G. Tellez1, A.M.M. Hamdy2 and B. M. Hargis1

1Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701

2Department of Animal Production, Faculty of Agriculture, Minia University, Egypt.

Summary

The gastrointestinal tract contains within it a microenvironment of bacteria that influence the host animal in many ways.  The microflora can metabolize several nutrients that the host can not digest and converts these to end products (such as short chain fatty acids), a process which has a direct impact on digestive physiology.  The microbiota directs the  assembly of the gut-associated lymphoid tissue, helps educate the immune system, affects the integrity of the intestinal mucosal barrier, modulates proliferation and differentiation of its epithelial lineages, regulates angiogenesis, modifies the activity of the enteric nervous system, and plays a key role in extracting and processing nutrients consumed in the diet.  Despite these important effects, the mechanisms by which the gut microbial community influences host biology remain almost entirely unknown.  Recent molecular-based investigations have confirmed the species diversity and metabolic complexity of gut microflora, though there is much work to be done in order to understand how they relate to each other as well as the host animal.  It is almost a century ago that Eli Metchnikoff proposed the revolutionary idea to consume viable bacteria to promote health.  Since that time, the area known as ‘probiotics’ has made dramatic progress, particularly during the past two decades.  The last 20 years have also seen the emergence of a new, related area of study –‘prebiotics’.  Utilization of these two ideas – providing live non-pathogenic bacteria as well as substrates for their growth – have potential to help optimize the health of animals by manipulating the gastrointestinal tract in positive ways.

Introduction

There is a tendency to regard all microorganisms as harmful; to equate bacteria with germs. Nothing could be further from the truth. The number of non pathogenic species far exceeds the number of pathogenic species and many of the known bacteria are in fact useful, even essential for the continued existence of life on earth.
One example of a beneficial group of microorganisms are those which inhabit the gastrointestinal tract (GIT) of animals.  The GIT  harbors an incredibly complex and abundant ensemble of microbes [1, 2,].  The intestine is in contact with components of this microflora from birth, yet little is known about their influence on normal development and physiology.  The GIT is more densely populated with microorganisms than any other organ and is an interface where the microflora may have a pronounced impact on animal and human biology [2].  The bacterial population of the human cecum and colon is numerically large with at least 1013 cfu/g [3]. Similar values have been reported in other omnivores such as pigs [4]. Bacteria comprise about 40–55% of solid stool matter [5].  Through out millions of years of evolution, animals have developed the means for supporting complex and dynamic consortia of microorganisms during their life cycle.  A transcendent view of vertebrate biology therefore requires an understanding of the contributions of these indigenous microbial communities to host development and adult physiology. The fragile composition of the gut microflora can be affected by various factors such as  age, diet, environment, stress and medication [6]. As with most complex ecosystems, it appears that the majority of species cannot be cultured when removed from their niches [6, 7].  Although a full definition of biodiversity awaits systematic application of molecular enumeration techniques, such as genotyping DNA encoding 16S rRNA [rDNA] genes [8, 9, 10]. More than 50 genera and at least 500–1,000 different species are distributed along the length of the gastrointestinal tract [11].  The dominant organisms in terms of numbers are anaerobes including bacteroides, bifidobacteria, eubacteria, streptococci, and lactobacilli, while others, such as enterobacteria, also may be found, usually in fewer numbers [11, 12].  Generally, bacteroides [including those that can utilize a wide range of polysaccharides] are most numerous and can comprise more than 30% of the total. Recent evaluation of the microflora ecology of the chicken intestine using 16s rDNA determined that Lactobacilli was the predominate organism in young birds with the population of Bifidobacterium dominating in older birds [13].

