Svetlana F. Lima

Prepartum and Postpartum Rumen Fluid Microbiomes: Characterization and Correlation with Production Traits in Dairy Cows

ABSTRACT Microbes present in the rumen of dairy cows are essential for degradation of cellulosic and nonstructural carbohydrates of plant origin. The prepartum and postpartum diets of high-producing dairy cows are substantially different, but in what ways the rumen microbiome changes in response and how those changes may influence production traits are not well elucidated. Here, we sequenced the 16S and 18S rRNA genes using the MiSeq platform to characterize the prepartum and postpartum rumen fluid microbiomes in 115 high-producing dairy cows, including both primiparous and multiparous animals. Discriminant analysis identified differences between the microbiomes of prepartum and postpartum samples and between primiparous and multiparous cows. 18S rRNA sequencing revealed an overwhelming dominance of the protozoan class Litostomatea, with over 90% of the eukaryotic microbial population belonging to that group. Additionally, fungi were relatively more prevalent and Litostomatea relatively less prevalent in prepartum samples than in postpartum ones. The core rumen microbiome (common to all samples) consisted of 64 bacterial taxa, of which members of the genus Prevotella were the most prevalent. The Chao1 richness index was greater for prepartum multiparous cows than for postpartum multiparous cows. Multivariable models identified bacterial taxa associated with increased or reduced milk production, and general linear models revealed that a metagenomically based prediction of productivity is highly associated with production of actual milk and milk components. In conclusion, the structure of the rumen fluid microbiome shifts between the prepartum and first-week postpartum periods, and its profile within the context of this study could be used to accurately predict production traits.

Altered microbiomes in bovine digital dermatitis lesions, and the gut as a pathogen reservoir.

Bovine digital dermatitis (DD) is the most important infectious disease associated with lameness in cattle worldwide. Since the disease was first described in 1974, a series of Treponema species concurrent with other microbes have been identified in DD lesions, suggesting a polymicrobial etiology. However, the pathogenesis of DD and the source of the causative microbes remain unclear. Here we characterized the microbiomes of healthy skin and skin lesions in dairy cows affected with different stages of DD and investigated the gut microbiome as a potential reservoir for microbes associated with this disease. Discriminant analysis revealed that the microbiomes of healthy skin, active DD lesions (ulcerative and chronic ulcerative) and inactive DD lesions (healing and chronic proliferative) are completely distinct. Treponema denticola, Treponema maltophilum, Treponema medium, Treponema putidum, Treponema phagedenis and Treponema paraluiscuniculi were all found to be present in greater relative abundance in active DD lesions when compared with healthy skin and inactive DD lesions, and these same Treponema species were nearly ubiquitously present in rumen and fecal microbiomes. The relative abundance of Candidatus Amoebophilus asiaticus, a bacterium not previously reported in DD lesions, was increased in both active and inactive lesions when compared with healthy skin. In conclusion, our data support the concept that DD is a polymicrobial disease, with active DD lesions having a markedly distinct microbiome dominated by T. denticola, T. maltophilum, T. medium, T. putidum, T. phagedenis and T. paraluiscuniculi. Furthermore, these Treponema species are nearly ubiquitously found in rumen and fecal microbiomes, suggesting that the gut is an important reservoir of microbes involved in DD pathogenesis. Additionally, the bacterium Candidatus Amoebophilus asiaticus was highly abundant in active and inactive DD lesions.

Subcutaneous Immunization with Inactivated Bacterial Components and Purified Protein of Escherichia coli, Fusobacterium necrophorum and Trueperella pyogenes Prevents Puerperal Metritis in Holstein Dairy Cows

