Mieke Stevens

Differential gene expression of the toll-like receptor-4 cascade and neutrophil function in early- and mid-lactating dairy cows

During early lactation, neutrophils display several reduced immune functions. Particularly, a delayed recruitment of neutrophils into the infected udder seems to be one of the underlying events involved in the severity of postpartum Escherichia coli intramammary infections. The purpose of this study was to analyze the effect of in vitro chemotaxis and diapedesis on the expression of toll-like receptor-4 (TLR4)-related genes in bovine blood neutrophils isolated from 10 early-lactating (EL) and 10 mid-lactating (ML) cows. Functional characterization of the neutrophil population was performed by measuring phagocytosis and production of reactive oxygen species (chemiluminescence). Messenger RNA was extracted from neutrophils, and the expression of TLR4 and associated genes in EL and ML cows was analyzed by reverse-transcription quantitative PCR. To study the effect of chemotaxis and diapedesis on the expression of genes of the TLR4 cascade, neutrophils were stimulated to (trans)migrate in response to C5a using in vitro models. Our salient findings were that both neutrophil migration in vitro and lactation stage induced significant changes in the expression of several genes of the TLR4 signaling cascade. Before migration, expression of TRAF6, ATF3, RELA, IL8, and C5aR were lower in EL than in ML cows. Diapedesis and chemotaxis induced an increase in expression of TLR4, ATF3, and IL8 in both EL and ML cows. Diapedesis resulted in a downregulation of Syk, a TLR4-associated gene, in ML cows. This study shows that the perturbations in neutrophil functions during EL are accompanied by modulation of TLR4 pathway genes. These data can contribute to the understanding of the mechanisms explaining the relationship between stage of lactation and risk of severe E. coli mastitis.

Variation of inflammatory dynamics and mediators in primiparous cows after intramammary challenge with Escherichia coli.

The objective of the current study was to investigate (i) the outcome of experimentally induced Escherichia coli mastitis in primiparous cows during early lactation in relation with production of eicosanoids and inflammatory indicators, and (ii) the validity of thermography to evaluate temperature changes on udder skin surface after experimentally induced E. coli mastitis. Nine primiparous Holstein Friesian cows were inoculated 24 ± 6 days (d) after parturition in both left quarters with E. coli P4 serotype O32:H37. Blood and milk samples were collected before and after challenge with E. coli. The infrared images were taken from the caudal view of the udder following challenge with E. coli. No relationship was detected between severity of mastitis and changes of thromboxane B2 (TXB2), leukotriene B4 (LTB4) and lipoxin A4 (LXA4). However, prostaglandin E2 (PGE2) was related to systemic disease severity during E. coli mastitis. Moreover, reduced somatic cell count (SCC), fewer circulating basophils, increased concentration of tumor necrosis factor-α (TNF-α) and higher milk sodium and lower milk potassium concentrations were related to systemic disease severity. The thermal camera was capable of detecting 2-3°C temperature changes on udder skin surface of cows inoculated with E. coli. Peak of udder skin temperature occurred after peak of rectal temperature and appearance of local signs of induced E. coli mastitis. Although infrared thermography was a successful method for detecting the changes in udder skin surface temperature following intramammary challenge with E. coli, it did not show to be a promising tool for early detection of mastitis.

An integrated view on how the management of the dry period length of lactating cows could affect mammary biology and defence

The dry period is necessary to facilitate cell turnover in the bovine mammary gland and to optimize milk production in the next lactation. An 8-week dry period has long been the golden standard of management for dairy cows. Genetic improvements and new management technologies have led to higher milk production and a need for re-evaluation of the dry period length. Over the last decade, dry period length has been proposed to be shortened or eliminated mainly from an economic point of view. However, the influence of modified dry period length on the immune defence of the bovine mammary gland and the occurrence of new intramammary infections has not yet been appreciated. The objective of this review is to discuss the bovine mammary gland biology, defence and systemic health when the dry period length is modified. Shortening or eliminating the dry period may minimize or remove the impact of milk accumulation at dry off, thereby lessening the immunodeficiency of the dam that is characteristic of this period. Composition of mammary secretions may change and the extent of tissue remodelling may be reduced when the dry period is reduced or eliminated. Additionally, impact of the dry period length on energy and nutritional status, and on hormonal and local regulatory factors, lead us to hypothesize that changing the dry period length might also affect the response to intramammary infection. It is concluded that there is a need to integrate mammary gland biology and defence mechanisms in studies dealing with modified dry period lengths.

Anaphylatoxin C5a-induced toll-like receptor 4 signaling in bovine neutrophils.

