Diseases Caused by Clostridium perfringens Toxins (Yellow Lamb Disease, Lamb Dysentery, Necrotic Enteritis, Enterotoxemia)

Francisco A. Uzal • Bradford P. Smith

Definition and Etiology

C. perfringens is a gram positive, toxin-producing, anaerobic, spore-forming rod that causes a variety of diseases in human beings and animals.

In addition to these six typing toxins, individual strains of C. perfringens may produce any of several other nontyped toxins, which may or may not contribute to the virulence of this microorganism, such as perfringolysin O and beta-2-toxin.²

C. perfringens enterotoxin is mainly responsible for the second most prevalent type of food poisoning in humans in the United States and also for a less prevalent antibiotic-associated diarrhea; its role in animal disease remains poorly determined.² Because the full name of this toxin is C. perfringens enterotoxin, the latter should be avoided to refer to other clostridial toxins produced in the intestine.

The different toxinotypes of C. perfringens cause diseases that are almost exclusively mediated by their powerful toxin arsenal.² Several diseases in many animal species have been ascribed to C. perfringens, but the ubiquitous nature of the organism and the fact that it rapidly overgrows into tissues after death makes the significance of its isolation questionable. This is particularly true for type A, which is the toxinotype most commonly found in the intestine of clinically healthy animals and in the environment.³

One additional potentially confusing bit of nomenclature concerns the term enterotoxemia.

Diseases produced by the major types of C. perfringens in livestock are discussed in the following paragraphs. Type E is only occasionally isolated from livestock, and its role in animal disease has not been confirmed. Likewise, the newly defined types F and G are not considered to have a significant role in diseases of livestock.¹ These three types are therefore not discussed here.

General Considerations on Diagnosis of Disease Caused by Clostridium perfringens

Meaningful diagnosis of C. perfringens as the cause of death or disease in an animal requires an integrative, open-minded approach. Type A is routinely isolated from soil and intes­tine of clinically normal animals. Types C and D are only rarely isolated from soil but can occasionally be isolated from asymptomatic animals. The isolation of C. perfringens from a necropsy specimen is therefore not by itself sufficient basis for the diagnosis. However, if some of its toxin is also demonstrated in gut contents and the history and lesions are compatible, a diagnosis of death from C. perfringens intoxication can be made. This probably does not apply to alpha-toxin, as this toxin is produced by all types of C. perfringens and it is very frequently found in the intestine of clinically normal animals.³

Typing of C. perfringens is mostly achieved by a multiplex PCR technique that detects the genes for toxin production in a clinical isolate.^1,5 In addition, alpha-, beta-, and epsilon-toxins can be detected with commercially available assays.

Clostridium perfringens Type A (Yellow Lamb Disease)

C. perfringens type A can be found in many soils and is a normal inhabitant of the gut in many species. Although all seven types of C. perfringens produce alpha-toxin, some type A strains may produce more than other types. The toxin is a phospholipase and causes lysis of RBCs, platelets, and leukocytes (equine and caprine RBCs are resistant to the hemolyzing effects of the toxin).⁷

The only type A disease on which there seems to be general agreement among several authors is the so-called yellow lamb disease (so named because of the icteric nature of necropsy specimens from such lambs). This is an uncommon disease characterized by widespread hemolysis, which leads to anemia, weakness, hemoglobinuria, and icterus. Affected animals have a high temperature and usually die within 6 to 12 hours of onset. Differential diagnoses for yellow lamb disease include other causes of hemolytic disease, such as leptospirosis and copper toxicosis. As with other type A infections, the diagnosis is always questionable because of the commensal nature of the organism and its rapid invasion after death. The finding of predominantly large Gram-positive rods in impression smears from intestinal mucosa and high counts (>10⁶ colony-forming units/g) of C. perfringens type A, lend support to the diagnosis.⁷

Although type A isolates have been suggested to be impli­cated in enteritis, abomasitis and enterotoxemia of cattle,⁸ definitive proof of the role C.

