Botulism

Botulism

N P J KRIEK AND M W ODENDAAL

Introduction

Botulism or ‘lamsiekte’ (literally, paralysis disease) in livestock is a non-febrile, highly fatal intoxication of cattle, sheep, goats, horses, mules, donkeys and, very rarely, pigs. It is characterized by partial or complete flaccid paralysis of the muscles of locomotion, mastication and deglutition. Botulism is an intoxication and not an infection, and in livestock it follows ingestion of decomposed organic matter, mostly of animal origin, such as carrion and putrid bones, but also of plant origin, containing toxin elaborated primarily by Clostridium botulinum type C or D, and more rarely type A or B. The toxin(s) are some of the most potent toxins known.69 Humans and other species of animals, such as dogs, mink, many species of domestic and wild birds (including turkeys, domestic fowls, water fowls, pheasants and ostriches), certain laboratory animals and fish species (e.g. salmonids) are also susceptible.105 Paradoxically, botulinum toxin, known as the most toxic natural product is currently being utilized increasingly in humans to treat a wide variety of cholinergic disorders varying from mild tics to severe forms of dystonia such as those found in advanced Parkinson’s disease.86, 95 It is expected that with genetic manipulation, the toxin may also be developed into an efficient carrier of oral medicines and vaccines.95

Botulism and its association with the ingestion of improperly preserved food has a long history in humans. The main sources of intoxication are meat and fish products, as well as non-acid vegetables such as beans, peas or beets. It was first described in humans in western Europe and Scandinavia104 by Kerner (1786–1862) and Van Ermengem (1851–1922). Kerner associated the occurrence of botulism, which was sometimes referred to as Kerner’s disease, with improperly prepared German sausages including ‘Schlackwurst, Leberwurst, Blutwurst’, and ‘Presskopf’. In 1912 Van Ermengem isolated the causative organism—a spore-forming, obligatory anaerobic bacterium — from spoiled meat and suggested that the bacterium be called Bacillus botulinus, the latter name being derived from ‘botulus’, the Latin word for sausage, and the disease became known as botulism. As the result of the work of Burke16 in 1919 and others, it was subsequently established that more than one type of the organism is responsible for the disease.

Seven toxin types of C. botulinum (designated from A to G) are currently recognized. Burke in 1919 was the first to recognize that different types of the organism exist, each with different toxins, and described C. botulinum types A and B.16 This was followed three years later by the discovery of C. botulinum type C from green fly larvae (Lucilia caesar), by Bengston in the USA while investigating a paralytic disease of chickens.5 During the same year Seddon in Australia found that a disease in cattle known as bulbar paralysis was caused by an organism which he called Bacillus parabotulinus. 92 Type C strains comprise two distinct subtypes designated C alpha (Bengston strain) and C beta (Seddon strain).44, 87 Type D was isolated by Du Toit and Robinson at the Onderstepoort Veterinary Institute in South Africa in 1928 from a cow that had died of a paralytic disease known in South Africa as ‘lamsiekte’.44 Bier isolated type E in 1936 and 1937 from outbreaks of botulism in humans in which fish was the source of the intoxication. Moller and Scheibel isolated and characterized type F from home-made liver paste associated with an outbreak of botulism in humans in 1960. In 1970 Gimenez and Cicarelli isolated type G from a soil sample, and it has since been associated with sudden deaths in humans in Argentina.107 This bacterium has recently been renamed C. argentinense.110

Botulism occurs in many countries of the world in situations where C. botulinum is present as a saprophyte in decaying organic material. The disease in cattle in South Africa was first recorded by Le Vaillant64 during the years 1780 to 1785 while he was travelling through the interior of the country. He considered it to be one of the four most destructive diseases of cattle in what is currently the Limpopo, Eastern and Western Cape provinces and, according to him, it appeared annually soon after the warm weather had set in and was particularly prevalent when grazing was poor and cattle showed a craving for bones.50

Figure 186.1 Poor condition and swollen carpal and tarsal joints of a bovine suffering from osteomalacia as a result of aphosphorosis

Figure 186.2 Historical photo of workers dosing bone meal to cattle on the farm Armoedsvlakte, South Africa, to prevent botulism

Both Commissioner De Mist in 1805 in his diary and Lichtenstein in 181271 recorded the occurrence of lamsiekte in several parts of the Cape Province. Hutcheon later encountered what he referred to as ‘Lam Ziekte’ in 1881 in the Bredasdorp and certain Kaffrarian districts. He commented on the higher prevalence of botulism in young, growing stock and lactating cows as compared to oxen and dry cows, and also that the prevalence of the disease was much higher when the vegetation was very low in feeding value than after good rains had fallen and the grass was lush. The prevalence of botulism usually decreased soon after the first rains of the season had fallen. Hutcheon54 noted the association of the disease with pica manifested by cattle in these localities, and related it to a deficiency of phosphorus in feed grown on soil deficient in lime and phosphorus (Figure 186.1). He ascribed the clinical signs to nutritional deficiency and maintained that they could be prevented by feeding the animals phosphate-rich supplements such as bran and bones.

This view was strongly supported by Borthwick9 who showed that regular feeding of bone meal greatly reduced the prevalence of lamsiekte in cattle (Figure 186.2). These views on the cause of botulism were not accepted at the time. This situation prevailed and losses occurred to such an extent that the disease threatened the future of cattle farming in these regions. Because of mounting pressure by farmers, Arnold Theiler was appointed in 1910 to investigate the cause of the disease.45 The farm Armoedsvlakte (meaning ‘poverty flats’) situated near the town of Vryburg in the western part of the North West Province was eventually purchased by the Government as a research station where much work on botulism and aphosphorosis was conducted.

In 1913 the investigations of Walker124 and Mitchell76demonstrated that botulism was neither infectious nor contagious and that it was a non-specific form of toxaemia that developed following the absorption of septic material. They succeeded in reproducing the disease by administering carcass material and crushed bones in various stages of decomposition, following the insistence of farmers that cattle contracted the disease as a result of the ingestion of stomach content, skins and carcass material of animals that had died of botulism.

In 1919 Theiler115 and his collaborators proved that botulism was produced in cattle by the ingestion of carcass material picked up from the veld. He also induced the disease experimentally in healthy animals by drenching them with decomposed carcass material collected at random from veld on which the disease occurred. In addition, Theiler reproduced botulism by drenching cattle with blowfly pupae collected in the vicinity of a botulism carcass; blowfly pupae obtained from animal carcasses in non-botulism areas failed to induce the disease. They concluded that the causal agent was a toxin present in decomposed animal matter in certain localities. Robinson finally identified the causative bacterium as a member of the botulinus group, C. botulinum type D.90, 117

Theiler explained the cause of botulism in range cattle by way of an aetiological chain composed of six elements, the removal of any one of which would prevent the occurrence of botulism under field conditions:

  • the presence of the toxicogenic saprophyte which produces the toxin during its multiplication in decaying organic matter,
  • the toxin,
  • the carcass material in which the saprophyte multiplies and in which the toxin is elaborated,
  • craving or pica which induces animals to devour bones (osteophagia) or other putrid carcass material that they would normally avoid, and
  • phosphorus-deficient vegetation growing in
  • phosphorus-deficient soil.

The role of C. botulinum as a cause of the disease in horses which in the USA is referred to as ‘forage poisoning’ was confirmed by Graham and Brueckner in 1919.43 In South Africa, Theiler and Robinson in 1927117 and Robinson in 193090 recorded several outbreaks of the disease in horses, mules and donkeys. Clostridium botulinum type C was isolated in South Africa from material associated with an outbreak of botulism in mules at the Onderstepoort Veterinary Institute.90

In southern Africa botulism is currently well controlled in livestock by vaccination, although sporadic outbreaks of the disease still occur. Botulism associated with pica as a consequence of aphosphorosis in certain parts of the country still accounts for most cases in South Africa, but occasional outbreaks in which aphosphorosis is not involved are on the increase. Common sources of botulinum toxin in the latter instances include chicken litter used as a feed supplement for ruminants because of its high nitrogen content, pollution of drinking water by the carcasses of animals that have drowned in water troughs, and roughage such as hay contaminated by the carcasses of small wild animals, such as rats and birds, or the feeding of spoiled lucerne (alfalfa) infected with the bacterium. It is not unusual for losses of between 100 and 500 cattle or sheep to be recorded in such outbreaks, particularly because they often occur in feedlots where large numbers of animals are fed contaminated feed or drink water from a common polluted source.62

Aetiology

Clostridium botulinum comprises a metabolically diverse group of Gram-positive bacilli that are anaerobic and form spores. They produce at least eight neurotoxins that are pharmacologically similar but serologically distinct from each other. Eight types of C. botulinum — types A, B, Calpha and C-beta, D, E, F and G — have been identified.103, 104 Clostridium botulinum type C-alpha produces C1, C2 and D toxins, and Type C-beta produces C2 toxin.56 The type of toxin produced is also determined by the specific phage with which the bacterium is infected.27–29, 55, 85 For example, C. botulinum type C can be cured of its prophage (a bacteriophage), after which it ceases to produce type C or D toxin. This non-toxigenic phage-sensitive strain of C. botulinum type C can be converted to another toxigenic species, Clostridium novyi type A, after infection by phage NA1 obtained from C. novyi type A.28 The production of botulinum toxins is not important for growth or the physiological wellbeing of the bacterium, as growth and toxin production can be manipulated independently of one another.93, 94

The species Clostridium botulinum is additionally divided into three groups on the basis of cultural and serological characteristics. The first group consists of all the type A strains and the proteolytic strains of types B and F; the second group includes strains of type E and the non-proteolytic (saccharolytic) strains of types B and F; and the third group contains all the non-proteolytic type C and D strains.17 A few strains of types C and D are mildly proteolytic but are nevertheless included in group three.104 The strains of C. botulinum type G, as described by Gimenez and Ciccarelli,42 are metabolically distinct and the organism has been renamed Clostridium argentinense as a result of these metabolic and cultural differences.110The three existing groups also differ in terms of their cell wall components.104

