A brief historical review of Type E botulism is presented. Emphasis is placed on the role of fish in the epidemiology. Experimental details are given of tests carried out on the spore resistance of these Type E strains in comparison with the other types of CI. botulinum. The data obtained indicate that they are more thermolabile than any of the other types, especially A or B. In addition, evidence is presented which shows that the Type E strains seem to be divisible into two groups on the basis of their spore stability. The importance of this thermolability is stressed in regard to isolations of the seemingly rare Type E from suspected foodstuffs or in routine surveys. Details are given also of experiments conducted on the Type E toxins. The effect of dextrose, certain peptones, and colony type on toxin production is discussed. Some experiments on the storage properties of the toxins are presented. Active immunization experiments on mice indicate that Type E toxoids are poor antigens. In only two groups did the mice exhibit demonstrable immunity. Even in these groups the level of immunity was exceedingly variable. By contrast, a Type A toxoid proved a good antigen, protecting mice to a uniformly high degree against the homologous toxin. Efforts to increase the antigenicity of Type E toxoids are discussed. In vitro cross-neutralization tests with four Type E toxins and their antitoxins are described. On the basis of these in vivo and in vitro tests, it is concluded that Type E toxins are not homogeneous: a conclusion supported by the evidence that some Type E toxins contain a chicken-lethal factor, while others do not. This evidence is discussed in relation to the problem of human immunization. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The endopeptidase activity assay developed for measurement of purified botulinum neurotoxin type A (BoNT/A) in clinical therapeutic preparations has been adopted to provide a specific measure of BoNT/A activity in culture supernatants of proteolytic C. botulinum type A. Electrophoretic studies and inhibition of BoNT/A activity by anti-A antibody confirmed the specificity of the assay. The minimum detection limit was 0.2 MLD50/ml indicating the assay as more sensitive than the standard mouse bioassay or any other in vitro assay available to date. Whilst the assay did not exhibit any cross reactions with non-proteolytic (saccharolytic) clostridia, proteolytic C. botulinum types B and F and C. sporogenes showed some cross reactions. The endopeptidase assay was used to investigate physiological aspects of BoNT/A production by proteolytic C. botulinum type A strain NCTC 7272. Growth studies at 15°C, 25°C and 37°C with strain NCTC 7272 demonstrated that the first appearance of BoNT/A (0.1-1.0 MLD50 ml) occurred during mid-late exponential or early stationary phase of growth. Extracellular BoNT/A formation was not proportional to viable count. Slightly more BoNT/A was detected at 25°C than 37° or 15°C. The results of BoNT/A formation by one of the growth curves at 25°C measured by the endopeptidase assay and mouse bioassays were very similar confirming the specificity of the assay. A simple method was developed to lyre the cells so that BoNT/A formation could be subsequently measured in the endopeptidase assay. The data obtained following lysis of cells and measurement of intracellular BoNT/A showed that both intracellular BoNT/A and total BoNT/A formation is not constitutive but are more closely proportional to viable count than extracellular BoNT/A. Release of BoNT/A from cells was not associated with autolysis. The conversion of BoNT/A from the single-chain to dichain form during growth has been measured. The use of the endopeptidase assay has been also exploited to study BoNT/A formation by this strain within the population of cells. There was only a four-fold difference in BoNT/A production by cells of strain NCTC 7272, and further work in this area is warranted. Attempts were made to use MAPs for the production of monoclonal antibodies to SNAP-25 following cleavage by BoNT/E. Whilst the outcome was unsuccessful, the soundness of the principle was demonstrated

