Frequency of non-aureus staphylococci and mammaliicocci species isolated from quarter clinical mastitis: a six-year retrospective study

Non-aureus staphylococci and mammaliicocci (NASM) are the most frequently isolated bacterial group from bovine milk samples. Most studies focus on subclinical mastitis caused by NASM, however NASM can cause clinical mastitis (CM) as well. We evaluated retrospective data from 6 years (2017–2022) to determine the species and frequency of NASM isolated from quarter bovine CM. The data comprised of microbiological results from quarter CM samples routinely submitted to Quality Milk Production Services (QMPS) , Cornell University, NY, US, for microbial identification by MALDI-TOF MS. A total of 9,909 microbiological results from 410 dairy herds were evaluated. Our results showed that 29 distinct NASM species were identified, with the 8 most prevalent NASM species being Staphylococcus chromogenes , S. haemolyticus , S. simulans , S. epidermidis , S. sciuri (now Mammaliicoc-cus sciuri ), S. agnetis/S. hyicus , S. borealis , and S. xy-losus . The NASM distribution remained similar among seasons, but the frequency of NASM CM cases was higher during the summer. Our results showed different patterns of variations in the isolation frequency over time, depending on the bacterial species: increasing or decreasing trends, cyclic fluctuations, and except for S. borealis , a significant seasonality effect for our study's most prevalent NASM was observed. This study showed that S. chromogenes remains the most frequent (43%) NASM species identified from bovine CM, followed by S. haemolyticus (18%), and S. simulans (12%).


INTRODUCTION
Non-aureus staphylococci and mammaliicocci (NASM) are the most frequently isolated bacterial group from bovine milk samples, comprising a heterogeneous group of numerous species (Condas et al., 2017a;Acharya et al., 2021;Taponen et al., 2022).NASM are Gram-positive bacteria, and they are known to be more commonly associated with subclinical mastitis (SCM) rather than clinical mastitis (CM) (Addis et al., 2023;Ruiz-Romero and Vargas-Bello-Pérez, 2023).Despite the high frequency of isolation, the epidemiology and the role of NASM as mastitis pathogens are not fully understood.
Based on the current literature, the impacts of NASM intramammary infection (IMI) on udder health seem inconsistent.Some studies report that NASM may offer cross-protection against other mastitis pathogens (De Buck et al., 2021;Souza et al., 2023) or are associated with increased milk production (Schukken et al., 2009).Contrarily, other studies describe NASM as persistent causes of mastitis (Vanderhaeghen et al., 2014;Acharya et al., 2021) with a negative impact on milk yield (Heikkilä et al., 2018).According to Condas et al. (2017b), discordant conclusions between studies have been described because NASM was generally considered as a single group of bacteria without differentiation at the species level.However, previous studies already described that species belonging to the NASM group have different behaviors (De Buck et al., 2021;Souza et al., 2022), which contribute to different mastitis outcomes.Evaluations of NASM at species-level can be helpful in Frequency of non-aureus staphylococci and mammaliicocci species isolated from quarter clinical mastitis: a six-year retrospective study G. Freu,123 G. Gioia, 4 B. Gross, 4 F. Biscarini, 5 P. Virkler, 6 R. Watters, 7 M. F. Addis,23 R. J. Franklin-Guild, 8 J. Runyan, 8 A. J. Masroure,23 V. Bronzo,23 M. V. dos Santos, 1 and P. Moroni234* understanding their distribution over time and their impact on dairy cows' udder health.
Specific NASM species can be isolated from diverse sources and may also affect the mammary gland differently.While some NASM may persist in the skin, teat canal, and udder, others seem to be transient residents of the udder from the environment (De Buck et al., 2021).Staphylococcus chromogenes and S. simulans seem to be the 2 most important mastitis-causing pathogens of the NASM group (Vanderhaeghen et al., 2014), considering they are described as the most frequently isolated pathogens from milk samples in previous reports (Acharya et al., 2021;Freu et al., 2023).On the other hand, S. warneri, S. xylosus, S. microti, S. haemolyticus, and S. succinus can act as IMI agents or as sample contaminants (Hamel et al., 2020).Several studies have been performed to identify risk factors associated with the prevalence of mastitis-causing NASM.Most NASM species isolation has been reported to occur at calving and during early lactation (Condas et al., 2017a), while from a herd perspective, farming systems (Petzer et al., 2022) and geographical regions have been reported to affect NASM species distribution (Condas et al., 2017a).
Despite significant scientific advances, NASM infections are still a matter of debate.While most studies focus on subclinical IMI caused by NASM, this bacterial group can also be an important cause of CM with considerable milk losses, up to 1.8 kg/d (Heikkilä et al., 2018).The aim of this study was to analyze retrospective data from Quality Milk Production Services (QMPS) to determine the species stratification and frequency of NASM species isolated from CM cases between 2017 to 2022 in 410 dairy herds.

