Genomic analysis of European bovine Staphylococcus aureus from clinical versus subclinical mastitis.

Intramammary infections (IMI) with Staphylococcus aureus are a common cause of bovine mastitis and can result in both clinical (CM) or subclinical mastitis (SCM). Although bacterial isolates of S. aureus differ in their virulence potential it is largely unclear which bacterial virulence factors are responsible for increased clinical severity. We performed a genome wide association study and used a generalized linear mixed model to investigate the correlation between gene carriage, lineage and clinical outcome of IMI in a collection of S. aureus isolates from cattle with CM (n = 125) and SCM (n = 151) from 11 European countries. An additional aim was to describe the genetic variation of bovine S. aureus in Europa. The dominant lineages in our collection were clonal complex (CC) 151 (81/276, 29.3%), CC97 (54/276, 19.6%), CC479 (32/276, 11.6%) and CC398 (19/276, 6.9%). Virulence and antimicrobial resistance (AMR) gene carriage was highly associated with CC. Among a selection of nine virulence and AMR genes, CC151, CC479 and CC133 carried more virulence genes than other CCs, and CC398 was associated with AMR gene carriage. Whereas CC151, CC97 were widespread in Europe, CC479, CC398 and CC8 were only found in specific countries. Compared to CC151, CC479 was associated with CM rather than SCM (OR 3.62; 95% CI 1.38-9.50) and the other CCs were not. Multiple genes were associated with CM, but due to the clustering within CC of carriage of these genes, it was not possible to differentiate between the effect of gene carriage and CC on clinical outcome of IMI. Nevertheless, this study demonstrates that characterization of S. aureus CC and virulence genes helps to predict the likelihood of the occurrence of CM following S. aureus IMI and highlights the potential benefit of diagnostics tools to identify S. aureus CC during bovine mastitis.

The presence of Mycoplasma bovis in colostrum.

In herds with Mycoplasma bovis circulation, colostrum is often considered infectious. However, in contrast to milk, the presence of M. bovis in colostrum was not previously evidenced. In this survey, the presence of M. bovis DNA was determined with real-time PCR in 368 colostrum samples from 17 herds, recently infected with M. bovis. Only 1.9% of the samples tested positive, with 13 herds having no positive samples and an overall within-herd prevalence of 3.2% (SD: 4.9%; Range: 0-30.0%). These results show that in infected herds M. bovis DNA can be retrieved in colostrum. To what extend colostrum is infectious remains to be determined.

Absence of zoonotic hepatitis E virus infection in Flemish dairy cows.

Recently, infectious HEV particles were discovered in milk and fecal samples of dairy cows in China. Given the recent increase of autochthonous HEV infections in Europe, we wanted to assess whether cows constitute an HEV reservoir in this region and hence may be responsible for the advance of HEV through consumption of cow produce. To verify the zoonotic risk cows potentially pose towards European consumers, we screened >10% of dairy milk farms in Flanders, Belgium for the presence of HEV. A quarter of these housed both cows and pigs, the latter a well-known reservoir for HEV. Milk and fecal samples were analyzed for the presence of HEV RNA and HEV-specific antibodies. Despite the fact that HEV is circulating amongst pig farms in Flanders and proof of active HEV infection in the pigs of at least one of the mixed farms included in our study, we could not detect any sign of active or past HEV infection in cows. The HEV prevalence in our study was 0%, with a 99.99% confidence interval (CI) for HEV RNA and anti-HEV antibody of [0%-2.30%] and [0%-4.23%] respectively. Our results suggest that, at least in Flanders, cows are not an HEV reservoir and hence do not pose a major health risk towards humans.

Pathogen-specific incidence rate of clinical mastitis in Flemish dairy herds, severity, and association with herd hygiene.

A one-year survey on clinical mastitis was conducted on 50 randomly selected commercial Flemish dairy herds to estimate the pathogen-specific incidence rate of clinical mastitis (IRCM). The severity of the cases and the potential associations with herd hygiene were studied. Participating producers sampled 845 cases and 692 dairy cows. The mean and median IRCM was estimated at 7.4 and 5.3 quarter cases per 10,000 cow-days at risk, respectively. A large between-herd variation was observed (range of 0-21.3). In general, the IRCM was lower in heifers compared with multiparous cows (2.9 vs. 11.0 quarter cases per 10,000 cow-days at risk). However, the overall IRCM in the first week after calving was higher in heifers compared with cows (43.4 vs. 31.6 quarter cases per 10,000 cow-days at risk). Streptococcus uberis (18.2% of the cases) and Escherichia coli (15.5%) were the most frequently isolated pathogens and no growth was observed in 19.9% of the cases. The majority of the cases (63.1%) were mild (only clots in milk). Moderate (hard quarter without general signs) and severe symptoms (systemic illness) were observed in 29.9 and 7.0% of the cases, respectively. Isolation of E. coli (vs. any other culture result) was more likely in moderate and severe cases compared with mild cases. Overall IRCM and E. coli IRCM were higher in dirty compared with clean herds based on udder hygiene scores (9.0 and 1.7 vs. 6.0 and 0.6 quarter cases per 10,000 cow-days at risk, respectively). This study broadens the knowledge on clinical mastitis in Flemish dairy herds and underlines the high risk of CM in early-lactation heifers, the role of the so-called environmental pathogens, and herd hygiene.