In this study we evaluate the efficacy of five vaccine formulations containing different combinations of proteins (FimH; leukotoxin, LKT; and pyolysin, PLO) and/or inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes) in preventing postpartum uterine diseases. Inactivated whole cells were produced using two genetically distinct strains of each bacterial species (E. coli, F. necrophorum, and T. pyogenes). FimH and PLO subunits were produced using recombinant protein expression, and LKT was recovered from culturing a wild F. necrophorum strain. Three subcutaneous vaccines were formulated: Vaccine 1 was composed of inactivated bacterial whole cells and proteins; Vaccine 2 was composed of proteins only; and Vaccine 3 was composed of inactivated bacterial whole cells only. Two intravaginal vaccines were formulated: Vaccine 4 was composed of inactivated bacterial whole cells and proteins; and Vaccine 5 was composed of PLO and LKT. To evaluate vaccine efficacy, a randomized clinical trial was conducted at a commercial dairy farm; 371 spring heifers were allocated randomly into one of six different treatments groups: control, Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5. Late pregnant heifers assigned to one of the vaccine groups were each vaccinated twice: at 230 and 260 days of pregnancy. When vaccines were evaluated grouped as subcutaneous and intravaginal, the subcutaneous ones were found to significantly reduce the incidence of puerperal metritis. Additionally, subcutaneous vaccination significantly reduced rectal temperature at 6±1 days in milk. Reproduction was improved for cows that received subcutaneous vaccines. In general, vaccination induced a significant increase in serum IgG titers against all antigens, with subcutaneous vaccination again being more effective. In conclusion, subcutaneous vaccination with inactivated bacterial components and/or protein subunits of E. coli, F. necrophorum and T. pyogenes can prevent puerperal metritis during the first lactation of dairy cows, leading to improved reproduction.

Uterine Microbiota Progression from Calving until Establishment of Metritis in Dairy Cows

ABSTRACT The objective of this study was to evaluate the progression of the uterine microbiota from calving until establishment of metritis. Uterine swabs (n = 72) collected at 0, 2, and 6 ± 2 days postpartum (dpp) from 12 metritic and 12 healthy cows were used for metagenomic sequencing of the 16S rRNA gene on the Illumina MiSeq platform. A heat map showed that uterine microbiota was established at calving. The microbiota changed rapidly from 0 to 6 ± 2 dpp, with a decrease in the abundance of Proteobacteria and an increase in the abundance of Bacteroidetes and Fusobacteria, which were dominant in metritic cows. Uterine microbiota composition was shared; however, metritic and healthy cows could be discriminated using relative abundance of bacterial genera at 0, 2, and 6 ± 2 dpp. Bacteroides was the main genus associated with metritis because it was the only genus that showed significantly greater abundance in cows with metritis. As the abundance of Bacteroides organisms increased, the uterine discharge score, a measure of uterine health, worsened. Fusobacterium was also an important genus associated with metritis because Fusobacterium abundance increased as Bacteroides abundance increased and the uterine discharge score worsened as the abundance increased. The correlation with uterine discharge score and the correlation with Bacteroides or Fusobacterium showed that other bacteria, such as Helcoccocus, Filifactor, and Porphyromonas, were also associated with metritis. There were also bacteria associated with uterine health, such as “Candidatus Blochmannia,” Escherichia, Sneathia, and Pedobacter.

Alterations to the Gut Microbiome Impair Bone Strength and Tissue Material Properties

Alterations in the gut microbiome have been associated with changes in bone mass and microstructure, but the effects of the microbiome on bone biomechanical properties are not known. Here we examined bone strength under two conditions of altered microbiota: (1) an inbred mouse strain known to develop an altered gut microbiome due to deficits in the immune system (the Toll‐like receptor 5–deficient mouse [TLR5KO]); and (2) disruption of the gut microbiota (ΔMicrobiota) through chronic treatment with selected antibiotics (ampicillin and neomycin). The bone phenotypes of TLR5KO and WT (C57Bl/6) mice were examined after disruption of the microbiota from 4 weeks to 16 weeks of age as well as without treatment (n = 7 to 16/group, 39 animals total). Femur bending strength was less in ΔMicrobiota mice than in untreated animals and the reduction in strength was not fully explained by differences in bone cross‐sectional geometry, implicating impaired bone tissue material properties. Small differences in whole‐bone bending strength were observed between WT and TLR5KO mice after accounting for differences in bone morphology. No differences in trabecular bone volume fraction were associated with genotype or disruption of gut microbiota. Treatment altered the gut microbiota by depleting organisms from the phyla Bacteroidetes and enriching for Proteobacteria, as determined from sequencing of fecal 16S rRNA genes. Differences in splenic immune cell populations were also observed; B and T cell populations were depleted in TLR5KO mice and in ΔMicrobiota mice (p < 0.001), suggesting an association between alterations in bone tissue material properties and immune cell populations. We conclude that alterations in the gut microbiota for extended periods during growth may lead to impaired whole‐bone mechanical properties in ways that are not explained by bone geometry.