Milk sample collection and analysis
As part of the herds' milking routine, CM cases were identified by examining the first milk strips and recorded by farm personnel.Cows with visible changes in the appearance of milk (presence of lumps, pus, clots, blood, or aqueous milk) and/or in the udder (edema, redness) were identified and milk samples from the affected mammary quarters were collected.
Quarter milk sample collection procedures were performed according to QMPS procedures (https: / / www .vet.cornell.edu/animal -health -diagnostic -center/ programs/ quality -milk -production/ services/ sample -submission), which are based on National Mastitis Council guidelines (NMC, 2017).Sample collection was performed by farm personnel or veterinarians who were well-acquainted with QMPS recommendations.Before milk collection, teat ends were cleaned and disinfected.The first milk strips were discarded and the milk was collected directly into a sterile tube.After milk collection, the samples were immediately transported in an isothermal box (≃ 4°C) to the laboratory.If milk samples were unable to be shipped within 24 h they were kept frozen at the farms (−20°C) and thawed at the laboratory for microbiological analysis.

Data set records
Retrospective data from 6 years (2017-2022) were used to determine the longitudinal trends of NASM isolated from quarter bovine CM.Data were obtained from the Ithaca and Warsaw QMPS laboratories, Cornell University, US.
The study used records from a total number of 304,753 quarter CM samples.Of this total, 43.81% (n = 133,503) had no bacterial growth, 31.39%(n = 95,666) had Gram-positive, and 18.03% (n = 54,941) had Gram-negative pathogens isolated.Other microorganisms (such as yeast and mold) were isolated in 2.58% (n = 7,876) of the CM samples, and contaminated samples comprised 2.43% (n = 7,401).Mixed culture (e.g., 2 different pathogens isolated from the same milk sample) was identified in 1.76% (n = 5,366) of the samples.Data about farm identification and sampling date were also available, while additional information on DIM, parity, CM severity, and treatment records were not available.
Only NASM-positive samples from CM cases were evaluated in this study.The initial data set was comprised by 11,735 NASM records.Duplicate entries and samples that were not mammary quarter clinical samples (i.e., composite samples) were removed.Mixed infections between NASM and/or other pathogens (such as other Gram-positive and Gram-negative bacte-ria, yeasts, mold, Bacillus sp.) were also removed from the data analysis, and only NASM pure cultures were considered in this study.Some NASM species have been recently reclassified as mammaliicoccal species (Madhaiyan et al., 2020).This applies in our study to S. sciuri, and S. vitulinus which are now Mammaliicoccus sciuri, and M. vitulinus, respectively.

