Biodiversity of Clostridium botulinum type E associated with a large outbreak of botulism in wildlife from Lake Erie and Lake Ontario

The genetic relatedness of Clostridium botulinum type E isolates associated with an outbreak of wildlife botulism was studied using random amplification of polymorphic DNA (RAPD). Specimens were collected from November 2000 to December 2008 during a large outbreak of botulism affecting birds and fish living in and around Lake Erie and Lake Ontario. In our present study, a total of 355 wildlife samples were tested for the presence of botulinum toxin and/or organisms. Type E botulinum toxin was detected in 110 samples from birds, 12 samples from fish, and 2 samples from mammals. Sediment samples from Lake Erie were also examined for the presence of C. botulinum. Fifteen of 17 sediment samples were positive for the presence of C. botulinum type E. Eighty-one C. botulinum isolates were obtained from plants, animals, and sediments; of these isolates, 44 C. botulinum isolates produced type E toxin, as determined by mouse bioassay, while the remaining 37 isolates were not toxic for mice. All toxin-producing isolates were typed by RAPD; that analysis showed 12 different RAPD types and multiple subtypes. Our study thus demonstrates that multiple genetically distinct strains of C. botulinum were involved in the present outbreak of wildlife botulism. We found that C. botulinum type E is present in the sediments of Lake Erie and that a large range of bird and fish species is affected.     

Clostridium botulinum type E in fish from the Great Lakes

Bott, Thomas L. (University of Wisconsin, Madison), Janet S. Deffner, Elizabeth McCoy, and E. M. Foster. Clostridium botulinum type E in fish from the Great Lakes. J. Bacteriol. 91:919-924. 1966.-The intestinal contents of more than 3,000 fish from Lakes Erie, Superior, Huron, and Michigan were examined for Clostridium botulinum type E. Demonstration of the organism was accomplished by identifying its toxin in liquid cultures inoculated with material from the alimentary tract. Incidence figures, expressed as per cent of the fish tested, were: Lake Erie, 1%; Lake Superior, 1%; Lake Huron, 4%; the main body of Lake Michigan, 9%; and Green Bay (on Lake Michigan), 57%. Thus, C. botulinum type E appears to be widely but unevenly distributed in the Great Lakes, and fish from all areas are potential carriers of it.     

Toxicity of Clostridium botulinum type E neurotoxin to Great Lakes fish: implications for avian botulism

Since 1999, large-scale mortalities of fish-eating birds have been observed on the Great Lakes, and more specifically on Lake Erie. Type E botulism has been established as the primary cause of death. The mechanism of type E botulism exposure in fish-eating birds is unclear. Given that these birds are thought to eat live fish exclusively, it seems likely that their prey play a key role in the process, but the role of fish as potential transport vectors of botulinum neurotoxin type E (BoNT/E) to birds has not been adequately investigated. Between June 2003 and April 2004 a methodological model for exposing fish to Clostridium botulinum was developed and used to compare the sensitivity of rainbow trout (Oncorhynchus mykiss), round goby (Neogobius melanostomas), walleye (Stizostedion vitreum), and yellow perch (Perca flavescens) to four doses (0, 800, 1,500, and 4,000 Mouse Lethal Doses) of Clostridium botulinum type E neurotoxin. Each fish species expressed unique changes in both behavior and skin pigmentation prior to death. Yellow perch survived significantly longer (P < 0.05) than the three other species at all toxin treatments. Results of this study suggest that live fish can represent a significant vector for transfer of BoNT/E to birds.     

Influence of Limnological Conditions on Clostridium Botulinum Type E Presence in Eastern Lake Erie Sediments (Great Lakes, USA)

