Integrity of prokaryotic mRNA isolated from complex samples for in vivo bacterial transcriptome analysis.

Even though several in vitro studies have focused on bacterial biology, the extent of such knowledge is not complete when considering an actual infection. As culture-independent microbiology methods such as high-throughput sequencing became available, important aspects of host-bacterium interactions will be elucidated. Based on microbiological relevance, we considered Bacteroides fragilis in a murine experimental infection as a model system to evaluate the in vivo bacterial transcriptome in host exudates. A disproportionate number of reads belonging to the host genome were retrieved in the first round of pyrosequencing, even after depletion of ribosomal RNA; the average number of reads related to the eukaryotic genome was 71.924-67.7%, whereas prokaryotic reads represented 34.338-32.3% in host exudates. Thus, different treatments were used to improve the prokaryotic RNA yield: i) centrifugation; ii) ultrasonic treatment; and iii) ultrasonic treatment followed by centrifugation. The latter treatment was found to be the most efficient in generating bacterial yields, as it resulted in a higher number of Bacteroides cells. However, the RNA extracted after this treatment was not of sufficient quality to be used in cDNA synthesis. Our results suggest that the methodology routinely used for RNA extraction in transcriptional analysis is not appropriate for in vivo studies in complex samples. Furthermore, the most efficient treatment for generating good bacterial cell yields was not suitable to retrieve high-quality RNA. Therefore, as an alternative methodological approach to enable in vivo studies on host-bacterium interactions, we advise increasing the sequencing depth despite the high costs.

Depressed DHEA and increased sickness response behaviors in lame dairy cows with inflammatory foot lesions.

Lameness is a multifactorial condition influenced by the environment, genetics, management and nutrition. Detection of lameness is subjective and currently limited to visual locomotion observations which lack reliability and sensitivity. The objective of this study was to search for potential biomarkers of inflammatory foot lesions that underlie most cases of lameness in dairy cows, with a focus on the sickness response and relevant endocrine, immune and behavioral changes. Serum and peripheral blood mononuclear cells (PBMC) were collected from eight sound and eight lame high-producing Holstein cows. Immune cell activation was investigated in PBMCs using a candidate gene approach in which the expression of pro-opiomelanocortin, interleukin-1beta, l-selectin, matrix metalloproteinase-9 and glucocorticoid receptor-alpha was measured via quantitative real time-RT-PCR. Endocrine changes were investigated by monitoring serum concentrations of cortisol and dehydroepiandrosterone (DHEA). Additionally, systematic behavioral observations were carried out to characterize a behavioral profile associated with a sickness response typical of this condition. Lame cows showed significantly lower eating (P=0.01) and ruminating (P=0.01) behaviors and higher incidence of self-grooming (P=0.04) compared to sound cows. Lame cows also showed a 23% decrease in serum DHEA (P=0.01) and 65% higher cortisol:DHEA ratio (P=0.06) compared to sound cows. However, no significant differences were found in candidate gene expression between lame and sound cows. In association with sickness behaviors, serum DHEA concentration and cortisol:DHEA ratio are promising objective indicators of inflammatory foot lesions in dairy cattle and may be useful as diagnostic targets for animals in need of treatment.

Analysis of the bovine neutrophil transcriptome during glucocorticoid treatment.

The objective of this study was to characterize a large portion of the bovine neutrophil transcriptome following treatment with the anti-inflammatory glucocorticoid dexamethasone (Dex). Total RNA was isolated from blood neutrophils of healthy cattle (5 castrated male Holsteins) immediately following cell purification (0 h) or after ex vivo aging for 4 h with or without added Dex. Additional neutrophils were cotreated with a glucocorticoid receptor (GR) antagonist (RU486) and Dex for 4 h. RNA was amplified, dye labeled (Cy3 or Cy5), and hybridized to a series of National Bovine Functional Genomics Consortium (NBFGC) microarrays. LOWESS data normalization followed by mixture model analyses showed that 11.15% of the spotted NBFGC cDNAs (2,036/18,263) were expressed in 4-h (untreated) neutrophils. Subsequent two-step mixed-model analysis detected (P < or = 0.05) 1,109 differentially expressed genes, of which contrast analysis indicated those that were independently responsive to aging (1,064), Dex (502), RU486 + Dex (141), or RU486 (357). In silico analysis revealed that 416 of the differentially expressed genes are unknown, 59 did not cluster well based on known function, and 634 clustered into 20 ontological categories. Independent validation of differential expression was done for 14 of the putatively Dex-responsive genes across these categories. Results showed that Dex induced rapid translocation of GR into the neutrophil nucleus and signaled dramatic alterations in expression of genes that delay apoptosis, enhance bactericidal activity, and promote tissue remodeling without inflammation or fibrosis. Thus these findings revealed hitherto unappreciated plasticity of blood neutrophils and potentially novel anti-inflammatory/wound-healing actions of glucocorticoids.

Macrophage lipid body induction by Chagas disease in vivo: putative intracellular domains for eicosanoid formation during infection.

Lipid bodies (LB), lipid-rich inclusions abundantly present in cells engaged in inflammation, are specialized intracellular domains involved in generating inflammatory mediators, the eicosanoids. Since the acute Trypanosoma cruzi infection triggers a potent inflammatory reaction characterized by a great increase of peripheral blood monocyte (PBM) and macrophage numbers, we investigated the LB occurrence in these cells. The experimental rat infection by T. cruzi (Y strain) induced significant increase of the LB numbers in peritoneal macrophages at day 6 and 12, accompanied by significant enhancement of Prostaglandin E(2) (PGE(2)) production, as measured by EIA. At day 12, ultrastructural analysis of the heart, a target organ of the disease, showed numerous macrophages with LB prominently increased in number (mean of 8.3 per section view, range of 1-25) compared to controls (mean of 2.6 per section view, range of 0-3) and size. PBM from all groups rarely showed LB. Our results demonstrate, for the first time, that T. cruzi infection in rats elicits important LB formation in inflammatory macrophages but not in PBM. The increase in LB numbers during infection positively correlates with increased generation of PGE(2), suggesting that LB may have a role in the heightened eicosanoid production observed during T. cruzi infection.