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.     

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

Fluid accumulation was induced in ligated intestinal loops of both pheasants and chickens injected with crude Clostridium botulinum C2 toxin. Necrosis of the epithelium and lamina propria of the duodenum and jejunum occurred in both species of bird after intraduodenal administration of crude C2 toxin. The severity of such reactions depended upon the dose and the period after administration of C2 toxin up to eight hours, and such reactions were suppressed specifically with rabbit anti-C2 toxin. These results confirm that C2 toxin is one of the causes of diarrhoea in avian botulism.     

Characterization of the neurotoxin produced by isolates associated with avian botulism

Several varieties of birds are affected by type C botulism. We conducted neutralization tests of culture supernatants of isolates from cases of avian botulism. Whereas the toxin produced by isolates derived from mammalian botulism was neutralized only with type C antitoxin, the toxins of all isolates related to avian botulism were neutralized with both type C and D antitoxins. An analysis of nucleotide sequences with several strains revealed that the neurotoxin gene in the isolates from avian botulism comprises two thirds of the type C neurotoxin gene and one third of the type D neurotoxin gene. This indicates that the neurotoxin of avian isolates is a mosaic of type C and D neurotoxins. We prepared three sets of primers to differentiate the gene for the mosaic form from the conserved genes of type C and D neurotoxins. The results of polymerase chain reaction with these primers indicated that all avian botulism-related isolates and specimens possess the gene for the mosaic form of the neurotoxin. The toxins purified from avian and mammalian isolates exhibited the same degree of lethality in mice, but the former showed greater toxicity to chickens than the latter. These results indicate that the mosaic neurotoxin is probably a pathogenic agent causing some forms of avian botulism.     

肉毒中毒症疫苗的研究现状

用于预防肉毒中毒症的传统疫苗有灭活菌苗和类毒素疫苗,近年来对于重组亚单位疫苗、重组嵌合体疫苗和DNA疫苗等研究成为热点,文章着重对这些新型疫苗的发展现状进行了综述,以期为相关研究提供借鉴。     

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.     

Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan

Clostridium botulinum types C and D are related to avian and mammalian botulism. Bovine botulism occurred at various farms from 2004 to 2007 in Japan. Since culture supernatants of isolates from cases of bovine botulism were neutralized completely and partially with type D and C antitoxins, respectively, we attempted to confirm the nucleotide sequences of the neurotoxin gene in isolates. The neurotoxin gene comprised two-thirds of the type D neurotoxin gene and one-third of the type C neurotoxin gene, indicating that the neurotoxin of bovine isolates is a mosaic of type D and C neurotoxins, D/C mosaic neurotoxin. We prepared four sets of primers to differentiate the genes of the mosaic and authentic forms with PCR. The results showed that all bovine botulism-related isolates possess the gene for the D/C mosaic form. Pulsed-field gel electrophoresis analysis demonstrated that isolates from bovine botulism which had occurred between 2004 and 2007 were genetically homologous, except for the isolate from one area. We further examined the biological and antigenic properties of the D/C mosaic neurotoxin, which was found to exhibit the highest lethal activity in mice compared with other types of neurotoxins. In the D/C mosaic neurotoxin, three epitopes recognized by monoclonal antibodies that specifically react to and neutralize the toxin were located in the carboxyl-terminal domain of the heavy chain. These results indicate that D/C mosaic neurotoxin is a pathogenic agent causing bovine botulism and has unique characteristics different from other type C and D neurotoxins.     

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.     

蓝马鸡和藏马鸡的血清酯酶同工酶酶谱

采用聚丙烯酰胺凝胶垂直平板电泳法对 1 9只蓝马鸡和 1 5只藏马鸡的血清酯酶同工酶酶谱进行了研究。结果发现 :(1 ) 2种马鸡的血清酯酶存在ES1 ,ES2和ES3 3种同工酶 ;(2 )蓝马鸡的ES1同工酶存在显现酶活性的ES1A (36 8% )和不显现酶活性的ES1O (63 2 % ) 2种表型 ,而藏马鸡全部为ES1O型 ;(3)ES2同工酶只有一条浓染的酯酶区带 ;(4)ES3同工酶存在ES3ABC ,ES3AB和ES3BC 3种表型 ,蓝马鸡和藏马鸡均以ES3ABC为优势表型 (分别为 68 4%和 60 0 % )。     

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.     

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.     

Detection of Clostridium botulinum types C and D toxin by ELISA

An ELISA to detect Clostridium botulinum type D toxin was developed using polyclonal antibodies to a semi-purified toxic complex of the neurotoxin. Sensitivity of the ELISA for detecting type C and type D toxin compared with mouse inoculation was 70% and specificity 96% on samples from animals with botulism diagnosed on clinical signs and herd history. However, both mouse inoculation and the ELISA failed to detect toxin in many animals with a presumptive diagnosis of botulism. Some cross-reaction was seen with Clostridium novyi type A, but not with other clostridial species. While the ELISA described here cannot replace mouse inoculation for the diagnosis of botulism, it is a useful additional test.     

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.     

