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.

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.

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.

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Peroxisomes are organelles that are essential for normal development in men and mice. In order to explore whether zebrafish could be used as a model system to study the role of peroxisomes, we examined their distribution pattern in developing and adult zebrafish and we tested different approaches to eliminate them during the first days after fertilization. In 4-day-old embryos, catalase-containing peroxisomes were obvious in the liver, the pronephric duct and the wall of the yolk sac, but transcripts for peroxisomal matrix and membrane proteins were also detected in the head region from 24 h post-fertilization. In adult zebrafish, catalase-containing peroxisomes remained prominent in the hepatocytes, the renal proximal tubules and the intestinal epithelium. Several peroxins, essential proteins for the biogenesis of peroxisomes, were targeted using knockdown approaches. Two morpholinos, blocking, respectively, splice sites in pex3 and pex13, only induced a short in frame deletion or insertion in the transcripts and did not result in the elimination of peroxisomes after injection into one-cell embryos. A morpholino blocking translation of pex13 was able to reduce the number of peroxisomes to variable extents. Finally, overexpression of a potential dominant negative fragment of Pex3p did not result in deletion of peroxisomes from developing zebrafish. We conclude that in zebrafish (1) peroxisomes, as visualized by DAB cytochemistry for catalase activity, are most conspicuous in the liver and renal tubular epithelium; this pattern is reminiscent of peroxisome occurrence in mammalian organs, (2) our approaches to eliminate these organelles during development by targeting peroxins were not successful.