Introduction
Post-weaning diarrhoea (PWD) is a significant enteric disease causing considerable economic losses for the pig industry. Among several etiological risk factors, enterotoxigenic Escherichia coli (ETEC) is considered to be a major cause, i.e. colibacillosis. The use of antibiotics at subtherapeutic concentrations was routinely used as growth promoters for several decades, but has since 1 January 2006 been banned in the European Union due to the increasing prevalence of resistance to antibiotics in pigs. The removal of in-feed antibiotics from piglet diets has negative economic consequences as it dramatically increases the rate of morbidity and mortality due to ETEC as well as the use of antibiotics for therapeutic purposes. The probably most used substitutes for in-feed antibiotics is the pharmacological levels (at 2,000 to 4,000 mg/kg) of zinc oxide (ZnO) during the first two weeks post-weaning to alleviate the diarrhea and growth check in post weaning piglets in many pig producing countries. Any reduction of dietary zinc will result in less excretion of zinc to the feces, and thereby in reduced contamination of the agricultural soil. Thus, alternatives to antibiotics and to the high levels of ZnO that can control ETEC infections in piglets postweaning will be of great advantage, also because of the risk of cross resistance between antibiotics and Zn or other heavy metals. A number of nutritional, genetic and management strategies have been reported in the literature as alternatives to in-feed antibiotics to prevent PWD, but, in most studies, the reduction of PWD has not been addressed as a primary response parameter (Lauridsen et al., 2017). The aim of this presentation is to evaluate the potential of commonly suggested dietary strategies for their effect on improving gut health, and thereby, for reducing post-weaning diarrhea (PWD) in pigs.
Pathogenesis of ETEC-post weaning diarrhea and its prevention by ZnO
Post-weaning diarrhea is a multifactorial disease, and its pathogenesis is still unclear. Frequently described, this is a condition of weaned pigs characterized by frequent discharge of watery faeces during the first two-three weeks after weaning. This condition is typically associated with faecal shedding of haemolytic enterotoxigenic E. coli (ETEC), proliferating in the ileum. The pathogenesis of ETEC-PWD can briefly be described as follows: 1) adherence of ETEC to specific receptors in the small intestine, 2) ETEC proliferation, 3) production of enterotoxins, and 4) hypersecretion of water and electrolytes to the lumen of the small intestine. The weaning process itself may due to external factors such as dietary changes, antibiotic use, and stressors, trigger inflammation and oxidative stress in the piglet gut, and this leads to a number of changes in the gut environment that favor the blooming of E. coli. An obvious solution to the treatment of PWD may therefore be the prevention of enteric inflammation and oxidative stress during the post weaning period. Other pathogenic bacteria may also be the cause of the PWD, but the presence of pathogens may in fact not necessarily be the cause of diarrhea in pigs. However, during the initial two weeks post weaning, where medical zinc is permitted for treating the PWD, the most frequent cause of diarrhea in pigs is the ETEC, and one may ask why ZnO has been identified as the most efficient alternative to antibiotics for the treatment of PWD in pigs.
Oral zinc supplementation is used for treating diarrhea in children in developing countries. A recent review concluded that in areas where the prevalence of zinc deficiency or the prevalence of malnutrition is high, Zn may be of benefit in children aged six months or more, while no benefit was obtained in children being less than 6 months and well-nourished (Lazzerini and Wanzira, 2016). In this review, several mechanisms of the action of zinc on acute diarrhea were addressed including its influence on more than 300 enzymes in the body; its promotion of immunity and resistance of mucosal layers and skin to infection, and development of the nervous system. Zinc is also important for antioxidant activity and it preserves thereby the cellular membrane activity. Within the gut, Zn restores mucosal barrier integrity and enterocyte brush-border enzyme activity, and upon intestinal pathogenic burden, it can promote the production of antibodies and circulating lymphocytes, as well as having a direct effect on ion channels, where it functions as a potassium channel blocker of adenosine 3-5-cyclic monophosphate-mediated chlorine secretion. Among studies included in this systematic review (Lazzerini and Wanzira, 2016), the most frequent Zn dose was 20 mg/day, and various types of Zn salts were used. The most frequently used medical zinc source for treating diarrhea in pigs is the ZnO. The Committee for Veterinary Medicinal Products (CVMP) of the European Medicines Agency (EMA) announced in December 2016 that in their opinion they found the risk/benefit balance was unfavourable for the therapeutic use of zinc oxide and that all products containing zinc oxide should have their marketing authorisations removed. However, as concluded by Zentek (2019), it can be assumed that in particular pharmacological zinc concentration in the feed and the subsequent significantly increased zinc concentrations in the digesta are causal for the occurrence of antimicrobial resistances.
