Background

Protozoan parasites are commonly found in the digestive tract of cattle, especially in the rumen and intestines, and have an important effect on their health. Ciliate species, mainly in the rumen, play an important role in rumen fermentation and the stability of rumen ecology [1]. On the contrary, protozoan parasites in the cattle gut are usually associated with intestinal diseases. Many intestinal protozoan parasites, such as Cryptosporidium, Eimeria, and Giardia, cause intestinal disease outbreaks in humans and animals [2, 3]. However, the controversial pathogenicity of some intestinal protozoan parasites, such as Blastocystis and nonpathogenic Entamoeba, is mainly observed in asymptomatic individuals [4]. Protozoan parasites interact with other gut microbes in symbiotic environments and co-evolve over time [5, 6]. At present, studies on the effects of parasites on calves mainly focus on the interaction between parasites and hosts, and there are few studies on the effects of parasites on the intestinal microflora of calves.

Intestinal microecology impacts cattle health and is correlated with nutrient metabolism, energy production, and the immune system [7]. Intestinal diseases are among the most important health problems in calves (0–1 year of age). Approximately 4–25% of calves in the United States die from diarrhea each year, causing tremendous economic losses to the cattle industry [8,9,10]. Although many factors cause calf diarrhea, disorders of the intestinal microbiome are the main manifestations of calf diarrhea [10,11,12]. Diverse gut microbiota prevent colonization by foreign pathogens and enhance the host immune system through interactions between antigens and immune cells during the early stages of life [8]. Studies have shown that restoring the intestinal microbial composition of diarrheal calves by fecal microbiota transplantation can ameliorate diarrhea in pre-weaning calves [13].

There are few effective drugs and vaccines for protozoan parasites such as Cryptosporidium and Giardia duodenalis [3, 14]. Changes in the intestinal bacteria may be related to various intestinal protozoan parasites, and understanding the effects of parasites on gut microbes could provide new insights into treating parasitic diseases [15,16,17]. In this study, parasites and other intestinal microecological elements in the posterior intestines of calves were characterized using metagenomic analysis. The association between intestinal protozoan parasites, other intestinal microbes, and calf diarrhea was also investigated, which provides a theoretical basis for maintaining calf intestinal health.

Methods

Animal management

Samples for this study were collected from a beef cattle farm in Henan Province, China. A total of 15 fresh stool samples were collected from four calves with bloody diarrhea (B1–B4), four calves with watery diarrhea (W1–W4), and seven calves with normal stools (N1–N7). All calves were 2–3 months old, and samples were collected by rectal sampling, stored in 2-ml cryotubes, transported on dry ice to the laboratory, and stored at −80 °C until use.

Sample preparation and Illumina sequencing

Total DNA from the fecal microbiota was extracted using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions (QIAamp Fast DNA Stool Mini Kit Handbook, www.qiagen.com/handbooks). The degree of degradation and potential contamination of DNA was analyzed by electrophoresis using 1% agarose gel. DNA purity was determined using a NanoPhotometer® spectrophotometer (IMPLEN, Westlake Village, CA, USA), and DNA concentration was measured using the Qubit® double-stranded DNA (dsDNA) Assay Kit on a Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). One microgram of the qualified DNA was used to construct the library. DNA samples were fragmented to 350 base pairs (bp) by sonication, and the DNA fragments were end-polished, A-tailed, and ligated with a full-length adaptor for Illumina sequencing with further polymerase chain reaction (PCR) amplification. The libraries were analyzed for size distribution using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified via real-time PCR. Libraries were sequenced using the Illumina PE150 platform (Illumina, Inc., San Diego, CA, USA).

Identification of intestinal microorganisms

Metagenomic analysis was carried out following previously published methods [18, 19]. The host sequence was removed from the raw data using Bowtie 2 (v2.4.5) [20] and assembled using MEGAHIT (v1.2.9) [21]. The sequences (≥ 500 bp) were used to predict the open reading frame using MetaGeneMark (v3.38) [22] and eliminate redundancy using CD-HIT (v4.5.8) [23]. Clean data from each sample were mapped to the initial gene catalog using Bowtie2. The corresponding relative abundance of each gene was calculated based on the following formula: Ai = Ci/∑ni=1 Ci (where Ni represents the number of reads mapped to each gene and Li represents the length of each gene; Ci = Ni/Li) [24]. The obtained genes were used to BLAST the sequences for bacteria, fungi, archaea, viruses, and intestinal protozoan parasites, which were extracted from the National Center for Biotechnology Information (NCBI) non-redundant (NR) database (https://www.ncbi.nlm.nih.gov) using DIAMOND software (v2.0.14) [25]. We used the lowest common ancestor (LCA) algorithm to obtain the number of genes and abundance information for each sample in each taxonomic hierarchy (kingdom, phylum, class, order, family, genus, and species) [26]. DIAMOND software was used to annotate the unigenes using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.kegg.jp/kegg/).

