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Mastitis affects a high proportion of dairy cows and is still one of the greatest challenges faced by the dairy industry. Staphylococcal bacteria remain the most important cause of mastitis worldwide. We investigated how distinct staphylococcal species evade some critical host defense mechanisms, which may dictate the establishment, severity, and persistence of infection and the outcome of possible therapeutic and prevention interventions. Thus, the present study investigated variations among distinct bovine-associated staphylococci in their capability to resist phagocytosis and to trigger respiratory burst activity of blood and milk polymorphonuclear neutrophil leukocytes (PMNL) in dairy cows. To do so, PMNL of 6 primiparous and 6 multiparous dairy cows were used. A collection of 38 non-aureus staphylococci (NAS) and 12 Staphylococcus aureus were included. The phagocytosis and intracellular reactive oxygen species (ROS) production by blood and milk PMNL were analyzed by flow cytometry. Phagocytosis, by both blood and milk PMNL, did not differ between S. aureus and NAS as a group, although within-NAS species differences were observed. Staphylococcus chromogenes (a so-called milk-adapted NAS species) better resisted phagocytosis by blood PMNL than the so-called environmental (i.e., Staphylococcus fleurettii) and opportunistic (i.e., Staphylococcus haemolyticus) NAS species. Otherwise, S. haemolyticus was better phagocytosed by blood PMNL than S. aureus, S. fleurettii, and S. chromogenes. No influence of the origin of the isolates within the staphylococci species in the resistance to phagocytosis by blood and milk PMNL was found. Overall, both S. aureus and NAS did not inhibit intracellular ROS production in blood and milk PMNL. Non-aureus staphylococci induced fewer ROS by milk PMNL than S. aureus, which was not true for blood PMNL, although species-specific differences in the intensity of ROS production were observed. Staphylococcus chromogenes induced more blood PMNL ROS than S. fleurettii and S. haemolyticus, and as much as S. aureus. Conversely, S. chromogenes induced fewer milk PMNL ROS than S. aureus. The origin of the isolates within the staphylococci species did not affect the ROS production by blood and milk PMNL. In conclusion, our study showed differences in staphylococci species in evading phagocytosis and triggering ROS production, which may explain the ability of some staphylococci species (i.e., S. aureus and S. chromogenes) to cause persistent infection and induce inflammation.

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 NAS-positive, 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).

This longitudinal study aimed to evaluate the impact of subclinical intramammary infection (IMI) with non-aureus staphylococcal (NAS) species in the first 18 d in milk (DIM) on the quarter milk somatic cell count (qSCC) and quarter milk yield (qMY) during the first 4 mo of lactation in Holstein Friesian heifers. Quarter milk samples were collected from 82 heifers from 1 to 4 DIM until 130 DIM on a biweekly (14 d) basis for determination of the qSCC; qMY data were available through the automatic milking systems. The quarter samples collected on the first (1–4 DIM) and second (15–18 DIM) sampling days were used for bacteriological culturing to determine the IMI status. In this study, 324 quarters from 82 heifers were enrolled, of which 68 were NAS-infected at the first sampling day. Only 16 (23.5%) of these quarters were still NAS-infected at the second sampling day, demonstrating the high spontaneous cure rate of these infections shortly after calving; 9 of these 16 cases were infected with the same NAS species. Interestingly, none of the NAS-infected quarters at the first sampling day acquired a new infection with a major pathogen at the second sampling day, whereas 2.3% of the noninfected quarters did. All 102 isolates phenotypically identified as NAS were further identified to the species level. Staphylococcus chromogenes was the most prevalent species on the first (29.4% of all NAS) and second (52.9%) sampling days. Quarters infected with Staph. chromogenes at the first sampling day had a significantly higher qSCC in later lactation than noninfected quarters, whereas this was not true for quarters infected with all other NAS species (i.e., as a group of species). The average daily qMY in the first 4 mo of lactation did not differ between noninfected quarters and quarters infected with Staph. chromogenes or all other NAS species at the first sampling day. Persistently NAS species-infected quarters in the first 18 DIM (i.e., infected with the same NAS species on the first and second sampling days) had the highest qSCC later in lactation, followed by quarters with a new NAS IMI (i.e., noninfected at the first sampling day and infected with NAS at the second sampling day). The qSCC from transiently NAS species-infected quarters (i.e., not infected with the same NAS species at the second sampling day) was not significantly higher in later lactation compared with that in noninfected quarters. The IMI status of quarters in the first 18 DIM, combining culture results at 1 to 4 and 15 to 18 DIM (new, persistent, and transient IMI), was not significantly associated with daily qMY in the first 4 mo after calving. In general, NAS should be considered minor pathogens with no adverse effect on daily qMY in quarters of heifers infected in the first 18 DIM and with a high spontaneous cure rate. Staphylococcus chromogenes was the most prevalent species, causing an increase in qSCC comparable to the level of quarters infected with a major pathogen; Staph. chromogenes caused most infections that persisted through at least the first 18 DIM.

