We characterized the temporal dynamics of the fecal microbiota of calves from two weeks of life to six months of age, in three commercial veal farms representative of management practices in the veal calf industry in France. The calves were mainly fed with milk replacers throughout the follow-up, and received several collective antibiotic treatments at therapeutic doses, most of them administered in the first weeks of fattening (Fig. 1). We did 16S rRNA gene sequencing to study the composition of the microbiota and quantitative PCR of the genus Escherichia as a proxy of E. coli to quantify the commensal populations of this bacteria of medical relevance. In two farms, we estimated the daily dose of milk powder recommended by the integrator to search for association with the relative abundances of genera. The most striking results of this study are (i) the homogenization of the fecal microbiota composition among calves, which began during the first month of life and was characterized by an increase of the α-diversity and of the proportion of the same dominant genera, (ii) the significant but limited effect of antibiotic treatments on the microbiota diversity and on the E. coli population size, and (iii) the significant associations of the estimated daily doses of milk powder with the relative abundances of some genera and with the farm level predicted number of E. coli / g from our model.
The development of the microbiota of these calves was characterized by the dominance of the Firmicutes and Bacteroidetes phyla (Fig. 3) and a switch of dominant genera, such as that of Lactobacillus and Bifidobacterium at early stages to Prevotella and Alloprevotella as the calves aged (Fig. 4), with a simultaneous increase in microbiota diversity (Fig. 6 and Additional file 7: Fig. S5a). These developmental features have already been described for calves with the same characteristics (age, sex, and breed) fed with milk replacers [12], as well as for females of the same breed in Canada [11, 12], USA [7, 41] and Japan [4] and for dairy calves of a different breed in Austria [5]. These shared findings suggest that the fecal microbiota of calves undergoes a predictable age-dependent trend that is common to distinct calf populations. The high heterogeneity of the microbiota composition at day 7 in the farms (corresponding to three weeks of age) could be attributable to the distinct origin of the calves, as they came from different dairy farms. Prior studies have noted the importance of exposure to dam’s bacterial communities and to environmental bacterial communities in influencing microbiota composition throughout the gut of the newly born calf [42, 43]. The transport from dairy farms could also be responsible of the high heterogeneity between calves at the beginning of the fattening, as its disrupting effect on gut microbiota has been reported in young beef cattle five days after their transport to feedlot [23]. From as soon as the end of the first month after arriving in the farms, massive and continuous bacterial successions (Fig. 5) gradually increased the similarity of the microbiota composition among calves on all farms, both in terms of presence and relative abundances of bacterial members, as it was shown by unweighted Unifrac distances and weighted Unifrac distances (Additional file 2: Fig. S2 and Fig. 2, respectively). This convergent pattern occurred in the absence of environmental or dietary change such as weaning, the calves being reared in dedicated closed buildings and drinking milk replacers throughout the fattening. This suggests that the influence of environmental factors and dietary factors on this trend of convergence is probably limited, and highlights the likely role of host physiology. Convergence related to age has also been observed in the ruminal microbiota of calves between one day and two years of life [10] and in both the ruminal and fecal microbiota of dairy cows receiving different diets before weaning [44]. The variability of the composition of fecal microbiota of pre-weaned dairy calves has been shown to be higher than fecal microbiota of lactating cows, suggesting the higher influence of the surrounding environment on calves compared to adults’ more mature microbiota [29, 44]. These studies suggest that such changes in composition are not restricted to the lower part of the gut and are not strongly driven by diet. An interesting hypothesis could be that the convergent stabilization of the microbiota composition over time (Fig. 2, Additional file 2: Fig. S2) may be linked to age-dependent shifts of the gut mucosal immune system, as it has been shown that the expression of Toll Like Receptors in both the rumen and colon changes as calves aged [45]. Constraints imposed by the gut environment and autochthonous microbiota on allochthonous bacterial settlement might become less permissive, resulting in more specific requirements as the calves aged.
The microbiota of calves in farms where collective antibiotic treatments were given in the previous fifteen days or during sampling underwent a reduction of microbiota diversity and E. coli number relative to calves of the same age that haven’t been exposed during the same period (Fig. 6b, Additional File 7: Fig. S5, Fig. 7c). We chose to take into account and pool in our analyses both long and short antibiotic treatments and molecules with different spectra to focus on common disruptive effects of antibiotics on microbial ecosystems. As all calves of the same farm received the same antibiotic treatment at the same time, the design of our study didn’t allow the analysis of each molecule’s specific effects. Indeed, we didn’t have negative controls in the same farm, so the results could have been biased by potentially environmental or diet variables, which could have modulated the relative abundances of some genera. The antibiotic-induced loss of diversity has already been reported in young beef cattle [23] as well as pre-weaned calves [28], and was often found associated with the depletion of beneficial bacteria and/or the increase of opportunistic pathogens [27, 28]. Antibiotic induced dysbiosis is also observed when pre-weaned calves are fed with low doses of antibiotic molecules [46].
