These authors contributed equally to this work.
The genus
Human babesiosis is induced by apicomplexan tick-transmitted haemoparasites, which are obligate intraerythrocytic protozoa of the genus
Regarding the apicomplexa parasites, host gut microbiota composition has helped to prevent/treat diseases, such as malaria [
Whether the
The collection and manipulation of small intestinal contents were approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. All procedures were conducted according to the Ethical Procedures and Guidelines of the People’s Republic of China. The study is reported following ARRIVE guidelines.
Twelve six-week-old male LVG Golden Syrian Hamsters that were bought from Chales River (Beijing, China) were randomly classified into 3 groups (T0, T1, and T2) and kept under pathogen-free conditions. The hamsters were given an ultraviolet-illuminated diet and distilled water at 25°C.
DNA extractions from intestinal contents were conducted using Cetyl Trimethyl Ammonium Bromide method, which is adequate for the extraction and purification of DNA from plants and plants derived food staff and is especially suitable for the elimination of polysaccharides and polyphenolic compounds. DNA concentration and purity were identified with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), which was diluted to 1 ng/μl with sterile water. 16S rRNA V4 region was amplified using a set of primers (515F and 806R). PCR amplification was conducted in a total of 50 μl reaction volume containing 25 μl of Phusion High-Fidelity PCR Master Mix (New England Biolabs, USA), 0.5 μm of each primer, and 10 ng templet DNA. The parameters of the PCR reaction included: initial denaturation at 98°C for 1 min, 30 cycles of denaturation at 98°C for the 30 sec, 50°C for 30 sec, and extension at 72°C for 90 sec, with a final extension at 72°C for 5 min. The PCR products were purified with a Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany).
Sequencing libraries were prepared with NEBNext Ultra IIDNA Library Prep Kit (E7645, New England Biolabs, USA) following the manufacturer’s instructions. The library quality was assessed by the Qubit2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. 250 bp paired-end reads were sequenced against libraries with the Illumina NovaSeq platform.
Sequenced reads were assigned to individual samples/groups based on the barcodes, which together with primer sequences were eliminated. A FLASH package (Ver1.2.11,
The phylogenetic association of each ASV and the different species among groups/samples were identified using QIIME2 software, which was also employed for multiple sequence alignments and normalization of the absolute abundance of ASVs. Normalized ASVs were employed to evaluate alpha diversity, based on 7 indices (observed ASVs, Chao1, Shannon, Simpson, Dominance, Good coverage, and Pielou). Among these indices, Observed ASVs, Chao1, and Dominance were chosen to assess community richness, while Shannon and Simpson were used to determining community diversity. Furthermore, Good’s coverage has deemed the parameter of sequencing depth; Pielou was employed to assess the species evenness.
Beta-diversity was identified based on the ASVs to assess the diversity of bacterial communities among samples and groups (T0, T1, and T2). Beta-diversity was evaluated by calculating weighted and unweighted UniFrac distances that show the distance between each pair of samples. Principle coordinate analysis (PCoA) was conducted using weighted and unweighted UniFrac distances to visualize the ordination and clustering among the samples.
QIIME2 was employed to determine multiple species in samples/groups and considerably different species were identified using MetaStat and
The datasets generated and analyzed during the current study are available in the National Genomics Data Center (NGDC) repository (
On day 0, hamsters in T1 and T2 were infected with
We obtained 12 intestinal contents from hamsters (4 control hamsters and 8 hamsters infected by
Alpha rarefaction values demonstrated that all samples attained saturation, which indicated that species number was not rise with sample size (
At the phylum, Bacteroidetes and Firmicutes comprised the main components of the gut microbiota in all groups. The relative abundance of Bacteroidetes rose from an average of 38% in the T0 group to 51.7% in the T1 group and 42% in the T2 group with the rise of the burden of parasite and the severity of disease, while the
At the family level, the most common component in all samples was Muribaculaceae, which rose from 31% in the T0 group to 45% in T1 and 36% in the T2 groups. The relative abundance of Helicobacteraceae and Clostridia UCG014 also demonstrated similar phenome. While Lachnospiraceae, Eubacteriaceae, and Desulfovibrionaceae were considerably decreased after
Alpha diversity was employed to assess the microbiota community in the samples. To evaluate the bacterial diversity, we used the Simpson diversity index of the alpha diversity parameter, implying the richness and relative abundance of ASVs. There is no significant difference among T0 (0.986±0.039), T1 (0.976±0.012), and T2 (0.989±0.045) groups (
For weighted UniFrac and unweighted UniFrac determining changes in species, the beta-diversity was distinct between the T2 group and T0. The
According to weighted and unweighted UniFrac distance matrixes, the similarity of microbiota composition was examined using PCoA analyses. Microbiota changed significantly as the disease progressed in hamsters, induced by
LEfSe analysis was employed to determine the biomarkers, which were bacterial taxa with substantial differences between groups (
Babesiosis is a common infectious disease worldwide, particularly in tropical and subtropical regions. The clinical symptoms of
Gut microbiota compositions in
Simultaneously, the families Lachnospiraceae, Ruminococcaceae, Eubacteriaceae, Desulfovibrionaceae, and Oscillospiraceae were substantially decreased after
We conducted the LEfSe analysis to determine the biomarkers with great differences between
To our best knowledge, this research is the first to evaluate the relationship between gut microbiota profile and
Bacterial community comparison of the groups. (A) alpha diversity analysis according to the Chao1 index; (B) alpha diversity analysis according to the observed amplicon sequence variant (ASV) index; (C) alpha diversity analysis according to the Shannon index; (D) alpha diversity analysis according to the Good’s coverage index.
