Arthropods to Eutherians: A Historical and Contemporary Comparison of Sparse Prenatal Microbial Communities Among Animalia Species.

Since the advent of next-generation sequencing, investigators worldwide have sought to discern whether a functional and biologically or clinically relevant prenatal microbiome exists. One line of research has led to the hypothesis that microbial DNA detected in utero/in ovo or prior to birth/hatching is a result of contamination and does not belong to viable and functional microbes. Many of these preliminary evaluations have been conducted in humans, mice, and nonhuman primates due to sample and specimen availability. However, a comprehensive review of the literature across animal species suggests organisms that maintain an obligate relationship with microbes may act as better models for interrogating the selective pressures placed on vertical microbial transfer over traditional laboratory species. To date, studies in humans and viviparous laboratory species have failed to illustrate the clear presence and transfer of functional microbes in utero. Until a ground truth regarding the status and relevance of prenatal microbes can be ascertained, it is salient to conduct parallel investigations into the prevalence of a functional prenatal microbiome across the developmental lifespan of multiple organisms in the kingdom Animalia. This comprehensive understanding is necessary not only to determine the role of vertically transmitted microbes and their products in early human health but also to understand their full One Health impact. This review is among the first to compile such comprehensive primary conclusions from the original investigator's conclusions, and hence collectively illustrates that prenatal microbial transfer is supported by experimental evidence arising from over a long and rigorous scientific history encompassing a breadth of species from kingdom Animalia.

Initiation of metformin in early pregnancy results in fetal bioaccumulation, growth restriction, and renal dysmorphology in a primate model.

BACKGROUND: In recent years, pragmatic metformin use in pregnancy has stretched to include prediabetes mellitus, type 2 diabetes mellitus, gestational diabetes mellitus, and (most recently) preeclampsia. However, with its expanded use, concerns of unintended harm have been raised. OBJECTIVE: This study developed an experimental primate model and applied ultrahigh performance liquid chromatography coupled to triple-quadrupole mass spectrometry for direct quantitation of maternal and fetal tissue metformin levels with detailed fetal biometry and histopathology. STUDY DESIGN: Within 30 days of confirmed conception (defined as early pregnancy), 13 time-bred (timed-mated breeding) Rhesus dams with pregnancies designated for fetal necropsy were initiated on twice-daily human dose-equivalent 10 mg/kg metformin or vehicle control. Pregnant dams were maintained as pairs and fed either a control chow or 36% fat Western-style diet. Metformin or placebo vehicle control was delivered in various treats while the animals were separated via a slide. A cesarean delivery was performed at gestational day 145, and amniotic fluid and blood were collected, and the fetus and placenta were delivered. The fetus was immediately necropsied by trained primate center personnel. All fetal organs were dissected, measured, sectioned, and processed per clinical standards. Fluid and tissue metformin levels were assayed using validated ultrahigh performance liquid chromatography coupled to triple-quadrupole mass spectrometry in selected reaction monitoring against standard curves. RESULTS: Among 13 pregnancies at gestational day 145 with fetal necropsy, 1 dam and its fetal tissues had detectable metformin levels despite being allocated to the vehicle control group (>1 µmol metformin/kg maternal weight or fetal or placental tissue), whereas a second fetus allocated to the vehicle control group had severe fetal growth restriction (birthweight of 248.32 g [<1%]) and was suspected of having a fetal congenital condition. After excluding these 2 fetal pregnancies from further analyses, 11 fetuses from dams initiated on either vehicle control (n=4: 3 female and 1 male fetuses) or 10 mg/kg metformin (n=7: 5 female and 2 male fetuses) were available for analyses. Among dams initiated on metformin at gestational day 30 (regardless of maternal diet), significant bioaccumulation within the fetal kidney (0.78-6.06 µmol/kg; mean of 2.48 µmol/kg), liver (0.16-0.73 µmol/kg; mean of 0.38 µmol/kg), fetal gut (0.28-1.22 µmol/kg; mean of 0.70 µmol/kg), amniotic fluid (0.43-3.33 µmol/L; mean of 1.88 µmol/L), placenta (0.16-1.00 µmol/kg; mean of 0.50 µmol/kg), fetal serum (0.00-0.66 µmol/L; mean of 0.23 µmol/L), and fetal urine (4.10-174.10 µmol/L; mean of 38.5 µmol/L) was observed, with fetal levels near biomolar equivalent to maternal levels (maternal serum: 0.18-0.86 µmol/L [mean of 0.46 µmol/L]; maternal urine: 42.60-254.00 µmol/L [mean of 149.30 µmol/L]). Western-style diet feeding neither accelerated nor reduced metformin bioaccumulations in maternal or fetal serum, urine, amniotic fluid, placenta, or fetal tissues. In these 11 animals, fetal bioaccumulation of metformin was associated with less fetal skeletal muscle (57% lower cross-sectional area of gastrocnemius) and decreased liver, heart, and retroperitoneal fat masses (P<.05), collectively driving lower delivery weight (P<.0001) without changing the crown-rump length. Sagittal sections of fetal kidneys demonstrated delayed maturation, with disorganized glomerular generations and increased cortical thickness. This renal dysmorphology was not accompanied by structural or functional changes indicative of renal insufficiency. CONCLUSION: Our study demonstrates fetal bioaccumulation of metformin with associated fetal growth restriction and renal dysmorphology after maternal initiation of the drug within 30 days of conception in primates. Given these results and the prevalence of metformin use during pregnancy, additional investigation of any potential immediate and enduring effects of prenatal metformin use is warranted.