The proteolytic strains of type A, B and F (Group 1) are motile, have peritrichous flagella, and are straight to slightly curved rods from 2 to 10 μm in length and 0,5 to 2 μm wide when cultured in peptone yeast glucose broth. The spores are oval and subterminal. In certain media with inadequate nutrients, they may increase in length to 20 to 45 μm. The cells normally occur singly, and less often in pairs or short chains. They retain the Gram stain very well, often only becoming Gram-negative with the commencement of sporulation. 104 Sporulation occurs readily on egg yolk agar plates after two days or on chopped meat agar slants incubated at 30 °C for one week.17 It appears that the highly toxigenic strains of all types tend to form fewer spores than less toxigenic strains.104 Group 2 strains include all the non-proteolytic strains of types B, E and F. The cells are motile, straight rods 1,7 to 15,7 μm long and 0,8 to 1,6 μm wide, occurring singly or in pairs. Spores are oval and subterminal and cause the cell to swell. Sporulation occurs readily on broth and solid media17 but the germination of spores in laboratory media is promoted by the addition of 0,1 per cent starch, which binds the long chain fatty acids capable of inhibiting spore germination. Clostridium botulinum type A spores are fully activated for germination by exposing them to a temperature of 80 °C for 10 to 20 minutes.104 Group 3 bacteria grown on proteose-yeast-peptone broth are straight rods, motile with peritrichous flagella, and with dimensions ranging between 3,0–22 × 0,5–2,4 μm. These cells occur singly or in pairs. The spores are oval and subterminal and cause swelling of the cell.17

With the exception of some strains of Group 3 organisms, C. botulinum is a strict anaerobe and even traces of oxygen in its environment inhibit its growth. These organisms are mesophiles and grow optimally at temperatures between 18 and 37 °C, and within a pH range between 7,0 and 7,6.8 Some of the non-proteolytic strains grow at relatively low temperatures (3 to 4 °C) whereas the proteolytic strains do not grow below 10 °C.51 The optimal temperature for toxin production is 25 to 30 °C. Non-proteolytic strains do not grow below pH5,0 to 5,2, whereas the proteolytic strains generally do not grow below pH 4,7 though type A has been shown to do so at pH 4,0.89

In pure culture, C. botulinum grows well on solid agar media such as horse blood agar, liver–veal–egg yolk agar and anaerobic egg yolk agar,51, 61 and in enriched, non-selective fluid media, including glucose-peptone broth, reinforced clostridial medium, trypticase-peptone-glucose-yeast extract- trypsin broth, or Robertson’s cooked meat medium.61, 123 A selective medium—C. botulinum isolation agar, containing cycloserine, sulfamethoxazole and trimethoprim as inhibitory agents — has been devised for the isolation of C. botulinum from human faecal material.21 Organic nutrient requirements for growth and toxin production of Groups 1 and 2 strains differ.128

After three days’ growth at 30 °C on horse blood agar, typical colonies of C. botulinum are smooth, circular with an irregular edge and about 3 mm in diameter, which may increase to 8mm following an extended period of incubation. Colonies are raised or flat, rough or smooth, and commonly show some spreading. Blood agar has no particular advantage as a culture medium but is a good general growth medium. Colonies have a similar appearance when grown on egg yolk agar, and the use of this substrate has the advantage that it allows detection of the lipase reaction. The lipolytic enzymes produced by C. botulinum cause the development of a precipitate in the medium underneath the colony and an iridescent film (pearly layer) covering the colony, both of which are due to the presence of free fatty acids.131 The lipolytic reaction can also be detected by scraping the colonies off the surface of an egg yolk agar plate and then flooding it with a copper sulphate solution which will stain the free fatty acid precipitates on the surface of the medium dark blue.51

The surface colonies on blood agar of the strains in Group 3 are beta-haemolytic, 1 to 5mm in diameter, circular to slightly irregular, flat to raised, and translucent to greyish-white, and have a slightly scalloped or lobated margin, a matt surface and a mottled internal structure.17, 103, 104 Subtle differences occur in the colony morphology of the different groups. They can also be distinguished from one another on the basis of differences in their motility, ability to ferment various sugars, fermentation products and ability to digest proteins, and in the production of enzymes.17

Strict anaerobic growth conditions are required to propagate the more toxigenic variants of these organisms. Exposing blood agar plates to air will inhibit their growth, while less toxigenic variants still grow on media that have been exposed to air.

Sporulation is one of the most important characteristics of the genus Clostridium and is a most efficient survival mechanism. The spores of C. botulinum are among the more resistant of the bacterial spores, thus allowing C. botulinum to survive some of the conventional procedures used for preservation of food that normally kill other bacteria. The spores of C. botulinum are resistant to ultraviolet light, alcohols, phenolic compounds, quaternary ammonium compounds and organic mercurials. Substances such as ethylene and propylene oxide and formaldehyde do kill the spores of C. botulinum, but not very rapidly. Spores are effectively destroyed by heating or radiation. The D-value (decimal reduction time) which is an indication of the thermal denaturation rate and a reflection of the number of minutes required at a specific temperature to reduce the viable count of a spore suspension to one tenth of its original value, is used to assess the resistance of spores to high temperatures. The D-value of types A, B and E spore suspensions ranges from 0,15 and 1,95 for temperatures between 80 and 121 °C.104 The heat resistance of spores is determined by various factors, such as the presence of fatty acids, calcium and iron, and the pH of the medium,104 while considerable variance in the D-value occurs after batches of spores of the same strain are grown in different media.104 It appears that spores from Group 1 are more resistant than those of Groups 2 and 3.

Apart from heat, radiation is used as a means of sterilizing food. To achieve indisputable microbial safety, radiation doses in the range of 35 to 45 kGy must be applied. This dosage is based on the 12D-concept; this is a dose high enough to destroy a theoretical population of 1 × 1012 C. botulinum spores per gram.4 However, organoleptic side-effects affecting the taste and odour of the food are produced by such high doses. Mild irradiation sensitizes bacterial spores to subsequent heat injury.

‘Botulinum neurotoxin’ refers to any one of seven substances, namely toxin types A, B, C1, D, E, F and G, that are produced by C. botulinum. The various toxins which are proteins have similar molecular weights and probably similar molecular structures. The eighth toxin, referred to as C2, is produced by type C and D strains but is not a neurotoxin. It is also referred to as ‘the botulinum binary toxin’ and has a unique structure and pharmacological action. It does not cause flaccid paralysis but it does act as an adenosine diphosphate ribosylating enzyme causing increased vascular permeability85 and necrosis in the epithelium and lamina propria of the duodenum and jejunum. 68 More details on the mechanism of action of the botulinum neurotoxins are available in a number of reviews. 11, 15, 48, 78, 79, 80, 93, 104, 105, 111 The toxins are relatively resistant to the action of mild chemical agents, but are susceptible to heat inactivation. High salt concentrations prevent toxin production.104

The molecular mass of the neurotoxins produced by types A to F varies from 150 000 to 160 000 Daltons. The toxins are synthesized as single polypeptide chains, but when released from the bacteria they are proteolytically cleaved to form two non-identical, protein fragments each consisting of a heavy chain or subunit with a molecular mass of 100 000 Daltons and a light chain or subunit with a molecular mass of 50 000 Daltons, linked by a disulphide bond.108 Type A and B neurotoxins contain at least one disulphide bond each, which on reduction causes loss of biological activity.93 However, botulinum toxin is moderately resistant to enzymic proteolysis under natural conditions but is inactivated by exposure to a temperature of 80 °C for 10 minutes.104

The botulinum toxin is synthesized intracellularly and is released into the culture supernatant when bacteria undergo autolysis after completion of growth cycles.93, 94 The concentration of free toxin may thus increase in the supernatant even after protein synthesis has stopped. It appears that botulinum neurotoxin may be synthesized as an inactive or less toxic precurser, which is then activated by selective cleavage by proteolytic enzymes. The ability of trypsin to activate botulinum neurotoxin has been demonstrated for types A to G.93

The potency of botulinum toxins, which may also be produced by bacteria grown on artificial media, is expressed as the LD50 (lethal dose in 50 per cent of animals) or MLD (minimum lethal dose) in mice. The L+ and the Ramon flocculation tests are used to determine the total amount of toxin present in the culture supernatant.57, 104 The MLD for mice of botulinum type A neurotoxin per kilogram body weight is 1,2 ng intraperitoneally (ip), for type B neurotoxin 1,2 to 2 ng ip, for C1 toxin 1,1 ng intravenously (iv), type C2 toxin 270 ng iv, type D toxin 0,4 ng ip, type E toxin 1,1 ng ip and type F 2,5 ng ip.41

Epidemiology

Botulism in animals has been described in cattle,23, 36, 47, 50, 74, 81 horses and foals,46, 72, 90, 113, 114 mules and donkeys,90 sheep and goats,116 pigs,3 water birds,49, 100 turkeys,98 ostriches and ducks,116 and dogs.2, 13, 20, 125

Among livestock, horses appear to be the most susceptible to the effects of the botulinum toxin. Amounts of toxin which are sublethal in mice, may rapidly cause the death of adult horses if administered intravenously.66 Pigs and carnivores, on the other hand, are very resistant to botulinum toxin and for all practical purposes are not susceptible to its effects. Clostridium botulinum type C1 toxin may be present in the livers of healthy pigs infected with C. botulinum type C without the pigs showing clinical signs.133 Cattle, sheep and goats appear to be equally susceptible to the effects of the toxin.116

There is an apparent geographic variation in the distribution of the different toxin types of C. botulinum. Toxin types C and D appear to be the exclusive cause of botulism of livestock in countries in the southern hemisphere, such as in Australia, South Africa and certain South American countries25, 116, 119 whereas type B is more often the cause of botulism in livestock in the USA and in Europe.14

Spores of C. botulinum occur commonly in soil, water, marine sediments and marine environments throughout the world.4, 53, 103, 104 Clostridium botulinum also appears to be a normal and innocuous inhabitant of the intestinal tract of horses, cattle and poultry where it multiplies and from which it is shed in large numbers in the faeces for up to eight weeks after primary infection.81

Outbreaks of botulism in livestock occur under a variety of conditions but can in general be subdivided into two main categories:

  • those associated with phosphorus deficiency and osteophagia in which the disease occurs sporadically over a relatively long period of time in animals (mainly cattle, but also to a lesser extent sheep and goats) maintained under extensive ranching conditions, and
  • secondly the form in cattle, sheep, goats and horses which is associated with the ingestion of toxic feed or water and which may result in the deaths of large numbers of animals over a short period of time.

