Freeze-dried fecal microorganisms as an effective biomaterial for the treatment of calves suffering from diarrhea

Freeze-dried fecal microorganisms as an effective biomaterial for the treatment of calves suffering from diarrhea | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Freeze-dried fecal microorganisms as an effective biomaterial for the treatment of calves suffering from diarrhea Tomonori Nochi, Jahidul Islam, Natsuki Ohtani, Yu Shimizu, Masae Tanimizu, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4168305/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fecal microbiota transplantation (FMT) is a therapeutic modality for treating neonatal calf diarrhea. Several practical barriers, including donor selection, fecal collection, and a limited timeframe for FMT, are the main constraints to using fresh feces for implementing on-farm FMT. We report the utility of FMT with pretreated ready-to-use frozen (F) or freeze-dried (FD) microorganisms for treating calf diarrhea. In total, 19 FMT (F-FMT, n = 10 and FD-FMT, n = 9) treatments were conducted. Both FMT treatments were 100% clinically effective; however, multi-omics analysis showed that FD-FMT was superior to F-FMT. Machine learning analysis with SourceTracker confirmed that donor microbiota was retained four times better in the recipient calves treated with FD-FMT than F-FMT. A predictive model based on receiver operating characteristic curve analysis and area under the curve showed that FD-FMT was more discriminative than F-FMT of the observed changes in microbiota and metabolites during disease recovery. These results provide new insights into establishing methods for preparing fecal microorganisms to increase the quality of FMT in animals and may contribute to FMT in humans. Biological sciences/Microbiology/Microbial communities/Microbiome Biological sciences/Microbiology/Microbial communities/Metagenomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Neonatal calf diarrhea is a serious health problem facing the livestock industry, which causes significant economic losses due to the increased morbidity and mortality rates 1 . A variety of viral and bacterial pathogens can cause calf diarrhea, including Rotavirus , coronavirus, bovine viral diarrhea virus (BVDV), bovine leukemia virus (BLV), Clostridium perfringens , Cryptosporidium parvum , Salmonellae , and Escherichia coli , and many of them are of zoonotic concern 2 . Antimicrobials are often prescribed for the treatment of calves with diarrhea 3 . However, indiscriminate and/or excessive use of these antimicrobials has led to the development of antimicrobial resistant (AMR) microorganisms, which has become a major global health problem 4 . Therefore, there is an urgent need to develop an alternative therapeutic for treating calf diarrhea to reduce the inappropriate use of the antimicrobials. Fecal microbiota transplantation (FMT) is a technique used to introduce the beneficial microorganisms prepared from feces of a healthy donor to improve the microbial environment of a diseased recipient 5 . We and other authors have demonstrated that FMT using fresh feces collected from healthy donors is effective in treating NCD 6 , 7 . However, veterinarians involved in FMT often face several practical challenges, such as selecting appropriate donors for FMT, confirming the absence of pathogens in the donor feces, testing for AMR microorganisms, preserving the fecal microorganisms under stable conditions, and effectively performing FMT within a short period of time to avoid the recipient’s health deterioration. Therefore, we have optimized a protocol that involves microbiota isolation from original feces content using a Nycodenz ® gradient system 8 . The optimized protocol was designed to preserve the microbial composition of donor-derived feces and its ecosystem by freezing or freeze-drying. Therefore, studies on FMT using frozen (F-FMT) and freeze-dried microorganisms (FD-FMT) are needed to investigate the colonization and maintenance of recipient gut microbiota, leading to diarrheal recovery in calves. Herein, we hypothesize that preparing donor microbiota for FD-FMT ensures transfer of microbiota to the recipient, which is associated with enrichment of keystone microbial taxa in the gut. Keystone microbial taxa are a clustered microbial community that ensures stable interactions between the microbiota and their metabolites 9 . Sequential samplings three-time intervals: before FMT, 1 day after FMT, and 7 days after FMT, will help understand the stepwise effects of F-FMT and FD-FMT and mechanisms of disease recovery. The objective of this study was to determine whether FMT using freeze-dried (FD) microbiota is effective in treating calf diarrhea, combining fecal metagenomics and metabolomics in a multiomics approach. Results Difference in microbiota between the frozen and freeze-dried microorganisms To confirm that the process used to prepare the fecal microorganisms separated from the donor’s feces did not alter the microbiota, frozen (F-FMT, n = 10) and freeze-dried (FD-FMT, n = 9) microorganisms were prepared for a total of 19 FMT studies (Fig. 1 and Supplementary Fig. 1). To demonstrate the FMT as a safe and virulence factor-free therapeutic, the fecal material obtained from the donor calf selected for FMT was initially confirmed to be pathogens-free (Fig. 2 A). The donor’s ages did not differ between the calves used for F-FMT (50.9 ± 30.06 d old) and those used for FD-FMT (40.78 ± 15.09 d old) (Fig. 2 B). The storage period starting from sample preparation to FMT was also identical between F-FMT (31.1 ± 16.68 d) and FD-FMT (39.45 ± 7.74 d) (Fig. 2 C). The Procrustes test 10 , which visualizes the superimposition of sample coordinates for ordination analysis used to compare the microbial taxonomic profiles before and after sample preparation in each season, showed that the microbial composition of the frozen and FD microorganisms had significantly correlated with that of the intact feces (R 2 = 0.6, p < 0.03 for frozen microorganisms, R 2 = 0.5, p < 0.02 for FD microorganisms) (Figs. 2 D and 2 E). No significant differences were observed in the alpha diversity parameters, including Shannon entropy, Pielou_eveness, observed OTUs, faith_PD (Fig. 2 F), and/or in the beta diversity based on weighted Uifrac distance and pairwise Permutational multivariate analysis of variance (PERMANOVA) before and after sample preparation or in both trials conducted between 2020–2021 and 2021–2022 (Fig. 2 G and Supplementary Table 1). Using BugBase 11 , a microbiome analysis tool that predicts the high-level phenotypes present in the microbiome samples, the proportion of each microbiome sample that includes the facultative anaerobic spp., Gram-negative spp., and gram-positive spp., and biofilm forming, and potentially pathogenic microorganisms was identical before and after sample preparation and between 2020–2021 and 2021–2022 (Fig. 2 H). However, the number of aerobic microorganisms was slightly reduced after samples preparation regardless of the experimental seasons (Fig. 2 H). The functional prediction of microbial communities in the isolated donor microbiota was carried out with Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) using the 16S rRNA gene sequencing data 12 . The results of the principal coordinates analysis (PCoA) and pairwise PERMANOVA test conducted on Bray–Curtis distance of the predicted gene family for the enzyme commission (EC) numbers confirmed that the frozen microorganisms and their respective intact feces did not significantly differ ( p = 0.585). The same phenomenon was also observed between the FD microorganisms and their respective intact feces ( p = 0.960) (Fig. 2 I and Supplementary Table 2). These results obtained by a metagenomic analysis indicated that the frozen and FD microorganisms were identical to their respective intact feces. Efficacy of F-FMT and FD-FMT in treatment of diarrhea in the recipient’s calves FMT studies were conducted with either the frozen or FD microorganisms in 2020–2021 and 2021–2022, respectively. There was no significant difference observed in ages of the recipient groups selected for two F-FMT and FD-FMT (Fig. 3 A). However, in context of donors, there was a significant difference observed between the donor and recipients in both F-FMT (50.9 ± 30.06 vs 20.9 ± 19.90; p < 0.05) and FD-FMT (40.78 ± 15.9 vs 16.11 ± 12.04, p < 0.05) (Fig. 3 B). In both trials of F-FMT and FD-FMT, they showed a complete reduction in the diarrheal scores (Fig. 3 C) and in the fecal water content of the 19 recipients (Fig. 3 D). The classical pathogen tests used for the detection of Rotavirus , coronavirus, E. coli , C. parvum , and C. perfringens in the recipient feces, revealed that C. parvum (11/19) and C. perfringens (8/19) pathogens were frequently detected regardless of the experimental seasons (Fig. 3 E). However, Rotavirus (4/19) and coccidia (0/19) were rarely detected. Consistent with the previous study 6 , C. perfringens was detected in feces of several cases (6/19) even after recovery from diarrhea, whereas C. parvum was barely detected in most cases (3/19), regardless of the used strategy of bacterial preparation from the donor feces (Fig. 3 E). In agreement with the previous study 6 , the blood tests used to measure the biochemical indicators demonstrated an increase in the total cholesterol level with F-FMT in 2020–2021 and FD-FMT in 2021–2022. The gamma-glutamyl transferase level decreased in both FMT treatments, whereas the other indicators [ i.e ., total protein, albumin, white blood cells, red blood cells, hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration] remained unchanged (Fig. 3 F). These results indicated that FMT was clinically effective in treating diarrhea in the recipient calves, regardless of how the donor feces were prepared (i.e., frozen or FD). Efficacy of F-FMT and FD-FMT in altering microbiome in the recipients F-FMT and FD-FMT showed similar clinical efficacy in treating diarrhea in the recipient calves; however, the differences between them were further evaluated to determine whether F-FMT or FD-FMT was superior in transforming the microbial community to the healthy conditions. Metagenomic analysis at the phylum level showed that the level of Bacteroidetes increased within 7 days after both F-FMT and FD-FMT, whereas the level of Proteobacteria decreased significantly (Fig. 4 A and Supplementary Fig. 2). At the family level, the levels of Paraprevotellaceae and Prevotellaceae belonging to the phylum Bacteroidetes increased significantly within 7 days after both F-FMT and FD-FMT, whereas that of Enterobacteriaceae belonging to the phylum Proteobacteria decreased significantly (Fig. 4 B and Supplementary Fig. 3). When considering the major genera, the levels of Fecalibacterium and Prevotella increased significantly within 7 days after both F-FMT and FD-FMT, whereas those of Bacteroides , Camphylobacter , and Veinollea decreased significantly (Fig. 4 C and Supplementary Fig. 4). These results confirmed that the relative abundance of the major microbial taxa displayed similar patterns of change in both FMT treatments, regardless of the differences in samples preparation. Linear discriminant analysis (LDA) of effect size (LEfSe) (LDA score > 2) was conducted to examine the overall bacterial features of those differentially represented before and after F-FMT and FD-FMT. Notable changes in microbial taxa were observed in FD-FMT compared with F-FMT (Fig. 4 D– 4 G). In F-FMT recipients, the phylum Proteobacteria and gram-negative genus Prevotella were enriched before FMT and after FMT (days 0 and 7) (Figs. 4 D and 4 E). By contrast, FD-FMT significantly changed the microbiota. Specifically, 16 microbial taxa were detected at different concentrations before FMT (day 0), which may represent the cause of pathogenesis; at the highest score obtained from Proteobacteria, a major phylum of gram-negative bacteria (Figs. 4 F and 4 G). After FMT (day 7), a consortium of 19 microbial taxa, including gram-positive and -negative bacteria, such as those belonging to the phylum Bacteroidetes, and several genera of Prevotella , Blautia , and Selenomonas , were enriched in FD-FMT (Figs. 4 E and 4 G). The alpha diversity of the microbiota in the recipients’ calves increased