Effects on poultry and livestock of feedcontamination with bacteria and fungi

K.G. Maciorowski a,1, P. Herrera a, F.T. Jones b,S.D. Pillai a, S.C Ricke a,∗,2

a Poultry Science Department, Texas A&M University, College Station,TX 77843-2472, United States

b Center of Excellence for Poultry Science, University of Arkansas,Fayetteville, AR 72701, United States

Abstract

Animal feed may serve as a carrier for a wide variety of microorganisms. The primary mode ofinoculation of feed materials is the transference of soil by wind, rain, mechanical agitation, or insectsto standing crops. Some of the microorganisms are adapted to the desiccated and relatively nutrient-poor conditions in soil and survive in similar niches on growing crops. Gastrointestinal pathogens canalso introduced into the food chain by animals defecating in the farm environment or by fertilizationof crops with manures. Other microorganisms are introduced during storage. In general, the amountof available water in the feed matrix determines whether a microorganism will grow or survive. Somemicroorganisms, primarily moulds, are adapted to the low amount of available moisture and growactively within stored seeds and grains. Others will produce spores or enter survival state until themoisture is high enough for bacterial action. There are numerous ways contaminating microorganismscan affect feed quality negatively including reducing dry matter and nutrients, causing musty orsour odours, causing caking of the feed and producing toxins. Finally, feed can act as a carrierfor animal and human pathogens. The type of feed, processing treatments and storage conditionscan all be factors that influence the population levels and types of microorganisms present. Theincidence and variation in the microflora found in animal feed and feed materials are reviewed.

∗ Corresponding author. Tel.: +1 479 575 4678; fax: +1 479 575 6936.E-mail address: sricke@uark.edu (S.C Ricke).

1 Current address: Department of Agriculture and Natural Resources, 1200 North DuPont Highway, DelawareState University, Dover, DE 19901, United States.

2 Current address: Department of Food Science, 2650 N. Young Ave., University of Arkansas, Fayetteville, AR72704, United States.

A select number of important human and animal pathogens are discussed. Finally there is a briefoverview over the detection, surveillance and management strategies of microbial contamination infeed and feed materials.© 2006 Elsevier B.V. All rights reserved.

Keywords: Animal feed; Poultry feed; Microbial diversity; Feed quality; Control

1. Introduction

The chemical and nutritional constituents of animal feeds are important for livestocknutrition and growth, but are only part of the animal feed matrix. From an ecologicalstandpoint, harvested grains are not only ingredients for livestock diets, but can act as sub-strate and transmission vectors for simple unicellular prokaryotic and eukaryotic organisms.Feeds may contain diverse microflora that is acquired from multiple environmental sources,including dust, soil, water, and insects. Feed materials may be inoculated at any time duringgrowing, harvesting, processing, storage and dispersal of the feed. The microflora found infeed materials come from a variety of ecological niches, such as soil and gastrointestinaltracts, and have to adapt to the conditions found in animal feed and feed components in orderto survive and/or grow. The microbial diversity found in different feeds is dependent on thewater activity, oxygen tension, pH and nutrient composition of the feed matrix. Microfloralgrowth is dependent on the moisture content of the feed material. Some microorganisms,primarily moulds, have adapted to conditions without free water and can actively growin stored grains. However, the majority of microorganisms must exercise various strate-gies to survive until there is sufficient water to support microbial activity. Microflora candecrease grain value through nutritional changes, physical damage, or the production oftoxins deleterious to animal health.

2. Bacterial ecology of raw feed ingredients

2.1. Origins of bacteria found in feed

Grains and oilseed crops possess a diverse microflora, with populations ranging from5 × 103 to 1.6 × 108 CFU/g, that are highly resistant to low moisture conditions (Richard-Molard, 1988; Multon, 1988). Plant material is primarily inoculated by dust generatedwhen soil is disturbed during mechanical harvesting, strong wind or rain. The soil envi-ronment is a collection of microhabitats comprised of clay particles, organic matter andaqueous domains that vary in pH, redox potential, ionic strength, nutrients, minerals andgas composition (Stotzky, 1997). This variability in soil microhabitats translates into adiverse microbial population, both anaerobic and aerobic. Bakken (1997) noted that thepredominant bacteria in soil are Gram-negative. In contrast, van Elsas and van Overbeek(1993) reported that Gram-positive corynebacteria outnumber Gram-negative bacteria. Themajor genera of soil bacteria have been reviewed by Alexander (1977), and quoted inBakken (1997), and have been expressed as the following proportions of viable, recover-

able bacteria in soil samples: Alcaligenes (0.02–0.12), Agrobacterium (<0.20), Arthrobac-ter (0.055–0.60), Bacillus (0.07–0.67), Flavobacterium (0.02–0.10), and Pseudomonas(0.03–0.15). The Gram-negative genera Achromobacter, Acinetobacter, Actinomycetes,Azotobacter, Moraxella, the Gram-positive genera Micrococcus, Mycobacterium, Strep-tomycetes, Streptococcus, and species of the order Cytophagaceae have also been detectedin soil (Christensen, 1977; Trevors and van Elsas, 1997). Limited nutrient turnover and thelow moisture content of soil restricts colonization by bacteria not adapted to these con-ditions. However, some bacteria, such as Enterobacter cloacae and Rhizobium spp., mayovercome this obstacle (Hinton and Bacon, 1995; van Veen et al., 1997). Insects, especiallyarthropods and arachnid mites, can also disseminate microorganisms across the surfaceof grain before harvest and in storage (Fleurat-Lessard, 1988; Multon, 1988; Poisson andCahagnier, 1988).

2.2. Origins and survival of pathogens in feed

When bacteria pathogenic either to animal or human hosts contaminate feed, it becomes apotential route of transmission of disease to both populations, and consequently of great con-cern to producers and consumers (Crump et al., 2002) As with natural bacterial contaminantsof feed, soil is the primary vector of inoculation. Soil mixed with animal faeces can con-taminate standing crops either by direct deposition or when used as fertilizer (Maciorowskiet al., 2004). Pathogenic bacteria found in fecal material are adapted to a markedly differentenvironment than found in soil. The animal intestine typically is characterized by a lowoxidation–reduction potential, sufficiently greater moisture and nutrient concentration thansoil. However, the intestinal environment is also highly competitive with limited residencetime for potential bacterial inhabitants. Unattached bacteria or bacteria that are sloughed offwith mucosal cells leave the intestinal environment and mix with soil bacteria. A portion ofthis population must then survive in the relatively desiccated and nutrient poor environmentuntil it may colonize another host. If the surviving bacteria is commensal inhabitant of thegut, such as nonpathogenic E. coli, their contribution to feed microflora may be of marginalconcern.

The mechanisms and maximum times of survival of intestinal and pathogenic bacteriaoutside their host is not completely understood. In a study involving air dried fecal material,E. coli could be isolated after 85 days of composting, whereas Salmonella spp. could notbe isolated after a maximum of 25 days (Dorn and Schleiff, 1997). In contrast, Temple etal. (1980) could consistently detect at least 103 E. coli and S. ser. Typhimurium in faecesfor 8 weeks after shallow burial in soil. Some anaerobic spore-bearing bacteria, such asClostridium spp., are able to exist as either vegetative cells or spores to become equallyproficient in surviving in both soil and gastrointestinal tracts (Haagsma, 1991).

Excrement from wild or domestic animals feeding on crops or on nearby garbage can be asource of bacterial contamination in crops. In numerous studies, Salmonella spp. have beenisolated from mice, rats, possums, skunks, raccoons, pigeons, and crows (Maciorowski etal., 2004; Crump et al., 2002; Tauxe, 2002). Predators, such as foxes and domestic cats, canconsume contaminated prey such as mice and insects and become vectors of contaminationthemselves. Houseflies and cockroaches feeding on fecal matter can act as both vectors andreservoirs for pathogens in the environment (Maciorowski et al., 2004).

2.3. Factors that influence feed bacterial diversity

Microbial populations present on grain and feed products, like those from soil, are alsodiverse and dependent upon the moisture content of feed. Herron et al. (1993) determinedthat Erwinia herbicola and Rahnella aquitilis were the dominant bacteria found on unhar-vested grasses, with lesser numbers of Serratia fonticola, Enterobacter cloacae, Hafniaalvei, and Escherichia coli. Lin et al. (1992) noted that, in fresh alfalfa with moisturecontent of 450 g/kg, enterobacteriaceae outnumbered yeasts and moulds.

