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

Abstract

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.

Keywords: bovine respiratory disease; lung ultrasound; nasopharyngeal microbiota.

Figure 1.

Nonmetric multidimensional scaling (NMDS) plots depicting Bray-Curtis dissimilarity between (A) calves with USS ≥3 and calves with USS = 0, (B) calves with USS = 0, USS = 3, and USS = 4, and (C) age categories. In panel A, black triangles represent calves with USS ≥3 and black circles represent calves with USS = 0. In panel B, black circles represent USS = 0, black triangles represent USS = 3, and black diamonds represent USS = 4. In panel C, open circles represent calves at 3 wk of age and open triangles represent calves at 6 wk of age. Analysis of beta diversity between groups was tested using a permutational multivariate ANOVA or an analysis of similarities. There were no statistically significant differences between calves with USS ≥3 and calves with USS = 0 (P = 0.74), between calves with USS = 0, USS = 3, and USS = 4 (P = 0.73), or between calves at 3 wk and 6 wk of age (P = 0.63). USS = lung ultrasound score; n = 48.

Figure 2.

Nonmetric multidimensional scaling (NMDS) plots depicting Bray-Curtis dissimilarity (A) between calves that are clinical respiratory score positive (CRS+) and calves that are clinical respiratory score negative (CRS−) and (C) between calves treated previously with antibiotics (ABC+) and calves not previously treated with antibiotics (ABC−). Ellipse plots (B, D) demonstrating the 95% CI and vectors for relative abundance of genera that were different between CRS+ vs. CRS− and ABC+ vs. ABC−, respectively. In panel A, open squares represent CRS+ and open circles represent ABC−. In panel B, the black ellipse represents the 95% CI of the Bray-Curtis dissimilarity metric among CRS+ and the open circle ellipse represents the 95% CI of the Bray-Curtis dissimilarity metric among CRS−. Vector labels: * = Stenotrophomonas spp., # = Pasteurella spp. In panel C, open circles represent ABC+ and black circles represent ABC−. In panel D, the black ellipse represents the 95% CI of the Bray-Curtis dissimilarity metric among ABC+ and the open circle ellipse represents the 95% CI of the Bray-Curtis dissimilarity metric among ABC−. Vectors labels: + = Sphingobacterium spp., * = Stenotrophomonas spp., S = Chryseobacterium spp., = = Psychrobacter spp. Analysis of beta diversity between groups was tested using a permutational multivariate ANOVA or an analysis of similarities. There was a tendency for a difference in beta diversity between CRS+ and CRS− calves (P = 0.06) and between ABC+ and ABC− calves (P = 0.05); n = 48.

Figure 3.

Mean percent relative abundance (RA) of 10 abundant phyla identified in the nasopharyngeal microbiota of calves with a lung ultrasound score (USS) ≥3 and calves with an USS = 0. “Others” category includes phyla with <1% mean RA (Fusobacteria, Cyanobacteria, Fibrobacteres, Spirochaetes, and a kingdom of unclassified bacteria). Blue = Tenericutes, red = Proteobacteria, gray = Bacteroidetes, yellow = Firmicutes, purple = Actinobacteria, green = others (n = 48).

Figure 4.

(A) Mean percent relative abundance (RA) of 16 abundant genera identified in the nasopharyngeal (NP) microbiota of calves with a lung ultrasound score (USS) ≥3 (n = 23) and calves with an USS = 0 (n = 25). “Others” category includes genera with <1% mean RA. (B) Mean percent RA of 16 abundant genera identified in the NP microbiota of calves of 3 wk (n = 13) and 6 wk of age (n = 35). (C) Mean percent RA of 16 abundant genera identified in the NP microbiota of CRS+ (n = 8) and CRS− calves (n = 40). CRS = clinical respiratory score. CRS+ includes calves with a CRS ≥2 for at least 2 parameters scored using the University of Wisconsin School of Veterinary Medicine Calf Health Scoring System (https://www.vetmed.wisc.edu/fapm/svm-dairy-apps/calf-health-scorer-chs/). CRS− includes calves with a CRS <2 for parameters scored. (D) Mean percent RA of 16 abundant genera identified in the NP microbiota of ABC+ (n = 7) and ABC− calves (n = 41). ABC = antibiotics administered in 30 d before enrollment. ABC+ includes calves administered antimicrobials by farm staff for treatment of respiratory disease in the 30 d before enrollment. ABC− includes calves not previously administered antimicrobials in the 30 d before enrollment.