Invasive slug populations (Arion vulgaris) as potential vectors for Clostridium botulinum

Abstract

BackgroundNorwegian meadows, including those for silage production, are recently found heavily invaded by the slug Arion vulgaris in exposed areas. As a consequence, large numbers of slugs might contaminate grass silage and cause a possible threat to animal feed quality and safety. It is well known that silage contaminated by mammalian or avian carcasses can lead to severe outbreaks of botulism among livestock. Invertebrates, especially fly-larvae (Diptera), are considered important in the transfer of Clostridium botulinum type C and its toxins among birds in wetlands. C. botulinum form highly resistant spores that could easily be consumed by the slugs during feeding. This study aimed to determine whether Arion vulgaris could hold viable C. botulinum and enrich them, which is essential knowledge for assessing the risk of botulism from slug-contaminated silage. Slug carcasses, slug feces and live slugs were tested by a quantitative real-time PCR (qPCR) method after being fed ≅ 5.8 × 104 CFU C. botulinum type C spores/slug.ResultsLow amounts of C. botulinum were detected by qPCR in six of 21 slug carcasses with an even spread throughout the 17 day long experiment. Declining amounts of C. botulinum were excreted in slug feces up to day four after the inoculated feed was given. C. botulinum was only quantified the first two days in the sampling of live slugs. The viability of C. botulinum was confirmed for all three sample types (slug carcasses, slug feces and live slugs) by visible growth in enrichment media combined with obtaining a higher quantification cycle (Cq) value than from the non-enriched samples.ConclusionsNeither dead nor live invasive Arion vulgaris slugs were shown to enrich Clostridium botulinum containing the neurotoxin type C gene in this study. Slugs excreted viable C. botulinum in their feces up to day four, but in rapidly decreasing numbers. Arion vulgaris appear not to support enrichment of C. botulinum type C.

Effect of invasive slug populations (Arion vulgaris) on grass silage. II: Microbiological quality and feed safety

“…C. botulinum type C was not detected in any silages, not surprisingly since the pH of all silages was below the minimum growth level of pH 5.1-5.4 (Lindström et al, 2010). A controlled laboratory study where A. vulgaris were fed with spores concluded that neither dead nor live slugs were shown to enrich C. botulinum type C, suggesting that slugs themselves are not a favorable growth substrate for C. botulinum type C (Gismervik et al, 2014). Based on these facts and given anaerobic ensiling conditions, there is so far little evidence of slugs constituting an animal health hazard due to infections or intoxication by botulinum C neurotoxin.…”

“…The tubes were heated in water bath for 10 min at 60 • C prior to incubation at 37 ± 1 • C in 72 ± 3 h. One milliliter of the lower parts of the cultures was transferred to 9 ml freshly made TPGY broth and incubated overnight at 37 ± 1 • C. The cultures were frozen (−70 • C) in parallel Eppendorf tubes until a real-time PCR detecting the BoNTC gene covering a chimeric C/D sequence (Takeda et al, 2005) was performed . DNA was extracted using Qiagen BioSprint 15 Blood kit (Qiagen, Hilden, Germany) and KingFisher mL for magnetic based separation (Thermo, Helsinki, Finland) as previously described (Gismervik et al, 2014). The extracted DNA was analyzed with CFX96 TM real-time PCR detection system (Bio-Rad Laboratories, Hercules, USA) using the PCR mixture described in Gismervik et al (2014).…”

“…DNA was extracted using Qiagen BioSprint 15 Blood kit (Qiagen, Hilden, Germany) and KingFisher mL for magnetic based separation (Thermo, Helsinki, Finland) as previously described (Gismervik et al, 2014). The extracted DNA was analyzed with CFX96 TM real-time PCR detection system (Bio-Rad Laboratories, Hercules, USA) using the PCR mixture described in Gismervik et al (2014). All samples and positive and negative controls were run in duplicate and both Cq values had to be below 40 to be considered as positive.…”

Invading slugs ( A rion vulgaris) can be vectors for L isteria monocytogenes

“…) or A. vulgaris with Clostridium botulinum spores (Gismervik et al . ). Both studies reported short excretion time (up to 3–4 days), indicating direct bacterial passage through the digestive system.…”

Towards the unravelling of the slug <i>A. ater–A. rufus</i> complex (Gastropoda Arionidae): new genetic approaches

“…Some of the species in this genus are highly invasive, for example Arion vulgaris (MoquinTandon 1855), which is one of the worst invading species in Europe (Gismervik et al, 2014). Key in any strategy for pest control and conservation purposes is species identification (Amstrong and Ball, 2005), and in the genus Arion it is important to distinguish between closely related alien and native species.…”