Made available in DSpace on 2014-06-11T19:27:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-06-26Bitstream added on 2014-06-13T19:55:42Z : No. of bitstreams: 1 nobrega_flc_me_araca.pdf: 216044 bytes, checksum: 97e7b77a880519d3b0dee0a840d76c6e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Foi avaliada a resposta imune de ovinos vacinados com toxóides botulínicos comerciais. Os animais dos Grupos (n=10) 1 e 2 foram vacinados com os toxóides bivalentes C e D, o Grupo (G) 3 recebeu a vacina polivalente contendo bacterinas e toxóides de outros Clostridium. e os pertencentes ao Grupo Controle não foram vacinados. O reforço vacinal foi realizado aos 42 dias após a dose inicial, e os níveis séricos de anticorpos contra as toxinas C e D foram avaliados pelo teste de ELISA indireto antes da vacinação, 15, 42, 90 e 180 dias após a primovacinação. Aos 15 dias da avaliação todos os grupos vacinados apresentaram um aumento significativo (p<0,05) nos títulos de anticorpos contra as toxinas C e D, com amplitude significativamente superior nos níveis de anticorpos contra a toxina C nos animais do G2 e contra a toxina D nos animais do G1. Aos 42 dias da vacinação primária houve uma queda significativa nos níveis de anticorpos contra a toxina C nos três grupos vacinados. Em relação à toxina D, somente o G1 apresentou queda nos níveis de anticorpos. Após o reforço vacinal dos grupos, quando avaliados aos 90 dias, observou-se novamente um aumento significativo nos níveis de anticorpos contra a toxina C nos três grupos, entretanto não houve variação nos níveis de anticorpos contra a toxina D. Ao longo dos 180 dias, o G2 apresentou, contra a toxina C, níveis de anticorpos superiores ao apresentado pelos outros grupos, porém contra a toxina D não houve variação significativa entre os grupos analisados após o reforço vacinal. / The immune response from sheep vaccinated against commercial botulinum toxoids was evaluated. The animals from Group (n=10) 1 and 2 were vaccinated with bivalents toxoids C and D, the Group (G) 3 received the polyvalent vaccine contained toxoids from other Clostridium and non-vaccinated animals were used as a control group. The booster dose was realized at 42 days after the initial dose and the antibodies levels against C and D toxins were evaluated by indirect ELISA, before of vaccination and 15, 42, 90 and 180 days after vaccination. At 15 days, the antibody level was significantly higher (P<0,05) against toxin C and D in all vaccinated groups, with antibodies levels against toxin C significantly higher (P<0,05) in the animals from G2 and against toxin D in the animals from G1. At 42 days of primary vaccination the antibodies levels against toxin C in the three vaccinated groups, while only the levels antibodies against toxin D from G1 showed a decrease. After the booster dose, at 90 days, a significantly higher in antibody levels against toxin C was observed, while no difference was observed against toxin D antibody levels. During 180 days, the G2 presented against toxin C antibody levels higher than presented by other groups, however against toxin D wasn’t significant variation between the groups after the booster dose.

Orientador: Iveraldo dos Santos Dutra / Banca: Tereza Cristina Cardoso da Silva / Banca: Luís Antonio Mathias / Resumo: Foi avaliada a resposta imune de ovinos vacinados com toxóides botulínicos comerciais. Os animais dos Grupos (n=10) 1 e 2 foram vacinados com os toxóides bivalentes C e D, o Grupo (G) 3 recebeu a vacina polivalente contendo bacterinas e toxóides de outros Clostridium. e os pertencentes ao Grupo Controle não foram vacinados. O reforço vacinal foi realizado aos 42 dias após a dose inicial, e os níveis séricos de anticorpos contra as toxinas C e D foram avaliados pelo teste de ELISA indireto antes da vacinação, 15, 42, 90 e 180 dias após a primovacinação. Aos 15 dias da avaliação todos os grupos vacinados apresentaram um aumento significativo (p<0,05) nos títulos de anticorpos contra as toxinas C e D, com amplitude significativamente superior nos níveis de anticorpos contra a toxina C nos animais do G2 e contra a toxina D nos animais do G1. Aos 42 dias da vacinação primária houve uma queda significativa nos níveis de anticorpos contra a toxina C nos três grupos vacinados. Em relação à toxina D, somente o G1 apresentou queda nos níveis de anticorpos. Após o reforço vacinal dos grupos, quando avaliados aos 90 dias, observou-se novamente um aumento significativo nos níveis de anticorpos contra a toxina C nos três grupos, entretanto não houve variação nos níveis de anticorpos contra a toxina D. Ao longo dos 180 dias, o G2 apresentou, contra a toxina C, níveis de anticorpos superiores ao apresentado pelos outros grupos, porém contra a toxina D não houve variação significativa entre os grupos analisados após o reforço vacinal. / Abstract: The immune response from sheep vaccinated against commercial botulinum toxoids was evaluated. The animals from Group (n=10) 1 and 2 were vaccinated with bivalents toxoids C and D, the Group (G) 3 received the polyvalent vaccine contained toxoids from other