Mycoplasma bovis is a major bovine pathogen associated with bovine respiratory disease complex and is responsible for substantial economic losses worldwide. M. bovis is also associated with other clinical presentations in cattle, including mastitis, otitis, arthritis, and reproductive disorders. To gain a better understanding of the genetic diversity of this pathogen, a multilocus sequence typing (MLST) scheme was developed and applied to the characterization of 137 M. bovis isolates from diverse geographical origins, obtained from healthy or clinically infected cattle. After in silico analysis, a final set of 7 housekeeping genes was selected (dnaA, metS, recA, tufA, atpA, rpoD, and tkt). MLST analysis demonstrated the presence of 35 different sequence types (STs) distributed in two main clonal complexes (CCs), defined at the double-locus variant level, namely, CC1, which included most of the British and German isolates, and CC2, which was a more heterogeneous and geographically distant group of isolates, including European, Asian, and Australian samples. Index of association analysis confirmed the clonal nature of the investigated M. bovis population, based on MLST data. This scheme has demonstrated high discriminatory power, with the analysis showing the presence of genetically distant and divergent clusters of isolates predominantly associated with geographical origins.M ycoplasma diseases cause substantial economic losses, particularly in intensively farmed cattle production systems worldwide, as a result of poor growth, morbidity, and deaths, as well as the costs associated with increased control and prophylactic measures. Mycoplasma bovis has increasingly been recognized as one of the main pathogens involved in the bovine respiratory disease complex, on its own or in association with other respiratory pathogens (1). M. bovis can also be found in association with mastitis, in which outbreaks can affect more than 20% of the cows in a herd, regardless of the stage of lactation, and infections are usually refractory to treatment. Arthritis and otitis have also been associated with M. bovis, usually appearing once pneumonia or mastitis is already established in the herd (1, 2). The control of M. bovis infections relies strongly on antimicrobial therapy, which has variable success rates in the field (3). Vaccination has been used in the early stages of cattle development and is mostly based on autogenous vaccines, which limits their use and the potential for widespread control of M. bovis infections (3).Taking into consideration the limited tools available for M. bovis disease management, the development of a dependable molecular typing scheme able to offer robust and reproducible epidemiological information would provide a valuable addition to control measures targeting this pathogen. M. bovis isolates have been characterized previously using multiple molecular typing methods, including amplified fragment length polymorphism (AFLP) analysis (4), random amplified polymorphic DNA (RAPD) analysis (5), pulsed-fiel...
The UK has approximately 10 million cattle (Veterinary Medicines Directorate 2011), and each year approximately 1.9 million of these cattle are affected by respiratory disease. A disease that is often complex, caused by viruses and/or bacteria, with as many as one-third of these infected with the Mycoplasma bovis pathogen (Nicholas and Ayling 2003). The welfare and economic effects of clinical cases of M. bovis on farming are therefore substantial and costs may be even more when morbidity associated with respiratory infection is considered. Apart from respiratory disease, M. bovis is also associated with other clinical signs, including mastitis, arthritis, meningitis, infertility, abortion and keratoconjunctivitis (Nicholas and Ayling 2003). Elimination of M. bovis is difficult and treatment with antimicrobials often has limited effect unless animals are treated early in the course of disease. In recent years, as much as 11 tonnes per annum of active antimicrobials have been sold for use in the UK cattle antimicrobial products, which includes all intramammary products (Veterinary Medicines Directorate 2011). With no commercial vaccines available in Europe, although use of autogenous vaccines has shown some success (Nicholas and others 2006), it is important to effectively target antimicrobial treatment to ensure prudent use of antimicrobials and to reduce the development of antimicrobial resistance. Between 2004 and 2009, in vitro minimum inhibition concentration (MIC) data for 45 M. bovis isolates have been determined for up to 13 antimicrobials.
dMycoplasma bovis isolates with decreased susceptibilities to tetracyclines are increasingly reported worldwide. The acquired molecular mechanisms associated with this phenomenon were investigated in 70 clinical isolates of M. bovis. Sequence analysis of the two 16S rRNA-encoding genes (rrs3 and rrs4 alleles) containing the primary binding pocket for tetracycline (Tet-1 site) was performed on isolates with tetracycline hydrochloride MICs of 0.125 to 16 g/ml. Mutations at positions A965T, A967T/C (Escherichia coli numbering) of helix 31, U1199C of helix 34, and G1058A/C were identified. Decreased susceptibilities to tetracycline (MICs, >2 g/ml) were associated with mutations present at two (A965 and A967) or three positions (A965, A967, and G1058) of the two rrs alleles. No tet(M), tet(O), or tet(L) determinants were found in the genome of any of the 70 M. bovis isolates. The data presented correlate (P < 0.0001) the mutations identified in the Tet-1 site of clinical isolates of M. bovis with decreased susceptibility to tetracycline. The bacterial pathogen Mycoplasma bovis causes a variety of clinical manifestations in cattle, including respiratory disease, mastitis, arthritis, and otitis, which result in substantial economic losses (1). The tetracyclines are among the few important antimicrobial agents that may be used to treat M. bovis infections (2).Tetracyclines are broad-spectrum antimicrobials that have been widely used in human and veterinary medicine (3). They inhibit protein synthesis by binding to the 30S ribosomal subunit and blocking an attachment of aminoacyl-tRNA to the A site (3). Resistance to tetracyclines is common in many bacterial species and may be achieved by (i) an energy-dependent efflux of the drug across the cell membrane mediated by efflux pumps, (ii) the presence of ribosomal protection proteins that confer tetracycline resistance, either by a reduction of the affinity of ribosomes to tetracyclines or by releasing the bound antimicrobial from the ribosome, (iii) the enzymatic inactivation of the drug, or (iv) the mutations in the 16S rRNA genes that affect the binding sites of tetracyclines (4).In Mollicutes, two mechanisms of resistance to tetracyclines have been identified so far, both of which are in Mollicutes species that infect humans. These include ribosomal protection by tet(M) determinants, described in naturally tetracycline-resistant strains of Mycoplasma hominis and Ureaplasma spp. (5, 6), as well as target modification with point mutation(s) in the 16S The aim of this study was to investigate the mechanisms associated with acquired decreased susceptibilities to tetracyclines in M. bovis isolates. ; 1980 [19]) were tested in this study, and their details are given in Table 1. Each isolate originated from different farms, was selected at random, and had no epidemiological link to other isolates unless indicated otherwise. The reference type strain M. bovis PG45 was obtained from the National Collection of Type Cultures, United Kingdom (strain NCTC10131, corresponding to ATC...
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