Occurrence of Bacillus sporothermodurans and other aerobic spore-forming species in feed concentrate for dairy cattle

Abstract

Aims: To determine the aerobic spore composition and presence of Bacillus sporothermodurans spores in feed concentrate for dairy cattle. Methods and Results: Six feed concentrate samples from ®ve different farms were analysed. High levels of spores (up to 10 6 spores g ±1 ) were found. Identi®cation of 100 selected isolates was obtained by a combination of fatty acid methyl esters analysis, ampli®ed ribosomal DNA restriction analysis and 16S rDNA sequencing. Ninety-seven isolates could be identi®ed to the species level or assigned to a phylogenetic species group. Most of the isolates obtained after a heat treatment of 10 min at 80°C were identi®ed as members of the B. subtilis group (32 isolates), B. pumilus (25 isolates), B. clausii (eight isolates) and B. licheniformis (eight isolates). The isolates with very heat-resistant spores, obtained after a heat treatment of 30 min at 100°C, were identi®ed as members of the B. subtilis group (®ve isolates), B. sporothermodurans (three isolates), B. amyloliquefaciens (one isolate), B. oleronius (one isolate) and B. pallidus (one isolate). Bacillus cereus was present in each feed concentrate sample and was isolated using a selective mannitol egg yolk polymyxin agar medium. Conclusions: Feed concentrate for dairy cattle contains known as well as as yet unknown species of Bacillus and related genera with properties relevant to the dairy sector. Signi®cance and Impact of the Study: The results formulate the hypothesis that feed concentrate can be a contamination source of spores, including those of B. sporothermodurans, for raw milk at the farm level.

Incidence and Diversity of Potentially Highly Heat-Resistant Spores Isolated at Dairy Farms

“…A polyphasic identification strategy similar to the one described by Vaerewijck et al (47) was applied. In the hierarchical approach, all isolates were initially screened by numerical analysis of their FAME profiles, resulting in clusters of closely related strains.…”

“…Soiling of the udder and teats is considered one of the most important factors in the contamination of raw milk by spores (49). High levels of aerobic spores, ranging from 10 to Ͼ10 5 CFU g Ϫ1 , were found in silage (41), and levels of 10 3 to 10 6 CFU g Ϫ1 were found in feed concentrate (47). When animals consume feed contaminated with spore-forming bacteria, large quantities of spores can be present in their feces which in turn can contaminate the udders and teats.…”

“…Occasionally, B. sporothermodurans spores were reported in feed concentrate, silage, soy, and pulp (9,37,47). However, to prevent spoilage of milk and dairy products by aerobic spore-forming bacteria, it is important to minimize the initial contamination.…”

Bacillus farraginis sp. nov., Bacillus fortis sp. nov. and Bacillus fordii sp. nov., isolated at dairy farms

“…The presence of B. sporothermodurans spores in feed concentrate, silage, soy and pulp was demonstrated for individual dairy farms (de Silva et al, 1998;Vaerewijck et al, 2001;Scheldeman et al, 2002). In order to prevent contamination of milk and dairy products with aerobic spore-forming bacteria, it is important to ensure that contamination of raw milk is minimized.…”

Relative Expression of Germination Genes SleB, GerAC, and gpr in Heat Treated Bacillus licheniformis and Bacillus subtilis

“…Low numbers of spores can be found in the raw milk of individual cows as well as in the bulk tanks of Californian dairies (Arechiga 2013 unpublished data). The presence of spores in the cows' milk is most likely attributed to the high number and large diversity of spores that are found in the pasture soil, as well as in the hay and feed concentrate of dairy cows (Christiansson et al,1998;Vaerewijck et al, 2001). …”