Prevalence of Clostridium botulinum types B, E and F in faecal samples from Swedish cattle

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Abstract

Faeces were collected from 60 cows at three slaughterhouses situated in southern and central Sweden. The faecal samples were collected during two sampling periods over the year, summer and winter. All samples were analysed for the presence of Clostridium botulinum spores, according to a combined selection and enrichment PCR procedure. One PCR assay was specific for part of the type B neurotoxin gene, while the other assay was specific for both type E and F neurotoxin genes. The prevalence of C. botulinum in Swedish cattle was established to be 73% for non-proteolytic type B and less than 5% for types E and F. Twenty-eight (64%) of the positive faecal samples had a spore load of less than 4 spores/g. Statistical analysis (ANOVA) showed that seasonal variation (summer and winter) had a significant effect on the prevalence of C. botulinum type B in cattle, whereas the effect of geographical location of rearing of the cattle (southern and central Sweden) was less significant.

Introduction

Increasing demand from consumers for convenient foods of high quality have resulted in the development of refrigerated processed foods with extended durability that require minimal preparation time and contain low concentrations of preservatives. The industrial development of such product is, however, overshadowed by concern over the possible presence of non-proteolytic strains of Clostridium botulinum. Spores present in the raw material used in refrigerated processed food with extended durability can survive the mild heat treatment employed and lead to germination and growth of the bacteria on account of the anaerobic atmosphere provided by the packaging of the food, and furthermore, to the production of the neurotoxin (Peck, 1997). Non-proteolytic strains of C. botulinum have an optimal growth temperature at 30 °C or less but are also known to grow and produce toxins of types B, E and F at temperatures as low as +3.0 °C (Graham et al., 1997). Botulinal neurotoxins are among the most potent biological toxins that have been identified in nature, causing death due to respiratory paralysis in humans and animals (Granum et al., 1995). Of the seven different serotypes of botulinal neurotoxins, types A, B, E and F are the only types reported to cause food-borne botulism in humans.

Surveys have been performed in Europe on the distribution of C. botulinum in the environment and in raw materials used for foodstuffs Huss, 1980, Hielm et al., 1998a, Hielm et al., 1998b, Braconnier et al., 2001. A recently performed survey in Sweden showed a high prevalence (62%) of C. botulinum type B in faeces from pigs using a combined selection and enrichment PCR procedure, selecting for non-proteolytic strains of types B, E and F (Dahlenborg et al., 2001). Furthermore, these results showed that both rearing conditions and seasonal variation had significant effects on the prevalence in pigs, whereas the effect of geographical location was less significant.

The literature contains only limited information on the occurrence of botulinal spores in faeces of healthy cattle, which can contaminate meat. In the present investigation, the prevalence of C. botulinum types B, E and F in Swedish cows was established using a combined selection and enrichment PCR procedure.

Section snippets

Spore preparation and culture conditions

Spores from non-proteolytic C. botulinum Eklund 2B were produced in a two-phase medium as described by Peck et al. (1992). During the production of spores, contamination was checked on blood agar plates (blood agar base (37 g/l; Lab M, Bury, UK) and citrated horse blood (4% (vol/vol), SVA, Uppsala, Sweden)) following aerobic and anaerobic incubation (Gas Pak Plus™ Anaerobic System, BBL®, Becton Dickinson Microbiology Systems) at 30 °C for 48 h. The spores were finally resuspended in water and

Detection using the combined selection and enrichment PCR procedure

Representative results from the two growth experiments on the cattle faeces homogenates inoculated with non-proteolytic type B spores, and after analysis with the modified combined selection and enrichment PCR procedure, are shown in Fig. 1. The detection limit for non-proteolytic C. botulinum type B was established to be 10 spores/g cattle faecal sample.

Prevalence of C. botulinum in cattle faecal samples

Of the 60 faecal samples collected from cattle in Sweden, 44 (73%) gave a positive PCR result with respect to the type B neurotoxin gene. No

Discussion

A more profound understanding of the occurrence and distribution of C. botulinum spores in the environment and in the raw materials used for foodstuffs is required in the future to maintain product safety in newly developed products, such as refrigerated processed foods with extended durability. However, the development of ready-to-eat foods is overshadowed by concern over the possible presence of non-proteolytic strains of C. botulinum. In Scandinavia, the incidence of the organism in

Acknowledgements

This work was financially supported by the Swedish Foundation for Strategic Research through a national, industry-orientated programme for research and postgraduate education, LiFT-Future Technologies for Food Production.

References (17)

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