close to the healthy conditions when FD-FMT (but not F-FMT) was applied (Fig. 4 H). In addition, the beta diversity of the microbiota showed a lack of difference in baseline in the recipient calves used for F-FMT and FD-FMT before treatment. Interestingly, significant differences were observed when comparing the donors and recipients calves before FMT in both treatments (Fig. 4 I and Supplementary Table 3). Moreover, no differences were observed between the donors and recipients’ calves 7 days after FD-FMT but not after F-FMT, confirming the possibility that the recipient calves can acquire the microbial composition of the donor within 7 days via FD-FMT (Supplementary Table 4). Further analysis of the extent of bacterial biogenesis after FMT using SourceTracker 13 showed that the average contribution of the donors to the development of microbiota of the recipient calves on day 1 and 7 was 5% and 11% for F-FMT; respectively, whereas it reached 10% and 40%; respectively, for FD-FMT (Fig. 4 J). Therefore, in the context of the donor’s microbiota engraftment, freeze-drying may be a superior method of microbial conditioning for FMT compared to just freezing. Efficacy of F-FMT and FD-FMT in altering the intestinal metabolites in the recipients To determine the exact effects of FD-FMT and F-FMT in treating diarrhea in the recipient calves, the fecal metabolites of the donors and the recipients before and 7 days after FMT were comprehensively analyzed using the capillary electrophoresis–Time-of-Flight Mass Spectrometry (CETOFMS). An interactive heatmap was provided to demonstrate all metabolites identified using Metaboanalyst 14 (Fig. 5 A). Principal component analysis (PCA) revealed significant differences in pretreatment (day 0) and posttreatment (day 7) recipients compared with the donors for F-FMT (Fig. 5 B and Supplementary Table 5) but only between the donors and pretransplant (day 0) recipients and not between donors and posttransplant (day 7) recipients for FD-FMT (Fig. 5 C and Supplementary Table 6). Procrustes analysis assessed similarity of metabolites between donors and recipients before (day 0) and after (day 7) FMT. In F-FMT, the Procrustes correlation was low ( R 2 = 0.2467) in donor vs. recipient on day 0 compared with donor vs. recipient on day 7 ( R 2 = 0.3748) (Supplementary Fig. 5A and 5B). However, in FD-FMT, the Procrustes correlation between donors and recipients was higher after FMT on day 7 than day 0 ( R 2 = 0.2036 vs. R 2 = 0.4334) (Supplementary Fig. 5C and 5D). Because overall Procrustes correlation between donors and recipients was higher in FD-FMT than F-FMT (Supplementary Figs. 5A–5D), recipient metabolites were potentially same as donor metabolites in FD-FMT. Notably, that the number of metabolites were reduced but not increased by FMT, which were greater for FD-FMT than F-FMT. Specifically, within 7 days after F-FMT or FD-FMT, 43 or 29 metabolites were upregulated and 35 or 62 were downregulated, respectively (Supplementary Tables 7 and 8). To identify the metabolic pathways that differed between the F-FMT and FD-FMT, KEGG enrichment analysis was performed based on total number of metabolites obtained from the fold-change (FC) analysis. KEGG enrichment analysis revealed that 38 metabolic pathways were involved in F-FMT and FD-FMT (Supplementary Fig. 6A and 6B and Supplementary Table 9). For F-FMT, the metabolites were mainly involved in purine metabolism, arginine biosynthesis, and pyrimidine metabolism ( P < 0.05) (Supplementary Table 9). For FD-FMT, the metabolites were primarily involved in glycine, serine, and threonine metabolism; aminoacyl-tRNA biosynthesis; histidine metabolism; glycerophospholipid metabolism; arginine and proline metabolism; alanine, aspartate and glutamate metabolism, and glutathione metabolism ( P < 0.05) (Supplementary Table 10). This study confirmed that the major metabolites in FD-FMT were involved in amino acid metabolism pathways. Next, the major metabolites detected in ATP-binding cassette (ABC) transporter, amino acid metabolism, lipid fatty acids, energy, polyamines, methylated compounds, and vitamins, short-chain fatty acids and bile acids were profiled in this study. Meanwhile, the levels of metabolites belonging to the ABC transporter metabolism, such as amino acids, were highly affected by FD-FMT rather than by F-FMT (Figs. 5 D– 5 F and Supplementary Figs. 7–10). During the diarrheal condition, high levels of several amino acids, such as arginine, proline, and histidine were abundantly present in the feces, which may represent a microbial dysbiosis 6 . Thus, FD-FMT displayed a clear understanding to retain the microbial symbiosis condition by enhancing amino acid utilizing bacteria in the gut. Furthermore, by using variable importance in projection (VIP) scores obtained from the partial least-squares discriminant analysis (PLS-DA), top 15 discriminating metabolites were found, in where in FD-FMT cases amino acid histidine, phenylalanine, cysteine, leucine, valine and isoleucine were found downregulated (Figs. 5 G and 5 H). Consequently, these results indicated that FD-FMT was more efficient than F-FMT in changing the metabolic environment in feces of the recipient calves within 7 days after treatment. Establishing the microbiota–metabolite correlation by FD-FMT during disease recovery To investigate whether the microbiota and metabolites were closely associated during disease recovery post F-FMT or FD-FMT, Pearson’s correlation analysis was conducted. Specifically, the levels of the 16 selected microbiotas categorized as residents or colonizers using Microbiome Multivariable Association with Linear Models (MaAsLin2) 15 in the Microbiomeanalyst 16 platform (Fig. 6 A and Supplementary Table 11). The 15 key metabolites shown in Figs. 5 G and 5 H were used for correlation analysis. Of the 240 (16 × 15) combinations, F-FMT showed five positive correlations before FMT (day 0) (Fig. 6 B) and three positive and two negative correlations 7 days after FMT (day 7) (Fig. 6 C). By contrast, of the 224 (16 × 14) combinations, FD-FMT displayed 14 positive and six negative correlations on day 0 (Fig. 6 D) and 17 positive and two negative correlations on day 7 (Fig. 6 E). For example, Campylobacteraceae and Enterobacteriaceae were positively correlated with choline ( P < 0.001 and P < 0.01), N 6-methyllysine ( P < 0.01 and P < 0.05), and succinic acid (both P < 0.001). Given that FD-FMT had a high number of correlations between metabolites and microbial taxa, these results indicate that compared with F-FMT, FD-FMT significantly changed the intestinal environment. To compare the predictive potential of F-FMT and FD-FMT in disease recovery through changes in microbiota and metabolites 7 days after FMT (day 7) vs. before FMT (day 0), a prediction model with area under the curve (AUC) of the receiver operating characteristic (ROC) was used 17 , using three feature sets: microbial taxa (n = 240), fecal metabolites (n = 596), and a combination of microbial taxa and fecal metabolites (n = 836). The AUC values of microbiota, metabolites, and microbiota + metabolites were 0.91, 0.78, and 0.76, respectively for F-FMT (Supplementary Fig. 11A), and 0.94, 0.81, and 0.81, respectively, for FD-FMT (Supplementary Fig. 11B). Thus, FD-FMT exhibited excellent discriminative power and superior performance in altering the microbiota and metabolites 7 days after FMT for diarrhea remission in calves (Fig. 6 F). Overall, despite the high functional similarity between F-FMT and FD-FMT, FD-FMT exhibited broader spectral performance and was superior to F-FMT in promoting changes in the microflora and metabolites. Discussion FMT is an excellent strategy for treating calf diarrhea without causing undesirable side effects 6 , 7 , 18 . Even for intractable diarrhea, FMT with fresh stool was 70% effective 6 . Therefore, there is a growing and urgent need to establish a method to prepare healthy donor-derived beneficial microorganisms disseminated in many areas without concerns for safety and stability 19 , 20 . In humans, especially those undergoing treatment for recurrent Clostridioides difficile infection, several protocols for preparing donor fecal material have been developed, such as frozen or FD fecal extracts, with ≥ 90% 21–23 efficacy rate. Moreover, different FMT protocols for screening and processing donor feces have been established in clinical practice 24 – 34 (Supplementary Table 12). However, only few studies have been conducted to establish a protocol for preparing donor feces in livestock production 35 – 37 (Supplementary Table 12). Therefore, this study focused on establishing a method for extracting and preserving fecal microbiota of calves for transplantation and analyzing its usefulness in effectively treating diarrhea. Specifically, Nycodenz ® —a nontoxic and nonionic water-soluble compound that can form self-density gradients—was used to isolate microorganisms from the feces of healthy calves on a large scale 8 . Notably, Nycodenz ® can isolate 10 10 viable bacteria per 2 g fresh feces in a preserved ecosystem, and this isolation procedure does not alter the composition of the original microflora concerning survival, distribution, and proportion 8 . Because the absence of common diarrhea-causing pathogens should be confirmed first, the protocol established in this study will be promising in eliminating the veterinarians’ concerns about donor selection and preparation and preservation of the microorganisms necessary for successful FMT. Developing donor-derived fecal microbiota products, rather than conditioning fresh feces each time on the farm, is an ideal strategy for preserving beneficial microorganisms and widely disseminating them to the farms for treating calves with diarrhea. Therefore, there are several advantages to optimizing the protocols for preserving microorganisms obtained from donor feces and using them later for transplantation. Increasing the stability of fecal microbiota will maintain the complex microbial network necessary to establish a microbial environment for recipient calves through transplantation and contribute to maximizing the efficacy of FMT to cure diarrhea. Among several possible procedures used for bacterial preservation that led to successful FMT, freeze-drying is strongly recommended throughout this study because it removes water from the microorganisms, temporarily stopping their metabolic activity and moving them into a dormant state. Microbial DNA is protected from hydrolytic damage and enzymatic degradation under FD conditions. Thus, the metabolic activity of FD microorganisms is quickly and appropriately resumed by rehydration 38 , 39 . By contrast, the freezing method impairs the intracellular metabolic activity of microbes and disrupts the mutualistic feeding mechanisms and ecological interactions in frozen samples, resulting in a significant reduction in microbial functionality compared with the original state. To improve the storage stability of frozen microorganisms, the use of cryoprotectants, such as glycerol and dimethyl sulfoxide, should be considered when preparing samples from donor feces 40 – 43 Nevertheless, glycerol is not recommended for lyophilization due to its high viscosity, resulting in a poorly dried and sticky product, which is unsuitable for rehydration and subsequent transplantation. Therefore, although cryoprotectants were not used in this study, future efforts should be made to select and optimize cryoprotectants that maintain/increase microbial survival in the dormant state for successful FMT using FD (not frozen) microorganisms. Herein, calves with diarrhea had more bacteria belonging to the phylum Proteobacteria, genus Enterobacteria and Galibacterium and were less likely to possess butyrate-producing bacteria, such as Bifidobacterium , Fecalibacterium , and Blautia . In addition, F-FMT and FD-FMT reduced Proteobacteria and improved diarrheal status, regardless of the nature of the microorganisms transplanted. Nonetheless, FD-FMT increased Gram-positive and -negative bacteria efficiently and in a balanced manner, thus creating a better microbial environment that led to diarrhea recovery in calves. Notably, FD-FMT facilitated enrichment of the genus Blautia and Selenomonas , which abundantly produce short-chain fatty acids and