Mycoplasma bovis subverts autophagy to promote intracellular replication in bovine mammary epithelial cells cultured in vitro.

Mycoplasma species are the smallest prokaryotes capable of self-replication. To investigate Mycoplasma induced autophagy in mammalian cells, Mycoplasma bovis (M. bovis) and bovine mammary epithelial cells (bMEC) were used in an in vitro infection model. Initially, intracellular M. bovis was enclosed within a membrane-like structure in bMEC, as viewed with transmission electron microscopy. In infected bMEC, increased LC3II was verified by Western blotting, RT-PCR and laser confocal microscopy, confirming autophagy at 1, 3 and 6 h post-infection (hpi), with a peak at 6 hpi. However, the M. bovis-induced autophagy flux was subsequently blocked. P62 degradation in infected bMEC was inhibited at 3, 6, 12 and 24 hpi, based on Western blotting and RT-PCR. Beclin1 expression decreased at 12 and 24 hpi. Furthermore, autophagosome maturation was subverted by M. bovis. Autophagosome acidification was inhibited by M. bovis infection, based on detection of mCherry-GFP-LC3 labeled autophagosomes; the decreases in protein levels of Lamp-2a indicate that the lysosomes were impaired by infection. In contrast, activation of autophagy (with rapamycin or HBSS) overcame the M. bovis-induced blockade in phagosome maturation by increasing delivery of M. bovis to the lysosome, with a concurrent decrease in intracellular M. bovis replication. In conclusion, although M. bovis infection induced autophagy in bMEC, the autophagy flux was subsequently impaired by inhibiting autophagosome maturation. Therefore, we conclude that M. bovis subverted autophagy to promote its intracellular replication in bMEC. These findings are the impetus for future studies to further characterize interactions between M. bovis and mammalian host cells.

Niacin stimulates EPH4EV mammary epithelial cell proliferation and mammary gland development in pubertal mice through activation of AKT/mTOR and ERK1/2 signaling pathways.

Previous studies have shown the effects of vitamins on the development of the mammary gland. However, the role of niacin in this process has not been reported. Therefore, the aim of this study is to investigate the effects of niacin on mammary gland development in pubertal mice and to use a mouse mammary epithelial cell line to study the underlying mechanism. The results showed that niacin could activate the AKT/mTOR and ERK signaling pathways and increase phosphorylation of 4EBP1 to promote the synthesis of cell proliferation markers, leading to the dissociation of the Rb-E2F1 complex in mMECs. In addition, 0.5% niacin promoted mammary duct development, increased the expression of cyclin D1/D3 and PCNA and activated Akt/mTOR and ERK1/2 in the mammary glands of pubertal mice. These results strongly suggest that niacin stimulates mammary gland development in pubertal mice through the Akt/mTOR and ERK1/2 signaling pathways.

Dry cow therapy and early lactation udder health problems-Associations and risk factors.

Mastitis remains the most expensive disease of dairy cows, and antibiotic dry cow therapy (DCT) at dry-off is an important part of mastitis control. Regardless of the infection status, blanket DCT is administered to all quarters of all cows, which is controversial due to the worldwide problem of antimicrobial resistance. Even though selective DCT of only infected cows is a more sustainable approach, choosing animals for treatment is not always straightforward. Our aim was to evaluate whether the herd-level DCT approach is associated with early lactation udder health problems, taking into account the cow characteristics. The information source was 2015-2017 Dairy Herd Improvement data with 7461 multiparous cows from 241 Finnish dairy herds. Information on the herd-level DCT approach was obtained from farmers' questionnaire responses in 2017, and the three different approaches were selective DCT, blanket DCT, and no DCT. The statistical tool for the data analysis was a generalized linear mixed model with a random herd effect for binary outcomes and a linear mixed model with a random herd effect for a continuous outcome. The two binary outcomes were the odds of having high milk somatic cell count (SCC ≥ 200 000 cells/mL) on the first test-day within 5-45 days in milk (DIM) and the odds of mastitis treatment in early lactation up to 45 DIM. The third outcome was the mean milk lnSCC (× 1000 cells/mL) within 120 DIM. Selective DCT was the prevailing treatment practice in our data. Blanket DCT was associated with lower SCC after calving. Cows more likely to have high SCC after calving were older cows, cows with high average SCC during the previous lactation, and cows with high milk yield near dry-off. A mastitis treatment in the early lactation was more likely if, during the previous lactation, the cow had high average SCC, high peak milk production, or high milk yield near dry-off. Our findings indicate that DCT is still effective in mastitis control. Cows with high milk yield, especially near dry-off, and cows with persistently high SCC require attention when considering next lactation udder health.

