CLOSTRIDIA IN DIARRHEAL DISEASES OF ANIMALS

By E. M. Baldwin, Jr. Omaha, Neb.,

and L. A. Griner

SchooE of Veterinary Medicine, Colorado Agrictiltural and Mechanical College, Fort Collins, Colo.

Clostridium perfringens is the only known species of the genus Clostridium that can be directly implicated as the cause of diarrhea in animals. The term enterotoxemia is used to designate the diseases caused by the various types of CZ. perfringens. Type C enterotoxemia of infant calves and lambs and Type D enterotoxemia of sheep are the only disease entities recognized in the United States. They will be subject to detailed discussion under separate headings following the literature review.

Review of Literature

Diarrhea is frequently a symptom of these diseases.

Cl. perfringens has been divided into six types, A, B, C, D, E. and F, on the basis of the toxins produced. From these six types, 15 soluble, antigenic toxic fractions have been identified and are designated by letters of the Greek al- phabet. OakIey and Warrack‘ and Van Heyningen2 have indicated that each of the six types of C1. perfringens produce one or more of the antigenic fractions. Generally, one of the fractions is produced in quantity and is termed the major toxin, whereas other fractions are termed minor.

The toxins of C1. perfringem Type A, of which alpha is the major toxin, are responsible for the production of gas gangrene in man and, occasionally, in domestic animals. Macrae, Murray, and Grant3 have described an entero- toxemia in young suckling calves characterized by sudden onset, diarrhea, and early mortality. Rose and Graham: in 1936, described an enterotoxemia jaundice of sheep and calves that they attributed to Cl. perfringens Type A.

Cl. perfringens Type B produces beta and epsilon antigens as the major toxins and is responsible for lamb dysentery, a highly fatal disease occurring during the first four days of life. In Great Britain, Gaiger and Dalling (1921), who first attributed the disease to a combination of the colon Bacillus and a mem- ber of the Welch group, later concluded that only the Welch organism was the etiological factor. This they called the “L.D. Bacillus” and showed that it differed essentially from the classical BaciZZus zjelchii. Wilson (1931) called this organism Type B in his classification. Hepple6 described a necrotic en- terotoxemia in a calf that he attributed to Type B. No report could be found of the isolation of Type B in the United States.

The major toxic antigen of Type C is beta toxin, a lethal and necrotizing toxin. McEwen and Roberts,6 in 1931, described a disease of sheep from Romney Marsh in England that they called “struck.” McEwen isolated a member of the Welch group that was designated Bacillus paludis. Later, B.

From cultures of fecal material, they isolated Type A.

168

Baldwin & Griner: Clostridia in Diarrheas 169 paludis became Type C in the Wilsdon classification of Clostridium welchii. Struck is an enterotoxemialike disease of mature sheep that has not been re- ported in this county. In an earlier publication Griner and Bracken' described an acute hemorrhagic enteritis in young calves, and Griner and Johnsons re- ported a similar disease in infant lambs, both caused by Type C. Heller,g in 1933, gave an account of an acute hemorrhagic enteritis in young shed-born lambs in California. Filtrates of intestinal contents were lethal to mice, but the toxic constituent was not identified. A recent report by Fiedl and Gibson'O incriminated C1. perfringens Type C as the etiological agent of an enterotoxemia of young suckling pigs. The disease was characterized by lassitude, inappe- tence, weakness, and diarrhea, with death occurring approximately 12 hours after the onset of symptoms.

It is generally distributed throughout the world. The primary toxic fraction of Type D is epsilon toxin, a lethal and necrotizing toxin that needs to be acti- vated by trypsin and other proteolytic enzymes. The minor toxins of Type D are alpha and theta.

Bosworth" has described a strain of Cl. perfringens isolated from a calf show- ing symptoms of an acute enterotoxemia. This strain has been classified as Type E, and it produced alpha toxin and a new toxin that he designated as iota.

