Clostridial diseases of small ruminants J. Glenn Songer

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J. Glenn Songer. Clostridial diseases of small ruminants. Veterinary Research, BioMed Central, 1998, 29 (3-4), pp.219-232. ￿hal-00902526￿

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Clostridial diseases of small ruminants

J. Glenn Songer

Department of Veterinary Science, University of Arizona, Tucson, AZ 85721, USA

(Received 20 October 1997; accepted 29 January 1998)

Abstract - Members of the genus Clo.stridium are extraordinarily diverse in their natural habi- tats, and, when introduced to animal hosts, a few produce acute and often fatal disease. In sheep and goats, as in many other species of domestic animals, pathogenesis is often mediated by one or more of the many toxic proteins produced by these organisms. Prevention and control strate- gies are frequently based upon amelioration, by immunoprophylaxis, of the effects of these molecules. In spite of their recognition for many years, clostridial diseases still present challenges to veterinary practitioners, diagnosticians and animal producers worldwide. © Inra/Elsevier, Pariss myonecrosis / enteritis / enterotoxemia / neuromuscular disease / bacterial toxins

Résumé - Clostridioses chez les petits ruminants. Les membres du genre Clostridium sont extraordinairement divers dans leur habitat naturel, et, lorsqu’ils ont pénétré chez leur hôte, cer- tains provoquent une maladie aiguë et souvent mortelle. Chez les moutons et les chèvres, comme dans beaucoup d’autres espèces animales domestiques, une ou plusieurs des nombreuses protéines toxiques produites par ces microorganismes est souvent à l’origine de la pathogenèse. La prévention et les stratégies de contrôle sont fréquemment basées sur l’amélioration, par immunoprophy- laxie, des effets de ces molécules. Malgré leur reconnaissance depuis de nombreuses années, les clostridioses présentent toujours des défis aux praticiens vétérinaires, aux personnes qui dia- gnostiquent les maladies, et aux producteurs, dans le monde entier. © Inra/Elsevier, Paris myonécrose / entérite / entérotoxémie / maladie neuromusculaire / toxine bactérienne

Tel.: (1) 520 621 2962; fax: (1) 520 621 63.66; e-mail: [email protected] 1. INTRODUCTION Hatheway, 1990; Rood and Cole, 1991), but a definitive role in pathogenesis has Clostridia are widely recognized as been demonstrated for only a few. The pathogens of humans, domestic animals species is divided into types on the basis of and wildlife (tables I and ll). The ready production of the four major toxins, a, (3, availability of inexpensive, efficacious r and t (table lll), as determined by in vivo immunoprophylactic products has not protection tests in guinea pigs or mice eliminated clostridial infections. Proven (Walker, 1990). a toxin is hemolytic, and putative virulence attributes mediate necrotizing and potently lethal (Rood and the pathogenesis of many types of infec- Cole, 1991), causing cytotoxicity through tions in myriad hosts. This review of hydrolysis of sphingomyelin and other clostridial disease in small ruminants will membrane phospholipids (Elder and cover muscle and soft tissue infections, Miles, 1957; Smith, 1979). Genes with intoxications and toxicoinfections, and significant sequence homology to cpa, the enteric infections. Other reviews (Smith, a toxin gene (Titball et al., 1989), can be 1979; McDonel, 1980) provide a broader found in other clostridia (Titball et al., context. 1993b).). Mucosal necrosis and, possibly, cen- tral nervous system (CNS) lesions are 2. MUSCLE AND SOFT TISSUE et caused by (3 toxin (Jolivet-Reynaud al.,., INFECTIONS 1986). The (3 toxin gene (cpb) has signif- icant sequence homology with a toxin, y toxin and leukocidin of Staphylococcus 2.1. Clostridium perfringens aureus (Hunter et al., 1993). The mouse minimum lethal dose (MLD) is about Clostridium perf’ringen.s is the most 500 ng per kg IV (Sakurai and Fujii, important cause of clostridial disease in 1987). Proteolysis of s prototoxin domestic animals (table 11). As many as (McDonel, 1986) converts it to toxin, 17 exotoxins of C. perlringens are resulting in a > 1 000-fold increase in tox- described in the literature (McDonel, 1986; icity (Bhown and Habeeb, 1977). ! £toxin is necrotizing (Buxton, 1978), and the five toxigenic types (Cole, 1995; Songer mouse MLD is about 300 ng (Sakurai and and Meer, 1996; Meer and Songer, 1997). 