Avian and fish botulism outbreaks have been recorded since 1999 in eastern Lake Erie. These outbreaks are caused by Clostridium botulinum type E, a toxin-producing bacteria that is found in anoxic substrates rich in organic material. We studied the environmental conditions present in eastern Lake Erie during 2002, a year when several botulism outbreaks were observed. We also tested for the presence of C. botulinum type E in lake sediments. Samples were taken at six stations from two sites of different depths in the Dunkirk (New York, USA) area. The depth of the sampling sites influenced physico-chemical and biological processes in the sediments. We used the quantitative polymerase chain reaction (Q-PCR) to quantify the levels of C. botulinum type E in the samples. Sediment samples contained a patchy distribution of type E spore concentrations (from not detectable to 5520 DNA copies/mg). Samples of benthic invertebrates tested positive for C. botulinum type E spores in tissues (Gammarus 2028 DNA copies/mg, oligochaetes 428 DNA copies/mg, chironomids 148 DNA copies/mg and dreissenid mussels 715 DNA copies/mg). Principal components analysis (PCA) from inshore stations indicated that a decrease in dissolved oxygen, pH and redox potential near the sediment was associated to an increase in specific conductance and the type E toxin gene in sediments. We also found that C. botulinum type E spores are present in sediments at different depths and at different times through the ice-free season.     

A relationship between avian carcasses and living invertebrates in the epizootiology of avian botulism.

A survey of the sources of Clostridium botulinum type C toxin possibly utilized as food by aquatic birds in an epizootic area of avian botulism in northern Utah showed that living aquatic and terrestrial invertebrates normally found in close association with dead, decomposing birds commonly carried the toxin. Of 461 samples associated with 21 species of avian carcasses, 198 were toxin-positive. Invertebrate species not normally scavengers of vertebrate tissues were less commonly and less highly toxic, particularly when captured 30 cm or more from a carcass; six of 237 samples of such aquatic invertebrates low-level toxin. Of the species tested, blow fly larvae (Calliphoridae) were the most consistently and highly toxic, although others, particularly adult and larval stages of several species of beetles (Coleoptera), contained toxin at levels probably significant in the epizootiology of the disease. An estimated 0.05 to 0.25 g of the most toxic fly larvae or 15 g of the most toxic beetles tested carried a mediam lethal dose for an adult mallard duck. Examination of stomach contents of aquatic birds dead of botulism showed that some had consumed invertebrates.     

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 by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France

The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France. A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C. botulinum spores per 25 g and 100% of samples inoculated with more than 30 C. botulinum spores per 25 g. The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did. The prevalence of C. botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C. botulinum most-probable-number counts, respectively, and is in the low range of C. botulinum contamination reported elsewhere. The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E. Type F was not detected. The high prevalence of C. botulinum type B in fish samples is relatively unusual compared with the high prevalence of C. botulinum type E reported in many worldwide and northern European surveys. However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.     

Possible Origin of the High Incidence of Clostridium botulinum Type E in an Inland Bay (Green Bay of Lake Michigan)

Bottom and shoreline sediments of Green Bay, northern Lake Michigan, and rivers of the Green Bay drainage basin, as well as soils of the surrounding land mass, were examined for Clostridium botulinum type E. Detection was based on identification of type E toxin in enrichment cultures and was influenced by many factors. Testing smaller amounts of sample in multiple cultures was more productive than examining large inocula in fewer cultures. Incubation at 30 C was unsatisfactory, but 14 days at 20 C or 7 days at 25 C gave good results. Mild heating (60 C for 30 min) of specimens reduced the incidence of positive findings. Freezing enrichment cultures prior to testing for toxicity eliminated many nonbotulinal toxic substances that killed mice. A control culture inoculated with type E spores was employed to show whether a specimen contained factors which could mask the presence of type E. Samples from 708 stations were tested in 2,446 cultures. Type E was found in nearly all underwater specimens of Green Bay and northern Lake Michigan but was present less frequently in samples taken along their shores. The incidence was still lower in the rivers emptying into Green Bay with the organism being rare on the shores of these rivers and in the soils of the land mass proper. Samples from the upper reaches of the rivers practically never contained type E. Runoff could deposit type E spores in Green Bay, but this is not considered to be the major factor in the high incidence of the organism. Multiplication in the bay itself is indicated.     

Occurrence of toxigenic Clostridium botulinum type C in the soil of wetlands in Saskatchewan

Mouse-lethal toxin identified as that of Clostridium botulinum type C by antitoxin neutralization was present in cultures of 38.0% of 326 soil samples collected from 28 wetlands in Saskatchewan. There was no difference in prevalence of toxicity between samples collected in spring and summer, and no relationship was evident between the occurrence of toxicity and water salinity, marsh type or water depth. There was a strong association between the prior occurrence of avian botulism in a marsh and the presence of toxin in cultures from soil; 59.2% of soil samples from marshes with a known history of botulism produced toxin, whereas only 6.2% of soil samples from marshes with no history of the disease produced toxin. Eight of the 10 soil samples collected from a marsh that had been dry for several years, and from another marsh that had not had a recognized outbreak of botulism for 11 yr produced toxin, indicating a long residual effect after a botulism outbreak. The results suggest that any wetland with a history of botulism is likely to suffer repeated occurrences because of heavy contamination of the soil with spores, and should be managed to control the disease.     

