Microbiology of animal feeds - J.P.F. D’MELLO

J.P.F. D’Mello
Formerly of the Scottish Agricultural College (SAC)
Edinburgh, United Kingdom

A wide range of microbes occurs naturally on, or as contaminants of forages, cereal grains, oilseed by-products and compound feeds. Beneficial effects can result from the occurrence of the lactic acid bacteria during the fermentation of forages during the process of ensilage. These bacteria favour the production of lactic acid, which helps in reducing the pH to around 4.0, thus preserving the forage for winter-feeding to ruminants. Lactic acid bacteria and yeast cultures have also been attributed with beneficial properties as feed probiotics for reducing scouring and increasing growth performance in farm animals. Animal feeds may become contaminated with harmful bacteria such as Salmonella, Listeria and E. coli. In the case of the latter organism, faecal sources and slurry have been identified as the primary routes of contamination both in pastures and compound feeds. Cereal grains and oilseed by-products are regularly contaminated with fungi occurring as plant pathogens or developing during storage. Major adverse effects arise in farm animals due to the production of mycotoxins by certain species and strains of these fungi. The use of fungicides and preservatives represents potential methods for reducing the prevalence of deleterious fungi. Extensive legislation exists for the control of a number of feed contaminants, particularly mycotoxins. These are reviewed together with current bovine spongiform encephalopathy (BSE) regulations.

1. INTRODUCTION

The microbiology of animal feeds emerged as an important issue in the wake of the Salmonella, E. coli O157, Campylobacter and BSE crises in the European Union (EU) and elsewhere and the momentum has been maintained by the foot and mouth epidemic of 2001/2 in the United Kingdom (UK). Fungal contamination of animal feeds is a regular occurrence on a worldwide scale and detrimental effects have been observed in all classes of farm animals due to the production of mycotoxins by certain species and strains of moulds. There are few grounds for complacency as regards animal feed safety and vigilance should now be the watchword for all those involved in the livestock industry. Nevertheless, it should be recognized that successful silage production depends upon the promotion of fermentation brought about by beneficial bacteria. This review will focus on several aspects of feed microbiology, including silage fermentation, fungal and bacterial contaminants, probiotics and legislation. Where relevant, comments on BSE legislation will be included to provide a more integrated picture of current developments and future strategy.

2. SILAGE MICROBIOLOGY

Successful preservation of high-moisture forage and other crops depends upon the controlling the activities of microbes, particularly bacteria. Conditions favouring optimal preservation include the rapid imposition and subsequent maintenance of anaerobic conditions at all stages in the process of ensilage. Under these conditions, lactic acid bacteria proliferate, using endogenous plant sugars to produce sufficient quantities of the acid to depress pH to around 4, the optimum value for successful preservation. Wet forages are difficult to preserve in this way and often provide ideal conditions for the growth of undesirable bacteria such as the Clostridia. The features of both types of bacteria are summarized in Table 1.

Species

The lactic acid bacteria (LAB) are of two types. The homofermentative species include Lactobacillus plantarum, Pediococcus pentosaceus and Enterococcus faecalis. The heterofermentative species comprise Lactobacillus brevis and Leuconostoc mesenteroides. The homofermentative group are more efficient in converting forage sugars into lactic acid (Table 1). Of these species L. plantarum is generally considered to be the most competitive, rapidly producing large amounts of lactic acid in freshly ensiled forage. LAB are essentially non-proteolytic organisms and are therefore considered to be beneficial in that they assist in conserving labile proteins and free amino acids in forage.

TABLE 1
Microbiology of ensilage¹

¹Compiled from McDonald et al. (1991).

Two groups of Clostridia are also recognized. The saccharolytic group includes Clostridium butyricum and C. tyrobutyricum. These species ferment residual sugars as well as lactic acid to butyric acid, causing a rise in pH. The proteolytic group includes C. bifermentans and C. sporogenes. These bacteria ferment amino acids to a number of different products (Table 1). The Clostridia thrive under high-moisture conditions and consequently the ensilage of wet forages is fraught with problems associated with adequate preservation and satisfactory voluntary intakes and performance of animals fed on such silages.

The Enterobacteria include Escherichia coli and Erwinia herbicola and are also considered to be undesirable in that they compete with LAB for plant sugars fermenting them to acetic acid, ethanol, CO2 and H2. They are also capable of catabolising amino acids to NH3 (Table 1).

Listeria monocytogenes is widely distributed in nature and also occurs in silage, particularly big bale silage. The increased incidence of listeriosis in sheep and cattle has been partly linked with the introduction of big bale silage. The relatively low density and limited fermentation characteristics of big bale silage and susceptibility of the bags to damage all favour the growth of L. monocytogenes. This organism is of particular significance because of it potential to contaminate animal products destined for human consumption.

