Developing Efficient Probiotics for Microbiota of Diarrhea-Resistant Livestock

Progress report for GS19-206

Project Type: Graduate Student
Funds awarded in 2019: $16,266.00
Projected End Date: 08/31/2021
Grant Recipient: University of Florida
Region: Southern
State: Florida
Graduate Student:
Major Professor:
Dr. Kwangcheol Jeong
University of Florida
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Project Information

Summary:

Calf diarrhea is one of the biggest challenges in both the dairy and beef industry worldwide. In the U.S., between 4 and 25 percent of calves die from diarrhea each year. The increased intestinal permeability and disturbance of gut microbiota are key factors leading to the pathogen-induced diarrhea. Brahman cattle contribute substantially to beef production in the southern regions of the US through crossbreeding, due to their heat tolerance and disease resistance. The heat stress can induce the damage of intestinal barrier dysfunction, but it is largely unclear whether the heat-tolerant Brahman calves have a more integrated intestinal epithelium that contributes to their resistance to diarrhea. Our preliminary studies found that fewer pathogens and mucin-degrading bacteria but more beneficial butyrate-producing commensals colonized in the gut of preweaning Brahman calves compared with Angus calves. Fecal microbiota transplantation from diarrhea-resistant livestock has been reported to relieve diarrhea of recipients. Here, we raised a hypothesis that gut microbiota of Brahman calves contributes to diarrhea resistance and strong intestinal mucus barrier through suppressing pathogenic bacteria and mucin-degrading bacteria. In this project, we first evaluated the differences in gut microbiota structure between Brahman and Angus cattle throughout the production lifecycle using a cohort multibreed Angus-Brahman (MAB) herd using the 16S rRNA gene sequencing. Consistent with preliminary studies, the beneficial butyrate-producing bacteria were enriched in cattle with more Brahman composition. Meanwhile, we collected the fecal samples from 91 3~5-week-old beef calves, and conducted 16S rRNA gene sequencing to understand the differences in gut microbiota composition between healthy and diarrheic calves. We found that the diarrheic cattle contained less diverse gut microbiota and harbored less relative abundance of butyrate-producing bacteria, such as Roseburia, Faecalibacterium, and Odoribacter, but higher abundant pathogens, including Campylobacter and Fusobacterium. Bacterial strains were isolated from  preweaning calves, and one of them were characterized as a new bacterial species as Streptococcus vicugnae. These results shed light to reducing the calf diarrhea by promoting the diversity of gut microbiota and developing probiotics from butyrate producers.

Project Objectives:
  • To compare the gut microbiota composition between disease-resistant Brahman cattle and fast-growing Angus cattle
  • To Characterizing the gut microbial composition and function between healthy and diarrheic calves
  • To isolate potential probiotic strains from diarrhea-resistant Brahman calves and test their antimicrobial activity
  • To evaluate the efficiency of potential probiotic strains on the reduction of diarrhea and investigate its functional mechanisms in terms of regulation on the gut microbiota structure and intestinal mucus barrier function

Cooperators

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Research

Materials and methods:

Evaluation of the breed effects on gut microbiota throughout the production life cycle of Multibreed Angus-Brahman (MAB) cattle.

The 16S rRNA gene amplicon sequencing data were previously obtained from the multibreed Angus-Brahman (MAB) population throughout life. Briefly, fecal samples from 239 preweaning calves, 195 postweaning calves, and 105 fattening calves were collected for conducting the 16S rRNA gene sequencing. Differences in Bray-Curtis distances among breed groups were analyzed using a permutational multivariate analysis of variance (PERMANOVA) with the beta-group-significance command. Breed group was set as fixed effect in the model. To evaluate the effect of breed composition on specific gut bacteria, multiple linear regression models were fitted using breed composition, age, and sex as explanatory variables, and log-transformed relative abundance of core bacterial taxa or IgG1 levels as responsive variables.

Characterizing the hindgut microbiota of healthy and diarrheic preweaning beef cattle

Animal management

The multibreed Angus-Brahman calves were naturally born on pasture at the Beef Research Unit (BRU) in Waldo, FL during the calving season. During the preweaning stage, calves were transferred to Santa Fe River Ranch Beef Unit, and were raised with their dams on the bahiagrass (Paspalum notatum) pastures.