Clostridium and non-vaccinated animals were used as a control group. The booster dose was realized at 42 days after the initial dose and the antibodies levels against C and D toxins were evaluated by indirect ELISA, before of vaccination and 15, 42, 90 and 180 days after vaccination. At 15 days, the antibody level was significantly higher (P<0,05) against toxin C and D in all vaccinated groups, with antibodies levels against toxin C significantly higher (P<0,05) in the animals from G2 and against toxin D in the animals from G1. At 42 days of primary vaccination the antibodies levels against toxin C in the three vaccinated groups, while only the levels antibodies against toxin D from G1 showed a decrease. After the booster dose, at 90 days, a significantly higher in antibody levels against toxin C was observed, while no difference was observed against toxin D antibody levels. During 180 days, the G2 presented against toxin C antibody levels higher than presented by other groups, however against toxin D wasn't significant variation between the groups after the booster dose. / Mestre

The objective of this study was to field-test the "microenvironment concept" of avian botulism epizootiology. The "microenvironment concept" hypothesizes that aquatic invertebrate carcasses may serve both as a substrate for toxin production by Clostridium botulinum type C in nature and as a vehicle for toxin transmission to waterbirds. This concept has become the generally accepted, but inadequately tested, working hypothesis of type C botulism epizootiology. This study attempted to experimentally induce type C botulism in sentinel flocks of mallard ducks (Anas platyrhynchos) and American coots (Fulica americana) on sewage oxidation ponds in northern Utah. The three experimental oxidation ponds were inoculated with Cl. botulinum type C (strain X220B2) endospores in June,l974. Aquatic invertebrate populations were monitored throughout the summer. Rotenone was used in August in two of the experimental ponds (one pond served as a control) to kill aquatic invertebrates and thereby provide a large amount of substrate for clostridial growth and toxin production. No botulism was detected among the sentinel birds even though they routinely ingested invertebrate carcasses. None of the samples of dead invertebrates collected from the experimental ponds contained detectable (in white mice) botulinum toxin. It was concluded that the "microenvironment concept," as it now stands, cannot always be a sufficient explanation of how type C botulism epizootics are initiated in nature. Microbiological experiments designed to determine why the invertebrate carcasses collected from the study ponds contained no botulinum toxin were started and are now ongoing. Early results indicate that Cl. botulinum (X220B2) cells may not normally be able to effectively compete (at least in terms of toxin production) with other microorganisms present in the sewage ponds. It is suggested that the initiation of a type C botulism epizootic in nature may require the alleviation of the inhibitory effects of other putrefactive microorganisms upon clostridial metabolism and/ or toxin. This hypothesis is highly speculative and requires much further experimentation.

The effect of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary-butyl hydroquinone (TBHQ) and 8- hydroxyquinoline (8-0HQ) upon cell growth and spore germination of Clostridium botulinum type 62A was studied using a complex liquid medium. The inhibition of spore germination was further investigated using a defined liquid medium containing L-alanine, L-lactic acid and NaHC0₃. Cell growth was monitored by optical density (600 nm) increase and germination was monitored either by optical density (600 nm) decrease or by increase in percent phase dark spores viewed under phase contrast microscopy. Strict anaerobic conditions were maintained throughout the study. As the concentration of each chemical was increased the rate of germination or growth decreased. This occurred for each pH tested. GHA, BHT, TBHQ and 8-0HQ were more effective inhibitors of growth and germination as the pH was lowered from 7.2 to 5.7. At pH 7.2 50 µg/ml BHA, 200 µg/ml TBHQ and 200 µg/ml 8-0HQ were the minimum concentrations necessary for 90% inhibition of germination. BHT (400 µg/ml) inhibited germination by 65% at pH 7.2. Minimum concentrations at which growth at. pH 7.2 was inhibited by 95% were 60 µg/ml BHA, 20 µg/ml BHT, 250 µg/ml TBHQ and 20 µg/ml 8-0HQ. Inhibition of growth and germination by each chemical was found to be reversible with the exception of the inhibition of growth by BHT. / M.S.