Clostridium perfringens Type B (Lamb Dysentery)

DEFINITION AND ETIOLOGY. Lamb dysentery is a disease of young lambs that has been described in Britain, South Africa, and the Middle East. Type B disease has not been reported in the American continent. Its clinical course and presentation are similar to those of necrotic enteritis caused by C. perfringens type C.^4,10 C. perfringens type B has been postulated, although not proved, to be involved in the pathogenesis of multiple sclerosis in humans.^11,12

CLINICAL SIGNS AND DIFFERENTIAL DIAGNOSIS. Affected lambs younger than 1 week of age become depressed and die. A yellowish diarrhea becomes brown from blood as the disease progresses. Morbidity rates may be high, and the mortality rate approaches 100%. The disease must be differentiated from types C and D enterotoxemia in lambs.¹⁰

PATHOPHYSIOLOGY. The pathogenesis of type B infections is not fully understood. On the basis of experimental infections, it was postulated that intestinal lesions are produced by beta­toxin, whereas lesions in the brain are produced by epsilon- toxin.¹³ Because epsilon-toxin requires trypsin for activation, whereas beta-toxin is degraded very quickly in the presence of trypsin and other proteases, it is thought that, depending on which of these toxins exerts its major action in a given case, clinical signs and lesions could be different.¹³

Gross and Microscopic Findings. Necropsy findings include necrotizing enteritis and ulcers (rarely perforating) in the small intestines and dehydration of the carcass and tissues. Focal symmetrical encephalomalacia is occationally observed.¹⁰

DIAGNOSIS.

TREATMENT, PREVENTION, AND CONTROL. Sanitation and the use of type B vaccine aid in prevention and control. Types B, C, and D toxoids cross-protect because of the overlap in toxin types. Treatment is usually unsuccessful because of the acute nature of the disease.¹⁰

Clostridium perfringens Type C (Necrotic Enteritis, Neonatal Hemorrhagic Enterotoxemia, Struck)

DEFINITION AND ETIOLOGY. C. perfringens type C elaborates the alpha-toxins, common to all types, and the beta-toxins. Beta-toxin is the main virulence of C. perfringens type C, and it is required for type C isolates to produce disease. The amount of this toxin produced varies greatly among isolates and determines the virulence of a type C strain.¹⁴

Necrotic enteritis is primarily a disease of neonates and occurs mainly in calves, lambs, foals, and piglets. A similar disease in adult sheep, known as struck, has a very limited geographic range in Great Britain.^15,16

CLINICAL SIGNS AND DIFFERENTIAL DIAGNOSIS. Affected animals may die acutely without diarrhea, but this is rare. The diarrhea may be yellow or, in more hemorrhagic cases, brownish. Gray-red streaks of fibrin and blood which look much like necrotic mucosa may be present in the stools. Foals with type C disease at first show acute abdominal pain, then explosive yellow diarrhea that becomes brown and hemorrhagic. Affected animals become dehydrated, anemic, weak and moribund, despite intensive therapy.

PATHOPHYSIOLOGY. The causative beta-toxin is readily destroyed by proteolytic enzymes such as trypsin present in the intestine. Neonates are especially predisposed to beta-toxin attack by the trypsin inhibitor effect of the colostrum, the function of which is to prevent proteolytic degradation of immunoglobulins. The scattered cases of necrotic enteritis in adult animals may be the result of diets containing trypsin inhibitors such a soy bean (high content), beans, peas, peanuts, or sweet potatoes and others. Type C disease has been reproduced experimentally in lambs and goats by administra­tion of type C cultures with soybean flour or other trypsin inhibitors.¹⁸,¹⁴

In humans, type C infection is known as enteritis necroticans, darmbrand, or pigbel, and it occurs sporadically in several Southeast Asian countries and occasionally in other parts of the world. Enteritis necroticans was highly prevalent in the 1960s in Papua New Guinea, where most cases were observed in malnourished children thought to be trypsin deficient.^15,19,20,21

Ingestion of a protein-rich diet into a protease deficient intestinal tract allows rapid growth of C. perfringens organisms, which produce large amounts of beta-toxin that is not destroyed by proteases. Beta-toxin results in necrosis and invasion of deeper intestinal layers. Death results from the direct effects of the severe diarrhea or may be caused by secondary bacteremia or toxemia from the compromised gut barrier.¹⁶

