Genetic characterization of Australian Mycoplasma bovis isolates through whole genome sequencing analysis

Parker, A.M.; Shukla, Ankit; House, John K.; Hazelton, M.S.; Bosward, Katrina L.; Kokotovic, Branko; Sheehy, Paul A.

doi:10.1016/j.vetmic.2016.10.010

Cited by 35 publications

(36 citation statements)

References 31 publications

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“…As suggested by the name, WGS involves sequencing the entire genome of selected isolates which can then be used for clinical diagnostics, disease outbreak investigation and controlling antimicrobial resistance. 77 90 Through SNP analysis it was suggested that a single strain existed, with comparative genomics revealing minimal variation between isolates from different anatomical locations, again consistent with previous findings using PFGE 66 and AFLP. 69 test kit by Biovet Inc (Quebec, Canada) is also commercially available for use on serum samples and has been used in studies evaluating inhouse assays.…”

Section: Whole Genome Sequencingsupporting

confidence: 83%

“…While the complete sequencing of these isolates has provided some insight into the content and dynamics of the organism as well as uncovering putative virulent genes, few studies have used WGS to compare the genetic diversity between a large number of isolates. A recent study examined 82 Australian M. bovis isolates collected over a 9 year period (2006–2015) from various geographical locations and bovine anatomical sites . Through SNP analysis it was suggested that a single strain existed, with comparative genomics revealing minimal variation between isolates from different anatomical locations, again consistent with previous findings using PFGE and AFLP .…”

Section: Molecular Diagnosissupporting

confidence: 71%

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A review of mycoplasma diagnostics in cattle

Parker

Sheehy

Hazelton

et al. 2018

Veterinary Internal Medicne

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Mycoplasma species have a global distribution causing serious diseases in cattle worldwide including mastitis, arthritis, pneumonia, otitis media and reproductive disorders. Mycoplasma species are typically highly contagious, are capable of causing severe disease, and are difficult infections to resolve requiring rapid and accurate diagnosis to prevent and control disease outbreaks. This review discusses the development and use of different diagnostic methods to identify Mycoplasma species relevant to cattle, with a particular focus on Mycoplasma bovis. Traditionally, the identification and diagnosis of mycoplasma has been performed via microbial culture. More recently, the use of polymerase chain reaction to detect Mycoplasma species from various bovine samples has increased. Polymerase chain reaction has a higher efficiency, specificity, and sensitivity for laboratory diagnosis when compared with conventional culture‐based methods. Several tools are now available for typing Mycoplasma spp. isolates, allowing for genetic characterization in disease outbreak investigations. Serological diagnosis through the use of indirect ELISA allows the detection of antimycoplasma antibodies in sera and milk, with their use demonstrated on individual animal samples as well as BTM samples. While each testing method has strengths and limitations, their combined use provides complementary information, which when interpreted in conjunction with clinical signs and herd history, facilitates pathogen detection, and characterization of the disease status of cattle populations.

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Section: Whole Genome Sequencingsupporting

confidence: 83%

Section: Molecular Diagnosissupporting

confidence: 71%

A review of mycoplasma diagnostics in cattle

Parker

Sheehy

Hazelton

et al. 2018

Veterinary Internal Medicne

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“…Reaction mixtures consisted of 0.5 mM of dNTPs, 5 mM of MgCl 2 , 0.5 U GoTaq polymerase, 1 μM of each primer set, 0.25 μM of each probe, 2.0 μL of 5x Buffer and 2 μL of DNA template in a final volume of 10 μL. Cycling conditions were 95°C for 60s, followed by 40 cycles of 95°C for 30s, 60°C for 30s and 72°C for 30s [28]. The assay was performed on a RotorGene TM 3000 RT-PCR System Thermocycler using the green, yellow and orange channels for M .…”

Section: Methodsmentioning

confidence: 99%

“…Reaction mixtures contained 0.25 mM dNTPs, 2.5 mM MgCl 2 , 1.5 U of GoTaq, 0.25 μM of each primer (Table 1), 8 μL of 5x Buffer and 5 μL DNA template in a final volume of 40 μL. Cycling conditions were 94°C for 5 min, followed by 35 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1 min, and a final extension of 72°C for 5 min [28]. The assay was performed on a Bio-Rad-T100 Thermocycler (Bio-Rad Laboratories Pty Ltd, Gladesville, NSW, Australia).…”