Cl. perfringens Type F is characterized by the ability of its spores to with- stand boiling for one to four hours. Alpha, beta, gamma, and nu toxins are produced by this type. In Europe, Type F is believed to produce enteritis necroticans,12 an acute toxemia fatal in man.

Cl. perfringens Type D is the cause of enterotoxemia in sheep and goats.

Hemorrhagic Enterotoxemia (Type C Enterotoxemia) Hemorrhagic enterotoxemia of calves and lambs has been ob-

served and confirmed in numerous areas in Colorado since 1951. Other states in which the disease has been confirmed in suckling calves are Montana, Ne- braska, and Wyoming.

Unconfirmed reports have been received from 10 states-California, Idaho, Illinois, Indiana, Iowa, Ohio, Oregon, South Dakota, Texas, and Utah. Type C toxins of Cl. perfringens have been found in newborn lambs in Colorado, Montana, and Wyoming.

It is believed that unfavorably cold, wet, windy weather a t calving time has some influence on the severity of outbreaks of the disease.

Species. Hemorrhagic enterotoxemia has been most frequently observed in beef breeds of cattle. In a few instances, however, it has occurred in dairy breeds. No record has been kept on the breeds of lambs in which the disease has occurred.

Field observations generally indicate that calves dying from hemorrhagic enterotoxemia are large, well-formed, and apparently vigor- ous animals. Stockmen who have experienced losses have confirmed these observations. They report that the disease most frequently attacks healthy, vigorous bull calves and that small, weak calves are rarely lost. It is generally agreed among cattlemen that calves dying of hemorrhagic enterotoxemia are

Distribution.

Symptomatology .

170 Annals New York Academy of Sciences from dams that produce an abundance of milk. There is no agreement that the disease occurs with greater frequency in calves from mature cows than in calves from heifers. Individual cows are reported to have lost their calves for two or three consecutive years.

Many stockmen are of the opinion that the incidence of the disease is greater than mortality figures indicate. They believe that calves develop subacute cases characterized by diarrhea from which they recover but remain unthrifty. This opinion is supported by serological studies in which the dams carried no antitoxin titer while their calves had titers ranging up to 16 units of Type C antitoxin. It is further coniirmed by the observation that in vaccinated herds the incidence of diarrhea and unthriftiness is markedly reduced below “normal.”

The symptoms displayed by individual calves vary with the degree of intoxi- cation. In many instances calves are found dead. Early in the disease, in- fected calves become listless and ceased nursing. These symptoms may be noticeable 12 to 24 hours prior to the onset of severe symptoms. Later they display evidence of acute colic such as uneasiness, straining, and kicking at the abdomen. Hemorrhagic diarrhea may or may not be observed, depending largely upon the severity and duration of illness. Calves become prostrate, develop opisthotonos, and tetanic and toxic spasms, followed by death. The course of clinical cases of the disease varies from 2 to 24 hours. Temperatures generally remain near normal but, terminally, become subnormal.

Few clinical symptoms other than early death are observed in lambs. Shiv- ering, bleating, and other evidence indicative of chilling or colic are observed. Few lambs show evidence of a hemorrhagic diarrhea. The lambs become weak, prostrated, and die.

Necropsy jwdings. Lesions appear to be consistently more severe in calves and are primarily hemorrhagic in character. The most marked pathological change is an extensive necrotic hemorrhagic enteritis of the jejunum and ileum. The lumen of the intestine contains considerable blood and necrotic mucous membrane. The intestinal contents are deep red, and portions of the large intestine may be hemorrhagic. Petechial or ecchymotic hemorrhages are con- sistently observed on the epicardium and thymus and, inconsistently, in the diaphragm, abomasum, and parietal pleura.