Analysis of genetic diversity in common loon Gavia immer using RAPD and mitochondrial RFLP techniques

We used random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) of mitochondrial cytochrome b (cyt b ) gene to evaluate the genetic diversity in common loon Gavia immer populations from two regions in the United States: New England (NE) and Michigan (MI). RAPD analysis with 18 primers showed 74% polymorphism in NE and 50% in MI loons (similarity coefficient F = 0.92). Although no population-specific markers were found, the frequencies of some RAPD bands varied between the two populations suggesting geographical differences. RFLP analyses with Bam HI enzyme and a 307-bp mitochondrial cyt b gene showed four haplotypes in the NE loon samples and two in the MI samples. The mtDNA haplotype diversity was 0.74 for NE and 0.51 for MI loons, supporting the RAPD data that NE loons have greater genetic diversity than MI loons.     

首次自肉毒中毒食品中分离的一株产生E型肉毒毒素的酪酸梭菌

对某卫生防疫站委托本研究室分离病原菌的一份引起肉毒中毒的食品一＂黄豆冬瓜酱＂进行检测和病原菌的分离,从中再次检出了Ｅ型肉毒毒素并分离到一产毒菌种,对该菌种的生物学及生化学特性进行检查,并检测其毒素基因（ＰＣＲ试验）。结果：该分离菌能产生Ｅ型肉毒毒素,ＰＣＲ检测结果也证明其具有Ｅ型肉毒神经毒素基因,但其多项生化特性与Ｅ型肉毒梭菌有明显差异,而与酪酸梭菌完全一致。结果说明该分离菌系产生Ｅ型肉毒毒素的酪酸梭菌,而非Ｅ型肉毒梭菌。由酪酸梭菌引起的食物中毒型肉毒中毒并从中毒食品中分离到该病原菌,这在国际上尚属首次报告。     

Habitat selection by loons in southcentral Alaska

Three species of loons nest in the Matanuska-Susitna Valley, an area 80 km north of Anchorage in southcentral Alaska. This is a region of intense change; its human population doubled between 1980–1990 to almost 36000 people. The Alaska Department of Fish and Game through its Loon Watch Program has monitored 150 to 200 lakes since 1984, half of which are used by loons. Common loons (Gavia immer) nested on lakes of at least 12 ha while Pacific loons (G. pacifica) used lakes as small as 4 ha. Red-throated loons (G. stellata) were found on even smaller ponds. On all waters used by reproductive loons, 89% were connected to other water bodies, 62% of the shore was appropriate for nesting and 85% of the area was <15 ft (4.6 m) deep. This littoral zone comprised habitat for three-spined stickleback fish (Gasterosteus aculeatus), the dominant food source for loons, and supported large macrophyte beds that were used by loons as chick-rearing areas. The effect of human presence on summer loon distribution was dependent on the willingness of humans to accommodate to the needs of loons.     

Occurrence of Escherichia coli and enterococci in Cladophora (Chlorophyta) in nearshore water and beach sand of Lake Michigan

Each summer, the nuisance green alga Cladophora (mostly Cladophora glomerata) amasses along Lake Michigan beaches, creating nearshore anoxia and unsightly, malodorous mats that can attract problem animals and detract from visitor enjoyment. Traditionally, elevated counts of Escherichia coli are presumed to indicate the presence of sewage, mostly derived from nearby point sources. The relationship between fecal indicator bacteria and Cladophora remains essentially unstudied. This investigation describes the local and regional density of Escherichia coli and enterococci in Cladophora mats along beaches in the four states (Wisconsin, Illinois, Indiana, and Michigan) bordering Lake Michigan. Samples of Cladophora strands collected from 10 beaches (n = 41) were assayed for concentrations of E. coli and enterococci during the summer of 2002. Both E. coli and enterococci were ubiquitous (up to 97% occurrence), with overall log mean densities (+/- standard errors) of 5.3 (+/- 4.8) and 4.8 (+/- 4.5) per g (dry weight). E. coli and enterococci were strongly correlated in southern Lake Michigan beaches (P < 0.001, R(2) = 0.73, n = 17) but not in northern beaches (P = 0.892, n = 16). Both E. coli and enterococci survived for over 6 months in sun-dried Cladophora mats stored at 4 degrees C; the residual bacteria in the dried alga readily grew upon rehydration. These findings suggest that Cladophora amassing along the beaches of Lake Michigan may be an important environmental source of indicator bacteria and call into question the reliability of E. coli and enterococci as indicators of water quality for freshwater recreational beaches.     