Section: Methodsmentioning

confidence: 99%

Comparison of culture and a multiplex probe PCR for identifying Mycoplasma species in bovine milk, semen and swab samples

et al. 2017

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Mycoplasma spp. are a major cause of mastitis, arthritis and pneumonia in cattle, and have been associated with reproductive disorders in cows. While culture is the traditional method of identification the use of PCR has become more common. Several investigators have developed PCR protocols to detect M. bovis in milk, yet few studies have evaluated other sample types or other important Mycoplasma species. Therefore the objective of this study was to develop a multiplex PCR assay to detect M. bovis, M. californicum and M. bovigenitalium, and evaluate its analytical performance against traditional culture of bovine milk, semen and swab samples. The PCR specificity was determined and the limit of detection evaluated in spiked milk, semen and swabs. The PCR was then compared to culture on 474 field samples from individual milk, bulk tank milk (BTM), semen and swab (vaginal, preputial, nose and eye) samples. Specificity analysis produced appropriate amplification for all M. bovis, M. californicum and M. bovigenitalium isolates. Amplification was not seen for any of the other Mollicutes or eubacterial isolates. The limit of detection of the PCR was best in milk, followed by semen and swabs. When all three Mycoplasma species were present in a sample, the limit of detection increased. When comparing culture and PCR, overall there was no significant difference in the proportion of culture and PCR positive samples. Culture could detect significantly more positive swab samples. No significant differences were identified for semen, individual milk or BTM samples. PCR identified five samples with two species present. Culture followed by 16S-23S rRNA sequencing did not enable identification of more than one species. Therefore, the superior method for identification of M. bovis, M. californicum and M. bovigenitalium may be dependent on the sample type being analysed, and whether the identification of multiple target species is required.

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“…However, no PAIs and secretory systems have been detected in any Mycoplasma species (Guo and Wei, 2012). Using the virulence factors database (VFDB), some virulence genes were identified in the M. bovis genome (Parker et al, 2016), but their impact on M. bovis virulence remains to be investigated.…”

Section: Introductionmentioning

confidence: 99%

Comparative Genomics of Mycoplasma bovis Strains Reveals That Decreased Virulence with Increasing Passages Might Correlate with Potential Virulence-Related Factors

Rasheed

Zhu

et al. 2017

Front. Cell. Infect. Microbiol.

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Mycoplasma bovis is an important cause of bovine respiratory disease worldwide. To understand its virulence mechanisms, we sequenced three attenuated M. bovis strains, P115, P150, and P180, which were passaged in vitro 115, 150, and 180 times, respectively, and exhibited progressively decreasing virulence. Comparative genomics was performed among the wild-type M. bovis HB0801 (P1) strain and the P115, P150, and P180 strains, and one 14.2-kb deleted region covering 14 genes was detected in the passaged strains. Additionally, 46 non-sense single-nucleotide polymorphisms and indels were detected, which confirmed that more passages result in more mutations. A subsequent collective bioinformatics analysis of paralogs, metabolic pathways, protein-protein interactions, secretory proteins, functionally conserved domains, and virulence-related factors identified 11 genes that likely contributed to the increased attenuation in the passaged strains. These genes encode ascorbate-specific phosphotransferase system enzyme IIB and IIA components, enolase, L-lactate dehydrogenase, pyruvate kinase, glycerol, and multiple sugar ATP-binding cassette transporters, ATP binding proteins, NADH dehydrogenase, phosphate acetyltransferase, transketolase, and a variable surface protein. Fifteen genes were shown to be enriched in 15 metabolic pathways, and they included the aforementioned genes encoding pyruvate kinase, transketolase, enolase, and L-lactate dehydrogenase. Hydrogen peroxide (H2O2) production in M. bovis strains representing seven passages from P1 to P180 decreased progressively with increasing numbers of passages and increased attenuation. However, eight mutants specific to eight individual genes within the 14.2-kb deleted region did not exhibit altered H2O2 production. These results enrich the M. bovis genomics database, and they increase our understanding of the mechanisms underlying M. bovis virulence.

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Genetic characterization of Australian Mycoplasma bovis isolates through whole genome sequencing analysis

Cited by 35 publications

References 31 publications

A review of mycoplasma diagnostics in cattle

A review of mycoplasma diagnostics in cattle

Comparison of culture and a multiplex probe PCR for identifying Mycoplasma species in bovine milk, semen and swab samples

Comparative Genomics of Mycoplasma bovis Strains Reveals That Decreased Virulence with Increasing Passages Might Correlate with Potential Virulence-Related Factors

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