PCR amplification and sequence analysis

Further specialization of parasites was accomplished using PCR. Giardia was identified based on the β-giardin (bg) gene [27], and Cryptosporidium [28], Eimeria [29], Blastocystis [30], and Entamoeba [31] were identified based on the small subunit (SSU) ribosomal RNA (rRNA) gene. After amplification, the DNA fragments were separated by agarose gel electrophoresis (1% agarose), stained with DNA Green (TIANDZ, Beijing, China), and observed using a Tanon 3500 gel imaging system (TANON, Shanghai, China). Amplified samples with the target band were selected as positive PCR production. Positive PCR amplicons with the target band were sequenced by SinoGenoMax (Beijing, China). Bidirectional sequencing was chosen to ensure the veracity of sequences. Phylogenetic analysis was conducted using MEGA 7.0 software (http://www.megasoftware.net/), choosing the maximum composite likelihood model, and bootstrap values were calculated by analyzing 1000 replicates.

Statistical analysis and visualization

Venn diagrams, alpha diversity (Chao1 and Shannon indices), and principal coordinates analysis (PCoA) based on the Bray–Curtis distance were calculated and plotted using Tutools (https://www.cloudtutu.com), and the ImageGP (https://416h86i955.zicp.fun/Cloud_Platform/front/#/) platform, and microbial features showing differential abundance were identified using linear discriminant analysis effect size (LEfSe) threshold criteria of linear discriminant analysis (LDA) score > 2 and P < 0.05 (http://huttenhower.sph.harvard.edu/galaxy/). To reveal the relationship between parasites and the microbial or KEGG ortholog group (KO), we calculated the pairwise Spearman’s rank correlation and removed coefficients below 0.7 with P > 0.05. We adjusted the P-value to avoid false positives using the Benjamin-Hochberg (BH) method from the ‘Hmisc’ package in R (v4.2.2) statistical software. Network analysis and visualization were conducted using Gephi (https://gephi.org/) and Cytoscape (https://cytoscape.org/).

Results

Species of intestinal protozoan parasites

The parasites identified using metagenomics belonged to the genera Giardia, Eimeria, Cryptosporidium, Blastocystis, and Entamoeba (Fig. 1A). Specific genetic information is shown in Additional file 3: Table S2. Not all positive samples were successfully amplified. Based on these results, the parasite species in calves were G. duodenalis assemblage E (Fig. 1B), Cryptosporidium bovis, Cryptosporidium ryanae (Fig. 1C), Eimeria bovis, Eimeria subspherica (Fig. 1D), Blastocystis (ST2 and ST10) (Fig. 1E), and Entamoeba bovis (Fig. 1F).

Fig. 1
figure 1

Intestinal parasitic protozoa detected in the calves. a Parasites annotated by metagenomic sequencing. b Phylogenetic relationships of Giardia duodenalis based on β-giardin (bg) nucleotide sequences. c Phylogenetic relationships of Eimeria based on the SSU rRNA gene. d Phylogenetic relationships of Cryptosporidium based on the SSU rRNA gene. e Phylogenetic relationships of Blastocystis based on the SSU rRNA gene. f Phylogenetic relationships of Entamoeba based on the SSU rRNA gene. Phylogenetic relationships were calculated using the maximum composite likelihood model. Percent bootstrap values greater than 50% from 1000 replicates are shown next to the branches. Triangles represent isolates detected in this study, and the name of samples which detected the parasite are written in parentheses

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Intestinal microbial imbalance in diarrheal calves

Comparing calves with watery diarrhea and calves with normal stools, the major intestinal microbiota of calves with bloody diarrhea changed greatly at the phylum and genus levels. At the phylum level, Firmicutes, Bacteroidetes, and Proteobacteria were the main phyla in the gut of the calves. Firmicutes and Bacteroidetes were mainly found in calves with watery diarrhea and calves with normal stools, but Proteobacteria were more abundant in calves with bloody diarrhea (Fig. 2A). At the genus level, Bacteroides were mainly found in calves with watery diarrhea and calves with normal stools and were lower in calves with bloody diarrhea (Fig. 2B).