1987). Ia of t toxin Fujii, Component Direct proof (i.e. in vivo studies with ADP-ribosylates globular skeletal muscle mutants) of a role in and nonmuscle actin (Stiles and Wilkins, isogenic pathogene- sis is lacking for all toxins of C. perfrin- 1986a, b; Vanderkerckhove et al., 1987); gens, with the of a and 0 toxins to sensitive cells and to the exception binding entry in histotoxic infections et is mediated Ib and (Awad a]., cytosol by (Stiles 1995). Results of studies of the direct Wilkins, 1986b; Considine and Simpson, effects in vivo of purified toxins, or of 1991; Rood and Cole, 1991). Physiologic studies, are com- effects include increased vascular vaccination/challenge perme- and there is little that the dermonecrosis and pelling, question ability, lethality other major, and probably minor, toxins (Bosworth, 1943; Craig and Miles, 1961).). are important in pathogenesis. 1 toxin is similar in structure and activity to C. spiroforme toxin and C2 toxin of C. Strains of type A are important causes botulinum types C and D (Perelle et al., of wound contamination, anaerobic cel- 1993). It is antigenically similar to an ADP lulitis and gas gangrene (Hatheway, 1990), ribosyltransferase produced by C. di!cile and a toxin plays a central role in patho- (Popoff et a]., 1988). genesis (Awad et al., 1995). Antibodies against native a toxin and against a genet- While not considered a major toxin in ically truncated C-terminal portion of the the classical sense, enterotoxin (CPE) is molecule (amino acids 247-370) protect often an important virulence attribute of mice against challenge with toxin or mul- C. McClane of C. et perfringens (McDonel, 1986; tiple LD50 perfjingens (Titball al.,., et al., 1988; Granum and Stewart, 1993). 1993a; Williamson and Titball, 1993). CPE production and sporulation are coreg- Type A, as well as other types of C. ulated, and toxin is released when the veg- cause enteric disease in etative cell is lysed. Proteolytic cleavage of perfringens, sheep, and are discussed below. CPE is followed by binding (via a C- terminal domain) and cytotoxicity (via a The key diagnostic components are N-terminal domain). Pore formation evaluation of clinical signs and lesions results in altered permeability, inhibition and bacteriologic culture; detection of tox- of macromolecular synthesis, cytoskele- ins is also important, but is rarely prac- tal disintegration and lysis (Hulkower et tised in many parts of the world (Walker, al., 1989; McClane and Wnek, 1990). CPE 1990; Carter and Chengappa, 1991). Cyto- and its gene (cpe) can occur in strains of all toxicity assays (Berry et al., 1988; Mahony et al., 1989) and immunoassays (McCiane wound, including those incurred through and Strouse, 1984; Harmon and Kautter, castration or docking. Umbilical infec- 1986; McClane and Snyder, 1987; Berry et tions are not uncommon in sheep (Timo- al., 1988; Cudjoe et al., 1991) for CPE ney et al., 1988). have been reported, as have immunoelec- Hemorrhage, edema and necrosis (Henderson, 1984), latex trophoresis develop rapidly as the infection spreads agglutination (Martin and Naylor, 1994), along muscular fascia] planes. Early immunodiffusion (Beh and 1978), Buttery, lesions are initially painful and warm, with and ELISA (Naylor et al., 1987; Martin pitting edema, but with time, the tissue et al., 1988; El-ldrissi and Ward, 1992; becomes crepitant and cold. Death fol- Holdsworth and Parratt, 1994) for entero- lows, often in less than 24 h. toxemia-associated toxins. Toxin detec- tion alone does not confirm the existence Toxic or potentially toxic products of C. of disease and failure to demonstrate tox- septicum include a toxin [oxygen-stable ins (particularly f3 toxin in gut contents), hemolysin (Ballard et al., 1992)], (3 toxin toxin can be expected owing to protease degra- (DNase, leukocidin), y dation. [hyaluronidase (Princewill and Oakley, 1976)!, 8 toxin (oxygen-labile hemolysin), Gene and PCR have been probes assays a neuraminidase and hemagglutinin reported (Havard et