EPIDEMIOLOGY. Neonates that ingest type C organisms during the first few days of colostrum feeding are at risk. They pick up the organism from an environment contaminated by a symptomatic or asymptomatic shedder, or from contaminated feed. Type C may be isolated from asymptomatic individuals on occasion but it is not usually considered a normal commensal as is type A. Once established on a premise, the disease may become endemic. In foals the disease is significantly associated with housing in a stall or dry-lot during the first few days of life, previous presence of livestock on the farm, low amounts of grass hay fed postpartum, being born on dirt, and having stock horse parentage (e.g., Quarter Horse, Paint).²²

GROSS AND MICROSCOPIC FINDINGS. Necrosis of the mucosa of the small intestine, especially the jejunum, is the consistent finding in all species. The large intestine may be similarly affected in horses, whereas in other species it may be normal except for intraluminal blood. The peritoneal cavity often contains excessive fluid, which clots when exposed to air because of a high content in protein. The mesenteric lymph nodes may be hemorrhagic. Microscopic study of affected gut shows necrosis and hemorrhage throughout the mucosa and submucosa, as well as mucosal and submucosal thrombosis. The tips of the necrotic villi and the surface of the colonic mucosa are covered with numerous large Gram-positive rods.^15,16

DIAGNOSIS. Although a presumptive diagnosis may be established on the basis of epidemiologic study and gross and microscopic pathologic study, coupled with isolation of C. perfringens type C from feces or intestinal content, confirmation of the diagnosis is based on demonstrating the beta-toxin in these specimens. Because this toxin is so sensitive to the action of proteases, failure to detect beta-toxin does not preclude a diagnosis of C. perfringens type C infection.^15,16

TREATMENT, PREVENTION, AND CONTROL. Once a case becomes clinically apparent, treatment generally is unsuccessful because of the fulminant nature of the disease. Foals can be treated with supportive intravenous broad-spectrum antibiotics (to cover Gram-negative and Gram-positive bacteremia caused by loss of gut integrity), oral metronidazole, fluids, plasma (intravenous and oral), withdrawal of milk for 24 hours, and intravenous C. perfringens type C antitoxin. Metronidazole (10 mg/kg PO or IV given twice daily) can be given to at-risk foals during an outbreak, beginning at 8 to 12 hours of age and continuing for 5 days. Toxoid has been administered to horses to control the disease on an apparently endemic property, with good results. Experimental oral treatment with trypsin has been used with apparent promising results. Vaccination is not routinely practiced, but it has proved to be effective in problem herds (J. Glenn Songer, personal communication). C. perfringens type C is a common component of multivalent vaccines. Because most cases of type C infection affect neonates, vaccination should be practiced on the dams. The primary vaccination series consists of two injections, approximately 1 month apart, with the final dose given roughly 2 weeks before parturition, and a yearly booster thereafter. Neonates should be vaccinated at 8 and 12 weeks of age on problem farms.⁴ Also see Chapter 48, Clostridium perfringens toxoids.

Clostridium perfringens Type D (Enterotoxemia, Overeating Disease, Pulpy Kidney Disease)

DEFINITION AND ETIOLOGY. Enterotoxemia caused by C. perfringens type D is a disease of major importance in sheep and goats, and of lesser importance in cattle. Ihis type is not a common soil organism, as is type A, but it may be isolated from the feces of a relatively small percentage of apparently normal sheep and goats, and less often, cattle. Most clinical disease occurs in animals fed a highly nutritious diet, especially grain-fed livestock. The disease occurs most frequently in animals older than 2 weeks, but rare cases have been seen in lambs as young as 1 day of age.^23,4

■ Clinical Signs and Epidemiology

Sheep. The sudden death of a well-fed, rapidly growing animal is the most common presentation of the acute form of entero toxemia. The disease may run its course in 30 to 90 minutes, with affected lambs showing ataxia, trembling, stiff limbs, opisthotonos, respiratory difficulty, convulsions, coma, and death. Diarrhea is rarely observed. At the onset of clinical signs, the animal may be hyperglycemic, and at death it may be glycosuric. The differential diagnoses should include other causes of neurologic signs such as listeriosis, enterotoxigenic E. coli infection, septicemia, polioencephalomalacia, acidosis, tetanus, and botulism, as well as causes of sudden death such as anthrax and black disease.⁴

