The aquaculture industry is expanding; yet it is challenged by disease outbreaks, many of which are caused by pathogenic bacteria. Such infections can be controlled by antibiotics, however, this contributes to the development and spread of antimicrobial resistance (AMR) and therefore alternative strategies for disease control must be developed. The purpose of this study was to investigate the antibacterial potential of the microbiome associated with the live-feed microalgae Isochrysis galbana. A GFP-tagged highly virulent strain of Vibrio anguillarum (strain 90-11-286) was incubated with different fractions of the algae microbiome and the fluorescence signal was followed to screen for its inhibition. The microalgal microbiome was able to inhibit V. anguillarum and was dominated by Alteromonadaceae and Rhodobacteraceae. When testing isolated pure bacterial cultures, only Phaeobacter could inhibit the pathogen in monoculture, however, given its low prevalence in the microbiome, it was not likely the cause of pathogen inhibition. Co-cultures of Halomonas and Ruegeria were capable of inhibiting the low virulent V. anguillarum strain NB10, however could not inhibit the high-virulent V. anguillarum strain 90-11-286. This study demonstrates that the native microbiomes of microalgae from aquaculture environments hold anti-pathogen potential and highlights the possible benefits of using mixtures of bacteria rather than pure cultures for pathogen suppression.

ABSTRACT Aquaculture is an essential food production sector for meeting the global demand for high-quality protein. However, the sector faces significant challenges from bacterial pathogens, particularly Vibrio anguillarum , which causes vibriosis in numerous commercially important fish species. Current disease management strategies rely heavily on antibiotics, leading to antimicrobial resistance and environmental concerns. Microalgal microbiomes represent promising alternatives for sustainable pathogen control, yet the molecular mechanisms underlying their inhibitory activity remain poorly understood. Here, we employed an integrated multi-omics approach to elucidate the mechanisms by which the microbiome of microalga Isochrysis galbana inhibits the highly virulent fish pathogen V. anguillarum strain 90-11-286. Using a GFP-based inhibition assay, we confirmed potent pathogen suppression by the algal microbiome, achieving complete inhibition at a 1:1000 ratio of pathogen to microbiome. Through 16S rRNA gene amplicon sequencing, metagenomics, metatranscriptomics, and metabolomics, we characterized community composition, genomic potential, gene expression patterns, and metabolite production during pathogen challenge. The inhibitory microbiome was dominated by Alteromonas macleodii and Vreelandella alkaliphila , with high-quality metagenome-assembled genomes revealing substantial secondary metabolite biosynthetic potential. Metatranscriptomic analysis revealed active expression of biosynthetic gene clusters encoding, for example, non-ribosomal peptide synthetases, particularly a siderophore gene cluster in V. alkaliphila . Metabolomic profiling confirmed the production of hydroxamate siderophores in the microbiome, including desferrioxamine analogues, proferrioxamine G1t, and tenacibactin D, which accumulated during pathogen inhibition, as well as 10 putative new compounds. Notably, siderophore production was constitutive rather than pathogen-induced, suggesting iron competition as the primary inhibitory mechanism. Our findings demonstrate that iron sequestration through siderophore production represents a key strategy for pathogen suppression in marine microbial communities. This work provides molecular evidence for microbiome-mediated disease control and establishes a foundation for developing rationally designed multi-strain probiotic consortia for sustainable aquaculture applications, offering an environmentally friendly alternative to antibiotic-based pathogen management strategies.

Tropodithietic acid (TDA) is a secondary metabolite with antimicrobial and iron-binding properties, produced by several marine Roseobacter group bacteria. Although classified as a siderophore, TDA is also produced under iron-replete conditions. To address this paradox, we fused the tdaCDE promoter region of Phaeobacter piscinae S26 with a promoter-less gfp reporter gene to monitor the transcription of TDA biosynthetic genes under different conditions and validated its accuracy using RT-qPCR. In both complex ($\frac{1}{2}$YTIO) and low-complexity (IOCGH) marine media, iron supplementation repressed tdaCDE transcription and reduced iron-chelation activity. However, TDA production responded differently depending on medium composition: iron suppressed TDA production in IOCGH but increased it in $\frac{1}{2}$YTIO. Antimicrobial activity of culture supernatants declined in late-stage iron-limited $\frac{1}{2}$YTIO-based cultures, even though tdaCDE transcription and iron-chelating activity increased. Acidification restored antimicrobial activity and increased detectable TDA levels, while several non-antimicrobial structural analogues-detected by LC-MS in non-acidified samples-diminished upon acidification, suggesting pH-driven interconversion. These findings reveal that TDA biosynthesis is regulated by iron availability but that the antimicrobial output of the producer depends on medium composition and pH-driven chemical transformations between TDA and its related structural analogues.