The lesions in lambs are spotted along the intestinal mucosa. Ecchymotic subserous hemorrhages may occur on the ansa spiralis, cecum, and small in- testine. The peritoneal cavity contains small quantities of a serosanguineous fluid. A mild fibrinous peritonitis covering the inflamed intestine may be ob- served. Serohemorrhagic lymphadenitis of the mesenteric lymph nodes is frequently found. Hemorrhages are occasionally observed in the thymus and diaphragm. The pericardium contains an excess of fluid, portions of which may be clotted. Moderate congestion and interstitial edema are observed in the lungs. The abomasum is frequently distended with large quantities of milk, only a poition of which may be coagulated. The mucous membrane of the abomasum is hyperemic and coated with a thick layer of tenacious mucus.

Histopathology. The most characteristic histopathological changes are ob- served in the large and small intestines. Sections of the small intestines of calves show extensive pathological changes, with the principal lesions occurring

Baldwin & Griner : Clostridia in Diarrheas 171

in the mucosa and submucosa. In some sections the necrotic process extends to the muscularis mucosa. Frequently the villi are completely destroyed, presumably by the necrotizing action of the toxin. The lumen of the intestine contains large quantities of erythrocytes, some of which are hemolyzed, frag- ments of necrotic villi, polymorphonuclear leukocytes, and many bacteria. Gram-positive bacilli, occurring singly or in short chains, are the dominant bacterial organisms. Varying degrees of hemorrhage, vascular congestion, edema, and distention of the lymphatics are noted in the muscularis and sub- serosa.

They vary from catarrhal enteritis and intense hyperemia of the mucosa to hemorrhage and necrosis of the mucosa and lamina propria. Histopathological changes in the abomasum are limited to catarrhal abomasitis, petechiasis of the mucosa, and edema of the submucosa. The mesenteric lymph nodes are edematous and hyperemic, the lymph spaces are filled with fluids and erythrocytes, and the lymph sinuses are distended. Subepicardial and focal myocardial hemorrhages may be noted in the heart. Liver sections are characterized by central congestion and cloudy swelling of the hepatic cords. Frequently the sinusoids are obliterated by the swollen parenchymal tissue. Considerable pigmentation of the cytoplasm may be observed. Microscopically, the kidneys show areas of hyperemia and hem- orrhage scattered throughout the parenchymal and stromal tissues. The epithelium of the convoluted tubules is swollen, and the cytoplasm shows con- siderable granulation.

Bacteriological and toxicoEogica1 findings. The dominant organism on direct smear of the intestinal content is a gram-positive, nonmotile, medium-sized bacillus, morphologically resembling CZ. perfringens. The organism, when incubated anaerobically on blood agar, produces a sharp hemolytic zone around each colony, a stormy fermentation in milk, and conforms to the typical carbo- hydrate reactions of Cl. perfringens.

Bacteria-free filtrates of intestinal contents from naturally occurring cases are lethal to mice when administered intravenously. Toxin present in intestinal filtrates can be neutralized by Types B and C anti- toxin. Such results indicate that beta toxin is present.

Strains of Cl. perfringens isolated from cases of hemorrhagic enterotoxemia in calves and lambs produce a lethal toxin when grown in modified Brewer’s medium as described by Vawter and Records.13 Filtrates contain a toxin which is neutralized by Types B and C antitoxin.

Symptoms are not frequently observed because of the rapid course of the disease. The principal findings are a history of sudden death and an acute hemorrhagic enteritis a t necropsy. Microscopic examination of a direct smear of the intestinal contents is helpful.

Laboratory diagnosis consists of the following procedure: The intestinal con- tents are diluted with equal parts of distilled water and filtered through a Seitz or comparable filter. The filtrate is then inoculated intravenously into Swiss mice weighing approximately 20 to 25 gm. The dosage is 0.2 ml. per mouse. If toxin is present, death usually occurs within 30 minutes, but may require 3 hours. Equal parts of toxic filtrate and antitoxin, diluted to contain approxi-

The intestinal lesions are less severe in lambs.

Bacteriology and toxicology.

Diagnosis.