The key difference between Zn and ZnO is that Zn is a chemical element (a transition metal), while ZnO is a chemical compound. One may ask why the ZnO (rather than other zinc sources) became the therapeutic source of zinc for treating diarrhea in pigs, when this is not a common Zn source for treating diarrhea in human. This can probably be ascribed to the first nutritional study using dietary ZnO for the prevention and treatment of PWD (Poulsen, 1995), and since its publication, supply of ZnO has received considerable attention, however, the exact modes of action remains to be elucidated. When addressing zinc replacement strategies it is important to note, that Zn can be applied at low dosages (around 150 ppm) to weaners as a nutritional component, while usage in high levels as zinc oxide (around 2,500 ppm) has been shown to be an effective strategy to prevent and control e.g. post-weaning diarrhoea problems (Poulsen, 2019). Hence, in the following, we will address potential dietary strategies for prevention of post diarrhea. In accordance with studies on Zn for children, the prevention by which ZnO have been reported to prevent PWD in pigs point towards both physiological and non-physiological mechanisms. A crucial response during the weaning transition is the feed intake of piglets, and enhanced feed intake of piglets provided dietary ZnO supplementation was in fact obtained (Broom et al., 2003). Improved physiological serum Zn status was observed in the study with weaned piglets in which beneficial effects of increased concentrations of ZnO on post-weaning diarrhea and growth of the piglets were obtained in piglets during the first two weeks after weaning (Poulsen, 1995). Other reports have found changes in some pancreatic enzymes and hormonal status (Hedemann et al., 2006). Furthermore, expression of genes and proteins related to energy and amino acids metabolism and in parallel accumulation of Zn in liver and pancreas in piglets post weaning after feeding high dietary ZnO for 4 weeks (Pieper et al., 2012), however, this may also lead to increased oxidative stress in these organs. Zn also has major influence on the innate and adaptive immune system (Bonaventura et al., 2015), as shown in pigs where dietary zinc level influenced immune responses of the weaned pig (Kloubert et al., 2018), and Zn enhances intestinal epithelial barrier function (Shao et al., 2017).
The non-physiological mechanisms by which ZnO have been reported to prevent PWD points towards its influence on the gastrointestinal microbiota. Reduced bacterial activity in the digesta from the gastrointestinal tract of pigs fed 2,500 mg/kg as ZnO compared with that in animals receiving 100 mg/kg ZnO, most likely reflecting a reduced load of bacteria present in the gastrointestinal tract of animals receiving the high ZnO level (Hojberg et al., 2005). Overall, the research on pigs indicate a major influence of ZnO dose on the gastrointestinal tract microbiota and show that, besides a potential promotion of feed intake, high dietary ZnO doses may further render more energy available for the host animal by generally suppressing the commensal gut microbiota. This has actually also been suggested as one of the working mechanisms behind the effect of antibiotic growth promoters (Collier et al., 2003).
With all these reported mechanisms by which zinc may prevent diarrhea in human and pigs, it is of course difficult to imagine a single food component or feed additive, which can replace zinc for its capability to prevent diarrhea. However, there are nutritional components and bioactive substances, which share some or even several of the modes of action by which Zn is suggested to prevent diarrhea.
Dietary strategies to prevent PWD
There are a number of potential dietary strategies, which have been proposed for preventing PWD, and some are in fact all-ready implemented in practice. Reduction of the dietary protein level is considered an effective strategy to prevent PWD. Since medicinal levels of ZnO have direct antimicrobial effects, potentially targeting specific pathogens such as E. coli strains, but also reduce the microbial load in general, and further exerts effects on host-immunity and epithelial barrier functions, such mechanisms are central to prevent progression of ETEC-infection of pigs. These will therefore also be keys to consider in order to identify Zn replacement strategies.
Restriction of protein level
There is a considerable body of research dating back to the 1950s and 1960s which has implicated an association between protein level and diarrhoea after weaning (Pluske, 2013), and as also stated in their review (Kil and Stein, 2010), crude protein is the most important nutrient associated with digestive disorders in the pig. Upon consumption of a meal, pH in the stomach is increased whereby proliferating of E. coli is facilitated. Feed ingredients such as soya bean meal, fishmeal and milk powder, which are typical sources of protein used in feed for weaned piglets, have a high buffering capacity, which also can increase stomach pH and thereby limit pepsin activity. Hence, excessive crude protein intake by weaned pigs can lead to increased microbial fermentation of undigested protein, and this is a contributing factor to development of PWD. One of the implemented strategies in practice to limit diarrhoea incidence is the use of low-protein diets, i.e. containing less than 18% crude protein. This strategy is not without challenges because reduction of the concentration of crude protein for weaned pigs may influence the growth performance because some of the indispensable amino acids may be present in concentrations below the requirement for maximum growth. However, the loss in growth of piglets on low-protein diets in comparison with piglets on diets with a normal protein content may be compensated on the longer term, as the lifetime performance in some studies did not differentiate (e.g. Wellock et al., 2009). Furthermore, crystalline amino acids (lysine, methionine, threonine, tryptophan, isoleucine and valine) in low-protein diets can be used to maintain the balance of the required amino acids for the weanling pigs and thereby pig performance (e.g. (Lordelo et al., 2008)), however, little is know regarding the requirement of specific amino acids during an infectious challenge, when the host immune responses are activated.
