We found significant differences in quarter milk SCC from quarters culture positive for different Staphylococcus species commonly associated with bovine IMI. The well-known major pathogen S. aureus was associated with the highest milk SCC, although the S. aureus isolates in our data were originally incorrectly identified as NAS and may thus have some special characteristics compared to the correctly identified S. aureus population. The NAS species S. agnetis/S. hyicus and S. simulans, earlier associated with clinical mastitis or more intense inflammation [5, 15,16,17], were found in our study to cause milk SCC statistically not significantly different from that caused by S. aureus. These species, as well as S. chromogenes, were mainly (86–88%) isolated from mastitic quarters, whereas a large proportion (30–50%) of other species were isolated from non-mastitic quarters. For example, 42% of S. epidermidis and 33% of S. haemolyticus originated from non-mastitic quarters, which decreased the overall mean SCC calculated for these species. However, SCC in mastitic samples positive for these two species was on the same level with the SCC of S. simulans positive samples and did not differ significantly from the SCC of S. agnetis/S. hyicus or S. simulans positive samples. Staphylococcus epidermidis and S. haemolyticus were associated with high SCC also in the study by Nyman et al. : the median SCC of milk samples from subclinical mastitis positive for S. epidermidis or S. haemolyticus was high and on the same level (from 1.1 to 2.0 million cells/mL) as the median SCCs of samples positive for S. simulans or S. hyicus. This may indicate that the species commonly isolated from healthy quarters mainly reside on teat skin or teat canal and lack effective virulence properties helping them to invade into the mammary gland. But once they manage to do so, they seem to cause an inflammatory reaction not very different from that of the NAS species predominantly associated with high SCC. Some strains may also be more virulent than other strains and one possible explanation is that the virulent strains cause IMI while the less virulent strains are controlled by the hosts’ immune defense already in the teat canal. Figure 1 shows that the biggest difference between the species is the proportion of isolates belonging to the SCC class 1 (non-mastitic). Distribution of isolates of different NAS species into the SCC classes illustrates better the effect of NAS species on SCC than the mean SCC. For example, 35.7% of S. aureus, 28.0% of S. agnetis/S. hyicus and 26.2% of S. simulans isolates belong to the two highest SCC classes, i.e., >500,000 cells/mL, while that proportion of all other species are < 20% (Table 2).
Supré et al.  followed quarters of 89 cows with diagnosed IMI on three herds with repeated sampling and found IMI by S. chromogenes, S. simulans and S. xylosus to increase milk SCC more than other NAS species isolated in quarter milk samples. The differences between SCC caused by these three NAS species and S. aureus infection were not statistically significantly different, although the actual geometric mean SCC of milk samples from S. aureus mastitis, 495,000 cells/mL, was higher than that of milk samples from S. chromogenes (226,000 cells/mL), S. simulans (130,000 cells/mL) or S. xylosus (85,000 cells/mL) mastitis. In contrast to the results of Supré et al. , S. chromogenes did not belong to the NAS species causing highest SCC in our study. Although most S. chromogenes were isolated from mastitic quarters, 80.4% were isolated from quarters with SCC ≤ 300,000 cells/mL (Table 2). Wuytack et al.  performed a cross-sectional sampling of approximately 25% of cows on eight herds. In this study, 39% of quarter milk samples originated from quarters with SCC ≤ 50,000 cells/mL and classified as non-mastitic. The NAS positive samples had a geometric mean SCC of 109,000 cells/mL, which is in agreement with the SCC from all samples in our study. The geometric mean of samples positive for S. chromogenes was 156,000 cells/mL and that of samples positive for S. haemolyticus 177,000 cells/mL . A similar SCC level, 144,000 cells/mL, in quarters of newly calved primiparous cows infected with S. chromogenes was reported by Valckenier et al. . Lower SCCs were reported by Condas et al. . The geometric mean SCC of NAS-positive quarters was 70,000 cells/mL. Staphylococcus agnetis, S. capitis, S. hyicus, S. gallinarum and S. simulans were the species which increased the SCC most and the geometric mean SCC of these species was significantly higher than that of the whole NAS group. The geometric mean SCCs of samples positive for these species varied from 81,000 cells/mL for quarters positive for S. agnetis to 123,000 cells/mL for samples positive for S. capitis. For comparison, the mean SCC for S. aureus positive samples was 174,000 cells/mL. Condas et al.  used a large data set from a sampling similar to that of Wuytack et al.  and likely including a large proportion of isolates originating from non-mastitic quarters.
Sampling strategy has much effect on the results. Higher SCCs are obtained when samples are taken from subclinical or clinical mastitis cases, compared to studies where lactating quarters are sampled without knowledge about the inflammation status. This kind of sampling increases the probability that some isolates originate from the teat skin or teat canal of non-mastitic cows, as shown by 29% of isolates originating from non-mastitic quarters in our study and 39% in the study by Wuytack et al. . Milk SCC varies a lot, from few thousands in a healthy quarter up to millions of cells/mL in a severely inflamed quarter. Mastitis is a dynamic phenomenon, and the infection status differs also during the IMI. Typically, the SCC is high at the beginning of a Staphylococcus IMI, but if the pathogen succeeds in avoiding the defensive reaction of the udder and the infection persists, the SCC usually decreases to a moderate but persistent level, with some variation .
In general, NAS cause subclinical or mild clinical mastitis, but differences between NAS species have been found in this respect. Some NAS species, in particular S. agnetis/S. hyicus and S. simulans, seem to be able to cause clinical mastitis more often than other NAS species [15,16,17, 20, 22]. In contrast, some NAS species, especially S. epidermidis, which is one of the most prevalent isolates from mastitis, has in experimental mastitis model shown to cause a milder inflammatory reaction than S. simulans . However, S. epidermidis was associated with high SCC in milk samples originating from subclinical mastitis . We found 42% of S. epidermidis isolates to originate from non-mastitic quarters but the SCC of mastitic S. epidermidis positive quarters not to be significantly different from the SCC caused by S. agnetis/S. hyicus or S. simulans. Unlike S. chromogenes, which typically affects heifers and primiparous cows and is considered part of bovine skin microbiota, S. epidermidis IMI is usually detected in multiparous cows [4, 18, 23]. Staphylococcus epidermidis is one of the most abundant bacterial colonisers of healthy human skin , but does it belong to bovine skin microbiota too? S. epidermidis has shown to be the NAS species commonly related to antimicrobial resistance and the species most commonly positive for the mecA gene encoding methicillin resistance [23, 25,26,27]. Persistent antimicrobial resistant S. epidermidis IMIs may cause a risk of emergence of antimicrobial-resistant staphylococci in the herd.
Following NAS species distribution in mastitis milk samples on herd level could help evaluation of transmission sources. For example, high presence of S. epidermidis may indicate transmission through milking equipment or possibly via milkers’ hands. Wuytack et al.  took swab samples from teat apices before and after milking. While most species were detected on teat skin both before and after milking, were S. epidermidis and S. agnetis detected only after milking, indicating infection from milking machine liners. However, strain typing is needed to ensure a common transmission source. In contrast, variation of different NAS species in milk samples indicates environmental transmission from manure, bedding, and other sources. At least 12 different NAS species have been isolated from bovine fecal samples , including species isolated frequently from milk samples. Laboratories using MALDI-TOF MS can already provide NAS speciation but this is not yet possible for laboratories that use commercial mastitis PCR tests. However, if the demand to add the most common NAS species in the tests’ selection of microbe species increases, the manufacturers of commercial mastitis PCR tests may become interested in providing such tests.