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 objectives of this study were (1) to determine the test characteristics and predictive values of cow-level milk somatic cell count (SCC) information from (multiple) test-day recordings before drying off to identify major-pathogen-infected cows at drying off; and (2) to explore to what extent (an estimate of) the herd prevalence of subclinical mastitis, milk yield level, and parity of the cows affects the estimates. In total, 550 cows from 15 commercial dairy herds with overall good udder health management were dried-off during a study period of 6 mo. Test-day SCC were available through milk recording and within 5 d before drying off cows were sampled for quarter-level bacteriological culturing serving as the gold standard. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) were calculated at different threshold values of SCC, ranging between 50,000 and 500,000 cells/mL, to detect major-pathogen-infected cows. At a commonly used threshold of 200,000 cells/mL, the Se and Sp of (a combination of) test-day SCC before drying off ranged between 37.6 and 57.6% and between 66.7 and 79.3%, respectively. Still, estimates were modified by the herd level prevalence of subclinical mastitis and the cow's milk yield and parity. For instance, at the 200,000 cells/mL threshold using the geometric mean SCC of the 3 last test-days, the overall Se, Sp, PPV, and NPV were 37.6, 79.3, 30.8, and 83.9%, respectively, whereas these were 27.8, 87.5, 21.7, and 90.6%, respectively, for heifers and 40.3, 73.5, 33.3, and 78.9%, respectively, for multiparous cows. In conclusion, test-day SCC records obtained via milk recording are reliable to detect dairy cows at drying off that are not infected with major pathogens as determined by bacteriological culture and could eventually facilitate implementation of selective dry cow therapy in commercial dairy herds. Because estimates of the herd-level prevalence of subclinical mastitis, milk yield level, and parity of the cows affect the estimates of the test characteristics and predictive values to some extent, one should consider taking these parameters into account when differentiating infected from uninfected cows based on SCC data.

In this study, we studied infection dynamics across the dry period using test-day somatic cell count (SCC) data from 739 Holstein cows from 33 randomly selected commercial dairy herds in Flanders, all of which applied blanket dry-cow therapy at dry-off. First, we determined infection dynamics, combining the last test-day SCC before dry-off and the first test-day SCC after calving. Next, we determined the effect of dry period infection dynamics, adjusting for the level of the second test-day SCC after calving, on the evolution of test-day SCC and milk yield (MY) and on clinical mastitis and culling hazard in the subsequent lactation. Using an SCC threshold of 200,000 cells/mL, 12.6% of the cows considered healthy before dry-off acquired a new intramammary infection (IMI) across the dry period, whereas 66.9% of the cows considered infected before dry-off cured from IMI. Infection dynamics across the dry period significantly affect a cow's SCC, clinical mastitis risk, and culling hazard in the subsequent lactation. Cows with a new IMI, a cured IMI, or a chronic IMI across the dry period had higher test-day SCC than healthy cows, and their test-day SCC evolved differently over time. This was not the case for test-day milk yield, for which no association with infection dynamics was detected. Furthermore, cows with a second test-day SCC

An experimental trial was conducted to explore the effect of vaccination with a polyvalent vaccine against mastitis (Startvac) on the early immune response after experimental intramammary challenge with a heterologous killed Staphylococcus aureus strain. The effect of vaccination on milk production, clinical signs, quarter milk somatic cell count, milk polymorphonuclear neutrophilic leukocyte (PMN) concentration and viability, the concentration of antigen-specific antibodies [slime associated antigenic complex (SAAC) and J5] and their IgG1 and IgG2 subtypes in both serum and whey, and the antigen-specific IFN-γ, IL-4, and IL-17 production by blood lymphocytes after in vitro stimulation with S. aureus and Escherichia coli extracts were determined. A cohort of 8 clinically healthy end-term cows and heifers were conveniently selected, of which half was vaccinated with Startvac at 45 and 10 d before the expected calving date and half served as nonvaccinated control animals. At 15 d in milk, 2 contralateral quarters of each of the 8 animals were challenged with 2×109 cfu/mL of the formaldehyde-killed S. aureusC195strain. The 2 other quarters were infused with phosphate-buffered saline and served as control quarters. The increase in both quarter milk somatic cell count and PMN concentration and the drop in milk production after S. aureus inoculation was less pronounced in the vaccinates than in the nonvaccinates, reflecting a less severe inflammatory response. No significant differences in PMN viability between vaccinates and nonvaccinates could be demonstrated. The serum SAAC- and J5-specific antibody concentration significantly increased across the dry period in the vaccinated animals only. The whey concentration of SAAC-specific antibodies was significantly higher in vaccinates than in nonvaccinates at both 15 and 17 d in milk, independent from the challenge status of the quarters. No significant differences in the whey J5-specific antibody concentration were observed. Vaccination with Startvac seems to primarily evoke a Th2 response for S. aureus characterized by a shift toward the IgG1 antibody subtype and accompanied by a less pronounced Th1 response. The type of response against E. coli was less clear, though a weak but significant shift toward the IgG2 antibody subtype after vaccination and high IFN-γ levels after in vitro stimulation suggest a Th1 response. The increased SAAC-specific antibody concentration in whey in vaccinates compared with nonvaccinates most probably triggers the opsonization of the inoculated S. aureus bacteria, resulting in a more efficient elimination of the bacteria from the mammary gland.