Statistical analysis
The microbiological results were stored in Excel (Microsoft Office, 2016) (Acharya et al., 2021).
For the analysis of trends, autocorrelations, and model-based pairwise correlations, only data on NASM species with more than 100 total counts (isolates) were retained; these were the following 8 NASM: S. chromogenes, S. haemolyticus, S. simulans, S. epidermidis, M. sciuri, S. agnetis/S.hyicus (MALDI-TOF MS could not differentiate between them), S. borealis, and S. xylosus.
To assess the presence of an increasing or decreasing trend, we used the non-parametric Jonckheere-Terpstra test (Terpstra 1952;Jonckheere 1954) for ordered data series.This test evaluates the null hypothesis of no ordering against the one-sided increasing (T1 ≤ T2 ... ≤ T2k) or decreasing (T1 ≥ T2 ≥ … Tk) alternatives.The Jonckheere-Terpstra test was conducted using 1000 permutations (reshuffling of the data series to construct the empirical null distribution).
To assess the presence of dependencies in the data series of NASM milk isolates over time, autocorrelations were calculated for each bacterial species, with a maximum lag of 18.The presence of autocorrelations was tested against specific ""white noise"" time series data (time series that show no autocorrelation), for which 95% of the autocorrelation values are expected to fall within ±

T
, where T is the length of the time series (Hyndman and Athanasopoulos 2014).
The effect of seasonality on the number of NASM cases was evaluated also by fitting linear regression models that included the effect of either the month of the year (12 classes) or the season (4 classes): where y ijk is the number of isolates for a specific NASM, μ i is the overall mean, lab j is the effect of laboratory (2 classes), month \ season k is the effect of the month (or the season) of the year (in classes), β 1 and β 2 are the corresponding regression coefficients, and e ijk are residuals.
Using equation ( 1), the residuals of the 2 models (one including month, and one including season) were used to estimate seasonality-adjusted pairwise correlations between NASM isolates, which were compared with the raw (non-model-adjusted) pairwise correlations between the same NASM.
The R environment for statistical computing was used to perform the additional data handling and statistical analysis (R Core Team, 2023).A statistical significance was assumed at P ≤ 0.05, and tendency of significance was considered if the P-value was between 0.05 and 0.10.

RESULTS
The initial data set comprised 11,735 microbiological records of NASM between 2017 and 2022.Initially, duplicate data and non-quarter clinical samples (i.e., composite samples) were removed (7.29%; n = 856/11,735).The data set comprised 10,879 microbiological records of NASM isolated from CM cases from 433 US dairy herds for a total of 7,899 cows.Of this total, 8.92% (n = 970/10,879) of the samples had mixed infections (2 different pathogens isolated from the same milk sample), and they were excluded from data analyses, and results were not reported.A total of 9,909 microbiological results from 410 dairy herds were evaluated.
Figure 1 shows the trend of NASM isolates from CM cases in the period 2017-2022.An obvious question to ask is whether there is an increasing or decreasing trend for any of these pathogens: results from the Jonckheere-Terpstra test show significant increasing trends (P < 0.05) for S. epidermidis and M. sciuri, and a significant decreasing trend for S. xylosus.Data on S. simulans showed a suggestive decreasing trend (P < 0.10) (Table 2).