Adverse effects of LPS on membrane proteins in lactating bovine mammary epithelial cells.

Mastitis causes a decrease in milk yield and abnormalities in milk components from dairy cows. Escherichia coli and the E. coli lipopolysaccharide (LPS) cell wall component directly downregulate milk production in bovine mammary epithelial cells (BMECs). However, the detailed mechanism by which this occurs in BMECs remains unclear. Various membrane proteins, such as immune sensors (Toll-like receptors, TLR), nutrient transporters (glucose transporter and aquaporin), and tight junction proteins (claudin and occludin) are involved in the onset of mastitis or milk production in BMECs. In this study, we investigated the influence of LPS on membrane proteins using an in vitro culture model. This mastitis model demonstrated a loss of glucose transporter-1 and aquaporin-3 at lateral membranes and a decrease in milk production in response to LPS treatment. LPS disrupted the tight junction barrier and caused compositional changes in localization of claudin-3 and claudin-4, although tight junctions were maintained to separate the apical membrane domains and the basolateral membrane domains. LPS did not significantly affect the expression level and subcellular localization of epidermal growth factor receptor in lactating BMECs with no detectable changes in MEK1/2-ERK1/2 signaling. In contrast, NFκB was concurrently activated with temporal translocation of TLR-4 in the apical membranes, whereas TLR-2 was not significantly influenced by LPS treatment. These findings indicate the importance of investigating the subcellular localization of membrane proteins to understand the molecular mechanism of LPS in milk production in mastitis.

Insulin regulates human mammosphere development and function.

Assessing the role of lactogenic hormones in human mammary gland development is limited due to issues accessing tissue samples and so development of a human in vitro three-dimensional mammosphere model with functions similar to secretory alveoli in the mammary gland can aid to overcome this shortfall. In this study, a mammosphere model has been characterised using human mammary epithelial cells grown on either mouse extracellular matrix or agarose and showed insulin is essential for formation of mammospheres. Insulin was shown to up-regulate extracellular matrix genes. Microarray analysis of these mammospheres revealed an up-regulation of differentiation, cell-cell junctions, and cytoskeleton organisation functions, suggesting mammosphere formation may be regulated through ILK signalling. Comparison of insulin and IGF-1 effects on mammosphere signalling showed that although IGF-1 could induce spherical structures, the cells did not polarise correctly as shown by the absence of up-regulation of polarisation genes and did not induce the expression of milk protein genes. This study demonstrated a major role for insulin in mammary acinar development for secretory differentiation and function indicating the potential for reduced lactational efficiency in women with obesity and gestational diabetes.

Longitudinal study on the effects of intramammary infection with non-aureus staphylococci on udder health and milk production in dairy heifers.