Mortality of young-of-the-year rainbow smelt (Osmerus mordax) in Lake Erie associated with the occurrence of Glugea hertwigi

An extensive mortality of young-of-the-year rainbow smelt (Osmerus mordax) occurred in Lake Erie during the fall of 1969. Dead and dying smelt were observed along the north shore from west of Long Point in the central basin to Port Maitland in the eastern basin. No other fish species was involved. Glugea hertwigi, a microsporidan parasite, was observed in 90% of the distressed smelt examined, and is believed to have been a major contributing factor to the mortality.     

Outbreaks of type C botulism in waterfowl in Japan

Four outbreaks of botulism in waterfowl were encountered over a five-year period of 1973 to 1977 in Japan. In all the outbreaks toxin was detected from all 12 sera, twenty-three of 24 gizzard contents from diseased or dead birds and one of three maggots. It was neutralized with Clostridium botulinum type C antitoxin serum, regardless of its origin. By using CO2 gas jet method, C. botulinum was isolated from four of 11 gizzards from diseased birds, five of 7 ones from dead birds, one of one maggot and one of one sludge sample, that is, eleven of 20 specimens in total. All 20 strains were identical with C. botulinum type C in biological properties. Most of the isolates showed a toxin titer ranging from 1,000 to 200,000 LD50 for mice. Four of them were identified as type C by mouse neutralization tests with antitoxin sera. The toxic suspensions of a strain 1-15 were administered orally to Chinese spot-billed ducks, which died when more than 200,000 LD50 mouse toxin was administered. Environmental conditions for occurrences of waterfowl botulism were discussed.     

Energy Density of Lake Whitefish Coregonus clupeaformis in Lakes Huron and Michigan

We collected lake whitefish Coregonus clupeaformis off Alpena and Tawas City, Michigan, USA in Lake Huron and off Muskegon, Michigan USA in Lake Michigan during 2002–2004. We determined energy density and percent dry weight for lake whitefish from both lakes and lipid content for Lake Michigan fish. Energy density increased with increasing fish weight up to 800 g, and then remained relatively constant with further increases in fish weight. Energy density, adjusted for weight, was lower in Lake Huron than in Lake Michigan for both small (≤800 g) and large fish (>800 g). Energy density did not differ seasonally for small or large lake whitefish or between adult male and female fish. Energy density was strongly correlated with percent dry weight and percent lipid content. Based on data from commercially caught lake whitefish, body condition was lower in Lake Huron than Lake Michigan during 1981–2003, indicating that the dissimilarity in body condition between the lakes could be long standing. Energy density and lipid content in 2002–2004 in Lake Michigan were lower than data for comparable sized fish collected in 1969–1971. Differences in energy density between lakes were attributed to variation in diet and prey energy content as well as factors that affect feeding rates such as lake whitefish density and prey abundance.     

DETECTION OF CLOSTRIDIUM BOTULINUM TYPE E TOXIN BY MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

Botulinum type E toxin is a well recognized causative agent of seafood botulism poisoning. Underprocessing or postretort recontamination of preserved seafoods has resulted in sporadic cases of botulism. Currently, laboratory mice are being used to detect this toxin. However, it requires three to six days to obtain final results. A rapid method using monoclonal antibody (Mab) enzyme immunoassay was therefore developed. Hybridomas secreting specific Mab against the type E epitope were generated by fusion of SP/20-Ag 14 myeloma cells with spleen cells from BALB/c mice immunized with botulinum type E neurotoxoid. Five potent, stable hybridomas were selected, cloned, propagated, and preserved in liquid nitrogen as cell lines. Immunoglobulin subisotyping showed these Mabs belonged to the IgG subclasses. No cross-reaction was observed with culture supernatants of C. botulinum types A, B, and F or with crude toxins extracts of type C and D. Large quantities of Mabs were produced in ascites fluids, harvested, and affinity purified. A Mab-based biotin-avidin amplified double sandwich enzyme-linked immunosorbent assay allowed detection of type E toxin in inoculated seafoods at levels equivalent to 1–10 MLDs/ml (5–10 pg/ml).     