al., 1992; Saito et al., (Gadalla and Collee, 1968), a chitinase 1992; Fach and Guillou, 1993; Daube et (Clarke and Tracey, 1956) and sialidase al., 1994; Kokai-Kun et al., 1994; Songer (Zenz et al., 1993). Unambiguous state- and Meer and Meer, 1996; Songer, 1997). ments about a role in pathogenesis can be In one study of more than 750 strains from made only for a toxin. Purified a toxin is bovine all with enterotoxemia, hybridized a cationic protein of about 48 kDa, which for a toxin and sialidase probes genes, is activated by proteolytic removal of a 4- most with a probe for the 0 toxin gene, a kDa carboxy-terminal fragment (Ballard et few with the probe for cpe, and none with al., 1993). The MLD is about 10 0 pg per probes for cpb, the E toxin gene (etx) and kg (Ballard et al., 1993) and death fol- the t toxin genes (iap and ibp) (Daube et lowing C..septicum challenge is delayed in PCR derived from the al., 1994). primers a toxin-immunized mice. Although a role sequences of cpa, cpb, etx, iap and ibp for potential virulence attributes other than have been successfully used to amplify a toxin has not been proven (Hatheway, toxin and genes (Fach et al., 1993; Songer 1990), it seems likely that, in combina- Meer, 1996; Meer and Songer, 1997). tion, they increase capillary permeability and cause myonecrosis and systemic tox- icity (Riddell et al., I 993). 2.2. Clostridium septicum The brief clinical course dictates a pref- erence for prevention rather than treat- Clo,stridium septicum is commonly ment. Antibody responses to somatic and found in soil and in the feces of domestic toxin antigens (Hjerpe, 1990; Gyles, 1993) animals (Princewill, 1985). Flukes can yield lifelong immunity (Green et al., carry spores into the livers of sheep 1987). Diagnosis of malignant edema is (Petrov et al., 1985), and iatrogenic infec- based upon clinical signs, gross and micro- tions occur (Harwood, 1984; Mullaney et scopic findings at post mortem, Gram- al., 1984), most commonly in horses. stains of direct smears and bacteriologic Wound infections by C. septicum are often culture (Carter, 1984). C. chauvoei infec- called malignant edema, and usually fol- tion should be ruled out (Carter and Chen- low direct contamination of a traumatic gappa, 1991) by use of a rapid method such as a fluorescent antibody test (Batty by fluke migration, and dissemination of a and Walker, 1963). toxin yields systemic effects with acute or peracute death (Elder and Miles, 1957). Its cardio-, neuro- histo- and hepatotoxic 2.3. Clostridium chauvoei effects produce edema, serosal effusion and focal hepatic necrosis (Elder and Aikat and CLostridiurn chauvoei causes blackleg, Miles, 1957; Dible, 1960; an emphysematous, necrotizing myositis Cotran, 1967; Rutter and Collee, 1967). (table I), which, in sheep, most often The name ’black disease’ derives from the characteristic of the underside of resembles malignant edema or gas gan- darkening grene. Affected animals may develop high the skin due to venous congestion. Roles fever, anorexia, depression and lameness, of other toxins, including f3 (lecithinase), with crepitant lesions, but sudden death y (necrotizing phospholipase D), 6 (oxy- is common. The central areas of lesions gen-labile hemolysin) and (lipase), are are dry and emphysematous, while the uncertain. C. uovyi type D, often referred periphery is often edematous, hemorrhagic to as C. haemolyticurn, causes bacillary and necrotic. Evidence of leukocytic infil- hemoglobinuria of cattle (Smith and tration is negligible (Timoney et al., 1988; Williams, 1984). The roles of a which Gyles, 1993). toxin, Typically, there is no effective treat- is and and necrotizing, hemolytic lethal, ment for C. uovyi infections, but effective a DNase which be (3 toxin, may responsi- prophylaxis with bacterinaoxoids or tox- ble for of muscle cell nuclei degeneration oids can be achieved (Timoney et al., (Ramachandran, 1969), have not been pre- 1988). cisely defined. Protection follows vaccination, and apparently arises from the immune 3. ENTERIC DISEASES response to a heat-labile, soluble antigen (Verpoort et al., 1966). Equine hyperim- mune serum and penicillin can be used for 3.1. Clostridium perfringens therapy and prophylaxis.

