Type C botulism in dogs from rural properties located in Goiânia, Brazil

Botulism is a disease usually fatal, caused by the ingestion of neurotoxins produced by Clostridium botulinum. In dogs, intoxication is caused by the ingestion of botulinum toxin type C, and animals often recover spontaneously. The present study describes the occurrence of type C botulism in two dogs domiciled on neighboring rural properties in the municipality of Goiânia, state of Goiás, Brazil, probably associated with ingestion of decomposing bovine carcass. Upon clinical evaluation, the dogs were alert in the lateral decubitus position with ascending flaccid paralysis, absence of eyelid reflexes, and reduced muscle tone. Due to their worsening clinical symptoms, the animals died within 12 h and 3 days after supportive treatment. Botulinum toxin type C was identified, in the serum and feces of both dogs, by seroneutralization in mice with homologous monovalent antitoxin. The results of the high-throughput gene sequencing showed that the abundance of C. botulinum in the fecal microbiota of one of the affected dogs was low (0.53%). In this way, the present study highlights the need of sanitary practices related to the appropriate collection and disposal of bovine carcasses in rural areas since they represent a risk factor for the occurrence of botulism in dogs domiciled on rural properties.

Is botulism type C transmissible to human by consumption of contaminated poultry meat? Analysis of a suspect outbreak in French Guyana.

Botulism type C was suspected in a 46-year old man after consumption of sick poultry from a flock where botulism type C was confirmed. The patient developed characteristic signs of botulism, but investigation of biological samples did not confirm the presence of Clostridium botulinum or botulinum toxin. Despite having classical botulism symptoms, the man recovered very quickly. This raises the question of botulism transmission to humans by ingestion of contaminated poultry.

Type C botulism outbreak in feedlot cattle fed contaminated corn silage.

A large outbreak of botulism in feedlot steers fed corn silage contaminated with Clostridium botulinum neurotoxin type C (BoNT/C) is reported occurring in Midwestern Brazil in August 2017. The onset of the outbreak occurred 15 days after 1700 steers started to be fed the contaminated corn silage. Affected steers were alert and afebrile with varying degrees of flaccid paralysis in various muscle groups. A total of 1100 steers were affected, 1090 of which died within four days. Ten steers recovered after treatment with antitoxin. No gross or microscopic lesions were found in affected steers. The diagnosis was based on epidemiological data, characteristic clinical signs, and positive mouse bioassay results. This outbreak is interesting due to the high number of fatally affected cattle and the on-site diagnostic approach. This case report demonstrates the difficulties in diagnosing and treating botulism in cattle.

Type C botulism in domestic chickens, dogs and black-pencilled marmoset (Callithrix penicillata) in Minas Gerais, Brazil.

Botulism is a well-known intoxication that affects humans and animals. The disease is endemic in cattle in Brazil and recently emerged as an important disease in commercial laying hens and broiler chickens in Europe. Dogs and other animal species can also be affected. Although antitoxins are commonly administered to humans diagnosed with botulism, in animals this is rarely the case and the treatment of botulism is still based only on support therapy. In the present work, we report an outbreak of type C botulism in Brazil that simultaneously affected domestic chickens, dogs and a black-pencilled marmoset (Callithrix penicillata). The successful use of Clostridium botulinum types C and D antitoxin for the treatment of an affected dog is also described.

Type C waterborne botulism outbreaks in buffaloes (Bubalus bubalis) in the Amazon region / Surtos de botulismo hídrico tipo C em búfalos (Bubalus bubalis) na região amazônica