Subacute and chronic cases may result in focal symmetric encephalomalacia (see Chapter 35) which causes affected lambs to be dull and unresponsive to normal environmental stimulation, and show respiratory difficulty. The major dif­ferential diagnosis is polioencephalomalacia.4

Morbidity rates are usually low, but the nature of the disease may be insidious with cases occurring over several days or weeks. Most affected animals die.⁴

Goats. The disease in goats may be acute, subacute or chronic; the acute form is very similar to the acute form described in sheep, whereas the chronic form is confined to the intestinal tract, mainly the colon, diarrhea being the main clinical sign. The subacute form of type D enterotoxemia in goats includes a combination of the neurologic and respiratory signs observed in the acute form of the disease and the diarrhea characteristic of the chronic form.⁴

As in sheep, morbidity rates are usually low, although outbreaks affecting a significant number of dairy goats have been reported. Lethality rates are also close to 100%.⁴

Cattle. Type D disease occurs rarely in cattle, and informa­tion about clinical signs and epidemiologic features in this species is scant. Experimental intravenous injection of type D epsilon-toxin and intraduodenal infusion of culture supernatant of C. perfringens type D in calves produced neurologic and respiratory signs similar to those described in the acute and subacute forms of the disease in sheep and goats.^24,25,26

PATHOPHYSIOLOGY. Some factor of overnutrition, often sudden access to heavy grain feeding or very rich pasture, provides substrate for rapid proliferation of the type D organism, which leads to elaboration of the epsilon-prototoxin. In vitro, production of epsilon-toxin by C. perfringens type D is enhanced in the absence of glucose. This seems similar to what occurs in vivo, when an animal is suddenly provided with excessive quantities of starch-rich food but adaptation of the ruminal flora is delayed. As a consequence of this, undigested starch reaches the intestine, and C. perfringens type D is likely to take advantage of it. The lack of digestion of starch would also be responsible for the absence of glucose in the small intestine, which enhances epsilon-toxin production.^27,4

Other factors that alter the intestinal environment (i.e., heavy parasite infestation) may also predispose sheep to type D enterotoxemia.^27,4 Although it has long been assumed that the same factors also predispose goats to type D enterotoxemia, little information is available in this regard, and cases of enterotoxemia have been reported in goats fed hay diets for several months.⁴

In the intestine, cleavage of the relatively inactive epsilon- prototoxin by proteases yields the fully active toxin. Epsilon­toxin thus increases intestinal permeability, facilitating its own absorption into the blood. Once in circulation, epsilon-toxin causes vascular damage with increased vascular permeability, which leads to edema in a variety of organs, notably the lungs and brain, and fluid accumulation in body cavities.^28,29 When affected animals survive 24 hours or more, the edema in the brain leads to parenchymal necrosis, the so-called focal symmetric encephalomalacia.3⁰ Epsilon-toxin crosses the blood-brain barrier and has also a direct effect on neurons and oligodendrocytes.²⁹

GROSS AND MICROSCOPIC FINDINGS

Sheep. In sheep that die of acute enterotoxemia, the carcass is usually in very good nutritional condition. Evidence of diarrhea around the rump may be observed, but this is rare. There is frequently an excess of yellowish pericardial, thoracic and abdominal fluid, which may contain fibrin strands; this fluid clots on exposure to air. Pulmonary congestion and edema and subserosal hemorrhages are evident. Usually there is no visible inflammation throughout the gastrointestinal tract, although the small and large intestine may be hyperemic and moderately distended with gas and liquid content. Softening of the renal parenchyma, so-called “pulpy kidney” may be present in a small number of cases, but it is of little, if any, diagnostic significance, inasmuch as it is probably consequence of accelerated autolysis and this change is never observed in freshly-dead carcasses.^27,3