Pelhate (1988) enumerated microbial populations in wheat containing 180 g moisture/kgand reported that, during the first 3 months of storage, bacteria of the genera Erwina,Enterobacter and Pseudomonas predominated. However, as storage continued and moisturelevels fell, microbial numbers decreased until bacteria could be isolated from less than 0.20of the samples. Furuta et al. (1980) detected total populations exceeding 105 organisms inpelleted alfalfa meal, while no aerobic bacteria could be detected in mineral supplementssuch as calcium or dicalcium carbonate, which contain virtually no free water.

Bacterial populations also vary by feed type. Bacterial populations on grains and seeds,can be divided into three different orders. Pseudomonas and Acetobacter spp. from the orderPseuomonodales and Streptomyces of the order Actinomycetales have been isolated fromfeed (Richard-Molard, 1988). The most diverse order on feeds, and most predominant in astudy by Lin et al. (1992), is the order Eubacteriales. Bacteria from at least five differentEubacteriales families have been reported in a review by Richard-Molard (1988), includingAchromobacteriaceae (Alcaligenes, Achromobacter, and Flavobacterium), Enterobacte-riaceae (Escherichia, Enterobacter, Paracolobactrum, Proteus, and Serratia), Micrococ-caceae (Micrococcus and Sarcina), Brevibacterium from Brevibacteriaceae, and Bacillaceae(Bacillus, Bacterium, and Clostridium). Enterobacteria represent a major portion of themicroflora adhering to standing grasses. Herron et al. (1993) estimated the numbers ofenterobacteria on unharvested grass to be approximately 104 CFU/g. McCapes et al. (1989)isolated E. coli from unprocessed poultry feed in 0.72–1.00 of the samples. Each feed typemay possess only a few members of order Eubacteriales. Hanis et al. (1988) detected Pseu-domonas in oat but not in wheat meal and Micrococcus spp. only in wheat meal. Generathat were common across substrates included Bacillus, Serratia, Clostridium, and moulds.

Microfloral populations in silage are predominately lactobacilli, with minor contribu-tions from Streptococcus, Leuconostoc, and Pediococcus spp. (ICMSF, 1998; Lin et al.,1992). The low pH produced by the lactobacilli fermentation inhibits the overgrowth ofother bacteria. Herron et al. (1993) determined that Enterobacteria greatly outnumber lac-tic acid bacteria on unharvested grasses. When the grass is harvested and placed undermoist anaerobic conditions, the lactobacilli proliferate, pH decreases, and the numbers ofenterobacteria rapidly decrease. If the conditions of ensiling are not optimal, the enterobac-teria can flourish, utilize available protein, increasing ammonia concentrations and pH, andproduce endotoxins.

The conditions under which ensiling is performed greatly influence the quality of theresulting feed. Allowing grass to wilt before ensilage slowed fermentation by the lactobacillias indicated by higher pH values and more residual sugars in the final product. It has beennoted that the practice of chopping maize or other forages might increase lactobacilli,pediococci, and leuconostoc populations by as much as 100-fold, either by allowing growth

in free plant juices or activation of previously nonculturable organisms by released plantenzymes (Lin et al., 1992; Pauly et al., 1999). Pauly et al. (1999) determined that silageproduced by precision chopping was more homogenous, had greater amounts of lacticacid, lower pH, and lower L. monocytogenes counts than courser, more heterogeneoussilages. Other studies have shown that silage made from forage collected by a precisionchop harvester, which produces a more homogenous product, had lower fungal spore counts,lower ammonium content, and higher energy content than silage made from forage collectedby a self-loading forage wagon.

2.4. Factors that influence survival of pathogens in feed

A variety of pathogenic microorganisms may survive in feeds. Lowering the pH of feeds(haylage, silage) or drying feeds (grains, meat meals) may considerably slow spoilage activ-ities and allow animal feed to be stored for extended periods of time. Ramırez et al. (2005)ensiled swine excreta, that contained greater than 106 CFU/g of Salmonella spp., with a mix-ture of sorghum and molasses for 11 days. After ensiling, the bacterium was not detected inany of the samples. McCaskey et al. (1996) inoculated mixtures of rumen contents, maize,alfalfa, poultry litter, cotton seed hulls, and peanut hulls with marker cultures of E. coli, S.typhimurium, and L. monocytogenes. The mixtures were ensiled and sampled every 5 for15 days for the presence of the three bacteria. E. coli and S. typhimurium dropped belowdetection limits by the 5th day; the Listeria disappeared on the 10th day. However, ensilingand drying may not completely eliminate pathogenic populations. Pathogens may utilizedsporulation cycles (Cl. botulinum and Cl. perfringens) or mechanisms to resist acid (L.monocytogenes) and/or desiccation (E. coli) to survive in stored animal feeds. These mech-anisms of resistance are a natural part of bacterial life cycles, as they must colonize animalintestines in order to continue their species.

3. Important bacterial pathogens found in feed

An overview of clinically important foodborne pathogens found in feed can be found inTable 1.

3.1. Clostridia

Two anaerobic Clostridia spp. are of major concern in feed: Cl. perfringens and Cl.botulinum. Cl. perfringens has been linked to bloat (gastric dilatation) in primates andnecrotic enteritis in poultry (Truscott and Al-Sheikhly, 1977; Bennett et al., 1980; Annett etal., 2002). Gangrenous dermatitis may result in a mortality rate of 1–2% per week in turkeys(Carr et al., 1996). It has been estimated that Cl. perfringens is the fourth most common food-borne illness in human, producing approximately 249,000 cases annually (Tauxe, 2002).Although commonly found in the intestine tract, is an opportunistic pathogen and can affectan animal whose normal intestinal microflora has been disrupted (Haagsma, 1991). Severalfactors have been implicated in outbreaks of necrotic enteritis including: diets high in wheat,barley, or fish meal, and damage to the gastrointestinal mucosa by high fiber litter or coccid-

Table 1Potential bacterial pathogens found in feed materials, the symptoms and/or disease they cause and notes regardingtheir ecology

Bacteria Symptoms/disease Notes

Cl. perfringens Gastric dilatation (primates), necroticenteritis (poultry), and gangrenousdermatitis (turkeys)

Can be a contaminant in inadequatelyensiled feed materials

Cl. botulinum Botulism Can be a contaminant in inadequatelyensiled feed materials. Produces siximmunologically distinct toxins

Listeria spp.(L. monocytogenes)

Septicemia, abortions, encephalitis,and eye infections

Can be a contaminant in inadequatelyensiled feed materials. Acid-resistantstrains can be found in high qualitysilage pH < 4

Escherichia coli Septicemia, cellulitis, swollen headsyndrome (poultry), and airsaculitis(poultry)

Has numerous pathways to surviveenvironmental stressors

Salmonella spp. Enteritis, diarrhea, and septicemia Has numerous pathways to surviveenvironmental stressors

iosis (Truscott and Al-Sheikhly, 1977; Annett et al., 2002) Cl. perfringens has been isolatedfrom faeces and in mixed animal feeds at populations ranging from 3 to 8000 organisms/g(Haagsma, 1991; Stutz and Lawton, 1984). In young chicks, Cl. perfringens can reach pop-ulations of 109 CFU/g cecal contents and cause growth depression (Barnes et al., 1972;Haagsma, 1991). Cl. perfringens may be isolated from pelleted feed due to its high resis-tance to heat in its spore state (Greenham et al., 1987). Zinc bacitracin is used to controlGram-positive growth and hinder Cl. perfringens sporulation (Elfadil et al., 1996c).