In sporadic outbreaks of botulism unassociated with aphosphorosis, C. botulinum-containing carcass material of mammals or birds may contaminate feed or water of domestic livestock. More rarely, spoiled vegetable matter such as grass hay, lucerne hay, grass clippings, silage, brewers’ grain and even kitchen refuse or ‘mynpap’ (porridge) obtained from the kitchen waste of mine hostels,23, 38, 47, 74, 96, 129 may serve as substrates for bacterial growth and toxin production. Three recent outbreaks in South Africa in farm animals exemplify this form of botulism. One occurred in sheep maintained under extensive farming conditions in the Upington district of the Northern Cape Province. It was only after hundreds of animals had died that the putrefying carcass of a sheep was found submerged in the water of a remote drinking trough on the farm. This led to the disease being finally diagnosed as botulism.63 The second outbreak occurred on a smallholding on which all the animals (cattle, sheep, donkeys, geese and chickens) except the pigs died of botulism as the result of the feeding of kitchen refuse which had been obtained from a large hostel and stored in metal drums before being fed to the animals.18 In the third outbreak, a field of lucerne had been sprayed with an insecticide in order to control an infestation of worms. Egrets, an insectivorous species of bird, had eaten the dead worms and in turn had died from insecticidal poisoning. When the lucerne was harvested for hay, some of the dead birds were baled in the lucerne. An outbreak of botulism occurred in the dairy cows fed the hay and the source of the toxin was only realized due to the presence of feathers in the lucerne hay.19

Chicken manure (especially broiler litter), which is often fed to ruminants as a supplement because of its high nitrogen content, frequently contains carcasses of dead chickens in which C. botulinum has multiplied and produced its toxins. Outbreaks have even occurred following the spread of chicken manure on pastures as a fertilizer or when it has been used as bedding material. Based on experimental data, it appears that the type of feed that animals are receiving, as well as a change in feed prior to their exposure to botulinum toxin, affects the dose of the toxin required to induce the disease in a specific species. In one study, goats on pasture as well as those being fed silage developed increased susceptibility to the effects of the toxin after their diet had been changed to hay and concentrated fodder.34

Probably the single most important factor responsible for botulism under extensive ranching conditions is the occurrence of osteophagia resulting from phosphorus-deficient grazing. Cattle, particularly, and sheep to a lesser extent, are prone to develop osteophagia as a result of phosphorus deficiency. Other species are not known to develop osteophagia. Economically serious outbreaks of botulism as a result of pica has been recorded in South Africa, Australia and several South American countries.25, 115, 119

Figure 186.3 Phosphorus content of vegetation during summer in South Africa

Figure 186.4 Phosphorus content of vegetation during winter in SouthAfrica

During spring and early summer in South Africa, the phosphorus content of the veld grasses is generally sufficient to prevent the development of osteophagia and the occurrence of botulism. However, when the grazing has matured, its phosphorus content decreases to such an extent that by late summer and winter (for a period ranging from five to nine months of the year)cattle grazing on it may manifest osteophagia (Figure 186.3 and Figure 186.4) unless the de?ciency is compensated for by supplying the animals with additional sources of phosphorus,for example in the for mof phosphate-containing licks.26 Deficiency of other dietary components, such as proteins, which often occurs concomitantly in such phosphorus-deficient grasses, may also cause animals to develop pica and hence predispose them to consume toxin-containing material. Growing, lactating, and pregnant cattle require more dietary phosphorus than another groups of cattle and these therefore are more prone to develop botulism. The requirement of phosphorus in these animals is such that the amount of phosphorus present in natural pastures in South Africa is often insufficient throughout the year.

In South Africa it appears that,apart from the carcass debris of domestic animals, the carcasses of small mammals such as mongooses and hares, birds and particularly tortoises are important sources of the toxin under ranching conditions.36 The carcasses of small mammals may remain toxic for up to 350 days but they usually lose their toxicity after four to six weeks, whereas tortoise carcasses retain their toxicity for up to 350 days on pasture and up to 600 days in the laboratory.36 Rain may leach much of the toxin from the carcass material of small mammals, whereas the shells and bones of tortoises retain the toxin for longer periods in wet weather. In Australia, botulism in cattle in phosphorus-de?cient areas is associated with the ingestion of carrion, chie?y dead rabbits containing the toxin.6 Prevailing climatic conditions affect toxin production. The toxin is readily produced in carcasses when there is suf?cient heat and moisture, but there is a drastic reduction in toxin production when the weather becomes cold and dry.65The production of botulinum toxin under optimal environmental conditions reaches its maximum within ?ve to ten days, whereafter it starts to decline. Type C botulinum toxin production in mouse carcasses under experimental conditions may reach concentrations of 2 × 10 5 to 2 × 10 6 mouse intraperitoneal MLD/g.102 The type of animal tissue and the micro?ora present in it appear to in?uence the amount of toxin produced by C. botulinum types C and E. Type C toxin is produced in much higher concentrations in chicken and mouse carcasses than in ?sh carcasses.102The micro climate within maggot-infested duck carcasses appears to be suitable for growth and toxin production byC.botulinum. 132

The occurrence of botulism under ranching conditions is increased by phosphorus deficiency in genetically improved, quicker-growing and higher-demanding breeds, overstocking, and inadequate removal of carcasses and carcass debris from pastures.25, 116 In addition, low-demanding cultivars of pasture grasses, such as Brachiaria decumbens in Brazil grown on phosphorus deficient soils, have a low phosphorus content which is insufficient for cattle maintenance and production during the rainy spring and summer seasons despite the luxurious pasture growth. This results in osteophagia and consequently to botulism in these seasons. The cultivation of large areas of the natural tree-savanna in central Brazil from the 1970s has led to severe losses in cattle as a result of botulism.24, 25 This is in contrast to the situation in South Africa, where most cases related to aphosphorosis occur during the dry winter months which prevail over a large part of the country.

Pigs are highly resistant to the effects of the botulinum toxin. The only recorded outbreak of botulism in this species of animal occurred in Australia and followed the ingestion of dead fish containing type C toxin.3

Toxicoinfectious botulism is a rare form of the disease in humans and animals which results when toxin is produced in vivo by C. botulinum growing in the body itself, rather than by the ingestion of toxin.104, 105 It may either follow wound infections or occur after ingested spores have germinated and proliferated vegetatively in the gastrointestinal tract. Wound botulism has been described in humans and is becoming a problem in drug addicts who use contaminated hypodermic needles. Only Group 1 strains of C. botulinum have been incriminated in this condition in humans.105 A similar form of wound botulism has been described in adult horses and young foals in which gastric ulcers, umbilical and lung abscesses, or necrotic wounds, such as those following castration, were the site of localization and proliferation of the organism.7, 113, 114 It appears that the growth of the organism in necrotic tissue in foals is potentiated by excessive amounts of corticosteroids contained in the fat of the milk of mares which are exposed to stress, such as that produced by excessively nutritious diets, the rearing of fastgrowing foals, or drastic changes in weather. Outbreaks often follow drastic changes in weather.113 Young foals afflicted in this way show the signs of the ‘shaker foal syndrome’, which are dysphagia, muscular tremors, weakness, recumbency and death.113 Both toxin type B and C of C. botulinum have been incriminated in horses.72, 113

Botulism in human infants is the result of the absorption of toxin elaborated by organisms, usually Group 1 strains, following their colonization of the intestinal tract.75, 105 A similar situation has been observed in pheasants, in which the crop was the site of colonization and toxin elaboration,22 as well as in broiler chickens, in which the caecum was the site of bacterial multiplication.77

Pathogenesis

A substantial quantity of botulinum toxin which is ingested is either not absorbed or is destroyed by digestive processes, including destructive processes by rumen bacteria in ruminants, so that the oral lethal dose is larger than the lethal dose of toxin administered or acquired by other routes. Small, non-lethal doses of toxin ingested over a period of time will also cause intoxication; the cumulative dose is, however, larger than that which induces botulism if it is received as a single dose. Animals may thus become poisoned by the ingestion of feed containing sublethal amounts of toxin if they are fed such material over a few successive days. How long the toxin remains in the intestinal tract or blood stream after ingestion is, however, unknown.34 The presence of complexing proteins of nucleotide sequences associated with the neurotoxin genes of serotypes A to F seems to allow absorption of the toxins from the intestinal tract.97 In the case of type C. botulinum toxin, a haemaglutinin appears to be important in the absorption of the toxin from the small intestine of guinea pigs.37

Following its absorption into the blood stream the botulinum toxin is disseminated throughout the body but, with the exception of type C2, it only has a pharmacological effect on peripheral nerve endings whose neurotransmitter is acetylcholine, 111 i.e. somatic motor nerves supplying skeletal muscles and efferent parasympathetic nerves. The central nervous system is protected by the blood–brain barrier. The flaccid paralysis caused by the toxin develops as a result of the inhibition of the release of acetylcholine at the neuromuscular junctions, with the result that no stimulus reaches the motor end-plates.104, 105, 111 The toxic effect of botulinum toxin is exerted by the dual action of its large subunit which carries the ligand and is responsible for the fixation of the toxin molecule to a specific tissue receptor on the terminal unmyelinated portions of nerve membranes, and the small subunit, a zinc endopeptidase possessing the pharmacological activity.91, 112 Following the binding of botulinum toxin to tissue receptors, it is internalized into the axoplasm where it displays its zinc-endopeptidase (or endoprotease) activity targeting the protein components of the neuro-exocytosis apparatus.78 The various types of botulinum toxin target different proteins in this apparatus. Types B, D, F and G specifically cleave VAMP/synaptobrevin in the synaptic vessel membrane; the A and E neurotoxins specifically cleave SNAP-25, a protein of the pre-synaptic membrane; and type C cleaves syntaxin, also a component of the nerve plasmalemma, probably with the aid of accessory factors.35 Each of the enzymes that target a specific molecule cleaves the molecule at a site that is specific and different for the individual toxin.78, 91 A progressively developing flaccid paralysis results; paralysis of the muscles of respiration being the eventual cause of death. Although toxin binds rapidly and irreversibly to tissue receptors it does not immediately cause paralysis.105 In experimentally isolated nerve-muscle preparations antitoxin has no protective action once this fixation has occurred.108 Botulinum toxin type C2 causes selective loss of non-muscle F-actin. It appears that the effect on actin, that supports the endothelial cell membrane configuration and integrity, is, at least in the lungs, to disrupt the intimal barrier function of endothelial cells.30 Other publications should be consulted for more detailed descriptions of the action of botulinum toxin.11, 15, 48, 78, 79, 80, 91, 93, 94, 105, 111