It is well known that signaling in neutrophils through both the complement component 5a (C5a) and C5a receptor (C5aR) and the toll-like receptor 4 (TLR4) pathways plays an essential role in innate defense. Neutrophil dysfunction, as seen during sepsis in severe mastitis during the periparturient period, is correlated with elevated concentrations of anaphylatoxin C5a. The aim of the current study was to elucidate the effect of C5a on TLR4 signaling in bovine neutrophils. Neutrophils were incubated with a high (but physiological) dose of purified C5a, and mRNA was extracted from neutrophils at different time points postincubation (PI). The incubation with C5a resulted in a biphasic C5aR expression profile, a phenomenon that might be explained by internalization (at 10 min PI) with subsequent reconstitution (starting at 40 min PI) of this receptor. The expression of TLR4, as well as its coreceptor, CD14, showed a similar biphasic change as observed with C5aR. In addition, changes in the mRNA expression levels of several genes belonging to the TLR4 pathway, such as TICAM-1, IKKα, and MAP3K7 were noted. The maximal expression of TLR4, CD14, and C5aR mRNA at 80 min PI was accompanied by a peak in IL8 mRNA, indicating that C5a is able to induce IL-8 production in neutrophils in vitro without the need of a costimulatory factor such as lipopolysaccharide. Moreover, a relatively constant expression of RELA was accompanied by increased expression of ATF3, an endogenous inhibitor of nuclear factor-κB mediated transcription, implying that C5a regulates TLR4 signaling and IL-8 synthesis independently. A significant time-dependent correlation was found between C5aR and TLR4, with the majority of the selected TLR4-dependent genes showing a significant correlation with C5aR at 80 min PI, when C5aR and TLR4 mRNA expression reached its maximum, suggesting crosstalk between both receptors. Taken together, this study showed that C5a is able to (1) alter the expression of genes belonging to the TLR4 pathway and (2) induce IL8 gene expression in bovine neutrophils. In addition, indications for cross-talk between complement activation and TLR4 signaling were found in the present study.

Differential microRNA expression analysis in blastocysts by whole mount in situ hybridization and reverse transcription quantitative polymerase chain reaction on laser capture microdissection samples

There is cumulating evidence that microRNAs (miRNAs) are important regulators of pluripotency and differentiation and, hence, of early lineage segregation in embryo development. To unravel the function of specific miRNAs, it is important not only to analyze miRNA expression in the entire blastocyst but also to determine the site and level of expression in the inner cell mass (ICM) versus trophectoderm (TE). A new strategy has been developed for miRNA expression analysis in ICM and TE using two complementary techniques. By whole mount in situ hybridization (WISH), it was visualized that bta-miR-155 is mainly expressed in the ICM. However, WISH does not provide quantitative data on expression differences between the two cell types. By reverse transcription quantitative polymerase chain reaction (RT-qPCR) on ICM and TE isolates taken from single blastocysts with laser capture microdissection (LCM), it was quantified that bta-miR-155 was 50-fold higher expressed in ICM than in TE. The possibility to quantify both miRNAs and messenger RNAs (mRNAs) in LCM samples offers the opportunity to analyze the expression of both miRNAs and potential targets in one sample. This article shows that a combination of WISH with LCM and subsequent RT-qPCR is a robust strategy to qualitatively and quantitatively analyze differential miRNA expression in discrete cell types of a single blastocyst.

Compromised neutrophil function and bovine E. coli mastitis: is C5a the missing link?

During early lactation, dairy cow are prone to developing severe mastitis in responses to intramammary Escherichia coli infections. These severe inflammatory responses have been correlated with reduced neutrophil function during the periparturient period. However, the causative mechanism of neutrophil dysfunction has not been elucidated. Studies in murine sepsis models have shown that during sepsis neutrophils are functionally paralysed due to the presence of high concentrations of complement factor 5a (C5a). In this review, we hypothesize that C5a as a critical early mediator in the development of severe E. coli mastitis. Furthermore, preliminary data suggest that crosstalk between C5a and TLR4 signalling in neutrophils may provide a positive feedback mechanism that may be involved in the pathogenesis of a severe mastitis response. Finally, we focus on the therapeutic potential of disrupting the C5a signalling pathway as an important strategy for treatment of severe E. coli mastitis in dairy cattle.