Bacteria adherence seems to be an essential first stage for the internalization of bacteria into the cytoplasm of the host cell, which is considered an important virulence strategy enabling bacteria to occupy a microenvironment separated from host defense mechanisms. Thus, this study aimed to explore the difference in the capacity of 4 bovine-associated staphylococci species or strains to adhere to and internalize into bovine mammary epithelial cells (MEC). Three different isolates of coagulase-negative staphylococci (CNS) were used: one strain of Staphylococcus fleurettii isolated from sawdust and considered an environmental opportunistic bacterium, and 2 dissimilar Staphylococcus chromogenes isolates, one cultured from a heifer's teat apex (Staph. chromogenes TA) and the other originating from a chronic intramammary infection (Staph. chromogenes IM). Also, one well-characterized strain of Staphylococcus aureus (Newbould 305) was used for comparison with a major mastitis pathogen. The CNS species and strains adhered to and internalized into MEC slower than did Staph. aureus. Still, we observed high variation in adhesion and internalization capacity among the different CNS, with Staph. chromogenes IM showing a greater ability to adhere to and internalize into MEC than the 2 CNS strains isolated from extramammary habitats. In conclusion, the 3 well-characterized bovine-associated CNS species and strains originating from distinct habitats showed clear differences in their capacity to adhere to and internalize into MEC. The observed differences might be related to their diversity in ecology and epidemiological behavior.

The main objective of this study was to quantify the (compound-specific) antimicrobial consumption (AMC) in adult cattle in a convenience sample of Flemish dairy herds. Antimicrobial consumption data were obtained between 2012 and 2013 by "garbage can audits" and expressed as antimicrobial treatment incidence (ATI), with the unit of the ATI being the number of defined daily doses animal (DDDA) used per 1,000 cow-days. Herds were stratified by DDDA into low-, medium-, and high-consuming herds to study the AMC per route of administration, and associations with parameters reflecting udder health, milk quality, and production performances were examined. The average ATI in adult dairy cattle for all compounds was 20.78 DDDA (per 1,000 cow-days). Large variation existed between herds (ranging from 8.68 to 41.62 DDDA). Fourth-generation cephalosporins were used most (4.99 DDDA), followed by penicillins (3.70 DDDA) and third-generation cephalosporins (2.95 DDDA). The average ATI of the critically important antimicrobials for human health (i.e., third- and fourth-generation cephalosporins and fluoroquinolones according to the World Organisation for Animal Health classification) was somewhat lower than the average ATI of the other antimicrobials (8.59 and 12.18 DDDA, respectively). The average ATI for intramammary treatment of (sub)clinical mastitis, for dry-cow therapy, and for systemically administered antimicrobials was 6.30, 6.89, and 7.44 DDDA, respectively. In low-consuming herds, most antimicrobials were being used for dry-cow therapy, whereas in high-consuming herds, most antimicrobials were being used as injectable or intramammary mastitis therapy. The incidence rate of treated mastitis was positively associated with ATI. Herds that applied blanket dry-cow therapy tended to have a higher ATI than herds in which cows were selectively dried off with long-acting antimicrobials. The ATI decreased with an increasing prevalence of primiparous cows.

Coagulase-negative staphylococci (CNS) are the main cause of bovine intramammary infections and are also abundantly present in extramammary habitats such as teat apices. Teat apex colonization (TAC) with CNS has already been explored in lactating dairy cows at the species level, whereas this is not true for dry cows and end-term heifers. Therefore, the aim of this observational study was to describe CNS TAC in nonlactating dairy cows and end-term heifers in Flemish dairy herds and to identify associated risk factors at the herd, cow, and quarter level. All CNS were molecularly identified to the species level using transfer RNA intergenic spacer PCR (tDNA-PCR) and sequencing of the 16S rRNA gene, allowing for species-specific statistical analyses using multivariable, multilevel logistic regression. Staphylococcus devriesei, Staphylococcus chromogenes, Staphylococcus haemolyticus, and Staphylococcus equorum were the most frequently isolated species. Staphylococcus chromogenes was the sole species colonizing teat apices of cows and heifers in all herds, whereas large between-herd differences were observed for the other species. Teat apices of red and white Holstein Friesians, of quarters dried off without an internal teat sealer, and swabbed in months with lower precipitation and higher ambient temperature were significantly more likely to be colonized by S. devriesei. Slightly dirty teat apices and teat apices swabbed in months with lower precipitation had higher odds of being colonized by S. chromogenes, whereas teat apices sampled in months with lower precipitation and higher ambient temperature were more likely to be colonized by S. haemolyticus. Dirty teat apices and teat apices swabbed in months with lower ambient temperature in combination with low precipitation had higher odds of being colonized by S. equorum. Diverse factors explaining CNS TAC, yet mostly related to humidity, ambient temperature, and hygiene, substantiate differences in epidemiological behavior and ecology between species.