Vibrio anguillarum remains a major problem in marine aquaculture, motivating the search for ecologically grounded microbial strategies for pathogen control. We previously showed that a coculture of Vreelandella alkaliphila D2 and Sulfitobacter pontiacus D3, isolated from Isochrysis galbana, inhibited V. anguillarum more strongly than either strain alone. Here, we demonstrate that this suppression is driven by desferrioxamines (DFO) produced by V. alkaliphila, enabling synergistic iron sequestration through siderophore sharing with S. pontiacus. Mass spectrometry imaging revealed multiple DFO analogues spatially colocalized within inhibition zones and confirmed xenosiderophore uptake of DFO analogues by Sulfitobacter. Bioassays showed iron-chelation activity in V. alkaliphila, while iron supplementation and DFO-B standards validated siderophore-mediated bacteriostatic inhibition. Untargeted metabolomics found no increase in siderophore abundance in coculture, indicating that enhanced inhibition results from shared iron chelation rather than elevated production. Genome mining identified a complete DFO biosynthetic gene cluster in V. alkaliphila and a desferrioxamine receptor in Sulfitobacter. Finally, in axenic I. galbana, Sulfitobacter, alone or with Vreelandella, maintained V. anguillarum at the level of inoculation, highlighting that the substrate has a marked influence on inhibitory activity. Together, these findings show how DFOs act as shared public goods that enable both cooperation and pathogen antagonism.

Tropodithietic acid (TDA) is the antimicrobial compound mediating the probiotic activity of Phaeobacter spp. against pathogenic vibrios in marine larviculture. In the related genera Tritonibacter, TDA is mainly produced during biofilm growth. The purpose of the study was to determine if biofilm formation increases expression of TDA biosynthesis genes in Phaeobacter piscinae and whether surface properties can promote this expression for aquaculture. Using a GFP reporter under the tdaCDE promoter and flow cytometry, TDA biosynthesis gene expression was two-fold higher in biofilms than in planktonic cells. Biofilm formation on four surfaces showed that the most hydrophobic material, PDMS, supported ∼30-fold higher early attachment than PMMA, COC and PP, while mature biovolume was similar across materials. The TDA gene expression to biovolume ratio increased as biofilms developed. P. piscinae biofilms inhibited Vibrio anguillarum, reducing counts to 105 CFU/ml compared to the control that was 108 CFU/ml. In the presence of V. anguillarum, PDMS biofilms showed 1.5-fold higher biovolume and 2.5-fold higher TDA biosynthesis gene expression, suggesting an antagonistic response. Thus, Phaeobacter is a more effective Vibrio antagonist in the biofilm state, supporting its potential as a probiotic for future aquaculture applications.

The interactions between microalgae and the bacteria living in the phycosphere are pivotal to the role they play in aquatic ecosystems. This study examines how two representatives of common phycosphere bacteria, Yoonia sp. TsM2\_T14\_4 (Rhodobacteraceae) and Maribacter sp. IgM3\_T14\_3 (Flavobacteriaceae), interact with three microalgal hosts: Isochrysis galbana , Tetraselmis suecica , and Conticribra weissflogii (formerly Thalassiosira weissflogii ) using dual transcriptomic analyses of both bacteria and microalgae. Bacterial transcriptomes differed significantly depending on microalgal host, with notable changes in carbohydrate metabolism among other COG categories. Yoonia sp. expressed genes involved in anoxygenic photosynthesis in co-culture with I. galbana , presumably due to its inability to utilize carbohydrates from this algal host, whereas Maribacter sp. expressed polysaccharide degradation genes in co-culture with C. weissflogii along with T9SS genes, which can be employed to secrete these hydrolytic enzymes. Specifically, a putative glucan endo-1,3-beta-D-glucosidase was highly expressed, an enzyme that can hydrolyze laminarin and curdlan. Maribacter sp. IgM3\_T14\_3 could utilize laminarin as a sole carbon source in laboratory settings, a polysaccharide commonly found in marine environments and produced by C. weissflogii . Surprisingly, microalgal transcriptomes remained largely unaltered in the presence of either of the bacteria compared to transcriptomes of axenic algal cultures. These findings highlight the adaptability of phycosphere bacteria to different microalgal hosts. Furthermore, it also indicates a commensalism between microalgae, Yoonia sp. and Maribacter sp., in which the bacteria adapt to and benefit from microalgal host exudates, whereas under the conditions employed here the microalgae are unaffected by the presence of these bacterial symbionts.