We conducted a longitudinal study to evaluate the effect of non-aureus staphylococci (NAS) causing subclinical intramammary infections (IMI) on quarter milk somatic cell count (qSCC) and quarter milk yield (qMY). In total, 324 quarters of 82 Holstein Friesian heifers were followed from calving to 130 d in milk (DIM) and were sampled 10 times each at 14-d intervals. The IMI status of each quarter was determined based on bacterial culture results at the current and previous or next sampling day, or both. The qSCC was determined on each sampling day and the average qMY on sampling day was available through stored daily milk weight data in the management program of the automatic milking system. A transient IMI (tIMI) was defined as a case where a specific pathogen was isolated from a quarter on only one sampling day and not on the previous or next sampling day. When the same bacterial strain, as defined by random amplification of polymorphic DNA-PCR, was isolated from the same quarter on multiple sampling days, it was defined as a persistent IMI (pIMI) status on those sampling days; a pIMI episode was defined as the combination of multiple consecutive pIMI statuses with the same bacterial strain on different sampling days. During this study, 142 subclinical IMI with NAS occurred in 116 different quarters from 64 animals, yielding in total 304 NAS isolates belonging to 17 different species. The prevalence of NAS was highest in the first 4 DIM. Overall, the predominant species was Staphylococcus chromogenes (52% of the isolates), followed by S. epidermidis (9.2%), S. xylosus (8.2%), and S. equorum (5.9%). Staphylococcus chromogenes was the only species for which an effect on qSCC and qMY could be analyzed separately; the other NAS species were considered as a group because of their low prevalence. Eighteen out of 40 IMI (45%) caused by S. chromogenes persisted over at least 2 sampling days, whereas only 10 of 102 (9.8%) IMI caused by other NAS species persisted for at least 2 sampling days. The average duration of pIMI episodes was 110.4 d for S. chromogenes and 70 d for the other NAS species. Remarkably, 17 of the 18 pIMI episodes with S. chromogenes started within the first 18 DIM. The qSCC was highest in quarters having a pIMI with a major pathogen, followed by quarters having a pIMI with S. chromogenes, and a pIMI with other NAS. Transient IMI with other NAS or with a major pathogen caused a small but significantly higher qSCC, whereas the qSCC in quarters having a tIMI with S. chromogenes was not statistically different compared with noninfected quarters. No significant differences in qMY were observed between quarters having a pIMI or tIMI with S. chromogenes or with the other NAS species compared with noninfected quarters, despite the higher qSCC. Quarters having a pIMI with major pathogens showed significantly lower daily milk production. Surprisingly, quarters that cured from an IMI with S. chromogenes had a significantly lower qMY than noninfected quarters.

Got Milk? Identifying and Characterizing Lactation Defects in Genetically-Engineered Mouse Models.

The ability to produce and expel milk is important for the health and survival of all mammals. Nevertheless, our understanding of the molecular events underlying the execution of this process remains incomplete. Whilst impaired mammary gland development and lactational competence remains the subject of focused investigations, defects in these events may also be an unintended consequence of genetic manipulation in rodent models. In this technical report, we outline established and emerging methods to characterize lactation phenotypes in genetically-engineered mouse models. We discuss important considerations of common models, optimized conditions for mating and the importance of litter size and standardization. Methods for quantifying milk production and quality, as well as protocols for wholemount preparation, immunohistochemistry and the preparation of RNA and protein lysates are provided. This review is intended to help guide researchers new to the field of mammary gland biology in the systematic analysis of lactation defects and in the preparation of samples for more focused mechanistic investigations.

TET2 directs mammary luminal cell differentiation and endocrine response.

Epigenetic regulation plays an important role in governing stem cell fate and tumorigenesis. Lost expression of a key DNA demethylation enzyme TET2 is associated with human cancers and has been linked to stem cell traits in vitro; however, whether and how TET2 regulates mammary stem cell fate and mammary tumorigenesis in vivo remains to be determined. Here, using our recently established mammary specific Tet2 deletion mouse model, the data reveals that TET2 plays a pivotal role in mammary gland development and luminal lineage commitment. We show that TET2 and FOXP1 form a chromatin complex that mediates demethylation of ESR1, GATA3, and FOXA1, three key genes that are known to coordinately orchestrate mammary luminal lineage specification and endocrine response, and also are often silenced by DNA methylation in aggressive breast cancers. Furthermore, Tet2 deletion-PyMT breast cancer mouse model exhibits enhanced mammary tumor development with deficient ERα expression that confers tamoxifen resistance in vivo. As a result, this study elucidates a role for TET2 in governing luminal cell differentiation and endocrine response that underlies breast cancer resistance to anti-estrogen treatments.

The Multi-Faced Role of PAPP-A in Post-Partum Breast Cancer: IGF-Signaling is Only the Beginning.