Freu et al.: NASM FREQUENCY FROM CLINICAL MASTITIS
In most cases, autocorrelations show a cyclic pattern: initial positive correlations are subsequently replaced by negative correlations and vice versa.These correlations are significantly different from white noise background (null hypothesis of absence of autocorrelation: blue dashed lines in Supplementary Figure 2) mainly at short time-lags, e.g., datapoints close together are correlated, while farther apart datapoints are not.Notable exceptions were S. chromogenes, which shows significant autocorrelations also at larger time-lags, and S. epidermidis which consistently showed positive correlations (consistent with the significantly increasing trend from the Jonckheere's test).
Considering the overall results during the studied period (2017 to 2022), S. chromogenes was the most frequently isolated NASM species, and remained the most frequently isolated species in all seasons (Table 3).In addition, S. haemolyticus, S. simulans, S. epidermidis, and M. sciuri remained the most frequent NASM species isolated from bovine CM cases according to seasonality.The highest number of CM cases caused by NASM was observed in summer (32.45% of the total samples), followed by fall (24.20%),spring (23.53%), and winter (19.82%;Table 3).A similar seasonality trend was observed for the total CM cases considering all pathogens.
The isolation frequency of the 8 most common NASM species varied between years and seasons (Supplementary Figure 1).S. chromogenes and S. simulans showed a heterogeneous isolation frequency during the years and seasons evaluated.Except in summer, S. haemolyticus showed an increase in the isolation frequency for all seasons in 2020 compared with the other evaluated years.A decrease in S. haemolyticus and an increase in M. sciuri and S. borealis isolation frequency was observed for all seasons in 2022 (Supplementary Figure 1).S. epidermidis showed an increase in the isolation frequency over time.Except in winter, the highest isolation frequency of S. agnetis/S.hyicus was observed for all seasons in 2020.
The effect of seasonality on the NASM isolates frequency was checked by modeling these as a function of either month of the year or season, for each NASM species.The pairwise correlations between isolate counts for the 8 NASM species with more than 100 total counts decreased when adjusting for month or season.For example, the correlation between S. chromogenes and S. agnetis/S.hyicus changed from 0.275 to 0.148 and 0.176; the correlations between S. haemolyticus and S. simulans changed from 0.295 to 0.229 and 0.233, considering the raw correlations and model-adjusted correlations for month and season, respectively.The effect of month or season on the isolate counts was significant (P < 0.05) for all species, except S. borealis and S. haemolyticus with the month model, while for M. sciuri with the month-model, the effect was suggestive (P < 0.10) (Table 4).