utilized amino acids for their metabolism. One possible consideration for why FD-FMT was far superior to F-FMT in improving the microflora can be attributed to the high frequency of Gram-negative bacteria preserved during sample preparation. Specifically, prediction strategy using BugBase showed that the freeze-drying procedure reduced the Gram-negative bacteria by 3.82% after sample preparation, whereas the frozen strategy reduced them by 29.81%. In addition, a highly significant correlation existed between indigenous and newly-colonized microbiota, and the metabolites produced by them, which were induced by FD-FMT rather than F-FMT, may have contributed to the development of a balanced microbial environment, indicating the possibility of competition and mutualistic feeding interactions among microorganisms, and that competition for resources may have a positive impact on the relative abundance of bacterial families created by FD-FMT in recipient calves. The establishment of fecal banks to store donor stool samples has advanced FMT technology, by ensuring timely and reliable availability—while maintaining safety and quality standards—and providing recipients with multiple options for selecting an appropriate donor. Several approved medical products consisting of donor stool are commercially available, particularly for treating recurrent C. difficile infection in humans, notably the BiomeBank licensed in Australia and REBYOTA licensed in the United States. These products contain frozen microorganisms and are transported under cold-chain control to the clinic for rectal administration to recipients in need 44 – 46 . Unlike fecal-derived microbial products for human use, there is an urgent need to develop new technologies for the stable preservation of fecal-derived microorganisms in livestock production, even where adequate cryopreservation facilities are not available. FD microbial preparations are the most appropriate materials stored stably in the field, and this convenience of storage can greatly expand the number of farms where FMT can be applied. Thus, unlike FMT for humans using frozen microorganisms, FMT for treating calf diarrhea can be widely implemented from a single donor with multiple recipients raised on multiple farms. In addition, the results of this study reveal that FD-FMT shows clear evidence of calf recovery from diarrhea, suggesting its potential use as a next-generation therapeutic agent. Future studies focusing on the impact of FD-FMT on the microbial community, removal of potential virulence factors and antibiotic resistance genes, and evaluation of the by-products obtained will provide the foundation necessary for the practical application of FD-FMT for treating calf diarrhea. Methods Development of the study This study was approved by the Ethics Committee of the respective institutions to determine the efficacy of prepared fecal microorganisms used for treating diarrhea in calves. This study was conducted using two experiments in the winter seasons from 2020 to 2021 (F-FMT) and from 2021 to 2022 (FD-FMT). In total 25 and 16 fecal samples were collected from healthy donor calves from four and three farms in 2020–2021 and 2021–2022, respectively. The fresh fecal samples were collected from the potential donors in the field and then stored immediately at − 80°C until the microorganisms become ready for transplantation. Microorganisms isolated from the fecal samples were frozen at − 80°C in the first year (2020–2021) or FD and stored at − 80°C until being used in the second year (2021–2022). A total of 19 FMTs were performed using either frozen microorganisms in 2020–2021 (n = 10) or FD microorganisms in 2021–2022 (n = 9). All FMT trials were conducted based on the individual donor-recipient pair, which was selected from the same farm to avoid the transmission of virulence factors. Experiment design for potential donor selection Optimal donors were selected from healthy calves to ensure the efficacy of FMT and avoid the risk of transmission of virulence factors during FMT. Therefore, before FMT, screening fecal and blood samples of potential donors for known pathogens (e.g., described below) is highly recommended to confirm that their fecal matter is safe to use for FMT. In this study, Rainbow Calf Scours® (Bio-X) was used to confirm the absence of Rotavirus , coronavirus, E. coli , C. parvum , and C. perfringens from feces of potential donors. The modified Wisconsin sugar centrifugal fraction method was conducted using a saturated sucrose solution to confirm the absence of protozoa and nematodes from feces of the potential donors. In addition, either nested-polymerase chain reaction (nested-PCR) or real time-PCR (RT-PCR) were conducted using whole blood-derived viral DNA or plasma-derived viral RNA at NDTS Co., Ltd., to confirm the absence of BLV and BVDV, respectively, from the blood of the optimal donors. The fecal materials were collected under aseptic conditions using sterile containers, gloves, and disposable sterile spoons from readily available commercial collection kits. Appropriate instructions were provided to the veterinarians to avoid contamination of fecal and blood samples with environmental residues or soil. Samples were delivered immediately to the microbiology laboratory to avoid substantial changes in the metabolic profiles and abundance of bacterial taxa 47 . Isolation of microorganisms for FMT using Nycodenz® Fecal samples (15–25 g) collected from potential donors were suspended in six volumes of phosphate-buffered saline (PBS) (1×) to ensure adequate bacterial density and separated using 80% (w/v) Nycodenz ® solution 8 . Initially, to prepare the fecal suspensions, the fecal samples were blended for 2 min using a standard commercial hand blender. The resulting slurries were passed through a nylon mesh (0.5 mm) to remove large debris, and 21 mL of the collected slurry was transferred to overlay 7 mL Nycodenz ® density gradient solution in a 50 mL glass tube. The overlayed fecal solution was centrifuged at 10,000 × g for 40 min at 4°C to enrich the bacterial fraction interface 8 . The upper layer of the fecal solution containing water-soluble debris was aspirated without disturbing the bacterial interface. Approximately 35–40 mL of the bacterial interface (e.g., fecal-derived microorganisms)-containing solution were collected from each donor sample. For F-FMT or FD-FMT, the fecal-derived microorganisms were either frozen only or freeze-dried. Selection of potential FMT recipients Recipient calves with diarrhea were selected by the veterinarians based on diarrheal consistency. Considering diarrheal score, fecal consistency was scored based on a scale of 1–4, where 1 = normal consistency, 2 = semi-formed or pasty, 3 = loose feces, and 4 = watery feces 6 . During selection and to ensure successful implantation, antibiotics were not used to treat recipient calves 1 week before FMT. To evaluate the efficacy of FMT, the presence of common pathogens ( i.e ., Rotavirus, coronavirus, BLV, BVDV, E. coli , C. parvum , C. perfringens , protozoa, and nematodes) in fecal and blood samples were initially investigated using commercialized pathogen kits before FMT (day 0) and after FMT at day 7. FMT procedure Donor samples, either frozen (F-FMT) or freeze-dried (FD-FMT) microorganisms, were delivered to the field on dry ice with a locally available transport system. F-FMT was thawed for 2 h in warm water at 37°C, whereas FD-FMT was suspended in 100 mL of sterile saline. To perform FMT with the microbiota adjusted to 100 mL, a catheter (60-cm long, 1-cm diameter) was inserted approximately 30 cm into the rectum of the recipient calf with diarrhea, approximately up to the second lumbar vertebra (Supplementary Fig. 11). Fecal bacterial DNA extraction, 16S rRNA sequencing, and taxonomic profiling Total bacterial DNA was extracted from the fecal samples and isolated from the bacterial interface using Norgen Stool DNA Isolation Kit, according to the manufacturer’s instructions. The quality and quantity of DNA were further assessed. The V3–V4 region of the bacterial 16S rRNA gene was amplified using the following primers: Forward primers mixed (5′-TGCTCTTCCGATCTGACNNNCCTACGGGNGGCWGCAG-3′, 5′-TGCTCTTCCGATCTGACNNNNCCTACGGGNGGCWGCAG-3′, 5′-TGCTCTTCCGATCTGACNNNNNCCTACGGGNGGCWGCAG-3′, and 5′-TGCTCTTCCGATCTGACNNNNNNCCTACGGGNGGCWGCAG-3′), and reverse primers mixed (5′-CGCTCTTCCGATCTCTGNNNGACTACHVGGGTATCTAATCC-3′, 5′-CGCTCTTCCGATCTCTGNNNNGACTACHVGGGTATCTAATCC-3′, 5′-CGCTCTTCCGATCTCTGNNNNNGACTACHVGGGTATCTAATCC-3′, and 5′-CGCTCTTCCGATCTCTGNNNNNNGACTACHVGGGTATCTAATCC-3′). The PCR fragments obtained from the first round of PCR were further amplified in the second round of PCR using the following primers: Forward primer (5′-CAAGCAGAAGACGGCATACGAGATxxxxxxxxxGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC-3′), and reverse primer (5′-AATGATACGGCGACCACCGAGATCTACACxxxxxACACTCTTTCCCTACACGACGCTCTTCCGATCTCTG-3′). All PCR products were sequenced using the MiSeq platform (Illumina) with MiSeq Reagent Kit v2 (500 cycles) 48 . After next-generation sequencing, the demultiplexed raw sequences were acquired from BaseSpace Sequence Hub (Illumina). The sequences were analyzed using QIIME 2 (version 2021.2) 49 , and metagenomics and functional prediction analyses were conducted based on the published methodologies 6 . Analyses of FMT-induced alterations in the recipient and metabolite profiles Based on the results obtained from 16S rRNA amplicon sequencing and using the freely available program SourceTracker, the efficacy of engraftment of donor-derived microorganisms in calf recipients with diarrhea post-FMT was investigated 13 , where a Bayesian algorithm was used to determine the percentage of microbial communities in the recipient calf that belonged to the donor microbiota. SourceTracker indicated coexistence rate of donor-derived microbiota. CE–TOFMS metabolomics analysis Using the fecal samples collected from the donors and recipients’ calves, a metabolomics analysis was conducted at the Human Metabolome Technologies, Inc. (Japan). The samples were analyzed using capillary electrophoresis coupled with time-of-flight mass spectrometry (CE–TOFMS, Agilent). The metabolite standards, instrumentation, and CE–TOFMS conditions were as described 50 . The metabolites of each reconstituted sample were separated in a fused silica capillary (i.d. 50 µm × 80 cm) (Agilent). The data acquisition had been carried out using an electrospray ionization cation and an anion full scan modes. In the positive mode, the capillary voltage was 30 kV, MS capillary voltage was 4,000 V, and the sample solution was injected for 10 s at 50 mbar. Meanwhile, in the negative mode, the capillary voltage was 30 kV, MS capillary voltage was 3,500 V, and the interjection time was 50 mbar for 22 s. Statistical analysis All error bars represent standard deviation. Statistical significance was assigned at P value < 0.05 and detected by GraphPad Prism 9.5.1 (GraphPad Software, San Diego, USA). The microbe–metabolite correlation heatmap was created by calculating Spearman’s correlation coefficients for each pairwise combination of microbial taxa abundance and metabolite intensity using the “corr.test” function in R package. Declarations Data availability All data generated or analyzed in this study are included in this article (and its supplementary information files). Acknowledgments We acknowledge Drs. Hidetoshi Kato, Takafumi Goto, Jun Kunisawa, and Hiromichi Ohtsuka for their practical advice related to this study. This study was primarily supported by three agencies; mainly a Livestock Promotional Subsidy from the Japan Racing Association (to T.N.), and by Grants-in-Aid for Scientific Research (A) 18H03969 and 22H00393 (to T.N.), and by Grants-in-Aid for Early-Career Scientists 20K15478 and 23K14062 (to J.I.). Author contributions J.I. contributed by designing the study, performed experiments, analyzed data, and wrote the manuscript. N.O., Y.S., M.T., Y.G., M.S., E.M., T.S., C.U., and H.T. contributed by performing FMT. A.M. and Y.Su. contributed by sequencing 16S rRNA genes. Y.Sa. contributed by making the analysis. H.Y. and N.A.K. contributed by providing comments on the manuscript. R.H. and M.F. contributed by supporting the experiments. T.N. contributed by designing the study and writing the manuscript. Competing interests The authors declare that they have no competing interests. References Wei, X. et al. 