Colonization begins at birth and is followed by progressive assembly of a complex and dynamic microbial society [14]. Assembly is presumably regulated by elaborate and combinatorial microbial–microbial and host–microbial interactions predicated on principles refined over the course of animal evolution.  Comparisons of rodents raised without exposure to any microorganisms to animals  that have assembled a microbiota since birth, or those that have been colonized with components of the microbiota during or after completion of postnatal development, has revealed a range of host functions affected by indigenous microbial communities. For example, the microbiota directs the assembly of the gut-associated lymphoid tissue [15] , helps educate the immune system [16, 17], affects the integrity of the intestinal mucosal barrier [18, 19, 20], modulates proliferation and differentiation of its epithelial lineages [21, 22], regulates angiogenesis [23], modifies the activity of the enteric nervous system [24], and plays a key role in extracting and processing nutrients consumed in the diet [25].  The microflora can metabolize proteins and protein degradation products, sulfur-containing compounds, and endogenous and exogenous glycoproteins [12]. Some organisms grow on intermediate products of fermentation such as H2, lactate, succinate, formate, and ethanol and convert these to end products including short chain fatty acids [SCFA], a process which has a direct impact on digestive physiology [26]. Although the mechanisms by which bacteria assert these effects on the gastrointestinal tract remain essentially unknown, research in this area is focusing on elucidating these mechanisms as well as manipulating the bacteria and the gastrointestinal environment towards achieving optimal health through probiotics and prebiotics.

The Role of Microorganisms on Digestive Physiology

Short Chain Fatty Acid Production

SCFA increase from undetectable levels in the ceca of day-of-hatch chicks to the highest concentration at day 15 of age as the enteric microflora becomes established [27].  The basic fermentative reaction in the human colon or chicken cecum is similar to that in obligate herbivores: hydrolysis of polysaccharides, oligosaccharides, and disaccharides to their constituent sugars, which are then fermented resulting in an increased biomass [28]. Carbohydrate hydrolysis is effected by a number of bacterial cell-associated and secreted hydrolases that can digest a range of carbohydrates which monogastric animals can not. Fermentation yields metabolizable energy for microbial growth and maintenance and also metabolic end products. Nitrogen for protein synthesis can come either from urea [via the urease reaction], undigested dietary protein, or endogenous secretions. The principal products are SCFA together with gases [CO2, CH4, and H2] and some heat [29].  Carbohydrates entering the large intestine can alter gut physiology in two ways: physical presence and fermentation. Effects of  SCFA can be divided into those occurring in the lumen and those arising from their uptake and metabolism by the cells of the large bowel wall.  SCFA are the principal luminal anions. They are relatively weak acids with pKa values of 4.8, and raising their concentrations through fermentation lowers digesta pH [29].  SCFA also serve as an important source of energy for the gut wall, providing up to 50 % of the daily energy requirements of colonocytes [28, 30]  Fermentable carbohydrates can alter the microbial ecology greatly by acting as substrates or supplying SCFA. Much attention has been directed toward the study of specific beneficial lactic acid bacteria, rather than the flora as a whole [30], however, the  SCFA have diverse functions with regards to host and microbial physiology.

Blood Flow and Muscular Activity

In vitro studies have shown that incubation with SCFA [as the sodium salts] at concentrations as low as 3 mM dilate precontracted colonic resistance arterioles in isolated human colonic segments [31].  Greater colonic blood flow has been observed with infusion of acetate, propionate, or butyrate [separately or as a mixture] into the denervated canine large bowel [32]. The mechanism of action of SCFA on blood flow does not involve either prostaglandins or α- or β-adrenoreceptor linked pathways [31].  The mechanisms of action may involve local neural networks as well as chemoreceptors together with direct effects on smooth muscle cells [33]. SCFA produced in the colon and entering the portal circulation seem to influence the upper gut musculature.  These actions are important for the maintenance of the function of the whole gastrointestinal system, not just the colon.  It is expected that greater blood flow enhances tissue oxygenation and transport of absorbed nutrients.

Enterocyte Proliferation

In rats, SCFA stimulate the growth of colorectal and ileal mucosal cells when they are delivered colorectally or intraperitoneally [34, 35].  In addition to promoting growth, the major SCFA (especially butyrate) appear to lower the risk of malignant transformation in the colon [36].  Secondary bile acids are cytotoxic, and in rats fed deoxycholate plus cholesterol, cell proliferation as measured by incorporation of [3H]thymidine was increased [37].  Some of the effects of SCFA may be due to low intra-colorectal pH rather than any specific SCFA. At a pH of 6, bile acids are largely protonated and insoluble and so would not be taken up by colonocytes [38].  Additionally, lower pH inhibits the bacterial conversion of primary to secondary bile acids [39, 40] and therefore lowers their carcinogenic potential.

