Outbreak of botulism in a dairy herd in Turkey

In this study, the clinical findings and results of haematological and biochemical analyses of 26 cattle with botulism were evaluated. The most important clinical signs in the affected cattle included: decreased appetite, ataxia, difficulty to rise, loss of tongue tone, salivation and bradycardia. A definitive diagnosis of botulism was based on demonstration of the preformed toxin in ruminal and intestinal contents and feed materials including poultry litter, by mouse inoculation test. This study is the first confirmation, by direct toxin isolation, of Clostridium botulinum type C and Clostridium botulinum type D in cattle, in Turkey.     

Investigation of serology for diagnosis of outbreaks of botulism in cattle

Serology has been used to diagnose retrospectively types C and D outbreaks of botulism in cattle in Australia and this study has investigated whether the approach would be applicable in England and Wales. Three hundred sera from routine surveillance submissions in England and Wales were used as a negative control population. Some stored sera were available from a small number of clinical cases of botulism and 125 samples were collected from cohort groups of clinical cases in four new outbreaks of botulism. Three of these outbreaks were identified as being caused by type D Clostridium botulinum toxin. Sera were tested by antibody ELISA in laboratories in Australia and Germany. There was no increase in the proportion of animals seropositive to type C or D antibody in the botulism-associated cattle. The proportion of samples which were seropositive to type D antibodies was <2% in both the negative control and outbreak populations. It was concluded that single time serology is unlikely to be helpful for retrospective diagnosis of outbreaks of type D botulism in England and Wales.     

Determination of the median toxic dose of type C botulinum toxin in lactating dairy cows.

Because of the difficulty in identifying botulinum toxin in cattle, it is hypothesized that cattle are sensitive to levels of toxin below the detection limits of current diagnostic techniques (the mouse protection bioassay and the immunostick enzyme-linked immunosorbent assay [ELISA] for type C botulinum toxin). Using an up-down method for toxicologic testing, the median toxic dose (MTD50) for cattle was determined. Four lactating Holstein cows were dosed at 0.125 or 0.25 ng/kg with Clostridium botulinum type C toxin and failed to develop clinical signs of botulism during the 7-day observation period. Three cows given 0.50 ng/kg of toxin developed clinical signs of botulism. From these results, the MTD50 was calculated at 0.388 ng/kg (3.88 mouse lethal doses/kg) using the trim-logit method. These results suggest that cattle are 12.88 times more sensitive to type C botulinum toxin than a mouse on a per kilogram weight basis. The mouse protection bioassay and the immunostick ELISA for type C botulinum toxin failed to identify the presence of the toxin in the serum, blood, and milk samples taken from all 7 animals.     

Use of enzyme-linked immunoassays for antibody to types C and D botulinum toxins for investigations of botulism in cattle

The development of specific enzyme-linked immunosorbent assays (ELISA) for antibody to types C and D Clostridium botulinum toxins for investigation of botulism in cattle is described. Partially purified type C and D toxins were used as antigens to develop these ELISAs. Specificity of the ELISAs was evaluated on sera from 333 adult beef and dairy cattle from areas with no history or evidence of botulism in animals or water birds. The test was also evaluated on sera from 41 herds that included herds vaccinated against botulism, confirmed botulism cases and herds from areas where the disease is considered endemic. The ELISAs detected the presence of antibody to botulinum toxins in samples from vaccinated cattle and both convalescent and clinically normal animals from unvaccinated herds with outbreaks of botulism. Antibody was also found in unvaccinated animals from herds in which there had been no diagnosed botulism cases in areas where botulism was considered endemic. Sera from some unvaccinated cattle with high ELISA reactivity was shown to be protective for mice in botulinum toxin neutralisation tests. The use of these tests in investigations of botulism in cattle is discussed.     

Evaluation of enzyme-linked immunosorbent assay for diagnosis of Clostridium perfringens enterotoxemias.

Two double sandwich enzyme-linked immunosorbent assays (ELISA) for Clostridium perfringens beta and epsilon toxins were assessed for routine diagnosis of enterotoxemias on intestinal contents of 151 sheep that died suddenly. Conventional tests (mouse assay and culture of organism) showed that 21 specimens were positive for Clostridium perfringens type C (beta toxin) and 39 were positive for Clostridium perfringens type D (epsilon toxin) enterotoxemias. Comparison of the ELISA results with conventional assays gave sensitivity and specificity rates respectively of 90.5% and 89.2% for beta toxin assay and 97.4% and 94.6% for epsilon toxin assay. With further refinement to improve the performance of the assay for beta toxin these tests could serve as a substitute for conventional tests in the laboratory diagnosis of Clostridium perfringens types B, C and D enterotoxemias.     