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Timing and delivery route effects of cecal microbiome transplants on Salmonella typhimurium infections in chickens: potential for in-hatchery delivery of microbial interventions. Anim Microbiome 5 ,11 (2023). Cheng, C. S. et al. Early intervention with faecal microbiota transplantation: an effective means to improve growth performance and the intestinal development of suckling piglets. Animal 13 , 533–541 (2019). Machiels BM et al. New Protocol for DNA Extraction of Stool. Biotechniques 28 , 286–290 (2000). Lewis, Z. T. et al. The impact of freeze-drying infant fecal samples on measures of their bacterial community profiles and milk-derived oligosaccharide content. PeerJ. 21, e1612, (2016). Wu, C. et al. The maintenance of microbial community in human fecal samples by a cost effective preservation buffer. Sci Rep 11 , 13453 (2021). Li, X. et al. Effects of stool sample preservation methods on gut microbiota biodiversity: New original data and systematic review with meta-analysis. Microbiol Spectr 11 , e0429722 (2023). Berland, M. et al. High engraftment capacity of frozen ready-to-use human fecal microbiota transplants assessed in germ-free mice. Sci Rep 11 , 4365 (2021). Bellali, S., Bou Khalil, J., Fontanini, A., Raoult, D. & Lagier, J. C. A new protectant medium preserving bacterial viability after freeze drying. Microbiol Res 236 , 126454 (2020). Yu, Y., Wang, W. & Zhang, F. The next generation fecal microbiota transplantation: to transplant bacteria or virome. Advanced Science 10 , e2301097 (2023). Tucker, E. C. et al. Stool donor screening within a therapeutic goods administration compliant donor screening program for fecal microbiota transplantation. JGH Open 7 , 172–177 (2023). Blair, H. A. RBX2660 (REBYOTA®) in preventing recurrence of Clostridioides difficile infection: a profile of its use in the USA. Drugs and Therapy Perspectives 39 , 331–338 (2023). Gorzelak, M. A. et al. Methods for improving human gut microbiome data by reducing variability through sample processing and storage of stool. PLOS One 10 , e0139529 (2015). Usami, K. et al. The gut microbiota induces Peyer’s-patch-dependent secretion of maternal IgA into milk. Cell Rep 36 , 109655 (2021). Bolyen, E. et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature Biotechnology 37 , 852–857 (2019). https://doi.org/10.1038/s41587-019-0209-9 Soga, T. et al. Simultaneous determination of anionic intermediates for Bacillus subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry. Anal Chem 74 , 2233–2239 (2002). Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4168305","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":289177422,"identity":"36de4ede-54a5-40cd-8508-8a9b3163eea3","order_by":0,"name":"Tomonori 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Association","correspondingAuthor":false,"prefix":"","firstName":"Hidekazu","middleName":"","lastName":"Tanaka","suffix":""}],"badges":[],"createdAt":"2024-03-26 08:35:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4168305/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4168305/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54446699,"identity":"ef612d58-bfa3-4d81-9811-3990dbd18660","added_by":"auto","created_at":"2024-04-10 16:22:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1043789,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the protocol steps. \u003c/strong\u003eFirst: Feces were homogenized with sterile phosphate buffer saline. Second: Fecal suspension was placed on top of the Nycodenz\u003csup\u003e®\u003c/sup\u003e (80%). Third: The feces suspension and Nycodenz\u003csup\u003e®\u003c/sup\u003e were centrifuged at 10,000 \u003cem\u003eg\u003c/em\u003e for 40 min at 4ºC, and then the microbiota-containing portions were collected. Four: Microbiota proceeded to either freeze or freeze-drying until its use for transplantation.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4168305/v1/6672b0e4f60249eaa0e2a6e3.jpg"},{"id":54446703,"identity":"862db8a6-36aa-4d23-95bb-de0c886ce73c","added_by":"auto","created_at":"2024-04-10 16:22:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1856153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrobial similarities of the donor’s microbiota to their respective intact feces.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Pathogen test for selected donor. \u003cstrong\u003eB\u003c/strong\u003e Donor age. \u003cstrong\u003eC\u003c/strong\u003e Shelf life of the donor. \u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eE \u003c/strong\u003eProcrustes analysis to assess the similarities of the isolated microbiota and its respective intact feces, which were calculated using the Bray­­–Curtis distance based on 16S rRNA gene sequence. The shown significance values were calculated using the \u003cem\u003eprotest \u003c/em\u003efunction from the vegan R package. \u003cstrong\u003eD \u003c/strong\u003eFrozen microbiota and intact feces, and \u003cstrong\u003eE\u003c/strong\u003e freeze-dried microbiota and its intact feces. \u003cstrong\u003eF\u003c/strong\u003e Alpha diversity indexes (means ± SD; n = 10 for F-FMT, and n = 9 for FD-FMT). \u003cstrong\u003eG\u003c/strong\u003e Beta diversity based on weighted UniFrac distances that were represented \u003cem\u003evia\u003c/em\u003e generating principal coordinate analysis (PCoA) plots at feature level through Quantitative Insights into Microbial Ecology 2 (QIIME2. \u003cstrong\u003eH \u003c/strong\u003ePhenotypic prediction using Bugbase (means ± s.d., n = 10, for F-FMT, and n = 9 for FD-FMT). Significance labels: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eI\u003c/strong\u003e The principal coordinates analysis (PCoA) plot based on Bray–Curtis distance of enzyme commission (EC) numbers obtained from the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) analysis (n = 10 for F-FMT, and n = 9 for FD-FMT) in QIIME2.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4168305/v1/490a710f02075603b3adfc56.jpg"},{"id":54446697,"identity":"6414b5b8-d7c0-4c0c-97ee-2021a1fe8136","added_by":"auto","created_at":"2024-04-10 16:22:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1897538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfficacy of frozen (F-FMT) and freeze-dried (FD-FMT) microbiota-based donor\u003c/strong\u003e.\u003cstrong\u003e A\u003c/strong\u003e Recipient age (means ± s.d; n = 10, for F-FMT, and n = 9, for FD-FMT). \u003cstrong\u003eB \u003c/strong\u003eComparison of ages between donor and recipients. Significance labels: **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01. \u003cstrong\u003eC\u003c/strong\u003e Diarrhea score (means ± s.d; n = 10, for F-FMT, and n = 9 for FD-FMT). Significance labels: **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003eD\u003c/strong\u003e Fecal water content (means ± s.d; n = 10 for F-FMT, and n = 9 for FD-FMT). Significance labels: *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003eE \u003c/strong\u003ePathogen results obtained from Rainbow kits. \u003cstrong\u003eF\u003c/strong\u003e Blood biochemical parameter (means ± s.d; n = 10 for F-FMT, and n = 9 for FD-FMT). Significance labels: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4168305/v1/d2d6010ad6d9924f1ea5e4e5.jpg"},{"id":54446701,"identity":"31d02dbb-ac46-4d53-bc5f-6bc402735177","added_by":"auto","created_at":"2024-04-10 16:22:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2370715,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the taxonomy and diversity of fecal microbiota according to FMT.\u003c/strong\u003e \u003cstrong\u003eA-C\u003c/strong\u003e Relative abundance of bacterial taxa based on \u003cstrong\u003eA \u003c/strong\u003ephylum \u003cstrong\u003eB \u003c/strong\u003egenus and \u003cstrong\u003eC \u003c/strong\u003efamily. The linear discriminant analysis effect size (LefSe) was performed \u003cem\u003evia \u003c/em\u003eGalaxy Project. The LDA scores were presented in the bar charts showing significant bacterial differences before (day 0) and after (day 7), while cladogram showed the most discriminative bacterial clades identified in F-FMT (\u003cstrong\u003eD, E\u003c/strong\u003e) and FD-FMT (\u003cstrong\u003eF\u003c/strong\u003e, \u003cstrong\u003eG\u003c/strong\u003e). \u003cstrong\u003eH\u003c/strong\u003e Alpha diversity indexes before and after FMT in F-FMT and FD-FMT (means ± s.d; n = 10 for F-FMT, and n = 9 for FD-FMT) demonstrating significance labels: *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05. \u003cstrong\u003eI\u003c/strong\u003e The principal coordinates analysis (PCoA) based on the weighted UniFrac distance before and after FMT in F-FMT and in FD-FMT. \u003cstrong\u003eJ \u003c/strong\u003eResults of Source tracker analysis showing the average contributions of the donor’s microbiota engraftment into the recipients both at day 1 and 7 in F-FMT and FD-FMT treatments. Source: donor microbiota (either frozen or freeze-donor) and recipient microbiota at day 0; sink: recipient microbiota at day 1 and 7. (n = 10 for F-FMT, and n = 9 for FD-FMT).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4168305/v1/cdd99dca4f71d3a7f908314b.jpg"},{"id":54446702,"identity":"201638e5-08dc-4f1a-9d24-87a720838ba0","added_by":"auto","created_at":"2024-04-10 16:22:48","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1819343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFecal metabolomics profile of F-FMT and FD-FMT. \u003c/strong\u003eThe fecal metabolomes of the calves were analyzed using capillary electrophoresis–time-of-flight mass spectrometry (CE-TOF-MS). \u003cstrong\u003eA\u003c/strong\u003e The abundant metabolites in the donor and recipients at day 0 and 7 are represented in a heatmap. \u003cstrong\u003eB, C\u003c/strong\u003e Principal component analysis (PCA) score plot from \u003cstrong\u003eB \u003c/strong\u003eF-FMT and \u003cstrong\u003eC \u003c/strong\u003eFD-FMT \u003cstrong\u003eD-F\u003c/strong\u003e Heat maps of the major metabolites that discriminate between the results before and after FMT in F-FMT and FD-FMT treatments. The major metabolites were obtained from \u003cstrong\u003eD \u003c/strong\u003eABC transporters, lipid, fatty acid metabolism, and energy metabolism. \u003cstrong\u003eE\u003c/strong\u003e Polyamines, methylated compounds, and vitamins. \u003cstrong\u003eF\u003c/strong\u003e Short-chain fatty acids and secondary bile acids. \u003cstrong\u003eG, H\u003c/strong\u003e Metabolites ranked by their contributions (the top 15) and are presented as variable importance in the projection (VIP) scores, which were identified by partial least-squares discriminant analysis (PLS-DA) in \u003cstrong\u003eG \u003c/strong\u003eF-FMT and \u003cstrong\u003eH \u003c/strong\u003eFD-FMT. The colored boxes on the right indicate the relative concentrations of the corresponding metabolite in each group (n = 10 for F-FMT, and n = 9 for FD-FMT).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4168305/v1/331e9a80312cec30ae545cbf.jpg"},{"id":54446700,"identity":"aa84bd58-9e7b-4348-96ca-d9c64cea6a60","added_by":"auto","created_at":"2024-04-10 16:22:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1717011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFecal microbiota–metabolite correlation in FD-FMT.