Milk microbiome in the first month of lactation and at weaning from ewes supplemented with zinc pre- and postpartum.

Mastitis is an important disease with economic and welfare implications in both clinical and subclinical states. The aim of this research was to sequence the hypervariable V4 region of the 16S rRNA gene to describe the microbial diversity and taxonomy of milk from clinically healthy ewes (Rambouillet, WF = 9; Hampshire, BF = 5). Experimental ewes represented a subset of a larger study assessing the impacts of divergent dietary zinc (Zn) concentrations [1 × National Academics of Sciences, Engineering, and Medicine (NASEM) recommendations = CON or 3 × NASEM recommendations = ZnTRT] throughout late gestation and lactation. Milk was collected at four periods during early lactation (18 to 24 h, 7 d, 14 d, and 21 d postpartum) and at weaning (84 ±â€…14 d postpartum). Somatic cell counts (SCC) were quantified, averaged, and classed (low: < 500 × 103; medium: 500 × 103 - 100 × 104; high: > 100 × 104 cells/mL). Milk samples (n = 67) were sequenced to identify bacteria and archaea; the most abundant phyla were Actinobacteria, Bacteroidetes, Cyanobacteria, Euryarchaeota, Firmicutes, Fusobacteria, Lentisphaerae, Proteobacteria, Spirochaetes, Tenericutes, Saccharibacteria TM7, and Verrucomicrobia. Mastitis pathogens were among the most relatively abundant genera, including Staphylococcus, Mannheimia, Corynebacterium, and Pseudomonas. Effects of breed, dietary Zn concentration, SCC class, and their two-way interactions on milk microbiome diversity and taxonomy were assessed within early lactation (using a repeated measures model) and weaning samples. Alpha-diversity metrics included Pielou's evenness, Faith's phylogenetic diversity, and Shannon's entropy indices. The main and interactive effects between Zn treatment, breed, SCC class, and period were variable in early lactation and not evident in weaning samples. Milk from BF ewes had increased Faith's phylogenetic diversity and Shannon's entropy, and differed in unweighted UniFrac composition (P ≤ 0.10). Milk from CON ewes had a reduced rate of composition change through early lactation (P = 0.02) indicating greater microbiome stability than ZnTRT ewe milk. These results support that milk is not sterile, and breed, dietary Zn concentration, and SCC class variably affect the milk microbiome. Findings from the current study provide important foundational insights into the effects of increased dietary Zn supplementation on longitudinal changes in the milk microbiome and associations with mammary gland health and mastitis.

Mastitis is an important disease with economic and welfare implications in both clinical and subclinical states. This research described the microbial diversity and taxonomy of milk collected from clinically healthy Rambouillet (WF; n = 9) and Hampshire (BF; n = 5) primiparous ewes in a longitudinal study involving differing dietary zinc concentrations [1 × National Academics of Sciences, Engineering, and Medicine (NASEM) recommendations, CON; 3 × NASEM recommendations, ZnTRT]. Milk was collected weekly during the first 3 wk of lactation and at weaning, and somatic cell counts (SCC) were classed (low, medium, high). Mastitis pathogens were among the most relatively abundant via amplicon sequencing, including Staphylococcus, Mannheimia, Corynebacterium, and Pseudomonas. Breed, zinc treatment, and SCC class effects on milk microbiome α-diversity and ß-diversity changes and taxonomy were assessed. These effects and their two-way interactions were limited but variable in early lactation samples and not evident in weaning samples. Notably, BF ewe milk samples had increased Faith's phylogenetic diversity and increased Shannon's entropy during early lactation, and CON ewe milk samples had a reduced rate of compositional change than ZnTRT samples. These results support the existence of a milk microbiome that is variably affected by breed, increased dietary zinc concentrations, and SCC class.

