Example of microbial contaminants of raw milk
Milk conservation and its organoleptic quality are greatly affected by different microbial contaminants (Table 1). These microorganisms are present either directly on the animal, in the farm environment or on the milking equipment. Industry requirements and country regulations require that the number of bacteria in raw milk be under a specific amount, often of 100 000 bacterial cells ml-1. Recent bacterial counts from a survey in Québec (Canada) found that most raw milk samples were under 50 000 bacterial cells ml-1 (http://www.lait.org/fichiers/RapportAnnuel/FPLQ-2010/controleQualite.pdf).
Contamination of raw milk by
2. The Clostridium
Bacteria from the genus
Cells from the genus
A group of clostria species had been recognized as important milk contaminant. Like most other
2.1.. Physiology and ecology of the
The different species of the genus
I: lactic acid + acetic acid → butyric acid + CO2
II: lactic acid → acetic acid + CO2 + 2H2
Lactic acid could come directly from the environment of the organism. Those pathways involve condensation of two pyruvate molecules, derived either from glucose or lactate (Figure 1).
Soil and decaying plants, including older plant parts in close contact with soil is the natural habitat of
When feed is contaminated by clostridial spores and ingested by the animal, spores could migrate through the rumen and concentrate in relation to total digesta volume. Digestion processes contribute to concentrate the number of spores, which could be quite high in cow manure. However, the enteric environment does not generally provide an adequate environment for the germination of spores of these species. It only plays a role in population diversity and transport of the organism to other environments.
Clostridial cells usually stop growing in presence of O2, but growth resumes when O2 concentration is under the physiological limit of the species. Oxygen sensibility depends on the metabolic level at time of exposure. Vegetative
Many clostridial species are specialized and need specific conditions to grow.
2.2.. Diversity of Clostridium at the farm level
On farm, many different environments exist thus leading to diversity of clostridial species. 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
3. Contamination of animal feeds and raw milk
Depending on climatic conditions, season, and specific feeding requirements, ruminant diet could include fresh forage during summer months or all year long under favourable climate, or be fed with hay, silage, and/or grains (including corn) during winter season in northern regions. Butyric acid spores could contaminate all of these feed types, but at different level and thus be part of a contamination cycle. Among clostridial species at farm level, species related to Cluster 1 are a major concern in relation with cheese making. However, some other species also need consideration for their potential in health problem namely
When silage is used as a feed source on a milk farm, silage conservation is of particular importance. Different events will take place that have relevance on the number of clostridial spores that could end up in raw milk in the bulk tank. During mowing and harvesting, contamination of crops by soil particules and manure aggregates will happen. During silage fermentation, butyric acid spores could germinate and grow if conditions are met. Animal will ingest the contaminated silage and spores will be released in feces. Clostridial spores will subsequently end up in manure, where population shift will happen before being applied again on crops. It could also contaminate teats, thus facilitating contamination of milk while milking the cows. These events can be considered as the spore cycle on a farm (Figure 3) (Pahlow et al., 2003).
3.1. The soil, reservoir of Clostridium and organic fertilizers
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. Even though, these different species could contaminate forage plant following harvesting and, cause hygienic problem in silage that could affect yield and health in some herds (see section 4.
The conservation method used to store forages has a direct impact on the subsequent number of spores. Hay will usually not allow germination of spores and subsequent development of the organism. Silage made from grasses or legumes could lead to important population of clostridial spores according to the ensilability of the crop. Lactic acid fermentation of whole corn silage is generally fast enough to ensure a good conservation. However, under aerobic instability environments,
3.2. Manure and slurries as organic fertilizers and their role in butyric acid spores contamination of soil and plants
Livestock manure (solid or liquid) is an important source of plant nutrients, and it can be valorized as fertilizers, especially with current high prices of mineral fertilizers. Concern has been expressed about the effect of manure application on soil properties and general hygienic quality of herbage over a number of years (Anderson & Christie, 1995). Contamination of herbage with manure (solid or liquid) by enterobacteria and clostridia may be important and represent a potential risk for silage making and health of the herd.