\u003c/strong\u003e \u003cstrong\u003eA \u003c/strong\u003ePearson’s correlation coefficients were calculated to assess the relationship between keystone microbial taxa from various families and major metabolites identified through variable importance in projection (VIP) score; \u003cstrong\u003eB–E \u003c/strong\u003epositive and negative correlations are depicted by color intensity, with red and green indicating positive and negative correlations, respectively. Significantly correlated keystone taxa and metabolites are labeled on the heatmap; heatmap illustrating the correlation between microbial taxa and metabolites \u003cstrong\u003eB\u003c/strong\u003e before FMT (day 0) and \u003cstrong\u003eC\u003c/strong\u003e after FMT (day 7) in F-FMT. Heatmap illustrating the correlation between microbial taxa and metabolites \u003cstrong\u003eD\u003c/strong\u003e before FMT (day 0) and \u003cstrong\u003eE\u003c/strong\u003e after FMT (day 7) in FD-FMT; \u003cstrong\u003eF\u003c/strong\u003e predictive potential of F-FMT and FD-FMT in calf diarrhea remission through microbiota–metabolite changes before (day 0) and after FMT (day 7). AUC results obtained by random forest are shown. The significance levels in the correlation test are presented as *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4168305/v1/9a1f662dd5194f9007408a0e.jpg"},{"id":58499233,"identity":"902c5506-927d-4b3d-ace6-badee92d9c86","added_by":"auto","created_at":"2024-06-17 12:59:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11594398,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4168305/v1/20d18596-7874-486f-9621-e53e24a7aa0e.pdf"},{"id":54446698,"identity":"60b78d47-df21-4fcf-aea2-0ee9b78244b4","added_by":"auto","created_at":"2024-04-10 16:22:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5779674,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureSupplementaryTable.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4168305/v1/11a5b958f2f4772e115d2289.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Freeze-dried fecal microorganisms as an effective biomaterial for the treatment of calves suffering from diarrhea","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeonatal calf diarrhea is a serious health problem facing the livestock industry, which causes significant economic losses due to the increased morbidity and mortality rates\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. A variety of viral and bacterial pathogens can cause calf diarrhea, including \u003cem\u003eRotavirus\u003c/em\u003e, coronavirus, bovine viral diarrhea virus (BVDV), bovine leukemia virus (BLV), \u003cem\u003eClostridium perfringens\u003c/em\u003e, \u003cem\u003eCryptosporidium parvum\u003c/em\u003e, \u003cem\u003eSalmonellae\u003c/em\u003e, and \u003cem\u003eEscherichia coli\u003c/em\u003e, and many of them are of zoonotic concern\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Antimicrobials are often prescribed for the treatment of calves with diarrhea\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, indiscriminate and/or excessive use of these antimicrobials has led to the development of antimicrobial resistant (AMR) microorganisms, which has become a major global health problem\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therefore, there is an urgent need to develop an alternative therapeutic for treating calf diarrhea to reduce the inappropriate use of the antimicrobials.\u003c/p\u003e \u003cp\u003eFecal microbiota transplantation (FMT) is a technique used to introduce the beneficial microorganisms prepared from feces of a healthy donor to improve the microbial environment of a diseased recipient\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. We and other authors have demonstrated that FMT using fresh feces collected from healthy donors is effective in treating NCD\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, veterinarians involved in FMT often face several practical challenges, such as selecting appropriate donors for FMT, confirming the absence of pathogens in the donor feces, testing for AMR microorganisms, preserving the fecal microorganisms under stable conditions, and effectively performing FMT within a short period of time to avoid the recipient\u0026rsquo;s health deterioration. Therefore, we have optimized a protocol that involves microbiota isolation from original feces content using a Nycodenz\u003cb\u003e\u0026reg;\u003c/b\u003e gradient system\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The optimized protocol was designed to preserve the microbial composition of donor-derived feces and its ecosystem by freezing or freeze-drying. Therefore, studies on FMT using frozen (F-FMT) and freeze-dried microorganisms (FD-FMT) are needed to investigate the colonization and maintenance of recipient gut microbiota, leading to diarrheal recovery in calves.\u003c/p\u003e \u003cp\u003eHerein, we hypothesize that preparing donor microbiota for FD-FMT ensures transfer of microbiota to the recipient, which is associated with enrichment of keystone microbial taxa in the gut. Keystone microbial taxa are a clustered microbial community that ensures stable interactions between the microbiota and their metabolites\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Sequential samplings three-time intervals: before FMT, 1 day after FMT, and 7 days after FMT, will help understand the stepwise effects of F-FMT and FD-FMT and mechanisms of disease recovery. The objective of this study was to determine whether FMT using freeze-dried (FD) microbiota is effective in treating calf diarrhea, combining fecal metagenomics and metabolomics in a multiomics approach.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eDifference in microbiota between the frozen and freeze-dried microorganisms\u003c/h2\u003e\n\u003cp\u003eTo confirm that the process used to prepare the fecal microorganisms separated from the donor\u0026rsquo;s feces did not alter the microbiota, frozen (F-FMT, n\u0026thinsp;=\u0026thinsp;10) and freeze-dried (FD-FMT, n\u0026thinsp;=\u0026thinsp;9) microorganisms were prepared for a total of 19 FMT studies (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Fig.\u0026nbsp;1). To demonstrate the FMT as a safe and virulence factor-free therapeutic, the fecal material obtained from the donor calf selected for FMT was initially confirmed to be pathogens-free (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The donor\u0026rsquo;s ages did not differ between the calves used for F-FMT (50.9\u0026thinsp;\u0026plusmn;\u0026thinsp;30.06 d old) and those used for FD-FMT (40.78\u0026thinsp;\u0026plusmn;\u0026thinsp;15.09 d old) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). The storage period starting from sample preparation to FMT was also identical between F-FMT (31.1\u0026thinsp;\u0026plusmn;\u0026thinsp;16.68 d) and FD-FMT (39.45\u0026thinsp;\u0026plusmn;\u0026thinsp;7.74 d) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). The Procrustes test\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, which visualizes the superimposition of sample coordinates for ordination analysis used to compare the microbial taxonomic profiles before and after sample preparation in each season, showed that the microbial composition of the frozen and FD microorganisms had significantly correlated with that of the intact feces (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.6, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.03 for frozen microorganisms, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.02 for FD microorganisms) (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). No significant differences were observed in the alpha diversity parameters, including Shannon entropy, Pielou_eveness, observed OTUs, faith_PD (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF), and/or in the beta diversity based on weighted Uifrac distance and pairwise Permutational multivariate analysis of variance (PERMANOVA) before and after sample preparation or in both trials conducted between 2020\u0026ndash;2021 and 2021\u0026ndash;2022 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG and Supplementary Table\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eUsing BugBase\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, a microbiome analysis tool that predicts the high-level phenotypes present in the microbiome samples, the proportion of each microbiome sample that includes the facultative anaerobic spp., Gram-negative spp., and gram-positive spp., and biofilm forming, and potentially pathogenic microorganisms was identical before and after sample preparation and between 2020\u0026ndash;2021 and 2021\u0026ndash;2022 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH). However, the number of aerobic microorganisms was slightly reduced after samples preparation regardless of the experimental seasons (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH). The functional prediction of microbial communities in the isolated donor microbiota was carried out with Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) using the 16S rRNA gene sequencing data\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The results of the principal coordinates analysis (PCoA) and pairwise PERMANOVA test conducted on Bray\u0026ndash;Curtis distance of the predicted gene family for the enzyme commission (EC) numbers confirmed that the frozen microorganisms and their respective intact feces did not significantly differ (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.585). The same phenomenon was also observed between the FD microorganisms and their respective intact feces (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.960) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI and Supplementary Table\u0026nbsp;2). These results obtained by a metagenomic analysis indicated that the frozen and FD microorganisms were identical to their respective intact feces.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eEfficacy of F-FMT and FD-FMT in treatment of diarrhea in the recipient\u0026rsquo;s calves\u003c/h2\u003e\n\u003cp\u003eFMT studies were conducted with either the frozen or FD microorganisms in 2020\u0026ndash;2021 and 2021\u0026ndash;2022, respectively. There was no significant difference observed in ages of the recipient groups selected for two F-FMT and FD-FMT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, in context of donors, there was a significant difference observed between the donor and recipients in both F-FMT (50.9\u0026thinsp;\u0026plusmn;\u0026thinsp;30.06 vs 20.9\u0026thinsp;\u0026plusmn;\u0026thinsp;19.90; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and FD-FMT (40.78\u0026thinsp;\u0026plusmn;\u0026thinsp;15.9 vs 16.11\u0026thinsp;\u0026plusmn;\u0026thinsp;12.04, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). In both trials of F-FMT and FD-FMT, they showed a complete reduction in the diarrheal scores (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC) and in the fecal water content of the 19 recipients (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). The classical pathogen tests used for the detection of \u003cem\u003eRotavirus\u003c/em\u003e, coronavirus, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eC. parvum\u003c/em\u003e, and \u003cem\u003eC. perfringens\u003c/em\u003e in the recipient feces, revealed that \u003cem\u003eC. parvum\u003c/em\u003e (11/19) and \u003cem\u003eC. perfringens\u003c/em\u003e (8/19) pathogens were frequently detected regardless of the experimental seasons (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). However, \u003cem\u003eRotavirus\u003c/em\u003e (4/19) and coccidia (0/19) were rarely detected. Consistent with the previous study\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eC. perfringens\u003c/em\u003e was detected in feces of several cases (6/19) even after recovery from diarrhea, whereas \u003cem\u003eC. parvum\u003c/em\u003e was barely detected in most cases (3/19), regardless of the used strategy of bacterial preparation from the donor feces (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). In agreement with the previous study\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, the blood tests used to measure the biochemical indicators demonstrated an increase in the total cholesterol level with F-FMT in 2020\u0026ndash;2021 and FD-FMT in 2021\u0026ndash;2022. The gamma-glutamyl transferase level decreased in both FMT treatments, whereas the other indicators [\u003cem\u003ei.e\u003c/em\u003e., total protein, albumin, white blood cells, red blood cells, hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration] remained unchanged (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). These results indicated that FMT was clinically effective in treating diarrhea in the recipient calves, regardless of how the donor feces were prepared (i.e., frozen or FD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eEfficacy of F-FMT and FD-FMT in altering microbiome in the recipients\u003c/h2\u003e\n\u003cp\u003eF-FMT and FD-FMT showed similar clinical efficacy in treating diarrhea in the recipient calves; however, the differences between them were further evaluated to determine whether F-FMT or FD-FMT was superior in transforming the microbial community to the healthy conditions. Metagenomic analysis at the phylum level showed that the level of Bacteroidetes increased within 7 days after both F-FMT and FD-FMT, whereas the level of Proteobacteria decreased significantly (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;2). At the family level, the levels of \u003cem\u003eParaprevotellaceae\u003c/em\u003e and \u003cem\u003ePrevotellaceae\u003c/em\u003e belonging to the phylum Bacteroidetes increased significantly within 7 days after both F-FMT and FD-FMT, whereas that of \u003cem\u003eEnterobacteriaceae\u003c/em\u003e belonging to the phylum Proteobacteria decreased significantly (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and Supplementary Fig.