Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of Moraxella bovoculi and Moraxella bovis isolates from cattle.
ABSTRACT: Infectious bovine keratoconjunctivitis (IBK) is an economically significant disease caused by Moraxella bovis. Moraxella bovoculi, although not reported to cause IBK, has been isolated from the eyes of cattle diagnosed with IBK. Identification of M. bovis and M. bovoculi can be performed using biochemical or DNA-based approaches, both of which may be time consuming and inconsistent between laboratories. We conducted a comparative evaluation of M. bovoculi and M. bovis identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a database provided by Bruker Daltonics (termed the BDAL database), the BDAL database supplemented with spectra generated in our study (termed the UNLVDC database), and with PCR-restriction-fragment length polymorphism (PCR-RFLP) typing. M. bovoculi ( n = 250) and M. bovis ( n = 18) isolates from cattle with or without IBK were used. MALDI-TOF MS using the UNLVDC database correctly identified 250 of 250 (100%) of M. bovoculi and 17 of 18 (94%) of M. bovis isolates. With the BDAL database, MALDI-TOF MS correctly identified 249 of 250 (99%) of M. bovoculi and 7 of 18 (39%) of M. bovis isolates. In comparison, the PCR-RFLP test correctly identified 210 of 250 (84%) of M. bovoculi and 12 of 18 (66%) of M. bovis isolates. Thus, MALDI-TOF MS with the UNLVDC database was the most effective identification methodology for M. bovis and M. bovoculi isolates from cattle.
Project description:<b>Introduction.</b> <i>Moraxella bovoculi</i> is frequently isolated from the eyes of cattle with infectious bovine keratoconjunctivitis (IBK; pinkeye). As with <i>M. bovis,</i> which has been causally linked to IBK, <i>M. bovoculi</i> expresses an RTX (repeats in the structural toxin) cytotoxin that is related to <i>M. bovis</i> cytotoxin. Pilin, another pathogenic factor in <i>M. bovis</i>, is required for corneal attachment. Seven antigenically distinct pilin serogroups have been described in <i>M. bovis</i>.<b>Hypothesis/Gap Statement.</b> Multiple different serogroups exist amongst type IV pilin encoded by <i>M. bovis</i>, however, it is not known whether <i>M. bovoculi</i> exhibits a similar degree of diversity in type IV pilin that it encodes.<b>Aim.</b> This study was done to characterize a structural pilin (PilA) encoded by <i>M. bovoculi</i> isolated from cases of IBK to determine if diversity exists amongst PilA sequences.<b>Methodology.</b> Ninety-four isolates of <i>M. bovoculi</i> collected between 2002 and 2017 from 23 counties throughout California and from five counties in four other Western states were evaluated.<b>Results.</b> DNA sequencing and determination of deduced amino acid sequences revealed ten (designated groups A through J) unique PilA sequences that were ~96.1-99.3 % identical. Pilin groups A and C matched previously reported putative PilA sequences from <i>M. bovoculi</i> isolated from IBK-affected cattle in the USA (Virginia, Nebraska, and Kansas) and Asia (Kazakhstan). The ten pilin sequences identified were only ~74-76 % identical to deduced amino acid sequences of putative pilin proteins identified from the previously reported whole-genome sequences of <i>M. bovoculi</i> derived from deep nasopharyngeal swabs of IBK-asymptomatic cattle.<b>Conclusions.</b> Compared to the diversity reported between structural pilin proteins amongst different serogroups of <i>M. bovis</i>, <i>M. bovoculi</i> PilA from geographically diverse isolates derived from IBK-affected cattle are more conserved.
Project description:The objective of this study was to evaluate the genetic diversity of Moraxella bovis and Moraxella bovoculi bacteria isolated from infectious bovine keratoconjunctivitis (IBK) outbreaks in the state of Rio Grande do Sul, Brazil. The genetic diversity among Moraxella spp. was evaluated by RAPD-PCR, JWP1-JWOPA07-PCR, ERIC-PCR and by sequencing the 16S-23S intergenic regions. Based on the dendrogram, two genetically differentiated clades were observed; 14 isolates were classified as M. bovis and 17 as M. bovoculi. Genetic distances between the M. bovis samples ranged from 0.0379 to 0.4285, while for M. bovoculi the dissimilarities ranged from zero to 0.7297. Alternatively, based on sequencing analyses of the 16S-23S intergenic region, M. bovis and M. bovoculi isolates were grouped into the same two different clades, but it was not possible to differentiate between isolates within clades. PCR techniques were demonstrated to be a satisfactory tool to unravel the genetic variability among Moraxella spp., while sequencing of the 16S-23S intergenic region was only able to differentiate two species of the Moraxella genus. Despite sampling geographically close regions, we demonstrate considerable genetic diversity in M. bovis and M. bovoculi strains and genetically distinct M. bovis strains co-infecting the same animal.
Project description:Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is one of the most common eye diseases in cattle. Several pathogens have been associated with IBK cases, however, Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and bovine herpesvirus type 1 (BHV-1) are most frequently observed. A multiplex real-time PCR assay using two reactions was developed for the detection and differentiation of these five pathogens. Detection sensitivities of the multiplex assays were compared to singleplex reactions testing for the same targets. Correlation coefficients (R<sup>2</sup>) of >0.99, and PCR efficiencies between 92 and 106% were demonstrated in all singleplex and multiplex real-time PCR reactions. The limits of detection (LOD) of multiplex assays for Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and BHV-1 were 19, 23, 25, 24 and 26 copies per reaction, respectively. No cross amplification was observed for specificity testing of 179 IBK positive clinical samples and 55 non-target clinical samples. Percentage of clinical samples positive for Mycoplasma bovoculi, Moraxella bovoculi, Moraxella bovis, BHV-1 and Mycoplasma bovis were 88.8% (159/179), 75.9% (136/179), 60.3% (108/179), 11.7% (21/179) and 10.0% (18/179), respectively. Moraxella bovis, Moraxella bovoculi and Mycoplasma bovoculi were more prevalent than Mycoplasma bovis and BHV-1 in IBK samples collected from animals in this study population. Our data indicates that the multiplex real-time PCR panel assay is highly sensitive and highly specific for the detection and differentiation of the five major pathogens associated with bovine pinkeye.
