Sources of Clostridia in Raw Milk on Farms

Abstract

A PCR-denaturing gradient gel electrophoresis (DGGE) method was used to examine on-farm sources of Clostridium cluster I strains in four dairy farms over 2 years. Conventional microbiological analysis was used in parallel to monitor size of clostridial populations present in various components of the milk production chain (soil, forage, grass silage, maize silage, dry hay, and raw milk). PCR amplification with Clostridium cluster I-specific 16S rRNA gene primers followed by DGGE separation yielded a total of 47 operational taxonomic units (OTUs), which varied greatly with respect to frequency of occurrence. Some OTUs were found only in forage, and forage profiles differed according to farm location (southern or northern Québec). More clostridial contamination was found in maize silage than in grass silage. Milk represented a potential environment for certain OTUs. No OTU was milk specific, indicating that OTUs originated from other environments. Most (83%) of the OTUs detected in raw milk were also found in grass or maize silage. Milk DGGE profiles differed according to farm and sampling year and fit into two distinct categories. One milk profile category was characterized by the presence of a few dominant OTUs, the presence of which appeared to be more related to farm management than to feed contamination. OTUs were more varied in the second profile category. The identities of certain OTUs frequently found in milk were resolved by cloning and sequencing. Clostridium disporicum was identified as an important member of clostridial populations transmitted to milk. Clostridium tyrobutyricum was consistently found in milk and was widespread in the other farm environments examined.

Genomic Sequence and Characterization of the Virulent Bacteriophage φCTP1 from Clostridium tyrobutyricum and Heterologous Expression of Its Endolysin

“…C. tyrobutyricum has also been isolated from spoiled processed cheese (24) and spoiled fruit pulp (5). The bacteria are also present in nonfood environments, such as soil, forage, silage, hay, and milk (14,39). The heat-resistant spores survive the treatments involved in pasteurization, processing, and canning, and even low numbers of spores can cause food spoilage (37).…”

Clostridial Spores in Animal Feeds and Milk

“…C. disporicum isolates are often present in swine manure and manure biofilm (Leung & Topp, 2001). (Julien et al, 2008). Operational taxonomic unit 32, 33, 35 and 38 are similar to C. tyrobutyricum sequences in gene banks; OTU 38 is also closely related to C. sporogenes, a specie close to C. tyrobutyricum; OTU 42, 45, 46 and 47 are closely related to Clostridium disporicum…”

“…Recent advances in molecular diversity techniques were applied to clostridial populations in order to follow distribution of different species. Diversity studies were performed in different environments of environment like soil, milk, plant surfaces, landfills, water treatment plants, and biogas fermenter (Herman et al, 1995;Julien et al, 2008;Klijn et al, 1995;Knabel et al, 1997;Van Dyke & McCarthy, 2002). For most of these studies, PCR primer sets designed specifically to amplify Clostridium species related to Cluster I (Collins et al, 1994) were used.…”

“…For most of these studies, PCR primer sets designed specifically to amplify Clostridium species related to Cluster I (Collins et al, 1994) were used. One of these studies reported diversity of clostridia species in four different environments on four milk farms in Quebec (Canada) from soil to the raw milk ( Figure 2) (Julien et al, 2008). Diversity patterns obtained following PCR-DGGE in fresh forage and stored feed samples are distinct from soil and milk samples.…”

“…Numbers of clostridial spores in the soil following plate counts on Reinforced Clostridial Agar (RCA) lead to mean counts around 4 log10 CFU g -1 soil (Julien et al, 2008). Considering the low selective capacity of this medium in relation with the high diversity of clostridial species that could be present in the soil environment (Figure 2), this number greatly overestimate the number of strictly "butyric acid" spores present in soil.…”