Botulism is a poisoning caused by botulinum neurotoxins (BoNTs). BoNTs serotypes C and D are involved in botulism outbreaks in cattle in several countries. Despite the high number of buffaloes worldwide, the real impact of botulism in buffaloes is not known, because it is not a notifiable disease in Brazil and only few studies have evaluated the occurrence of the disease in buffaloes. Those studies did not conduct diagnostic tests to confirm the presence of BoNTs. The objective of the present study was to describe three outbreaks of botulism in buffaloes in the Brazilian Amazon region considering epidemiological and clinical data as well as laboratory diagnosis to confirm the presence of BoNTs. The results of the bioassay were negative in the tissues and in feed samples, but positive for BoNT C in water samples. Confirmation of the occurrence of botulism in buffaloes allows the implementation of preventive strategies in susceptible herds. Waterborne botulism in buffaloes is prevented by ensuring the constant circulation of water collections and restricting the presence of dead animals and bones in order to prevent the accumulation of organic matter and the development of anaerobic conditions, which might favor the replication of Clostridium botulinum. Another measure that can be adopted is the shading of the pasture, in order to maintain the thermal comfort for the buffaloes and to avoid the excess of permanence of them in the water pools.(AU)

Botulismo é uma intoxicação causada por neurotoxinas botulínicas (BoNTs). Os sorotipos C e D de BoNTs estão envolvidos em surtos de botulismo em bovídeos em vários países. Apesar do elevado número de búfalos em todo o mundo, o real impacto do botulismo em búfalos não é conhecido; pois não é uma doença de notificação obrigatória no Brasil e poucos estudos avaliaram a incidência desta doença em búfalos. Além disso, estes estudos não realizaram testes diagnósticos para confirmar a presença de BoNTs. O objetivo do presente estudo foi descrever três surtos de botulismo em búfalos na região amazônica brasileira, considerando dados epidemiológicos e clínicos, bem como o diagnóstico laboratorial para confirmar a presença de BoNTs. Os resultados do bioensaio em camundongos foram negativos em todos os tecidos e nas amostras de alimentos testados; no entanto foram positivos para BoNT C nas amostras de água. A confirmação da ocorrência de botulismo em búfalos permite a implementação de estratégias preventivas nos rebanhos. O botulismo hídrico nos búfalos pode ser prevenido assegurando-se que coleções de água fossem mantidas limpas, sem a presença de animais mortos e ossadas no seu interior e não permitindo o acúmulo de matéria orgânica e condições de anaerobiose favoráveis à multiplicação de Clostridium. botulinum. Outra medida que pode ser adotada é o sombreamento das pastagens, a fim de manter o conforto térmico dos búfalos e assim evitar o excesso de sua permanência dentro das fontes de água.(AU)

New insight in the epidemiology of avian botulism outbreaks: necrophagous flies as vectors of Clostridium botulinum type C/D.

Avian botulism outbreaks spread through the bird carcass-maggot cycle, in which Clostridium botulinum and blowflies interact to ensure their reproduction in a mutualistic relationship where neurotoxin/spore-bearing maggot is one of the keystones. Here we investigated the hypothesis that adult blowflies may also play a significant role in botulism outbreaks by carrying C. botulinum cells between carcasses. We carried out a field experiment placing bird carcasses free of C. botulinum type C/D in containers only accessible to necrophagous flying insects in wetlands where avian botulism outbreaks were occurring and in control sites. Additionally, we performed laboratory trials to evaluate if blowflies may carry C. botulinum type C/D and for how long. Maggots bearing C. botulinum type C/D developed in 27.5% of carcasses placed in wetlands during botulism outbreaks. Calliphoridae flies in laboratory trials were able to transfer C. botulinum between two points and excreted it in their spots for up to 24 h after an infective feeding. Our results confirm that adult necrophagous flies play a role in the spreading of botulism outbreaks, which have implications in the epidemiology of this disease.

Chronic botulism in a Saxony dairy farm: sources, predisposing factors, development of the disease and treatment possibilities.

