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

doi:10.21203/rs.3.rs-4168305/v1

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

https://doi.org/10.21203/rs.3.rs-4168305/v1

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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

Neonatal calf diarrhea is a serious health problem facing the livestock industry, which causes significant economic losses due to the increased morbidity and mortality rates¹. 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 concern². Antimicrobials are often prescribed for the treatment of calves with diarrhea³. 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 problem⁴. 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 recipient⁵. 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 system⁸. 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 metabolites⁹. 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.

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. 2A). 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. 2B). 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. 2C). The Procrustes test¹⁰, 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 (R² = 0.6, p < 0.03 for frozen microorganisms, R² = 0.5, p < 0.02 for FD microorganisms) (Figs. 2D and 2E). No significant differences were observed in the alpha diversity parameters, including Shannon entropy, Pielou_eveness, observed OTUs, faith_PD (Fig. 2F), 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. 2G and Supplementary Table 1).

Using BugBase¹¹, 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. 2H). However, the number of aerobic microorganisms was slightly reduced after samples preparation regardless of the experimental seasons (Fig. 2H). 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 data¹². 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. 2I 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. 3A). 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. 3B). In both trials of F-FMT and FD-FMT, they showed a complete reduction in the diarrheal scores (Fig. 3C) and in the fecal water content of the 19 recipients (Fig. 3D). 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. 3E). However, Rotavirus (4/19) and coccidia (0/19) were rarely detected. Consistent with the previous study⁶, 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. 3E). In agreement with the previous study⁶, 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. 3F). 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. 4A 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. 4B 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. 4C 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. 4D–4G). In F-FMT recipients, the phylum Proteobacteria and gram-negative genus Prevotella were enriched before FMT and after FMT (days 0 and 7) (Figs. 4D and 4E). 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. 4F and 4G). 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. 4E and 4G). 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. 4H). 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. 4I 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 SourceTracker¹³ 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. 4J). 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 Metaboanalyst¹⁴ (Fig. 5A). Principal component analysis (PCA) revealed significant differences in pretreatment (day 0) and posttreatment (day 7) recipients compared with the donors for F-FMT (Fig. 5B 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. 5C 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 (R² = 0.2467) in donor vs. recipient on day 0 compared with donor vs. recipient on day 7 (R² = 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 (R² = 0.2036 vs. R² = 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. 5D–5F 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 dysbiosis⁶. 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. 5G and 5H). 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 Microbiomeanalyst¹⁶ platform (Fig. 6A and Supplementary Table 11). The 15 key metabolites shown in Figs. 5G and 5H were used for correlation analysis. Of the 240 (16 × 15) combinations, F-FMT showed five positive correlations before FMT (day 0) (Fig. 6B) and three positive and two negative correlations 7 days after FMT (day 7) (Fig. 6C). By contrast, of the 224 (16 × 14) combinations, FD-FMT displayed 14 positive and six negative correlations on day 0 (Fig. 6D) and 17 positive and two negative correlations on day 7 (Fig. 6E). For example, Campylobacteraceae and Enterobacteriaceae were positively correlated with choline (P < 0.001 and P < 0.01), N6-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 used¹⁷, 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. 6F). 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.

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% effective⁶. 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 scale⁸. Notably, Nycodenz® can isolate 10¹⁰ 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 proportion⁸. 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.

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 taxa⁴⁷.

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® solution⁸. 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 interface⁸. 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 feces⁶. 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 methodologies⁶.

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 investigated¹³, 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 described⁵⁰. 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.

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.

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

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Abstract

Figures

Introduction

Results

Difference in microbiota between the frozen and freeze-dried microorganisms

Efficacy of F-FMT and FD-FMT in treatment of diarrhea in the recipient’s calves

Efficacy of F-FMT and FD-FMT in altering microbiome in the recipients

Efficacy of F-FMT and FD-FMT in altering the intestinal metabolites in the recipients

Establishing the microbiota–metabolite correlation by FD-FMT during disease recovery

Discussion

Methods

Development of the study

Experiment design for potential donor selection

Isolation of microorganisms for FMT using Nycodenz®

Selection of potential FMT recipients

FMT procedure

Fecal bacterial DNA extraction, 16S rRNA sequencing, and taxonomic profiling

Analyses of FMT-induced alterations in the recipient and metabolite profiles

CE–TOFMS metabolomics analysis

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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