Insulin-like growth factor (IGF) signaling and control of local bioavailability of free IGF by the IGF binding proteins (IGFBP) are important regulators of both mammary development and breast cancer. A recent genome-wide association study (GWAS) identified small nucleotide polymorphisms that reduce the expression of IGFBP-5 as a risk factor of developing breast cancer. This observation suggests that genetic alterations leading to a decreased level of IGFBP-5 may also contribute to breast cancer. In the current review, we focus on Pregnancy-Associated Plasma Protein A (PAPP-A), a protease involved in the degradation of IGFBP-5. PAPP-A is overexpressed in the majority of breast cancers but its role in cancer has only begun to be explored. More specifically, this review aims at highlighting the role of post-partum involution in the oncogenic function of PAPP-A. Notably, we summarize recent studies indicating that PAPP-A plays a role not only in the degradation of IGFBP-5 but also in the deposition of collagen and activation of the collagen receptor discoidin 2 (DDR2) during post-partum involution. Finally, considering the immunosuppressive microenvironment of post-partum involution, we also discuss the unexpected finding made in Ewing Sarcoma that PAPP-A plays a role in immune evasion. While the immunosuppressive role of PAPP-A in breast cancer remains to be determined, collectively these studies highlight the multifaced role of PAPP-A in cancer that extends well beyond its effect on IGF-signaling.

Investigation of dairy cow performance in different udder health groups defined based on a combination of somatic cell count and differential somatic cell count.

Mastitis is still the costliest disease in milk production. In particular, its subclinical form, which may spread unnoticeably within a herd, is a major challenge. Somatic cell count (SCC) is broadly used as an indicator for mastitis and thus the basis for udder health management programmes, e.g. through dairy herd improvement (DHI) testing. Since recently, differential somatic cell count (DSCC, representing the combined proportion of polymorphonuclear neutrophils and lymphocytes as a percentage of total SCC) is available in addition. Our study was aimed to investigate dairy cow performance in four newly defined udder health groups (UHG) based on SCC and DSCC results from DHI testing. In total, 961,835 test-day results generated in Austria, China, Estonia, Germany, and Spain between January 2019 and March 2020 were available for data analyses. Cows were categorised into four UHG depending on test day SCC and DSCC results (UHG A: healthy/normal, ≤200,000 cells/mL and ≤65 %; B: suspicious, ≤200,000 cells/mL and >65 %; C: mastitis, >200,000 cells/mL and >65 %; D: chronic/persistent mastitis, >200,000 cells/mL and ≤65 %). Linear mixed effect models were used to compare the performance of cows between the UHG based on the parameters milk weight, energy-corrected milk, fat, protein, lactose, and estimated milk value. Highest performance was found for cows in UHG A and ranged between 21.4 (Austria) and 38.3 kg per cow and day (Spain). Interestingly, cows in group B were significantly less productive (0.9-2.4% less daily milk production) compared to those in group A. Cows in groups C (6.0-9.8% less daily production compared to group A) and D were, as expected, even less productive with a particularly significant drop for cows in group D (17.5-38.5% less daily production). These trends could be observed in all countries involved in this study. Proportions of cows in the four different UHG differed between countries, changed slightly within countries depending on season, differed depending on parity and days in milk, and were seen to vary hugely between herds. In conclusion, this study demonstrates changes in performance of dairy cows depending on their udder health status as defined based on the combination of SCC and DSCC. In particular cows in UHG B and D are of interest as they cannot be identified working with SCC only. Nevertheless, the actual udder health management measures that could be taken based on the new UHG still require further investigation.

Incidence of milk leakage after dry-off in European dairy herds, related risk factors, and its role in new intramammary infections.

The incidence of milk leakage (ML) after dry-off (DO) and related risk factors was studied in 1,175 dairy cows from 41 commercial herds in 8 European countries: Belgium, Czech Republic, Denmark, France, Germany, Italy, the Netherlands, and Spain. Milk leakage was assessed twice for 30 s each during 3 visits at 20 to 24 h, 30 to 34 h, and 48 to 52 h after DO. Information related to dry-cow management and udder health was collected at herd and cow level, including individual somatic cell count (ISCC) from test-day controls and occurrence of clinical mastitis cases from DO until 30 d in lactation. Mixed-effect logistic regression analyses were used to identify possible risk factors for ML and to study the association between ML and new intramammary infections. Intramammary infections were defined as clinical mastitis cases during the dry period and in the first 30 d in lactation or a rise in ISCC from before to after the dry period (threshold: 200,000 cells/mL) or both. Milk leakage was observed in 24.5% of the cows between 20 and 52 h after DO, where the herd incidence varied between 0.0 and 77.8%. The reduction in number of milkings in the weeks before DO had statistically significant effect on the ML incidence. When the milking frequency was reduced from 3 times/d to 2 or maintained at twice a day, cows had 11 (95% CI = 3.43-35.46) or 9 (95% CI = 1.85-48.22) times higher odds of leaking milk, respectively, compared with cows where the milking frequency was reduced from twice to once a day. Also, the milk production 24 h before DO was associated with ML incidence. Hence, cows with a milk production between 13 and 21 L or above 21 L had 2.3 (95% CI = 1.48-3.53) and 3.1 (95% CI = 1.79-5.3) times higher odds of leaking milk, respectively, compared with cows with a milk production below 13 L. A higher ML incidence was present in the group of cows with an average ISCC in the last 3 mo before DO ≥200,000 cells/mL (odds ratio = 1.7; 95% CI = 1.13-2.41) compared with cows with an average ISCC <100,000 cells/mL. Quarters with ML tended to have 2.0 times higher odds of developing clinical mastitis compared with quarters not leaking milk. Cows with ML tended to have 1.5 times higher odds of intramammary infections (i.e., an increase of ISCC or clinical mastitis) compared with cows without ML.

