Characterization of the Fecal Bacterial Microbiota of Healthy and Diarrheic Dairy Calves

Gomez, D.E.; Arroyo, L.G.; Costa, M.C.; Viel, L.; Weese, J.S.

The development of intestinal microbiota in neonatal calves is a dynamic and complex process influenced by external and internal factors that affect intestinal microbial succession. 30 External factors include microbial load in the environment, delivery mode, 34 type of colostrum, 35 type of feeding (raw milk versus pasteurized milk versus milk replacer), 29 housing, 36 and administration of probiotics, prebiotics, or antibiotics. [37] [38] [39] Individual factors that can influence gut microbiota include nutritional state, functional immaturity of the immune system, intestinal pH, peristalsis, bile acids, bacterial mucosal receptors, and microbial interactions. 40 Therefore, management factors are a likely explanation for the interfarm differences that were noted, but further study of factors that influence both the individual calf and farm-level microbiota is needed. In humans and most other studied species, a small number of phyla tend to account for the majority of the intestinal microbiota. Firmicutes tends to be the dominant phylum in most animal species, 12, 15, 41, 42 as was the case here. The predominance of Firmicutes, Actinobacteria, and Proteobacteria noted in these calves is also consistent with previous studies in infants, 41 foals, 42 piglets, 15 and calves. 12, 35 The predominance of Actinobacteria is presumably a reflection of the early colonization of the gut in neonates. Bovine milk contains complex nondigestible oligosaccharides 43 that promote proliferation of specific gut microbes, especially Bifidobacterium spp. from the Actinobacteria phyla. 44 Although it is difficult to define normal calf microbiota, general trends can be inferred from previous studies. From a phylum-level perspective, the fecal microbiota of healthy Holstein calves during the neonatal period was dominated by Firmicutes, with a relative abundance ranging from 64 to 82%, followed by Bacteroidetes (8-24%), Proteobacteria (4-10%), Fusobacteria (1-6%), and Actinobacteria (1-2%). 12 At the genus level, several studies identified higher abundance of Bacteroides and Clostridium spp. in the feces of healthy calves. [27] [28] [29] In addition, in pre-weaned Holstein calves relative abundance of Faecalibacterium prausnitzii, a microorganism that belongs to the phylum Firmicutes, was higher during the first weeks of life and this high abundance was associated with decreased incidence of diarrhea. 12 Our results differed with the aforementioned studies in several aspects. The phylum Bacteroidetes accounted for <1% of the total sequences identified in healthy and diarrheic calves, something that could be accounted for, at least in part, by methodology, because it is well established that differences in Bacteroidetes abundance can be found with Illumina vs 454 sequencing likely due to the choice of different primers (V4-V5 vs V4, respectively). 45 Proteobacteria, especially members of the Enterobacteriaceae family, were enriched in feces of healthy calves from F2, and in feces of diarrheic calves from both F1 and F2. The increase in diarrheic calves is not surprising as this phylum often is associated with intestinal dysbiosis, 46 but the difference between farms was interesting. A recent study of nursing calves from 5 beef farms identified some farms with high relative abundances of Proteobacteria, 47 suggesting that higher Proteobacteria levels could be a farm-associated effect, perhaps from management practices. This could be of concern because recent stu