Bioactive feed components and feed additives - and their mode of action
As stated above, the various possible modes of action by which a given dietary strategy; a feed additive, or a bioactive feed component may prevent progression of ETEC-infection of pigs, are related to the gut microbiota and or to the host physiology. Thus, targeted strategies for prevention of ETEC-infection can be ascribed according to the certain step of the pathogenesis as described below, and examples of feed additives and bioactive components playing the mode of action is given in brackets:
1) Protecting the gut from ETEC adhesion and colonization by reducing the sensitivity of fimbrial receptors on the porcine enterocytes and/or blocking the fimbriae of ETEC (e.g. specific immunoglobulins).
2) Inhibiting the growth of ETEC in the gut due to bactericidal or bacteriostatic effects (e.g., organic acids, medium-chain fatty acids, fermented liquid feed, antimicrobial peptides, bacteriophages, lysozomes).
3) Maintaining a balanced intestinal microbiota (e.g., probiotics, prebiotics).
4) Improving the host immune functions (systemically and locally) including prevention of excessive inflammation (e.g., immunoglobulins, fatty acids, vitamins, trace minerals).
5) Preventing disruption of intestinal mucosal integrity and/or improving the morphology of the small intestinal epithelium (e.g., specific egg yolk antibodies, bacteriophages).
In the following, the mechanisms by which some of the above-mentioned examples of feed additives considered capable of promoting gut health of pigs and thereby prevent ETEC infection are described (Lauridsen et al., 2019):
One of the main parameters influencing bacterial growth is pH, and therefore reducing the luminal pH, i.e. by addition of organic acids or other acidifiers, has an antibacterial effect (Canibe et al., 2005). Lactic acid bacteria are able to grow at relatively low pH, and are therefore more resistant to organic acids than, for example, enterobacteria. Among organic acids, butyric acid has been in focus, especially in human but also in pig research, due to the multiple ways by which this compound affect the host. Fermented liquid feed (FLF) is another feeding strategy to reduce the luminal pH (of the stomach). FLF is prepared by mixing water or another liquid, e.g., whey, with feed and thereafter incubation of this mixture (for a certain period of time, at a certain temperature). As fermentation progresses, lactic acid bacteria proliferate, resulting in high concentration of especially lactic acid and low pH, leading to reduced numbers of coliform bacteria (Canibe and Jensen, 2012). Thereby a reduced load of E. coli reaches the ileum, protecting proliferation of pathogens, and maintaining a stable bacterial community.
Antimicrobial lipids such as fatty acids and monoglycerides are promising antibacterial agents that destabilize bacterial cell membranes, causing a wide range of direct and indirect inhibitory effects. Besides, medium-chained fatty acids (MCFA) are an immediate energy source for the host and its immune cells, and improve intestinal integrity during inflammatory conditions. Combining MCFA with organic acids are some of the most recent initiatives to identify alternatives to antibiotics (Ferrara et al., 2017), however, more in vivo studies are needed to document the efficacy of these feed additives for preventing PWD as well as solving the challenges of reduced palatability of some MCFA (Lauridsen, 2020). In this context, encapsulation can be used to allow the organic acids (including short-chain fatty acids) to reach the distal small intestine without being absorbed and thereby exert their antibacterial effect at the site of interest. Mono- and diglycerides are produced upon enzymatic hydrolysis of dietary triglycerides, and research has suggested that these fatty acids and monoglycerides exert antibacterial effects against infectious pathogens (Zentek et al., 2011). Using these agents as therapeutics requires improvement of the delivery in order to be applied in vivo.
Bacteriophages are bacteria-targeting viruses, common in all natural environments and very specific as each type generally attacks specific bacterial species. Several studies have evaluated the antimicrobial ability of phages targeting E. coli, including the fimbriae types F4 and F18, most commonly associated with piglet PWD. The use of phages is still limited in controlling feed borne pathogens in pigs, and more knowledge is needed to understand essential challenges, including phage resistance, phage-host interactions as well as unwanted perturbations of the gut microbiota.