Coagulase-negative staphylococci (CNS) are frequently isolated from quarters with subclinical mastitis, teat apices, and the cows' environment. Virulence, ecology, epidemiological behavior, and effect on udder health vary between different CNS species. Staphylococcus chromogenes, Staph. simulans, and Staph. xylosus are frequently present in milk and have a more substantial effect on quarter milk somatic cell count than other species. Therefore, these species are considered the "more relevant" CNS. As species-specific factors associated with CNS intramammary infection (IMI) have not yet been identified and susceptibility for IMI differs between cows and quarters, this study aimed to identify predictors for CNS IMI at the cow and quarter level (some of them changing over time) with a specific focus on the aforementioned more relevant CNS. Precise data were available from a longitudinal study (3,052 observations from 344 quarters from 86 dairy cows belonging to 3 commercial dairy herds). All CNS were molecularly identified to the species level, and multivariable, multilevel logistic regression models taking into account the longitudinal nature of the data, were fit to study the likelihood of infection. Staphylococcus chromogenes, Staph. xylosus, and Staph. cohnii were the most frequently isolated species from CNS IMI in older cows, whereas Staph. chromogenes, Staph. xylosus, and Staph. simulans were the main species found in IMI in heifers. Quarters from heifers (as opposed to multiparous cows), from heifers and multiparous cows in third or fourth month in lactation (as opposed to early lactation

Coagulase-negative staphylococci (CNS) are a group of bacteria classified as either minor mastitis pathogens or commensal microbiota. Recent research suggests species- and even strain-related epidemiological and genetic differences within the large CNS group. The current pilot study investigated in 2 experiments whether a mouse mastitis model validated for bovine Staphylococcus aureus can be used to explore further differences between CNS species and strains. In a first dose titration experiment, a low inoculum dose of S. aureus Newbould 305 (positive control) was compared with increasing inoculum doses of a Staphylococcus chromogenes strain originating from a chronic bovine intramammary infection to a sham-inoculated mammary glands (negative control). In contrast to the high bacterial growth following inoculation with S. aureus, S. chromogenes was retrieved in very low levels at 24 h postinduction (p.i.). In a second experiment, the inflammation inflicted by 3 CNS strains was studied in mice. The host immune response induced by the S. chromogenes intramammary strain was compared with the one induced by a Staphylococcus fleurettii strain originating from cow bedding sawdust and by a S. chromogenes strain originating from a teat apex of a heifer. As expected, at 28 and 48 h p.i., low bacterial growth and local neutrophil influx in the mammary gland were induced by all CNS strains. As hypothesized, bacterial growth p.i. was the lowest for S. fleurettii compared with that induced by the 2 S. chromogenes strains, and the overall immune response established by the 3 CNS strains was less pronounced compared with the one induced by S. aureus. Proinflammatory cytokine profiling revealed that S. aureus locally induced IL-6 and IL-1β but not TNF-α, whereas, overall, CNS-inoculated glands lacked a strong cytokine host response but also induced IL-1β locally. Compared with both other CNS strains, S. chromogenes from the teat apex inflicted a more variable IL-1β response characterized by a more intense local reaction in several mice. This pilot study suggests that an intraductal mouse model can mimic bovine CNS mastitis and has potential as a complementary in vivo tool for future CNS mastitis research. Furthermore, it indicates that epidemiologically different bovine CNS species or strains induce a differential host innate immune response in the murine mammary gland.

Heifer mastitis is a well-known problem, with several pathogens being involved. Several generic risk factors associated with the likelihood of intramammary infections (IMI) in fresh dairy heifers have been identified before. Yet, a need exists to identify pathogen groupspecific factors, as the effect of (groups of) pathogens on udder health and milk yield is different. The aim of the present study was to identify pathogen groupspecific risk factors for IMI in heifers participating in a prepartum antimicrobial treatment trial, allowing us to test the hypothesis that different factors are of importance between treated and untreated control heifers as well. Data from a clinical trial in which end-term heifers were treated systemically (over 3 consecutive days) 2 wk before calving with penethamate hydriodide (n = 76) or remained untreated (n = 73), were available. Several potential risk factors at the herd, heifer, and quarter level were recorded in the first 3 d in milk. Quarters from untreated heifers supplemented with ≥4 mg of selenium/d prepartum were significantly less likely to be infected with coagulase-negative staphylococci (CNS), whereas quarters were more likely to be infected with CNS when assistance during calving was needed. Udder edema before calving significantly decreased the odds of IMI with major pathogens. In treated heifers, no factors were detected that were associated with the likelihood of CNS IMI, whereas quarters from heifers were significantly more likely to be infected with major pathogens when they were housed in the calving pen more than 1 d and when they had been in contact with the lactating cows before calving. The risk factors for IMI that were identified in treated heifers were different than those in untreated heifers, independent of the pathogen group that was considered. It looks as if prepartum treatment not only changed the likelihood of infection, but also the factors that were associated with infection. However, except for treated heifers with an IMI with major pathogens, only a small proportion of the variation could be explained in the final models. Therefore, factors other than those that were studied could explain the likelihood of infection.

