Direct and culture-enriched 16S rRNA sequencing of cecal content of healthy horses and horses with typhlocolitis

Abstract

Next generation sequencing has demonstrated that alpha diversity of the fecal microbiota is significantly altered in horses with typhlocolitis. The objective of this study was to evaluate the bacterial composition of the cecum content of horses with and without typhlocolitis through direct and culture-enriched 16S gene sequencing of six healthy horses and six horses with acute typhlocolitis; a case-control study design. Cecal content was collected after euthanasia. An aliquot was used for direct 16S gene sequencing. Another was serially diluted with brain heart infusion (BHI) and plated onto five different agar media. All culture medias, except for MacConkey, were incubated anaerobically. Bacterial colonies were harvested in bulk and used for DNA extraction, 16S PCR amplification, and sequenced using the Illumina MiSeq platform. Predominant phyla in healthy and diseased horses were Firmicutes, followed by Bacteroidetes in all cultured medias, except for MacConkey agar, in which Proteobacteria was the dominant phylum. Greater bacterial richness was identified in sequenced cecal contents as compared to cultured plates (P < 0.05). Culture-enriched molecular profiling combined with 16S rRNA gene sequencing offer an alternative method for the study of the gut microbiota of horses. For direct cecum content 16S gene amplification, the alpha diversity indices were lower in diarrheic horses compared to healthy horses (P < 0.05). A higher relative abundance of Fusobacteriota was found in 2/6 samples from diarrheic horses. The role of Fusobacteriota in equine colitis deserves investigation.

Relationship between the components of mare breast milk and foal gut microbiome: shaping gut microbiome development after birth

“…This suggests that these bacteria may have been acquired temporarily from the environment rather than in utero during pregnancy (Zakia et al. 2023 ). After birth, a variety of external routes of microbial transmission to the foal are possible, including ingestion of bacteria from the mother’s skin, udder, vagina, and hairs through licking the mother’s body (Garber et al.…”

Current Understanding of Equine Gut Dysbiosis and Microbiota Manipulation Techniques: Comparison with Current Knowledge in Other Species

“…Several studies have explored microbial alterations in horses with diarrhea/colitis [ 59 , 72 , 89 , 93 , 94 , 95 , 96 ], generally reporting decreased diversity and altered bacterial composition. Decreased abundances in bacteria of the phylum Firmicutes have been reported, and Lactobacillus was more abundant in healthy horses in the study by Zakia et al [ 90 ], whereas other studies reported increased abundance in diarrheic horses [ 89 , 96 ]. These discrepancies across studies may reflect the lack of absolute quantification inherent to amplicon sequencing technologies.…”

“…Many diseases have been associated with alterations in the gut microbiota, including diarrhea, colitis, equine metabolic syndrome and colic [ 89 , 90 , 91 , 92 ]. It remains unclear if the composition and diversity of the intestinal microbiota influence the susceptibility to developing specific diseases.…”

“…Unconventional Clostridia species have been suggested to cause diarrhea in horses [ 99 ]. Clostridium sensu stricto was increased three-fold in the cecal content of horses with diarrhea compared to healthy horses [ 90 ]. Still, another study found no significant difference in the prevalence of Clostridium innocuum [ 100 ].…”

“…It is, however, possible to highlight certain phyla and bacterial families that should be present in a healthy equine microbiota of the colon and cecum. When looking at the relative abundance of bacterial communities, Bacteroidetes and Firmicutes are the phyla with the highest abundance in most studies, followed by Proteobacteria and Verrucomicrobia [ 54 , 89 , 90 , 93 , 94 , 95 ]. Their order of importance tends to vary from one study to another, but current research on horse microbiomes concludes that all those phyla are an important factor for the gut’s health [ 16 , 54 , 89 , 90 , 91 , 93 , 94 , 95 , 101 ].…”