The aim of this study is to investigate Clostridium botulinum at a Saxony dairy farm with 159 cows and 18 heifers. The animals exhibited clinical symptoms of chronic botulism. To determine the source of the infection, feces, blood, organs, and gastrointestinal fluids of dead or euthanized cows; as well as soil, water, silage and manure were tested for C. botulinum spores and BoNTs using ELISA. BoNT/C and C. botulinum type C were detected in 53% and 3% of tested animals, respectively, while BoNT/D and C. botulinum type D were detected in 18% of the animals. C. botulinum also was detected in organs, gastrointestinal fluids, drinking water and manure. To evaluate possible treatments, animals were given Jerusalem artichoke syrup (JAS), Botulism vaccine (formalinised aluminum hydroxide gel adsorbed toxoid of C. botulinum types C and D) or a suspension of Enterococcus faecalis. After four weeks treatment with JAS, BoNT/C and C. botulinum type C were not detected in feces. In contrast, BoNT/D and C. botulinum type D were not significantly influenced by the JAS treatment. Vaccination with botulism vaccine and the E. faecalis suspension significantly decreased BoNT/D and C. botulinum type D. A significant increase of Enterococci was detected in animals treated with E. faecalis. Interestingly, there was a negative correlation between the detection of both BoNT and C. botulinum with the concentration of Enterococci in feces. Although C. botulinum C and D antibodies increased significantly (p < 0.0001) after vaccination with the botulism vaccine, the reduction of C. botulinum and BoNT in feces did not result in recovery of the animals because they were deficient of trace elements [manganese (Mn), cobalt (Co), copper (Cu) and selenium (Se)]. Animals treated with trace elements recovered. It appears that intestinal microbiota dysbiosis and trace element deficiency could explain the extensive emergence of chronic Botulism.

Purification, crystallization and preliminary X-ray analysis of an HA17-HA70 (HA2-HA3) complex from Clostridium botulinum type C progenitor toxin.

The haemagglutinin (HA) complex of Clostridium botulinum type C toxin is composed of three types of subcomponents: HA33, HA17 and HA70 (also known as HA1, HA2 and HA3, respectively). Here, a 260 kDa HA17-HA70 complex was crystallized. His-tagged HA17 and maltose-binding-protein-tagged HA70 were expressed in Escherichia coli and their complex was affinity-purified using a combination of amylose resin chromatography and nickel-nitrilotriacetic acid agarose chromatography. Diffraction data were collected to 8.0 Šresolution and the crystal belonged to the tetragonal space group P4(1)2(1)2. The molecular-replacement solution indicated that one molecule of HA17 was bound to each HA70 monomer.

An atypical Clostridium strain related to the Clostridium botulinum group III strain isolated from a human blood culture.

A nontoxigenic strain isolated from a fatal human case of bacterial sepsis was identified as a Clostridium strain from Clostridium botulinum group III, based on the phenotypic characters and 16S rRNA gene sequence, and was found to be related to the mosaic C. botulinum D/C strain according to a multilocus sequence analysis of 5 housekeeping genes.

Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D and their mosaic variants C-D and D-C in a multicenter collaborative trial.

Two real-time PCR arrays based on the GeneDisc(®) cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated with avian and mammalian botulism. The GeneDisc(®) arrays developed as part of the DG Home funded European project 'AnibioThreat' were highly sensitive and specific when tested on pure isolates and naturally contaminated samples (mostly clinical specimen from avian origin). Results of the multicenter collaborative trial involving eight laboratories in five European Countries (two laboratories in France, Italy and The Netherlands, one laboratory in Denmark and Sweden), using DNA extracts issued from 33 pure isolates and 48 naturally contaminated samples associated with animal botulism cases, demonstrated the robustness of these tests. Results showed a concordance among the eight laboratories of 99.4%-100% for both arrays. The reproducibility of the tests was high with a relative standard deviation ranging from 1.1% to 7.1%. Considering the high level of agreement achieved between the laboratories these PCR arrays constitute robust and suitable tools for rapid detection of C. botulinum types C, D and mosaic types C-D and D-C. These are the first tests for C. botulinum C and D that have been evaluated in a European multicenter collaborative trial.

Environmental factors influencing the prevalence of a Clostridium botulinum type C/D mosaic strain in nonpermanent Mediterranean wetlands.