Cl. botulinum, being a obligate anaerobe, has been associated primarily with contam-inated carion, haylage, silage (Notermans et al., 1981; Neill et al., 1989; Weinberg andMuck, 1996; Ortolani et al., 1997; Pauly et al., 1999), or sandy pastures contaminated withspores (MacKay and Berkhoff, 1982). If forage is too moist before ensiling, fermentationby lactobacilli may be delayed allowing for secondary fermentation by Clostridia spp. tooccur (Weinberg and Muck, 1996). The usual etiology of botulism is from the ingestion oftoxin produced in feed, Cl. botulinum may colonize young or immunocompromised animals(MacKay and Berkhoff, 1982; Kelly et al., 1984). Cl. botulinum strains may produce anyof eight immunologically distinct toxins (A–F), three of which (B–D) are associated withbotulism in animals (Schoenbaum et al., 2000). Cl. botulinum type B and/or C have beenisolated from damp alfalfa hay, hay cubes, brewer’s grains, and mixed poultry feed (Dohmset al., 1982; Haagsma et al., 1990; Kinde et al., 1991; Wichtel and Whitlock, 1991). Due tothe ability of Clostridium spp. to persist in soil, fertilizing fields with contaminated manuremay create a reservoir for Cl. botulinum in the farm environment (Notermans et al., 1981).

3.2. Listeria

Listeria spp. are invasive microorganisms that have been reported to cause abortions,encephalitis and septicemia in ruminants (Johnson et al., 1996; Woo-Sam, 1999). Eye

infections have been linked to the direct inoculation of the eye with Listeria monocyto-genes in silage (Nightingale et al., 2004). L. monocytogenes can compete in environmentswith low oxygen and high moisture concentrations and is a potential pathogen in silages.Isolates of L. monocytogenes have been linked between clinically affected ruminants andconsumed silage by phage typing, molecular footprinting and ribotyping (Vazquez-Bolandet al., 1992; Wiedman et al., 1996; Wiedmann et al., 1997; Nightingale et al., 2004). Lis-teria spp. infections are associated with poorly fermented silage with a pH > 4 (Ryser etal., 1997; Woo-Sam, 1999): Fenlon (1985) isolated L. monocytogenes in 0.44 of mouldysilage samples. Populations near the surface of silage may contain 12,000 organisms/g.Silage linked to listeriosis in sheep has been noted to contain 106 cells/g (Fenlon, 1986;Fernandez-Garayzabal et al., 1992). Even adequately fermented silage may support thegrowth of acid-resistant Listeria spp. (Husu et al., 1990). Ryser et al. (1997) isolated thefive ribotypes of L. monocytogenes (including one linked to food-borne illness) from 0.08of high-quality maize silage with pH values <4. Recent reports suggest the acid-adaptedL. monocytogenes also exhibit increase resistance to thermal and chemical stressors. Thus,Listeria spp. may become a greater problem to the animal feed industry in the future.

Listeria spp., even though they do not possess a spore cycle, may survive for as long as10–12 years (Woolford, 1990). L. monocytogenes has been shown to grow on maize andhas been implicated in the infection of over 1500 people from improperly canned maize(Aureli et al., 2000). The epidemiology of listeriosis may be complicated by infected animalscontaminating the farm environment by shedding the bacterium in their faeces (Husu, 1990;Nightingale et al., 2004). Not only is L. monocytogenes shed by clinically affected animals,apparently healthy animals maybe latent carriers. Several studies have shown that the L.monocytogenes can be detected in up to 0.50 of fecal samples collected from animals (cattle,sheep, goats, pigs, and poultry) with no clinical manifestations of listeriosis (Nightingale etal., 2004). It is not know whether carriers act as a reservoir for the bacterium or are activelyinvolved in its transmission. However, enumeration and ribotyping of the L. monocytogenesstrains isolated from the soil, feeds and faeces on cattle farms with clinically affected animalssuggested that the animals acquire the pathogen from feed, which then multiplies in theanimal, is shed in the faeces, mixed with soil, which then acts as a source of contaminationfor the farm. The presence of L. monocytogenes in the feed of small ruminants (sheep andgoats) is enough to cause clinical disease. However, contributing factors such as pregnancy,postpartum immune deficiency, or immunosuppressive factors in silage are necessary forcausing the disease in exposed cattle (Grønstøl, 1980; Nightingale et al., 2004).

3.3. E. coli

E. coli strains are a normal component of animal and human intestinal microflora, and thusserve as an indicator of fecal contamination in feed (Geornaras et al., 2001). Whereas mostcases of human colibacillosis are mild or self-limiting, outbreaks of E. coli strain O157:H7have gained recent public attention as it is a potentially lethal foodborne pathogen (Tauxe,2002). Different strains of E. coli can cause a wide variety of disease syndromes in animalsincluding septicemia, swollen head syndrome, cellulitis, and airsacculitis (Geornaras et al.,2001; Gomis et al., 2003; Miller et al., 2004). Some serotypes of E. coli (O2, O(21, 83),O78, and O115), have been linked to avian cellulitis (Peighambari et al., 1995; Elfadil et

al., 1996a,b; Norton, 1997). Cellulitis may accompany abdominal abrasions or scratches,lack of adequate cleaning in laying operations or excess temperature or humidity at hatch(which may not allow proper healing of the navel). These conditions allow E. coli to colonizethe subcutaneous regions of birds, leading to the annual condemnation of 700,000 birds atslaughter (Messier et al., 1993). The prevalence of E. coli in dairy cattle herds has beenrelated to the feeding of maize silage (Herriott et al., 1998). Grain feeding has been suggestedto promote increases in acid resistant E. coli in cattle (Diez-Gonzalez et al., 1998), while hayfeeding and abrupt dietary changes have been linked to longer durations of E. coli O157:H7shedding (Hovde et al., 1999). However, an environmental link (more frequent reinfectionin grain fed animals compared to pasture) cannot be dismissed as a factor (Hovde et al.,1999).

E. coli is commonly found in animal feed, indicating that fertilization or contaminationwith wildlife faeces may potentially be major routes of pathogen transmission. Lynn et al.(1998) isolated E. coli from 0.30 of a variety of cattle feeds. It was detected in approximately0.30 of dry oilseeds and grains, and 0.50 of dry forages, versus only 0.11 of wet forages.E. coli may be more prevalent in dry matrices due to survival mechanisms, such as theaccumulation of trehalose or sucrose to maintain membrane integrity (Billi et al., 2000).Riordan et al. (2000) noted that E. coli O157:H7, once exposed to one type of stress (acid)may be more resistant to other stresses as well (heat), implicating a survival state that theorganism may adopt when the cell is threatened. Bass et al. (1999) screened 100 pathogenicavian E. coli isolates for antibiotic resistance. The isolates exhibited multiple antibioticresistance to �-lactams, chloramphenicols, tetracyclines, and aminoglycosides. The isolateswere then screened for markers of class one integrons, intI and qacEΔ1. Sixty-three isolateswere positive for these markers. Further molecular analysis revealed that these isolatescontained elements of the transposon Tn21, a cassette of multiple antibiotic resistancegenes found in clinically important human and animal strains of E. coli and Salmonellaspp.

3.4. Salmonella

Nontyphoid Salmonella spp. are estimated to be the third most common cause of humanfood borne illness in the United States, causing approximately 1.3 million cases annually(Tauxe, 2002). Being ubiquitous in nature, the bacterium can quickly be spread verticallyand horizontally throughout a poultry flock or livestock herd. Humans can be exposed tothe bacteria by consuming improperly prepared eggs, meat or milk from infected animalsor from foods contaminated by the faeces of infected animals (Patrick et al., 2004). Humanoutbreaks of Salmonella enterica serovars Hadar, Heidelberg, Virchow, and Agona in cattleand chickens have been traced to contaminated bone, meat and fish meal (Crump et al., 2002).The serovars causing human foodborne illness have evolved over time, with outbreaks ofS. enterica ser. Agona in the 1970s, S. enterica ser. Enteritidis in the 1980s, and S. entericaTyphimurium DT104 in the 1990s (Crump et al., 2002; Tauxe, 2002; Patrick et al., 2004). S.enterica ser. Agona became a major public health risk in the United States in the 1970s andbecame the eighth most common isolate in cases of human salmonellosis by 1972 (Crumpet al., 2002). Epidemiology investigations revealed that people became ill after consumingcontaminated poultry. The ultimate source of the epidemic was traced back to a poultry

raising facility in Mississippi that had used feed derived from fish meal imported fromPeru. It has been estimated that S. enterica ser. Agona has cause over 1 million cases ofhuman illness since its introduction into the American food chain.