Probiotics are, by definition, live microorganisms that, when administered in adequate amounts, confer health benefits to the host, and are one of the functional foods that link diet and health. Prebiotics (primarily carbohydrates that resist digestion in the ileum) are defined as selectively fermentable components inducing specific changes in composition and/or activity of the gastrointestinal microbiota and conferring host well-being and health benefits. During the past years, many studies on pre- and probiotics (or their combination also known as synbiotics) have been carried out in pigs. These studies have shown a broad range of beneficial effects in terms of pathogen inhibition and its consequences, including immunological development and fortification of intestinal barrier functions. Because prebiotics are readily available substrates for probiotics, prebiotics may improve the survival of concurrently administered probiotic strains. Studies have shown potential capacity of probiotics in terms of immunomodulatory activities, but contrasting effects can also be obtained, which is probably due to differences with respect to the probiotic strain used, experimental settings, diets, initial microbiota colonization, administration route, time and frequency of administration of the probiotic strain and sampling for analysis (Roselli et al., 2017).
Passive immunization, i.e. the administration of antibodies (immunoglobulins) for protecting the host against infections, is not a new idea and seems to constitute a real and widely applicable alternative to antibiotics in modern animal production (Hedegaard and Heegaard, 2016). Oral administration of specific chicken Igϒ has been shown to be effective against a variety of intestinal pathogens, including ETEC (Diraviyam et al., 2014). Use of blood plasma or purified porcine immunoglobulin G from pooled natural pig plasma is also of interest as an immune-enhancing technology. Yeast derivatives based on Saccharomyces cerevisiae, where the bioactive components are mannans and β-glucans, may be immunomodulatory and can prevent colonization of pathogenic E. coli. However, verification is needed on the exact composition and dosage of the bioactive components of the yeast derivatives, as well as use of antibodies, specifically when to apply and dose required.
Other active ingredients such as vitamins, and trace elements and antimicrobial peptides (AMPs), have a major influence on the immune system. As recently reviewed (Lauridsen et al. 2021), vitamins have been much less considered in relation to gut health and function of pigs although they share a lot of similar functions with Zn, and several recent studies in relation to human intestinal health and disease have been published. Once the immune system is activated, the nutrient partitioning is altered and partly directed to produce immune molecules and inflammatory responses. Reduction of inflammatory responses (for instance by acetylsalicylic acid and vitamin E) and oxidative stress reactions (by using antioxidative enzymes and vitamins) during the activation of the immune cells may protect against severity of infectious disease, including disruption of epithelial barrier function and mucosal injury, and eventual subsequent septic shock due to toxins entering the system. Thus, the support on the immune system via vitamins and trace elements (selenium, copper, and zinc) is important, and any transient deficiency during the weaning due to low feed intake or malnutrition should be avoided. Furthermore, some vitamins, trace elements, clay, and carbohydrates etc. exert specific actions on intestinal barrier integrity by influencing (size and/or activity) of goblet cells of the ileum and hence protecting the epithelial barrier function.
During recent years, blends or combinations of the above-mentioned feed additives have been investigated in relation to gut health effects in pigs, which seems a reasonable strategy as one molecule cannot prevent e.g. progression of E. coli infection at all steps. In this context, algae, plant components (e.g. dried plant material), plant extracts and essential oils, berries and fruit extracts have gained interest in terms of potential benefits on gut health in pigs. These ‘food’- related components may encompass a natural cocktail of antibacterial and immunomodulatory agents. However, the proposed bioactivity of the given cocktail, and its stability in feeds, as well as sensory acceptance, should be investigated further. Likewise, dairy-based products gained increasing interest as a potential nutritional tool to prevent piglets from developing diarrhoea due to ETEC (Sugiharto et al., 2015). Bovine colostrum, milk and milk fractions such as whey and casein contain several bioactive components with antimicrobial and immunomodulatory properties, but the knowledge related to the application of the dairy-based products to prevent ETEC infection and post-weaning diarrhoea is very limited.
Conclusion and perspectives
Although the mechanisms behind the success of high dietary ZnO levels in terms of limiting PWD in pigs are still not clear, they are considered to be related both to an impact on the gut microbiota and to the physiological effects of zinc on the animal. Protein restriction is crucial for prevention of PWD, and in addition, there is however, there is probably not a single feed additive, which can replace ZnO for its capability to prevent PWD. However, many potential feed additives share several of the mechanisms by which Zn may influence the ETEC infection and development of diarrhea. The ideal feed additive for enhancing gut health of pigs is probably a cocktail of various antibacterial and immunomodulatory agents targeting the specific challenges of the microbiome-host interaction as E. coli infection progresses in piglets post-weaning.
Presented at the 2021 Animal Nutrition Conference of Canada. For information on the next edition, click here. 