DISCUSSION
NASM are the most frequently isolated bacterial group from bovine milk samples.Most of previous studies focused on the role of NASM causing SCM, however NASM can also be associated with CM.This study analyzed retrospective data from 6 years (2017-2022) to determine the speciation and frequency of NASM isolated from bovine CM.Our results showed 29 NASM species were identified from CM cases.The 8 most prevalent NASM accounted for 95% of the total isolated species, which included S. chromogenes, S. haemolyticus, S. simulans, S. epidermidis, M. sciuri, S. agnetis/S.hyicus, S. borealis, and S. xylosus.These results are consistent with previous reports evaluating NASM from CM and SCM cases (Condas et al., 2017a;De Buck et al., 2021;Kurban et al., 2022).
Similarly with previous studies, S. chromogenes is the primary NASM species isolated from CM in dairy cows (Addis et al., 2023;Freu et al., 2023).In our study, S. chromogenes was isolated in approximately 43% of all NASM isolates, which is similar to other studies carried out in North America (48 to 49%; Condas et al., 2017b;a;Jenkins et al., 2019) and Europe (41%; De Visscher et al., 2016) evaluating NASM from CM and SCM cases.The higher isolation frequency of S. chromogenes may be a result of the higher ability of this pathogen to successfully colonize the mammary gland compared with other NASM species (De Visscher et al., 2016;Hamel et al., 2020).
After S. chromogenes, S. haemolyticus was the second most frequently isolated NASM species from CM cases in our study.In agreement with our findings, S. haemolyticus was the second most frequently found pathogen in a US dry-cow treatment clinical trial (Jenkins et al., 2019).Although S. haemolyticus has been described as an environmental pathogen that can act as sample contaminant (Hamel et al., 2020), it has the ability to colonize the skin and teat apex and consequently cause IMI (Taponen et al., 2022).Our results supported the idea of S. haemolyticus as an opportunistic pathogen for the mammary gland as only CM cases were evaluated.
We observed a reduction in S. haemolyticus isolation and an increase in the frequency of S. borealis isolation in 2022 compared with previous years.This reduction occurred because of the MALDI Biotyper System library update at the AHDC on November 30, 2021 which began identifying a novel species of S. borealis.Accordingly, isolates identified as S. haemolyticus here may include some isolates of S. borealis as MALDI-TOF   MS did not differentiate them before the library update (Pain et al., 2020).S. borealis appears to be an emerging mastitis pathogen that needs to be studied and monitored in future research.It is important to mention that the MALDI Biotyper System was originally developed for human clinical microbiology and recently has begun to be used for identification of mastitis isolates (Tomazi et al., 2014).The veterinary spectra database has been constantly updated, and it is possible that new species can emerge or may benefit from a spectrum library expansion.
S. simulans was the third most prevalent NASM species in our study, and this pathogen was also a frequent NASM species isolated in previous reports evaluating quarter milk samples suspected of SCM (Nyman et al., 2018) or in a mastitis prevalence study (Taponen et al., 2022).Previously, S. chromogenes and S. simulans were associated with IMI in > 90% of cases when assessing repeated NASM species detection in milk samples to differentiate between IMI and contamination (Hamel et al., 2020).These 2 species accounted for more than 54% of the total NASM isolates of CM cases in our study.S. chromogenes and S. simulans have been reported as host-adapted pathogens (Piessens et al., 2011;Fry et al., 2014;De Visscher et al., 2016), with adverse impacts on udder health (Supré et al., 2011) and may cause persistent IMI (Piessens et al., 2011;Nyman et al., 2018), which may partially explain the high isolation frequency of both pathogens in our study.
Despite being isolated in lower frequency than S. chromogenes, S. haemolyticus, and S. simulans, other NASM species observed in our study were S. epider-midis, M. sciuri, S. agnetis/S.hyicus, and S. xylosus.Previous studies reported this latter group of NASM as important mastitis causes, although with varying isolation frequency (Jenkins et al., 2019;Taponen et al., 2022;Addis et al., 2023).For example, in a Finnish mastitis prevalence study, S. epidermidis was the most prevalent, while M. sciuri was one of the less frequently isolated NASM species (Taponen et al., 2022).In a US clinical trial study, S. epidermidis and M. sciuri have been described as the most prevalent NASM species, and S. agnetis/S.hyicus and S. xylosus occurred at frequencies less than 1% (Jenkins et al., 2019).It is important to mention that although S. epidermidis was the fourth most frequently isolated microorganism in our study, this pathogen has been previously reported as the main NASM species isolated from bovine milk samples in many studies (Mahmmod et al., 2018;Nyman et al., 2018;Taponen et al., 2022).Also S. epidermidis has been described to be more prevalent in SCM than CM (Persson Waller et al., 2011).
The varying NASM species isolation frequency observed among different studies may be attributed to the sampling strategy, mastitis presentation form, geographic region, management factors, herd size, housing systems, and methodology of bacterial identification used (Condas et al., 2017a;De Visscher et al., 2017;Petzer et al., 2022).Regarding the sampling strategy, e.g., herd, cow, or quarter-level sampling as well as the sampling frequency and sampling method used (Friman et al., 2017) could result in differences in mastitis outcomes.In general, composite samples have a low sensitivity, caused by the dilution of bacterial numbers by milk from uninfected quarters (Adkins and Middleton, 2018).The sampling at mammary quarter level can be considered one of the strengths of our study.
Once this study focused on CM caused by NASM, we were not able to identify differences between NASM species isolated from CM and SCM cases.No differences between mastitis occurrence (CM and SCM) for S. chromogenes, S. simulans, and S. haemolyticus were found by Persson Waller et al. (2011).A recent retrospective investigation showed that S. chromogenes, S. haemolyticus, S. epidermidis, M. sciuri, S. xylosus were the 5 most prevalent NASM species isolates from both CM and SCM cases (Addis et al., 2023).This suggests that other factors besides the mastitis presentation form may influence the distribution of NASM species.
