The purpose of this prospective case-control study was to describe bacterial communities in the nasopharynx (NP) of preweaned dairy calves with and without ultrasonographic lung lesions. Additional objectives included evaluating the effects of previous antibiotic therapy and age on the NP microbiota composition. A total of 257 Holstein heifer calves were enrolled into a separate study investigating the genomics of resistance to bovine respiratory disease (BRD) over a 4-week follow up period. Calves were examined twice using thoracic ultrasound and clinical respiratory score (early exam: 4 weeks old; late exam: 7 weeks old). From this population, case and age-matched controls were selected to undergo deep NP swabbing for the current study. Cases were defined by the presence of significant ultrasonographic lung lesions (≥ 1 lobe completely consolidated). Controls were defined by the lack of these lesions.
The NP swabs were taken at the time of exam and calf information was collected from farm management software. Swabs were placed in phosphate buffer solution, and stored at -80°C. Following DNA extraction, the V4 region of the 16S rRNA gene was amplified using PCR. Libraries were sequenced using the MiSeq platform. The generated sequences were processed through Mothur 1.36.1 and outputted taxonomy and diversity data were analyzed in RStudio. Diversity data was analyzed using t-tests and general linear models. Comparisons of relative abundance (RA) of genera between groups were performed using Kruskal Wallis tests. Multiple linear regression was used to investigate the impact of time point of exam and antibiotic treatment, in the month prior to examination, on RA of genera.
In total, 50 swabs (1:1 case to control) were collected. Two swabs were lost during transport, therefore, 48 swabs (cases = 23, controls = 25) obtained from 44 calves, were used for analysis. Thirty-five (73%) swabs were collected during the late exam. Overall, the most common genera were Acinetobacter sp, Escherichia sp, Mycoplasma sp, Pasteurella sp and Psychrobacter sp. Alpha diversity was not different between cases and controls (P = 0.78), nor between early and late time points (P = 0.37). The RA of Mycoplasma sp. was higher in controls (P = 0.03) and the RA of Pasteurella sp. tended to be higher in controls (P = 0.08). The RA of both Mycoplasma sp. and Pasteurella sp. was not affected by exam time point or antibiotic treatment (P > 0.75 and P > 0.12, respectively).
To the authors’ knowledge, this is the first study to evaluate the community dynamics of the NP microbiota from calves with and without ultrasonographic lung lesions. We did not expect to identify a higher RA of Mycoplasma sp. in the control calves, as previous studies have shown a higher RA of Mycoplasma sp. in calves with BRD compared to healthy calves. Future studies are needed to better understand this discrepancy, including metagenomics studies to infer speciation and determine pathogenicity of the identified Mycoplasma genus. It would also be important to know if the duration of ultrasound lesions affected the RA of pathogenic bacteria. This study helps to demonstrate the complexities of identifying NP microbiota changes associated with disease status. This may impact future attempts to utilize NP microbiota characteristics in diagnosis and treatment of BRD.
Clinical instructor and PhD student
University of Wisconsin-Madison
I completed my DVM at Tufts University Cummings School of Veterinary Medicine in 2012 and then completed an internship in food animal medicine and surgery at Colorado State University in 2013. I finished my residency in large animal internal medicine at the University of Wisconsin and achieved Diplomate certification in 2016. I am currently pursuing a PhD at the University of Wisconsin while working as a clinical instructor in the Large Animal Hospital.
Thursday, June 14
11:00 AM – 11:15 AM
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