Validation of tDNA-intergenic spacer PCR for species level identification of bovine coagulase-negative staphylococci

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A Staphylococcus xylosus isolate with a new mecC allotype

Recently, a novel variant of mecA known as mecC (mecA(LGA251)) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified a Staphylococcus xylosus isolate that harbors a new allotype of the mecC gene, mecC1. Whole-genome sequencing revealed that mecC1 forms part of a class E mec complex (mecI-mecR1-mecC1-blaZ) located at the orfX locus as part of a likely staphylococcal cassette chromosome mec element (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec

Prevalence of intramammary infection in Dutch dairy herds

A Survey was carried out in 2003 in 49 dairy herds to determine the overall and pathogen-specific prevalence of intramammary infection (IMI) in Dutch dairy herds, and to compare the distribution with four Studies performed from 1973 to 1985 in The Netherlands. Herds were randomly selected stratified over the 12 Dutch provinces, had at least 40 lactating cows and participated in the Dutch milk recording system. Quarter milk samples were collected from all 408 cows with a somatic cell count (SCC) >= 250 000 cells/ml and 145 heifers with SCC >= 150 000 cells/ml at the last milk test before the farm visit. Additionally, samples were collected from 519 (approximately 25%) of the remaining low-SCC cows and heifers with a SCC at the last milk test before the farm visit of <250 000 and <150 000 cells/ml, respectively. Bacterial growth occurred in 37.3% of milk samples of high-SCC cows and in 21.1% of low-SCC cows. Coagulase-negative staphylococci (CNS) were the most frequently isolated group Of bacteria (10.8% of quarters) and were found in all herds. Prevalence of Staphylococcus aureus IMI was lower in 2003 than in 1973, respectively 1.8% and 6.2% of quarters. Prevalence of Streptococcus uberis and Str. dysgalactiae IMI was almost the same in the five samplings during the 30-year period, at 1.1-1.7 and 0.9-1.5%., respectively. Str. agalactiae was not found in this study. Prevalence of CNS IMI was higher in lactating heifers, while prevalence Of Str. uberis, Str. dysgalactiae and penicillin-resistant Staph. aureus IMI was higher in older cows. Because distribution of pathogens changes over time, herd-level samples for bacteriological Culturing must be taken regularly to monitor udder health. Additionally, national mastitis prevalence studies give important information through monitoring the national udder health status

Effect of prepartum dry cow antibiotic treatment in dairy heifers on udder health and milk production

A high percentage of heifers calve with intramammary infections. One of the measures available to control intramammary infections is treatment with antibiotics before calving. In this study, the effects of prepartum treatment of nonlactating heifers with a 600-mg cloxacillin dry cow treatment on the prevalence of culture-positive milk samples at calving and 10 to 14 d in milk (DIM), the incidence of clinical mastitis, somatic cell count (SCC), and milk production during first lactation were quantified. A total of 184 heifers on 13 dairy farms were treated with antibiotics 8 to 10 wk before the expected calving date. Another 185 heifers served as untreated controls. Coagulase-negative staphylococci were the most frequently isolated group of bacteria in the treatment and control groups at calving (32 and 42%), and at 10 to 14 DIM (15 and 19%), respectively. The prevalence of minor pathogens at calving was lower in the treatment group compared with the control group (34 and 43%, respectively). Staphylococcus aureus was the most frequently isolated major pathogen in the treated and control heifers at calving (4 and 5%) and at 10 to 14 DIM (2 and 3%), respectively. The prevalence of major pathogens was lower in the treated heifers at 10 to 14 DIM compared with the control group (4 and 6%, respectively). Cumulative incidence risk of clinical mastitis during the lactation was 9 and 18% in the treatment and control groups, respectively. Treatment with cloxacillin 8 to 10 wk before calving resulted in a lower prevalence of culture-positive milk samples at calving and lower quarter milk SCC in early lactation [30,000 +/- 4,600 (standard deviation) cells/mL in treated heifers versus 40,000 +/- 4,600 cells/mL in control heifers], and was associated with lower average test-day SCC (55,000 +/- 1,400 cells/mL in treated heifers versus 71,000 +/- 1,500 cells/mL in control heifers) and lower incidence of clinical mastitis throughout lactation. The improved udder health resulted in a higher average test-day milk production in the first lactation (24.5 +/- 3.2 kg in treated heifers versus 23.6 +/- 3.1 kg in control heifers). Dairy farms with heifer mastitis problems need to analyze their mastitis management. Prepartum treatment of heifers with dry cow antibiotics may be helpful by decreasing the prevalence of mastitis-causing pathogens at calving and at 10 to 14 DIM

