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Introduction
The microbial community of the gastrointestinal system of cattle remains understudied. Due to its influence on nutrient absorption, productivity, potential reservoir of human and animal pathogens, as well as overall animal health, there is a need to better understand bovine gut microbial communities1. Recently, high-throughput sequencing using 16S rRNA amplicons has provided deeper information on the fecal bovine microbiota composition, and the results obtained to date indicate a high diversity2.
The central part of the Chihuahuan Desert in Mexico has a high diversity of wildlife3,4. The bovine (Bos taurus) has been raised as grazing livestock since its introduction at the end of the 16th century, being the most important economic activity in this area5. However, this activity is the main reason of ecological deterioration which affects wildlife; for example, this ruminant species competes for forage resources with endemic animal species (i.e., Gopherus flavomarginatus, Bolson tortoise)3. Cattle grazing also exerts strong pressure on plant populations, modifying their cover; this may increase soil erosion susceptibility in this desert3,6.
The cattle gut microbiome has many microbial species that play an important role in health and productivity7,8. These microbes are essential for the fermentation of consumed plant matter that is converted into energy for the host9. However, bovines asymptomatically transport bacterial species that are potential pathogens to wildlife as Escherichia coli, Campylobacter spp., Salmonella spp. and Listeria spp.6,10. In recent years, the extensive use of land for agriculture has increased the densities of cattle populations creating positive correlations with pathogenic infections by fecal bacteria11. Though, knowledge about bovine fecal bacterial diversity under grazing management systems is relatively scarce12. This study aimed to explore for the first time the diversity and abundance of fecal bacteria from bovines under grazing-marginal conditions in the Mapimi Biosphere Reserve, center of the Chihuahuan desert, using next-generation sequencing (16S rRNA).
Material and methods
All the methods and activities of this study were in strict accordance with accepted guidelines for ethical use, care and welfare of animals in research at international13 and national14 levels, with institutional approval reference number UJED-FCB-2018-07.
Study area
The study was developed in the Mohovano de las Lilas locality, northeast of the Mapimi Biosphere Reserve in Mexico (26°00’ and 26°10’N, 104°10’ and 103°20’W) in the center of the Chihuahuan desert. This area has warm, very arid climate, with an average annual temperature of 25.5 ° C, and an average annual precipitation of 264 mm. The predominant vegetation is rosette and microphile scrub, as well as halophyte, and gypsophila plants15.
Field work
In July 2018, three fresh fecal samples were collected from three healthy male bovines. From each fecal sample, 0.25 g was collected from the center of the sample and deposited it in BashingBead™ cell lysis tubes (Zymo Research Corp.) adding 750 μL of lysing/stabilizing solution. Each tube was processed in a TerraLyzer™ cellular disruptor (Zymo Research Corp.) during 20 sec according to the equipment specifications.
Laboratory work
DNA was extracted from the samples using the Xpedition™ Soil/Fecal DNA MiniPrep kit (Zymo Research Corp.) in a laminar UV flow hood in sterile conditions. The amount of DNA obtained was measured in a Qubit™ fluorometer (Invitrogen). Then, the V3-V4 region of the 16S rRNA gene was amplified using the following primers16: S-D-Bact-0341-b-S-17, 5´-CCTACGGGNGGCWGCAG-3´ and S-D-Bact-0785-a-A-21, 5´-GACTACHVGGGTATCTAATCC-3´. The step after sequencing was realized using a Illumina protocol17,18 and thereafter, the samples was sequenced in MiSeq of 2 × 250 paired final. The complete sequencing process is available in García-De la Peña et al19.
Data availability
The files used in this study were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA614584).
Bioinformatic analysis
The DNA sequences were analyzed using Quantitative Insights into Microbial Ecology bioinformatics software (QIIME)20. Both forward and reverse sequences were assembled using the PEAR program21 considering Q30 the quality criterion (one false base for every 1,000 bases). Chimeric sequences were discarded with USEARCH22. Then, the operational taxonomic units (OTUs) were selected with the UCLUST method22 at 97 % similarity; a representative sequence for each OTU was obtained, and the taxonomy was assigned using EzBioCloud database as reference23. A simple random rarefaction process was performed24 in order to obtain a standardized file for all samples. The relative abundance for the phylum and family levels were represented as stacked bar plots using R, and genus level was visualized as a heatmap using Morpheus software (Morpheus, https://software.broadinstitute.org/morpheus); hierarchical clustering (average linkage method with Euclidean distance) was used to visualize samples dendrogram25.
Results and discussion
In this study, the average number of sequences assembled was 155,915. A mean ± sd of 109,814 ± 16,686 bacterial sequences were obtained after taxonomic designation. The average number of OTUs with a 97 % of similarity was 6,661 ± 431 (Table 1).
Table 1 Fecal sequences information of Bos taurus at the Mohovano de las Lilas locality, Mapimi Biosphere Reserve, Mexico
| Sample | Total | Assembled | Discarded | BS | BSS | OTUs |
|---|---|---|---|---|---|---|
| 1 | 322,428 | 138,862 | 183,566 | 131,275 | 98,084 | 6,293 |
| 2 | 223,470 | 145,379 | 78,091 | 136,807 | 102,441 | 6,556 |
| 3 | 305,380 | 183,506 | 121,874 | 173,177 | 128,916 | 7,135 |
| Mean | 283,759 | 155,915 | 127,843 | 147 086 | 109 814 | 6,661 |
BS= bacteria sequences after taxonomical designation, BSS= bacteria sequences after singletons removal; OTUs= operational taxonomic units.
