Non-aureus staphylococci (NAS) are predominantly isolated from bovine milk samples of quarters suffering from subclinical mastitis. They are also abundantly present on dairy cows' teat apices and can be recovered from bovine fecal samples, as recently described. Differences in ecology, epidemiology, effect on udder health, and virulence or protective traits have been reported among the species within this group. The objectives of this study were (1) to describe the species-specific distribution of NAS in 3 bovine-associated habitats, namely quarter milk, teat apices, and rectal feces, and (2) to evaluate the virulence potential of NAS by comparing their distribution in contrasting milk sample strata and the presence of selected virulence genes. A cross-sectional, systematic sampling procedure was followed in 8 dairy herds that participated in the local Dairy Herd Improvement program in Flanders, Belgium. Quarter milk samples (n = 573) were collected from 144 lactating cows in 8 herds. In 5 of the 8 herds, teat apex swabs (n = 192) were taken from 15 lactating cows, before and after milking, and from 18 dry cows. In the same 5 herds, rectal feces were sampled from 80 lactating cows (n = 80), taking into account that a cow could only serve as the source of one type of sample. In addition, milk samples of all clinical mastitis cases were continuously collected during the 1-yr study period from March 2017 to March 2018 in the 8 herds. In total, 1,676 Staphylococcus isolates were phenotypically identified and subjected to MALDI-TOF mass spectrometry. Thirty-three, 98, and 28% of all quarter milk, teat apex, and rectal fecal samples were NASpositive, respectively, reaffirming the presence of NAS in rectal feces. The overall predominant species in the 3 habitats combined were Staphylococcus haemolyticus, Staphylococcus chromogenes, and Staphylococcus hominis. Four, 16, and 12% of the healthy quarters (quarter milk somatic cell count ≤50,000 cells/mL of milk), quarters with subclinical mastitis (quarter milk somatic cell count >50,000 cells/mL of milk), and quarters with clinical mastitis, respectively, were NAS-positive, suggesting that the potential to cause (mild) clinical mastitis is present among NAS. This was substantiated by comparing the presence of virulence genes of NAS isolates originating from contrasting milk sample strata (healthy quarters and quarters with clinical mastitis).

The aims of this study were to determine whether non-aureus staphylococci (NAS) are present in rectal feces of healthy dairy cows, and if so, to delineate species to which they belong and to study several phenotypic and genotypic traits as a first step toward determining the potential impact of fecal shedding of NAS on bovine udder health. Fecal samples were aseptically collected from the rectum of 25 randomly selected clinically healthy dairy cows in a commercial dairy herd using an automated milking system. Fecal NAS were isolated and then identified at the species level using transfer RNA-intergenic spacer PCR and sequencing of the 16S rRNA housekeeping gene. Strain typing was performed using random amplification of polymorphic DNA (RAPD)-PCR. The antimicrobial resistance profiles, biofilm formation, and growth and inhibitory characteristics of all NAS isolates were evaluated. Half of the cows were shedding NAS, resulting in 31 NAS isolates belonging to 11 different species. The most prevalent species were Staphylococcus rostri (23%, n = 7), Staphylococcus cohnii (16%, n = 5), and Staphylococcus haemolyticus (13%, n = 4) with all Staphylococcus agnetis, Staphylococcus chromogenes, and Staph. rostri isolates belonging to the same strain according to RAPD banding patterns. Acquired antimicrobial resistance was observed in 28 of the 31 NAS isolates, mainly due to β-lactamase production. Most of the isolates (84%, n = 27) had a weak biofilm-forming potential, but only 2 contained the bap gene. The ica and aap genes were not detected in any of the isolates. In vitro growth of Staphylococcus aureus and Streptococcus dysgalactiae was inhibited by Staph. agnetis isolates, and Staph. chromogenes isolates were able to inhibit the growth of Strep. dysgalactiae and Streptococcus uberis.All fecal isolates were able to grow when oxygen and iron were limitedly available, mimicking the growth conditions in the mammary gland.

The presence of non-aureus staphylococci (NAS) in bovine rectal feces has recently been described. Similar to other mastitis causing pathogens, shedding of NAS in the environment could result in intramammary infection. The objective of this study was to investigate whether NAS strains present in feces can cause intramammary infection, likely via teat apex colonization. During a cross-sectional study in 5 dairy herds, samples were collected from the habitats quarter milk, teat apices, and rectal feces from 25%, 10%, and 25% of the lactating cows, respectively, with a cow serving as the source of one type of sample only. Samples from clinical mastitis cases were continuously collected during the 1-year study period as well. The 6 most prevalent NAS species, Staphylococcus (S.) chromogenes, S. cohnii, S. devriesei, S. equorum, S. haemolyticus, and S. hominis, were further subtyped by random amplification of polymorphic deoxyribonucleic acid polymerase chain reaction (RAPD-PCR), when the same NAS species was present in the same herd in the three habitats. For S. chromogenes, S. cohnii, S. devriesei, and S. haemolyticus, the same RAPD type was found in rectal feces, teat apices, and quarter milk, indicating that fecal NAS can infect the mammary gland. For S. hominis and S. equorum, we were unable to confirm the presence of the same RAPD types in the three habitats.

DO, where the herd incidence varied between 0.0 and 77.8%. The reduction in number of milkings in the weeks before DO had statistically significant effect on the ML incidence. When the milking frequency was reduced from 3 times/d to 2 or maintained at twice a day, cows had 11 (95% CI = 3.43-35.46) or 9 (95% CI = 1.85-48.22) times higher odds of leaking milk, respectively, compared with cows where the milking frequency was reduced from twice to once a day. Also, the milk production 24 h before DO was associated with ML incidence. Hence, cows with a milk production between 13 and 21 L or above 21 L had 2.3 (95% CI = 1.48-3.53) and 3.1 (95% CI = 1.79-5.3) times higher odds of leaking milk, respectively, compared with cows with a milk production below 13 L. A higher ML incidence was present in the group of cows with an average ISCC in the last 3 mo before DO ≥200,000 cells/mL (odds ratio = 1.7; 95% CI = 1.13-2.41) compared with cows with an average ISCC <100,000 cells/mL. Quarters with ML tended to have 2.0 times higher odds of developing clinical mastitis compared with quarters not leaking milk. Cows with ML tended to have 1.5 times higher odds of intramammary infections (i.e., an increase of ISCC or clinical mastitis) compared with cows without ML.

No abstract