Protective effect of botulinum C/D mosaic toxoid against avian botulism

Avian botulism is a paralytic disease caused by a toxin produced by Clostridium botulinum type C. Since type C isolates from cases of avian botulism produced a neurotoxin consisting of a mosaic form of parts of type C and D neurotoxins, we examined the antitoxin titers in the convalescent sera of botulism-affected birds which belonged to family Anatidae. ELISA using the C/D mosaic neurotoxin as an antigen revealed that the antibody was detected in the sera at 2 weeks, but not at 5 weeks after the onset, suggesting that the antibody only appeared for a short period in the convalescent phase. However, we failed to detect the antibody titers with anti-chicken IgG instead of anti-duck IgG. We therefore examine the immunological properties of IgG among different families and species. The results revealed that different species of IgG in the same family exhibited strong cross-reactivity. Ducks immunized once with the toxoid together with a commercial oil-adjuvanted vaccine were found to develop sufficient antibody to protect against a challenge with a lethal toxin dose. The ELISA titers did not correspond to the neutralization titers in the sera of immunized ducks at the early stage during immunization. These findings suggest that the neutralizing titer was more useful than the ELISA titer for evaluating the protection against the toxin, but the ELISA technique may be applicable for detecting the occurrence of botulism.     

Detection of Clostridium perfringens epsilon toxin by ELISA

An enzyme-linked immunosorbent assay (ELISA) has been developed as an alternative to neutralisation tests in mice to detect Clostridium perfringens type D epsilon toxin in the intestinal contents of animals which have died from suspected enterotoxaemia. The test was sensitive and quantitative and gave excellent agreement with the mouse protection test.     

Comparative immunogenicity of two bivalent botulinum vaccines

OBJECTIVE: To compare the ability of a new single-dose botulinum vaccine containing a non-mineral oil adjuvant with a single dose of a conventional botulinum vaccine product to produce antibody to Clostridium botulinum types C and D in cattle in Northern Australia. DESIGN AND PROCEDURE: One hundred and fifty Brahman steer weaners were randomly divided into two groups receiving either a single dose of CSL Bivalent Botulinum vaccine or Websters Singvac. Blood samples were collected at 0, 8 and 24 weeks and tested by antibody ELISA. The final samples were also tested by the toxin neutralisation test, to test titres of neutralising antibody. RESULTS: Six months after inoculation, cattle vaccinated with Websters Singvac had ELISA antibody response twice that of CSL conventional product. However, this difference was only evident for neutralising antibody to type C botulinum toxin. Both products produced similar titres of type D neutralising antibody after a single dose. CONCLUSION: Websters' Singvac produces a greater neutralising antibody response to type C botulism upon single inoculation than a conventional vaccine. The product produces an equivalent neutralising antibody response to type D.     

Type C botulism in cattle being fed ensiled poultry litter

A botulinum toxin from ensiled poultry litter which caused a major outbreak of bovine botulism was characterised as type C1. The litter produced transient ataxia when fed to two experimental calves and the clinical signs were accompanied by a transient appearance of serum toxin. Type C1 toxin was demonstrated in muscle tissues which had been taken during the outbreak from an affected animal with high circulating serum toxin, and held frozen for seven months. Clostridium botulinum type C organisms were demonstrated in faeces from another affected animal and also in kidney tissue from a third animal. These observations have implications for the diagnosis and management of future outbreaks of botulism and for the potential health risk from the meat of affected animals.     

Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum

Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities.     

Susceptibility of mink to Clostridium botulinum type E toxin

Experiments aiming at elucidation of the toxicity of Clostridium botulinum type E for mink are described. The observations indicate that amounts in the order of 2 x10^s intraperitoneal MLD (mice) or approximately 200 MLD per g of type E toxin will kill a mink after oral administration. The symptoms observed in the animals were atypical as there was an unusually short period between administration of the toxin and the onset of symptoms and deaths of the animals. Similar results were obtained when Clostridium botulinum type E toxin was fed to Swiss mice. When mice were protected by subcutaneous injections of type E antitoxin prior to feeding the animals survived without showing any symptoms.Subcutaneous injection of type E toxin in amounts of the order of 2 x10^s intraperitoneal MLD (mice) killed mink, and typical symptoms of botulism were observed. This quantity corresponds to ap-proximately 2 intraperitoneal MLD (mice) per g.Comparison is made with previous observations obtained in similar experiments made with Clostridium botulinum type C toxin. It is shown that mink arc substantially less susceptible to type E than to type C toxin when the toxins are given by mouth. On this basis previous results in reports on outbreaks of botulism in mink caused by Clostridium botulinum type E may be regarded as questionable.     