The effect of the antibiotic treatments was limited relative to the longitudinal changes (Additional file 6: Tables S2, S3 and S4), which is consistent with findings in dairy pre-weaned calves which received enrofloxacin and tulathromycin metaphylactic treatments [47] and beef cattle which received oxytetracycline or tulathromycin injection [23]. These findings can be explained by the existence of a natural resistome independently of any antibiotic treatment, carried by some abundant families in the fecal microbiota of pre-weaned calves, as it has been recently shown [3]. Genera of these antibiotic-resistance genes carrying families were found as dominant in the feces of the veal calves, such as Anaerostipes, Blautia and Roseburia (Lachnospiraceae family), Enterococcus (Enterococcaceae family), Faecalibacterium and Pseudoflavonifractor (Ruminococcaceae family), Bacteroides (Bacteroidaceae family) and Streptococcus (Streptococcaceae). Members of the Enterobacteriaceae family, such as E. coli, were also found to be a major reservoir of antibiotic resistance gene within the microbiota resistome.
Two recent studies found that the resistome of fecal microbiota in pre-weaned dairy calves was composed of resistance conferring genes against tetracycline, sulfonamides, trimethoprime, β-lactams and macrolides [3, 29]. These results suggest the existence of a natural resilience of fecal bacterial communities to collective antibiotic treatments in veal farms, the antibiotics used to treat the calves in our study belonged to these classes. None of these studies reported the presence of mcr-1 gene, which confers resistance to colistin, another molecule used to treat calves, in microbial communities, although another study detected the gene in calves’ commensal E. coli [32]. Moreover, it has been shown that this natural resistome was shaped by the bacterial phylogeny of the fecal microbiome and decreases as calf aged, one of the main driver been the decrease in abundance of the Enterobacteriaceae family, in which 90% of the members were classified as E. coli [3]. It is well-known that commensal E. coli populations of veal calves harbor high levels of antibiotic resistance genes [32, 48], and that these genes are diverse. Antibiotic treatments may have promoted the increase in numbers of specific pre-existing E. coli strains in the beginning of the fattening period, as the extended treatments during the first month did not result in massive depletion of E. coli population.
Among others, we found a possible link between E. coli population dynamics and milk replacer, which is reconstituted from dry milk powder and is rich in lactose (Fig. 7d) [49]. Lactose allows the growth of the vast majority of E. coli strains [50]. It has been shown that the lag time and generation time of E. coli strains, which depend on metabolic efficiency and are crucial for gut colonization and persistence [51, 52], are influenced by the type and abundance of available nutrients in the habitat [53]. Our findings are consistent with another study in which the fecal microbiota of Simmental calves was followed during their first three months of life, although only six calves were included in the study and the sampling was sparse [5]. The relative abundance of the genus Escherichia was found to be maximal during the milk-feeding period and decreased before weaning. Associations between the estimated dose of milk powder and four other genera were also found. The concomitant fluctuations of this lactose rich source and the relative abundances of Enterococcus and Mitsuokella genera strongly suggest the direct role of host diet on members of the fecal microbiota, although the β-galactosidase enzyme that enables lactose utilization was not found in two of them. The discrepancy between the associations found for the Megasphaera and Dialister genera, and the absence of the β-galactosidase enzyme sequence in the genome of known members of these genera could be explained by the utilization of another nutrient present in the milk powder by the members of these taxa, or a small redundancy of carbon sources among members of these genera.
Our study had some limitations. First, we followed the fecal microbiota of calves reared in commercial veal farms, so the calves were not randomly assigned to the different farms (nor antibiotic treatments) and neither the environmental nor diet variables could be controlled as in a randomized trial. Nevertheless, one of the aim of this study was to characterize the fecal microbiota of calves reared under common veal farm practices. These three farms, in which field studies had already been made [32], were representative of management practices in the veal calf industry in France. Second, calves were only sampled after seven days spent in farms, so no information regarding their microbiota composition before any antibiotic treatment was available. They were also sampled on a monthly basis, whereas high-frequency sampling has been recommended in early life microbiome studies in infants [54]. Furthermore, sampling was performed independently of antibiotic treatment. Hence, some short-term and mid-term age- and antibiotic-associated changes may have been missed. Third, although we tracked the dynamics of microbiota using 16S rRNA gene sequencing and E. coli qPCR, we only focused on specific features of this complex ecosystem and may have missed specific patterns at other levels. For example, we had no information concerning the dynamics of fecal bacterial loads, which has been shown to vary in newborn calves [1, 2, 17] and be linked with microbiota composition [55]. We also have no information concerning changes at the species level, although genus-level metabolic diversification has been shown within the microbiota [56, 57], nor intra-species level, as the similar absence of a link between nutritional preferences and phylogenetic distance has been shown within E. coli species [50].
Nevertheless, veal calves as studied in this work have relevant attributes to explore the microbiota maturation process. First, batches are composed of male calves of the same age and breed (usually Holsteins), so there is high genetic and physiological homogeneity among them. Second, they share the same living environment and diet, which are not subject to major changes, as they are reared in dedicated closed buildings in which the conditions are stable and controlled to optimize their growth. Moreover, they do not experience any drastic shift in their diet, as it remains predominantly composed of milk replacers during the six months of fattening. Third, the systematic administration of antibiotics at therapeutic doses to all members of the batch is common practice to prevent the spread of infectious diseases [31]. As healthy young subjects sharing similar controlled conditions over a long time period and who experience common antibiotic exposure, veal calves represent a unique chance to disentangle the driving factors of microbiota assembly in real conditions.