MALDI-TOF MS biomarker detection models to distinguish RTX toxin phenotypes of Moraxella bovoculi strains are enhanced using calcium chloride supplemented agar

Authors: HILLE, MATTHEW - University Of Nebraska; Clawson, Michael - Mike; Dickey, Aaron; LOWERY, JUSTIN - University Of Nebraska; LOY, JOHN - University Of Nebraska

Submitted to: Frontiers in Cellular and Infection Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2021
Publication Date: 3/16/2021
Citation: Hille, M.M., Clawson, M.L., Dickey, A.M., Lowery, J.H., Loy, J.D. 2021. MALDI-TOF MS biomarker detection models to distinguish RTX toxin phenotypes of Moraxella bovoculi strains are enhanced using calcium chloride supplemented agar. Frontiers in Cellular and Infection Microbiology. 11. Article 632647. https://doi.org/10.3389/fcimb.2021.632647.
DOI: https://doi.org/10.3389/fcimb.2021.632647

Interpretive Summary: Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is the most common eye disease of cattle. IBK primarily affects calves and can cause substantial pain and blindness in severe cases. Over ten million calves are infected each year in the United States. In addition to an animal well-being issue, IBK imparts a large economic toll from the treatment, prevention, and deleterious effects of the disease. Moraxella bovoculi is a species of bacteria that is associated with IBK. There are different strains of M. bovoculi, including those that either have, or do not have a toxin (known as a repeats-in-toxin or RTX). This toxin is one of the most potent bacterial virulence factors that cause IBK. Strains that have the toxin secrete it, resulting in substantial damage to the eyes and facilitating bacterial invasion of damaged eye tissue. In this manuscript, we report a test that can rapidly and cost effectively detect whether or not M. bovoculi strains have the toxin in minutes. In addition to identifying strain carriers of the toxin, the test can be used in conjunction with existing diagnostic to direct treatment plans of affected animals, and could be used for the development of efficacious vaccines against M. bovoculi at the strain level.

Technical Abstract: Moraxella bovoculi is the bacterium most commonly cultured from ocular lesions of cattle with infectious bovine keratoconjunctivitis, also known as bovine pinkeye. Some strains of M. bovoculi contain operons encoding for a repeats-in-toxin (RTX) toxins, which is a known virulence factor of multiple veterinary pathogens. We explored the utility of MALDI-TOF MS and biomarker detection models to classify the presence or absence of an RTX phenotype in M. bovoculi. Ninety strains that had undergone whole genome sequencing were classified by the presence or absence of complete RTX operons and confirmed with a visual assessment of hemolysis on blood agar. Strains were grown on Tryptic Soy Agar (TSA) with 5% sheep blood, TSA with 5% bovine blood that was supplemented with 10% fetal bovine serum, 10 mmol/ CaCl2, or both. The formulations were designed to determine the influence of growth media on toxin production or activity, as calcium ions are required for toxin secretion and activity. Mass spectra was obtained for strains grown on each agar formulation and biomarker models were developed using ClinProTools 3.0 software. The most accurate model was developed using spectra from strains grown on TSA with 5% bovine blood and supplemented with CaCl2, which had a sensitivity and specificity of 93.3% and 73.3%, respectively, regarding RTX phenotype classification. The same biomarker model algorithm developed from strains grown on TSA with 5% sheep blood had a substantially lower sensitivity and specificity of 68.0% and 52.0%, respectively. Our results indicate that MALDI-TOF MS biomarker models can accurately classify strains of M. bovoculi regarding the presence or absence of RTX toxin operons and that agar media modifications improve the accuracy of these models.