A total of 258 bovine-associated Staphylococcus aureus isolates from the United States, Chile, and the United Kingdom, plus the reference isolate S. aureus Newbould 305 (NCIMB 702892), were analyzed by multilocus sequence typing (MLST). A collection of previously characterized United Kingdom isolates were also included in the analysis. The results demonstrated that MLST is suitable for the differentiation of bovine S. aureus isolates from various sites (milk, teat skin, milking machine unit liners, hands, and bedding) and countries. The theory of the host specificity of S. aureus is supported by the detection of a previously undescribed clonal complex that comprised 87.4% of the isolates studied, with representatives from all geographic locations investigated. This suggests that a single clonal group has achieved a widespread distribution and is responsible for the majority of infections. Some sequence types (STs; ST25, ST115, ST124, and ST126) demonstrated site specificity, as they were significantly (P < 0.05) associated with milk or teat skin.Staphylococcus aureus is a major cause of bovine mastitis and is spread from cow to cow (skin or milk) via the milking machine (35, 57). Environmental spread may also occur, since strains of S. aureus have been isolated from the environment of dairy farms and from other species that are present on dairy farms (32,39).A number of studies have identified potential sources of the pathogen and have investigated strain-specific differences (19, 40). The major potential sources identified were milk, body sites, and, to a lesser extent, the environment (40). Studies investigating the global population structure of bovine S. aureus suggest that a relatively few specialized clones are responsible for the majority of intramammary infections (IMIs) (26, 55), although some authors did not report between-farm genetic homogeneity (25,46). Most of these studies have used techniques such as phage typing (19, 40) and pulsed-field gel electrophoresis (PFGE) (7, 25) to compare isolates. These methods lack intercenter reproducibility (55). Library typing systems such as binary typing (BT) (53) and multilocus sequence typing (MLST) (31) have been developed to overcome these problems by producing results that are repeatable between laboratories and over time.The aim of this study was to investigate the effectiveness of MLST as a method for the typing of S. aureus isolates of bovine origin from a number of distinct geographical sources. A collection of isolates previously characterized by phage typing (19) and by PFGE and binary typing (55) was used to compare these methods to MLST. The data were then used in a preliminary analysis of the evolutionary and population biology of S. aureus isolates of bovine origin.
A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.
An outbreak of Staphylococcus aureus intramammary infections on an organic dairy farm was monitored for 10 months. Environmental and milk samples were collected from a total of 26 cows and a group of 21 purchased heifers about to be introduced into the milking herd. There was variation in the rate of isolation of S. aureus (9.5 to 43.8%) from individual mammary quarters, although no S. aureus isolates were detected in the milk samples collected from the heifers. One hundred ninety-one S. aureus isolates were detected from cow milk samples (n ؍ 182), milking machine clusters (n ؍ 4), farm personnel (n ؍ 4), and the environment (n ؍ 1). Multilocus sequence typing (MLST) had a typeability of 100% when it was applied to the 191 isolates. Among the 191 isolates there was limited strain diversity, with seven sequence types (STs) dominated by two strains with closely related STs that differed at a single locus. Within individual mammary quarters there were naturally occurring dual infections, although this was identified in only 0.4% of milk samples. Different strains were associated with variable persistence within quarters. MLST is clearly a very suitable tool for the differentiation and analysis of S. aureus populations detected on dairy cattle farms.Mastitis is the most frequently occurring health problem in organic dairy cattle herds, with Staphylococcus aureus and Streptococcus uberis being the most frequently isolated pathogens (41, 43). Control of contagious mastitis is more difficult on organic dairy farms than on conventional dairy farms because the routine use of antibiotic dry cow therapy (DCT) at the end of lactation is not permitted (12) and DCT is currently the most effective method of reducing contagious pathogen infections (32).The lactating mammary gland is the primary reservoir of S. aureus (5, 24), although environmental S. aureus isolates have been detected on heifer body sites and in the dairy farm environment (20,29). While nonmilk isolates may not form a large reservoir of infection (29), their presence may improve the ability of S. aureus to circumvent control mechanisms and persist within a herd, and their importance can be investigated through strain typing.Molecular epidemiological investigations of S. aureus isolated from intramammary infections (IMIs) on individual dairy farms have identified clonal populations characterized by dominant strains and low strain diversity (16, 49). Strain dominance is likely to be associated with virulence and persistence within the mammary gland (31). Although some studies have reported the genetic diversity of S. aureus on individual farms (15, 34), these contrasting results may be due to variations in sampling and typing protocols or to differences in herd sizes and management techniques.A range of different typing techniques have been employed to understand the epidemiology of S. aureus infections. Random amplified polymorphic DNA analysis, ribotyping, biotyping, pulsed-field gel electrophoresis, and binary typing are the major techniques previous...
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