In the subacute and chronic cases, the gross lesions are similar to those in the acute form, but herniation of the cerebel­lar vermis or focal symmetrical encephalomalacia, or both, may be seen in the brain.^27,3

Microscopic changes in the brain of sheep with type D enterotoxemia are observed in approximately 90% of acute and subacute cases and consist of intramural proteinaceous edema that is perivascular, vascular, or both. Microscopic lesions of focal symmetrical encephalomalacia can be observed in subacute and chronic cases.^27,3

Goats. Gross lesions in the acute form of the disease are similar to those in sheep, with the exception that brain lesions are very rare. In subacute cases, there is also mesocolonic edema and necrohemorrhagic colitis, which in some cases may extend to the small intestine. Chronic cases show colitis or enterocolitis with usually no involvement of organs outside 273

the gastrointestinal tract.²,³

Microscopic changes are usually not seen in acute cases, except for very rare occasions in which perivascular edema of the brain similar to that described for sheep is seen. In the subacute and chronic forms, the main microscopic lesions consist of fibrinonecrotizing colitis, and, less frequently, 273

enterocolitis.^27,3

Cattle. Type D enterotoxemia occurs very rarely in cattle, and the few cases documented share most clinical and pathologi­cal characteristics with the disease in sheep.^27,3

DIAGNOSIS. In sheep, a presumptive diagnosis of type D enterotoxemia can be based on clinical history, including sudden access to a diet high in carbohydrates, clinical signs, and gross postmortem lesions. Hyperglycemia and glycosuria can be diagnostically useful when present, although this is neither pathognomonic nor consistent. In freshly dead animals, large numbers of Gram-positive rods found in smears of intestinal mucosa support a diagnosis of enterotoxemia. Isolation of C. perfringens type D from small or large intestinal content is also supportive of this diagnosis; however, because this microorgan­ism can be found in a relatively small percentage of clinically normal animals, isolation alone is not diagnostic.Confirmation of the diagnosis should be based on one or more of the fol­lowing: (1) perivascular or mural vascular edema in the brain; (2) focal symmetrical encephalomalacia; and (3) demonstration of epsilon-toxin in intestinal content.

In goats with acute and subacutely disease, the same criteria mentioned for sheep apply, although perivascular edema in the brain is rarely seen. Necrotizing colitis and, less frequently, enterocolitis seen in some acute and subacute cases supports but does not confirm a diagnosis. Confirmation of any of the forms of the disease should be based on detection of epsilon­toxin in intestinal content. In the rare cases in which perivascular edema is seen in the brain, this is considered a diagnostic finding. The diagnostic criteria for cattle have not been established, although it is usually assumed that they are probably similar to those reported for sheep.^27,3

TREATMENT, PREVENTION, AND CONTROL. If initiated at the first suspicion of overeating disease, type D antitoxin and oral antibiotics (sulfa) may have dramatic results.Vaccination is also recommended in an outbreak, but owners should be cautioned that antibody titers take between 12 and 14 days to appear, so deaths are expected to continue occurring for a while. The use of antitoxin provides passive immunity while active immunity from vaccination has time to occur.

The diet should be adjusted downward (more grass hay, less grain and legumes) in outbreaks to try to minimize the substrate, especially starch, that reaches the bacteria. Lambs on rich pasture should be moved to poorer pasture or corralled and fed hay until several days after they have been vaccinated twice.

Sheep and goats brought into a feedlot should be vaccinated before arrival, and should have the concentrate ration increased slowly to minimize microfloral disruptions.

Vaccination with type D toxoid is effective in preventing disease. Two doses should be given 4 to 6 weeks apart, to lambs and kids of approximately 2 to 3 months of age, which is when colostral antibody titers fall below protective levels. This should be followed with an annual booster to sheep and revaccination of goats every 3 to 4 months. The annual booster is recom­mended a week or two before heavy grain feeding or exposure to rich pasture begins or, in dams, 2 to 3 weeks before delivery. The aluminum hydroxide adjuvanted vaccines may cause raised subcutaneous lumps (most noticeable on goats) that may go on to abscess.²,³