Longitudinal metagenomic profiling of bovine milk to assess the impact of intramammary treatment using a third-generation cephalosporin

Antimicrobial usage in food animals has a direct impact on human health, and approximately 80% of the antibiotics prescribed in the dairy industry are used to treat bovine mastitis. Here we provide a longitudinal description of the changes in the microbiome of milk that are associated with mastitis and antimicrobial therapy. Next-generation sequencing, 16 S rRNA gene quantitative real-time PCR, and aerobic culturing were applied to assess the effect of disease and antibiotic therapy on the milk microbiome. Cows diagnosed with clinical mastitis associated with Gram-negative pathogens or negative aerobic culture were randomly allocated into 5 days of Ceftiofur intramammary treatment or remained as untreated controls. Serial milk samples were collected from the affected quarter and the ipsilateral healthy quarter of the same animal. Milk from the mastitic quarter had a higher bacterial load and reduced microbial diversity compared to healthy milk. Resolution of the disease was accompanied by increases in diversity indexes and a decrease in pathogen relative abundance. Escherichia coli-associated mastitic milk samples had a remarkably distinct bacterial profile, dominated by Enterobacteriaceae, when compared to healthy milk. However, no differences were observed in culture-negative mastitis samples when compared to healthy milk. Antimicrobial treatment had no significant effect on clinical cure, bacteriological cure, pathogen clearance rate or bacterial load.

The effect of injectable trace minerals (selenium, copper, zinc, and manganese) on peripheral blood leukocyte activity and serum superoxide dismutase activity of lactating Holstein cows.

The objective of this study was to evaluate the effect of subcutaneous supplementation of 300 mg of zinc, 50 mg of manganese, 25 mg of selenium, and 75 mg of copper on peripheral blood leukocyte activity and serum β-hydroxybutyrate (BHBA) concentrations at 10 ± 2 days in milk (DIM), and on serum superoxide dismutase (SOD) activity during the transition period and subsequent lactation of multiparous Holstein cows. A total of 250 multiparous cows were randomly allocated into one of two treatments groups, namely, trace mineral supplemented (TMS) or control. Cows in the TMS group were injected at 230 and 260 days of gestation, and 35 days postpartum. Serum SOD activity was measured at enrollment, and 10, 60 and 100 DIM. Serum BHBA concentration and leukocyte function were assessed at 10 DIM. Overall serum SOD activity for TMS and control was 16.01 and 12.71 U/mL, respectively. The interaction between treatment and time of serum collection was significant. Additionally, overall serum SOD activity was 12.85 and 14.78 U/mL for cows diagnosed with mastitis and unaffected cows, respectively. Treatment did not affect leukocyte function. For parity >2, TMS cows had lower serum BHBA concentrations than control cows; BHBA concentrations were 0.41 and 0.27 mmol/L for control and TMS cows, respectively. In conclusion, cows diagnosed with mastitis had decreased serum SOD activity, and trace mineral supplementation increased serum SOD activity although leukocyte function was not affected by supplementation.