Mucin Production

Evidence has been presented that mucus production and release is stimulated locally by endogenous production of SCFA by gut microflora [41, 42, 43].  Additionally, some studies have been completed evaluating the influence of specific beneficial or probiotic organisms on mucin production.  In vitro studies with Lactobacillus plantarum 299v suggest that the ability of organisms to inhibit adherence of attaching and effacing organisms to intestinal epithelial cells is mediated through their ability to increase expression of MUC2 and MUC3 intestinal mucins [44, 45]. The benefits of probiotics mediated through intestinal mucin upregulation may have broader applicability than enteropathogen intervention in poultry. Several investigators have shown that the increase in mucin production following probiotic administration inhibited replication, disease symptoms and shedding of rotavirus in humans [46, 47, 48, 49]. In the proximal colon, an increase in the butyrate concentration altered crypt depth and the number of mucus-containing cells; the increase in butyrate was highly correlated with the number of neutral-mucin-containing cells [50, 51].

Probiotic, Prebiotic and Synbiotic

The use of lactic acid bacteria as feed supplements goes back to pre-Christian times when fermented milks were consumed by humans. It was not until last century that Eli Metchnikoff, working at the Pasteur Institute in Paris, evaluated the subject from a  scientific basis.  Metchnikoff, documented a direct link between human longevity, and the necessity of maintaining a healthy balance of the beneficial and pathological microorganisms residing in the human gut. Metchnikoff's 1908 Nobel Prize in physiology had been awarded for his discovery of phagocytes and other immune system components, but his accurate description of vital elements in the body's intestinal flora is equally notable. He developed and prescribed to his patients bacteriotherapy, i.e. the use of lactic acid bacteria in dietary regimens [52].  In support of this he cited the observation that Bulgarian peasants consumed large quantities of soured milk and also lived long lives. He had no doubt about the causal relationship, and subsequent events have, in part, confirmed his thesis. He isolated what he called the 'Bulgarian bacillus' from soured milk and used this in subsequent trials. This organism was probably what became known as Lactobacillus bulgaricus and is now called L. delbrueckii subsp bulgaricus which is one of the organisms used to ferment milk and produce yogurt [52]. After Metchnikoff's death in 1916 the center of activity moved to the USA. Knowledge available at that time suggested the use of L. acidophilus and many trials were carried out using this organism. Encouraging results were obtained especially in the relief of chronic constipation [53].  In the late 1940's interest in the gut microflora was stimulated by two research developments.  Firstly, the finding that antibiotics included in the feed of farm animals promoted their growth. A desire to discover the mechanism of this effect led to increased study of the composition of the gut microflora and the way in which it might be affecting the host animal. Secondly, the more ready availability of germ-free animals provided a technique for testing the effect that the newly discovered intestinal inhabitants were having on the host. This increased knowledge also showed that L. acidophilus was not the only Lactobacillus in the intestine and a wide range of different organisms came to be studied and later used in probiotic preparations [54].

A probiotic is defined as a live microbial food supplement which benefits the host by improving its intestinal microbial balance [55]. The presence of normal gut microflora may improve the metabolism of the host bird in various ways, including absorptive capacity [56], protein metabolism [57], energy metabolism and fiber digestion [58], energy conversion [59] and gut maturation [60].  Balanced colonic microflora and immunostimulation are major functional effects attributed to the consumption of probiotics [55]. Many probiotic effects are mediated through immune regulation, particularly through balance control of proinflammatory and anti-inflammatory cytokines [61, 62]. However, probiotics can only be effective if the requirements for their growth are present in the GIT.  The concept of prebiotics is relatively new; it developed in response to the notion that nondigestible food ingredients (e.g. nondigestible oligosaccharides) are selectively fermented by one or more bacteria known to have positive effects on gut physiology. Bacteria fed by a preferential food substrate have a proliferative advantage over other bacteria [63]. Some prebiotics have shown to selectively stimulate the growth of endogenous lactic acid bacteria and Bifidobacteria in the gut to improve the health of the host [63]. Probiotic numbers have been enhanced by prebiotics that selectively stimulate the growth and/or activity of one or a limited number of bacterial species already resident in the large intestine, and, thus, improve host health [12].  In this way, prebiotics selectively modify the colonic microflora and can potentially influence gut metabolism [63]. However, the bacterial nutrient package will not be advantageous without the presence of the targeted, beneficial bacteria and likewise the live microbial product will not succeed if the environment into which it is introduced is unfavorable [64]. The concept of synbiotic has been proposed recently to characterize foods with both prebiotic and probiotic properties as health enhancing functional foods [42].