Figure 186.5 Botulisminanox. Note protrusion of the tongue

Figure 186.6 Cow unable to rise voluntarily even when ‘stimulated’ by biting its tail

Clinical signs in cattle exposed to type B toxin appear to differ from those caused by the C and D toxins and are associated with paralysis of the cervical portion of the oesophagus. It would seem that this effect results because smaller amounts of acetylcholine are secreted by the nerve endings of the parasympathetic nerves, which innervate the affected portion of the oesophagus. This has led to the suggestion that if cattle are exposed to relatively small amounts of toxin, the parasympathetic motor end-plates are selectively affected because of their smaller reserves of acetylcholine.14

The chronic effects of botulinum toxin are comparable to surgical denervation, and complete recovery depends on the formation of new neuromuscular junctions.104

Clinical signs

The clinical signs caused by intoxication with types C and D in cattle vary considerably and depend on the quantity of toxin that has been ingested. The disease has arbitrarily been divided by Theiler and his collaborators116 into peracute, acute, subacute and chronic forms based upon the onset, the sequence of development of paralysis in different muscle groups and severity of the clinical signs. Range cattle suffering from phosphorus deficiency that contract botulism by ingesting toxic material lying in pastures, most commonly develop the subacute to chronic form of the disease; a few may even be so mildly affected that they escape notice. More acute forms are rare under such circumstances but they do occur. A feature of botulism which, apart from anything else, is of diagnostic significance, is that the case fatality rate is invariably high; most affected animals will die, but if an animal does recover it is most likely to have suffered from subacute or chronic disease. In sporadic outbreaks unassociated with osteophagia, such as those following the ingestion of a toxic batch of hay or kitchen refuse, new clinical cases are detected over a period of days. At the beginning of an outbreak cattle usually die acutely, but as the outbreak progresses, the duration of clinical signs tends to increase, until at the end of the outbreak a few lightly affected animals may even recover fully.

Figure 186.7 Typical posture of a heifer suffering from the advanced stage sof botulism

Figure 186.8 Boer goat suffering from botulism. Note the protrusion of the tongue

Figure 186.9 Typical stance of a Dorper sheep suffering from botulism

The incubation period inexperimental cases varies from about 18 hours to 16 days but in natural disease in cattle it usually varies from two to six days but deaths may occur for up to 17 days after animals have had access to toxic feed. 116,119 The duration of illness may be directly correlated with the length of the incubation period; it may be as short as 24 hours in peracute cases, and up to seven days or longer in more chronic cases. There is also an intermittent form of botulism in which generally mild clinical signs are manifested at intervals of a few days with periods of remission.

Affected animals are afebrile and typically manifest partial or complete flaccid paralysis of the muscles of locomotion, mastication and deglutition. The paresis, which progresses to paralysis, usually commences in the hind quarters and then spreads progressively forwards, affecting in turn the fore limbs, the neck and head, the rapidity of succession decreasing from the peracute to the chronic cases. Sensory perception and reflex actions of the eyes are maintained for a considerable time after skeletal muscular paralysis has set in, and animals generally appear mentally alert. Hypersensitivity and what may be perceived as aggressiveness may occasionally be manifested. In some cases, which are considered to be atypical, the muscles of mastication and deglutition, but not those of the locomotor system are paralysed.

The appetite is mostly unaffected, animals will prehend and masticate food for as long as they are able. A few animals may be constipated or, more exceptionally, suffer from diarrhoea. Ruminal tympany often develops in animals that lie in awkward positions in the terminal stage of the disease.

The naturally acquired, peracute disease is seldom encountered; some animals suffering from it may die suddenly without clinical signs having been noticed. In this form, paralysis of the muscles of locomotion, mastication and deglutition is usually marked and affected animals soon become prostrate and are more or less completely paralysed. At first they assume a sternal lying position frequently with the neck turned back and the head resting on the flank. In the later stages they become laterally recumbent. Chewing and swallowing movements are seldom possible, the tongue often protrudes from the mouth (Figure 186.5), the lower jaw is slack and there is profuse salivation.

In acute lamsiekte the clinical signs generally resemble those of the peracute disease, but the course of the disease is more protracted. Although most animals lie down during the initial stages of the disease, others are able to remain standing. At first, affected animals are able to rise without assistance and, when disturbed, move about sluggishly and with a stiff gait. As the disease progresses they are unable to rise voluntarily (Figure 186.6) and even when lifted to their feet, they are seldom able to remain standing when all support is removed, and usually drop down helplessly. Initially the head is held upright, but in the later stages the neck is doubled back with the head resting on the flank (Figure 186.7). However, if the head is manually raised, it drops back involuntarily towards the flank when released. Impairment of the muscles of mastication and deglutition is usually pronounced, causing boluses of grass to accumulate in the back of the mouth. Paresis and paralysis of the tongue also set in at this stage; in the latter instance the tip of the tongue either lies on the incisors or protrudes from the mouth. In some cases paralysis of the tongue is not complete and the animal may still be able to retract it into the mouth after it has been physically extracted.116 In these cases the mouth can be manually opened with relative ease. In addition, if, when examining a suspected case, the clinician stands behind the head of the animal to one side of the neck, grasps the horns or ears firmly in both hands and shakes the head of the animal vigorously, the slack lower jaw meeting the upper jaw often causes a rather characteristic ‘plop’ sound to be emitted. Some degree of paresis, or paralysis of the tail is also usually evident.

In the subacute disease the clinical signs differ from those of acute lamsiekte only in degree and in the period required for their evolution. Affected animals walk with a stiff, sluggish gait, and usually prefer to lie down. As the disease progresses, it becomes more and more difficult for them to rise voluntarily, and when complete paralysis has set in, rising is no longer possible.

The duration of the clinical signs in chronic lamsiekte is longer than seven days. In the early stages affected animals usually eat well, but they generally appear tucked up and have staring coats. Swallowing and chewing are not always affected. Paralysis of the legs develops to a variable extent, and although animals prefer to lie down, they generally are able to rise without assistance. There is rapid loss of condition. The severity of the clinical signs progresses until prostration and, in most cases, death supervene.

The signs in cattle affected by type B toxin generally develop within three days of their being exposed to toxic material, usually brewers’ grain or grass silage.83 These differ markedly from those caused by type C and D toxin. In general the disease is characterized by anorexia, profuse salivation, regurgitation of feed and water following their ingestion, and dehydration. The animals manifest a decreased tonus of skeletal muscles but they are not ataxic, nor is the tail or the tongue paralysed. The initial signs are associated with reduced appetite, reduction in milk production, and a decrease in the consistency of the faeces which sometimes progresses to a frank diarrhoea. Pronounced salivation and regurgitation of feed and water are the main characteristics of this type of intoxication, the latter often resulting in inhalation of the regurgitated material and the development of a foreign body pneumonia. Severely affected animals usually die, while those that are mildly affected recover uneventfully after a period which may be as long as three to six weeks.14

In both sheep and goats the clinical signs are similar to those in cattle (Figure 186.8), except that the muscles of mastication and swallowing are not as obviously affected. In peracute cases, animals may die without signs having been observed or within four hours of their commencement.120 Affected sheep are listless, show muscular incoordination, weakness of neck muscles and torticollis, walk with a wobbly gait, are disinclined to drink water, and, infrequently, salivate excessively. When driven, they lag behind the rest of the flock. Characteristic features are that sheep and goats develop an arched back with a drooping tail, head and neck (Figure 186.9)59, 104 and manifest an abdominal type of respiration and frequent urination, while sheep often, but not invariably, show a fine trembling of the tail stump. If an affected sheep is grasped by the ears and the head shaken up or down, as in cattle, the lower jaw may strike the upper jaw to emit a clapping sound. Respiratory distress may be evident. The prognosis for the disease is extremely poor, but a few may recover without specific antiserum treatment.

In adult horses and foals a similar neuromuscular paralytic syndrome is evident in those animals suffering from the toxicoinfectious syndrome caused by type B toxin. This disease referred to in foals as the ‘shaker foal syndrome’, occurs sporadically, most frequently in animals between two and four weeks of age, although adult animals are as susceptible to the effects of the toxin. Some foals may be found dead without having been noticed to be sick while others live for 24 to 40 hours before they die. They are often found in the prone position and are unable to rise. When assisted to their feet they may remain standing for a few minutes and walk with a stiff, stilted gait only to fall to the ground again after manifesting severe trembling of the muscles of the legs. When down they are alert and the muscle tremors cease. As the disease progresses, they are only able to stand for very short periods, but eventually cannot do so at all even when assisted. Affected foals also have difficulty in swallowing, with the result that they eat with difficulty and milk may run out of the sides of their mouths while they are suckling. They periodically attempt to urinate but void only small amounts, and usually become constipated. The pupils of the eyes are dilated and the pupillary light reflex is decreased. As the disease progresses, respiratory distress becomes pronounced and breathing becomes abdominal in type, and there is an increased heart rate. The temperature of the affected animals remains normal during the course of the disease. The mortality rate is usually in excess of 90 per cent, and in nonfatal cases or those in which treatment is effective, it may take up to three weeks for the muscular weakness to disappear. In foals that survive for a number of days, signs of an aspiration pneumonia may develop.64, 72, 113 The clinical signs in adult horses affected by type B toxin are similar to those in foals.113 Affected horses are initially lethargic and have a stilted gait, after which the typical signs develop within a matter of six to eight hours. Animals that become recumbent cannot retain a position of sternal recumbency due to the profound weakness caused by the flaccid paralysis.129

With certain exceptions, clinical signs in horses affected by type C toxin are similar to those in horses suffering from type B intoxication. Animals become ill within three days of ingesting feed containing type C toxin. Most die within 48 to 72 hours, the mortality rate being in excess of 80 per cent. The initial signs of intoxication, which may precede the development of overt clinical signs by 24 hours, are mild and characterized by an asymmetric decrease in palpebral tone. As the condition progresses, horses intermittently stand with slight carpal flexion while muscle fasciculations may be palpated in the triceps area. They retain their appetite during the course of the intoxication and, contrary to animals suffering from the effects of type B toxin, have no difficulty in swallowing. They do become constipated, however.66 Within 24 hours these signs develop into severe muscular tremors of the fore and hind legs.

