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.

Eutrophication and bacterial pathogens as risk factors for avian botulism outbreaks in wetlands receiving effluents from urban wastewater treatment plants.

Due to the scarcity of water resources in the "Mancha Húmeda" Biosphere Reserve, the use of treated wastewater has been proposed as a solution for the conservation of natural threatened floodplain wetlands. In addition, wastewater treatment plants of many villages pour their effluent into nearby natural lakes. We hypothesized that certain avian pathogens present in wastewater may cause avian mortalities which would trigger avian botulism outbreaks. With the aim of testing our hypothesis, 24 locations distributed in three wetlands, two that receive wastewater effluents and one serving as a control, were monitored during a year. Sediment, water, water bird feces, and invertebrates were collected for the detection of putative avian pathogenic Escherichia coli (APEC), Salmonella spp., Clostridium perfringens type A, and Clostridium botulinum type C/D. Also, water and sediment physicochemical properties were determined. Overall, APEC, C. perfringens, and C. botulinum were significantly more prevalent in samples belonging to the wetlands which receive wastewater. The occurrence of a botulism outbreak in one of the studied wetlands coincided with high water temperatures and sediment 5-day biochemical oxygen demand (BOD5), a decrease in water redox potential, chlorophyll a, and sulfate levels, and an increase in water inorganic carbon levels. The presence of C. botulinum in bird feces before the onset of the outbreak indicates that carrier birds exist and highlights the risk of botulinum toxin production in their carcasses if they die by other causes such as bacterial diseases, which are more probable in wastewater wetlands.

The same clade of Clostridium botulinum strains is causing avian botulism in southern and northern Europe.

Avian botulism is a paralytic disease caused by Clostridium botulinum-produced botulinum neurotoxins (BoNTs), most commonly of type C/D. It is a serious disease of waterbirds and poultry flocks in many countries in Europe. The objective of this study was to compare the genetic relatedness of avian C. botulinum strains isolated in Spain with strains isolated in Sweden using pulsed-field gel electrophoresis (PFGE). Fifteen strains were isolated from Spanish waterbirds using an immunomagnetic separation technique. Isolates were characterized by PCR, and all were identified as the genospecies Clostridium novyi sensu lato and eight harboured the gene coding for the BoNT type C/D. PFGE analysis of the strains revealed four highly similar pulsotypes, out of which two contained strains from both countries. It also showed that outbreaks in wild and domestic birds can be caused by the same strains. These results support a clonal spreading of the mosaic C. botulinum type C/D through Europe and give relevant information for future epidemiological studies.

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.