Development of a Highly Sensitive and Specific Assay to Detect Staphylococcus aureus in Bovine Mastitic Milk

Graber, H.U.; Casey, Michael G.; Naskova, J.; Steiner, Adrian; Schaeren, W.

doi:10.3168/jds.2006-902

Cited by 83 publications

(90 citation statements)

References 21 publications

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“…The nuc gene is highly specific for Staph. aureus (Brakstad et al, 1992;Graber et al, 2007) and may, therefore, be used for definitive identification of suspicious colonies (Syring et al, 2012).…”

Section: Clinical Outcomes and Molecular Genotyping Of Staphylococcusmentioning

confidence: 99%

Clinical outcomes and molecular genotyping of Staphylococcus aureus isolated from milk samples of dairy primiparous Mediterranean buffaloes (Bubalus bubalis)

Guccione

Cosandey

Pesce

et al. 2014

Journal of Dairy Science

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Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows and in Mediterranean buffaloes. Genotype B (GTB) is contagious in dairy cows and may occur in up to 87% of cows of a dairy herd. It was the aim of this study to evaluate genotypes present, clinical outcomes, and prevalence of Staph. aureus in milk samples of primiparous Mediterranean dairy buffaloes. Two hundred composite milk samples originating from 40 primiparous buffaloes were collected from May to June 2012, at d 10, 30, 60, 90, and 150 d in milk (DIM) to perform somatic cell counts and bacteriological cultures. Daily milk yields were recorded. Before parturition until 40 to 50 DIM, all primiparous animals were housed separated from the pluriparous animals. Milking was performed in the same milking parlor, but the primiparous animals were milked first. After 50 DIM, the primiparous were mixed with the pluriparous animals, including the milking procedure. Individual quarter samples were collected from each animal, and aliquots of 1 mL were mixed and used for molecular identification and genotyping of Staph. aureus. The identification of Staph. aureus was performed verifying the presence of nuc gene by nuc gene PCR. All the nuc-positive isolates were subjected to genotype analysis by means of PCR amplification of the 16S-23S rRNA intergenic spacer region and analyzed by a miniaturized electrophoresis system. Of all 200 composite samples, 41 (20.5%) were positive for Staph. aureus, and no genotype other than GTB was identified. The prevalence of samples positive for Staph. aureus was 0% at 10 DIM and increased to a maximum of 22/40 (55%) at 90 DIM. During the period of interest, 14 buffaloes tested positive for Staph. aureus once, 6 were positive twice, and 5 were positive 3 times, whereas 15 animals were negative at every sampling. At 90 and 150 DIM, 7 (17.5%) and 3 buffaloes (7.5%), respectively, showed clinical mastitis (CM), and only 1 (2.5%) showed CM at both samplings. At 60, 90, and 150 DIM, 1 buffalo was found with subclinical mastitis at each sampling. At 30, 60, 90, and 150 DIM, 2.5 (1/40), 22.5 (9/40), 35 (14/40), and 10% (4/40) were considered affected by intramammary infection, respectively. Buffaloes with CM caused by Staph. aureus had statistically significantly higher mean somatic cell count values (6.06 ± 0.29, Log 10 cells/mL ± standard deviation) and statistically significantly lower mean daily milk yields (7.15 ± 1.49, liters/animal per day) than healthy animals (4.69 ± 0.23 and 13.87 ± 2.64, respectively), buffaloes with IMI (4.82 ± 0.23 and 11.16 ± 1.80, respectively), or with subclinical mastitis (5.47 ± 0.10 and 10.33 ± 0.68, respectively). Based on our knowledge, this is the first time that Staph. aureus GTB has been identified in milk samples of dairy Mediterranean buffaloes.

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Section: Clinical Outcomes and Molecular Genotyping Of Staphylococcusmentioning

confidence: 99%

Clinical outcomes and molecular genotyping of Staphylococcus aureus isolated from milk samples of dairy primiparous Mediterranean buffaloes (Bubalus bubalis)

Guccione

Cosandey

Pesce

et al. 2014

Journal of Dairy Science

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“…The nuc gene is highly specific for Staph. aureus (Brakstad et al, 1992;Graber et al, 2007) and was therefore used for definitive identification. All nuc-positive isolates (i.e., Staph.…”

