Whole genome sequencing of Moraxella bovoculi reveals high genetic diversity and evidence for interspecies recombination at multiple loci

Authors: Dickey, Aaron; Schuller, Genevieve - Gennie; LOY, JOHN - UNIVERSITY OF NEBRASKA; Clawson, Michael - Mike

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/29/2018
Publication Date: 12/17/2018
Citation: Dickey, A.M., Schuller, G., Loy, J.D., Clawson, M.L. 2018. Whole genome sequencing of Moraxella bovoculi reveals high genetic diversity and evidence for interspecies recombination at multiple loci. PLoS One. 13(12):e0209113. https://doi.org/10.1371/journal.pone.0209113.
DOI: https://doi.org/10.1371/journal.pone.0209113

Interpretive Summary: Infectious Bovine Keratoconjunctivitis (IBK), also known as “pinkeye” affects up to 10% of beef cattle in the United States. The disease is a significant animal health and welfare concern, as it can result in eye ulceration, substantial pain, and blindness in severe cases. IBK can be successfully treated with the antibiotics tulathromycin and oxytetracycline, however, we, and others have found microbes with resistance genes to both of those antibiotics in cattle. Thus, there is a real need for efficacious vaccines that protect cattle from pinkeye and thus reduce the need for antibiotic treatments. There are two bacterial species of Moraxella that associate with IBK. One is Moraxella bovis, which is known to cause the disease. The other is Moraxella bovoculi, which was only recently discovered, and although a single strain has been experimentally shown not to cause IBK, M. bovoculi is isolated from IBK positive eyes much more frequently then M. bovis, suggesting that some strains may have an important biological role in IBK. We previously showed that there are two genetically distinct strain types, or genotypes (genotypes 1 and 2) of M. bovoculi in cattle that appear to vary in their pathogenic potential, and possibly their niche distributions. We also identified a single genotype 1 isolate with resistance genes to tulathromycin, and oxytetracycline. In this study, the genomes of 243 M. bovoculi isolates were sequenced and analyzed. We show for the first time that isolates of either genotype can be isolated from the eyes of cattle without signs of IBK. We further show that genotype 1 M. bovoculi have a high frequency of pathogenesis genes and overall diversity, and that they are commonly found in IBK positive eyes. We additionally detected genotype 1 isolates with multiple antibiotic resistance genes, including those encoding resistance to tulathromycin and oxytetracycline, in support of our previous observation. Additional studies will be required to determine if genotype 2 M. bovoculi are statistically less frequently present in IBK eyes then genotype 1, and if there are significant frequency differences between pathogen gene contents between genotype 1 and 2 strains. However, the results of this study confirm that there are distinctly different strains of M. bovoculi in the eyes of cattle that can differ by their complement of virulence factors and antibiotic resistance genes. Vaccines that target M. bovoculi may have to be designed to account for extensive genetic diversity that exists between strains.

Technical Abstract: Moraxella bovoculi is frequently cultured from the ocular secretions and conjunctiva of cattle with Infectious Bovine Keratoconjunctivitis (IBK). Previous work has shown that single nucleotide polymorphism (SNP) diversity in this species is quite high with 81,284 SNPs identified in eight genomes representing two distinct genotypes isolated from IBK affected eyes (genotype 1) and the nasopharynx of cattle without clinical IBK signs (genotype 2), respectively. The goals of this study were to identify SNPs from a collection of geographically diverse and epidemiologically unlinked M. bovoculi strains from the eyes of IBK positive cattle (n=183) and another from the eyes of cattle (most from a single population at a single time-point) without signs of IBK (n=63) and to characterize the genetic diversity. Strains of both genotypes were identified from the eyes of cattle without IBK signs. Only genotype 1 strains were identified from IBK affected eyes, however, these strains were isolated before the discovery of genotype 2, and the protocol for their isolation would have preferentially selected genotype 1 M. bovoculi. The core genome comprised ~74% of the whole and contained >127,000 filtered SNPs. More than 80% of these characterize diversity within genotype 1 while 23,611 SNPs (~18%) delimit the two major genotypes. Genotype 2 strains lacked a repeats-in-toxin (RTX) putative pathogenesis factor and any of ten antibiotic resistance genes carried within a genomic island. Within genotype 1, prevalence of these elements was 0.85 and 0.12 respectively in strains from eyes that were IBK positive. Recombination appears to be an important source of genetic diversity for genotype 1 and undermines the utility of ribosomal-locus-based species identification. The extremely high genetic diversity in genotype 1 presents a challenge to the development of an efficacious vaccine directed against them, however, several low-diversity pilin-like genes were identified. Finally, the genotype-defining SNPs described in this study are a resource that can facilitate the development of more accurate M. bovoculi diagnostic tests.