Fig. 2
figure 2

Comparison of gut microbes among calves with bloody diarrhea, watery diarrhea, and normal stools. a Relative abundance (%) at the phylum level. b Relative abundance (%) at the genus level. c PCoA of gut microbes based on the Bray–Curtis distance. d α-diversity (Chao1 and Shannon index) of gut microbes. Boxes of α-diversity denote the interquartile range (IQR) between the first and third quartiles (the 25th and 75th percentiles, respectively), and the line inside denotes the median. Whiskers denote the lowest and highest values within 1.5 times and the IQR from the first and third quartiles, respectively

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The intestinal microbes of calves with diarrhea show disorders, especially calves with bloody diarrhea. The PCoA results show that calves with watery diarrhea and calves with normal stools have comparable intestinal microbiota compositions, which differ significantly from calves with bloody diarrhea (Fig. 2C). The diversity of microbes declined with an increase in the degree of diarrhea, while in calves with bloody diarrhea, it was significantly reduced (Fig. 2D).

The taxa that most likely explain the differences between calves with bloody diarrhea, calves with watery diarrhea, and calves with normal stools were defined by LEfSe. One hundred thirty-six biomarkers were detected at the genus level (LDA > 2, P < 0.05). Thirty-two biomarkers significantly enriched in calves with bloody diarrhea, including various fungi and opportunistic pathogens, such as Escherichia, Streptococcus, Salmonella, and Shigella. Thirty-two biomarkers in calves with watery diarrhea, mainly Bacteroidetes, Firmicutes, and Proteobacteria, and 71 biomarkers were significantly enriched in calves with normal stool and mainly belonged to Firmicutes (Additional file 1: Fig. S1).

Relationship between parasites, other microbes, and calf diarrhea

Cryptosporidium, Eimeria, and Giardia were distributed in both diarrheal and healthy calves and were more abundant in diarrheal cattle and lower in calves with normal stools. However, Blastocystis and Entamoeba were found only in calves with normal stools, with a higher abundance than that of the other parasites (Additional file 1: Fig. S2).

The network analysis results showed that the parasites were associated with various other gut microbes (Fig. 3). In total, five protozoan parasites formed two distinct networks, indicating that protozoan parasites have different mutual regulatory relationships with other intestinal microbes. Blastocystis and Entamoeba were related to the diversity of the intestinal bacteria, archaea, fungi, and viruses in calves. All 494 genera of intestinal microbes were positively associated with Blastocystis and Entamoeba; about 81.2% (401/494) were bacteria, and the others were archaea (41/494), fungi (44/494), and viruses (8/494). Interestingly, Blastocystis and Entamoeba were significantly positively correlated with various archaea, including many methanogens: Methanothermus, Methanothermococcus, Methanothermobacter, Methanosalsum, and Methanolacinia. One hundred sixty-one intestinal microbes were associated with Eimeria, Cryptosporidium, and Giardia, including 62 genera of bacteria, 97 fungi, and two viruses. Most of the bacteria were negatively correlated with Eimeria, Cryptosporidium, and Giardia, and all the fungi and viruses were positively correlated with them, meaning that these protozoan parasites may be related to the imbalance of other gut microbes.

Fig. 3
figure 3

Relationship between protozoan parasites and calf microbiome. Network relationships between protozoan parasites and gut microbes at the genus level. The relationship was calculated using the pairwise Spearman rank correlation, the removed coefficient was below 0.7, P > 0.05, and the P-value was adjusted to avoid false positives using the BH method

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Key microbes associated with protozoan parasites and the health of calves

Twenty-seven biomarkers were not only significantly enriched in calves with bloody diarrhea, watery diarrhea, and normal stools, but were also associated with Eimeria, Cryptosporidium, and Giardia. In addition, all intestinal microorganisms associated with Blastocystis and Entamoeba were not significantly enriched in calves with varying degrees of diarrhea. Among the biomarkers, seven genera of bacteria were negatively correlated with Eimeria, which were mainly enriched in the intestines of calves with normal stools, namely, Dietzia, Flavonifractor, Gemmiger, Intestinimonas, Lachnoclostridium, Negativibacillus, and Ruthenibacterium. All 18 genera of fungi were positively correlated with Eimeria, Cryptosporidium, and Giardia were enriched in the intestines of calves with bloody diarrhea, including Blastocladiella, Diaporthe, Diplocarpon, Endocarpon, Fomitiporia, Gaeumannomyces, Gelatoporia, Hydnomerulius, Kalmanozyma, Leucosporidium, Magnaporthe, Magnaporthiopsis, Melampsora, Meyerozyma, Sclerotinia, Setosphaeria, Spiromyces, and Trichophyton. The two viruses, Alphaentomopoxvirus and Pandoravirus, were enriched in the intestines of calves with bloody diarrhea (Fig. 4).

