastic bag was then suspended under the prepucial orifice with adhesive tape to collect urine samples as they were voided. The urine was injected intravenously into mice in 0.1 cc. and 0.2 cc. quantities to determine the presence of a lethal factor. Samples of jugular vein blood were collected in vials containing ammonium-potassium oxalate crystals prior to and 0.5, 3, and 12 hours post injection. The blood was used for total and differential leukocyte counts and packed cell volume determinations (micro-hematocrit). All blood studies were done according to the Manual of Clinical Lab- oratory Methods.* To test for the presence Of toxin in the calves' blood, 10 cc. samples were drawn from the left jugular vein prior to and at 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, and 60 minutes post injection. Later in the experiment, ammonium- potassium oxalate was used as an anticoagulant to eliminate the formation of fibrin clots in the serum. The samples were immediately taken to the laboratory and centrifuged at 1350 rpm. for two minutes and the serum removed. The serum (0.2 cc.) was then injected intravenously into 17-20 *0. E. Hepler, Charles C. Thomas Publisher, Spring- field, Illinois. 43 Gm. white mice, three for each sample. This was done to determine how long an MLD of toxin for a mouse remained in the blood stream of the calf. The five goats used in this project were of the Toggenburg breed and varied in weight from 140 to 160 pounds. They were also housed in the basement of the large animal clinic. PART IV RESULTS AND DISCUSSION Typing of toxin: Three days post injection of the toxin incubated with the different types of sera, the following results were observed. TABLE I. Typing of toxinl' Inoculum used Mouse Inoculated l 2 3 4 Control Test fluid + saline + + + + A Test fluid + Sera A + + + + B Test fluid + Sera B O 0 + 0 C Test fluid + Sera C 0 0 0 O D Test fluid + Sera D + + + + "Bos— worth" Test fluid + Sera E + + + + mouse lived mouse died within 3 days . for procedure, see page 39 of Materials and Methods. 0.9 cc. of test fluid was combined with 0.3 cc. Of sera incubated and 0.3 cc. of this mixture was inoculated intravenously into the lateral coccygeal vein Of mice. FJ+<®O mm «P \N O 67 The appearance of a lethal factor in the urine near the two to three hour collection is quite significant as a possible route of toxin excretion. This lethal factor could be neutralized or reduced in potency by combining the urine with Clostridium perfringens antitoxin. No reduction in potency was noted when the urine was diluted with saline. It should be noted that antiserum protected the mice to some degree in the one case in which it was tried. There- fore, it appears that the toxin is excreted by the kidneys in a relatively unchanged form, two to three hours post in- jection. PART V SUMMARY AND CONCLUSIONS In preliminary work on this problem, it was deter- mined that Clostridium perfringens type C toxin would kill goats and mice but not calves. The routes of administra- tion tried in calves were intravenous, oral, and direct injection into the intestinal tract. However, if the toxin was combined with trypsin, death could be produced. The symptoms shown upon injection of toxin into calves were as follows: coughing, lacrimation, colic, urination, slight respiratory distress, temperature increase of one degree, heart and respiratory rate doubled, decrease in packed cell volume, increase in the total leukocyte count, and an inverse relationship between the percentage of neu- trOphils and lymphocytes. Blood samples drawn from the jugular vein of calves after injection of 15,000 mouse MLD/100 pounds, showed the toxin to be removed from the blood stream within twenty-five minutes of its injection. A substance lethal to mice appearing in urine samples collected two to three hours after toxin injection, suggests that the toxin is excreted in a relatively unchanged form. 68 10. 11. 12. BIBLIOGRAPHY Baldwin, E.: Clostridial Enterotoxemia. Vet. Med., 54. (1959): 123. Barner, R. D.: Hemorrhagic Enterotoxemia. Dept. of Surg. and Med., College of Vet. Med., M.S.U., E. Lansing, Mich. Class Handout. 1961. Beck, C. C., Ellis, D. 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Seven Products to Combat Clostridium perfringens. Haver-Lockhart Lab. "Messenger," Sept.-Oct., (1957). Use of Antibiotics Against Clostridium perfringens. Annual Review of Micro., 7, (1953): 236. MICHIGAN STATE UNIVERSITY LIBRARIES I III III III III IIIIIIIIIII 3 129 3178 3008 i I I | l