ARTICLE https://doi.org/10.1038/s41467-020-20389-5 OPEN Longitudinal evaluation of fecal microbiota transplantation for ameliorating calf diarrhea and improving growth performance Hyun Sik Kim1,5, Tae Woong Whon1,3,5, Hojun Sung1, Yun-Seok Jeong1, Eun Sung Jung 2, Na-Ri Shin1,4, ✉ Dong-Wook Hyun1, Pil Soo Kim1, June-Young Lee1, Choong Hwan Lee2 & Jin-Woo Bae 1 1234567890():,; Calf diarrhea is associated with enteric infections, and also provokes the overuse of anti- biotics. Therefore, proper treatment of diarrhea represents a therapeutic challenge in live- stock production and public health concerns. Here, we describe the ability of a fecal microbiota transplantation (FMT), to ameliorate diarrhea and restore gut microbial compo- sition in 57 growing calves. We conduct multi-omics analysis of 450 longitudinally collected fecal samples and find that FMT-induced alterations in the gut microbiota (an increase in the family Porphyromonadaceae) and metabolomic profile (a reduction in fecal amino acid con- centration) strongly correlate with the remission of diarrhea. During the continuous follow-up study over 24 months, we find that FMT improves the growth performance of the cattle. This first FMT trial in ruminants suggest that FMT is capable of ameliorating diarrhea in pre- weaning calves with alterations in their gut microbiota, and that FMT may have a potential role in the improvement of growth performance. 1 Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University, Seoul 02447, Republic of Korea. 2 Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea. 3Present address: Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea. 4Present address: Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup-Si, Jeollabuk-Do 56212, Republic of Korea. 5These authors contributed equally: Hyun Sik Kim, Tae Woong Whon. ✉ email: [email protected] NATURE COMMUNICATIONS | (2021) 12:161 | https://doi.org/10.1038/s41467-020-20389-5 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-20389-5 he beef industry has made great improvements in animal samples. Finally, to determine whether the mature gut microbiota Tfacilities, feeding, welfare, breeding, herd management, and instilled by FMT affects the development or growth of calves, the use of biopharmaceuticals. However, the prevention their gut microbiota, serum metabolic markers, and body mass and timely treatment of calf diarrhea still represent a challenge1. gain were followed during the fattening period. According to the results of the U.S. National Animal Health Monitoring System study, published in 2018 (Calf Note 203), 39% of calf mortality is caused by diarrhea during the first 3 weeks of Results life. The causes of calf diarrhea that have been identified are FMT ameliorates diarrhea in preweaning calves. FMT has been infectious agents, including viruses (for example, the rotavirus applied to monogastric animals, including humans21,25 and group, bovine viral diarrhea virus, and bovine coronavirus), swine22,23. However, to our knowledge, no study to date has bacteria (for example, Salmonella spp. and Escherichia coli), and assessed the use of FMT in ruminants. Prior to assessing the protozoa (for example, Eimeria zuernii), and non-infectious fac- efficacy of FMT for the treatment of calf diarrhea, we performed a tors, such as stress, diet, and humidity2,3. Given that most of the preliminary trial to verify the safety of oral FMT (Supplementary financial loss in beef production is attributable to calf morbidity Table 1). We collected a fecal sample from a healthy donor and and mortality, a better understanding of the etiology and pre- orally administered six randomly selected recipient calves, vention of diarrhea in preweaning calves is essential to maximize regardless of the presence of diarrhea, with 0.5 mg/ml fecal beef production and profitability. concentration twice a day (Supplementary Fig. 1a, b; see Antibiotics have been widely used to treat or prevent diarrhea “Methods”). We then closely observed these calves for 16 days and promote growth in livestock4–6. Recently, however, accu- and found no signs of abnormal behavior or acute illness. Next, mulating evidence has shown that the use of antibiotics in animal we performed 16S rRNA gene amplicon sequencing (hereafter husbandry is associated with many adverse effects. The emer- referred to as a metataxonomic analysis) of fecal samples col- gence of antibiotic-resistant bacteria and antibiotic residues in lected per rectum 0, 1, 2, 4, 8, and 16 days post-FMT (Supple- meat are recognized as major problems7–9. Due to their potential mentary Fig. 1c) to determine the temporal changes in the calf gut to kill both pathogenic