Fungi occur in silages as yeasts and moulds. The yeasts include species of Candida, Saccharomyces and Torulopsis. The moulds associated with silages include various species of Aspergillus, Penicillium and Fusarium. The occurrence of these fungi is of particular concern due to their potential to produce harmful mycotoxins.

Modulation of microbial activity

The activities of bacteria may be controlled to facilitate preservation of ensiled forages. Wilting the crop before ensiling is a common method of restricting fermentation, allowing the growth of LAB but inhibiting the activities of undesirable organisms such as Clostridia and Enterobacteria. An added benefit of wilting is that effluent production is reduced, thus minimising environmental pollution from this source.

Additives may be used to control or stimulate fermentation by bacteria in ensiled forages. Formic acid is widely used to restrict fermentation by artificially reducing pH to below 4.0. Commercial products are available containing, for example, 85 percent formic acid which is applied during the harvesting of the forage. However, even at the recommended rates of application, the bacteria are still able to ferment sugars to lactic acid. Stimulation of silage bacteria may be brought about in two ways. Addition of molasses to the forage prior to ensiling ensures sufficient substrate for LAB thus causing the desired reduction in pH values. More recently, however, it has been suggested that a limiting factor is the relatively low concentrations of LAB in forage crops at harvest. Consequently, a number of commercial inoculants containing freeze-dried cultures of homofermentative LAB have become available for application to harvested forage. Efficacy of this procedure depends upon the inoculation rate as well as the presence of adequate concentrations of sugars.

3. FUNGAL CONTAMINATION OF CONCENTRATES AND FORAGES

Fungal contamination of cereal grains, oil-seed meals and forages continues to represent a major animal health risk throughout the world and particularly in the humid tropics. The risks arise primarily from the ability of particular species and strains of fungi to produce harmful compounds known as mycotoxins. These substances arise from the secondary metabolism of fungi in response to a wide range of genetic and environmental factors.

Mycotoxin contamination of forages and cereals frequently occurs in the field following infection of plants with specific pathogenic fungi or with symbiotic endophytes. In addition, contamination may occur during processing and storage of harvested products and feed whenever environmental conditions are appropriate for spoilage fungi. Moisture content and ambient temperature are key factors affecting fungal colonisation of and mycotoxin production in concentrates and compound feeds.

Inhalation of fungal spores or consumption of mycelia may cause conditions collectively known as ‘mycoses’. An example of such a condition is mycotic abortion arising from the systemic transmission and delivery of fungal material to the placenta.

Classification

It is conventional to subdivide toxigenic fungi into ‘field’ (or plant pathogenic) and ‘storage’ (or saprophytic/spoilage) organisms. Claviceps, Neotyphodium Fusarium and Alternaria are classical representatives of field fungi while Aspergillus and Penicillium exemplify storage organisms. Mycotoxigenic species may be further distinguished on the basis of geographical prevalence, reflecting specific environmental requirements for growth and secondary metabolism. Thus, Aspergillus flavus, Aspergillus parasiticus and Aspergillus ochraceus readily proliferate under warm, humid conditions whereas P. expansum and P. verrucosum are essentially temperate fungi. Consequently, the Aspergillus mycotoxins predominate in plant products emanating from the tropics and other warm regions, while the Penicillium mycotoxins occur widely in temperate foods, particularly cereal grains. Fusarium fungi are more ubiquitous, but even this genus contains toxigenic species which are almost exclusively associated with cereals from tropical and sub-tropical countries. The distribution of the important fungi in concentrates and forages is shown in Table 2 which also provides a list of the major mycotoxins.

The Aspergillus genus dominates all other fungi in respect of mycotoxin production in cereals and oilseeds. For example, Dhand et al. (1998) observed that Aspergillus was the most significant genus in dairy and other feeds in the tropics. Three species are responsible for virtually all mycotoxin production by this genus: Aspergillus flavus, Aspergillus parasiticus and Aspergillus ochraceus. A. flavus, and A. parasiticus synthesize the aflatoxins, while A. ochraceus produces the ochratoxins.

Aspergillus species

The aflatoxins include aflatoxin B1, B2, G1 and G2 (AFB1, AFB2, AFG1 and AFG2, respectively). In addition, aflatoxin M1 (AFM1) may well occur in the milk of dairy cows consuming AFB1contaminated feeds. The aflatoxigenic Aspergilli are generally regarded as storage fungi, proliferating under conditions of relatively high moisture/humidity and temperature. Aflatoxin contamination is, therefore, almost exclusively confined to tropical feeds such oilseed by-products derived from groundnuts, cottonseed and palm kernel. Aflatoxin contamination of maize is also an important problem in warm humid regions where A. flavus may infect the crop prior to harvest and remain viable during storage.