\u0026nbsp;3). When considering the major genera, the levels of \u003cem\u003eFecalibacterium\u003c/em\u003e and \u003cem\u003ePrevotella\u003c/em\u003e increased significantly within 7 days after both F-FMT and FD-FMT, whereas those of \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eCamphylobacter\u003c/em\u003e, and \u003cem\u003eVeinollea\u003c/em\u003e decreased significantly (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;4). These results confirmed that the relative abundance of the major microbial taxa displayed similar patterns of change in both FMT treatments, regardless of the differences in samples preparation.\u003c/p\u003e\n\u003cp\u003eLinear discriminant analysis (LDA) of effect size (LEfSe) (LDA score\u0026thinsp;\u0026gt;\u0026thinsp;2) was conducted to examine the overall bacterial features of those differentially represented before and after F-FMT and FD-FMT. Notable changes in microbial taxa were observed in FD-FMT compared with F-FMT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). In F-FMT recipients, the phylum Proteobacteria and gram-negative genus \u003cem\u003ePrevotella\u003c/em\u003e were enriched before FMT and after FMT (days 0 and 7) (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). By contrast, FD-FMT significantly changed the microbiota. Specifically, 16 microbial taxa were detected at different concentrations before FMT (day 0), which may represent the cause of pathogenesis; at the highest score obtained from Proteobacteria, a major phylum of gram-negative bacteria (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). After FMT (day 7), a consortium of 19 microbial taxa, including gram-positive and -negative bacteria, such as those belonging to the phylum Bacteroidetes, and several genera of \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eBlautia\u003c/em\u003e, and \u003cem\u003eSelenomonas\u003c/em\u003e, were enriched in FD-FMT (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). The alpha diversity of the microbiota in the recipients\u0026rsquo; calves increased close to the healthy conditions when FD-FMT (but not F-FMT) was applied (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH). In addition, the beta diversity of the microbiota showed a lack of difference in baseline in the recipient calves used for F-FMT and FD-FMT before treatment. Interestingly, significant differences were observed when comparing the donors and recipients calves before FMT in both treatments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI and Supplementary Table\u0026nbsp;3). Moreover, no differences were observed between the donors and recipients\u0026rsquo; calves 7 days after FD-FMT but not after F-FMT, confirming the possibility that the recipient calves can acquire the microbial composition of the donor within 7 days \u003cem\u003evia\u003c/em\u003e FD-FMT (Supplementary Table\u0026nbsp;4). Further analysis of the extent of bacterial biogenesis after FMT using SourceTracker\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e showed that the average contribution of the donors to the development of microbiota of the recipient calves on day 1 and 7 was 5% and 11% for F-FMT; respectively, whereas it reached 10% and 40%; respectively, for FD-FMT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Therefore, in the context of the donor\u0026rsquo;s microbiota engraftment, freeze-drying may be a superior method of microbial conditioning for FMT compared to just freezing.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eEfficacy of F-FMT and FD-FMT in altering the intestinal metabolites in the recipients\u003c/h2\u003e\n\u003cp\u003eTo determine the exact effects of FD-FMT and F-FMT in treating diarrhea in the recipient calves, the fecal metabolites of the donors and the recipients before and 7 days after FMT were comprehensively analyzed using the capillary electrophoresis\u0026ndash;Time-of-Flight Mass Spectrometry (CETOFMS). An interactive heatmap was provided to demonstrate all metabolites identified using Metaboanalyst\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Principal component analysis (PCA) revealed significant differences in pretreatment (day 0) and posttreatment (day 7) recipients compared with the donors for F-FMT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB and Supplementary Table\u0026nbsp;5) but only between the donors and pretransplant (day 0) recipients and not between donors and posttransplant (day 7) recipients for FD-FMT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC and Supplementary Table\u0026nbsp;6). Procrustes analysis assessed similarity of metabolites between donors and recipients before (day 0) and after (day 7) FMT. In F-FMT, the Procrustes correlation was low (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.2467) in donor vs. recipient on day 0 compared with donor vs. recipient on day 7 (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.3748) (Supplementary Fig.\u0026nbsp;5A and 5B). However, in FD-FMT, the Procrustes correlation between donors and recipients was higher after FMT on day 7 than day 0 (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.2036 vs. \u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.4334) (Supplementary Fig.\u0026nbsp;5C and 5D). Because overall Procrustes correlation between donors and recipients was higher in FD-FMT than F-FMT (Supplementary Figs.\u0026nbsp;5A\u0026ndash;5D), recipient metabolites were potentially same as donor metabolites in FD-FMT. Notably, that the number of metabolites were reduced but not increased by FMT, which were greater for FD-FMT than F-FMT. Specifically, within 7 days after F-FMT or FD-FMT, 43 or 29 metabolites were upregulated and 35 or 62 were downregulated, respectively (Supplementary Tables\u0026nbsp;7 and 8). To identify the metabolic pathways that differed between the F-FMT and FD-FMT, KEGG enrichment analysis was performed based on total number of metabolites obtained from the fold-change (FC) analysis. KEGG enrichment analysis revealed that 38 metabolic pathways were involved in F-FMT and FD-FMT (Supplementary Fig.\u0026nbsp;6A and 6B and Supplementary Table\u0026nbsp;9).\u003c/p\u003e\n\u003cp\u003eFor F-FMT, the metabolites were mainly involved in purine metabolism, arginine biosynthesis, and pyrimidine metabolism (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Supplementary Table\u0026nbsp;9). For FD-FMT, the metabolites were primarily involved in glycine, serine, and threonine metabolism; aminoacyl-tRNA biosynthesis; histidine metabolism; glycerophospholipid metabolism; arginine and proline metabolism; alanine, aspartate and glutamate metabolism, and glutathione metabolism (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Supplementary Table\u0026nbsp;10). This study confirmed that the major metabolites in FD-FMT were involved in amino acid metabolism pathways. Next, the major metabolites detected in ATP-binding cassette (ABC) transporter, amino acid metabolism, lipid fatty acids, energy, polyamines, methylated compounds, and vitamins, short-chain fatty acids and bile acids were profiled in this study. Meanwhile, the levels of metabolites belonging to the ABC transporter metabolism, such as amino acids, were highly affected by FD-FMT rather than by F-FMT (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF and Supplementary Figs.\u0026nbsp;7\u0026ndash;10). During the diarrheal condition, high levels of several amino acids, such as arginine, proline, and histidine were abundantly present in the feces, which may represent a microbial dysbiosis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Thus, FD-FMT displayed a clear understanding to retain the microbial symbiosis condition by enhancing amino acid utilizing bacteria in the gut. Furthermore, by using variable importance in projection (VIP) scores obtained from the partial least-squares discriminant analysis (PLS-DA), top 15 discriminating metabolites were found, in where in FD-FMT cases amino acid histidine, phenylalanine, cysteine, leucine, valine and isoleucine were found downregulated (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH). Consequently, these results indicated that FD-FMT was more efficient than F-FMT in changing the metabolic environment in feces of the recipient calves within 7 days after treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eEstablishing the microbiota\u0026ndash;metabolite correlation by FD-FMT during disease recovery\u003c/h2\u003e\n\u003cp\u003eTo investigate whether the microbiota and metabolites were closely associated during disease recovery post F-FMT or FD-FMT, Pearson\u0026rsquo;s correlation analysis was conducted. Specifically, the levels of the 16 selected microbiotas categorized as residents or colonizers using Microbiome Multivariable Association with Linear Models (MaAsLin2)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e in the Microbiomeanalyst\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e platform (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA and Supplementary Table\u0026nbsp;11). The 15 key metabolites shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH were used for correlation analysis. Of the 240 (16 \u0026times; 15) combinations, F-FMT showed five positive correlations before FMT (day 0) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB) and three positive and two negative correlations 7 days after FMT (day 7) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). By contrast, of the 224 (16 \u0026times; 14) combinations, FD-FMT displayed 14 positive and six negative correlations on day 0 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD) and 17 positive and two negative correlations on day 7 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). For example, \u003cem\u003eCampylobacteraceae\u003c/em\u003e and \u003cem\u003eEnterobacteriaceae\u003c/em\u003e were positively correlated with choline (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), \u003cem\u003eN\u003c/em\u003e6-methyllysine (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and succinic acid (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003cp\u003eGiven that FD-FMT had a high number of correlations between metabolites and microbial taxa, these results indicate that compared with F-FMT, FD-FMT significantly changed the intestinal environment. To compare the predictive potential of F-FMT and FD-FMT in disease recovery through changes in microbiota and metabolites 7 days after FMT (day 7) vs. before FMT (day 0), a prediction model with area under the curve (AUC) of the receiver operating characteristic (ROC) was used\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, using three feature sets: microbial taxa (n\u0026thinsp;=\u0026thinsp;240), fecal metabolites (n\u0026thinsp;=\u0026thinsp;596), and a combination of microbial taxa and fecal metabolites (n\u0026thinsp;=\u0026thinsp;836). The AUC values of microbiota, metabolites, and microbiota\u0026thinsp;+\u0026thinsp;metabolites were 0.91, 0.78, and 0.76, respectively for F-FMT (Supplementary Fig.\u0026nbsp;11A), and 0.94, 0.81, and 0.81, respectively, for FD-FMT (Supplementary Fig.\u0026nbsp;11B). Thus, FD-FMT exhibited excellent discriminative power and superior performance in altering the microbiota and metabolites 7 days after FMT for diarrhea remission in calves (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). Overall, despite the high functional similarity between F-FMT and FD-FMT, FD-FMT exhibited broader spectral performance and was superior to F-FMT in promoting changes in the microflora and metabolites.