Foodborne Salmonella spp. contamination in feeds detection methodologies and controlmeasures has been extensively reviewed elsewhere and will not be discussed here (Williams,1981a,b,c; Ricke et al., 1998, 2004). The physiology of Salmonella spp. lends itself quitewell to the contamination and survival on a wide range of feeds and feed ingredients. Somefeeds, such as bone, meat, and fish meal, have been associated with high rates, in somesurveys ranging from 0.17 to 0.43, of Salmonella contamination (Stott et al., 1975; Furutaet al., 1980; Maciorowski et al., 2004). Salmonella spp. are organisms that have developeddiverse mechanisms to survive outside of their preferred niche, the intestine, as reviewedby Foster and Spector (1995). The pathogen may utilize a starvation stress response (SSR)to resist low concentrations of available carbon, nitrogen, and phosphorus (Harder andDijkhuizen, 1983). Salmonella spp. may utilize a stationary-phase acid tolerance response(ATR) in order to survive transient low pH levels, genes for catalase (katE and katG) totolerate peroxide, 10 different proteins for superoxide radicals and mechanisms involvingglutamate, K+ ions, proline, or trehalose to maintain osmotic pressures (Foster and Spector,1995). Salmonella spp. have found to be particularly resistant to dehydration. Survival ofthe bacterium in meat and bone meal, dry milk and poultry feed has been shown to beinversely related to moisture content, with greater survivability seen at a water activity of0.43 and 0.52 than at 0.75 (Juven et al., 2004). These adaptations have allowed Salmonellaspp. to be potentially resistant to environmental stress, to survive in multiple environmentsand, ultimately, to infect human hosts.

3.5. Other pathogens

Other pathogens may also be present in feeds and grains, though their transmissionroutes are less clear. Campylobacter spp. are estimated to be the second most commoncause of human foodborne illness in the United States and maybe responsible for up to 4million infections per year (Hingley, 1999; Tauxe, 2002). Due to rapid horizontal transmis-sion throughout flocks, the route of vertical transmission is largely unclear (Shanker et al.,1990; Humphrey et al., 1993). The primary route of inoculation appears to be direct contactwith contaminated faeces or drinking water at the breeding or hatchery facility (Pearsonet al., 1993; Humphrey et al., 1993; Weijtens et al., 1993). Old and emerging pathogenssuch as Bacillus anthracis, Mycobacterium spp., Newcastle disease virus, the protozoanToxoplasma gondii, the nematode Trichinella spirillis and the agent causing bovine spongi-form encephalopathy may be isolated from forages or meat meals (Tauxe, 2002; Hinton,2000; Crump et al., 2002). However, these pathogens are not considered to be major threatsto human health due to intensive hygiene, surveillance and vaccination programmes inplace.

Feed, therefore, may contain a diverse microflora originating from soil that is char-acterized by its ability to survive under conditions of desiccation and a wide range ofmicroenvironments. The dissemination of bacteria may be assisted by boring insects thatinvade seeds. Low water activity limits bacterial grown in stored grain. When animal feedis stored under moist, anaerobic conditions such as silage or haylage, bacterial diversity

is inhibited by low pH. However, bacteria in feed have received relatively little attentioncompared to moulds and fungi.

4. Incidence and importance of mycoflora in feeds

4.1. Origins of feed mycoflora

Mycoflora (moulds) can also be present in feed and present a potential threat to feedquality and seed survival. Moulds may cause a decrease in seed germination, musty orsour odours, dry matter and nutrient loss, caking, mycotoxin formation and, ultimately, areduction in feed monetary value (Beuchat, 1978; Sauer et al., 1992).

Different populations of moulds may be found in growing versus stored grain and can bedivided into two large groups: field fungi and storage fungi. Field fungi may include the non-pathogenic Absidia, Alternaria, Chaetomium, Cladosporium, Diplodia, Phaeoramularia,and Rhizopus spp. and the pathogenic Drechslera (Helminthosporium) and Fusarium spp.which invade kernels or seeds of plants while they are still growing (Wallace and Sinha,1975; Sauer et al., 1992; Miller, 1995). Some moulds, such as Aspergillis flavus, can be botha plant pathogen, causing kernel rot in maize and a storage fungi (Placinta et al., 1999). Thegrowth of mycoflora on crops is highly dependent on climatic conditions, e.g. rainfall andtemperature (Miller, 1995; Bohra and Purohit, 2003). A. flavus and Aspergillus parasiticusgrow best and produce aflatoxin at temperatures greater than 21 ◦C (Thompson and Henke,2000). Fungal invasion is enhanced when the crops are stressed, such as during drought orinsect infestation. Field fungi are characterized by requirements for a high moisture content(greater than 200 g/kg), and thus are vulnerable to drying post-harvest (Sauer et al., 1992).Though some mycelium may remain dormant in feeds after harvesting, most die duringstorage or international transport (Wallace and Sinha, 1975; Sauer et al., 1992).

An estimated 1 in 3000 dried seeds may contain moulds that have adapted to growthin environments with low concentrations of available water. These xerophilic moulds,which are rarely isolated from growing grains, include member of the genera of Can-dida, Hansenula, and Penicillium roquefortin when the moisture contents of grains rangearound 190 g/kg (Clarke and Hill, 1981). When the water activity of the grain decreases torange from 0.68 to 0.80, the Aspergillus and Penicillium spp. predominate, with minor con-tributions from Absidia and Mucor spp. (Ito et al., 1973; Wallace and Sinha, 1975; Beuchat,1978; Clarke and Hill, 1981; El-Kady and Youssef, 1993). Like their bacterial counterparts,these moulds may be disseminated by grain-feeding insects and mites (Sauer et al., 1992;Miller, 1995).

The same fungi responsible for contaminating field and stored grains are, not surprisingly,also reported in animal feed and feed ingredients. Moreno Romo and Fernandez (1986) iso-lated both field (Cladosporium and Fusarium spp.) and storage fungi (Aspergillus, Mucor,and Penicillium spp.) in commercial mixed poultry feeds, though storage genera were foundwith a greater incidence. Aspergillus, Fusarium and Penicillium spp. have also been detectedin fish meal and rabbit feeds, with one study reporting a high incidence of Aphanocladiumalbum (Moharram et al., 1989; El-Kady and Youssef, 1993; Abarca et al., 1994; Bragulat etal., 1995). Pedersen (1992) noted that insect excrement, exuviate (cast skins), as well as the

ability of insects to pierce the protective wax coating of kernels, may alter grain environmentand encourage mould infestation. Intact fungal chlamydospores have been observed insideinsect fecal pellets using electron microscopy (Curl et al., 1983). Aspergillus, Fusarium,Mucor, and Penicillium spp. have even been isolated from poultry litter, although the routeof expose was uncertain. The mycoflora could have been indigenous to the litter, inoculatedby spilled feed, insect vectors or fungal spores surviving passage through poultry intestinaltracts.

4.2. Factors that influence feed mycoflora ecology

Total mould enumerations in poultry feed in a study by Tabib et al. (1981) rangedfrom 5 to 1.2 × 106 CFU/g. Total mycofloral activity may also be measured by respiratoryproduction of carbon dioxide, as 90% of the respiration in maize with 150–160 g moisture/kgmay be due to fungi (Bartov and Paster, 1986; Sauer et al., 1992). Mould growth hasbeen associated primarily with the moisture content of feed ingredients (Thompson andHenke, 2000), but may be also positively associated with zinc concentrations in feed andthe surface area available for mycofloral attack (Jones and Hamilton, 1987). Jones andHamilton (1987) suggested that increased fungal activity may be due to increased aeration,since fungal activity was greatly increased in pan feeders as opposed to tube feeders inpoultry production.

4.3. Mycoflora and feed quality

Mycofloral activity may decrease the quality of grain by several methods includingrespiratory heating and reducing nutrient content. Mycofloral respiration and growth byA. glaucus, A. candidus, and A. flavus may raise the temperature of grain within a silo to55 ◦C, maintaining that temperature for weeks (Sauer et al., 1992). This temperature over aprolonged period of time is sufficient enough to blacken soybeans and may damage aminoacids through Maillard reactions (Anderson-Hafermann et al., 1993; Johnson et al., 1998;Erickson et al., 1999a,b; Birlouez-Aragon et al., 2001). Grains that contain excess moistureare vulnerable to advanced mycofloral attack, which produces musty and caked feed that canblock augers and automatic feeding equipment (Sauer et al., 1992). Some storage moulds,such as Penicillium chrysogenum, Aspergillus flavus, and Rhizopus chizopodifarmis, areproteolytic, and may decrease dry matter digestibility, amino acid, vitamin and fat contentsin feed (Bartov et al., 1982; Megalla et al., 1990; Russell et al., 1991).