Master of Science / Department of Horticulture, Forestry, and Recreation Resources / David A. Haukos / There is a wide variety of diseases that affect wild migratory birds. Occurrence, causes, and impacts of disease outbreaks in wild bird populations are rarely studied beyond documentation of large epizootic events. The relationships between the wildlife-livestock-human interface is rapidly blurring together. Global interests in avian diseases increased around 1990 as a result of the prevalence of zoonosis and potential threat to domestic livestock. A central disease reporting protocol does not exist in many states, which has led to a lack of available historical knowledge of disease occurrence that could be used to predict and manage future outbreaks. Due to changes of abundance and distribution of the migrant populations of Ross’s goose (Chen rossii) and Snow goose (C. caerulescens), geese are increasing their stopover duration in Kansas potentially increasing risk of disease outbreaks. We compiled historic records of wild waterfowl disease events in Kansas from 1967-2014 and related the frequency of events with indices of light geese abundance from 1970-2014. We found 32 reports spanning 16 counties consisting of the diseases avian cholera, avian botulism, aspergillosis, renal coccidiosis, west Nile, aflatoxicosis, and mycotoxicosis. Using a retrospective survey, we found there was a significant relationship between population densities of light geese in Kansas during the Mid-Winter Waterfowl Inventory and occurrence of avian cholera. Efforts to increase the understanding of relationships between disease outbreaks and host species will improve management of future disease outbreaks. Understanding factors known to facilitate wild waterfowl disease events (e.g., environmental, species, and individual), may assist in disease identification and determine a disease management course of action. This course of action is predetermined in a disease management plan. Disease management plans should be developed at the state and station level; incorporating planning, response, disease control, and surveillance and monitoring schemes to build upon the centralized disease database and to promote future disease understanding.

The neuroparalytic syndrome called botulism is caused by the neurotoxins produced by bacteria in the genus Clostridium. There are seven toxigenic types of C. botulinum (A to G) based on antigenically distinct toxins produced by different strains of the organism. Animal botulism is caused by C. botulinum type C and D neurotoxins and has a severe economic impact on cattle farming in South Africa and neighbouring countries. Current treatment regimes include the use of acetylcholine for symptomatic treatment, but this is unfortunately very seldom successful. All indications are that there is no cure for this disease and that effective control can only be achieved through development of efficacious vaccines. The botulinum vaccine currently in use in South Africa contains an adjuvanted toxoid form of the type C and o neurotoxins. However, this bivalent vaccine relies on problematic anaerobic cultivation of the Clostridium bacterium followed by isolation, purification and inactivation of the toxin by treatment with formalin. Apart from the fastidious growth requirements of this organism, it has been reported that the production of toxin by these cells declines rapidly and eventually ceases, following laboratory passaging of the bacterial cultures. In addition, improper inactivation of the toxins may also lead to the demise of animals following vaccination. Thus, there exists a great need for a safe, effective and inexpensive vaccine against botulism. To investigate the potential of types C and D botulinum neurotoxins as efficacious recombinant vaccine candidates against botulism, full-length copies of the genes were obtained by polymerase chain reaction (PCR) amplification from bacteriophage DNA isolated from Clostridium botulinum type C (Stockholm) and D (South Africa) cultures. The full-length genes were cloned and subsequently sequenced to verify their integrity. By making use of PCR-based site-directed mutagenesis procedures, three amino acid mutations were introduced in the zinc-binding motif of the respective neurotoxins. Mutation of this domain has previously been reported to successfully detoxify type C neurotoxin. The wild-type and mutant genes were subsequently expressed in insect cells using the BAC-to-BAC™ baculovirus system. Although, unique protein bands corresponding to the size of the neurotoxins could not be seen in Coomassie brilliant blue-stained gels, Western blot analysis indicated immunoreactive material for wild-type and mutant type C corresponding to the size of the type C toxin light chain. However, there was no conclusive evidence to support the successful expression of the full-length wild-type and mutant type D genes. / Dissertation (MSc (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

This thesis investigates how the Bear River marsh’s protection became a national interest and a cause for conservation in the Progressive Era. The thesis documents how the marsh declined because of irrigation development culminating with an outbreak of avian botulism in 1910, and traces the long process to protect the marshland. The research focused on examining local water development patterns of the nineteenth and early twentieth centuries, ornithological research in the 1910s, and the national sportsmen’s conservation movement of the 1920s. Upon examination of these events, it becomes clear that a coalition of ornithologists, sportsmen, and policy makers worked together to institute change that affected the marsh. Through showing how groups came together in the past to work for conservation, this research highlights the important role that a coalition of groups can have in reshaping how a landscape is viewed and managed.