Yellow perch genetic structure and habitat use among connected habitats in eastern Lake Michigan

Maintenance of genetic and phenotypic diversity is widely recognized as an important conservation priority, yet managers often lack basic information about spatial patterns of population structure and its relationship with habitat heterogeneity and species movement within it. To address this knowledge gap, we focused on the economically and ecologically prominent yellow perch (Perca flavescens). In the Lake Michigan basin, yellow perch reside in nearshore Lake Michigan, including drowned river mouths (DRMs)—protected, lake‐like habitats that link tributaries to Lake Michigan. The goal of this study was to examine the extent that population structure is associated with Great Lakes connected habitats (i.e., DRMs) in a mobile fish species using yellow perch as a model. Specifically, we tested whether DRMs and eastern Lake Michigan constitute distinct genetic stocks of yellow perch, and if so, whether those stocks migrate between the two connected habitats throughout the year. To do so, we genotyped yellow perch at 14 microsatellite loci collected from 10 DRMs in both deep and littoral habitats during spring, summer, and autumn and two nearshore sites in Lake Michigan (spring and autumn) during 2015–2016 and supplemented our sampling with fish collected in 2013. We found that yellow perch from littoral‐DRM habitats were genetically distinct from fish captured in nearshore Lake Michigan. Our data also suggested that Lake Michigan yellow perch likely use deep‐DRM habitats during autumn. Further, we found genetic structuring among DRMs. These patterns support hypotheses of fishery managers that yellow perch seasonally migrate to and from Lake Michigan, yet, interestingly, these fish do not appear to interbreed with littoral fish despite occupying the same DRM. We recommend that fisheries managers account for this complex population structure and movement when setting fishing regulations and assessing the effects of harvest in Lake Michigan.     

Quantitative effects of carbohydrates and aromatic amino acids on Clostridium botulinum toxin gene expression using a rapid competitive RT/PCR assay

A rapid competitive RT/PCR assay was developed to determine the effects of nutrients on Clostridium botulinum type E toxin gene expression. The type E strain (EVH) was grown in a nutrient-rich broth containing 1% glucose (base medium). Toxin gene expression was quantified at both mid and late exponential phases of growth. It was found that toxin encoding mRNA levels were highly growth phase dependent with elevated levels found in late exponential phase compared to mid exponential phase. Changing the carbohydrate source had a smaller effect on toxin encoding mRNA levels but as earlier results have suggested, toxin encoding mRNA levels show a strong correlation with type E growth rate. The results have important implications for the food industry whereby risk of type E botulism could be correlated to the nutrient composition of the contaminated food or assessed from C. botulinum growth rates in challenged foodstuffs.     

The nucleotide and deduced amino acid sequences of EcoRI fragment containing the 5'-terminal region of Clostridium botulinum type E toxin gene cloned from Mashike, Iwanai and Otaru strains

Chromosomal DNAs were extracted from toxigenic three Clostridium botulinum type E strains isolated from food-borne botulism. After digestion by EcoRI, the fragments were cloned into Escherichia coli by using bacteriophage lambda gt11 and screened with monoclonal antibody recognizing the light chain component of botulinum type E toxin. The fragments (about 1 kbp size) cloned from each strain were recloned into a plasmid vector pUC118. The E. coli cells transformed with the recombinant plasmids produced 33 kDa protein with or without IPTG (isopropyl-beta-D-thiogalactopyranoside) which reacted with the monoclonal antibody. The nucleotide sequences of the cloned EcoRI fragments from the three type E strains were identical and contain the 5'-terminal region of the type E toxin gene. It was also found that there exist several highly homologous nucleotide sequences among the botulinum types A, C and E, and tetanus toxin genes in both translated and untranslated regions.