Role of Microorganisms in Poultry Production

The GIT serves as the interface between diet and the metabolic events that sustain life. In poultry, intestinal villi, which play a crucial role in digestion and absorption of nutrients, are underdeveloped at hatch [65] and maximum absorption capacity is attained by 10 days of age [66].  Understanding and optimizing the maturation and development of the intestine in poultry will improve feed efficiency, growth and overall health of the bird. In the immediate post hatch period birds must undergo the transition from energy supplied by the endogenous nutrients of the yolk to exogenous carbohydrate-rich feed. During that critical time dramatic changes occur both in the intestinal size and morphology [65]. Maturational changes also affect the epithelial cell membranes, a major mechanical interface between the internal environment of the host and the luminal contents [67]. Studies on nutrition and metabolism during the early phase of growth in chicks may help in optimizing nutritional management for maximum growth [68]. By dietary means it is possible to affect the development of the gut and the competitiveness of both beneficial and harmful bacteria, which can alter not only gut dynamics, but also many physiologic processes due to the end products metabolized by symbiotic gut microflora. Additives such as probiotics and prebiotics are now extensively used throughout the world. The chemical nature of these additives are well understood, but the manner by which they benefit the animal is not  [69].

Probiotics as an Alternative to Antibiotics for Control of Bacterial Pathogens and improved performance  in Poultry.

Bacterial antimicrobial resistance in both the medical and agricultural fields has become a serious problem worldwide. Antibiotic resistant strains of bacteria are an increasing threat to animal and human health, with resistance mechanisms having been identified and described for all known antimicrobials currently available for clinical use. There is currently increased public and scientific interest regarding the administration of therapeutic and sub-therapeutic antimicrobials to animals, due primarily to the emergence and dissemination of multiple antibiotic resistant zoonotic bacterial pathogens [70].  Social pressures have lead to creation of regulations to restrict antibiotic use in poultry and livestock production.  There is a need to evaluate potential antibiotic alternatives to improve disease resistance in high intensity food animal production.  Nutritional approaches to counteract the debilitating effects of stress and infection may provide producers with useful alternatives to antibiotics.  Improving the disease resistance of animals grown without antibiotics will not only benefit the animals’ health, welfare, and production efficiency  but is also a key strategy in the effort to improve the microbiological safety of poultry products [70].  During the last four years, our laboratory has worked toward the identification of probiotic candidates for poultry which can actually displace Salmonella and other enteric pathogens which have colonized the gastrointestinal tract of chicks and poults.  Published studies [71] indicated that after screening more than 8 million enteric organisms for competition in vitro, 36 organisms were identified that had the ability to exclude Salmonella in neonatal poultry.  Further screening allowed the identification of 11 lactic acid bacteria (of the genus or related to Lactobacillus in the product FM-B11TM (Floramax)) that were even more efficacious in the treatment of Salmonella infected chicks and poults [72].  In laboratory challenge studies, 80-90% reductions in Salmonella recovery rates from challenged chicks treated with Floramax probiotic culture were typical.  By selecting Salmonella infected flocks pre-slaughter, we have demonstrated that treating such flocks with Floramax, approximately two weeks prior to slaughter, can markedly reduce environmental Salmonella recovery in commercial turkey and broiler operations [72].  Treatment of idiopathic enteritis in commercial poults with Floramax also compared favorably to selected antibiotic therapy in recent studies [73].  Large scale commercial trials have indicated that appropriate administration of this probiotic mixture to turkeys increased body weight gain at processing by approximately 194 g with over 120 flocks evaluated [74], with similar performance gains observed in more limited commercial trials with broilers.  Administration of dietary lactose at a very low concentration [0.1%] greatly enhanced the growth rates of probiotic-treated turkeys under commercial conditions and furthered reduced total production costs [74].  These data indicate that selection of therapeutically efficacious probiotic cultures with marked performance benefits in poultry is possible, and that defined cultures can sometimes provide an attractive alternative to conventional antimicrobial therapy. 