Genetic parameters of weeping teats in Italian Saanen and Alpine dairy goats and their relationship with milk production and somatic cell score.

This paper reports a quantitative genetics analysis of weeping teats (WT), an abnormality of the mammary gland in goats. Weeping teats are characterized by milk oozing out of the teat or by the presence of multiple cysts near its base. This abnormality has been routinely recorded in Italian Alpine and Saanen goats since 2000 using a score of 0 or 1 (0 = defect not present, 1 = defect present). No information is available on the genetic background of WT or its relationship with production or other udder-related traits. Data obtained by the Italian National Sheep and Goat Breeders Association (Rome, Italy) were used to estimate both heritability of WT and its genetic correlation with milk yield, somatic cell score, and udder traits. The final data set used in the analysis included 2,178 Saanen and 2,309 Alpine primiparous goats kidding from 2009 to 2014. The pedigree data included 7,333 Saanen and 7,421 Alpines, respectively. A threshold-linear multivariate animal model was used to estimate variance and covariance components. A genealogical data analysis was also implemented, including genealogical data completeness, inbreeding, and identification of possible most recent common ancestors. On average, around 4 and 13% of primiparous Saanen and Alpine females kidding from 2009 to 2014 showed mammary gland abnormality, respectively. Weeping teats heritability was 0.27 and 0.26 for Saanen and Alpine, respectively. Genetic correlations between milk production or somatic cell score ranged from -0.16 in Saanen to 0.43 in Alpine, but the standard error of the estimates was very large. Positive genetic correlations were observed among WT and teat characteristics in both Saanen and Alpine. The average inbreeding of abnormality carriers was 2.4 and 5.1 for Saanen and Alpine, respectively. The genealogical data analysis identified 4 common ancestors of affected does in Saanen and 2 in Alpine. These results indicate that WT have a possible genetic background. A genome-wide association study might help in understanding the polygenic or monogenic determination of this abnormality.

Estrogen exacerbates mammary involution through neutrophil-dependent and -independent mechanism.

There is strong evidence that the pro-inflammatory microenvironment during post-partum mammary involution promotes parity-associated breast cancer. Estrogen exposure during mammary involution drives tumor growth through neutrophils' activity. However, how estrogen and neutrophils influence mammary involution are unknown. Combined analysis of transcriptomic, protein, and immunohistochemical data in BALB/c mice showed that estrogen promotes involution by exacerbating inflammation, cell death and adipocytes repopulation. Remarkably, 88% of estrogen-regulated genes in mammary tissue were mediated through neutrophils, which were recruited through estrogen-induced CXCR2 signalling in an autocrine fashion. While neutrophils mediate estrogen-induced inflammation and adipocytes repopulation, estrogen-induced mammary cell death was via lysosome-mediated programmed cell death through upregulation of cathepsin B, Tnf and Bid in a neutrophil-independent manner. Notably, these multifaceted effects of estrogen are mostly mediated by ERα and unique to the phase of mammary involution. These findings are important for the development of intervention strategies for parity-associated breast cancer.

The Synergism of PGN, LTA and LPS in Inducing Transcriptome Changes, Inflammatory Responses and a Decrease in Lactation as Well as the Associated Epigenetic Mechanisms in Bovine Mammary Epithelial Cells.