Prevalence and herd-level risk factors for intramammary infection with coagulase-negative staphylococci in Dutch dairy herds

International audienc

Application of Pulsed-Field Gel Electrophoresis and Binary Typing as Tools in Veterinary Clinical Microbiology and Molecular Epidemiologic Analysis of Bovine and Human Staphylococcus aureus Isolates

Thirty-eight bovine mammary Staphylococcus aureus isolates from diverse clinical, temporal, and geographical origins were genotyped by pulsed-field gel electrophoresis (PFGE) after SmaI digestion of prokaryotic DNA and by means of binary typing using 15 strain-specific DNA probes. Seven pulsed-field types and four subtypes were identified, as were 16 binary types. Concordant delineation of genetic relatedness was documented by both techniques, yet based on practical and epidemiological considerations, binary typing was the preferable method. Genotypes of bovine isolates were compared to 55 previously characterized human S. aureus isolates through cluster analysis of binary types. Genetic clusters containing strains of both human and bovine origin were found, but bacterial genotypes were predominantly associated with a single host species. Binary typing proved an excellent tool for comparison of S. aureus strains, including methicillin-resistant S. aureus, derived from different host species and from different databases. For 28 bovine S. aureus isolates, detailed clinical observations in vivo were compared to strain typing results in vitro. Associations were found between distinct genotypes and severity of disease, suggesting strain-specific bacterial virulence. Circumstantial evidence furthermore supports strain-specific routes of bacterial dissemination. We conclude that PFGE and binary typing can be successfully applied for genetic analysis of S. aureus isolates from bovine mammary secretions. Binary typing in particular is a robust and simple method and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovine S. aureus

Screening of bovine coagulase-negative staphylococci from milk for superantigen encoding genes

A collection of 102 coagulase-negative staphylococci (CNS), isolated from cases of subclinical and clinical bovine mastitis and belonging to 10 different species, were screened by PCR for the presence of genes encoding enterotoxins and enterotoxin-like toxins (sea, seb, sec, sed, see, seg, seh, sei, sej, selk sell selm, seln, selo, selp, selq and selu), toxic shock syndrome toxin-1 (tst), and exfoliative toxins A and B (eta and etb). No toxin gene sequences were amplified from any of the isolates, indicating that superantigens encoded by genes detectable by the PCR tests used were not involved in the development of subclinical and clinical mastitis in cattle infected with the CNS isolates tested

Staphylococcus devriesei sp. nov., isolated from teat apices and milk of dairy cows

Ten non-motile, Gram-stain-positive, coagulase-negative staphylococci were isolated from bovine milk and teat apices. All isolates were catalase-positive, with anteiso-C-15:0, iso-C-15:0, anteiso-C-17:0, iso-C-17:0 and C-18:0 as predominant fatty acids and diphosphatidylglycerol and phosphatidylglycerol as major polar lipids. The results of sequence analysis of the 16S rRNA gene and four housekeeping genes (rpoB, hsp60, tuf and dnaJ) in combination with tRNA-intergenic spacer length analysis showed that the isolates form a separate branch within the genus Staphylococcus. Based on 16S rRNA gene sequencing, the phylogenetically most closely related species are Staphylococcus haemolyticus, S. hominis and S. lugdunensis, with >98.7% sequence similarity. The DNA G+C content varies from 33.3 to 33.7 mol%, and DNA-DNA hybridization with the nearest neighbours, based on 16S rRNA gene sequences, confirmed that the isolates represent a novel Staphylococcus species. All isolates induced a small zone of complete haemolysis on Columbia agar with 5% sheep blood and exhibited a homogeneous biochemical fingerprint that is discriminative from the phylogenetically most closely related species. Based on these results, it is proposed to classify the ten isolates as Staphylococcus devriesei sp. nov., with strain KS-SP 60(T) (=LMG 25332(T) =CCUG 58238(T)) as the type strain