Manure contains different microorganisms, including enterobacteria such as
Several clostridia species had been identified in manure. They were mainly proteolytic, because this environment contains high level of proteins and amino acids. Polysaccharides degraders Clostridia are also present because of high concentration of polysaccharides like cellulose and lignin (Table 2).
|Clostridia group||Example of species|
|Polysaccharides degrader||Clostridium cellulolyticum|
Aeration of liquid manure may be performed on some farms before spreading on fields. Aeration has a positive effect on the number of different undesirable microorganisms by reducing number of enteric microbes like
Fertilisation with aerated liquid manure act positively in overall quality of silage over un-aerated liquid manure (Heinonen-Tanski et al., 1998). However, clostridial spores do not seems to be affected by this treatment. Inhibition of clostridial population in silage will be more easily controlled by lactic acid fermentation (Langó & Heinonen-Tanski, 1995).
Time after spreading of livestock manures is important to allow stabilization of microbial population and thus reduction of undesirable microorganisms in silage environment. Ultraviolet radiation, water stress and competition with normal epiphytic population are the principal factors contributing to lower enterobacteria population after spreading. Östing & Lindgren (1991) and Davies et al. (1996) studied the impact of timing application on crops and silage harvesting prepared from manured forage stands. In their study, enterobacteria and coliform populations diminished after a seven weeks period following manure dressing (Figure 4). At that time, coliforms were below detection level. They also observed the impact of wilting on corresponding cell numbers (Figure 4).
Clostridia spores in the soil seem to vary with forage stands following application of organic fertilizers. Figure 5 shows difference in number of soil clostridia from two different crops stands fertilized with the same four fertilizers: beef cow solid manure, beef cow liquid manure, paper sludge and mineral. Soil under alfalfa showed a clostridial population that was one order of magnitude inferior to the similar conditions under timothy.
Aggregates of farmyard manure present on the soil or on shoots could be picked up at harvesting (Rammer et al., 1994). In the silo, these aggregates create small pockets where fermentation is less efficient and thus lead to higher pH. Microorganisms developing in and around those pockets will differ from those growing only on nutrients from the crops. Enterobacteria and clostridia are present in higher number in these pockets.
Rapid development of the biogas industry will lead to high volume of digestates that will eventually be applied on crops as organic fertilizers. Clostridia are responsible for part of the microbial processes taking place during that kind of anaerobic fermentation and, at present, few studies had been conducted to study their diversity.
Regulatory offices will also require evaluation of pathogen microorganisms and coliforms before allowing spreading permits. Moreover, milk quality agencies will need to conduct investigations on the bacterial spores in relation with animal feed quality, either in pasture or silage.
Even if application of organic fertilizers do not seem to affect soil microbial population (Anderson & Christie, 1998; Rammer et al., 1994), incorporation of organic matters will positively influence soil microbial population (Janvier et al., 2007).
In the short term, application of animal manure will increase the number of clostridial spores on the crop (Pahlow et al., 2003). Care should be taken to uniformly apply organic fertilizers to avoid entry of manure aggregates with forages while harvesting and subsequently contaminate silage. Interval between application and harvest should also be determined to minimize entry of aggregates in the silo via the harvesting machineries.
3.3.. Contamination of the plant
Plants are in contact with clostridial spores at different moments during their growth. After seed germination, the fragile root and shoot epidermal cells are directly in contact with soil and could easily collect spores, which could then bind to microsites along surfaces and subsequently be incorporated inside cells layers as the plant grow. Clostridial spores could also penetrate root following breakage of the outside layer by mechanical action or when ruminant are grazing. Above grounds structures could also be contaminated with clostridial spores by action of raindrops on the soil and by wind dispersion of soil.
Natural plant contamination by butyric acid spores is generally below detection level when using plate counts techniques (log10 <2). The presence of dead and decaying tissue near ground level could lead to higher counts, around 10 to 100 spores g-1 plant tissue. Counts on corn stalk, leaves, silk and maturing kernel differ by several order of magnitude (Table 3) following plate counts on RCA medium using the techniquedevelop by Jonsson, (Jonsson, 1990). These organisms do not constitute a true epiphytic bacterial population because they develop on decaying matter, not directly from plant exudates.