4.4. Mycotoxins and their health effects

Disease caused by mycoflora is primarily attributable to the consumption of toxins pro-duced by the moulds. Agriculturally important mycotoxins, the fungi that produce them andthe disease syndromes they cause are presented in Table 2. However, Miller et al. (2004)recounted a case of aspergillosis in captive 2-week-old mallards. Due to poor hygienicpractices and ventilation, Aspergillis was allowed to flourish in the housing facility. Fungalspores contaminated all spaces of the facility including litter and feed. Dust stirred up bythe movement of the ducklings was inhaled, introducing the spores into the lungs where

Table 2Mycotoxins, the fungal agents producing them and their toxic properties

Toxin Fungal agents Toxicity

Aflatoxin (B1, B2, G1, G2) Aspergillus spp. (A. flavus, A.parasiticus)

Hepatotoxic

Ochratoxin A Aspergillus spp. (A. ochraceus),Penicilluim spp. (P. verrucosum)

Nephratoxic, carcinogenic,teratogenic, neurotoxic, immunotoxic

Trichothecenes (T2 toxin,diacetoxyscirpenol,4-deoxynivalenol(vomitoxin), nivalenol)

Fusarium spp., Myrothecium spp.,Stachybotrys spp., Trichoderma spp.Cephalosporium spp., Trichotheciumspp., Verticimonosporium spp.

Nausea, feed refusal, mucousmembrane irritant, neurotoxic

Fumonisins Fusarium spp. (F. verticillioidesNirenberg, F. proliferatumNirenberg)

Hepatoxic, nephrotoxic, pulmonaryedema (swine),leukoencephalomalacia (horses),hepatocarcinoma (rats)

Zearalenone Fusarium spp. (F. graminearum, F.culmorum, F. sacchari)

Estrogenic, infertility, prolapseduterus, reduced milk production

they began growing. Sporadic outbreaks of aspergillosis in wild birds have been reported,however these incidents are preceded by some immunocompromising insult.

Mycotoxin production in feeds has been extensively studied. Mycotoxins are a broadclass of secondary metabolites produced by many genera of fungi; however the majorityof the clinically important ones are primarily produced by the genera Aspergillus, Penicil-lium, and Fusarium (Table 2). Animals consuming mycotoxins may suffer from symptomsranging from decreased growth rates, hepatic and nephritic toxicities, reproductive failures,neurological degeneration and death (Burditt et al., 1983; Livesey, 1994; Bohra and Purohit,2003). The severity of mycotoxicosis differs by toxin and the species exposed. For example,pigs are less sensitive to the effects of aflatoxin B1 than poultry, which in turn are over 20times as resistant compared to rabbits (El-Daraway and Marai, 1994; Miller, 1995). Simi-larly, cattle are more resistant to the effects of deoxynivalenol than pigs, due to degradationof the toxin in the rumen. Aflatoxicol, a toxic metabolite of aflatoxin, has been isolatedfrom all tissues of laying hens consuming aflatoxin B contaminated feed, including eggs(Trucksess et al., 1983). Metabolites of mycotoxins have been recovered from the meat andmilk of cattle who have consumed contaminated feed (Phillips et al., 2002).

Mycotoxins have been detected in most grains and feed ingredients (Bohra and Purohit,2003). Small grains (such as wheat, sorghum, oats, rye, barley, and rice) are less susceptibleto mycotoxin formation than large grains (such as maize) in the field. Mahmoud (1993)reported that soybean meal is resistant to mycotoxin formation, although El-Kady andYoussef (1993) detected aflatoxin in 0.35 of soybean seed samples. Aflatoxin production hasbeen linked to factors which promote storage fungi growth, including long feed residencetimes in broiler houses, high zinc concentrations in feed, and high relative humidity inhouses (Jones et al., 1982, 1984). The incidence of caked feed in delivery trucks, storagebins, conveyors and feeding troughs has also been linked to the presence of mycotoxinsin feed (Hamilton, 1975). Mycotoxins are often found in relatively high concentrations offoliage waste; contaminated maize screenings such as cobs, broken kernels, and stalks cancontain ten times the levels of fumonisin that found in whole kernels (Bennett et al., 1996). It

is often the case that multiple mycotoxins are be discovered in contaminated feed material,either due to multiple moulds growing on the grain or one mould producing several toxins(Miller, 1995).

5. Methods of detection of microbial contamination

5.1. Presence of indicator organisms

Given the difficulty of monitoring the myriad of microorganisms which may contaminateanimal feeds, tracking a variety of indicator organisms have been proposed as a means tomonitor overall hygiene and fecal contamination. Toranzos and McFeters (1997) reviewedseveral indicators of fecal contamination, including total coliforms (Gram-negative, non-sporeforming rods that ferment lactose and produce acid and gas within 48 h at 35 ◦C),fecal coliforms (coliforms that produce gas and acid at 44.5 ◦C), and E. coli. Sulphite-reducing clostridia (Cl. perfringens and Cl. welchii), Pseudomonas spp., or a ratio of fecalcoliforms/fecal streptococci may also be used. In a series of studies Maciorowski et al.(2002a,b) examined the utility of standard low nutrient agar medium and detergent-basedextraction to enumerate bacterial populations from animal and poultry feeds. The low nutri-ent agar medium is widely used for the assessment of microbial quality in public watersources (Reasoner and Geldreich, 1985; Horgan et al., 1999). In general, Maciorowski etal. (2002a,b) concluded that use of the low nutrient medium for the microbial enumerationsof fresh and stored animal feeds produced comparable results compared to the more tra-ditional bacterial plating media. However, detergent-based extraction techniques to detachmicroorganisms did not improve recovery of microorganisms from feeds.

Male specific RNA coliphage may also be used as an indicator of fecal contaminationin environmental samples (Sobsey et al., 1995; Straub et al., 1995; Woody and Cliver,1995; Ashelford et al., 1999). Animal feeds with varying types and levels of microbialcontamination were investigated by Maciorowski et al. (2001a) to determine if male specificor somatic bacteriophages could be detected and characterized by their nucleic acid content(RNA or DNA) as a potential fecal indicator in feed. Using a two-step enrichment approachand E. coli as the host, phages were detected in a wide variety of feed ingredients andcommercial dietary mixes. Coliphages, both male specific and somatic, were detected in atleast one sample of every feed matrix tested, and were considered to possibly be ubiquitousthroughout feed. However, no clear associations could be detected between the incidenceof RNA or DNA phages and either fecal coliforms or Salmonella spp.

Fecal indicators may not necessarily correlate with the indicated presence of pathogens.In dried foods, Silliker and Gabis (1976) detected fecal coliforms in over 1800 samplesthat were negative for Salmonella spp. Likewise, Salmonella spp. were found in 59 samplesthat were negative for fecal coliforms. Ammonia and anionic organic compounds in litterappear to be toxic to indicator organisms, complicating the task of tracing fecal contami-nation (Gupta et al., 1997). Some epiphytic plant bacteria, notably Erwina herbicola andPseudomonas syringe can produce antibiotics or inhibitors against E. coli (Volksch et al.,1996), complicating detection in moist feeds. In short, monitoring pathogens either in feedmixtures or feed ingredients will probably require direct detection of the pathogen.