Restlessness manifested by the horses striking the ground with their fore legs and lying down for short periods, is apparent. Muscle tremors disappear after the animals have been in the prone position for a short period and it may erroneously be assumed that they have recovered. As the intoxication progresses, affected animals become sternally recumbent and lie with the neck extended and the muzzle resting on the ground. Terminally, horses assume a position of lateral recumbency while paddling their legs rhythmically. Mydriasis and slow pupillary light reflexes are seen in few of the affected animals.

Pathology

There are no macroscopic or microscopic lesions that can be regarded as characteristic of, or specific for botulism. There may be a catarrhal enteritis and petechiae in the small intestines and the endocardium. Chronically affected animals may be constipated and manifest signs of ruminal and colonic stasis and fatty changes in the liver.50 Excess pericardial fluid and pulmonary oedema may also be present.119 In sheep that have died as a consequence of types C and D intoxication, extra-cardiac macroscopic lesions may be so extensive that their occurrence is suggestive of a heart failure syndrome.121 These lesions include mild to moderate accumulations of transudate in the body cavities, marked pulmonary oedema and congestion, the presence of thick white foam in the trachea, multiple epicardial petechiae and ecchymoses, myocardial pallor of the inner half of the ventricular walls, mild swelling of the liver and nephrosis.

Foreign bodies reflecting the existence of pica, such as bones, tortoise shell, bits of wood, stones, fragments of china and pieces of iron, may be found in the rumen or reticulum in animals on veld grazing. However, no indications of pica will be evident in animals in which the toxin is ingested in prepared feed or water.50

At necropsy the carcasses of horses that have died of toxicoinfectious botulism contain few characteristic lesions. The presence of excess pericardial fluid containing variable amounts of fibrin strands is a consistent finding. Focal areas of necrosis or abscesses may occur in the liver, navel, lungs, skin or muscles. In many cases gastric ulcers are present at the margo plicatus and occasionally at the pylorus. In addition, severe pulmonary oedema, intestinal stasis, and distention of the urinary bladder are present.113

The presence of extensive, intermuscular oedema in the neck region, suspected to be by caused type C2 toxin ingested concurrently with type C1 toxin, has been reported in horses that died of botulism.66 In some animals the oedema may extend caudally as far as the lumbar region.

Diagnosis

Botulism is often difficult to diagnose. A presumptive diagnosis is made on the basis of the history, clinical signs and negative post-mortem examination. Factors in the history of the outbreak which support a diagnosis of botulism are lack of or inadequate immunization against botulism and, in the case of range animals, the presence of osteophagia. The possibility of other diseases causing similar clinical signs to those of botulism should be systematically excluded. A useful diagnostic is that the number of cases diminishes drastically in affected herds or flocks following administration of primary and secondary doses of vaccine within a month of each other. In addition, the response of sick animals to botulism antiserum therapy may also be of some assistance.

A definitive diagnosis of botulism is dependent on the demonstration of botulinum toxin in specimens taken from diseased animals or those that have died, and in the suspected source of the toxin if this is still available. Although determining the presence of botulinum toxin in specimens submitted for the purpose is the most reliable way to confirm a diagnosis, many of the available test systems lack sensitivity and it is often very difficult to confirm the diagnosis of botulism.

Specimens collected for the diagnosis of botulism in live animals should consist of serum and faeces. Specimens (5 to 20 ml) should be taken of the rumen content and the contents of the small and large intestines from dead ruminants. In horses, specimens of the caecal contents as well as from any necrotic lesion present in the gastrointestinal tract are required. From all animal species 5 to 20 ml of serum and pericardial, thoracic and peritoneal transudates should be submitted. If available, specimens from several animals should be collected. Specimens of what is suspected to be the source of the toxin, such as feed concentrate, grass, silage, carcasses or parts of carrion, drinking water, mud or soil should also be submitted. Since the toxin is often unevenly distributed in feed, representative samples for analysis should be taken from various portions of it. After being collected and packed in sealed containers, the specimens should be deep-frozen if possible. If specimens cannot be frozen, they should at least be kept refrigerated and submitted packed in ice to a laboratory.61

Although its use is sometimes advocated, isolation of the bacterium from the intestinal tract provides equivocal results since these bacteria may also be isolated from healthy animals. The specimens (feed and tissues such as liver or intestinal lesions) for the isolation of the organism should first be inoculated in cooked meat broth and trypticasepeptone- glucose-yeast extract broth with trypsin (TPGYT), and incubated under anaerobic conditions at 26 and 35 °C respectively for five days. After this initial incubation period, if no growth is observed the cultures should be incubated for a further five days. Subcultures should be examined to determine the type of organism and toxin according to published methodology.17, 52, 61, 109

To determine the presence of botulinum toxin in tissue and feed specimens, the toxin is extracted in a gelatinephosphate buffer solution which is divided into at least four separate portions, each being subjected to different treatments consisting of heat, the addition of trypsin or polyvalent or monovalent C. botulinum antiserum. An amount of 0,5 to 1 ml of each portion is injected intraperitoneally into at least two mice which are then observed for up to ten days for signs of botulism.61 In this way the presence of botulinum toxin and the determination of the toxin type in a specimen can be achieved.8, 61, 106 Serum collected from horses affected with toxicoinfectious botulism invariably tests negatively since the concentration of toxin in the serum is generally so low that the toxin cannot be detected by conventional methods.113 The toxicity of feed samples may also be determined by test-feeding the sample to specifically immunized and unimmunized cattle, sheep or laboratory animals.119 More recently, an enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against toxin types C and D in sera obtained from unvaccinated cattle suspected to be suffering from botulism.60

Other methods for the detection of the presence of the botulinum toxin in various types of specimens include an immunodiffusion method,33 radioimmunoassay,12 various immunochemical assays,1, 20, 39, 40, 67, 82 and the temperature- induced microcomplement fixation test.25, 126, 127 Except for the microcomplement fixation test which is highly sensitive, the sensitivity of these other tests does not exceed that of mouse bioassay. A rapid in vitro test utilizing the enzymatic activities of the individual toxins, has been developed for the detection of type B neurotoxin from proteolytic strains of C. botulinum in food products. This test has a sensitivity greater than that obtained when using the mouse bioassay, and can identify the presence of the toxin in a number of food products including pâté, cheese, fish, mince meat and sausage.130

Differential diagnosis

The two most important differential diagnoses for botulism in South Africa are diplodiosis (a mycotoxicosis caused by Diplodia maydis poisoning) in cattle, and krimpsiekte (a plant poisoning caused by members of the Crassulaceae containing bufadienolides) in sheep and goats. In the latter the clinical signs of paralysis, salivation, retention of food in the pharynx and the typical posture, and the lack of specific macro- and histopathological changes render the two conditions indistinguishable.62

In ruminants other important differential diagnoses that may be considered include polioencephalomalacia and conditions associated with hypocalcaemia and hypomagnesaemia, nutritional or plant toxin-induced myopathies such as ionophore poisoning and poisoning with Geigeria spp. In cattle ingestion of the toxin of the fungus Aspergillus clavatus, organophosphate and lead poisoning, and infectious diseases such as rabies, listeriosis, bovine ephemeral fever, sporadic bovine encephalomyelitis, thrombotic meningoencephalitis, cerebral babesiosis and cerebral theileriosis, as well as non-specific bacterial meningitis and encephalitides, may also mimic some of the clinical signs of botulism.23 In sheep the possibility of tick paralysis caused either by Ixodes rubicundus or Rhipicephalus evertsi evertsi, focal symmetrical encephalomalacia, twin lamb disease (domsiekte) and Coenurus cerebralis infection should also be considered. In the case of tick paralysis, removal of the ticks is followed by recovery from the paralysis. In horses infection with equine herpesvirus 1, and various viral encephalitides should be considered.66

Control

There is no specific treatment of diseased animals suffering from botulism, apart from the administration of hyperimmune serum specific for the toxin type involved, which may assist in the recovery of animals still in the early stages of the disease. The antiserum should be prepared and standardized according to international convention.1, 10 In South Africa these sera are available from Onderstepoort Biological Products which markets types C and D antisera separately. As the type of C. botulinum responsible for the disease in an animal or group of animals is generally not known until some time period has elapsed (if ever), it is the general practice in South Africa to mix together antisera containing types C and D antitoxin immediately before administration, which is carried out by the intravenous route. As the antisera are expensive they are generally used selectively for the treatment of valuable stock.

The commercially available antisera contain a minimum of 1 000 international units per millilitre, and are prepared from hyperimmunized horses. Cattle and horses are treated with 5 ml each of types C and D antiserum intravenously, sheep with 1 to 2ml of each type, and poultry and other bird species with 0,5 ml type C intravenously. This treatment may be repeated within 24 hours. Response to treatment varies; in some animals beneficial effects may occur within minutes,88 while in others this only occurs after five days.14 Sometimes no beneficial effects become evident. The possibility of the development of anaphylactic shock in animals that are receiving antiserum therapy is significant and should be anticipated. The use of antiserum in horses has been shown to reduce mortality rates from 80 to 30 per cent.113 In horses suffering from type C intoxication, treatment with the specific antiserum will improve the prognosis if it is instituted early in the course of the disease, before they become recumbent. Following the administration of a five day course of the antitoxin, one or two relapses may occur, often at intervals of five to eight days, after which the treatment regimen should be repeated. Horses that survive often develop a severe atrophy of the suprascapular and the gluteal muscles which becomes apparent when the neurological signs of the disease have disappeared, and may persist for up to five months.66

Good nursing is essential when treating animals suffering from botulism. Dehydration, if present, should be corrected but it must always be borne in mind that the muscles of deglutination may be affected, and great care must be exercised when administering fluids by the oral route in order to prevent the possible development of ‘dosing pneumonia’. In both cattle and sheep, acid-base imbalance often occurs and should be corrected. The administration of a mild laxative to horses may be indicated in order to evacuate the colon and prevent impaction. Recumbent animals may require some sort of support system that will keep them in a position of sternal recumbency; this is particularly important in ruminants in order to prevent the formation of ruminal tympany.

The prevention of the form of botulism in range cattle predisposed to osteophagia is three-pronged: vaccination, correction of the phosphorus deficiency and removal of the source of the intoxication, e.g. carcass debris (when this is practically feasible).