Clastridiurre perfringens type A causes enterotoxemia, or yellow lamb disease, 2.4. Clostridium novyi which occurs primarily in the western US (McGowan et al., 1958). Depression, ane- Clostridium novyi type C is nontoxi- mia, icterus and hemoglobinuria, are fol- and avirulent, but strains of A genic type lowed by death after a clinical course of cause in humans and wound gas gangrene 6-12 h, and large numbers of C. perfrin- infections in animals, and the hallmark gen.s are found in intestinal contents. A lesion is edema. with ’Bighead’ rapidly similar condition occurs in goats (Russell, spreading edema of the head, neck and 1970), and type A probably also causes cranial thorax, occurs in rams fol- young tympany, sometimes accompanied invasion C. A by lowing by novyi type of sub- hemorrhagic, necrotic abomasitis in calves. cutaneous tissues damaged by fighting bacilli are demonstrable on and Gram-positive (Sterne Batty, 1975). the mucosa and in the submucosa and a Infectious necrotic hepatitis (’black dis- toxin is found in intestinal contents ease’) of sheep and cattle is the result of C. (Roeder et al., 1988). Intravascular hemol- novyi type B infection. Dormant spores ysis, capillary endothelial damage, platelet germinate in liver tissue, often damaged aggregation, shock and cardiac effects in natural infections are predictable systemic gest toxemia, and enteric lesions, dysen- actions of a hemolytic toxin (Stevens et tery and diarrhea are often absent (Sterne al., 1988; Timoney et a]., 1988). Chy- and Thomson, 1963). Similarities of cpb, motrypsin resistance of a toxin from the (3 toxin gene, to the genes for staphy- enterotoxemia isolates may allow accu- lococcal a and y toxins and leukocidin mulation in the gut and entry to circula- (Hunter et al., 1993), strengthen sugges- tion (Ginter et al., 1995). tions that (3 toxin may affect the CNS et al., 1986; McDonel, C. B is iso- (Jolivet-Reynaud perfrihgens type frequently 1986). However, hemorrhagic enterotox- lated from cases of in newborn dysentery emia has not been reproduced in lambs by lambs II) and enteritis (table hemorrhagic inoculation with cell-free culture super- in Disease is more goats (Frank, 1956). natant fluid (Niilo, 1986). common in the UK, South Africa and the Middle East than in the US (Timoney et Enterotoxemia (’overeating’) in sheep al., 1988). In lambs, inappetence, abdom- of all ages except newborns is caused by inal pain and bloody diarrhea are followed C. perfrivgetes type D (table II) (Timoney by recumbency and coma. Lesions con- et al., 1988). Lambs 3-10 weeks old, suck- sist primarily of hemorrhagic enteritis, ling heavily lactating ewes, are commonly with evidence of enterotoxemia (Frank, affected, as are feedlot animals up to 100 1956). Chronic disease in older lambs months of age. Upsets in the gut flora, fol- (’pine’) is characterized by chronic lowing sudden changes to a rich diet, con- abdominal pain without diarrhea. Patho- tinuous feeding of concentrates (Popoff, genesis of type B infections may be due to 1984), and the presence of excess dietary additive or synergistic effects of a, p and starch in the small intestine are often c toxins. involved. e toxin facilitates its own absorp- tion (Niilo, 1993), resulting in toxemia Neonates of most are species highly with little or no enteritis. Some animals to infection C. susceptible by perfringen.s dullness, retraction