Selective use of antibiotics in neonatal calf diarrhoea and its influence on fecal microbiota

Objectives: Waste milk (WM) is a major by-product of the dairy industry that includes low-quality colostrum, transitional milk, milk from cows administered veterinary drugs for the treatment of mastitis and/or other diseases, milk with high somatic cell counts (SCC), and milk that cannot be marketed. The large amount of discarded WM generates environmental pollution and represents the loss of a valuable resource that may serve as a good feed source for dairy calves because of its high nutrient content. However, using WM for feeding calves might expose new-born calves to infectious agents. Moreover, residual antimicrobials may have a negative influence on the animals’ gut microbiota in terms of selection of antimicrobial-resistant microorganisms as well as in terms of interference with its proper maturation, by acting on specific taxonomic groups or by inducing long-term dysbiosis. The constant antibiotic pressure exerted on the calf microbiota by these residues may interfere with its physiological development by selectively inhibiting specific phylogenetic subgroups and increasing the selection and transfer of antibiotic resistance genes to the gut microbiota. Therefore, despite the apparent economic advantages for the farmer, WM might impair the correct physiological and immunological functionality of the calf gut and favor the selection of antibiotic resistance traits. We assessed the impact of WM on the correct development of the calf intestinal microbiome by analyzing the effect on intestinal health and on the fecal microbiota of calves fed with WM in the first weeks of life. Materials and methods: WM was assessed for nutritional content and antimicrobial residue concentration by mass spectrometry. The trial, carried out over 8 weeks, included 12 Italian Friesian male calves. After 3 days of colostrum, 6 calves were fed Waste Milk (WM group) and 6 calves were fed Bulk Tank Milk (BM group) for 2 weeks. Then, for 6 weeks, all 12 calves received a weaning diet of milk substitute and starter feed. Every week for the first 2 weeks, and every 2 weeks for the remaining 6, we subjected all calves to clinical examination and collected rectal swabs to investigate the fecal microbiota composition by 16S rRNA gene analysis. Research protocols were approved by the University of Milan (protocol number 78_2018). Results: The composition on WM and BM in terms of SCC, fat, protein, lactose, and microbial inhibitors content was as follows. WM: 450,000 cells/ml; 3.7%; 3.6%; 4.7%; present. BM: 284,000 cells/ml; 4.23%; 3.60%; 4.97%; absent. At the end of the trial, the two groups showed statistically significant differences in both clinical terms, including reduced weight gain and increased occurrence of diarrhea events in the WM group, and in terms of fecal microbiota composition. Almost all WM calves (5/6) developed diarrhea in the first 2 weeks (vs 1/6 BM calves). In the following 6 weeks, only 1 episode of diarrhea occurred in 1 WM calf. WM calves’ body weight was significantly lower than BM calves along the trial. The 16S rRNA gene analysis indicated a sharp reduction in the fecal microbiota alpha-diversity of WM vs BM calves, most significant at Wk4 (p < 0.02), two weeks after exposure to WM. Beta-diver- sity of the fecal microbiota was significant between time-points (p = 0.0069505). As for the alpha-diversity results, significant differences were observed between WM and BM calves at Wk4 (p < 0.05). Based on the normalized relative OTU levels, WM and BM calves showed significant differences at all time-points. At the end of the trial, Bacteroidetes, Firmicutes, and Saccharibacteria decreased while Chlamydiae increased. Significant changes were also observed in 7 classes, 8 orders, 19 families, 47 genera. Among the most relevant findings was the general decrease of beneficial taxa, like Faecalibacterium, vs an increase in other taxa and potential pathogens, includ- ing Campylobacter, Pseudomonas, and Chlamydophila. Lactobacillus and Lachnoclostridium increased, but since the first was present in the milk substitute, its higher abundance in WM calves might indicate a lower microbiome resilience. Conclusion: In conclusion, these results suggest that adding to the risk of increasing antibiotic resistance, feeding pre-weaned calves with WM is related to a higher incidence of calf diarrhea and relevant changes in the fecal microbiota composition.