Mastitis is usually caused by a variety of pathogenic bacteria that include both Gram-positive and Gram-negative bacteria. Lipopolysaccharide (LPS) is the pathogen-associated molecular pattern (PAMP) of Gram-negative bacteria, and peptidoglycan (PGN) and lipoteichoic acid (LTA) are those of Gram-positive bacteria. The effects of LPS, PGN and/or LTA on inflammatory response and lactation in bovine mammary epithelial cells (BMECs) are well studied, but the epigenetic mechanisms of their effects received less attention. Furthermore, since the three PAMPs are often simultaneously present in the udder of cows with mastitis, it has implications in practice to study their additive effects. The results show that co-stimulation of bovine mammary epithelial cells with PGN, LTA, and LPS induced a higher number of differentially expressed genes (DEGs) and greater expressions of inflammatory factors including interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor-α (TNF-α), chemokine (C-X-C motif) ligand (CXCL)1, and CXCL6. In addition, co-stimulation further increased DNA hypomethylation compared with sole LPS stimulation. Co-stimulation greatly decreased casein expression but did not further decrease histone acetylation levels and affect the activity of histone acetyltransferase (HAT) and histone deacetylase (HDAC), compared with sole LPS stimulation. Collectively, this study demonstrated that PGN, LTA, and LPS had an additive effect on inducing transcriptome changes and inflammatory responses in BMECs, probably through inducing a greater decrease in DNA methylation. Co-stimulation with PGN, LTA, and LPS decreased casein expression to a greater degree, but it might not be linked to histone acetylation and HAT and HDAC activity.

PGN and LTA from Staphylococcus aureus Induced Inflammation and Decreased Lactation through Regulating DNA Methylation and Histone H3 Acetylation in Bovine Mammary Epithelial Cells.

Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are the most common pathogens of mastitis, and S. aureus generally causes subclinical mastitis which is more persistent and resistant to treatment. Peptidoglycan (PGN) and lipoteichoic acid (LTA) are cell wall components of S. aureus. Although the roles of PGN and LTA in causing inflammation are well studied, the epigenetic mechanisms of the effects of PGN and LTA on the inflammation and lactation remain poorly understood. This study characterized the gene expression profiling by RNA sequencing and investigated DNA methylation and histone acetylation in relation to inflammation and lactation in the immortalized bovine mammary epithelial cell line (MAC-T). The cells were cultured for 24 h with neither PGN nor LTA (CON), PGN (30 µg/mL), LTA (30 µg/mL), and PGN (30 µg/mL) + LTA (30 µg/mL), respectively. The number of differentially expressed genes (DEGs) and the expression of proinflammatory factors including interleukin (IL)-1ß, IL-6, IL-8, chemokine (C-X-C motif) ligand (CXCL)1, and CXCL6 of the treatments increased in the following order: CON < PGN < LTA < PGN + LTA, and the DEGs mainly enriched on the cytokine-cytokine receptor interaction and chemokine signaling pathway. LTA and PGN + LTA induced hypomethylation of global DNA by suppressing DNA methyltransferase (DNMT) activity. PGN and LTA, alone or combined, decreased the mRNA expression of casein genes (CSN1S1, CSN2, and CSN3) and the expression of two caseins (CSN2 and CSN3), and reduced histone H3 acetylation by suppressing histone acetyltransferase (HAT) activity and promoting histone deacetylase (HDAC) activity. Collectively, this study revealed that PGN and LTA induced inflammation probably due to decreasing DNA methylation through regulating DNMT activity, and decreased lactation possibly through reducing histone H3 acetylation by regulating HAT and HDAC activity in bovine mammary epithelial cells.

The effect of low intensity laser irradiation of inflamed udders on the efficacy of antibiotic treatment of clinical mastitis in dairy cows.

