Type C botulism in young dogs

A diffuse lower motor neurone paralysis developed in a 6-month-old male Australian cattle dog pup 4 days after it had eaten the carcase of a rotting duck in Centennial Park, Sydney. Two other dogs which ate smaller portions of the same carcase were less severely affected. Clostridium botulinum type C was isolated from and C. botulinum type C toxin was detected in faeces from the severely affected dog. The serum contained 25 LD50 of toxin/ml. The high C. botulinum count and toxin level in the faeces declined progressively during the ensuing weeks, but 114 days after ingesting the carcase C. botulinum type C was still present in faeces and a low toxin titre persisted. Soil, mud and water samples in the area of the duck ponds in the park contained C. botulinum type C spores. Spores and high toxin titres were also found in the intestine of the carcases of 2 birds in the area.     

The occurrence of Clostridium botulinum type E in Finnish trout farms and the prevention of toxin formation in fresh-salted vacuum-packed trout fillets.

The occurrence of C. botulinum on two Finnish rainbow trout farms were studied. C. botulinum type E toxin was detected from samples of fish intestines in 10% and 4% of the samples and in 0% and 100% of dam bottom sediments, respectively. The toxin formation of inoculated C. botulinum type E in three different brands of commercial fresh-salted vacuum-packed trout fillets was also investigated. In the brand with a salt concentration of 2.7% (a w = 0.96) and no nitrate, the toxin was formed in two weeks at 10 and 20 degrees C. If the pH of the product was lowed to 4.90 and NaCl concentration increased to 5.67% (a w = 0.94%) no toxin was formed even without the use of nitrate. By adding 0.1% of sodium nitrate to the curing solution (0.046% nitrate in the product) the toxin formation was brought to an end. According to the results of the study C. botulinum type E presents a potential health risk for man as well as for fish also in Finland. Trout products must be manufactured and stored in such a way that the possibility of toxin formation is eliminated.     

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.     

The occurrence of Clostridium botulinum at aquatic sites in and around Auckland and other urban areas of the North Island

Samples of bottom muds from lakes and waterways in the Auckland area were inoculated into bottles of cooked meat medium. After incubation, the media were tested for production of Clostridium botulinum toxin which was detected with samples from 11 of the 20 sites examined. Although toxins produced from all positive samples were neutralized by specific antisera of both type C and D toxins, it is likely that C. botulinum type Calpha is the only type present in the Auckland area. All samples from other urban areas of the North Island gave negative results.     

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.     

Emergence of suspected type D botulism in ruminants in England and Wales (2001 to 2009), associated with exposure to broiler litter

Scanning surveillance by the Veterinary Laboratories Agency revealed the emergence of suspected botulism in ruminants in 2003, presented as flaccid paralysis. From 2003 to 2009, 168 cattle and 19 sheep incidents were recorded, with mortality between 5 and 80 per cent. All sheep incidents and 95 per cent of cattle incidents had proximity to broiler litter. From July 2006, the gut contents collected from 74 affected cattle and 10 affected sheep were tested for Clostridium botulinum toxins using mice bioassays and for organisms by culture. Type D toxin was identified in 32 per cent of cattle and 18 per cent of sheep samples. C botulinum type D organisms were identified in 40 per cent of cattle and 30 per cent of sheep samples, but broth from one sample reacted with C and D antisera. Type C botulism has previously been reported more commonly than type D in the UK and has been associated with the use of poultry litter as fertiliser, bedding or feed. The almost exclusive association with C botulinum type D toxins or organisms in the gut contents in this survey suggests a change in the source or epidemiology of botulism in the UK. The source of C botulinum type D was uncertain. Broilers may carry C botulinum type D in their gut flora subclinically. The emergence of a new type D strain, or changes in broiler husbandry and nutrition, medication and other enteric infections may have affected colonisation with C botulinum. Further investigation of poultry and farm environments for sources of type D awaits the development of tests for C botulinum toxins that do not require the use of mice.     

Diagnosis of botulism types C and D in cattle by a monoclonal antibody-based sandwich ELISA

This study was undertaken to evaluate two monoclonal antibody-based sandwich ELISAs (sELISAs) for the detection of Clostridium botulinum neurotoxins (BoNTs) types C and D from culture-enriched intestinal content samples from cattle. To validate the diagnostic significance of the presence of cultivable, toxin-producing C botulinum in the intestines of cattle, samples from both suspect and non-suspect botulism cases were examined. BoNT was detected by both sELISAs in a greater number of suspect animals than by direct testing of uncultured samples by mouse bioassay. One sELISA detected two BoNT C and one BoNT Group III mosaic isoform in three animals that were missed by the other, and both sELISAs failed to identify samples from two mouse bioassay-positive BoNT C animals. BoNT D was also detected in one non-suspect sample by one of the sELISAs.     