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFMT is an excellent strategy for treating calf diarrhea without causing undesirable side effects\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Even for intractable diarrhea, FMT with fresh stool was 70% effective\u003csup\u003e6\u003c/sup\u003e. Therefore, there is a growing and urgent need to establish a method to prepare healthy donor-derived beneficial microorganisms disseminated in many areas without concerns for safety and stability\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In humans, especially those undergoing treatment for recurrent \u003cem\u003eClostridioides difficile\u003c/em\u003e infection, several protocols for preparing donor fecal material have been developed, such as frozen or FD fecal extracts, with \u0026ge;\u0026thinsp;90%\u003csup\u003e21\u0026ndash;23\u003c/sup\u003e efficacy rate. Moreover, different FMT protocols for screening and processing donor feces have been established in clinical practice\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28 CR29 CR30 CR31 CR32 CR33\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (Supplementary Table\u0026nbsp;12). However, only few studies have been conducted to establish a protocol for preparing donor feces in livestock production\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e (Supplementary Table\u0026nbsp;12). Therefore, this study focused on establishing a method for extracting and preserving fecal microbiota of calves for transplantation and analyzing its usefulness in effectively treating diarrhea. Specifically, Nycodenz\u003cb\u003e\u0026reg;\u003c/b\u003e\u0026mdash;a nontoxic and nonionic water-soluble compound that can form self-density gradients\u0026mdash;was used to isolate microorganisms from the feces of healthy calves on a large scale\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Notably, Nycodenz\u003cb\u003e\u0026reg;\u003c/b\u003e can isolate 10\u003csup\u003e10\u003c/sup\u003e viable bacteria per 2 g fresh feces in a preserved ecosystem, and this isolation procedure does not alter the composition of the original microflora concerning survival, distribution, and proportion\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Because the absence of common diarrhea-causing pathogens should be confirmed first, the protocol established in this study will be promising in eliminating the veterinarians\u0026rsquo; concerns about donor selection and preparation and preservation of the microorganisms necessary for successful FMT.\u003c/p\u003e \u003cp\u003eDeveloping donor-derived fecal microbiota products, rather than conditioning fresh feces each time on the farm, is an ideal strategy for preserving beneficial microorganisms and widely disseminating them to the farms for treating calves with diarrhea. Therefore, there are several advantages to optimizing the protocols for preserving microorganisms obtained from donor feces and using them later for transplantation. Increasing the stability of fecal microbiota will maintain the complex microbial network necessary to establish a microbial environment for recipient calves through transplantation and contribute to maximizing the efficacy of FMT to cure diarrhea. Among several possible procedures used for bacterial preservation that led to successful FMT, freeze-drying is strongly recommended throughout this study because it removes water from the microorganisms, temporarily stopping their metabolic activity and moving them into a dormant state. Microbial DNA is protected from hydrolytic damage and enzymatic degradation under FD conditions. Thus, the metabolic activity of FD microorganisms is quickly and appropriately resumed by rehydration\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. By contrast, the freezing method impairs the intracellular metabolic activity of microbes and disrupts the mutualistic feeding mechanisms and ecological interactions in frozen samples, resulting in a significant reduction in microbial functionality compared with the original state. To improve the storage stability of frozen microorganisms, the use of cryoprotectants, such as glycerol and dimethyl sulfoxide, should be considered when preparing samples from donor feces\u003csup\u003e\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Nevertheless, glycerol is not recommended for lyophilization due to its high viscosity, resulting in a poorly dried and sticky product, which is unsuitable for rehydration and subsequent transplantation. Therefore, although cryoprotectants were not used in this study, future efforts should be made to select and optimize cryoprotectants that maintain/increase microbial survival in the dormant state for successful FMT using FD (not frozen) microorganisms.\u003c/p\u003e \u003cp\u003eHerein, calves with diarrhea had more bacteria belonging to the phylum Proteobacteria, genus \u003cem\u003eEnterobacteria\u003c/em\u003e and \u003cem\u003eGalibacterium\u003c/em\u003e and were less likely to possess butyrate-producing bacteria, such as \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eFecalibacterium\u003c/em\u003e, and \u003cem\u003eBlautia\u003c/em\u003e. In addition, F-FMT and FD-FMT reduced Proteobacteria and improved diarrheal status, regardless of the nature of the microorganisms transplanted. Nonetheless, FD-FMT increased Gram-positive and -negative bacteria efficiently and in a balanced manner, thus creating a better microbial environment that led to diarrhea recovery in calves. Notably, FD-FMT facilitated enrichment of the genus \u003cem\u003eBlautia\u003c/em\u003e and \u003cem\u003eSelenomonas\u003c/em\u003e, which abundantly produce short-chain fatty acids and utilized amino acids for their metabolism. One possible consideration for why FD-FMT was far superior to F-FMT in improving the microflora can be attributed to the high frequency of Gram-negative bacteria preserved during sample preparation. Specifically, prediction strategy using BugBase showed that the freeze-drying procedure reduced the Gram-negative bacteria by 3.82% after sample preparation, whereas the frozen strategy reduced them by 29.81%. In addition, a highly significant correlation existed between indigenous and newly-colonized microbiota, and the metabolites produced by them, which were induced by FD-FMT rather than F-FMT, may have contributed to the development of a balanced microbial environment, indicating the possibility of competition and mutualistic feeding interactions among microorganisms, and that competition for resources may have a positive impact on the relative abundance of bacterial families created by FD-FMT in recipient calves.\u003c/p\u003e \u003cp\u003eThe establishment of fecal banks to store donor stool samples has advanced FMT technology, by ensuring timely and reliable availability\u0026mdash;while maintaining safety and quality standards\u0026mdash;and providing recipients with multiple options for selecting an appropriate donor. Several approved medical products consisting of donor stool are commercially available, particularly for treating recurrent \u003cem\u003eC. difficile\u003c/em\u003e infection in humans, notably the BiomeBank licensed in Australia and REBYOTA licensed in the United States. These products contain frozen microorganisms and are transported under cold-chain control to the clinic for rectal administration to recipients in need\u003csup\u003e\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Unlike fecal-derived microbial products for human use, there is an urgent need to develop new technologies for the stable preservation of fecal-derived microorganisms in livestock production, even where adequate cryopreservation facilities are not available. FD microbial preparations are the most appropriate materials stored stably in the field, and this convenience of storage can greatly expand the number of farms where FMT can be applied. Thus, unlike FMT for humans using frozen microorganisms, FMT for treating calf diarrhea can be widely implemented from a single donor with multiple recipients raised on multiple farms. In addition, the results of this study reveal that FD-FMT shows clear evidence of calf recovery from diarrhea, suggesting its potential use as a next-generation therapeutic agent. Future studies focusing on the impact of FD-FMT on the microbial community, removal of potential virulence factors and antibiotic resistance genes, and evaluation of the by-products obtained will provide the foundation necessary for the practical application of FD-FMT for treating calf diarrhea.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eDevelopment of the study\u003c/h2\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the respective institutions to determine the efficacy of prepared fecal microorganisms used for treating diarrhea in calves. This study was conducted using two experiments in the winter seasons from 2020 to 2021 (F-FMT) and from 2021 to 2022 (FD-FMT). In total 25 and 16 fecal samples were collected from healthy donor calves from four and three farms in 2020\u0026ndash;2021 and 2021\u0026ndash;2022, respectively. The fresh fecal samples were collected from the potential donors in the field and then stored immediately at \u0026minus;\u0026thinsp;80\u0026deg;C until the microorganisms become ready for transplantation. Microorganisms isolated from the fecal samples were frozen at \u0026minus;\u0026thinsp;80\u0026deg;C in the first year (2020\u0026ndash;2021) or FD and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until being used in the second year (2021\u0026ndash;2022). A total of 19 FMTs were performed using either frozen microorganisms in 2020\u0026ndash;2021 (n\u0026thinsp;=\u0026thinsp;10) or FD microorganisms in 2021\u0026ndash;2022 (n\u0026thinsp;=\u0026thinsp;9). All FMT trials were conducted based on the individual donor-recipient pair, which was selected from the same farm to avoid the transmission of virulence factors.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eExperiment design for potential donor selection\u003c/h2\u003e\n\u003cp\u003eOptimal donors were selected from healthy calves to ensure the efficacy of FMT and avoid the risk of transmission of virulence factors during FMT. Therefore, before FMT, screening fecal and blood samples of potential donors for known pathogens (e.g., described below) is highly recommended to confirm that their fecal matter is safe to use for FMT. In this study, Rainbow Calf Scours\u0026reg; (Bio-X) was used to confirm the absence of \u003cem\u003eRotavirus\u003c/em\u003e, coronavirus, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eC. parvum\u003c/em\u003e, and \u003cem\u003eC. perfringens\u003c/em\u003e from feces of potential donors. The modified Wisconsin sugar centrifugal fraction method was conducted using a saturated sucrose solution to confirm the absence of protozoa and nematodes from feces of the potential donors. In addition, either nested-polymerase chain reaction (nested-PCR) or real time-PCR (RT-PCR) were conducted using whole blood-derived viral DNA or plasma-derived viral RNA at NDTS Co., Ltd., to confirm the absence of BLV and BVDV, respectively, from the blood of the optimal donors. The fecal materials were collected under aseptic conditions using sterile containers, gloves, and disposable sterile spoons from readily available commercial collection kits. Appropriate instructions were provided to the veterinarians to avoid contamination of fecal and blood samples with environmental residues or soil. Samples were delivered immediately to the microbiology laboratory to avoid substantial changes in the metabolic profiles and abundance of bacterial taxa\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eIsolation of microorganisms for FMT using Nycodenz\u0026reg;\u003c/h2\u003e\n\u003cp\u003eFecal samples (15\u0026ndash;25 g) collected from potential donors were suspended in six volumes of phosphate-buffered saline (PBS) (1\u0026times;) to ensure adequate bacterial density and separated using 80% (w/v) Nycodenz\u003cstrong\u003e\u0026reg;\u003c/strong\u003e solution\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Initially, to prepare the fecal suspensions, the fecal samples were blended for 2 min using a standard commercial hand blender. The resulting slurries were passed through a nylon mesh (0.5 mm) to remove large debris, and 21 mL of the collected slurry was transferred to overlay 7 mL Nycodenz\u003cstrong\u003e\u0026reg;\u003c/strong\u003e density gradient solution in a 50 mL glass tube. The overlayed fecal solution was centrifuged at 10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 40 min at 4\u0026deg;C to enrich the bacterial fraction interface\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The upper layer of the fecal solution containing water-soluble debris was aspirated without disturbing the bacterial interface. Approximately 35\u0026ndash;40 mL of the bacterial interface (e.g., fecal-derived microorganisms)-containing solution were collected from each donor sample. For F-FMT or FD-FMT, the fecal-derived microorganisms were either frozen only or freeze-dried.