The aim of the study was to evaluate the effect of STP-99 laser irradiation applied locally to inflamed cow udders on the efficacy of clinical mastitis treatment with either intramammary infusions of antibiotic products or systemic injections of antibiotics. Examinations were carried out on 124 milking dairy cows suffering from clinical, bacterial mastitis. Cows with signs of local acute inflammation were treated with approved intramammary antibiotic products at labeled doses as control. The exposed cows received the same antibiotic treatment but were also subjected to irradiation of the inflamed udders with a laser for 5 consecutive days (2 minutes a day). Cows with local and systemic signs of mastitis were treated with either intramuscular injections of approved antibiotics in label doses alone (controls), or with the same intramuscular treatment protocol and laser irradiation of inflamed glands for 5 consecutive days (2 minutes a day). The recovery rate after intramammary treatment with antibiotics was 43.7%. Irradiation with laser significantly (P < 0.05%) increased the recovery rate by 31.2%. The recovery rate in the cow cohort receiving systemic treatment with antibiotics was 46.7%. The laser irradiation resulted in a 16.6% increase in recovery. Supportive treatment with laser irradiation increased recovery rates by 24.2%.

Efficacy of cabergoline in a double-blind randomized clinical trial on milk leakage reduction at drying-off and new intramammary infections across the dry period and postcalving.

The abrupt cessation of milking at dry-off may induce milk leakage, which may increase the risk of new intramammary infections (IMI). This study assessed the efficacy of 1 i.m. injection of 5.6 mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France) at drying-off on milk leakage after dry-off and new IMI across the dry period and postcalving compared with a placebo (negative control) and an intramammary antibiotic treatment (positive control) under field conditions. The study was a double-blind, randomized, 3-arm, multicenter, clinical trial performed under Good Clinical Practice conditions. Data from 900 dairy cows of various breeds from 63 farms in France, Germany, and Hungary were analyzed. Only quarters with no bacterial growth at drying-off and a cow somatic cell count ≤200,000 cells/mL were included. Quarters infected with major or minor pathogens or cows with high somatic cell count at time of inclusion were excluded. Cows that qualified for the study were visited 7 times in total before and after drying-off and after calving. Presence (yes/no) of milk leakage was recorded on the day after dry-off. A new infected quarter (new IMI) was defined as one with a major pathogen present in any one of the 2 postcalving samples. Two mixed logistic regression models were fitted to the data to evaluate the efficacy of cabergoline in the reduction of milk leakage and new IMI. One i.m. injection of cabergoline at drying-off significantly reduced the incidence of milk leakage the day after dry-off compared with both placebo and antibiotic treatment. Cabergoline-treated cows significantly reduced the risk of new IMI by major pathogens across the dry period and postcalving by 21% when compared with placebo cows (20.5 vs. 26.0%, respectively). However, when milk leakage was added to the model, the significance of cabergoline was reduced. We interpreted this to show that milk leakage is an intervening variable between treatment with cabergoline and lower risk of new IMI. The antibiotic treatment significantly decreased the odds of new IMI compared with both cabergoline and placebo. However, because several countries are currently disallowing the preventive use of antibiotics at dry-off in noninfected quarters, the dry-off facilitator cabergoline may therefore be of particular value to reduce the risk of new IMI across the dry period.

Review: Proteomic approaches to control lactational parameters in dairy cows.

The use of a proteomic approach to investigate changes in the milk proteome is growing and has parralleled the increasing technological developments in proteomics moving from early investigation using a gel-based two-dimensional separation approach to more quantitative method of current focus applying chromatography and mass spectrometry. Proteomic approaches to investigate lactational performance have made substantial findings especially in the alterations in lactation during mastitis. An experimental model of Streptococcus uberis infection of the mammary gland has been used as a means to determine change not only in the milk proteome, but also in the peptidome and in the metabolome caused by the infection. Examination of the peptidome, that is the peptides of less than 25 kDa in molecular weight, demonstrated an increase in small peptides most of which were casein degradation products but also included small bioactive peptides such as mammary-associated serum amyloid A3 (MSAA3). The peptidome has also been shown to differ depending on the causative bacteria of naturally occuring mastitis. The use of a non-gel-based relative quantitative proteomic methodology has revealed major changes in the protein component of milk in mastitis. The S. uberis infection lead to increases in the concentrations of proteins such as cathelicidins, haptoglobin, MSAA3 and decreases milk content of proteins such as xanthine oxidase, butyrophilin and ß-1,4-galactosyltransferase. Analysis of all protein change data identified the acute phase, coagulation and complement pathways as well as proteins related to bile acid metabolism as being most modified. Examination of the small molecular weight organic molecules of milk using a metabolomic approach identified an increase in the content in milk during mastitis of bile acids such as taurochenodeoxycholic acid. Notable changes were also found in metabolites responding to infection of the mammary gland. Carbohydrate and nucleic acid metabolites were reduced, whereas lipid and nitrogen containing metabolites were increased. The latter included increases in amino acids along with di and tri peptides, likely to be the result of casein degradation. The use of proteomics and other omic technology is in its infancy in investigation of lactational parameters, but can already provide additional insight into the changes involved in disease and will have further value in physiological and nutritional investigation of lactation.