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.     

Tonsils--place of botulinum toxin production: results of routine laboratory diagnosis in farm animals

Since a case of a veterinarian was reported, who was likely to be infected/intoxicated by Clostridium botulinum during the handling of a diseased animal, tonsils in animals were tested for botulinum neurotoxin and bacterial forms of neurotoxic Clostridium botulinum during routine botulism laboratory examinations including standard samples (intestinal tract and liver) from 48 cattle, 11 horses, and 14 goats. Ten out of 60 samples from tonsils contained free botulinum toxin, and 12 out of 59 were positive for live toxin producing bacteria. In 32 out of 162 intestinal samples toxin was detected. Toxin producing bacteria were found in 37 samples. Eight of 56 liver samples contained free toxin, and 15 out of 43 toxigenic bacteria. Samples from 10 slaughter pigs were all negative, whereas from slaughter cattle tonsils had a high incidence of toxin (7 of 10) or toxigenic bacteria (2 of 8). The results are discussed in the context of effects on animal health and botulism as zoonosis.     

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.     

聚合酶链反应（PCR）鉴定猪肉方法的建立及应用

依据猪小卫星ＤＮＡ序列设计了１对引物,建立了ＰＣＲ鉴定生、熟猪肉的方法。应用本方法特异地扩增出预期的猪３０７ｂｐ的ＤＮＡ片段,其检测敏感度对生猪肉达３８．６ｆｇＤＮＡ,对熟猪肉和高压猪肉为０．３７５ｆｇ全基因组ＤＮＡ。运用该引物仅可扩增生猪ＤＮＡ的单一片段,而对马、牛、羊等１２种动物肉的ＤＮＡ扩增则呈阴性。应用本法对６３份生、熟猪肉进行鉴定,正确率为１００％,对各种样品检测,均可在６ｈ内完成。     

Two cases of equine grass sickness with evidence for soil-borne origin involving botulinum neurotoxin

Botulism is caused by different types of Clostridium botulinum, a soil bacterium. Equine grass sickness (equine dysautonomia) is suspected of being a clinical form of this disease. On a stud where this disease occurred twice within 8 months, grass and soil samples and necropsy specimens of one horse were tested for the presence of bacterial forms and toxin of C. botulinum. Different types and type mixtures (A-E) of C. botulinum and botulinum neurotoxin were found. For the first time, it has been shown that green grass blades contain botulinum toxin. The results support the hypothesis that equine grass sickness is a clinical form of botulism, a soil-borne disease.     

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.     

Two Cases of Equine Grass Sickness with Evidence for Soil‐borne Origin Involving Botulinum Neurotoxin

Botulism is caused by different types of Clostridium botulinum, a soil bacterium. Equine grass sickness (equine dysautonomia) is suspected of being a clinical form of this disease. On a stud where this disease occurred twice within 8 months, grass and soil samples and necropsy specimens of one horse were tested for the presence of bacterial forms and toxin of C. botulinum. Different types and type mixtures (A–E) of C. botulinum and botulinum neurotoxin were found. For the first time, it has been shown that green grass blades contain botulinum toxin. The results support the hypothesis that equine grass sickness is a clinical form of botulism, a soil‐borne disease.     

Evaluation of immunochromatographic test of Shiga toxin 2e in enrichment cultures of swine edema disease clinical samples

To simplify the diagnosis of swine edema disease, overnight culture supernatants of swine clinical samples were assayed using immunochromatographic test strips we developed previously. Small-intestinal contents, mesenteric lymph nodes, and fecal samples were cultured in casamino acid-yeast extract broth overnight, after which supernatants were loaded onto immunochromatographic test strips to determine whether they could detect Shiga toxin 2e (Stx2e). Among 23 clinical samples in which PCR-identified stx2e-positive E. coli were isolated, samples from seven of ten small-intestinal contents, one of three mesenteric lymph nodes and six of ten fecal samples showed Stx2e-positive reactions in the protein-based immunochromatographic test. Additionally, one small-intestinal content sample, in which stx2e-positive E. coli were not isolated, showed an Stx2e-positive reaction. Furthermore, the immunochromatographic test results of the samples were associated with the toxin concentration determined by sandwich ELISA and cytotoxicity assay results on Vero cells. The toxin concentration range of the samples with positive and negative reactions were 2.1–196.2 ng/ml and 0–12.8 ng/ml, respectively. The sensitivity and specificity of this immunochromatographic test strip calculated from all clinical samples analyzed in this study were 60.9% and 94.4%, respectively. Our immunochromatographic test strip has strong potential for simple and accurate diagnosis for edema disease by detecting toxin expression, complementing the PCR method.     

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.     