Over billions of years, marine microorganisms evolved a vast genetic potential to produce the molecules we denote as natural products or secondary metabolites. While these molecules show promise as therapeutics, their ecological roles, beyond those as antimicrobials, are receiving increasing attention. This review examines recent advances in our understanding of the ecological functions of marine microbial natural products and highlights both known and emerging roles. We summarize the involvement of these natural products in biological, ecological, and biogeochemical processes in the oceans; outline how their production may profoundly affect the producing organism; and discuss how the presence of natural product-producing microorganisms may affect microbiome composition and function. Despite progress, knowledge about the ecological roles of marine microbial natural products remains limited, and we also discuss challenges and opportunities in this field, including promising new technologies that could provide novel insights.

The ongoing seventh cholera pandemic, which began in 1961, poses an escalating threat to public health. There is a need for new cholera control measures, particularly ones that can be produced at low cost, for the one billion people living in cholera-endemic regions. Orally delivered VHHs, functioning as target-binding proteins, have been proposed as a potential approach to control gastrointestinal pathogens. Here, we describe the development of an orally deliverable bivalent VHH construct that binds to the B-pentamer of cholera toxin, showing that it inhibits toxin activity in a murine challenge model. Infant mice given the bivalent VHH prior to V. cholerae infection exhibit a significant reduction in cholera toxin-associated intestinal fluid secretion and diarrhoea. In addition, the bivalent VHH reduces V. cholerae colonization levels in the small intestine by a factor of 10. This cholera toxin-binding protein holds promise for protecting against severe diarrhoea associated with cholera.

Phaeobacter are marine alphaprotebacteria capable of producing a potent antibacterial compound, tropodithietic acid. Here we demonstrate that they are part of the microbiome of marine bryozoans where they during warmer months reach 105 CFU/g. The levels exhibited a bimodal fluctuation, in both bryozoans and seawater across seasons. However, the population of Phaeobacter sp. was already established in the bryozoans prior to the peak in seawater and did not accumulate as a function of filter feeding on phytoplankton biomass, suggesting that the seawater population is likely seeded from the bryozoan-associated Phaeobacter sp. population rather than the opposite. By comparing whole-genome sequences of more than 100 bryozoan-associated Phaeobacter isolates sampled over a 12-year period, we found that all belonged to the same novel species and no systematic genetic changes occurred within it over the 12 year sampling period despite the fact that the population oscillated from below the limit of detection and across five orders of magnitude to 5.2 Log10 CFU g-1 bryozoan within individual years and hence were subject to drift. All isolates had the genetic capacity to produce tropodithietic acid (TDA) and the algicidal compounds, roseobacticides. The genes encoding the enzymes for TDA biosynthesis remained stable over time, indicating a conserved phenotype important in the ecophysiology of the bacteria. TDA biosynthetic genes were actively transcribed within the bryozoan host further corroborating the notion that the secondary metabolites of this novel host-associated Phaeobacter sp. may be central to its role within the bryozoan microbiome.