and beneficial microbes, the use of wide- microbiota in response to the FMT treatment. Principal coordi- spectrum antibiotics can promote the colonization of the gut by nate analysis (PCoA) of the weighted UniFrac distance matrix pathogenic microbes, which can cause disease10–12. Thus, anti- revealed that the intestinal microbial changes in the recipient’s biotic therapy of diarrheic calves often leads to the recurrence of calves were influenced by the microbial composition of the serious diarrhea within days of starting it. The presence of an administered feces more than by the feed pellets or maternal milk imbalance in the intestinal microbial community, followed by the (Supplementary Fig. 1d, f). Concordant with the shift in the administration of antibiotics, is capable of activating immune structure of the microbial population in the recipient’s calves, the responses, inflammation, and peristalsis in the host gut, which number of amplicon sequence variants (ASVs) shared with the causes diarrhea13,14. Importantly, a combination of recurrent donor’s calf was highest 16 days after the start of treatment diarrhea and antibiotic abuse in preweaning calves may result in (Supplementary Fig. 1e). We estimated if microbiota in the gut of the immaturity of the ruminal and intestinal microbiota, which the recipient’s calves originated from the gut of the donor’s calf, has permanent negative effects on the digestion and absorption of or if they were derived from some other external source at during dietary components during the fattening period15–17. Given that the trial. To demonstrate this capability, we used SourceTracker antibiotics also have detrimental impacts on ecology and food analysis to quantify the proportions of the different microbial safety, their use should be reduced in farming, and alternatives to samples (sources) in a target microbial community (sink). Sour- antibiotic treatment identified. ceTracker analysis showed that the mean contributions of the Several lines of evidence suggest that reconstitution of a healthy donor feces to the gut bacterial community of the recipient’s microbial community is an effective method of preventing or calves increased during the 16 days after the initiation of treat- treating gastrointestinal disorders18,19. In particular, fecal ment in all the calves and that there was no contribution from microbiota transplantation (FMT, instillation of the feces from a external sources other than the donor feces (Supplementary healthy donor into the gastrointestinal tract of a recipient patient) Fig. 2a, b). These results enabled us to expect that oral FMT is is a highly effective therapy for Clostridioides difficile infection capable of affecting the structure of the intestinal microbiota in (CDI), irritable bowel syndrome, and inflammatory bowel disease young ruminants without having negative effects on calf mor- (IBD)20,21. Microbial controls for the prevention or treatment of bidity or mortality. gastrointestinal diseases of economic animals as well as human To further investigate the effects of FMT on the gut microbiota patients have been applied. Recent studies have indicated that the of diarrheic calves, we enlarged both the donor and recipient microbiota-derived bacteriocin (i.e., gassericin A) targets the host cohorts of calves. Healthy calves (n = 6) that would provide the intestinal epithelium and confers resistance to diarrhea in early fecal samples were rigorously selected according to the inclusion weaned piglets, and FMT has the potential to prevent necrotizing and exclusion criteria shown in Table 1. We orally treated the enterocolitis in preterm piglets22,23. In addition, the transmission diarrheic recipient calves with 5 g feces as a bolus (0.1 g/ml feces) of rumen fluid or cud from healthy donor cattle to sick recipient five times in total (FMT, n = 20; Fig. 1a). This total quantity of cattle was practiced prior to any understanding of the rumen feces was 12.5 times that used in the preliminary trial. Age- microflora. Indeed, rumen transformation (the introduction of a matched diarrheic calves treated with saline (CON, n = 14) or wide range of rumen microorganisms, including bacteria, proto- antibiotics (ABX, n = 23) were included as negative and zoa, fungi, and archaea) has been used to treat animals exposed to treatment controls, respectively. We then characterized the botanical toxins24. severity of diarrhea in the calves on a daily basis and collected In the present study, we evaluated the therapeutic effect of fecal samples per rectum 0, 2, 4, 8, 16, 32, and 48 days after the FMT in calves with diarrhea. We collected fecal samples from treatments commenced.