5.2. Molecular approaches to microbial diversity characterization

Researchers have investigated microbial population behavior in complex environmentsby examining DNA sequences that are conserved across pathogen species, plasmids, or byusing restriction enzymes to characterize genomes based on sequences vulnerable to diges-tion. Borneman et al. (1996) used small subunit (ss) rRNA genes to examine microbialpopulation diversity in pasture soil. Hurek et al. (1997) used a combination of 16S rDNAanalysis and BOX-PCR (targeting repetitive intergenic sequence elements of Streptococcusspp.) to investigate nitrogen-fixing Azoarcus spp. in soil. Restriction fragment length poly-morphism (RFLP) analysis, in which population diversity is measured by variations in thelength of an amplified 16S rDNA gene, has been used in environmental samples such asbioreactor sludge, termite guts, and seagrass (Halophila stipulacea), and to determine bac-terial population differences in cow versus human fecal populations (Weidner et al., 1996;Liu et al., 1997; Bernhard and Field, 2000). If specific microorganisms could be trackedthroughout the environment, a clearer understanding of transmission cycles may lead tonew advances in slowing disease spread.

Molecular analysis has also been applied as a surveillance method to detect microbialcontamination in animal feed samples (Maciorowski et al., 2005). These techniques have anadvantage over accepted methods involving the isolation of bacteria by enrichment, selectiveculturing, and biochemical or serological identification is that molecular assays are morerapid. Traditional culturing methods can take up to 5–7 days to produce results versus24–36 h for molecular analysis. Thus, a feed mill that produces several tons of feed per hourcan monitor its production in a more timely manner. There are numerous commercial PCRkits available. A sampling of PCR kits for the detection of Salmonella spp. include: BAXTM

(Qualicon, Wilmington, DE.), ProbeliaTM (Sanofi Diagnosic Pasteur, Marnes La Coquette,France), and TaqManTM (PE Applied Biosystems, Foster City, CA). All of these assayshave reported greater than 95% accuracy in detecting the bacterium in various matrices.

Part of the difficulty with molecular assays lies with the problem of extracting andrecovering representative samples from feeds for molecular analyses. Several methods forthe extraction of microbial nucleic acids have been tested to create a sampling strategy thatmay be used to isolate and amplify microbial DNA from animal feeds using polymerasechain reaction (PCR). A detergent-based initial detachment of microorganisms from feedsfollowed by the modified procedure of Widmer et al. (1996) was found to be the mostrobust extraction technique for removing inhibitory compounds (Maciorowski et al., 2001b,2002c,d). However, the extraction requires an overnight incubation step, which complicatesits use as a rapid method. Incubation at lower temperatures (−70 ◦C instead of −20 ◦C) maybe able to shorten this incubation. As more commercial assays become available standardprotocols for extraction, enrichment and PCR can be more systematically evaluated todetermine the efficacy, detection limits, and the minimum enrichment time required forroutine analyses of animal feeds.

5.3. Detection of mycotoxins in feed

Methods for the detection of mycotoxins in grains include: high performance liquid chro-matography, gas chromatography, thin layer chromatography, mass spectrometry, enzyme

linked immunosorbent assay (ELISA), and molecular biological analysis (Placinta et al.,1999; Yoshizawa et al., 2004). The ongoing goal is to produce assays that are rapid, accurate,can handle high through-put, and require the minimal amount of equipment.

Chromatographic methods, such as high performance liquid chromatography (HPLC),gas chromatography (GC), and thin layer chromatography, can be used to isolate myco-toxins from other components in feed samples (Eke et al., 2004; Yoshizawa et al., 2004).Chromatography can be coupled to a ultraviolet, fluorescence, flame ionization, or electroncapture detector or to a mass spectrometer to definitely detect and quantify mycotoxins.Separation and/or detection of mycotoxins can be enhanced by chemical derivatizationwhere the toxin is conjugated to another compound that alters its physiochemical prop-erties. In general, these methods are sensitive and able to detect and quantify of multiplemycotoxins in a biological samples. However, these methods are not suited for rapid analy-sis, as considerable sample preparation is usually needed to remove contaminants that couldinterfere with the assay (Eke et al., 2004). Sample extraction and clean-up procedures caninclude liquid-liquid separation, solid-phase extraction, gel permeation and immunoaffin-ity chromatography. Eke et al. (2004) utilized columns consisting of layers of ammoniumsulfate, celite, alumina, and C18 silica to clean up acetonitrile/water extracts of maize forthe detection of multiple trichothecenes by flame ionization and mass selective detection.However, these columns were still unable to eliminate the interfering effects of the highprotein content found in samples of maize gluten feed. Furthermore, these techniques useexpensive machinery, requiring extensive maintenance and trained personnel to keep themat their optimal operation. Conventional analytic methods for the detection of zearalenonerequire the use of chlorinated solvents which can sometimes give inaccurate results. Urracaet al. (2004) optimized a method of pressurized liquid extraction to overcome these short-comings. They determined that optimal extraction conditions were obtained using a 50:50methanol/acetonitrile solvent at 50 ◦C and 1500 psi. This procedure produced extracts withrecovery rates greater than 90% with no contaminates that interfered with quantificationby a fluorescence detector. The process can be automated and is relatively inexpensive andrapid when compared with other extraction procedures.

Immunoassays have the advantage of being rapid to perform, require minimal equipmentand training to perform well. By careful selection of specific monoclonal antibodies, theassays can be made both sensitive and specific. Yoshizawa et al. developed three ELISAkits for the detection of deoxynivalenol (DON), nivalenol (NIV) and T-2 toxin (T-2) inwheat using monoclonal antibodies. Extraction was a single step procedure using of 85%acetonitrile in water. The samples required no clean up, but require mild acetylation in orderto measure total trichothecene concentration. Recovery of the toxin from wheat sampleswas above 90%. The assays were found to have detection limits of 80 ng/g for DON and NIVand 30 ng/g of T-2. The results were found to correlate well with the results of GC–MS, wasrelatively rapid (<2 h), and could handle 42 sample per assay. Shim et al. (2004) developed afluorescence polarization immunoassay for the detection of ochratoxin A (OTA). This assayis a competitive immunoassay that measures the change in the polarity of a fluorescein-OTA conjugate when bound to an OTA-specific monoclonal antibody. The assay had aconcentration range of 5–200 ng/ml OTA, with a limit of detection of 3 ng/ml. No crossreactivity with other mycotoxins, patulin, zearalonone, T-2, and aflatoxin B1, was observed.Extraction of OTA was a single step procedure using either ethanol or methanol and requiring

no clean up; recovery of OTA from spiked barely samples were above 90%. When the assaywas tested with naturally contaminated barley samples, the results correlated well with acompetitive indirect ELISA used as a confirmatory assay.

Only recently has molecular biological analysis been applied to the detection of myco-toxin producing fungi in feed. Patino et al. (2004) developed a PCR detection assay to detectfumonisin-producing strains of Fusarium verticilliodes. They produced two sets of primersfrom derived the intergenic spacer regions of rDNA. One set of primers was species specificfor F. verticilliodes; the other set was specific for both the species and for fumonisin produc-ers. The assay was then used to analyze 54 strains of F. verticilliodes isolated from maizeand bananas. The strains isolated from maize were found to produce the toxin fumonisinB1; whereas, the strains recovered from bananas did not. The strains isolated from maizewere amplified by both sets of primers. The strain derived from banana were amplified byonly the species-specific primers but not by the primers specific for fumonisin production.When the PCR assay was used on other species of Fusarium, neither set of primers producedany amplification fragments. Edwards et al. (2001) developed a competitive PCR assay toquantify the number of trichothecene-producing Fusarium ssp. in winter wheat. They useda primer specific to the Tri5 gene encoding trichodiene synthase, an enzyme the catalyzethe first step in the production of trichothecenes. They then used this assay to ascertain therelationship between the amount of PCR product, concentration of deoxyvalenol (DON),and the number of infected spikelets in samples of winter wheat experimentally infectedwith three strains of fusarium. It was determined that the amount of PCR product corre-lated well with concentrations of DON found in the grain samples. However, there was nocorrelation between PCR product and the number of visibly infected spikelets.