Vaccination against botulism has been effectively used in South Africa since 1938.73 Commercially produced vaccines are available, and are produced by Onderstepoort Biological Products. They contain toxoids of types C and D which are adsorbed onto aluminium hydroxide gel in order to enhance their immunogenicity.31 The initial vaccination administration should be followed by a second one four to seven weeks later. Thereafter it is given as an annual booster, timed, if possible, to precede the commencement of the season in which botulism is most prevalent. An experimental vaccine consisting of a water-in-oil emulsion of type C and D toxoids which is administered intramuscularly has been found to induce superior immune responses in cattle when compared to those produced by the aluminium hydroxide-based vaccine.58 It is, however, not commercially available. If the regimen for the active immunization of animals recommended by the manufacturer of the vaccine is not followed, inadequate or only partial protection of the animals may result, and some of them may manifest typical clinical signs of the disease should they subsequently contract it.

As botulism in animals is not always an easy disease to diagnose or to confirm by laboratory means, particularly in the early stages of an outbreak, it is often advisable to recommend that all the animals in the group containing suspected cases be vaccinated as soon as possible.

A combined vaccine for botulism and blackquarter is also available commercially from Onderstepoort Biological Products for use where cattle are to be immunized against both these diseases.

Correction of phosphorus deficiency in cattle is essential in areas where botulism in range animals occurs and, apart from the role it plays in reducing the prevalence of the disease, it has many other beneficial effects, including increased productivity. There are several methods of administering additional phosporus to animals, the most frequently used being licks containing phosphate or bone meal or the addition of phosphates, in the correct formulation, to drinking water.73

The removal of the sources of the intoxication and making them unavailable to animals also helps to reduce the prevalence of botulism. Where cattle are maintained under extensive systems of management are concerned, the source is usually carcass debris. It is perhaps not always practically feasible to remove all of it from large properties, but attempts should be made to remove as much of it as possible. If this is not done, it may be found that in the long term outbreaks of botulism on a property are self-perpetuating. Possible sources of the toxin should be destroyed, buried deeply or placed so that they are unavailable to cattle. If burburied or placed in, for example, a wired-off enclosure it should be remembered that dogs or wild animals, such as hyenas, may dig them up or drag them away to make them available to the cattle once again.

It is imperative that ruminants be vaccinated against botulism before they are fed poultry litter for the first time. Thereafter they should be immunized regularly. In South Africa it is required by law that poultry litter sold commercially be adequately sterilized. Nevertheless it is still essential that ruminants receiving it be adequately protected against botulism by immunization.

Other methods for the control of botulism, particularly that which occurs in sporadic outbreak form, consist of, for example, the prevention of contamination of hay silage and drinking water. Spoilt hay or other feedstuffs, or those contaminated by the carcasses of small animals should not be fed to animals. When silage is prepared, attention should be paid to attaining the correct pH; no production of botulism toxin occurs at a pH of less than 5,3 and where the water activity is less than 0,94.

Foals suckled bydamsvaccinated against botulism are protected by passive, colostrum-derived immunity during the period in which they are most likely to contract toxico-infectious botulism. It is recommended that mares be vaccinated (taking cognizance of the toxin type most likely to be involved) on two occasions at an interval of six weeks, the second being administered two to four weeks before parturition.118