Intensive fish rearing in aquaculture is challenged by infectious diseases, and although vaccines have been successfully developed for mature fish, alternative disease control measures are needed for fish larvae and juveniles. Probiotics offer a promising alternative to antibiotics, with the potential to reduce the risk of antibiotic resistance. Probiotics are typically isolated and used as pure cultures; however, in natural environments, it is the concerted effort of the complex microbiome that keeps pathogens at bay. Here, we developed an in vitro assay to evaluate the anti-pathogen efficacy of mixed algal microbiomes from the live feed microalgae Tetraselmis suecica and Isochrysis galbana. The inhibition of a green fluorescent protein (GFP)-tagged Vibrio anguillarum, a key fish pathogen, by microbial communities was measured and quantified as reduction in fluorescence. The Isochrysis galbana microbiome was more inhibitory to V. anguillarum than the Tetraselmis suecica microbiome. During co-culture with the pathogen, the bacterial density of the Isochrysis microbiomes increased, while the diversity was reduced as determined by metataxonomic analyses. Bacteria isolated from the fully inhibitory microbiomes were members of Alteromonadaceae, Halomonadaceae, Rhodobacteraceae, Vibrionaceae, Flavobacteriaceae, and Erythrobacteraceae. Although some strains individually inhibited the pathogen, these were not the key members of the microbiome, and enhanced inhibition was observed when Sulfitobacter pontiacus D3 and Vreelandella alkaliphila D2 were co-cultured, even though neither was inhibitory as monocultures. Thus, this study demonstrates that microbial communities derived from natural algal microbiomes can have anti-pathogen effects, and that bacterial co-cultures may offer synergistic advantages over monocultures as probiotics, highlighting their promise for aquaculture health strategies.IMPORTANCEAquaculture is the fastest-growing food protein-producing sector, and sustainable disease control measures are required. Probiotics have gained interest as a promising solution for combating fish pathogens, and using mixtures of microorganisms rather than pure cultures may represent a more stable pathogen control. We developed an assay using green fluorescent protein (GFP) tagging of a fish pathogen, enabling the quantitative assessment of the anti-pathogen effects of complex microbiomes. We show that the efficiency of pathogen suppression can be increased with co-cultures compared to monocultures, thus emphasizing the potential in using mixtures of bacteria as probiotics.

Bacterial enteric pathogens are major contributors to the global burden of diarrheal diseases and the associated consequences for human health including malnutrition, growth stunting, morbidity, and mortality. While mortality from diarrhea has decreased, incidence remains high, and better interventions for preventing disease are needed. Single-domain antibodies (i.e., VHHs), functioning as target-binding proteins in the gastrointestinal tract, have been proposed as a potential approach to mitigate bacterial pathogenesis. Here, we describe a mitigation strategy where precision binding of a bivalent VHH to the receptor-binding B-pentamer of heat-labile enterotoxin aggregates the AB5 toxin and impairs enterotoxigenic Escherichia coli colonization in a flow chamber model simulating the human intestine. The VHH construct also binds to the structurally similar cholera toxin and effectively abrogates its intestinal cell cytotoxicity in vitro. Based on these results, we believe that targeting virulence could emerge as a new strategy for the management of bacterial enteric pathogens, supporting gut health in at-risk populations alongside vaccination campaigns or in populations without access to vaccines.

Two Gram-negative, motile, rod-shaped bacteria, designated as C3_T13_0T and A36a-5aT, were isolated from marine environments collected off the coast of Crete, Greece, and from a biofilm in Jyllinge Harbor in Roskilde Fjord, Denmark, respectively. 16S rRNA gene phylogeny revealed that the two strains belong to the genus Phaeobacter. The major cellular fatty acids (>5%) were C18 : 1  ω7c for strain A36a-5aT and C18 : 1  ω7c, C18 : 2  ω7,13 and C16 : 0 for strain C3_T13_0T. The major respiratory quinone was ubiquinone-10, and the predominant polar lipids were phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine for both strains. C3_T13_0T and A36a-5aT both produce the antimicrobial compound tropodithietic acid. The C3_T13_0T strain grows at temperatures between 8 and 37 °C with an optimum of 25 °C, whereas A36a-5aT grows between 5 and 37 °C with an optimum of 30 °C. The salinity range for growth was 2-9 % NaCl (w/v) for C3_T13_0T with an optimum of 3-4% and 0-10% NaCl (w/v) for A36a-5aT with an optimum of 2-6%. The genomes of the two strains shared an average nucleotide identity (ANI) of 84% and had lower than 87% ANI when compared with genomes of Phaeobacter spp. type strains. The genome of C3_T13_0T was 4,432,895 bp with a G+C content of 55.96 mol%, and the genome of A36a-5aT was 4,050,005 bp with a G+C content of 61.37 mol%. Based on the phylogenetic and phenotypic analyses presented here, the two isolates C3_T13_0T (=DSM 118664T=LMG 33738T) and A36a-5aT (=DSM 118663T=LMG 33737T) are considered to represent two novel species belonging to the genus Phaeobacter, for which the names Phaeobacter cretensis sp. nov. and Phaeobacter bryozoorum sp. nov. are proposed, respectively.