6. Strategies for the control of microbial contamination in feed

6.1. Control of bacterial contamination

An overview of several methods used to control bacterial contamination of feeds maybe found in Table 3. Several strategies that have been tried to overcome feed degrada-tion including: shortening storage time to prevent browning and caking of the feed, and

Table 3Control methods for bacterial feed contaminants, modes of action and notes

Control strategy Notes

Rapid drying Reduces the amount of available waterShorter storage time Reduces the amount of caking, mustiness, and browning of feedZinc bacitracin Controls Gram-positive growth and hinder Clostridia spp. sporolationMineral acids, short-chain fatty

acids, isopropyl alcohol,aldehydes, trisodium phosphate

Added to feed as disinfectants

Bacteriophages Kills actively growing bacteria in feedPropionic acid producing bacteria Added to silage to aid in acid productionBacteriocin producing bacteria Added to silage to control pathogens

supplementation with soybean oil to overcome fat losses (Bartov et al., 1982). To preventovergrowth by storage microorganisms, rapid drying has been widely used to preservegrain (ICMSF, 1998). Zinc bacitracin can be added to feed to control Gram-positive bacte-rial growth and hinder Cl. perfringens sporolation (Elfadil et al., 1996c). Other disinfectingagents have been added to feeds in order to control pathogens. Some of the compounds testedinclude: mineral acids (such as hydrochloric, sulfuric, and nitric acid), short-chain fatty acids(like propionic, butyric, and lactic acid), isopropyl alcohol, aldehydes, and trisodium phos-phate (Maciorowski et al., 2004). However, the antimicrobial activity of these disinfectantsare inhibited by the high concentrations of organic matter in feed. Furthermore, some ofthese compounds are corrosive and/or toxic at higher concentrations, thus limiting their usein processed feeds.

Ensilage has long been used as a method of feed preservation. The fermentation processinhibits the growth of bacteria due to the rapid decrease of pH in the feed matrix. There havebeen numerous attempts to optimize the process in order to better prevent contaminationby spoilage and/or pathogenic microflora. Research has focused on screening, breed, orgenetically manipulated Lactobacillus spp. and Pediococcus spp. to produce strains thatwill optimal for ensile specific feed materials (Weinberg and Muck, 1996). Co-inoculationof lactic and propionic acid producing bacteria are being used to enhance the reduction offeed pH. Bacteria that produce bacteriocin-like compounds are also being introduced intosilage to control potential pathogens and spoilage bacteria, such as L. monocytogenes.

Lactobacillus spp. have also been used as probiotic agents to modify the gastrointestinalenvironment in order to prevent the overgrowth of pathogens. Murry et al. (2004) reportedthe effects of Lactobacillus salivarius and Lactobacillus plantarum on the growth of E. coli,S. enterica ser. Typhimurium, and Clostridia perfringens in poultry feed media. Poultry feedmedia was created by blending maize/soybean-based starter and grower diets, filtering theresulting broth, and adding 1.2% agar. Plates of the media were inoculated with a singlestreak of either strain of lactobacilli and incubated for 48 h at 37 ◦C. The plates were thenspread plated with the pathogens and incubated for another 24 h. Growth inhibition zoneswere then measured and compared with control plates. It was determined that all threepathogens were inhibited by the lactobacilli. The ranking of observed growth inhibitionwas E. coli > Cl. perfringens > S. typhimurium. When the pH and organic acids profiles ofthe poultry feed media were measured, it was found that the plates inoculated with eitherlactobacilli had lower pH values and a greater concentration of acetic and lactic acids. Thesein vitro findings suggest that supplementation of feed with these lactobacilli strains wouldbe beneficial in control the growth of these pathogens in poultry feed.

Mikkelsen et al. (2004) reported that the physical properties of feed affected gastrointesti-nal conditions in pigs. Finely and coarsely ground barley/wheat/soy feeds were producedand fed to Landrace × Yorkshire pigs. It was observed that the pigs consuming the coarselyground feed had significantly higher concentrations of anaerobic bacteria, higher levels ofgastric ATP, increased concentrations of several organic acids, and a lower gastric pH thanthose pigs receiving the finely ground feed. These findings were all evidence of increasedbacterial fermentation in the stomachs of the pigs eating the coarse feed. When the stomachcontents from both groups were inoculated with S. enterica serovar Typhimurium and thebacterial counts quantified over time, a greater reduction of bacterial numbers was observedin the stomach contents of the pigs consuming the coarse diet. It was known that pigs fed the

coarsely ground meal had greater gastric water retention and slower gastric passage ratesthan those fed the finely ground diet. It was hypothesized that these conditions were thoughtto be more conducive to greater bacterial fermentation and increased production of organicacids. The higher levels of propionic, butyric, and lactic acids in the gastrointestinal tractsof these animals exerted increased antimicrobial action against the bacteria. However, thepigs consuming the coarsely ground diet exhibited significantly better feed conversion rateswhen compared to those receiving the finely ground feed.

Processing of individual feed components into a combined ration represents anotherstep where feed can be contaminated. A survey conducted in 1993 by the Food and DrugAdministration (FDA) of 78 rendering plants producing animal protein-based feed and 46feed mills making vegetable protein-based feed found that S. enterica could be detected in0.56 of the 101 samples of animal-based feeds and 0.36 of the 50 samples of the vegetable-based feeds tested (Crump et al., 2002). Another survey performed by the FDA in 1997found that approximately 0.16 of compound feeds were contaminated with Salmonella spp.(Davies and Wray, 1997). Identification of the areas in the feed mill and the processesprone to contamination is a necessary prerequisite to correcting the problem. When nineanimal feedmills in the United Kingdom were sampled for Salmonella contamination, itwas determined that the intake pits, where newly arrived feed was delivered and the coolerdownstream of the pelleting machine had the highest rates of contamination. Other sitessuch as storage bins, grinders and loading docks exhibited lower rates of contamination.Bird droppings found at the intake pits and loading docks were deemed potential sources ofcontamination. Methods of repelling birds from the area and rapid removal of spilled feedwere required to contain this problem.

Heat treatment, usually during conditioning, pelleting or extrusion, has been shown tobe an effective way of reducing pathogen and toxin levels (Stott et al., 1975; Furuta et al.,1980; Davies and Wray, 1997; Ekperigin et al., 1990). Reductions in bacterial contamina-tion by heat is dependent on the temperature, treatment time and the moisture content of thefeed (Stott et al., 1975; Maciorowski et al., 2004). Furuta et al. (1980) reported that 15 s at70–80 ◦C could reduce the load of coliforms in poultry feed mash a thousand fold. Whereas,Ekperigin et al. (1990) reported that the optimal temperature, time and moisture contentfor conditioning feed in order to reduce populations of E. coli and Salmonella spp. were85.7 ◦C, 4.1 min, and 145 g moisture/kg. However, Cl. perfringens can survive pelletingat 90 ◦C due to the heat resistance of its spores (Greenham et al., 1987; Maciorowski etal., 2004). In addition, the agent that causes bovine spongiform encephalopathy is resistantto temperatures that would denature most viruses and bacteria. The cooling apparatus justdownstream of the conditioner/pelleter is another potential site of contamination (Daviesand Wray, 1997). The most highly contaminated site was found to be the feed inlet. Themost likely time period for the cooler to become contaminated is the first few minutes afterthe system goes online when the heater is not yet at operational temperature. Proceduresto reduce potential contamination at this point involve allowing the heater and coolers tocome to operational temperatures before processing commences and to clean and micro-biologically sample the cooler inlet between shifts. The air source used by the pelletingmachine has also been implicated as a source of potential contamination (Jones and Ricke,1994). Air inlets should be situated away from areas where dust may be generated, such asingredient receiving areas and loading docks.

The use of ionizing radiation has been investigated as a method of eliminating microbialcontamination in feed (Maciorowski et al., 2004). Levels of radiation need to eliminate vari-ous contaminants range from 1 kGy (1 Gy = 100 rad) for eukaryotic parasites, to 10–35 kGyfor vegetative bacteria, and up to 50 kGy of viruses. However, radiation levels of 10 kGycan destroy thiamin and riboflavin and 35 kGy can destroy amino acids, thus negativelyaffecting feed quality.

Finally, feed stored at the farm, feedlot and other sites must be protected from microbialrecontamination. This should involve maintaining clean facilities and equipment and havingadequate personnel hygiene procedures in place. The contamination of feed and equipmentwith Campylobacter spp. has been implicated in the spread of the bacterium on the farm. Thedisinfection of boots has been effective in reducing transmission between poultry houses(Shanker et al., 1990; Humphrey et al., 1993). E. coli O157:H7 has been shown to persist inwater troughs for long periods of time (Tauxe, 2002). Programs to control rodents, cats wildanimals and birds are also necessary to prevent them gaining access to the feed and contam-inating it with their faeces (Davies and Wray, 1997; Schoenbaum et al., 2000; Tauxe, 2002).