References

  1. APPLETON, N., WILTON-SMITH, P., HAMBLETON, P., MODI, N., GATLEY, S. & MELLING, J., 1986. In vitro assays for botulinum toxin and antitoxins. Development in Biological Standardization, 64, 141–145.
  2. BARSANTI, J.A., WALSER, M., HATHEWAY, L.L., BOWEN, J.M. & CROWELL, W., 1978. Type C botulism in American foxhounds. Journal of the American Veterinary Medical Association, 172, 809–813.
  3. BEIERS, P.R. & SIMMONS, G.C., 1967. Botulism in pigs. Australian Veterinary Journal, 43, 270–271.
  4. BEKKER, V., 1989. The occurrence of Clostridium botulinum types A, B and E in South African habitats. M. Sc. thesis, Department of Microbiology and Plant Pathology, Faculty of Agriculture, University of Pretoria.
  5. BENGSTON, I.A., 1922. Preliminary note on a toxin producing anaerobe isolated from the larvae of Lucilia caesar. Public Health Report, 37, 164.
  6. BENNETTS, H.W. & HALL, H.T.B., 1938. Botulism of sheep and cattle in Western Australia: Its cause and its prevention by vaccination. Australian Veterinary Journal, 14, 105–119.
  7. BERNARD, W., DIVERS, T.J., WHITLOCK, R.H., MESSICK, J. & TULLENERS, E., 1987. Botulism as a sequel to open castration in a horse. Journal of the American Veterinary Medical Association, 191, 73–74.
  8. BISPING, W. & AMTSBERG, G., 1988. Colour Atlas for the Diagnosis of Bacterial Pathogens in Animals. Berlin & Hamburg: Paul Parey Scientific Publishers.
  9. BORTHWICK, J.D., 1896. Annual Report of the College of Veterinary Surgeons. Cape, 1895.
  10. BOWMER, E.J., 1963. Preparation and assay of the International standards for Clostridium botulinum types A, B, C, D and E antitoxins. Bulletin of the World Health Organization, 29, 701–709.
  11. BOROFF, D.A. & DASGUPTA, B.R., 1971. Botulinum toxin. In: kadis, s., montie, t.c. & ajl, s.j., (eds). Microbial Toxins: Bacterial Protein Toxins. Volume IIA, 1–63. London & New York: Academic Press.
  12. BOROFF, D.A. & SHU-CHEN, G., 1973. Radioimmunoassay for type A toxin of Clostridium botulinum. Applied Microbiology, 25, 545–549.
  13. BORST, G.H.A., LAMBERS, G.M. & HAAGSMA, J., 1986. Botulismus type C bij de hond. Tijdschrift voor Diergeneeskunde, 111, 1104–1105.
  14. BREUKINK, H.J., WAGENAAR, G., WENSING, TH., NOTERMANS, S. & POULOS, P.W., 1978. Voedselvergiftiging bij runderen veroorzaakt door het eten van bierbostel besmet met Clostridium botulinum type B. Tijdschrift voor Diergeneeskunde, 103, 303–311.
  15. BRIN, M.F., 1997. Botulinum toxin: Chemistry, pharmacology, toxicology and immunology. Muscle and Nerve (supplement), 6, S144–168.
  16. BURKE, G.S., 1919. Notes on Bacillus botulinus. Journal of Bacteriology, 4, 555–565.
  17. CATO, E.P., GEORGE, W.L. & FINEGOLD, S.M., 1986. Genus Clostridium Prazmowski 1880. In: sneath, p.h.a., mair, n.s., sharpe, m.e. & holt, j.g., (ed.). Bergey’s Manual of Systematic Bacteriology. Vol. II, 1141–1200. Baltimore & London: Williams & Wilkens.
  18. COETZER, J.A.W., 1993. Faculty of Veterinary Science, University of Pretoria, South Africa. Personal communication.
  19. COETZER, J.A.W. & KELLERMAN, T.S., 1993. Faculty of Veterinary Science, University of Pretoria, South Africa. Personal communication.
  20. DEZFULIAN, M. & BARTLETT, J.G., 1984. Detection of Clostridium botulinum type A toxin by enzyme-linked immunosorbent assay with antibodies produced in immunologically tolerant animals. Journal of Clinical Microbiology, 19, 645–648.
  21. DEZFULIAN, M., MCCROSKEY, L.M., HATHEWAY, C.L. & DOWELL, V.R., 1981. Selective medium for isolation of Clostridium botulinum from human feces. Journal of Clinical Microbiology, 13, 526–531.
  22. DINTER, A. & KULL, K.E., 1954. Über einen Ausbruch des Botulismus sanenkuken. Nordisk Veterinaermedicin, 6, 866–872.
  23. DIVERS, T.J., BARTHOLOMEW, R.C., MESSICK, J.B., WHITLOCK, R.H. & SWEENEY, R.W., 1986. Clostridium botulinum type B toxicosis in a herd of cattle and a group of mules. Journal of the American Veterinary Medical Association, 188, 382–386.
  24. DÖBEREINER, J., 1999. Embrapa-CNPAB/PSA, Km 47, Seropédica, Rio de Janeiro 23851-970, Brazil. Personal communication.
  25. DÖBEREINER, J., TOKARNIA, C.H., LANGENEGGER, J. & DUTRA, I.S., 1992. Epizootic botulism of cattle in Brazil. Deutsche Tierärtzliche Wochenschrift, 99, 188–190.
  26. DU TOIT, P.J., LOUW, J.G. & MALAN, A.I., 1940. A study of the mineral content and feeding value of natural pastures in the Union of South Africa (Final Report). Onderstepoort Journal of Veterinary Science and Animal Industry, 14, 123–327.
  27. EKLUND, M.W. & POYSKY, F.T., 1974. Interconversion of type C and D strains of Clostridium botulinum by specific bacteriophages. Applied Microbiology, 27, 251–258.
  28. EKLUND, M.W., POYSKY, F.T., MEYERS, J.A. & PELROY, G.A., 1974. Interspecies conversion of Clostridium botulinum type C to Clostridium novyi type A by bacteriophage. Science, 186, 456–458.
  29. EKLUND, M.W., POYSKY, F.T., REED, S.M. & SMITH, C.A., 1971. Bacteriophage and the toxigenicity of Clostridium botulinum type C. Science, 172, 480–482.
  30. EMERT, L., DUNCKER, H.R., BRUCKNER, H., GRIMMINGER, F., HANSEN, T., ROSSIG, R., AKTORIES, K. & SEEGER, W., 1997. Ultrastructural changes of lung capillary endothelium in response to botulinum C2 toxin. Journal of Applied Physiology, 82, 382–388.
  31. ERASMUS, B.J., CAMERON, C.M., HUNTER, P., CILLIERS, J.A., OBEREM, P.T., STOLTSZ, W.H. & DE WAAL, D.T., 1990. Onderstepoort Vaccines. Booklet issued by the Department of Agriculture and Development and obtained from the Directorate Agricultural Information, Private Bag X144, Pretoria 0001.
  32. FARROW, B.R.H., MURRELL, W.G., REVINGTON, M.L., STEWART, B.J. & ZUBER, R.M., 1983. Type C botulism in young dogs. Australian Veterinary Journal, 60, 374–377.
  33. FERREIRA, J.L., HAMDY, M.K., ZAPATKA, F.A. & HERBERT, W.O., 1981. Immunodiffusion method detection of type A Clostridium botulinum. Applied and Environmental Microbiology, 42, 1057–1061.
  34. fjolstad, m., 1973. The effects of Clostridium botulinum type C13 given orally to goats. Acta Veterinaria Scandinavica, 14, 69–80.
  35. FORAN, P., LAWRENCE, G.W., SHONE, C.C., FOSTER, K.A. & DOLLY, J.O., 1996. Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: Correlation with its blockade of catecholamine release. Biochemistry, 35, 2630–2636.
  36. FOURIE, J.M., 1946. The persistence of botulism toxin in carcass material, with special reference to that in tortoises. Journal of the South African Veterinary Medical Association, 17, 85–87.
  37. FUJINAGA, Y., INOUE, K., WATANABE, S., YOKOTA, K., HIRAI, Y., NAGAMACHI, E. & OGUMA, K., 1997. The haemagglutinin of Clostridium botulinum tye C progenitor toxin plays an essential role in binding of toxin to the epithelial cells of guinea pig small intestine, leading to the efficient absorption of the toxin. Microbiology, 143, 3841–3847.
  38. GIBSON, L.A.S., 1986. Botulism in dairy cows. The Veterinary Record, 118, 309.
  39. GIBSON, A.M., MODI, N.K., ROBERTS, T.A., HAMBLETON, P. & MELLING, J., 1988. Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system. Journal of Applied Bacteriology, 64, 285–291.
  40. GIBSON, A.M., MODI, N.K., ROBERTS, T.A., SHONE, C.C., HAMBLETON, P. & MELLING, J., 1987. Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system. Journal of Applied Bacteriology, 63, 217–226.
  41. GILL, M., 1987. Bacterial toxins. In: laskin, a.i. & lechevalier, h.a., (eds). CRC Handbook of Microbiology. 2nd edn. Vol. VIII. Toxins and Enzymes. Florida: CRC Press, Inc.
  42. GIMENEZ, D.F. & CICCARELLI, A.S., 1970. Another type of C. botulinum. Zentralblat für Bacteriologie, I. Abteil Original, 215, 221.
  43. GRAHAM, R. & BREUCKNER, R.L., 1919. Studies in forage poisoning. The relation of B. botulinus to forage poisoning or cerebrospinal meningitis in horses. Journal of Bacteriology, 4, 1–21.
  44. GUNNISON, J.B. & MEYER, K.F., 1929. Cultural study of an international collection of Clostridium botulinum and C. parabotulism. Journal of Infectious Disease, 45, 119–134.
  45. GUTSCHE, T., 1979. There was a Man. The Life and Times of Sir Arnold Theiler K.C.M.G. of Onderstepoort. Cape Town: Howard Timmins.
  46. HAAGSMA, J., HAESEBROUCK, F., DEVRIESE, L. & BERTELS, G., 1990. An outbreak of botulism type B in horses. The Veterinary Record, 127, 206.
  47. HAAGSMA, J. & TER LAAK, E.A., 1978. Een geval van botulismus type B bij runderen, veroorzaakt door de voedering van kuilgras. Tijdschrift voor Diergeneeskunde, 103, 910–912.
  48. HABERMANN, E. & DREYER, F., 1986. Clostridial neurotoxins: Handling and action at the cellular and molecular level. Current Topics in Microbiology and Immunology, 129, 93–179.
  49. HAY, C.M.E., VAN DER MADE, H.N. & KNOETZE, P.C., 1973. Isolation of Clostridium botulinum type C from an outbreak of botulism in wild geese. Journal of the South African Veterinary Association, 44, 53–56.
  50. HENNING, M.W., 1956. Animal Diseases in South Africa. 3rd edn. South Africa: Central News Agency.
  51. HOBBS, G., CROWTHER, J.S., NEAVES, P., GIBBS, P.A. & JARVIS, B., 1982. Detection and isolation of Clostridium botulinum. In: corry, j.e.l., roberts, d. & skinner, f.a., (eds). Isolation and Identification Methods for Food Poisoning Organisms. London & New York: Academic Press.
  52. HOLDEMAN, L.V., CATO, E.P. & MOORE, W.E.C., 1977. Anaerobic Laboratory Manual. 4th edn. V.P.I. Anaerobic Laboratory, Virginia Polytechnic Institute and State University, Virginia.
  53. HUSS, H.H., 1980. Distribution of Clostridium botulinum. Applied and Environmental Microbiology, 39, 764.
  54. HUTCHEON, D., 1881, 1885, 1896. Lamsiekte. Annual Reports of the College of Veterinary Surgeons, Cape, 1880, 1884, and 1894.
  55. IIDA, H., OGUMA, K. & INOUE, K., 1974. Phage-conversion of toxigenicity in Clostridium botulinum types C and D. Japanese Journal of Medical Science and Biology, 27, 101–102.
  56. JANSEN, B.C., 1971A. The toxic antigenic factors produced by Clostridium botulinum types C and D. Onderstepoort Journal of Veterinary Research, 38, 93–98.
  57. JANSEN, B.C., 1971B. The quantitative determination of the toxic factors produced by Clostridium botulinum (Van Ermengem, 1886) types C and D. Onderstepoort Journal of Veterinary Research, 38, 177–180.
  58. JANSEN, B.C., KNOETZE, P.C. & VISSER, F., 1976. The antibody response of cattle to Clostridium botulinum types C and D toxoids. Onderstepoort Journal of Veterinary Research, 43, 165–174.
  59. JENSEN, R. & SWIFT, B.L., 1982. Diseases of Sheep. 2nd edn. Philadelphia: Lea & Febiger.
  60. JUBB, T.F., ELLIS, T.M. & GREGORY, A.R., 1993. Diagnosis of botulism in cattle using ELISA to detect antibody to botulism toxins. Australian Veterinary Journal, 70, 226–227.
  61. KAUTTER, D.A., LYNT, R.K. & SOLOMON, H.M., 1984. Clostridium botulinum. In: Bacteriological Analytical Manual of the Division of Microbiology Centre for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 6th edn., chapter 18, 18.01-18.10. Virginia: Association of Official Analytical Chemists.
  62. KELLERMAN, T.S., 1993. Veterinary Research Institute, Private Bag X5, Onderstepoort, South Africa. Personal communication.
  63. KELLERMAN, T.S. & COETZER, J.A.W., 1993. Veterinary Research Institute, Private Bag X5, Onderstepoort, South Africa. Personal communication.
  64. KELLY, A.P., JONES, R.T., GILLICK, J.C. & SIMS, L.D., 1984. Outbreak of botulism in horses. Equine Veterinary Journal, 16, 519–521.
  65. KIMBERLING, C.V., 1988. Jensen and Swift’s Diseases of Sheep. 3rd edn. Philadelphia: Lea & Febiger.