A total of 17 phyla, 24 classes, 33 orders, 50 families, 281 genera, and 297 species were determined. The most abundant phyla (Figure 1) were Firmicutes (
Figure 1 Relative abundance (%) of fecal bacteria taxa (phylum level) from three samples of Bos taurus at the Mohovano de las Lilas locality. Only the first 10 more abundant phyla are shown
At family level, Ruminococcaceae (
Figure 2 Relative abundance (%) of fecal bacteria taxa (family level) from three samples of Bos taurus at the Mohovano de las Lilas locality. Only the first 10 more abundant families are shown
From 281 classified genera found in this study, 36.6 % have a taxonomic name; this percentage is higher than the reported by Kim and Wells44 in feces of cattle where only 110 genera were classified, and about 41 % of the total sequences couldn’t be assigned to a known genus (Figure 3). Nevertheless, the results showed here increase the number of genera of the B. taurus fecal microbiota previously reported 12,45,46, who confirmed that the fecal bacterial microbiota is extremely diverse in cattle, and has not yet fully described. Sporobacter was the most abundant genus found in the fecal samples of cattle in this study. This genus was reported in alpaca47, deer sika28, horse48, donkey49, and the Bolson tortoises Gopherus flavomarginatus19. This genus is related to digestion of plant ligno-cellulosic matter; however relatively little is known about the role of this bacteria in the degradation process50. Durso et al51 reported Faecalibacterium, Ruminococcus, Roseburia, and Clostridium as important components of the fecal bovine microbiota. These genera were also determined in the present study. According to some studies52,53 these bacteria constitute 50 to 70 % of the total number of microorganisms in the digestive system of ruminants. These animals have specific gut microbial taxa as they are dependent on these bacteria to extract energy and nutrients from food54, besides having specialized anatomical and physiological adaptations to the cellulolytic fermentation of low nutrition - high fiber vegetal material55. The presence of other bacterial genera reported in this study could be the result of environmental and genetic factors, age, breed, diet, phylogeny, among others56,57,58. Recently56,59,60, was demonstrated that herbivorous animals have the most diverse microbiota since they depend on microbial metabolic pathways to maximize energy and nutrient extraction from feeding61.
Figure 3 Heatmap of Bos taurus fecal bacteria sample at genus level at the Mohovano de las Lilas locality. Only the first 40 more abundant genera are shown
Although the gut microbiome usually remains stable over time assisting as a defense system against pathogens and other disease-causing agents in the host, the disturbance of this community can lead to animal disease62,63. In the present study, the samples collected were obtained from apparently healthy bovines. However, bacteria considered of veterinary importance were found in these animals; this could be a potential health risk because they are carriers of these microorganisms. For example, Campylobacter, Clostridium, Corynebacterium and Fusobacterium were found in the fecal samples. These genera have been associated with cattle disease. Campylobacter has been reported as a cause of infertility and abortion in ruminants64,65; also represents a critical threat to public health, because it can be transmitted from cattle to humans66,67,68. Clostridium has been reported causing diseases and death in ruminants, especially in cattle; examples are respiratory diseases69, botulism70 and the blackleg71. Corynebacterium has been reported in beef and dairy cattle associated with renal disease72, mastitis73,74, and tuberculosis75,76,77; also, it is considered as an important emergent pathogen for humans78. Finally, Fusobacterium was reported by others79-82, causing abscesses in cattle. It is important to develop other studies that provide information on the pathogenicity and dynamics of these potential pathogens in the bovines of the Mapimi reserve.
At species level, Pseudobacteroides cellulosolvens and Campylobacter fetus were registered in the present study. Pseudobacteroides cellulosolvens is anaerobic bacteria that degrade plant cell wall polysaccharides and cellulosic, being capable of using cellulose or cellobiose as a sole carbon source83. Campylobacter fetus is a relevant species; the main reservoirs of this bacteria are both the intestinal and the genital tracts in cattle and sheep64,65. This species causes spontaneous abortion and infertility in cattle, while it is also an opportunistic pathogen to humans84.
Due to the free-grazing management in the Mapimi Reserve, the bovine feces remain over the soil until natural processes degrade them. Consequently, native fauna can be in contact with these feces, increasing the probability of interspecific transmission of some bacteria85. Although it has been previously reported that there are no evidence of cross-parasite infection between cattle and mule deer in the Mapimi Biosphere Reserve86, it is important to clarify whether this same scenario occurs for bacteria. McAllister and Topp87 estimate that about 77 % of the pathogens that usually infect livestock can also affect wildlife. However, also wildlife is considered an important source of microorganisms that could cause infectious diseases to domestic animals and humans88,89. For these reasons, it is important to develop studies focused on risk management at the interface of domestic species and native fauna, considering the implications for the transmission of microorganisms with pathogenic potential88,89. This information could lead to establish microbiological control strategies for wild fauna populations and livestock within the area.
Conclusions and implications
Information about the bovine fecal microbiota under extensive grazing conditions is scarce. From economic, ecological and health perspectives, it is crucial to determine the bacterial diversity -from phyla to species-, in the intestine of domestic ruminants. The present study is the first insight into the fecal bacterial composition of bovines in the Mapimi Biosphere Reserve in Mexico using next generation sequencing. This information significantly expands the knowledge about the composition and abundance of bacteria that are part of the microbiological community of the bovine intestine. In this case, the approach was through the analysis of feces in free grazing cattle. Although a large number of bacterial taxa were reported from the collected samples, it was not possible to determine the genus or species of some bacteria, so it is still necessary to go further into the taxonomy using specific molecular markers. However, the results obtained in the present study could be used as a bacteriological baseline for monitoring the grazing bovine intestinal health status, and to trace possible interactions with the fecal microbiota of native roaming wildlife in the area. Finally, it is important to emphasize that the next generation massive sequencing is a very effective technique that simplifies the analysis of complete bacterial communities; therefore, complementary studies on the microbiota in this and other bovine populations in Mexico are warranted.