The bovine colostrum microbiome and its association with clinical mastitis

In an effort to characterize colostrum microbial diversity and its potential associations with early-lactation clinical mastitis, we used high-throughput sequencing of the 16S rRNA gene to investigate the bovine colostrum microbiome. A prospective observational study was conducted that included 70 Holstein cows; colostrum samples were collected from all 4 mammary gland quarters. Colostrum samples were categorized according to whether the quarter was diagnosed (CMC) or not diagnosed (NCMC) with clinical mastitis during the first 30 d postpartum. Colostrum samples were dominated by Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Fusobacteria, and Tenericutes phyla, with the 6 most common taxa [order (o), family (f), and genus (g)] being g_Staphylococcus, g_Prevotella, f_Ruminococcaceae, o_Bacteroidales, o_Clostridiales, and g_Pseudomonas. The colostrum microbiota of primiparous cows was significantly richer (higher number of bacterial species) than that of multiparous cows, and differences in colostrum taxonomic structure between parities were also observed. The microbial community of NCMC samples of primiparous cows was significantly more diverse than that of CMC samples, and the relative abundances of the Tenericutes and Fusobacteria phyla as well as the Mycoplasma and Fusobacterium genera were significantly higher in NCMC than in CMC samples of primiparous cows. The colostrum core microbiome, defined as the bacterial taxa common to all colostrum samples examined, was composed of 20 taxa and included bacterial genera already known to be associated with mastitis (e.g., Staphylococcus, Mycoplasma, and Streptococcus spp.). Our results indicate that the colostrum microbiome of primiparous cows differs from that of multiparous cows, and it harbors some diversity and taxonomic markers of mammary gland health specific to primiparous cows only.

The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.

The upper respiratory tract (URT) hosts a complex microbial community of commensal microorganisms and potential pathogens. Analyzing the composition and nature of the healthy URT microbiota and how it changes over time will contribute to a better understanding of the pathogenesis of pneumonia and otitis. A longitudinal study was conducted including 174 Holstein calves that were divided in four groups: healthy calves, calves diagnosed with pneumonia, otitis or both diseases. Deep pharyngeal swabs were collected on days 3, 14, 28, and 35 of life, and next-generation sequencing of the 16S rRNA gene as well as quantitative PCR was performed. The URT of Holstein dairy calves aged 3 to 35 days revealed to host a highly diverse bacterial community. The relative abundances of the bacterial genera Mannheimia, Moraxella, and Mycoplasma were significantly higher in diseased versus healthy animals, and the total bacterial load of newborn calves at day 3 was higher for animals that developed pneumonia than for healthy animals. Our results corroborate the existing knowledge that species of Mannheimia and Mycoplasma are important pathogens in pneumonia and otitis. Furthermore, they suggest that species of Moraxella can potentially cause the same disorders (pneumonia and otitis), and that high neonatal bacterial load is a key contributor to the development of pneumonia.

Ingestion of Milk Containing Very Low Concentration of Antimicrobials: Longitudinal Effect on Fecal Microbiota Composition in Preweaned Calves

Although antimicrobial drugs are central to combat disease in modern medicine, the use of these drugs can have undesired consequences for human and animal health. One consequence is the post-therapy excretion of pharmacological agents, such as the elimination of drug residues at very low concentrations in the milk of lactating mammals. Limited information is currently available on the impact from the exposure of the gut microbiota to drug residues using in vivo natural models. The objective of our study was to address this knowledge gap and evaluate the effect on the fecal microbiota composition from feeding preweaned dairy calves raw milk with residual concentrations of ampicillin, ceftiofur, penicillin, and oxytetracycline from birth to weaning. At birth, thirty calves were randomly assigned to a controlled feeding trial where: 15 calves were fed raw milk with no drug residues (NR), and 15 calves were fed raw milk with drug residues (DR) by adding ceftiofur, penicillin, ampicillin, and oxytetracycline at final concentrations in the milk of 0.1, 0.005, 0.01, and 0.3 μg/ml, respectively. Fecal samples were rectally collected from each calf once a week starting at birth, prior to the first feeding in the trial (pre-treatment), until 6 weeks of age. Sequencing of the microbial 16S rRNA genes was conducted using the Illumina MiSeq, which provides a high resolution of the microbiota down to the genus level. Discriminant analysis showed that, except for pre-treatment samples, calves fed milk with drug residues and calves fed milk without drug residues easily discriminated at the genus level on their weekly microbial profile. However, analysis comparing the abundance of taxon between NR and DR showed significant differences only at the genus levels, and not at the phylum, class, order or family levels. These results suggest that although drug residues can result in clear discriminate gut microbial communities, they do not result in disruption of taxonomic levels above the genus.