Between 1978 and 2008, 13 avian botulism outbreaks were recorded in the wetlands of Mancha Húmeda (central Spain). These outbreaks caused the deaths of around 20,000 birds from over 50 species, including globally endangered white-headed ducks (Oxyura leucoceophala). Here, a significant association was found between the number of dead birds recorded in each botulism outbreak and the mean temperature in July (always >26°C). The presence of Clostridium botulinum type C/D in wetland sediments was detected by real-time PCR (quantitative PCR [qPCR]) in 5.8% of 207 samples collected between 2005 and 2008. Low concentrations of Cl(-) and high organic matter content in sediments were significantly associated with the presence of C. botulinum. Seventy-five digestive tracts of birds found dead during botulism outbreaks were analyzed; C. botulinum was present in 38.7% of them. The prevalence of C. botulinum was 18.2% (n = 22 pools) in aquatic invertebrates (Chironomidae and Corixidae families) and 33.3% (n = 18 pools) in necrophagous invertebrates (Sarcophagidae and Calliphoridae families), including two pools of adult necrophagous flies collected around bird carcasses. The presence of the bacteria in the adult fly form opens up new perspectives in the epidemiology of avian botulism, since these flies may be transporting C. botulinum from one carcass to another.

Type C botulism in a commercial turkey farm: a case report.

Botulism is an intoxication caused by exotoxins of Clostridium botulinum. The case of botulism described here occurred on a commercial meat turkey farm with two houses. Toms and hens were maintained in two separate houses, toms in house A and hens in house B. At 10 wk of age, an increase in mortality was observed in the toms located in house A. Clinically the animals presented with paralysis of the legs, wings, and neck. Affected birds were sitting and reluctant to move. Necropsy failed to find any specific lesions. In liver, heart, muscles, crop, and gizzard as well as in intestinal contents, DNA of C. botulinum type C was detected by PCR. The result was confirmed by a mouse lethality neutralization test. During the 2 wk after the onset of the clinical signs the mortality was about 12%. The hens kept in house B did not show any symptoms and remained healthy. Investigations of environmental samples to detect the source of the toxin were not successful. After 2 wk clinical signs and mortality abated. At 16 wk of age, toms again showed the same clinical signs accompanied by raised mortality. Again C. botulinum toxin type C was detected. Within 2 wk the total mortality reached roughly 50%. Based on the "precautionary principle" and in agreement with the local authorities, the birds were euthanatized using CO2 in order to not compromise food safety.

Molecular characterization and comparison of Clostridium botulinum type C avian strains.

Type C botulinum neurotoxin (BoNT/C)-producing Clostridium botulinum causes animal botulism worldwide and has become a serious problem in poultry flocks and waterfowl in Sweden. The objectives of the present study were to isolate, characterize and subtype C. botulinum type C avian isolates in order to increase the knowledge of the genetic diversity. Isolates from 13 birds were identified by 16S rRNA sequencing and BoNT/C gene detection by real-time polymerase chain reaction (PCR). Conventional PCR was used to distinguish a chimeric BoNTC/D gene, often associated with avian botulism, from the BoNT/C gene. The isolates analysed all contained the gene coding for a chimeric toxin type C/D. Two fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA analysis (RAPD), were optimized and used to investigate the epidemiological relatedness among the strains. The isolates were divided into three different pulsotypes based upon their restriction profiles for SmaI and SalI. The RAPD system proved to be as discriminative as PFGE. This study reveals a small genetic diversity among Swedish type C strains, with a high similarity between strains from broilers and herring gulls.

Optimization of polymerase chain reaction for detection of Clostridium botulinum type C and D in bovine samples.