Post-weaning diarrhoea (PWD) in piglets presents a widespread problem in industrial pig production and is often caused by enterotoxigenic E. coli (ETEC) strains. Current solutions, such as antibiotics and medicinal zinc oxide, are unsustainable and are increasingly being prohibited, resulting in a dire need for novel solutions. Thus, in this study, we propose and evaluate a protein-based feed additive, comprising two bivalent heavy chain variable domain (VHH) constructs (VHH-(GGGGS)3-VHH, BL1.2 and BL2.2) as an alternative solution to manage PWD. We demonstrate in vitro that these constructs bind to ETEC toxins and fimbriae, whilst they do no affect bacterial growth rate. Furthermore, in a pig study, we show that oral administration of these constructs after ETEC challenge reduced ETEC proliferation when compared to challenged control piglets (1-2 log10 units difference in gene copies and bacterial count/g faeces across day 2–7) and resulted in week 1 enrichment of three bacterial families (Prevotellaceae (estimate: 1.12 ± 0.25, q = 0.0054), Lactobacillaceae (estimate: 2.86 ± 0.52, q = 0.0012), and Ruminococcaceae (estimate: 0.66 ± 0.18, q = 0.049)) within the gut microbiota that appeared later in challenged control piglets, thus pointing to an earlier transition towards a more mature gut microbiota. These data suggest that such VHH constructs may find utility in industrial pig production as a feed additive for tackling ETEC and reducing the risk of PWD in piglet populations.

Many bacteria co-exist and produce antibiotics, yet we know little about how they cope and occupy the same niche. The purpose of the present study was to determine if and how two potent antibiotic-producing marine bacteria influence the secondary metabolome of each other. We established an agar- and broth-based system allowing co-existence of a Phaeobacter species and Pseudoalteromonas piscicida that, respectively, produce tropodithietic acid (TDA) and bromoalterochromides (BACs). Co-culturing of Phaeobacter sp. strain A36a-5a on Marine Agar with P. piscicida strain B39bio caused a reduction of TDA production in the Phaeobacter colony. We constructed a transcriptional gene reporter fusion in the tdaC gene in the TDA biosynthetic pathway in Phaeobacter and demonstrated that the reduction of TDA by P. piscicida was due to the suppression of the TDA biosynthesis. A stable liquid co-cultivation system was developed, and the expression of tdaC in Phaeobacter was reduced eightfold lower (per cell) in the co-culture compared to the monoculture. Mass spectrometry imaging of co-cultured colonies revealed a reduction of TDA and indicated that BACs diffused into the Phaeobacter colony. BACs were purified from Pseudoalteromonas; however, when added as pure compounds or a mixture they did not influence TDA production. In co-culture, the metabolome was dominated by Pseudoalteromonas features indicating that production of other Phaeobacter compounds besides TDA was reduced. In conclusion, co-existence of two antibiotic-producing bacteria may be allowed by one causing reduction in the antagonistic potential of the other. The reduction (here of TDA) was not caused by degradation but by a yet uncharacterized mechanism allowing Pseudoalteromonas to reduce expression of the TDA biosynthetic pathway. IMPORTANCE The drug potential of antimicrobial secondary metabolites has been the main driver of research into these compounds. However, in recent years, their natural role in microbial systems and microbiomes has become important to determine the assembly and development of microbiomes. Herein, we demonstrate that two potent antibiotic-producing bacteria can co-exist, and one mechanism allowing the co-existence is the specific reduction of antibiotic production in one bacterium by the other. Understanding the molecular mechanisms in complex interactions provides insights for applied uses, such as when developing TDA-producing bacteria for use as biocontrol in aquaculture.