of the head, C and display type (MacKinnon, 1989) (table ll), and convulsions (Niilo, 1993; colonization in advance of normal intesti- opisthotonus Popoff, 1984), but sudden death is com- nal flora or alteration of flora by dietary mon. Degeneration and necrosis in the are factors in changes significant patho- CNS is typical (Buxton and Morgan, et al., 1988). In lambs, genesis (Timoney 1976), and focal encephalomalacia is a type C infection resembles lamb dysen- chronic neurological manifestation of non- and be nervous tery, may accompanied by fatal disease (Griner, 1961; Buxton and and signs, including tetany opisthotonus. Morgan, 1976). The extent of incoordina- Peracute death, without other occasionally tion and convulsions is directly related to clinical is not but the signs, uncommon, the severity of lesions (Griner, 1961). Peri- clinical course may also extend to several toneal and pericardial effusions are typical ewes and other adult days. Young sheep in sheep, and glycosuria is pathognomonic can also C enterotoxemia, a develop type (Gardner, 1973; Niilo, 1993). The com- condition known as ’struck’, in which the mon name ’pulpy kidney’ derives from clinical disease occurs so that it rapidly the post mortem of hyperemic, often that the animal has been autolysis suggests toxin-damaged tissue. struck by lightning. Mucosal damage, per- haps caused by poor quality feed, facili- Goats develop catarrhal, fibrinous, or tates abomasal and small intestinal mul- hemorrhagic enterocolitis. The condition tiplication of organisms, with resulting is often chronic, and pulpy kidney is mucosal necrosis. Fluid accumulation in absent (von Rotz et al., 1984; Blackwell the peritoneum and thoracic cavity sug- and Butler, 1992). C. perfringens type E is an apparently 3.2. Clostridium septicum uncommon cause of enterotoxemia of lambs (table II), and recent isolates have Clostridium septicum also causes been obtained from calves with hemor- enteric infections (Schamber et al., 1986) rhagic enteritis, in the western and mid- (table I). The organism penetrates the lin- western US (Meer and Songer, 1997). ing of the abomasum of sheep, producing However, type E remains of uncertain braxy, a fatal bacteremia (Saunders, 1986). overall importance in animal disease. Mortality rates are high in yearling sheep in the UK, Norway and Iceland, and cases have been in Australia An increasing body of evidence sug- reported Europe, et and the US. The gests a role for enterotoxigenic strains, (Ellis al., 1983) patho- particularly of type A, in the etiology of genesis of C. septicum-infection is not diarrheal conditions in several animal well understood, but impaired mucosal species (Estrada-Correa and Taylor, 1989; function may follow ingestion of frozen feed. The then and Niilo, 1993). In one study, CPE production organism multiplies was observed in 12 % of isolates from cat- disseminates, producing local lesions and tle, sheep and chickens with enteritis toxemia (Saunders, 1986; Schamber et al., (Niilo, 1978), and in another, genotyping 1986). Edema, hemorrhage, and necrosis revealed that about 5 % of isolates are occur in the abomasum and proximal small enterotoxigenic, with most of these being intestine (Ellis et al., 1983). The patho- type A (Songer and Meer, 1996; Meer and genesis is not well-understood, but a toxin Songer, 1997). is probably of primary importance.