Botulism of aquatic birds at the Stary u Pohorelic pond (Breclav District)

In the years 1981 and 1982 when a mass mortality of wild and domestic water birds was observed on the Stary pond, eleven diseases or just died birds were examined by the neutralization test for botulotoxin and four sludge samples for the presence of Clostridium botulinum. C. Botulinum toxin of C type was detected in four wild ducks (Anas platyrhynchos) from September 1981 and July 1982 and in one gull (Larus ridibundus) and July 1982. The highest titres (of mice i. p. LD50/g) of botulotoxin in ducks were 10(6.3) in the intestinal contents, 10(6.1) in the liver tissue and 10(5.1) in the stomach contents. In one duck we detected, besides the C type, a smaller botulotoxin amount of A type. Therefore we can recommend a complete serotypifying for the cases of botulism in water birds. Out of four sludge samples collected in September 1982 one sample was negative, whereas in three samples the C botulotoxin was demonstrated after propagation and one C. botulinum strain of C type was isolated.     

Evaluation of conventional and radiometric fecal culture and a commercial DNA probe for diagnosis of Mycobacterium paratuberculosis infections in cattle.

Radiometric (RCM) and conventional fecal culture (HEY) and a commercial polymerase chain reaction/DNA probe were evaluated as diagnostic tests for subclinical paratuberculosis in dairy cattle using fecal specimens from a repository of paratuberculosis specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis, by conventional or radiometric culture, from fecal samples or internal organs of dairy cattle without diarrhea or chronic weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. Among 182 infected cattle, RCM and HEY fecal culture and the DNA probe had test sensitivities of 54.4%, 45.1% and 33.5%, respectively. Fecal samples from only 111 of the M. paratuberculosis-infected cows tested positive by at least one of the three tests and these cows were designated as fecal shedders; the remaining 71 were considered to have prepatent infections. Among the 111 M. paratuberculosis fecal shedders, RCM, HEY and the probe detected the organism in 89.2%, 73.8% and 55.0% of the fecal specimens, respectively. Herd prevalence significantly affected the sensitivity of all three diagnostic tests (p less than 0.05) but only affected the fecal shedder detection efficiency of the DNA probe (p less than 0.01). No positive DNA probe results were found on 100 randomly selected fecal samples from cows in four certified paratuberculosis-free herds, thus the DNA probe was 100% specific. Probe analyses could be performed in 24 h or less. Time to complete the culture-based tests was 12 wk for HEY and 7 wk for RCM.(ABSTRACT TRUNCATED AT 250 WORDS)     

The prevalence of verotoxins, Escherichia coli O157:H7, and Salmonella in the feces and rumen of cattle at processing.

Fecal samples collected from cattle at processing during a 1-year period were tested for verotoxins (VT1, VT2), Escherichia coli O157:H7, and Salmonella. Verotoxins were detected in 42.6% (95% CI, 39.8% to 45.4%), E. coli O157:H7 in 7.5% (95% CI, 6.1% to 9.1%), and Salmonella in 0.08% (95% CI, 0.004% to 0.5%) of the fecal samples. In yearling cattle, the median within-lot prevalence (percentage of positive samples within a lot) was 40% (range, 0% to 100%) for verotoxins and 0% for E. coli O157:H7 (range, 0% to 100%) and Salmonella (range, 0% to 17%). One or more fecal samples were positive for verotoxins in 80.4% (95% CI, 72.8% to 86.4%) of the lots of yearling cattle, whereas E. coli O157:H7 were detected in 33.6% (95% CI, 26.0% to 42.0%) of the lots. In cull cows, the median within-lot prevalence was 50% (range, 0% to 100%) for verotoxins and 0% (range, 0% to 100%) for E. coli O157:H7 and Salmonella (range, 0% to 0%). Verotoxins were detected in one or more fecal samples from 78.0% (95% CI, 70.4% to 84.2%) of the lots of cull cows, whereas E. coli O157:H7 were detected in only 6.0% (95% CI, 3.0% to 11.4%) of the lots of cull cows. The prevalence of verotoxins in fecal samples was lower in yearling cattle than in cull cows, whereas the prevalence of E. coli O157:H7 in fecal samples was higher in yearling cattle than in cull cows. The prevalence of E. coli O157:H7 in fecal samples was highest in the summer months. Rumen fill, body condition score, sex, type of cattle (dairy, beef), and distance travelled to the plant were not associated with the fecal prevalence of verotoxins or E. coli O157:H7. The prevalence of verotoxins in fecal samples of cull cows was associated with the source of the cattle. It was highest in cows from the auction market (52%) and farm/ranch (47%) and lowest in cows from the feedlot (31%). In rumen samples, the prevalence of verotoxins was 6.4% (95% CI, 4.2% to 9.4%), and it was 0.8% (95% CI, 0.2% to 2.3%) for E. coli O157:H7, and 0.3% (95% CI, 0.007% to 1.5%) for Salmonella.