  66. KINDE, H., BETTEY, R.L., ARDANS, A., GALEY, F.D., DAFT, B.M., WALKER, R.L., EKLUND, M.W. & BYRD, J.W., 1991. Clostridium botulinum type C intoxication associated with consumption of processed alfalfa hay cubes in horses. Journal of the American Veterinary Medical Association, 199, 742–746.
  67. KOZAKI, S., DUFRENNE, J., HAGENAARS, A.M. & NOTERMANS, S., 1979. Enzyme linked immunosorbent assay (ELISA) for detection of Clostridium botulinum type B toxin. Japanese Journal of Medical Science and Biology, 32, 199–205.
  68. KURAZONO, H., HOSOKAWA, M., MATSUDA, H. & SAKAGUCHI, G., 1987. Fluid accumulation in the ligated intestinal loop and histopathological changes of the intestinal mucosa caused by Clostridium botulinum C2 toxin in the pheasant and chicken. Research in Veterinary Science, 42, 349–353.
  69. LAMANNA, C. 1959. The most poisonous poison. Science, 130, 763–772.
  70. LE VALLIANT, M., 1796. Travels into the Interior Parts of Africa. Vol. II.
  71. LICHTENSTEIN, 1812. Travels in Southern Africa in the Years 1803–1806, (English edition). Van Riebeeck Society.
  72. mackay, r.j. & berkhoff, g.a., 1982. Type C toxicoinfectious botulism in a foal. Journal of the American Veterinary Medical Association, 180, 163–164.
  73. MASON, J.H., STEYN, H.P. & BISSCHOP, J.H.R., 1938. The immunization of bovines against lamsiekte. Journal of the South African Veterinary Medical Association, 9, 65–70.
  74. MCLOUGHLIN, M.F., MCILLROY, S.G. & NEILL, S.D., 1988. A major outbreak of botulism in cattle being fed ensiled poultry litter. The Veterinary Record, 122, 579–581.
  75. MILLS, D.C. & ARNON, S.S., 1987. The large intestine as the site of Clostridium botulinum colonization in human infant botulism. The Journal of Infectious Diseases, 156, 997–998.
  76. MITCHELL, D.T., 1913. Lamsiekte. Second Report of the Director of Veterinary Research, Union of South Africa.
  77. MIYAZAKI, S. & SAKAGUCHI, G., 1978. Experimental botulism in chickens: The caecum as the site of production and absorption of botulinum toxin. Japanese Journal of Medical Science and Biology, 31, 1–15.
  78. MONTECUCCO, C. & SCHIAVO, G., 1995. Structure and function of tetanus and botulinum neurotoxins. Quarterly Review of Biophysics, 28, 423– 427.
  79. MONTECUCCO, C., SCHIAVO, G. & ROSSETTO, O., 1996. The mechanism of action of tetanus and botulinum neurotoxins. Archives of Toxicology (supplement), 18, 342–354.
  80. MONTECUCCO, C., SCHIAVO, G., TUGNOLI, V. & DE GRANDIS, D., 1996. Botulinum neurotoxins: mechanism of action and therapeutic applications. Molecular Medicine Today, 2, 418–424.
  81. NOTERMANS, S., BREUKINK, H.J., WENSING, TH. & WAGENAAR, G., 1978. Voorkomen van Clostridium botulinum in pensinhoud en faeces van runderen gevoederd met bostel welke natuurlijk besmet was met Clostridium botulinum. Tijdschrift voor Diergeneeskunde, 103, 1327– 1333.
  82. NOTERMANS, S., HAGENAARS, A.M. & KOZAKI, S., 1971. The enzyme-linked immunosorbent assay (ELISA) for the detection and determination of Clostridium botulinum toxins A, B and E. Methods in Enzymology, 84, 223–238.
  83. NOTERMANS, S., HAVELAAR, A.H., DUFRENNE, J.B. & OOSTERIEM, J., 1985. Voorkomen van Clostridium botulinum op rund vee houderij bedrijven. Tijdschrift voor Diergeneeskunde, 110, 5, 175–180.
  84. OGUMA, K. & IIDA, H., 1979. High and low toxin production by a non-toxigenic strain of Clostridium botulinum type C following infection with type C phages of different passage history. Journal of General Microbiology, 112, 203–206.
  85. OHISHI, I., 1983. Response of mouse intestinal loop to botulinum C2 toxin: Enterotoxic activity induced by cooperation of nonlinked protein components. Infection and Immunity, 40, 691–695.
  86. PACCHETTI, C., ALBANI, G., MARTIGNONI, E., GODI, L., ALFONSI, E. & NAPPI, G., 1995. ‘Off’ painful dystonia in Parkinson’s disease treated with botulinum toxin. Movement Disorders, 10, 333–336.
  87. PFENNIGER, W., 1924. Toxico-immunologic and serologic relationship of Bacillus parabotulinus type C and B. parabotulinus, ‘Seddon’. Journal of Infectious Disease, 34, 348–352.
  88. PROZESKY, L., 1993. Faculty of Veterinary Science, P.O. Onderstepoort, South Africa. Personal communication.
  89. RAATJES, G.J.M. & SMELT, J.P.P.M., 1979. Clostridium botulinum can grow and form toxin at pH values lower than 4,6. Nature, 281, 398–399.
  90. ROBINSON, E.M., 1930. The bacteria of the Clostridium botulinum C and D types. Sixteenth Report of the Director of Veterinary Services. Union of South Africa.
  91. SCHIAVO, G., BENFENAT, F., POULAIN, B., ROSSETTO, O., POLVERINO DE LAURETO, P., DASGUPTA, B.R. & MONTECUCCO, C., 1992. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature, 359, 832–835.
  92. SEDDON, H.R., 1922. The specific identity of Bacillus parabotulinus. Journal of Comparative Pathology and Therapy, 35, 275–280.
  93. SIMPSON, L.L., 1981. The origin, structure, and pharmacological activity of botulinum toxin. Pharmacological Reviews, 33, 155–188.
  94. SIMPSON, L.L., 1986. Molecular pharmacology of botulinum toxin and tetanus toxin. Annual Review of Pharmacology and Toxicology, 26, 427–453.
  95. SIMPSON, L.L., 1999. Botulism toxin: Potent poison, potent medicine. Hospital Practice (Office edition), 34, 87–91.
  96. SMART, J.L., JONES, T.O., CLEGG, F.G. & MCMURTRY, M.J., 1987. Poultry waste associated type C botulism in cattle. Epidemiology of Infection, 98, 73–79.
  97. SINGH, B.R., LI, B. & READ, D., 1995. Botulinum versus tetanus neurotoxins: Why is botulinum neurotoxin but not tetanus neurotoxin a food poison? Toxicon, 33, 1541–1547.
  98. SMART, J.L., LAING, P.W. & WINKLER, C.E., 1983. Type C botulism in intensively farmed turkeys. The Veterinary Record, 113, 198–200.
  99. SMITH, G.R. & MORYSON, C.J., 1977. A comparison of the distribution of Clostridium botulinum in soil and lake mud. Journal of Hygiene, Cambridge, 78, 39–41.
  100. SMITH, G.R., OLIPHANT, J.C. & EVANS, E.D., 1983. Diagnosis of botulism in water birds. The Veterinary Record, 112, 457–458.
  101. SMITH, G.R. & TURNER, A., 1987. Factors affecting the toxicity of rotting carcasses containing Clostridium botulinum type C. Epidemiology of Infection, 98, 345–351.
  102. SMITH, G.R. & TURNER, A., 1989. The production of Clostridium botulinum toxin in mammalian, avian and piscine carrion. Epidemiology of Infection, 102, 467–471.
  103. SMITH, L.D. & WILLIAMS, B.L., 1984. C. botulinum. In: The Pathogenic Anaerobic Bacteria. 3rd edn. Springfield, Illinois: Charles C. Thomas.
  104. SMITH, L.D.S, 1977. Botulism: The Organism, its Toxins, the Disease. Springfield, Illinois: Charles C. Thomas.
  105. SMITH, L.D.S. & SUGIYAMA, H., 1988. Botulism: The Organism, its Toxin, the Disease. 2nd edn. Springfield, Illinois: Charles C. Thomas.
  106. SOLBERG, M., POST, L.S., FURGANG, D. & GRAHAM, C., 1985. Bovine serum eliminates rapid nonspecific toxic reactions during bioassay of stored fish for Clostridium botulinum toxin. Applied and Environmental Microbiology, 49, 644–649.
  107. SONNABEND, O., SONNABEND, W., HEINZLE, R., SIGRIST, T., DIRNHOFER, R. & KRECH, U., 1981. Isolation of Clostridium botulinum type G and identification of type G botulinal toxin in humans: Report of five sudden unexpected deaths. The Journal of Infectious Disease, 143, 22–27.
  108. STEPHEN, J. & PIETROWSKI, R.A., 1986. Bacterial Toxins. 2nd edn. Washington: American Society for Microbiology.
  109. STERNE, M. & BATTY, I., 1975. Pathogenic Clostridia. Boston & London: Butterworths.
  110. SUEN, J.C., HATHEWAY, C.L., STEIGERWALT, A.G. & BRENNER, D.J., 1988. Clostridium argentinense sp. nov.: A genetically homogeneous group composed of all strains of C. botulinum toxin type G, and some nontoxigenic strains previously identified as C. subterminale or C. hastiforme. International Journal of Systematic Bacteriology, 38, 375– 381.
  111. SUGIYAMA, H., 1980. Clostridium botulinum neurotoxin. Microbiological Reviews, 44, 419–448.
  112. SUGIYAMA, H., 1986. Clostridium botulinum. In: gyles, c.l. & thoen, c.o., (eds). Pathogenesis of Bacterial Infections in Animals. Iowa: Iowa State University Press.
  113. SWERCZEK, T.W., 1980A. Toxicoinfectious botulism in foals and adult horses. Journal of the American Veterinary Medical Association, 176, 217–220.
  114. SWERCZEK, T.W., 1980B. Experimentally induced toxicoinfectious botulism in horses and foals. American Journal of Veterinary Research, 41, 348–350.
  115. THEILER, A., 1920. Cause and prevention of lamsiekte. Journal of the Department of Agriculture, South Africa, June 1920.
  116. THEILER, A., 1927. Lamsiekte (parabotulism) in cattle in South Africa. Eleventh and Twelfth Reports of the Director of Veterinary Education and Research, Part II, 821–1361.
  117. THEILER, A. & ROBINSON, E.M., 1927. Der Botulismes der Haustiere. Zeitschrift für Infektioneskrankheiten, 31, 165.
  118. THOMAS, R.J., ROSENTHAL, D.V. & ROGERS, R.J., 1988. A Clostridium botulinum type B vaccine for prevention of shaker foal syndrome. Australian Veterinary Journal, 65, 78–80.
  119. TRUEMAN, K.F., BOCK, R.E., THOMAS, R.J., TAYLOR, J.D., GREEN, P.A., ROEGER, H.M. & KETTERER, P.J., 1992. Suspected botulism in three intensively managed Australian cattle herds. The Veterinary Record, 130, 398–400
  120. VAN DER LUGT, J., 1993. Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort, South Africa. Personal communication.
  121. VAN DER LUGT, J.J., DE WET, S.C., BASTIANELLO, S.S., KELLERMANT, T.S. & VAN JAARSVELD, L.P., 1995. Two outbreaks of Type C and Type D botulism in sheep and goats in South Africa. Journal of the South African Veterinary Association, 66, 77–82.
  122. VAN ERMENGEM, E., 1912. Botulismus. Handbuch der pathologische Mikrobiologie, Vol. IV. Kolle & Wasserman.
  123. VARNAM, A.H. & EVANS, M.G., 1991. Foodborne Pathogens. An Illustrated Text. London: Wolfe Publishing Ltd.
  124. WALKER, J., 1913. Investigations into the disease lamsiekte in cattle. Second Report of the Director of Veterinary Research, Union of South Africa.
  125. WALLACE, V. & MCDOWELL, D.M., 1986. Botulism in a dog—first confirmed case in New Zealand. New Zealand Veterinary Journal, 34, 149–150.
  126. WEISS, H.E., RADEMACHER, G., DOLL K. & DIRKSEN, G., 1990. Schnelldiagnose des Botulinus beim Rind mittels Mikro-Wärmekomplementbindungsreaktion. Deutsche Tierärtzliche Wochenschrift, 97, 398–400.
  127. WEISS, H.E. & WEISS, H., 1988. Nachweiss von Clostridium botulinum Toxin mittels Mikro-Wärmekomplementbindungsreaktion. Tierärtzliche Unschau, 43, 117–126.
  128. WHITMER, M.E. & JOHNSON, E.A., 1988. Development of improved defined media for Clostridium botulinum serotypes A, B and E. Applied and Environmental Microbiology, 54, 753–759.
  129. WICHTEL, J.J. & WHITLOCK, R.H., 1991. Botulism associated with feeding alfalfa hay to horses. Journal of the American Veterinary Medical Association, 199, 471–472.
  130. WICTOME, M., NEWTON, K.A., JAMESON, K., DUNNIGAN, P., CLARKE, S., GAZE, J., TAUK, A., FOSTER, K.A. & SHONE, C.C., 1999. Development of in vitro assays for the detection of botulinum toxins in foods. Fems Immunology and Medical Microbiology, 24, 319–323.
  131. WILLIS, A.T. & HOBBS, G., 1958. A medium for the identification of clostridia producing opalescence in egg-yolk emulsions. Journal of Pathology and Bacteriology, 75, 299–305.
  132. WOBESER, G. & GALMUT, E.A., 1984. Internal temperature of decomposing duck carcasses in relation to botulism. Journal of Wildlife Diseases, 20, 267–271.
  133. YAMAKAWA, K., KAMIYA, S., YOSHIMURA, K. & NAKAMURA, S., 1992. Clostridium botulinum type C in healthy swine in Japan. Microbiology and Immunology, 36, 29–34.