Sample collection

Fecal samples were collected from 91 beef calves ranging in age from 21 days to 35 days as previously described. Briefly, each fecal sample was collected from the rectal-anal junction (RAJ) using two sterile cotton swabs. Swabs with fecal samples were placed in a 15 mL conical tube on ice and were transported to the laboratory within 1 hour for further processing. Each swab sample was resuspended in mixture of 2 mL of Luria-Bertani (LB) broth and 2 mL of 30% glycerol. The fecal solution was then split into four 2 mL tubes and frozen in an ultra-low freezer at -80°C.

Observation of fecal samples 

The morphology of feces was observed to detect whether the feces are watery or bloody. The calves that had normal solid feces were considered healthy calves. The calves that had had feces that are either watery, or pale in color and wet, or bloody were considered abnormal calves.

16S rRNA gene amplicon sequencing

Fecal samples were thawed on ice and homogenized. Then 1 mL of each sample was used for DNA extraction using QIAamp PowerFecal DNA kit according to the manufacturer’s instructions (Qiagen, USA). To understand bacterial community, a dual-index sequencing strategy was used (REF). Briefly, the V4 region of the 16S rRNA gene was amplified by polymerase chain reaction (PCR) with dual-index primers. The PCR amplification reaction consisted of 1 µL forward index primer (10 mM), 1 µL reverse index primer (10 mM), 1 µL 10 ng/µL DNA template, and 17 µL Pfx AccuPrime master mix (Invitrogen, USA). Amplification was initiated with denaturation for 5 min at 95 ℃, followed by 30 cycles of 95 ℃ for 30 s, annealing at 55 ℃ for 30 sec and extension at 72 ℃ for 1 min, with a final elongation for 5 min at 72 ℃. The amplicons were purified and normalized using the SequalPrep plate normalization kit (Invitrogen, USA). The same amount of barcoded V4 amplicons from each sample were pooled to construct the DNA library.

Bioinformatic analysis for 16S rRNA gene amplicon sequencing

The 16S amplicon sequencing data were analyzed with version 2 of the Quantitative Insights into Microbial Ecology (QIIME 2) pipeline. Briefly, paired-end raw reads were imported, and the quality of the initial bases was evaluated according to the Interactive Quality Plot. The sequence quality control was performed with the Divisive Amplicon Denoising Algorithm (DADA2) pipeline implemented in QIIME 2, including steps for filtering low quality reads, denoising reads, merging the paired-end reads, and removing chimeric reads. The phylogenetic tree was generated using the align-to-tree-mafft-fasttree pipeline from the q2-phylogeny plugin of QIIME 2. The sequencing depth was normalized to 10,080 sequences per sample. The Shannon index and Bray-Curtis distance were measured by the core-metrics-phylogenetic method. All amplicon sequence variants (ASVs) were classified into the bacterial taxonomy using the q2-feature-classifier plugin of QIIME 2 and the SILVA 138 database (https://www.arb-silva.de/documentation/release-1381/).

Statistical analysis

The alpha diversity of gut microbiota between healthy and abnormal calves was compared using the Student’s t test. Differences in Bray-Curtis distances between healthy and normal calves were analyzed using a permutational multivariate analysis of variance (PERMANOVA) with the beta-group-significance command.

Research results and discussion:

The difference in gut microbiota structure between Angus and Brahman throughout the production lifecycle 

Figure 1

Gut microbiota structure was significantly influenced by breed composition in preweaning (Fig. 1A), postweaning (Fig. 1B), and fattening (Fig. 1C) stages, showing greater dissimilarity with increasing genetic distance, regardless of the growth stage.  Breed composition effects on gut microbiota, analyzed with combined microbiota of all three stages together, showed that the gut microbiota structure of calves was significantly different among the six BGs (Fig. 1D, p = 0.001). The greatest difference in microbiota structure was observed between BG1 and BG6 (p = 0.015), the calves of which had the greatest genetic distances; this indicates that the effects of host genetics are not specific to certain growth stages, but are universal throughout life.
To identify specific bacterial genera affected by breed composition, associations between breed composition and the log10 transformed relative abundance of core bacterial taxa were evaluated using multiple linear regression models that included the explanatory variables of age, sex, and breed composition. At the genus level, the relative abundances of 36 (52.2%) out of 69, 32 (40%) out of 80, and 31 (37.3%) out of 83 core bacterial genera were significantly associated or showed tendency with breed composition in preweaning, postweaning and fattening calves, respectively (Fig. 1E). Among the bacterial genera, the relative abundance of Oscillospira, Roseburia and Sutterella showed positive associations with Brahman composition throughout life (Fig. 2E). Interestingly, Oscillospira (h2 = 0.46) and Sutterella (h2 = 0.42) showed relatively high heritability estimates (Fig. 1F), which indicates their colonization is dramatically influenced by host genetics,while Roseburia (h2 = 0.21) seems to be more susceptible to environmental conditions.