The aim of this review is to assess the effect of coagulase-negative staphylococci (CNS) species on udder health and milk yield in ruminants, and to evaluate the capacity of CNS to cause persistent intramammary infections (IMI). Furthermore, the literature on factors suspected of playing a role in the pathogenicity of IMI-associated CNS, such as biofilm formation and the presence of various putative virulence genes, is discussed. The focus is on the 5 CNS species that have been most frequently identified as causing bovine IMI using reliable molecular identification methods (Staphylococcus chromogenes, Staphylococcus simulans, Staphylococcus haemolyticus, Staphylococcus xylosus, and Staphylococcus epidermidis). Although the effect on somatic cell count and milk production is accepted to be generally limited or nonexistent for CNS as a group, indications are that the typical effects differ between CNS species and perhaps even strains. It has also become clear that many CNS species can cause persistent IMI, contrary to what has long been believed. However, this trait appears to be quite complicated, being partly strain dependent and partly dependent on the host's immunity. Consistent definitions of persistence and more uniform methods for testing this phenomenon will benefit future research. The factors explaining the anticipated differences in pathogenic behavior appear to be more difficult to evaluate. Biofilm formation and the presence of various staphylococcal virulence factors do not seem to (directly) influence the effect of CNS on IMI but the available information is indirect or insufficient to draw consistent conclusions. Future studies on the effect, persistence, and virulence of the different CNS species associated with IMI would benefit from using larger and perhaps even shared strain collections and from adjusting study designs to a common framework, as the large variation currently existing therein is a major problem. Also within-species variation should be investigated.

Associations between herd management practices and both bacterial counts (BC) and coliform counts (CC) from 254 and 242 dairy herds in Flanders (Belgium), respectively, were studied. Data were analyzed using multivariable, multilevel linear regression analysis, allowing variance components analyses. Both BC and CC fluctuated throughout the year, although the milk quality parameters followed an opposite pattern. Bacterial count values decreased with each increase of the cleaning frequency of the cubicles (once per week, once per day, twice per day, or more than twice per day) between January and March. Herds with a conventional milking parlor had substantially lower BC than herds where the cows were milked using an automatic milking system. Lower BC were observed when the milking parlor was equipped with an automatic cluster removal system, when premilking teat disinfection was applied, when the dry cows were supplemented with a mix of minerals and vitamins, and when the teats were prepared either first wet and dried or via an automatic milking system. Milking cows with a high-pipeline milking parlor setup or with an automatic milking system was associated with substantially higher CC values. Herds where prepartum heifers were often treated with antimicrobials before calving had a lower CC than farms where heifers were either not or only rarely treated. Most variation in BC and CC resided at the herd level rather than at the observation level, indicating that management is important in the control of both BC and CC. Still, only a small proportion of the total variance was explained by factors capturing information related to the milking, herd health, and dry cow management, which suggests that the bacteriological milk quality and, in particular, CC is primarily driven by other factors than the ones included in this study.

The objective of this study was to further scrutinize the previously found positive association between intramammary infection (IMI) caused by coagulase-negative staphylococci (CNS) in early lactating heifers and test-day daily milk yield (MY) throughout first lactation, with a specific focus on the effect of the heifers' genetically determined milk production levels and the incidence of clinical mastitis. Two precise longitudinal data sets were analyzed using a series of statistical models including potential confounding and intermediate variables. The final database included the IMI status at calving, composite milk somatic cell count (SCC) and MY records at test day up to 285 d in milk (DIM), farmer-recorded clinical mastitis (CM) cases between 14 and 285 DIM, estimated new IMI incidence based on a SCC threshold of 100,000 cells/mL between 14 and 285 DIM, DIM, average 305-d MY at the herd level, and the heifers' genetic merit for MY from 240 dairy heifers from 29 dairy herds. Seventy-one (29.6%) early lactating heifers were noninfected, 108 heifers (45.0%) were CNS infected, and 61 heifers (25.4%) were infected with any major pathogen. The positive effect of CNS IMI in early lactation on test-day MY was estimated at 1.32 kg/d using a first basic mixed regression model. Correcting for the confounder genetic merit for MY reduced this effect to 1.17 kg. Interestingly, taking into account the confounding effect of herd resulted in an increase of the estimate from 1.32 to 2.2 kg/d. The positive effect of CNS IMI in early lactation on MY after correcting the model for both confounders was estimated at 2.05 kg/d. Heifers infected with CNS in the first DIM tended to have fewer CM cases throughout lactation compared with the noninfected herd mates. Including the intermediate variable CM in the model explained 0.16 kg/d of the corrected effect of 2.05 kg/d. Inclusion of test-day SCC, another intermediate variable, however, increased the estimate by 0.11 kg/d. With an appropriate correction for several confounders and biologically understood intermediate variables such as CM, test-day SCC, and new IMI based on SCC threshold of 100,000 cells/mL, an unexplained test-day MY difference between CNS-infected and noninfected heifers of 2.0 kg/d remained.