Botulism is a rare but serious paralytic illness caused by a nerve toxin that is produced by the bacterium Clostridium botulinum. The economic, medical and alimentary consequences can be catastrophic in case of an epizooty. A polymerase chain reaction (PCR)-based assay was developed for the detection of C. botulinum toxigenic strains type C and D in bovine samples. This assay has proved to be less expensive, faster and simpler to use than the mouse bioassay, the current reference method for diagnosis of C. botulinum toxigenic strains. Three pairs of primers were designed, one for global detection of C. botulinum types C and D (primer pair Y), and two strain-specific pairs specifically designed for types C (primer pair VC) and D (primer pair VD). The PCR amplification conditions were optimized and evaluated on 13 bovine and two duck samples that had been previously tested by the mouse bioassay. In order to assess the impact of sample treatment, both DNA extracted from crude samples and three different enrichment broths (TYG, CMM, CMM followed by TYG) were tested. A 100% sensitivity was observed when samples were enriched for 5 days in CMM followed by 1 day in TYG broth. False-negative results were encountered when C. botulinum was screened for in crude samples. These findings indicate that the current PCR is a reliable method for the detection of C. botulinum toxigenic strains type C and D in bovine samples but only after proper enrichment in CMM and TYG broth.

Esporos e toxinas de Clostridium botulinum dos tipos C e D em cacimbas no Vale do Araguaia, Goiás / Spores and toxins of Clostridium botulinum in raining ponds used as water supply for cattle in pastures in the Araguaia Valley, State of Goiás, Brazil

Foram avaliadas a ocorrência e distribuição de esporos e toxinas de Clostridium botulinum tipos C e D em 300 cacimbas empregadas como bebedouro de bovinos em 130 propriedades rurais localizadas em 12 municípios do Vale do Araguaia, Estado de Goiás. A presença de esporos foi determinada indiretamente pelo cultivo em meio de cultura, seguido da inoculação e neutralização em camundongo das amostras de sedimento do interior das cacimbas, e do solo superficial e fezes de bovinos, coletadas ao seu redor. A presença de toxina foi avaliada diretamente pela inoculação em camundongo do sedimento filtrado das cacimbas, também seguida da neutralização em camundongo com antitoxinas C e D. A presença de esporos de C. botulinum foi significativamente maior (p<0,05) nas fezes de bovinos (31 por cento), quando comparadas com os resultados das amostras de solo superficial (19 por cento) e dos sedimentos (10 por cento). Foram detectadas toxinas botulínicas dos tipos C, D, ou classificadas como pertencentes ao complexo CD, em seis amostras (2 por cento) das 300 cacimbas. Das 130 propriedades trabalhadas, em 122(93,85 por cento) foram encontrados esporos ou toxinas de Clostridium botulinum em pelo menos uma das variáveis pesquisadas, enquanto somente 8(6,15 por cento) não apresentaram qualquer contaminação A idade e profundidade das cacimbas estiveram associadas com a freqüência de detecção de esporos e toxinas. Assim, quanto mais velhas e rasas, maior a freqüência do isolamento de esporos e toxinas. A contaminação das cacimbas do Vale do Araguaia goiano com esporos e toxinas do Clostridium botulinum tipos C e D demonstra o risco potencial permanente e crescente para a ocorrência da intoxicação botulínica de origem hídrica nos bovinos.

The occurrence and distribution of Clostridium botulinum spores and toxins type C and D in 300 ponds, used by cattle for drinking on 130 farms located in 12 municipalities of the Rio Araguaia valley, State of Goiás, Brazil, was evaluated. The presence of spores was determined indirectly by cultivation in culture medium, followed by inoculation and neutralization in mice of samples of the sediment from the bottom of the raining ponds, from superficial soil and from cattle feces collected to its circuit. The toxin presence was evaluated directly by inoculation in mice of the filtered sediment of the ponds, followed by the neutralization in mice with antitoxins C and D. The presence of C. botulinum spores was significantly more frequent (p<0,05) in the cattle feces (31 percent), when compared with the results of the superficial soil samples (19 percent) and the sediments (10 percent). Botulinum toxins of type C and D or classified as belonging to the CD compound were detected in 6 samples (2 percent) of the 300 ponds. Of the 130 worked farms, in 122 (93,85 percent) ponds Clostridium botulinum spores or toxins were found in at least one of the researched variables, whilst ponds on only 8 (6,15 percent) farms did not present any contamination. Age and depth of the ponds were associated with the frequency of detection of botulinum spores and toxins. The older and shallower the ponds were, the larger was the frequency of isolation of the spores and toxins. The contamination of the ponds in the Araguaia valley with Clostridium botulinum spores and toxins type C and D demonstrates the permanent and growing potential risk for the occurrence of botulism in cattle through drinking water.