6.2. Fungal and mycotoxin control

Processing can also potentially redistribute the concentration of pathogens and toxin incontaminated grains. Brera et al. (2004) measured the amounts of fumonisin B1 in variousfractions of dry milled maize. The naturally contaminated maize contained a toxin concen-tration of 4.54 mg/kg. The highest contaminated fractions were germ and bran, with 8.92and 7.08 mg/kg, respectively. Fractions of large and small grits and polenta had relativelylow concentrations of the toxin (<0.60 mg/kg). Animal feed flour, a combination of germ,bran, small grits, and foliage waste, such as cobs, broken kernels and stalks, was determinedto have a toxin concentration of 9.36 mg/kg. The higher concentrations of fumonisin B1found in the germ, bran, and animal feed flour were due to localization of mould infestationto the external portions of the kernel. In addition, the high fat content of the germ makesit a favorable area for mould growth. Similar results were observed in maize that was wetmilled (Bennett et al., 1996). When naturally contaminated maize, having a fumonisin B1concentration of 15 �g/g, was milled, the germ, bran and gluten exhibited concentrations of3.1, 5.7, and 5.8 �g/g, respectively. The toxin concentration in the steepwater used duringprocessing was 2.1 �g/g. In contrast, the starch produced by wet milling had a level of<0.1 �g fumonisin B1/g.

Attempts to control mycoflora in crops and stored feed materials have proven difficult dueto ubiquitous nature of moulds and the variation of mycotoxin chemical structure. A reviewof some of the methods used to control mycotoxin concentrations in feed can be found inTable 4. Research into breeding crops that are resistant to infection by field fungi have todate been disappointing (Miller, 1995). As fungal infestation of grain is heterogeneouslydispersed in feed, attempts to screen affected grains/seeds by color, density or size havebeen used in an attempt to remove them from the feed supply. However, it has been notedthat mycoflora can colonize crops and produce mycotoxins without causing visible damage(Patino et al., 2004). Conversely, signs of fungal infestation do not necessarily correlatewith the presence of mycotoxin. Broken maize kernels can have ten times higher levels offumonisin B1 as compared to intact grain (Bennett et al., 1996). However, visibly undamaged

Table 4Strategies of controlling mycotoxins in feed

Control strategies Mode of action

Screening Screening of infested grains/seeds by sorting by color, density, size,or fluorescence (black light). Broken grain particles can havehigher levels of mycotoxins as compared to intact grain

Propionic acid Added to stored grain to prevent mould growth. Chemicallydenatures mycotoxins

UV irradiation Irradiation of grain prior to storage to kill fungi. Physicallydeactivates mycotoxins

Heat treatment Denaturation of mycotoxins by heating during cooking, extrusion,and/or pelleting. Some mycotoxin resist denaturation even attemperature approaching 260 ◦C

Calcium hydroxide, sodiumbisulphate, hydrogen peroxide,sodium hypochlorite

Chemical inactivation of mycotoxins

Aluminosilicate clays Binds mycotoxin in the gastrointestinal tract to prevent adsorptionAmmoniation Uses ammonium hydroxide or gaseous sammonia to denature

mycotoxins under alkaline conditionNixtamalization Traditional method of controlling mycotoxins in maize by boiling

and soaking in calcium oxide (lime) to produce tortilla flourOzonation Gaseous or aqueous ozone is passed through grains to cause

oxidative deactivation of mycotoxins

kernels of maize can contain greater than 10 �g fumonisin B1/g. It has been observed thatsome kernels of maize infected with A. flavus fluoresce under UV light (Thompson andHenke, 2000). Ranchers and wildlife managers have attempted to use this “blacklight” test asan inexpensive and rapid method of toxin screening. Thompson and Henke (2000) evaluatedthis method to determine if there was a relationship between aflatoxin concentration and thepercentage of fluorescing kernels. It was determined that this method had a false negativerate of 23%. Further, the test was only accurate at predicting the presence of the toxin atconcentration above 700 �g/kg. Some mycotoxins, such as deoxynivalenol, are associatedwith hull or the outer surfaces of the grain, thus removing the outer layers would reducethe amount of the toxin. House et al. (2003) showed that pearling naturally contaminatedbarley for 15 s left 85% of the grain mass while reducing 66% of the deoxynivalenol.

As with bacterial contaminants, heat treatment has been utilized to denaturation mycotox-ins during cooking, extrusion and/or pelleting (Bohra and Purohit, 2003; Buser and Abbas,2002). Buser and Abbas (2002) reported that heat denaturation of aflatoxin in cottonseedwas both time and temperature dependent, with an estimated 47% reduction at 160 ◦C.However, some mycotoxins, such as zearalenone, resist denaturation by heat (Urraca et al.,2004). Irradiation with ultraviolet light can be used to kill fungi prior to storage and deac-tivates mycotoxins. Phyllosilicate clays, an anti-caking material, added at 5 g/kg poultrydiet has been shown to prevent aflatoxicosis at high doses of the toxin (7500 ppb) withoutaffecting adsorption of other nutrients (Phillips et al., 2002). Propionic acid has been addedto stored grain to prevent mould growth and to chemically denature mycotoxins. Feed hasbeen treated with numerous chemical agents including calcium hydroxide, sodium bisul-phate, hydrogen peroxide, and sodium hypochlorite have been used to inactivate mycotoxins

(Bohra and Purohit, 2003). Nixtamalization is a traditional method practiced in Mexico todetoxifying aflatoxin in corn by boiling and soaking in calcium oxide (lime) to producetortilla flour. Ammonia, in the form of ammonium hydroxide or gaseous ammonia has beenused to denature mycotoxins under alkaline condition. Gaseous or aqueous ozone has beenpassed through grains to cause oxidative deactivation of mycotoxins (Prudente and King,2002). While ozone works on a wide variety of mycotoxins, it is expensive to produce,cannot be stored, and is a hazardous material requiring extensive safety precautions.

7. Summary

Feed may serve as a substrate for a wide variety of microorganisms. Some of themicroflora are adapted to the desiccated conditions in soil and are transferred by insects,dust, and wind to similar niches in feed. Some bacteria are adapted to a niche where theyare capable of degrading organic matter and/or exist in a survival state until the moisture ishigh enough for bacterial action. While other microorganisms, primarily moulds, activelygrow within stored seeds and use seed nutrients and the low amount of available mois-ture as substrates. Growth rates are markedly decreased compared to other niches (suchas intestinal tracts), but a wide variety of microbial species can survive on grain particles.Preventing microbial contamination of animal feeds will require more representative androbust detection approaches that can assess microbial diversity. Molecular methods holdpromise as a viable technology to accomplish this but will require considerable optimizationand standardization before routine use can become a reality in the animal feed industry.

There is evidence that controlling microbial contamination in livestock feed producessignificant health benefits in both human and animal populations. In 1991 Sweden starteda Hazard Analysis and Critical Control Point (HAACP) program to monitor S. entericacontamination in animal feed as part of a comprehensive “farm to fork” Salmonella spp.control system (Crump et al., 2002). Over 7000 sample of feed are tested annually. Detectionof a positive feed sample initiates more extensive testing and corrective measures. The resultsof an integrated surveillance program covering animal feed, livestock health, processedfoods and carcasses and human health suggest that the HAACP program has been successful.S. enterica has been virtually eliminated from animal feed and in meat. In addition the annualrates of human salmonellosis have dropped from 14 cases per 100,000 people in 1991 to 8cases per 100,000 in 2000.

Acknowledgements

This research was supported by the Texas Higher Education Coordinating Board’sAdvanced Technology Program (Grant #999902-165) and the Research Enhancement Pro-gram grant of the Texas Agricultural Experiment Station of the Texas A&M UniversitySystem (Grant #2-102). This research was also supported by Hatch grant H8311 adminis-tered by the Texas Agricultural Experiment Station. K.G.M. was supported by an EndowedGraduate Fellowship from Pilgrim’s Pride, Inc., Pittsburg, TX, and the Heep FoundationInternship. P.H. is supported by USDA-NRI Grant 2004-04571.

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