TABLE 2
Toxigenic fungi of concentrates and forages¹

¹Adapted from D’Mello (2000).

As stated above, A. ochraceus produces the ochratoxins. However it shares this property with at least two Penicillium species. Ochratoxin A (OTA) and ochratoxin B are two forms that occur naturally as contaminants, with OTA being more ubiquitous, occurring predominantly in cereal grains and in the tissues of animals given contaminated feed. Another mycotoxin, citrinin, often co-occurs with OTA, particularly in wheat.

Fusarium species

The important Fusarium fungi of cereals include: F. graminearum, F. culmorum, F. sporotrichioides, F. poae and F. moniliforme (Table 2). These species produce a wide range of mycotoxins. Of particular significance in animal health are the trichothecenes, zearalenone and the fumonisins. The trichothecenes are sub-divided into four basic groups, with types A and B representing the most important members. Type A trichothecenes include T-2 toxin, HT-2 toxin, neosolaniol and diacetoxyscirpenol. Type B trichothecenes include deoxynivalenol (also known as vomitoxin), nivalenol and fusarenon-X. The production of the two types of trichothecenes is characteristic for a particular Fusarium species. However, a common feature of the secondary metabolism of these fungi is their ability to synthesize zearalenone which, consequently, occurs as a co-contaminant with certain trichothecenes. The fumonisins are synthesized by a distinct group of Fusarium species (Table 2). Three members of this group (fumonisin B1, B2 and B3) often occur together in maize.

Virtually all the toxigenic species of Fusarium described above are also major pathogens of cereal plants, causing diseases such as head blight in wheat and barley and ear rot in maize. Harvested grain from diseased crops is, therefore, likely to be contaminated with the appropriate mycotoxins, and this is supported by ample evidence.

Neotyphodium coenophialum

The endophytic fungus Neotyphodium coenophialum occurs in close association with perennial tall fescue, while another related fungus, N. lolii, may be present in perennial ryegrass (D’Mello, 2000). Ergopeptine alkaloids, mainly ergovaline, occur in N. coenophialuminfected tall fescue, while the indole isoprenoid lolitrem alkaloids, particularly lolitrem B are found in N. lolii-infected perennial ryegrass. The ergopeptine alkaloids reduce growth, reproductive performance and milk production in cattle, while the lolitrem compounds induce neurological effects in ruminants.

Phomopsis leptostromiformis

In Australia, lupin stubble is valued as fodder for sheep, but infection with the fungus Phomopsis leptostromiformis is a major limiting factor due to toxicity arising from the production phomopsins by the fungus. Mature or senescing parts of the plant, including stems, pods and seeds are particularly prone to infection. Phomopsin A is considered to be the primary toxin, causing effects such as ill-thrift, liver damage, photosensitization and reduced reproductive performance in sheep (D’Mello and Macdonald, 1998).

Pithomyces chartarum

Pithomyces chartarum is a ubiquitous saprophyte of pastures with the capacity to synthesize sporidesmin A, a compound causing facial eczema and liver damage in sheep.

Use of organic acids

Fungal contamination of feeds, particularly high-moisture cereal grains may be prevented by the use of organic acids prior to storage. Propionic acid has emerged as an effective agent for this purpose. However, mixtures of organic acids are now commercially available. The efficacy of BASF mixtures has recently been undertaken in two studies conducted at the Scottish Agricultural College (SAC) and published by Blanchard et al. (2001). The preservatives tested included Lupro-Mix® NC (containing a mixture of 38 percent propionic acid, 34 percent formic acid, 8 percent ammonia and 20 percent water) and Lupro-Grain® (a mixture of 92 percent, propionic acid, 4 percent ammonia and 4 percent propandiol). The efficacy was compared with propionic acid at similar levels of addition (0.10, 0.25 and 0.50 percent). In the first investigation, the three preservatives were tested against 6 species of storage fungi using a purified medium (potato dextrose agar, PDA) for culture of these moulds. The minimum inhibition concentration (MIC) was then determined for the test products. As shown in Table 3, propionic acid emerged as the most effective and consistent preservative, with Lupro-Mix® being more effective than Lupro-Grain® in controlling Penicillium expansum. In contrast, Lupro-Grain® was more effective than Lupro-Mix® for the suppression of Aspergillus flavus and A. parasiticus, while both were equally efficient against A. ochraceus and P. verrucosum and P. roquefortii. P. verrucosum was more effectively controlled by the four test compounds than the other storage fungi under the conditions of this investigation.