The prevalence of diarrheic calves in the early preweaning MAB calves

To detect the diarrheic rate in young MAB calves, the morphology of feces collected from 91 3~5-week-old calves was observed and recorded. Among the 91 fecal samples, 74 were normal solid, 6 were mild watery with pale color, 8 contained blood, 2 were severe water, and 1 was severe bloody.

The microbiota diversity between healthy and abnormal feces

Figure 2

To detect whether healthy calves contained more diverse gut microbiota, the bacterial richness reflected by the number of ASVs and bacterial evenness reflected by Shannon index were compared between calves that had normal solid feces and those that had abnormal feces.  As shown in Fig. 2, the number of ASVs was higher in healthy calves compared to abnormal calves (Fig. 2A, P = 0.017), and the several watery feces and severe bloody feces contained the lowest number of ASVs compared with normal solid feces, mild watery, and mild bloody feces (Fig. 2B). Although the Shannon index did not show a significant difference between healthy and abnormal calves, the severe water feces and severe bloody feces showed a lower Shannon index compared with normal solid feces. These data suggest that a more diverse gut microbiota is beneficial to prevent diarrhea. 

The distinct microbiota composition in abnormal feces compared with healthy feces 

Figure 3

To characterize the gut microbiota between diarrheic and healthy calves, we compared the microbiota composition based on the morphology of feces. The principal coordinates analysis (PCoA) plot based on the Bray-Curtis distance did not show a significant difference between healthy calves and calves with abnormal feces (Fig. 3A, P = 0.214). However, a separation between the severe watery or severe bloody feces with normal solid feces along the Axis 2 was observed (Fig. 3B).  More specifically, pathogenic bacteria Fusobacteria, Campylobacter, Tyzzerella,  Veillonella were enriched in either severe watery feces or severe bloody feces, while potential butyrate-producers Oscillospirace, which had higher relative abundance in disease-resistant Brahman cattle, were more abundant in healthy calves. These data support the isolation of potential butyrate-producing bacteria to relieve diarrhea. 

 

Participation Summary

Educational & Outreach Activities

1 Journal articles
1 Training the undergraduate about understanding the gut microbiota between healthy and diarrheic calves

Participation Summary

6 Ag professionals participated
Education/outreach description:

With the support of the SARE grant, we have published a peer-reviewed article on a high-impact Nature Publishing Group Journal ISME J (Impact factor: 9. 18), which title is “Host genetics exerts lifelong effects upon hindgut microbiota and its association with bovine growth and immunity”. The paper reported the difference in gut microbiota between disease-resistant Brahman cattle and fast-growing Angus cattle throughout production lifecycle, and the connections between specific commensal bacteria and animal health. We have also trained one undergraduate student for observation of fecal samples to characterize diarrhea and analyze the high-throughput microbiota data using advanced bioinformatic tools. We plan to present our findings in the EPI research day early next year. We also would like to share our research results with farmers and the industry community during Field Day events once Cov1d-19 is controlled well. 

Project Outcomes

1 New working collaboration
Project outcomes:

We have published a research article on the high-impact Nature Publishing Group journal ISME J. We are still in the middle of isolating the bacteria strains from healthy calves to inhibit the pathogens colonized in diarrhea calves. Meanwhile, other mitigation strategies, including diet manipulation, farm management that will enhance the gut microbiota diversity and increase the abundance of identified benefit commensals can also be applied to relieve diarrhea on the farm. We plan to transfer our research findings to application in the near future to reduce the economic cost caused by diarrhea.  

Knowledge Gained:

With the support of the SARE grant, we have extensively understood the gut microbiota between disease-resistant Brahman cattle and fast-growing Angus cattle across different growth stages, which provide informative evidence showing how animal breeding influences animal growth and immunity throughout modulating the gut microbiota. Furthermore, we characterize the microbiota structure between healthy and diarrheic calves according to their fecal morphology and identify specific bacteria that are potential for combat diarrhea.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.