Clostridium botulinum type D intoxication in a dairy herd in Ontario

Thirty-four Holstein cows died after exposure to Clostridium botulinum type D toxin, presumably from contaminated haylage. The presence of type D toxin in ruminal contents was confirmed by mouse inoculation. This is the first confirmation by direct toxin isolation of C. botulinum type D toxin in cattle in North America.     

A major outbreak of botulism in cattle being fed ensiled poultry litter

Eighty of a group of 150 housed beef cattle showed classical signs of botulism after eating a batch of ensiled poultry litter. Sixty-eight of the animals died and Clostridium botulinum type C toxin was detected in 18 of 22 sera examined. C botulinum organisms were isolated from the ensiled litter and type C toxin was demonstrated in samples of decomposed poultry carcases present in the litter. This outbreak of bovine botulism was the most serious to have been recorded in Europe and was the first associated with feeding ensiled poultry litter.     

Thirteen cases of botulism in horses fed big bale silage

An outbreak of pharyngeal and limb paresis involving four horses and nine ponies in the south east of England is described. Nine of the animals died or were destroyed on humane grounds. The clinical features suggested a diagnosis of botulism and mouse innoculation tests confirmed the presence of type B toxin in the serum of one case. All animals were fed big bale silage. It is describe how, in plastic wrapped silage manufacture, conditions of fermentation may be inadequate to prevent the growth of Clostridium botulism. Examination of a sample of silage fed to the affected horses suggested that this was probably the source of the toxin.     

Fatal Clostridium botulinum toxicosis in eleven Holstein cattle fed round bale barley haylage.

Twenty-two lactating Holstein cattle in Tennessee had clinical signs of intoxication with preformed Clostridium botulinum toxin. These signs included weakness, paralysis of the tongue and chest muscles, abdominal breathing, and, in 11 of the 22 cows, death. Differential diagnoses included hypocalcemia, hypomagnesemia, carbohydrate overload, and several toxicoses including mycotoxin, lead, nitrate, organophosphate, atropine or atropine-like alkaloid, and botulism. A diagnosis of botulism by the ingestion of preformed C. botulinum type B toxin was made by eliminating these other diseases, by finding C. botulinum type B spores in 3 bales of round bale barley haylage fed to these cattle, and by isolating preformed type B toxin from 1 of the 3 bales. Confirmation of the toxin type was made by demonstrating mouse lethality by intraperitoneal injection of specimen extracts with neutralization by C. botulinum type B antitoxin. The haylage, harvested green and encased in black plastic bags to facilitate fermentation, was presumably contaminated by the botulinum toxin when fermentation failed to produce enough acid to lower the pH to 4.5, the pH below which C. botulinum growth is inhibited. Farmers and ranchers who use round hay balers to produce haylage should be alert to this potential problem.     

Botulism in chickens associated with elevated iron levels

Clostridium botulinum type C toxicosis was diagnosed by the mouse inoculation test in two outbreaks of botulism in commercial broiler and roaster chickens. One case involved 7-wk-old commercial roaster chickens, and the other involved 15-day-old commercial broiler chickens. A definitive point source for preformed C. botulinum exotoxin was not identified in either case investigation. Elevated iron concentrations in the drinking water and/or feed may have presented a significant risk factor that may have resulted in intestinal proliferation of C. botulinum and subsequent botulism.     

Susceptibility of Foxes to Clostridium Botulinum Type C and E Toxins

Investigations were performed to determine the exact susceptibility of foxes to Clostridium botulinum type C and E toxins.Doses of 5 mill. MLD type C toxin mixed with the feed did not cause symptoms of botulism in either cubs or adult foxes. Subcutaneous injections of 300,100(0 MLD or more were fatal to cubs, while 750,000 MLD caused the death of all adults.Regarding type E toxin, doses of 1 mill. MLD affected neither cubs nor adults on oral administration. Subcutaneously injected doses of 5,000 MLD or more killed all cubs, while 10,000 MLD was required to produce lethal effect on adult animals.The conclusion made is that foxes are highly resistant to both type C and E Clostridium botulinum toxins following oral application. It is further revealed that foxes are 60–70 times more susceptible to type E than to type C toxin when injected subcutaneously.     

Presumptive diagnosis of Clostridium botulinum type D intoxication in a herd of feedlot cattle

Fifty-two feedlot cattle exhibited clinical signs suggestive of botulism. Clostridium botulinum type D organisms were recovered from ruminal fluid of 4 of the 5 affected animals tested and were isolated from bakery waste fed to the cattle. Clostridium botulinum type D has not been reported previously in Canadian cattle.