TABLE 3
Minimum inhibition concentrations of three test products against six storage fungi inoculated on purified substrate (potato dextrose agar)¹

¹ Taken from Blanchard et al. (2001)

In the second trial, moist sterilized barley grain was inoculated with A. parasiticus and treated with 0.10, 0.25 and 0.50 percent of the three test products. In contrast to the findings of the first study, Lupro-Grain® emerged as the most effective product with the lowest MIC value (Table 4). Substrate type influenced the efficacy of test compounds against A. parasiticus. Discrepancies may have been caused by differences in water activity (i.e. moisture content) or in buffering capacity of the two substrates. Clearly much more work needs to be done to fully assess the efficacy of these preservatives in other cereals and in compound feeds.

4. SALMONELLA SPECIES

Many species of Salmonella have been implicated in diseases of farm animals. Of these, S. typhimurium is universally distributed while S. enteriditis has emerged as a regular pathogen of poultry and contaminant of eggs and chicken meat. Animal feeds are thought to be an important source of these bacteria. Meat and bone meal and fisheal are frequently contaminated with Salmonella. Intensive pasture utilisation provides an additional source through contamination of faeces from infected animals. Furthermore, the practice of spreading cattle slurry on to pastures in conventional and organic farms is another potentially significant source of infection.

TABLE 4
Minimum inhibition concentration of three test products against A. parasiticus inoculated on to barley grain^1,2

¹ Taken from Blanchard et al. (2001).
² 100g sterilised barley grain used with moisture content adjusted to 28 percent

In many parts of the world poultry manure is used as a feed for ruminants. For example in the USA, two poultry waste products are available for such use: dried poultry waste and dried poultry litter (Jeffrey et al., 1998). Dried poultry waste represents undiluted excreta generally derived from caged layer flocks, whereas dried poultry litter is a mixture of excreta and litter. These products are heated to reduce bacterial contamination but are, nevertheless, not sterile. It is reassuring to note that in the trail of Jeffrey et al. (1998) that all samples of processed poultry litter collected from 13 dairy farms were totally free of Salmonella contamination even though virtually all samples contained Enterobacteriacea, non-glucose fermenting Gram-negative and Gram-positive bacteria.

5. ESCHERICHIA COLII INCLUDING E.COLI O157

It is widely recognized that cattle feeds contain E. coli through contamination with faeces. There is particular concern over the occurrence of E. coli O157 since this form has been definitively linked with specific outbreaks of illness in humans.

The application of slurry on to pastures means that there is potential for the transfer of faecal E. coli to grazing animals, a practice that has caused some disquiet among those concerned with food safety. Hancock et al. (1998) maintain that it is important to distinguish the notion of ‘reservoir’ from that of an incidental host as regards the source of E. coli O157. They were not able to support the thesis that cattle are the reservoir species of this form of the bacterium. The Pennington Group (1997) also accepted that impact of spreading slurry on land still requires evaluation but proceeded to recommend the introduction of precautionary measures with this practice. Thus, the contamination of pasture grass with E. coli O157 from slurry remains a contentious issue.

Other forms of E. coli occur extensively in cattle feeds. Thus, Lynn et al. (1998) reported that just over 30 percent of cattle feed samples collected from 13 dairies and four feed mills in USA were found to be contaminated with E. coli, although E. coli O157 was not detected in any of these samples. In five dairies, concentrations of E. coli exceeded 1000 colony forming units/g feed. These authors suggested that attention should focus on the replication of E. coli in moist feeds and duration of storage in feed bunks. Other studies in the USA also demonstrated the occurrence of E. coli (non-O157) in cattle feeds. Jeffrey et al. (1998) reported that 13 non-O157 E. coli bacteria were isolated from 52 samples of dried poultry litter destined for dairy feeds.

7. FOOT AND MOUTH VIRUS

Foot and mouth disease has been a major problem in the UK during 2001/2, with devastating effects on the livestock industry. The disease is spread by inhalation and by ingestion of contaminated materials. The outbreak in the UK has been tentatively attributed to the feeding of catering waste to pigs. While this association may never be confirmed, it is clear that in the intensive rearing of animals due recognition should be given to the need to correctly process animal feeds. However, in the EU, the feeding of catering waste containing meat products is banned under a recent directive. Thus, in theory at least, this source of the foot and mouth virus should be excluded from livestock farms.