172 Annals New York Academy of Sciences mately 200 beta units, are mixed and allowed to stand for one hour a t room temperature. Mice are inoculated intravenously with the neutralized mixture, using a dosage of 0.4 ml. per mouse. Control mice are inoculated as before with filtrate only. Death of the control mice and survival of the mice receiving the filtrate antitoxin mixture identifies the intestinal toxin to be the beta fraction of C1. perjringeizs Type C. If the toxin is not neutralized, further tests are necessary. As an alternative to filtration, the diluted intestinal contents may be centrifuged for one-half hour a t 3,000 rpm, and the supernatant used. In such an event it is not advisable to rely upon mouse deaths occurring after three hours.

If toxin is not detected in intestinal contents, cultural methods may be em- ployed in an effort to isolate C1. perfringens. Cultures are typed by the toxin- neutralization test.

Cl. perfringens Type C antitoxin may be used under field conditions as an aid in establishing a diagnosis. The therapeutic and prophylactic effectiveness of antitoxin has been demonstrated by Griner and Johnson8 and Griner and Ba1d~in.I~

Control. Immunological studies conducted by Griner and Baldwin14 demon- strated that the disease could be controlled by biological products. A whole- culture toxoid was effective prophylactically when administered to the preg- nant cow between the third and sixth month of gestation. The active immunity stimulated in the cow was passed via the colostrum to the offspring and provided the offspring with adequate passive immunity during the period of greatest danger. CZ. perjripzgens Type C antitoxin, derived from hyperim- munized horses, was demonstrated to be effective prophylactically when ad- ministered to the newborn shortly after birth. It was also effective therapeuti- cally when used in large doses and administered early in the course of the disease.

Ovine Enterotoxemia ( T y p e D Enterotoxemia) Distribution. This disease entity is also known as overeating disease, pulpy

kidney disease, braxylike disease, and apoplexylike disease. Type D entero- toxemia is widespread throughout the world’s sheep raising industry. Little is known of the disease in goats and, therefore, the following discussion will exclude this species.

It is generally agreed that the most aggressive and vigorous lambs are the most likely victims. The inciting cause is thought to be the ingestion of large quantities of highly nutri- tive feedstuffs, which results in an indigestion and a static condition of the in- testinal tract. Under such conditions i t is assumed that, should the animal in question be harboring a toxogenic strain of Cl. perfringens Type D, the or- ganisms are provided with a suitable environment and grow rapidly, producing toxin. Under laboratory conditions it is definitely known that toxin can be produced only if the selected media contains nutritives that induce rapid growth. Usually 500 mouse MLD per cc. are found in the contents of the intestinal tract in the region of the jejunum in lambs which have succumbed to

All ages of sheep are susceptible to enterotoxemia.

Baldwin & Griner: Clostridia in Diarrheas 173 enterotoxemia. Baldwin et ~ 1 . ' ~ demonstrated that lambs weighing approxi- mately 100 pounds were killed by the intravenous administration of 2,000 mouse MLD .

Suckling lambs two months of age or less are frequently victims of entero- toxemia. This is particularly the case in Australia and New Zealand, as well as in the United States. The incidence of the disease has been observed to be greater in single rather than twin lambs, presumably because of the more abun- dant supply of milk from the ewe. Active immunization of the ewe during pregnancy by means of Cl. perfringens Type D toxoid is successful in providing the suckling lamb with passive protection for a period up to two months. Antitoxin is readily transferred via the dam's colostrum and milk to the off- spring.

Enterotoxemia losses are common among pastured lambs, particularly dur- ing the spring of the year when the pasture is rich in feed value. Any sudden change from poor to rich pasture is apt to precipitate losses. The chronic form of enterotoxemia, characterized principally by a profuse diarrhea and inappe- tence, is quite commonly observed in outbreaks occurring in pastured animals.

