The nasopharyngeal microbiota of preweaned dairy calves with and without ultrasonographic lung lesions.

The primary objective of this cross-sectional study was to identify associations between the diversity and composition of the nasopharyngeal (NP) microbiota and pneumonia status, as diagnosed by ultrasonography (US), in preweaned dairy calves. Characteristics of the NP microbiota were compared between calves with and without pneumonia, as diagnosed by US. Secondary objectives were to compare the composition of the NP microbiota between calves by age, clinical respiratory score (CRS), and previous antibiotic therapy. Holstein heifer calves (n = 50) from a southern Wisconsin dairy were enrolled at either 3 or 6 wk of age; 4 calves were sampled at both time points. Antibiotic treatment history was also collected for the 30 d before enrollment. For the purpose of this study, pneumonia was defined as having lobar pneumonia, as diagnosed by US, in at least 1 lung lobe. Following examination by CRS and US, a deep nasopharyngeal swab was obtained for 16S rRNA amplicon sequencing. Alpha diversity was reduced in calves that were CRS positive, and beta diversity tended to be different in calves previously treated with antibiotics and in calves that were CRS positive. Microbial diversity was not different between calves with and without pneumonia. The most dominant genus identified was Mycoplasma spp.; however, there was no association between relative abundance (RA) and pneumonia status. The median RA of Mycoplasma spp. was increased by 25 (95% confidence interval, CI: 3, 40) in calves at 3 wk of age compared with 6 wk of age. The median RA of Pasteurella spp. was increased by 1.5 (95% CI: 0.1, 3) in calves with pneumonia, as diagnosed by US, compared with calves without pneumonia. Additionally, Pasteurella spp. was increased by 2.3 (95% CI: 0, 9) in CRS-positive calves compared with CRS-negative calves. The median RA of Psychrobacter spp. was increased by 2 (95% CI: 0, 12) and median RA of Chryseobacterium spp. was increased by 0.15 (95% CI: 0, 2) in calves that were not treated previously with antibiotics compared with calves previously treated with antibiotics.

Influence of sampling technique and bedding type on the milk microbiota: Results of a pilot study.

The objective of this pilot study was to evaluate the influence of sampling technique and exposure to different bedding types on the milk microbiome of healthy primiparous cows. Primiparous Holstein cows (n = 20) with no history of clinical mastitis or monthly somatic cell counts >150,000 cells/mL were selected for this study. From each enrolled cow, a composite milk sample was aseptically collected from all 4 mammary quarters (individual quarter somatic cell counts <100,000 cells/mL), 1 individual quarter milk sample was collected using conventional aseptic technique, and 2 individual quarter milk samples were collected directly from the gland cistern using a needle and vacuum tube. All milk samples were cultured using standard milk microbiological techniques and DNA was extracted. Extracted DNA was subjected to PCR and next-generation sequencing for microbiota determination. All samples yielded relatively little total DNA. Amplification of PCR was successful in 45, 40, and 83% of composite, conventional, and cisternal samples, respectively. Bacteria were successfully cultured from 35% of composite milk samples but from none of the quarter milk samples collected using conventional or cisternal sampling techniques. Bacterial DNA sequences were assigned to operational taxonomic units (OTU) based on 97% sequence similarity, and bacterial richness and diversity were determined. Most samples were dominated by low-prevalence OTU and of the 4,051 identified OTU, only 14 were prevalent at more than 1% each. These included bacteria typically recovered from environmental sources. Chao richness was greatest in composite samples and was 636, 347, and 356 for composite, conventional quarter, and cisternal milk samples, respectively. Shannon diversity was similar among sample types and ranged from 3.88 (quarter) to 4.17 (composite). Richness and diversity did not differ by bedding type among cisternal samples, but the power of this pilot study was limited due to small sample size. Despite the small sample size, for milk samples collected from the gland cistern, overall bacterial community composition differed among bedding types. These results demonstrate that sampling technique and bedding type may be associated with the microbiota detected in bovine milk, and we suggest that these variables should be considered in designing and reporting studies about the milk microbiota.