Although many studies have examined the relation between a wide range of factors and quarter milk somatic cell count (qSCC), including physical characteristics of the teat and changes in teat tissue due to milking, the effect of short-term, milking-induced changes in teat dimensions on somatic cell count has not yet been investigated. To identify teat dimensions and milking-induced changes in teat dimensions associated with qSCC, we conducted a longitudinal study (n herds =6, n cows =72, n measurements =12). Parity, stage of lactation, teat barrel diameter, and changes in teat barrel diameter during milking were identified as factors associated with qSCC. Teats with wider barrels had higher qSCC. Negative changes in the diameter of the teat barrel during milking (i.e., thinner teats postmilking compared with premilking) were associated with lower qSCC, whereas positive changes (i.e., thicker teats postmilking compared with premilking) were associated with higher qSCC. Selection toward more optimal teat characteristics may therefore result in improved milk quality and udder health. However, a threshold might exist for the maximum reduction in teat barrel diameter below which udder health is negatively influenced. If so, changes in teat barrel diameter might serve as an indicator for suboptimal milking and incorrect choice of teatcup liner or milking machine settings and thus help improve management of the herd.

Coagulase-negative staphylococci (CNS) are the most commonly isolated bacteria from goat milk, but they have often been identified with phenotypic methods, which may have resulted in misclassification. The aims of this paper were to assess the amount of misclassification of a phenotypic test for identifying CNS species from goat milk compared with transfer RNA intergenic spacer PCR (tDNA-PCR) followed by capillary electrophoresis, and to apply the tDNA-PCR technique on different capillary electrophoresis equipment. Milk samples were collected from 416 does in 5 Californian dairy goat herds on 3 occasions during lactation. In total, 219 CNS isolates were identified at the species level with tDNA-PCR and subjected to the API 20 Staph identification test kit (API Staph; bioMérieux, Durham, NC). If the same species was isolated multiple times from the same udder gland, only the first isolate was used for further analyses, resulting in 115 unique CNS isolates. According to the tDNA-PCR test, the most prevalent CNS species were Staphylococcus epidermidis, Staphylococcus caprae, and Staphylococcus simulans. Typeability with API staph was low (72%). Although the API Staph test was capable of identifying the majority of Staph. epidermidis and Staph. caprae isolates, sensitivity for identification of Staph. simulans was low. The true positive fraction was high for the 3 most prevalent species. It was concluded that the overall performance of API Staph in differentiating CNS species from goat milk was moderate to low, mainly because of the low typeability, and that genotypic methods such as tDNA-PCR are preferred.

A longitudinal study in 3 dairy herds was conducted to profile the distribution of coagulase-negative Staphylococcus (CNS) species causing bovine intramammary infection (IMI) using molecular identification and to gain more insight in the pathogenic potential of CNS as a group and of the most prevalent species causing IMI. Monthly milk samples from 25 cows in each herd as well as samples from clinical mastitis were collected over a 13-mo period. Coagulase-negative staphylococci were identified to the species level using transfer-RNA intergenic spacer PCR. The distribution of CNS causing IMI was highly herd-dependent, but overall, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus cohnii, and Staphylococcus simulans were the most prevalent. No CNS species were found to cause clinical mastitis. The effect of the most prevalent species on the quarter milk somatic cell count (SCC) was analyzed using a linear mixed model, showing that Staph. chromogenes, Staph. simulans, and Staph. xylosus induced an increase in the SCC that is comparable with that of Staphylococcus aureus. Almost all CNS species were able to cause persistent IMI, with Staph. chromogenes causing the most persistent infections. In conclusion, accurate species identification cannot be ignored when studying the effect of CNS on udder health, as the effect on SCC differs between species and species distribution is herd-specific. Staphylococcus chromogenes, Staph. simulans, and Staph. xylosus seem to be the more important species and deserve special attention in further studies. Reasons for herd dependency and possible cow-and quarter-level risk factors should be examined in detail for the different species, eventually leading to cost-benefit analyses for management changes and, if needed, treatment recommendations.

