Genotype classification of Moraxella bovis using MALDI-TOF MS profiles

Olson, Hannah G.; Loy, John Dustin; Clawson, Marion; Wynn, Emily L.; Hille, Matthew M.

doi:10.3389/fmicb.2022.1057621

Cited by 4 publications

(3 citation statements)

References 27 publications

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“…The multiplex PCR of Moraxella spp. further confirmed the presence of M. bovis and M. bovoculi known to cause IBK (Irby and Angelos 2018;Karthik et al 2022;Olson et al 2022) in the studied samples. The amplification graphs showed a threshold cycle of 22, and there was a tendency to increase the value of the curve from cycle 20-22 to 30 due to the accumulation of amplification products in the positive control and the studied samples.…”

Section: Discussionsupporting

confidence: 81%

Metagenomic Analysis and Identification of Epizootic Strains of the Causative Agent of Infectious Bovine Keratoconjunctivitis in Kazakhstan

2023

Int J Vet Sci

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This research aimed to identify the causative agent of infectious bovine keratoconjunctivitis (IBK) Moraxella strains and differentiate them using 16S rRNA sequencing and PCR. The 16S ribosomal RNA (16S rRNA) gene sequencing method was used to determine the microbiome. This is due to the high commonness of this gene in the genomes of all prokaryotes, which allows selecting universal primers to strengthen areas of interest and reduce the cost and time of research. PCR-RT was used for the identification of Moraxella strains. In this study, we identified the causative agents Moraxella bovis and Moraxella bovoculi based on the metagenomics analysis of infected cattle in the Republic of Kazakhstan. We visualized the obtained data using Krona's diagram and EDGE bioinformatics software. We differentiated M. bovis and M. bovoculi from other cultures of Moraxella spp. using PCR-RT. For the first time in Kazakhstan, strains that cause IBK were identified using metagenomic analysis. This raises the diagnosis of IBK, a widespread infectious cattle disease, to a genetic level. Our research will serve as the foundation for the further scientific study of the accompanying microorganisms, followed by recommendations for herd welfare and the prevention of economic losses associated with the death and therapeutic manipulations of sick animals.

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Section: Discussionsupporting

confidence: 81%

Metagenomic Analysis and Identification of Epizootic Strains of the Causative Agent of Infectious Bovine Keratoconjunctivitis in Kazakhstan

2023

Int J Vet Sci

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“…Several molecular studies have investigated the 3′ region of the cytotoxin gene in M. bovis , M. bovoculi , and M. ovis [ 16 ]. Techniques such as matrix-assisted laser desorption ionization–time of flight have been employed for biomarker determination [ 17 , 18 ], and PCR-Restriction Fragment Length Polymorphism analysis has been utilized for differentiation in IBK [ 19 ]. A mRT-PCR protocol has also been developed to detect five pathogens [ 20 ].…”

Section: Discussionmentioning

confidence: 99%

Development and evaluation of a multiplex polymerase chain reaction in real-time for differential diagnosis of Moraxella-induced keratoconjunctivitis in livestock

Strochkov,

Sattarova,

Boranbayeva

et al. 2023

Vet World

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Background and Aim: Infectious bovine keratoconjunctivitis (IBK) is a prevalent ocular disease that affects livestock, leading to substantial economic losses due to reduced production and culling of infected animals. Moraxella spp. is common bacterial pathogens that can cause keratoconjunctivitis in livestock. Therefore, rapid and accurate diagnosis is crucial for effective treatment and disease control. This study aimed to develop a multiplex real-time polymerase chain reaction (mRT-PCR) assay for the detection and differentiation of Moraxella bovoculi, Moraxella ovis, and Moraxella bovis. Materials and Methods: Three reference strains of Moraxella as positive controls and 36 lacrimal swab samples collected from cattle were used to evaluate the developed mRT-PCR assay DNA extraction that was performed using the RIBO-sorb DNA/RNA extraction kit. Primers and probes were designed using the SpeciesPrimer pipeline. The annealing temperature, primer and probe concentrations, and sensitivity and specificity of the assay were optimized. Results: An mRT-PCR assay was developed to detect pathogens associated with IBK in cattle on the basis of optimized parameters. The specificity and sensitivity of this assay were confirmed using samples containing individual pathogens (O – M. ovis, B – M. bovis, and BO – M. bovoculi), combinations of two pathogens (O-B, B-BO, and O-BO), and when the DNA of all three pathogens was present in a single reaction (O-B-BO). The analytical sensitivity of mRT-PCR for detecting M. ovis and M. bovoculi DNA was 21 copies or 50 fg per reaction, whereas that for M. bovis was 210 copies or 500 fg per reaction. In addition, this assay has been tested on samples isolated from the affected eyes of cattle in the Akmola region of the Republic of Kazakhstan. Conclusion: For the first time in the Republic of Kazakhstan, the proposed mRT-PCR assay for the simultaneous detection of three Moraxella spp. pathogens has been developed. This assay exhibits the required specificity and high sensitivity for mRT-PCR, facilitating the timely implementation of effective measures for disease control and the prevention of economic losses. These losses are linked to a reduction in livestock breeding value, a reduction in meat and milk production, a reduction in the reproductive performance of heifers, resulting in fewer offspring, as well as costs related to the treatment of affected animals. Keywords: Moraxella bovis, Moraxella bovoculi, Moraxella ovis, Moraxella spp., multiplex real-time polymerase chain reaction, Pinkeye.

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“…6 Moraxella could not be speciated in that study; hence, different species of Moraxella may have been statistically different between samples, or a difference could possibly even be seen at the level of genotype. 7,12,15 Including primers in our proposed assay to genotype M. bovis and M. bovoculi would allow for a more accurate evaluation of the data. Further testing is warranted, in addition to challenge studies, to determine the role, if any, of M oculi in pinkeye.…”

mentioning

confidence: 99%

Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs

Wilkes,

Kattoor,

Weng

et al. 2023

J VET Diagn Invest

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Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species of Moraxella: M. bovis and M. bovoculi. A third novel Moraxella spp., designated tentatively as M. oculi, has been identified from the eyes of cattle with and without pinkeye. These 3 Moraxella spp. can be found in various combinations within the same clinical sample, making speciation of this genus directly from a sample impossible with Sanger sequencing. Assessing Moraxella diversity found in IBK- and non-IBK–affected cattle eyes, independent of culture, may provide additional information about IBK by avoiding the selectivity bias of culturing. We developed a targeted NGS panel to detect and speciate these 3 Moraxella spp. directly from bovine ocular swabs. Our targeted panel amplifies bacterial essential genes and the 16S-23S ribosomal RNA intergenic spacer region (ITS) of the 3 Moraxella spp. and speciates based on these sequences. Our panel was able to differentiate the 3 species directly from DNA extracted from 13 swabs (6 from healthy animals, 7 from animals with IBK), and every swab except one (clinically healthy eye) had the 3 Moraxella spp. Targeted NGS with sequencing of Moraxella spp. housekeeping genes appears to be a suitable method for speciation of Moraxella directly from ocular swabs.

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Genotype classification of Moraxella bovis using MALDI-TOF MS profiles

Cited by 4 publications

References 27 publications

Metagenomic Analysis and Identification of Epizootic Strains of the Causative Agent of Infectious Bovine Keratoconjunctivitis in Kazakhstan

Metagenomic Analysis and Identification of Epizootic Strains of the Causative Agent of Infectious Bovine Keratoconjunctivitis in Kazakhstan

Development and evaluation of a multiplex polymerase chain reaction in real-time for differential diagnosis of Moraxella-induced keratoconjunctivitis in livestock

Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs

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