CPE is when weakly immunogenic 4. NEUROTOXIC DISEASES administered via the intestinal tract. Dis- ease gives rise to serum antibodies in sheep and other domestic species, but anti- bodies produced following parenteral inoc- 4.1. Clostridium botulinum ulation are not protective (Niilo and Cho, 1985; Estrada-Correa and Taylor, 1989). Botulism, caused by C. botulinum, is The best target for immunoprophylaxis an intoxication with any of seven neuro- may be the toxin’s membrane binding toxins, which results in neuroparalysis event (Hanna et al., 1989; Mietzner et al., (Smith and Sugiyama, 1988; Rocke, 1993) 1992). (table I). Botulinum toxins share the abil- ity to block acetylcholine release from cholinergic nerve endings (Simpson, Immunoprophylaxis is a control mea- 1981), but are serologically distinct. sure of paramount importance, due to the C2 toxin is not neurotoxic, but has ADP-ribo- rapid and frequently fatal course of dis- sylating activity similar to t, toxins of C. ease caused by the various types of C. per- perfringens and C. spiroforme (Ohishi fringens. Lambs born to ewes vaccinated 1983; Simpson, 1989). against types B, C or D are protected against dysentery (Smith and Matsuoka, Singular names, including loin disease 1959; Kennedy et al., 1977; Odendaal et and lamziekte (cattle), limberneck and al., 1989), and may be immunized at 3 western duck sickness (waterfowl), and days of age (Kennedy et al., 1977). Ente- spinal typhus and shaker foal syndrome rocolitis, but not toxemia, may occur in (horses), have been applied to the various vaccinated goats (Blackwell et al., 1991; conditions affecting animals. The disease Blackwell and Butler, 1992). in sheep can arise from many sources (Smith and Sugiyama, 1988). Phosphorus case fatality rate of at least 50 % (Timoney deficiency may encourage pica, leading et al., 1988). to of botulinum toxin-con- consumption Strains of C. tetani which do not pro- carcasses, and death due to taining duce tetanus toxin are avirulent, and botulism. Clinical signs include anorexia, widespread vaccination with toxoid has ataxia, in swal- incoordination, difficulty dramatically lessened the impact of tetanus and excessive salivation. Flaccid lowing on animal Passive immunity the production. paralysis, affecting respiratory system, acquired from the dam protects for 2-3 causes death of the animal. eventually months. Attention to apparent wounds, and administration of penicillin to halt Dogma states that enough toxin to production of toxin and antitoxin to neu- immunize is more than enough to kill tralize preformed toxin, are useful thera- (Timoney et al., 1988; Rocke, 1993), but toxoids of botulinum toxin are used for peutics. immunoprophylaxis (Jansen et al., 1976; The past decade has brought many Johnston and Whitlock, 1987; Smith and advances in the understanding of the Sugiyama, 1988). Polyvalent antitoxins nature and mechanism of action of tetanus can be effective for therapy. and botulinum neurotoxins. These molecules are two-chain polypeptides of 150 000. Blockade of neurotransmitter Mr release in the CNS (glycine and GABA 4.2. Clostridium tetani by tetanus toxin and acetylcholine by botulinum toxins) causes spastic (in Tetanus, caused by C. tetani, usually tetanus) or flaccid (in botulism) paralysis. to on nerve ter- follows contamination of a wound or the Binding specific receptors minals is followed internalization of umbilicus by soil, but nonaseptic surgery, by toxin into neurons et al., 1996). docking and castrating, and ear tagging (Poulain The chain of the toxin molecule, may also be initiating factors. The wound light which has may be trivial, but necrosis is usually zinc-dependent endopeptidase Paiva et al., 1993; Li et al., required to provide conditions of lowered activity (de is translocated from the endosomal oxygen and allow germination of spores. 1994), to the where it The incubation period varies with the tox- compartment cytosol, attacks one of three inogenicity of the strain, rate of transfer specifically synaptic of toxin to the target tissues, and the rela- proteins (VAMP/synaptobrevin, syntaxin, or which mediate tive susceptibility of the host, and may SNAP-25) exocytosis of neurotransmitters et al., 1997; range from 24 h to 2 weeks (Kryzha- (Comille novsky, 1981; Wellh6ner, 1982). Toxin Galli et al., 1994). A second, recently is transported retrograde, moving intraax- reported, inhibitory mechanism does not involve but result from onally via the peripheral motor nerve end- proteolysis, may activation of neuronal ings. It binds to presynaptic axonal termi- transglutaminases nals, resulting in hyperactivity of motor (Deloye et al., 1997; Poulain, 1994). neurons. Clinical signs include muscular tremor and increased stimulus response, impaired muscle function in the head and 5. CONCLUSION neck, and difficulty in chewing and swal- lowing due to trismus. Spasms give way to Control by vaccination has decreased permanent rigidity, respiration becomes the incidence, and perhaps also the visi- increasingly difficult, and death follows bility, of clostridial diseases in domestic in a few days to less than 2 weeks, with a animals. This seems particularly true of enteric diseases. Renewed interest in providing a better sense of the importance mechanisms of pathogenesis has yielded of various clostridial diseases. new information about clostridia, and par- ticularly in the mode of action of their tox- ins. 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