8. BOVINE SPONGIFORM ENCEPHALOPATHY (BSE)

The prion proteins of processed animal proteins have recently emerged as important feed contaminants implicated in the development of BSE in cattle. Prion proteins are normal animal tissue components with the capacity to transform into agents causing fatal neurological syndromes in a wide range of species. The significance of prions has been highlighted following the emergence of bovine spongiform encephalopathy (BSE) as a major disease of cattle in the United Kingdom. The onset of this disease was attributed to the feeding of cattle with meat-and-bone meal prepared from carcasses of scrapie-infected sheep. The latter disease is also caused by prion proteins as is the human equivalent, new variant Creutzfeldt-Jakob disease (vCJD). The incidence of vCJD in humans has been linked with the consumption of BSE-contaminated beef. It is this association that has led to extensive and stringent legislation in the European Union concerning the use of specified animal products in livestock feeding.

9. PROBIOTICS

A number of microbial products added to the feed may exert beneficial effects on livestock health and production. These products are not attributed with specific nutritional roles and are given the term ‘probiotics’. Lactobacillus acidophilus, for example, appears to be endowed with the ability to reduce scouring and increase liveweight gain in calves, but the effects are not consistent between trials. Yeast cultures may also be used as probiotics (Wallace and Newbold, 1992). It should be stressed that the mechanisms of action of probiotics have yet to be elucidated and there is a need for critical cost-benefit analysis of the various products as they become available for commercial use.

10. LEGISLATION

It might be expected that bacterial contamination of feeds and forages should be comprehensively regulated by statutory measures. However, in the United Kingdom, regulations only exist for Salmonella contamination. For example, under UK regulations positive identification of Salmonella in feeds must be reported to a ‘veterinary officer of the Minister’ (HMSO, 1989). Current evidence regarding E. coli reservoirs is not sufficiently convincing to warrant prohibition of the spreading of slurry or manure on to pasture. It is difficult to see how faecal sources of this bacterium in ruminant feeds and pastures can be reduced by the introduction of regulations. Any introduction of such legislation will require prior evaluation of the critical control points involved in the initial contamination and subsequent spread of E. coli on farms.

The acute toxicity of fungal secondary compounds and in particular the carcinogenicity of certain mycotoxins has led to the establishment of regulatory and advisory directives for primary feed ingredients and complete rations. Table 5 is designed to illustrate the type of directives in place in the United Kingdom, European Union and in North America. As can be seen, extensive regulations exist for the aflatoxins, but for ochratoxin A and deoxynivalenol only advisory directives have been proposed, unsupported by legislative measures. Of particular concern is the absence of statutory regulations for the carcinogenic fumonisins.

Animal feeds have been subject to additional stringent controls in the aftermath of the BSE crisis in the United Kingdom and elsewhere in Europe. In the UK, it has been illegal to feed ruminants with any form of mammalian protein since November 1994. The feeding of mammalian meat-and-bone meal (mMBM) to any farmed livestock has been prohibited since April 1996. The latter ban was introduced to avoid inadvertent feeding of mMBM to ruminants. Surveillance results of animal feeds indicate widespread compliance with these regulations, with 99.7 percent of feeds found to be negative for prohibited proteins. EU-wide feed legislation is also in place regarding BSE control of feeds. Thus, the Regulation (EC) No. 1774/2002 of the European Parliament and of the Council of 3 October 2002 requires Member States that only materials derived from animal declared fit for human consumption following veterinary inspection may be used for the production of feeds. It imposes the exclusion of dead animals and other condemned materials from the feed chain, the complete separation during collection, transport, storage, handling and processing of animal waste not intended for animal feed or human food and the complete separation of plants dedicated to feed production from plants processing other animal waste destined for destruction. It also bans intra-species recycling and it sets out clear rules on what must and may be done with the excluded animal materials, imposing strict identification and traceability system requiring certain products such as meat and bone meal and fats destined for destruction to be permanently marked to avoid possible fraud and risk of diversion of unauthorized products into food and feed. The new Regulation requires the complete disposal, by incineration or landfill after appropriate heat treatment, of Category 1 materials [i.e., animal by-products presenting highest risk such as transmissible spongiform encephalopathies (TSEs) or scrapie]. Category 2 materials (including animal by-products presenting a risk of contamination with other animal diseases) may be recycled for uses other than feed after appropriate treatment (e.g. biogas, composting, oleo-chemical products, etc). Finally, only category 3 materials (i.e., by-products derived from healthy animals slaughtered for human consumption) may be used in the production of feed following appropriate treatment in approved processing plants.

TABLE 5
Examples of worldwide guidelines and regulations for fungal secondary compounds (mycotoxins)¹

¹Adapted from D’Mello (2000).

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