Microalgal microbiomes play vital roles in the growth and health of their host, however, their composition and functions remain only partially characterized, especially across microalgal phyla. In this study, a natural seawater microbiome was introduced to three distinct, axenic species of microalgae, the haptophyte Isochrysis galbana, the chlorophyte Tetraselmis suecica, and the diatom Conticribra weissflogii (previously Thalassiosira ), and its divergence and assembly was monitored over 49 days using 16S rRNA amplicon and metagenomic analyses. The microbiomes had a high degree of host specificity in terms of taxonomic composition and potential functions, including CAZymes profiles. Rhodobacteraceae and Flavobacteriaceae families were abundant across all microalgal hosts, but I .galbana microbiomes diverged further from T. suecica and C. weissflogii microbiomes. I .galbana microbiomes had a much higher relative abundance of Flavobacteriaceae, whereas the two other algal microbiomes had higher relative abundances of Rhodobacteraceae. This could be due to the mixotrophic nature of I. galbana affecting the carbohydrate composition available to the microbiomes, which was supported by the CAZymes profile of I. galbana microbiomes diverging further from those of T. suecica and C. weissflogii microbiomes. Finally, the presence of denitrification and other anaerobic pathways was found exclusively in microbiomes of C. weissflogii potentially resulting from anoxic microenvironments in aggregates formed by this diatom during the experiment. These results underline the deterministic role of the microalgal host species on microbiome composition and functional profiles along with other factors, such as trophic mode of the microalgal host.Importance As the main primary producers of the oceans, microalgae serve as cornerstones of the ecosystems they are part of. Additionally, they are increasingly used for biotechnological purposes such as the production of nutraceuticals, pigments, and antioxidants. Since the bacterial microbiomes of microalgae can affect their hosts in beneficial and detrimental ways, understanding these microbiomes is crucial to both ecological and applied roles of microalgae. The present study advances the understanding of microalgal microbiome assembly, composition, and functionality across microalgal phyla, which may inform modeling and engineering of microalgal microbiomes for biotechnological purposes.

In nature, secondary metabolites mediate interactions between microorganisms residing in complex microbial communities. However, the degree to which community dynamics can be linked to secondary metabolite potential remains largely unknown. In this study, we address the relationship between community succession and secondary metabolism variation. We used 16S and 18S rRNA gene and adenylation domain amplicon sequencing, genome-resolved metagenomics, and untargeted metabolomics to track the taxons, biosynthetic gene clusters, and metabolome dynamics in situ of microorganisms during marine biofilm succession over 113 days. Two phases were identified during the community succession, with a clear shift around Day 29, where the alkaloid secondary metabolites, pseudanes, were also detected. The microbial secondary metabolite potential changed between the phases, and only a few community members, including Myxococotta spp., were responsible for the majority of the biosynthetic gene cluster potential in the early succession phase. In the late phase, bryozoans and benthic copepods were detected, and the microbial nonribosomal peptide potential drastically decreased in association with a reduction in the relative abundance of the prolific secondary metabolite producers. Conclusively, this study provides evidence that the early succession of the marine biofilm community favors prokaryotes with high nonribosomal peptide synthetase potential. In contrast, the late succession is dominated by multicellular eukaryotes and a reduction in bacterial nonribosomal peptide synthetase potential.

The globally important production of fish and shellfish in aquaculture is challenged by disease outbreaks caused by pathogens such as Vibrio crassostreae . These outbreaks not only lead to substantial economic loss and environmental damage, but treatment with antibiotics can also lead to antibiotic resistance affecting human health. Here, we evaluated the potential of probiotic bacteria, specifically the newly identified strain Phaeobacter piscinae S26, to counteract these threats in a sustainable manner. Through a systematic assessment of the antagonistic effect of P. piscinae S26 against V. crassostreae DMC-1, particularly within the context of algal feed systems, the study demonstrates the effectiveness of P. piscinae S26 as probiotic and thereby provides a strategic pathway for addressing disease outbreaks in aquaculture. This finding has the potential of significantly contributing to the long-term stability of the industry, highlighting the potential of probiotics as an efficient and environmentally conscious approach to safeguarding aquaculture productivity against the adverse impact of pathogens.

Despite the broad clinical usage of microbial secondary metabolites with antibiotic activity, little is known about their role in natural microbiomes. Here, we studied the effect of production of the antibiotic tropodithietic acid (TDA) on the producing strain, Phaeobacter piscinae S26, a member of the Roseobacter group.

Single-domain antibodies (sdAbs) are exceptionally stable fragments derived from the antigen-binding domains of immunoglobulins. They can withstand extreme pH, high temperature, and proteolysis, making them suitable for controlling gastrointestinal (GI) infections in humans and animals. sdAbs may function in their native soluble form, although different derived protein formats and the use of delivery vehicles can be useful for improved oral delivery. We discuss selected examples of the use of orally delivered sdAbs for protecting humans and animals against GI infections caused by pathogenic bacteria, viruses, and parasites. We finally provide perspectives on how sdAbs may be applied industrially and what challenges should be overcome for orally delivered sdAbs to reach the market.