Natural Clostridium botulinum type C toxicosis in a group of cats.

Clinical signs of botulism were observed in a group of eight cats, four of which died, after being fed pelican carrion. Clostridium botulinum type C was isolated from one cat. The microorganism and its toxin were found in the pelican. This is apparently the first report of natural botulism in cats.

Type C botulism due to toxic feed affecting 52,000 farmed foxes and minks in Finland.

The largest reported outbreak of type C botulism in fur production animals is described. Epidemiological investigation of 117 out of 157 (response rate, 74.5%) farms revealed that 44,130 animals died or were euthanized, while 8,033 animals with milder symptoms recovered. The overall death rate in all animals at risk was 21.7%. The death rates were significantly higher in blue and shadow foxes (24.2 and 27.8%, respectively) than in silver and blue silver foxes and minks (below 4%). All minks had been immunized against botulinum toxin type C. Deaths were associated with feed manufactured by a local processor, 83 of whose customer farms (70.9%) reported dead or sick animals. Five feedlots out of 19 delivered to the farms on the day preceding the onset of the outbreak (day 2) were associated with a death rate higher than 40%. These feedlots consisted of fresh feed processed on day 2 and feed processed 1 day earlier (day 1). In laboratory analysis, the day 2 feed contained botulinum toxin type C (>600 minimum lethal doses/g), while the day 1 feed did not contain toxin. Toxin was not detected in feed raw-material samples. Clostridium botulinum type C was detected by PCR in some feed components and in feed. However, as the feed temperature was continuously 8 degrees C or below and the pH was continuously 5.6 or below according to the manufacturer, it seems unlikely that spore germination and toxin formation occurred during overnight storage. Hence, the events leading to toxin formation were not determined.

Prevalence of neurotoxic Clostridium botulinum type C in the gastrointestinal tracts of tilapia (Oreochromis mossambicus) in the Salton Sea.

Tilapia (Oreochromis mossambicus) have been implicated as the source of type C toxin in avian botulism outbreaks in pelicans (Pelecanus erythrorhynchos, Pelecanus occidentalis californicus) at the Salton Sea in southern California (USA). We collected sick, dead, and healthy fish from various sites throughout the Sea during the summers of 1999 through 2001 and tested them for the presence of Clostridium botulinum type C cells by polymerase chain reaction targeting the C(1) neurotoxin gene. Four of 96 (4%), 57 of 664 (9%), and five of 355 (1%) tilapia tested were positive for C. botulinum type C toxin gene in 1999, 2000, and 2001, respectively. The total number of positive fish was significantly greater in 2000 than in 2001 (P<0.0001). No difference in numbers of positives was detected between sick and dead fish compared with live fish. In 2000, no significant relationships were revealed among the variables studied, such as location and date of collection.

Detection of Clostridium botulinum type C cells in the gastrointestinal tracts of Mozambique Tilapia (Oreochromis mossambicus) by polymerase chain reaction.

We established a method of directly detecting Clostridium botulinum type C cells, while minimizing spore detection, in the intestinal contents of Mozambique tilapia (Oreochromis mossambicus). This technique involved extraction of predominantly cellular DNA from tilapia intestinal tracts and used a polymerase chain reaction assay to detect presence of type C1 toxin gene. We consistently detected C. botulinum type C cells in tilapia gastrointestinal contents at a level of 7.5 x 104 cells per 0.25 g material or 1.9 x 103 cells. This technique is useful for determining prevalence of the potentially active organisms within a given population of fish and may be adapted to other types of C. botulinum and vertebrate populations as well.