|Corn structure||Spore counts (log10 CFU g-1 fresh weight)|
|Leaves||2 to 3|
|Silk||2.3 to 3.6|
|Kernel||3.4 to 3.6|
Silage is a conservation method that gained in popularity in the last half century all over the world (Wilkinson et al., 2003). This technique is use to store forage or other fermentable crops to feed ruminants. In livestock production, forage silage as a feed varied from 40 to 100% of the diet. The goal of silage making is to preserve the nutritional value of the forage while limiting lost of dry matter. The ensiling process to make silage is well described elsewhere and won’t be presented in details here (McDonald et al., 1991; Pahlow et al., 2003; Rooke & Hatfield, 2003). From a microbiological point of view,
Making silage is an ecological microbial process, and control of
DM = 450 − 80 x (water soluble carbohydrates / buffering capacity)
According to this relation, sugars and buffering capacity determine dry matter content needed to ensure a good conservation. Sugars and buffering capacity vary with many factors but still fairly related to species as presented in Figure 6. Simulation model (Leibensperger & Pitt, 1987) showed that clostridial silages are favoured by lower sugar-buffering capacity ratios, lower dry matter content, lower initial population of lactic acid bacteria, and high initial temperatures, and high initial pH.
Conditions in the silo could favour germination of butyric acid spores and development of vegetative cells of those organisms. Population as high as 9 log10 CFU g-1 silage could be observed, but generally their number averaged between 3 to 5 log10 CFU g-1 silage. Counts of clostridial spores in the silo could be highly variable. Two grass silage ensiled in clamp silo showed differences in number of clostridial spores in relation with depth. High spore concentration was observed in the first 50 cm (Spoelstra, 1990). Until recently, high concentration of butyric bacteria spores was associated with anaerobic instability of silage due to a lack of lactic acid to lower the pH during the primary fermentation phase or to a low concentration of nitrate (Kaiser et al., 2002). Visser et al., (2007a) reported high concentration of butyric bacteria spores in association with aerobic instability. These observations were also observed by Borreani and Tabacco (2008, 2010).
Butyric silage must be avoided as silage ingestion by the animal could be reduced and hygienic status might be a problem. Lactic acid molecule used for the production of butyric acid by clostridia contributes to raise the pH of the silage, as lactic acid (pKa= 3.86) is a stronger acid than butyric acid (pKa= 4.82). Consequently, this environment is less inhibitory for many other microorganisms. Moreover, as the pH raises, proteolytic clostridia species establish themselves and will degrade protein to amino acids and to ammonia (silage clostridia of group 1). These biochemical modifications will reduce the nutritional value of the feed.
Clostridia are strongly inhibited by the presence of nitrite (Spoelstra, 1985). Nitrite is an intermediate in the reduction of nitrate to ammonia and nitric oxide. High concentration of nitrate in grass forage may result from intensive grassland fertilization with nitrogen. Value could reach 1 to 8 g kg-1 DM (up to 30 g kg DM-1) (Spoelstra, 1985). Even if high N fertilization contribute to lower the amounts of fermentable carbohydrates in relation with protein and thus the buffering capacity, high nitrite concentration might be beneficial. Within few hours after ensiling, bacteria and plant start to reduce nitrates leading to nitrite and nitric oxide accumulation during the first week. This period is crucial for the silage quality as it corresponds to time where germination conditions are optimal for clostridial spores, because pH has not reach anaerobic stability level.
Interestingly, clostridia are also able to reduce nitrate to ammonium. Experimentation with irradiate grass inoculate with
Enterobacteria could have positive effect on
3.5. Contamination of milk
High number of butyric spores has an indirect role in cheese quality as their presence in some type of cheese may cause a defect called “late blowing”. Chemical composition of raw milk does not present a favourable environment for clostridial spore germination, so acting as a transition medium for these spores. Cheese fermentation by lactic acid bacteria allows accumulation of lactic acid that will induce germination of spores. Favorable conditions for late blowing are mainly observed in hard and semi-hard cheeses, including Emmentaler, Gouda, Edamer, Comté, generally shortly aged and of high pH. Dryer cheese, like Parmesan and Chedar are generally less affected.
Late blowing symptoms present appearance and gustative changes in the cheese, following fermentation of glucose and xylose to butyric acid and gas, mainly hydrogen and CO2. Gas production will show balloon-like expansion (Figure 7) and a rancid distasteful consistence is obtained (Innocente & Corradini, 1996). Four clostridial species are frequently observed in late blowing cheese:
Level of contamination by butyric acid spores in milk could be controlled before and after milking. As mentioned in previous sections, high contamination level of butyric acid spores in silage could be an important factor and several cheese producers required that their milk suppliers feed no silage to their milking cows. This should also applies for corn silage as high counts of clostridial spores were observed in aerobic deteriorated silage (Vissers et al., 2007a). Good management of silage constitute the first step in controlling subsequent contamination steps. In order to prevent development of butyric acid spores in silage, care should be taken to reach an excellent conservation of the silage in order to reduce contamination of butyric acid spores in raw milk via dirty teats (Table 4).