Purpose and scope Each year thousands of western waterfowl succumb to disease, predators, mechanical injury and other decimating factors . Based on a review of records it is conservatively estimated that an average of 25,000 ducks have succumbed to botulism on western marsh areas annually. In a recent study, the United States Fish and Wildlife Service valued each duck and goose at $8.00 (McLeod, 1950). Applying this value to the estimated annual numerical loss, a total of $200,000 has been lost each season in mortality of western waterfowl from botulism. Control of this malady would reduce annual waterfowl and monetary losses. Prevention and cure are the only means of controlling botulism in wild ducks. At present, no economical preventative measure exists and control is based on curing stricken birds. The purpose of this study was to evaluate the cost of treatment and rate of recovery of birds stricken with botulism when treated by selected methods . The 4 treatments selected for evaluation were: (1) hospital inoculation, (2) fresh water, (3) field inoculation, and (4) no treatment or control . Research included a comprehensive evaluation of factors such as age, sex, species, body condition, degree of affliction, reaction to various amounts of antitoxin, and reaction t o selected treatment methods, thought to be pertinent in botulism control. This study was conducted during botulism outbreaks of 1953 and 1954, and was confined to state-owned marshlands of Utah. Study areas Three major areas provided sick birds used in this research: (1) Ogden Bay Refuge, (2) Farmington Bay Refuge, and (3) the Public Shooting Grounds . These man-made marsh areas are located on the saline flat lands adjacent to Great Salt Lake. The majority, 1,979 or 89.3 percent, of sick birds were taken from Ogden Bay Refuge on the Weber River Delta. This state-owned waterfowl refuge contains approximately 13,700 acres of diversified habitat . Excellent conditions for the production and existence of Clostridium botulinum, type c, the causative agent of botulism, were apparently present throughout the area. Farmington Bay Refuge, approximately 20 miles south of Ogden Bay Refuge in Davis County, Utah, was dried for improvements in 1953, but was traversed regularly during the 1954 season. In preparation for the hunting season, water was diverted into the north lake of Farmington Bay on October 1, 1953. A two-man crew picked up and disposed of approximately 2,000 dead ducks from approximately 20 acres of the reflooded marsh on October 6, 1953. This was the most serious outbreak of the study and indicated the rapidity with which sickness advanced. Few sick birds were noted, which indicated that the crisis had passed. Farmington Bay Refuge provided 121 of the 2,214 ducks treated during 1953 and 1954. Not more than 12 sick or dead birds were seen on the Public Shooting Grounds, 8 miles west of Corinne, Utah, in 1953. During the 1953 season, 3 sick birds were transported from the area to Ogden Bay Refuge for treatment. In 1954 this state-owned shooting area supplied lll sick birds for treatment. Other areas were observed during the study but did not provide sick birds for treatment. These areas consisted of : (1) State -owned lands beyond Bear River Migratory Bird Refuge, and (2) Smith and Utah Lakes west of Provo, Utah.