Comparisons Between  Genotypic 16S rRNA , MIDI, and Biolog Identifications of FM-B11 Lactic Acid Bacteria

As a blend of facultative and obligate bacteria, the composition of lactic acid bacteria poses a unique problem for microbial identification. The identification techniques of choice for facultative anaerobes are biochemical analyses, but the standard identification system for lactic acid bacteria is cellular fatty acid profiling. However, these phenotypic methods can yield variable results. Genotypic methods that rely on comparisons of 16S rRNA sequences from unknown bacteria are proving to be valuable for use in a wide range of genera and are not sensitive to variable culture conditions. Genotypic 16S rRNA identification of organisms from probiotic cultures may be more reliable than the current standard microbial techniques applied separately to different microbial groups. Although there are many new experimental molecular identification techniques, such as microarray hybridization and matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry, sequence analysis of 16S rRNA is the predominant molecular technology currently available for microbial identification [75]. The 16S rRNA sequences of many bacteria species in a population may be analysed simultaneously with denaturing gradient gel electrophoresis or random fragment length polymorphism analyses. The detailed information needed to identify each species represented in the complex microbial population of a competitive exclusion product can only be fully obtained from the 16S rRNA at the level of the nucleotide sequence. As an example, we have devised an identification scheme using the MIDI System ID from two different private laboratories [Micro Test Lab Inc., Agawam, MA 01001, USA; and Microbial ID Inc., Newark, DE 19713, USA] the Biolog ID System [Biolog, Inc., Hayward, CA 94545, USA]  and the 16S rRNA Sequence Analyses [Microbial ID Inc., Newark, DE 19713, USA] for identification of the individual component bacteria present in the commercial probiotic Floramax (Table 1).  The results of the that study show that the complex populations of bacteria present in Floramax are not easy to accurately identify, especially with phenotypic techniques. Genotypic identification by 16S rRNA gene sequence analysis has potential to improve the accuracy of bacterial identification, especially as the contents of sequence databases become more comprehensive. Conventional technologies can detect human pathogens, because they are well-established in comparative databases, but emerging and opportunistic pathogens are not. These results support a suggestion by the MIDI company [76] to use 16S rRNA sequence analysis to identify obligate and facultative anaerobes, such as those in FM-B11TM (Floramax). Though ambiguity exists between different methods of identification of non-pathogenic probiotic bacteria, identification of known pathogens is much more consistent.  Therefore, the use of fully defined cultures for competitive exclusion or probiotic use are still inherently safer than undefined cultures or those where organisms are identified after the culture has been produced.

Conclusions

The interest in digestive physiology and the role of microorganisms has generated data whereby human and animal well being can be enhanced and the risk of disease reduced.  New molecular techniques that allow an accurate assessment of the flora composition  resulting in improved strategies for elucidating mechanisms.  Given the recent international legislation and domestic consumer pressures to withdraw growth-promoting antibiotics and limit antibiotics available for treatment of bacterial infections, both probiotics and prebiotics can offer alternative options.  New advances in the application of synbiotics (compatible probiotics and prebiotics), are directed to produce significant changes in gut physiology  and provide even higher levels of health as well as increasing performance parameters. 

Metchnikoff founded the research field of probiotics, aimed at modulating the intestinal microflora.  However, other parts of the body containing endogenous microflora or problems relating to the immune system may also be candidates for probiotic therapy.  Research has shown that probiotics have potential for human health issues such as: vaginal candidiosis [77]; dental caries [78,79]; allergies [80]; autoimmune diseases [81]; urogenital infections [82]; atopic diseases [83]; rheumatoid arthritis [84]; and respiratory infections [85].  Current research is still heavily biased toward gastrointestinal applications for probiotics, such as: chronic constipation [53]; chronic diarrhea  [86]; inflammatory bowel disease [87]; irritable bowel syndrome [88]; and food allergy [89], but the possibilities for impacting many areas of health are numerous.  Much research has been completed in efforts to understand and apply the natural benefits of non-pathogenic bacteria, but there is much still to do.

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