Section: Rs-pcrmentioning

confidence: 99%

Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer

Boss

Cosandey

Luini

et al. 2016

Journal of Dairy Science

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Staphylococcus aureus is globally one of the most important pathogens causing contagious mastitis in cattle. Previous studies using ribosomal spacer (RS)-PCR, however, demonstrated in Swiss cows that Staph. aureus isolated from bovine intramammary infections are genetically heterogeneous, with Staph. aureus genotype B (GTB) and GTC being the most prominent genotypes. Furthermore, Staph. aureus GTB was found to be contagious, whereas Staph. aureus GTC and all the remaining genotypes were involved in individual cow disease. In addition to RS-PCR, other methods for subtyping Staph. aureus are known, including spa typing and multilocus sequence typing (MLST). They are based on sequencing the spa and various housekeeping genes, respectively. The aim of the present study was to compare the 3 analytic methods using 456 strains of Staph. aureus isolated from milk of bovine intramammary infections and bulk tanks obtained from 12 European countries. Furthermore, the phylogeny of animal Staph. aureus was inferred and the zoonotic transfer of Staph. aureus between cattle and humans was studied. The analyzed strains could be grouped into 6 genotypic clusters, with CLB, CLC, and CLR being the most prominent ones. Comparing the 3 subtyping methods, RS-PCR showed the highest resolution, followed by spa typing and MLST. We found associations among the methods but in many cases they were unsatisfactory except for CLB and CLC. Cluster CLB was positive for clonal complex (CC)8 in 99% of the cases and typically positive for t2953; it is the cattle-adapted form of CC8. Cluster CLC was always positive for t529 and typically positive for CC705. For CLR and the remaining subtypes, links among the 3 methods were generally poor. Bovine Staph. aureus is highly clonal and a few clones predominate. Animal Staph. aureus always evolve from human strains, such that every human strain may be the ancestor of a novel animal-adapted strain. The zoonotic transfer of IMI-and milk-associated strains of Staph. aureus between cattle and humans seems to be very limited and different hosts are not considered as a source for mutual, spontaneous infections. Spillover events, however, may happen.

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“…Based on these genome-wide comparisons, primer sets were designed to amplify a partial segment of the aroD gene for S. hyicus and S. agnetis (Table 3). Standard multiplex PCR, including sets of aroD gene primers to identify S. hyicus and S. agnetis, respectively, and a nuc gene primer set to identify S. aureus (35), was performed using the following thermocycler conditions: 94°C for 15 min, followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The run was completed at 72°C for 5 min and then held at 4°C.…”

Section: Confirmation Of Species Identification By Housekeeping Gene mentioning

confidence: 99%

Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis

et al. 2017

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Staphylococcus hyicus and Staphylococcus agnetis are two coagulasevariable staphylococcal species that can be isolated from bovine milk and are difficult to differentiate. The objectives of this study were to characterize isolates of bovine milk origin from a collection that had previously been characterized as coagulase-positive S. hyicus based on phenotypic species identification methods and to develop a PCR-based method for differentiating S. hyicus, S. agnetis, and Staphylococcus aureus. Isolates (n ϭ 62) were selected from a previous study in which milk samples were collected from cows on 15 dairy herds. Isolates were coagulase tested and identified to the species level using housekeeping gene sequencing. A multiplex PCR to differentiate S. hyicus, S. agnetis, and S. aureus was developed. Pulsed-field gel electrophoresis was conducted to strain type the isolates. Based on gene sequencing, 44/62 of the isolates were determined to be either S. agnetis (n ϭ 43) or S. hyicus (n ϭ 1). Overall, 88% (37/42) of coagulase-positive S. agnetis isolates were found to be coagulase positive at 4 h. The herd-level prevalence of coagulasepositive S. agnetis ranged from 0 to 2.17%. Strain typing identified 23 different strains. Six strains were identified more than once and from multiple cows within the herd. Three strains were isolated from cows at more than one time point, with 41 to 264 days between samplings. These data suggest that S. agnetis is likely more prevalent on dairy farms than S. hyicus. Also, some S. agnetis isolates in this study appeared to be contagious and associated with persistent infections.KEYWORDS Staphylococcus, cattle, milk S taphylococci are the most common bacteria isolated from the cow's mammary gland and are frequently associated with subclinical or mild clinical mastitis (1-3). Staphylococci can be differentiated into groups using the coagulase test. Hemolytic coagulase-positive isolates are often presumptively identified as Staphylococcus aureus, a so-called "major" contagious mastitis pathogen, and coagulase-negative staphylococci are frequently grouped together as "minor" pathogens (4).Currently, there are two coagulase-variable staphylococcal species that have been associated with bovine mastitis, Staphylococcus hyicus and Staphylococcus agnetis. Staphylococcus hyicus was the first staphylococcal species to be described as coagulase variable, with coagulase production being found in approximately 24 to 56% of strains (5). The coagulation reaction of S. hyicus was described as often having weak activity,

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Development of a Highly Sensitive and Specific Assay to Detect Staphylococcus aureus in Bovine Mastitic Milk

Cited by 83 publications

References 21 publications

Clinical outcomes and molecular genotyping of Staphylococcus aureus isolated from milk samples of dairy primiparous Mediterranean buffaloes (Bubalus bubalis)

Clinical outcomes and molecular genotyping of Staphylococcus aureus isolated from milk samples of dairy primiparous Mediterranean buffaloes (Bubalus bubalis)

Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer

Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis

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