Animal behaviour must also be considered as part hygiene program on the farm. Some cows prefer to lie down on dirty patches. Teats of these cows are generally more heavily contaminated with feces conducting to contamination of milk (Visser et al., 2007b).
After milk being collected on farms, milk could be process differently according to different regulations dictated by countries. Pasteurisation has no effect on clostridia, as their spores are heat tolerant. Other techniques should be use to remove butyric spores.
In Europe,, most countries allow the utilization of lysozyme or nisin. Lysozymes are acid hydrolase enzymes produced by animal cells, like lymphocyte or egg white cells that hydrolyse bacterial cell wall. Lysozymes from egg white (0.5 % dry weight) are able to kill most vegetative clostridial cells, but have no direct action on the spores (Wasserfall & Teuber, 1979). Lysozymes are classified within the Class I enzyme and are therefore considered acceptable as food additive. Adding egg white lysozymes in cheese is allowed in several countries (European Communities Directive no. 95/2/EC).
Nisin are antibacterial peptides and classified as lantibiotic, producted by different species of the Order Lactobacilliales. This family of bioactive peptides have the capacity to kill bacteria via pore formation in cell-wall following binding with Lipid II (Hsu et al., 2004) in different pathogen, like listeria and clostridia. Similar to lysozymes, nisin mode of action also target vegetative cells and has no effect on their spores. Addition of Nisin E234 (from
New filtration and centrifugation technique now allow removal of bacteria and bacterial spores in milk (Su & Ingham, 2000). Bactofugation use high-speed centrifugation technique that could often be performed at pasteurization temperature. Bactofugation will remove 86 to 92 % or aerobic spores and between 91 and 97 % of anaerobic spores.
|Lower butyric acid spores contamination at ensiling||Set mowing cutting height above 7 cm to minimize soil entry in the silo.|
Limit manure aggregates in the silo.
Silage additive may help under sub-optimal conditions.
|Develop an efficient lactic fermentation||Wilt to recommended level in accordance to the silo type.|
Fill and close rapidly the silo to restrict infiltration of air in the silo.
Adding a lactic inoculant will contribute to faster the lactic fermentation.
|Ensure good aerobic stability||All previous actions to minimize development of butyric bacteria will help.|
Ensure high compaction and well sealed silo.
Never wilt over 500 g kg-1DM
Silage inoculants may help.
Silage removal at feed out should be adapted to the type of silo, the herd size and the season.
|Good milking hygiene||Routinely practice good hygiene in milking parlours with excellent teat cleaning before milking the cows|
Microfiltration technique use ceramic microfiltration membranes that are able to retain suspended particles, microorganisms and fat. Protein, carbohydrates, minerals and acids contain in the milk will pass through. Microfiltration will remove more than 99 % of the aerobic and anaerobic spores of milk. Both centrifugation and microfiltration will modify milk composition and are mainly use for commercial cheese manufacturing, since only skim milk could be use and organoleptic characteristics of raw milk are modified.
Considering other clostridia species in silage,
Types C and D are generally associated with animal botulism. Five factors are required for an animal to suffer from botulism: presence of viable organism, an environment that will support growth and toxin production, ingestion of toxin, absorption of toxin, and susceptibility of the host (Holdeman, 1970). The toxin acts by inhibiting production of acetylcholine in nerve cells connected to muscles, leading to paralysis of the lung or other locomotor muscles. Most cases are related to ingestion of contaminated foods.
Important outbreaks of botulism in cattle had increased over the past decades (Lindström et al., 2010). This trend may be explained by the growing use of plastic-packaged forage silage, allowing the growth and toxin production from
In the vast majority of cattle botulism outbreaks, the source of the neurotoxin is the feed. Contamination routes differ according to
Cattle environment introduce important risk of contamination of milk by botulism spores, but studies on the prevalence and contamination level of raw milk are scarce. Milk counts showed that numbers were generally under detection level (2040 spores L-1). Milk products, like cheese, may have contamination level around 10 spores g-1 cheese (Franciosa et al., 1999). Cheeses that may be contaminated are the same as the ones with butyric acid spores.