Simple Summary Post-weaning diarrhoea (PWD) is a major challenge in pig production, which results in the high use of ZnO for its prevention and the utilisation of antibiotics for its treatment. There is a global agenda to decrease the use of antibiotics in order to reduce the risk of antimicrobial resistance (AMR); moreover, from June 2022, administering medical levels of ZnO has been banned in many countries due to its negative impact on the environment and AMR. Many feeding strategies and feed additives have been found to be tools to reduce PWD risk, which affect the gut microbiota and the host through different modes of action. Many feeding strategies and additives show positive but variable effects on PWD. A combination of such interventions tailored according to the specific conditions is most probably the best strategy. Abstract In many countries, medical levels of zinc (typically as zinc oxide) are added to piglet diets in the first two weeks post-weaning to prevent the development of post-weaning diarrhoea (PWD). However, high levels of zinc constitute an environmental polluting agent, and may contribute to the development and/or maintenance of antimicrobial resistance (AMR) among bacteria. Consequently, the EU banned administering medical levels of zinc in pig diets as of June 2022. However, this may result in an increased use of antibiotic therapeutics to combat PWD and thereby an increased risk of further AMR development. The search for alternative measures against PWD with a minimum use of antibiotics and in the absence of medical levels of zinc has therefore been intensified over recent years, and feed-related measures, including feed ingredients, feed additives, and feeding strategies, are being intensively investigated. Furthermore, management strategies have been developed and are undoubtedly relevant; however, these will not be addressed in this review. Here, feed measures (and vaccines) are addressed, these being probiotics, prebiotics, synbiotics, postbiotics, proteobiotics, plants and plant extracts (in particular essential oils and tannins), macroalgae (particularly macroalgae-derived polysaccharides), dietary fibre, antimicrobial peptides, specific amino acids, dietary fatty acids, milk replacers, milk components, creep feed, vaccines, bacteriophages, and single-domain antibodies (nanobodies). The list covers measures with a rather long history and others that require significant development before their eventual use can be extended. To assess the potential of feed-related measures in combating PWD, the literature reviewed here has focused on studies reporting parameters of PWD (i.e., faeces score and/or faeces dry matter content during the first two weeks post-weaning). Although the impact on PWD (or related parameters) of the investigated measures may often be inconsistent, many studies do report positive effects. However, several studies have shown that control pigs do not suffer from diarrhoea, making it difficult to evaluate the biological and practical relevance of these improvements. From the reviewed literature, it is not possible to rank the efficacy of the various measures, and the efficacy most probably depends on a range of factors related to animal genetics and health status, additive doses used, composition of the feed, etc. We conclude that a combination of various measures is probably most recommendable in most situations. However, in this respect, it should be considered that combining strategies may lead to additive (e.g., synbiotics), synergistic (e.g., plant materials), or antagonistic (e.g., algae compounds) effects, requiring detailed knowledge on the modes of action in order to design effective strategies.

/pdf/review-on-preventive-measures-to-reduce-post-weaning-2t7l840x.pdf

Review on Preventive Measures to Reduce Post-Weaning Diarrhoea in Piglets

工程枯草芽孢杆菌通过Nrf2-Keap1途径缓解ETEC K88感染仔猪导致的肠道氧化损伤

Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties. In this study, we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32 (WB800-KR32) using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli (ETEC) K88 in weaned piglets. Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet. The feed of the control group (CON) was infused with normal sterilized saline; meanwhile, the ETEC, ETEC+WB800, and ETEC+WB800-KR32 groups were orally administered normal sterilized saline, 5×1010 CFU (CFU: colony forming units) WB800, and 5×1010 CFU WB800-KR32, respectively, on Days 1–14 and all infused with ETEC K88 1×1010 CFU on Days 15–17. The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance, improved the mucosal activity of antioxidant enzyme (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) and decreased the content of malondialdehyde (MDA). More importantly, WB800-KR32 downregulated genes involved in antioxidant defense (GPx and SOD1). Interestingly, WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum. WB800-KR32 markedly changed the richness estimators (Ace and Chao) of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces. The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway, providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.

Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet

Last several years, a rapid increase in drug resistance to traditional antibiotics has driven the emergence and development of antimicrobial peptides (AMPs). AMPs have also gained considerable attention from scientists due to their high potency in combatting infectious pathogens. A subset of analogues and their derivatives with specific targets have been successfully designed based on natural peptide patterns. In this review, scientific knowledge on the mechanisms of action related to biological activity and structure-activity relationship (SAR) of AMPs are summarized, and the biological applications in several important fields are critically discussed. SAR shows that the positive charge, secondary structure, special amino acid residues, hydrophobicity, and helicity of AMPs are closely related to their biological activities. The combination of nanotechnology, bioinformatics, and genetic engineering can accelerate to achieve the application of AMPs as effective, safe, economical, and nonresistant antimicrobial agents in medicine, the food and feed industries, and agriculture in coming years. Given the intense interest in AMPs, further investigations are needed in the future to evaluate the specific structure and function that make their use favorable in several industries. This review may provide a comprehensive reference for future studies on chemical modifications, mechanistic exploration, and applications of AMPs.

Industrial application of antimicrobial peptides based on their biological activity and structure-activity relationship

Antimicrobial peptides (AMPs) serve as alternative candidates for antibiotics and have attracted the attention of a wide range of industries for various purposes, including the prevention and treatment of piglet diarrhea in the swine industry. Escherichia coli, Salmonella, and Clostridium perfringens are the most common pathogens causing piglet diarrhea. In this study, the antimicrobial peptide gloverin2 (BMGlv2), derived from Bombyx mandarina, was explored to determine the efficient prevention effect on bacterial piglet diarrhea. BMGlv2 was heterologously expressed in Trichoderma reesei Tu6, and its antimicrobial properties against the three bacteria were characterized. The results showed that the minimum inhibitory concentrations of the peptide against E. coli ATCC 25922, S. derby ATCC 13076, and C. perfringens CVCC 2032 were 43.75, 43.75, and 21.86 μg/mL, respectively. The antimicrobial activity of BMGlv2 was not severely affected by high temperature, salt ions, and digestive enzymes. It had low hemolytic activity against rabbit red blood cells, indicating its safety for use as a feed additive. Furthermore, the measurements of the leakage of bacterial cell contents and scanning electron microscopy of C. perfringens CVCC 2032 indicated that BMGlv2 exerted antimicrobial activity by destroying the cell membrane. Overall, this study showed the heterologous expression of the antimicrobial peptide BMGlv2 in T. reesei and verified its antimicrobial properties against three common pathogenic bacteria associated with piglet diarrhea, which can provide a reference for the applications of AMPs as an alternative product in industrial agriculture.

/pdf/characterization-of-recombinant-antimicrobial-peptide-bmglv2-1sc5ijrn.pdf

Characterization of Recombinant Antimicrobial Peptide BMGlv2 Heterologously Expressed in Trichoderma reesei

Diarrhea is a serious issue among livestock, and pathogens and viral infections are the main causes of diarrhea, especially in weaned piglets. Additionally, overuse of antibiotics can lead to severe environment pollution. Therefore, our objective was to study a 32-residue engineered antimicrobial peptide, KR-32, that displays high antimicrobial activity with minimal hemolytic activity and cytotoxicity. Minimum inhibitory concentration and scanning electron microscopy results indicated that antimicrobial peptide KR-32 exerted its antimicrobial activity by destroying the cell membrane integrity. Piglets with clinical diarrhea were divided into three groups and treated with saline (control), KR-32 for 3 days. The antimicrobial peptide KR-32 alleviated the diarrhea of the weaned piglets by reducing the diarrheal rate and index, and it significantly decreased the IL-6 and TNF-α levels compared with the control group levels. To further study the inflammation of the piglets, we tested the effects of KR-32 on IPEC-J2 in vitro. We found that KR-32 attenuated lipopolysaccharides (LPS)-induced inflammation by activating the STAT-1 signaling pathway. In addition, KR-32 significantly reduced pro-inflammation factors IL-6, IL-8 and TNF-α compared with the LPS-treated group. LPS-induced inflammation also affected intestinal barrier functions, but KR-32 enhanced the intestinal barrier by increasing expression of TJ proteins (claudin-1 and occludin) in IPEC-J2. In conclusion, our research reveals AMP KR-32 may have potential application for the control of infectious disease in livestock, and furthermore, AMP KR-32 is a potent antibacterial agent.

Antibacterial, Cytotoxicity and Mechanism of the Antimicrobial Peptide KR-32 in Weaning Piglets

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Antibacterial, Cytotoxicity and Mechanism of the Antimicrobial Peptide KR-32 in Weaning Piglets