Corrigendum to "Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk, rectal feces, and teat apices" (J. Dairy Sci. 105:9995-10006).

Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with ferritin iron supplementation. OnlyStaphylococcus hominis strains originating from quarter milk were able to significantly utilize ferritin as an iron source to reverse the growth inhibition caused by chelating agent 2,2'-bipyridyl in varying degrees. Both S. chromogenes strains (IM and TA) and all S. hominis strains were unable to significantly use lactoferrin as an iron source for growth recovery.

Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk, rectal feces, and teat apices.

Staphylococcus hominis, a member of the non-aureus staphylococci (NAS) group, is part of the human and animal microbiota. Although it has been isolated from multiple bovine-associated habitats, its relevance as a cause of bovine mastitis is currently not well described. To successfully colonize and proliferate in the bovine mammary gland, a bacterial species must be able to acquire iron from host iron-binding proteins. The aims of this study were (1) to assess the genetic diversity of S. hominis isolated from bovine quarter milk, rectal feces, and teat apices, and (2) to investigate the capacity of bovine S. hominis isolates belonging to these different habitats to utilize ferritin and lactoferrin as iron sources. To expand on an available collection of bovine S. hominis isolates (2 from quarter milk, 8 from rectal feces, and 19 from teat apices) from one commercial dairy herd, a subsequent single cross-sectional quarter milk sampling (n = 360) was performed on all lactating cows (n = 90) of the same herd. In total, 514 NAS isolates were recovered and identified by MALDI-TOF mass spectrometry; the 6 most prevalent NAS species were S. cohnii (33.9%), S. sciuri (16.7%), S. haemolyticus (16.3%), S. xylosus (9.6%), S. equorum (9.4%), and S. hominis (3.5%). A random amplified polymorphic DNA (RAPD) analysis was performed on 46 S. hominis isolates (19 from quarter milk, 8 from rectal feces, and 19 from teat apices). Eighteen distinct RAPD fingerprint groups were distinguished although we were unable to detect the presence of the same RAPD type in all 3 habitats. One S. hominis isolate of a distinct RAPD type unique to a specific habitat (8 from quarter milk, 3 from rectal feces, and 4 from teat apices) along with the quality control strain Staphylococcus aureus ATCC 25923 and 2 well-studied Staphylococcus chromogenes isolates ("IM" and "TA") were included in the phenotypical iron test. All isolates were grown in 4 types of media: iron-rich tryptic soy broth, iron-rich tryptic soy broth deferrated by 2,2'-bipyridyl, and deferrated tryptic soy broth supplemented with human recombinant lactoferrin or equine spleen-derived ferritin. The growth of the different strains was modified by the medium in which they were grown. Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with iron supplementation. Staphylococcus hominis strains from all 3 habitats were able to significantly utilize ferritin but not lactoferrin as an iron source to reverse the growth inhibition, in varying degrees, caused by the chelating agent 2,2'-bipyridyl.

The effect of intramammary infection in early lactation with non-aureus staphylococci in general and Staphylococcus chromogenes specifically on quarter milk somatic cell count and quarter milk yield.

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.

Mastitis prevention and control practices and mastitis treatment strategies associated with the consumption of (critically important) antimicrobials on dairy herds in Flanders, Belgium.

The main objectives of this study were to evaluate to what extent variations in herd-level antimicrobial consumption (AMC) can be explained by differences in management practices that are consistently effective in the prevention of (sub)clinical mastitis, on the one hand, and by differences in mastitis treatment strategies, on the other hand. Antimicrobial consumption data were obtained during 2012 and 2013 by "garbage can audits" and expressed as antimicrobial treatment incidences (ATI) for all compounds combined (total ATI) and for the critically important antimicrobials for human health separately. Data on mastitis prevention and control practices were obtained via face-to-face interviews performed during herd visits in March 2013. Some management practices and treatment strategies related to udder health were associated with the total AMC. However, the results demonstrated that implementing effective udder health management practices does not necessarily imply a low AMC and vice versa. Herds participating in a veterinary herd health management program and herds selectively drying off cows used fewer antimicrobials compared with herds not participating in such a program or applying blanket dry-cow therapy. Moreover, herds treating (some) (sub)clinical mastitis cases with intramammary homeopathic substances consumed fewer antimicrobials than herds not applying such homeopathic treatments. Besides these factors, no other direct association was found between effective udder health management practices on the one hand and AMC on the other hand. Also, the use of critically important antimicrobials was only associated with the way in which subclinical mastitis cases were treated. The latter indicates that the AMC of critically important antimicrobials is potentially driven by factors other than those included in this study such as those related to the "mindset" of the veterinarians and their farmers. Future research should therefore aim to unravel the reasoning of vets and their farmers behind the use of those critically important antimicrobials for the treatment of mastitis and other diseases.

Pathogen group-specific risk factors for intramammary infection in treated and untreated dairy heifers participating in a prepartum antimicrobial treatment trial.

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 group-specific 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 group-specific 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.

Manageable risk factors associated with bacterial and coliform counts in unpasteurized bulk milk in Flemish dairy herds.

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.

Characterization of coagulase-negative staphylococcus species from cows' milk and environment based on bap, icaA, and mecA genes and phenotypic susceptibility to antimicrobials and teat dips.