Fig. 4
figure 4

Microbes that not only are associated with parasites but also vary significantly in different degrees of diarrhea

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Relationship between parasites and other gut microbial function

Functional annotation based on the KEGG database showed that the intestinal microorganisms of calves had abundant functional genes (Additional file 1: Fig. S3). The functional categories identified included Metabolism, Cellular Processes, Environmental Information Processing, Genetic Information Processing, Human Diseases, and Organismal Systems. PCoA results showed that, at the KO level, the samples of calves with watery diarrhea and normal stools showed a clear distance from calves with bloody diarrhea. This suggests that severe diarrhea could affect gut microbial function (Additional file 1: Fig. S4).

The functions of other gut microbes were related to intestinal parasites. A total of 611 KOs were found to be associated with parasites by calculating their correlation coefficients (Additional file 2: Table S1), and 87 KOs were disease-related (Fig. 5A). The genes annotated as 87 KOs were aligned with the NCBI NR database to trace the possible integration of the bacteria. Sixty-six KOs were successfully annotated, most of which were from fungi (Fig. 5B).

Fig. 5
figure 5

Key pathway associated with protozoan parasites and calf diarrhea. a Disease- and parasite-related KOs. b Carriers of KOs associated with diseases and parasites

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Discussion

Intestinal protozoan parasitic diseases are prevalent in ruminants, and infections with intestinal protozoan parasites in cattle are associated with outbreaks of diarrhea, mainly in calves, leading to economic losses for agricultural producers [32, 33]. However, the relationship between parasites and calf gut microbes remains unclear. Here, we have shown the relationship between parasites, gut microbes, and diarrhea.

The abundance of parasites found in this study was associated with calf diarrhea. Eimeria, Cryptosporidium, and Giardia were detected in both diarrheal and normal calves, but in higher abundance in diarrheal calves. Previous studies have shown similar results; the oocysts/cyst per gram of feces of Eimeria, Cryptosporidium, and Giardia in diarrheal animals were positively higher than in healthy animals [34,35,36]. Nevertheless, Blastocystis and Entamoeba were found only in healthy calves, which may indicate a positive association with intestinal health. The pathogenicity of Blastocystis and Entamoeba in cattle is controversial; however, Blastocystis and Entamoeba are highly prevalent in cattle and are mostly detected in healthy cattle [37, 38], which is consistent with the results of this study.

Calf diarrhea was often characterized by an imbalance in intestinal flora [10,11,12], and this study also revealed intestinal microbial imbalance in calves with diarrhea. Moreover, Eimeria, Cryptosporidium, and Giardia intestinalis were related to an imbalance in gut microbes. Previous studies have also shown negative effects of these parasites on gut microbes. For example, Eimeria damaged the chicken intestinal barrier and reduced the abundance of probiotic bacteria [16]. Cryptosporidium infection decreased bacterial diversity [39], and Giardia infection was associated with significant dysbiosis within the murine foregut and hindgut [15]. By contrast, Blastocystis and Entamoeba in this study were associated with the diversity of other intestinal microorganisms. The same phenomenon has been observed in humans, where it was found that Blastocystis was associated with both higher richness and higher evenness of the gut bacterial microbiota, whereas Entamoeba was associated only with higher richness, all of which have a role in maintaining intestinal health [17]. It is worth noting that Blastocystis and Entamoeba were significantly positively correlated with various methanogenic archaea. The possible use of methanogens as probiotics has received particular attention in humans [40, 41]; therefore, the positive relationship between Blastocystis, Entamoeba, and methanogenic archaea may be beneficial for gut health. The relationship between archaea and parasites in animals has received little attention, and more research is needed to uncover this phenomenon.

Little attention has been paid to the relationship between fungi and parasites in cattle. In this study, many fungi that were significantly enriched in calves with bloody diarrhea were positively correlated with Eimeria, Cryptosporidium, and Giardia. The parasites and fungi may play a synergistic role in intestinal disease in calves. Previous studies have found that higher fungal abundance is associated with intestinal disease, with a higher prevalence and abundance in patients with inflammatory bowel disease [42], which enhances inflammation by preventing intestinal healing [43, 44]. Fungi are neglected microorganisms that influence intestinal diseases in cattle, and further studies on the interactions between fungi and parasites are needed.

Conclusion

In conclusion, this study revealed the relationship between protozoan parasites and the calf microbiome. Eimeria, Cryptosporidium, and Giardia are associated with calf diarrhea and intestinal microbial disorders. By contrast, Blastocystis and Entamoeba play positive roles in maintaining intestinal health and microbial diversity. In addition, many fungi have a potential synergistic relationship with Eimeria, Cryptosporidium, and Giardia; on the contrary, archaea were only positively correlated with Blastocystis and Entamoeba. Due to the limited collection from the same farm, more extensive sampling in the future will clearly help produce a better association between protozoan parasites and intestinal health. As we described only the association of protozoan parasites, microbiome, and health of calves in this study, further work, including intervention studies, will be needed to fully elucidate the role of the protozoan parasites in the intestinal environment.