Project description: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 putative 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.
Project description:Moraxella bovoculi strain KZ-1 was isolated from cattle that had symptoms of infectious bovine keratoconjunctivitis (IBK) in northern Kazakhstan. Here, we report the draft genome sequence of this strain.
Project description:BACKGROUND:Infectious bovine keratoconjunctivitis (IBK) in beef cattle has major welfare and production implications. Effective vaccination against IBK would also reduce antibiotic use in beef production. OBJECTIVE/HYPOTHESIS:To evaluate the efficacy of a conditionally licensed commercial IBK vaccine containing Moraxella bovoculi bacterin. Primary working hypothesis was that animals vaccinated with 2 doses of the commercial M. bovoculi vaccine would have a lower risk of disease. ANIMALS:Spring born calves at a university cow-calf herd. After excluding animals with ocular lesions, calves eligible for prevention assessment in 2017 and 2018 were 163 (81 vaccinated, 82 unvaccinated) and 207 (105 vaccinated, 102 unvaccinated). One hundred sixty two and two hundred and six calves completed the follow-up period in 2017 and 2018, respectively. METHODS:A randomized controlled trial. The trial design was a 2-arm parallel trial with a 1:1 allocation ratio. RESULTS:In both years, calves receiving the vaccine had more IBK. This effect was small. The pooled risk ratio was 1.30 (95% confidence interval 0.84-2.01). The pooled unadjusted difference in mean weight (kg) at weaning was -0.88 (95% confidence interval-7.2-5.43). CONCLUSIONS AND CLINICAL IMPORTANCE:We were unable to document that the M. bovoculi bacterin vaccine had a protective effect for the incidence of IBK in our single herd in a 2-year study.
Project description:In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.
Project description:<i>Moraxella bovoculi</i> is the bacterium most often cultured from ocular lesions of cattle with infectious bovine keratoconjunctivitis, also known as bovine pinkeye. Some strains of <i>M. bovoculi</i> contain operons encoding for a repeats-in-toxin (RTX) toxin, 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 <i>M. bovoculi</i>. 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/LCaCl<sub>2</sub>, 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 were 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 CaCl<sub>2</sub>, 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 <i>M. bovoculi</i> regarding the presence or absence of RTX toxin operons and that agar media modifications improve the accuracy of these models.
Project description:<h4>Background</h4>Mycoplasma bovis is an important pathogen of cattle worldwide. Many different clinical manifestations of infection can occur, including respiratory disease, arthritis, and mastitis, causing heavy losses to beef and dairy industries. Because Mycoplasma species are slow-growing and fastidious, traditional identification methods are not cost- or time-effective, and improved methods are sought to streamline laboratory processes. High-resolution melting PCR (HRM-PCR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are 2 relatively recent tools that are rapid and inexpensive to use; we tested 9 isolates of M. bovis using both assays. The HRM-PCR assay used universal mycoplasma primers for the 16S-23S intergenic spacer region (IGSR).<h4>Results</h4>The resulting melting profiles of the field isolates were indistinguishable from the reference strain, indicating accurate identification. For the MALDI-TOF MS, each M. bovis isolate was accurately identified. Mycoplasma arginini and Mycoplasma alkalescens isolates did not identify as M. bovis when tested by either assay.<h4>Conclusions</h4>Our work shows that either assay could be used to identify unknown M. bovis isolates. For future work, the MALDI-TOF MS library should be expanded to include more mycoplasmas, and the HRM-PCR assay should be tested on additional mycoplasmas to ensure that the melting profiles are sufficiently distinctive.
Project description:Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) recently emerged as a technology for the identification of bacteria. In this study, we aimed to evaluate its applicability to human and ruminant mycoplasmal identification, which can be demanding and time-consuming when using phenotypic or molecular methods. In addition, MALDI-TOF MS was tested as a subtyping tool for certain species. A total of 29 main spectra (MSP) from 10 human and 13 ruminant mycoplasma (sub)species were included in a mycoplasma MSP database to complete the Bruker MALDI Biotyper database. After broth culture and protein extraction, MALDI-TOF MS was applied for the identification of 119 human and 143 ruminant clinical isolates that were previously identified by antigenic or molecular methods and for subcultures of 73 ruminant clinical specimens that potentially contained several mycoplasma species. MALDI-TOF MS resulted in accurate (sub)species-level identification with a score of ?1.700 for 96% (251/262) of the isolates. The phylogenetically closest (sub)species were unequivocally distinguished. Although mixtures of the strains were reliably detected up to a certain cellular ratio, only the predominant species was identified from the cultures of polymicrobial clinical specimens. For typing purposes, MALDI-TOF MS proved to cluster Mycoplasma bovis and Mycoplasma agalactiae isolates by their year of isolation and genome profiles, respectively, and Mycoplasma pneumoniae isolates by their adhesin P1 type. In conclusion, MALDI-TOF MS is a rapid, reliable, and cost-effective method for the routine identification of high-density growing mycoplasmal species and shows promising prospects for its capacity for strain typing.