Antimicrobial consumption on dairy herds and its association with antimicrobial inhibition zone diameters of non-aureus staphylococci and Staphylococcus aureus isolated from subclinical mastitis

The main objectives of this study were to quantify the consumption of antimicrobials on a convenience sample of dairy herds and to determine the association between herd-level antimicrobial consumption and inhibition zone diameters (IZD) of non-aureus staphylococci and Staphylococcus aureus isolates from subclinical mastitis cases. Also, the association between the IZD of non-aureus staphylococci and Staph. aureus isolates within a herd was studied. Antimicrobial consumption data on 56 Flemish dairy farms were obtained between 2013 and 2014 by so-called garbage can audits and expressed as antimicrobial treatment incidence (ATI), with the unit of ATI being the number of defined daily doses animal (DDDA) used per 1,000 cow-days. The average total ATI in adult dairy cattle for all active substances was 18.73 DDDA per 1,000 cow-days and ranged from 6.28 to 42.13 DDDA between herds. The ATI of critically important (for human health) antimicrobials was 6.91 DDDA per 1,000 cow-days; that is, 37% of total antimicrobial consumption. The average ATI for intramammary therapy of (sub)clinical mastitis, intramammary dry-cow therapy, and systemically administered therapy was 5.20, 6.70, and 6.73 DDDA, respectively. The IZD of 239 non-aureus staphylococci and 88 Staph. aureus isolates originating from milk samples from cows with subclinical mastitis collected on selected dairy herds were determined using Kirby-Bauer disk diffusion and ranged between 6 and 42 mm. Because only a limited number of clinical breakpoints (Clinical and Laboratory Standards Institute) and epidemiological cut-off values (European Committee on Antimicrobial Susceptibility Testing) are available for mastitis-causing bacteria in bovine, IZD were used as a proxy for antimicrobial resistance. Inhibition zone diameters of non-aureus staphylococci for cefquinome, a critically important β-lactam antibiotic, were negatively associated with the ATI of critically important β-lactam for systemically administered therapy and positively with the ATI for intramammary therapy of (sub)clinical mastitis of critically important β-lactam antimicrobials. Only for neomycin was a positive association between the IZD of non-aureus staphylococci and Staph. aureus isolates within the same herd observed.

REVIEW ARTICLE: An integrated view on how the management of the dry period length of lactating cows could affect mammary biology and defence

The dry period is necessary to facilitate cell turnover in the bovine mammary gland and to optimize milk production in the next lactation. An 8-week dry period has long been the golden standard of management for dairy cows. Genetic improvements and new management technologies have led to higher milk production and a need for re-evaluation of the dry period length. Over the last decade, dry period length has been proposed to be shortened or eliminated mainly from an economic point of view. However, the influence of modified dry period length on the immune defence of the bovine mammary gland and the occurrence of new intramammary infections has not yet been appreciated. The objective of this review is to discuss the bovine mammary gland biology, defence and systemic health when the dry period length is modified. Shortening or eliminating the dry period may minimize or remove the impact of milk accumulation at dry off, thereby lessening the immunodeficiency of the dam that is characteristic of this period. Composition of mammary secretions may change and the extent of tissue remodelling may be reduced when the dry period is reduced or eliminated. Additionally, impact of the dry period length on energy and nutritional status, and on hormonal and local regulatory factors, lead us to hypothesize that changing the dry period length might also affect the response to intramammary infection. It is concluded that there is a need to integrate mammary gland biology and defence mechanisms in studies dealing with modified dry period lengths.

REVIEW ARTICLE: An integrated view on how the management of the dry period length of lactating cows could affect mammary biology and defence: Dry period length and bovine mammary defence

The dry period is necessary to facilitate cell turnover in the bovine mammary gland and to optimize milk production in the next lactation. An 8-week dry period has long been the golden standard of management for dairy cows. Genetic improvements and new management technologies have led to higher milk production and a need for re-evaluation of the dry period length. Over the last decade, dry period length has been proposed to be shortened or eliminated mainly from an economic point of view. However, the influence of modified dry period length on the immune defence of the bovine mammary gland and the occurrence of new intramammary infections has not yet been appreciated. The objective of this review is to discuss the bovine mammary gland biology, defence and systemic health when the dry period length is modified. Shortening or eliminating the dry period may minimize or remove the impact of milk accumulation at dry off, thereby lessening the immunodeficiency of the dam that is characteristic of this period. Composition of mammary secretions may change and the extent of tissue remodelling may be reduced when the dry period is reduced or eliminated. Additionally, impact of the dry period length on energy and nutritional status, and on hormonal and local regulatory factors, lead us to hypothesize that changing the dry period length might also affect the response to intramammary infection. It is concluded that there is a need to integrate mammary gland biology and defence mechanisms in studies dealing with modified dry period lengths.