Does swab type matter? Comparing methods for Mannheimia haemolytica recovery and upper respiratory microbiome characterization in feedlot cattle

Abstract

Background: Bovine respiratory disease (BRD) is caused by interactions among host, environment, and pathogens. One standard method for antemortem pathogen identification in cattle with BRD is deep-guarded nasopharyngeal swabbing, which is challenging, costly, and waste generating. The objective was to compare the ability to recover Mannheimia haemolytica and compare microbial community structure using 29.5 inch (74.9 cm) deep-guarded nasopharyngeal swabs, 16 inch (40.6 cm) unguarded proctology swabs, or 6 inch (15.2 cm) unguarded nasal swabs when characterized using culture, real time-qPCR, and 16S rRNA gene sequencing. Samples for aerobic culture, qPCR, and 16S rRNA gene sequencing were collected from the upper respiratory tract of cattle 2 weeks after feedlot arrival.

Results: There was high concordance of culture and qPCR results for all swab types (results for 77% and 81% of sampled animals completely across all 3 swab types for culture and qPCR respectively). Microbial communities were highly similar among samples collected with different swab types, and differences identified relative to treatment for BRD were also similar. Positive qPCR results for M. haemolytica were highly concordant (81% agreed completely), but samples collected by deep-guarded swabbing had lower amounts of Mh DNA identified (Kruskal-Wallis analysis of variance on ranks, P < 0.05; Dunn-test for pairwise comparison with Benjamini-Hochberg correction, P < 0.05) and lower frequency of positive compared to nasal and proctology swabs (McNemar's Chi-square test, P < 0.05).

Conclusions: Though differences existed among different types of swabs collected from individual cattle, nasal swabs and proctology swabs offer comparable results to deep-guarded nasopharyngeal swabs when identifying and characterizing M. haemolytica by culture, 16S rRNA gene sequencing, and qPCR.

Keywords: 16S rRNA gene sequencing; Antimicrobial resistance; Bovine respiratory disease; Culture; Disease surveillance; Metagenomics; qPCR.

Fig. 1

Antimicrobial resistance patterns of M. haemolytica isolated from each calf separated by swab type. Each isolate is identified by the calf from which it was isolated. Blank lines indicate calves that did not have M. haemolytica isolated from that swab, though it was M. haemolytica positive via (an)other swab type(s). S susceptible, I/R intermediate or resistant, CEFT ceftiofur, DANO danofloxacin, ENRO enrofloxacin, GAMI gamithromycin, PEN penicillin, SPEC spectinomycin, TET tetracycline, TILD tildipirosin, TILM tilmicosin, TUL tulathromycin

Fig. 2

Box and whisker of log(ng Mh DNA) per ng DNA added vs. swab type. (Kruskal–Wallis test, P < 0.05; *pairwise Dunn test with Benjamini–Hochberg correction, P < 0.05). Key: DG = double guarded swab, NS = nasal swab, and PS = proctology swab

Fig. 3

Box and whisker of log(ng Mh DNA) per ng DNA added vs. swab type, separated by BRD treatment (Kruskal–Wallis test, P > 0.05). Key: DG = double guarded swab, NS = nasal swab, and PS = proctology swab, BRD:Y = BRD positive, BRD:N = BRD negative

Fig. 4

Box and whisker of log(ng Mh DNA) per ng DNA added vs. culture results (Wilcoxon rank-sum test, P < 0.05)

Fig. 5

Box and whisker plots of log(ng Mh DNA) per ng DNA added vs sampling group, separated by Swab Type (Wilcoxon rank-sum test, P < 0.05)

Fig. 6

A Principal coordinates analysis (PcoA) of generalized UniFrac values illustrating differences in microbial community composition between samples collected with each swab type. The PCoA demonstrates clustering of ASVs from microbial communities collected with DG swabs, NS, or PS. Shaded areas represent 95% confidence ellipses for each swab type. Microbial community composition differed significantly between each community type (pairwise PERMANOVA with Benajmin-Hochberg correction, P < 0.005). B Barplot showing the relative abundances of the six phyla representing greater than 1% of the whole community illustrating the variation in microbial community structure across all samples. Error bars on the barplot demonstrate the standard error of the mean relative abundance for each of the six phyla when sampled using DG swabs, NS, or PS. Significant differences between relative abundances as collected with each swab type are illustrated by different letters (Pairwise Wilcoxon rank-sum test with Benjamini–Hochberg correction, P < 0.05)

Fig. 7

Bar plot illustrating the mean relative abundance of microbial orders within BRD negative or positive animals as sampled with DG swab, NS, or PS. Abundances were normalized to the total number of CSS-normalized ASVs within each sample. The 8 most abundant orders are displayed in the legend

Fig. 8

Bar plot demonstrating differences in the relative abundance of each of the 6 most abundant phyla in BRD negative and BRD positive animals as collected with DG swabs, nasal swabs, or proctology swabs. Error bars display the standard error of the mean. Significant differences among relative abundances within each phylum are noted with an asterisk (Kruskal–Wallis analysis of variance by ranks, P < 0.05)

Fig. 9

Bar plot demonstrating differences in the relative abundance of microbial taxa of interest within BRD negative and BRD positive animals as collected with DG swabs, nasal swabs, or proctology swabs. Error bars display the standard error of the mean. Significant differences among relative abundances within each phylum and swab type are noted with an asterisk (Kruskal–Wallis analysis of variance by ranks, P < 0.05). Note the difference in the relative abundance scale for 4A and 4B

Fig. 10

Bar plot showing the relative abundances among all classified taxa for five Pasteurellaceae genera and unassigned Pasteurellaceae ASVs within M. haemolytica culture-negative and culture-positive animals, as collected with DG swabs, nasal swabs, or proctology swabs. Error bars demonstrate the standard error of the mean relative abundance of Pasteurellaceae. The six most abundant genera across all samples are displayed in the legend. Abbreviations: un., unclassified