The composition of the microbiota in the full-term fetal gut and amniotic fluid: a bovine caesarean section study

The fetal development of the intestinal immune system is stimulated by the maternal microbiota, but it is still unclear whether viable bacteria exist in the healthy fetus. Analysis of such low microbial biomass environments are challenging due to contamination issues. The aims of the current study were to assess the bacterial load and characterize the bacterial composition of the amniotic fluid and meconium of full-term calves, leading to a better knowledge of prenatal bacterial seeding of the fetal intestine. Amniotic fluid and rectal meconium samples were collected during and immediately after elective caesarean section, performed in 25 Belgian Blue cow-calf couples. The samples were analyzed by qPCR, bacterial culture using GAM agar and 16S rRNA gene amplicon sequencing. To minimize the effects of contaminants, we included multiple technical controls and stringently filtered the 16S rRNA gene sequencing data to exclude putative contaminant sequences. The meconium samples contained a significantly higher amount of bacterial DNA than the negative controls and 5 of 24 samples contained culturable bacteria. In the amniotic fluid, the amount of bacterial DNA was not significantly different from the negative controls and all samples were culture negative. Bacterial sequences were identified in both sample types and were primarily of phyla Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria, with some individual variation. We conclude that most calves encounter in utero maternal-fetal transmission of bacterial DNA, but the amount of bacterial DNA is low and viable bacteria are rare.


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Characterizing the very first intestinal bacteria is essential for a better understanding of the co-

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The cows' owners were informed about the study and gave their written consent. were housed for an elective C-section.

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In total 25 Belgian Blue cows and their calves (7 males and 18 females) were sampled from 88 November 2017 until March 2019. The cows were housed in tie-stalls at the facility for 9.5 d 89 ± 5.8 (mean ± standard deviation) prior to C-section and had ad libitum access to hay and water.

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During this period, rectal temperature was measured twice daily, and calving indicators such 91 as udder distension, teat filling, pelvic ligament relaxation, vaginal discharge, vulvar edema, 92 and behavioral changes were monitored every two hours by graduate veterinary students.

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Prior to elective C-section, in cows that exhibited a drop in temperature, cervical dilation was 94 assessed by manual palpation. The vulvar region was cleaned with iodine soap and water. A 95 gloved hand was inserted vaginally and the opening of the portio vaginalis cervicis was 96 estimated. For the present study, elective C-section was performed when the cow had a minimal 97 cervical dilation of eight centimeters, with no rupture of the fetal membranes prior to surgery.

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All cows were healthy according to their vital parameters (heart rate, temperature, respiratory 99 rate) and there was no clinical evidence of intrauterine infection or contamination. Prior to surgery, the cows were restrained in a standing position in a surgery crush specifically 102 designed for cattle. C-section procedure was done as described by Kolkman et al. (2007). was gently introduced in the rectum and the swab was exposed. Samples were taken in 119 duplicate, one stored immediately at -80 °C with no additives, and the other stored at -80 °C in 120 a sterile 2 ml cryovial containing 1 ml of a 30 % glycerol solution, prepared by diluting glycerol 121 (≥99 %, G2025, Sigma-Aldrich (Merck), Overijse, Belgium), in ultra-pure, nuclease-free water 122 (W4502, Sigma-Aldrich (Merck), Overijse, Belgium) to a final 30 % concentration.

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Negative field controls were processed in the surgery room, using the same sampling     The processed data was in silico filtered to remove ASVs which represented probable 216 contaminants, as described previously (Husso et al., 2020). Briefly, an ASV was removed if its 217 prevalence in actual samples was ≤2× its prevalence in instrument controls (DNA extracted 218 from empty sampling instruments only handled aseptically in a laminar flood cabinet) or its 219 prevalence in field controls (empty sampling instruments exposed to the surgery room 220 environment), and if its mean relative abundance in actual samples was ≤10× its mean 221 abundance in instrument controls or its mean abundance in field controls. The filtering was 222 performed separately for meconium and amnion samples. If less than 500 reads remained after 223 the decontamination, as was the case for six meconium samples, the sample was removed from 224 further analyses.  The 16S rRNA gene copy numbers in amniotic fluid and meconium samples were assessed by 248 qPCR (Fig. 1). To minimize variation between qPCR runs, the copy numbers were normalized Decontamination of the 16S rRNA gene amplicon sequencing data 266 We performed a stringent filtering of the 16S rRNA gene sequencing data to remove the 267 amplicon sequence variants (ASVs) potentially stemming from reagent contaminants and 268 environmental bacterial DNA (Table 1).

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The decontamination was based on the prevalence and relative abundance of each ASV in the  Bacteroidetes and Actinobacteria phyla, with individual variation (Fig. 2). In both sample 279 types, the inter-individual variation increased at lower taxonomic levels (Supplementary Table   280 1 and Fig. 3) Sphingomonas and Acinetobacter were the most prevalent genera (Supplementary Table 1).

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The observed composition and abundances for the commercial standard matched the expected 287 composition provided by the manufacturer. types in more detail (Fig. 3).  Comparison of amniotic fluid and meconium microbiota composition 316 The microbiota composition was significantly different for amniotic fluid and meconium 317 samples at the ASV (PERMANOVA, P < 0.001, R2 = 0.049, Fig. 5 A), and genus level 318 (PERMANOVA, P < 0.001, R2 = 0.062, Fig. 5 B).     Table 2. 351 Both gram negative and gram-positive strains were isolated from the samples.

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In the present study, we sampled amniotic fluid during, and the corresponding calves intestinal secretions, it is unexpected that the two would harbor completely different bacteria.

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However, the amniotic fluid microbiota may fluctuate dynamically over time, while meconium 480 represents a more stable collection of substances accumulated over the gestation period.

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Moreover, fetal excretion in the amniotic fluid is limited in cattle. Meconium is usually 482 expelled only after birth, and fetal urine largely accumulates in the allantoic cavity, between 483 chorion and amnion, rather than in the amniotic cavity (Bongso and Basrur, 1976).

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The less permeable synepitheliochorial placenta evidenced by three distinct layers may restrict In conclusion, we found that the meconium of full-term calves delivered by elective caesarean

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The cows' owners were informed about the study and gave their written consent for their cows' 516 participation.