It has been reported that NASM has the ability to survive in different environments and opportunistically cause mastitis (Ruiz-Romero et al., 2023).Piessens et al. (2011) emphasized that the distribution of NASM species can be herd-specific.Therefore herd-specific factors e.g., poor housing conditions, management, and milking procedures can also influence species-specific  prevalence.Finally, concerning bacterial identification, phenotypical tests are not able to make a reliable distinction between NASM species (Tomazi et al., 2014).Molecular methods are increasingly replacing conventional microbiological culture (different selective media and biochemical methods) in mastitis diagnostics.The proteomics analysis by MALDI TOF -MS has been described as an accurate, fast, and cost-effective tool that allows species-level identification for many pathogens isolated from milk samples (Kurban et al., 2022).Using longitudinal data, trends between consecutive datapoints are increasing or decreasing and can show a cyclic pattern.Autocorrelations within the count series for each bacterial species can help assess the presence of such patterns in the data.When evaluating longitudinal data, an interesting observation to make is the trends over time, i.e., how the number of mastitis cases according to each pathogen have changed over time.Recently research with NASM strongly suggests that  Staphylococci not identified at species-level by MALDI-TOF MS. strain differences within species should no longer be ignored (Wuytack et al., 2020;De Buck et al., 2021), and these differences could help us understand the trends over time (increase, decrease, or cyclic pattern) observed for specific NASM species in our study.In this sense, although not evaluated here, the strain's virulence profile and mastitis severity could also affect the differences in mastitis outcome (De Buck et al., 2021;Souza et al., 2022).The ability of NASM to cause mild CM was demonstrated by Wuytack et al. (2020), but this last study also reported NASM in teat apices, and rectal feces samples in the same herds.It is possible that the mastitis-causing pathogen would remain a predominant factor relating to mastitis severity (Fredebeul-Krein et al., 2022).In a previous study, the increased numbers of toxin and host immune evasion genes specifically were associated with more severe disease outcomes (Naushad et al., 2019).Later, De Buck et al. (2021) reported that the development of disease and interactions of virulence factors with the host are complex and determined by the interaction of genes rather than the presence of a specific virulence gene.Differences in the virulence capacity of NASM species found in mastitis seem to be present, although a clear and consistent understanding is yet to be established.
Although previous studies consider NASM as a minor, commensal, or contaminants pathogen (Friman et al., 2017;Hamel et al., 2020;Wuytack et al., 2020), the results of our study suggest that the role of NASM as a cause of CM should not be ignored.Further studies evaluating the differences between NASM species isolated from CM and SCM are recommended.
An increase in M. sciuri isolation frequency was observed in 2022 compared with previous years and across all seasons.The Jonckheere-Terpstra test confirmed this increase isolation frequency of M. sciuri.This increase in the M. sciuri isolation frequency was mainly seen in 3 farms involved in our study (F14, n = 26 cases; F194, n = 40 cases; and F334, n = 21).These 3 farms accounted for > 65% of the M. sciuri CM cases recorded in 2022.Most CM cases by M. sciuri occurred during the winter for F14 (17/26; 65.4%), during the summer for F194 (22/40; 55.0%) and during the fall for F334 (11/21; 52.4%).M. sciuri has been previously described by its environmental nature (De Visscher et al., 2016).Therefore, farm management seems to play an important role in M. sciuri infections.Recently M. sciuri has emerged as a pathogen of global interest, carrying a broad repertoire of antimicrobial resistance genes (Moura et al., 2023).A better understanding of M. sciuri behavior as a cause of CM and its impact on udder health is needed.The One Health perspective should be considered as this pathogen is spreading at the human-animal-environment interface (Moura et al., 2023).
Our findings showed a large variability in the isolation frequency among the 8 most prevalent NASM according to seasonality and years, but with a significant seasonality effect (except for S. borealis).A seasonal effect on NASM species identification from bulk milk samples has been previously reported (De Visscher et al., 2017), but contrasts with an earlier study by Gillespie et al. (2012), which investigated Staphylococcus spp. in bulk tank milk samples from East Tennessee dairy farms and did not observe any seasonal effect.The variability between and within the seasons observed in our study can be partially attributed to the Staphylococcus group's ability to survive in different environments and opportunistically cause mastitis.In our study, summer showed the highest number of CM cases by NASM compared with fall, spring, and winter.Considering all pathogens, this same seasonality trend was observed for the total CM cases.Acharya et al. (2021) reported higher odds of isolating NASM from milk in the spring and summer months compared with the winter months but these authors do not discuss this result.We can speculate that summer weather conditions (e.g., heat and humidity) can create an environment conducive to bacterial growth and proliferation.These factors, associated with cow's heat stress, can affect the cow's immune system (Gupta et al., 2023), leading to an increased susceptibility to IMI, whereby NASM species can act as opportunistic pathogens.It is essential to mention that NASM species affect the mammary gland differently depending on its pathogenicity, virulence, ecology, host adaptation, and antimicrobial resistance profiles (De Buck et al., 2021).Differences among species should be further explored, and NASM behavior according to seasonality needs to be considered.
The primary limitation of this study is that it was based on passive surveillance data, as the decision for samples being submitted to the laboratory depended on a veterinarian's or farmer's choice.Data on herds (e.g., management, housing system, and bedding type), cows (parity, days in milk, and treatment history), and mastitis history (severity) were not available, which could help understand the NASM species distribution.A second aspect is that our data can also be comprised of results from field studies on CM cases or from onfarm surveillance.A subset of samples may present a selection bias, as it may originate from cows with a history of treatment failure or be related to a specific mastitis control program used on the studied farms.The third consideration is the MALDI Biotyper library, which, before the database expansion, may have underestimated (e.g., S. borealis) or overestimated (e.g.,