The objectives of the study were to define the sensitivity and specificity of the California Mastitis Test (CMT) in determining the presence of intramammary infection in postpartum dairy goats and to determine whether antibiotic therapy increased bacteriological cure rate and lowered somatic cell count (SCC) compared with untreated controls. A CMT was performed and milk samples were collected for bacteriology from 211 glands of 106 does between 0 and 10 d after kidding. From a population of 3,239 glands from goats in 4 commercial herds; goats with one or both glands with a CMT score of >1 and from which bacteria were isolated were either assigned to be treated with 3 intramammary infusions at 12-h intervals of 75 mg of sodium ampicillin and 250 mg of sodium cloxacillin (n = 57 glands) or left as untreated controls (n = 49 glands). Milk samples were collected again 14 +/- 3 and 21 +/- 3 d later for bacteriology and SCC determination. Composite milk yield, goat SCC, length of lactation, and survival data were collected. A partial budget was constructed to assess the cost effectiveness of treatment. At a cut point of greater than trace, the sensitivity, specificity, and positive and negative predictive values of the CMT were 0.74, 0.74, 0.42, and 0.92, respectively. Treatment increased the bacteriological cure rate compared with no treatment [30/57 (53%) vs. 6/49 (12%)], but there was a pathogen by treatment interaction whereby treatment increased cure proportion in glands infected with minor, but not major, pathogens. Treatment reduced the foremilk gland-level SCC [1,595 (95% CI = 1,106-2,300) vs. 3,028 (95% CI = 2,091-4,385) geometric mean (x 1,000) cells/mL] but not the SCC at goat level [1,596 (95% CI = 1,219-2,090) vs. 1,488 (95% CI = 1,132-1,955) geometric mean (x 1,000) cells/mL] compared with no treatment. Milk yield, risk of removal from the herd; and length of lactation were not altered by treatment. Treatment resulted in a loss of NZ$20.39/doe. It was concluded that use of the CMT as a screening test resulted in a higher likelihood of finding a gland that would be infected than selecting a gland at random. Treatment increased bacteriological cure rate and reduced SCC at gland level compared with no treatment. However; at goat level, milk yield, SCC, and survival were not altered, resulting in no economic benefit of treatment.

Intramammary infections (IMI) in recently calved dairy heifers are more common than was formerly believed but their relevance for future performance has been studied only rarely. In the present study, the association between the IMI status of fresh heifers and their subsequent udder health, milk production, and culling in first lactation was explored. Quarter milk samples were collected between 1 and 4 d in milk (DIM) and between 5 and 8 DIM from 191 dairy heifers in 20 dairy herds for bacteriological culturing and somatic cell count (SCC) analysis. Monthly milk recording data including composite milk SCC and test-day milk yield (MY) were obtained for the first 285 DIM or until culling. Farmer-recorded clinical mastitis cases were available. Data were analyzed using mixed models and survival analysis. Approximately 80% of the fresh heifers (79.8%) had at least one culture-positive quarter. Coagulase-negative staphylococci (CNS) were the most frequently isolated pathogens (72%), followed by esculin-positive streptococci (4.6%) and Staphylococcus aureus (3.5%). Overall geometric mean SCC at quarter level decreased between the first and second samplings from 348,000 to 116,000 cells/mL. Heifers infected with CNS had an intermediate average test-day SCC (84,000 cells/mL) during the first 285 DIM compared with noninfected heifers (53,000 cells/mL) and heifers infected with major pathogens (195,000 cells/mL). Heifers infected with major pathogens had a much lower average daily MY (18.3 kg) during first lactation compared with noninfected animals (21.3 kg). That CNS-infected heifers out-produced their noninfected counterparts could be at least partially explained by their significantly lower incidence of clinical mastitis (incidence risk 3.6 vs. 21.0%) during first lactation compared with noninfected heifers. We conclude that although CNS cause the majority of IMI in heifers around calving, they should not be a reason for serious concern.

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.

Polymorphonuclear neutrophilic leukocytes (PMNL) play an important role in the first line cell-mediated immune defense of the body in general and of the mammary gland against mastitis pathogens in particular. Reduced viability of PMNL close to parturition may explain the high incidence of infectious diseases and the high prevalence of intramammary infections (IMI) in periparturient dairy heifers. Apoptosis of blood PMNL 1 wk before the expected calving date and of blood and milk PMNL at 1 to 4 d in milk was determined using flow cytometry. Information on heifer and gland characteristics was collected before calving and in early lactation. Data were analyzed using multivariable, multilevel regression analysis. Supplementation of a commercial mineral/vitamin mix before calving was associated with less blood (14.4 +/- 2.9 vs. 22.4 +/- 2.1%) and milk PMNL apoptosis (19.0 +/- 1.1 vs. 26.4 +/- 0.9%) near calving, presumably related to higher blood selenium concentrations. Both blood and milk PMNL apoptosis showed seasonal variation with the highest proportion of apoptotic cells between January and March (32.0 +/- 6.1 and 34.6 +/- 2.7%, respectively) and April and June (31.3 +/- 5.7 and 37.8 +/- 2.3%, respectively). Heifers losing 0.25 points or more of their body condition in the periparturient period had higher proportions of apoptotic blood PMNL in early lactation compared with heifers losing less than 0.25 points (24.0 +/- 2.8 vs. 16.6 +/- 1.7%). Milk PMNL apoptosis was less pronounced in quarters having teat orifices colonized with non-aureus staphylococci before calving (18.9 +/- 1.0 vs. 29.4 +/- 1.0%). The variation in blood PMNL apoptosis before and after calving mainly resided at the heifer level (71.4 and 98.4% of the total variation, respectively), whereas the variation in milk PMNL apoptosis mainly resided at the heifer (45.7% of the total variation) and quarter levels (45.5% of the total variation). These data imply that the impaired blood and milk PMNL viability in periparturient heifers can be reduced by optimization of certain heifer management practices such as supplementation of minerals/vitamins, and pasture and feeding strategies.