Effect of intramammary infection with non-aureus staphylococci in early lactation in dairy heifers on quarter somatic cell count and quarter milk yield during the first 4 months of lactation.

A longitudinal study was conducted to assess to what extent intramammary infection (IMI) with non-aureus staphylococci (NAS) within the first 4 d after calving in dairy heifers affects quarter milk yield (qMY) and quarter milk somatic cell count (qSCC) during the first 4 mo of lactation. In total, 324 quarters from 82 Holstein Friesian heifers from 3 commercial dairy herds equipped with an automatic milking system were included and followed from calving up to 4 mo in lactation. The automatic milking system allowed us to precisely determine the daily qMY. A milk sample from each quarter was collected in early lactation (between 1 and 4 d in milk) for bacteriological culturing and measurement of the qSCC. Subsequently, milk samples were taken on a biweekly basis for measurement of the qSCC. The milk prolactin level in early lactation was measured, and the relation with NAS IMI was determined. Overall, NAS IMI in early lactation caused only a slight but significant increase in qSCC compared with milk from noninfected quarters during the first 4 mo in lactation, whereas no significant difference in daily qMY was present between NAS-infected and noninfected quarters. The milk prolactin level in early lactation did not differ between NAS-infected and noninfected quarters either. Our data suggest that IMI with NAS (as a group) present shortly after calving do not have an adverse effect on later production. The milk prolactin concentrations were not dissimilar between NAS-infected and noninfected quarters and thus cannot explain why NAS-infected quarters do not produce less than noninfected quarters.

Randomized, controlled, superiority study of extended duration of therapy with an intramammary antibiotic for treatment of clinical mastitis.

The objectives of this study were to evaluate the efficacy of therapy of clinical mastitis following intramammary infusion of 2 durations of intramammary antibiotic therapy and to analyze clinical and microbiology data at enrollment or within 4 d of commencement of treatment as potential predictors of subsequent clinical and bacteriological cure. A total of 304 glands were randomly assigned to intramammary treatment with a combination of amoxicillin, clavulanic acid, and prednisolone either 3 times at 12-h intervals (3×12, n = 156) or 5 times at 12-h intervals (5×12 hourly, n = 148). At enrollment (d 0), assessments were made of the cow's demeanor, rectal temperature, teat-end hyperkeratosis, presence of heat or swelling of the gland, presence of clots in the milk, and California Mastitis Test (CMT) score. Cows were revisited on d 4, 14, and 21 and assessed for rectal temperature, the presence of clots in milk, heat or swelling of the gland, and CMT score. Milk samples were collected from enrolled glands for bacteriology (d 0, 4, 14, and 21) and for somatic cell count (d 14 and 21). Data were analyzed using generalized linear models. Treatment group was the key independent variable, but herd was included in the models. Other potentially confounding variables were included where associated at the bivariate level. A second series of analyses were undertaken to predict likelihood of clinical and bacteriological cure using the clinical signs cow age and days in milk, as well as bacteriological results at d 0 and 4. Although we found a higher clinical failure rate in the 3×12 than 5×12 hourly treatment group (28.2 ± 5.0 vs. 13.4 ± 3.6%, respectively), we noted no difference in bacteriological cure percentage (73.3 ± 7.8 vs. 72.0 ± 7.4%, respectively). The presence of heat or swelling at d 4, days in milk at initiation of treatment, age of the cow, and change in CMT score between d 0 and 4 were predictive of clinical and bacteriological cure. We concluded that increasing the duration of treatment resulted in significantly fewer clinical failures, but had no effect on cure proportion, somatic cell count, or new infection rate. Knowledge of cow age and days in milk and clinical signs were predictive of clinical and bacteriological cure proportion and may provide guidance for producers, under veterinary direction, as to when to extend or recommence therapy.