Calf rumen microbiome from birth to weaning and shared microbial properties to the maternal rumen microbiome.

Optimization of host performance in cattle may be achieved through programming of the rumen microbiome. Thus, understanding maternal influences on the development of the calf rumen microbiome is critical. We hypothesized that there exists a shared microbial profile between the cow and calf rumen microbiomes from birth through weaning. Specifically, our objective was to relate the calf's meconium and rumen fluid microbiomes in early life to that of the cow rumen fluid prior to parturition and at weaning. Rumen fluid was collected from multiparous Angus crossbred cows (n = 10) prior to parturition and at weaning. Immediately following the parturition, meconium and rumen fluid were collected from the calf. Rumen fluid was collected again from the calf on day 2, day 28, and at weaning. The rumen fluid microbial profile and subsequent volatile fatty acid (VFA) profile were characterized using 16S rRNA sequencing and gas liquid chromatography, respectively. Microbial data was analyzed using QIIME2 and the GLM procedure of SAS was used to analyze the VFA profile. Alpha diversity was similar in the early gut microbiome (meconium, rumen fluid at birth and day 2; q ≥ 0.12) and between the cow and calf at weaning (q ≥ 0.06). Microbial composition, determined by beta diversity, differed in the early rumen microbiome (rumen fluid at birth, day 2, and day 28; q ≤ 0.04), and VFA profiles complimented these results. There were similarities in composition between meconium, rumen fluid at birth, and rumen fluid from the cow at weaning (q ≥ 0.09). These data indicate successive development of the rumen microbiome and stabilization over time. Similarities between meconium and rumen fluid at birth potentially indicates in utero colonization of the calf gastrointestinal tract. Similarities in composition between the early calf rumen microbiome and the cow at weaning prompt an interesting comparison and area for future consideration in terms of identifying at what stage of gestation might colonization begin. Overall, this study provides insight into similarities between the cow and calf microbiomes and may be helpful in developing hypotheses for the pathway of colonization and programming potential in the early gut.

Developmental programming has highlighted important influences of maternal factors on offspring development. Recent research indicates a programming potential of the rumen microbiome and understanding this role as well as how inoculation occurs may allow beef producers to optimize management practices of gestating cows such that offspring performance is improved via the rumen microbiome. To investigate this, rumen fluid samples were collected from mature cows immediately prior to calving, from their calf immediately after calving with a meconium sample, day 2, and day 28 as well as collected from both dam and calf at weaning. The rumen and meconium microbiome of the newborn calf were similar to each other as well as to the cow rumen microbiome at weaning, although not to the cow rumen microbiome immediately prior to calving. The shared microbiome of the early calf gut highlight a common source of inoculation. The similarities with the cow rumen at weaning could indicate initiation of colonization occurs early in gestation. Results indicate there are shared microbial properties between the cow and calf rumen microbiome. This further supports the opportunity to alter the calf rumen microbiome to improve productivity through the management of the cow during gestation.

Effects of management strategies during early lactation and weaning on etiological agents of ovine subclinical mastitis and antimicrobial susceptibility of milk-derived bacterial isolates.

Subclinical mastitis is a common intramammary disease in sheep production systems. Expenses associated with compromised animal performance, therapeutic interventions, and decreased ewe longevity make efforts to minimize its prevalence worthwhile. The objectives of this study were to 1) quantify the prevalence of subclinical mastitis throughout lactation, 2) evaluate the impact of bedding treatments on subclinical mastitis during early lactation, 3) evaluate the efficacy of prophylaxis and feed restriction during weaning on subclinical mastitis cure rates, and 4) identify levels and types of antimicrobial resistance in milk-derived bacteria. Ewe milk samples were collected at days 1, 2, and 28 post-partum, weaning, and 3-d post-weaning for bacterial identification via culture-based methods. Staphylococcus spp. and Streptococcus spp. isolates were subjected to in vitro antimicrobial susceptibility testing. The overall prevalence of subclinical mastitis defined by culture growth ranged between 22% and 66% and differences were observed between post-weaning and days 1 and 28 milk samples. Commonly isolated bacteria include coagulase-negative staphylococci (CoNS; 59%), Bacillus spp. (35%), Mannheimia haemolytica (10%), Staphylococcus aureus (8%), Streptococcus spp. (5%), and Corynebacterium spp. (5%). Early milk samples (days 1 and 2) were compared between jug bedding treatment: jugs were recently vacated, cleaned, and dusted with barn lime before adding fresh straw (CLEAN) or jugs were previously vacated and fresh straw was added atop soiled bedding (SOILED). Jug bedding treatment did not affect the prevalence of subclinical mastitis, though CoNS had greater sulfadimethoxine resistance in SOILED isolates than CLEAN isolates (P = 0.03). Three different weaning treatments were used: ewes were injected with penicillin at weaning (PENN), ewes had restricted feed access 48 h prior to and 72 h post-weaning (FAST), or a combination of these treatments (COMBO). Weaning treatment did not affect the prevalence of subclinical mastitis or cure rate from weaning to 3-d post-weaning, though all PENN and no FAST milk S. aureus isolates were resistant against tetracycline (P = 0.08). Subclinical mastitis prevalence tended to decrease from weaning to post-weaning (P = 0.08). These data show that subclinical mastitis is common throughout lactation and the levels of antimicrobial resistance of bacteria isolated from ewe milk are generally low against commonly used antimicrobials.