Feeder lambs are subject to losses from enterotoxemia because of the desire of their owners to fatten the lambs as quickly as pxsible by feeding large quantities of grain concentrates. Losses in nonimmunized animals not in- frequently reach alarming proportions (5 to 20 per cent). Unfortunately the animal owner cannot predict which animals are more prone to succumb to this disease. For example, heavy losses may occur when one group of lambs is fed less than a pound of grain per day, whereas the next group may suffer no losses when fed twice that quantity. Prior to the use of biologics to control enterotoxemia losses, the best managed group of feeder lambs was expected to experience a loss of 3 to 5 per cent during a fattening period of three months. It is now common practice to immunize feeder lambs with a whole-culture toxoid, one injection of which provides adquate protection against losses. Approximately 3 million doses of this biologic are used annually. A specific antitoxin is also used widely to stop losses occurring in nonimmunized feeder lambs. Prior to the advent of effective immunizing agents in 1949, the only method of controlling an outbreak of enterotoxemia was to reduce drastically the intake of feed concentrates, thus increasing the length of time and cost necessary to produce a marketable fat animal.

The extent of such losses is not known in this country. Serological evidence indicates that subclinical enterotoxemia does occur and, logically, if this be the case, the older the animal the greater the likelihood of natural active immunity.

Acute enterotoxemia is characterized principally by a rapid progressive weakness accompanied by signs of central nervous system disturbance. Listlessness, incoordination, salivation, dyspnea, opisthotonus, convulsions, and coma are the outstanding symptoms. Death generally occurs within four to eight hours after the onset of symptoms. Occasional animals may die in less than one hour. Mortality of affected animals approaches 100 per cent. Specific antitoxin therapy is effective in approximately 50 per cent

Mature sheep occasionally are affected with enterotoxemia.

Symptomafology.

174 Annals New York Academy of Sciences of the cases if it is initiated early in the course of symptoms. Since death occurs rapidly there is little opportunity for diarrhea to occur in the average case. Animals that live 24 hours or more generally develop a profuse diarrhea.

Chronic enterotoxemia is characterized by a watery diarrhea and inappetence, as well as the sequelae of these symptoms. Nervous symptoms are infrequent. Occasional animals recover and can be demonstrated to contain circulating antitoxin.

Animals that die of the acute form of enterotoxemia will generally exhibit the following gross pathology: cardiac hemorrhages most frequently located subendocardially, congestion of the mesenteric blood vessels, petechial and ecchymotic hemorrhages on the thymus, congestion and swelling of the kidneys, congestion of the abomasum and small intestine, and large blotchy-type hemorrhages in the muscles, diaphragm, small intestine, perito- neum, and pleura. The kidneys degenerate into a pulpy mass a few hours after death, which characteristic gives rise to the name “pulpy kidney disease”. Not infrequently, only minor pathological changes are observed.

Chronic enterotoxemia is characterized principally by a mild to moderate catarrhal gastroenteritis involving the abomasum and small intestine. Other gross pathology is frequently absent except for secondary complications such as pneumonia.

A specific toxoid and antitoxin are available to control losses from enterotoxemia. Both have stood the trial of time and have been generally accepted by the sheep industry as being effective products.

ATecropsyjindi~zgs.

Control.

Discussion Cl. perfringeits Types C and D are the only recognized causes of specific dis-

ease entities in this country. The enterotoxemias caused by them are respon- sible for an undetermined but unquestionably large loss to the livestock in- dustry annually. Particular emphasis has been placed upon the acute form of these diseases, but what of the subacute and chronic enterotoxemia charac- terized principally by diarrhea? It is possible that loss from these less dramatic forms of enterotoxemia may also be of considerable economic importance. This is only one of many fields of investigation open for exploration.

The study of enterotoxemia as caused by CI. perfriqens and possibly other bacteria is in its infancy. There exists little doubt that knowledge gained in the future will reveal that this entity is of greater importance and of wider scope than heretofore recognized.