The aim of this study was to investigate whether the main coagulase-negative staphylococci (CNS) species involved in bovine intramammary infections (IMI) possess specific characteristics that promote colonization of the udder. Virulence markers associated with biofilm formation, antimicrobial resistance, and biocide tolerance were compared between typically contagious CNS species (Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus simulans) and those rarely causing IMI (Staphylococcus sciuri, Staphylococcus equorum, and others) to find possible associations with pathogenicity. Coagulase-negative staphylococci isolates (n=366) belonging to 22 different species were analyzed by PCR for the presence of the biofilm-associated genes bap and icaA, and the methicillin resistance gene mecA. A selection of 82 isolates was additionally tested for their susceptibility to 5 antibiotics and 2 commercial teat dip products. Minimum inhibitory concentrations of antimicrobials were determined by Etest (AB bioMérieux, Marcy l'Etoile, France), and a microdilution method was optimized to determine minimum biocidal concentrations of teat dips. The bap, icaA, and mecA genes were detected significantly more in isolates from CNS species typically living in the cows' environment than in isolates from IMI-causing species. Antimicrobial resistance was mainly against erythromycin (23%) or oxacillin (16%), and was detected more often in the environmental species. The isolates least susceptible to the teat dips belonged to the IMI-causing species Staph. chromogenes and Staph. simulans. We concluded that carriage of biofilm genes and antimicrobial resistance were not associated with the ability to colonize the mammary gland because free-living CNS species constituted a more significant reservoir of biofilm and resistance determinants than did IMI-causing species. In contrast, increased tolerance to biocides may favor the establishment of bovine IMI by some CNS species.

The effect of marine algae in the ration of high-yielding dairy cows during transition on metabolic parameters in serum and follicular fluid around parturition.

Sixteen Holstein cows were assigned to 2 groups to evaluate the caloric and metabolic effect of feeding marine algae (ALG) from 3 wk prepartum until 12 wk postpartum. Milk production characteristics and the profiles of hormones and metabolites in the serum were monitored from -7 to 46 d in milk (DIM) and in follicular fluid (FF) from 14 to 46 DIM. All cows received a corn- and grass silage-based partially mixed ration supplemented with concentrate and protein supplement. In the diet of the ALG group, 2 kg of the concentrate was replaced by a concentrate containing ALG (44 g/d of docosahexaenoic acid). Diets were isocaloric (net energy basis) and equal in intestinal digestible protein. The ALG diet increased milk yield (41.2 vs. 38.2 kg/d) and decreased milk fat yield (1.181 vs. 1.493 kg/d) and milk fat content (31.6 vs. 40.7 g/kg). Protein yield (1.336 vs. 1.301 kg/d) was not affected but a tendency toward decreased milk protein content (32.8 vs. 34.7 g/kg) was observed. Marine algae supplementation increased the ß-hydroxybutyric acid (BHBA) concentration in FF of the ALG cows compared with that in the controls (0.992 vs. 0.718 mmol/L). The total protein concentration in FF was decreased in ALG (62.9 vs. 67.6 g/L). Plasma and serum metabolites did not significantly differ between treatments except for a tendency toward a lower concentration of urea in the serum of the control compared with ALG (4.69 vs. 5.13 mmol/L). Based on metabolizable energy calculations, a daily energy-sparing effect of 3.48 Mcal was obtained due to milk fat depression (MFD). The concomitant increase in milk yield suggests that at least part of this spared energy is used to stimulate milk production. Theoretically, 3.48 Mcal of ME could lead to an increase in milk yield of 7.43 kg/d, which is higher than the observed 3 kg/d. However, when evaluating nutrient requirements during MFD in early lactation, we calculated that increased milk production is caused by a propionate-saving effect of 2.71 mol in the udder when milk fat is depressed. Concurrent increased BHBA concentrations in FF in the ALG group cannot be attributed to a worsened energy status of the animals because all other indicators contradict any change in energy balance, indicating that BHBA might not be an appropriate metabolic parameter to estimate the energy balance in early lactating dairy cows during MFD.

Pathogen group specific risk factors at herd, heifer and quarter levels for intramammary infections in early lactating dairy heifers.

Risk factors for intramammary infections caused by coagulase-negative staphylococci, contagious major pathogens and environmental major pathogens in early lactating heifers were evaluated at the herd, heifer and quarter levels. In total, 764 quarters of 191 dairy heifers in 20 randomly selected farms in Flanders (Belgium) were sampled. Quarter milk samples were collected between 1 and 4 days in milk and between 5 and 8 days in milk for bacteriological culture. Data were analyzed using multivariable, multilevel logistic regression analysis. Higher average herd milk somatic cell count (>200,000 cells/mL), not having an effective fly control strategy, contact with lactating cows prior to calving and moderate to severe udder edema prior to calving increased the odds of intramammary infections caused by contagious major pathogens. Poor heifer hygiene and lack of mineral/vitamin supplementation prior to calving were risk factors for intramammary infection caused by environmental major pathogens. Teat apex colonization with coagulase-negative staphylococci prior to calving seemed to protect quarters against intramammary infections caused by major pathogens. Poor heifer hygiene before calving, a non-clipped udder and not practicing of teat dipping prior to calving increased the odds of intramammary infection with coagulase-negative staphylococci. Although management is important in the prevention and control of intramammary infections in early lactating heifers, most variation in the prevalence of intramammary infections resided at the heifer and quarter levels, indicating that the susceptibility for intramammary infections around calving is mainly determined by heifer and quarter characteristics.

Heifer and quarter characteristics associated with periparturient blood and milk neutrophil apoptosis in healthy heifers and in heifers with subclinical mastitis.

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.