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eSelection of potential FMT recipients\u003c/h2\u003e\n\u003cp\u003eRecipient calves with diarrhea were selected by the veterinarians based on diarrheal consistency. Considering diarrheal score, fecal consistency was scored based on a scale of 1\u0026ndash;4, where 1\u0026thinsp;=\u0026thinsp;normal consistency, 2\u0026thinsp;=\u0026thinsp;semi-formed or pasty, 3\u0026thinsp;=\u0026thinsp;loose feces, and 4\u0026thinsp;=\u0026thinsp;watery feces\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. During selection and to ensure successful implantation, antibiotics were not used to treat recipient calves 1 week before FMT. To evaluate the efficacy of FMT, the presence of common pathogens (\u003cem\u003ei.e\u003c/em\u003e., Rotavirus, coronavirus, BLV, BVDV, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eC. parvum\u003c/em\u003e, \u003cem\u003eC. perfringens\u003c/em\u003e, protozoa, and nematodes) in fecal and blood samples were initially investigated using commercialized pathogen kits before FMT (day 0) and after FMT at day 7.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eFMT procedure\u003c/h2\u003e\n\u003cp\u003eDonor samples, either frozen (F-FMT) or freeze-dried (FD-FMT) microorganisms, were delivered to the field on dry ice with a locally available transport system. F-FMT was thawed for 2 h in warm water at 37\u0026deg;C, whereas FD-FMT was suspended in 100 mL of sterile saline. To perform FMT with the microbiota adjusted to 100 mL, a catheter (60-cm long, 1-cm diameter) was inserted approximately 30 cm into the rectum of the recipient calf with diarrhea, approximately up to the second lumbar vertebra (Supplementary Fig.\u0026nbsp;11).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eFecal bacterial DNA extraction, 16S rRNA sequencing, and taxonomic profiling\u003c/h2\u003e\n\u003cp\u003eTotal bacterial DNA was extracted from the fecal samples and isolated from the bacterial interface using Norgen Stool DNA Isolation Kit, according to the manufacturer\u0026rsquo;s instructions. The quality and quantity of DNA were further assessed. The V3\u0026ndash;V4 region of the bacterial 16S rRNA gene was amplified using the following primers:\u003c/p\u003e\n\u003cp\u003eForward primers mixed (5\u0026prime;-TGCTCTTCCGATCTGACNNNCCTACGGGNGGCWGCAG-3\u0026prime;, 5\u0026prime;-TGCTCTTCCGATCTGACNNNNCCTACGGGNGGCWGCAG-3\u0026prime;, 5\u0026prime;-TGCTCTTCCGATCTGACNNNNNCCTACGGGNGGCWGCAG-3\u0026prime;, and 5\u0026prime;-TGCTCTTCCGATCTGACNNNNNNCCTACGGGNGGCWGCAG-3\u0026prime;), and reverse primers mixed (5\u0026prime;-CGCTCTTCCGATCTCTGNNNGACTACHVGGGTATCTAATCC-3\u0026prime;, 5\u0026prime;-CGCTCTTCCGATCTCTGNNNNGACTACHVGGGTATCTAATCC-3\u0026prime;, 5\u0026prime;-CGCTCTTCCGATCTCTGNNNNNGACTACHVGGGTATCTAATCC-3\u0026prime;, and 5\u0026prime;-CGCTCTTCCGATCTCTGNNNNNNGACTACHVGGGTATCTAATCC-3\u0026prime;).\u003c/p\u003e\n\u003cp\u003eThe PCR fragments obtained from the first round of PCR were further amplified in the second round of PCR using the following primers:\u003c/p\u003e\n\u003cp\u003eForward primer (5\u0026prime;-CAAGCAGAAGACGGCATACGAGATxxxxxxxxxGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC-3\u0026prime;), and reverse primer (5\u0026prime;-AATGATACGGCGACCACCGAGATCTACACxxxxxACACTCTTTCCCTACACGACGCTCTTCCGATCTCTG-3\u0026prime;). All PCR products were sequenced using the MiSeq platform (Illumina) with MiSeq Reagent Kit v2 (500 cycles)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. After next-generation sequencing, the demultiplexed raw sequences were acquired from BaseSpace Sequence Hub (Illumina). The sequences were analyzed using QIIME 2 (version 2021.2)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, and metagenomics and functional prediction analyses were conducted based on the published methodologies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eAnalyses of FMT-induced alterations in the recipient and metabolite profiles\u003c/h2\u003e\n\u003cp\u003eBased on the results obtained from 16S rRNA amplicon sequencing and using the freely available program SourceTracker, the efficacy of engraftment of donor-derived microorganisms in calf recipients with diarrhea post-FMT was investigated\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, where a Bayesian algorithm was used to determine the percentage of microbial communities in the recipient calf that belonged to the donor microbiota. SourceTracker indicated coexistence rate of donor-derived microbiota.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eCE\u0026ndash;TOFMS metabolomics analysis\u003c/h2\u003e\n\u003cp\u003eUsing the fecal samples collected from the donors and recipients\u0026rsquo; calves, a metabolomics analysis was conducted at the Human Metabolome Technologies, Inc. (Japan). The samples were analyzed using capillary electrophoresis coupled with time-of-flight mass spectrometry (CE\u0026ndash;TOFMS, Agilent). The metabolite standards, instrumentation, and CE\u0026ndash;TOFMS conditions were as described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The metabolites of each reconstituted sample were separated in a fused silica capillary (i.d. 50 \u0026micro;m \u0026times; 80 cm) (Agilent). The data acquisition had been carried out using an electrospray ionization cation and an anion full scan modes. In the positive mode, the capillary voltage was 30 kV, MS capillary voltage was 4,000 V, and the sample solution was injected for 10 s at 50 mbar. Meanwhile, in the negative mode, the capillary voltage was 30 kV, MS capillary voltage was 3,500 V, and the interjection time was 50 mbar for 22 s.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eAll error bars represent standard deviation. Statistical significance was assigned at \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and detected by GraphPad Prism 9.5.1 (GraphPad Software, San Diego, USA). The microbe\u0026ndash;metabolite correlation heatmap was created by calculating Spearman\u0026rsquo;s correlation coefficients for each pairwise combination of microbial taxa abundance and metabolite intensity using the \u0026ldquo;corr.test\u0026rdquo; function in R package.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed in this study are included in this article (and its supplementary information files).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Drs. Hidetoshi Kato, Takafumi Goto, Jun Kunisawa, and Hiromichi Ohtsuka for their practical advice related to this study. This study was primarily supported by three agencies; mainly a Livestock Promotional Subsidy from the Japan Racing Association (to T.N.), and by Grants-in-Aid for Scientific Research (A) 18H03969 and 22H00393 (to T.N.), and by Grants-in-Aid for Early-Career Scientists 20K15478 and 23K14062 (to J.I.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.I. contributed by designing the study, performed experiments, analyzed data, and wrote the manuscript. N.O., Y.S., M.T., Y.G., M.S., E.M., T.S., C.U., and H.T. contributed by performing FMT. A.M. and Y.Su. contributed by sequencing 16S rRNA genes. Y.Sa. contributed by making the analysis. H.Y. and N.A.K. contributed by providing comments on the manuscript. R.H. and M.F. contributed by supporting the experiments. T.N. contributed by designing the study and writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWei, X. \u003cem\u003eet al.\u003c/em\u003e Detection of infectious agents causing neonatal calf diarrhea on two large dairy farms in Yangxin Country, Shandong Province, China. \u003cem\u003eFront Vet Sci\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e,589126. (2021).\u003c/li\u003e\n\u003cli\u003eCho, Y. il \u0026amp; Yoon, K. J. An overview of calf diarrhea - infectious etiology, diagnosis, and intervention. \u003cem\u003eJ Vet Sci\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1\u0026ndash;17 (2014).\u003c/li\u003e\n\u003cli\u003eConstable, P. D. 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A. \u003cem\u003eet al.\u003c/em\u003e Methods for improving human gut microbiome data by reducing variability through sample processing and storage of stool. \u003cem\u003ePLOS One\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e0139529 (2015).\u003c/li\u003e\n\u003cli\u003eUsami, K. \u003cem\u003eet al.\u003c/em\u003e The gut microbiota induces Peyer\u0026rsquo;s-patch-dependent secretion of maternal IgA into milk. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 109655 (2021).\u003c/li\u003e\n\u003cli\u003eBolyen, E. \u003cem\u003eet al.\u003c/em\u003e Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. \u003cem\u003eNature Biotechnology\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 852\u0026ndash;857 (2019). https://doi.org/10.1038/s41587-019-0209-9\u003c/li\u003e\n\u003cli\u003eSoga, T. \u003cem\u003eet al.\u003c/em\u003e Simultaneous determination of anionic intermediates for \u003cem\u003eBacillus subtilis\u003c/em\u003e metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry. \u003cem\u003eAnal Chem\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 2233\u0026ndash;2239 (2002).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4168305/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4168305/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFecal microbiota transplantation (FMT) is a therapeutic modality for treating neonatal calf diarrhea. Several practical barriers, including donor selection, fecal collection, and a limited timeframe for FMT, are the main constraints to using fresh feces for implementing on-farm FMT. We report the utility of FMT with pretreated ready-to-use frozen (F) or freeze-dried (FD) microorganisms for treating calf diarrhea. In total, 19 FMT (F-FMT, n\u0026thinsp;=\u0026thinsp;10 and FD-FMT, n\u0026thinsp;=\u0026thinsp;9) treatments were conducted. Both FMT treatments were 100% clinically effective; however, multi-omics analysis showed that FD-FMT was superior to F-FMT. Machine learning analysis with SourceTracker confirmed that donor microbiota was retained four times better in the recipient calves treated with FD-FMT than F-FMT. A predictive model based on receiver operating characteristic curve analysis and area under the curve showed that FD-FMT was more discriminative than F-FMT of the observed changes in microbiota and metabolites during disease recovery. These results provide new insights into establishing methods for preparing fecal microorganisms to increase the quality of FMT in animals and may contribute to FMT in humans.\u003c/p\u003e","manuscriptTitle":"Freeze-dried fecal microorganisms as an effective biomaterial for the treatment of calves suffering from diarrhea","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-10 16:22:41","doi":"10.21203/rs.3.rs-4168305/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f68689d7-249c-4b2b-9515-dd24271f02e2","owner":[],"postedDate":"April 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30447505,"name":"Biological sciences/Microbiology/Microbial communities/Microbiome"},{"id":30447506,"name":"Biological sciences/Microbiology/Microbial communities/Metagenomics"}],"tags":[],"updatedAt":"2024-06-17T12:51:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-10 16:22:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4168305","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4168305","identity":"rs-4168305","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}