The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.

Natural transference of maternal microbes to the neonate, especially at birth via the vaginal canal, has recently been recognized in humans and cows; however, its microbial influence on calf health has not yet been documented. We compared the bacterial communities in vaginal and fecal samples from 81 pregnant dairy cows versus those in nasopharyngeal and fecal samples collected at 3, 14 and 35 days of life from their respective progeny. The microbiota of the calf upper respiratory tract (URT), regardless of calf age, was found to be highly similar to the maternal vaginal microbiota. Calf fecal microbiota clustered closely to the maternal fecal microbiota, progressing toward an adult-like state over the first 35 days when relative abundances of taxa were considered. Sixty-four, 65 and 87% of the detected OTUs were shared between cow and calf fecal microbiota at days 3, 14 and 35 respectively, whereas 73, 76 and 87% were shared between maternal vaginal microbiome and calf URT microbiota at days 3, 14 and 35, respectively. Bacteroidetes, Ruminococcus, Clostridium, and Blautia were the top four genera identified in maternal and calf fecal samples. Mannheimia, Moraxella, Bacteroides, Streptococcus and Pseudomonas were the top five genera identified in maternal vaginal and calf URT samples. Mannheimia was relatively more abundant in the vaginal microbiota of cows whose progeny were diagnosed with respiratory and middle ear disease. Our results indicate that maternal vaginal microbiota potentially influences the initial bacterial colonization of the calf URT, and that might have an important impact on the health of the calf respiratory tract and middle ear.

Evaluation of milk sample fractions for characterization of milk microbiota from healthy and clinical mastitis cows.

Amplicon sequencing technique has been increasingly applied to the clinical setting as a sensitive diagnostic tool. Therefore, it is of great importance to develop a DNA extraction method that accurate isolates DNA from complex host-associated microbiota. Given the multifactorial etiology of clinical mastitis and the diversified lifestyle of bacterial species harboring in milk, here four distinct milk sample fractions: raw whole milk, milk fat, casein-pellet, and casein-pellet + fat from healthy cows and cows with clinical mastitis, were subjected to bead-beating DNA extraction, followed by high-throughput sequencing. We aimed to identify the best approach for characterization of the milk microbiota and detection of mastitis pathogens (Klebsiella spp., Streptococcus spp. and Escherichia coli). DNA from each milk fraction tested was extracted by two commercial kits, which include physical, mechanical and chemical lysis; in total 280 DNA samples from 35 cows were analyzed. Milk-health-status were categorized into four groups (healthy group; E. coli-mastitis group; Klebsiella spp.-mastitis group; and Streptococcus spp.-mastitis group). Bacterial phyla and families were described for each milk-health-status group across milk sample fractions and DNA extraction kits. For the mastitis groups the relative abundance of f__Enterobacteriaceae and f__Streptococcaceae were compared to determine the efficacy of procedures in detecting the mastitis pathogens. The four milk fractions used allowed efficiently and uniformly detection of the causative agent of mastitis. Only 27% of the families detected in healthy milk were shared among the samples extracted from all fractions of milk samples; followed by 3, 4, and 12% for the samples from E. coli-mastitis, Klebsiella spp.-mastitis and Streptococcus spp-mastitis, respectively. However, the shared families comprised a mean relative abundance greater than 85%, regardless of milk-health-status, milk fraction and DNA isolation method. Taxonomic data at the family level showed that sequences from mastitis milk samples cultured positive for E. coli and Klebsiella spp. were predominantly affiliated with f__Enterobacteriaceae, while for Streptococcus spp. were dominated by f__Streptococcacea, followed by f__Pseudomonadaceae and f__Enterococcaceae. Microbial community analysis revealed that most of the microbial community composition corresponded to milk bacterial species irrespective of the DNA isolation method and milk fraction evaluated.

Microbiota of cow's milk; distinguishing healthy, sub-clinically and clinically diseased quarters.

The objective of this study was to use pyrosequencing of the 16S rRNA genes to describe the microbial diversity of bovine milk samples derived from clinically unaffected quarters across a range of somatic cell counts (SCC) values or from clinical mastitis, culture negative quarters. The obtained microbiota profiles were used to distinguish healthy, subclinically and clinically affected quarters. Two dairy farms were used for the collection of milk samples. A total of 177 samples were used. Fifty samples derived from healthy, culture negative quarters with a SCC of less than 20,000 cells/ml (group 1); 34 samples derived from healthy, culture negative quarters, with a SCC ranging from 21,000 to 50,000 cells/ml (group 2); 26 samples derived from healthy, culture negative quarters with a SCC greater than 50,000 cells/ml (group 3); 34 samples derived from healthy, culture positive quarters, with a SCC greater than 400,000 (group 4, subclinical); and 33 samples derived from clinical mastitis, culture negative quarters (group 5, clinical). Bacterial DNA was isolated from these samples and the 16S rRNA genes were individually amplified and pyrosequenced. All samples analyzed revealed great microbial diversity. Four bacterial genera were present in every sample obtained from healthy quarters (Faecalibacterium spp., unclassified Lachnospiraceae, Propionibacterium spp. and Aeribacillus spp.). Discriminant analysis models showed that samples derived from healthy quarters were easily discriminated based on their microbiota profiles from samples derived from clinical mastitis, culture negative quarters; that was also the case for samples obtained from different farms. Staphylococcus spp. and Streptococcus spp. were among the most prevalent genera in all groups while a general multivariable linear model revealed that Sphingobacterium and Streptococcus prevalences were associated with increased 10 log SCC. Conversely, Nocardiodes and Paenibacillus were negatively correlated, and a higher percentage of the genera was associated with a lower 10 log SCC.

Fecal microbial diversity in pre-weaned dairy calves as described by pyrosequencing of metagenomic 16S rDNA. Associations of Faecalibacterium species with health and growth.

In this study, we use barcoded pyrosequencing of the 16S rRNA gene to characterize the fecal microbiota of neonatal calves and identify possible relationships of certain microbiota profiles with health and weight gain. Fecal samples were obtained weekly from 61 calves from birth until weaning (seventh week of the calves' life). Firmicutes was the most prevalent phylum, with a prevalence ranging from 63.84% to 81.90%, followed by Bacteroidetes (8.36% to 23.93%), Proteobacteria (3.72% to 9.75%), Fusobacteria (0.76% to 5.67%), and Actinobacteria (1.02% to 2.35%). Chao1 index gradually increased from the first to the seventh postnatal week. Chao1 index was lower during the third, fourth, and fifth week of life in calves that suffered from pneumonia and were treated with antibiotics. Diarrhea incidence during the first four weeks of the calves' life was also associated with a reduction of microbial diversity during the third week of life. Increased fecal microbial diversity after the second week of life was associated with higher weight gain. Using discriminant analysis we were able to show differences in the microbiota profiles between different weeks of life, between high and low weight gain groups of calves, and between calves affected and not affected with diarrhea during the first four weeks life. The prevalence of Faecalibacterium spp. in the first week of life was associated with weight gain and the incidence of diarrhea, with higher prevalence being associated with higher weight gain and less diarrhea. Representative sequences from Faecalibacterium spp. were closely affiliated to Faecalibacterium prausnitzii. Results presented here provide new information regarding the intestinal microbiota of neonatal calves and its association with health and growth. Fecal microbial diversity was associated with calf age, disease status and growth rates. Results suggesting a possible beneficial effect of Faecalibacterium spp. on health and growth are promising.