Summary The genus Clostridium contains only one organism directly associated with

diarrheal disease : Clostridium perfringem (welchii) . Cl. perfrimgem is classified into six types, each type producing a different

toxin pattern. The toxins are antigenically distinct and may be coinmon to more than one type. In the United States Types C and D have been incrimi- nated as the cause of distinct animal diseases associated with diarrhea.

Hemorrhagic enterotoxemia, a disease of suckling calves and lambs, is caused by the toxins of Cl. perfringens Type C . In this disease death may occur within

Baldwin & Griner: Clostridia in Diarrheas 175 2 to 24 hours after the onset of symptoms. The longer the course of the dis- ease the more likely the development of diarrhea, which is frequently hemor- rhagic. Chronic nonfatal cases characterized priiicipally by diarrhea probably occur according to field observations and serological evidence. Beta toxin, the major toxin of Cl. perfringens Type C can be readily demonstrated in the jejunem and ileum of animals dead of the disease. Beta toxin is necrotizing and lethal. The necrotizing effect of the toxin is evidenced by extensive severe necrosis of the intestinal mucosa.

Ovine enterotoxemia is a disease of sheep of all ages, caused by the toxins of Cl. perfringens Type D. The course of the acute form of the disease isusually four to eight hours and is characterized by severe nervous symptoms, prostra- tion, coma, and death. The chronic type is characterized principally by diar- rhea and inappetence. The major toxin of CZ. perfringens Type D is termed epsilon and it is necrotizing and lethal. The lesions i t produces in the small intestine are less intense than those due to beta toxin.

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2. VAN HEYNINGEN, W. E. 1950. Bacterial Toxins. Blackwell. Oxford, England. 3. MACRAE, D. R., E. G. MURRAY & J. G. GRANT. 1943. Enterotoxemia in young suckled

calves. Vet. Record. 66: 203-204. 4. ROSE, A. L. & E. GRAHAM. 1936. Enterotoxemia jaundice of sheep and cattle. Aus-

tralian Vet. J. 12: 212. 5. HEPPLE, J. R. Necrotic enterotoxemia in a calf due to Clostridium welchii type

B. Vet. Record. 64: 633-634. 6. MCEWEN, A. D. & R. S. ROBERTS. Struck: enteritis and peritonitis of sheep

caused by a bacterial toxin from the alimentary canal. J. Comp. Pathol. Therap.

7. GRINER, L. A. & F. K. BRACKEN. 1953. Clostridium perfringens (Type C) in acute hemorrhagic enteritis of calves. J. Am. Vet. Med. Assoc. 122: 99-102.

8. GRINER, L. A. & H. W. JOHNSON. 1954. Clostridium pwfringensType C in Hemor- rhagic enterotoxemia of Jambs. J. Am. Vet. Med. Assoc. 126: 125-127.

9. HELLER, H. H. The apparent cause of an infectious enteritis of very young lambs. J. Bacteriol. 25: 91-92.

10. FIEDL, H. I. & E. A. GIBSON. 1955. Studies on piglet mortality. 2. Clostridium wekhii infection. Vet. Record. 67:.

11. BOSWORTH, T. J. 1943. A new type of toxin produced by Clostridiztm welcizii. J. Comp. Pathol. Therap. 53: 245-255.

12. OAKLEY, C . L., H. E. Ross, G. P. RIGHT, M. G. MACFARLANE, W. S. GORDON & W. E. VAN HEYNINGEN. Discussion on clostridial toxins in relation to type spec- ificity for different species of host.

13. RECORDS, E. & L. R. VAWTER. Bacillary hemoglobinuria of cattle and sheep. Univ. Nevada Agr. Exptl. Stat. Bull. 173.

14. GRINER, L. A. & E. M. BALDWIN. Further work on hemorrhagic enterotoxemia of infant calves and lambs.

15. BALBWIN, E. M., L. D. FREDRICK & J. D. RAY. 1948. The control of ovine enterotox- emia by the use of CEostridizim perfringens Type D bacterin. Am. J. Vet. Research. 9: 296-303.

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