CONCLUSION
Twenty-nine NASM species were identified from bovine CM cases, with S. chromogenes, S. haemolyticus, S. simulans, S. epidermidis, M. sciuri, S. agnetis/S.hyicus, S. borealis, and S. xylosus being the 8 most prevalent NASM species.S. chromogenes was the most frequent NASM species isolated from bovine CM.A seasonality effect for our study's most prevalent NASM was observed.A variation in the isolation frequency was observed between years and seasons for our study's 8 most prevalent NASM.
, and M. vitulinus were referred as S. sciuri, and S. vitulinus in the Bruker Biotyper® library;.3 MALDI-TOF MS could not differentiate them.4Staphylococcinot identified at species-level by MALDI-TOF MS.

Figure 1 .
Figure 1.: Number of NASM species isolated from clinical mastitis in 410 dairy herds over time (year, month) in the period 2017-2022.Only pathogens with >100 total counts were included.

Freu
et al.: NASM FREQUENCY FROM CLINICAL MASTITIS

Freu
et al.: NASM FREQUENCY FROM CLINICAL MASTITIS

Freu
et al.: NASM FREQUENCY FROM CLINICAL MASTITISS.haemolyticus) some NASM species identified before 2022, although it still provided valuable information at NASM species level.Despite all these limitations, this study provides an understanding of NASM species distribution over time and valuable insights to enhance CM control strategies.Using NASM distribution data dairy herds could: a) identify which species require more or less attention, b) work to improve the milking routine (e.g., pre-and post-milking teat disinfection) or herd management, and c) reduce the reservoir of infection (e.g., strategic treatment and culling).

Table 1 .
Freu et al.: NASM FREQUENCY FROM CLINICAL MASTITIS Distribution of NASM species isolated from bovine clinical mastitis from 410 dairy herds during 2017 to 2022 NASM 1

Table 2 .
Jonckheere non-parametrical test for increasing or decreasing trends in the number of detected NASM from clinical mastitis samples from 2017 to 2022.Trends with P-values <0.05 was conventionally considered significant (presence of trend) 1Non-aureus staphylococci and mammaliicocci;.2 MALDI-TOF MS could not differentiate them.3 M. sciuri was referred as S. sciuri in the Bruker Biotyper® library.

Table 3 .
Distribution of NASM species isolated from bovine clinical mastitis from 410 dairy herds during 2017 to 2022, according to the seasonality 2M. sciuri, and M. vitulinus were referred as S. sciuri, and S. vitulinus in the Bruker Biotyper® library;.3MALDI-TOF MS could not differentiate them.4

Table 4 : F-statistic and P-values from linear regression models of counts as a function of either month of the year (12 classes) or season (4 classes)
2MALDI-TOF MS could not differentiate them.3M.sciuri was referred as S. sciuri in the Bruker Biotyper® library.