This study was financially supported by the Science Fund for Creative Research Groups of Gansu Province (22JR5RA024), the Natural Science Foundation of Gansu (22JR5RA031), Gansu Natural Science Foundation (21JR11RA130), the Science and Technology Development Guiding Program of Lanzhou (2019ZD-55), Cuiying Scientific and Technological Program of Lanzhou University Second Hospital (CY2019-BJ05), the National Science Foundation of China (grant no. 31972701), ASTIP (grant no. CAAS-ASTIP-2016-LVRI), and NBCIS (grant no. CARS-37), the Leading Fund of Lanzhou Veterinary Research Institute (LVRI-SZJJ-202105), and the hatching program of SKLVEB (SKLVEB2021CGQD02).
The authors declare no competing financial interests.
Data curation: Wang Y, Yueli N
Formal analysis: Zhang S
Funding acquisition: Guan G
Investigation: Wang Y
Methodology: Zhang S, Wang J, Li X
Supervision: You C, Zhang D, Guan G
Writing – original draft: Zhang S, Wang J
Microscopy of
Microbial composition changes. Relative abundance of bacterial phyla and families in hamsters. Each bar suggests the top 10 most abundant phyla and families, while others imply unclassified and low-abundance ASVs. (A) bacterial community composition at the phylum; (B) taxonomic heatmap of the 3 groups at the phylum level; (C) bacterial community composition at the families; (D) taxonomic heatmap of the 3 groups at the families.
Operational taxonomic units (ASVs) analysis in the different samples.
Principal coordinates study based on weighted (A) and unweighted (B) UniFrac distance matrixes of microbiota profiles of hamsters infected by
Biomarkers in different groups. (A) cladogram of the most differentially abundant bacterial taxa in the 2 groups (T0 vs T1); (B), bacterial taxa that significantly differentiated in T0 vs T1 groups were determined by LEfSe analysis; (C), cladogram of the most differentially abundant bacterial taxa in the 2 groups (T0 vs T2); (D), bacterial taxa that significantly differentiated in T0 vs T2 groups were identified by LEfSe analysis.
Basic information of the sequence data from infected and uninfected hamster small intestinal samples
Group | Sample name | Qualified tags | Nochime tags | Average length (nt) | Q20 | Q30 | GC (%) | Effective tags (%) | ASV |
---|---|---|---|---|---|---|---|---|---|
T0 | T0S1 | 101,060 | 68,583 | 417 | 96.92 | 91.17 | 54.2 | 66.7 | 608 |
T0S2 | 100,352 | 68,754 | 417 | 96.79 | 90.8 | 54.4 | 67.0 | 667 | |
T0S3 | 107,321 | 70,960 | 415 | 97.01 | 91.3 | 54.2 | 64.9 | 650 | |
T0S4 | 101,936 | 64,287 | 415 | 97.01 | 91.36 | 54.4 | 61.4 | 679 | |
| |||||||||
T1 | T1S1 | 104,339 | 70,300 | 418 | 96.73 | 90.81 | 54.3 | 66.7 | 588 |
T1S2 | 108,420 | 71,697 | 419 | 96.56 | 89.93 | 54.1 | 64.9 | 740 | |
T1S3 | 110,857 | 73,670 | 419 | 96.99 | 90.98 | 53.8 | 64.4 | 722 | |
T1S4 | 107,882 | 78,769 | 418 | 96.83 | 91.05 | 54.2 | 72.1 | 599 | |
| |||||||||
T2 | T2S1 | 90,804 | 61,839 | 418 | 96.67 | 90.55 | 53.9 | 66.1 | 445 |
T2S2 | 109,550 | 75,387 | 416 | 97.1 | 91.53 | 53.1 | 67.3 | 555 | |
T2S3 | 104,938 | 76,067 | 413 | 97.17 | 91.69 | 52.9 | 71.3 | 608 | |
T2S4 | 103,767 | 75,601 | 418 | 96.84 | 91.06 | 53.8 | 70.8 | 541 |
Alpha diversity indexes for the microbial communities detected in the hamster samples
Group | Sample name | Chao1 | Dominance | Goods coverage | Observed ASVs | Pielou_e | Shannon | Simpson |
---|---|---|---|---|---|---|---|---|
T0 | T0S1 | 608 | 0.016 | 1 | 608 | 0.8 | 7.399 | 0.984 |
T0S2 | 668 | 0.013 | 1 | 667 | 0.814 | 7.638 | 0.987 | |
T0S3 | 650.091 | 0.018 | 1 | 650 | 0.796 | 7.439 | 0.982 | |
T0S4 | 678.125 | 0.009 | 1 | 678 | 0.843 | 7.928 | 0.991 | |
| ||||||||
T1 | T1S1 | 588.214 | 0.01 | 1 | 588 | 0.826 | 7.601 | 0.99 |
T1S2 | 740.3 | 0.007 | 1 | 740 | 0.86 | 8.199 | 0.993 | |
T1S3 | 722.25 | 0.008 | 1 | 722 | 0.842 | 7.996 | 0.992 | |
T1S4 | 599.077 | 0.017 | 1 | 599 | 0.786 | 7.252 | 0.983 | |
| ||||||||
T2 | T2S1 | 445 | 0.018 | 1 | 445 | 0.803 | 7.064 | 0.982 |
T2S2 | 555.273 | 0.026 | 1 | 555 | 0.776 | 7.078 | 0.974 | |
T2S3 | 609.667 | 0.04 | 1 | 608 | 0.747 | 6.906 | 0.96 | |
T2S4 | 541.333 | 0.012 | 1 | 541 | 0.819 | 7.434 | 0.988 |