Decreased insulin sensitivity (IS) in dairy cows supports milk yield but increases the risk for metabolic and reproductive disorders. Although several inducers of decreased IS are known, it is unclear to what extent it is congenitally determined. The main aim was to investigate differences in IS between neonatal calves of the Belgian Blue (BB) breed, reared for beef production, and the Holstein-Friesian (HF) breed, reared for milk yield. Additionally, a small number of East Flemish (EF) calves, a local dual-purpose breed, were compared with the 2 other breeds. Ten BB, 12 HF, and 4 EF calves with similar age, ration, and housing were selected. In the intravenous glucose tolerance test, blood samples were taken at regular intervals after an intravenous glucose bolus of 150 mg/kg. Area under the curve (AUC), peak concentration, and elimination rate of insulin and glucose were computed. The quantitative insulin sensitivity check index (QUICKI) and revised QUICKI were computed using basal glucose, insulin, and nonesterified fatty acid concentrations. In the intravenous insulin tolerance test, blood samples were obtained from 4 calves of each breed at regular times after an intravenous insulin challenge of 0.05 IU/kg. Based on the decline in glucose concentrations relative to basal levels, the insulin-stimulated blood glucose response was computed. Basal insulin concentrations were higher in HF (1.58 +/- 0.40 microU/mL) than in BB calves (0.35 +/- 0.09 mmol/L). Compared with BB calves, HF and EF calves had higher basal glucose concentrations (4.40 +/- 0.16 vs. 5.70 +/- 0.35 and 5.81 +/- 0.13 mmol/L, respectively), insulin peak concentrations (4.62 +/- 1.09 vs. 9.70 +/- 1.45 and 16.44 +/- 5.58 microU/mL, respectively), insulin AUC (86.71 +/- 18.81 vs. 222.65 +/- 45.00 and 293.69 +/- 109.22 microU/mL.min, respectively), and glucose AUC (256.22 +/- 17.53 vs. 335.66 +/- 18.74 and 321.03 +/- 10.05 mmol/L min, respectively). Glucose elimination rates were lower in HF (1.37 +/- 0.22%/min) than in BB calves (2.35 +/- 0.25%/min). The QUICKI was lower in HF and EF than in BB calves (0.52 +/- 0.039 and 0.57 +/- 0.068 vs. 0.76 +/- 0.038, respectively), and the revised QUICKI was lower in HF (0.86 +/- 0.11) than in BB calves (1.59 +/- 0.17). The insulin-stimulated blood glucose response did not differ between breeds. Because management differences were negligible, our results suggest breed-specific differences in glucose partitioning and IS. These findings may reflect different rearing purposes of the breeds, although extrapolation of the data to adult animals should be done cautiously.

The aim of the present study was to develop a flow cytometric procedure for the quantification of the proportion of viable, apoptotic, and necrotic polymorphonuclear neutrophilic leukocytes (PMNL) isolated from both low- and high-somatic-cell-count quarter milk samples. Milk PMNL were differentiated from other cells by indirect fluorescent labeling using a primary anti-bovine granulocyte monoclonal antibody (CH138A) and an Alexa 647-labeled secondary antibody. Polymorphonuclear neutrophilic leukocytes were identified flow cytometrically based on their cytoplasmic granularity and CH138A-positivity. Additional labeling with annexin-V-fluorescein isothiocyanate and propidium iodide was used to determine milk PMNL viability. Thirty milk samples were run in parallel to assess the repeatability of the immunoassay and 6 repeated measurements per sample were performed to assess the instrument stability. Fluorescence microscopic verification of the CH138A staining pattern showed both a high sensitivity (90.9%) and specificity (92.3%). The combination of the side-scatter properties of granulated PMNL and CH138A-Alexa 647 positivity allows the distinction of labeled PMNL from other milk cells and particles that may bind nonspecifically, and from autofluorescent particles present in milk. Quantification of the proportion of PMNL and viable, apoptotic, and necrotic subpopulations in parallel samples gave repeatable results with concordance correlation coefficients varying between 0.93 and 0.99. The average coefficient of variation for repeated measurements in identical samples ranged between 4.2 and 9.7%. In conclusion, this is the first flow cytometric method suited for the simultaneous quantification of viable, apoptotic, and necrotic bovine milk PMNL in a straightforward manner.