Subclinical mastitis is a common intramammary disease in livestock. Expenses associated with compromised animal performance, therapeutic interventions, and decreased ewe longevity make minimizing its prevalence worthwhile. The objectives of this study were to quantify the prevalence of subclinical mastitis, evaluate the impact of bedding treatments on subclinical mastitis, evaluate the efficacy of weaning treatments, and identify levels of antimicrobial resistance in milk-derived bacteria. The overall prevalence of subclinical mastitis was 45%. Common bacteria included coagulase-negative staphylococci (CoNS), Bacillus spp., Mannheimia haemolytica, Staphylococcus aureus, Corynebacterium spp., and Streptococcus spp. Early lactation milk samples were compared between jug bedding treatments: jugs were cleaned before adding fresh straw (CLEAN) or jugs had fresh straw added atop soiled bedding (SOILED). Jug bedding treatment did not affect the prevalence of subclinical mastitis, though did affect CoNS resistance to sulfadimethoxine. Three different weaning treatments were used: ewes were administered penicillin at weaning, ewes had restricted feed access at weaning, or a combination of the two treatments. Weaning treatment did not affect the prevalence of subclinical mastitis, though subclinical mastitis prevalence decreased post-weaning. Our data show that subclinical mastitis is generally prevalent throughout lactation, and the levels of antimicrobial resistance of bacteria isolated from ewe milk are generally low.

Comparing the maternal-fetal microbiome of humans and cattle: a translational assessment of the reproductive, placental, and fetal gut microbiomes.

Despite differences in gut physiology and morphology, both humans and cattle require a functional gut microbiome in early life. Evidence suggests that both species acquire gut microbes prior to birth, likely from a maternal source, indicating the use of similar mechanisms and timing for fetal gut colonization. Unlike mouse models, cattle share a similar gestation length, parity, and placental microbiome characteristics to humans. The large size of calves allow for contamination-protected sampling of the gut, vagina, and uterus, which would typically require invasive procedures in human cohorts. The ruminant placenta also exhibits a larger degree of separation between maternal and fetal physiology, necessitating a direct and explicit route by which microbes may access the fetal gut. These and other features permit cattle to act as a translational model for early gut colonization. However, cattle do not share similar placental morphology, gut function, or early immune system interactions with humans, creating barriers to their use as a biomedical model. Identifying similarities and differences between humans and cattle may outline the most important functions of the placental and fetal gut microbiomes, indicate the source of these microbes, and highlight the role of maternal or environmental influences upon fetal health across species.

Late Gestation Maternal Feed Restriction Decreases Microbial Diversity of the Placenta While Mineral Supplementation Improves Richness of the Fetal Gut Microbiome in Cattle.

Feed intake restriction impacts both humans and ruminants in late gestation, although it is unknown whether this adverse maternal environment influences the microbiome of the reproductive tract, and through it, the colonization of the fetal gut. A 2 × 2 factorial design including a 70% feed intake restriction (feed restricted 'FR' or control diets 'CON') and mineral supplementation (unsupplemented 'S-' or supplemented 'S+') was used to analyze these effects in multiparous cows (n = 27). Vaginal swabs were obtained 60, 30, and 10 days prior to the estimated calving date, along with neonatal rumen fluid and meconium. Placental tissues and efficiency measurements were collected. Microbial DNA was extracted for 16S sequencing of the V4 region. Feed restriction decreased the diversity of the placental microbiome, but not the vagina, while mineral supplementation had little impact on these microbial communities. Mineral supplementation did improve the richness and diversity of the fetal gut microbiomes in relation to reproductive microbes. These differences within the placental microbiome may influence individual health and performance. Adequate maternal nutrition and supplementation yielded the greatest placental efficiency, which may aid in the establishment of a healthy placental microbiome and fetal microbial colonization.