Alternative vaccination against equine botulism (BoNT/C)

REASON FOR PERFORMING STUDY: In Europe the incidence of botulism in horses has increased in the last decade due to the growing popularity of haylage feeding. Recombinant vaccines are safer and less expensive to produce and are generally better tolerated than toxoids. OBJECTIVES: To investigate whether the recombinant C-terminal half of the heavy chain of the botulinum neurotoxin C (Hc BoNT/C) in combination with an immunstimulatory adjuvant is an appropriate vaccine candidate for horses by testing its efficacy to induce neutralising antibodies and by comparing its immunogenic properties and adverse reactions to a commercial toxoid vaccine. Formation of oedema and local pain reactions were assessed. ELISA and Western blot assay against Hc BoNT/C and testing of neutralising antibody induction in a mouse protection assay were used to evaluate the immune response. RESULTS: With the recombinant vaccine, only minor local swelling with full recovery after 5 days was noted after brisket injections. The toxoid vaccine produced local, painful reactions with longer recovery periods of up to 2 weeks. Horses vaccinated with either vaccine induced neutralising antibodies after the second booster vaccination, while seroconversion on ELISA and Western blot to Hc BoNT/C was apparent after the first recombinant vaccination, and at various time points in the vaccination schedule in horses that received commercial toxoid vaccine. CONCLUSION: The recombinant vaccine showed fewer adverse reactions compared to the only commercially available vaccine but induced similar concentrations of neutralising antibodies. There was no correlation between the serological response to Hc BoNT/C and the neutralising capacity of serum. POTENTIAL RELEVANCE: Recombinant Hc BoNT/C is an appropriate vaccine candidate to stimulate production of neutralising antibodies against botulinum neurotoxin C in horses and creates only minor local reactions at the injection site.     

Changes in haematological profile of common pheasant (Phasianus colchicus) induced by transit to pheasantry

The aim of this study was to assess haematological changes in hand-reared pheasants (Phasianus colchicus) transported from intensive housing facilities to a pheasantry. Selected haematological parameters were monitored in a group of 100 pheasants (50 males and 50 females) aged of 9 weeks that were transported for 4 hours by a covered lorry in crates, with a total body weight of 12 +/- 0.5 kg per crate (Group C12 - floor space: 290 cm2/kg) and with a total body weight of 18 +/- 0.5 kg per crate (Group C18 - floor space: 195 cm2/kg). Blood samples were taken from 10 randomly selected males and 10 females before transport (CON group) and 20 hours after transport (C12 and C18 groups). Examinations consisted in determining the total erythrocyte and leukocyte counts, haematocrit values, haemoglobin levels and differential leukocyte counts, whereby the proportions of heterophil, basophil and eosinophil granulocytes, lymphocytes and monocytes of the total leukocytes were computed. The changes in the parameters of red blood cell count were manifested by an increase (P < 0.01) in the haemoglobin level, MCH (mean cell haemoglobin) and MCHC (mean cell haemoglobin concentration) values and a decrease (P < 0.01) in the total erythrocyte count and haematocrit level in both C12 and C18 pheasants, when compared with the control group of non-transported pheasants. C18 pheasants exhibited also a significant increase (P < 0.05) in MCV (mean cell value) value. When analyzing differential leukocyte counts, C18 pheasants showed a decrease (P < 0.01) in heterophil counts and H/L ratio, whereas values in C12 pheasants did not differ from the non-transported control group. Individual counts of lymphocytes were decreased (P < 0.05) in C12 pheasants, whereas basophil counts were increased (P < 0.01) in both C12 and C18 pheasants. Total leukocyte count was decreased (P < 0.01) in C12 and C18 pheasants. In conclusion, the specific requirements of pheasants, as primarily wild animals, for the density in crates should be respected during transportation and they should be transported at lower densities than other poultry species, at least 290 cm2/kg live weight should be provided.     

蓝马鸡和藏马鸡血液蛋白谱的研究

采用聚丙烯酰胺凝胶电泳法对蓝马鸡和藏马鸡的血红蛋白、红细胞蛋白质、血清前白蛋白、白蛋白和运铁蛋白的蛋白谱进行了研究。结果发现 ,蓝马鸡和藏马鸡的血红蛋白 (HB)为由A1 和A2 2条区带组成的A1 A2 型 ;红细胞蛋白质 (EP)、血清白蛋白 (ALB)和运铁蛋白 (TF)分别呈单一的AA ,CC和FS。血清前白蛋白有Pr 1和Pr 2这 2种蛋白质组成     

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.     

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.     

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.     

Outbreak of type C botulism in captive wild birds

In late summer 2010, an outbreak of type C botulism affected the birds kept in a dam at a southern Brazilian zoo. A total of 14(10 black-necked swans, Cygnus melancoryphus; 3 Muscovy ducks, Cairina moschata; and 1 fulvous whistling-duck, Dendrocygna bicolor) out of 100 birds died after showing flaccid paralysis of the skeletal muscles characterized by general locomotion deficit, flight and swimming disorders, dropped neck, and severe dyspnea. Carcasses of dead birds (some infested by larvae of sarcophagus fly) scattered in the bird enclosure, and oxygen-free, organically rich mud and/or shallow standing waters present at the edges of the weir were identified as possible toxin sources. Postmortem examinations revealed no significant pathological changes. Epidemiologic and clinical findings indicated the diagnosis of type C botulism toxin, which was confirmed by mouse bioassay and seroneutralization.     

Avian botulism and the high prevalence of Clostridium botulinum in the Norflok Broads.

An account is given of a severe outbreak of type C botulism in waterfowl that occurred on the Norfolk Broads during the exceptionally warm summer of 1975. Forty-five mud samples were collected from 22 well distributed aquatic sites representing a considerable proportion of the total number of Broads. All samples except one (ie, 97-8 per cent) were shown to contain Clostridium botulinum and 58 per cent contained more than one type of the organism. Types B, C and E were demonstrated in 62-2 per cent, 51-1 per cent and 60 per cent of samples respectively. Recent surveys, made by identical methods, of aquatic environments in the London area and the Camargue (France) showed prevalences of Cl botulinum of 72-5 per cent and 4-5 per cent respectively. It seems likely that the Norfolk Broads will continue to present a risk to waterfowl from botulism in future hot summers.