Further evidence for the existence of environmental and host-associated species of coagulase-negative staphylococci in dairy cattle.

Coagulase-negative staphylococci (CNS) are abundantly present in the dairy farm environment and on bovine skin and mucosae. They are also the most prevalent bacteria causing bovine intramammary infections (IMI). Reservoirs and transmission routes of CNS are not yet fully unraveled. The objectives of this study were to explore the distribution of CNS in parlor-related extramammary niches and to compare it to the distributions of CNS causing IMI in those herds. Niches that were targeted in this study were cows' teat apices, milking machine unit liners, and milker's skin or gloves. Each of the three herds had its own CNS microbiota in those niches. The most prevalent species in the parlor-related extramammary niches were Staphylococcus cohnii, S. fleurettii, and S. equorum in the first, second, and third herd, respectively, whereas S. haemolyticus and S. sciuri were found in all herds. S. cohnii and S. fleurettii, as well as S. haemolyticus, which was present in each herd, were also frequently found in milk samples. By contrast, S. chromogenes, S. simulans, and S. xylosus favored the mammary gland, whereas S. equorum was more common in the parlor-associated niches. Within each herd, species distribution was similar between teat apices and milking machine unit liners. In conclusion, some of the extramammary niches related to the milking process might act as infection sources for IMI-causing CNS. This study provides further evidence that the group of CNS species is comprised of environmental, opportunistic and host-adapted species which differ in ecology.

Staphylococcus agnetis sp. nov., a coagulase-variable species from bovine subclinical and mild clinical mastitis.

Thirteen Gram-positive-staining coagulase-variable staphylococci were isolated from subclinical and mild clinical mastitic bovine milk (n=12) and a teat apex (n=1). The results of sequence analysis of the 16S rRNA gene and two housekeeping genes, rpoB and tuf, and DNA fingerprinting with amplified fragment length polymorphism (AFLP) analysis showed that the isolates formed a separate branch within the genus Staphylococcus. The phylogenetically most closely related species were Staphylococcus hyicus and Staphylococcus chromogenes. DNA-DNA hybridization with S. hyicus DSM 20459(T) and S. chromogenes DSM 20674(T) confirmed that the isolates belonged to a separate species. The predominant fatty acids were i-C(15:0), ai-C(15:0), i-C(17:0) and C(20:0) and the peptidoglycan type was A3α L-Lys-Gly(5). Based on the results of genotypic and phenotypic analyses, it is proposed that the thirteen isolates represent a novel species, for which the name Staphylococcus agnetis sp. nov. is proposed. Strain 6-4(T) (=DSM 23656(T)=CCUG 59809(T)) is the type strain.

Validation of amplified fragment length polymorphism genotyping for species identification of bovine associated coagulase-negative staphylococci.

In many countries, coagulase-negative staphylococci (CNS) are currently the most common cause of intramammary infection in lactating cows. In order to elucidate the importance of various CNS species in udder health and milk quality, further research conducted on the species level is required. Phenotypic identification of CNS species appears to be unreliable and more accurate and reproducible genotypic methods are needed. In the current study, use of amplified fragment length polymorphism (AFLP) genotyping was validated for species identification of bovine associated CNS. An initial reference library was generated with AFLP fingerprints of 52 different CNS type and reference strains. Next, 247 bovine CNS field isolates with known species identity were analyzed. These field isolates had been previously identified by gene sequencing and were randomly divided into two subsets, i.e. a training set and a validation set. The training set was identified against the initial reference library containing only type and reference strains, which resulted in a typeability of 80.5%. Accuracy of the AFLP identifications, being the correspondence with gene sequencing results, was 95.0%. Fingerprints of the training set were then added to the initial library and identification of the validation set was done by means of this extended library. By adding bovine CNS to the library, performance of the AFLP identification method improved considerably. Final typeability and accuracy were 98.4% and 99.2%, respectively. Numerical analysis of AFLP fingerprints proves to be an accurate genotypic method for identification of CNS from bovine origin. The constructed AFLP library provides a useful identification tool for field studies on the subject of CNS.

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.